International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: https://www.tandfonline.com/loi/ljfp20

Evaluation of Phenolic Content, Antioxidant

Activity, and Nutritional Composition of Cordia
evolutior (Clarke) Gamble

Karuppusamy Arunachalam & Thangaraj Parimelazhagan

To cite this article: Karuppusamy Arunachalam & Thangaraj Parimelazhagan (2014)

Evaluation of Phenolic Content, Antioxidant Activity, and Nutritional Composition of Cordia
evolutior (Clarke) Gamble, International Journal of Food Properties, 17:1, 226-238, DOI:
10.1080/10942912.2011.619294

To link to this article: https://doi.org/10.1080/10942912.2011.619294

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Apr 2013.
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International Journal of Food Properties, 17:226–238, 2014
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942912.2011.619294

EVALUATION OF PHENOLIC CONTENT, ANTIOXIDANT

ACTIVITY, AND NUTRITIONAL COMPOSITION
OF CORDIA EVOLUTIOR (CLARKE) GAMBLE

Karuppusamy Arunachalam and Thangaraj Parimelazhagan

Department of Botany, Bioprospecting Laboratory, Bharathiar University,
Coimbatore, Tamil Nadu, India

The objectives of this study were to determine phenolic content, antioxidant activity of
methanolic extracts from different parts of Cordia evolutior (leaf, bark, and fruit), and nutri-
tion composition. The leaf extract showed the highest total phenolic content (25.40 ± 0.34 mg
GAE/g extract) and total flavonoid content (69.70 ± 3.37mg RE/g extract) accompanied
with the best antioxidant activity through all antioxidant assays. The fruit proximate com-
positions of crude protein, ash, carbohydrate, fiber, and fat were analyzed. Macro-nutrient
contents were found to be higher in the fruit when compared to micronutrients. The anal-
ysis also showed the presence of almost all of the essential and non-essential amino acids.
Linolenic acid content was higher than stearic acid among the fatty acids in the fruit.

Keywords: Cordia evolutior, Antioxidant activity, Phenolic content, Anti-radical power,

Nutrition composition.

INTRODUCTION
Living organisms require an ample amount of oxygen for their metabolism and
energy production. However, free radicals are produced during the energy production
process[1] as the unavoidable consequence of respiration in aerobic organisms. Free radicals
are unstable species that react rapidly and destructively with biomolecules, such as protein,
lipid, DNA, and RNA, in the body. Uncontrolled generation of free radicals is associated
with lipid and protein peroxidation, resulting in cell structural damage, tissue injury, or
gene mutation, and ultimately leads to the development of various health disorders, such as
Alzheimer’s disease, cancer, atherosclerosis, diabetes mellitus, hypertension, and ageing.[2]
These protective effects are considered to be related to the various antioxidants contained
in fruits and vegetables.[3] On the other hand, lipid auto-oxidation, which is initiated by
free radicals, also results in food quality deterioration.[4] Antioxidants play an important
role in defending the body against free radicals damage. Antioxidants refer to a group of
compounds that are able to delay or inhibit the oxidation of lipids or other biomolecules
and, thus, prevent or repair the damage of the body cells that is caused by oxygen.[5,6]
They work by preventing the formation of new free radical species, converting existing
free radicals into less harmful molecules and preventing radical-chained reactions.[7] For

Received 6 July 2011; accepted 21 August 2011.

Address correspondence to Thangaraj Parimelazhagan, Department of Botany, Bioprospecting Laboratory,
Bharathiar University, Coimbatore, Tamil Nadu 641 046, India. E-mail: drparimel@gmail.com

226
 ANTIOXIDANT ACTIVITY OF C. EVOLUTIOR (CLARKE) GAMBLE 227

instance, phenolic compounds, such as quercetin and ellagic acid, are good antioxidants
that are able to protect body cells from injuries caused by reactive oxygen and nitrogen
species.[8]
Cordia evolutior belongs to the family Cordiaceae. The fruit is edible, which is tradi-
tionally used by the tribes in the Anamalai hills, Coimbatore district, Western Ghats, Tamil
Nadu.[9] The plant bark and leaves are bitter, acrid, thermogeneic, anthelmintic, digestive,
carminative, diuretic, anaphrodisiac, depurative, vulnerary, and stomachic. They are useful
in cephalalgia, gastropathy, anorexia, flatulence, helminthiasis, strangury, indolent ulcers,
leprosy, skin diseases, hysteria, and epilepsy. The main objectives of the study were to
determine the phenolic content and antioxidant activities of methanolic extracts from dif-
ferent parts of C. evolutior. Therefore, the edible part of the fruit was evaluated by its
nutrition composition.

