Beni-Suef University Journal of Basic and Applied Sciences 6 (2017) 10–14

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Beni-Suef University
Journal of Basic and Applied Sciences
journal homepage: www.elsevier.com/locate/bjbas

In vitro antioxidant activity, total phenolic and flavonoid contents of

ethanol extract of stem and leaf of Grewia carpinifolia
Olamide E. Adebiyi a,b,⇑, Funsho O. Olayemi b, Tan Ning-Hua a, Zeng Guang-Zhi a
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, China
b
Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Nigeria

a r t i c l e i n f o a b s t r a c t

Article history: Free radicals are reactive molecules involved in many physiological processes and have been associated
Received 7 October 2016 with many diseases, such as cancer, arthritis and liver injury. As a result, there is need to explore sub-
Received in revised form 19 December 2016 stances with free radical scavenging and or antioxidant activity. The present study was designed to eval-
Accepted 21 December 2016
uate the free radical scavenging activity of ethanol extract of leaf and stem of Grewia carpinifolia using
Available online 4 January 2017
various in vitro models. Ascorbic acid was used as the reference in the study. 1,1-Diphenyl-2-picryl
hydroxyl (DPPH) quenching assay, 2,20 -azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS) cation
Keywords:
decolorization test, ferric reducing antioxidant power (FRAP) Assay systems were selected for the present
Grewia carpinifolia
Antioxidant activity
experiment. The ability of the extracts to inhibit lipid oxidation was measured using Thiobarbituric Acid
DPPH Reactive Substances (TBARS) assay. The extracts were used at 0.2, 0.4, 0.6, 0.8 and 1 mg/ml concentra-
ABTS tions and radical scavenging activity was determined in terms of inhibition percentage. The IC50 (concen-
FRAP tration required for 50% inhibition) was also calculated for each radical. The study revealed that Grewia
carpinifolia has a high radical scavenging activity in the various radical systems. The total phenolic con-
tent was 19.08 ± 1.21 mg gallic acid equivalent (GAE)/g extract and 14.85 ± 1.09 mg GAE/g extract for the
leaf and stem respectively while the flavonoid content was 9.00 ± 0.13 and 13.22 ± 1.53 mg quercetin/g
extract. The antioxidant activity of Grewia carpinifolia extract may be due to the high level of flavonoids
and phenols in the plant.
Ó 2016 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction et al., 2003). The growing need to complement these endogenous

Antioxidants are substances that significantly delay or inhibit
oxidation of an oxidizable substrate when present at low concen-
trations in comparison with those of the substrate (Halliwell and
Gutteridge, 1999). The activities of free radicals have been impli-
cated in aging, destruction of DNA, obstruction of arteries, cancer,
strokes, cardiac and central nervous system (CNS) disorders which
have led to an increase in the investigation of substances that can
protect against these reactive oxygen species and thus may play a
role in disease prevention (Rakesh et al., 2010; Kapadiya et al.,
2016).
Endogenous antioxidants are synthesized within the system of
living organisms and repair free radical damage internally by initi-
ating cell regeneration while exogenous antioxidants which are
derived from sources outside the living systems such as diets
(Jaouad and Torsten, 2010) stimulate cell repair externally (Wolfe
⇑ Corresponding author at: Department of Veterinary Physiology, Biochemistry
and Pharmacology, University of Ibadan, Nigeria.
E-mail address: olamideadebiyi24@gmail.com (O.E. Adebiyi).
antioxidants has led to an increased supplementation by exoge-
nous sources. At present, there are keen interests and widespread
researches on exogenous antioxidants from natural sources per-
haps, due to the fact that they are less expensive, readily available
and believed to have lesser side effects when compared to their
synthetic counterparts (Tadhani et al., 2007).
Grewia carpinifolia belongs to the family, Tiliaceae and it is
extensively found in tropical climates, they are also found in Asia
and North America (Goyal, 2012). For instance, in Côte d’Ivoire
its leaves are consumed as vegetables (Herzog et al., 1993). The leaf
and stem of the plant have been reported to have anxiolytic and
anti-parasitic (Goyal, 2012; Adebiyi et al., 2015) amongst other
medicinal properties. In addition, preliminary phytochemical
screening has indicated the presence of tannins, saponins, flavo-
noids, glycosides, phenols and steroids in the leaf and stem of G.
carpinifolia (Adebiyi et al., 2016). However, despite the widespread
folklore uses of Grewia carpinifolia in the management of various
diseases, apart from the report on the CNS activity of the plant
(Adebiyi et al., 2016), there is no documented report to evaluate
and provide information on the antioxidant activity of this plant.

