334
The Molluscicidal Activity of Coumarins from Ethulia conyzoides and of Dicumarol M. M. Kady 1.5 Leon Brimer ', P. Furu . Else Lemmich 1,2, H. Marie Nielsen2, Susanne T. Thiilborg 1,?, Ole Thastrup3, and S. Brogger Christensen2'" l Danish Bilharziasis Laboratory. DK-2920 Charlottenlund. Denmark
Received: September 16, 1991
Abstract The molluscicidal principles of Ethulia conyzoides were identified as ethuliacoumarin A (1) and isoethuliacoumarin A (2). Ethuliacoumarin A possessed an LC90 between 19 and 23.5 ppm depending on the age of the snail against Biomphalaria glabrata, and between
12 and 15 ppm against Ijulinus truncatus. In addition,
ethuliacoumarin A was found to be cercaricidal at 25 ppm and ovicidal. Ethuliacoumarin has the structural requirements considered essential for anticoagulant activity. Consequently the anticoagulant dicumarol (4) was tested and found to be molluscicidal in the range from 2.5 to 10 ppm. In contrast, the coumarin anticoagulant warfarin (3) did not show molluscicidal activity.
Key words Ethulia conyzoides, molluscicidal activity, ovicidal activity, cercaricidal activity, ethuliacoumarin A, isoethuliacoumarin A, dicumarol, warfarin.
Introduction WHO has given plant molluscicides a high priority as tools for the integrated control of the disease schistosomiasis (1—3) because of their low cost (4) and rapid biodegradability (5, 6). At present three natural products are considered as very promising: the endod berries (Phytolacca dodecandra), the pods of Swartzia madagascariensis, and the pods of Tetrapleura tetraptera (7). Since the active principles in all of these products are saponins, the possibility exists that should snails develop resistance
against one of these products they may be resistant towards all three of the molluscicides. Thus, there is still a major need for the development of other molluscicides of plant origin.
During the course of our work on the molluscicidal properties of some medicinal plants, we noticed the reported significant molluscicidal activities of extracts of aerial parts of Ethulia conyzoides L. (Asteraceae) (8). E. conyzoides, growing wild in Egypt, has for centuries been used in folk medicine as an anthelmintic and for abdominal
disorders (9). A major secondary metabolite, ethulia-
coumarin A (1), has been established to possess anthelmintical properties whereas isoethuliacoumarin A (2) was reported to be inactive (10). In the present report, we describe the molluscicidal effects of the plant as caused by ethuliacoumarin A (1) as well as isoethuliacoumarin A
(2). In addition, we want to report the anticoagulant dicumarol (4) as a potent molluscicide.
Materials and Methods Materials Ethulia con yzoides L. was collected on canal banks near Mansoura, Egypt, in the summer 1988. The material was identified in the Department of Botany, Faculty of Agriculture,
Mansoura University. A voucher specimen is kept at the Department of Plant Protection, Mansoura University. Dicumarol and Warfarin® were obtained from DAK. Copenhagen and Sigma, St. Louis. respectively. Laboratory cultures of Biomphalaria glabrata
Say (of Puerto Rican origin) and Bulinus truncatus Audoin (of Egyptian origin) were obtained from laboratory colonies maintained for 12 years at the Danish Bilharziasis Laboratory. The snails were reared in dechlorinated tap water at pH 7.4 at 26 2°C, exposed to artificial illumination for 9 hours daily, and fed with food prepared from ground dried lettuce, fish food flakes. agar, and water. Snail egg masses were collected on polyethylene bags placed in stock aquaria.
The Schistosoma rnansoni cercariae of Puerto Rican origin were recovered from infected B. glabrata for subsequent cercaricidal tests.
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Department of Organic Chemistry, Royal Danish School of Pharmacy. Universitetsparken 2. DK-2 100 Copenhagen 0. Denmark Symbion, Haraldsgade, DK-2100 Copenhagen 0. Denmark Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, BUlowsvej 13. DK-1870 Frederiksberg, Denmark Present address: Pesticide Division, Faculty of Agriculture. Mansoura University. Mansoura. Egypt ' Address for correspondence
Planta Med. 58 (1992) 335
The MolluscicidalActivity of Coumarins from Ethulia conyzoides and of Dicamarol H OH
HO.)CH3
HQcH3 JCH3 H2 O—O 1 Ethutiacoumarin A
HO. CH2
tality of 10 to 15% was seen in DMSO. After an exposure period of 24 hours the snails were washed with clean water and allowed a
24 hour recovery period. Mortality was recorded and the data
were analyzed by the probit method (16). Niclosamide
(iXH32
(Bayluscide) was used as a reference molluscicide (LC50 0.024 ppm and 0.032 ppm against B. glabrata and B. truncates, respectively).