MATERIALS AND METHODS

The chemicals used in this study were of analytical reagent grade and include:
methanol, chloroform, Tween 20, and trichloroacetic acid; ammonium thiocyanate
(99.99%) and linoleic acid; gallic acid, β-carotene (Type I synthetic, 95%) and (+) α-
tocopherol, iron (II) chloride-80 mesh 98%, ethylenediaminetetra acetic acid (EDTA),
1,1-diphenyl-2-picrylhydrazyl (DPPH), and Lascorbic acid 99%; Folin-Ciocalteu phenol
reagent, hydrochloric acid (37%), aluminium trichloride (AlCl3 ), and dimethyl sulphoxide
(DMSO) (Merck, Darmstadt, Germany); thiobarbituric acid and buffer solution (phos-
phates) pH 7.00 ± 0.02, 20◦ C.

Sample Preparation
Cordia evolutior (Clarke) Gamble plant was collected from Anamalai Hills,
Coimbatore District, Tamil Nadu. The collected plant material was identified and their
authenticity was confirmed by comparing the voucher specimen at the herbarium of
Botanical Survey of India, Southern Circle Coimbatore, Tamilnadu. C. evolutior plants
were cleaned and separated into three different parts, namely, leaf, bark, and fruit. All parts
were individually freeze-dried (Model Number: LMF–25, Shanghai Lyomac Mechanical
Technology Co., Ltd., Shanghai, China (Mainland).

Methanol Extraction
Different parts of C. evolutior were, respectively, pulverized into fine powder using
a willy mill and mixed with methanol at the ratio of 1:10 (w/v). Then these mixtures
were manually swirled for 15 min and left in a sonicator (Power sonic 505, HwaShin
Technology Co., Seoul, Korea) for 60 min. After that, these mixtures were individually
filtered through Whatman filter paper No. 1, and the entire extraction process was repeated
twice on the residue obtained from the filtration process. The filtrates were individually
pooled and methanol was removed from the filtrates under reduced pressure (Rotavapor
R210, Buchi, Postfach, Flawil, Switzerland). Finally, C. evolutior extracts were cooled
in a dessicator for 30 min before the yield of each extract was calculated. The methano-
lic extracts from different parts of C. evolutior were kept at −80◦ C prior to further
analyses.
228 ARUNACHALAM AND PARIMELAZHAGAN

Total Phenolic Content (TPC)

TPC of C. evolutior extracts were determined using Folin-Ciocalteu assay.[10] Briefly,
100 mg of C. evolutior methanolic extracts were individually dissolved in 10 mL of
methanol. Then, 0.1 mL of these solutions was mixed with 2.5 mL of 10-fold diluted Folin-
Ciocalteau reagent, and 2.0 mL of 7.5% sodium carbonate (Na2 CO3 ). After incubation at
40◦ C for 30 min, the absorbance of the reaction mixtures was measured at 760 nm by using
a spectrophotometer (Pharmaspec UV-1700, Shimadzu, Kyoto, Japan). The equation of the
gallic acid calibration curve was Y = 8.2313, X − 0.078 (where X was concentration of
garlic acid equivalents (GAE) expressed as milligrams GAE per gram of dried extract and
Y was measured absorbance), and the correlation coefficient was R2 = 0.9971.

Total Flavonoid Content (TFC)

TFC was determined by the aluminium calorimetric method[11] using Rutin as a stan-
dard. Briefly, the test samples were individually dissolved in DMSO. Then, the sample
solution (150 μL) was mixed with 150 μl of 2% AlCl3 . After 10 min of incubation at
ambient temperature, the absorbance of the supernatant was measured at 435 nm by using
a spectrophotometer (Pharmaspec UV-1700, Shimadzu, Kyoto, Japan). Rutin was chosen
as a standard (the concentration range: 0.005 to 0.1 mg/mL) and the total flavonoid content
was expressed as milligram RE per g of dry extracts. The absorbance at 415 nm = 0.0097
rutin (mg/mL) + 0.0127, R2 = 0.9995.

