See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/254355770

Antioxidant Potential of Water Hyacinth ( Eichornia crassipes ): In

Vitro Antioxidant Activity and Phenolic Composition

Article  in  Journal of Aquatic Food Product Technology · January 2012

DOI: 10.1080/10498850.2011.621582

CITATIONS READS

3 710

3 authors, including:

Surendraraj Alagarsamy Sabeena Farvin

Kuwait Institute for Scientific Research Technical University of Denmark
20 PUBLICATIONS   93 CITATIONS    39 PUBLICATIONS   794 CITATIONS   

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Sabeena Farvin on 05 April 2015.

The user has requested enhancement of the downloaded file.

This article was downloaded by: [DTU Library]
On: 23 February 2015, At: 22:44
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Aquatic Food Product

Technology
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/wafp20

Antioxidant Potential of Water

Hyacinth (Eichornia crassipes): In Vitro
Antioxidant Activity and Phenolic
Composition
a b c
A. Surendraraj , K. H. Sabeena Farvin & R. Anandan
a
Institute of Food and Dairy Technology , TANUVAS , Chennai , India
b
Division of Industrial Food Research, National Institute of Food
(DTU-Food), Technical University of Denmark , Lyngby , Denmark
c
Biochemistry and Nutrition Division, Central Institute of Fisheries
Technology , Cochin , India
Accepted author version posted online: 17 Jan 2012.Published
online: 10 Jan 2013.

To cite this article: A. Surendraraj , K. H. Sabeena Farvin & R. Anandan (2013) Antioxidant Potential
of Water Hyacinth (Eichornia crassipes): In Vitro Antioxidant Activity and Phenolic Composition,
Journal of Aquatic Food Product Technology, 22:1, 11-26, DOI: 10.1080/10498850.2011.621582

To link to this article: http://dx.doi.org/10.1080/10498850.2011.621582

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
 Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions
Downloaded by [DTU Library] at 22:44 23 February 2015
 Journal of Aquatic Food Product Technology, 22:11–26, 2013
Copyright © Taylor & Francis Group, LLC
ISSN: 1049-8850 print/1547-0636 online
DOI: 10.1080/10498850.2011.621582

Antioxidant Potential of Water Hyacinth

(Eichornia crassipes): In Vitro Antioxidant Activity
and Phenolic Composition

A. Surendraraj,1 K. H. Sabeena Farvin,2 and R. Anandan3

Downloaded by [DTU Library] at 22:44 23 February 2015

1
Institute of Food and Dairy Technology, TANUVAS, Chennai, India
2
Division of Industrial Food Research, National Institute of Food (DTU-Food),
Technical University of Denmark, Lyngby, Denmark
3
Biochemistry and Nutrition Division, Central Institute
of Fisheries Technology, Cochin, India

The aims of the present study were (a) to extract and quantify the main phenolic acids and tocopherols
from the petiole, leaves, and flowers of Eichornia crassipes; (b) to evaluate the antioxidant capacity of
the extracts in four in vitro systems (1,1-diphenyl-2-pycryl-hydrazyl [DPPH] radical scavenging ability,
iron chelating activity, reducing power, and prevention of oxidation in a liposome model system); and
(c) its effectiveness in retarding lipid peroxidation in fish oil by accelerated stability test. Significant
differences were observed in total and individual phenolic contents and in the antioxidant activities of
extracts from the various parts of E. crassipes. Out of the 11 phenolic acids analyzed, ethanolic extracts
contained high amounts of gallic, protocatechuic, gentisic, and p-hydroxybenzoic acid, whereas, water
extracts contained less amounts of a varied number of phenolic acids. Ethanolic extracts of flower,
which contained the highest total phenolic content, were found to have high DPPH radical scavenging
activity and reducing power. However, ethanolic extracts of leaf exerted a high Fe2+ chelating activity
and also inhibited lipid peroxidation process both in liposomes and fish oil. Our results demonstrate that
E. crassipes, an underutilized aquatic weed, could be a potential natural antioxidant source for food,
feed, and pharmaceutical applications.

Keywords: aquatic weed, natural antioxidants, phenolic compounds, tocopherols

INTRODUCTION

Lipid peroxidation is one of the most important causes of quality deterioration in foods, leading to
the loss of functional properties and nutritional value (Yen et al., 1999). Oxidized polyunsaturated
fatty acids in raw or processed foods also contribute to carcinogenesis, arteriosclerosis, and the aging

The authors acknowledge Dr. D. Ramasamy, Professor and Head, Institute of Food and Dairy Technology, TANUVAS,
for providing the necessary facilities for this work. The authors are thankful to Dr. Charlotte Jacobsen, Head of Seafood
Research, National Food Institute, Technical University of Denmark, for support in carrying out the accelerated stability test
in fish oil.
Correspondence should be addressed to A. Surendraraj, Institute of Food and Dairy Technology, TANUVAS, Alamathi
P.O., Red Hills (via), Chennai– 600 052, India. E-mail: asurendraraj@rediffmail.com
11
 12 SURENDRARAJ, FARVIN, AND ANANDAN

process in humans (Yagi, 1987). Generally, synthetic antioxidants such as butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), propyl gallate, and tertiary butyl hydroquinone (TBHQ)
are added to control the lipid oxidation process in foods (Sobedio et al., 1991). However, the use
of synthetic antioxidants has limitations due to health risks and toxic effects (Linderschmidt et al.,
1986). Moreover, consumers are increasingly avoiding foods prepared with preservatives of chem-
ical origin; therefore, the importance of exploiting natural antioxidants from various sources and
replacing synthetic antioxidants with natural ingredients has attracted increasing attention. As plants
produce antioxidants to control the oxidative stress caused by sunlight and oxygen, they became a
source of useful new compounds with antioxidant activity. The commercial utilization of plants
as sources of antioxidants to enhance health and food preservation is of current interest (Olukemi
et al., 2005). Epidemiological studies have shown positive associations between the consumption of
phenolic-rich foods or beverages and the prevention of diseases (Scalbert and Williamson, 2000).
These effects have been attributed to antioxidant components such as plant phenolics including
Downloaded by [DTU Library] at 22:44 23 February 2015

