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Antioxidant Activity and Protective effect of Banana Peel against Oxidative Hemolysis of Human Erythrocyte at Different Stages of Ripening

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Abstract

Phytochemicals such as polyphenols and carotenoids are gaining importance because of their contribution to human health and their multiple biological effects such as antioxidant, antimutagenic, anticarcinogenic, and cytoprotective activities and their therapeutic properties. Banana peel is a major by-product in pulp industry and it contains various bioactive compounds like polyphenols, carotenoids, and others. In the present study, effect of ripening, solvent polarity on the content of bioactive compounds of crude banana peel and the protective effect of peel extracts of unripe, ripe, and leaky ripe banana fruit on hydrogen peroxide-induced hemolysis and their antioxidant capacity were investigated. Banana (Musa paradisica) peel at different stages of ripening (unripe, ripe, leaky ripe) were treated with 70% acetone, which were partitioned in order of polarity with water, ethyl acetate, chloroform (CHCl₃), and hexane sequentially. The antioxidant activity of the samples was evaluated by the red cell hemolysis assay, free radical scavenging (1,1-diphenyl-2-picrylhydrazyl free radical elimination) and superoxide dismutase activities. The Folin-Ciocalteu's reagent assay was used to estimate the phenolic content of extracts. The findings of this investigation suggest that the unripe banana peel sample had higher antioxidant potency than ripe and leaky ripe. Further on fractionation, ethyl acetate and water soluble fractions of unripe peel displayed high antioxidant activity than CHCl₃ and hexane fraction, respectively. A positive correlation between free radical scavenging capacity and the content of phenolic compound were found in unripe, ripe, and leaky ripe stages of banana peel.
Antioxidant Activity and Protective effect of Banana Peel
against Oxidative Hemolysis of Human Erythrocyte
at Different Stages of Ripening
Shanthy Sundaram &Shadma Anjum &
Priyanka Dwivedi &Gyanendra Kumar Rai
Received: 31 August 2010 /Accepted: 10 February 2011 /
Published online: 3 March 2011
#Springer Science+Business Media, LLC 2011
Abstract Phytochemicals such as polyphenols and carotenoids are gaining importance
because of their contribution to human health and their multiple biological effects such as
antioxidant, antimutagenic, anticarcinogenic, and cytoprotective activities and their
therapeutic properties. Banana peel is a major by-product in pulp industry and it contains
various bioactive compounds like polyphenols, carotenoids, and others. In the present
study, effect of ripening, solvent polarity on the content of bioactive compounds of crude
banana peel and the protective effect of peel extracts of unripe, ripe, and leaky ripe banana
fruit on hydrogen peroxide-induced hemolysis and their antioxidant capacity were
investigated. Banana (Musa paradisica) peel at different stages of ripening (unripe, ripe,
leaky ripe) were treated with 70% acetone, which were partitioned in order of polarity with
water, ethyl acetate, chloroform (CHCl
3
), and hexane sequentially. The antioxidant activity
of the samples was evaluated by the red cell hemolysis assay, free radical scavenging
(1,1-diphenyl-2-picrylhydrazyl free radical elimination) and superoxide dismutase activi-
ties. The FolinCiocalteu's reagent assay was used to estimate the phenolic content of
extracts. The findings of this investigation suggest that the unripe banana peel sample had
higher antioxidant potency than ripe and leaky ripe. Further on fractionation, ethyl acetate
and water soluble fractions of unripe peel displayed high antioxidant activity than CHCl
3
and hexane fraction, respectively. A positive correlation between free radical scavenging
capacity and the content of phenolic compound were found in unripe, ripe, and leaky ripe
stages of banana peel.
Keywords Musa paradisica .Erythrocyte lysis .Superoxide dismutase .Radical scavenging
activity .Phenolic content .Ripening
Appl Biochem Biotechnol (2011) 164:11921206
DOI 10.1007/s12010-011-9205-3
S. Sundaram (*):S. Anjum :P. Dwivedi
Centre for Biotechnology, University of Allahabad, Allahabad 211002, India
e-mail: shanthy_s@rediffmail.com
S. Anjum :G. K. Rai
Centre of Food Technology, University of Allahabad, Allahabad 211002, India
Introduction
Banana is the second largest producer after citrus fruit account for only around 16% of
global world product. India is contributing 27% of the world for banana production [1].