DPPH Scavenging Activity

DPPH scavenging activity of C. evolutior extracts was determined according to the
method described by Singh et al.,[12] with slight modifications. In brief, 0.1 mL C. evolutior
extract at various concentrations was added, respectively, to 0.49 mL of methanol and
0.39 mL of DPPH methanolic solution (4 mg/100 mL). Then, the mixtures were vortexed
vigorously and allowed to stand in the dark for 60 min. Finally, the absorbance of these
mixtures was measured by using a spectrophotometer (Pharmaspec UV-1700, Shimadzu,
Kyoto, Japan) at 515 nm. The sample concentration providing 50% of radical scaveng-
ing activity (IC50 ) was obtained through interpolation of linear regression analysis. The
lower IC50 indicates higher radical scavenging activity and vice versa. Ascorbic acid and
α-tocopherol were used as standards.

Hydroxyl Radical Scavenging Activity

Hydroxyl radical scavenging activity of C. evolutior methanolic extracts was deter-
mined by using an electron spin resonance spectrometer (JEOL-JES-FA100, Tokyo, Japan).
Hydroxyl radical was generated through a Fenton reaction, with 5,5-dimethyl N-oxide
pyroline (DMPO) as the trapping agent. The reaction mixture contained 40 μL of DMPO
(400 mM), 37.5 μL of FeSO4 (0.4 mM), 112.5 μL of EDTA (0.1 mM), 60 μL of the sample
or blank, and 150 μL of H2 O2 (2.0 mM). The electron spin resonance (ESR) measurement
was conducted 1 min after preparing each reaction mixture at room temperature. The con-
dition of ESR measurement was as follows: sweeping field, 336.45 ± 5 mT; microwave
power, 8 mw; mod width, 0.1 mT; sweep time, 2 min; time constant, 0.1 s; and ampli-
tude, 160. Dimethyl sulphoxide (DMSO) was used as the standard in this experiment and
 ANTIOXIDANT ACTIVITY OF C. EVOLUTIOR (CLARKE) GAMBLE 229

hydroxyl radical scavenging activity of C. evolutior extracts were expressed in gram DMSO
equivalent per gram of sample (g DMSOE/g sample).

β-Carotene Bleaching Activity

β-Carotene bleaching activity of C. evolutior extracts was determined according to
the method of Wettasinghe and Shahidi.[13] In brief, 3 mL of β-carotene solution (5 mg
β-carotene/50 mL chloroform) were added to 40 mg of linoleic acid and 400 mg of Tween
20. Then, the mixture was mixed well and dried under a stream of nitrogen. Immediately,
100 mL of distilled water were added to the dried mixture to form a β-carotene-linoleic acid
emulsion. In order to determine the β-carotene bleaching activity of the extract, 1.5 mL
of emulsion were respectively added to 20 μL of C. evolutior extracts. After that, these
mixtures were incubated in a water bath at 50◦ C for 60 min. Finally, the absorbances
of the reaction mixtures were read at 470 nm by using a UV–visible spectrophotometer
(Pharmaspec UV-1700, Shimadzu, Kyoto, Japan). Alpha-tocopherol was used as a standard
in this experiment. Antioxidant activity (AA%) of C. evolutior extracts were calculated by
using the following equation:

AA% = 100(DRc − DRs )DRc,

whereby AA = antioxidant activity; DRC = degradation rate of the control: [(a/b)/60];

DRS = degradation rate of the sample: [(a/b)/60]; a = initial absorbance of the sample;
b = absorbance after 60 min of incubation.

Total Antioxidant Activity Assay

Ferric thiocyanate (FTC) test. A FTC test on C. evolutior extracts was conducted
according to the method described by Kikuzaki and Nakatani.[14] In this study, 4 mg of
C. evolutior extracts were individually dissolved in 4 mL of methanol. Then, the extract
solutions were respectively mixed with linoleic acid (4.1 mL), 0.05 M phosphate buffer
pH 7.0 (8 mL), and distilled water (3.9 mL). These mixtures were then kept in screw-
cap containers at 40◦ C in the dark. In order to determine the FTC values, 0.1 mL of
these mixtures was respectively added into 9.7 mL of 75% ethanol and 0.1 mL of 30%
ammonium thiocyanate. Precisely 3 min after the addition of 0.1 mL of ferrous chloride
solution (in 3.5% HCl) to the reaction mixture, the absorbance of the samples was read at
500 nm by using a spectrophotometer (Pharmaspec UV-1700, Shimadzu, Kyoto, Japan).
This procedure was repeated every 24 h until the control sample reached its maximum
absorbance value. Ascorbic acid and α-tocopherol were used as standard antioxidants in
this test.
Thiobarbituric acid (TBA) test. A TBA test[15] was conducted instantly after
the control sample from the FTC test reached its maximum absorbance value. In brief,
1.0 mL of 20% aqueous trichloroacetic acid and 2.0 mL of 0.67% aqueous thiobarbituric
acid were added to 2 mL of sample solutions acquired from the FTC test. The mixtures were
then placed in a boiling water bath for 10 min. After cooling under running tap water, the
mixtures were centrifuged at 3000 rpm for 30 min. Finally, the absorbance of supernatants
at 532 nm was measured by using a spectrophotometer (Pharmaspec UV-1700, Shimadzu,
Kyoto, Japan).
230 ARUNACHALAM AND PARIMELAZHAGAN

