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Phytochemical and Antioxidant Effect of Spathodea campanulata leaf Extracts

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Aim: Spathodea campanulata is a medicinal plant useful in traditional medicine for the treatment and prevention of some diseases of bacterial and non microbial origins. As a result of this, it becomes very important to investigate the phytochemical and antioxidant (in vitro and in vivo) activities of the plant leaf extracts by chemical methods to ascertain its potential role in folklore medicine. Study Design: In vitro and in vivo by chemical methods. Methodology: 1.5 kg each of S. campanulata air dried leaves ground to powder was extracted separately with ethanol, methanol and petroleum ether at room temperature (25±2°C). Results: The leaf extracts showed qualitatively the presence of saponin, steroid, flavonoids, glycoside, alkaloids, phenol, tannin, terpenoids, phlobatanin and antraquinone. Amount of quantitative phytochemicals screened from the extracts was more in ethanol followed by aqueous, methanol and was least in petroleum ether. Valuable in vitro antioxidant activities were exhibited by the aqueous, ethanol, methanol and petroleum ether extracts in free radical (DPPH), hydroxyl scavenging activities and ferric reducing antioxidant properties. Decrease in values was observed in the in vivo antioxidant assay of glutathione and catalase levels in group of mice infected with Salmonella typhi for three days while there was increase in lipid peroxidation on comparison with negative control value. However improvement in enzymatic antioxidant levels of mice was observed when treated with the plant ethanol leaf extract. The recorded data in the study proposed the use of leaf extract of S. campanulata in traditional medicine hence its inhibition potentials and barrier to generation of free radicals.
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*Corresponding author: E-mail: akharaiyifc@gmail.com;
International Journal of Biochemistry Research
& Review
7(3): 148-159, 2015, Article no.IJBcRR.2015.064
ISSN: 2231-086X
SCIENCEDOMAIN international
www.sciencedomain.org
Phytochemical and Antioxidant Effect of
Spathodea campanulata leaf Extracts
Akharaiyi Fred Coolborn
1*
, Boboye Bolatito
2
, Akpambang Victoria Omolara
3
and F. C. Adetuyi
2
1
Department of Biological Sciences, Afe Babalola University, P.M.B 5454, Ado Ekiti, Ekiti State,
Nigeria.
2
Department of Microbiology, Federal University of Technology, P.M.B 704, Akure, Ondo State,
Nigeria.
3
Department of Chemistry, Federal University of Technology, P.M.B 704, Akure, Ondo State, Nigeria.
Authors’ contributions
This work was carried out in collaboration between all authors. Author AFC designed the study, wrote
the first draft of the manuscript, carried out all laboratories work and performed the statistical analysis.
Authors BB and FCA wrote the protocol, supervised the work and managed the analyses of the study.
Author AVO managed the literature searches and edited the manuscript. All authors read and
approved the final manuscript.
Article Information
DOI: 10.9734/IJBcRR/2015/16371
Editor(s):
(1)
Alexander A. Kamnev, Inst. of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences,
Saratov, Russia.
Reviewers:
(1)
Anonymous, Mahasarakham University, Thailand.
(2)
H.P. Singh, Department of Hill Area Tea Science, Institute of Himalayan Bioresource Technology (IHBT), Council of
Scientific and Industrial Research (CSIR), Palampur, Himachal Pradesh, India.
Complete Peer review History:
http://www.sciencedomain.org/review-history.php?iid=1038&id=3&aid=9111
Received 29
th
January 2015
Accepted 25
th
March 2015
Published 4
th
May 2015
ABSTRACT
Aim:
Spathodea campanulata is a medicinal plant useful in traditional medicine for the treatment
and prevention of some diseases of bacterial and non microbial origins. As a result of this, it
becomes very important to investigate the phytochemical and antioxidant (in vitro and in vivo)
activities of the plant leaf extracts by chemical methods to ascertain its potential role in folklore
medicine.
Study Design: In vitro and in vivo by chemical methods.
Methodology: 1.5 kg each of S. campanulata air dried leaves ground to powder was extracted
separately with ethanol, methanol and petroleum ether at room temperature (25±2°C).
Original Research Article
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
149
Results:
The leaf extracts showed qualitatively the presence of saponin, steroid, flavonoids,
glycoside, alkaloids, phenol, tannin, terpenoids, phlobatanin and antraquinone. Amount of
quantitative phytochemicals screened from the extracts was more in ethanol followed by aqueous,
methanol and was least in petroleum ether. Valuable in vitro antioxidant activities were exhibited by
the aqueous, ethanol, methanol and petroleum ether extracts in free radical (DPPH), hydroxyl
scavenging activities and ferric reducing antioxidant properties. Decrease in values was observed
in the in vivo antioxidant assay of glutathione and catalase levels in group of mice infected with
Salmonella typhi for three days while there was increase in lipid peroxidation on comparison with
negative control value. However improvement in enzymatic antioxidant levels of mice was
observed when treated with the plant ethanol leaf extract. The recorded data in the study proposed
the use of leaf extract of S. campanulata in traditional medicine hence its inhibition potentials and
barrier to generation of free radicals.
