European Journal of Medicinal 2(2): 132-139, 2012

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Larvicidal and Antifungal Properties of Picralima nitida () Leaf Extracts

Peace M. E.Ubulom 1, N. G. Imandeh 2, Chinweizu E. Udobi 3* and Ibrahim IIya 4

1Faculty of Pharmacy, University of Uyo, . 2Department of Zoology, University of Jos, Nigeria. 3College of Science & Technology, Kaduna Polytechnic, Kaduna, Nigeria. 4Department of Medicinal Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria.

Received 20 th September 2011 Research Article Accepted 29 th December 2011 Online Ready 28 th February 2012

ABSTRACT

The larvicidal and antifungal activities of ethanolic and aqueous leaf extracts of Picralima nitida were evaluated in static bioassays on 4 th instar larvae of Anopheles gambiae and three fungal species: Aspergillus flavus, Candida albicans and Microsporum canis. All extractions were done using distilled water and 50% ethanol. Larvicidal assays were carried out at extract concentrations of 0.15, 0.30, 0.45, 0.60 and 0.75% w/v, for 72h. For the antifungal studies extract concentrations used were 200, 100, 50 and 25mg/ml. At the end of larvicidal assay the highest concentration recorded mortality of 57.60% and 38.40% for ethanolic and aqueous leaf extracts respectively. 72h LC 50 values obtained from Probit analysis, using SPSS version 17 were 0.660% and 1.057% w/v for ethanolic and aqueous leaf extracts respectively. Larvae in the control experiments registered no death throughout the period of experiment, rather they were actively wriggling and some even metamorphosed into pupae. For the antifungal studies the agar well diffusion technique was employed. Antifungal effects were determined using measurements of inhibition zone diameter (IZD). Results obtained revealed that both the aqueous and ethanolic leaf extracts exerted antifungal effect on A. flavus and C. albicans, but no antifungal effect was exhibited against M. canis, at the extract concentrations used in this study, rather a steady growth in the test plates seeded with M. canis was observed. The same was applicable with the negative controls. The drug, ketoconazole exerted antifungal effect on all test organisms. Phytochemical screening of the leaf revealed the presence of

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*Corresponding author: Email: [email protected];

European Journal of Medicinal Plants, 2(2): 132-139, 2012

alkaloids, cardiac glycosides, saponins and terpenes. The leaf of P. nitida possesses larvicidal and antifungal potential and therefore warrants a more thorough exploitation.

Keywords: Larvicidal; antifungal; ethanolic; aqueous; leaf extracts; Picralima nitida.

1. INTRODUCTION

All over the world especially in South of the Sahara, vector-borne diseases remains a cause for serious concern. Mosquito vectors for instance constitute a major public health menace. The mosquito, Anopheles gambiae, has been incriminated with several disease- causing organisms such as Plasmodium spp, responsible for the notorious malaria scourge. According to Alabi (2010), “malaria fever is one of the deadliest diseases ravaging Africa. Annually, millions of people and man-hours with attendant economic implications are lost to this pandemic”. A.gambiae is also a vector of the filarial nematode, responsible for filariasis.

Different strategies have been devised to curb disease transmission by these vectors but these have suffered certain limitations. These limitations have necessitated the search for environmentally safe, degradable, affordable and target-specific compounds against these insect-vectors. The search for such compounds has been directed to the plant kingdom (Mathur, 2003). Larviciding is a preferred option in vector control because larvae occur in specific areas and can thus be more easily controlled. Treatments provide control before the biting adults appear and disperse from the breeding sites.

Phytomedicines have also shown great promises in the treatment of intractable infectious diseases (Firenzuoli and Gori, 2007). Several medicinal plants have been screened for their activity on different species of microorganisms. The antimicrobial activity of ethanolic and aqueous extracts of Sida acuta on microorganisms from skin infections has been documented by Ekpo and Etim (2009). Ubulom et al. (2011) have reported the antifungal property of aqueous and ethanolic seed extracts of P. nitida on A. flavus, C. albicans and M. canis.