Proximate Composition and Minerals

The proximate composition of C. evolutior fruit was determined by AOAC.[16]
Moisture, crude fat, protein, dietary fiber, and ash were determined according to the
Association of Official Analytical Chemists standard method. The protein content of the
samples was calculated using the factor N × 6.25. Carbohydrate was estimated by sub-
tracting the sum of moisture, protein, fat, fiber, and ash content from 100 g. Calcium,
potassium, and sodium were estimated using a flame photometer (EEI 100, GmbH,
Hamburg, Germany). Iron and magnesium were determined by use of an absorption spec-
trophotometer (Baird Alpha 2AA, Baird Corporation, Bedford, UK). Gross energy was
determined using bomb calorimetry. The minerals of C. evolutior fruits were measured by
wet digestion method. Ashes of C. evolutior were added to HCl:H2 O (1:3) and dried in a
water bath. Sample solutions were subsequently analyzed by an inductively coupled plasma
atomic emission spectroscope (ICP-AES, Thermo Jarrell Ash, Franklin, MA, USA).

Amino Acid Analysis

Amino acids were determined as per the method of Ishida et al.[17] First, 100 mg
of finely homogenized sample was taken in a borosil test tube. Then, 10 mL 6 N HCl
was added into the test tube. The tubes were sealed after filling with nitrogen gas and the
contents of the tube were digested by keeping at 120◦ C for 24 h in an oven. The test tubes
were cooled and then the contents were filtered using Whatmann No. 1 filter paper. The
test tubes were rinsed with distilled water and then filtered. The filtrate was evaporated in
a vaccum flash evaporator. Deionized water was added into the tubes and the evaporation
was allowed to continue until the contents are acid free. The amino acids were dissolved in
0.05 N HCl. Samples were filtered through a membrane filter of 0.45 μM, and 20 μL of this
was injected into an amino acid analyzer (Shimadzu LC-10AS HPLC, McKinley Scientific,
Sparta, NJ, USA) equipped with a cation exchange column packed with a strongly acidic
cation exchange resin, i.e., styrene di-vinyl benzene co-polymer with sulphonic group. The
mobile phase of the system consists of two buffers: buffer A and buffer B. A gradient
system was followed for the effective separation of amino acids. The oven temperature was
maintained at 60◦ C. The total run was programmed for 60 min. The amino acid analysis
was done with a non-switching flow method and fluorescence detection after post-column
derivatization with ortho-phthaladehyde or o-phthalaldehyde. In the case of proline and
hydroxyl proline, imino group is converted to amino group with hypochlorite. Amino acid
standard was also run to calculate the concentration of amino acids in the sample. The
amount of each amino acid is expressed as g/100 g protein. The essential amino acids
score was calculated with reference to the FAO/WHO reference amino acid pattern:

(FAO/WHO) Amino acid score = (Test amino acid/Reference amino acid) × 100.

Analysis of Fatty Acids

Fatty acid compositions of C. evolutior fruit was determined by the AOAC.[18] For
lipid extraction, 50 g of fruits were homogenized in 150 mL of chloroform:methanol (1:2,
v/v) mixture and reacted at 100◦ C for 30 min filling N2 , and then filtrated through Whatman
No. 1 filter paper. Extracted lipid from each sample was saponified in 0.5 N NaOH
methanolic solution and esterified in BF3 -methanol. Fatty acids were determined using a
 ANTIOXIDANT ACTIVITY OF C. EVOLUTIOR (CLARKE) GAMBLE 231

gas chromatography (Varian star 3600; Varian Inc., Walnutcreek, CA, USA) equipped with
a flame ionization detector and Omagawax 205 fused-silica bond capillary column (30 m ×
0.32 mm, i.d. × 0.25 μm film thickness). The temperatures of the oven, injection port, and
detector were 140, 250, and 260◦ C, respectively, and nitrogen flow rate was 50 mL/min.