Keywords: Spathodea campanulata; leaf extracts, phytochemical; antioxidant; inhibition.
1. INTRODUCTION
Medicinal plants are used for the ailment of
several microbial and non-microbial originated
diseases due to their valuable effects in health
care. Several plants have therapeutic and
pharmaceutical effects for antimicrobial,
antioxidant, anti-infectious and anti-tumour
activities [1,2]. In plants, the synthesized
aromatic substances (metabolites) are used as
defensive weapons against predation by
microorganisms, insects and herbivores. These
defensive molecules give plants their medicinal
values which are appreciated by human beings
because of their importance in health care of
individuals and communities [3]. Reports have
shown the capability of plant phytochemicals to
elicit various physiological responses [4]. A great
number of plants worldwide showed a strong
antioxidant activity and a powerful scavenger
activity against free radicals [5,6].
Antioxidants are radical scavengers which
protect the human body against free radicals that
may cause pathological conditions such as
ischemia, anaemia, asthma, arthritis,
inflammation, neuro-degeneration, Parkinson’s
diseases, ageing process and perhaps
dementias [7]. Flavonoids and flavones are
widely distributed secondary metabolites with
antioxidant and antiradical properties [8]. Many
human diseases are caused or negatively
affected by free radicals. The natural defence of
human organs against free radicals is not always
sufficient mainly due to the significant exposition
to free radicals from external sources in the
modern world [9], therefore, supplements from
natural sources as endowed in medicinal plants
and fruits are of necessity.
Plant and plant products are being used as a
source of medicine due to their potent antioxidant
activities, no side effects and of economic
viability [10]. Flavonoids and phenolic
compounds widely distributed in plants have
been reported to exert multiple biological effects,
including antioxidant, free radical scavenging
abilities, anti-inflammatory, anti-carcinogenic etc
[11]. Phytoantioxidants are readily available, less
toxic, serving as food and the medicinal
components have been suggested to reduce
threat of wide range of ROS [12].
Spathodea campanulata P. Beauv species is
belonging to the Bignoniaceae family. The
flowers are employed as diuretic and anti-
inflammatory, while the leaves are used against
kidney diseases, urethra inflammations and as
an antidote against animal poisons. The stem
bark preparations are employed against enemas,
fungus skin diseases, herpes, stomach aches
and diarrhoea [13].
In this study, we will investigate the
phytochemicals in quality and quantity using
chemical methods, in vitro and in vivo
antioxidants of the plant extracts by chemical
methods and animal model for its validity in
folklore medicine.
2. MATERIALS AND METHODS
2.1 Collection of Plant Samples and
Extracts Preparations
Healthy looking leaves of S. campanulata was
collected from forest in Akure, Ondo State,
Nigeria and identified in Department of Forestry
and Wood Technology, Federal University of
Technology, Akure, Nigeria. The voucher number
of the plant AF 1504 was deposited in the
herbarium. The leaves were air dried for 3 weeks
at room temperature of 25±2°C in laboratory and
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
150
ground to powder with a mechanical grinder. 1.5
kg each of the powders obtained was extracted
separately with ethanol, methanol and petroleum
ether at room temperature (25±2°C). The
resulting crude extracts were filtered with triple
layered sterile muslin cloth and concentrated
using a rotary evaporator (RE -52 A Union
Laboratories, England) at 40-45°C. The water
extract was evaporated in a shaker water bath
regulated at 50°C. The concentrated extracts
were contained in plastic containers, labeled
accordingly as aqueous extract (AE), ethanol
extract (EE), methanol extract (ME) and
petroleum ether extract (PE) and were used for
the phytochemical and antioxidant activities.
2.2 Qualitative Phytochemical Determi-
nation
2.2.1 Alkaloids test
Five grams each of plant extract was stirred with
5 ml of 1% aqueous hydrochloric acid on a steam
bath. One millilitre of the filtrate was treated with
few drops of Draggendoff’s reagent. Blue-black
turbidity serves as preliminary evidence of
alkaloids [14].
2.1.2 Saponin test
Five grams of each extract was shaken with
distilled water in a test tube. Frothing which
persists on warming was taken as preliminary
evidence of the presence of saponins [14].
2.2.3. Tannins test
Five grams of each extract was stirred with 100
ml distilled water and filtered. Ferric chloride
reagent was added to the filtrate. A blue-black or
blue green precipitate determines the presence
of Tannins [14].
