Journal of Ethnopharmacology, 29 (1990) 233-237 Elsevier ScientificPublishersIrelandLtd.
233
Short communication
TRYPANOCIDAL EFFECT OF AN AQUEOUS ACALYPHA HISPIDA LEAVES
E.O. OKANLA’,
J.A. OWOYALEb
OF
and J.A. AKINYANJU’
Departments of “Biological Sciences Kwara State (Niger&d (Accepted
EXTRACT
and bChemistry,
University
of Ilorin, P.M.B.
1515, Ilorin_
January 4, 1990)
Introduction There is an urgent need for new drugs to combat early and late stage infections of both Trypanosoma brucei rhodesiense and T. b. gambiense. At the present time, suramin is the drug of choice for early-stage T.b. rhodesiense and pentamidine for early-stage T. b. gambiense. For late-stage infections with central nervous system involvement, melasoprol is used. None of the drugs is ideal (World Health Organization, 19811 since all produce undesirable side effects (Fairlamb et al., 1977; Basson et al., 197’71 and attempts to counteract the toxic effects with dimercaprol also neutralize the therapeutic effect of the drugs (Maegraith, 19761. Plant extracts have been used for the treatment of different ailments since antiquity. The role that cinchona extract played in the chemotherapy of malaria is significant and has led to the production of a variety of synthetic drugs. One avenue of search for drugs against African trypanosomes may therefore be the screening of plant extracts for anti-trypanosome activity. Akinyanju et al. (19861 reported the activity of a crude aqueous extract of the leaves of Acalypha torta against some microorganisms. We now report the activity of A. hispida leaf extracts in laboratory rats against T. b. brucei a morphologically indistinguishable relative of T. b. rhodesiense, and T.b. gambiense (Schmidt and Roberts, 19771. Materials and methods Plant material Leaves of Acalypha hispida Brum. (Euphorbiaceael were collected at the Parks and Gardens Unit of the University of Ilorin during the months of Published and Printed in Ireland
234
January and February. These were authenticated by Dr. Zac Gbile of the Forest Research Institute of Nigeria, Ibadan, Nigeria. Herbarium specimens were deposited at the Botany Unit of the Department of Biological Sciences, University of Ilorin. Animals
Male and female albino rats (Wistar stock) weighing 84-100 g were purchased from the Post Graduate Research Centre, University Teaching Hospital, Ibadan. The animals were maintained in plastic cages and fed a commercial feed (Ladokun & Sons Livestock Feeds Ltd., Ibadan, Nigeria1 with drinking water freely allowed. Parasites
A monomorphic strain of Trypanosoma brucei was used in this experiment. This strain, Nsukka/62/NITR/8/18 Clone B (81, was obtained from the Post Graduate Research Centre, University of Ibadan. Preparation Acalypha
of extracts hispida leaf extracts
were prepared by boiling 50 g of leaves dried at room temperature in 200 ml of distilled water for 4 h. The aqueous extract was then filtered through a Whatman No. 1 filter paper. The reddish-brown filtrate obtained was concentrated by heating on a boiling water bath and the concentrated material was then air dried to a powdery state. The extract yield was 12.4% w/w in terms of the dry starting material.
Screening
for anti-trypanosome
activity
A total of 48 animals in 16 groups of 3 animals each were used in this experiment. All animals were infected with T. brucei by intraperitoneal injection of 1 x lo3 organisms in 0.1 ml of 0.85% saline solution containing 1% normal rat serum. The animals were sampled every day thereafter by microscopic examination of a drop of tail vein blood. Treatment was administered on the first day that the animals became positive. Animals in groups 1, 2 and 3 respectively received a single 50-mg dose of the extract orally, intramuscularly or intravenously. Groups 4, 5 and 6 received two 50-mg daily doses by the same routes while groups 7, 8 and 9 were given three 50mg daily doses by the same routes. Three groups (10, 11 and 121 were given a single 150-mg dose each, while physiological saline alone was administered to groups 13, 14 and 15. Animals in group 16 were infected but not treated. Following treatment, all animals were examined for parasitemia on a daily basis as described above. Results
The results obtained are presented in Table 1. Acalypha hispida leaf extracts showed trypanocicadal activities with all regimens and routes of
235
TABLE 1 EFFECTS
OF
TRYPANOSO~A
Treatment and dosage (days of treatment)
A
CRUDE
EXTRACT OF ACALYPHA HISPIDA INFECTION IN ALBINO RATS
LEAVES
ON
BRUCEI BRUCEI
Group
Route
Mean days parasitefree
‘I-Day survival
Mean days to death
Extract, 50 mg (x 1)
1 2 3
p.0. i.m. iv.