2 Isoethujiacoumarin A
Ovicidal activity Egg masses of an age ofO to 1 day or 5 days were
3 wartarin
4 Dicumarol
Fig. 1 The structures of the molluscicidal coumarins ethuliacoumarin A (1), isoethuliacoumarin A (2), dicumarol (4), and warfarin (3). Notice that the absolute configurations of ethuliacoumarin A and isoethuliacoumarin A are opposite to those previously drawn.
Isolation of molluscicidal metctholites Comminuted air-dried aerial parts (200 g) of El con yzoides were extracted with ethanol at room temperature for 24 hours. The residue obtained after concentration in vacuo (17 g) was partitioned between water and toluene. The aqueous phase was extracted with ethyl acetate and the three phases were concentrated in vacuo. Only the residue from the toluene phase (9g) possessed a molluscicidal activity, which did not decline after decolorizing with activated charcoal. The residue was chromatographed over acid-washed, activated silica gel 60 (Merck.
60—200 mesh) containing 10% of water using hexane-ethyl acetate (9: 1) to which increasing amounts of ethyl acetate were added as an eluent. Three fractions showed strong molluscicidal properties. The major active fraction was concentrated in vacuo to
give 1.33g of colourless crystals, m.p. 59—60°C (hexane), [a) ÷41.9° (c 0.10, CHCI3). The UV, IR, and 'H-NMR spectra matched those described for ethuliacoumarin A (1). m.p. 60—63 °C, [al° ÷28° (c 0.1, CHCI3) (11). The second active fraction was concentrated to give 0.34g of colourless crystals, m.p. 155—157°C (hexane), [a]: —22.7° (c 0.40, CHCI3). The UV, IR, and 'H-NMR
matched those described for isoethuliacoumarin A (2). m.p.
156—157°C (hexane), [aj°: —20.4°(c 1.61, CHCI3)(12). Shortage of material in the third active fraction prevented further studies of these principles.
Molluscicidal activity Molluscicidal activity of the isolated coumarins was assessed against B. glabrata and B. truncatus according to the WHO provisional technique (13) modified according to Duncan and Sturrock (14). Comparative toxicity tests on newly hatched (1—24 hours old), juveniles (3—5mm diameter), and young mature (8—10 mm diameter) snails, were conducted as recommended in the WHO memorandum (15). Ethanol was used as solvent for the stock solutions of ethuliacoumarin A (1) and isoethuliacoumarin A
(2) whereas the stock solutions of warfarin (3) and dicumarol (4) were prepared using dimethyl sulfoxide (DMSO) as solvent. The
resulting concentration of DMSO in all tests was 1% v/v. Five young mature or ten juvenile snails were exposed in 200 ml disposable plastic containers, while 20 newly hatched snails were exposed in 30m1 disposable plastic petri dishes. All test solutions
were prepared with dechlorinated water in twofold serial dilutions. Four replicates were used for each concentration. Controls with the same dilutions of the organic solvents were included. Ethanol in the concentrations used was not toxic whereas a mor-
of exposure the egg masses were washed, placed in clean water, and the number of dead embryos or hatched snails counted using a binocular microscope.
Cercaricidal activity Cercariae of S. mansoni freshly shed from B.
glabrata under strong light were collected in dechlorinated tap
water. The suspensions were diluted to contain 5 to 10 cercariae per ml and the diluted suspensions were added to solutions of ethuliacoumarin A yielding final concentrations of 20. 25. 30. 35, and 40 ppm. Two controls were included, a suspension of cercariae in water and a suspension of cercariae in water containing
40il of ethanol (95%). The cercariae were observed using a binocular microscope until 5 hours after addition to the solution of ethuliacoumarin A and considered dead when totally immobile.