flavonoids, ascorbic acid, tocopherols, and carotenoids (Rice-Evans et al., 1996; Torel et al., 1996;
Cotelle et al., 1996; Cos et al., 1998).
Water hyacinth (Eichornia crassipes) is a free floating tropical aquatic weed, which has become
naturalized in many warm areas of the world. It reproduces sexually by seeds and is one of the most
productive plants on earth; thus it is considered the world’s worst aquatic plant (Center et al., 1999).
This aquatic plant causes severe environmental and economic problems in India and other tropical
countries as it forms dense mats that interfere with navigation, recreation, irrigation, and power
generation (Chillers, 1991). In the past, these plants were used for feed formulations (Edwards et al.,
1985) and phytoremediation of metal contaminated waters (Agunbiade et al., 2009). The methanolic
extracts of the plants were reported to have anti-inflammatory and anti-ulcerogenic effects in rats
(Sanitha, 2005). Recently, extracts of the plants have shown to exert antitumor activity in mice
models (Ali et al., 2009). However, the antioxidant activities of these plants are not yet studied
in detail, and only one report is available in the literature about the radical scavenging activity of
E. crassipes (Chantiratikul et al., 2009). Moreover, for exploring the antioxidant potential of the
plant extracts, a single test is insufficient to identify the different mechanisms involved. Therefore,
it could be of significance to examine the utilization of this waste aquatic weed as a source of
natural antioxidants for retarding lipid peroxidation. The aims of the present study were: (a) to
extract and quantify the phenolic fractions from the petiole, leaves, and flowers of E. crassipes; (b)
to evaluate the antioxidant capacity of the extracts in four in vitro systems (1,1-diphenyl-2-pycryl-
hydrazyl [DPPH] radical scavenging ability, iron chelating activity, reducing power, and prevention
of oxidation in a liposome model system); and (c) to evaluate the effectiveness of these extracts
in retarding lipid peroxidation in fish oil by accelerated stability test. In addition, the tocopherol
content of the extracts was also determined to investigate if this could explain the difference in their
antioxidant activity. The information gained may help in understanding the antioxidant compounds
present in these aquatic weeds to promote the potential application of these compounds as natural
antioxidants in foods, pharmaceuticals, and in health-promoting functional foods.

MATERIALS AND METHODS

Materials
The petiole, leaves, and flowers of water hyacinth (E. crassipes) were collected from the water
bodies in and around Chennai, Tamil Nadu, India (June 2009). Cod liver oil was kindly donated
from Maritex A/S (Sortland, Norway). The peroxide value (PV) and anisidine value (AV) of the
oil used was 0.11 ± 0.07 meq/kg and 0.47 ± 0.04, respectively. L-α-phosphatidyl choline, DPPH,
thiobarbituric acid, ascorbic acid, and ethylene diamine tetra acetic acid (EDTA) were obtained from
Sigma (St. Louis, MO, USA). All other reagents were of analytical grade.
 ANTIOXIDANT ACTIVITY OF WATER HYACINTH 13

Preparation of Extract
The petiole, leaves, and flowers of E. crassipes were washed with tap water and dried in shade. The
dried samples were powdered by using a kitchen blender. The material which had passed through
an 80 mesh sieve was retained for use and stored at −40◦ C until extraction. For the preparation
of ethanolic extract, 5 g of powdered sample was extracted with 50 mL of 96% ethanol at room
temperature overnight and centrifuged at 2,800 rpm for 10 min. The supernatant was collected in a
separate bottle, and the residue was re-extracted three times under the same conditions as mentioned
above. The combined filtrate was evaporated in a rotary evaporator at 40◦ C. In the case of water
extract, 5 g of powdered sample was extracted with 100 mL of distilled water overnight at room
temperature and centrifuged at 2,800 rpm for 10 min. The supernatant was filtered through ordinary
filter paper to remove any floating materials. The residue was re-extracted three times under the
same conditions, and the combined extracts were freeze dried rather than evaporated. These extracts
were stored at −80◦ C until analysis. The extracts were re-dissolved in corresponding solvents (water
Downloaded by [DTU Library] at 22:44 23 February 2015

or 96% ethanol) for analysis.

Determination of Total Phenolic Content

Total phenolic content (TPC) in the extracts was determined according to the method of Singleton
and Rossi (1965). An aliquot (100 μL) of extract was mixed with 0.75 mL of Folin-Ciocalteu
reagent (diluted 10-fold with distilled water) and kept at room temperature for 5 min. Sodium bicar-
bonate (6%, 0.75 mL) was added to the mixture and incubated at room temperature for 90 min. The
absorbance was measured at 725 nm. A standard curve was plotted using different concentrations of
gallic acid, and the amount of total phenolics was calculated as gallic acid equivalents in mg/100 g
of dried material.

Identification of Phenolic Acids by High Performance Liquid Chromatography

Identification of phenolic acid was done using a modified method of Onyeneho and Hettiarachchy
(1993). The extract was passed through a 0.45 μm filter and injected into reverse phase high perfor-
mance liquid chromatography (HPLC) equipped with a diode array detector (Agilent Technologies,
Rising Sun, MD, USA). The column used was Zorbax Eclipse® XDB C8 analytical column
(250 mm × 4.6 mm; Agilent Technologies) with a 5 μm packing material. Elution was performed
with an isocratic mixture of methanol and 10 mM ammonium acetate buffer, pH 5.4 (12:88 v/v), at
a flow of 1 mL/min. Detection was done using a diode array detector with a reference wavelength
of 280 nm. Retention times and peak areas were monitored, and individual phenolic acids were
identified by the retention time of authentic standards.

Determination of Tocopherol Content

Tocopherol content in the ethanolic extracts was determined using HPLC (Waters, Milford, MA,
USA), equipped with a fluorescence detector. Two g of the ethanolic extract was extracted with
3 mL chloroform: methanol (1:1) mixture, and the chloroform phase was evaporated under nitro-
gen. The residue obtained was re-dissolved in 1 mL of n-heptane and injected into a Spherisorb
s5w column (250 mm × 4.6 mm) (Waters). Elution was performed with an isocratic mixture of
n-heptane/2-propanol (100: 0.4; v/v) at a flow rate of 1 mL/min. Detection was done with excita-
tion at 290 nm and emission at 330 nm (AOCS, 1992). Results are expressed in μg tocopherol per
g of extract.
 14 SURENDRARAJ, FARVIN, AND ANANDAN

DPPH Radical Scavenging Activity

The scavenging effect on α,α-diphenyl-β-picrylhydrazyl (DPPH) free radical was measured by the
method of Shimada et al. (1992) with slight modification. DPPH solution (1 mL, 0.1 mM in 95%
ethanol) was mixed with 1 mL of extract (at concentrations of 10, 100, 200, 500, or 1,000 μg/mL).
The mixture was shaken and kept at room temperature for 30 min. Absorbance of the resulting
solution was measured at 517 nm. For blank, 1 mL of distilled water was used instead of the sample,
and sample control was also made for each fraction by mixing 1 mL of sample with 1 mL of 95%
ethanol. BHT at a concentration of 200 μg/mL was used as a reference. Radical scavenging capacity
was calculated as follows:

1 − (Abs. of sample − Abs. of sample control) × 100.

