International Journal of Food Properties

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

Major Polyphenolics in Pineapple Peels and their

Antioxidant Interactions

Ti Li, Peiyi Shen, Wei Liu, Chengmei Liu, Ruihong Liang, Na Yan & Jun Chen

To cite this article: Ti Li, Peiyi Shen, Wei Liu, Chengmei Liu, Ruihong Liang, Na Yan & Jun Chen
(2014) Major Polyphenolics in Pineapple Peels and their Antioxidant Interactions, International
Journal of Food Properties, 17:8, 1805-1817, DOI: 10.1080/10942912.2012.732168

To link to this article: https://doi.org/10.1080/10942912.2012.732168

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International Journal of Food Properties, 17:1805–1817, 2014
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942912.2012.732168

MAJOR POLYPHENOLICS IN PINEAPPLE PEELS

AND THEIR ANTIOXIDANT INTERACTIONS

Ti Li, Peiyi Shen, Wei Liu, Chengmei Liu, Ruihong Liang,

Na Yan, and Jun Chen
State Key Laboratory of Food Science and Technology, Nanchang University,
Nanchang, China

Major polyphenolic compounds in pineapple peels were identified and quantified. The
antioxidant capacities of pineapple peel extracts and these polyphenolic compounds were
determined using DPPH q scavenging capacity and phosphomolybdenum method. Effects of
these polyphenolics’ interactions on their antioxidant capacity were also evaluated. Gallic
acid (31.76 mg/100 g dry extracts), catechin (58.51 mg/100 g), epicatechin (50.00 mg/100 g),
and ferulic acid (19.50 mg/100 g) were found to be the main polyphenolics in pineap-
ple peels. The IC50 for DPPH q scavenging assay of the extracts was 1.13 mg/ml and total
antioxidant capacity was 0.037 g ascorbic acid equivalents/g. The order of DPPH q scav-
enging capacity of per mole of these polyphenolic compounds present in pineapple peels
was gallic acid > epicatechin = catechin > ferulic acid, but it was different when using
phosphomolybdenum method the order of which was epicatechin. > catechin > gallic acid =
ferulic acid. Results of polyphenolics’ interactions indicated no synergistic effects. In the
combinations of ferulic acid-epicatechin and ferulic acid-gallic acid, additive effects were
found using both antioxidant activity assays.

Keywords: Pineapple peels, Polyphenols, DPPH q scavenging capacity,

Phosphomolybdenum method, Interaction.

INTRODUCTION
Polyphenolic compounds are secondary plant metabolites that exist ubiquitously in
the plant kingdom where they have a wide range of different structures[1] and physiologi-
cal properties, such as anti-allergenic, anti-artherogenic, anti-inflammatory, anti-microbial,
antioxidant, anti-thrombotic, cardioprotective, and vasodilatory effects.[2−6] The inter-
est in polyphenolic antioxidants has increased remarkably in the last decade because of
their capacity in scavenging free radicals associated with various diseases. Phenolic acids,
flavonoids, and tannins are regarded as the main dietary phenolic compounds. Of these,
phenolic acids consist of two subgroups, i.e., the hydroxybenzoic and hydroxycinnamic
acids.[7] Flavonoids constitute the largest group of plant phenolics, accounting for over half

Received 8 June 2012; accepted 17 September 2012.

Address correspondence to Chengmei Liu, State Key Laboratory of Food Science and Technology,
Nanchang University, 235 Nanjing East Road, Nanchang 330047, Jiangxi, China. E-mail: chengmeiliu@yahoo.
com.cn
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/
ljfp.

