Effect of Extraction Procedures, Genotypes and Screening Methods to Measure the Antioxidant Potential and Phenolic Content of Orange-fleshed Sweetpotatoes (Ipomoea batatas L.)
Jace D. Everette
The antioxidant activities and phenolic contents of the five orange-fleshed sweetpotato genotypes namely SP-122, SP-129, SP-115, SP-323 and SP-425 in relation to the two extraction methods (hydrophilic and lipophilic fraction) were examined. The antioxidant capacity was investigated with three different screening methods: the 2, 2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Oxygen Radical Absorbance Capacity (ORAC). It was found that the antioxidant activity of sweetpotato extracts in hydrophilic fraction have a significant antioxidant effect when tested by each method. There was a relationship between total polyphenol content and antioxidant function in case of ABTS (r = 0.59) and ORAC (r = 0.35). The hydrophilic ABTS values correlate significantly with the hydrophilic DPPH values (r = 0.84) and the hydrophilic ORAC values correlate reasonably well with the hydrophilic ABTS values (r = 0.85). In case of the hydrophilic DPPH values and hydrophilic ORAC values also showed a strong correlation (r = 0.87). However, antioxidant activities with the lipophilic extracts were not significantly correlated. Among the sweetpotato genotypes studied, the SP-129 had significantly higher antioxidant activity in ORAC and ABTS methods. In case of DPPH methods, the genotypes SP-425 showed higher activity compared to others studied; however, phenolic content of all genotypes was ranged 3.5-4.8 mg TAE/g dry weight. Among the methods examined, ABTS proved the best for antioxidant determination in orange-fleshed sweetpotatoes followed by ORAC method. The information provided by this research will also facilitate the genetic and chemical breeding study for improvement of the desired quality criteria of orange fleshed sweetpotatoes as well as other produces.
to cite this article:
Jace D. Everette and Shahidul Islam, 2012. Effect of Extraction Procedures, Genotypes and Screening Methods to Measure the Antioxidant Potential and Phenolic Content of Orange-fleshed Sweetpotatoes (Ipomoea batatas L.). American Journal of Food Technology, 7: 50-61.
Received: August 04, 2011;
Accepted: November 08, 2011;
Published: December 20, 2011
The sweetpotato [Ipomoea batatas L. Lam.] is the seventh most important
food crop in the world (FAO, 1997) and is among the crops
selected by the U.S. National Aeronautics and Space Administration to be grown
in a controlled ecological life support system as a primary food source (Hoff
et al., 1982). These food crops have a high content of phenolic antioxidants,
especially caffeic and chlorogenic acids (Teow et al.,
2007; Islam, 2006, 2008a,
2009). Orange-fleshed sweet potatoes are also rich in
carotenoids (Teow et al., 2007; Yoshimoto
et al., 2003). Purple-fleshed sweet potatoes also have high anthocyanin
content (Yang and Gadi, 2008). Sweet potatoes have also
been shown to have antidiabetic (Kusano et al., 2001;
Matsui et al., 2002; Islam,
2006) and antimicrobial (Islam, 2008b) properties.
Sweetpotato cultivars whose roots are used for a beverage, a paste, a powder,
an alcohol drink and a natural colorant have been developed in this decade (Yoshimoto,
2001; Islam, 2006).
Phenolic compounds are a diverse group of secondary metabolites present in
higher plants that play important roles in the structure of plants and are involved
in a number of metabolic pathways (Harbone,1980; Prior
and Cao, 1999). Plant phenolics, because of their diversity and extensive
distribution, can be argued to be an important group of natural antioxidants
and contribute to organoleptic and nutritional qualities of fruit and vegetables.
