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In vivo activity of an extract of Pavetta owariensis bark on experimental Schistosoma mansoni infection in mice
Journal of Ethnopharmacology,
18 (1986) 187-192 Elsevier Scientific Publishers Ireland Ltd.
187
IN VIVO ACTIVITY OF AN EXTRACT OF PA VETTA 0 WARZENSZS BARK ON EXPERIMENTAL SCZ-ZZSTOSOMAMANSONZ INFECTION IN MICE
ALIOU M. BALD@,
E. VAN MARCKb and M. VANHAELENa
aLaboratoire de Pharmacognosie, Znstitut de Pharmacie, Universite Libre de Bruxelles, B205-4, Boulevard du Triomphe, 1050 Bruxelles and bLaboratory of Histopathology and Experimental Schistosomiasis, Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerpen (Belgium) (Accepted
September 24, 1986)
Summary Mice were experimentally infected with Schistosoma manspi. Comparisons were carried out of total body weight, weight of liver, spleen and left lung, number of adult worms and eggs, and size of liver granulomas between an untreated group and a group treated only with an alcoholic extract of Pauetta owariensis. In the treated group, liver granulomas were smaller for both the acute and chronic infections, indicating a modulation of the granulomatous reaction. A reduction of worm burden, especially of male worms was observed after treatment of chronically infected mice with the same extract.
Introduction Since the introduction of tartar emetic by Christopherson (1918), progress in the chemotherapy of schistosomiasis has been remarkable, particularly in the last 10 years. At present, only three drugs are in use: metriphonate, oxamniquine and praziquantel (Pellegrino et al., 1977; Webbe and James, 1977; Gigase and Demedts, 1986). Since it is possible to obtain new chemotherapeutic agents from natural sources, it seems advisable to study the numerous plants used for the treatment of schistosomiasis in traditional medicine in Africa and elsewhere. This study reports the activity of extracts of Pauetta owariensis P. Beauv. (Rubiaceae), a well-known anthelminthic used in Guinean traditional medicine, on experimental Schistosoma manioni infection. The “white bark” and “red bark” varieties of this plant are used without any distinction by traditional healers. However, in the Guinean flora the “white bark” variety is more widespread. 01986 EIsevier Scientific Publishers Ireland Ltd. 0378-8741/86/$02.45 Published and Printed in Ireland
188
Materials and methods The present study was performed with the stem bark of trees growing in Seredou (Guinea-Conakry). Samples were collected from this region in January 1983 for the “red bark” variety and in April 1985 for the “white bark” variety; they were taxonomically identified by the Department of Botany in the Research Center of Medicinal Plants - SQredou. Voucher specimens have been deposited at the herbarium of the Center. Preparation of the extract The barks were dried in the shade, powdered and percolated with 70% ethanol. Each ethanolic extract was concentrated in vacua below 50°C to dryness. Extract 1 (yield 8.9% w/w dry weight) was prepared from the “white bark” variety and Extract 2 (yield 9.6% w/w dry weight) from mixing (1: 1 w/w) “white bark” and “red bark” varieties. Each ethanolic extract was then reconstituted as an aqueous suspension so that 1 ml was equivalent to 16 mg of the dried starting material. Animals Six-week-old outbred female Swiss mice, weighing about 20 g, were used and were given free access to food (standardized diet of mouse pellets, Hope Farms, Woerden, The Netherlands) and water for the duration of the experiment. Parasite A Puerto-Rican strain of Schistosoma mansoni maintained in the laboratory since 1975 in mice and Biomphalaria glabrata snails (albino PuertoRican strain) were employed. Experimental procedure Twenty mice (Sl--520) were infected transcutaneously using the ring method of Smithers and Terry (1965) with 300 cercariae for the acute infection group and 21 other mice (S21-S41) were infected with 30 cercariae for the chronic infection group. In the acute infection group, treatment was started 35 days after infection. One milliliter of Extract 1 was given by gavage to each mouse (Sll--520) daily for 10 days. Ten mice were left untreated (Sl-SlO). In the chronic infection group, treatment started 81 days after infection by administering to each mouse (S31--541) by gavage daily, 1 ml of Extract 2 for 13 days. Ten infected mice (S21-S30) were left untreated. All mice were killed with an overdose of a heparinized sodium pentobar-