0.3 2.0 5.0
313 313 313
7.0 7.0 9.7
Extract, 50 mg (x 2)
4 5 6
p.0. i.m. i.v.
0.6 2.0 1.0
313 013 313
7.0 6.0 7.0
Extract, 50 mg (x 3)
7 8 9
p.0. i.m. i.v.
1.0 3.0 3.0
313 313 313
7.0 8.3 8.0
Extract, 150 mg (x 1)
10 11 12
p.0. i.m. iv.
0.P 0.0” 0.6
O/3 O/3 013
3.0 3.0 5.0
Normal saline, 0.1 ml lx 3)
13 14 15
p.0. i.m. i.v.
0.0 0.0 0.0
013 013 O/3
5.0 5.0 5.0
Untreated
16
-
0.0
o/3
5.0
“Parasites were cieared from blood but the animals died on the same day.
administration. The least activity was observed in the cases of animals treated orally irrespective of the number of doses given. Intramuscular and intravenous injections of the extract appeared to be the most effective. In the animals treated intravenously, a single injection of 50 mg of the extract appears to be the best since maximum activity was observed with minimum toxicity as evidenced by the long average number of days during which the animals were parasite-free after treatment. Infected animals receiving a single dose of 150 mg died much earlier than those receiving single or multiple doses of 50 mg. Discussion
This study has demonstrated the activity of an aqueous extract of A. hisbrucei in rats. From the results presented, many questions need to be addressed. While it is obvious that the extract
pdda leaves against T. brucei
236
kills the parasites (a dersirable effect) and can kill the host (a most undesirable situation), any judgement about the toxicity of the active ingredient in this extract on the host should be deferred until a purified fraction or a pure compound can be tested. The authors are optimistic that the toxicity will be reduced but it is unlikely that it can be eliminated completely. Currently used drugs in the treatment of African trypanosomiasis are very toxic to the host. Clearly, just the demonstration of activity suggests optimism even when toxicity is apparent (Jaffe et al., 19’701. Another concern is the fact that, in all cases, treated animals in which parasite counts had dropped to O/ml soon became parasitemic and died. Post-chemotherapy recurrence of parasitemia in animals infected with African trypanosomes is a well-known phenomenon (Basson et al., 1977; Evans and Brightman, 19801. Suramin, a very important drug in the treatment of African trypanosomiasis, is not very effective in late infections. This is thought to happen because trypanosomes may have infested the brain and thereby escape the action of the drug. Abolarin et al. (19721 have shown that after infected animals have been treated with salicylhydroxamic acid (SHAM) + glycerol, parasites are cleared from the blood, but remain in the choroid plexus of the brain from where they later disseminate. Clarkson and Brohn (19761 reported that animals infected with T.b. rhodesiense and treated with SHAM + glycerol became aparasitemic in about 24 h. Parasite dissemination started after 6 days and death eventually occurred. The maximum number of days during which the treated animals in our experiment were free of parasites was 5 days. The authors believe that the results of this experiment are encouraging and work is in progress to test purified fractions of this extract with the hope that some fractions will be less toxic and more effective. Acknowledgements
This study was supported by University of Ilorin Senate Research Grant No. 8.184.46. We acknowledge the contributions of Miss. M.A. Adewoyin, Miss M.I. Jospeh and Miss A.O. Arukhe, who at different times did preliminary work on this study. References Abolarin, M.O., Evans, D.A., Tovey, sickness. Trypanosome developing
D.G. and Ormerod, in choroid plexus
W.E. (19721 Cryptic stage of sleepingepithelial cells. British Medical Journal
285, 1380 - 1382. Akinyanju, J.A., Owoyale, J.A. and Okanla, E.O. (1986) Antimicrobial effect of a leaf extract of Acalypha torta In: A. Sofowora (Ed.), The State of Medicinal Plants Research in Nigeria, Proceedings of a Workshop on Medicinal Plants, University of Ife, Nigeria, pp. 247-251. Basson, W., Page, M.L. and Myburgh, D.P. (1977) Human trypanosomiasis in Southern Africa. South African Medical Journal 51 (21, 453-457. Clarkson, A.B. and Brohn, F.H. (1976) Trypanosomiasis: An approach to chemotherapy by the inhibition of carbohydrate catabolism. Science 194, 204 - 205.