Statistical Analyses
Data was reported as mean ± standard deviation from triplicate determination.
Analysis of variance (ANOVA) accompanied with the least significant difference (LSD) and
Tukey tests (SPSS for Windows, Version 15, SPSS Inc., Chicago, IL, USA) were conducted
to identify the significant difference between samples (p < 0.05).

RESULTS AND DISCUSSION

Extraction Yield, Total Phenolic Content, and Total Flavonoid Content
Table 1 presents the yield, total phenolic content, and total flavonoid content of
C. evolutior methanolic extracts. The yield of the extracts varied from 13.66 to 23.78%.
Among all tested extracts, the highest and the lowest yields were respectively obtained
from the bark and fruit of C. evolutior (p < 0.05). The yield of methanolic extracts from
different parts of C. evolutior is presented in the following order: bark > leaf > fruit (p <
0.05). The content of extractable phenolic compounds in the C. evolutior extracts was deter-
mined through a linear gallic acid standard curve (y = 8.2313x + 0.078; r2 = 0.9971).
The total phenolic content of C. evolutior extracts varied from 2.85 to 25.40 mg GAE/g
extract. The highest content of total phenolic compounds was detected in the C. evolutior
leaf extract (25.40 mg GAE/g extract), whereas the lowest content was measured in the
fruit extract (2.85 mg GAE/g extract) (p < 0.05). Total phenolic content of C. evolutior
extracts is arranged in the following descending order: leaf > bark > fruit (p < 0.05). This
finding is in agreement with some previous studies, which reported that total phenolic con-
tent of leaf extract was higher than other parts of the plant for Beta vulgaris (blood turnip),
Petroselinum crispum (parsley), and Coriandrum sativum (Chinese parsley).[19,20] Higher
amounts of phenolics in leaflets in comparison to that of roots may be attributed to the
presence or absence of light that affects the phenolic contents of organs. Generally, there is
a rise in total phenolics in plants grown in the sunny situations relative to the shady ones,
but it can be seen at the intra-individual level by comparing plant parts exposed to differ-
ent amounts of light.[21,22] This suggests that leaf might be the part that is rich in phenolic
compounds in many plants. Several studies have revealed that the phenolic content in the
plants are associated with their antioxidant activities,[23] probably due to their redox prop-
erties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen
quenchers.[24]

Table 1 Extraction yield, total phenolic content, and total flavonoid content of C. evolutior extracts (n = 3).

Part of Extraction yield Total phenolic content Total flavonoid content

C. evolutior (% w/w) (mg GAE/g extract) (μg RE/g extract)

Leaf 16.29 ± 2.15a 25.40 ± 0.34a 69.70 ± 3.37a

Bark 23.78 ± 2.09b 10.25 ± 0.40b 9.68 ± 0.74b
Fruit 13.66 ± 0.52c 2.85 ± 0.21c 2.52 ± 0.74c

Values with different superscript letters within the same column are statistically different (p < 0.05).
232 ARUNACHALAM AND PARIMELAZHAGAN

Flavonoids are the most common and widely distributed group of plant phenolic com-
pounds, which usually are very effective antioxidants.[25] In this study, the total flavonoid
content of methanolic extracts from different parts of C. evolutior was evaluated by alu-
minium colorimetric assay. The total flavonoid content of C. evolutior extracts was varied
considerably from 2.52 to 69.70 μg RE/g extract. The data presented in Table 1 indicates
that the highest flavonoid content of 69.70 μg RE/g extract was observed in the extract of
leaf and the lowest content was observed in the extract of the fruit (2.52 μg RE/g extract)
(p < 0.05). Total flavonoid content of C. evolutior extracts is arranged in the following
sequence: leaf > bark > fruit (p < 0.05). The total flavonoid content of C. evolutior extracts
is well in correspondence to the total phenolic content (r = 0.9836). This indicates that the
flavonoids are the major phenolic compounds present in C. evolutior plant.