Results Molluscicidal activity
The molluscicidal activity of ethuliacoumarin A against different stages of B. glabrata and B. truncates is given in Table 1. B. glabrata is more tolerant to the molluscicide than is B. truncates. Newly hatched snails are approximately 1.3 or two times more susceptible than young mature snails (8 to 10 mm) of B. glabrata orB. truncatus, respectively. The molluscicidal effects of four
coumarins all fulfilling the structural requirements for
coumarin anticoagulant activity in mammalia are given in
Table 2. It is remarkable that warfarin in contrast to
dicumarol as well as ethuliacoumarin A and isoethuliacoumarin A is inactive. Table 1
Molluscicidal activity of ethuliacou mann A against Biomphalaria glabrata and Bulinus truncatus at different ages. Age
Newly hatched (1—24h) Juveniles (3—5 mm) Young mature
B. truncatus
B. glabarata LC90(ppm)
LC50(ppm)
LC(ppm)
1.9
6 1.8
12 1.8
LC50(ppm) 15
1.8
17
1.8
20.5 2.1
20 0.9
23.5 2.3
19
8.8
1.5
11
1.1
12.5 0.9
15
1.5
(8—10mm) The molluscicidal activity of ethuliacoumarin A was determined as recommended by WHO (13) modified accordingto Ref. (14). A stock solution of the coumarin in ethanol was diluted with dechlorinated tap water in twofold serial dilutions. The maximum concentration of ethanol in the test solution was 1%. Five or ten snails were exposed to the coumarin for 24 hours whereupon the snails were washed and observed for additional 24 hours. Each experiment was performed in quadruplicate. The range gives the 95% confidence limit as determined by the probit method (16).
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exposed to serial dilutions of ethuliacoumarin A in 30m1 petridishes. Masses with 10 to 15 eggs or 20 to 30 eggs from B. truncatus or B. glabrata, respectively, were used. After 24 hours
336 Planta Med. 58(1992)
M. M. Kadyetal.
isoethuliacoumar;n A on B. glabrata.
Coumarin tested
Table 3 The cercaricidal effects of ethuliacoumarin A on S. mansoni. Dependency on time and concentration.
Mortality (%) at a concentration of [mg/mi (ppm)l 25 10 2.5 1.0 0.25 Controla
5
nt.
n.t.
50
5 0
20
12 3 12 3
100
0 95 0
n.t.
U
100
nt.
nt.
n.t.
nt.
0
25b 100b
Wart arm
Dicumarol EthuliacoumarinA lsoethuliacoumarinA
0
The experiments were performed as described in Table 1. except that the stock solutions of warfarin and dicumarol were prepared in DMSO. The experiments were performed using 20 snails at each concentration. a In the cases of warfarin and dicumarol the control was a 1 % aqueous solution of DMSO, in the case of ethuliacoumarin A and isoethuliacoumarin A the control was a 1 % aqueous solution of ethanol. Saturated solution of the coumarin.
Ovicidal activity
The ovicidal data for ethuliacoumarin A
Concentration (ppm)
0.25
Mobility of cercariae at indicated time Time (hours) 0.5 1.0 2.0 3.0 4.0 5.0
NE NE NE NE NE NE ME ME ME ME ME IM ME ME ME ME ME IM
Control 20 25 30 35
NE NE
40
ME ME
NE NE
NE NE
NE NE
ME ME ME
IM
IM
Twenty to 40 cercariae were incubated up till 5 hours with a solution of the coumarin prepared by dilution of an ethanolic stock solution Each experiment was performed in triplicate. NE: no effect: ME: moderate effect: lM: immobile.
days later than those of the control group and that an increasing proportion of the embryos showed deformities. Some of the embryos remained moving inside the egg
capsules for a period of up till 30 days before they B. truncatus are depicted in Figure 2. The eggs of eventually died. against newly laid and older egg masses of B. glabrata and
B. glabrata are somewhat more susceptible than those of B.
truncatus and in both cases 5 day old eggs were more
Cercaricidal activity
tolerant than newly laid eggs. In addition, it was observed
that eggs exposed to the coumarin hatched one to three
tration. At 40 ppm all cercariae were immobile after 1
against eggs from Biomphalaria glabrata
hour, whereas some were still alive after 3 hours at
LJ
Newe
at •0
0
As is evident from Table 3, the period of time from exposure of the cercariae of S. inansoni to ethuliacoumarin A until immobility is dependent on the concen25 ppm. The locomotory tail was detached from a greater number of cercariae at higher concentrations. No cercariae were affected in either of the two controls or at a concentration of ethuliacoumarin A of 20 ppm. Discussion
0,
0 E
Ethuliacoumarin A, isolated from air-dried aerial parts of E. con yzoides in a yield of 0.7%, exhibits significant molluscicidal activity against B. glabrata as well as B. truncatus (Table 1) although the latter is a little more
Ui
susceptible. Different susceptibilities of two snail species to molluscicides have previously been observed for other molagainst eggs from Biomphalaria glabrata
luscicides and snails (13). The most potent saponins in endod are more efficient molluscicides (LC90 approximately
,_ at
00)
3 ppm) but the ovicidal activity (LC90 500 ppm) is much lower than that of ethuliacoumarin A (17, 18). This ability
-
of ethuliacoumarin A to prevent egg hatching is of im-
7C
portance in controlling snail populations and consequently
.