P. nitida (: Apocynaceae) is a medicinal plant, commonly referred to as Akuamma plant. Many herbalists have claimed to use the leaves, roots, seeds or stem bark for the treatment of various fevers, hypertension, jaundice, gastrointestinal disorders and malaria (Iwu, 1993 & Etukudo, 2003). Its biological activities have also been reported by researchers such as Iroegbu and Nkere (2005) and Inya-Agha et al., (2006). However, no scientific document has been encountered on the larvicidal as well as the antifungal effect of aqueous and ethanolic leaf extracts of P. nitida on the mosquito and fungal species used in this study. This study was thus aimed at investigating the lethality/toxicity of the aqueous and ethanolic leaf extracts of P. nitida to fourth instar larvae of A. gambiae as well as the antifungal effect of these extracts on A. flavus, C. albicans and M. canis.

2. EXPERIMENTAL DETAILS

2.1 Collection of Plant Materials

The leaves of P. nitida used in this study were collected from Anua Obio in Uyo Local Government Area of Akwa Ibom State, Nigeria. Identification was done by the Department of

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European Journal of Medicinal Plants, 2(2): 132-139, 2012

Botany and Ecological Studies, University of Uyo, Nigeria and a voucher specimen with herbarium number: Ubulom UUH 875 (Uyo) was deposited in the herbarium of the Department.

2.2 Extraction Procedure

The plant leaves were first air-dried on laboratory tables at room temperature (28 + 2°C). This was followed by pulverization using the crusher machine (Atlas exclusive, ALZICO Ltd, Type YL 112 M-4) in the pilot plant unit of National Institute for Pharmaceutical Research and Development (NIPRD) Abuja. 500g each of the pulverized leaves were macerated separately in distilled water and 50% ethanol for 72h, with periodic stirring. Each extract was filtered repeatedly using muslin cloth, non-absorbent cotton wool and Whatman No. 1 filter paper. This was done to get rid of the marc. The aqueous filtrate was concentrated using a lyophiliser (Aqua Lyovac GT2, Germany), while the ethanolic filtrate was first concentrated in vacuo at 40 °C using a rotary evaporator (Bibby Sterlin Ltd, England, RE. 200), after which it was freeze-dried using the aforementioned lyophiliser.

2.3 Phytochemical Screening

The leaf extract of P. nitida was screened for its phytochemical components using the methods described by Harborne (1984), Evans (2002) and Sofowora (2006). The plant metabolites that were tested for were alkaloids, anthraquinones (free and combined), cardiac glycosides, flavonoids, saponins, phlobatannins, tannins and terpenes.

2.3.1 Test organisms

Fourth instar larvae of A. gambiae used in this investigation were provided by National Arbovirus and Vectors Research Centre (NAVRC), Enugu, Nigeria. The fungal species ( A. flavus , C. albicans and M. canis) were obtained from the Department of Microbiology, University of Uyo, Nigeria. These fungal specimens were separately plated out on sterilized Sabouraud Dextrose Agar (Biomark). They were purified after isolation through repeated subculturing and characterized using the methods of Collins and Lyne (1970) and Cruickshank et al., (1975). They were subsequently stored in agar slants in the refrigerator at 4 °C, prior to experiment reported in this study.

2.3.2 Larvicidal assay

The larvicidal activity of ethanolic and aqueous extracts of the leaf of P. nitida was evaluated in static bioassay, on fourth instar larvae of A. gambiae, for 72h (WHO (2005). Stock solution of each extract was prepared and in both cases (aqueous and ethanolic), the leaf extracts of P. nitida were first solubilised using the solvent dimethyl sulphoxide (DMSO). Sterile distilled water was further added to obtain a final volume of 100ml and this was mixed thoroughly and it formed the stock solution. The stock solution was heated in a water bath for 2minutes at a temperature of 40 °C, and then allowed to cool. This was done in order to reactivate phytochemicals that may have been inactivated due to excessive cooling as encountered in refrigerating and freeze-drying. From this stock solution of the extract, graded concentrations of ethanolic and aqueous leaf extracts were prepared to obtain 0.15, 0.30, 0.45, 0.60 and 0.75% w/v concentrations of each extract. Twenty five larvae were exposed to each bioassay medium in plastic assay cups, containing nutrients (a pinch each of fine quaker

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oats). Each extract concentration had five replicates. The control which was also replicated had 25 larvae (per replicate) immersed in 100ml distilled water, to which larvae food had been introduced. Both the test and control set ups were maintained at room temperature (28 + 2°C). Observations were made at 24, 48 and 72h and larvicidal activity of each extract was determined, by counting the number of dead larvae each day, until the end of the experiment. Larvae were considered dead when no mobility was observed and no response to stimulus with a Pasteur pipette.

