Journal of Ethnopharmacology 111 (2007) 464–467
Evaluation of antiplasmodial activity of ethanolic seed extract of Picralima nitida Jude E. Okokon a,∗ , B.S. Antia b , A.C. Igboasoiyi c , E.E. Essien c , H.O.C. Mbagwu a a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria b Department of Chemistry, University of Uyo, Uyo, Nigeria c Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria Received 19 October 2006; received in revised form 29 November 2006; accepted 8 December 2006 Available online 20 December 2006
Abstract The in vivo antiplasmodial activity of the ethanol seed extract of Picralima nitida grown particularly for the leaf and seed in Niger Delta region of Nigeria was evaluated in Plasmodium berghei berghei infected mice. Picralima nitida (35–115 mg/kg day) exhibited significant (P < 0.05) blood schizonticidal activity both in 4-day early infection test and in established infection with a considerable mean survival time though not comparable to that of the standard drug, chloroquine, 5 mg/kg day. The seed extract possesses significant (P < 0.05) antiplasmodial activity which correlate with it reported in vitro activity. © 2007 Published by Elsevier Ireland Ltd. Keywords: Malaria; Antiplasmodial; Picralima nitida
1. Introduction Malaria is still a major health problem in Nigeria and other tropical countries where transmission of the disease is rarely control. Poor and inadequate drainage system in most towns and villages has provided a safe breeding environment for the vectors, thus causing repeated infection on the population at a very alarming rate. Drug resistance, fake drug syndrome and high cost of newer effective drugs have been the major factors affecting the poor populace, thus making their choice of herbal remedies inevitable and economical. Herbal preparations are greatly patronise in the treatment of malaria in Nigeria and is available at roadsides of most urban cities eventhough there is little or, if any, scientific evidence of their efficacy. The aim of this work was to evaluate the antiplasmodial activity of Picralima nitida in vivo. Picralima nitida (Staph) Th & HD (Apocynaceae) is a glaborous shrub of 3–10 m high, occuring in African forest region from Ivory coast to Zaire and Uganda (Ajanohoun et al., 1996). Ethnobotanically, the stem bark is used to pre-
∗
Corresponding author. Tel.: +234 802 3453678. E-mail address:
[email protected] (J.E. Okokon).
0378-8741/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2006.12.016
pare remedies to treat malaria and sexual impotence, while the fruits are used for dysmenorrhoea and gastrointestinal disorder (Ajanohoun et al., 1996). The various part of the plant have been reported to be effective antipyretic, antihypertensive, hypoglycaemic and antitussive (Oliver, 1960; Dalziel, 1961; Ayensu, 1978). The plants seed have been reported to contain alkaloids like akuammine, akuammicine, akuammidine, picratidine, akuammigine, pseudoakuammigine, picraline and picralicine (Guyledouble et al., 1964; Moller et al., 1972; Arens et al., 1982; Ezekwesili, 1983; Ansa et al., 1990). The plant have been reported to have antiinflammatory and analgesic (Duwiejua et al., 2002), antidiarrhoeal (Kouitcheu et al., 2006), antimicrobial (Fakeye et al., 2000; Nkere and Iroegbu, 2005) in vitro antiplasmodial (Iwu and Klayman, 1992; Iwu et al., 1992a; Kapadia et al., 1993; Ezeamuzie et al., 1994; Francois et al., 1996), hypoglycaemic (Inya-Agha, 1999; InyaAgha et al., 2006), antileishmanial (Iwu et al., 1991; Iwu et al., 1992b), antitrypanosomial (Wosu and Ibe, 1989). However, there is apparently no scientific report on the in vivo antiplasmodial potential of the ethanolic seed extract of Picralima nitida in Plasmodium berghei infection in mice. The aim of this work was to evaluate the antiplasmodial activity of Picralima nitida in vivo and correlate with its reported in vitro activity.
J.E. Okokon et al. / Journal of Ethnopharmacology 111 (2007) 464–467
2. Materials and methods 2.1. Plant materials Fresh seeds of Picralima nitida were collected in August, 2006 from a forest in Ubulu, in Oru west LGA of Imo State, Nigeria. The plant was identified and authenticated by Dr. Margaret Bassey, a taxonomist in the Department of Botany, University of Uyo, Uyo, Nigeria. Hebarium specimen was deposited at Faculty of Pharmacy Hebarium. The fresh seeds (2 kg) of the plant were dried on 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 2.98% (w/w). The extract was stored in a refrigerator at 4 ◦ C until used for experiment reported in this study. 2.2. 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. 2.3. 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 Plasmodium berghei berghei parasitized erythrocytes per ml. This was prepared by determining both the percentage parasitaemia and the erythrocytes 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 Plasmodium berghei beghei parasitized red blood cells.