6C
having the potential of reducing schistosomiasis trans-
..
50 •
40
9 9
ill. •.i. 15
12
mission.
.
21
18
24
-.. 27
33
30
36
39
I
42
Concentration in mg/mi (ppm) Fig. 2
In addition to being molluscicidal as well as ovicidal, ethuliacoumarin A is also cercaricidal (Table 3). Within the limits of molluscicidal concentrations all cercariae were immobilized in 4 hours.
Ovicidal activity of ethuliacoumarin A (1). Newly laid (0—1 day old) or old (5 days old) eggs from B. truncatus (10— 15) or from B. glabrata (20—30) were exposed to the coumarin in the given concentrations for 24 hours. After 24 hours the eggs were washed, placed in clean water and observed daily.
Ethuliacoumarin A as well as isoethulia-
coumarin A (Table 2) are 3-substituted 4-hydroxycoumarins in which the side chain has a potential carbonyl function in position 3'. This structural unit has been suggested to be the pharmacophore of some anticoagulants in-
cluding dicumarol and warfarin (19). This observation
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Table 2 Molluscicidal effect of warfarin, dicumarol, ethuliacoumarin A. and
mania Med. 58(7992) 337
The Molluscicidal Activity of Coumarins from Ethulia conyi.oides and of Dicumarol
prompted us to test the molluscicidal activities of the latter
References
two compounds. Dicumarol was found to be potent the solubility in water (Table 2).
In conclusion, the molluscicidal activity of ethuliacoumarin A and isoethuliacoumarin A have led to the discovery that some coumarins fulfilling the structural requirements suggested to be essential for anticoagulant and K-vitamin antagonistic activities are potent molluscicides. This finding opens a broader range of coumarins to be investigated for use as molluscicides than the furanocoumarins (20—22).
In addition to controlling the life cycle of the intermediate hosts. ethuliacoumarin A also interferes with the life cycle of the parasitic trematodes by killing the cercariae. the larval stage that are infective for humans. The fact that the herb E. con yzoides is growing fertile on the banks of canals, which might be infested with snails.
makes this species a potential tool in the control of schistosomiasis. The possible anticoagulant effects ofethuliacournarin A and isoethuliacoumarin A, however, make ecotoxicological studies necessary before extended field applications of the plant extracts.
Acknowledgements M. M. K. is grateful to the Danish International Development Agency (Danida) for a visiting fellowship. S. B. C. is
supported by a grant from the Alfred Benzon Foundation. Mrs. Rita lierk-Hansen and Susanne Kronborg (DBL) are thanked for technical assistance.
Ileyneman. D. (1984) in: Phytolacca dodecandra (Endod), (Lemma. A., Heyneman. D., Silangwa, S. M.. eds.). p. ix, Tycooly mt. PubI. l.td., Dublin.
2 Anonymous (1987) in: F.ndod II (Phytolacca dodecandra), (Makhubu. L., Lemma, A., Heyneman, D., eds.), p. 131, Council of
International Pubi. Aff.. New York. Kloos, H., McCullough. F. S. (1987) in: Plant Molluscicides. (MoLt, K. C.. ed). p.45. John Wiley and Sons Ltd.. New York. l.emma, A., Gull, P., Duncan. J., Mazengia. B.. (1978) Proceedings of the International Conference on Schistosomiasis, Cairo. p.415. Msangi. A. S., Zeller. C.(1965) Proc. East African Acad. 3.52. Lemma. .'\.. Yau, P.1974) Ethiopian Med. J. 13. 115. Kloos. II.. McCullough. F. S. (1987) in: Plant Molluscicides, (Mott. K. F., ed). John Wiley & Sons l.td.. New York, p. 45. Ahmed. F. (I. M.. Bashir, .\. K., Kheir. Y. M. F. (1984) Planta Med. 74. (1962) in: The Medicinal and Watt. J. M., Brevor-Brandwijk. M Poisonous Plants of Southern and Eastern Africa, 2nd. edn., C. S. Livingstone Ltd, l.ondon. p. 228. Mahmoud, Z. F.. Sarg. T. M., Amer. M. E.. Khafagy. S. M. (1983) Pharmazie 38. 486. Bohlmann, F.. Zdero. C. (1977) Phytochemistry 16, 1092. 2 Balbaa, S. I.. Ilalim. A. F., Halaweish. F. 1., Bohlmann, F. (1980) Phytochemistry 19, 1519. Who (1 961) WId. HIth. Org. Techn. Rep. Ser. 214. 30. Duncan, J., Sturrock, R. F. (1987) in Ref(7), p. 257. ° WHO (1965) Bull. WId. HIth. Org. 33. 567. Lichtfield, J. T., Wilcoxon. F. (1949) J. Pharmacol. Exp. Therap. 96. 99. Lemma, A. (1970) Hull. WId. 111th, Org. 42, 597.