http://dx.doi.org/10.1016/j.bjbas.2016.12.003
2314-8535/Ó 2016 Beni-Suef University. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 O.E. Adebiyi et al. / Beni-Suef University Journal of Basic and Applied Sciences 6 (2017) 10–14
Thus, the aim of this study was to evaluate the in vitro antioxidant
activity of ethanol extract of Grewia carpinifolia leaf and stem.
2. Materials and method
2.1. Plant material
The leaf and stem of Grewia carpinifolia were collected in
September, 2015 (during the rainy season) from the Botanical Gar-
den of the University of Ibadan, Nigeria and identified at the For-
estry Research Institute of Nigeria where a herbarium specimen
was deposited (voucher number FHI 109693).
2.1.1. Preparation of extract
Plant materials were washed with distilled water and dried at
room temperature to constant weights. The dried plant materials
were ground separately to powder. 1 kg of each ground plant
materials was soaked separately in ethanol for 48 h on an orbital
shaker. Extracts were filtered using a Buckner funnel and What-
man No 1 filter paper. Each filtrate was concentrated to dryness
under reduced pressure at 40 °C using a rotary evaporator.
2.2. Chemicals
2,20 -azinobis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS),
1,1-Diphenyl-2-picrylhydrazyl (DPPH), 3-(2-pyridyl)-5,6-diphe
nyl-1,2,4-triazine-40 ,400 -disulfonic acid, potassium ferricyanide;
catechin, ascorbic acid, catechin, tannic acid, quercetin, Folin–Cio
calteus’s phenol reagent and sodium carbonate and FeCl3 were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). All the
chemicals used including the solvents, were of analytical grade.
2.3. Determination of total phenolics
Using modified Folin–Ciocalteu method (Kaur and Kapoor,
2002), total phenol contents in the extracts were determined.
50 lL aliquots of 12.5, 25, 50, 100, 200, and 400 l g/ml methanolic
gallic acid solutions were mixed with 100 lL Folin–Ciocalteu
reagent (diluted ten-fold) and 100 lL (75 g/L) sodium carbonate.
The mixture was incubated at 25 °C for 30 min, the quantitative
phenolic estimation was performed at 765 nm. The calibration
curve was constructed by plotting the absorbance against concen-
tration. A similar procedure was adopted for the test samples as
described above. Total phenolic content was expressed as mil-
ligrams of gallic acid equivalent (GAE) per g of extract using the
following equation based on the calibration curve: y = 0.0003x
+ 0.0716, R2 = 0.9365, where x was the absorbance and y was the
tannic acid equivalent (mg/g).
2.4. Determination of total flavonoids
Estimation of the total flavonoids in the plant extracts was car-
ried out using the method of Ordonez et al. (2006). 0.5 ml of 2%
AlCl3 ethanol solution was added to 0.5 ml of sample. After one
hour at room temperature, the absorbance was measured at
420 nm. Extract samples were evaluated at a final concentration
of 1 mg/ml. Total flavonoid content was calculated as quercetin
(mg/g) using the following equation based on the calibration
curve: y = 0.0255x, R2 = 0.9812, where x was the absorbance and
y was the quercetin equivalent (mg/g).
11
2.5. In vitro antioxidant activity
2.5.1. ABTS radical scavenging activity
ABTS radical-scavenging activity of the extract was determined
according to method described by Re et al. (1999). The ABTS radical
cation (ABTS+) was produced by the reaction between 5 ml of