Antiradical Activity
Table 2 shows DPPH and hydroxyl radicals scavenging activity of C. evolutior
methanolic extracts. In general, IC50 values of all tested samples through a DPPH scav-
enging activity test were ranging from 1.40 to 26.41 mg/mL and the DPPH scavenging
activity is arranged in the following descending order: ascorbic acid (IC50 = 0.02 mg/mL)
> α-tocopherol (IC50 = 0.06 mg/mL) > leaf > bark > fruit (p < 0.05). In this study,
DPPH scavenging activity of C. evolutior extracts shows a similar trend with the result of
total phenolic content (r = 0.9228) and total flavonoid content (r = 0.8478), indicating that
DPPH radical scavenging activity of C. evolutior extracts is highly related to the amount of
phenolic compounds, especially flavonoids that are present in the extracts.
Meanwhile, hydroxyl radical scavenging activities of C. evolutior extracts were rang-
ing from 38.37 to 145.90 g DMSOE/g extract (Table 2). Among all tested extracts, leaf
extract exhibited the strongest hydroxyl radical scavenging activity (145.90 g DMSOE/g
extract), while fruit extract showed the least antiradical activity (38.37 g DMSOE/g extract)
(p < 0.05). Hydroxyl scavenging activity of C. evolutior extracts is presented in the follow-
ing descending order: leaf > bark > fruit (p < 0.05). A correlation test shows that hydroxyl
radical scavenging activity of C. evolutior extracts is well correlated with its total phenolic
(r = 0.9171) and total flavonoid (r = 0.9512) contents, supporting the former statement on
the contribution of phenolic compounds (particularly flavonoids) in antiradical activity of
C. evolutior extracts.

β-Carotene Bleaching (BCB) Activity

Antioxidant activity of C. evolutior extracts as measured by bleaching of β-
carotene was determined through the interpolation of a linear α-tocopherol standard curve
(y = 16.784x + 3.1533; r2 = 0.9708) and expressed in mg alpha-tocopherol equivalent
(Teq)/g extract. In the BCB assay, linoleic acid produces hydroperoxides during incubation

Table 2 DPPH and hydroxyl radicals scavenging activity of C. evolutior methanolic extracts (n = 3).

C. evolutior DPPH radical scavenging Hydroxyl radical scavenging

extracts activity (IC50 [mg/mL]) activity (g DMSOE/g extract)

Leaf 1.40 ± 0.01a 145.90 ± 22.04a

Bark 2.12 ± 0.22b 54.57 ± 0.35b
Fruit 26.41 ± 2.83c 38.37 ± 2.42c

Values with different superscript letters within the same column are statistically different (p < 0.05).
 ANTIOXIDANT ACTIVITY OF C. EVOLUTIOR (CLARKE) GAMBLE 233

Figure 1 Antioxidant activity of Cordia evolutior extracts through BCB assay.

at 50◦ C. The presence of hydroperoxides cause rapid discoloration of β-carotene.[26]

However, hydroperoxides formed in this system can be neutralized by the antioxidants
from the extracts.
A variation in antioxidant activity of C. evolutior extracts ranging from 0.51 to
4.43 mg Teq/g extract was observed. Figure 1 shows that the C. evolutior leaf extract
again exhibited the highest antioxidant activity (4.43 mg Teq/g extract) through BCB assay,
while fruit extract showed the least antioxidant activity (0.51 mg Teq/g extract) towards the
bleaching of β-carotene. Antioxidant activity of C. evolutior extracts through BCB assay is
arranged in the following order: leaf > bark > fruit (p < 0.05). This result is in agreement
with the previous study by Elzaawely et al.,[27] who reported that the leaf extract of Alpinia
zerumbet exhibited higher inhibitory activity towards β-carotene oxidation than other parts
of the plant. On the other hand, bark extract of C. evolutior also showed good antioxidant
activity in reducing the oxidation of β-carotene in this study (p < 0.05). As discussed pre-
viously, high antioxidant activity of C. evolutior leaf and bark extracts might be due to its
high phenolic content, likewise flavonoids, in particular.

