Antimalarial potential of the ethanolic leaf extract of Pseudocedrala kotschyi

Akuodor Godwin Christian, Ajoku Gloria Ahunna, Ezeunala Mercy Nwakaego, Chilaka Kingsley Chimsorom, Asika Ebere ChileDepartment of Pharmacology, College of Medical Sciences, University of Calabar, NigeriaDepartment of Microbiology, Human Virology and Biotechnology, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, NigeriaDepartment of Pharmacology and Therapeutics, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, NigeriaDepartment of Pharmacology and Therapeutics, Faculty of Clinical Medicine, Ebonyi State University, Abakaliki, Nigeria

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Antimalarial potential of the ethanolic leaf extract of Pseudocedrala kotschyi

Akuodor Godwin Christian1*, Ajoku Gloria Ahunna2, Ezeunala Mercy Nwakaego2, Chilaka Kingsley Chimsorom3, Asika Ebere Chile4
1Department of Pharmacology, College of Medical Sciences, University of Calabar, Nigeria
2Department of Microbiology, Human Virology and Biotechnology, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria
3Department of Pharmacology and Therapeutics, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Nigeria
4Department of Pharmacology and Therapeutics, Faculty of Clinical Medicine, Ebonyi State University, Abakaliki, Nigeria

ARTICLE INFO ABSTRACT

Article history:

Received in revised form 18 July 2014 Accepted 15 August 2014

Available online 20 December 2014 Keywords:

Antimalaria

Pseudocedrala kotschyi

Leaf

Ethanol

In vivo

Mice Objective: To establish the efficacy of Pseudocedrala kotschyi (P. kotschyi) for the treatment of acute malaria attack used in Nigeria. Methods: The ethanolic leaf extract was investigated for antimalarial activity against Plasmodium berghei berghei (P. berghei berghei) in mice. Fourday suppressive, curative effect against established infection and prophylactic models of antiplasmodial studies were used. Results: The leaf extract of P. kotschyi (100-400 mg/kg b.w. p.o.) exhibited significant dose dependent activity against the parasite in the suppressive and curative, and also had repository activity. The antimalarial effect of P. kotschyi is comparable to that of chloroquine. The ethanolic leaf extract also prolonged the survival time of the infected mice. The LD50of the plant extract was established to be ≥5 000 mg/kg b.w. p.o. in mice. Conclusion: The results showed that the leaf extract has potential antiplasmodial activity, which can be exploited in malaria therapy.

*Corresponding author: Akuodor GC, Department of Pharmacology, College of Medical Sciences, University of Calabar, Nigeria.

Tel: +2348036725237

E-mail: goddyakuodor@yahoo.com

1. Introduction

Malaria remains the world most devastating human parasitic infection, afflicting more than 500 million people each year[1]. Mortality currently estimated at over a million people per year, has risen in recent years, probably due to increasing resistance to antimlarial medicines[2].

The constant evolution of the malaria parasite has rendered the cheapest and most widely available antimalarial treatments ineffective, especially with the recent reports about the increasing resistance of Plasmodium falciparum (P. falciparum) to artemisininbased compounds[3-5].

However, there is deep concern that this parasite will soon develop total resistance to such orthodox treatments. This has led researchers to look for other alternatives, one of which is evaluation of medicinal plants. Therefore, there is an urgent need to explore and utilize the naturally endowed rich biodiversity of communities through research that could translate to benefits for mankind. Hence, such studies on medicinal and beneficial plants could provide useful leads for the synthesis of important active compounds.

Pseudocedrela kotschyi (P. kotschyi) is one of such medicinal plants whose therapeutic value no doubt has a folkloric background. The plant is found abundantly in the moisture of heavy soils. It has been used in the treatment of various diseases by traditional healers. Various extracts of the plant have been reported to possess antidiabetic, antiepileptic, analgesic, antipyretic and antimicrobial properties[6-9].

The aim of the current study therefore is to investigate the ethanol leaf extract of P. kotschyi for antimalarial potency.

2. Material and methods

2.1. Collection of plant material

Fresh leaves of P. kotschyi were collected in the month ofApril, 2010 from chaza village, Suleja, Niger State, Nigeria and duly authenticated in the Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria by Dr. (Mrs) Jemilat A. Ibrahim. A specimen of the plant with voucher number (NIPRD/H/6542) was subsequently deposited at the herbarium of NIPRD for reference. The international plant name index is Meliaceae Pseudocedrela kotschyi. Bot. Jahrb. Syst. 22 (1): 154, 1895 (19 Nov. 1895) (IK).

