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Bioequivalence & Bioavailability International Journal
Bioprospective Screening of Antibacterial and Phytochemical Activity of
Caesalpinia Pulcherrima (Pride of Barbados) on Selected Clinical Isolate Bioeq uiv & Bioavailab Int J
Bioprospective Screening of Antibacterial and Phytochemical
Activity of Caesalpinia Pulcherrima (Pride of Barbados) on
Selected Clinical Isolate
OludareTemitope Osuntokun1*, Julianah JU2 and Thonda OA3
1Department of Microbiology, Adekunle Ajasin University, Nigeria
2Department of Microbiology, Adekunle Ajasin University, Nigeria
3Department of Biological Science, Kings University, Nigeria
*Corresponding author: Oludaretemitope Osuntokun, Department of Microbiology, Faculty of Science, Adekunle Ajasin
University, Akungba Akoko, PMB 001, Ondo State, Nigeria, Tel: 8063813635; E-mail: osuntokun4m@yahoo.com
Abstract
This research work was carried out to evaluate the antibacterial and Phytochemical activities of Caesalpiniapulcherrima
leaf and stem bark on some clinical isolates. The plant parts were collected from St. Benedict Catholic Church premises,
Owena Barracks, Akure, Ondo State. Crude ethyl acetate extracts of Caesalpinia pulcherrima leaves and stem bark was
assayed for its antibacterial activity against Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa,
Bacillus subtilis, Escherichia coli, and Salmonella typhi. using agar well diffusion method. Ofloxacin was used as standard
control. The ethyl acetate extract of Caesalpiniapulcherrima leaves inhibited the growth of Escherichia coli, Staphylococcus
aureus, and Salmonellatyphi with 12mm, 10mm and 12mm zone of inhibition at 100mg/ml concentration and 4mm, 4mm
and 6mm zone of inhibition at 12.5mg/ml concentration respectively but ineffective against Klebsiella pneumonia and
Pseudomonas aeruginosa. It also inhibits the growth of Bacillus substilis with 7mm zone of inhibition at 100mg/ml
concentration and 3.0mm zone of inhibition at 25mg/ml concentration.
The ethyl acetate extract of the stem bark inhibited the growth of Escherichiacoli, with 12mm and 4mm zone of inhibition
at 100mg/ml and 25mg/ml concentration and Staphylococcus and Salmonellatyphi with 10mm and 15mm inhibition
zones at 100mg/ml concentration and 4mm and 4mm zone of inhibition at 12.5mg/ml respectively, but were ineffective
against other isolates. Both qualitative and quantitative phytochemical evaluations were also carried out on both the
leaves and stem bark of Caesalpinia pulcherrima and results revealed the presence of several phytoconstituents such as
alkaloids, cardiac glycoside, steroids, anthraquinones, phenols, tannins, saponins, flavonoids and reducing sugar with the
bark possessing more of the phytochemicals. It has been reported in the literature that these species exhibit a wide range
Research Article
Volume 1 Issue 3
Received Date: November 02, 2017
Published Date: December 29, 2017
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
2
of pharmacological properties, including antiulcer, anticancer, anti-diabetic, anti-inflammatory, antimicrobial, and anti-
rheumatic activities that have proven to be efficacious in ethno-medicinal practices. This work is aimed at; investigating
the antibacterial and phytochemical activity of Caesalpinia pulcherrima against selected bacteria species.
Keywords: Caesalpinia pulcherrima leaf; Stem bark Antibacterial; Phytochemical activity
Introduction
Caesalpinia pulchirrima (Fabaceae) is native to tropics
and subtropics area of the Americas. This plant is widely
distributed in Bangladesh and India. It is a common
medicinal plant in India, Taiwan and south East Asian and
African countries [1].
Caesalpinia pulchirrima is a striking ornamental plant,
widely grown in domestic and public gardens and has a
beautiful inflorescence in yellow, red and orange.
Caesalpinia pulchirrima species is ash rub growing to 3 m
tall. The leaves are bi pinnate, 20-40 cm long, bearing 3-
10 pairs of pinnae, each with 6-10 pairs of leaflets 15-
25 mm long and 10-15 mm broad. The flowers are borne
in racemes up to 20 cm long, each flower with five yellow,
orange or red petals. The fruit is a pod6-12 cm long.
Flowers are red or yellow, fragrant. Flowering season of
this plant start from September to November and fruits
from March to April [2].