1805
1806 LI ET AL.

of the 8000 naturally occurring polyphenolic compounds.[8] Variations in substitution pat-

terns to ring C result in the major flavonoid classes, i.e., flavonols, flavones, flavanones, fla-
vanols (or catechins), isoflavones, flavanonols, and anthocyanidins.[9] Lately, researches on
antioxidant capacity of polyphenols from different sources have emerged in large numbers
measured by different assays, including DPPH q scavenging capacity, phosphomolybdenum
method, β-carotene-linoleate model system, ABTS radical-scavenging ability, superoxide
anion radical scavenging activity, etc.[10−13]
Pineapples are one of the leading commercial fruits of the tropics around the world
and the major producing areas are Southeast Asia and Latin America.[14] The canning
industry is producing large quantities of solid and liquid wastes, which leads to a seri-
ous environmental problem for disposal.[15] As waste products of the pineapple cannery,
pineapple peel waste could be a potential source for the extraction of beneficial bioactive
compounds. One alternative use for value addition of pineapple peels is the isolation of
bromelain.[16] So far, there have been reports of nutrient analyses of pineapples, includ-
ing fiber, protein, carotenoid, mineral composition, total polyphenol content, etc.[17,18]
However, only a few researches have concentrated on the antioxidant capacity of pineap-
ples. de Oliveira et al.[13] has studied the total phenolic content and antioxidant activities
of methanolic extracts of pineapple (Ananas comosus) residues (including pulp, seeds, and
peels from a local juice factory) using DPPH q and superoxide anion scavenging activity.
The research of total polyphenols of pineapple (Calendar, India) extracted with different
solvents was reported by Hossain et al.[10] and the results showed that the polyphenolic
contents of the extracts were found to be highest in methanol corresponding to the high-
est antioxidant properties. However, to the best of the authors knowledge, there has
been no report regarding the types of polyphenolic compounds in pineapple peels and
their antioxidant interactions are also not known, both of which are need to be further
explored. Therefore, in the present work, pineapple peels were employed as materials
and the major polyphenolic compounds and their antioxidant interactions have been stud-
ied. The composition and contents of polyphenols in pineapple peels were determined by
high performance liquid chromatography-mass spectroscopy (HPLC-MS). The antioxidant
capacities of pineapple peel extracts (PPE) and these major polyphenolics were mea-
sured and compared using DPPH q scavenging capacity and phosphomolybdenum method.
Furthermore, the effects of the polyphenol-polyphenol interactions on their antioxidant
capacity were evaluated.

MATERIALS AND METHODS

Materials
For this experiment we used the Bali variety which is one of the most popular pineap-
ple cultivars grown in Hainan, China. Bali pineapples were collected from Wanning, Hainan
province of China from January to March, 2011 and purchased from a local market in
Nanchang, Jiangxi, China. The peel of pineapples is deep yellow when ripe.
DPPH q (2,2’-diphenyl-2-picrylhydrazyl radical) was purchased from Solarbio
(Beijing, China). Standards of (+) –catechin (≥ 98%, 20 mg), (–) –epicatechin (≥ 98%,
20 mg), gallic acid (99.1%, 100 mg), ferulic acid (99.5%, 50 mg) and ascorbic acid (100%,
100 mg) were obtained from National Institutes for Food and Drug Control (Beijing,
China). Methanol and acetonitrile were of chromatographic grade and the other reagents
were of analytical grade.
 POLYPHENOLICS IN PINEAPPLE 1807

Extraction of Polyphenols from Pineapple Peels

Sample preparation and extraction were processed according to the method described
by de Oliveira et al.[13] The peels of pineapple were removed and rinsed with water before
being processed. The pineapple peels were then dried in a ventilated oven at 60◦ C for
48 h and ground to a fine powder. Extraction of 25 g of pineapple peels were carried out
by reflux, first with 150 ml of n-hexane to remove non-polar compounds and then with
150 ml methanol for 4 h at 60◦ C. Supernatants were decanted and filtered through Whatman
filter paper followed by concentration and drying in a vacuum rotary evaporator (RE 2000,
Rongya, Shanghai) at 50 ± 10◦ C, and the extracts were kept under nitrogen in a refrigerator
for 1 h.