2.2.4 Phlobatannins test
Disposition of red precipitate when an aqueous
extract of the test samples was boiled with 1%
hydrochloric acid determines the presence of
phlobatannins [14].
2.2.5 Flavonoids test
Five millilitres of diluted ammonia solution was
added to aqueous filtrate of the test samples
followed by the addition of concentrated H
2
SO
4
.
A yellow coloration observation determines the
presence of flavonoids [14].
2.2.6 Cardiac glycosides (keller-killiani test)
Five grams of each extract was dissolved in 2 ml
of glacial acetic acid containing a drop of ferric
chloride solution. This was under laid with 1 ml
concentrated H
2
SO
4
. A brown ring of the
interface indicates a deoxy-sugar characteristic
of cardenolides. A violet ring may appear below
the brown ring, while in the acetic acid layer, a
green ring may form just gradually spread
throughout this layer [14].
2.2.7 Anthraquinones test
A total of 0.5 g of the extract was shaken with
100 ml of benzene and filtered. Five millilitres of
10% ammonia solution was added to the filtrate.
The mixtures were shaken and the presence of
pink, red or violet colour in the lower phase of the
ammonia indicates the presence of free
anthraquinones [15].
2.2.8 Terpenoids (Salkowski test)
Five millilitres of each extract was mixed in 2 ml
of chloroform, and 3 ml of concentrated H
2
SO
4
was carefully added to form a layer. A reddish
brown colouration of the inter face was formed to
show positive results for the presence of
terpenoids [14].
2.3 Quantitative Phytochemical Determi-
nation
2.3.1 Determination of total phenol contents
The total phenolic content of the extracts was
determined using a modified Folin-Ciocalteu
method [16,17]. 200 μl of sample was mixed with
2.6 ml of distilled water, 200 μl of Folin-
Ciocalteu’s phenol reagent was added to each
tube. The content was vortexed and incubated
for 5 min. Then 2 ml of 7% Na
2
CO
3
was added to
each tube. The content in the tube was vortexed
and incubated for 2 h with intermediate shaker.
The absorbance of samples was in
spectrophotometer at 752 nm. Total phenol
contents were expressed as milligrams of Gallic
acid per gram of dry extract.
2.3.2 Determination of total flavonoids
content
The content of flavonoids was determined using
quercetin as a reference compound. Stock
solution (0.50 µl) of each extract was mixed with
50 µl of aluminium trichloride and potassium
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
151
acetate. The absorption at 415 nm was read after
30 minutes at room temperature. Standard
quercetin solution was prepared from 0.01 g
quercetin dissolved in 20 ml of ethanol. All
determinations were carried out in duplicate. The
amount of flavonoids in extracts was expressed
as quercetin equivalent (QE) /gram dry weight
[18,19]
2.3.3 Saponin determination
Spectrophotometric method of [20] was used. 2 g
of finely ground sample was weight into a 250 ml
beaker and 100 ml of Isobutyl alcohol was
added. The mixture was shaken in a shaker
water bath for 5h to ensure uniformity in the
mixture. The mixture was filtered with No 1
Whatman filter paper into 100 ml beaker
containing 20 ml of 40% saturated solution of
magnesium carbonate (MgCO
3
). The mixture
obtained was again filtered with filter paper to
obtain a clean colourless solution. 1ml of the
colourless solution was pipetted into 50ml
volumetric flask, 2 ml of 5% ferric chloride
(FeCl
3
) solution was added and made up to the
mark with distilled water. It was allowed to stand
for 30minutes for colour development. The
absorbance was read against blank at 380 nm.
2.3.4 Tannin determination
0.2 g of finely ground sample was weighed into a
500 ml sample bottle. 100 ml of 70% aqueous
acetone was added and properly covered. The
bottles were kept in shaker water bath for 2 h at
30°C. Each solution was then centrifuged and
the sediment was stored in ice. 0.2 ml of each
solution was pipitted into test tubes and 0.8 ml of
distilled water was added. Standard tannin acid
solutions were prepared from a 0.5 mg/ml of the
stock and the solution made up to 1 ml with
distilled water. 0.5 ml of Folin-ciocateau reagent
was added to both sample and standard followed
by 2.5 ml of 20% Na
2
CO
3
the solutions were then
shaken vigorously and allowed to incubate for 40
minutes at room temperature, its absorbance
was then read at 725 nm against a reagent blank
concentration of the same solution from a
standard tannic acid curve was prepared [15].
