CHAPTER THREE
METHODOLOGY
3.1 Study Design
An experimental design was adopted to investigate the phytochemical composition and
antibacterial activity of unripe Musa sapientum (banana) fruit peel.
3.2 Sample Collection
Fresh unripe Musa sapientum fruits were collected in the morning from the Tombo
community, located in the Western Area of Sierra Leone. The samples were taken to the
Botany Department at Fourah Bay College, University of Sierra Leone, for identification
and taxonomic authentication.
3.3 Study Site
The study was conducted at the Pharmaceutical Science Laboratory, College of Medicine
and Allied Health Sciences (COMAHS), University of Sierra Leone, and the
Microbiology Laboratory at the Pharmacy Board of Sierra Leone.
3.4 Study Duration
The study was carried out from January to August 2025.
3.5 Reagents and Instruments
Reagents:
- Ethanol (95%)
- Distilled water
- Mayer’s reagent
- Acetic acid
- Copper acetate
- Hydrochloric acid (1% HCl)
- Ferric chloride (5% FeCl₃)
- Sulfuric acid (H₂SO₄)
- Olive oil
- Mueller-Hinton agar
- Nutrient agar
- Molisch’s reagent
- Black seed oil
Instruments:
- Beakers
- Test tubes
- Measuring cylinders
- Water bath
- Hot plate
- Whatman filter paper
- Funnels
- Mortar and pestle (or blender)
- Droppers
- Refrigerator
- Autoclave
- Incubator
- Petri dishes
- Inoculating loop/needle
- Micropipette or sterile pipette
- Sterile cotton swabs
- Cork borer or sterile tips
- Bunsen burner
- Rotary evaporator
- Vortex mixer
3.6 Extraction Medium
The solvents used for extraction were ethanol (95%) and distilled water.
3.7 Test Methods
3.7.1 Extraction of Plant Material
The collected banana peels were washed thoroughly with tap water, followed by rinsing
with distilled water. The peels were sliced into smaller pieces and air-dried in a shaded
area at room temperature for seven days. Once fully dried, the peels were ground into
powder using a mortar and pestle and stored in airtight containers.
**Aqueous Extract:** 10 g of powdered peel was mixed with 100 ml of distilled water in
a conical flask. The mixture was stirred intermittently and left to stand for 72 hours. After
filtration using Whatman filter paper, the filtrate was concentrated with a rotary
evaporator at a temperature below 45°C to obtain the crude aqueous extract.
**Ethanolic Extract:** Similarly, 10 g of powdered peel was macerated in 100 ml of
95% ethanol, stirred intermittently, and allowed to stand for 72 hours. The extract was
filtered and concentrated using a rotary evaporator at a temperature below 45°C.
3.7.2 Phytochemical Screening
Standard qualitative tests were conducted to detect the presence of major phytochemicals
in the extracts:
- **Alkaloids:** Presence confirmed by creamy white precipitate upon addition of HCl
and Mayer’s reagent.
- **Flavonoids:** Pale brown color developed after adding ammonia solution and HCl.
- **Tannins:** A dirty-green precipitate was observed after adding ferric chloride.
- **Glycosides:** Oil layer appeared after mixing extract with acetic acid and
concentrated sulfuric acid.
- **Saponins:** Stable emulsion formed upon vigorous shaking with olive oil.
- **Phenols:** Bluish-black coloration observed upon addition of ferric chloride
solution.
- **Steroids:** Red coloration developed upon adding concentrated sulfuric acid.
- **Resins:** Reddish-brown precipitate formed upon mixing with copper acetate.
- **Carbohydrates:** A reddish ring formed when Molisch’s reagent and sulfuric acid
were added and heated.
3.7.3 Antibacterial Activity
Bacterial strains used included: Staphylococcus aureus, Streptococcus pyogenes,
Klebsiella pneumoniae, and Escherichia coli. These were obtained from the Microbiology
Laboratory at the Pharmacy Board of Sierra Leone.
3.7.4 Sub-Culturing of Bacteria
Nutrient agar plates were prepared and streaked with bacterial isolates using inoculating
loops. Plates were incubated at 37°C for 24 hours to confirm viability.
3.7.5 Preparation of Bacterial Suspension
A 0.9% sodium chloride solution was prepared. Colonies of bacterial isolates were
suspended in the saline solution and vortexed until uniform turbidity was observed.
3.7.6 Preparation of Culture Medium
Mueller-Hinton agar (7 g in 250 ml distilled water) was prepared, sterilized at 121°C for
15 minutes in an autoclave, cooled to 45°C, and poured into sterile Petri dishes to
solidify.
3.7.7 Antibacterial Susceptibility Testing
The agar well diffusion method was employed. Agar plates were swabbed with bacterial
suspensions. Wells (7 mm diameter) were bored into the agar, and 50 μl of each extract
was introduced into each well. Plates were left for 20 minutes and incubated at 37°C for
24 hours. The zones of inhibition were measured in millimeters and recorded. Black seed
oil and distilled water were used as positive and negative controls, respectively.