ABTS stock solution and 5 ml of 2.45 mM potassium persulfate
(K2S2O8) solution, stored in the dark at room temperature for
16 h. Before use, this solution was diluted with water to get an
absorbance of 0.700 ± 0.020 at 734 nm and equilibrated at 30 °C.
The plant extract at various concentrations were diluted with
dimethyl sulfoxide (DMSO) to get sample solution. 5 lL of sample
solution was homogenized with 195 lL ABTS + solution, the mix-
ture was incubated at room temperature for 6 min and its absor-
bance was recorded at 734 nm. Blanks were run in each assay.
ABTS scavenging ability was expressed as IC50 (lg/ml) and the
inhibition percentage calculated using the following formula:
ABTS scavenging activityð%Þ ¼ ðA0  A1 Þ=A0  100
where A0 is the absorbance of the control, and
A1 is the absorbance of the sample.
2.5.2. DPPH radical scavenging activity
This test was measured as described by Blois (1958). DPPH was
dissolved in methanol to a 0.025 g/L. The plant extract at various
concentrations was diluted with dimethyl sulfoxide (DMSO) to
get sample solution. 5 lL of the sample solution in a 96-well plate
following which 195 lL DPPH working solution was added to each
well. After a 20 min reaction at room temperature, the absorbance
of the solution was measured at 515 nm. The free radical scaveng-
ing activity of each fraction was determined by comparing its
absorbance with that of a blank solution (no sample). The ability
to scavenge the DPPH radical was expressed as percentage inhibi-
tion and calculated using the following equation: The results
DPPH scavenging activityð%Þ ¼ ðA0  A1 Þ=A0  100
where A0 is the absorbance of the control and A1 is the absorbance
of the sample.
2.5.3. Ferric reducing antioxidant power (FRAP) assay
The ability to reduce ferric ions was measured using the method
described by Benzie and Strain (1996). The FRAP reagent was pro-
duced just before use by mixing 300 mM sodium acetate buffer
(pH 3.6), 10.0 mM TPTZ (tripyridyl triazine) solution and
20.0 mM FeCl3.6H2O solution in a ratio of 10:1:1 in volume. Sam-
ples at different concentrations (100, 200, 300, 400 and 500 lg/
ml) were then added to 3 ml of FRAP reagent and the reaction mix-
ture was incubated at 37 °C for 30 min. The increase in absorbance
at 593 nm was measured. Fresh working solutions of FeSO4 were
used for calibration. The antioxidant capacity based on the ability
to reduce ferric ions of sample was calculated from the linear cal-
ibration curve and expressed as mmol FeSO4 equivalents per gram
of sample. The optical density was read at 593 nm.
2.5.4. Thiobarbituric acid reactive substances (TBARS) assay
A mixture of 1 ml plant extract, 4 ml 99.5% ethanol, 4.1 ml 2.5%
linoleic acid in 99.5% ethanol, 8.0 ml 0.02 M phosphate buffer (pH
7.0) and 3.9 ml distilled water was placed in an oven at 40 °C in the
dark for 1 h. TBARS assay was conducted according to methods
described (Kikuzaki and Nakatani, 1993). 2 ml of 20% trichloroace-
tic acid and 2 ml of 0.67% of thiobarbituric acid was added to 1 ml
of sample solution. The mixture was placed in boiling water bath
for 10mins. It was centrifuged after cooling at 3000 rpm for
20 min. The absorbance of the supernatant was measured at
552 nm and recorded again after the maximum was reached.
Antioxidant activity was described by percentage inhibition as
12 O.E. Adebiyi et al. / Beni-Suef University Journal of Basic and Applied Sciences 6 (2017) 10–14