Phenolic compounds exist universally in most of the vegetables, which are also
rich sources of natural antioxidants (Peluso et al.,
1995; Chuda et al., 1996; Shimozono
et al., 1996; Kaul and Khanduja, 1998; Yoshimoto
et al., 1999a,b; Yoshimoto,
2001; Yoshimoto et al., 2003; Murayama
et al., 2002; Islam et al., 2002a,b,
Islam, 2006, 2008a). Dietary
antioxidants have attracted special attention because they can protect the human
body from oxidative stress, which may cause many diseases including cancer,
aging and cardiovascular diseases (Stevens et al.,
1995; Hagerman et al., 1998; Kaul
and Khanduja, 1998; Prior et al., 1998; Robards
et al., 1999; Yoshimoto et al., 1999a,
b; Yoshimoto, 2001; Yoshimoto
et al., 2003; Islam et al., 2005,
2008a, 2009). Therefore, sweetpotato may become
an excellent source material for biologically active compounds.
Oxidative stress is known to cause chronic diseases such as cancer, Parkinsons
disease, Alzheimers dementia, heart disease, arthritis and many other
ailments (Waris and Ahsan, 2006). Oxidative stress occurs
as a result of free radicals such as peroxyl, hydroxyl and superoxide radicals
(Buechter, 1988). Antioxidants found in fruits and vegetables
neutralize free radicals and promote health (Ames et al.,
1993). These antioxidants include anthocyanins, polyphenols, carotenoids
and flavonoids (Woolfe, 1992; Islam,
2006, 2008b). Therefore, consumption of fruits and
vegetables is necessary to combat oxidative stress and prevent degenerative
Studies have been done which compared the antioxidant content of white, orange,
pink, red and purple-fleshed sweet potatoes. These studies indicated that purple
fleshed sweet potatoes had the highest overall antioxidant content and color
intensity was indicative of antioxidant capacity White fleshed sweet potatoes
displayed antioxidant activity that was less than the darker fleshed varieties
(Teow et al., 2007). Studies on other foods have
also shown that the more highly pigmented varieties are also richer in antioxidant
content (Awika et al., 2003; Bao
et al., 2005). The purpose of this study is to determine the variation
in extraction methods in relation to antioxidant capacity among five genotypes
of orange fleshed sweetpotatoes. Also, to find out the suitable antioxidant
assays method(s) for orange fleshed sweetpotatoes.
MATERIALS AND METHODS
Chemicals: Trolox (2, 5, 7, 8-tetramethylchroman-2-carboxylic acid),
tannic acid, 2, 2-diphenyl-1-picryl hydrazyl (DPPH), 2, 2-azinobis(3-ethyl-benzothiazoline-6-sulfonic
acid) (ABTS), methyl-β-cyclodextrin and sodium persulfate, were purchased
from Sigma-Aldrich (St. Louis, MO, USA). 2, 2Azobis (2-amidino propane)
dihydrochloride (AAPH) and fluorescein were obtained from ACROS organics (New
Jersey, USA). Folin-Ciocalteau phenol reagent was purchased from MP Biomedicals
All other reagents used were of analytical or HPLC grades.
Preparation of sweetpotato samples for analysis: The experiment was
conducted during 2009 to 2011 at the University of Arkansas at Pine Bluff, USA.
Trials were carried out over 3 years using 25 sweetpotato genotypes grown to
screen for suitable genotypes with desireable characteristics. Five orange fleshed
genotypes were chosen for further study for biochemical characteristics such
as SP-122, SP-129, SP-115, SP-323 and SP-425 of orange-fleshed sweet potato
were tested. These genotypes had similar color intensity. The sweetpotato storage
roots were washed with tap water and stored at -80°C for 24 h prior to freeze
drying. The bulbs were manually cut into small pieces and freeze dried for 60
h in a Millrock MD Freeze-Dryer at -54°C and 96 mT. The samples were then
manually ground into a fine powder and stored at -20°C prior to extraction.
Lipophilic and hydrophilic extracts methods: Five grams of freeze-dried
powder were stirred for 2 min in 25 mL of hexane and the mixture was filtered
using a Büchner funnel. The hexane extraction was repeated twice and the
combined lipophilic extracts were evaporated to dryness at 50°C using a
rotary evaporator. The dried hexane extract was re-dissolved in 50 mL of 50%
acetone/50% water containing 7% methylated β-cyclodextrin. The remaining
residue was then extracted twice with 25 mL of acidified methanol (80% methanol/
7% acetic acid). The combined hydrophilic extracts were made to 50 mL with acidified
methanol. This method of extraction was developed by Teow
et al. (2007).
