Antimicrobial Activities of the Extracts and Fractions of Allanblackia floribunda

INTRODUCTION

Allanblackia floribunda Oliver (Clausiaceae), commonly known as Tallow tree reproduces by seeds. Its occurrence is limited to tropical Africa, but centred mostly on the lowland rainforests (Van Rompaey, 2003).

The nuts of the plant produce fine oil taken by the members of local communities in Tanzania to relieve rheumatism (Anonymous, 2004). Also, in Cameroon, the stem bark of the plant mixed with Capsicum frutescens or Solanum anguivi is used for the treatment of cough (Betti, 2004). In Gabon, the bark is pounded and rubbed on the body to relieve painful conditions. There also, a decoction is taken for dysentery and as a mouthwash for toothache and, in Côte D`Ivoire, for stomach pains (Steentoft, 1988). In Ghana, the bark is used for medicinal purposes such as toothache, diarrhoea and as a pain reliever (Abiww, 1990). The bark decoction of the stem and root is also used in Central African Republic and West Africa to treat toothache, dysentery and as an analgesic (Lewis and Elvin-Lewis, 1977).

In Akwa Ibom State of Nigeria, the leaves as well as the bark of the stem and root of the plant are used by the local communities to treat dysentery, diarrhoea, skin diseases and some other microbial diseases.

The fruits and the seed kernels produce a hard white fat (Cunningham, 1993). The use of the fat in soap production has been suggested (Foma and Abdala, 1985). A new prenlated xanthone known as Allanxanthone A and some other known xanthones have been identified from the stem bark of A. floribunda and have also been shown to have cytotoxic activity against KB cell line (Nkengfack et al., 2002).

This study aimed at evaluating the leaves, stem bark and root bark of A. floribunda for antimicrobial activities against Staphylococcus aureus NCIB 8588, Bacillus subtilis NCIB 3610, Escherichia coli NCIB 86, Proteus vulgaris NCIB 67, Pseudomonas aeruginosa NCIB 950, Klebsiella pneumoniae NCIB 418, Candida albicans and Aspergilus flavus, in order to validate the ethnobotanical use of this plant for microbial diseases.

MATERIALS AND METHODS

Extraction: The fresh leaves (1 kg), stem bark (1 kg) and root bark (1 kg) of Allanblackia floribunda were collected in June, 2006, air-dried for a week and reduced to powder. The powder (300 g) was macerated in 1 L of EtOH-H₂O (1:1) for 72 h. The liquid extract obtained was concentrated to dryness in vacuo at 40°C to yield dry ethanol extract (40-50 g).

Phytochemical screening: Applying the methods of Sofowora (1993) and Trease and Evans (2002), the dry ethanol extract of each part was subjected to phytochemical screening to reveal the presence of its secondary metabolites.

Partition chromatography: The dry crude ethanol extract (30 g) of each plant part was dissolved in 40 mL of distilled water and successively partitioned with n-hexane (50 mL x3), chloroform (50 mL x3), ethyl acetate (50 mL x3) and n-butanol (50 mL x3) to yield their respective fractions. The fractions were separately concentrated to dryness in vacuo to give dry residues.

Test organisms: The bacteria used in this study were typed cultures obtained from the Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, while the fungi were clinical isolates collected from the same source. The bacteria: Staphylococcus aureus NCIB 8588, Bacillus subtilis NCIB 3610, Escherichia coli NCIB 86, Proteus vulgaris NCIB 67, Pseudomonas aeruginosa NCIB 950 and Klebsiella pneumoniae NCIB 418 were sustained on nutrient agar (Oxoid) slant at 4°C prior to use. However, the fungi Candida albicans and Aspergilus flavus were sustained on Sabouraud`s Dextrose Agar (Oxoid) slants at 4°C before use.

Antimicrobial test: The dry ethanol extract and the dry fractions were evaluated against the test microorganisms using agar-gel diffusion method described by Alade and Irobi (1993). The ethanol extract and the fractions were redissolved in Dimethyl sulphoxide (DMSO). The ethanol extract and the fractions were tested at concentration levels of 40 and 80 mg mL^-1. Fixed volumes (150 μL) of the ethanol extract, fractions and DMSO were separately introduced into equidistant wells bored on the surface of the agar and Sabouraud`s plates, which had been previously inoculated with one of the test organisms. A well containing a standard drug, chloramphenicol was made in the bacteria plates, while the fungal plates had a hole containing Nystatin as standard drug.

The bacteria were incubated at 37°C for 24 h, while the fungi were incubated at 25°C for seven days. The presence of zones of inhibition surrounding the wells was taken as an evidence of antimicrobial activity.