0 Lemma, A., Brody. G., Newell, G. W., Parkhurst, H. M., Skinner. W. A. (1972)J. Parasit. 58, 104. ° Chmielewska, I., Cieslak, J. (1958) Tetrahedron 4. 135. 20 Schonberg, A.. Latif. N. (1954) J. Am. Chem. Soc. 76, 6208. 21 Adesina, S. K., Adewunnii, C. 0. (1985) Fitoterapia 56, 289. 22 Adewunmi, C. 0., t)elIe Monache, F. (1989) Fitoterapia 60, 79.
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whereas warfarin showed no activity within the range of
The Molluscicidal Activity of Coumarins from Ethulia conyzoides and of Dicumarol M. M. Kady 1.5 Leon Brimer ', P. Furu . Else Lemmich 1,2, H. Marie Nielsen2, Susanne T. Thiilborg 1,?, Ole Thastrup3, and S. Brogger Christensen2'" l Danish Bilharziasis Laboratory. DK-2920 Charlottenlund. Denmark
Received: September 16, 1991
Abstract The molluscicidal principles of Ethulia conyzoides were identified as ethuliacoumarin A (1) and isoethuliacoumarin A (2). Ethuliacoumarin A possessed an LC90 between 19 and 23.5 ppm depending on the age of the snail against Biomphalaria glabrata, and between
12 and 15 ppm against Ijulinus truncatus. In addition,
ethuliacoumarin A was found to be cercaricidal at 25 ppm and ovicidal. Ethuliacoumarin has the structural requirements considered essential for anticoagulant activity. Consequently the anticoagulant dicumarol (4) was tested and found to be molluscicidal in the range from 2.5 to 10 ppm. In contrast, the coumarin anticoagulant warfarin (3) did not show molluscicidal activity.
Key words Ethulia conyzoides, molluscicidal activity, ovicidal activity, cercaricidal activity, ethuliacoumarin A, isoethuliacoumarin A, dicumarol, warfarin.
Introduction WHO has given plant molluscicides a high priority as tools for the integrated control of the disease schistosomiasis (1—3) because of their low cost (4) and rapid biodegradability (5, 6). At present three natural products are considered as very promising: the endod berries (Phytolacca dodecandra), the pods of Swartzia madagascariensis, and the pods of Tetrapleura tetraptera (7). Since the active principles in all of these products are saponins, the possibility exists that should snails develop resistance
against one of these products they may be resistant towards all three of the molluscicides. Thus, there is still a major need for the development of other molluscicides of plant origin.
During the course of our work on the molluscicidal properties of some medicinal plants, we noticed the reported significant molluscicidal activities of extracts of aerial parts of Ethulia conyzoides L. (Asteraceae) (8). E. conyzoides, growing wild in Egypt, has for centuries been used in folk medicine as an anthelmintic and for abdominal
disorders (9). A major secondary metabolite, ethulia-
coumarin A (1), has been established to possess anthelmintical properties whereas isoethuliacoumarin A (2) was reported to be inactive (10). In the present report, we describe the molluscicidal effects of the plant as caused by ethuliacoumarin A (1) as well as isoethuliacoumarin A
(2). In addition, we want to report the anticoagulant dicumarol (4) as a potent molluscicide.