189
bital solution, 3 days (48 days post-infection for the acute infection group) and 14 days (108 days post-infection for the chronic infection group) after the end of the treatment. The animals were perfused with saline via the left heart ventricle after trans-section of the portal vein and adult worms were recovered on a mesh screen and counted. In addition, the following parameters were determined: total body weight just before sacrifice, weight of liver, spleen and left lung, total number of eggs (viable or not) from the intestines, liver and lung. After digestion of weighed tissue specimens in 5% KOH (2 ml per lung and 10 ml per liver or intestine) for 4 h at 37”C, the total volume was determined and S. mansoni eggs were counted in three samples of 0.1 ml. Pathological examination Liver, spleen, kidney and lung samples were fixed in 10% formalin, embedded in paraffin, and sectioned (5 pm thickness). The sections were stained with hematoxylin-eosin. The size of the liver granulomas was measured with a calibrated ocular, each value for an individual animal being the mean of 100-200 measurements of 2 diameters (Boros et al., 1983). Statistical methods The 95% confidence intervals for means were calculated from the t value of the Student’s t-distribution. Significant difference was accepted when the calculated P values were smaller than 0.05. Results Acute infection One mouse in the untreated group died before the date of sacrifice. A statistically significant difference (P = 0.02) was observed between the treated and untreated groups with respect to the size of liver granulomas. Mean values are summarized in Table 1. The other parameters did not differ significantly (P > 0.05). The anatomopathological examination of the specimens of both the treated and the untreated groups showed the typical aspects of a recent Schistosoma munsoni infection in livers, while kidneys and lungs were normal. Histopathology of the spleen did not show any difference. Chronic infee tion Significant differences between the treated and the untreated groups were recorded for the following parameters: number of total adult worms (P = 0.04), male worms (P = 0.04) (Table 2), and granuloma size (P = 0.04) (Table 2). The other parameters were not significantly different.
190 TABLE 1 COMPARISON OF PARAMETERS BETWEEN UNTREATED AND ORALLY TREATED (1 ml Extract l/animal once daily) S. MANSOZVZ-INFECI’ED MICE (acute infection) Meant
Liver granuloma size (mm) Total body weight (g) Spleen weight (g) Left lung weight (g) Liver weight (g) Total worm count Eggs/g liver tissue Eggs/intestine
S.E.M.
Untreated group (N= 9)
Treated group (N= 10)
0.35 27.0 0.15 0.13 2.10 41 3097 16967
0.29 27.5 0.09 0.19 1.98 45 4477 13050
+ 0.04 ? 1.8 ? 0.08 + 0.03 + 0.34 ? 13 ? 439 + 8321
k 0.06* + 2.8 2 0.03 + 0.12 + 0.41 ? 31 + 2536 2 11183
*Statistically significant from untreated group, P = 0.02.
Discussion In acute infection, the only parameter which was significantly different between the treated and the untreated mice was granuloma size (Table l), TABLE 2 COMPARISON OF PARAMETERS BETWEEN UNTREATED AND ORALLY TREATED (1 ml Extract B/animal once daily) S. MANSONZ-INFECTED MICE (chronic infection) Mean +_S.E.M.
Liver granuloma size (mm) Total body weight (g) Spleen weight (g) Left lung weight (g) Liver weight (g) Total worm count (g) Male worm count (g) Eggs/g liver tissue Eggs/intestine
Untreated group (N = 10)
Treated group (N= 11)
0.32 32.4 0.29 0.16 2.16 8.90 8.00 1525 2588
0.24 34.2 0.25 0.13 2.19 4.21 3.64 732 1493
t 0.04 t 3.2 r 0.07 2 0.05 + 0.52 t 4.66 t 4.60 r 1022 ? 1554
2 0.02* 5 3.3 + 0.08 2 0.04 + 0.30 ? 4.63** + 4.07** + 878 + 2308
*Significantly different from the untreated group, P = 0.04.Note that 6/11 of treated mice were without granuloma formation. **Significantly different from the untreated group, P = 0.04. Note that one animal of the treated group and two animals of the untreated group presented with a singleaex infection (female).
