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PHYTOTHERAPY RESEARCH Phytother. Res. 21, 366–368 (2007) Published online 18 January 2007 in Wiley InterScience J. E. OKOKON ET AL. (www.interscience.wiley.com) DOI: 10.1002/ptr.2059
Antiplasmodial Activity of Setaria megaphylla Jude E. Okokon1*, Peace M. E. Ubulom2 and Aniekan E. Udokpoh2 1 2
Pharmacology and Toxicology Department, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria
The antimalarial activity of an ethanol leaf extract of Setaria megaphylla was studied in vivo in mice infected with Plasmodium berghei berghei during early and established infections. Setaria megaphylla (100–300 mg/ kg/day) exhibited a significant (p < 0.05) blood schizonticidal activity in 4-day early infection and in established infection with a significant (p < 0.05) mean survival time comparable to that of the standard drug, chloroquine, 5 mg/kg/day. The leaf extract possesses a promising antiplasmodial activity in vivo which can be exploited in malaria therapy. Copyright © 2007 John Wiley & Sons, Ltd. Keywords: Setaria megaphylla; malaria; Plasmodium berghei berghei.
INTRODUCTION Setaria megaphylla (Steud) Dur & Schinz (Poaceae), also called broad leafed brittle grass, is a tall, robust, tufted, perennial grass used mainly as pasture grass. It occurs in tropical and subtropical areas of Africa, America and India where there is high rainfall (Van Oudtshoorn, 1999; Lowe, 1989). The plant is used traditionally by the Ibibios ethnic group in Akwa Ibom State, Nigeria in the treatment of various ailments including haemorrhoids, urethritis and diabetes. The plant has also been reported to possess antiplasmodial activity in vitro (Clarkson et al., 2004). The ethanol leaf extract (LD50 is reported to be 2.4 ± 0.5 g/kg) contains flavonoid, carbohydrate, terpenes, saponins, tannins, anthraquinones and cardiac glycosides and has hypoglycaemic and antidiabetic activity (Okokon and Antia, 2006). The present study was aimed at evaluating in vivo the antiplasmodial activity of the leaf extract against Plasmodium berghei infection in mice.
Animals Albino Swiss mice (21–28 g) of either sex were obtained from the University of Uyo animal house. They were maintained on standard animal pellets and water ad libitum. Permission and approval for animal studies were obtained from the College of Health Sciences Animal Ethics Committee, University of Uyo. Parasite inoculation. The chloroquine-sensitive Plasmodium berghei berghei was obtained from National Institute of Medical Research, Lagos, Nigeria and maintained in mice. The inoculum consisted of 5 × 107 P. berghei berghei parasitized erythrocytes/mL. This was prepared by determining both the percentage parasitaemia and the erythrocyte count of the donor mouse and diluting the blood with isotonic saline in proportions indicated by both determinations. Each mouse was inoculated on day 0, intraperitoneally, with 0.2 mL of infected blood containing about 1 × 107 P. berghei berghei parasitized red blood cells.
Plant materials. Fresh leaves of Setaria megaphylla were collected in November, 2005 at Anwa forest in Uruan, Akwa Ibom State, Nigeria. The plant was identified and authenticated by Dr Margaret Bassey, a taxonomist in the Department of Botany, University of Uyo, Uyo. Nigeria. A herbarium specimen was deposited at Faculty of Pharmacy Herbarium with voucher no. FPHUU 221. The fresh leaves (2 kg) of the plant were dried on a laboratory table for 2 weeks and reduced to powder. The powder (100 g) was macerated in 95% ethanol (300 mL) for 72 h. The liquid filtrate obtained was concentrated in vacuo at 40 °C. The yield was 2.08% w/w. The extract was stored in a refrigerator at 4 °C until used for experiment reported in this study.
