In vitro Study on the Antimicrobial Activity and Phytochemical Analysis of Ethanolic Extracts of the Mesocarp of Voacanga africana

MATERIALS AND METHODS

Collection of Plant Materials
Ripe fruits of the plant were harvested from the wild and identified as V. africana Stapf. by a plant Taxonomist, at the Department of Biology, Federal University of Technology, Owerri, Imo State, Nigeria. The study was carried out from November, 2007 to March, 2009. The fruits were slit open and seeds extricated. The mesocarp were air-dried for 14 days, pulverized and stored in air-tight sterile bottle.

Test Organism
Clinical isolates of the Bacteria-E. coli, P. aeruginosa, S. marcescens, S. aureus and the test Fungi-C. albicans, were collected from the Department of Microbiology, Federal Medical Centre, Owerri, Nigeria while the other test fungi-A. flavus, A. niger, A. solani and R. stolonifer, were collected from the Plant Pathology Laboratory, National Root Crop Research Institute, Umudike, Abia State, Nigeria. They were separately sub-cultured and the pure culture resub-cultured on Nutrient Agar and Sabouraud Dextrose Agar media, respectively and stored at 4^oC for further studies.

Extraction of Active Principles
Cold and hot absolute ethanol were used in the extractions according to the methods described by Biyiti et al. (1988), Harborne (1973) and Ogbonna et al. (2007), respectively.

In the cold percolation, 20 g of the dried, blended mesocarp were weighed out, transferred into a beaker and 100 mL of absolute alcohol added. The mixture were agitated and allowed to extract at laboratory temperature for 48 h. The mixture was then filtered in a flask, using Whatman’s No. 1 filter paper (Ogbonna et al., 2007). The filtrate was evaporated at 40°C on a hot plate till supernatant. The concentrated extracts were allowed to cool and stored in a sterile bottle. The hot ethanol extraction (Soxhlet), 20 g of the dried powdered mesocarp were fed into the Soxhlet extractor and extracted for 24 h at 80°C in 200 mL of absolute ethanol. The extracts were allowed to cool and stored at 4°C in a sterile bottle.

Phytochemical Screening
Standard screening procedures were followed as described by Trease and Evans (1983) and Ayoola et al. (2008).

Test for Reducing Sugars (Fehling’s Test)
The aqueous ethanol extract (0.5 g in 5 mL of water) was added to boiling Fehling’s solution (A and B) in a test tube. The solution was observed for a colour reaction.

Test for Anthraquinones
The 0.5 g of the extract was boiled with 10 mL of sulphuric acid (H₂SO₄) and filtered while hot. The filtrate was shaken with 5 mL of chloroform. The chloroform layer was pipetted into another test tube and 1 mL of dilute ammonia was added. The resulting solution was observed for colour changes.

Test for Saponins
To 0.5 g of extract was added 5 mL of distilled water in a test tube. The solution was shaken vigourously and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigourously after which it was observed for the formation of an emulsion.

Tannins
Sample of the powdered mesocarp (3 g) was boiled in 5 mL of distilled water for 3 min on a hot plate. The mixture was filtered while hot and the resulting filtrate was used to carry out ferric chloride test. 1.0 g sample of the filtrate was weighed into a beaker and 10 mL of distilled water added. This was boiled for 5 min. Two drops of 5% ferric chloride (FeCl₂) was then added. Production of greenish precipitate indicated the presence of tannins (Trease and Evans, 1983).

Test for Alkaloids
The 0.5 g of extract was diluted to 10 mL with acid alcohol, boiled and filtered. To 5 mL of the filtrate was added 2 mL of dilute ammonia. Five milliliter of chloroform was added and shaken gently to extract the alkaloidal base. The chloroform layer was extracted with 10 mL of acetic acid. This was divided into two portions. Mayer’s reagent was added to one portion and Draggendorff’s reagent to the other. The formation of a cream (with Mayer’s reagent) or reddish brown precipitate (with Draggendorff’s reagent) was regarded as positive for the presence of alkaloids.

Test for Cardiac Glycosides
To 0.5 g of extract diluted to 5 mL in water was added 2 mL of glacial acetic acid containing one drop of ferric chloride solution. This was underlayed with 1 mL of concentrated sulphuric acid. A brown ring at the interface indicated the presence of a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer a greenish ring may form just above the brown ring and gradually spread throughout this layer.

Microbial Susceptibility Test
Agar-well diffusion method was used in the investigation as described by Boakye-Yiadom (1979) and Radhika et al. (2008). Five wells, 8 mm each were made on solidified nutrient agar and sabouraud dextrose agar media plates, respectively with the aid of a sterile cork borer. 0.2ml of the log phase culture of the test microbes: E. coli, P. aeruginosa, Serratia marcescen and S. aureus were seeded on the surface of the nutrient agar medium while C. albicans, A. flavus, A. niger, A. solani and R. stolonifer were seeded on the sabouraud dextrose agar medium, using swab stick. The cut agar discs were removed with the aid of sterile forceps. Concentrations of 25, 50, 100, 150, 200, 250 and 500 mg mL^-1 of the extracts were separately introduced into separate cavities. Three control holes were set up, one, empty, one filled with gentamycin and the other filled with clotrimazole, to serve as positive control for the bacteria and fungi, respectively.

The plates were incubated at 37°C for 24 h and 15 days, respectively for the bacteria and fungal cultures. The observed zones of inhibition were measured using transparent metric ruler.