Measured antioxidant activity in the ORAC assay: The procedure for the
ORAC assay was established by Prior (2003). Trolox was
dissolved in a phosphate buffer (0.075 M, pH 7.4). The phosphate buffer was
used as a blank and solvent used to make necessary dilutions. Fluorescence measurements
were taken using a Synergy HT Multi-Mode microplate reader (BioTek Instruments,
Inc. Winooski, Vermont). The lipophilic fractions were diluted 10-fold and the
hydrophilic fractions were diluted 100-fold. For the diluted samples, 20 μL
were added to 200 μL fluorescein (incubated at 37°C). Then, 20 μL
AAPH were added rapidly to each well via a multi-channel pipette. Immediately
after addition of AAPH, the plate was agitated for 1 sec before the first reading.
Readings were taken for 2 h at 1 min intervals. Excitation and emission wavelengths
were set at 480 and 520 nm, respectively. The area under curves was measured
using Image J software. Data were expressed as μmoles Trolox equivalent
per gram dry weight (μmol TE -1 dw).
Measured radical scavenging activity in the ABTS assay: The radical-scavenging
activity of the extracts was measured by following the procedure reported by
Walker and Everette (2009). Stock solutions of ABTS
(5.00x10-4 M) and sodium persulfate (6.89 x10-3 M) in
PBS (pH = 8.0) were prepared. The ABTS radical cation solution was prepared
by adding 1 mL of sodium persulfate to 99 mL of stock ABTS. The solution was
stored for 16 h. This produced a solution of ABTS+ which gave an
absorbance of approximately 0.85 at 734 nm. A 10 mM stock solution of Trolox
was prepared for every sample tested. Dilutions were prepared for each sample
tested. Dilution strength was dependent upon each extracts relative antioxidant
capacity. For each dilution, 20 μL were added to 2.5 mL of ABTS.+
solution and incubated in a dry bath at 37°C for 30 min. Absorbances were
measured at 734 nm on an ASYS UVM 340 plate reader. TEAC values were measured
by comparing the slopes of sample plots compared to that of Trolox.
Measured radical scavenging activity in the DPPH assay: The radical-scavenging
activity of the extracts was measured according to a slightly modified method
described by Brand-Williams et al. (1995). A stock
solution of DPPH (6 mM) was prepared by dissolving 0.0263 g in 10 mL of ethanol
(or methanol). The stock solution is diluted to prepare a 60 μM working
solution. Again, a 10 mM stock solution of Trolox was prepared for every sample
tested. Dilutions were prepared for each sample tested. Dilution strength was
dependent upon each extracts relative antioxidant capacity. For each dilution,
20 μL were added to 2.5 mL of DPPH. solution and incubated in
a dry bath at 37°C for 30 min. Absorbances were measured at 520 nm on an
ASYS UVM 340 plate reader. TEAC values were measured by comparing the slope
of sample plots to the slope of Trolox. Antioxidant activity was reported as
μmoles Trolox equivalent per gram dry weight sample (μmol TE g-1
Determination of total phenols: Total phenolic content of each extract
was measured using a slightly modified method reported by Singleton
et al. (1999). The extracts were diluted with distilled water, followed
by the addition of 0.25 mL of Folin-Ciacalteau reagent (1 N). Then, 1.25 mL
of sodium carbonate is added to each dilution and incubated at room temperature
for 40 min. The following dilutions were made for each sample: 1:100, 1:50,
1:33, 1:25 and 1:20. The absorbance was measured at 725 nm on an ASYS UVM 340
plate reader. Distilled water was measured as the blank. Tannic acid was used
as a standard. Total phenolic content was expressed as milligrams Tannic acid
equivalents per gram dry weight sample (mg TAE g-1 dw). TAE were
measured by comparing the slope of samples to the slope tannic acid.
Statistical analysis: The data were analyzed as a combined series of
CRDs for laboratory experiments. Data were subjected to analysis of variance
(ANOVA). Results are presented as means of four individual experiments. Pearson
correlations and t-tests were performed in Microsoft Excel. Group differences
were evaluated using t-tests with p<0.05 considered to be an indication of
a statistically significant difference.