Total Antioxidant Activity: Ferric Reducing Antioxidant Power (FRAP)

and TBA Tests
Figure 2 shows the hydroperoxides inhibitory activity of C. evolutior extracts through
FTC test. As shown in Fig. 2, almost all C. evolutior extracts (except for fruit) significantly
retarded the formation of hydroperoxides in the linoleic acid emulsion system throughout
the incubation period as compared to the control sample (p < 0.05). From the third day
onward, the absorbance value of the control was higher (p < 0.05) than other samples
(except for fruit) and reached its maximum absorbance on the ninth day of incubation. The
overall inhibitory activity of C. evolutior extracts against hydroperoxides formation can be
established in the following descending order: ascorbic acid > leaf > bark > fruit > α-
tocopherol > control. The fruit extract showed greater absorbance value (p < 0.05) than
234 ARUNACHALAM AND PARIMELAZHAGAN

Figure 2 Hydroperoxides inhibitory activity of C. evolutior extracts through ferric thiocyanate test.

the control sample at the end point of the incubation period (ninth day), indicating that
C. evolutior fruit extract might possess pro-oxidative properties that enhance the autox-
idation of linoleic acid in the emulsion system and increase the generation of reactive
substances.
The C. evolutior leaf extract exhibited the highest hydroperoxides inhibitory activ-
ity that was superior to ascorbic acid, α-tocopherol, as well as other tested C. evolutior
extracts (p < 0.05). This result suggested that the leaf extract might contain primary
antioxidant compounds, which are able to react aggressively with free radicals, particularly
hydroxyl radicals, thereby terminating the radical-chained reaction and retard the formation
of hydroperoxides.[28] After the control sample reached its maximum absorbance value in
the FTC test, a TBA test was subsequently conducted on the samples. This test measures the
thiobarbituric acid reactive substances content at a later stage of lipid oxidation, involving
the quantification of the secondary products formed from lipid oxidation. In this test, low

Figure 3 Thiobarbituric acid reactive substances inhibitory activity of C. evolutior extracts measured by
thiobarbituric acid test.
 ANTIOXIDANT ACTIVITY OF C. EVOLUTIOR (CLARKE) GAMBLE 235

absorbance value indicates higher thiobarbituric acid reactive substances inhibitory activ-
ity. Figure 3 shows that the fruit extract exhibited the weakest thiobarbituric acid reactive
substances inhibitory activity, while the leaf extract possessed the strongest activity (p <
0.05). The strength of thiobarbituric acid reactive substances inhibitory activity of leaf and
bark extracts was similar to ascorbic acid (p > 0.05) but superior to α-tocopherol (p <
0.05).
Thiobarbituric acid reactive substances inhibitory activity of all tested samples is pre-
sented in the following descending order: leaf > bark > ascorbic acid > α-tocopherol >
fruit > control (p < 0.05). The trend of thiobarbituric acid reactive substances inhibitory
activity of C. evolutior extracts is rather similar to the trend of the FTC test in this study.
This suggests that reduction of thiobarbituric acid reactive substances content in leaf and
bark samples might due to the lower hydroperoxides accumulation in the respective sam-
ples, previously. Besides, secondary antioxidant compounds that might present in these
extracts may also contribute to the inhibition of hydroperoxides decomposition in these
samples.

Proximate Composition
Proximate compositions of C. evolutior fruit are shown in Table 3. The crude protein
was found to be high (23.28%) compared to fat (3.86%) ash content (10.47%), carbohydrate
(11.32%) and fiber (5.56%). Plant food that provides more than 12% of its calorific value
from protein are considered to be a good source of protein. Furthermore, adults, pregnant
and lactating mothers required 34–56 g, 13–19 g, and 71 g of protein daily, respectively.[29]
In accordance with these, the fruit selected for the present study, C. evolutior fruit, might
serve as an efficient source of protein-rich food.

Minerals
The mineral contents of C. evolutior fruit are shown in Table 3. It has been found that
the fruit was rich in Ca, K, and Mg. The values for the Ca, K, and Mg were 1,774, 1,322,
and 361 mg/100 g, respectively. Calcium has been reported to be effective in the building

Table 3 Proximate composition, minerals, and fatty acids of the C. evolutior fruit.

Proximate composition (%) Minerals (mg/100 g) Fatty acids composition (%)

Moisture 6.95 ± 0.03 Ca 1774.82 ± 95.86 Caprylic acid 0.07 ± 0.011

Crude lipid 3.86 ± 0.04 K 1322.82 ± 35.81 Capric acid 0.90 ± 0.01
Crude protein 23.28 ± 0.37 Mg 361.51 ± 1.82 Myristic acid 0.76 ± 0.01
Crude ash 10.47 ± 0.35 Fe 16.81 ± 0.55 Palmitic acid 11.78 ± 0.07
Carbohydrate 11.32 ± 0.23 Na 14.62 ± 6.10 Palmitoleic acid 2.07 ± 0.29
Fiber 5.56 ± 0.05 Zn 4.12 ± 1.12 Stearic acid 4.34 ± 0.01
Cu 0.75 ± 0.03 Oleic acid 08.46 ± 0.34
Linoleic acid 30.39 ± 0.93
Linolenic acid 33.14 ± 1.04
Arachidic acid 0.21 ± 0.01
Behenic acid 1.46 ± 0.16
Others 6.41 ± 0.09

Results were mean ± SD (n = 3).

