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Polyphenols in Barringtonia racemosa and their protection against oxidation

of LDL, serum and haemoglobin

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 Food Chemistry 146 (2014) 85–93

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Polyphenols in Barringtonia racemosa and their protection against

oxidation of LDL, serum and haemoglobin q
Kin Weng Kong a, Sarni Mat-Junit a,b, Amin Ismail c, Norhaniza Aminudin b,d, Azlina Abdul-Aziz a,b,⇑
a
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
University of Malaya Centre for Proteomics Research, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
c
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
d
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

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

Article history: The polyphenolic profiles and antioxidant activities of the water extracts of Barringtonia racemosa shoots
Received 9 May 2013 (leaves and stems) were explored. Two methods, freeze drying and air drying, for preparation of the
Received in revised form 21 August 2013 shoots, were also compared. Freeze drying was better as air drying caused 5–41% reduction of polyphe-
Accepted 3 September 2013
nols. Three phenolic acids and three flavonoids were identified, using UHPLC. The descending order of
Available online 11 September 2013
polyphenols in the leaves and stems was gallic acid > ellagic acid > quercetin > protocatechuic
acid > rutin > kaempferol. In vitro antioxidant analyses were performed using biological samples. In the
Keywords:
LDL oxidation assay, B. racemosa leaf extract (IC50 = 73.0 lg/ml) was better than stem extract (IC50 = 226 -
Natural products
Extraction
lg/ml) at inhibiting the formation of TBARS and lipid hydroperoxides. Similar trends were observed for
Barringtonia racemosa serum and haemoglobin oxidation. B. racemosa leaf extract was better than its stem extract in delaying
Polyphenols the time required to oxidise haemoglobin to methaemoglobin. The high polyphenolic content of B. race-
Lipid peroxidation mosa shoots could have contributed towards their antioxidative effects.
Haemoglobin oxidation Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
UHPLC

1. Introduction
Barringtonia racemosa (L.) Spreng is a widely-grown plant
belonging to the Lecythidaceae family. Its leaves are used to reduce
high blood pressure and as a depurative, whereas the pounded
leaves, roots and barks are used to reduce itchiness and chicken
pox (Ong & Nordiana, 1999; Orwa, Mutua, Kindt, Jamnadass, & Si-
mons, 2009). In Malaysia, the shoots of B. racemosa are usually con-
sumed as a salad. A recent study has reported B. racemosa to have
high antioxidant activities (Kong, Mat-Junit, Aminudin, Ismail, &
Abdul-Aziz, 2012) and its high phenolic content, in addition to
the presence of diterpines, triterpenoids, steroids and saponins, is
postulated to contribute towards the antioxidant activities (Dera-
niyagala, Ratnasooriya, & Goonasekara, 2003).
Amongst the phenolic compounds that have been reported in
the leaves of B. racemosa include gallic acid, ferulic acid, naringin,
rutin, luteolin, kaempferol, protocatechuic acid, ellagic acid and
q assays as they can provide details on the ability of antioxidants
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike License, which permits non-
commercial use, distribution, and reproduction in any medium, provided the
original author and source are credited.
⇑ Corresponding author at: Department of Molecular Medicine, Faculty of
Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: +603
79674915; fax: +603 79674957.
E-mail address: azlina_aziz@um.edu.my (A. Abdul-Aziz).
quercetin (Hussin et al., 2009; Kong et al., 2012). However, details
on the presence of free and bound polyphenols in the shoots of B.
racemosa are not available. Ultra-high performance liquid chroma-
tography (UHPLC) is an improved chromatographic system with
high sensitivity, resolution and rapid separation, which can be used
for the analysis of polyphenolic compounds in plants. UHPLC has
significantly shortened the elution times for polyphenolic com-
pounds, providing a rapid analytical method. In this study, a UHPLC
system was utilised to analyse polyphenols in the shoots of B.
racemosa.
Polyphenols are antioxidants that can reduce the susceptibility
of biological molecules to oxidants. Various antioxidant assays are
used for estimation of the antioxidant capacities of plants. In addi-
tion to the established chemical antioxidant assays, in vitro biolog-
ical antioxidant assays are also useful, especially in the initial
evaluation of the antioxidant properties of plant materials, before
proceeding with more invasive in vivo models. Biological antioxi-
dant assays can be a more useful approach compared to chemical
to prevent oxidation of biological materials such as lipids, DNA
and proteins. In this study, isolated serum, LDL and haemoglobin
were used as the biological models to measure the ability of the
water extracts of the leaves and stems of B. racemosa to prevent
their oxidation. We hope this current study can shed more light

