- Email: [email protected]
Inhibition of Trypanosoma cruzi growth by medical plant extracts
Fitoterapia 73 (2002) 569–575
Inhibition of Trypanosoma cruzi growth by medical plant extracts ´ c, G.R. Schinellaa,b,*, H.A. Tourniera,b, J.M. Prietoc, J.L. Rıos a a,b H. Buschiazzo , A. Zaidenberg a
´ ´ Facultad de Ciencias Medicas, ´ Catedra de Farmacologıa, Universidad Nacional de La Plata, Calles 60 y 120, La Plata 1900, Argentina b ´ de Investigaciones Cientıficas ´ Comision de la Provincia de Buenos Aires, La Plata, Argentina c ` ` ` Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Valencia, Spain Received 21 June 2002; accepted 1 August 2002
Abstract This study describes the screening of extracts obtained from 18 plants and two fungi used in the Chinese and Mediterranean traditional medicines on epimastigote forms of Trypanosoma cruzi. The extracts were tested against epimastigote of T. cruzi Bra C15C2 clone in vitro at 27 8C and at a concentration of 250 mgyml in axenic culture. Angelica dahurica, A. pubescens, A. sinensis, Astragalus membranaceus, Coptis chinensis, Haplophyllum hispanicum, Phellodendron amurense, Poria cocos, Ranunculus sceleratus and Scutellaria baicalensis showed significant effects against the parasite with a percentage of growth inhibition between 20 and 100%. C. chinensis and R. sceleratus showed the greatest activity with IC50 values of 1.7 mgyml for C. chinensis and 10.7 mgyml for R. sceleratus. These activities are greater than that of allopurinol. C. chinesis and R. sceleratus extracts did not show cytotoxic effects on rat polimorphonuclear cells using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide and lactic dehydrogenase assays. These results allowed us to suggest that R. sceleratus and C. chinensis could be a source of new compounds clinically active against T. cruzi. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Antiprotozoal; Trypanosoma cruzi; Natural products; Coptis chinensis; Ranunculus sceleratus
*Corresponding author. Fax: q54-221-423-6710. E-mail address: [email protected] (G.R. Schinella). 0367-326X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 0 2 . 0 0 2 4 6 - 0
570
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
1. Introduction Trypanosoma cruzi is the etiologic agent of Chagas disease, that affects approximately 20 million people from Southern California to Argentina and Chile w1x and this is, after malaria, the most prevalent vector-borne illness in Latin America. All the drugs recommended for the treatment of Chagas disease have serious limitations including limited effectiveness and important drug-related side effects. There are some drugs of promise for the treatment of this disease w2,3x but the absence of compounds with a selective cytotoxic activity against the parasite makes the search for new compounds an urgent priority. One of the approaches of the antiparasitic chemotherapy relies on testing the biological activity of natural products w4,5x. Extracts derived particularly from plants offer novel possibilities to obtain new compounds active against T. cruzi. In the course of a screening program for potential antiprotozoal drugs, we assessed the effect of extracts obtained from 18 plants and two fungi which are used in traditional medicine in China and the Mediterranean area, against epimastigotes of T. cruzi in axenic cultures. 2. Experimental 2.1. Plant material The botanical species assayed are detailed in Table 1. Ranunculus sceleratus, Scrophularia auriculata, Inula viscosa, Helichrysum italicum and Haplophyllum Table 1 Botanical species Name
Organ
Extract
Yield (% wyw)
Angelica dahurica Bertham et Hoocker f. A. pubescens Maxim. F. A. sinensis (Oliv.) Diels Astragalus membranaceus Bunge Atractylodes macrocephala Koidzumi Codonopsis pilosula (Franch) Nannfeldt Coptis chinensis Franch Curcuma aromatica Salisb. Forsythia suspensa (Thunberg) Vahl. Haplophyllum hispanicum Spach Helichrysum italicum (L.) Moench Inula viscosa (L.) Aiton Lentinus edodes Berk Paeonia lactiflora Pallas Phellodendron amurense Ruprecht Poria cocos (Schw.) Wolf Ranunculus sceleratus L. Rehmania glutinosa (Gaaertnet) Liboschitz Scrophularia auriculata L. Scutellaria baicalensis Georgi