237
Evans, D.A. and Brightman, C.A.J. (19801 Pleomorphism and the problem of recrudescent parasitemia following treatment with salicylhydroxamic acid (SHAM) in African trypanosomiasis. Transactions of the Royal Society for Tropical Medicine and Hygiene 74, 601-604. Fairlamb, A.H., Opperdoes, F.R. and Borst, P. (19771 New approach to screening drugs for activity against African trypanosomes. Nature 256, 270 - 271. Jaffe, J.J., McCormack, J.J. and Meymerian, E. (19701 Trypanocidal properties of 5’.O-sulfamayladenosine, a close structural analog of nucleocidin. Experimental Parasitology, 28, 535-543. Maegraith, B.G. (19761 Trypanosomiases. In: B. Maegraith (Ed), Clinical Tropical Diseases, Blackwell Scientific Publications, Oxford, pp. 438 - 463. Schmidt, G.D. and Roberts, L.S. (19771 Order Kinetoplastida: Trypanosomes and their kin. In: G.D. Schmidt and L.S. Roberts (Ed.), Foundations of Parasitology. C.V. Mosby Company, St. Louis, MD, pp. 52-78. World Health Organization (1981) Report of the Second Scientific Working Group on African Trypanosomiases. World Health Organization, Geneva (TDR/TRY-SWG (2)/81.3).
233
Short communication
TRYPANOCIDAL EFFECT OF AN AQUEOUS ACALYPHA HISPIDA LEAVES
E.O. OKANLA’,
J.A. OWOYALEb
OF
and J.A. AKINYANJU’
Departments of “Biological Sciences Kwara State (Niger&d (Accepted
EXTRACT
and bChemistry,
University
of Ilorin, P.M.B.
1515, Ilorin_
January 4, 1990)
Introduction There is an urgent need for new drugs to combat early and late stage infections of both Trypanosoma brucei rhodesiense and T. b. gambiense. At the present time, suramin is the drug of choice for early-stage T.b. rhodesiense and pentamidine for early-stage T. b. gambiense. For late-stage infections with central nervous system involvement, melasoprol is used. None of the drugs is ideal (World Health Organization, 19811 since all produce undesirable side effects (Fairlamb et al., 1977; Basson et al., 197’71 and attempts to counteract the toxic effects with dimercaprol also neutralize the therapeutic effect of the drugs (Maegraith, 19761. Plant extracts have been used for the treatment of different ailments since antiquity. The role that cinchona extract played in the chemotherapy of malaria is significant and has led to the production of a variety of synthetic drugs. One avenue of search for drugs against African trypanosomes may therefore be the screening of plant extracts for anti-trypanosome activity. Akinyanju et al. (19861 reported the activity of a crude aqueous extract of the leaves of Acalypha torta against some microorganisms. We now report the activity of A. hispida leaf extracts in laboratory rats against T. b. brucei a morphologically indistinguishable relative of T. b. rhodesiense, and T.b. gambiense (Schmidt and Roberts, 19771. Materials and methods Plant material Leaves of Acalypha hispida Brum. (Euphorbiaceael were collected at the Parks and Gardens Unit of the University of Ilorin during the months of Published and Printed in Ireland
234
January and February. These were authenticated by Dr. Zac Gbile of the Forest Research Institute of Nigeria, Ibadan, Nigeria. Herbarium specimens were deposited at the Botany Unit of the Department of Biological Sciences, University of Ilorin. Animals
Male and female albino rats (Wistar stock) weighing 84-100 g were purchased from the Post Graduate Research Centre, University Teaching Hospital, Ibadan. The animals were maintained in plastic cages and fed a commercial feed (Ladokun & Sons Livestock Feeds Ltd., Ibadan, Nigeria1 with drinking water freely allowed. Parasites
A monomorphic strain of Trypanosoma brucei was used in this experiment. This strain, Nsukka/62/NITR/8/18 Clone B (81, was obtained from the Post Graduate Research Centre, University of Ibadan. Preparation Acalypha
of extracts hispida leaf extracts
were prepared by boiling 50 g of leaves dried at room temperature in 200 ml of distilled water for 4 h. The aqueous extract was then filtered through a Whatman No. 1 filter paper. The reddish-brown filtrate obtained was concentrated by heating on a boiling water bath and the concentrated material was then air dried to a powdery state. The extract yield was 12.4% w/w in terms of the dry starting material.