0308-8146/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.09.012
86 K.W. Kong et al. / Food Chemistry 146 (2014) 85–93
on the ability of B. racemosa extracts to act as anti-oxidative agents
based on biological materials.
This study is a continuation of our previous work, focusing on
the aqueous extracts of B. racemosa which, when compared to
the ethanol, ethyl acetate and hexane extracts, possess the highest
phenolic content and antioxidant activities, (Kong et al., 2012). We
quantified the polyphenols, both in the free and bound forms, to
better understand the nature of their structure in the plant, which
would shed some light on their bioavailability and subsequent bio-
activity. In addition, we tested the effects of two drying methods
(freeze drying vs. air drying) on the polyphenolic content of the
plant. An optimal drying method is important to prevent losses
of polyphenols during processing.
2. Materials and methods
2.1. Analytical reagents and chemicals
HPLC-grade and other analytical grade chemicals and reagents
were obtained from general suppliers. Diethyldithiocarbamic acid
(DETC) sodium salt and all polyphenolic standards were of HPLC
grade (purity > 95%) and were obtained from Sigma–Aldrich Chem-
ical Co. (St. Louis, MO).
2.2. Sampling and sample preparation
The shoots of B. racemosa were collected from the state of Ke-
dah, in northern Peninsular Malaysia. The leaves and stems of
the shoots were separated and dried using two methods: freeze
drying and air drying. For freeze drying, samples were allowed to
freeze at 80 °C for 3 days before lyophilisation in a freeze dryer.
Air drying was performed under a running fan at room tempera-
ture for 7 days. The dried stems and leaves were subsequently
ground into powder and sieved via a 1-mm mesh. All prepared
samples were stored at 20 °C until further analyses.
2.3. Extraction of polyphenols
Two grams of either freeze-dried or air-dried samples were ex-
tracted with 40 ml of water. Each extraction was performed three
times, in an incubator shaker (Innova 4300; New Brunswick Scien-
tific, New Jersey, USA) at 200 rpm and 30 °C for 24 h. The extract
was later centrifuged (Jouan CR3i multifunction centrifuge; Ther-
mo Scientific, Waltham, NJ) at 1389g for 5 min at 4 °C. The super-
natant was filtered through Whatman No. 4. paper and the filtrate
was lyophilised. The dried powder was weighed and pooled to-
gether and stored at 20 °C until further analyses.
2.4. Preparation of soluble free and bound polyphenols
Free polyphenols (X) were estimated by dissolving the dried
powder in water containing 20 mM DETC sodium salt prior to
UHPLC analysis. Free and bound polyphenols (Y) were estimated
by subjecting the dried powder to acid hydrolysis to break the link-
ages between phenolic compounds and other organic compounds,
such as glucose, tartaric acid and terpenes. Hydrolysis conditions
were modified from the method described by Aziz, Edwards, Lean,
and Crozier (1998). The crude extract (5 mg) was mixed with 2 ml
of 1.2 N HCl containing 20 mM DETC sodium salt in a hydrolysis
vial. The hydrolysis was performed in a heating module (Reacti-
Therm Heating/Stirring Module No. 18971; Pierce, Rockford, IL)
at 90 °C for 2 h. The hydrolysate was then diluted to 1 mg ex-
tract/ml with water containing 20 mM DETC sodium salt prior to
chromatographic analysis.
2.5. Ultra-high performance liquid chromatography (UHPLC) analysis
All samples were filtered through 0.20-lm PTFE membrane fil-
ters prior to chromatographic analysis. Separation of polyphenols
was performed on a UHPLC system (Agilent Technologies 1290
Infinity, Waldbronn, Germany) (Kong et al., 2012). The stationary
phase consisted of an Agilent Zorbax Eclipse Plus C18
(50  2.1 mm, 1.8 lm) column and 5 ll of the sample were in-
jected into the system. A binary mobile phase made up of 0.1% tri-
fluoroacetic acid (TFA) (A) and acetonitrile (B), with the flow rate
adjusted to 0.6 ml/min, was employed. Separation of polyphenols
was achieved using a linear gradient system: 5–15% B in 6 min;
15–25% B in 3 min; 25–60% B in 3 min; 60–80% B in 0.6 min; 80–
100% B in 0.8 min. Absorption spectra were monitored in the re-
gion of 200–500 nm throughout the analysis. The polyphenolic
compounds were detected at 280 and 325 nm by the diode array
detector. All polyphenolic standards were prepared in 50% metha-
nol containing 20 mM DETC sodium salt.
2.6. Identification and quantification of polyphenolic compounds
Phenolic acids and flavonoids were identified by comparing the
retention time (tR) and absorption spectra of the samples with
those of authentic standards. External standard was used to plot
the calibration curve (0–80 lg/ml). Results were expressed as lg/
g dry weight (dw) of sample. The percentage of free and bound
polyphenols was calculated.
The limit of detection (LOD) and limit of quantification (LOQ)
were determined as described by the guidelines from the Interna-
tional Conference on Harmonization (ICH) (1996). Three calibra-
tion curves were plotted using three sets of polyphenolic
standards, injected at concentrations ranging from 2.5 to 20 lg/
ml. The equations of the calibration curves were then derived.
Mean of the slopes (S) and standard deviation of the intercepts
(r) were calculated. LOD and LOQ were subsequently estimated
according to the following formulae:
LOD ¼ 3:3  r=S
LOQ ¼ 10  r=S
2.7. Copper-mediated serum oxidation assay
The in vitro antioxidant assays were conducted only on the
freeze dried samples, as it was shown to be a better drying method
for polyphenols compared to the air drying method. The serum oxi-
dation assay was modified from the method of Bem et al. (2008),
using serum from healthy volunteers. A 0.4-ml solution of serum
(final concentration of 25%) was treated with 70 ll of the aqueous
extracts of B. racemosa leaf, stem or gallic acid (0–1000 lg/ml) be-
fore incubation at 37 °C for 30 min. Oxidation was initiated with
CuSO4 (100 lM) followed by a 24-h incubation at 37 °C (Maxi-
shake model SBD50 BIO; Heto, Allerod, Denmark). Then, 33.3 ll
EDTA (27 mM) were added. Thiobarbituric acid reactive substances
(TBARS) were measured as described in the following section. Re-
sults were expressed as nmol TBARS/ml serum.
2.8. Measurement of TBARS
TBARS were measured following the method of Buege and Aust
(1978) with some modifications. Two hundred microlitres of the
reaction mixture from the serum oxidation assay were treated
with 0.8 ml of TBA: TCA: HCl (1:1:1) reagent (0.37% TBA, 15%
TCA, 0.25 N HCl). The mixture was heated at 90 °C for 20 min and
cooled at room temperature for 10 min before centrifugation at
3000g for 10 min. Absorbance of the supernatant was measured
 K.W. Kong et al. / Food Chemistry 146 (2014) 85–93 87