Its various parts have been used for cure of a number
of disorders including pyrexia, menoxenia, wheezing,
bronchitis, and malarial infection [3]. Traditionally leaves
of Caesalpinia pulchirrimaare used as purgative, tonic,
antipyretic, emmenagogue, whereas roots have folkloric
use in convulsion, intermittent fever, lungs and skin
diseases [4]. Flavonoids are polyphenolic compounds,
widely distributed in the plant kingdom. They are
reported to exhibit various pharmacological activities
such as CNS, cardiotonic, lipid lowering, anti-oxidant,
Hepato protective and hypoglycemic activities [5]. The
Caesalpinia pulchirrima possesses various bioactive
compounds such as steroid, reducing sugar, triterpenoids,
sugar, alkaloids, phenolic compounds, flavonoids,
catechins, saponins, tannins, anthraquinones and amino
acid.
The leaves of the plant Caesalpinia pulchirrima are
reported to contain hydrocyanic acid, tannins and benzoic
acid. The plant contains various phyto active consituents
such as glycosides, rotenids, is flavones, flavonone,
chalcones, flavanols, flavones and sterols [1]. Root of
Caesalpinia pulchirrima showed the presence of
diterpenoids, isovouacapenol C and pulcherrimin A. The
stem contains peltogynoidsbhonducellin, 6-
methoxypulcherrimin and homomisoflavonoids. The
flavonoids are polyphenolic compounds and reported to
exhibit various pharmacological activities such as CNS
activity, cardiotonic activity, lipid lowering activity,
antioxidant activity, hepatoprotective activity,
hypoglycemic activity and so on [5]. These active
constituents and the above mention activities in turn
appear to correlate with some other biological activities.
Survey revealed that the different parts of
Caesalpiniapulchirrima have been screened for various
pharmacological activities but anti-diabetic and
antioxidant activities were not investigated in Caesalpinia
pulchirrima flowers (Figure 1).
Figure 1: Caesalpinia pulcherrima (Pride of Barbados
plant).
Caesalpinia pulcherrima also known as Pride of
Barbados plant is a perennial large shrub or small tree
found throughout the world. It has several medicinal
properties, used in treatment of ulcer, fever, tumors,
asthma, cholera, for abortion, promotes menstrual flow,
as a purgative or the watery evacuation of the bowels, for
producing energy, to relieve chest affections, widely used
for the cure of bronchitis, for malarial fevers and so many
others [3]. Ceasalpiniapulcherrima also has uses in the
folk medicine: the stem is used as an abortifacient and
emmenagogue, while decoctions of the roots and bark are
used as a febrifuge and to treat liver disorders as well as
ulcers from mouth and throat. Previous studies on this
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
3
plant have resulted in the isolation of several
diterpenoids [6,7], flavonoids, peltogynoids, and
homomisoflavonoids. Some of the constituents were
found to possess antitumor, antimicrobial properties. The
infusion of the leaves or the bark is used to prevent
recurrence like malaria, promote menstrual flow, and
work as a purgative and for producing energy. Due to the
presence of different chemical compounds, the bark part
of this plant may possess some pharmacological activities.
A combination of the roots, bark, and leaves may be
boiled into a medicinal tea, which is given to patients as a
treatment for fever, jaundice, kidney disease, and
gastrointestinal disorders. Gargling with the tea is also
said to treat sores in the mouth or throat. Disorders from
the respiratory system can be treated by giving the
patients the seed of the plant to chew. The root also
contains toxins that are sometimes used by folk doctors to
induce abortion.
A liquid extracted from the flowers of the plant is often
used topically as an eye wash or applied to the body as an
insecticide. The liquid is sometimes consumed to treat a
variety of other conditions. Patients with severe
gastrointestinal disorders, including dysentery or severe
diarrhea, may also be given the fruit of the plant, which is
said to have astringent properties, to eat. These
properties help the plant to dry out the intestinal tract.
The plant is known, however, to be an antiseptic and an
anti-inflammatory. These qualities may make it useful in
the treatment of gastrointestinal disorders and internal or
external wound.
Preliminary medical studies have also indicated that
Caesalpiniapulcherrima may also assist in weight loss.
Mice given enzymes that are found in this plant were able
to lose weight at a faster rate than the mice in the control
group. Despite its potential medicinal uses
Materials and Methods
Plant Sample
Collection, Source and Identification of Plant
Materials: The leaves and barks of Caesalpinia
pulcherrima were used for this study. The leaves and stem
bark were collected in November 2016 from St. Benedict
Catholic Church compound in 32 Artillery Regiment
Owena Cantonment Army Barracks, Akure, Ondo State
and were authenticated at the department of Plant
Science and biotechnology, Adekunle Ajasin University,
Akungba Akoko, Ondo State (Figure 2).
Figure 2: Site of plant collection.