Root Root Wood Root Root Root Root Rhizome Fruit Aerial parts Aerial parts Aerial parts Sclerotium Root Root bark Sclerotium Aerial parts Root Aerial parts Root
Ethanol 70% Ethanol 70% Ethanol 70% Ethanol 70% Aqueous Aqueous Methanol Aqueous Ethanol 70% Methanol Methanol Methanol Aqueous Aqueous Methanol Ethanol 70% Methanol Ethanol 70% Methanol Methanol
10.5 22.5 27.0 16.5 32.5 25.0 17.5 8.0 11.5 8.7 11.2 16.7 8.0 7.0 19.5 1.4 5.9 19.0 6.9 22.0
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
571
hispanicum were collected at Pinedo, Riola, Torrent, Chiva and El Vedat de Torrent (Valencia, Spain), respectively. Specimens of these plants were deposited in the herbarium of the Department of Pharmacology, Faculty of Pharmacy, University of Valencia. The rest of the species were commercial samples obtained from Asia Natural Products S.A. Co., Amposta (Tarragona, Spain). 2.2. Extraction Extraction was performed by stirring plant material (50 g) with the solvent specified in Table 1 at 40 8C. The solvents were removed under vacuum and the residues and water extracts were lyophilised and dissolved in dimethylsulfoxide (DMSO). The percentage yields are indicated in Table 1. 2.3. Chemicals The extraction was performed with ethanol or methanol PA (Merck). Water extracts were obtained with bi-distilled water. The culture medium base used was Medium 199 (Gibco BRL, Life Technologies, NY). The chemicals for the assays were of analytical grade (Sigma Co., St. Louis). 2.4. Animals Male Wistar rats (250–300 g) were used. They were kept in standard environmental conditions and fed with rodent diet with tap water ad libitum. 2.5. In vitro assays T. cruzi epimastigotes (clone Bra C15C2) w6x were cultured in F29 media supplemented with 10% (vyv) heat-inactivated fetal calf serum at 27 8C, with an inoculum of 5=105 cellsyml w7x. Plant extracts were added at a final concentration of 250 mgyml. All assays were carried out in triplicate. Final DMSO concentration was less than 0.5%. Parasites were counted after 72 h of contact with the samples in a Neubauer chamber and stained with Wrigth-Giemsa. The activity of the extracts was assessed by comparison with the negative control (DMSO). Allopurinol was used as a reference drug. 2.6. Cytotoxicity studies The plant extracts cytotoxicity was assessed on elicited peritoneal leukocytes obtained from rats w8x with a viability greater than 95% determined by the trypan blue exclusion test. The cell viability was assessed using two criteria. In the first one, the leakage of the cytoplasmic marker enzyme lactate dehydrogenase (LDH) was determined in supernatant of the incubation of polymorphonuclear cells (PMN) in the presence of extracts (200 mgyml) or vehicle. A commercial kit was used (Boehringer–
572
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
Mannheim, Argentina Lot.yCh.-B: 69685401). The activity of the extracts was assessed by comparison with a positive control (0.2% vyv Triton X100). As a second criteria, we measured the capacity of PMN mitochondrial dehydrogenase enzymes to convert the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan, which is assessed spectrophotometrically at 490 nm w9x. Chlropromazine (200 mM) was used as a reference compound (positive control). 