DPPH radical scavenging capacity (%) =
Abs. blank
Downloaded by [DTU Library] at 22:44 23 February 2015

Iron (Fe2+ ) Chelating Activity

The Fe2+ chelating activity of the extracts was estimated by the modified method of Dinis et al.
(1992). To 1 ml of the extract (at a concentration of 10, 100, 200, 500, or 1,000 μg/mL), 2.7 mL
of deionised water and 0.1 ml of 2 mM ferrous chloride was added. After 3 min, the reaction was
stopped by the addition of 5 mM ferrozine (0.2 mL). The mixture was shaken vigorously and left
at room temperature for 10 min. Absorbance of the resulting solution was measured at 562 nm.
A blank was run in the same way by using distilled water instead of sample. Sample control was
made for each extract without adding ferrozine. For comparison of metal chelating activity, known
chelator EDTA at a concentration of 200 μg/mL was used as reference. The chelating capacity was
calculated as follows:

1 − (Abs. of sample − Abs. of sample control) × 100.

Iron chelating activity (%) =
Abs. of blank

Reducing Power
The reducing power was measured according to the method of Oyaizu (1986) with some modifica-
tions. To 1 mL of extract (at a concentration of 10, 100, 200, 500, or 1,000 μg/mL), 1 mL 0.2 M
phosphate buffer (pH 6.6) and 1 mL of 1% potassium ferric cyanide were added. The mixture was
incubated at 50◦ C for 20 min, and 1 mL of 10% TCA was added to this reaction mixture. An aliquot
of 2 mL from the incubation mixture was mixed with 2 mL of distilled water and 0.4 mL of 0.1%
ferric chloride in test tubes. After 10 min, the absorbance was measured at 700 nm. Ascorbic acid at
a concentration of 200 μg/mL was used as a reference as it has good reducing property. Increased
absorbance (A700 ) of the reaction mixture indicated increased reducing power.

Inhibition of Lipid Peroxidation in a Liposome Model System

Liposomes were prepared from soybean phosphatidylcholine according to the method of Farvin
et al. (2010). Lipid oxidation was performed in a model system containing 0.1 mg of phosphatidyl-
choline liposomes per mL of phosphate buffered saline (PBS; 3.4 mM Na2 HPO4 -NaH2 PO4 , 0.15 M
NaCl, pH 7.0) and extracts at concentrations of 10, 100, 200, 500, and 1,000 μg/mL. Lipid oxida-
tion was initiated by iron redox cycling using 50 μM FeCl3 and 100 μM ascorbate. The order of
addition was buffer, extracts, liposome, ferric chloride, and ascorbic acid. The reactants were mixed
by vortexing for 2 s and incubated at 37◦ C in a water bath for 1 h. The liposome assay solution
with distilled water instead of sample was used as control. BHT at a concentration of 200 μg/mL
 ANTIOXIDANT ACTIVITY OF WATER HYACINTH 15

was also made in a similar manner for comparison. Lipid oxidation was measured by determin-
ing the concentrations of thiobarbituric acid reactive substance (TBARS) formed according to the
method of Buege and Aust (1978). Aliquots (0.25 mL) of liposome suspensions were sampled into
test tubes and made up to 1 mL by adding distilled water (0.75 mL). Then, 2 mL of TBA reagent
(3.75 g/L TBA; 150 g/L trichloroacetic acid; HCl 0.25 mol/L; 0.1 g/L BHT) was added. The
tubes were closed and heated in boiling water bath for 15 min, immediately cooled, and centrifuged
(1,500 × g; 10 min). A reagent blank was prepared in the same manner as mentioned above with
distilled water instead of sample. The absorbance at 532 nm of the supernatant was read against a
blank.

Accelerated Stability Test (OxiPres)

The antioxidant effects of the extracts were determined in fish oil by measuring the oxidative stabil-
ity as the induction period at 100◦ C in an OxiPres (MikroLab, Århus, Denmark). Five g of fish oil
Downloaded by [DTU Library] at 22:44 23 February 2015

with added extracts (1,000 μg/mL) were placed in the OxiPres reaction vessel at 100◦ C and with an
oxygen pressure of 5 bar. The drop in oxygen pressure in the reaction vessels as a result of oxygen
consumption was recorded by using a picolog recorder. A control with fish oil without the addition
of any extract and a reference with fish oil added with 200 μg/mL of BHT were also made. The
induction period was determined in duplicate as the crossing point of the tangents to the curve.

Statistical Analysis
The data for total phenolic contents were subjected to one-way analysis of variance, and all
the other data obtained were analyzed by two-way analysis of variance. The statistical compar-
isons among the samples were performed with Bonferroni multiple comparison test using the
statistical package program Graphpad prism 4 (Graphpad Software Inc., San Diego, CA, USA).
A p-value <0.05 was considered as statistically significant. Data of various antioxidant assays, total
and individual phenolic contents, and tocopherol were subjected to multivariate analysis. A princi-
pal component analysis (PCA) was performed using Unscrambler version 7.6 SR-1 (Camo, Oslo,
Norway). All variables were weighted (1/standard deviation), and the models were validated using
full cross-validation.

