Evaluation of antioxidant activity and chemical analysis of the leaf of Telfairia occidentalis

Abstract

The methanol extract of Telfairia occidentalis leaf and n-hexane, ethyl acetate, butanol fractions were evaluated for their free radical scavenging activity, with DPPH assay. n-hexane fraction had the highest activity exhibiting an IC 50 of 78.50µg/ml, comparable to that of the commercial antioxidant BHT. Chromatographic analysis showed the presence of three components in n-hexane fraction (2H 1 , 2H 3 and 2H 4) with Rf values of 0.86, 0.71 and 0.55, respectively. Two components were seperated in ethyl acetate fraction (2E 1 and 2E 5) with Rf values of 0.85 and 0.25, respectively, while the three components of butanol fraction (2B 1 , 2B 2 , and 2B 3) have Rf values of 0.90, 0.88 and 0.83 respectively. In addition, total phenolic content of T. occidentialis was determined as catechin equivalents. The n-hexane fraction which had the highest DPPH free radical scavenging activity also had the highest total flavonoid contents. The high flavonoid content was responsible for the antioxidant and free radical scavenging activities of Telfairia occidentalis leaf.

Full text

Nigerian Journal Of Pharmaceutical and Applied Science Research 1(1): 21-28, December 2011
Evaluation of Antioxidant Activity And Chemical Analysis Of The Leaf Of T. occidentalis.Nkereuwem et al. Page 21
Evaluation of Antioxidant Activity And Chemical Analysis Of The Leaf Of Telfairia occidentalis
Nkereuwem, A. O., Eseyin, O. A., Udobre S. A. and Ebong, A.
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Uyo,
Nigeria.
*Corresponding Author
E-mail: nkereuwemanthony@yahoo.com
ABSTRACT
The methanol extract of Telfairia occidentalis leaf and n-hexane, ethyl acetate, butanol
fractions were evaluated for their free radical scavenging activity, with DPPH assay. n-hexane
fraction had the highest activity exhibiting an IC50 of 78.50µg/ml, comparable to that of the
commercial antioxidant BHT. Chromatographic analysis showed the presence of three components
in n-hexane fraction (2H1, 2H3 and 2H4) with Rf values of 0.86, 0.71 and 0.55, respectively. Two
components were seperated in ethyl acetate fraction (2E1 and 2E5) with Rf values of 0.85 and 0.25,
respectively, while the three components of butanol fraction (2B1, 2B2, and 2B3) have Rf values of
0.90, 0.88 and 0.83 respectively. In addition, total phenolic content of T. occidentialis was
determined as catechin equivalents. The n-hexane fraction which had the highest DPPH free radical
scavenging activity also had the highest total flavonoid contents. The high flavonoid content was
responsible for the antioxidant and free radical scavenging activities of Telfairia occidentalis leaf.
KEY WORDS: Antioxidants, DPPH, free radical scavenging activity, flavoniods.
INTRODUCTION
Reactive oxygen species (ROS), including
free radicals are reported to cause damage of
biological system, and to be involved in aging
and in the pathogenesis of some diseases
such as arthritis, atherosclerosis, diabetes and
cancer (Ames, 1983; Feher et al., 1987;
Aruoma, 1998). Almost all organisms possess
antioxidants and repair systems that evolved
to protect them against oxidative damage,
these systems are insufficient to prevent
them entirely. However, antioxidants may be
used to help human body to reduce oxidative
damage (Yang et al., 2002). Plants contain
different natural products, which have a
remarkable role in the traditional medicine in
different
countries. Nowadays the prevention of many
diseases has been associated with the
ingestion of different plants rich in natural
antioxidants (Johnson, 2001; Virgilli et al.,
2001; Adedapo et al., 2008). In recent years,
there has been a particular interest in the
antioxidant and health benefit of
phytochemicals in food and vegetables. This
was as a result of their potential effects on
human health (Wei and Shiow, 2001). Many
researchers especially in the filed of medical
science have observed free radical scavenging
ability and antioxidant property in Telfairia