Screening
for anti-trypanosome
activity
A total of 48 animals in 16 groups of 3 animals each were used in this experiment. All animals were infected with T. brucei by intraperitoneal injection of 1 x lo3 organisms in 0.1 ml of 0.85% saline solution containing 1% normal rat serum. The animals were sampled every day thereafter by microscopic examination of a drop of tail vein blood. Treatment was administered on the first day that the animals became positive. Animals in groups 1, 2 and 3 respectively received a single 50-mg dose of the extract orally, intramuscularly or intravenously. Groups 4, 5 and 6 received two 50-mg daily doses by the same routes while groups 7, 8 and 9 were given three 50mg daily doses by the same routes. Three groups (10, 11 and 121 were given a single 150-mg dose each, while physiological saline alone was administered to groups 13, 14 and 15. Animals in group 16 were infected but not treated. Following treatment, all animals were examined for parasitemia on a daily basis as described above. Results
The results obtained are presented in Table 1. Acalypha hispida leaf extracts showed trypanocicadal activities with all regimens and routes of
235
TABLE 1 EFFECTS
OF
TRYPANOSO~A
Treatment and dosage (days of treatment)
A
CRUDE
EXTRACT OF ACALYPHA HISPIDA INFECTION IN ALBINO RATS
LEAVES
ON
BRUCEI BRUCEI
Group
Route
Mean days parasitefree
‘I-Day survival
Mean days to death
Extract, 50 mg (x 1)
1 2 3
p.0. i.m. iv.
0.3 2.0 5.0
313 313 313
7.0 7.0 9.7
Extract, 50 mg (x 2)
4 5 6
p.0. i.m. i.v.
0.6 2.0 1.0
313 013 313
7.0 6.0 7.0
Extract, 50 mg (x 3)
7 8 9
p.0. i.m. i.v.
1.0 3.0 3.0
313 313 313
7.0 8.3 8.0
Extract, 150 mg (x 1)
10 11 12
p.0. i.m. iv.
0.P 0.0” 0.6
O/3 O/3 013
3.0 3.0 5.0
Normal saline, 0.1 ml lx 3)
13 14 15
p.0. i.m. i.v.
0.0 0.0 0.0
013 013 O/3
5.0 5.0 5.0
Untreated
16
-
0.0
o/3
5.0
“Parasites were cieared from blood but the animals died on the same day.