Determination of Total Polyphenolic Contents (TPC)

Total polyphenolic content of PPE was determined according to the method of de
Oliveira et al.[13] with minor modifications. 0.25 mg extracts were dissolved in 0.5 ml
milliQ purified water and 0.50 ml aliquots or deionized water (control) were mixed by man-
ual shaking, for 10–15 s, with 2.50 ml of Folin-Ciocalteau reagent. After 5–8 min, 7.50 ml
of saturated sodium carbonate solution was added and solution diluted to 50 ml with deion-
ized water. The reaction mixture was kept in dark for 2 h and its absorbance was measured
at 760 nm against water in a UV-Vis spectrophotometer (T6, Puxi, China). The concen-
tration of the total polyphenols was determined as mg of gallic aid equivalents by using
an equation obtained from gallic acid calibration curve. The TPC was calculated as mg of
gallic acid equivalents/100 g fresh pineapple peels and mg of gallic acid equivalents/g dry
pineapple peels powders.

Identification and Quantification of Major Polyphenolic Compounds

HPLC-MS analysis of PPE were performed on an Agilent 1200 series HPLC system
(Agilent Technologies, USA), equipped with an autosampler, binary pump, degasser, and
a UV-Vis diode array detection (DAD) connected directly to the mass detector (Agilent
G6430A mass hunter) with an Electrospray ionization (ESI) source. HPLC analysis was
performed according to the method described by Kammerer et al.[19] PPE (20 µL) was
injected into an Elipse XDB C-18 column (5 µm, 250 × 4.6 mm, i.d., Agilent Technologies,
USA). The HPLC conditions for the polyphenols were as follows:
System 1: The mobile phase consisted of 2% (v/v) acetic acid in water (eluent A)
and 0.5% acetic acid in water and acetonitrile (50:50, v/v; eluent B) using a gradient pro-
gram as follows: from 10–24% B (20 min), from 24–30% B (20 min), from 30–55% B
(20 min), from 55–100% B (15 min), 100% B isocratic (8 min), from 100–10% B (2 min).
Simultaneous monitoring was performed at 280 nm at a flow rate of 1.0 mL/min.
System 2: The mobile phase consisted of the same eluents as described for system
1 using a gradient program as follows: from 10–15% B (10 min), 15% B isocratic (3 min),
from 15–25% B (7 min), from 25–55% B (30 min), from 55–100% B (1 min), 100% B
isocratic (5 min), from 100–10% B (0.1 min). Simultaneous monitoring was performed at
320 nm at a flow rate of 1.0 mL/min.
Mass spectra were obtained by electrospray-ionization in negative mode. The ESI
parameters were drying gas (N2 ) flow and temperature, 10 L/min and 350◦ C, respectively;
1808 LI ET AL.

nebulizer pressure, 30 psi, and capillary voltage, 4000 V. Scan range of the mass spectrome-
try was m/z 50–1500. The contents of polyphenolic compounds were calculated from their
areas with regression equations from standard curves obtained from HPLC analysis, and
are expressed as mg per 100 g dry peel powders.

Antioxidant Capacity
DPPH q scavenging capacity. The DPPH q scavenging capacities of PPE and the
pure polyphenolic compounds were measured according to the method of Hossain et al.,[10]
with some modifications. An aliquot of 0.1 ml of PPE (0.25–1.50 mg/mL) and pure
polyephenolic compounds (25–150 µM) at six different concentrations was mixed with
3.9 ml of 100 µM DPPH q solution. The reaction mixture was incubated for 30 min in
the darkness at room temperature. The absorbance of resulting solution was measured at
517 nm with a spectrophotometer (T6, Puxi, China). The control was prepared as above
without any extracts and methanol was used for the baseline correction. The radical scav-
enging capacity of the test samples was measured as a decrease in the absorbance of DPPH q
and was calculated by using the following equation:

Inhibition (%) = [(Acontrol − Asample )/Acontrol ] × 100

All samples were run in triplicate. The results were expressed as IC50 , and obtained
by plotting the remaining percentage of DPPH q against the sample concentration to obtain
the amount of antioxidant necessary to decrease the initial DPPH q concentration by 50%.
The lower value of IC50 indicates higher antioxidant activity.

