Parasitol Res (2012) 110:521–526 DOI 10.1007/s00436-011-2516-z
ORIGINAL PAPER
Antitrypanosomal activity of some medicinal plants from Nigerian ethnomedicine Oyindamola O. Abiodun & Grace O. Gbotosho & Edith O. Ajaiyeoba & Reto Brun & Ayoade M. Oduola
Received: 19 April 2011 / Accepted: 22 June 2011 / Published online: 26 July 2011 # Springer-Verlag 2011
Abstract Human African trypanosomiasis is a neglected tropical disease with complex clinical presentation, diagnosis, and difficult treatment. The available drugs for the treatment of trypanosomiasis are old, expensive, and less effective, associated with severe adverse reactions and face the problem of drug resistance. This situation underlines the urgent need for the development of new, effective, cheap, and safe drugs for the treatment of trypanosomiasis. The search for new antitrypanosomal agents in this study is based on ethnomedicine. In vitro antitrypanosomal activity of 36 plant extracts from 10 plant species from Nigerian
O. O. Abiodun (*) Department of Pharmacology and Therapeutics, University of Ibadan, Ibadan, Nigeria e-mail:
[email protected] G. O. Gbotosho Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Ibadan, Nigeria E. O. Ajaiyeoba Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria R. Brun Parasite Chemotherapy Unit, Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland A. M. Oduola Strategic and Discovery Research, Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization, 1211 Geneva 27, Switzerland
ethnomedicine was evaluated against bloodstream forms of Trypanosoma brucei rhodesiense STIB 900. Cytotoxic activity was determined against mammalian L6 cells. Alamar blue assay was used to measure the endpoint of both antitrypanosomal and toxicity assays. The ethyl acetate extract of leaves of Ocimum gratissimum Linn. (Labiatae) showed the highest antitrypanosomal activity (IC50 of 2.08±0.01 μg/ml) and a high selective index of 29. Furthermore, the hexane, ethyl acetate, or methanol extracts of Trema orientalis (L.) Blume (Ulmaceae), Pericopsis laxiflora (Benth. ex Baker) Meeuwen, Jatropha curcas Linn. (Euphorbiaceae), Terminalia catappa Linn. (Combretaceae), and Vitex doniana Sweet (Verbenaceae) displayed remarkable antitrypanosomal activity (IC50 2.1– 17.2 μg/ml) with high selectivity indices (20–80) for trypanosomes. The antitrypanosomal activity of T. catappa and T. orientalis against T. brucei rhodesiense (STIB 900) is being reported for the first time in Nigerian ethnomedicine, and these plants could be a potential source of antitrypanosomal agents.
Introduction Human African trypanosomiasis (HAT) is caused by a protozoan parasite of the genus Trypanosoma and transmitted by tsetse flies (Glossina morsitans). Trypanosoma brucei rhodesiense causes the acute disease in East Africa, while Trypanosoma brucei gambiense causes chronic disease in West and Central Africa. The two forms of HAT are characterized by an initial hemolymphatic stage, followed by a second central nervous infection when the parasites crossed the blood–brain barrier. However, in T. brucei rhodesiense infection, there is early invasion of the central nervous system. Trypanosomiasis is a major health concern in many
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African countries where about 50 million people are at the risk of infection (Févre et al. 2006). Prevalence is strongly dependent on control measures, which are often neglected during periods of political instability, thus leading to resurgence (Brun et al. 2010). In the absence of effective vector control to reduce the number of flies in existing foci, chemotherapy remains the mainstay of control of trypanosomiasis. However, the available drugs for the treatment of trypanosomiasis are old, expensive, complicated to administer, less effective, cause severe adverse reactions, and face the problem of drug resistance (Hotez et al. 2007; WHO 2009). This situation underlines the urgent need for the development of new, effective, cheap, and safe drugs for the treatment of trypanosomiasis. To date, there is no drug of plant origin that is available for the treatment of trypanosomiasis, however some plants have been shown to possess considerable antitrypanosomal activities (Hoet et al. 2004; Atawodi 2005; Shuaibu et al. 2008). Also, approximately 80% of the world population uses traditional medicine, primarily based on natural products (Macía et al. 2005). This inspired us to further intensify the search for antitrypanosomal agents from the plant compendium. Thus the present study investigates the in vitro antitrypanosomal and cytotoxic activities of some plants used for the treatment of febrile illnesses in Nigerian ethnomedicine.