191
although values for spleen weight and number of eggs in the intestine were on the verge of significance. Such non-significant differences could indicate true activity which could be improved by changing the treatment schedule (increase of dosage, increase of duration of treatment and/or period between treatment and sacrifice). With oltipraz, a slow acting drug, approximately 2 months are required before its full schistosomicidal effect becomes evident (Bueding et al., 1982). It is also well known that the amount of praziquantel required to achieve parasite reductions of at least 95% depends on the host species, on the dosage, on the routes and on the schedules of administration (Giinnert and Andrews, 1977; Pellegrino et al., 1977). Considering these results and in order to assess possible differences of biological activity between the two plant varieties, a red/white mix was used for the treatment of the chronic infection. In the chronic study, treatment gave rise to a single-sex infection in l/11 mice and only dead worms were found in the livers of 4/11 animals. Significant differences were recorded when comparing liver granuloma sizes (Table 2) and total number of adult schistososomes. Male worms could be more sensitive to the drug than female. Comparisons of the number of eggs per gram of liver tissue showed a difference on the verge of significance, with lower numbers in the treated group (P = 0.08). The activity of the extracts was thus more striking in chronic infection than in acute infection. This fact could be related either to the greater sensitivity of older worms or to the slightly different treatment schedule or to the use of the “red bark” variety. Ethanol extracts of Puuetta owariensis in mice with an experimental Schistosomiasis mansoni infection clearly reduced liver granuloma size. This modulation of the liver granuloma may be regarded as beneficial since liver granulomas are mainly responsible for the morbidity of schistosomiasis. Chemical identification of the biologically active constituents of the plant are in progress. Acknowledgements This study has been realized through a W.H.O. fellowship for the first author (A.B.). This paper does not necessarily reflect the views of the W.H.O. Sincere thanks are due to M.L. Kestens, B. SC. for help with the statistical analyses. Technical assistance of Mrs. Vanhove-Vereecken and of Mrs. De Maesschalck-Penne is acknowledged. Secretarial assistance by Miss G. Verhulst is greatly appreciated. References Boros, D.L., Lande, M.A. and Carrick, Jr., L. (1983) The influence of host and parasitic factors on Schistosoma mansoni egg-induced tissue fibrosis. Contributions to Microbiology and Immunology 7, 230-236. Bueding, E., Dolan, P. and Leroy, J.P. (1982) The antischistosomal activity of oltipraz. Research Communications in Chemical Pathology and Pharmacology 37 (2), 293-303.
192 Christopherson, J.B. (1918) The successful use of antimony in bilharziasis. Lancet 325327. Gigase, P.L. and Demedts, P. (1986) Drugs used in the treatment of schistosomiasis. Antimicrobial Agents Annual 1, 295-303. Giinnert, R. and Andrews, P. (1977) Praziquantel, a new broad-spectrum antischistosomal agent. Zeitschrift fiir Parasitenkunde 52, 129-150. Pellegrino, J., Lima-Costa, F.F., Carlos, M.A. and Mello, R.T. (1977) Experimental chemotherapy of Schistosoma mansoni. XIII. Activity of praziquantel, an isoquinolinepyrazino-derivative, on mice, hamsters and cebus monkeys. Zeitschrift fiir Parasitenkunde 52,151-168. Smithers, S.R. and Terry, R.J. (1965) The infection of laboratory hosts with cercariae of S. mansoni and the recovery of adult worms. Parasitology 55, 695-700. Webbe, G. and James, C. (1977) A comparison of the susceptibility to praziquantel of Schistosoma haematobium, S. japonicum, S. mansoni, S. intercalatum and S. matthei in hamsters. Zeitschrift fiir Parasitenkunde 52.169-177.