465
4), thin films were made from the tail blood of each mouse and the parasitaemia level was determined by counting the number of parasitised erythrocytes out of 200 erythrocytes in random fields of the microscope. Average percentage chemosuppression was calculated as A−B × 100, A where A is the average percentage parasitaemia in the negative control A group and B is the average percentage parasitaemia in the test group. 2.6. Evaluation of schizontocidal activity established infection (curative or Rane test) Evaluation of 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 Plasmodium 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 Picralima nitida seed extract (35, 70, 115 mg/kg day) in acqeous solution, 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 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 (days 0–27). 2.7. Statistical analysis Data obtained from the study were analyzed statistically using Student’s test and values of P < 0.05 were considered significant. 3. Results
2.4. Determination of LD50 3.1. Acute toxicity The LD50 of the extract was determined using albino mice by intraperitoneal (i.p.) route using the method of Lorke (1983). 2.5. Evaluation of schizontocidal activity on early infection (4-day test) Schizontocidal 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 Plasmodium berghei berghei parasitized erythrocytes. The animals were divided into five groups of five mice each and orally administered, shortly after inoculation with 35, 70 and 115 mg/kg day doses of the Picralima nitida seed extract in acqeous solution, chloroquine 5 mg/kg day and an equivalent volume of distilled dater (negative control) for 4 consecutive days (days 0–3). On the fifth day (day
The mice were treated intraperitoneally with a single dose of 1–5 g/kg of Picralima nitida seed extract after being starved for 24 h. The route was chosen because of its sensitivity and rapid results. Picralima nitida seed extract (1–5 g/kg) produced physical signs of toxicity 30 min to 1 h after administration. These signs include writhing, decreased motor activity, gasping, decreased respiratory and body/limb tone and death. The intensities of these effects were proportional to the dose administered. All the animals treated with 3 and 4 g/kg of the extract died and the LD50 was calculated to be 387.29 mg/kg. 3.2. 4-Day test Ethanolic seed extract of Picralima nitida produced a dose dependent chemosuppressive effect at various doses employed
466
J.E. Okokon et al. / Journal of Ethnopharmacology 111 (2007) 464–467
Table 1 Antiplasmodial activity of Picralima nitida seed extract during 4-day test Drug/extract
Dose (mg/kg day)
Picralima nitida extract
35 70 115 5 0.2 ml
Chloroquine (standard) Distilled water (control)
Average (%) parasitaemia 15.33 13.13 12.0 4.33 44.5
± ± ± ± ±
1.25* 1.69* 1.41* 1.24* 3.26
Average (%) suppression 65.5 70.4 73.0 90.2 –
Data are expressed as mean ± S.D. for five animals per group. * P < 0.05 when compared to control.
4. Discussion
Fig. 1. Effect of Picralima nitida on established infection.
in this study. The chemosuppression were 65.5, 70.4 and 73.0%, respectively for 35, 70, and 115 mg/kg day doses. The chemosupression produced by the extract were significant (P < 0.05) compared to control and uncomparable to that of the standard drug (chloroquine 5 mg/kg day) with a chemosuppression of 90.2% (Table 1). 3.3. 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 positive control (chloroquine). On day 7, the average percentage parasitaemia for the groups were 45.0, 27.0, 25.0, 9.0 and 75.0% for 35, 70, 115 mg/kg day of the extract, chloroquine and control groups, respectively (Fig. 1). The mean survival times (m.s.t.) of the extract treated groups were significantly (P < 0.05) longer than that of control and were comparable to that of the standard drug, chloroquine. The values are given in Table 2.