2.7. Statistical analysis Data were expressed as mean"S.D. Statistical analysis was performed by oneway analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons. Differences were considered significant at P-0.05. The inhibitory concentration 50% (IC50) was calculated from the concentrationyeffect regression line. In each case, an appropriate range of 4–5 concentrations was used. 3. Results and discussion In this work, we assessed the anti-trypanosomal activity of extracts obtained from plants and fungus commonly used in the Chinese and Mediterranean medicines. The species were tested against epimastigotes of T. cruzi in axenic cultures. Although the model used neglects the different sensitivity of the other stages of T. cruzi, it is very useful to identify active compounds against the parasite w4x. The anti-trypanosomal activities of the different extracts are summarised in Table 2. When tested at a concentration of 250 mgyml, Coptis chinensis, H. hispanicum, Phellodendron amurense, R. sceleratus and Scutellaria baicalensis were the most active extracts with percentages of inhibition higher than 50%. Some of these plants were already known to have pharmacological properties. We have previously demonstrated that the methanol extracts of C. chinensis and S. baicalensis, plants used in the traditional Chinese medicine, had anti-inflammatory and antioxidant activities in different models w10,11x. On the other hand, another report demonstrated antitrypanosomal effects of S. baicalensis and Coptis japonica on related parasites like bloodstream forms of Trypanosoma brucei rhodesiense in vitro w12x. R. sceleratus and H. hispanicum are species used in phytotherapy in the Mediterranean area. Ranunculus species have been studied in the phytochemical and biological areas but the knowledge of their pharmacological actions is scanty. R. sceleratus particularly was demonstrated to have anti-inflammatory w13x and antifungal activities w14x. Extracts of H. hispanicum were also shown to have antiinflammatory activity w15x but to our knowledge, this is the first description of H. hispanicum and R. sceleratus activities against some specie of the Tripanosomatide family. An important criterion in the search of active compounds against T. cruzi with therapeutic perspectives, is to determine if they show toxic effects on mammalian host cells. For that purpose, two bioassays were used to determine the cytotoxicity
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
573
Table 2 Effect of plant extracts on the growth of epimastigote forms of Trypanosoma cruzi Botanical name Angelica dahurica A. pubescens A. sinensis Astragalus membranaceus Atractyloides macrocephala Codonopsis pilosula Coptis chinensis Curcuma aromatica Forsythia suspensa Haplophyllum hispanicum Helichrysum italicum Inula viscosa Lentinus edodes Paeonia lactiflora Phellodendron amurense Poria cocos Ranunculus sceleratus Rehmania glutinosa Scrophularia auriculata Scutellaria baicalensis Allopurinol Control (DMSO)
Parasites =10y6 a
14.5"1.0** 14.9"4.2** 19.4"0.5** 13.5"2.2** 23.0"1.0 ns 20.8"4.3 ns 0.0"0.0** 21.4"7.1 ns 19.1"4.0 ns 8.5"1.3** 23.0"3.0 ns 27.5"5.8 ns 26.8"2.6 ns 27.9"2.6 ns 11.3"1.4** 16.8"1.3** 0.7"0.3** 24.5"2.1 ns 23.3"1.8 ns 7.5"0.4** 5.4"0.5** 24.0"3.7
% Inhibition 40 38 19 44 4 3 100 11 20 65 4 0 0 0 53 30 97 0 3 69 78 –
a Values represent the parasite numbery5 ml of culture medium and are the mean"S.D. of triplicate determination. **Values are significantly different from those of the control (P-0.01).
Table 3 Cytotoxic effects of plant extracts on elicited rat peritoneal leukocytes Cytotoxicity assays
Control (y) C. chinensis H. hispanicum R. sceleratus S. baicalensis Control (q)
LDH
MTT
16.3"7.3a 13.7"5.5 ns 16.7"3.2 ns 18.7"6.7 ns 28.7"7.4 ns 140.3"2.5**
100b 124"22 ns 116"23 ns 108"14 ns 123"22 ns 69"6**
a
LDH activity is expressed as international unitsyl. Values mean% of cell viability. ** Values are significantly different from those of the negative control group (P-0.01) and are the mean of triplicate determinations. b
of C. chinensis, H. hispanicum, R. sceleratus and S. baicalensis extracts on elicited rat peritoneal leukocytes. MTT and LDH assays assess the metabolic activity and cell membrane damage, respectively. None of the extracts induced lactic dehydro-
574
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
Fig. 1. Dose-dependent inhibition of the growth of Trypanosoma Cruzi by Ranunculus sceleratus and Coptis chinensis extracts.