Evaluation of Total Antioxidant Capacity (TAC) by

Phosphomolybdenum Method
The total antioxidant capacities of PPE and the pure polyphenolic compounds were
evaluated by the method of Prieto et al.[20] 0.1 ml of each sample solution (1 mg/ml) and
ascorbic acid were combined with 1 ml of reagent (0.6 M sulphuric acid, 28 mM sodium
phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a
boiling water bath at 95◦ C for 90 min. After the samples had cooled to room temperature,
the absorbance of aqueous solution each was measured at 695 nm against blank using a
UV-Vis spectrophotometer (T6, Puxi, China). A typical blank solution contained 1 ml of
reagent solution and the appropriate volume of the same solvent used for the sample and
it was incubated under the same conditions. The experiment was performed in triplicates.
The antioxidant activity is expressed as equivalents of ascorbic acid (g/g of extract). The
higher value of antioxidant activity indicates higher total antioxidant activity.

Antioxidant Interactions of Polyphenolics

In order to evaluate the possible interactions among the polyphenolic compounds of
pineapple peels, combinations of two and all of them together were measured using DPPH q
scavenging capacity and phosphomolybdenum method. To determine the synergistic,
antagonistic, or additive effect for DPPH q scavenging capacity and TAC, the following
equations described by Hidalgo et al.[21] were used:
 POLYPHENOLICS IN PINEAPPLE 1809

Difference in DPPH• scavenging capacity (%) =

 
100 − mixture IC50 × 200/ (A IC50 + B IC50 ) ;

where mixture IC50 is the result obtained experimentally for the mixture of two
polyphenolics (A and B), and A IC50 and B IC50 are the IC50 measured individually for
each compound.

Difference in TAC (%) = [mixture TAC value × 100/( A TAC value × A concentration

+ B TAC value × B concentration)] − 100;

where mixture TAC value is the result obtained experimentally for the mixture of two
polyphenolics (A and B), and A TAC value and B TAC value are the TAC value mea-
sured individually for each compound. Positive values (significant difference was found
between theoretical and real values at P < 0.05) are considered as synergistic effects, neg-
ative values (significant difference was found between theoretical and real values at P <
0.05) are antagonistic effects, and additive effects are defined when there are no significant
difference between theoretical and real values.[21,22]

Statistical Analysis
The results were reported as means ± standard deviation (SD) of at least three mea-
surements. One way analysis of variance (ANOVA) was used to compare the means, and the
least significant difference (LSD) test showed the values statistically different. Differences
were considered significant at P < 0.05. All statistical analyses were performed with
SPSS 17.0.

RESULTS AND DISCUSSION

Extraction and Composition of Polyphenolic Compounds from
Pineapple Peels
Polyphenols extracted with different solvents showed different yields. Hossain
et al.[10] had studied the effect of solvents on the polyphenols extraction in pineapple
(Calendar, India) and the results indicated that the yield was found to be highest in methanol
(21.50%) followed by ethyl acetate (4.90%) and water extract (4.30%). Furthermore, the
extraction of phenolic compounds from the fruits was commonly achieved with methanol
or aqueous methanol.[23] Therefore, methanol was chosen for polyphenol extraction in this
research and the yield of pineapple (Bali, China) peels extracts was 24.95% which was a
little higher than that reported by Hossain et al.,[10] but was lower than 30.2% reported
by de Oliveira et al.[13] However, the phenolic content of 31.98 mg gallic acid equivalents
(GAE)/g extracts in this study was less than 51.1 mg caffeic acid equivalents/g pineapple
extracts reported by Hossain et al.[10] but much higher than 9.1 mg GAE/g dry extracts
reported by de Oliveira et al.[13] The TPC of pineapples (Calendar, India) and pineapple
(Ananas comosus) residues were 10.99 mg caffeic acid equivalents/g fresh weight (FW)
and 2.75 mg GAE/g dry weight (DW), respectively.[10,13] In this study, it was found that
the TPC of pineapple peels was 7.98 mg GAE/g DW (148.91 mg GAE/100 g of FW).
1810 LI ET AL.