Nigerian Journal Of Pharmaceutical and Applied Science Research 1(1): 21-28, December 2011
Evaluation of Antioxidant Activity And Chemical Analysis Of The Leaf Of T. occidentalis.Nkereuwem et al. Page 22
occidentalis (Oboh and Akindahunsi, 2004;
Oboh et al., 2007; Iweala and obidina, 2009,
Kayode et al., 2009, kayode et al., 2010). The
hypoglyceamic properties of the plant have
also been reported (Aderibigbe et al., 1999;
Eseyin et al., 2000; Eseyin et al., 2005; Eseyin
et al, 2007; Eseyin et al., 2010).
The antioxidant effect is mainly due to
compents, such as flavoniods. Antioxidants
can interfere with the oxidation process by
reacting with free radicals, chelating catalytic
metals, acting as oxygen scavengers (Shahidi
and Wanasundara, 1992) and preventing lipid
auto-oxidation (Brand-Williams et al., 1995;
Bondet et al., 1997). Research work had
justified the use of the leaf of Telfairia
occidentialis in Nigeria in the treatment of
certain disease in which the participation of
reactive oxygen species (ROS) have been
implicated. This could be as a result of an
antioxidant and free radical scavenging ability
(Kayode and Kayode, 2011). Much have been
reported on the various medicinal properties
of the leaf extract of Telfairia occidentalis but
little or no report have been published on the
active pharmacological constituents isolated
from this very important plant. In this
research work, attempt were made to isolate
some constituents using column and thin
layer chromatographic methods.
MATERIALS AND METHODS
Collection of plant materials Plant material
Telfairia occidentalis leaf used for this work
was obtained from a local market in Uyo,
Akwa Ibom State, Nigeria in March, 2010 and
authenticated by Dr. Mrs. Margaret Bassey, a
taxonomist in Botany Department, University
of Uyo, Uyo, Akwa Ibom State, Nigeria. The
leaves were cleaned and dried in the shade,
then powered to 40 mesh and stored in an
airtight container at 25oc.
Extraction and isolation of the chemical
constituents
Air dried powered leaf of Telfairia
occidentalis (731g) was exhaustively extracted
with a total volume of 2.5L of methanol in a
soxhlet apparatus using continuous
extraction. Evaporation and concentration of
the solvent afforded the methanol extract
(198.3g). This was further extracted
successively with a total volume of 2.5L of the
following solvent: n-hexane, ethyl acetate and
butanol. Evaporation and concentration of
the solvents afforded the n-hexane fraction
(HF,93.89g), ethyl acetate fraction (EF,
22.43g) and butanol fraction (BF, 13.23g).
Phytochemical test was carried out on the
crude extract and the fractions.
The fractions were fractionated on Silica gel
(absorbent) (60-120µg mesh) as follows:
n-hexane fraction: Silica gel (148.3g) was
used to fractionate 1.0gm of the HF using n-
hexane, chloroform, methanol and water as
solvent in that order.
Ethyl acetate fraction: Silica gel (93.6g) was
used to separate 2.0g of the EAF using
benzene, chloroform, ethyl acetate, methanol
and water as solvent in that order.
Butanol fraction: Silica gel (114.2g) was used
to separate 2.7g of the BF using benzene,
ethyl acetate, methanol and water as solvent
in that order.
New solvents were introduced gradually in
these ratios 80:20; 50:50; 20:80 to prevent
cracking. In each chromatographic analysis,
15ml was collected in each test tube and the
solvent allowed to evaporate at room
temperature. Thin layer (TLC)