Preparation of Plants Extract
Caesapinia pulcherrima leaves and barks were rinsed
with clean water to remove dirt and other particles and
were dried at room temperature for two weeks. The dried
leaves and barks were crushed manually into powdered
form with the help of a mortar and pestle. 300g of each of
the sample were soaked in 900ml of ethyl acetate for 7
days respectively. After which it was filtered with No1
what man filter paper. The extract was evaporated to
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
4
dryness by rotary evaporator to get a viscous mass. The
viscous mass was then poured into a glass petri dishes
and air dried and weighed to get a dried extract. The
extract was weighed to be 18g for ethyl acetate extract of
Caesalpinia pulcherrima bark 13g for ethyl acetate extract
of Caesalpinia pulcherrima leaves. This extract was used
for pharmacological screening. The extract was then
prepared for each solvent extract by dissolving 1g of
extract in 2.5ml of DMSO and 7.5ml of distilled water [8].
Extraction of Plant
Rotary evaporator was used in the removal of solvent
of extraction from the extracts. The boiling point of ethyl
acetate is 77o C respectively, the set temperature was also
set on the rotary evaporator for each solvent. After
evaporation, the extract were poured into different Petri
dishes and air dried.
Test Organisms
The sensitivity of the following bacteria to extracts of
Caesalpiniapulcherrima was assayed: Staphylococcus
aureus, Bacillus subtilis, Escherichia coli, Pseudomonas
aeruginosa, Salmonella typhi, and Klebsiella pneumonia.
These organisms were collected from the Microbiology
laboratory of AdekunleAjasin University, Akungba Akoko,
and Ondo state. The organisms were maintained by sub
culturing onto nutrient agar slants and grown at 37°C for
24hours and was kept in the refrigerator until when
needed [8].
Antibacterial Assay
The extracts of the leaves and bark of Caesalpinia
pulcherrima were tested for antibacterial activity against
the test organisms employing the Agar well diffusion
method [9].
The test bacterium was spread on the surface of the
Mueller Hinton Agar plates and the plates were allowed to
solidify. Wells were bored on the plates using a sterile
cork borer of 6mm diameter. A stock solution of 100
mg/ml of each extract obtained by dissolving the extract
in sterile distilled water, varying concentrations of the
extracts were prepared to obtain 50, 25 and 12.5mg/ml of
each extract. The wells were filled with 0.1ml of the
extract containing different concentration and antibiotics
(Ofloxacin) used for control were introduced into the
wells. The plates containing the extract were left on the
bench for 2hours to allow the extract diffuse into the agar
before incubation of the plates at 37°C for 18-24hours.
The positive control consisted of Ofloxacin, the standard
antibacterial drug at concentration of 50mg/ml. After
24hours, the plates were examined for clear zones around
the well indicating the antibacterial activity of the extract
[8].
Phytochemical Screening
Qualitative Phytochemical Analysis of
Caesalpinia Pulcherrima
Test for Reducing Sugars: One milliliter of the plant
filtrate was mixed with Fehling A and Fehling B
separately; a brown colour with Fehling B and a green
colour with Fehling A indicate the presence of reducing
sugars.
Test for Alkaloids
TLC method 1: Solvent system: Chloroform: methanol:
25% ammonia (8:2:0.5). Spots can be detected after
spraying with Dragendorff reagent. Orange spot shows is
a positive result.
TLC method 2: Wet the powdered test samples with a
half diluted NH4OH and lixiviated with EtOAc for 24hr at
room temperature. Separate the organic phase from the
acidified filtrate and basify with NH4OH (pH 11-12). Then
extract it with chloroform (3X), condense by evaporation
and use for chromatography. Separate the alkaloid spots
using the solvent mixture chloroform and methanol
(15:1). Spray the spots with Dragendorff’s reagent. An
orange spot show is a positive result [10].
Test for Anthraquinone
a) Borntrager’s test Heat about 50mg of extract with 1ml
10% ferric chloride solution and 1ml of concentrated
hydrochloric acid. Cool the extract and filter. Shake the
filtrate with equal amount of diethyl ether. Further
extract the ether extract with strong ammonia. Pink or
deep red coloration of aqueous layer [11].
b) Borntrager’s test Add 1 ml of dilute (10 %) ammonia to
2 ml of chloroform extract. A pink-red color in the
ammoniacal (lower) layer [12].
Test for Cardiac Glycosides
Kellar- Kiliani test: Dissolve 50 mg of methanolic extract
in 2 ml of chloroform. Add H2SO4 to form a layer. Brown
ring at interphase shows is a positive result [12].
TLC method: Extract the powdered test samples with
70% EtOH on rotary shaker (180 thaws/min) for 10hr.