Fig. 1. UHPLC chromatograms of freeze dried B. racemosa shoot extracts. (a) non-hydrolysed leaf extract, (b) hydrolysed leaf extract, (c) non-hydrolysed stem extract, (d)
hydrolysed stem extract, and (e) standard mix detected at 280 nm. Gallic acid (1), protocatechuic acid (2), rutin (3), ellagic acid (4), quercetin (5) and kaempferol (6).

at 532 nm (Varian Cary 50 Conc, Melbourne, Australia). TBARS 2.9. Copper-mediated LDL oxidation assay
were calculated using a malondialdehyde–thiobarbituric acid
(MDA–TBA) complex molar extinction coefficient of Isolation of LDL was conducted through a heparin–citrate buffer
1.56  105 M1 cm1. precipitation method previously developed by Wieland and Seidel
88 K.W. Kong et al. / Food Chemistry 146 (2014) 85–93

Table 1
Profiles of the chromatographic parameters for phenolic acids and flavonoids in the shoots of B. racemosa.

Peak Polyphenolic tR ± SD kmax kmax reported Regression equation for LOD and S r LOD (ng/ LOQ (ng/
no. compound (min) (nm) (nm) LOQ ml) ml)
1 Gallic acid 1.65 ± 0.01 272 271a y = 10.35x  8.917, r2 = 0.968 9.63 0.393 135 408
y = 9.294x  9.359, r2 = 0.975
y = 9.246x  9.701, r2 = 0.970
2 Protocatechuic acid 2.73 ± 0.02 260; 296 269; 294b y = 11.85x  0.169, r2 = 0.999 11.7 0.510 144 438
y = 11.56x  1.043, r2 = 0.999
y = 11.55x  1.061, r2 = 0.999
3 Rutin 9.34 ± 0.07 258; 356 255; 354a y = 5.883x  2.883, r2 = 0.997 5.75 0.994 571 1730
y = 5.705x  2.161, r2 = 0.986
y = 5.647  0.918, r2 = 0.998
4 Ellagic acid 10.43 ± 0.03 254; 364 254; 365a y = 12.35x  1.627, r2 = 1.000 36.0 13.0 229 695
y = 12.59x  2.271, r2 = 1.000
y = 12.89  3.360, r2 = 1.000
5 Quercetin 11.40 ± 0.01 256; 372 255; 367a y = 9.573x  27.86, r2 = 0.986 9.18 2.60 933 2830
y = 8.953x  23.16, r2 = 0.986
y = 9.011  23.60, r2 = 0.986
6 Kaempferol 12.00 ± 0.04 266; 366 265; 364a y = 3.269x  2.492, r2 = 0.993 2.76 1.52 1820 5500
y = 2.489x + 0.121, r2 = 1.000
y = 2.507x + 0.144, r2 = 0.999

tR, Retention time; LOD, limit of detection at 280 nm; LOQ, limit of quantification at 280 nm; S, mean of the slopes; r, standard deviation of the intercepts; r2, coefficient of
determination.
a
Abad – García et al. (2009).
b
Fischer, Carle, and Kammerer (2011)

