CHAPTER II. MATERIALS AND METHODS
II.1. Collection and identification of plant materials
The fruits and twigs of Ficus ovata were collected
from Mount Kala, Centre region of Cameroon. The plant was identified at the
Cameroon National Herbarium, Yaounde, where a voucher specimen was conserved
under the reference number 26996SRF/Cam. The collected plant parts
were separated from undesirable materials. They were dried under the
shade separately. The plant parts were ground into tiny debris with
the help of a suitable grinder, kept in a cool and dry place until analysis
commenced.
Preparation of extracts
We weight 125g of each plant part and macerated separately in
1L of ethanol 95% or 1L of ethanol: water solution in the ratio 1:1 for
48hours. The whole mixture was successively filtered through a piece of clean,
white cotton material. The filtrates (ethanolic and hydroethanolic extracts of
the twigs and fruits) obtained were evaporated to dryness in a drying room. We
obtained four extracts as shown below (figure 9).
Grinding and drying under the shade
Maceration with ethanol or hydroethanolic (1:1) solvents for
48H,
Filtration
Residue
Filtrate
Solvent
Evaporation
FOHF (1:1) Extract
FOEF
Extract
Twigs and Fruits of Ficus ovata (moraceae)
FOHT Extract
FOET
Extract
37°C
Figure 9 :
protocol for the extraction by maceration in ethanol and
hydroethanol (1:1)
FOHT: Ficus Ovata Hydroethanolic Twigs
FOHF: Ficus Ovata Hydroethanolic Fruits
FOET: Ficus Ovata Ethanolic Twigs
FOHF: Ficus Ovata Ethanolic Fruits
II.2. In vitro study
II.2.1. Phytochemical screening of the extracts
In view of determining the different secondary metabolites
responsible for the biological activity of the plant, extracts underwent
phytochemical screening as follows;
1- Test for Phenol was done by dissolving 250
mg of each extracts in 4 ml of distilled water and the content heated for 15
minutes. After cooling and filtration 2 drops of freshly prepared ferric
cyanate solution (1 ml FeCl3 1% and 1ml KFe (CN)6) was
added to 1 ml of each filtrate. The presence of a greenish-blue coloration
indicated the presence of polyphenols (Harbone, 1976).
2- Test for Alkaloids (Mayer test)
was done by heating 100 mg of the each extracts in 2 ml of
H2SO4 2% for 2 minutes after which the content was
filtered. Few drops of the Mayer reagent (1.358g HgCl2+500 ml
H2O and 0.8g KI +200 ml H2O) were added in 1ml of the
filtrate and the presence of a white precipitate or turbid solution was an
indicator of the presence of alkaloids (Odebeyi and Sofowora,
1978).
3- Test for Saponines was done by
adding 250 mg of the extract in 5 ml of distilled water. After vigorous
homogenisation the mixture was heated to boil, the appearence of foam that
persisted 20 minutes after cooling was an indicative of the presence of
saponines (Wall et al., 1954).
4- Test for Tannins was done by adding 100 mg
of each extracts in 2 ml of distilled water followed by heating in a water bath
and then filtering. Few drops of 3% ferric chloride were added in 1 ml of
filtrate and the observation of a blue-black or greenish-dark coloration
indicated the presence of tannins (Trease and Evans, 1989).
5- Test for phlobatannins was done by
dissolving 100 mg of each extract in 2 ml of distilled water. After filtration,
to 0.5 ml of each filtrate was added 1 ml of hydrochloric acid 1% and the
deposit of a red precipitate was an indicator of the presence of phlobatannins
(Trease and Evans, 1989).
6- Test for glycosides was done by dissolving
100mg of each extract in 5 ml of HCl then neutralised by 5 ml of 5% caustic
soda (soude). Drops of Fehling's solution [A (40 g of CuSO4 5
H2O per litre) + B (160 g of tartrate double of sodium and potassium
+ 130 g of NaOH per litre)] were added one after another and the appearance of
a red precipitate was an indicative of the presence of glycosides
(Trease and Evans, 1978).
7- Test for flavonoids was done by dissolving
250 mg of each extract in 5 ml of sodium hydroxide 1N.The observation of a
yellow solution is a preliminary evidence of the presence of
flavonoids and the decolourisation of the yellow colour observe on the addition
of a few drops of concentrated HCl confirms its presence (Trease and
Evans, 1978).
Preparation of variable concentration of
extracts
The mother solution (5 mg/ml) was prepared by dissolving 100
mg of the pure extract with 20 ml of water. After vigorous homogenising, the
following daughter solutions were prepared. It is from this working solution
that the test below was carried on.
Table V: Preparation of working
solution of our extract
|
Tubes
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
|
Concentration (mg/ml)
|
5
|
2.5
|
1.5
|
0.75
|
0.5
|
0.25
|
0.05
|
0.025
|
0.012
|
|
Volume of extract (ml)
|
5
|
2.5
|
1.5
|
0.75
|
0.5
|
0.25
|
0.05
|
0.025
|
0.012
|
|
Volume of solvent (ml)
|
0
|
2.5
|
3.5
|
4.25
|
4.5
|
4.75
|
4.95
|
4.975
|
0.980
|
|
Final volume (ml)
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
5
|
| 