Materials and methods Plant collection and authentication All the plant species except Cassia siamea were collected between January and March 2006, in Ibadan, Oyo State Nigeria. C. siamea stem bark was collected in Otu in Oyo State of Nigeria in January 2002. These 10 plant species from eight families were identified and authenticated by Mr. Osiyemi and late Mr. Usang Felix of the Forestry Research Institute of Nigeria, Ibadan where voucher specimens were deposited (Table 1). Extraction of plant material Plant parts were air-dried, powdered, and extracted three times at 5 min interval sequentially with hexane, ethyl acetate, and methanol using an accelerated solvent extractor (ASE 200 Dionex Corporation, Switzerland), at a temperature of 70°C and pressure 120.0 bar. The extracts were filtered and solvents removed using a rotary evaporator under reduced temperature and pressure. Plant extracts were stored at−20°C till needed for analysis. In total 36 extracts from 10 plant species were prepared.
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Preparation of stock solutions of plant extracts and standard drugs Stock solutions (10 mg/ml) of plant extracts and standard antitrypanocidal drug melarsoprol were prepared in dimethyl sulfoxide (DMSO) and diluted with culture media on the day of experiment to the desired starting concentration. The DMSO concentration never exceeded 0.5% and did not inhibit the parasite growth. In vitro antitrypanosomal activity In vitro antitrypanosomal activity was determined using the Alamar Blue assay (Räz et al. 1997). Briefly, T. brucei rhodesiense (STIB 900) were cultivated and maintained in minimum essential medium as previously described (Baltz et al. 1985) with 0.2 mM 2-mercaptoethanol, 1 mM Na pyruvate, and 15% heat-inactivated horse serum. Serial drug dilutions of plant extracts and melarsoprol ranging from 450 to 0.62 μg/ml and 90 to 0.13 μg/ml were prepared in a 96-well plate respectively. Bloodstream forms of T. brucei rhodesiense (STIB 900) from axenic culture (4×104/ml) were added to each well of the 96-well plate and incubated at 37°C under 5% CO2 for 69 h. Thereafter, resazurin (0.125 mg/ml) was added to each well and the plate was further incubated for additional 2– 4 h. Fluorescence development in assay plate was measured using Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) by using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. Fluorescence development measured was expressed as the percentage of the control. Data were transferred into the graphic program Softmax Pro (Molecular Devices Cooperation) which calculated 50% inhibitory concentration (IC50) values from the sigmoidal inhibition curves. Determination of cytotoxic activity of plant extracts Rat skeletal myoblast L6 cell maintained in RPMI 1640 (Roswell Park Memorial Institute medium) media containing 10% fetal calf serum and 1.7 μM L-glutamate was used to assess cytotoxic activity of the plant extracts. The cytotoxic activity of plant extracts was determined according to previously described methods (Ahmed et al. 1994). Briefly, L6 cells were seeded in 96-well microtiter plates at a density of 4×103 L6 cells/well in culture medium and exposed to serial drug or plant extract dilutions. The assay plates were incubated for 70 h at 37°C in 5% CO2. After 70 h of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. Then resazurin at 0.125 mg/ml was added to each well and the plates were further incubated for 2 h. The
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Table 1 In vitro antitrypanosomal activity and cytotoxicity of plants from Nigerian ethnomedicine Plant parts
Plant parts
Herbarium number
Mean IC50 ±SEM (μg/ml) T. brucei rhodesiense (STIB 900)
Rat skeletal myoblast cells (L6)
Selectivity index (L6)/STIB 900
10.26±3.52
226.46±5.91
22.07
3.50±0.59
32.53±3.30
9.29
11.70±3.61
409.02±0.59
34.96
Hexane extract
13.46±2.70
52.62±2.54
3.91
Ethyl acetate extract
13.87±2.57
46.16±2.64
3.33
Methanol extract
20.35±6.67
129.38±4.04
6.36
10.71±3.97
400.02±6.15
38.81
7.36±0.50
85.48±3.13
11.61
23.07±7.55
>450
19.51
Hexane extract
10.92±1.98
431.35±3.21
38.22
Ethyl acetate extract
17.04±1.13
122.6±0.70
8.81
Methanol extract
40.98±2.50
>450
10.98
T. orientalis (L.) Blume (Ulmaceae)
L
FHI 107813
Hexane extract Ethyl acetate extract Methanol extract T. orientalis (L.) Blume (Ulmaceae)