18 (1986) 187-192 Elsevier Scientific Publishers Ireland Ltd.
187
IN VIVO ACTIVITY OF AN EXTRACT OF PA VETTA 0 WARZENSZS BARK ON EXPERIMENTAL SCZ-ZZSTOSOMAMANSONZ INFECTION IN MICE
ALIOU M. BALD@,
E. VAN MARCKb and M. VANHAELENa
aLaboratoire de Pharmacognosie, Znstitut de Pharmacie, Universite Libre de Bruxelles, B205-4, Boulevard du Triomphe, 1050 Bruxelles and bLaboratory of Histopathology and Experimental Schistosomiasis, Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerpen (Belgium) (Accepted
September 24, 1986)
Summary Mice were experimentally infected with Schistosoma manspi. Comparisons were carried out of total body weight, weight of liver, spleen and left lung, number of adult worms and eggs, and size of liver granulomas between an untreated group and a group treated only with an alcoholic extract of Pauetta owariensis. In the treated group, liver granulomas were smaller for both the acute and chronic infections, indicating a modulation of the granulomatous reaction. A reduction of worm burden, especially of male worms was observed after treatment of chronically infected mice with the same extract.
Introduction Since the introduction of tartar emetic by Christopherson (1918), progress in the chemotherapy of schistosomiasis has been remarkable, particularly in the last 10 years. At present, only three drugs are in use: metriphonate, oxamniquine and praziquantel (Pellegrino et al., 1977; Webbe and James, 1977; Gigase and Demedts, 1986). Since it is possible to obtain new chemotherapeutic agents from natural sources, it seems advisable to study the numerous plants used for the treatment of schistosomiasis in traditional medicine in Africa and elsewhere. This study reports the activity of extracts of Pauetta owariensis P. Beauv. (Rubiaceae), a well-known anthelminthic used in Guinean traditional medicine, on experimental Schistosoma manioni infection. The “white bark” and “red bark” varieties of this plant are used without any distinction by traditional healers. However, in the Guinean flora the “white bark” variety is more widespread. 01986 EIsevier Scientific Publishers Ireland Ltd. 0378-8741/86/$02.45 Published and Printed in Ireland
188
Materials and methods The present study was performed with the stem bark of trees growing in Seredou (Guinea-Conakry). Samples were collected from this region in January 1983 for the “red bark” variety and in April 1985 for the “white bark” variety; they were taxonomically identified by the Department of Botany in the Research Center of Medicinal Plants - SQredou. Voucher specimens have been deposited at the herbarium of the Center. Preparation of the extract The barks were dried in the shade, powdered and percolated with 70% ethanol. Each ethanolic extract was concentrated in vacua below 50°C to dryness. Extract 1 (yield 8.9% w/w dry weight) was prepared from the “white bark” variety and Extract 2 (yield 9.6% w/w dry weight) from mixing (1: 1 w/w) “white bark” and “red bark” varieties. Each ethanolic extract was then reconstituted as an aqueous suspension so that 1 ml was equivalent to 16 mg of the dried starting material. Animals Six-week-old outbred female Swiss mice, weighing about 20 g, were used and were given free access to food (standardized diet of mouse pellets, Hope Farms, Woerden, The Netherlands) and water for the duration of the experiment. Parasite A Puerto-Rican strain of Schistosoma mansoni maintained in the laboratory since 1975 in mice and Biomphalaria glabrata snails (albino PuertoRican strain) were employed. Experimental procedure Twenty mice (Sl--520) were infected transcutaneously using the ring method of Smithers and Terry (1965) with 300 cercariae for the acute infection group and 21 other mice (S21-S41) were infected with 30 cercariae for the chronic infection group. In the acute infection group, treatment was started 35 days after infection. One milliliter of Extract 1 was given by gavage to each mouse (Sll--520) daily for 10 days. Ten mice were left untreated (Sl-SlO). In the chronic infection group, treatment started 81 days after infection by administering to each mouse (S31--541) by gavage daily, 1 ml of Extract 2 for 13 days. Ten infected mice (S21-S30) were left untreated. All mice were killed with an overdose of a heparinized sodium pentobar-