RESULTS AND DISCUSSION

Extraction Yield, Total Phenolic Content, and Tocopherol Content

Because of safety concerns regarding the use of some organic solvent extracts in foods, we selected
approved food grade solvents such as water and ethanol for extraction of antioxidant compounds.
Table 1 shows the extraction yields of the water and ethanolic extracts of different parts of E. cras-
sipes. Water extracts showed higher yield when compared to ethanolic extracts. The extraction yield
of water extracts ranged from 21.3 to 25.5%, while the ethanolic extracts ranged from 7.9 to 10.1%.
Extraction yield of flowers was found to be the highest among the samples. This shows that there
was an increase in yield with increased solvent polarity (polarity index of water and ethanol are
9 and 5.2, respectively), indicating that more polar compounds were found in these samples. Water
hyacinth was reported to be one of the rich sources of cysteine and SH containing proteins (Bodo
et al., 2004). Polar solvents like water can also extract sugars, proteins, glycosides, organic acids,
tannins, salts, and mucus. This might be the reason for the higher extraction yield of water extracts.
Considerable variations in the extraction yield found among the different parts might be attributed
to the polarities of different compounds present in these plant parts.
 16 SURENDRARAJ, FARVIN, AND ANANDAN

TABLE 1
The extraction yield of water and ethanolic extracts of E. crassipes

Yield (%)

Plant part Ethanolic extract (EE) Water extract (WE)

Petiole 9.5 21.3

Leaf 7.9 22.2
Flower 10.1 25.5

The total phenolic content of different parts of E. crassipes ranged from 83.9 ± 11.4 to
665.9 ± 71.1 mg gallic acid equivalent (GAE)/100 g of dried sample (Figure 1). Flowers of E. cras-
sipes generally contained more phenolic compounds than leaves and petiole. The ethanolic extract of
Downloaded by [DTU Library] at 22:44 23 February 2015

flower contained significantly (p < 0.001) higher quantity of TPC when compared to other extracts.
Though not significantly (p > 0.05) different, the water extracts of petiole and leaves contained
more TPC when compared to corresponding ethanolic extracts. Earlier, Chantiratikul et al. (2009)
reported a TPC of 62.4 ± 0.1 and 39.5 ± 0.3 mg GAE/g for the acetone/methanol (1:1) extracts
of leaves and petiole of E. crassipes, respectively. The direct comparison of our results with their
study is difficult as the extraction solvents, units, and parts of the plants are different.
The tocopherol content in the ethanolic extracts of E. crassipes is shown in Table 2. The
tocopherol homologues ranged from 1.16 ± 0.17 to 13.42 ± 1.64 μg/g extract. The ethanolic
extracts of petiole contained significantly (p < 0.001) higher content of α-tocopherol, whereas
leaves contained significantly (p < 0 .001) higher δ-tocopherol among the extracts. There was no
β-tocopherol in any of the extracts, and γ-tocopherol was found only in the petiole.

Identification and Quantification of Phenolic Acids

The major phenolic acids in the extracts were identified by HPLC (Table 3). In general, ethanolic
extracts showed fewer of the tested phenolic acids. A total of 11 phenolic compounds including

Water Ethanolic

800
TPC (mg GAE/100g of dried powder)

700

600

500

400

300

200

100

0
Petiole Leaf Flower

FIGURE 1 Total phenolic content of ethanolic and water extracts of petiole, leaves, and flowers of E. crassipes.
Results are average of triplicate determination ± standard deviation (EE: ethanolic extract; WE: water extracts).
 ANTIOXIDANT ACTIVITY OF WATER HYACINTH 17

TABLE 2
The tocopherol content (μg/g extract) in ethanolic extracts of different parts of E. crassipes. Results
are average of triplicate determination on the same samples ± standard deviation. The letters a, b, c
represent significant difference between samples, and x, y, z represent significant difference in
different tocopherol homologues.

Samples α-tocopherol β-tocopherol γ-tocopherol δ-tocopherol

Petiole 13.42 ± 1.64c,z − 1.16 ± 0.17x 2.52 ± 0.01a,y

Leaf 8.22 ± 0.08b,y − − 6.16 ± 0.77b,x
Flower 2.38 ± 0.01a,x − − 2.27 ± 0.01a,x
Downloaded by [DTU Library] at 22:44 23 February 2015

6 benzoic phenolic types (gallic, protocatechuic, gentisic, p-hydroxybenzoic, vanillic, and sali-
cyclic acid) and 5 cinnamic phenolic types (chlorogenic, syringic, caffeic, p-coumaric, and ferulic
acid) were analyzed. The ethanolic extracts were found to contain only four benzoic type phenolic
acids—namely, gallic, protocatechuic, gentisic, and p-hydroxybenzoic acid; however, the amount
of which was significantly (p < 0.001) higher than that present in water extracts. Ethanolic extracts
of flowers contained significantly (p < 0.001) higher levels of gentisic, protocatechuic acids, and
p-hydroxybenzoic acid when compared to leaves and petiole. Even though water extracts contained
lower amounts of phenolic acids when compared to ethanolic extracts, they contained a varied num-
ber of different phenolic acids. In addition to gallic, protocatechuic, gentisic, and p-hydroxybenzoic
acid, all water extracts contained chlorogenic, vanillic, and caffeic acids in low levels. P-coumaric
acid was found only in water extracts of leaves. The presence of flavanoids such as naringenin,
keapferol, myricetin, rutin, and vanillin from the leaves and petiole of E. crassipes has been reported
(Chantiratikul et al., 2009). Most of the compounds which we identified were phenolic acids, and
it should be noted that so far no literature is available on the differential phenolic composition of
E. crassipes plant parts. Hence, direct comparison of our results on phenolic acid constituents to
other studies is not possible.

Antioxidant Activities in In Vitro Systems

DPPH Radical Scavenging Activity
The DPPH radical scavenging activity showed a concentration dependency and increased with
increase in concentration, except for water extracts of flower which showed a steady state after 500
μg/mL (Figure 2). The ethanolic extracts of flower showed significantly (p < 0.001) higher DPPH
radical activity (79.36 ± 0.61%) at the highest concentration (1,000 μg/mL) tested and was as
efficient as BHT (79.86 ± 1.01%) at 200 μg/mL concentration. This was followed by the ethanolic
and water extracts of leaves. Petiole was shown to have the lowest DPPH radical scavenging activity
among the tested extracts, which might be due to the low content of TPC. Earlier, Chantiratikul et al.
(2009) also reported good DPPH radical scavenging activity for the acetone/methanol extracts of
leaves as opposed to the petiole.
Phenolic compounds are generally very active in scavenging DPPH free radicals due to their
fast electron transfer process, while hydrogen atom abstraction becomes a marginal reaction path
(Foti et al., 2004). In the present study, it was observed that the extracts containing high levels of
total phenolic content (ethanolic extract of flowers and water and ethanolic extracts of leaves) were
also potent DPPH radical scavengers, suggesting that phenolic compounds may be the principle
constituents responsible for antiradical properties of the extracts. However, water extracts of flower
with a good amount of TPC did not show high DPPH radical scavenging activity. One of the reasons
 Downloaded by [DTU Library] at 22:44 23 February 2015

18
TABLE 3
The major phenolic acids in the extracts of different parts of E. crassipes identified by HPLC (mg/g extract). Results are average of triplicate determination on the same
samples ± standard deviation. The letters a, b, c represent significant difference between samples, and w, x, y, z represent significant difference between phenolic acids.