2.3.5 Alkaloid determination
5g sample was weighed into a 250 ml beaker
and 200 ml of 10% acetic acid in ethanol was
added and allowed to stand for 4minutes. It was
filtered and the extract was 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 was
completed. The whole solution was allowed to
settle and the precipitate was collected and
washed with dilute ammonium hydroxide and
then filtered. The residue is then considered as
alkaloid which was dried and weighed. [21]
Alkaloids (%). = W
3
– W
2
x 100
W
1
2.4. In vitro Antioxidant Screening
2.4.1 Ferric Reducing Antioxidant Property
The method of [9] was adopted but with little
modifications. 0.1 g each aqueous, methanol,
ethanol and petroleum ether extract of each (0.1
g) were dissolved in 20 ml of water and filtered.
The filtrate (2.5 ml) was taken and 2.5 ml of
phosphate buffer (pH 6.6) and 2.5 ml of
potassium ferrocyanide were added. The
mixtures was incubated at a temperature of 50
o
C. 10% Trichloroacetic acid was added, followed
by the addition of 5 ml of distilled water and 1 ml
of 0.1% ferric chloride. All determinations were
carried out in duplicate. The absorbance of the
standard and the samples were read in
spectrophotometer at 700 nm wavelength
against reagent blank.
2.4.2 Free Radical Scavenging
The method used was almost the same as used
by [22,23] but was modified in details. An aliquot
of 0.5 ml of 0.1 mm 1, 1- diphenyl 1-2
picrylhdrazyl (DPPH) radical (Sigma Aldrich, St.
Louis, USA) in the concentration of 0.05 mg/ml.
Aqueous, methanol, ethanol and petroleum ether
extracts each at a concentration of 20 mg/ml
were placed in cuvettes. The reaction was mixed
at room temperature and kept for 20 minutes.
Absorbance was read with a spectrophotometer
at a wavelength of 520 mm. The absorbance of
the DPPH radical solution containing the plant
extract was expressed as mg of L-ascorbic
(sigma Chemical Co, St. Louis, USA) per 1 g of
dry plant material. Calibration was used in such
cases, where the plant extracts were replaced
with a freshly prepared solution of ascorbic acid
in deionised water (concentration from 0 to 1.6
mg/ml – 100 mg/ml). All determinations were
replicated. The experiment was performed in
triplicate. The percentage of the DPPH free
radical was calculated using the following
equation:
DPPH scavenging effect (%) = A
o
– A
1
× 100
A
o
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
152
Where A
0
was the absorbance of the control, and
A
1
was the absorbance in the presence of the
extract or positive control.
2.4.3 Hydroxyl radical scavenging assay
The capacity to scavenge hydroxyl radicals was
measured according to the method proposed by
[24] with modification. The hydroxyl radicals are
generated by iron-ascorbate- EDTA-H
2
O
2
, which
then react with deoxyribose to form thiobarbituric
acid reactive substances (TBARS). This
substances yield pink chromogen at low pH while
heating with trichloroacetic acid (TBA). The
reaction mixture contained 4mM deoxyribose, 0.3
mM ferric chloride, 0.2 mM EDTA, 0.2 mM
ascorbic acid, 2 mM H
2
O
2
and various
concentrations of extracts. The tubes were
capped tightly and incubated for 30 min at 37°C.
Then 0.4 ml of 5% TBA and 0.4ml of 1% TBA
were added to the reaction mixture which was
kept in a boiling water bath for 20 min. The
intensity of pink chromogen was measured
spectrophotometrically at 532 nm against the
blank sample. Ascorbic acid was used as a
positive control. All tests were performed in
triplicate. The hydroxyl radical scavenging
activity of leaves extract was reported as %
inhibition of deoxyribose degradation and
calculated using the following equation:
% Inhibition = A
o
– A
1
× 100
A
o
Where A
0
was the absorbance of the control and
A
1
was the absorbance in the presence of the
extract or positive control.
2.4.4 In vivo antioxidant screening
The antioxidant assay was performed with liver
tissues of experimental Swiss albino mice which
were anesthetized with chloroform soaked in
cotton wool. The mice were dissected openly and
the liver was repeatedly washed with ice-cold
saline until uniformly pale and was immediately
removed. The liver were homogenized with 4
volume of ice-cold 0.1 M potassium phosphate
buffer (pH = 7.4) containing 1.15% (w/v) KCl.
The homogenate was centrifuged at 10000 g for
60 min. The supernatant was used for the study.