(1983). Five millilitres of serum were vortexed with 50 ml of hep-
arin–citrate buffer (0.064 M trisodium citrate, 50000 IU/l heparin,
pH 5.05) and incubated for 10 min at room temperature. The ser-
um was centrifuged at 1000g for 10 min to precipitate the insolu-
ble lipoproteins. The sediment was resuspended in 1 ml of 10 mM
phosphate-buffered saline (PBS, pH 7.4). Protein content of the LDL
suspension was measured using bovine serum albumin as standard
(Lowry, Rosebrough, Farr, & Randall, 1951).
Copper-mediated LDL oxidation assay was initiated by incubat-
ing a solution of LDL (8 mg/ml of protein) with 70 ll of B. racemosa
leaf extract, stem extract or gallic acid (0–1000 lg/ml) for 30 min
at 37 °C. Then, 33.3 ll of 50 lM CuSO4 were added. The mixture
was incubated at 37 °C for 24 h. After that, 33.3 ll EDTA (27 mM)
were added. The concentration of TBARS was measured as previ-
ously described and results were expressed as nmol TBARS/g LDL
protein. A positive control group with copper-induced oxidation
but without sample treatment was prepared. A negative control
group without induction of oxidation and sample treatment was
also analysed in parallel.
The same experiment as above, but using a lower concentration
of LDL suspension (4 mg/ml protein), was repeated for the deter-
mination of lipid hydroperoxides (LHP), another by-product of li-
pid peroxidation.
2.10. Measurement of lipid hydroperoxides in LDL oxidation
LHP was measured according to the method of Nourooz-Zadeh,
Tajaddini-Sarmadi, Ling, and Wolff (1996) with slight modifica-
tions. An aliquot of the treated LDL (0.1 ml) was added with
0.9 ml of Fox reagent containing 250 lM ammonium sulphate,
250 lM iron (II) sulphate, 100 lM xylenol orange, 25 mM H2SO4
and 4 mM butylated hydroxytoluene in 90% (v/v) methanol. The
mixture was then incubated for 30 min at 37 °C and centrifuged
at 3000g for 10 min. Absorbance of the mixture was measured
at 560 nm. The difference between means of initial absorbance
and the absorbance after incubation was calculated. The LHP con-
tent was determined using a molar absorption coefficient of
4.3  104 M1 cm1. Results were expressed as lmol LHP/g LDL
protein.
2.11. Nitrite-induced haemoglobin oxidation assay
The haemoglobin oxidation assay was modified from the meth-
od of Marouf, Zalzala, Al-Khalifa, Aziz, and Hussain (2011). Erythro-
cytes from healthy volunteers were washed 3 times with 10 mM
PBS and lysed in a hypotonic solution (5 mM PBS) for 1 h at 4 °C.
Then, the solution was centrifuged at 1000g for 10 min and the
supernatant was collected. The haemolysate (0.75 ml) was mixed
with 0.5 ml of B. racemosa leaf extract, stem extract or gallic acid
(0–1000 lg/ml). Subsequently, 50 ll of freshly prepared sodium
nitrite (0.65 mM) were added to induce oxidation of haemoglobin
(Hb) to methaemoglobin (MetHb). The formation of MetHb was
monitored at 631 nm every 5 min up to 30 min. The amount of
MetHb was determined using a molar extinction coefficient of
3.7 mM1 cm1.
2.12. Statistical analysis
Data were expressed as means ± standard deviation of triplicate
analyses. Data were statistically analysed using the SPSS statistical
software, version 15 (SPSS Inc, Chicago, IL). Independent t-test was
used for comparison of means between groups. One-way analysis
of variance (ANOVA) and Tukey’s Honestly Significant Different
test was used to compare means among groups. The level of signif-
icance was set at p < 0.05. Graph Pad Prism Version 5.1 software
(GraphPad Software Inc., San Diego, CA) was used to predict the
time needed to convert 50% of the Hb to MetHb, using a non-linear
regression model.
3. Results and discussion
3.1. Identification of chromatographic peaks for polyphenols
Fig. 1 shows the UHPLC chromatograms of the leaf and stem ex-
tracts of the shoots of B. racemosa, prepared through the freeze dry-
ing method. Six polyphenolic compounds were identified,
consisting of three phenolic acids and three flavonoids. The pheno-
lic acids were gallic acid, protocatechuic acid and ellagic acid, while
the flavonoids were rutin, quercetin and kaempferol. Hussin et al.
 K.W. Kong et al. / Food Chemistry 146 (2014) 85–93 89

a b

c d

e f

Fig. 2. Basic chemical structure and UV–Vis spectra of polyphenolic compounds.