In this study acute toxicity and evaluation of in vivo antiplasmodial potential of ethanolic seed extract of Picralima nitida were carried out. The extract was found to be moderately toxic (Homburger, 1989) with a LD50 value of 387.29 mg/kg. In vitro antimalarial potential of the various parts (leaf, roots, seed, stembark and fruit-rind) have been reported (Iwu and Klayman, 1992; Iwu et al., 1992a,b; Kapadia et al., 1993; Ezeamuzie et al., 1994; Francois et al., 1996). Various degrees of the antiplasmodial activity of the parts on Plasmodium falciparum were reported. The activity of the various parts have been linked to the indole alkaloids present in this plants (Iwu and Klayman, 1992; Iwu et al., 1992a,b; Kapadia et al., 1993). Akuammine, an indole alkaloid in the seed has been reported to be responsible for the antiplasmodial activity in the seed (Kapadia et al., 1993). However, alkaloids like cryptolepine from Cryptolepis sanguinolenta, use to treat malaria in Ghana, is highly active against human malaria parasite in vitro but not in vivo (Kirby et al., 1995). The result of this study shows that the ethanolic leaf extract possesses a significant in vivo antiplasmodial activity in both early and established infections in addition to the reported in vitro activity. Though this activity was uncomparable to that of the standard drug perhaps due to the crude nature of the extract. The mechanism of action of the extract needs to be elucidated although some plants are known to exert antiplasmodial action either by causing red cell oxidation (Etkin, 1997) or by protein synthesis inhibition (Kirby et al., 1989). The extract could have elicited its action through any of the above-mentioned mechanism or by some other means yet to be determined. 5. Conclusion The results of this study shows that the seed extract possesses in vivo antiplasmodial activity in addition to the in vitro action.
Table 2 Mean survival time of mice receiving various doses of ethanolic seed extract of Picralima nitida
Acknowledgement
Drug/extract
Dose (mg/kg day)
Mean survival time (day)
The authors are grateful to Mr. Nsikan Malachy for his technical assistance.
Picralima nitida extract
35 70 115 5 0.2 ml
14.0 16.0 21.6 28.0 9.0
Chloroquine (standard) Distilled water (control)
± ± ± ± ±
0.81* 0.88* 5.90* 0.00* 0.48
Data are expressed as mean ± S.D. for five animals per group. * P < 0.05 when compared to control.
References Ajanohoun, J.E., Aboubakar, N., Diamante, K., Ebot, M.E., Ekpere, J.A., EnowOrock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu, K.J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiale, A.L., Mbome, I.L., Mubiru, N.K., Nancy, W.L., Nkongmeneck, B., Satabie, B., Sofowora, A., Tamze, V., Wirmum,
J.E. Okokon et al. / Journal of Ethnopharmacology 111 (2007) 464–467 C.K., 1996. Contribution to ethnobotanical and floristic studies in Cmeroun. Traditional medicine and Pharmacopoeia. Technical and Research Commission of the Organisation of African Unity. OAU/STRC, pp. 60– 61. Ansa, A.R., Kapadia, G.J., Lloyd, H.A., Sokoloski, E.A., 1990. Picratidine: a new indole alkaloid from Picralima nitida seeds. Journal of Natural Products. Lloydia 53, 975–977. Arens, H., Borbe, H.O., Ulbrich, B., Stoeckigt, J., 1982. Detection of pericine a new central nervous system active indole alkaloid from Picralima nitida cell suspension culture by opiate receptor binding studies. Planta Medica 46, 210–214. Ayensu, E.S., 1978. Medicinal Plants in West Africa. Reference Publications Inc., Algonac, Michigan, p. 330. Dalziel, J.M., 1961.The Useful Plants of West Tropical Africa. The Crown Agents for Overseas Government and Administration, London. Duwiejua, M., Woode, E., Obiri, D.D., 2002. Pseudo-akuammigine, an alkaloid from Picralima nitida seeds, has anti-inflammatory and analgesic actions in rats. Journal of Ethnopharmacology 81, 73–79. Etkin, N.L., 1997. Antimalarial plants used by Hausa in Northern Nigeria. Tropical Doctor 27, 12–16. Ezekwesili, J.O., 1983. Biostudies of Picralima nitida (Staph) Th & Hel. Dur. M.Sc. Thesis. University of Nigeria, Nsukka, Nigeria. Ezeamuzie, K., Ojinnaka, M.C., Uzogara, E.O., Orji, S.E., 1994. Antiinflammatory, antipyretic and antimalarial activities of West African medicinal plant, Picralima nitida. African Journal of Medicine and Medical Sciences 23, 85–90. Fakeye, T.O., Itiola, O.A., Odetola, H.A., 2000. Evaluation of antimicribial property of stembark of Picralima nitida. Phytotherapy Research 14, 368– 370. Francois, G., Ake, A.L., Holenz, J., Bringmann, G., 1996. Constituents of Picralima nitida display pronounced inhibitory activities against asexual erythrocytic forms of Plasmodium falciparum. Journal of Ethnopharmacology 54, 165–168. Guyledouble, L., Oliver, M., Quirin, T., 1964. Alkaloids of Picralima nitida. VIII. Studies of leaves and roots. Isolation of two new alkaloids—picraphylline and picracine. Annals of Pharmacotherapy, France 27, 463–468. Homburger, F., 1989. In Vivo Testing in the Study of Toxicity and Safety Evaluation. In: Marquis, J.K. (Ed.), A Guide to General Toxicology, 2nd edn. Karger, New York. Inya-Agha, S.I., 1999. The hypoglycaemic properties of Picralima nitida. Nigerian Journal Natural Product and Medicine 3, 66–67. Inya-Agha, S.I., Ezea, S.C., Odukoya, O.A., 2006. Evaluation of Picralima nitida hypoglycaemic activity, toxicity and analytical standards. Planta Medica doi:10.55/S-2006-949825.