Extracts Gallic Protocatechuic Gentisic p-Hydroxybenzoic Chlorogenic Vanillic Syringic Caffeic Salicylic p-Coumaric Ferulic

Petiole EE 3.15 ± 0.01b,x 22.33 ± 0.1b,y 38.27 ± 1.94b,z 7.56 ± 1.21b,x − − − − − − −

Leaf EE 4.08 ± 0.01b,x 27.04 ± 0.34b,y 49.93 ± 0.17b,z 8.52 ± 0.85b,x − − − − − − −
Flower EE 6.75 ± 0.03b,w 53.94 ± 0.89c,y 137.26 ± 3.75c,z 20.34 ± 3.44c,x − − − − − − −
Petiole WE 1.57 ± 0.01a,y 1.76 ± 0.01a,y 6.34 ± 0.30a,z 1.03 ± 0.01a,x 0.40 ± 0.01a,x 0.43 ± 0.01a,x − 0.22 ± 0.01a,x − − −
Leaf WE 3.74 ± 0.05b,y 2.81 ± 0.02 a,y 5.87 ± 0.01a,z 5.30 ± 0.03b,z 0.53 ± 0.01a,x 0.68 ± 0.01a,x − 0.41 ± 0.01a,x − 0.28 ± 0.01x −
Flower WE 1.67 ± 0.01a,y 2.03 ± 0.05a,y 7.04 ± 1.02a,z 1.55 ± 0.04a,x 0.62 ± 0.01a,x 0.99 ± 0.01a,x − 0.38 ± 0.01a,x − − −
 ANTIOXIDANT ACTIVITY OF WATER HYACINTH 19

Petiole WE Leaf WE Flower WE Petiole EE

Leaf EE Flower EE
90

80

70
DPPH Radical scavenging (%)

60

50

40

30
Downloaded by [DTU Library] at 22:44 23 February 2015

20

10

0
10 100 200 500 1000
–10
Concentration in ug/ml

FIGURE 2 The DPPH radical scavenging activity of water and ethanolic extracts of petiole, leaves, and flowers of
E. crassipes. Results are average of triplicate determination ± standard deviation (EE: ethanolic extract; WE: water
extracts).

for high TPC of these samples might be due to the color reaction of proteins in the water extracts
with Folin-Ciocalteu reagent, which leads to overestimation of total phenolic compounds.

Reducing Power
The reducing property indicates that the antioxidant compounds are electron donors capable of
reducing the oxidized intermediates of the lipid peroxidation process (Yen and Chen, 1995). In the
present study, it is clear that the reducing power (as indicated by the absorbance at 700 nm) was
a function of concentration and increased with increase in concentration (Figure 3). None of the
extracts had as high reducing power as ascorbic acid, which showed a reducing power of 2.28 ± 0.28
(at 200 μg/mL concentration). The order of the reducing power was leaf WE > flower WE > leaf
EE > flower WE> petiole WE > petiole EE. It was observed that the extracts containing high levels
of total phenolics were also potent in reducing ferric iron, suggesting that polyphenols may be the
principal constituents responsible for these properties of the extracts.

Fe2+ Chelating Activity

Transition metals, such as Fe2+ and Cu2+ , can catalyze the generation of reactive oxygen species
such as hydroxyl radical (·OH) and superoxide anion (O2 − ), (Stohs and Bagachi, 1995). In partic-
ular, Fe2+ generates ·OH by the Fenton reaction, by which the lipid peroxidation chain reaction
is accelerated. Moreover, Fe2+ catalyses the breakdown of lipid peroxides, which leads to the
formation of volatile oxidation products responsible for off-flavor formation. Therefore, the chela-
tion of metal ions often reduces lipid oxidation. At the highest concentration (1,000 μg/mL) tested,
almost all extracts showed ≥90% Fe2+ chelating activity, and there was no significant difference
 20 SURENDRARAJ, FARVIN, AND ANANDAN

Petiole WE Leaf WE Flower WE

Petiole EE Leaf EE Flower EE

0,4
Reducing power (Abs at 700 nm)

0,35

0,3

0,25

0,2

0,15

0,1
Downloaded by [DTU Library] at 22:44 23 February 2015

0,05

0
10 100 200 500 1000
Concentration in ug/ml

FIGURE 3 The reducing power of water and ethanolic extracts of petiole, leaves, and flowers of E. crassipes. Results
are average of triplicate determination ± standard deviation (EE: ethanolic extract; WE: water extracts).

Petiole WE Leaf WE Flower WE

Petiole EE Leaf EE Flower EE
100
90
80
Fe2+ Chelating activity (%)

70
60
50
40
30
20
10
0
10 100 200 500 1000
Concentration in ug/ml

FIGURE 4 The Fe2+ chelating capacity of water and ethanolic extracts of petiole, leaves, and flowers of E. cras-
sipes. Results are average of triplicate determination ± standard deviation (EE: ethanolic extract; WE: water extracts)
(color figure available online).

(p > 0.05) between the extracts (Figure 4). The Fe2+ chelating activity was found to be concen-
tration dependent in the case of water extracts and increased with an increase in concentration.
In the case of ethanolic extracts, there was no significant difference in the different concentrations
tested. Water extracts of petiole showed the highest Fe2+ chelating activity (94.76 ± 0.55%) at
1,000 μg/mL concentration, which was as effective as EDTA (93.72 ± 0.15 %) at 200 μg/mL
concentration.
 ANTIOXIDANT ACTIVITY OF WATER HYACINTH 21

Metal binding ability of E. crassipes is well-known and is used in the bioremediation of metal
contaminated waters. Agunbiade et al. (2009) have reported that leaves and shoots of E. crassipes
accumulate more metals than in roots. The higher Fe2+ chelating activity of the ethanolic extracts
in all concentrations tested may be due to the presence of specific phenolic compounds, which have
been reported to be good chelating agents (Robards et al., 1999). The catechol derivatives such as
protocatechuic acid and catechin have been reported to be strong metal chelaters (Ueda et al., 1996).
The high metal chelating activity of ethanolic extracts may thus be due to their very high content of
protocatechuic acid.