(2009) detected six polyphenols in the leaves of B. racemosa, con-
sisting of gallic acid, rutin, kaempferol, ferulic acid, naringin and
luteolin. We did not detect the presence of ferulic acid, naringin
and luteolin and this could be due to variation in the extraction
method (Ignat, Volf, & Popa, 2011).
Due to variation in the absorption spectra of the polyphenolic
compounds and to ensure maximum detection, two wavelengths,
280 and 325 nm, were utilised to observe the separated polyphe-
nols. The kmax of all the polyphenols in this study corresponded
to the kmax reported from the literature (Table 1). Identification
of the polyphenolic compounds was done by comparing the reten-
tion times (tR) of the sample peaks (Fig. 1(a–d)) with those of
authentic standards (Fig. 1(e)). For further validation, the UV–Vis
spectrum and the kmax of the eluted peaks generated from the
diode array detector were compared with the spectrum of the
authentic standards (Fig. 2).
Fig. 2 shows the UV–Vis spectra of the polyphenolic compounds
detected in the plant sample. Gallic acid, protocatechuic acid and
ellagic acid had UV–vis spectra analogous to hydroxybenzoic acids,
due to the presence of benzoyl groups that formed a chromophore
with absorption spectra ranging from 255 to 280 nm (Abad-García,
Berrueta, Garmón-Lobato, Gallo, & Vicente, 2009). The flavonols
quercetin and kaempferol gave an intense band I at 347–370 nm
and band II at 250–267 nm, due to the substitution of hydroxyl
group at carbon 3 of the C ring (Abad-García et al., 2009). Rutin,
which is a glycoside of quercetin, gave the same intense bands I
or II as its aglycone (quercetin) (Abad-García et al., 2009). The
LOD and LOQ of each polyphenolic compounds were calculated
and tabulated in Table 1.
3.2. Quantification of free and bound polyphenols
Quantification of the polyphenols in the leaves and stems of B.
racemosa is presented in Table 2. Overall, the leaves have higher
amounts of polyphenolic compounds than the stems. In addition,
90 K.W. Kong et al. / Food Chemistry 146 (2014) 85–93

Table 2
Quantification of polyphenols in the shoots of B. racemosa prepared by freeze drying or air drying.

Freeze dried Air dried

Free (lg/g dw) Free + Bound (lg/g dw) Free (%) Bound (%) Free (lg/g dw) Free + Bound (lg/g dw) Free (%) Bound (%)
Leaf
Gallic acid 453 ± 7.49a 2200 ± 6.69b 20.56 79.44 727 ± 11.8c 2270 ± 1.28b 32.06 67.94
Protocatechuic acid ND 573 ± 2.73a 100 ND 545 ± 4.09b – 100
Rutin 547 ± 16.45 ND 100 ND ND – –
Ellagic acid 250 ± 6.48a 1450 ± 21.7b 17.25 82.75 443 ± 24.5c 1140 ± 6.00d 38.73 61.27
Quercetin ND 931 ± 3.53a 100 ND 647 ± 18.7b – 100
Kaempferol ND 443 ± 7.77a 100 ND 260 ± 10.7b – 100
Stem
Gallic acid 278 ± 12.9a 1720 ± 3.77b 16.6 83.84 480 ± 1.54c 1430 ± 25.7d 33.55 66.45
Protocatechuic acid ND 210 ± 1.43a 100 94.6 ± 1.32b 229 ± 6.89c 41.35 58.65
Rutin ND ND ND ND – –
Ellagic acid 180 ± 12.1a 782 ± 21.0b 23.05 76.95 106 ± 4.96c 545 ± 9.41d 19.54 80.46
Quercetin ND ND ND ND – –
Kaempferol ND ND ND ND – –

Results are means ± standard deviations of triplicate analyses (lg/g dry weight, dw). Values with different lower case letters indicated significant difference (p < 0.05) within
the same compound. ND, not detected.

