African Journal of Food Science and Technology (ISSN: 2141-5455) Vol. 2(2) pp.

022-029, February, 2011

Available online http://www.interesjournals.org/AJFST
Copyright © 2011 International Research Journals

Full Length Research Paper

Total antioxidant activity, phenolic, flavonoid and

ascorbic acid contents of Nigerian vegetables.
Olajire A. A1* and Azeez L2
1*
Industrial and Environmental Chemistry Unit, Department of Pure and Applied Chemistry,
Ladoke Akintola University of Technology, Ogbomoso, Nigeria
2
Industrial and Environmental Chemistry Unit, Department of Chemical Sciences, Fountain University,
Osogbo, Nigeria
Accepted 17 January, 2011

Antioxidant activities, total phenolic, flavonoid and ascorbic acid contents of different vegetables
commonly consumed in Nigeria were determined. The antioxidant activities of vegetables ranged from
22.15% (Talinum triangulare) to 92.30% (Capsicum frutesceus). Solanum macrocarpon, with the lowest
IC50, is the most potent vegetable of the samples analyzed, that could scavenge most free radicals; while
Cucumis sativus, with the highest IC50, is the least potent. Total phenolic, flavonoid and ascorbic acid
– 1 – 1
contents of vegetables ranged from 22.1 to 370.68 mg quercetin g ; 10.23 to 215.39 mg quercetin g
– 1
and between 16.67 to 150.67 mg ascorbic acid g , respectively. A high and significant correlation
existed between antioxidant activity and total phenolic content of vegetables (r2 = 0.861, p < 0.05),
indicating that total phenolic content is the major contributor to the antioxidant activity of vegetables.
However, flavonoids, which belong to the phenolic compounds, were not significantly correlated with
2 2
antioxidant activity (r = 0.143, p < 0.05). Ascorbic acid fairly correlated (r = 0.546, p < 0.05) with
2
antioxidant and phenolic content (r = 0.591, p < 0.05).

Keywords: Antioxidant activity, total phenolics, total flavonoids, ascorbic acid, vegetables,

INTRODUCTION

Our body is exposed to a large number of foreign Kim et al, 2009). Natural antioxidants are classified
chemicals everyday (Santhakumari et al, 2003). The according to their mechanism of action as chain-breaking
most of which are man-made and our inability to properly antioxidants which scavenge free radicals or inhibit the
metabolize them negatively affects our health by the initiation step or interrupt the propagation step of
generation of free radicals. Free radicals are also oxidation of lipid and as preventive antioxidants which
generated during normal metabolism of aerobic cells slow the rate of oxidation by several actions but do not
(Carmen and Florin, 2009; Ghaseme et al, 2009; Li et al, convert free radicals (Ou et al, 2002; Thaipong et al,
2008; Hunag et al, 2005; Zaporozhets et al, 2004; 2006; Ebrahinzadeh et al, 2008; Semalty et al, 2009; El-
Odukoya et al, 2007). The oxygen consumption inherent Qudah, 2008; Hodzic et al, 2008; Othman et al, 2007;
in cells growth leads to the generation of series of oxygen Temraz and Hel-Tantawy, 2008; Ahmad and Beigh,
free radicals. Highly active free radicals and their 2008). However; there have been concerns about
uncontrolled production are responsible for numerous synthetic antioxidants such as butylated hydroxyanisole
pathological processes such as cell tumour (prostate and (BHA) and butylated hydroxytoluene (BHT) because of
colon cancers) and coronary heart diseases (Karadenz et their possible activity as promoters of carcinogenesis
al, 2005; Barros et al, 2007; Chanwitheesuk et al, 2005; (Rahman et al, 2008). There is growing interest toward
Marinova et al, 2005; Jagadish et al, 2009). natural antioxidants from herbal sources (Larson, 1998;
Antioxidants can significantly delay or prevent the Gazzani et al, 1988; Velioglu et al, 1988).
oxidation of easily oxidizable substances (Atrooz, 2009; Epidemiological and in vitro studies on medicinal plants
and vegetables strongly have supported the idea that
plant constituents with antioxidant activity are capable of
*Corresponding author Email: olajireaa@yahoo.com exerting protective effects against oxidative stress in
 Olajire and Azeez 023