Antioxidant Activity in the Liposome Model System

The liposome system allows the lipid-soluble components to be present in the lipid phase without
the presence of a co-solvent, while the water soluble antioxidants can be added to the aqueous phase
Downloaded by [DTU Library] at 22:44 23 February 2015

of the liposome. Figure 5 depicts the effect of different concentrations of extracts on phosphatidyl-
choline liposome oxidation, induced by ferric/ascorbic acid. Even though almost all extracts showed
>40% inhibition, the ethanolic extracts of leaves and water extracts of flowers were significantly
(p < 0.01) better in preventing TBARS formation compared to other extracts. At the highest con-
centration (1,000 μg/mL) tested, all extracts except ethanolic extracts of flowers (significant at p <
0.01) showed an effective inhibition of lipid peroxidation as BHT (57.8% at a concentration of
200 μg/mL). In spite of the higher DPPH radical scavenging activity, Fe2+ chelating activity, and
reducing power, ethanolic extracts of flowers showed the least inhibitory effect on the Fe2+ -induced
lipid peroxidation in the liposome model system when compared to other extracts.
Phenolic compounds are known to be effective antioxidants in lipid systems with a high con-
tent of polyunsaturated fatty acids as they easily transfer a hydrogen atom to lipid peroxyl radicals

Petiole WE Leaf WE Flower WE

Petiole EE Leaf EE Flower EE
80

70
% Inhibition of TBARS formation

60

50

40

30

20

10

0
10 100 200 500 1000
Concentration in ug/ml

FIGURE 5 The inhibition of lipid peroxidation in liposome model system by the water and ethanolic extracts of
petiole, leaves, and flowers of E. crassipes. Results are average of triplicate determination ± standard deviation (EE:
ethanolic extract; WE: water extracts).
 22 SURENDRARAJ, FARVIN, AND ANANDAN

(LOO•) and form the aryloxyl radical (ArO•). This does not have the capacity to act as a chain
carrier and hence couples with another radical, thus quenching the radical process (Ruberto et al.,
2001). Even though the extracts containing high levels of total phenolic content were also potent
in retarding lipid peroxidation, some parts which contained very low levels of total phenolics,
such as petiole, also showed good inhibition, suggesting that both phenolic compounds and some
other constituents in the extracts were responsible for the antioxidant activity. Earlier, Bodo et al.
(2004) reported compounds such as glutathione, cysteine, and SH-containing derivatives as the
antioxidant components in E. crassipes. Thus, some of these co-extracted compounds also might
have contributed to the overall antioxidant activity of the extracts.

Accelerated Stability Test (OxiPres)

Results from the accelerated stability test are shown in Table 4. The oxidative stability of fish oil
was significantly (p < 0.001) higher for the sample with BHT, when compared to control, since
Downloaded by [DTU Library] at 22:44 23 February 2015

the time for drop in oxygen pressure (induction time) was longer than the control. None of the
extracts was as effective as BHT in this system. All the ethanolic extracts and water extracts, except
the water extracts of flower, showed significantly (p < 0.001) higher oxidative stability when com-
pared to control. Leaves showed the highest induction period, both in water and ethanolic extracts,
followed by petiole. Interestingly, the water extracts of flowers, in spite of higher TPC and higher
inhibition of lipid peroxidation in liposomes, showed poor performance in this system and showed
a significantly (p < 0.001) lower induction period than that of the control. This might be due to the
instability of these extracts under extreme temperature and pressure conditions. Other reasons for
this observation could be linked to the structure of different model systems, the location, and orien-
tation of the tested antioxidants in different model systems which have a great impact on antioxidant
activity. Earlier, Xu and Chang (2009) also reported significant reduction of total phenolics, indi-
vidual phenolic acids, and the antioxidant activity of different bean varieties by thermal and high
pressure processing. The tocopherol derivatives and related isoprenoids extracted into ethanolic
extracts in addition to the phenolics also might have contributed to antioxidant activity in these
extracts.

Correlation Between Phenolic Contents and Antioxidant Activity

To give an overview of the results and to further evaluate the similarities and differences among
different parts of E. crassipes, a PCA was made on the antioxidant assays, tocoperol, phenolic

TABLE 4
The induction time of different extracts evaluated in fish oil by accelerated stability
test. Results are average of triplicate determination ± standard deviation. The letters
a, b indicate significant difference between the water and ethanolic extracts. The
symbol ∗∗∗ represents p < 0.001, significantly different from control.

Induction time in minutes

Sample Water extract Ethanolic extract

Petiole ∗∗∗ 238 ± 2.0a ∗∗∗ 253 ± 3.0b

Leaf ∗∗∗ 261.5 ± 1.5a ∗∗∗ 282 ± 2.0b

Flower ∗∗∗ 195 ± 10.0 a ∗∗∗ 246 ± 6.0b

Control (Fish oil without adding any extract) 215 ± 5.5

BHT (200 μg/g) ∗∗∗ 402.5 ± 2.5
 ANTIOXIDANT ACTIVITY OF WATER HYACINTH 23

compounds, and TPC data. The first two principle components (PC1 and PC2) explain 93% and 5%
of the total variation in the data set, respectively (Figures 6a and 6b). The scores plot from the PCA
analysis gave information about the similarities in antioxidant assays and phenolics between the
different sample codes (Figure 6b). The scores plot showed a clustering of different sample codes
suggesting similarity between ethanolic and water extracts of leaves and petiole while the ethanolic
and water extracts of flowers are not similar.
The loadings plot visualizes correlations between the measured variables (antioxidant assays,
tocopherols, TPC, and individual phenolic compounds; Figure 6a). The variations in TPC, DPPH
radical scavenging activity, and reducing power were well explained by PC1. Since TPC, DPPH
radical scavenging, and reducing power were closely loaded on PC1 (Figure 6a), it indicates that
TPC and these two antioxidant properties were highly and positively correlated with each other.
Accordingly, in the scores plot, flower ethanolic extracts with highest TPC, DPPH radical scaveng-
ing activity, and reducing power were located farthest to the right along PC1 (Figure 6b). On the
Downloaded by [DTU Library] at 22:44 23 February 2015