Polyphenols content in pineapple (Bali, China) peels was lower than some fruits
which have been studied for their high content of polyphenols, including grapes, apples,
and teas. Researches indicated that TPC were 201.0 and 296.3 mg GAE/g FW of red
grape and apple, respectively.[24] For black tea, its TPC ranged from 62 to 107 mg GAE/g
DW.[25] However, TPC of pineapple peels is higher than that of banana (Musa Paradasiaca)
(72.2 mg GAE/100 g FW),[26] and it was close to that of starfruit (acidic) (142.9 mg
GAE/100 g FW).[27]
In order to give a full picture of the qualification and quantification of polyphenolic
constituents in pineapple peels, the polyphenolic acids and flavonoids in the extracts were
determined by HPLC-MS (Figure 1) and the results are presented in Table 1. Four peaks
in the chromatograms were identified as gallic acid, catechin, epicatechin and ferulic
acid by comparing their retention times and pseudomolecular ion [M-H]− with authen-
tic standards. In details, gallic acid was identified with ESI-MS pseudomolecular and
fragment ions at m/z 169.0 [M-H]− and 125.0 [M-COOH]− , respectively. Catechin and
epicatechin, with the same pseudomolecular ions m/z (289.1 [M-H]− ), and fragment ions
m/z 245 (a loss of a –CH2 CHOH-), were identified by comparing with the authentic stan-
dards for their retention times. Ferulic acid was identified with 192.9 [M-H]− and 133.8
[M-H-COO-CH3-]− .
To conduct the quantification for each phenolics, a calibration was constructed
by measuring six different concentrations of the standard solution. The content of each
phenolic compound could be found in Table 1 which shows that catechin and epicatechin

Table 1 Identification of polyphenolic compounds in pineapple peels by HPLC–ESIMS.

Other
Molecular [M-H]− fragment ions Tentative
Rt/min weight (m/z) (m/z) identification Molecular structure Contenta

2.602 170 169.0 125 Gallic acid HO

31.76 ± 2.28a
O
HO
OH

HO

16.118 290 289.1 245, 205 Catechin OH

58.51 ± 3.59b
OH

HO O

OH
OH

24.447 290 289.1 245, 205 Epicatechin OH

50.00 ± 4.39c
OH

HO O

OH
OH

24.504 194 192.9 133.8 Ferulic acid HO 19.50 ± 1.93d

OH
OH3C

All values are expressed as means ± standard deviation.

a Results expressed: as mg/100 g dry peel. different letters in this column mean significant differences

(p < 0.05).
 POLYPHENOLICS IN PINEAPPLE 1811

A.

B.

Figure 1 HPLC chromatograph of pineapple peel extracts detected at (A) 280 nm and (B) 320 nm. Peak numbers
refer to Table 1.

were the two major monomeric polyphenolics in pineapple peels, followed by gallic acid
and ferulic acid.
It has been reported that some fruits and teas are particularly rich in flavanol
monomers (catechins).[28] For examples, research on composition and content of grape
(Narince) showed that major polyphenolic compounds were gallic acid, (+)-catechin,
(−)-epicatechin, and rutin.[29] The main polyphenolic compounds in apples are chloro-
genic acid, (+)-catechin, and (−)-epicatechin.[30] These researches as well as the authors
results indicate that catechin and epicatechin exist widely in high amount in some fruits
with high antioxidant capacity.

Antioxidant Capacity of PPE

The antioxidant capacity of PPE was measured by DPPH q scavenging capacity and
phosphomolybdenum method. Our results found that the IC50 value of PPE was 1.13 mg/ml
1812 LI ET AL.

Table 2 Antioxidant capacity of PPE and the major polyphenolic compounds.

Sample PPE Catechin Epicatechin Gallic acid Ferulic acid

IC50a 1.13 ± 0.03 107.39 ± 8.52a 103.24 ± 9.37a 87.07 ± 4.23b 139.70 ± 10.57c
TACb 0.037 ± 0.003 1.754 ± 0.105a 1.930 ± 0.037b 0.692 ± 0.014c 0.622 ± 0.053c

All values are expressed as means ± standard deviation.

a Results expressed: as mg/ml for PPE; as µM for catechin, epicatechin, gallic acid, and ferulic acid measured

by DPPH q scavenging assay;

b TAC; Results expressed: as g ascorbic acid equivalents/g for PPE; as mol ascorbic acid equivalents/mol for

catechin, epicatechin, gallic acid, and ferulic acid measured by phosphomolybdenum method.
ab Different letters in each row (except for PPE) mean significant differences (p < 0.05).

