3 MATERIALS AND METHODS
3.1 Chemicals and reagents
Folin Ciocalteu’s Phenol reagent, acetic acid, Wagners
reagent, iron(III) chloride, hydrochloric acid, sodium
carbonate, ammonia solution, gallic acid, quercitin,
methanol, and diethyl ether were purchased from
Merck Ltd. All the chemicals and reagents were
of analytical grade and were used without further
purification. Reagents used for standard preparations
are quercetin and gallic acid were purchased from
Sigma-Aldrich.
3.2 Equipment
Sohxlet assembly, Rotary Evaporator (R-200, Buchi
Larbortechnik), Analytical Balance CITIZEN scale
CX-220 (CITIZEN Private Ltd), 20∼325 Mesh
Grinder Vertical hammer mill pulverizer, Beckman
Coulter DU 720 UV–Visible spectrophotometer (700
series (6584) R), Calibrated micropipettes used to
accurate measurement and transfer.
3.3 Sample collection
Senna didymobtrya roots were collected from natural
geographical landscapes of West Uyoma sub-location,
Siaya County, Kenya.They were identified and authenticated at the Department of Biological sciences, Moi
University where voucher specimen (SD 2018/03)
were preserved for future reference.
3.4 Preparation of Senna didymobtrya roots extract
The collected roots were washed several times with
distilled water to remove dust. They were dried at
room temperature under shade for 3 weeks, chopped
into small pieces (approximately one centimetre) and
pulverized using a laboratory mill.
The extraction was carried out according to the
method described by Kigondu, Rukunga, Keriko,
Tonui, and Gathirwa (2009) with slight modifications.
Using an electric analytical beam balance, for each
extraction solvent 50 g of powdered dried plant material was weighed, placed in Soxhlet apparatus, and
separately extracted with 250 mL of diethyl ether,
methanol, and distilled water solvents for 48 hours.
The extracted sub samples were transferred in a conical
flask, and then placed in a rotary vacuum evaporator
in a water bath at 40
◦ C to recover the solvent and concentrate the crude extract. The semi-solid extracts were
placed in sterile beakers and were placed in a desiccator containing anhydrous sodium sulfate for 24 hours
for complete evaporation of the solvent. The total yield
of the solid crude extracts were weighed and put in a
tightly screwed capped glass containers and stored in
the refrigerator at 4
◦ C prior to use in the phytochemical screening and determination of total phenol and
flavonoid content.
Yield of the extract obtained was calculated as
follows:
Extractive yield value =
Weight of concentrated extract
Weight of plant dried powder
× 100
3.5 Qualitative phytochemical analysis
The phytochemical analysis of the extracts for identification of bioactive chemical constituents was done
using standard procedures by Trease and Evans (1989)
and Sofowara (1983).
Tannins
A sample of 0.5 g was put in a test tube and 20 mL
of distilled water was added and heated to boiling.
The mixture was then filtered and 0.1% of FeCl 3 was
added to the filtrate and observations made. A brownish green colour or a blue black colouration indicate
the presence of tannins.
Saponins
The crude solvent extract was mixed with 5 mL of
water and vigorously shaken. The formation of a stable
form indicates the presence of saponins.
Flavonoids
About 1 g of the plant extract was mixed with a few
fragments of magnesium ribbon (0.5 g) and a few drops
of concentrated hydrochloric acid were added. Development of a pink or magenta red colour after 3 minutes
indicates the presence of flavonoids.
Terpenoids
The solvent extracts of the plant material were taken in
a clean test tube, 2 mL of chloroform was added, the
test tube was vigorously shaken, and then evaporated
to dryness. To this, 2 mL of concentrated sulfuric acid
was added and heated for about 2 minutes. A grayish
colour indicates the presence of terpenoids.
Glycosides
Keller–Kilani test
The solvent plant material extract was mixed with 2
mL of glacial acetic acid containing 1–2 drops of 2%
solution of FeCl 3 , the mixture was then poured into
a test tube containing 2 mL of concentrated sulfuric
acid. A brown ring at the interface of the two solutions
indicates the presence of cardiac glycoside.
Alkaloids
The crude extract was mixed with 1% of HCl in a test
tube. The test tube was then heated gently and a few
drops of Mayers and Wagners reagents were added
by the side of the test tube. A resulting precipitate
confirms the presence of alkaloids.
Steroids
Liebermann’s Burchard reaction
About 2 g of the solvent extract was put in a test tube
and 10 mL of chloroform was added and filtered. The
filtrate, 2 mL, was mixed with 2 mL of a mixture of
acetic acid and concentrated sulfuric acid.A blue green
ring indicates the presence of steroids.
Phenols
The plant extract was put in a test tube and treated
with a few drops of 2% FeCl 3 . A blue green or black
colouration indicates the presence of phenols.