236 ARUNACHALAM AND PARIMELAZHAGAN

of skeletal structures and muscle functioning, while magnesium is important in the ionic
balance and enzyme co-factors.[30] It has been shown that C. evolutior fruit also had some
minerals, such as Ca, K, and Mg, which were beneficial to the human body so that they
were thought to be used as food materials useful in health.

Aminoacid Analysis
The aminoacid composition of the C. evolutior fruit sample along with the essen-
tial amino acid requirements pattern suggested by FAO/WHO is shown in Table 4. The
amino acid profile of the sample revealed that the proteins of the fruit contained ade-
quate levels of amino acid as compared with FAO/WHO. The amount of amino acids,
histidine (6.8 g/100 g protein), leucine (9.7 g/100 g protein), iso leucine (7.8 g/100 g
protein), phenylalanine + tyrocine (9.6 g/100 g protein), threonine (7.2 g/100 g pro-
tein), tryptophan (1.8 g/100 g protein), and valine (8.2 g/100 g protein), were found to
be higher, whereas the amino acid lysine (1.2 g/100 g protein), methonine + cysteine
(g/100 g protein), recorded a lower level but appreciable content than the reference pat-
tern of FAO/WHO. Amino acids in the form of proteins make up the greatest portion
of our body weight. Apart from this, cysteine was found to have the ability to quench
DPPH radicals.[31] Arginine plays vital roles in nutrition and metabolism. It is classified
as an essential amino acid in infants and young children and as a conditionally essential
amino acid in adults at times of trauma or disease. The chemical score and amino acid
index are widely used for screening potential protein foods. Amino acid deficiency can be
met by consuming large amounts of legumes, or by taking a mixture of legumes, or by
employing the complimentarily that exists between high sulphur amino acid cereals and
legumes.

Fatty Acids
Fatty acids compositions of C. evolutior fruit are shown in Table 3. A total of 11 fatty
acids ranging from C8 to C22 were identified. In C. evolutior fruit, the higher content of
unsaturated fatty acids, such as linoleic acid (30%) and linolenic acid (33%), rather than that
of saturated fatty acids, such as palmitic acid (11%) and steric acid (4%), were recorded.

Table 4 Amino acid profile of C. evolutior fruit.

Amino acids (g/100 g protein) C. evolutior fruit Reference patterna

Histidine 6.8 1.9

Lysine 3.2 5.8
Leucine 9.7 6.6
Isoleucine 7.8 2.8
Methionine + Cysteine 2.2 2.5
Phenylalanine + Tyrosine 9.6 6.3
Threonine 7.2 3.4
Tryptophan 1.8 1.1
Valine 8.2 3.5

Amino acid reference pattern of protein for diets of children 2–5 years old. Values are % of protein. Each amino
acid in the reference pattern was presumed to score a value equaling 100. Values for each cultivar are expressed
relatively to the reference pattern.
a Source: FAO/WHO (1985).
 ANTIOXIDANT ACTIVITY OF C. EVOLUTIOR (CLARKE) GAMBLE 237

The fatty acid composition of a diet alters the composition of membrane phospholipids,
which in turn changes membrane functions.[32] The plant contains unsaturated fatty acids
of physiologic and nutritional significance, such as oleic and linoleic acids, which could be
contributory to the nutritional needs of the consumers.

CONCLUSION
This study clearly demonstrated that high antioxidant activity was observed in the
leaf and bark extracts of Cordia evolutior as compared to other tested extracts. Many
plant-based foods are good sources of phytochemical antioxidants and exhibit cardio-health
promotion properties. Phytochemical antioxidants from fruits and leaves can significantly
inhibit the development of cardiovascular disease. The C. evolutior fruit proximate compo-
sitions of crude protein, carbohydrate, and fiber contents were presented at a better level.
Macro-nutrient contents were found to be higher in the fruit when compared to micronu-
trients. The fruit nutrition composition findings lead to use as a dietary supplement or as a
functional ingredient in nutraceutical and pharmaceutical products.

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