RESULTS AND DISCUSSION
Total phenols: The phenolic content of each extract was also evaluated
(Fig. 1). The hydrophilic extract for SP115 demonstrated the
most total phenols (4.8 mg TAE g-1 dw) whereas the SP-323 hydrophilic
extract demonstrated the least (3.5 mg TAE g-1 DW). However, statistical
analysis indicated the five varieties were not significantly different from
one another (p>0.05). The lipophilic extract for SP-129 contained the most
phenols (0.64 mg TAE g-1 DW) whereas the SP-122 lipophilic extract
contained the least (0.37 mg TAE g-1 DW). Similar to the hydrophilic
extracts, statistical analysis indicated the three varieties were not significantly
different from one another (p>0.05). Several authors reported that highest
phenolic content is typically an indication of highest antioxidant activity
(Islam et al., 2003a, 2011;
Islam, 2006, 2008a). The SP-129
extracts repeatedly displayed higher antioxidant activity than the other two
varieties. However, the hydrophilic SP-129 extract contained a lower amount
of phenols than the other two hydrophilic extracts for SP-122 and SP-115. Therefore,
the phenolic content results suggest the three varieties have very similar amounts
of polyphenols. Polyphenols constitute one of the most numerous and ubiquitous
groups of plant metabolics and are an integral part of the human diet. Orange
fleshed sweetpotatoes are known to possess a variety of antioxidant properties
(Yoshinaga et al., 1999; Yoshimoto
et al., 2001).
Antioxidant capacity in ABTS, DPPH and ORAC assays: The antioxidant
activity of five varieties of orange fleshed sweetpotatoes was evaluated in
three antioxidant assays.
|| Total phenolic content of sweet potato extracts (Bars indicate
||Antioxidant activity of sweet potato extracts towards the
ABTS radical cation (Bars indicate standard error
Although, we have found a good correlation among all the methods used here
for assessing antioxidant capacity, using more than one antioxidant assay is
strongly recommended a single method will provide basic information about antioxidant
properties, but a combination of methods describes the antioxidant properties
of the sample in more detail. The ABTS radical scavenging capacity was examined
and both fractions of SP-129 followed by SP-115 demonstrated the highest activity
of the genotypes studied (p<0.05) (Fig. 2). The DPPH radical
scavenging capacity was measured for the five genotypes studied (Fig.
||Antioxidant activity of hydrophilic sweet potato extracts
towards the DPPH radical (Bars indicate standard error)
The genotypes SP-425 showed the most activity followed by SP-129. The results
suggested that the lipophilic extracts were not active in the DPPH assay for
orange colored sweetpotatoes. There were no significant differences among SP-122,
SP-115 and SP-323 (p>0.05). In the ORAC assay, the antioxidant activity measured
was again comparable to the other two assays. Further, the rank order for the
hydrophilic ORAC results indicated SP-129 had the highest activity while SP-323
had the lowest. All five genotypes had statistically different (p<0.05) activities
from each other (Fig. 4). The lipophilic ORAC results indicated
SP-129 was also the most active variety and SP-122 was the least active. In
addition, statistical analysis indicated SP-129 was significantly different
from the other four varieties (p<0.05); however, SP-122 and SP-115 were not
significantly different from each other (p>0.05). The antioxidant activities
of the hydrophilic and lipophilic extracts ranged from 45.41 to 23.44 μmol
TE g-1 dw and 1.23 to 0.723 μmol TE g-1 dw, respectively
(Fig. 4). The study showed SP-129 demonstrated the greatest
antioxidant activity among the five genotypes tested in all antioxidant models
in both hydrophilic and lipophilic fractions. As expected, the hydrophilic fractions
displayed considerably more activity than the lipophilic. The fact that SP-129
displayed considerably more activity than SP-122 and SP-115 which had similar
color intensity indicated that considerable variation may occur among orange-fleshed
sweet potatoes of similar color intensity.