biological systems (Cao et al, 1996; Block and Patterson, Chemicals

1992; Ness and Powles 1997).
Standards: BHA (butylated hydroxyanisol), α-tocopherol, L-
Vegetables and fruits contain high concentration of
ascorbic acid, Quercetin, Folin-ciocalteu’s phenol, 2,2-diphenyl-1-
numerous redox-active antioxidants such as polyphenols, picrylhydrazyl (DPPH) were all purchased from Sigma-Aldrich,
carotenoids, ascorbic acids, tocopherol and flavonoids Germany. Sodium carbonate, Aluminium chloride, 2, 6-
which fight against hazardous oxidative damage of plant dichlorophenolindophenol and methanol were purchased from BDH
cells (Odukoya, 2007; Karadenz et al, 2005; Ou et al, Poole, England. All the chemicals used were of analytical grade.
2002; El-Qudah, 2008). In animals, antioxidants Deionized distilled water (ddH2 O) was used throughout the
experiment. Jenway 6405 UV-Visible Spectrophotometer by Buch
production is much more limited and generation of free Scientific Inc.USA was used for analysis.
radicals during metabolism beyond the antioxidant
capacity has been implicated in the pathogenesis of most
diseases. Thus, the consumption of dietary antioxidants Extraction
from vegetables and fruits is beneficial in preventing
these diseases (Sumazian et al, 2010; Faujam et al, The samples were cut into pieces and lyophilized with Lyotrap
freeze drying machine (LTE Scientific Ltd UK) to remove the
2009; Magdalena et al, 2009). moisture content. Lyophilization was used to give the samples
Owing to the relationship between free radical uniform moisture removal and submit the products for analysis in
scavenging capacity of vegetables and fruits, many similar form. Resulting dried samples were powdered using
analytical methodologies have been published for the Moelinux blender.
determination of antioxidant ability. Phenolics in fruits Precisely, 1g of ground lyophilized sample was weighed and
extracted twice with a total volume of 100 mL of 70% aqueous
have been monitored by HPLC (Tung et al, 2007) or
methanol. The mixture was shaken on an orbital shaker (Stuart
colorimetrically using Folio-ciocalteu reagent (Faujam et SSLI, Barlword Scientific Ltd Britain) for 75 min at 300 rpm and then
al, 2009; Magdalena et al, 2009). Several assays have filtered through Whatman No. 4 filter paper. The combined
been used to evaluate total antioxidant capacity of foods methanolic extract was then evaporated at 40o C using rotary
and food products including spectrophotometric methods evaporator (R205D, Shensung Biological Science & Technology,
using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (Ghaseme et China) to dryness and then dissolved in absolute methanol for
analysis. The plant’s parts used in this study and their uses are
al, 2009; Li et al, 2008; Odukoya et al, 2007; Jagadish et given in Table 1.
al, 2009; Atrooz, 2009; Kim et al, 2009; Semalty et al,
2009; Ahmad and Beigh, 2008); 2, 2’-azinobis (3-
ethylbenzothiazoline-6-sulfonic acid (ABTS+) (Ou et al, DPPH Radical Assay
2002; Thaipong et al, 2006); ferric reducing power
(FRAP) (Atrooz, 2009; Kim et al, 2009; Ou et al, 2002; The hydrogen atom or electron donating abilities of the
corresponding extracts and some pure compounds were measured
Thaipong et al, 2006); oxygen radical absorbance from the bleaching of the purple-coloured methanolic solution of 2,
capacity (ORAC) (Atrooz, 2009; Kim et al, 2009; Ou et al, 2-diphenyl-1-picrylhydrazyl (DPPH) as shown in the equation
2002; Thaipong et al, 2006; Ebrahinzadeh et al, 2008; below.
Semalty et al, 2009; El-Qudah, 2008); the β-carotene
linoleate model (Barros et al, 2007); voltammetry and
amperometic methods (Magdalena et al, 2009). N N
.
Our objectives were to (1) determine the total N
AH
NH
+ A
.
O2N NO2 +
antioxidant activity, phenolic, flavonoid and ascorbic acid O2N NO2