Add 70% lead acetate to the filtrate and centrifuge at
5000rpm/10 min. Further centrifuge the supernatant by
adding 6.3% Na2CO3 at 10000 rpm/10min. Dry the
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
5
retained supernatant and re-dissolved in chloroform and
use for chromatography. Separate the glycosides using
EtOAc-MeOH-H2O (80:10:10) solvent mixture. The color
and hRf values of these spots can be recorded under
ultraviolet (UV254 nm) light [10].
Test for Flavonoid
Shinoda test: To 2-3ml of methanolic extract, add a piece
of magnesium ribbon and 1ml of concentrated
hydrochloric acid. Pink red or red coloration of the
solution, shows is a positive result [11].
TLC method: Extract 1g powdered test samples with
10ml methanol on water bath (60°C/ 5min). Condense the
filtrate by evaporation, and add a mixture of water and
EtOAc (10:1 m L), and mix thoroughly. Retain the EtOAc
phase and use for chromatography. Separate the
flavonoid spots using chloroform and methanol (19:1)
solvent mixture. The color and hRf values of these spots
can be recorded under ultraviolet (UV254nm) light [10].
Test for Phenol
Phenol test Spot the extract on a filter paper. Add a drop
of phosphomolybdic acid reagent and expose to ammonia
vapors. Blue coloration of the spot, shows is a positive
result [11].
Test for Saponin
Frothing test / Foam test: Add 0.5ml of filtrate with 5ml
of distilled water and shake well. Persistence of frothing
shows is a positive result [13].
TLC method: Extract two grams of powdered test
samples with 10 ml 70% EtOH by refluxing for 10 min.
condense the filtrate, enrich with saturated n-Bu OH, and
mix thoroughly. Retain the butanol, condense and use for
chromatography. Separate the saponins using chloroform,
glacial acetic acid, methanol and water (64:34:12:8)
solvent mixture. Expose the chromatogram to the iodine
vapors. The colour (yellow) and hRf values of these spots
were recorded by exposing chromatogram to the iodine
vapours [10].
Test for Steroid
TLC method Extract two grams of powdered test
samples with 10ml methanol in water bath (80°C/15
min). Use the condensed filtrate for chromatography.
The sterols can be separated using chloroform, glacial
acetic acid, methanol and water (64:34:12:8) solvent
mixture. The color and hRf values of these spots can be
recorded under visible light after spraying the plates with
anisaldehyde- sulphuric acid reagent and heating
(100°C/6 min)The color (Greenish black to Pinkish black)
and hRf values of these spots can be recorded under
visible light [10].
Test for Tannin
Braemer’s test 10% alcoholic ferric chloride will be
added to 2-3ml of methanolic extract (1:1)Dark blue or
greenish grey coloration of the solution [11,13].
Quantitative Method of Analysis of
Caesalpinia Pulcherrima
Saponins
About 20grams each of dried plant samples were
ground and, put into a conical flask after which 100 ml of
20 % aqueous ethanol were added. The mixture was
heated using a hot water bath. At about 55°C, for 4 hour
with continuous stirring, after which the mixture were
filtered and the residue re-extracted with a further 200 ml
of 20% ethanol. The combined extracts were reduced to
40 ml over a water bath at about 90°C. The concentrate
was transferred into a 250ml separate funnel and 20ml of
diethyl ether were added and then shaken vigorously. The
aqueous layer was recovered while the ether layer was
discarded. The purification process was repeated three
times. 60ml of n-butanol were added. The combined n-
butanol extracts were washed twice with 10m1 of 5%
aqueous sodium chloride. The remaining solution was
heated in a water bath. After evaporation, the samples
were dried in the oven to a constant weight; the saponin
content was calculated as percentage of the starting
material.
Flavonoids
About 10 g of the plant sample were extracted
repeatedly with 100 ml of 80% aqueous methanol, at
room temperature. The whole solution was filtered
through what man filter paper No 42. The filtrate were
later transferred into a crucible and evaporated into
dryness over a water bath; the dry content was weighed
to a constant weigh.
Tannins
About 500 mg of the plant sample were weighed into a
50 ml plastic bottle. 50 ml of distilled water was added
and shaken for 1 hour on a mechanical shaker. This was
filtered into a 50 ml volumetric flask and made up to the
marked level. Then, 5 ml of the filtrate was transferred
into a test tube and mixed with 2 ml of 0.1 M FeCl in 0.1 M
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
6
Hcl and 0.008 M potassium Ferro-cyanide. The
absorbance was measured at 120nm within 10 minutes.
The tannins content was calculated using a standard
curve of extract.