2.2. Extraction of plant material

The leaves were cleaned and taken to the laboratory where they were cut into pieces and air-dried at room temperature for 7 d and ground to powder using mortar and pestle. Three hundred and fifty grams of the grounded leaf powder was then macerated in 1.5 L of ethanol for 24 h and filtered. The filtrate was dried on a water bath at reduced temperature to recover the extract and the yield calculated to be 38 g (11% w/w). The extract was stored in an airtight container and used for the study.

2.3. Phytochemical analysis

The phytochemical screening of ethanol extract of P. kotschyi leaf was carried out to determine the presence of the following compounds; alkaloids, tannins, saponins, terpenoides, flavonoids, steroids and cardiac glycosides, carbohydrates, reducing sugar, phlobatannins and anthraquinones using standard procedures[10-12].

2.4. Experimental animals

Swiss albino mice (18-22 g) of both sexes were used for the study. The mice were bred and kept in the Animal House, Department of Pharmacology, College of Medical Sciences, University of Calabar, Nigeria.

The animals were housed in cages at room temperature and moisture, under naturally illuminated environment of 12:12 h dark/light cycle. They were fed on standard diet and had free access to water. Treatment of the animals was in accordance with the Principles of Laboratory Animal Care (Revised)[13].

2.5. Acute toxicity of the extract

The lethal dose (LD50) of the ethanol leaf extract of P. kotschyi was determine in mice using the method as described by Lorke[14] with slight modification. Mice of both sexes were fasted overnight for the toxicity test. The study was done in two phases. In the first phase, 3 groups of 3 mice per cage were administered 10, 100 and 1 000 mg/kg of the extract p.o. The mice were observed for signs of toxicity and mortality for the first 4 h and 24 h. In the second phase, another 3 groups of 3 mice in each cage were further administered 1 600, 2 900 and 5 000 mg/kg of the leaf extract. The mice were also observed for signs of toxicity and mortality at regular intervals for 24 h, 48 h and 72 h respectively.

2.6. Malaria parasites

The chloroquine-sensitive Plasmodium berghei berghei (P. berghei berghei) (NK65) used to assess the antimalarial activity of P. kotschyi leaf was obtained from National Institute for Medical Research (NIMR) Lagos, and kept at the Department of Pharmacology, College of Medical Sciences, University of Calaba, Nigeria. The parasites were maintained by continuous re-infestation in mice.

2.7. Inocula

Parasitized erythrocytes were obtained from a donor infected mouse by cardiac puncture. This was prepared by determining percentage parasitaemia and the erythrocytes count of the donor mouse and diluting them with normal saline in proportions indicated by both determinations[15]. Each mouse was inoculated intraperitoneally with infected blood suspension (0.2 mL) containing 1×107P. berghei berghei parasitized red blood cells.

2.8. Suppressive test

A 4--day suppressive test as described by Akuodor et al[16], Mbah et al[17] Was employed for the study. Thirty Swiss albino mice of both sexes weighing (18-22 g) were passaged intraperitonealy with standard inocula of P. berghei containing 1×107infected erythrocytes. Four hours after inoculation, the infected mice were randomly divided into 5 groups of 6 mice per cage and treated for four consecutive days (D0-D3). Group 1 received 0.2 mL of normal saline (Drug-free control). Group 2, 3 and 4 received 100, 200 and 400 mg/kg of the ethanol leaf extract respectively, while group 5 received 10 mg/kg of Chloroquine diphosphate. All doses were administered orally. On the fifth day (D4), thin films were made from the tail blood of each mouse.

The films were fixed with methanol, stained with 10%Giemsa and parasite density determined by microscopically (Olympus CX 21, Japan) counting the parasitized red blood cells on at least 1 000 red blood cells in 10 different fields[15].

2.9. Repository test

This was assessed by using the method described by[18,19]. Thirty Swiss albino mice of both sexes weighing (18-22 g) were randomly divided into 5 groups of 6 mice per cage for ethanol leaf extract of P. kotschyi. The mice were orally administered with various doses of the extract (50, 100 and 200 mg/kg), 10 mg/kg chloroquine (reference group) and 0.2 mL normal saline (control group) for three consecutive days.