Nigerian Journal Of Pharmaceutical and Applied Science Research 1(1): 21-28, December 2011
Evaluation of Antioxidant Activity And Chemical Analysis Of The Leaf Of T. occidentalis.Nkereuwem et al. Page 23
chromatographic analysis was carried out on
these eluents and the Rf of each eluent was
determined. Eluent with identical Rf values
were pooled together to obtain the following
pooled fractions:
HF: 2H1, 2H3, 2H4 with Rf values 0.86, 0.71
and 0.55, respectively.
EAF: 2E1 and 2E5 with Rf values 0.85 and 0.25,
respectively.
BF, 2B1, 2B2 and 2B3 with Rf values 0.90, 0.88
and 0.83, respectively. The TLC plates of these
fractions were sprayed with the following
colour reagents:
Conc. sulphuric acid: A light yellow or pale
orange colouration confirmed the presence of
steroids.
Ferric chloride: A blue colouration confirmed
the presence of tannins.
Dragendorff’s solution: A reddish-brown
colouration confirmed the presence of
alkaloids.
Determination of Total Phenolic Content
(TPC) of Extract and Fractions
Total phenolics was quantified and
expressed as gallic acid equivalent according
to a method proposed by Singleton and Rossi
(1999). 1ml of Folin-Ciocalteu’s reagent,
previously diluted (1:20), was added to 1ml of
samples (250µg/ml) and mixed thoroughly. To
the mixture, 4ml of sodium carbonate (75g/L)
and 10ml of distilled water were added and
mixed well. The mixture was allowed to stand
for 2hr at room temperature. Contents were
then centrifuged at 2000g for 5min and the
absorbance of the supernatant was taken at
760nm. A standard curve was obtained using
various concentrations of gallic acid. Results
were expressed as percentage of Gallic Acid
Equivalents (GAE) per 100gram of fresh mass.
Determination of Total Flavonoids Contents
(TFC) of extract and Fractions
Total flavoniod contents was
measured by aluminum chloride colourimetric
assay based on the method modified by
Marinova, Ribarova and Atanasova (2005). To
0.1ml of extracts in a 10ml volumetric flask,
distilled water was added to make the volume
to 5ml and 0.3ml 5% NaNO3 was added to
this. 3ml of 10% AlCl3 was then added
5minutes later. After 6 minutes, 2ml of 1M
NaOH was added and the absorbance
measured at 510nm. Cathecol was used as a
standard.
DPPH free radical scavenging assay
Free radical scavenging activities was
determined using the DPPH free radical
method. Various concentrations of the
samples were added to 3ml of daily-prepared
methanol DPPH solution (0.1nm). The mixture
was shaken and left to stand at room
temperature in the dark. After 30min,
absorbance was measured at 517nm against a
blank (containing all reagents except the test
samples). Assays were carried out in
triplicates. The concentrations of the samples
that gave 50% inhibition of DPPH (IC50) were
obtained from the graph of 1% (inhibition
percentage) versus concentration of the
sample in µg/ml. The percentage inhibition of
DPPH (1%) was calculated using the equation.
1% = (A blank – Asample/Ablank) x 100
Where Ablank is the absorbance of the blank
solution
Asample is the absorbance of the test sample.
RESULTS AND DISCUSSION

Nigerian Journal Of Pharmaceutical and Applied Science Research 1(1): 21-28, December 2011
Evaluation of Antioxidant Activity And Chemical Analysis Of The Leaf Of T. occidentalis.Nkereuwem et al. Page 24
Results of Phytochemical Analysis of Crude
Extract and n-Hexane, Ethyl acetate, Butanol
Fractions and Chromatography analysis for
steroids, tannins and alkaloids are shown in
tables 1 and 2. While the results of total
Phenolic Content (TPC) and Total Flavonoid
Content (TFC), and DPPH free radical
scavenging activity (IC50) for methanol
extract and fractions are shown in tables 3
and 4
.
Table 1: Results of Phytochemical analysis of ME, HF, EAF and BF
S/N
Phytochemical test
Crude extract
n
-
hexane
fraction
Ethyl acetate
fraction
Butanol
fraction
1
Tannin Test with
Fe2Cl3
Present
Present
Present
Present
2
Saponnin test
Present
Present
Present
Present
3
Alkaloid
Prese
nt
Present
Present
Present
4
Test for Steriods
Salkowski
Test
Lieberman-
Burchard
test
Present
Present
Present
Present
Present
Terpenes Present
Present
Present
5
Flavonoids
Present
Present
Present
Present
6
Glycoside
Present
Present
Pres
ent
Present
DISCUSSION
Currently, there is an increasing demand to
evaluate the antioxidant properties from
plants (Pratt, 1992). In this research work, the
methanolic crude extract, fractions and
separated constituents were analysed for
their antioxidant activity and the
phytochemical(s) responsible for this effect.
Researchers have observed free radical
scavenging ability and antioxidant property in
Telfairia occidentialis (Oboh and Akindahunsi,
2004; Oboh et al., 2006; Iweala and Obida,
2009; Kayode et al., 2010).
The results of free radical scavenging activity
of Telfairia occidentalis are shown in table 5.
The crude methanolic extract presented a
significant free radical scavenging activity,
with an IC50 of 31.25µg/ml. Comparison of the
obtained IC50 data (table 5) indicated a potent
activity for the HF (IC50 = 78.50µg/ml) and a
moderate free radical scavenging effect for
EAF (IC50=86.30µg/ml) and BF (IC50=
142.40µg/ml). The fractions were purified by
column and thin layer chromatography on
silica gel to afford the constituents. N-hexane
fraction gave three components 2H1, 2H3 and
2H4 with IC50 value of 32.5 µg/ml, 70.40 µg/ml
and 100.50 µg/ml respectively. Ethyl acetate
fraction gave two components 2E1 and 2E5
with IC50 value of 36.50µg/ml and
82.20µg/ml respectively. Butanol fraction
gave three components 2B1, 2B2 and 2B3.
The DPPH free radical scavenging activity of
the constituents from BF were very low.