Minimum Inhibitory Concentration of the Extracts
Determination of the Minimum Inhibitory Concentrations (MIC) followed the methods of Egorov (1985) and Radhika et al. (2008). Extracts concentrations of 10, 15, 25, 50, 100, 125, 150, 200, 250 and 500 mg mL^-1 were used in the exercise. The lowest concentration of each of the extracts in each treatment, showing zero growth after 24 h for the bacteria and 15 days for the fungi, were recorded as the MIC values. Percentage (%) inhibition was calculated as extract concentration/concentration of standard (antibiotic e.g., Gentamycin) multiplied by 100.

Minimum Cidal/Static Concentration
The determination of the minimum bactericidal cum fungicidal concentrations of the extracts, was done according to the procedure described by Rotimi et al. (1988) and Alade and Irobi (1993). The innoculum from the culture tubes containing different concentrations of the extracts, showing no visible growth of the organisms from the MIC test, were subcultured in sterile nutrient agar and incubated at 37°C for 24 h and 15 days, respectively for the bacteria and fungi. The lowest concentration of the extracts without any growth was noted as the minimum cidal concentration (MBC/MFC).

RESULTS AND DISCUSSION

The phytochemical screening test, showed the presence of some active principles; Alkaloids, Anthranoids, Anthraquinone, Cardiac glycoside, Saponins, Starch and Tannins. Phenol and Phlobtanin were absent (Table 1). At the expiration of the incubation periods of 24 h and 15 days, respectively, for the bacteria and fungi sets. The zones of inhibition of E. coli, P. aeruginosa, S. marcescen and S. aureus, C. albicans, A. flavus, A. niger, A. solani and R. stolonifer were determined and the results are shown in Table 2. The antimicrobial sensitivity test, using Agar-Well Diffusion technique, showed that there was no inhibition on the growth of A. flavus, A. niger and P. aeruginosa, by the extracts. However, other test microbes were susceptible to the extracts. With mean inhibition diameter ranging from 6.5-10 mm in the hot ethanolic extract and 6.5-8.5 mm in the cold extract (Table 2). The minimum inhibitory concentration of the extracts against the test organisms susceptible to it range from 25-100 mg mL^-1 in hot ethanol extract and 50-200 mg mL^-1 in the cold extract (Table 3).

The choice of absolute ethanol as the extraction agent was due to the fact that it was readily available and cheap to procure. The vapourization temperature (78°C) is blow the thermolabile limit for most protein (Adebayo, 1990). Some fruits mesocarp contained oil and fatty acids that were not soluble in water (Ogbonna et al., 2007). The low pH of between 4.7 and 4.9 showed that the extracts are weak acid medium, suggesting that the active principles in the extracts are potent in acid medium.

Table 1:	Phytochemical analysis of the mesocarp extracts of V. africana
+ve = Present, -ve = Absent

Table 2:	Sensitivity test for the bacteria and fungal species on the mesocarp extracts of V. africana (Percentage inhibition in bracket)
HEE: Hot ethanol extract, CEE: Cold ethanol extract, EH: Empty hole, GH: Gentamicin hole, CH: Clotrimazole hole

Table 3:	Minimum inhibitory concentration of the mesocarp extracts of V. africana

This is in consonance with the report of Nwadiaro and Nwachukwu (2007), on the extracts of Cymbopogon citratus, Ceiba pentandra and Loranthus bengwelensis. The extracts had antibacterial activity against, E. coli, S. marcescens and S. aureus. It also demonstrated antifungal activity against A. solani, C. albican and R. stolonifer. Thus; suggesting that the mesocarp extracts of V. africana has a broad spectrum antimicrobial property. The antibacterial and the antimycotic potency may be due to the presence of some active principles: Alkaloids, Anthranoids, Anthraquinone, Glycosides, Saponins, Starch and Tannins. This is in consonance with the report of Ebena et al. (1991) on the extracts of Garcinia kola, Boreria ocymoides, Kola nitida and Citrus aurantifolia, Nwadiaro and Nwachukwu (2007) on the extracts of C. citratus, C. pentandra and L. bengwelensis, Duru et al. (2009), on bark extracts of V. africana and Deepthi et al. (2008), on the extracts of Alstonia scholaris.

The sensitivity test result, showed that the extracts were less potent than the standard antibiotics; Gentamicin and Clotrimazole, used in the study. The hot and the cold fractions were apparently not equipotent, The hot extraction method had an advantage over the cold method since it inhibited S. marcescens at same concentration. At 100 mg mL^-1 extract concentration, the HEE inhibition values were 30.76, 37.50, 39.47, 30 and 20.97% against E. coli, S. marcescens, S. aureus, A. solani and R. stolonifer, respectively while that of CEE were 28.84, 00, 34.21, 26.67 and 0% E. coli, S. Vmarcescens, S. aureus, A. solani and R. stolonifer, respectively. However, they were equipotent against C. albicans, 25.92% for both HEE and CEE treatments.

Generally, the less potency, of the extracts, relative to the standard antibiotics may be due to the fact that it was crude and required further purification.

The results also showed that the extracts did not elicit antimicrobial activity against P. aeruginosa, A. flavus and A. niger. These effects could be either due to cultural composition of the media or the biochemical composition of the target structure of the organism Ogbonna et al. (2007). It is also possible, that the insensitivity of the non-susceptible test microbes, on the extracts could be due to short period of exposure, on the part of the test bacteria or the concentration quotient was minimal for cidal activity on the part of the test fungi.

The result obtained in this study had shown that the mesocarp of V. africana, which hitherto, waste in our forest contain medicinally useful phyto-chemicals, such as Alkaloids, anthranoids, anthraquinones, glycosides, saponins, starch and tannins. These substances are antimicrobial and could be extracted for disease therapy, pharmaceutical exploits, researches in Microbiology, Biotechnology and general medicine.