Materials and Methods Materials Ethulia con yzoides L. was collected on canal banks near Mansoura, Egypt, in the summer 1988. The material was identified in the Department of Botany, Faculty of Agriculture,
Mansoura University. A voucher specimen is kept at the Department of Plant Protection, Mansoura University. Dicumarol and Warfarin® were obtained from DAK. Copenhagen and Sigma, St. Louis. respectively. Laboratory cultures of Biomphalaria glabrata
Say (of Puerto Rican origin) and Bulinus truncatus Audoin (of Egyptian origin) were obtained from laboratory colonies maintained for 12 years at the Danish Bilharziasis Laboratory. The snails were reared in dechlorinated tap water at pH 7.4 at 26 2°C, exposed to artificial illumination for 9 hours daily, and fed with food prepared from ground dried lettuce, fish food flakes. agar, and water. Snail egg masses were collected on polyethylene bags placed in stock aquaria.
The Schistosoma rnansoni cercariae of Puerto Rican origin were recovered from infected B. glabrata for subsequent cercaricidal tests.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Department of Organic Chemistry, Royal Danish School of Pharmacy. Universitetsparken 2. DK-2 100 Copenhagen 0. Denmark Symbion, Haraldsgade, DK-2100 Copenhagen 0. Denmark Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, BUlowsvej 13. DK-1870 Frederiksberg, Denmark Present address: Pesticide Division, Faculty of Agriculture. Mansoura University. Mansoura. Egypt ' Address for correspondence
Planta Med. 58 (1992) 335
The MolluscicidalActivity of Coumarins from Ethulia conyzoides and of Dicamarol H OH
HO.)CH3
HQcH3 JCH3 H2 O—O 1 Ethutiacoumarin A
HO. CH2
tality of 10 to 15% was seen in DMSO. After an exposure period of 24 hours the snails were washed with clean water and allowed a
24 hour recovery period. Mortality was recorded and the data
were analyzed by the probit method (16). Niclosamide
(iXH32
(Bayluscide) was used as a reference molluscicide (LC50 0.024 ppm and 0.032 ppm against B. glabrata and B. truncates, respectively).
2 Isoethujiacoumarin A
Ovicidal activity Egg masses of an age ofO to 1 day or 5 days were
3 wartarin
4 Dicumarol
Fig. 1 The structures of the molluscicidal coumarins ethuliacoumarin A (1), isoethuliacoumarin A (2), dicumarol (4), and warfarin (3). Notice that the absolute configurations of ethuliacoumarin A and isoethuliacoumarin A are opposite to those previously drawn.
Isolation of molluscicidal metctholites Comminuted air-dried aerial parts (200 g) of El con yzoides were extracted with ethanol at room temperature for 24 hours. The residue obtained after concentration in vacuo (17 g) was partitioned between water and toluene. The aqueous phase was extracted with ethyl acetate and the three phases were concentrated in vacuo. Only the residue from the toluene phase (9g) possessed a molluscicidal activity, which did not decline after decolorizing with activated charcoal. The residue was chromatographed over acid-washed, activated silica gel 60 (Merck.
60—200 mesh) containing 10% of water using hexane-ethyl acetate (9: 1) to which increasing amounts of ethyl acetate were added as an eluent. Three fractions showed strong molluscicidal properties. The major active fraction was concentrated in vacuo to
give 1.33g of colourless crystals, m.p. 59—60°C (hexane), [a) ÷41.9° (c 0.10, CHCI3). The UV, IR, and 'H-NMR spectra matched those described for ethuliacoumarin A (1). m.p. 60—63 °C, [al° ÷28° (c 0.1, CHCI3) (11). The second active fraction was concentrated to give 0.34g of colourless crystals, m.p. 155—157°C (hexane), [a]: —22.7° (c 0.40, CHCI3). The UV, IR, and 'H-NMR
matched those described for isoethuliacoumarin A (2). m.p.
156—157°C (hexane), [aj°: —20.4°(c 1.61, CHCI3)(12). Shortage of material in the third active fraction prevented further studies of these principles.
Molluscicidal activity Molluscicidal activity of the isolated coumarins was assessed against B. glabrata and B. truncatus according to the WHO provisional technique (13) modified according to Duncan and Sturrock (14). Comparative toxicity tests on newly hatched (1—24 hours old), juveniles (3—5mm diameter), and young mature (8—10 mm diameter) snails, were conducted as recommended in the WHO memorandum (15). Ethanol was used as solvent for the stock solutions of ethuliacoumarin A (1) and isoethuliacoumarin A
(2) whereas the stock solutions of warfarin (3) and dicumarol (4) were prepared using dimethyl sulfoxide (DMSO) as solvent. The
resulting concentration of DMSO in all tests was 1% v/v. Five young mature or ten juvenile snails were exposed in 200 ml disposable plastic containers, while 20 newly hatched snails were exposed in 30m1 disposable plastic petri dishes. All test solutions
were prepared with dechlorinated water in twofold serial dilutions. Four replicates were used for each concentration. Controls with the same dilutions of the organic solvents were included. Ethanol in the concentrations used was not toxic whereas a mor-
of exposure the egg masses were washed, placed in clean water, and the number of dead embryos or hatched snails counted using a binocular microscope.