There are many reports attempting to rank the antioxidant properties of different
plant materials using different methods (Pellegrini et
al., 2003; Prior et al., 2003; Huang
et al., 2002, 2005) including ORAC (Ou
et al., 2002a), DPPH (Islam et al., 2009),
ABTS (Walker and Everette, 2009). The antioxidant capacity
assays measure the combined effect of many antioxidants present in the sample
which are able to scavenge free radicals generated in the assays. Interactions
between antioxidant are also reflected in the assay value. Both ORAC and ABTS
are indicators of the free-radical scavenging ability of antioxidant against
peroxyl radical, using the same mechanism of hydrogen atom transfer.
||Measurement of sweet potato extracts oxygen radical absorbance
capacity (Bars indicate standard error)
|| Correlation between Total phenolics and different antioxidant
methods (n = 20)
On the other hand, ORAC measures the meta-chelating properties of antioxidant
thereby expressing a radical prevention ability of the sample (Huang
et al., 2005). Nevertheless in contrast to Ou
et al. (2002b) and Huang et al. (2002),
who did not find any general agreement among the antioxidant methods used, our
data correlated well. A slight difference among the results obtained by the
three assays might relate to the different conditions of measurements and the
sensitivity of the assays.
Correlations: There was a direct relationship between total polyphenol
contents antioxidant function in case of ABTS (r = 0.59) and ORAC (r = 0.35)
|| Correlation between hydrophilic ABTS values and hydrophilic
|| Correlation between hydrophilic ORAC values and hydrophilic
|| Correlation between hydrophilic DPPH values and hydrophilic
These data are in accordance witht hat of other authors who have shown that
high total polyphenol content increases antioxidant function and that there
is a liner correlation between polyphenol and antioxidant activity (Gorinstein
et al., 2003; Islam et al., 2003a;
Islam, 2006, 2008b). The three
different antioxidant methods used in this study to examine antioxidant activity
were compared. The antioxidant activity seen for SP-129 was the highest in all
three assays. As shown in Fig. 6 and 7,
the hydrophilic ABTS values correlate reasonably well with the hydrophilic DPPH
values (r = 0.84) and the hydrophilic ORAC values correlate reasonably well
with the hydrophilic ABTS values (r = 0.85). In Fig. 8, the
hydrophilic DPPH values and hydrophilic ORAC values showed a significant correlation
(r = 0.87). However, antioxidant activity with the lipophilic extracts correlated
poorly when compared in all methods studied.
The antioxidant properties depend on several structural features of the molecule
of polyphenols in its base structure and are primarily attributed to the high
reactivity of hydroxyl substituents. The B-ring hydroxyl configuration is the
most significant determinant of scavenging of ROS. 30, 40-di OH (catechol) structure
in the B-ring strongly enhances lipid peroxidation inhibition. A free 3-hy-droxyl
group and 30, 40-catechol (dihydroxy) structure, a C2-C3 double bond and a 4-oxo
group on the C ring endow the flavonoid with potent antioxidant function. The
superiority of quercetin in inhibit- ing both metal and nonmetal-induced oxidative
damage is partially ascribed to its free 3-OH substituent which is thought to
increase the stability of the flavonoid radical (Davies,
2000). Fenton-induced oxidation is strongly inhibited by flavonoids with
catechol, 4-oxo and 50-OH arrangements (Cao et al.,
1997; Cheng and Breen, 2000; Yoshimoto
et al., 1999a; Yoshimoto, 2001; Islam,
2006, 2008a, 2009) which
is superior to isoforms that lack these features. Future research should include
the use of in vivo techniques such as molecular marker analysis. Additional
research on polyphenolic should include a standardization of methods for quantification,
evaluation of physiological activities and bioavailability. The interactions
of the various polyphenolics with pharmaceutical should also be emphasized in
future studies. The results may help in future conventional and chemical breeding
program for specific constituents to enhance or reduce the bioactive phytochemicals
and for improvement of the desired quality criteria of orange fleshed sweetpotatoes
as well as other produces.
The authors would like to thank Dr. Richard B. Walker, Professor of Biochemistry,
University of Arkansas at Pine Bluff for his help and valuable comments. The
author also thankful to Mr. Z. Adam and L. Anderson for their assistance during
the period of this study. This work has been funded by United State Department
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