Nigerian Journal Of Pharmaceutical and Applied Science Research 1(1): 21-28, December 2011
Evaluation of Antioxidant Activity And Chemical Analysis Of The Leaf Of T. occidentalis.Nkereuwem et al. Page 25
Table 2: Chromatography analysis for steroids, tannins and alkaloids
Isolated
Compounds
Conc. H
2
SO
4
(Steroid)
Fe
2
Cl
2
(Tannins)
Dragendorff’s
soln
(Alkaloids)
2H
1
Present
Present
Present
2H
3
Present
Present
Present
2H
4
Present
Present
Present
2E
1
Present
Present
Present
2E
5
Present
Present
Present
2B
1
Absent
Present
Absent
2B
2
Presen
t
Absent
Present
2B
3
Present
Absent
Present
2B
5
Present
Present
Present
Table 3: Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of Extracts and Fractions
Sample
s
MF
HF
EAF
BF
2H
1
2H
3
2H
4
2E
1
2E
5
2B
1
2B
2
2B
3
TPC
(%)
0.09
50
0.07
00
0.072
0
0.06
00
0.01
32
0.01
24
0.09
0
0.07
20
0.05
40
0.01
90
0.07
40
0.05
20
TFC (%)
0.01
75
0.01
95
0.013
5
0.01
65
0.01
95
0.01
10
0.00
60
0.00
30
0.00
15
Very low conc.
Table 4: DPPH free radical scavenging activity (IC50) for methanol extract and fractions.
Sample
IC50 (

g/ml) (95% confidence limit)
Methanol extract (ME)
31.25(29.50
-
33.86)
n
-
Hexane fraction (HF)
78.50(74.59
-
82.41)
Ethyl acetate fraction (EAF)
86.30(81.93
-
90.62)
Butanol fraction (BF)
142.40(135.28
-
149.52)
2H
1
32.50(30.87
-
34.13)
2H
3
70.40(66.88
-
73.92
)
2H
4
100.50(95.47
-
105.53)
2E
1
82.20(78.09
-
86.31)
2E
5
36.50(34.67
-
38.33)
The result of total flavonoid content (TFC) is
shown in Table 4. The crude methanolic
extract had a high TFC (0.0175%). In the
fractions: HF had a high TFC (0.019%)
followed by BF (0.016%) and EAF (0.013%). In
the separated constituents of HF, 2H1 had the
highest TFC (0.019%) followed by 2H3
(0.011%) and 2H4 (0.006%). In EAF, 2E1 was
higher (0.003%) than 2E5 (0.0015).
Constituents from BF had very low TFC.
The result of total phenolic content (TPC)
(table 3) indicated higest TPC in EAF (0.072%)

Nigerian Journal Of Pharmaceutical and Applied Science Research 1(1): 21-28, December 2011
Evaluation of Antioxidant Activity And Chemical Analysis Of The Leaf Of T. occidentalis.Nkereuwem et al. Page 26
followed by HF (0.07%) and BF (0.060%). In
the separated constituents of HF the TPC was
2H4 had the highest (0.090%) then 2H1
(0.013%) and 2H3 (0.012%). In EAF, 2E1 had a
higher TPC (0.072%) than 2E5 (0.0540%). In
BF, 2B2 had the highest TPC (0.074%) followed
by 2B3 (0.052%) and 2B1 (0.019%).
Several studies have demonstrated that
plants are natural antioxidant sources due
mainly to the presence of flavonoids, which
act by reducing free radicals (Wilson, 1988;
Miller 1996; Pietta, 2000; Geber et al., 2002;
Matteo and Esposito, 2003; Behera et al.,
2006). The n-hexane fraction (HF) indicated a
high DPPH free radical scavenging activity
which was contributed by component 2H1.
The TPC was highest in HF and in the
separated constituent 2H1. Comparing the BF,
though the TPC was high in the separated
constituents, it had little or no DPPH free
radical activity which inferres that TPC had no
effect on the DPPH free radical scavenging
activity. It can therefore be inferred from the
work that flavonoid contents of the leaves of
Telfairia occidentalis was responsible for its
DPPH free radical scavenging activity.
Researchers have justified the use of the leaf
of Telfairia occidentalis in Nigeria in the
treatment of certain diseases like diabetes,
cholesterolaemia, liver problems etc in which
the participation of reactive oxygen species
have been implicated. This could be as a
result of the antioxidant and free radical
scavenging ability (Kayode and Kayode, 2011)
which might have been attributable to the
high flavonoid content of Telfairia occidentalis
leaf.
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