2.4.4.1 Determination of lipid peroxidation (LPO)
Determination of the antioxidant status was
carried out by measuring the level of its lipid
peroxidation with the method of [25]. Mice liver
microsome (2 mg/ ml) was mixed with 0.1 ml of
FeSO
4
(26% mM), 0.1 ml of ascorbate (0.13
mM), 0.1 ml of the sample in 150 mM KCl/Tris-
HCl buffer solution (pH = 7.4). The mixture was
incubated at 37°C for 60 min in a water bath;
0.75 ml of 2 M trichloroacetic acid/1.7 M HCl was
added to stop the reaction, then tubes were
centrifuged (4000 rpm, 10 min) and 0.5 ml of the
supernatant was mixed with 0.15 ml TBA and
was heated at 95°C for 10 min. The level of
malondialdehyde was determined by measuring
the absorbance at 532 nm. The percent of lipid
peroxidation inhibition was calculated using the
Equation:
%I = (A
0
- A
1
/ A
0
) × 100
Here, A
0
is the absorbance of the control
reaction; A
1
is the absorbance in the presence of
the agents.
2.4.4.2 Determination of glutathione (GSH)
The amount of non enzymatic antioxidant system
(reduced glutathione: GSH) was by the methods
of [26]. The principle was based on the
determination of reduced glutathione in each
dilution by the measurement of the absorbance
of colored solution developed within 5 min of the
generation of Elman’s reagent at 430 nm
wavelength.
2.4.4.3 Determination of catalase (CAT)
The enzymatic lipid peroxidation (Catalase: CAT)
was determined by the method of [27]. The
principle was made use of here to monitoring the
rate of enzyme catalyzed decomposition of
hydrogen peroxide (H
2
O
2
) using Potassium
tetraoxomanganate VII (KMnO
4
). 50 microlitre of
liver homogenate was added to a test tube. H
2
O
2
was then added to the tube and incubated on ice
for 3 min. H
2
SO
4
was used to stop the reaction.
Finally, KMnO
4
was added and the absorbance
recorded at 480 nm. In this assay,
1 unit of enzyme activity =
K
0.00693
Where
K = S
O
×
2.3
S
2
t
Where S
o
= Absorbance of standard-absorbance,
S
2
= Absorbance of standard-absorbance of
sample. T = Time interval. The measured
activities were normalized with the protein
content of each sample.
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
153
2.5 Experimental Animals
Apparently healthy Swiss albino mice of between
23-35 g were used. The animals were contained
in a cage and maintained under standard
laboratory conditions. They were given rodent
pellets (Vital feeds) and water ad libitum. They
were acclimatized for 2 weeks and were fasted
over night with free access to water prior the
experiments. The animals were conducted in
compliance with NIH Guide for Care and Use of
Laboratory Animals. Before the experiment, the
mice were divided into seven groups of five mice
per group.
2.5.1 Administration of crude extracts of S.
campanulata
A total of 35 mice were used. They were divided
into seven groups of five mice each. While group
1 served as negative control which was only
allowed to normal mouse feed and water, group
two served as positive control and was
administered with 10
7
Cfu/ml of Salmonella typhi.
To groups 3, 4, 5, 6 and 7, previously
administered with 10
7
Cfu/ml of S. typhi for 3
days were co administered with 0.5 ml each of
varying doses of ethanol leaf extracts at
concentrations of 200, 400, 800, 1000 and 2000
mg/kg
bw
orally as a single daily dose using wash
bottle whose dispenser was directly laid on the
mice throat. After 7 days of treatment each
mouse was sacrificed and livers were obtained
for in vivo antioxidant activity.
2.6 Statistical Analysis
The results were expressed as mean ± standard
deviation (SD) and were subjected to one way
analysis of variance (ANOVA). The least
significant difference (LSD) was performed for
the pair wise mean comparisons, to determine
the significant treatment dose at 95% level of
confidence. Values were considered statistically
significant at (P<0.05).
3. RESULTS
3.1 Qualitative phytochemical screening
Qualitatively identified phytochemical in leaf
extract of S. campanulata are saponin, steroids,
glycosides, alkaloids, phenols, terpenoids,
tannins, anthraquinones, phlobatannin and
flavonoids. However, saponin, glycosides,
phenols and flavonoids were found to be more in
aqueous and ethanol extracts than methanol and
petroleum ether extracts (Table 1).