genase release and did not affect the activity of the mitochondrial succinate dehydrogenase (Table 3). Since C. chinensis and R. sceleratus extracts showed a higher antiparasitic activity than that obtained with the reference drug, they were tested at various concentrations. Fig. 1 shows a dose-response relationship obtained for both extracts. The IC50 values of 1.7 mgyml (r 2s0.9858, P-0.01) for C. chinensis and 10.7 mgyml (r 2s 0.9992, P-0.01) for R. sceleratus reflect the important inhibitory activity of both extracts. Other dose-dependent effects observed with C. chinensis and R. sceleratus were the morphological changes in the epimastigote form of T. cruzi. Inoculation of these extracts at concentration of 250 mgyml produced total lysis of the parasites. In the range of 10–30 mgyml C. chinensis produced vacuolar degeneration and R. sceleratus produced internal granulations. Taken together, the high potency, 5–25 times higher than the values taken as reference in these kind of assays w4x, and the absence of citotoxicity in the model employed, allows us to suggest that R. sceleratus and C. chinensis extracts constitute an important source of new compounds with potential therapeutic applications. Acknowledgments This work was supported by grants from the Ministry of Health of the Province of Buenos Aires and FEMEBA Foundation. We thank Asia Natural Products Co. ´ C. Abel for the for supplying some samples. The authors are grateful to Marıa language revision of the manuscript.
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
575
References w1 x w2 x w3 x w4 x w5 x w6 x w7 x w8 x w9 x w10x w11x w12x w13x w14x w15x
Urbina JA. Mem Inst Oswaldo Cruz 1999;94(I):349. Stoppani AOM. Medicina (Buenos Aires) 1999;59(II):147. Croft SL. Mem Inst Oswaldo Cruz 1999;94:215. ´ Sepulveda-Boza S, Cassels BK. Planta Med 1996;62:98. Traub-Cseko YM, Momen H. Parasitol Today 1995;11:315. Pan SC. Bull World Health Organ 1982;60:101. ´ G, Buschiazzo H. Pharmacol Toxicol 1999;84:98. Zaidenberg A, Tournier H, Schinella G, Marın Moroney MA, Alcaraz MJ, Forder RA, Carey F, Hoult JRS. J Pharm Pharmacol 1988;40:787. Mosmann T. J Immunol Methods 1983;65:55. ´ ´ ˜ S, Rıos ´ JL. Fitoterapia 2001;72:221. Cuellar MJ, Giner RM, Recio MC, Manez ´ JL. Life Sci Schinella GR, Tournier HA, Prieto JM, Mordujovich de Buschiazzo P, Rıos 2002;70:1023. Yabu Y, Nose M, Koide T, Ohta N, Ogihara Y. Southeast Asian J Trop Med Public Health 1998;29:599. ´ ´ ˜ S, Cerda´ S, et al. Phytother Res 1998;12:18. Cuellar MJ, Giner RM, Recio MC, Just MJ, Manez Misra SB, Dixit SN. Experientia 1978;34:1442. ´ ˜ S, Massmanian A, Waterman PG, et al. Z Naturforsch Prieto JM, Recio MC, Giner RM, Manez 1996;51c:618.
Inhibition of Trypanosoma cruzi growth by medical plant extracts ´ c, G.R. Schinellaa,b,*, H.A. Tourniera,b, J.M. Prietoc, J.L. Rıos a a,b H. Buschiazzo , A. Zaidenberg a
´ ´ Facultad de Ciencias Medicas, ´ Catedra de Farmacologıa, Universidad Nacional de La Plata, Calles 60 y 120, La Plata 1900, Argentina b ´ de Investigaciones Cientıficas ´ Comision de la Provincia de Buenos Aires, La Plata, Argentina c ` ` ` Departament de Farmacologia, Facultat de Farmacia, Universitat de Valencia, Valencia, Spain Received 21 June 2002; accepted 1 August 2002
Abstract This study describes the screening of extracts obtained from 18 plants and two fungi used in the Chinese and Mediterranean traditional medicines on epimastigote forms of Trypanosoma cruzi. The extracts were tested against epimastigote of T. cruzi Bra C15C2 clone in vitro at 27 8C and at a concentration of 250 mgyml in axenic culture. Angelica dahurica, A. pubescens, A. sinensis, Astragalus membranaceus, Coptis chinensis, Haplophyllum hispanicum, Phellodendron amurense, Poria cocos, Ranunculus sceleratus and Scutellaria baicalensis showed significant effects against the parasite with a percentage of growth inhibition between 20 and 100%. C. chinensis and R. sceleratus showed the greatest activity with IC50 values of 1.7 mgyml for C. chinensis and 10.7 mgyml for R. sceleratus. These activities are greater than that of allopurinol. C. chinesis and R. sceleratus extracts did not show cytotoxic effects on rat polimorphonuclear cells using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide and lactic dehydrogenase assays. These results allowed us to suggest that R. sceleratus and C. chinensis could be a source of new compounds clinically active against T. cruzi. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Antiprotozoal; Trypanosoma cruzi; Natural products; Coptis chinensis; Ranunculus sceleratus