P. laxiflora (Benth. ex Baker) Meeuwen (Leguminose)
B
L
FHI 107813
FHI 107814
Hexane extract Ethyl acetate extract Methanol extract V. doniana Sweet. (Verbenaceae)
V. doniana Sweet. (Verbenaceae)
L
B
FHI 108354
FHI 108354
Hexane extract
6.58±1.05
ND
NA
11.79±2.05
335.47±10.47
29.34
58.16±3.62
>450
7.74
Hexane extract
9.67±1.20
414.76±10.55
64.65
Ethyl acetate extract
4.72±0.06
126.45±4.45
26.5
26.35±1.86
>450
17.08
17.24±2.79
276.75±3.51
25.03
7.80±1.83
159.92±2.99
20.5
13.91±2.21
377.59±6.84
27.14
Hexane extract
17.48±0.05
161.45±5.97
19.44
Ethyl acetate extract
10.56±0.78
77.69±3.0
8.41
Methanol extract
22.57±0.47
427.54±3.5
18.94
Hexane extract
12.56±0.09
169.08±10.16
12.11
Ethyl acetate extract
42.49±0.99
>450
10.59
30.37±4.40
>450
14.82
Hexane extract
9.91±2.13
14.19±0.35
1.43
Ethyl acetate extract
8.70±2.94
65.79±0.27
13.66
54.24±3.39
>450
8.3
Hexane extract
2.38±0.13
34.66±4.05
14.56
Ethyl acetate extract
2.08±0.01
60.14±3.75
28.91
Methanol extract
5.45±1.14
424.20±5.24
76.89
Hexane extract
25.88±4.17
50.40±0.59
1.95
Ethyl acetate extract
10.19±1.74
14.00±1.22
1.46
Methanol extract
46.50±4.11
105.92±3.87
2.22
Ethyl acetate extract Methanol extract J. curcas Linn. (Euphorbiaceae)
L
FHI 108355
Methanol extract T. catappa Linn. (Combretaceae)
L
FHI 107812
Hexane extract Ethyl acetate extract Methanol extract P. amarus Schum & Thonn (Euphorbiaceae)
I. cylindrica P. Beauv. (Poaceae)
L
L
FHI 108356
FHI 107172
Methanol extract E. hirta Linn. (Euphorbiaceae)
L
FHI 108351
Methanol extract O. gratissimum Linn. (Labiatae)
C. siamea Fabaceae
L
L
FHI 108353
FHI 106558
Melasoprol Podophyllotoxin
1.65×10−3 ±0.53×10−3
ND
NA
ND
0.52×10−5 ±0.2×10−5
NA
The IC50 values given are means±standard error of two (cytotoxicity assay) or three (antitrypanosomal assay) independent assays, each assay was run in duplicate SEM standard error, L leaves, B stem bark, ND not done, NA not available
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assay plates were evaluated as described for T. brucei rhodesiense assay. Podophyllotoxin was used as a positive reference. Classification of activity Antitrypanosomal activity of plant extracts was classified as active (IC50 30 μg/ml). For the purpose of this study, an extract lacked toxicity to L6 cells by displaying an IC50 value >90 μg/ml.
Results and discussion The 10 plant species, the part of plants extracted, and percentage yields after extraction are shown in Table 1. The IC50 of plant extracts against T. brucei rhodesiense (STIB 900) bloodstream forms, mammalian L6 cells, and selective indices are presented in Table 1. Of the 36 plant extracts tested against the T. brucei rhodesiense (STIB 900) bloodstream forms, 11 were active (30 μg/ml). The ethyl acetate and hexane extract of leaves of Ocimum gratissimum showed the highest activity against T. brucei rhodesiense (STIB 900) bloodstream forms (IC50 = 2.08 ± 0.01 μg/ml and 2.38 ± 0.13 μg/ml, respectively). This was followed by the ethyl acetate extract of leaves of Trema orientalis (IC50 = 3.50 ± 0.59 μg/ml). Furthermore, the IC50 values of plant extracts against mammalian L6 cell were very high for 24 out of the 35 plants extracts evaluated (IC50 >90 μg/ml), signifying low toxicity. The least toxic extracts were ethyl acetate and methanol extract of leaves or stem bark of Vitex doniana, Pericopsis laxiflora, Jatropha curcas, Imperata cylindrica, and Euphorbia hirta with IC50 value >450 μg/ml (Table 1). In contrast, the remaining extracts displayed IC50 20). The highest SI values (SI=80)
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was obtained from methanol extract of leaves of O. gratissimum. Likewise, hexane, ethyl acetate, or methanol extracts of leaves or stem bark of O. gratissimum, T. orientalis, P. laxiflora, J. curcas, Terminalia catappa, and V. doniana are of particular interest associating antitrypanosomal activity (IC50 2.1–17.2 μg/ml) with high selectivity indices (20–77). Antimalarial and cytotoxicity activities of some of the plants included in this study have been described previously (Abiodun et al. 2010, 2011). The antitrypanosomal activity of the following plants T. catappa, T. orientalis, I. cylindrica P. Beauv. (Poaceae) and Phyllanthus amarus Schum & Thonn (Euphorbiaceae) evaluated in this study against T. brucei rhodesiense is being reported for the first time in Nigerian