other hand, ethanolic and water extracts of leaves and petiole with rather low levels of TPC, as
well as weak scavenging activity against DPPH and reducing power, are situated on the left side
along the PC1. The four benzoic type phenolic acids (protocatechuic, gallic, p-hydroxy benzoic,
and gentisic acids) were placed on the right corner between Fe2+ chelating activity and DPPH in
the loadings plot, which corresponds to ethanolic extracts of flowers containing higher quantities of
these phenolics in the scores plot. This shows that the phenolics are positively correlated with Fe2+
chelating activity and DPPH (Figures 6a and 6b).
The variations in the Fe2+ chelating activity were mainly explained by PC2. In the scores plot,
almost all the ethanolic extracts appeared in the positive part of PC2 due to their relatively high
Fe2+ chelating activity in all the concentrations tested; whereas water extracts, which showed rel-
atively high Fe2+ chelating activity only in the higher concentrations, were located in the negative
part of the PC2 (Figure 6b). In addition, Fe2+ chelating capacity loaded heavily on the second
component in the loadings plot where TPC has less loading, which illustrates well that no clear
correlation exists between TPC and chelating activity. The three phenolic compounds—caffeic,
chlorogenic, and vanillic—are grouped together and are placed in the negative part of PC2 as they
are only present in water extracts. These three phenolic acids are placed opposite to Fe2+ chelating
activity in the loadings plot indicating that the phenolic acids are negatively correlated with Fe2+
chelating activity (Figure 6a). Earlier, Chen and Ahn (1998) also reported that caffeic acid was
a weak chelator. It could not chelate Fe2+ tightly, and it probably formed a temporary complex
with Fe2+ .
The inhibition of TBARS formation in liposome assay was not well-explained (< 50% explained)
by this model. The extension of induction time in fish oil (IT oil) by accelerated stability was
placed in the positive part of PC2 where all the ethanolic extracts were loaded (Figure 6a).
This is in agreement with the raw data that all the ethanolic extracts showed higher induc-
tion time when compared to the water extracts. Moreover, induction times are placed close
to alpha and delta tocopherols in the correlation loadings, suggesting that these are positively
correlated.
Similar to our study, many studies have reported positive correlations between total phenolic
content and radical scavenging activity and reducing power of plant extracts (Jiang et al., 2009;
Robards et al., 1999). Hence, phenolic compounds appear to be the major contributors to radical
scavenging and reducing properties of these extracts. However, some other active components in
different extracts may have synergistic or antagonistic effects on the scavenging activity, which
can give contradictory results requiring further characterization of these extracts. Interestingly,
Fe2+ chelating activity correlated neither with TPC nor with other antioxidant activities. A similar
trend was reported for malting barley extracts by having poor correlation of Fe2+ chelating activity
with both TPC and other antioxidant activities (Zhao et al., 2008).
 24 SURENDRARAJ, FARVIN, AND ANANDAN

(a)
PC2 Correlation Loadings (X)
1.0
δ -toc RP-10
Fe2+Chel-200
RP-100 Fe2+Chel-100
Fe2+Chel-10
IT oil
DPPH-10
Photocatechnic
α-toc Gallic
Hydroxybenzoic
Gentisic
Inh-TBARS100ug DPPH-1000
0.5

Inh-TBARS10
γ -toc DPPH-50 0
DPPH-100
Fe2+Chel-1000
RP-200 DPPH-200

0 RP-1000 RP-500
TPC
Inh-TBARS500ug
Inh-TBARS1000u
Coumaric
Downloaded by [DTU Library] at 22:44 23 February 2015

Inh-TBARS200ug
Fe2+Chel-500

–0.5

Caffeic
Chlorogenic
Valnitic
–1.0
PC1

–1.0 –0.8 –0.6 –0.4 –0.2 0 0.2 0.4 0.6 0.8 1.0
, X-expl: 93%,5%

(b)
PC2 Scores
100

Leaf EE
50
flower EE
Petiole EE

Petiole WE Leaf WE

–50

flower WE
–100
PC1
–200 –150 –100 –50 0 50 100 150 200 250 300 350 400 450
X-expl: 93%,5%

FIGURE 6 The PCA model on antioxidant assays and phenolic composition results measured in different parts
of E. crassipes: (a) correlation loadings of water and ethanolic extracts of different parts of E. crassipes showing
PC1 and PC2; (b) score plot of water and ethanolic extracts of different parts of E. crassipes showing PC1 and PC2.
EE represents ethanolic extract and WE represents water extracts (color figure available online).
 ANTIOXIDANT ACTIVITY OF WATER HYACINTH 25

CONCLUSION

The results of this experiment demonstrated that different parts of E. crassipes contained different
levels of total and individual phenolics and possessed diverse antioxidant properties. In general, a
high phenolic content correlated with various antioxidant activities studied, indicating that polyphe-
nols are one of the active components in these extracts. However, some parts with low total phenolic
content also showed antioxidative effects in some of the assays, indicating that some other co-
extracted active compounds—such as tocopherols in ethanolic extracts and proteins or peptides
in water extracts—may also contribute to the overall antioxidant properties which need further
investigation. All the ethanolic extracts, especially those of leaves, were able to protect highly
unsaturated fish oils from oxidation, suggesting their potential as natural antioxidants in functional
foods and pharmaceuticals. However, further research is required for the characterization of free,
esterified, glycoside, and ester-bound phenolic acids as well as flavonoids present in these extracts.
Downloaded by [DTU Library] at 22:44 23 February 2015

Furthermore, studies of antioxidative effects of these extracts in real food systems and in in vivo
studies are also necessary.