Table 1. Qualitative phytochemical screening
of plant extracts
Secondary
Metabolites
AE
EE
PE
Saponin ++
+++
++ +
Steroids +
+
+ +
Flavonoids + +
++
++ ++
Tannins +
++
+ +
Glycosides ++ ++ ++ ++
Alkaloids +
++
+ +
Phenols ++
++
+ +
Terpenoids ++
++
+ +
Phlobatannin + + - -
Anthraquinones +
+
- -
Legend: AE = Aqueous extract, EE = Ethanol extract,
ME = Methanol extract, PE = Petroleum ether extract,
+ = positive, - = negative
3.2 Quantitative Phytochemical
Ethanol extract quantified highest amounts of
phytochemical where saponin was 3.98 ± 0.3
mg/ml, flavonoids (1.32±0.2 mg/ml), Tannin
(0.55±0.6 mg/ml), alkaloids (2.74±0.4%) and
phenols (0.70±0.6 mg/ml of tannic acid
equivalent (TAE). In aqueous extract saponin
was 3.92 + 0.1 mg/ml, flavonoids with a value of
0.56±0.2 mg/ml, 0.22±0.4 mg/ml of tannins,
1.46±0.01% of alkaloids and 0.40±0.1 mg/ml
TAE of phenols. Related phytochemical values
were observed between methanol and petroleum
ether extracts where saponin was 1.14±0.3
mg/ml and 0.83±2.0 mg/ml in methanol and
petroleum ether extracts respectively. Flavonoids
values in methanol extract was 0.24±0.1 and
0.17±0.1 mg/ml in petroleum ether extract, tannin
value in methanol extract was 0.18±1.2 and
0.15±2.4 mg/ml in petroleum ether extract;
alkaloids was 0.93±1.0% in methanol extract and
0.71±2.1 % in petroleum ether extract, phenols
values in methanol extract was 0.38±0.1 and
0.25±0.1 mg/ml TAE in petroleum ether extract
(Fig. 1).
3.3 In vitro Antioxidant Activity
The in vitro antioxidant assay of the aqueous,
ethanol, methanol and petroleum ether extracts
in free radical (DPPH) scavenging, ferric
reducing antioxidant property and hydroxyl
scavenging activities is presented in Table 2.
Free radical scavenging (DPPH) activities in
aqueous, ethanol, methanol and petroleum ether
leaf extracts was 1.44±0.5, 1.57±1.4, 0.40±1.4
and 0.30±0.1 (mg of ascorbic acid/1g dry plant
material) respectively. Ferric reducing antioxidant
property in that order was 1.19±0.4, 1.69±0.18,
1.74±0.5 and 2.84±1.8 (mg of ascorbic acid/1g
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
154
dry plant material) respectively. The hydroxyl
scavenging activity of the aqueous, ethanol,
methanol and petroleum ether extracts was 0.65
+ 0.90, 0.43±0.50, 0.89±0.54 and 0.87±0.42 (mg
of ascorbic acid/1g dry plant material)
respectively.
3.4 In vivo Antioxidant Activity
Table 3 illustrates the in vivo efficacy of S.
campanulata ethanol leaf extracts on the
enzymatic antioxidant in mice. A significant LPO
level increase (P<0.05) in the positive control
was observed when compared with the negative
control values. However, decrease in LPO level
was observed in bacterial/extract treated mice
alongside extract concnetrations. While this
decrease in LPO values, increased values were
observed in GSH and CAT levels in the extract
treated groups of mice alongside extract
concentrations.
4. DISCUSSION
Novel traditional technologies such as infusion,
decoction and concoction with water solutions to
high polar solvents such as ethanol and
methanol have been used to improve herbal
therapy in traditional medicines. Practices in
using one or more of the mentioned methods
have helped in providing lasting solutions for
prevention and cure of deleterious diseases in
the traditional way.
4.1 Qualitative Phytochemical Screening
From the phytochemical results, it is evident that
extraction of bioactive compounds from leaves of
S. campanulata have notable potentials to
strengthen the available methods to address
health problems in urban and rural areas of
developing countries where orthodox or modern
medicine are not afforded by many individuals.
The chemical methods employed for the
qualitative secondary metabolites identified
saponin, alkaloids, phenols and flavonoids
among others in the plant’s leaf. However, these
phytochemical have been reported to be highly of
therapeutic importance [9,28]. Medicinal plants
management in quality and quantity of
administration could provide effective health care
as a challenge under the best economic
circumstance. In the world’s poorest countries,
where infectious diseases are rife and resources
limited, such challenges can assume over
whelming proportions, hence the resurgence in
the use of herbal preparations to treat diseases
[29]. Therefore, plants evolutions in
phytochemical and antioxidant properties are
important values in prediction of potential drugs
or herbal preparation able to be effective in
management of diseases most especially by
those that can accept their innumerable values
for alternative therapy.