contents of commonly consumed vegetables in Nigeria

and identify which of these vegetables has the highest NO2 NO2
free radical scavenging activity; (2) to determine level of Purple Bleached
correlation of these measured parameters.
One mL of various concentrations of the extracts in methanol was
added to 4 mL of 0.1 mmol L–1 methanolic solution of DPPH. A
MATERIALS AND METHODS blank probe was obtained by mixing 4 mL of 0.1 mmol L–1
methanolic solution of DPPH and 200 µL of deionized distilled water
(ddH2O). After 30 min. of incubation in the dark at room
Sampling procedures temperature, the absorbance was read at 517 nm against the
prepared blank. Inhibition of free radicals by DPPH in percent (I %)
We used in this study fifteen vegetables, Vernonia amygdalina, was calculated using this formula:
Brassica oleracea, Cucumis sativus, Murraya koenigii, Telfaria
occidentalis, Basella alba, Amaranth caudatus, Corchorus olitorius,  ( Ablank − Asample) 
Ocinum gratissimum, Capsicum frutesceus, Spinacia oleracea, I (%) =   x 100 (1)
 Ablank 
Talinum triangulare, Solanum macrocarpon, Allium cepa and
Lycopersicon esculentum bought from various markets in Osogbo where Ablank is the absorbanceof the control reaction( containingall
and identified by Dr Awodoyin from Botany Department (Fountain reagentsexcept the test compundand Asample is the absorbanceof the
University, Osogbo).
test compound.
 024 Afr. J. Food Sci.Technol.

Table 1: Plants parts used in this study and their uses

Botanical name Vernacular/Common Part used for this Uses

name study

Vernonia amygdalina Ewuro/ Bitter leaf Leaves Soup making

Brssica oleracea Cabbage Leaves Eaten raw as salad
Cucumis sativus Cucumber Leaves Eaten raw as salad
Murraya koenigii Curry leaf Leaves As condiment
Telfaria occidentalis Ugu/Pumpkin leaf Leaves and stem Soup making
Basella alba Amunututu/Green leaf Leaves and stem Soup making
Amaranth caudatus Tete/ Green Amaranth Leaves and stem Soup making
Corchorus olitorius Ewedu/ Jute mallow Leaves and stem Soup making
Ocinum gratissimum Efinrin/ Mint leaf Leaves As condiment and herb
Capsicum frutesceus Ata rodo/Red pepper Fruit Soup making
Spinacia oleracea Soko/Spinach Leaves and stem Leaves used in soup making
Talinum triangulare Gbure/Water leaf Leaves and stem Leaves used in soup making
Solanum macrocarpon Gbagba/Egg plant leaf Leaves and stem Leaves used in soup making
Allium cepa Alubosa pupa/Onion Bulb Eaten raw and soup making
Lycopersicon Tomato Fruit Eaten raw and soup making
esculentum

L-ascorbic acid, Quercetin, BHA and α-tocopherol were used as Statistical Analysis
standard controls. IC50 values denote the concentration of sample
which is required to scavenge 50% of DPPH free radicals. Experimental results were expressed as mean ± standard deviation.
All measurements were replicated three times. The data were
correlated using Pearson correlation coefficient at p < 0.05. The
Total phenolics, total flavonoids and ascorbic acids IC50 values were calculated using linear regression analysis.

