Plant material
Fresh leaves of Senna
occidentalis (L.) were collected from botanical garden of
University of Ibadan, Ibadan. Specimens were identified by Mr. Donatus Eratus
and authenticated by Dr. Ayodele of the Department of Botany, University of
Ibadan, Oyo State, Nigeria. The leaves were chopped into pieces using knife and
then air dried under shade for 10 days and grounded into mesh size and kept in a
non-absorptive nylon for subsequent use.
Preparation of extracts
Dried and milled leaf materials were extracted successively with
Soxhlet extractor at temperature of 80 °C. Each of the solvent; hexane,
ethylacetate and methanol were allowed to remain in contact with the plant
material for 12 h; the extracts were evaporated to dryness using rotary
evaporator.
Phytochemical analysis
The extracts were analyzed for the presence of alkaloids, resins,
tannins, saponins, flavonoids, glycosides, phenols, anthraquinones, cardiac
glycosides, steroids, phlobatannins, reducing sugars [22–25].
Test for alkaloids
0.2 g of extracts was shaken with 1 % HCl for two minutes. The
mixture was filtered and drops of Dragendorff’s reagent added. Formation of a
precipitate indicated the presence of alkaloids.
Test for saponins
0.2 g of extracts was shaken with 5 ml of distilled water in a test
tube. Frothing which persists on warming was taken as evidence for the presence
of saponins.
Test for tannins
0.2 g of extracts was stirred with distilled water and filtered.
Ferric chloride was added to the filtrate. A blue-black, green or blue-green
precipitate was taken as an evidence for the presence of tannins.
Test for steroids (Salkowski’s test)
0.2 g of the extracts was dissolved in 2 ml of chloroform.
Concentrated sulphuric acid was carefully added to form a lower layer. A
reddish-brown colour at the interphase indicated the deoxy sugar characteristics
of cardenolides.
Test for cardiac-active glycoside (keller-killani test)
0.2 g of the extracts was dissolved in 2 ml of glacial acetic acid
containing one drop of ferric chloride solution followed by the addition of 1 ml
of concentrated sulphuric acid. A brown ring at the interface confirmed the
presence of cardiac glycoside.
Test for reducing sugars
0.2 g of the extracts was shaken with distilled water and filtered.
The filtrate was boiled with drops of Fehling’s solution A and B for two
minutes. An orange precipitate on boiling with the Fehling’s solution indicated
the presence of reducing sugars.
Test for flavonoids
A little amount of magnesium powder and few drops of concentrated
hydrochloric acid were added to 3 ml of the extracts. A red or intense red
colouration indicated the presence of flavonones.
Test for resins
5 ml of copper acetate solution was added to 5 ml of the extracts.
The resulting solution was shaken vigorously and allowed to separate. A green
coloured solution is an evidence of the presence of resin.
Test for anthraquinones
0.2 g of the extracts was shaken with 4 ml of benzene. The mixture
was filtered and 2 ml of 10 % ammonia solution was added to the filtrate. The
mixture was shaken and the presence of pink, red or violet colour in the
ammoniacal (Lower) phase indicated the presence of free anthraquinones.
Test for phenols
0.2 g of extracts was dissolved in Ferric chloride solution. A
green or dirty green precipitate indicated the presence of phenolic
compound.
Test for phlobatannins
The extracts (0.5 g) was dissolved in distilled water and filtered.
The filtrate was boiled with 2 % HCl solution. Red precipitate shows the
presence of Phlobatannins.
Test for glycosides
The extracts was hydrolyzed with HCl solution and neutralized with
NaOH solution. A few drops of Fehlings solution A and B were added. Red
precipitate indicates the presence of glycosides.
Scavenging of hydrogen peroxide
The ability of the extracts to scavenge hydrogen peroxide was
determined according to the method by Nabavi et
al., 2008a and 2008b [26, 27]. A
solution of hydrogen peroxide (2 mM) was prepared in phosphate buffer (pH 7.4).
The concentration of hydrogen peroxide was determined by absorption at 285 nm
using a UV/Vis spectrophotometer. The samples at ‘1 mg/ml, 0.5 mg/ml,
0.25 mg/ml, 0.125 mg/ml and 0.0625 mg/ml’ were added to
H2O2. The decrease in
absorbance of H2O2 at 285 nm was
measured spectrophotometrically after ten minutes (10 min) against a blank
solution containing the test sample in phosphate buffer solution (PBS) without
H2O2 and blank solution
containing phosphate buffer without hydrogen peroxide (control). All the tests
were performed in triplicate. The percentage of hydrogen peroxide scavenged by
the extracts was calculated as follows:
$$ \%\kern0.5em Scavenged\kern0.5em \left[{H}_2{O}_2\right]=\left({A}_c-{A}_s\right)/{A}_c\Big]\times 100 $$
(1)
Where Ac is the absorbance of the control
and As the absorbance in the presence of the sample of
extract and standard [25,
26]. The values of % inhibition
were obtained from Eq. 1. For the 50 %
Inhibitory Concentration (IC50) evaluation of the
extract, graphs showing the concentration of the test samples (hexane extract,
ethyl acetate extract, methanol extract and the alpha tocopherol) versus %
Inhibition (% H2O2 reduction) were
plotted.
Preparation of graded concentration of the samples
1000 mg of each sample was weighed and dissolved into 5 ml of the
solvent of extraction in order to obtain proper dissolution. From the 200 mg/ml
solution, 2.5 ml was taken into another sample bottle and 2.5 ml of solvent was
added to give 100 mg/ml. From this, 2.5 ml is taken into another sample bottle
and 2.5 ml of solvent was added to give 50 mg/ml solution. From the 50 mg/ml
solution, 2.5 ml was taken into another sample bottle and 2.5 ml of solvent
added to give 25 mg/ml solution. Similar procedure was followed to obtain the
12.5 mg/ml and 6.25 mg/ml concentrations, using dry filter paper dispersion
method.
Organisms
Bacteria- S.a
: Staphylococcus aureus,
E.c
: Eshericha coli,
B.sab
: Bacillus subtilis,
Ps. a
: Pseudomonas aeruguinosa,
Sal. t
: Salmonella typhi,
Klebs
: Klebsiellae pneumoniae.,
Fungi-
C. a
: Candida albicans,
A. n
: Aspergillus niger,
Pen
: Penicillum notatum,
Rhi
. : Rhizopus stolonifer.
-ve: Negative control; Methanol for methanolic
extract, ethylacetate for ethylacetate extract and hexane for hexane
extract.
+ve: Positive control; Gentamicin 10 μg/ml (bacterial) and
Tiocosnazole 70 % (fungi).
Antimicrobial screening
The microbes used were suspended in suitable nutrition media and
was poured into a sterile petri-dish and allowed to incubate for 24 h at 37 °C.
Suitably cut circular filter paper pieces were immersed in the antibiotic
solutions of both test samples and standards. Then the pieces were placed on the
nutritional microbial media all over with suitable gaps in between and incubated
again.
After 24 h of incubation, the plates were removed and the diameter
of the zone of inhibition of test and standard samples were measured in
millimeters.
By comparing the areas of zone of inhibition of test extracts with
standard, the concentration and potency of test samples were determined.