Fig. 1. Quantitative estimation of secondary metabolites from the leaf extracts
Legend: AE = Aqueous extract, EE = Ethanol extract, ME = Methanol extract, PE = Petroleum ether extract
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
155
Table 2. In vitro antioxidant
Plant Name
FRAS DPPH (mg of ascorbic acid/1 g
dry plant material)
FRAP (mg of ascorbic acid/1g dry plant
material)
Hydroxyl radical scavenging assay
AE EE ME PE
AE EE ME PE
AE EE ME PE
S. campnulata 1.44 + 0.5 1.57+ 1.4 0.40 + 1.4 0.30 + 0.1 1.19 + 0.4 1.69 + 0.18 1.74 + 0.5 2.48 + 1.8
0.65+ 0.9 0.43 + 0.50 0.86 +0.54 0.87+0.42
Legend: AE = Aqueous extract, EE = Ethanol extract, ME = Methanol extract, PE = Petroleum ether extract
Table 3. In vivo antioxidant assay
Group
LPO(µM/g)
GSH(µM/g)
CAT(µM/g)
Control (-) 98.94±6.66 34.76±1.40 70.58±6.66
Control(+) 138.40±2.10 22.34±2.00 47.36±3.76
200 mg/kg
bw
128.76±1.16 25.19±1.76 48.10±1.01
400 mg/kg
bw
127.63±1.14 26.30±2.14 48.56±2.44
800 mg/kg
bw
124.71±2.65
26.46±2.11
53.38±1.13
1000 mg/kg
bw
124.03±3.14 27.19±1.36 55.24±1.41
2000 mg/kg
bw
122.01±1.92 28.63±1.31 58.27±1.34
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
156
4.2 In vitro Antioxidant Activity
The antioxidant screening result of the extracts
showed that it has appreciable amount of
bioactive compounds. This is in correlation with
some studies elsewhere that medicinal plants
used in traditional medicine and healing are one
of the sources of antioxidants. The antioxidant
activities of the test plant aqueous, ethanol,
methanol and petroleum ether leaf extracts
investigated with DPPH free radical scavenging
assay showed valuable results which contributed
to the interest of screening further the plants
extract for more medical related indices to
evaluate its therapeutic value. Evidence has it
that DPPH free radical scavenging by
antioxidants is due to their hydrogen denoting
ability [30]. The antioxidant properties
determined showed that the solvents were able
to extract substances with antioxidants potency.
However, the petroleum extract was less efficient
in the extraction of the substances with
antioxidants to other employed solvents. The
reducing power of the extracts increased with
ethanol extract than other solvents and variations
in reducing power ability was noticed among the
extracts. Hence the leaf extracts of S.
campanulata exhibited exciting free radical
scavenging activity; the plant extracts may be
useful in reducing harmful effects of free radicals
in the maintenance of health and management of
cancer, aging process, Parkinson’s diseases and
perhaps dementias.
Ferric reducing antioxidant property (FRAP) is
used for the determination of reducing power of
various samples which is shown by colour
change of test samples solutions from yellow to
blue and green in proportionate to the reducing
power of various samples. The presence of
antioxidants in the test sample solutions would
result in reducing Fe
3+
to Fe
2+
by denoting an
electron by the extracts. The extracts with
reducing power revealed that they are electron
donors, reduced the oxidized intermediates and
act as primary antioxidant substrates [31]. Colour
change from yellow to blue and green was
dependant on the reducing power of each
compound present and the chemical solvents
used. This reaction of ferric form in ferric
tripyridyl triazine complex changes to ferrous
form showing the various shades of green and
blue which were of different reaction time and
colour. The changes in colour were however
observed by measuring at wavelength of 500nm.
Hence the compounds present in the extracts
differ in types and quantity; different results were
of course envisaged. The compounds with
reducing potential will react with potassium
hexacyanoferrate(III) to form potassium
hexacyanoferrate(II), which then reacted with
ferric chloride to form ferric
hexacyanoferrate(II) complex that is green in
colour. The ferric reducing potential of the plant
extracts could be due to their reducing power
ability by the bioactive compounds in the
extracts. Similar result of ferric reducing
potentials of plant extracts was recorded by [32,
33]. The FRAP results further confirmed the
antioxidant properties of the plants extracts
observed in the DPPH assay. The correlation
between reducing power and the DPPH values of
the plant extracts in this study is assumed to be
due to the same mechanism on which the
employed methods of antioxidant assay rely.
From the correlation in results, it could be
justified that the plant extracts tested can act as
electron donors and react with free radicals
conversion to more stable products, thus
breaking the radical chain reaction and
preventing cardiovascular or carcinogenic related
diseases.