On the fourth day, the mice were passaged intraperitoneally with standard inocula of P. berghei berghei containing 1x107infected erythrocytes. Seventy-two hours later (D7), thin films were made from the tail blood of each mouse. The films were fixed with methanol, stained with 10% Giemsa and parasitaemia level assessed by microscopically (Olympus CX 21, Japan) counting the parasitized red blood cells on at least 1 000 red blood cells in 10 different fields[15].

2.10. Curative test

On the first day (D0), thirty Swiss albino mice were passaged intraperitonealy with standard inocula of 1×107P. berghei berghei infected erythrocytes. Seventytwo hours after, the mice were randomly divided into 5 groups of 6 mice per cage. Group 1 received 0.2 mL of normal saline (Drug-free control). Group 2, 3 and 4 received 100, 200 and 400 mg/kg of the ethanol leaf extract respectively, while group 5 received 10 mg/kg of Chloroquine diphosphate. All doses were administered orally. Treatment continued daily until the seventh day when thin films were made from the tail blood of each mouse.

The films were fixed with methanol, stained with Giemsa and parasitemia density examined by microscopically counting the parasitized red blood cells on at least 1 000 red blood cells in 10 different fields[20,21].

The mean survival time of each group was determined by finding the average survival time (days) of the mice in each group over a period of 28 days (D0-D27).

2.11. Statistical analysis

Results obtained were expressed as mean±S.E.M. The significance of difference between the controls and treated groups were determined using one-way analysis of variance (ANOVA. P<0.05 were considered to be statistically significant[22].

3. Results

3.1. Phytochemical test

Results obtained from the phytochemical screening of the ethanol leaf extract of P. kotschyi showed the presence of alkaloids, tannins, saponins, terpenoides, flavonoids, steroids, cardiac glycosides, carbohydrates, reducing sugar, while phlobatannins and anthraquinones were absent.

3.2. Acute toxicity test

There was no mortality recorded in the mice upon oral administration even at doses as high as 5 000 mg/kg. This indicates that the experimental doses used are relatively safe.

3.3. Suppressive test

The ethanol leaf extract of P. kotschyi demonstrated dose dependent schizonticidal activity at the doses employed in the study. The extract at 400 mg/kg caused 91% suppression as against 90% and 79% suppression induced by 200 and 100 mg/kg respectively. The standard drug chloroquine however caused 94% suppression (Table 1).

Table 1.Suppressive effect of ethanol leaf extract of P. kotchyi against P. berghei berghei in mice.

D5= Day five, *significantly different from control at P<0.05 (n=6).

Table 2.Repository effect of ethanol leaf extract of P. kotchyi against P. berghei berghei in mice.

D7= Day seven, *significantly different from control at P<0.05 (n=6).

3.4. Repository test

The ethanol leaf extract of P. kotschyi exhibited significant(P<0.05) dose dependent reduction in parasitemia density of 75%, 81% and 90% at 100, 200 and 400 mg/kg respectively, whereas chloroquine treated group caused 94% reduction in parasitemia density in the test (Table 2).

Table 3.Curative effect of ethanol leaf extract of P. kotchyi against P. berghei berghei in mice. Mean parasitemia density

D3=Day three, D7=Day seven, *significantly different from control at P<0.05 (n=6). All mice treated with chloroquine survived until day 28.

3.5. Curative test

There was a dose dependent reduction in the level of parasitemia in the treated group unlike in the saline control group in which there a consistent increase in the blood parasite density.

The mean survival time also increased dose dependently. Death was observed in the control group on day 8 and by day 11, all mice in the group died (mean survival time of 9 days). On the other hand, mice in the group that received 100, 200 and 400 mg/kg survived beyond 21 days. Chloroquine treated group survived the 28 days duration of observation (Table 3).

4. Discussion

Phytochemicals constitute an integral part of medicinal plants and are responsible for their numerous bioactivities. Numerous plants containing a wide variety of phytochemicals as their bioactive principle have antiplasmodial activities[23,24]. Although the mechanism of the action of the leaf extract has not been evaluated in the present study, some of the constituents detected have however been implicated in antiplasmodial activities by different mechanisms. The antiplasmodial activity of Berlina grandiflora has been tracked to the alkaloids, flavonoids and terpenoids contained in the plant[20].