189
bital solution, 3 days (48 days post-infection for the acute infection group) and 14 days (108 days post-infection for the chronic infection group) after the end of the treatment. The animals were perfused with saline via the left heart ventricle after trans-section of the portal vein and adult worms were recovered on a mesh screen and counted. In addition, the following parameters were determined: total body weight just before sacrifice, weight of liver, spleen and left lung, total number of eggs (viable or not) from the intestines, liver and lung. After digestion of weighed tissue specimens in 5% KOH (2 ml per lung and 10 ml per liver or intestine) for 4 h at 37”C, the total volume was determined and S. mansoni eggs were counted in three samples of 0.1 ml. Pathological examination Liver, spleen, kidney and lung samples were fixed in 10% formalin, embedded in paraffin, and sectioned (5 pm thickness). The sections were stained with hematoxylin-eosin. The size of the liver granulomas was measured with a calibrated ocular, each value for an individual animal being the mean of 100-200 measurements of 2 diameters (Boros et al., 1983). Statistical methods The 95% confidence intervals for means were calculated from the t value of the Student’s t-distribution. Significant difference was accepted when the calculated P values were smaller than 0.05. Results Acute infection One mouse in the untreated group died before the date of sacrifice. A statistically significant difference (P = 0.02) was observed between the treated and untreated groups with respect to the size of liver granulomas. Mean values are summarized in Table 1. The other parameters did not differ significantly (P > 0.05). The anatomopathological examination of the specimens of both the treated and the untreated groups showed the typical aspects of a recent Schistosoma munsoni infection in livers, while kidneys and lungs were normal. Histopathology of the spleen did not show any difference. Chronic infee tion Significant differences between the treated and the untreated groups were recorded for the following parameters: number of total adult worms (P = 0.04), male worms (P = 0.04) (Table 2), and granuloma size (P = 0.04) (Table 2). The other parameters were not significantly different.
190 TABLE 1 COMPARISON OF PARAMETERS BETWEEN UNTREATED AND ORALLY TREATED (1 ml Extract l/animal once daily) S. MANSOZVZ-INFECI’ED MICE (acute infection) Meant
Liver granuloma size (mm) Total body weight (g) Spleen weight (g) Left lung weight (g) Liver weight (g) Total worm count Eggs/g liver tissue Eggs/intestine
S.E.M.
Untreated group (N= 9)
Treated group (N= 10)
0.35 27.0 0.15 0.13 2.10 41 3097 16967
0.29 27.5 0.09 0.19 1.98 45 4477 13050
+ 0.04 ? 1.8 ? 0.08 + 0.03 + 0.34 ? 13 ? 439 + 8321
k 0.06* + 2.8 2 0.03 + 0.12 + 0.41 ? 31 + 2536 2 11183
*Statistically significant from untreated group, P = 0.02.
Discussion In acute infection, the only parameter which was significantly different between the treated and the untreated mice was granuloma size (Table l), TABLE 2 COMPARISON OF PARAMETERS BETWEEN UNTREATED AND ORALLY TREATED (1 ml Extract B/animal once daily) S. MANSONZ-INFECTED MICE (chronic infection) Mean +_S.E.M.
Liver granuloma size (mm) Total body weight (g) Spleen weight (g) Left lung weight (g) Liver weight (g) Total worm count (g) Male worm count (g) Eggs/g liver tissue Eggs/intestine
Untreated group (N = 10)
Treated group (N= 11)
0.32 32.4 0.29 0.16 2.16 8.90 8.00 1525 2588
0.24 34.2 0.25 0.13 2.19 4.21 3.64 732 1493
t 0.04 t 3.2 r 0.07 2 0.05 + 0.52 t 4.66 t 4.60 r 1022 ? 1554
2 0.02* 5 3.3 + 0.08 2 0.04 + 0.30 ? 4.63** + 4.07** + 878 + 2308
*Significantly different from the untreated group, P = 0.04.Note that 6/11 of treated mice were without granuloma formation. **Significantly different from the untreated group, P = 0.04. Note that one animal of the treated group and two animals of the untreated group presented with a singleaex infection (female).