4.3 In vivo Antioxidant Activity
Administration of S. typhi at dose concentration
of 10
7
Cfu/ml daily for three days showed
negative reactions in the in vivo antioxidant
assay on lipid peroxidation (LPO), Glutathione
(GSH) and Catalase (CAT). Effects proving these
results are the higher value observed in the
positive control than the negative control in LPO
and the lesser values in GSH and CAT in the
negative control than the positive control. In such
process where higher value of LPO in positive
control than negative control, non-enzymatic lipid
peroxidation or enzymatic peroxidation would
have occurred because when lipids is oxidized
without the release of energy, unsaturated lipid
go rancid as a result of deterioration when
directly reacted with molecular oxygen on
catalyses by free radicals which must have been
generated in the mice. The increase in lipid
peroxidation in the positive control and
bacteria/extract treated mice than the negative
control group could likely be the increase in free
radicals developed during abnormal metabolic
activities and the imbalances in antioxidant
defense causing an oxidative stress which could
have decreased glutathione level leading to
oxidative damage and resulting to an increase in
lipid peroxidation level. This is in agreement with
[34]. Increased lipid peroxidation is a highly
destructive process that induces a plethora of
Coolborn et al.; IJBcRR, 7(3): 148-159, 2015; Article no.IJBcRR.2015.064
157
structural and functional alterations of cellular
membranes and involves oxidation of fatty acids
[35]. Though the reduction in values for the
period of pre-treatment with extracts were not
same with the negative control, values were
comparable, signifying that if pre-treatment is
extended the tendency of values becoming equal
with the negative control is predictable.
Glutathione (GSH) is the major compound in the
intracellular redox status regulation and it is
considered as an important cofactor in many
metabolic reactions [36]. The observed elevation
of GSH in liver of mice after extract pre-treatment
for seven days may be due to a compensatory
response induced by imbalance in the redox
status of the cell as a result of excessive
hydrogen peroxide (H
2
O
2
) production and
progressive decrease of glutathione peroxide.
This result is in agreement with [37,32,33] who
observed that enzyme activity of glutathione
peroxide (GSH-Px) in liver was significantly
increased in extract treated mice after injury with
toxic substances. Such inhibitory effect could
underlay the efficacy of the leaf extracts of S.
campanulata in this study. Increase in GSH value
in the bacteria/extract treated mice was also on
dose dependant. This is in agreement with the
results obtained by [38-40] who assumed that
GSH correlates in a time and dose dependant
fashion with an increase in levels of extracts.
Catalase (CAT) is metallo proteins and
accomplished their antioxidant functions by
enzymatically detoxifying the peroxide (OH,
H
2
O
2
) and superoxide anion. The CAT increase
and LPO decrease in the bacteria/extract treated
group, is an indication of effectiveness of the
plant antioxidant. This manifestation is evident
because catalase converts harmful hydrogen
peroxide into water and oxygen which in that
order protects the liver tissue from highly reactive
hydroxyl radicals able to be generated by S. typhi
infection at a concentration of 10
7
Cfu/ml for the
three days induction. The reduction in the
activities of this enzyme as observed in the
positive control mice may results in number of
deleterious effects due to accumulation of highly
toxic metabolites and hydrogen peroxide on S.
typhi infection. This is liable to induce oxidative
stress in the cells [41]. The co-administration of
the extracts in various concentrations increased
the activities of catalase in mice preventing the
accumulated excess free radicals thereby
protecting the liver from intoxication induced by
S. typhi infection.
The in vivo antioxidant assay showed that S.
typhi at concentration of 10
7
Cfu/ml induced
oxidative stress in mice by decreasing
endogenous antioxidant defence system of ferric
reducing with an increase in lipid peroxidation in
liver which is likely liable to cell death in the mice
liver. Conclusively, it is evidenced that the leaf
extracts of S. campanulata could be of barrier to
generation of free radicals. The appreciable
phytochemicals; in vitro and in vivo antioxidants
status has established the potential value this
plant could play in medicine. It is therefore
suggested that its employment in traditional
medicine be encouraged.
5. CONCLUSION
Valuable phytochemical screened from S.
campanulata were of notable potentials to
strengthen the available methods to address
health problems where orthodox or modern
medicine are not afforded by many individuals.
The antioxidants potential was encouraging as
observed in the animal model study. The
recorded data in the study proposed the use of
leaf extract of S. campanulata in traditional
medicine hence its inhibition potentials and
barrier to generation of free radicals.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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... Catalase (CAT) is one of the important metalloproteins in the supportive team of defence against reactive oxygen species (ROS). [32] Widely distributed in all animal tissues, it is a protective enzyme tissue from reactive hydroxyl radicals which accomplished their antioxidant functions by enzymatically detoxifying the peroxide (OH, H2O2) and superoxide anion. [29,32] This haemoprotein containing four haem groups, that catalyses the decomposition of H2O2 to water and O2 and thus, protects the cell from oxidative damage by H2O2 and OH. ...
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... However, in alloxan-induced diabetic rats the extract significantly reduced blood glucose levels with the highest effect at 400 mg/kg comparable to those of control and chlorpropamide (Tanayen et al., 2014) Antioxidant, Antimicrobial, Analgesic, Anti-inflammatory, Antimalarial, Anticonvulsant, Wound Healing, Hepatoprotective (Coolborn et al., 2015;Rajesh et al., 2010;Ilodigwe and Akah, 2009; ...
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