(Table 2). Hossain et al.[10] has reported that 0.25 mg/ml methanolic extracts of pineapple
(Calendar, India) could inhibit nearly 90% of DPPH q, while we found that only 19.75%
of DPPH q was scavenged by 0.25 mg/ml of PPE. Moreover, it has also been reported
0.1 mg/ml pineapple (Ananas comosus) residues extracts quenched approximately 17%
of DPPH q.[13] The main reasons accounting for this could be differences of the phenolic
contents in the extracts and pineapples’ locations, prevailing climatic conditions, as well as
postharvest handling.
There are wide variations between the antioxidant capacities of different fruits
or vegetables. In the present article, the IC50 value of pineapple (Bali, China) peels
was 1.13 mg/mL, which was higher than that of red grape pomace (Nerod Avola)
(0.039 mg/ml),[31] but lower than that of apple pomace (Reinders) extract (6.33 mg/ml).[32]
The TAC of PPE was measured spectrophotometrically through phosphomoly-
bdenum method, which is based on the reduction of Mo (VI) to Mo (V) by the antioxidant
agents.[33] One gram of PPE exhibited TAC equivalent to 0.037 g ascorbic acid (Table 2)
which was much lower than 0.34 g ascorbic acid/g pineapple extracts (Calendar, India)
reported by Hossainet al.,[10] but close to 0.042 g ascorbic acid/g grape (Vitis vinifera)
seed extract.[34]

Antioxidant Capacity of Pure Polyphenolic Compounds

DPPH q scavenging capacity and phosphomolybdenum method were also used to eval-
uate the antioxidant of the four polyphenolic compounds found in pineapple peels. DPPH q
scavenging capacities of the two main polyphenolics in pineapple peels — catechin and
epicatechin, were nearly the same (Table 2). Catechin and epicatechin are two flavonoid
stereoisomers, so they may share the same level of DPPH q scavenging capacity.
Gallic acid was found to be the most active DPPH q radical scavenger while ferulic
acid was the least active scavenger (Table 2). DPPH q has a single electron and the method
has been widely used to evaluate the hydrogen-donating ability of compounds by accepting
an electron or hydrogen radical.[35] Gallic acid is one of the hydroxybenzoic acids with
the C6 –C1 structure, while ferulic acid belongs to hydroxycinnamic acids with a three-
carbon side chain (C6 –C3 ).[36] The antioxidant activity of polyphenolic acids increases
with increasing degree of hydroxylation, as in the case of the trihydroxylated gallic acid,[7]
which exhibited higher antioxidant capacity than ferulic acid with only one hydroxyl group.
The order of DPPH q scavenging capacities of the four polyphenolic compounds
present in pineapple peels was in the order gallic acid > epicatechin = catechin > ferulic
acid (Table 2). Yilmaz and Toledo[37] have studied the proton-donating activity of catechin,
 POLYPHENOLICS IN PINEAPPLE 1813

epicatechin, and gallic acid by the galvinoxyl method. The result indicated 2.9, 3.1, and
3.2 protons were available for antioxidative donation per mole of catechin, epicatechin,
and gallic acid, respectively. In this research, the order of DPPH q scavenging capacity of the
four compounds is in general agreement with that expressed in terms of protons available
for antioxidative donation, which could be found structurally.
However, when assayed by phosphomolybdenum method, the order of TAC of
these polyphenolic compounds was epicatechin > catechin > gallic acid = ferulic acid,
which differed from that of the DPPH q scavenging capacity. Table 2 showed that flavanols
(catechin and epicatechin) exhibited higher TAC than phenolic acids (gallic acid and ferulic
acid) and among these, epicatechin exhibited the highest antioxidant capacity (1.930 mol
ascorbic acid equivalents/mol). It is important to realize that the TAC of the four pure
compounds determined by phosphomolybdenum method may be different from a proton
donation as determined by DPPH q scavenging assay and a further study is required to better
understand the mechanisms involved in these differences.