191
although values for spleen weight and number of eggs in the intestine were on the verge of significance. Such non-significant differences could indicate true activity which could be improved by changing the treatment schedule (increase of dosage, increase of duration of treatment and/or period between treatment and sacrifice). With oltipraz, a slow acting drug, approximately 2 months are required before its full schistosomicidal effect becomes evident (Bueding et al., 1982). It is also well known that the amount of praziquantel required to achieve parasite reductions of at least 95% depends on the host species, on the dosage, on the routes and on the schedules of administration (Giinnert and Andrews, 1977; Pellegrino et al., 1977). Considering these results and in order to assess possible differences of biological activity between the two plant varieties, a red/white mix was used for the treatment of the chronic infection. In the chronic study, treatment gave rise to a single-sex infection in l/11 mice and only dead worms were found in the livers of 4/11 animals. Significant differences were recorded when comparing liver granuloma sizes (Table 2) and total number of adult schistososomes. Male worms could be more sensitive to the drug than female. Comparisons of the number of eggs per gram of liver tissue showed a difference on the verge of significance, with lower numbers in the treated group (P = 0.08). The activity of the extracts was thus more striking in chronic infection than in acute infection. This fact could be related either to the greater sensitivity of older worms or to the slightly different treatment schedule or to the use of the “red bark” variety. Ethanol extracts of Puuetta owariensis in mice with an experimental Schistosomiasis mansoni infection clearly reduced liver granuloma size. This modulation of the liver granuloma may be regarded as beneficial since liver granulomas are mainly responsible for the morbidity of schistosomiasis. Chemical identification of the biologically active constituents of the plant are in progress. Acknowledgements This study has been realized through a W.H.O. fellowship for the first author (A.B.). This paper does not necessarily reflect the views of the W.H.O. Sincere thanks are due to M.L. Kestens, B. SC. for help with the statistical analyses. Technical assistance of Mrs. Vanhove-Vereecken and of Mrs. De Maesschalck-Penne is acknowledged. Secretarial assistance by Miss G. Verhulst is greatly appreciated. References Boros, D.L., Lande, M.A. and Carrick, Jr., L. (1983) The influence of host and parasitic factors on Schistosoma mansoni egg-induced tissue fibrosis. Contributions to Microbiology and Immunology 7, 230-236. Bueding, E., Dolan, P. and Leroy, J.P. (1982) The antischistosomal activity of oltipraz. Research Communications in Chemical Pathology and Pharmacology 37 (2), 293-303.
192 Christopherson, J.B. (1918) The successful use of antimony in bilharziasis. Lancet 325327. Gigase, P.L. and Demedts, P. (1986) Drugs used in the treatment of schistosomiasis. Antimicrobial Agents Annual 1, 295-303. Giinnert, R. and Andrews, P. (1977) Praziquantel, a new broad-spectrum antischistosomal agent. Zeitschrift fiir Parasitenkunde 52, 129-150. Pellegrino, J., Lima-Costa, F.F., Carlos, M.A. and Mello, R.T. (1977) Experimental chemotherapy of Schistosoma mansoni. XIII. Activity of praziquantel, an isoquinolinepyrazino-derivative, on mice, hamsters and cebus monkeys. Zeitschrift fiir Parasitenkunde 52,151-168. Smithers, S.R. and Terry, R.J. (1965) The infection of laboratory hosts with cercariae of S. mansoni and the recovery of adult worms. Parasitology 55, 695-700. Webbe, G. and James, C. (1977) A comparison of the susceptibility to praziquantel of Schistosoma haematobium, S. japonicum, S. mansoni, S. intercalatum and S. matthei in hamsters. Zeitschrift fiir Parasitenkunde 52.169-177.