Cercaricidal activity Cercariae of S. mansoni freshly shed from B.
glabrata under strong light were collected in dechlorinated tap
water. The suspensions were diluted to contain 5 to 10 cercariae per ml and the diluted suspensions were added to solutions of ethuliacoumarin A yielding final concentrations of 20. 25. 30. 35, and 40 ppm. Two controls were included, a suspension of cercariae in water and a suspension of cercariae in water containing
40il of ethanol (95%). The cercariae were observed using a binocular microscope until 5 hours after addition to the solution of ethuliacoumarin A and considered dead when totally immobile.
Results Molluscicidal activity
The molluscicidal activity of ethuliacoumarin A against different stages of B. glabrata and B. truncates is given in Table 1. B. glabrata is more tolerant to the molluscicide than is B. truncates. Newly hatched snails are approximately 1.3 or two times more susceptible than young mature snails (8 to 10 mm) of B. glabrata orB. truncatus, respectively. The molluscicidal effects of four
coumarins all fulfilling the structural requirements for
coumarin anticoagulant activity in mammalia are given in
Table 2. It is remarkable that warfarin in contrast to
dicumarol as well as ethuliacoumarin A and isoethuliacoumarin A is inactive. Table 1
Molluscicidal activity of ethuliacou mann A against Biomphalaria glabrata and Bulinus truncatus at different ages. Age
Newly hatched (1—24h) Juveniles (3—5 mm) Young mature
B. truncatus
B. glabarata LC90(ppm)
LC50(ppm)
LC(ppm)
1.9
6 1.8
12 1.8
LC50(ppm) 15
1.8
17
1.8
20.5 2.1
20 0.9
23.5 2.3
19
8.8
1.5
11
1.1
12.5 0.9
15
1.5
(8—10mm) The molluscicidal activity of ethuliacoumarin A was determined as recommended by WHO (13) modified accordingto Ref. (14). A stock solution of the coumarin in ethanol was diluted with dechlorinated tap water in twofold serial dilutions. The maximum concentration of ethanol in the test solution was 1%. Five or ten snails were exposed to the coumarin for 24 hours whereupon the snails were washed and observed for additional 24 hours. Each experiment was performed in quadruplicate. The range gives the 95% confidence limit as determined by the probit method (16).
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exposed to serial dilutions of ethuliacoumarin A in 30m1 petridishes. Masses with 10 to 15 eggs or 20 to 30 eggs from B. truncatus or B. glabrata, respectively, were used. After 24 hours
336 Planta Med. 58(1992)
M. M. Kadyetal.
isoethuliacoumar;n A on B. glabrata.
Coumarin tested
Table 3 The cercaricidal effects of ethuliacoumarin A on S. mansoni. Dependency on time and concentration.
Mortality (%) at a concentration of [mg/mi (ppm)l 25 10 2.5 1.0 0.25 Controla
5
nt.
n.t.
50
5 0
20
12 3 12 3
100
0 95 0
n.t.
U
100
nt.
nt.
n.t.
nt.
0
25b 100b
Wart arm
Dicumarol EthuliacoumarinA lsoethuliacoumarinA
0
The experiments were performed as described in Table 1. except that the stock solutions of warfarin and dicumarol were prepared in DMSO. The experiments were performed using 20 snails at each concentration. a In the cases of warfarin and dicumarol the control was a 1 % aqueous solution of DMSO, in the case of ethuliacoumarin A and isoethuliacoumarin A the control was a 1 % aqueous solution of ethanol. Saturated solution of the coumarin.