Effect of Interactions of Four Polyphenolics on Their Antioxidant

Capacity
The antioxidant capacities of combinations of two polyphenolic compounds with the
molar ratio of 1:1 obtained from DPPH q scavenging capacity and phosphomolybdenum
method were compared with their theoretical values. In this way, any statistically signif-
icant effects resulting from these combinations could be established whether synergistic,
antagonistic or additive. Antioxidant capacities of two phenolics combined in a mix-
ture were reported by Heo et al.,[22] including catechin, chlorogenic acid, and quercetin,
etc. Hidalgoet al.[21] has studied the effects of flavonoid-flavonoid interactions on their
antioxidant activity by mixing two kinds of flavonoids at different molar ratios. All of the
selected phenolics are very common in fruits. Combinations of two phenolics were chosen
since they may provide basic information to determine what type of interaction contributes
to TAC in the complicated model mixtures of phenolics.
The results of DPPH q scavenging capacities are summarized in Table 3, and no syn-
ergistic effect was revealed for the different polyphenolics combinations. When ferulic
acid was mixed with epicatechin or gallic acid, the statistic analysis suggested that there
was an additive effect. Antagonistic effects were found in the combinations of catechin-
epicatechin, catechin-gallic acid, catechin-ferulic acid, and epicatechin-gallic acid. It has
been reported that when catechin combined with epicatechin, there was an antagonis-
tic effect using DPPH q scavenging assay and the difference was –14.1%.[21] The results
indicated that this difference was –22.16%.

Table 3 Antioxidant activity of polyphenolics combinations measured by DPPH q scavenging assay.

Combinations (1:1) CAT + ECAT CAT + GA CAT + FA EPCAT + GA EPCAT + FA GA + FA

IC50 (µM) 128.65 ± 3.75 121.19 ± 2.88 141.55 ± 5.72 113.08 ± 5.15 135.13 ± 6.49 127.79 ± 4.62
Difference in −22.16 ± 5.35a −24.64 ± 8.72a −14.57 ± 8.05a −18.83 ± 4.58a −11.28 ± 5.52 −12.70 ± 6.39
DPPH qScavenging
capacity (%)

Catechin (CAT), epicatechin (ECAT), gallic acid (GA), and ferulic acid (FA).
All values are expressed as means ± standard deviation.
a Within rows indicates significant difference was found between theoretical and real values at P < 0.05.
1814 LI ET AL.

Table 4 Theoretical and real values of total antioxidant capacity of polyphenolics combinations measured by
phosphomolybdenum method.

Combinations(1:1) CAT + ECAT CAT + GA CAT + FA EPCAT + GA EPCAT + FA GA + FA

Theoretical valuea 1.932 1.201 1.225 1.383 1.396 0.664

Real valuea 1.842 ± 0.14 1.223 ± 0.31 1.188 ± 0.21 1.311 ± 0.35 1.276 ± 0.33 0.657 ± 0.29

Catechin (CAT), epicatechin (ECAT), gallic acid (GA), and ferulic acid (FA).All values are expressed as means
± standard deviation.
a Results expressed: as mol ascorbic acid equivalents/mol; the statistical analysis in each column indicates there

is no significant difference between the theoretical and real values at P < 0.05.