*Corresponding author. Fax: q54-221-423-6710. E-mail address: [email protected] (G.R. Schinella). 0367-326X/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 0 2 . 0 0 2 4 6 - 0
570
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
1. Introduction Trypanosoma cruzi is the etiologic agent of Chagas disease, that affects approximately 20 million people from Southern California to Argentina and Chile w1x and this is, after malaria, the most prevalent vector-borne illness in Latin America. All the drugs recommended for the treatment of Chagas disease have serious limitations including limited effectiveness and important drug-related side effects. There are some drugs of promise for the treatment of this disease w2,3x but the absence of compounds with a selective cytotoxic activity against the parasite makes the search for new compounds an urgent priority. One of the approaches of the antiparasitic chemotherapy relies on testing the biological activity of natural products w4,5x. Extracts derived particularly from plants offer novel possibilities to obtain new compounds active against T. cruzi. In the course of a screening program for potential antiprotozoal drugs, we assessed the effect of extracts obtained from 18 plants and two fungi which are used in traditional medicine in China and the Mediterranean area, against epimastigotes of T. cruzi in axenic cultures. 2. Experimental 2.1. Plant material The botanical species assayed are detailed in Table 1. Ranunculus sceleratus, Scrophularia auriculata, Inula viscosa, Helichrysum italicum and Haplophyllum Table 1 Botanical species Name
Organ
Extract
Yield (% wyw)
Angelica dahurica Bertham et Hoocker f. A. pubescens Maxim. F. A. sinensis (Oliv.) Diels Astragalus membranaceus Bunge Atractylodes macrocephala Koidzumi Codonopsis pilosula (Franch) Nannfeldt Coptis chinensis Franch Curcuma aromatica Salisb. Forsythia suspensa (Thunberg) Vahl. Haplophyllum hispanicum Spach Helichrysum italicum (L.) Moench Inula viscosa (L.) Aiton Lentinus edodes Berk Paeonia lactiflora Pallas Phellodendron amurense Ruprecht Poria cocos (Schw.) Wolf Ranunculus sceleratus L. Rehmania glutinosa (Gaaertnet) Liboschitz Scrophularia auriculata L. Scutellaria baicalensis Georgi
Root Root Wood Root Root Root Root Rhizome Fruit Aerial parts Aerial parts Aerial parts Sclerotium Root Root bark Sclerotium Aerial parts Root Aerial parts Root
Ethanol 70% Ethanol 70% Ethanol 70% Ethanol 70% Aqueous Aqueous Methanol Aqueous Ethanol 70% Methanol Methanol Methanol Aqueous Aqueous Methanol Ethanol 70% Methanol Ethanol 70% Methanol Methanol
10.5 22.5 27.0 16.5 32.5 25.0 17.5 8.0 11.5 8.7 11.2 16.7 8.0 7.0 19.5 1.4 5.9 19.0 6.9 22.0
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
571
hispanicum were collected at Pinedo, Riola, Torrent, Chiva and El Vedat de Torrent (Valencia, Spain), respectively. Specimens of these plants were deposited in the herbarium of the Department of Pharmacology, Faculty of Pharmacy, University of Valencia. The rest of the species were commercial samples obtained from Asia Natural Products S.A. Co., Amposta (Tarragona, Spain). 2.2. Extraction Extraction was performed by stirring plant material (50 g) with the solvent specified in Table 1 at 40 8C. The solvents were removed under vacuum and the residues and water extracts were lyophilised and dissolved in dimethylsulfoxide (DMSO). The percentage yields are indicated in Table 1. 2.3. Chemicals The extraction was performed with ethanol or methanol PA (Merck). Water extracts were obtained with bi-distilled water. The culture medium base used was Medium 199 (Gibco BRL, Life Technologies, NY). The chemicals for the assays were of analytical grade (Sigma Co., St. Louis). 