% inhibition ¼ ðA0  A1 Þ=A0  100

where A0 is the absorbance of the control and A1 is the absorbance

of the sample.
Ascorbic acid was used as the reference and all analyses were
done in triplicate.

2.6. Statistical analysis

The experimental results were expressed as mean ± standard

error of mean (SEM) of three replicates. Where applicable, the data
were subjected to one way analysis of variance (ANOVA) and the
differences between samples were determined by Duncan’s Multi-
ple Range test using Graph Pad Prism software version 5.0 (San
Diego, USA). P Values 6 0.05 were regarded as significant. Fig. 1. ABTS radical scavenging activity.

3. Results

The total phenolic contents in the examined leaves and stem of

G. carpinifolia extracts were 19.08 and 14.85 mg GAE/g respec-
tively. The leaf had a higher concentration of phenols but total fla-
vonoids were greater in the stem extract (Table 1).
In Fig. 1, G. carpinifolia extract was found to be effective in scav-
enging the ABTS radical. The percentage inhibition of this radical
was concentration-dependent. At 0.5 mg/ml, the inhibition of the
leaf and stem extract was 58.15% and 70.25% respectively and that
of ascorbic acid was 80.02%. The IC50 (concentration required for
50% inhibition) of ascorbic acid was 0.31 mg/ml while that of the
leaf and stem extract was 0.32 and 1.98 mg/ml respectively.
The DPPH method revealed that the scavenging of the free rad-
icals was found to be 28.56, 43.60, 56.85, 58.90 and 72.60% at 0.2, Fig. 2. DPPH radical scavenging activity.
0.4, 0.6, 0.8 and 1 mg/ml respectively for the ethanol extract of G.
carpinifolia leaf. The percentage inhibition of the DPPH radical by
the extract of G. carpinifolia stem was 11.85, 19.11, 22.81, 35.07
and 62.12% at 0.2, 0.4, 0.6, 0.8 and 1 mg/ml respectively (Fig. 2).
In the DPPH assay, the IC50 of ascorbic acid was 0.18 mg/ml while
that of the leaf and stem extract was 0.39 and 0.81 mg/ml
respectively.
Antioxidant activities of the ethanolic extracts of the plants as
determined by the FRAP assay are depicted in Fig. 3. In the FRAP
assay the absorbance of G. carpinifolia was found to be 0.11, 0.15,
0.22, 0.68, 2.77 and that of the stem was 0.10, 0.14, 0.22, 0.35,
2.57 at 0.2, 0.4, 0.6, 0.8 and 1 mg/ml respectively. The reference;
ascorbic acid had an absorbance of 4.00 at the maximum dosage
of 1 mg/ml (Fig. 3)
The percentage inhibition using the TBARS assay ranged from
23.05 to 84.56% for the leaf extract while that of the stem was
Fig. 3. FRAP assay.
between 20.4 and 75.15% at the minimum and maximum concen-
tration respectively. The IC50 of ascorbic acid was 0.38 mg/ml while
that of the leaf and stem extract was 0.24 and 0.30 mg/ml respec-
4. Discussion
tively. The IC50 of the leaf and stem extract of G. carpinifolia in the
TBARS assay was lower than that of ascorbic acid (see Fig. 4).
Plants have diverse groups of phenolic compounds, such as sim-
ple phenolics, phenolic acids, anthocyanins, hydroxycinnamic acid
Table 1 derivatives and flavonoids. All these phenolic classes have gained
Polyphenol contents of the ethanol extracts of the leaf and stems of Grewia carpinifolia extensive attention because of their physiological functions,
(n = 3, X ± SEM). including free radical scavenging, anti-mutagenic, anti-
Phenolics Leaf Stem carcinogenic and anti-inflammatory effects (Manthey, 2000;
Total phenola 19.08 ± 1.21* 14.85 ± 1.09
Bandoniene and Murkovic, 2002).
Total Flavonoidsb 9.00 ± 0.13 13.22 ± 1.53* According to Pietta (2000), the antioxidant activity of phenolics
a
is largely due to their redox properties which make them act as
Expressed as mg of gallic acid equivalent (GAE)/g of dry plant material.
b reducing agents, hydrogen donors, singlet oxygen quenchers and
Expressed as mg quercetin/g of dry plant material.
*
Indicates that this value is significantly different from the other at P < 0.05. as well as potential metal chelators. In this study, a considerable
 O.E. Adebiyi et al. / Beni-Suef University Journal of Basic and Applied Sciences 6 (2017) 10–14
Fig. 4. TBARS assay.
high level of phenolics was observed in the ethanol extracts of the
leaf and stem of Grewia carpinifolia. This may explain the wide-
spread folklore use of the plant.
The ABTS.+ assay has been employed as an index that reveals
the antioxidant activity of test samples (Wu et al., 2006). The scav-
enging of the ABTS+ radical by the stem extracts in this study was
found to be as high as 100%; same as the standard at the highest
concentration of 1 mg/ml. This shows that Grewia carpinifolia
extract presents a good ability to scavenge the ABTS radical. In