Evaluation of schizonticidal activity on early infection (4 day test). The schizonticidal activity of the extract was evaluated using the method described by Knight and Peters (1980). Each mouse was inoculated on the first day (day 0), intraperitoneally, with 0.2 mL of infected blood containing about 1 × 107 P. berghei berghei parasitized erythrocytes. The animals were divided into five groups of five mice each and orally administered, shortly after inoculation with 100, 200 and 300 mg/kg/ day doses of the S. megaphylla extract, chloroquine 5 mg/kg/day and an equivalent volume of distilled water (negative control) for 4 consecutive days (day 0 to day 3). On the fifth day (day 4), thin films were made from the tail blood of each mouse and the parasitaemia level was determined by counting the number of parasitized erythrocytes out of 200 erythrocytes in random fields of the microscope. The average percentage chemosuppression was calculated as
* Correspondence to: Jude E. Okokon, Pharmacology and Toxicology Department, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria. E-mail: judeefi
[email protected]
⎛ A − B⎞ 100 ⎜ ⎟ ⎝ A ⎠
MATERIALS AND METHODS
Copyright © 2007 John Wiley & Sons, Ltd. Copyright © 2007 John Wiley & Sons, Ltd.
Received 18 September 2006 Phytother. Res. 21, 366–368 (2007) Revised DOI: 25 October 2006 10.1002/ptr Accepted 25 October 2006
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ANTIPLASMODIAL ACTIVITY OF SETARIA MEGAPHYLLA
Table 1. Antiplasmodial activity of S. megaphylla during 4-day test
Drug/Extract
S. megaphylla extract
Chloroquine (standard) Distilled water (control)
Dose (mg/kg/day) 100 200 300 5 0.2 mL
Average (%) parasitaemia
Average (%) suppression
± ± ± ± ±
62.89 76.44 85.70 88.19 –
16.59 10.53 6.39 5.28 44.71
0.33a 0.46a 0.38a 0.11a 2.04
Data are expressed as mean ± SD for five animals per group. a p < 0.05 when compared with control.
where A is the average percentage parasitaemia in the negative control group and B, average percentage parasitaemia in the test group. Evaluation of schizonticidal activity established infection (curative or Rane test). Evaluation of the curative potential of the extract was done using a method similar to that described by Ryley and Peters (1970). The mice were injected intraperitoneally with standard inoculum of 1 × 107 P. berghei berghei infected erythrocytes on the first day (day 0). Seventy-two hours later, the mice were divided into five groups of five mice each. The groups were orally administered with S. megaphylla extract (100, 200, 300 mg/kg/day), chloroquine (5 mg/ kg) was given to the positive control group and an equal volume of distilled water to the negative control group. The drug/extract was given once daily for 5 days. Thin films stained with Giemsa stain were prepared from the tail blood of each mouse daily for 5 days to monitor the parasitaemia level. The mean survival time for each group was determined arithmetically by finding the average survival time (days) of the mice (post inoculation) in each group over a period of 28 days (day 0–27). Statistical analysis. Data obtained from the study were analysed statistically using Student’s t-test and values of p < 0.05 were considered significant.
RESULTS
Figure 1. Effect of S. megaphylla on established infection (curative test).
Table 2. Mean survival time of mice receiving various doses of ethanol extract of S. megaphylla
Drug/Extract
S. megaphylla extract
Chloroquine (standard) Distilled water (control)
Dose (mg/kg/day) 100 200 300 5 0.2 mL
Mean survival time (day) 11.66 17.66 28.0 28.0 10.33
± ± ± ± ±
1.24a 1.42a 0.00a 0.00a 0.94
Data are expressed as mean ± SD for five animals per group. a p < 0.05 when compared with control.
4-day test The ethanol root extract of S. megaphylla produced a dose dependent chemosuppressive effect at the various doses employed in this study. The chemosuppressions were 62.89%, 76.44% and 85.70%, respectively, for 100, 200 and 300 mg/kg/day doses. The chemosuppressions produced by the extract were significant (p < 0.05) compared with the control and comparable to that of the standard drug (chloroquine 5 mg/kg/day) with a chemosuppression of 88.8% (Table 1).