The reason accounting for these antagonistic effects of DPPH q scavenging capac-
ity may be that a hydrogen-bonding between polyphenolics occurs in these interactions,
decreasing the availability of the hydroxyl groups, which may in turn reduce the possibility
of interaction with the DPPH q.[21] In this study, it was found that catechin and epicatechin
had nearly the same level of DPPH q scavenging capacity. However, when they were paired
with the other two phenolic acids respectively, different extents of antagonistic effects were
found. To explain the observed variability in inhibition of DPPH q radicals between the two
combinations, the steric structure difference between these two isomers could be responsi-
ble for the different interactions in these mixtures. In contrast with catechin, epicatechin
possesses higher steric hindrance, which may decrease the hydrogen-bonding between
polyphenolics. Moreover, the two additive effects were found in the interactions of ferulic
acid with epicatechin or gallic acid. So the small number of hydroxyl group of ferulic acid
may account for the small quantity of hydrogen-bonding with other polyphenolics.
The results of interactions between the two polyphenolic compounds measured by
phosphomolybdenum method also indicated that there was no synergistic effect. However,
all the six combinations showed additive effects (Table 4), which was different from DPPH q
scavenging capacity. Although there was a slight decrease in the combination of catechin-
epicatechin when comparing with summation of their antioxidant capacities, no significant
difference was found between theoretical and real values. When gallic acid was mixed with
ferulic acid, the real value was nearly the same as the theoretical value. It was obvious
that additive effects were found in the combinations of ferulic acid-epicatechin and ferulic
acid-gallic acid using both antioxidant assays.
To further investigate the interactions of two polyphenolics by phosphomolybdenum
method, various molar ratios were used according to Hidalgo et al.[21] in the model mix-
tures. The results (Table 5) indicated that there was no significant synergistic/antagonistic
but an additive effect between the real and theoretical values. Typical example could be
found when epicatechin mixed with ferulic acid with the molar ratio of 0.5:1.5 (mol:mol).
The real value was only 0.06% higher than its theoretical value. This suggested that TAC
was equal to the summation of individual antioxidant capacity of polyphenolics. Finally,
the antioxidant capacity of the four polyphenolics mixed at the concentration according
to their molar ratio in pineapple peels was determined by phosphomolybdenum method.
The results showed the real value was only 6.77% lower than its theoretical value which
indicated an additive effect on their antioxidant capacity.

CONCLUSIONS
Pineapple (Bali, China) peels were extracted with methanol and the extraction yield
was 24.95% with the polyphenols purity of 31.98 mg gallic acid equivalents/g. The major
 POLYPHENOLICS IN PINEAPPLE 1815

Table 5 Theoretical values (TV) and real values (RV) of total antioxidant activitya of polyphenolics combinations
measured by phosphomolybdenum method.

Molar ratiob 0.5:1.0 0.5:1.5 1.0:0.5 1.5:0.5

Combinations TV RV TV RV TV RV TV RV

CAT + ECAT 2.978 2.697 ± 0.022 4.037 4.161 ± 0.038 2.806 2.415 ± 0.177 3.682 3.269 ± 0.122
CAT + GA 2.076 2.028 ± 0.014 2.952 2.740 ± 0.086 1.524 1.585 ± 0.056 1.849 1.939 ± 0.014
CAT + FA 1.562 1.730 ± 0.029 1.849 1.771 ± 0.018 2.095 1.925 ± 0.022 2.971 2.718 ± 0.042
EPCAT + GA 2.432 2.308 ± 0.089 3.486 3.258 ± 0.170 1.702 1.903 ± 0.061 2.027 1.967 ± 0.091
EPCAT + FA 1.740 1.927 ± 0.058 2.083 2.084 ± 0.050 2.450 2.588 ± 0.058 3.504 3.859 ± 0.110
GA + FA 1.010 1.143 ± 0.009 1.352 1.509 ± 0.019 0.991 1.127 ± 0.015 1.316 1.365 ± 0.057

Catechin (CAT), epicatechin (ECAT), gallic acid (GA), and ferulic acid (FA).
a Results expressed: as mol ascorbic acid equivalents; the statistical analysis indicates there is no significant

difference between the theoretical and real values of each combination at P < 0.05.
b Polyphenolic compounds were mixed at different molar ratios (mol:mol).

polyphenolic compounds existing in pineapple peels were catechin (58.51 mg/100 g dry
extracts), epicatechin (50.00 mg/100 g), gallic acid (31.76 mg/100 g), and ferulic acid
(19.50 mg/100 g). These four polyphenolic compounds exhibited their antioxidant capac-
ities with structure-activity relationships. Results of polyphenolics interactions indicated
no synergistic effects. However, a more detailed study using other experimental models
is required in order to better understand the antioxidant mechanisms involved in these
interactions.

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