REFERENCES

Agunbiade, F. O., Olu-Owolabi, B. I., and Adebowale, K. O. 2009. Phytoremediation potential of Eichornia crassipes in
metal-contaminated coastal water. Bioresour. Technol. 100: 4521–4526.
Ali, H., Patel, M., Ganesh, N., and Ahi, J. 2009. The world’s worst aquatic plant as a safe cancer medicine “antitumor activity
on melanoma induced mouse by Eichornia crassipes: in vivo studies.” J. Pharm. Res. 2: 1365–1366.
AOCS. 1992. AOCS Official Method Ce 8-89. Champaign, IL: Author.
Bodo, R., Azzouz, A., and Hausler, R. 2004. Antioxidative activity of water hyacinth components. Plant Sci. 166:
893–899.
Buege, J. A., and Aust, S. D. 1978. Microsomal lipid peroxidation. In: Methods in Enzymology, Biomembranes. Part
C: Biological Oxidations. Vol. 52. Fleisher, S., and Packer, L. (Eds.). London, United Kingdom: Academic Press. pp.
302–310.
Center, T. D., Dray, F. A., Jubinsky, G. P., and Grodowitz, M. J. 1999. Biological control of water hyacinth under condition
of maintenance management: Can herbicides and insects be integrated? Environ. Manage. 23: 241–256.
Chantiratikul, P., Meechai, P., and Nakbanpote, W. 2009. Antioxidant activity and phenolic content of extracts from Salvinia
molesta and E. crassipes. Res. J. Biol. Sci. 4: 1113–1117.
Chen, X., and Ahn, D. U. 1998. Antioxidant activities of six natural phenolics against lipid oxidation induced by Fe2+ or
ultraviolet light. J. Am. Oil Chem. Soci. 75: 1717–1721.
Chillers, C. J. 1991. Biological control of water hyacinth, Eichornia crassipes (Pontederiaceae) in South Africa. Agric.
Ecosys. Environ. 37: 207–217.
Cos, P., Ying, L., Calomme, M., Hu, J. P., Cimanga, K., Van Poel, B., Pieters, L., Vlietinck, A. J., and Vanden Berghe,
D. 1998. Structure activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide
scavengers. J. Nat. Prod. 61: 71–76.
Cotelle, N., Bemier, J. L., Catteau, J. P., Pommery, J., Wallet, J. C., and Gaydou, E. M. 1996. Antioxidant properties of
hydroxylflavones. Free. Rad. Biol. Med. 20: 35–43.
Dinis, T. C. P., Madeira, V. M. C., and Almeida, L. M. 1994. Action of phenolic derivatives (acetaminophen, salicylate,
and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem.
Biophys. 315: 161–169.
Edwards, P., Kamal, M., and Wee, K. L. 1985. Incorporation of composted and dried water hyacinth in pelleted feed for the
tilapia Oreochromis niloticus (Peters). Aquacul. Fisheries Managt. 1: 233–248.
Farvin, K. H. S., Nielsen, N. S., and Jacobsen, C. 2010. Antioxidant activity of potato peel extracts in a fish-rapeseed oil
mixture and in oil-in-water emulsions. J. Am. Oil. Chem. Soc. 87: 1319–1332.
Foti, M. C., Daquino, C., and Geraci, C. 2004. Electron-transfer reaction of cinnamic acids and their methyl esters with the
DPPH radical in alcoholic solutions. J. Org. Chem. 69: 2309–2314.
Jiang, S., Wang, C., Chen, Z., Chen, M., Wang, Y., Liu, C., Zhou, Q., and Li, Z. 2009. Antioxidant properties of the extract
and subfractions from old leaves of Toona sinensis roem (Meliaceae). J. Food Biochem. 33: 425–441.
 26 SURENDRARAJ, FARVIN, AND ANANDAN

Linderschmidt, R., Trylka, A., Goad, M., and Witschi, H. 1986. The effects of dietary butylated hydroxytoluene on liver and
colon tumor development in mice. Toxicol. 38: 151–160.
Olukemi, O. A., Olukemi, I. O., Oluwatoyin, S. M., Austin, A. O., Mansurat, L. B., and Olufunmilola, T. I. 2005. Antioxidant
activity of Nigerian dietary spices. Electron. J. Environ. Agric. Food. Chem. 4: 1086–1093.
Onyeneho, S. N., and Hettiarachchy, N. S. 1993. Antioxidant activity, fatty acid and phenolic acid composition of potato
peels. J. Sci. Food. Agric. 62: 345–350.
Oyaizu, M. 1986. Studies on products of browning reactions: Antioxidative activities of browning products of browning
reaction prepared from glucosamine. Jpn. J. Nutr. 44: 307–315.
Rice-Evans, C. A., Miller, N. J., and Paganga, G. 1996. Structure antioxidant activity relationships of flavonoids and
phenolics acids. Free. Radical. Biol. Med. 20: 933–956.
Robards, K., Prenzler, P., Tucker, P., Swatsitang, G., and Glover, W. 1999. Phenolic compounds and their roles in oxidation
process in fruits. Food Chem. 66: 401–436.
Ruberto, G., Baratta, M. T., Biondi, D. M., and Amico, V. 2001. Antioxidant activity of extracts of marine algal genus
Cystoseira in a micellar model system. J. Appl. Phycol. 13: 403–407.
Sanitha, B. 2005. Phytochemical screening and pharmacological evaluation of Eichhornia crassipes (Martius) Solms
Downloaded by [DTU Library] at 22:44 23 February 2015

laubach. MFSc dissertation, CIFE, Mumbai, India.

Scalbert, A., and Williamson, G. 2000. Dietary intake and bioavailability of polyphenols. J. Nutr. 130: 2073S–2085S.
Shimada, K., Fujikawa, K., Yahara, K., and Namkamura, T. 1992. Antioxidative properties of xanthan on the autooxidation
of soyabean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40: 945–948
Singleton, V. L., and Rossi, J. A. 1965. Colourimetry of total phenolics with phosphomolybdic-phosphotungstic acid
reagents. Am. J. Enol. Vitic. 16: 144–158.
Sobedio, J. L., Kaitaramita, J., Grandgiral, A., and Malkkl, Y. 1991. Quality assessment of industrial pre-fried french fries.
J. Am. Oil. Chem. Soc. 68: 299–302.
Stohs, S. J., and Bagachi, D. 1995. Oxidative mechanisms in the toxicity of metal ions. Free. Rad. Biol. Med. 18: 321–336.
Torel, J., Cillard, J., and Cillard, P. 1996. Antioxidant activity of flavonoids and reactivity with peroxy radical. Phytochem.
25: 383–385.
Ueda, J., Saito, N., Shimazu, Y., and Ozawa, T. 1996. Comparison of scavenging abilities of antioxidants against hydroxyl
radicals. Arch. Biochem. Biophys.333: 377–384.
Xu, B., and Chang, S. K. C. 2009. Total phenolic, phenolic acid, anthocyanin, flavan-3-ol, and flavonol profiles and
antioxidant properties of pinto and black beans (Phaseolus vulgaris L.) as affected by thermal processing. J. Agric. Food
Chem. 57: 4754–4764.
Yagi, K. 1987. Lipid peroxides and human diseases. Chem. Phys. Lipids 45: 337–351.
Yen, G. C., and Chen, H. Y. 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agri.
Food. Chem. 43: 27–32.
Yen, G. C., Chen, H. Y., and Lee, C. E. 1999. Measurement of antioxidative activity in metal ion-induced lipid peroxidation
systems. J. Sci. Food. Agric. 79: 1213–1217.
Zhao, H., Fan, W., Dong, J., Lu, J., Chen, J., Shan, L., Lin, Y., and Kong, W. 2008. Evaluation of antioxidant activities and
total phenolic contents of typical malting barley varieties. Food Chem. 107: 296–304.

View publication stats