In vitro anthelmintic activity of stem and root barks of Alstonia boonei De Wild

Alstonia boonei De Wild ethanol extracts of the stem bark (ABSB) and root bark (ABRB) were evaluated for possible anthelmintic activity. Three different concentrations of each extract (50, 100 and 150 mg/ml) were evaluated for in vitro anthelmintic activity by determining the effects of the extracts on the paralysis and death times of Pheretima posthuma. Mebendazole (MBZ) 15 mg/mg was used as reference anthelmintic. ABSB and ABRB demonstrated a concentration dependent anthelmintic activity with a reduction in paralytic and death times upon increase in the concentration of the extracts. ABSB revealed better anthelmintic activity than ABRB at all concentrations tested. ABSB also revealed a significant paralytic time (p<0.01) at 150 mg/ml with reference to MBZ. Phytochemical screening revealed the presence of alkaloids, tannins, flavonoids saponins and glycosides in ABSB and ABRB.


INTRODUCTION
Medicinal plants have been used over the decades to manage helminth infections.However, scientific evaluation of some of these traditional treatments has not been conducted so as to validate their usage.With the current increase in anthelmintic resistance amongst both human and farm animals (Vercruysse et al., 2011), it is imperative that the traditional usage of some of these medicinal plants be validated and novel molecules isolated in order to curb the menace.The use of medicinal plant as anthelmintic has been practised in many indigenous cultures for centuries.Studies have shown that in many developing countries, ethno medicine is still the primary treatment option for many parasitic diseases (Tanner et al., 2011;Gazzinelli et al., 2012).Alstonia boonei De Wild (Apocynaceae) is an indigenous African tree mostly found in the evergreen rain forest of tropical West Africa (Gosse et al., 1999).
The plant is well known by almost all traditional healers practising along the west coast of Africa (Adotey, 2012).The bark of the plant is known to possess antirheumatic, anti-inflammatory, anthelmintic and antidiabetic properties (Hadi and Bremner, 2001).Traditionally, a cold infusion of the fresh or dried bark is used as an anthelmintic and also to expel other intestinal parasites in children (Adotey, 2012).Studies conducted on the methanol extracts of the stem bark have demonstrated potent *Corresponding author.E-mail: j.a.apenteng@gmail.comor japenteng@central.edu.gh.Tel: +233249449249.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License anti-inflammatory, analgesic and antipyretic properties (Olajide et al., 2000).Aqueous and ethanol extracts of the stem bark have also demonstrated antimicrobial and anthelmintic activities (Adomi, 2006;Danquah et al., 2012).The stem bark which is mostly used for traditional treatment of malaria is also known to possess very potent antioxidant compounds (Akinmoladun et al., 2007).
Extensive research conducted on this plant has resulted in the identification of various bioactive compounds of diverse pharmacological activity, including; Boonein: a 9 carbon terpenoid lactone possibly known to be the precursor in indole alkaloid biogenesis (Marini-Bottelo et al., 1983).Although much research has been conducted on this plant, there is limited information on the anti-infective potentials of the various parts of the plant.Current anti-infective information available is mostly related to the stem bark of the plant.In the face of high levels of microbial resistance to antibiotics as well as other anti-infective agents, it is important that new compounds be identified and developed.This study therefore aimed to determine and compare the anthelmintic potential of the stem and root barks of A. boonei in order to support its traditional uses.

Collection of plant
The stem and root bark of A. boonei were identified and collected in the month of March, 2015 from the Aburi Botanic Gardens in the Eastern Region of Ghana.The plant samples were authenticated by Mr. Albert Asiedu Prempeh, a botanist and curator of the garden.Voucher specimens CUC/DPS/2015/A018 and CUC/DPS/2015/A019 were deposited in the Pharmacognosy Section of the Department of Pharmaceutical Science, Central University College.The samples were washed with distilled water to get rid of debris and then air dried for 3 weeks at room temperature (25 to 28°C).

Extraction of plant
The extraction process was carried out using the method described by Adu et al. (2015) with slight modifications.The dried plant samples were milled into coarse powder using laboratory mill equipment.A quantity of 250 g of both samples was weighed and extracted using 70% v/v ethanol by cold maceration for 72 h.The supernatant and the bulk extract was then filtered using a filter paper with the aid of a vacuum pump.The extracts obtained were concentrated using a rotary evaporator (Buchi, Germany, R210) at 40°C.The concentrates were later dried in a hot air oven at 40°C to obtain the solid extract.The extracts were then stored at 4°C until needed.