administration. The least activity was observed in the cases of animals treated orally irrespective of the number of doses given. Intramuscular and intravenous injections of the extract appeared to be the most effective. In the animals treated intravenously, a single injection of 50 mg of the extract appears to be the best since maximum activity was observed with minimum toxicity as evidenced by the long average number of days during which the animals were parasite-free after treatment. Infected animals receiving a single dose of 150 mg died much earlier than those receiving single or multiple doses of 50 mg. Discussion
This study has demonstrated the activity of an aqueous extract of A. hisbrucei in rats. From the results presented, many questions need to be addressed. While it is obvious that the extract
pdda leaves against T. brucei
236
kills the parasites (a dersirable effect) and can kill the host (a most undesirable situation), any judgement about the toxicity of the active ingredient in this extract on the host should be deferred until a purified fraction or a pure compound can be tested. The authors are optimistic that the toxicity will be reduced but it is unlikely that it can be eliminated completely. Currently used drugs in the treatment of African trypanosomiasis are very toxic to the host. Clearly, just the demonstration of activity suggests optimism even when toxicity is apparent (Jaffe et al., 19’701. Another concern is the fact that, in all cases, treated animals in which parasite counts had dropped to O/ml soon became parasitemic and died. Post-chemotherapy recurrence of parasitemia in animals infected with African trypanosomes is a well-known phenomenon (Basson et al., 1977; Evans and Brightman, 19801. Suramin, a very important drug in the treatment of African trypanosomiasis, is not very effective in late infections. This is thought to happen because trypanosomes may have infested the brain and thereby escape the action of the drug. Abolarin et al. (19721 have shown that after infected animals have been treated with salicylhydroxamic acid (SHAM) + glycerol, parasites are cleared from the blood, but remain in the choroid plexus of the brain from where they later disseminate. Clarkson and Brohn (19761 reported that animals infected with T.b. rhodesiense and treated with SHAM + glycerol became aparasitemic in about 24 h. Parasite dissemination started after 6 days and death eventually occurred. The maximum number of days during which the treated animals in our experiment were free of parasites was 5 days. The authors believe that the results of this experiment are encouraging and work is in progress to test purified fractions of this extract with the hope that some fractions will be less toxic and more effective. Acknowledgements
This study was supported by University of Ilorin Senate Research Grant No. 8.184.46. We acknowledge the contributions of Miss. M.A. Adewoyin, Miss M.I. Jospeh and Miss A.O. Arukhe, who at different times did preliminary work on this study. References Abolarin, M.O., Evans, D.A., Tovey, sickness. Trypanosome developing
D.G. and Ormerod, in choroid plexus
W.E. (19721 Cryptic stage of sleepingepithelial cells. British Medical Journal
285, 1380 - 1382. Akinyanju, J.A., Owoyale, J.A. and Okanla, E.O. (1986) Antimicrobial effect of a leaf extract of Acalypha torta In: A. Sofowora (Ed.), The State of Medicinal Plants Research in Nigeria, Proceedings of a Workshop on Medicinal Plants, University of Ife, Nigeria, pp. 247-251. Basson, W., Page, M.L. and Myburgh, D.P. (1977) Human trypanosomiasis in Southern Africa. South African Medical Journal 51 (21, 453-457. Clarkson, A.B. and Brohn, F.H. (1976) Trypanosomiasis: An approach to chemotherapy by the inhibition of carbohydrate catabolism. Science 194, 204 - 205.
237
Evans, D.A. and Brightman, C.A.J. (19801 Pleomorphism and the problem of recrudescent parasitemia following treatment with salicylhydroxamic acid (SHAM) in African trypanosomiasis. Transactions of the Royal Society for Tropical Medicine and Hygiene 74, 601-604. Fairlamb, A.H., Opperdoes, F.R. and Borst, P. (19771 New approach to screening drugs for activity against African trypanosomes. Nature 256, 270 - 271. Jaffe, J.J., McCormack, J.J. and Meymerian, E. (19701 Trypanocidal properties of 5’.O-sulfamayladenosine, a close structural analog of nucleocidin. Experimental Parasitology, 28, 535-543. Maegraith, B.G. (19761 Trypanosomiases. In: B. Maegraith (Ed), Clinical Tropical Diseases, Blackwell Scientific Publications, Oxford, pp. 438 - 463. Schmidt, G.D. and Roberts, L.S. (19771 Order Kinetoplastida: Trypanosomes and their kin. In: G.D. Schmidt and L.S. Roberts (Ed.), Foundations of Parasitology. C.V. Mosby Company, St. Louis, MD, pp. 52-78. World Health Organization (1981) Report of the Second Scientific Working Group on African Trypanosomiases. World Health Organization, Geneva (TDR/TRY-SWG (2)/81.3).