2.3.3 Screening of extracts for antifungal activity

The agar well diffusion technique (Ubulom, et al., 2010) was employed in the evaluation of antifungal activity of the leaf extracts of P. nitida. Standardised inoculum (1 x 10 6cfu/ml of each test fungus was spread on to sterile Sabouraud Dextrose Agar (SDA) plates. The plates were allowed to dry and a sterile cork borer (6mm diameter) was used to bore wells in the agar plates. Stock solutions of both the aqueous and ethanolic leaf extracts were prepared and in both cases DMSO was used to achieve solubilisation as aforementioned in larvicidal assay. From the stock solutions, graded concentrations of the extracts were prepared by serial dilution to obtain 200,100, 50 and 25mg/ml extract concentrations of both aqueous and ethanolic leaf extracts. Each extract concentration was separately introduced in triplicate wells into SDA plates already seeded with the standardized inoculum (1 x 10 6cfu/ml) of the test fungal cells. The plates were allowed to stand for 1 hr for diffusion to take place. Positive control was set up using separate plates. In this case, the assay consisted of each test organism and the drug, ketoconazole, at a concentration of 30mg/ml. There was also the negative control which consisted in each case of the test fungal species and two drops of 20% DMSO in distilled water in a final volume of 10ml. The control experiments were also replicated thrice. All plates (test and control) were incubated at room temperature (28 + 2°C) for 48h. The external diameters of visible zones of growth inhibition were measured after incubation. The mean inhibition zone diameter (IZD) was determined in each case.

2.4 Statistical Analysis

Data obtained from this study were analysed using Microsoft excel® and SPSS version 17.

3. RESULTS AND DISCUSSION

The yield values of the aqueous and ethanolic leaf extracts of P. nitida were 5.26 and 3.52% w/w respectively. The constituent compounds detected by phytochemical screening of the crude leaf extract were alkaloids, cardiac glycosides, saponins and terpenes.

There was reduction in activity (e.g. wriggling/motility) of larvae exposed to the different test solutions. This was more apparent as concentrations of extract increased. The results obtained showed a time and concentration/dose dependent increase in larvicidal activity (Table 1). No mortality was observed in the controls, rather larvae in the control set up were agile and activity wriggling, throughout the duration of the experiment. Some larvae in the controls even metamorphosed into pupae. The 72h median lethal concentration values (72hLC 50 ) were determined by probit analysis using the method described by Finney, 1971. SPSS version 17 was employed in the analysis.

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Table 1. Bioassay results showing effects of leaf extracts of P. nitida on larvae of A. gambiae

Extract Log. No. of Ethanolic extract Aqueous extract Conc. Conc. Larvae % mortality Probit % Probit (%w/v) exposed mortality mortality mortality Control 0.00 125 0.00 0.00 0.00 0.00 0.15 2.18 125 0.00 0.00 0.00 0.00 0.30 2.48 125 9.60 3.72 0.00 0.00 0.45 2.65 125 21.60 4.23 10.40 3.72 0.60 2.78 125 46.40 4.90 22.40 4.23 0.75 2.88 125 57.60 5.20 38.40 4.69

Table 2. Observations recorded showing the 72hLC 50 values for ethanolic and aqueous leaf extracts of P. nitida assayed against A. gambiae larvae

Extract P. nitida/A. gambiae Regression equation

72hLC 50 value 95% conf. Interval (% w/v) (% w/v) Ethanolic leaf 0.660 0.608 – 0.733 Y =- 11.444 + 4.059 X extract Aqueous leaf 1.057 - Y =- 9.181 + 3.036 X extract

The fungal species used in this study demonstrated variable sensitivity to the leaf extracts of P. nitida . Observations made revealed that both the aqueous and ethanolic leaf extracts of P. nitida did not at any concentration reported inhibit the growth of M. canis (Table 3).