the present study, there was increased scavenging activity of the
DPPH radicals with increasing concentration of the plant extracts
which may indicate an increased ability to donate hydrogen ions
resulting in a lighter solution which is proportional to the number
of electrons gained (Silva et al., 2005; Villaño et al., 2007). There-
fore, it may be postulated that Grewia carpinifolia, has DPPH scav-
enging activity, by reducing the radical to corresponding hydrazine
as a result of its hydrogen ion-donating ability. However, the scav-
enging of the DPPH radical by the extracts was found to be much
lower than that of ABTS+. Stereo selectivity of the radicals and
the solubility of extracts in diverse solvent systems are some fac-
tors that have been reported to affect the capacity of extracts to
react and quench different radicals (Yu et al., 2002). Wang et al.
(1998) found that some compounds which have ABTS+ scavenging
activity did not show DPPH scavenging activity. This is however
not the case in this study. This further showed the capability of
Grewia carpinifolia extracts to scavenge different free radicals in
different systems, indicating that they may be useful therapeutic
agents for treating radical-related pathological damage.
The FRAP assay measures the reducing potential of an antioxi-
dant reacting with a ferric tripyridyltriazine (Fe3+-TPTZ) complex
and produce a coloured ferrous tripyridyltriazine (Fe2+-TPTZ)
(Benzie and Strain, 1996). Generally, the reducing properties are
linked with the presence of compounds which exert their action
by breaking free radical chain by donating a hydrogen atom (Duh
et al., 1999). In the present study as shown in Fig. 3, the absorbance
of G. carpinifolia clearly increased, due to the formation of the Fe2+-
TPTZ complex with increasing concentration as seen also in the ref-
erence antioxidant. Hence the plant should be able to donate elec-
trons to free radicals stable in the actual biological and food
systems. The leaf and stem extracts of G. carpinifolia showed a con-
siderable antioxidant effect in all the assays. The leaf showed a
higher antioxidant activity in all the assays with the exception of
the ABTS assay where the stem extract had a higher activity at
all the tested concentration. The high percentage inhibition
recorded in the TBARS assay might indicate the ability of the
extract to inhibit linoleic acid peroxidation.
Previous reports on the antioxidant activity of G. carpinifolia are
little or none in the literature; this made the comparison of our
13
results with those of previous studies very difficult. However, our
result in the present study is similar to several other documenta-
tions on the antioxidant activities of other Grewia species such as
G. asiatica which is native to Asia and has many pharmacological
activities including antioxidant properties (Zia-Ul-Haq et al.,
2013). Recently, Rehman et al. (2013) also demonstrated that G.
optiva possesses high antioxidant activity and contains several sec-
ondary metabolites such as flavonoids, alkaloids, terpenoids, tan-
nins and saponins. The antihyperglycemic effect of G. asiatica
fruit extract has also been linked to its improvement of the pancre-
atic b-cells as well as its antioxidant effects (Khattab et al., 2015).
G. tiliaefolia has similarly been stated to have the ability to scav-
enge free radicals in both in vivo and in vitro models (Selvam
et al., 2010)
The present study has revealed that the ethanol extract of G.
carpinifolia contains substantial amount of phenolics and thus,
can be inferred that these phenolics are responsible for its marked
antioxidant activity as assayed through various in vitro models
used in this study. This is consistent with several reports that have
shown close relationship between total phenolic contents and
antioxidative activity of fruits, plants and vegetables (Albayrak
et al., 2010; Biju et al., 2014; Baba and Malik, 2015). Therefore, G.
carpinifolia leaf and stem extracts have considerable antioxidant
properties and the consumption of this under-exploited plant
may play a role in preventing human diseases in which free radi-
cals are involved, such as cancer, cardiovascular disease, and pre-
mature aging. However, further investigations on the in vivo
antioxidant activity and the different antioxidant mechanisms
are warranted.
Conflict of interest
None declared.
Acknowledgements
The authors are grateful to OWSD (Organisation for Women in
Science in Developing Countries) and SIDA (Swedish International
Cooperation Agency) for the fellowship award (#3240274098) to
OEA.
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