467
Iwu, M.M., Jackson, J.E., Tally, J.D., Klayman, D.L., 1991. Antileishmanial activity of medicinal plants used in Nigerian traditional medicine. American Journal of Tropical Medicine and Hygiene 45, 208. Iwu, M.M., Klayman, D.L., Bass, G.T., Andersen, S.T., Schuster, B.G., 1992a. The antimalarial activity of indole alkaloids from Picralima nitida: structure–activity relationships. American Journal of Tropical Medicine and Hygiene 47, 179. Iwu, M.M., Jackson, E., Tally, J.D., Klayman, D.L., 1992b. Evaluation of plants extract for antileishmanial activity using a mechanism based radiorespirometric microtechnique (RAM). Plant Medica 58, 436–441. Iwu, M.M., Klayman, D.L., 1992. Evaluation of the in vitro antimalarial activity of Picralima nitida extracts. Journal of Pharmacology 36, 133–135. Kapadia, G.T., Angerhofer, C.K., Ansa, A.R., 1993. Akuammine: an antimalarial indolemonoterpene alkaloid of Picralima nitida seeds. Planta Medica 59, 565–566. Kirby, G.C., Noamese, B.K., Paine, A., Warhurst, D.C., Philipson, J.D., 1995. In vitro and in vivo antimalarial activity of crytolepine, a plant-derived indoloquinolone. Phytotherapy Research 9, 359–363. Kirby, G.C., O’Neil, M.J., Philipson, J.D., Warhurst, D.C., 1989. In vitro studies on the mode of action of quassinoids with activity against chloroquine resistant Plasmodium falciparum. Biochemistry and Pharmacology 38, 4367–4374. Knight, D.J., Peters, W., 1980. The antimalarial action of N-benzyloxy dihydrotriazines. The action of cycolguanil (BRL50216) against rodent malaria and studies on its mode of action. Annals of Tropical Medicine and Parasitology 74, 393–404. Kouitcheu, M.L., Penlap, B.V., Kouam, J., Ngadjui, B.T., Fomum, Z.T., Etoa, F.X., 2006. Evaluation of antidiarrhoeal activity of the fruit-rind of Picralima nitida (Apocynaceae). African Journal of Traditional, Complementary and Alternative Medicine 3, 66–73. Lorke, D., 1983. A new approach to practical acute toxicity test. Archives of Toxicology 54, 275–286. Moller, B.L., Seedorff, L., Nartey, F., 1972. Alkaloids of Picralima nitida. Phytochemistry 11, 2620–2621. Nkere, C.K., Iroegbu, C.U., 2005. Antibacterial screening of the roots, seed and stembark extracts of Picralima nitida. African Journal of Biotechnology 4, 522–526. Oliver, B., 1960. Encyclopedia of Medicinal Plants. College of Arts, Science and Tech. Ibadan. Ryley, J.F., Peters, W., 1970. The antimalarial activity of some quinone esters. Annals of Tropical Medicine and Parasitology 84, 209–222. Wosu, L.O., Ibe, C.C., 1989. Use of extract of Picralima nitida bark in the treatment of experimental trypanosomiasis: a preliminary study. Journal of Ethnopharmacology 25, 263–268.