Table 3. Observations recorded showing Inhibitory effect of the leaf extract of P. nitida on A. flavus, C. Albicans and M. canis

Extract Inhibition zone diameters (mm) (mg/ml) Aqueous Leaf Extract Ethanolic Leaf Extract A. C. M. A. C. M. flavus albicans canis flavus albicans canis 200 9.67+0.67 9.00+0.58 - 13.00+ 0.58 11.00+0.58 - 100 7.00+0.58 6.33+0.33 - 11.00+0.00 8.67+0.33 - 50 3.67+0.33 - - 9.00+0.58 6.00+0.58 - 25 ------Positive 30.00+0.00 25.33+0.33 31.67+0.88 30.00+0.58 26.00+0.58 31.67+0.88 control Negative ------control Key: Values are expressed as mean + SEM (n=3) Positive control = ketoconazole (30mg/ml); Negative control = Test organism, minus extract solution

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25 and 50mg/ml of the aqueous leaf extract did not inhibit the growth of C. albicans, but mean values of 6.33 + 0.33 and 9.00 + 0.58mm were recorded for extract concentrations 100 and 200mg/ml respectively. For A. flavus, growth inhibition with the aqueous leaf extract was observed from 50mg/ml of extract (Table 3). The ethanolic leaf extract did not inhibit the growth of A. flavus and C. albicans at 25mg/ml. inhibition was observed from 50mg/ml and the range of inhibition was 9-13mm for A. flavus and 6-11mm for C. albicans (Table 3).

Generally, the zone of inhibition increased with increase in concentration of extract. Inhibition of the test fungal species was observed in cases all with the use of the drug, ketoconazole (Table 3) while, negative controls gave a steady increase in fungal growth.

The yield value (% w/v) was higher for the aqueous than the ethanolic leaf extract. This suggests that the leaf of P. nitida has a higher proportion of water-soluble components. The phytochemical compounds detected in the leaf of P. nitida have been reported to have biological activity (Leven et al., 1979; Lee, 2000; Okeke et al., 2001; Wiesman and Chapagain, 2006). These phytochemical may have performed alone or in synergy. Mortality of larvae of A. gambiae was due to a combination of factors such as the lethality of the extracts/test solutions and the fact that larvae of this mosquito species often prefer clean water bodies as their habitat (Service, 2008). Their immersion in the test solutions thus amounted to an unfavourable environment for them. It represented a milieu that was very different from their natural habitat. This certainly also posed a threat to their continued survival. This is further substantiated by the fact that in the control experiments no mortality was recorded (Table1). The increase in larval mortality with increase in extract concentration and increase in exposure time reported in this study corroborates the reports of Obomanu et al. (2006).

Fungal species used in this study exhibited varying degrees of susceptibility to the leaf extracts of P. nitida. This observed difference in susceptibility could be attributed to the inherent resistance factors of the test organisms among other factors (Ekpo and Etim, 2009). For instance the leaf extract (aqueous and ethanolic) did not (at any of the concentrations used in this study) inhibit the growth of M. canis, but did inhibit the growth of A. flavus and C. albicans, with differing values of inhibition zone diameter. Studies by Ubulom et al. (2011) revealed that both the aqueous and ethanolic seed extracts of P. nitida inhibited the growth of M. canis. In their phytochemical screening of the seeds they detected the presence of flavonoids and tannins. These photochemicals were not detected in the leaves. Tannins were reported to possess antifungal property by Baba-Moussa et al., (1999). Also, Reyes- Chilpa et al. (2009) reported that flavonoids possess antifungal property. Thus, the absence of flavonoids and tannins in the leaves of P. nitida may have been the reason for the absence of inhibitory effect of the leaf extracts on M. canis. However, further investigation is required to substantiate this view.

Although the results obtained from bioassays involving the fungal species and the extracts did not compare favourably with results obtained from assays using the standard drug, ketoconazole, the leaf extract of P. nitida is still a promising antifungal agent. The lower values obtained with the extracts could be attributed to the fact that the extracts used were in the crude state/form. The isolation and characterization of the active principle(s) for further investigations would greatly improve both the intensity and spectrum of activity.

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4. CONCLUSION

Results obtained from this study have confirmed the larvicidal effect of the crude leaf extracts of P. nitida on A. gambiae and its antifungal effect on A. flavus and C. albicans.

ACKNOWLEDGMENTS

Authors appreciate the assistance received from the Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria and the Entomology Unit of National Arbovirus and Vectors Research Centre (NAVRC), Enugu, Nigeria. Authors are also appreciative of the technical assistance received from Pharmaceutical Microbiology and Parasitology Unit, Faculty of Pharmacy, University of Uyo, Nigeria.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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