Over the years therapeutic manipulations in folk Medicine and Orthodox medical
practices, has been dependent on pharmacopoeic formulations from plants. These
plants, generally called Medicinal Plants, have been found to contain active
secondary metabolites called Phytochemicals (Okwu and Josiah,
2006; Chukwuedo et al., 2007; Ogbonna
et al., 2007; Ojo and Jide-Ojo, 2007; Nwadiaro
and Nwachukwu, 2007; Deepthi et al., 2008;
Oigiangbe et al., 2007; Duru
et al., 2009).
Medicinal plants constitutes, the flora of wild and cultivated species of trees, shrubs, herbs and lianas. These groups of plants belong to different taxonomic families, among which is the Apocynaceae, which harbor the shrub-V. africana Stapf.
Voacanga africana is an abundant, deciduous, mesophytic, sap-woody, perennial, aborescent shrub of the primary and secondary forest, within the Tropical Rain Forest especially in Nigeria and the Guinea Savannah woodland belt. A mature V. africana crop is not more than 10 m tall, lowly branched, stem, with smooth, grayish white bark. Slash exudes milky latex.
The leaves and roots decoction of this plant had been implicated in folk medicine
for the treatment of malaria, diarrhea, infant convulsion, insane persons and
heart arches (Burkill, 1995). This stimulated interest
to further investigate this plant, with a view to determining the antimicrobial
activity of the fruit mesocarp extracts in in vitro culture as well as
the phytochemical composition of the extracts.
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.
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 4oC
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.
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 Whatmans 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.
Standard screening procedures were followed as described by Trease
and Evans (1983) and Ayoola et al. (2008).
Test for Reducing Sugars (Fehlings Test)
The aqueous ethanol extract (0.5 g in 5 mL of water) was added to boiling
Fehlings 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.
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. Mayers reagent was added to one portion and Draggendorffs
reagent to the other. The formation of a cream (with Mayers reagent) or
reddish brown precipitate (with Draggendorffs 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
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.
|| Phytochemical analysis of the mesocarp extracts of V.
|+ve = Present, -ve = Absent
||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
|| 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.