Ovicidal activity
The ovicidal data for ethuliacoumarin A
Concentration (ppm)
0.25
Mobility of cercariae at indicated time Time (hours) 0.5 1.0 2.0 3.0 4.0 5.0
NE NE NE NE NE NE ME ME ME ME ME IM ME ME ME ME ME IM
Control 20 25 30 35
NE NE
40
ME ME
NE NE
NE NE
NE NE
ME ME ME
IM
IM
Twenty to 40 cercariae were incubated up till 5 hours with a solution of the coumarin prepared by dilution of an ethanolic stock solution Each experiment was performed in triplicate. NE: no effect: ME: moderate effect: lM: immobile.
days later than those of the control group and that an increasing proportion of the embryos showed deformities. Some of the embryos remained moving inside the egg
capsules for a period of up till 30 days before they B. truncatus are depicted in Figure 2. The eggs of eventually died. against newly laid and older egg masses of B. glabrata and
B. glabrata are somewhat more susceptible than those of B.
truncatus and in both cases 5 day old eggs were more
Cercaricidal activity
tolerant than newly laid eggs. In addition, it was observed
that eggs exposed to the coumarin hatched one to three
tration. At 40 ppm all cercariae were immobile after 1
against eggs from Biomphalaria glabrata
hour, whereas some were still alive after 3 hours at
LJ
Newe
at •0
0
As is evident from Table 3, the period of time from exposure of the cercariae of S. inansoni to ethuliacoumarin A until immobility is dependent on the concen25 ppm. The locomotory tail was detached from a greater number of cercariae at higher concentrations. No cercariae were affected in either of the two controls or at a concentration of ethuliacoumarin A of 20 ppm. Discussion
0,
0 E
Ethuliacoumarin A, isolated from air-dried aerial parts of E. con yzoides in a yield of 0.7%, exhibits significant molluscicidal activity against B. glabrata as well as B. truncatus (Table 1) although the latter is a little more
Ui
susceptible. Different susceptibilities of two snail species to molluscicides have previously been observed for other molagainst eggs from Biomphalaria glabrata
luscicides and snails (13). The most potent saponins in endod are more efficient molluscicides (LC90 approximately
,_ at
00)
3 ppm) but the ovicidal activity (LC90 500 ppm) is much lower than that of ethuliacoumarin A (17, 18). This ability
-
of ethuliacoumarin A to prevent egg hatching is of im-
7C
portance in controlling snail populations and consequently
.
6C
having the potential of reducing schistosomiasis trans-
..
50 •
40
9 9
ill. •.i. 15
12
mission.
.
21
18
24
-.. 27
33
30
36
39
I
42
Concentration in mg/mi (ppm) Fig. 2
In addition to being molluscicidal as well as ovicidal, ethuliacoumarin A is also cercaricidal (Table 3). Within the limits of molluscicidal concentrations all cercariae were immobilized in 4 hours.
Ovicidal activity of ethuliacoumarin A (1). Newly laid (0—1 day old) or old (5 days old) eggs from B. truncatus (10— 15) or from B. glabrata (20—30) were exposed to the coumarin in the given concentrations for 24 hours. After 24 hours the eggs were washed, placed in clean water and observed daily.
Ethuliacoumarin A as well as isoethulia-
coumarin A (Table 2) are 3-substituted 4-hydroxycoumarins in which the side chain has a potential carbonyl function in position 3'. This structural unit has been suggested to be the pharmacophore of some anticoagulants in-
cluding dicumarol and warfarin (19). This observation
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Table 2 Molluscicidal effect of warfarin, dicumarol, ethuliacoumarin A. and
mania Med. 58(7992) 337
The Molluscicidal Activity of Coumarins from Ethulia conyi.oides and of Dicumarol
prompted us to test the molluscicidal activities of the latter
References
two compounds. Dicumarol was found to be potent the solubility in water (Table 2).
In conclusion, the molluscicidal activity of ethuliacoumarin A and isoethuliacoumarin A have led to the discovery that some coumarins fulfilling the structural requirements suggested to be essential for anticoagulant and K-vitamin antagonistic activities are potent molluscicides. This finding opens a broader range of coumarins to be investigated for use as molluscicides than the furanocoumarins (20—22).
In addition to controlling the life cycle of the intermediate hosts. ethuliacoumarin A also interferes with the life cycle of the parasitic trematodes by killing the cercariae. the larval stage that are infective for humans. The fact that the herb E. con yzoides is growing fertile on the banks of canals, which might be infested with snails.
makes this species a potential tool in the control of schistosomiasis. The possible anticoagulant effects ofethuliacournarin A and isoethuliacoumarin A, however, make ecotoxicological studies necessary before extended field applications of the plant extracts.
Acknowledgements M. M. K. is grateful to the Danish International Development Agency (Danida) for a visiting fellowship. S. B. C. is
supported by a grant from the Alfred Benzon Foundation. Mrs. Rita lierk-Hansen and Susanne Kronborg (DBL) are thanked for technical assistance.
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whereas warfarin showed no activity within the range of