Alkaloids
Five grams of the plant sample were weighed into a
250ml beaker and 200ml of 10% acetic acid in ethanol
was then be added, the reaction mixture were covered
and allowed to stand for 4 hour. This was filtered and the
extract will be concentrated on a water bath to one-
quarter of the original volume. Concentrated ammonium
hydroxide was added drop-wise to the extract until the
precipitation is complete. The whole solution were
allowed to settle and the precipitate was collected,
washed with dilute ammonium hydroxide and then
filtered; the residue being the alkaloid, which was dried
and weighed to a constant mass.
Results
Table 1 present a summary of the antibacterial assay of
ethyl acetate extract of Caesalpinia pulcherrima leaves on
test bacterial species with of loxacin as antibiotics. A total
of six assays were performed. The initial concentration of
the Ethyl acetate extract of the leaves of Caesalpinia
pulcherrima was 100mg/ml with 12.0mm zone of
inhibition and 4.0mm and 6.0mm zone of inhibition at
concentration of 12.5mg/ml inhibits Escherichia coli and
Salmonella typhi. Staphyloccocus aureus was also found to
be very susceptible to this extract with 10mm zone of
inhibition at 100mg/ml and 4mm at 12.5mg/ml. Bacillus
substilis was found to be the least susceptible with 7mm
at 100mg/ml and 3mm at 25mg/ml. Pseudomonas
aeruginosa and Klebsiella pneumonia was not inhibited by
these extracts at any concentration used in this study.
In Table 1; the Ethyl acetate extract of the barks of
Caesalpiniapulcherrima shows that, Salmonella typhi and
Escherichia coli was observed to be the most susceptible
organism with 15.00mm and 10.00mm zone of inhibition
at 100mg/ml and 4.0mm and 4.00mm zone of inhibition
at concentration of 12.5mg/ml and 25mg/ml respectively.
Staphylococcus aureus was found to be the least
susceptible organism with 10.00mm at 100mg/ml and
4.00mm at 12.5mg/ml concentration. Bacillus substilis,
Klebsiella pneumonia and Pseudomonas aeruginosa was
found to be non-susceptible to the ethyl acetate extract of
the bark of Caesalpiniapulcherrima with no zones of
inhibition at any concentration (Figures 3 & 4) (Table 2).
Test organisms
Inhibition Zones diameter (mm)
100
mg/ml
50
mg/ml
25
mg/ml
12.5
mg/ml
Escherichia coli
12
9
6
4
Bacillus subtilis
7
5
3
0
Pseudomonas
aeruginosa
9
5
4
0
Staphylococcus
aureus
10
7
5
4
Klebsiella
pneumonia
10
8
5
0
Salmonella typhi
12
10
8
6
Table 1: Antibacterial activity of caesalpiniapulcherrima
leaf on selected clinical organisms.
Figure 3: Showing antibacterial activity of
Ceasalpiniapulcherrima leaf against Salmonella typh.
Test organisms
Inhibition Zones diameter
(mm)
100
mg/ml
25
mg/ml
12.5
mg/ml
E. coli
12
4
3
Bacillus subtilis
10
6
3
Pseudomonas
aeruginosa
8
4
1
Staphylococcus aureus
10
6
4
Klebsiella pneumonia
12
7
4
Salmonella typhi
15
10
4
Table 2: Antibacterial Activity of Caesalpinia Pulcherrima
Stem bark On Selected Clinical Organism.
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
7
Figure 4: Showing antibacterial activity of
Ceasalpiniapulcherrima stem bark against Escherichia coli.
The crude methanol, ethyl acetate, N-hexane, Dichloro-
methane extract of the leaf and stem bark of
Caesalpiniapulcherrima were qualitatively and
quantitatively tested for the presence of the components,
such as alkaloids, cardiac glycoside, steroids,
anthraquinine, phenol, tannins, saponin, flavonoids,
reducing sugars, oxalate and phytate and the results were
given in Table 3 below.
Table 3, represent a summary of the qualitative
phytochemical analysis of methanolic extract of
Caesalpiniapulcherrima leaf and stem bark for the
presence of alkaloids, cardiac glycoside, steroids,
anthraquinones, phenol, tannins, saponins, flavonoids,
and reducing sugar. The methanolic extract of the leaves
of Caesalpiniapulcherrima possess alkaloids,
anthraquinones, tannins, saponins, and reducing sugar,
but doesn’t possess cardiac glycoside, steroids, phenols,
and flavonoids. The bark possess alkaloids, cardiac
glycosides, tannins, saponins and reducing sugar, whereas
the methanolic extract of the bark doesn’t possess
steroids, anthraquinones, phenols, and flavonoids.