ethnomedicine. All the three extracts of T. catappa displayed great selectivity for T. brucei rhodesiense over L6 cells. Previous reports of in vitro antitrypanosomal activity of other species of the genus Terminalia, Terminalia avicennioides, had been reported (Bizimana et al. 2006; Shuaibu et al. 2008). Flavonoid glycosides, squalene, ursolic acid, 2α, 3β, 23-trihydroxyurs-12-en-28-oic and hydrolysable tannins were isolated from the leaves of T. catappa (Fan et al. 2004; Chen and Li 2006; Lin et al. 2001). Similarly, hydrolyzable tannins such as ellagic acid, flavogallonic acid, punicalagin, and terchebulin isolated from stem bark of T. avicennioides showed in vitro antitrypanosomal activity with no cytotoxic effect on a mammalian cell line (Shuaibu et al. 2008). Furthermore, all the hexane and methanol extracts of the leaves of T. orientalis showed great selectivity for T. brucei rhodesiense over L6 cells Phytochemical investigation of the trunk and root bark of T. orientalis afforded compounds such as xanthones, secoiridoids, ursane derivatives, triterpenes phytosteroids, dihydrophenanthrene, and phenyldihydroisocoumarin (Tchamo et al. 2001; Dijoux-Franca et al. 2001). Antitrypanosomal activity of xanthones isolated from the root bark of Garcinia livingstonei has been reported. Also, one of the dimeric xanthone garcilivin A showed a higher and nonselective antiparasitic activity and cytotoxicity (Sordat-Diserens et al.1992; Mbwambo et al. 2006). seco-Iridoids isolated from the wood bark of Calycophyllum spruceanum showed weak in vitro activity against trypomastigote forms of Trypanosoma cruzi (Cardona Zuleta et al. 2003). Triterpenes and phytosterols from Strychnos spinosa showed in vitro antitrypanosomal activity (Hoet et al. 2007). Only the hexane extract of I. cylindrica showed antitrypanosomal activity but lacked strong selectivity for T. brucei rhodesiense over L6 cells. Phytochemical investigation of the aerial parts of I. cylindrica afforded four methoxylated flavonoids, β-sitosterol-3-0-β-D-glucopyranosyl-6′-tetradecanoate, 3-hydroxy-4-methoxybenzaldehyde, together with daucosterol, β-sitosterol, and αamyrin (Mohamed et al. 2009). All extracts of P. amarus
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showed moderate antitrypanosomal activity. Ethyl acetate extract exhibited a nonspecific activity against T. brucei rhodesiense and mammalian L6 cells, while hexane and methanol showed borderline selectivity. Antitrypanosomal activity and nonspecific cytotoxicity in neoplastic and primary cell cultures of extracts and compounds (justicidin B and piscatorin) from Phyllanthus piscatorum were previously reported (Gertsch et al. 2003). In vitro antitrypanosomal activity of some of the plants evaluated in this study has been previously reported. The antitrypanosomal activity of these plants was confirmed in this study. This was the case for P. laxiflora (Benth. ex Baker) Meeuwen (Leguminose) (Hoet et al. 2004), C. siamea Fabaceae, J. curcas Linn. (Euphorbiaceae) (Bacchi 2002), V. doniana Sweet (Verbenaceae) (Atawodi 2005), and O. gratissimum Linn. (Labiatae) (Luize et al. 2005; Adamu et al. 2009). In contrast, Bawm and co-workers reported that E. hirta Linn. (Euphorbiaceae) lacked antitrypanosomal activity against Trypanosoma evansi (Bawm et al. 2010). However, activity was observed in this study. The difference in the antitrypanosomal activity observed in this study and the previous study may be as a result of many factors such as the specie of the parasite used, season, age, intraspecies variation, soil, climate, methods used for extraction, and bioassay (Muregi et al. 2003).
Conclusion The antitrypanosomal activity of T. catappa, T. orientalis, I. cylindrica, and P. amarus against T. brucei rhodesiense (STIB 900) is being reported for the first time in Nigerian ethnomedicine, and these plants could be a potential source of antitrypanosomal agents. Investigations to identify the active antitrypanosomal compounds from these plants are in progress. It is hoped that a lead compound may be identified that could be developed as antitrypanosomal agent. Acknowledgments Oyindamola Abiodun was supported at Swiss Tropical and Public Health Institute by a training fellowship from the Medicines for Malaria Venture. Declarations of interest Nil.
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