2.4. Animals Male Wistar rats (250–300 g) were used. They were kept in standard environmental conditions and fed with rodent diet with tap water ad libitum. 2.5. In vitro assays T. cruzi epimastigotes (clone Bra C15C2) w6x were cultured in F29 media supplemented with 10% (vyv) heat-inactivated fetal calf serum at 27 8C, with an inoculum of 5=105 cellsyml w7x. Plant extracts were added at a final concentration of 250 mgyml. All assays were carried out in triplicate. Final DMSO concentration was less than 0.5%. Parasites were counted after 72 h of contact with the samples in a Neubauer chamber and stained with Wrigth-Giemsa. The activity of the extracts was assessed by comparison with the negative control (DMSO). Allopurinol was used as a reference drug. 2.6. Cytotoxicity studies The plant extracts cytotoxicity was assessed on elicited peritoneal leukocytes obtained from rats w8x with a viability greater than 95% determined by the trypan blue exclusion test. The cell viability was assessed using two criteria. In the first one, the leakage of the cytoplasmic marker enzyme lactate dehydrogenase (LDH) was determined in supernatant of the incubation of polymorphonuclear cells (PMN) in the presence of extracts (200 mgyml) or vehicle. A commercial kit was used (Boehringer–
572
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
Mannheim, Argentina Lot.yCh.-B: 69685401). The activity of the extracts was assessed by comparison with a positive control (0.2% vyv Triton X100). As a second criteria, we measured the capacity of PMN mitochondrial dehydrogenase enzymes to convert the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan, which is assessed spectrophotometrically at 490 nm w9x. Chlropromazine (200 mM) was used as a reference compound (positive control). 2.7. Statistical analysis Data were expressed as mean"S.D. Statistical analysis was performed by oneway analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons. Differences were considered significant at P-0.05. The inhibitory concentration 50% (IC50) was calculated from the concentrationyeffect regression line. In each case, an appropriate range of 4–5 concentrations was used. 3. Results and discussion In this work, we assessed the anti-trypanosomal activity of extracts obtained from plants and fungus commonly used in the Chinese and Mediterranean medicines. The species were tested against epimastigotes of T. cruzi in axenic cultures. Although the model used neglects the different sensitivity of the other stages of T. cruzi, it is very useful to identify active compounds against the parasite w4x. The anti-trypanosomal activities of the different extracts are summarised in Table 2. When tested at a concentration of 250 mgyml, Coptis chinensis, H. hispanicum, Phellodendron amurense, R. sceleratus and Scutellaria baicalensis were the most active extracts with percentages of inhibition higher than 50%. Some of these plants were already known to have pharmacological properties. We have previously demonstrated that the methanol extracts of C. chinensis and S. baicalensis, plants used in the traditional Chinese medicine, had anti-inflammatory and antioxidant activities in different models w10,11x. On the other hand, another report demonstrated antitrypanosomal effects of S. baicalensis and Coptis japonica on related parasites like bloodstream forms of Trypanosoma brucei rhodesiense in vitro w12x. R. sceleratus and H. hispanicum are species used in phytotherapy in the Mediterranean area. Ranunculus species have been studied in the phytochemical and biological areas but the knowledge of their pharmacological actions is scanty. R. sceleratus particularly was demonstrated to have anti-inflammatory w13x and antifungal activities w14x. Extracts of H. hispanicum were also shown to have antiinflammatory activity w15x but to our knowledge, this is the first description of H. hispanicum and R. sceleratus activities against some specie of the Tripanosomatide family. An important criterion in the search of active compounds against T. cruzi with therapeutic perspectives, is to determine if they show toxic effects on mammalian host cells. For that purpose, two bioassays were used to determine the cytotoxicity