The ethanol leaf extract of P. kotschyi has been shown to possess schzonticidal activity in vivo following oral administration to infected mice. The extract demonstrated good schozonticidal activity against early infections at the various doses employed in the study.

In the established infection, the ethanol leaf extract at various doses showed significant dose dependant schizonticidal activity. The observed antimalarial activity of the leaf extract is consistent with the traditional use of the plant as herbal medication against the disease and indicative of its potential as a chemotherapeutic antimalarial agent. The plant extract has a noteworthy antimalarial activity as the mean survival time values which at doses used were twice or more than that of control group.

In this study, chloroquine was used as the standard antimalarial drug. Chloroquine has been used for curative, suppressive and prophylactic antiplasmodial activities. In early and established infection, chloroquine interrupts with the heme polymerization by forming a FP-chloroquine complex. This complex is responsible for the disruption of the parasite’s cell membrane function and ultimately leads to auto digestion. Though, chloroquine exhibited higher suppressive, prophylactic and curative antiplasmodial activities by the extent of inhibition of parasitemia, the leaf extract of P. kotschyi also showed similar antiplasmodial activities.

P. berghei berghei has been used in studying the activity of potential antiplasmodials in vivo in rodents[25,26], and it produces diseases similar to those of human plasmodial infection[27,28]. Agents with suppressive activity against P. berghei berghei were known for antiplasmodial activity[29]. Earlier studies on extracts of P. kotschyi showed that it possess analgesic and antipyretic properties[7,8]. Agents with such properties are known to produce additional remedy to malaria patients[30].

In conclusion, the extract has demonstrated significant antimalarial activity as seen in its ability to suppress P. berghei berghei infection in the three models evaluated. The findings lend pharmacological support to the folkloric use of the plant in the treatment of malaria.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgement

The authors are grateful to Dr. (Mrs) Jemilat A. Ibrahim of the Department of Medicinal Plant Research and TraditionalMedicine, NIPRD, Abuja, Federal Capital Territory, Nigeria, for her botanical assistance.

Reference

[1] WHO. The World Health Organization World Malaria Report 2005. Geneva: World Health Organization; 2005.

[2] World Health Organization. World Malaria Report 2011: the successes, existing challenges and the way forward. Geneva: World Health Organization; 2011.

[3] Htut ZW. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 2009; 361: 1807-1808.

[4] Rogers WO, Sem R, Tero T, Chim P, Lim P, Muth S, et al. Failure of artesunate-mefloquine combination therapy for uncompleted Plasmodium falciparum malaria in South Cambodia. Malaria J 2009; 8(10).

[5] Cui L, Wang Z, Miao J, Miao M, Chandra R, Jiang H, et al. Mechanism of in vitro resistance to dihydroartemisinin in Plasmoduim falciparum. Mol Microbiol 2012; 86(1): 111-128.

[6] Georgewill UO, Georgewill OA. Effect of extract of Pseudocedrel kotschyi on blood glucose concentration of alloxan induced diabetic albino rats. Eastern J Med 2009; 14: 17-19.

[7] Anuka JA, Ijezie DO, Ezebnik ON. Investigation of pharmacological actions of the extract of the extract Pseudocedrela kotschyi in laboratory animals. ABSTRACTS of the proceedings of XXVIIth Annual Regional Conference

[8] Akuodor GC, Essien AD, Essiet GA, Essien David-Oku, Akpan JL, Udoh FV. Evaluation of antipyretic potential of Pseudocedrela kotschyi Schweint. Harms (Meliaceae). Eur J Med Plants 2013; 3(1): 105-113.

[9] Ayo RG, Audu OT, Ndukwe GI, Ogunshola AM. Antimicrobial activity of leaves of Pseudocedrela kotschyi (Schweint.) Harms Afr J Biotechnol 2010; 9 (45): 7733-7737.

[10] Mukherjee PR. Quality Control of herbal Drugs, an Approach to Evaluation of Botanicals. 13th Edn. New Delhi: Busines Horizones Publisher; 2006, p. 419-459.

[11] Oloyode OI. Chemical profile of unripe pulp of Carica papaya. Pak J Nutr 2005; 4: 379-381.