Total phenolics were determined using Folin-Ciocalteu method of

Jagadish et al., (2009) with slight modification. The methanolic
RESULTS AND DISCUSSION
extracts (0.5 mL) were added to a 25 mL volumetric flask filled with
10 mL ddH2 O and 2.5 mL of 0.2 N Folin-Ciocalteu phenol reagent.
A reagent blank using ddH2O instead of sample was prepared. After Total antioxidant activity
5 min., 2 mL of 2% Na2CO3 solution were added with mixing. The
solution was diluted to the volume (25 mL) with ddH2O and then The antioxidant studies of different vegetables in Nigeria
allowed to stand for 90 min., and the absorbance was measured at have been done. This study focused on total antioxidant
780 nm versus the prepared blank. Quercetin was used as
standard for the calibration curve. Total phenolic contents were
activity in Nigerian local vegetables (Table 2). Total
calculated as mg quercetin g –1 dry weight of sample. antioxidant activity of the vegetables ranged from 22.15%
The AlCl3 method (Jagadish et al, 2009) was used for the for Talinum triangulare to 92.30% for Capsicum
determination of the total flavonoid content of the sample extracts. frutesceus. Solanum macrocarpon is the most potent
The methanolic extracts (1.5mL) was added to 10 mL volumetric vegetable of all that could scavenge most free radicals as
flask filled with 5 mL ddH2O and 0.3 mL 5% NaNO2 and mixed. A shown by the lowest IC50 value while Cucumis sativus
reagent blank using ddH2 O instead of sample was prepared. After 5
min., 1.5ml of 2% methanolic AlCl3 solution was added. Two mL of with the highest IC50, is the least potent (Table 2). The
1 mol dm–3 NaOH was added 5 min. later and then the volume was IC50 of Solanum macrocarpon (6.21 mg mL–1) compared
–1
made up to 10 mL with ddH2 O. The mixture was vigorously shaken to standards: L-ascorbic acid (IC50 = 2.60 mg mL ),
–1
on orbital shaker for 5 min. at 200 rpm and after 10 min. of Quercetin (IC50 = 1.31 mg mL ), α-tocopherol (IC50
incubation the absorbance was read at 367nm. Flavonoid contents =13.20 mg mL–1) and BHA (IC50 = 3.36 mg mL–1), shows
were calculated using a standard calibration curve, prepared from
that it can scavenge more free radicals than α-tocopherol.
Quercetin. The flavonoid contents were expressed as mg quercetin
g–1 of extract. The total antioxidant activity obtained in this study were
Ascorbic acid was determined using the method described by comparable with those obtained by Marinova et al.,
Barros et al (2007). The methanolic extract was diluted with 10 mL (2005) but higher than that of Odukoya et al (2007). This
of 0.5% oxalic acid and the mixture was shaken for 45 min. on could be due to methods used for the analysis and the
orbital shaker at 200 rpm at room temperature and filtered through medium of extraction as pointed out by Li et al., (2008).
Whatman No. 4 filter paper. Precisely 1 mL of the filtrate was mixed
with 9 mL of 0.1mol L–1 of 2, 6-dichlorophenolindophenol. A reagent
blank using ddH2 O instead of sample was prepared. The Relevant antioxidant activities
absorbance was read within 30 min at 515 nm against the prepared
blank. The ascorbic acid content was calculated using the Relevant antioxidant compounds such as total phenolic,
calibration curve, prepared from L-ascorbic acid. total flavonoid and total ascorbic acid contents were
 Olajire and Azeez 025

Table 2: Antioxidant activity, flavonoid, phenolics and ascorbic acid contents of the vegetables studied
d
Botanical % Yield Flavonoid Phenolic DPPH Ascorbic IC50
a a b c
name/Standards content content antioxidant acid

Vernonia amygdalina 33 216.33±2.89 238.4±5.24 66.73±0.3 98.81±1.54 12.37

Brssica oleracea 63 19.29±2.2 22.1±2.95 31.88±0.27 18.32±1.2 49.62
Cucumis sativus 80 62.43±5.1 101.33±13.05 28.19±0.13 16.67±0.14 71.14
Murraya koenigii 26 243.59±4.44 327.43±7.65 88.43±0.05 150.67±3.21 7.35
Telfaria occidentalis 36 117.25±2.11 251.85±12.83 77.02±0.08 150.34±1.41 11.67
Basella alba 42 26.53±3.57 81.11±6.55 30.49±0.05 47.74±0.4 34.45
Amaranth caudatus 20 69.67±1.15 186.67±67 32.71±2.62 60.81±2.14 15.81
Corchorus olitorius 30 81.38±0.07 200.03±16.07 63.34±0.11 93.93±0.44 11.84
Ocinum gratissimum 25 105.2±5.66 252.2±4.1 72.11±0.04 69.34±2.41 8.67
Capsicum frutesceus 54 24.78±4.2 370.38±6.42 92.30±0.14 135.61±3.11 14.04
Spinacia oleracea 29 139.63±2.71 204.70±5.22 57.3±0.49 38.21±0.04 12.63
Talinum triangulare 36 81.48±6.41 49.26±4.76 22.15±0.17 29.27±1.2 40.51
Solanum macrocarpon 20 215.39±15.5 256.67±13.34 80.59±0.9 111.2±0.01 6.21
Allium cepa 72 10.23±1.93 225.93±8.48 76.92±0.3 35.52±0.02 23.41
Lycopersicon esculentum 48 12.62±0.14 246.88±5.93 70.39±0.08 37.67±0.51 17.05
α-tocopherol 19.07 13.20
L-Ascorbic acid 96.26 2.60
Quercetin 95.24 1.31
BHA 89.28 3.36

Each value is expressed as mean ± standard deviation (n=3); amg quercetin/g of extract; b% of methanolic radical
scavenging activity; cmg ascorbic acid/g of extract; dmg /mL of effective concentration at which 50% of DPPH radicals are
scavenged;

120
Antioxidant Activity

100

80

60

40

20

0
0 100 200 300 400
Total Phenolics

Figure 1. Correlation between antioxidant activity and total phenolics, (r2 = 0.861).