Phytochemical screening
Phytochemical tests for the presence of some plant secondary metabolites were performed on the powdered plant samples for the presence of tannins, alkaloids, flavonoids and glycosides (Trease and Evans, 2002).

Experimental organism
Adult Indian earthworms (Pheretima posthuma) which have anatomical resemblance to human intestinal roundworms were obtained from the nursery of a vegetable farm near Central University College, Accra, Ghana.Normal saline solution (0.9%) was used to wash the worms to remove all debris.

In vitro anthelmintic activity evaluation
The experiment was performed with slight modifications to the method described by Bhawar et al. (2009).Earthworms of lengths 3.0 to 6.0 cm were employed.Extract concentrations of 50, 100 and 150 mg/ml were prepared using distilled water.Mebendazole (MBZ) at a concentration of 15 mg/ml was used as the reference standard.Normal saline solution (0.9%) was used as a negative control.

Experimental procedure
Five earthworms were placed in each Petri dish into which the various extracts concentrations and reference drug were added.Observations were made for the time taken by the various extract concentrations to cause paralysis and death of the individual worms.Paralysis was observed when no movement was seen in the worms unless when shaken vigorously.Death was denoted by a lost in motility of the worms even upon pricking with a pin and placement in 50°C warm water, coupled with a fading away of body colour.Solutions within which worms demonstrated vigorous motility and life after ≥360 min of exposure where classified as not exerting anthelmintic activity (Na= No activity).

Statistical analysis
Results were presented as mean ± standard deviation (N=5).Analysis was done using GraphPad prism version 5 (GraphPad Software, San Diego, CA, USA) by two way analysis of variance (ANOVA) followed by bonferonni post-test analysis which recognises *p<0.05,**p<0.01,and ***p<0.001as statistically significant.

Anthelmintic activity
The extracts demonstrated some level of anthelmintic activity at the concentrations tested.ABSB demonstrated more potent activity than ABRB in a concentration dependent manner with shorter paralysis and death times as compared to ABRB (Tables 2 and 3).

DISCUSSION
The results from the anthelmintic bioassay revealed that both extracts ABSB and ABRB have anthelmintic activity.These results correlate with research conducted by Danquah et al. (2012) reporting the possible anthelmintic potentials of the stem bark extracts of A. boonei.The ability of plants to exhibit anthelmintic activity has largely been attributed to the presence of tannins.Tannins are believed to exert anthelmintic activity by interfering with the energy generation of the helminth parasite by uncoupling oxidative phosphorylation or by binding to free proteins in the gastrointestinal tract of the helminth.This eventually results in death of the parasite (Danquah et al., 2012;Adu et al., 2015;Olusegun-Joseph et al., 2012).The results from the phytochemical screening revealed the presence of tannins in both ABSB and ABRB; this could have been responsible for the anthelmintic activity.Studies conducted by Mute (2009) also reported the role of alkaloids in anthelmintic activity by causing paralysis of worms through their action on the central nervous system of the helminth.This therefore implies that the presence of alkaloids in the extract also contributed to their anthelmintic activity.
The results obtained however revealed that the anthelmintic activity was concentration dependent for both extracts with the higher concentrations demonstrating better anthelmintic activities (Tables 2 and  3).ABSB demonstrated better anthelmintic activity than ABRB.ABSB also demonstrated significant paralytic times (p<0.05) at 100 mg/ml and (p<0.01) at 150 mg/ml with reference to MBZ.This could therefore imply that the stem bark of the plant possesses more bioactive compounds responsible for anthelmintic activity than the root bark, hence the higher activity.These findings support the folkloric use of A. boonei as an anthelmintic agent.

Conclusion
Ethanol extracts of stem and root barks of A. boonei possess anthelmintic properties.The stem bark exerts better anthelmintic activity than the root bark.

Table 2 .
Paralysis time of ABSB and ABSB against P. posthuma.

Table 3 .
Death time of ABSB and ABRB against P. posthuma .