the amounts of bound phenolics were approximately 20% more
than the free phenolics.
The polyphenols in the leaves of B. racemosa in descending or-
der were gallic acid > ellagic acid > quercetin > protocatechuic
acid > rutin > kaempferol. In contrast, only three polyphenols were
detected in the stems, in the order of gallic acid > ellagic acid > pro-
tocatechuic acid. A previous study reported the leaves of B. racemo-
sa, extracted with acidified methanol, to contain 172 lg/g dw of
gallic acid, 59.1 lg/g dw of rutin and 5.75 lg/g dw of kaempferol
which were lower than our values (Hussin et al., 2009). In addition
to the extraction method, the differences in polyphenolic content
may have also been due to variation in pedoclimatic and agro-
nomic conditions (Manach, Scalbert, Morand, Rémésy, & Jiménez,
2004).
In plants, phenolic acids are usually coupled with the cell wall
complexes or form ester and glycosidic linkages with organic com-
pounds, such as glucose, quinic, maleic and tartaric acid and terp-
enes (Chew, Khoo, Amin, Azrina, & Lau, 2011). Flavonoids can occur
in plants as both aglycones and glycosides, with the latter in higher
amounts (Sakakibara, Honda, Nakagawa, Ashida, & Kanazawa,
2003). Acid hydrolysis functions to degrade the ester and glyco-
sidic bonds of polyphenolic compounds, providing a rapid estima-
tion of the amounts of free and bound polyphenols in plant
samples.
Tannin is an important chemical constituent in B. racemosa
(Bandaranayake, 2002). The hydrolysable tannins are complexes
of hydroxybenzoic acids, which can be classified into gallotannins
and ellagitannins, derived from the glucose esters of gallic acid and
ellagic acid, respectively (Ignat et al., 2011). Our results showed
that there was more bound gallic acid and ellagic acid, compared
to the aglycone forms, indicating that most of these acids are in
the form of hydrolysable tannins. Quercetin and kaempferol only
existed in the plant in their conjugated forms and not as aglycone
(Table 2). Rutin is a conjugated form of quercetin; hence part of the
conjugated quercetin may include rutin as well.
In this study, two methods for preparation of the shoots of B.
racemosa were tested, either freeze drying or air drying. The former
method gave a higher content of polyphenolic compounds com-
pared to the latter (Table 2). Generally, there was an approximately
5–41% reduction (p < 0.05) in the free and bound polyphenols in the
air dried samples, compared to the freeze dried samples. In the air
drying method, the shoots were dried at room temperature to min-
imise degradation of polyphenols. However, lower temperature was
associated with prolonged drying time, whereas freeze drying pro-
vided a more rapid drying process (López et al., 2010). Other studies
have reported contrasting results. Korus (2011) reported lower
amounts of phenolic compounds in air-dried kale leaves at 55 °C
compared to freeze-dried samples. In contrast, Katsube, Tsurunaga,
Sugiyama, Furuno, and Yamasaki (2009) showed no significant dif-
ference in the phenolic content of air-dried mulberry leaves (tem-
perature below 60 °C) with freeze-dried leaves. Nonetheless, a
previous kinetics study reported that extended drying time led to
a noticeable deterioration of phenolic compounds (López et al.,
2010). Our study showed that freeze drying is a better method for
preparation of polyphenols from the shoots of B. racemosa.
Levels of gallic acid, protocatechuic acid, ellagic acid, quercetin
and kaempferol in B. racemosa leaves are comparable if not higher
than several teas, raspberry, carob and vegetables such as lettuce,
leek, onion, endive, celery and curry kale (Hertog, Hollman, &
Venema, 1992; Manach et al., 2004; Sakakibara et al., 2003). Nev-
ertheless, the efficiency of aglycones derivation and their deterio-
ration are essential issues that need to be taken into
consideration to obtain more accurate quantitative data.
3.3. Serum oxidation
Higher levels of TBARS indicated higher levels of lipid oxidation.
At lower concentrations (25–50 lg/ml), the aqueous extract of B.
racemosa leaf showed a concentration-dependent decrease in
TBARS formation, although at higher concentrations, inhibition
was not significantly different (Fig. 3(a)). On the other hand, the
aqueous extract of B. racemosa stem was less effective in prevent-
ing lipid peroxidation and did not show significant changes in
TBARS formation over the concentration range used in this study.
The higher ability of the leaf extract to prevent serum oxidation
compared to the stems is likely due to its higher polyphenolic con-
tent (Table 2). In addition to the four polyphenols that were found
in both the leaves and the stems, the leaves also contained querce-
tin and kaempferol, which were not detected in the stems. Poly-
phenols, particularly the free forms, have the structural features
required for radical scavenging and metal chelation, hence are
good antioxidants and inhibitors of lipid peroxidation (Fraga, Gall-
eano, Verstraeten, & Oteiza, 2010).
When the values of TBARS were expressed as percentage of
inhibition, at the highest concentration (1000 lg/ml), B. racemosa
leaf extract inhibited 46% of TBARS as opposed to 19% for its stem
extract. Gallic acid however did not show much difference in inhib-
iting lipid peroxidation at the range of concentrations used in this
study. Although the TBARS values seemed to show an increasing
trend at lower concentrations of gallic acid (25–100 lg/ml) and
 K.W. Kong et al. / Food Chemistry 146 (2014) 85–93 91

a 6

5
j

ij
ghi
4 hi
fghi efghi efghi defgh
efghi
TBARS (nmol/ml serum)
cdefgh
bcdefg bcdef bcdefg
3
abcde abcde abcd
ab abc
2 a a

Treatment (µg/ml)

b 250

h
200
gh
gh g

150 g

f
ef
100 de
de
de bcde bcde
cde abcd abcde
abcd
50 abc
ab ab
a

Treatment (µg/ml)

c 1.2

gh gh h gh
fgh efg
1.0 e ef

d d
d cd bcd
LHP (µmol/g LDL protein)