RESULTS

The extracts of the leaves, stem bark and root bark showed various classes of compounds (Table 1) inherent in the plant. The extracts of the three parts contained tannins and cardiac glycosides in high concentration, while flavonoids and terpenes occurred in moderate concentration. Anthraquinones, phlobatannins and alkaloids were absent in all the parts, while saponins were abundant in the leaves and absent in others.

Table 1:	Phytochemical screening results of the plant parts of Allanblackia floribunda

+++: High concentration; ++: Moderate concentration; - : Absent

The extracts of the leaves, stem bark and root bark exhibited varying degrees of antimicrobial effects, with leaves showing the highest antibacterial activity and the root bark, the least (Table 2). It is noteworthy that none of the extracts was effective against the test fungi. Also, E. coli was resistant to all the extracts. The inhibitory effect of the leaf extract against Ps. aeruginosa was much better than that of Chloramphenicol. The purification of the extracts by partition chromatography showed some improvement on the antimicrobial activity of all the extracts. For instance, the ethyl acetate fraction of the leaf extract elicited an improvement on the activity of the extract, though fungal activity was still lacking. With the exception of aqueous fraction, all the other fractions generally showed improved activity than the stem bark extract. This improved activity included fungal effect against C. albicans. However, n-hexane fraction gave the highest activity. This pattern is similar for root bark extract.

DISCUSSION

The result of this study showed that the extracts and fractions of the leaves, stem bark and root bark of Allanblackia floribunda gave good inhibitory effects against all the test microorganisms except A. flavus.

The result also revealed that the leaf extract gave more significant inhibitory effects than those of stem bark and root bark. However, all the extracts exhibited only antibacterial effects.

The fractions obtained from the extracts through partition chromatography gave improved activity against test organisms, including activity against C. albicans which the extracts failed to inhibit. A similar result has been observed for the leaves of Heinsia crinita (Ajibesin et al., 2003). This suggests that antimicrobial activity was increased by purification of active constituents of the plant. Generally, all the extracts and the fractions failed to inhibit A. flavus. However, aqueous fraction did not show significant inhibitory effect against all the test organisms except B. subtilis. This may be due to the fact that virtually all the antimicrobial principles had been extracted from the aqueous phase during fractionation. The best inhibitory effect was elicited by the leaf extract against Ps. aeruginosa, while the least was by ethyl acetate fraction of the stem bark against B. subtilis. Furthermore, the antimicrobial activities of the extracts and the fractions were dose-dependent.

The presence of terpenes observed in the phytochemical screening may be responsible for the enhanced effect of n-hexane fraction, since terpenes are non-polar compounds located in non-polar fractions. In a similar fashion, tannins, flavonoids and saponins may account for the improved activity of the ethyl acetate and butanol fractions. The antimicrobial activity of phenolics (tannins, flavonoids) and saponins has been established in some plants (Burapadaja and Bunchoo, 1995; Adesina et al., 2000; Binutu and Cordell, 2000; Pistelli et al., 2002; Mandal et al., 2005).

Table 2:	Antimicrobial activity of the extracts and fractions of the plant parts on the test organisms

L1 = Leaf ethanol extractB1 = Stem bark ethanol extractR1 = Root ethanol extract L2 = Leaf n-hexane fractionB2 = Stem bark n-hexane fractionR2 = Root n-hexane fraction L3 = Leaf chloroform fractionB3 = Stem bark chloroform fractionR3 = Root chloroform fraction L4 = Leaf ethyl acetate fractionB4 = Stem bark ethyl acetate fractionR4 = Root ethyl acetate fraction L5 = Leaf butanol fractionB5 = Stem bark butanol fractionR5 = Root butanol fraction L6 = Leaf aqueous fractionB6 = Stem bark aqueous fractionR6 = Root aqueous fraction A = 80 mg mL^-1Values are mean±SD (n = 4).- = No zone of inhibition. B= 40 mg mL^-1*: p<0.01 with respect to control C = Chloramphenicol (2 μg mL^-1) N = Nystatin (2 μg mL^-1) NA = Not applicable DM = DMSO

The extracts of the plant and their fractions gave significant zones of inhibition against the test organisms, thereby validating the ethnobotanical claims on this plant as remedy for the treatment of infections and diseases caused by these organisms. This result corroborates the reports on the validation of claims from ethnobotanical survey on other plants used to treat microbial diseases (Rajakaruna et al., 2002; Khan and Omoloso, 2002; Idowu et al., 2005; Rene et al., 2006; Ekpo et al., 2007). From the result, the plant was found to be effective against bacterial infections, but weak against fungal diseases. Further, the leaves whose extract and fractions exhibited the best activity are recommended for use in preference to other parts of the plant. Since activity improved with purification, this encourages further investigation to be carried out on the active fractions of the plant in order to isolate and characterize the active constituents responsible for the improved activity recorded in this study. The active constituents if isolated may show much better activity, leading to the production of effective antibiotics.