successfully analyzed from local Nigerian vegetables. works have been done on the effects of phenolic
The different antioxidant activities of the vegetables can compounds on total antioxidants (Li et al, 2008;
be ascribed to their total phenolic concentrations. When Magdalena et al, 2009; Ghaseme et al, 2009; Jagadish et
comparing the data in Table 2, Capsicum frutesceus had al, 2009; Atrooz, 2009; Kim et al, 2009; Semalty et al,
the highest phenolic content (370.68 mg quercetin g–1) 2009 and Ebrahinzadeh et al, 2008), and correlations
followed by Murraya koenigii, Solanum macrocarpon, between phenolic compounds and total antioxidants (Bin
Ocinum gratissimum, Telfaria occidentalis, Lycopersicon Li et al, 2008; Barros et al, 2007; Chanwitheesuk et al,
esculentum, Vernonia amygdalina, Allium cepa, Spinacia 2005). This same trend was also obtained in our study.
2
olerace, Corchorus olitoriusa, Amaranth caudatus, There was a good linear correlation (r = 0.861, p < 0.05)
Cucumis sativus, Basella alba, Talinum triangulare and between the total phenolic content and the scavenging of
Brassica oleracea, this later had the least phenolic DPPH radical in each extract (Figure 1). These results
content (22.1 mg quercetin g–1). Several comprehensive indicated that the radical scavenging capacity of each
026 Afr. J. Food Sci.Technol.

400
350

Total Phenolics 300

250
200
150
100
50
0
0 50 100 150 200 250 300
Flavonoids content
Figure 2. Correlation between total phenol and flavonoid content, (r2 = 0.1477).

100
90
Antioxidant Activity

80
70
60
50
40
30
20
10
0
0 50 100 150 200 250 300
Flavonoids content
Figure 3. Correlation between antioxidant activity and flavonoid content, (r2 = 0.1373).

extract might be mostly related to their concentration of are flavonoids which possess biological activities such as
phenolic hydroxyl group. The antiradical activity of anti-inflammatory, anti-carcinogenic and anti-
phenolic compounds depends on their molecular atherosclerotic acitivities. There was no correlation
structure, on the availability of phenolic hydrogens and on between total flavonoids and radical scavenging activity,
the possibility for stabilization of the resulting phenoxyl (r2 = 0.143) as shown in Figure 3. This lack of relationship
radicals formed by hydrogen donation (Catherine et al, is in agreement with other reports (Heinonen et al., 1998;
1996; Ramarathnam et al, 1997). Flavonoids which Anagnostopoulou et al., 2006; Nickavar et al., 2007);
belong to the phenolic compounds, poorly correlated (r2 = which indicates that flavonoids did not contribute to
0.145, p < 0.05) with phenolic content of the vegetables antioxidant activity of vegetables.
analyzed (Figure 2). Ascorbic acid contents of vegetables analysed are given
Flavonoid contents of the vegetables are shown in Table in Table 2. Murraya koenigii had the highest value of
2. Solanum macrocarpon had the highest value of 215.39 150.67 mg ascorbic acid g–1 and Cucumis sativus had the
–1
mg quercetin g and Allium cepa had the lowest value of lowest value of 16.67 mg ascorbic acid g–1. The values
–1
10.23 mg quercetin g . Among the phenolic compounds are in agreement with values obtained by Sumazian et
 Olajire and Azeez 027

100
90
Antioxidant Activity 80
70
60
50
40
30
20
10
0
0 50 100 150 200
Ascorbic acid content

Figure 4. Correlation between antioxidant activity and ascorbic acid, (r2 = 0.546).

400
350
Total Phenolics

300
250
200
150
100
50
0
0 50 100 150 200
Ascorbic acid content
Figure 5. Correlation between total phenolics and ascorbic acid, (r2 = 0.581).

al., (2010) and Ahmad and Hussain Beigh (2008), but contribution to the total antioxidant activities of
higher than what were obtained by Okiei et al., (2009). vegetables.
There is no correlation between total ascorbic acid and
total antioxidant activities (r2 = 0.546, p < 0.05; Figure 4)
and phenolic content (r2 = 0.591, p < 0.05; Figure 5). CONCLUSION
According to Bahorun et al. (2004), it is normal when total
ascorbic acid do not correlate with the total antioxidant The antioxidant capacities, total phenolic, flavonoid and
activities since total ascorbic acid made little or no ascorbic acid contents of fifteen vegetables commonly
consumed in Nigeria were evaluated. Some of the
028 Afr. J. Food Sci.Technol.

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