0.8 abc bcd abc

ab abc ab
a

0.6

0.4

0.2

0.0

Treatment (µg/ml)

Fig. 3. The effects of BLE, BSE and GA on TBARS produced from (a) serum oxidation, (b) LDL oxidation. The effects of BLE, BSE and GA on (c) lipid hydroperoxides produced
from LDL oxidation. Values with different lower case letters are significantly different (p < 0.05). Control (Cu2+), negative control group without sample and Cu2+-induced
oxidation; Control (+Cu2+), positive control group without sample but with Cu2+-induced oxidation; BLE, B. racemosa leaf extract; BSE, B. racemosa stem extract; GA, gallic acid.

lower TBARS at higher gallic acid concentrations (250–1000 lg/ We had initially reported the presence of gallic acid, proto-
ml), this was not statistically significant. catechuic acid, ellagic acid, quercetin and kaempferol in the shoots
92 K.W. Kong et al. / Food Chemistry 146 (2014) 85–93

Table 3
The effect of the freeze dried shoots of B. racemosa on nitrite – induced methaemoglobin formation.

Treatment (lg/ml) MetHb at 30 min (lM) Inhibition of MetHb at 30 min (%) Time taken to form 50% MetHb (min)
C(NO
2) 1.38 ± 1.55a
99.5 ± 0.53g
–
C(+NO
2) 292 ± 18.0g 0.00 ± 6.15a 2.79
BLE 25 273 ± 0.79fg 6.69 ± 0.27ab 2.50
BLE 50 248 ± 27.7ef 15.3 ± 9.46bc 12.00
BLE 100 99.4 ± 0.15cd 66.0 ± 0.05de 34.50
BLE 250 72.5 ± 16.5bc 75.2 ± 5.63ef 29.45
BLE 500 59.0 ± 4.43b 79.5 ± 1.51f 107.10
BLE 1000 7467 ± 12.2bc 74.5 ± 4.16ef 117.40
BSE 25 248 ± 0.41ef 15.2 ± 0.14bc 7.70
BSE 50 290 ± 8.71g 0.73 ± 2.98a 1.32
BSE 100 289 ± 14.4g 1.14 ± 4.94a 5.44
BSE 250 215 ± 31.2e 26.5 ± 10.67c 6.68
BSE 500 59.8 ± 7.16b 79.6 ± 2.45f 32.88
BSE 1000 63.9 ± 0.93bc 78.1 ± 0.32ef 205.00
GA 25 77.8 ± 6.74bc 73.4 ± 2.30ef 70.24
GA 50 81.2 ± 0.71bc 72.2 ± 0.24ef 67.76
GA 100 91.7 ± 4.29bcd 68.6 ± 1.47def 52.29
GA 250 98.4 ± 7.53cd 66.4 ± 2.57de 64.37
GA 500 126 ± 1.11d 56.8 ± 0.38d 27.73
GA 1000 268 ± 2.46fg 8.37 ± 0.84ab 6.17

Results are expressed as means ± standard deviations (n = 3). Values with different lower case letters (a–g) within the same column are significantly different (p < 0.05).
MetHb, methaemoglobin; Control (NO  
2 ), negative control group without sample and without NO2 – induced oxidation; Control (+NO2 ), positive control group without
sample but with NO 2 – induced oxidation; BLE, B. racemosa leaf extract; BSE, B. racemosa stem extract; GA, gallic acid.