[12] Sumitra C, Jigna P, Nehal K. Evaluation of antibacterial activity and phytochemical analysis of Bauhinia variegate L. Bark. Afr J Biomed Res 2006; 9: 53-56.

[13] NIH. Guide for the car and use of Laboratory animal (Revised). Washington: NIH Publication; 1985, p. 83-23.

[14] Lorke D. A new approach for acute toxicity testing. Arch Toxicol 1983; 54: 275-287.

[15] Akuodor GC, Amos GM, Essien AD, Essien David-Oku, Akpan JL, Ezeokpo BC. Antimalarial potency of the leaf extract of Aspilia Africana (pers) C.D. Adams. Asian Pac J Trop Med 2012; 5: 126-129.

[16] Akuodor GC, Maryam IdrisiUsman, Theresa C Ugwu, Akpan JL, Ghasi SI, Osunkwo UA. In vivo schizontal activity of ethanolic leaf extract of Gongronema latifolium on Plasmodium berghei berghei in mice. Ibnosina J Med Biomed Sci 2010; 2(3): 118-124.

[17] Mbah CC, Akuodor GC, Anyalewechi NA, Iwuanyanwu TC, Osunkwo UA. In vivo antiplasmodial activities of aqueous extract of Bridelia ferruginea stem bark against Plasmodium berghei berghei in mice. Pharm Biol 2012; 50(2): 188-194.

[18] Adzu B, Salawu OA. Screening Diospyros mespiliformis extract for antimalaria potency. Int J Biol Chem Sci 2009; 3(2): 271-276.

[19] Awe SO, Makinde JM. Evaluation of the antimalarial activity of Morinda lucida using both in vivo and in vitro techniques. West Afr J Pharmacol Drug Res 1997; 13: 39-44.

[20] Akuodor GC, Anyalewechi NA, Ikoro NC, Akpan JL, Megwas UA, Iwuanyanwu TC, et al. Evaluation of antiplasmodial activity of Berlina grandiflora leaf extract against Plasmodium berghei in mice. Afr J Microbiol Res 2010; 4(21): 2211-2214.

[21] Chandel S, Bagai U. Antiplasmodial activity of Ajuga bracteosa against Plasmodial berghei infected BALB/c mice. Indian J Med Res 2010; 131: 440-444.

[22] Betty K, Stern J. Essential medical statistics. 2nd edition. Massachusetts: Blackwell Sciences Ltd; 2003.

[23] Matur BM, Mathew T, Ifeanyi CIC. Analysis of the phytochemical and in vivo antimalarial properties of Phyllanthus fraternus Webster extract. New York Sci J 2009; 2: 12-19.

[24] Alshawsh SM, Mothana RA, Al-Shamahy HA, Alsllami SF, Lindequist U. Assessment of antimalarial activity against Plasmodium falciparum and phytochemical screening of some Yemeni medicinal plants. eCAM 2007; 6: 453-456.

[25] Thomas AM, Van Der Wel AM, Thoma AW, Janse CJ, Water AP. Transfection systems for animal models of malaria. Parasitol Today 1998; 14: 248-249.

[26] Pedroni HC, Bettoni CC, Spalding SM, Costa TD. Plasmodium berghei Development of an irreversible experimental malaria model in wistar rats. Exp Parasitol 2006; 113: 193-196.

[27] English MC, Waruiri C, Lightowler C, Murphy SA, Kirigha G, Marsh K. Hyponatraemia and dehydration in severe malaria. Arch Dis Childhood 1996; 74: 201-205.

[28] Kumar KA, Singn S, Babu PP. Studies on the glycoprotein modification in erythrocyte membrane during experimental cerebral malaria. Exp Parasitol 2006; 114: 173-179.

[29] Elufioye TO, Agbedahunsi JM. Antimalarial activities of Tithonia diversifolia (Asteraceae) and Crossopteryx febrifuga (Rubiaceae) on mice in vivo. J Ethnopharmacol 2004; 93: 167-171.

[30] Addae-kyereme J, Croft SL, Kendrick H, Wright CW. Antiplasmodial activities of some Ghanian plants traditionally used for fever/malaria treatment and some alkaloids isolated from Pleiocarpa mutica in vivo antimalarial activity of Pleiocarpine. J Ethnopharmacol 2001; 76: 99-103.

Document heading 10.1016/S2221-6189(14)60077-9

28 March 2014