152
3.1 Chemicals and reagents
Folin Ciocalteu’s Phenol reagent, acetic acid, Wagners
reagent, iron(III) chloride, hydrochloric acid, sodium
carbonate, ammonia solution, gallic acid, quercitin,
methanol, and diethyl ether were purchased from
Merck Ltd. All the chemicals and reagents were
of analytical grade and were used without further
purification. Reagents used for standard preparations
are quercetin and gallic acid were purchased from
Sigma-Aldrich.
3.2 Equipment
Sohxlet assembly, Rotary Evaporator (R-200, Buchi
Larbortechnik), Analytical Balance CITIZEN scale
CX-220 (CITIZEN Private Ltd), 20∼325 Mesh
Grinder Vertical hammer mill pulverizer, Beckman
Coulter DU 720 UV–Visible spectrophotometer (700
series (6584) R), Calibrated micropipettes used to
accurate measurement and transfer.
3.3 Sample collection
Senna didymobtrya roots were collected from natural
geographical landscapes of West Uyoma sub-location,
Siaya County, Kenya.They were identified and authenticated at the Department of Biological sciences, Moi
University where voucher specimen (SD 2018/03)
were preserved for future reference.
3.4 Preparation of Senna didymobtrya roots extract
The collected roots were washed several times with
distilled water to remove dust. They were dried at
room temperature under shade for 3 weeks, chopped
into small pieces (approximately one centimetre) and
pulverized using a laboratory mill.
The extraction was carried out according to the
method described by Kigondu, Rukunga, Keriko,
Tonui, and Gathirwa (2009) with slight modifications.
Using an electric analytical beam balance, for each
extraction solvent 50 g of powdered dried plant material was weighed, placed in Soxhlet apparatus, and
separately extracted with 250 mL of diethyl ether,
methanol, and distilled water solvents for 48 hours.
The extracted sub samples were transferred in a conical
flask, and then placed in a rotary vacuum evaporator
in a water bath at 40
◦ C to recover the solvent and concentrate the crude extract. The semi-solid extracts were
placed in sterile beakers and were placed in a desiccator containing anhydrous sodium sulfate for 24 hours
for complete evaporation of the solvent. The total yield
of the solid crude extracts were weighed and put in a
tightly screwed capped glass containers and stored in
the refrigerator at 4
◦ C prior to use in the phytochemical screening and determination of total phenol and
flavonoid content.
Yield of the extract obtained was calculated as
follows:
Extractive yield value =
Weight of concentrated extract
Weight of plant dried powder
× 100
3.5 Qualitative phytochemical analysis
The phytochemical analysis of the extracts for identification of bioactive chemical constituents was done
using standard procedures by Trease and Evans (1989)
and Sofowara (1983).
Tannins
A sample of 0.5 g was put in a test tube and 20 mL
of distilled water was added and heated to boiling.
The mixture was then filtered and 0.1% of FeCl 3 was
added to the filtrate and observations made. A brownish green colour or a blue black colouration indicate
the presence of tannins.
Saponins
The crude solvent extract was mixed with 5 mL of
water and vigorously shaken. The formation of a stable
form indicates the presence of saponins.
Flavonoids
About 1 g of the plant extract was mixed with a few
fragments of magnesium ribbon (0.5 g) and a few drops
of concentrated hydrochloric acid were added. Development of a pink or magenta red colour after 3 minutes
indicates the presence of flavonoids.
Terpenoids
The solvent extracts of the plant material were taken in
a clean test tube, 2 mL of chloroform was added, the
test tube was vigorously shaken, and then evaporated
to dryness. To this, 2 mL of concentrated sulfuric acid
was added and heated for about 2 minutes. A grayish
colour indicates the presence of terpenoids.
Glycosides
Keller–Kilani test
The solvent plant material extract was mixed with 2
mL of glacial acetic acid containing 1–2 drops of 2%
solution of FeCl 3 , the mixture was then poured into
a test tube containing 2 mL of concentrated sulfuric
acid. A brown ring at the interface of the two solutions
indicates the presence of cardiac glycoside.
Alkaloids
The crude extract was mixed with 1% of HCl in a test
tube. The test tube was then heated gently and a few
drops of Mayers and Wagners reagents were added
by the side of the test tube. A resulting precipitate
confirms the presence of alkaloids.
Steroids
Liebermann’s Burchard reaction
About 2 g of the solvent extract was put in a test tube
and 10 mL of chloroform was added and filtered. The
filtrate, 2 mL, was mixed with 2 mL of a mixture of
acetic acid and concentrated sulfuric acid.A blue green
ring indicates the presence of steroids.
Phenols
The plant extract was put in a test tube and treated
with a few drops of 2% FeCl 3 . A blue green or black
colouration indicates the presence of phenols.
152