On day 7, the average percentage parasitaemia for the groups were 30.0%, 28.0%, 7.0%, 6.0% and 73.0% for 100, 200, 300 mg/kg/day of the extract, chloroquine and control groups, respectively (Fig. 1). The mean survival time (m-s.t.) of the extract treated groups were significantly (p < 0.05) longer than that of the control and was comparable to that of the standard drug, chloroquine. The values are given in Table 2.
DISCUSSION Curative test On established infection, it was observed that there was a daily increase in parasitaemia of the control group. However, there was a daily reduction in the parasitaemia levels of the extract treated group as well as that of the positive control (chloroquine). Copyright © 2007 John Wiley & Sons, Ltd.
In this study, preliminary phytochemical screening and acute toxicity studies as well as evaluation of antiplasmodial activity of ethanol root extract of S. megaphylla were carried out. The leaf was found to contain terpenes and flavonoids which have been reported to be responsible for the antimalarial activities of plants (Philipson Phytother. Res. 21, 366–368 (2007) DOI: 10.1002/ptr
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and Wright, 1991; Christensen and Kharazmi, 2001). These compounds could have elicited the observed antiplasmodial activity either singly or in synergy with each other. The results also indicated that the root extract possesses blood schizonticidal activity as evident from the chemosuppression obtained during the 4-day early infection test. The activity of the extract was comparable to that of the standard drug, chloroquine (5 mg/kg/ day) at the doses studied. On established infection, the extract exhibited a significant curative activity though not as much as the standard drug, chloroquine. The curative effect of the extract was further demonstrated in the significant mean survival time of the extracttreated groups compared with control. The chloroquine and extract doses employed in this study could not offer 100% parasitaemia clearance in the infected mice, the crude nature of the extract. This
activity could be improved by further purification of the drug and/or the extract.
CONCLUSION The results of this study correlate well with that reported in vitro by Clarkson et al. (2004) and confirms the traditional usage of this plant as malaria remedy. Therefore, it would be interesting if the active principle could be isolated, identified and characterized from this promising medicinal plant. Acknowledgement The authors are grateful to Mr Nsikan Malachy for his technical assistance.
REFERENCES Christensen SB, Kharazmi A. 2001. Antimalarial natural products. Isolation, characterization and biological properties. In Bioactive Compounds from Natural Sources: Isolation, Characterization and Biological Properties, Tringali C (ed.). Taylor & Francis: London, 379–432. Clarkson C, Maharay VJ, Crouch NR et al. 2004. In vitro antiplasmodial activity of medicinal plants native to or naturalized in South Africa. J Ethnopharmacol 92: 177–191. Knight DJ, Peters W. 1980. The antimalarial action of Nbenzyloxy dihydrotriazines. The action of clociguanil (BRL50216) against rodent malaria and studies on its mode of action. Ann Trop Med Parasitol 74: 393–404.
Copyright © 2007 John Wiley & Sons, Ltd.
Lowe J. 1989. The Flora of Nigeria Grasses, 2nd edn. Ibadan University Press: Ibadan, Nigeria. Okokon JE, Antia BS. 2006. Hypoglycaemic and antidiabetic activity of ethanolic leaf extract of Setaria megaphylla on normal and alloxan diabetic rats. J Nat Remedy (In Press). Philipson JD, Wright CW. 1991. Antiprotozoal compounds from plants sources. Planta Med 57: 553–559. Ryley JF, Peters W. 1970. The antimalarial activity of some quinone esters. Ann Trop Med Parasitol 84: 209–222. Van Oudtshoorn FP. 1999. Guide to Grasses of South Africa. Briza Publications. Cape Town.
Phytother. Res. 21, 366–368 (2007) DOI: 10.1002/ptr