of B. racemosa (Kong et al., 2012). In this study, we reported the
additional presence of rutin and further quantified the amounts
of the polyphenols and performed further validation studies to
confirm the identification of the polyphenols. Our previous study
also reported significant levels of ascorbic acid in the leaf water ex-
tracts, hence together with polyphenols, they could be the major
compounds contributing towards preventing serum oxidation. Re-
sults are in agreement with previous studies that described the
ability of hydrophilic antioxidants including curcumin and Trolox
to inhibit serum lipid peroxidation, in fact better than the lipo-
philic antioxidant a-tocopherol (Jalali-Khanabadi, Mozaffari-Khos-
ravi, & Parsaeyan, 2010; Schnitzer et al., 1998). Additionally,
mutual synergistic effects of different polyphenolic compounds
and other non-polyphenolic compounds can also enhance the anti-
oxidative effect (Dai & Mumper, 2010).
3.4. LDL oxidation
Fig. 3(b) shows the results for the LDL oxidation assay. There
was a concentration-dependent decrease in TBARS in LDL treated
with B. racemosa leaf and stem extracts, indicating the extracts
could significantly inhibit copper-mediated LDL oxidation. Lower
concentrations of B. racemosa leaf extract (IC50 = 73.0 lg/ml) were
adequate to inhibit 50% of TBARS formation compared to its stem
extract (IC50 = 226 lg/ml), implying the former to be a more effec-
tive inhibitor of LDL oxidation. Polyphenols such as ellagic acid,
gallic acid and protocatechuic acid, which were present in the
leaves, have been reported to be able to inhibit lipid peroxidation,
while specifically, ellagic acid has been shown to inhibit LDL oxida-
tion (Anderson et al., 2001; Hseu et al., 2008). The positive control,
gallic acid, showed almost constant effect at all concentrations
tested with TBARS similar to that of the negative control (without
Cu2+). This observation could be due to the high reactivity of gallic
acid as a pure compound whereby low concentrations were al-
ready sufficient to inhibit reactivity of the copper (Cu2+) ions.
In addition to MDA, LHP, the intermediate product of lipid per-
oxidation, were also measured. We hypothesised that the plant ex-
tracts could have also interfered with the propagation of LHP and
hence the chain reaction of lipid peroxidation. Interestingly, a sim-
ilar trend to TBARS formation was found (Fig. 3(c)). Analyses
showed that B. racemosa leaf extract inhibited approximately 20%
of LHP at concentrations above 100 lg/ml while B. racemosa stem
extract required a higher concentration (P500 lg/ml) to achieve
similar inhibition. Gallic acid was more potent, requiring a lower
concentration (50 lg/ml) to reach similar inhibition of LHP produc-
tion. These results demonstrate that in addition to preventing the
formation of MDA, B. racemosa extracts are also able to inhibit
the formation of LHP.
3.5. Haemoglobin oxidation
During redox reaction, Hb enhances the reduction of nitrite
(NO 2+
2 ) to nitric oxide (NO), converting ferrohaem (Fe ) to ferri-
3+
haem (Fe ), thus causing the formation of MetHb. MetHb-medi-
ated LDL oxidation has been postulated to promote
atherosclerosis (Umbreit, 2007). In this study, the effect of B. race-
mosa leaf extract, stem extract and gallic acid on Hb oxidation was
measured via a NO2-induced MetHb formation method (Table 3). B.
racemosa leaf extract showed a concentration-dependent increase
in the inhibition of MetHb formation and the highest inhibition
was seen at 500 lg/ml (79.51%). B. racemosa stem extract showed
a slightly different pattern of inhibition, with lower inhibition of
MetHb formation at low concentrations (25–100 lg/ml), after
which there was a considerable leap in the inhibition of MetHb for-
mation at concentrations above 250 lg/ml. Silibinin, a flavonoid,
was reported to show similar dose–response relationship in Hb
oxidation (Marouf et al., 2011). A threefold lower concentration
of B. racemosa leaf extract (116 lg/ml) was needed to inhibit
approximately 50% of MetHb formation compared to its stem ex-
tract (385 lg/ml).
Gallic acid on the other hand showed pro-oxidant activities by
increasing MetHb formation, particularly at high concentrations.
High concentrations of gallic acid may increase NO production,
NO may form nitrite (NO 2 ) via auto-oxidation, further reacting
with Hb, leading to formation of MetHb and NO (Umbreit, 2007).
This implies that lower concentrations would be more biologically
relevant, especially for pure compounds. In another study, gallic
acid, at a concentration of 50 lg/ml prevented lipid peroxidation
of erythrocytes and did not exhibit pro-oxidant effects (Hseu
 K.W. Kong et al. / Food Chemistry 146 (2014) 85–93
et al., 2008). This supports our observation that gallic acid is pro-
tective at low concentrations.
Overall, B. racemosa leaf and stem extracts could delay the time
to achieve maximal MetHb formation as well as the time needed to
achieve 50% formation of MetHb. B. racemosa leaf extract was bet-
ter than stem extract in protecting and delaying the oxidation of
Hb to MetHb and was especially protective at concentrations above
100 lg/ml whereas B. racemosa stem extract was protective at con-
centrations above 500 lg/ml. A similar observation was also re-
ported by Sulaiman and Hussain (2011), whereby Hb treated
with anthocyanin delayed the formation of MetHb. On the other
hand, gallic acid, at a concentration of 1000 lg/ml acted as
pro-oxidant, showing increased production of MetHb with in-
creased incubation time.
4. Conclusion
This study showed that freeze drying was a better method for
the preparation of the shoots of B. racemosa as oppose to air drying,
as the former method could reduce the degradation of polyphe-
nols. Using UHPLC analyses, we reported gallic acid and ellagic acid
as the main polyphenols in the leaves. This study also provides
in vitro evidence on the ability of the aqueous extracts of B. racemo-
sa to provide protection against oxidation of biological compo-
nents, including serum, LDL and Hb. The presence of polyphenols
in the shoots could play a major role in the observed protective ef-
fect against oxidative damage. There is a great potential for B. race-
mosa leaves to be developed as protective agents against oxidative
stress-related diseases.
Acknowledgements
This research project was funded by the following research
grants: RG340/11HTM, RG458/12HTM, H-20001-00-E000009 and
PV061/2012A from University of Malaya, Kuala Lumpur, Malaysia.
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