In Table 4, the ethyl acetate extract of
Caesalpiniapulcherrimabark possess all the
phytoconstituent except for steroids where it either
possess it or not. The ethyl acetate extract of the leaves of
Caesalpiniapulcherrima possess cardiac glycosides,
anthraquinones, phenols, tannins and saponnins but
doesn’t possess alkaloids, steroids, flavonoids and
reducing sugar.
Table 5, shows qualitative phytochemical analysis of
Dichloromethane extract of Caesalpiniapulcherrima leaf
and stem bark for the presence of the 9
phytoconstituents. The leaves and stem bark possess
alkaloids, steroids, phenols, saponins, flavonoids and
reducing sugar. The bark doesn’t contain cardiac
glycosides and the leaves also do not contain
anthraquinones and tannins phytoconstituents.
Sample
Alkaloid
Cardiac Glycoside
Steroids
Anthraquinone
Phenols
Tannins
Saponin
Flavonoids
Reducing Sugar
Leaves
+ve
-ve
-ve
+ve
-ve
+ve
+ve
-ve
+ve
Barks
+ve
+ve
-ve
-ve
-ve
+ve
+ve
-ve
+ve
Keys: +ve = Presence, -ve = Absence, ND= Not d
Table 3: Qualitative Phytochemical Analysis of Caesalpinia
Pulcherima leaf And Stem Bark Extract (Methanol) %.
Sample
Alkaloid
Cardiac Glycoside
Steroids
Phenols
Tannins
Saponin
Flavonoids
Reducing Sugar
Leaves
-ve
+ve
-ve
+ve
+ve
+ve
-ve
-ve
Barks
+ve
+ve
-+ve
+ve
+ve
+ve
+ve
+ve
Keys: +ve = Presence, -ve = Absence, ND= Not detected.
Table 4: Qualitative Phytochemical Analysis of
Caesalpiniapulcherrima Leaf and Stem Bark Extract (Ethyl
Acetate) %
Sample
Alkaloid
Cardiac Glycoside
Steroids
Anthraquinone
Phenols
Tannins
Saponin
Flavonoids
Reducing Sugar
Leaves
+ve
+ve
+ve
-ve
+ve
-ve
+ve
+ve
+ve
Barks
+ve
-ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
Keys: +ve = Presence, -ve = Absence, ND= Not detected.
Table 5: Qualitative Phytochemical Analysis of
Caesalpiniapulcherrima Leaf and Stem Barks Extract
(Dichloro Methane) %.
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
8
In Table 6, the qualitative phytochemical analysis of N-
Hexane extract of the leaves of Caesalpiniapulcherrima do
not possess alkaloids, steroids, flavonoids and reducing
sugar but possesses cardiac glycosides, anthraquinones,
phenols, tannins, and saponins. The N-Hexane extract of
the stem bark of caesalpiniapulcherrima contains all the
phytoconstituents except for alkaloids and reducing sugar
where it either possesses it or not.
Table 7, present a summary of the quantitative
phytochemical analysis of methanolic extract of
Caesalpiniapulcherrima leaf and stem bark for the
presence of alkaloids, oxalate, phytate, phenol, tannins,
saponins, and flavonoids. The methanolic extract of the
leaves of Caesalpiniapulcherrima reveals the presence of
all the phytoconstituents to be in trace amount, with
alkaloids and oxalate to be in lowest amount and the
others to be in high amount. The phytochemicals present
in the bark are all in the trace form except for flavonoids
that is not detected at all.
Table 8, shows quantitative result of the plants using
ethyl acetate as solvent. The leaves of
Caesalpiniapulcherrima reveals the presence of alkaloids,
oxalates, phytates, phenols, tannins in high amount with
phytate having the highest amount as 31.49 , flavonoids in
moderate amount with 7.23 and saponins in lowest
amount with 5.75. Whereas, the bark depicts the presence
of constituents like alkaloids, oxalate, phytate, phenol,
tannins in high form with tannins being the highest of
them all in quantity with 36.10 and saponin was present
in moderate form while flavonoids was in trace form.
Sample
Alkaloid
Cardiac Glycoside
Steroids
Anthraquinone
Phenols
Tannins
Saponin
Flavonoids
Reducing Sugar
Leaves
-ve
+ve
-ve
+ve
+ve
+ve
+ve
-ve
-ve
Barks
-+ve
+ve
+ve
+ve
+ve
+ve
+ve
+ve
-+ve
Keys: +ve = Presence, -ve = Absence, ND= Not detected
Table 6: Qualitative Phytochemical Analysis of
Caesalpiniapulcherrima Leaf and Stem Barks Extract
(Dichloro Methane) %.