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
573
Table 2 Effect of plant extracts on the growth of epimastigote forms of Trypanosoma cruzi Botanical name Angelica dahurica A. pubescens A. sinensis Astragalus membranaceus Atractyloides macrocephala Codonopsis pilosula Coptis chinensis Curcuma aromatica Forsythia suspensa Haplophyllum hispanicum Helichrysum italicum Inula viscosa Lentinus edodes Paeonia lactiflora Phellodendron amurense Poria cocos Ranunculus sceleratus Rehmania glutinosa Scrophularia auriculata Scutellaria baicalensis Allopurinol Control (DMSO)
Parasites =10y6 a
14.5"1.0** 14.9"4.2** 19.4"0.5** 13.5"2.2** 23.0"1.0 ns 20.8"4.3 ns 0.0"0.0** 21.4"7.1 ns 19.1"4.0 ns 8.5"1.3** 23.0"3.0 ns 27.5"5.8 ns 26.8"2.6 ns 27.9"2.6 ns 11.3"1.4** 16.8"1.3** 0.7"0.3** 24.5"2.1 ns 23.3"1.8 ns 7.5"0.4** 5.4"0.5** 24.0"3.7
% Inhibition 40 38 19 44 4 3 100 11 20 65 4 0 0 0 53 30 97 0 3 69 78 –
a Values represent the parasite numbery5 ml of culture medium and are the mean"S.D. of triplicate determination. **Values are significantly different from those of the control (P-0.01).
Table 3 Cytotoxic effects of plant extracts on elicited rat peritoneal leukocytes Cytotoxicity assays
Control (y) C. chinensis H. hispanicum R. sceleratus S. baicalensis Control (q)
LDH
MTT
16.3"7.3a 13.7"5.5 ns 16.7"3.2 ns 18.7"6.7 ns 28.7"7.4 ns 140.3"2.5**
100b 124"22 ns 116"23 ns 108"14 ns 123"22 ns 69"6**
a
LDH activity is expressed as international unitsyl. Values mean% of cell viability. ** Values are significantly different from those of the negative control group (P-0.01) and are the mean of triplicate determinations. b
of C. chinensis, H. hispanicum, R. sceleratus and S. baicalensis extracts on elicited rat peritoneal leukocytes. MTT and LDH assays assess the metabolic activity and cell membrane damage, respectively. None of the extracts induced lactic dehydro-
574
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
Fig. 1. Dose-dependent inhibition of the growth of Trypanosoma Cruzi by Ranunculus sceleratus and Coptis chinensis extracts.
genase release and did not affect the activity of the mitochondrial succinate dehydrogenase (Table 3). Since C. chinensis and R. sceleratus extracts showed a higher antiparasitic activity than that obtained with the reference drug, they were tested at various concentrations. Fig. 1 shows a dose-response relationship obtained for both extracts. The IC50 values of 1.7 mgyml (r 2s0.9858, P-0.01) for C. chinensis and 10.7 mgyml (r 2s 0.9992, P-0.01) for R. sceleratus reflect the important inhibitory activity of both extracts. Other dose-dependent effects observed with C. chinensis and R. sceleratus were the morphological changes in the epimastigote form of T. cruzi. Inoculation of these extracts at concentration of 250 mgyml produced total lysis of the parasites. In the range of 10–30 mgyml C. chinensis produced vacuolar degeneration and R. sceleratus produced internal granulations. Taken together, the high potency, 5–25 times higher than the values taken as reference in these kind of assays w4x, and the absence of citotoxicity in the model employed, allows us to suggest that R. sceleratus and C. chinensis extracts constitute an important source of new compounds with potential therapeutic applications. Acknowledgments This work was supported by grants from the Ministry of Health of the Province of Buenos Aires and FEMEBA Foundation. We thank Asia Natural Products Co. ´ C. Abel for the for supplying some samples. The authors are grateful to Marıa language revision of the manuscript.
G.R. Schinella et al. / Fitoterapia 73 (2002) 569–575
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References w1 x w2 x w3 x w4 x w5 x w6 x w7 x w8 x w9 x w10x w11x w12x w13x w14x w15x
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