Sample
Alkaloid
Oxalate
Phytate
Phenol
Tannins
Saponin
Flavonoids
Leaves
3.23
1.25
4.25
4.31
4.36
4.37
5.2
Barks
2.2
2.1
2.32
2.37
2.3
2.25
ND
Table 7: Quantitative Phytochemical Analysis of
Caesalpinia Pulcherrima Leaf and Stem Bark Extract
(Methanol) %.
Sample
Alkaloid
Oxalate
Phytate
Phenol
Tannins
Saponin
Flavonoids
Leaves
20
26.19
31.49
22.08
25.07
5.75
7.23
Barks
19.82
24.77
29.49
24.21
36.1
6.53
4.78
Table 8: Quantitative Phytochemical Analysis of
Caesalpiniapilcherrima Leaf and Stem Bark Extract (Ethyl
Acetate) %.
Table 9, reveals the quantitative phytochemical results
for Caesalpiniapulcherrima leaves and stem bark using N-
Hexane as extracting solvent. It reveals the presence of
alkaloids and flavonoids in moderate amount with 12.00
and 7.23 respectively and oxalate and saponins was
present in trace amount with 2.19 and 5.75 respectively,
whereas, phytate, phenol and tannins were present in
high amount with 20.49, 22.08 and 25.07 respectively in
the leaves. The bark shows that most of the
phytochemicals were present in high amount, except for
saponins and flavonoids which was present in small or
trace amount with 6.53 and 4.78 respectively.
Sample
Alkaloid
Oxalate
Phytate
Phenol
Tannins
Saponins
Flavonoids
Leaves
12
2.19
20.49
22.08
25.07
5.75
7.23
Barks
19.82
14.77
12.49
20.21
13.1
6.53
4.78
Table 9: Quantitative Phytochemical Analysis of
Caesalpiniapulcherrima Leaf and Stem Bark Extract (N-
Hexane) %.
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
9
Discussion
The present study carried out on the leaves and stem
bark of Caesalpiniapulcherrima extracts revealed the
possession of medicinal activities. Studies with reference
to their specific antibacterial activity had been done to
negligible extent. The screening of leaf and bark of
Caesalpiniapulcherrima for antibacterial activity was
carried out by agar well diffusion method. The selection of
this plant is based on its use in folk medicine. In the
present study, six bacterial strains were used which are
responsible for various minor or major infections in
humans. They are Staphylococcus aureus, Bacillussubtilis,
Escherichia coli, Pseudomonas aeruginosa, Salmonella
typhi and Klebsiellapneumoniae. All the extracts of leaves
and stem bark of Caesalpiniapulcherrima had shown
potent antibacterial activity. The extracts of leaves of
Caesalpiniapulcherrima showed higher activity compared
to the stem bark.
Ethyl acetate extracts of Caesalpiniapulcherrima stem
bark were found to have higher inhibitory activity against
Salmonella typhi, with 15mm zone of inhibition at
100mg/ml and 4mm zone of inhibition at 12.5mg/ml, and
Staphyloccocusaureus being the least susceptible
organism, with 10mm zone of inhibition at 100mg/ml
concentration and 4mm zone of inhibition at 12.5mg/ml
concentration. The stem bark was also found to have high
inhibitory activity to Escherichia coli, with 12mm zone of
inhibition at concentration of 100mg/ml and 4mm zone
of inhibition at 25mg/ml. For the extract to have
inhibitory activity against these test organisms, it might
be due to certain bioactive compounds such as alkaloid,
flavonoids and tannins present in the stem bark extract of
Caesalpiniapulcherrima. This could also be as a result that
the extracting solvent (ethyl acetate) used was able to
extract certain bioactive compound that was active
against the test organisms. Bacillus substilis, Pseudomonas
aeruginosa and Klebsiella pneumonia were found not to be
susceptible to ethyl acetate extract of
Caesalpiniapulcherrima stem bark. This might be because
the extracting solvent used was unable to extract the
bioactive component necessary for the inhibition of the
organisms [14].
Ethyl acetate extract of the Caesalpiniapulcherrima
leaves exhibited higher inhibitory activity against all the
tested organisms except Bacillus substilis and Klebsiella
pneumonia, with Escherichia coli and Salmonella typhi
having the highest zone of inhibition of 12mm at
100mg/ml, 4mm and 6mm zone of inhibition at
12.5mg/ml concentration, followed by
Staphyloccocusaureus, with 10mm zone of inhibition at
100mg/ml and 4mm zone of inhibition at 12.5mg/ml
concentration. Whereas, Bacillus substilis was found to be
the least susceptible organism with 7mm and 3mm zones
of inhibition at 100mg/ml and 25mg/ml concentrations
respectively. This might be that some bioactive
compounds present in the leaf extract, possess
antibacterial activities against the test organisms.
Bacillussubstilis and Klebsiellapneumonia that was
resistant to Caesalpiniapulcherrima leaf extract could be
due to the absence of compounds necessary to inhibit the
growth of these organisms in the extract. The absence of
these compounds might be because the solvent used in
the study was unable to extract all necessary bioactive
compounds from the leaf. Also concentration would have
been more effective enough to inhibit the growth of all the
microorganisms probably if much concentration was used
[14]. It was reported that the ethyl acetate extracts of
leaves of Caesalpinia Pulcherrima possess antimicrobial
activity against Escherichia coli, Bacillussubtilis and
Staphylococcus aureus [15]. The results of antibacterial
activity of crude extracts of Caesalpiniapulcherrima were
summarized in (Tables 1 & 2) respectively.
However, the crude methanol, ethyl acetate, N-hexane,
Dichloro-methane was qualitatively and quantitatively
tested for the presence of steroids, glycosides, alkaloids,
flavonoids, anthraquinones, tannins, oxalate, phytate and
phenols. The methanol, ethyl acetate and n-hexane extract
of leaf of Caesalpiniapulcherrima does not possess
steroids and flavonoids phytoconstituents. Methanol,
ethyl acetate, dichloromethane & n-hexane extracts
possess tannins and saponin whereas methanol &
dichloromethane extracts possess reducing sugar. The
stem bark possesses alkaloids and reducing sugar in
methanol, ethyl acetate, dichloromethane, n-hexane
extracts. Ethyl acetate and n-hexane extracts of the bark
possess all the phytoconstituent. Whereas methanol
extracts does not possess phenols and flavonoids.
Therefore, the detected different bioactive compounds
in the different crude may be responsible for the
antibacterial activity of plant crude extracts. It is reported
that Saponins are bioactive chemical constituents which
are involved in plant disease resistance because of their
antimicrobial activity. Tannins are phenolic compound
and their derivatives are also considered as primary
antioxidants or free radical scavengers [16]. Flavonoids
groups exhibited a wide range of biological activities such
as antioxidant, anti-inflammatory, antimicrobial,
anticancer and anti-allergic [17]. The presence of
qualitative phytoconstituents was reported in (Tables 4-
6).
Bioequivalence & Bioavailability International Journal
OludareTemitope Osuntokun, et al. Bioprospective Screening of Antibacterial and
Phytochemical Activity of Caesalpinia Pulcherrima (Pride of Barbados) on Selected
Clinical Isolate. Bioequiv & Bioavailab Int J 2017, 1(3): 000118.
Copyright© OludareTemitope Osuntokun, et al.
10
In the same way, the quantitative phytochemical
analysis revealed the presence of most of the constituents
tested for in different amounts. Most of the
phytoconstituents were present in high quantity, some in
moderate amounts and others in small or trace amount
[18]. History of use of herbal medicine in the treatment of
diseases can be identified with the history of medicine
and with the history of civilization itself. All parts of
plants were used in Ayurveda, Unani and Homeopathic
systems of medicine for the treatment of various human
diseases [19-24].
Conclusion
The results revealed that the crude extracts of
Caesalpiniapulcherrima contain certain constituents like
alkaloids, cardiac glycosides, tannins, steroids,
anthraquinones, phenols, saponnins, flavonoids and
reducing sugar which could make the plant useful in
treating different ailments and have potential to provide
useful drug for human use. The present study exhibited
the antibacterial effect of various extracts of
Caesalpiniapulcherrima. The inhibitory effect of the
extracts justified the medicinal use of
Caesalpiniapulcherrima. Hence, it is apparent that this
plant has been found to possess effective antibacterial
substances against a wide range of microorganisms. The
plant can also be further explored for its activity against
wide spectrum of microbes and can be developed into
powerful antibiotics.
Recommendation
I recommend a continuous use of the
Caesalpiniapulcherrima (pride of Barbados) plant, as the
plant can be used against malaria, as an anti-
inflammatory, an anti-microbial, against staph infections,
and is said to kill cancer cells. It is also an ornamental
plant that can be used for decorative purposes in gardens
and as houseplant.
Acknowledgements
The authors wish to express their appreciation to all
the technical staffs of the laboratory unit of Both the
Department of Microbiology, Faculty of Science, Adekunle
Ajasin University, Akungba Akoko, Ondo State,
Department of Microbiology, Faculty of Science and
Department of Pharmaceutical Science (Natural product
chemistry), Faculty of Pharmacy, Obafemi Awolowo
University, Ile Ife, Osun State, Nigeria for their support
and all the technical assistance rendered during the
course of this research work.
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