From an environmental perspective, it is vital that plant byproducts produced by the agro-
food industry be reused. Peels of a variety of fruits and plants are gaining attention as a
natural source of polyphenols and bioactive compounds, which possesses various beneficial
effects on human health. Apple pomace has been shown to be a good source of polyphenols
and exhibits strong antioxidant and anti-proliferative activity [2]. Mango peels contain a
good content of polyphenols and dietary fiber [3]. Grape pomace, a by-product of wine
industry, is a good source of anthocyanins, catechins, flavanoids, phenolic acids, and
dietary fiber. Tomato peels are reported to be a good source of carotenoids .There is very
little or negligible work on the physiological and biochemical evaluation of edible (pulp)
and non-edible portion (peel) of banana fruit [4].The main by-product of the banana
processing industry is the peel, accounting for 30% of the fruit, which constitutes
environmental hazard [5].High dietary fiber content of banana peels makes them promising
for a variety of applications in nutraceutical and medicinal application. By doing this, the
aim is to prevent oxidative stress related disorder in human. The exact mechanism behind
the aging is not clearly known but there is sufficient evidence suggesting that aging could
be caused by accumulation of reactive oxygen species [6]. Free radical damages the
structural and functional components of the cell such as lipid, protein, carbohydrates, DNA,
and RNA. Banana peel contains high content of micronutrient compared to fruit pulp [7]. It
attracts great attention because of their nutritional and antioxidant properties, especially the
compounds, ascorbate, catechin, gallocatechin, and dopamine. Due to the importance of
these compounds, it is necessary to understand its initial production and losses during fruit
development, ripening, and maturation. The changes in enzymatic and nonenzymatic
components of the plant antioxidant defenses have been previously described during fruit
ripening in several plants. For example, Jime'nez et al. [8] reported that the levels of
glutathione (GSH) and ascorbate (AsA) in tomato increased during the ripening process.
However, a different result has been shown by Wang and Jiao [9], who found that GSH,
AsA, and related enzyme should decline with ripening of blackberry fruit.
The present investigation was undertaken to evaluate the antioxidant activity of banana
fruit peel with the aim of exploiting the potential value of the waste banana peels and the
role of these antioxidant systems in the degradation of membranes during maturation,
ripening, and senescence of banana fruit peel. In the present studies, we have attempted to
determine the changes in the antioxidant systems at different stages of ripening of the
banana peel (Musa paradisica) and special emphasis was laid on maximum extraction with
solvents of varying polarity. In addition, superoxide dismutase (SOD) and 2,2-diphenyl-
1-picryl hydrazyl (DPPH) activities were examined after in vitro incubation of the extract
with fresh human erythrocyte.
Materials and Methods
Chemicals and Reagents
DPPH, gallic acid, methionine, nitroblue tetrazolium (NBT), riboflavin, ascorbic acid
(vitamin C) were purchased from Sigma-Aldrich. All other reagents and chemicals of
analytical grade were procured from local sources and milli-Q quality water was used.
Appl Biochem Biotechnol (2011) 164:11921206 1193
Sample Collection and Extraction
Banana (M. paradisica, local name Bhusawal keli) fruits were purchased from local
market of Kaushambi, Uttar Pradesh, India, at different stages of ripening without any
ethylene and stored at 20 °C for 24 h before being extracted. Harvesting and
determination of maturity was carried out by skilled workers, combining the following
techniques: day count, chromacity, firmness, pH, and flavor of fruits; the mature young
fruits were harvested carefully when peel tightly intact with edible part of fruit is in
fully green stages and left for 1 day at room temperature .The banana fruits were peeled
to get peel with firm texture and no smell. Some of the fruits were left for another
8 days at room temperature to ripen and their flesh became a soft and developed a
typical banana flavor. Peel separated from the flesh part of banana fruit with yellow
stages without brown spotting. Whereas overripe stages have a strong smell with leaky
edible portion when fruits are left for another 3 days (in this stages peel were yellow
brown). The whole fruit of banana was washed thoroughly under running tap water,
dried on paper towel. The peel tissues of fresh unripe green, ripe yellow, and leaky ripe
brown with traces of yellow banana fruit (200 g) were heated in 750 ml of distilled
water for 5 min. Banana peel was homogenized with 70% acetone twice at room
temperature using pre-chilled pestle and mortar for 48 h under shaking conditions using
an electrical shaker. The combined extracts of 70% acetone extracts were then filtered
and centrifuged at 4 °C in Beckman refrigerated centrifuge machine for 15 min at
15,000×g. The supernatant was concentrated to 150 ml. This aqueous extract was
partitioned into a chloroform (CHCl
3
) and water, using a separator and then extracted
with aqueous saturated ethyl acetate. Fractionate and 70% acetone extract were
concentrated using a rotary evaporator with the water bath set at 40 °C. The percentage
yieldofextractsrangedfrom7%to19%w/w. The antioxidant activities of hexane,
CHCl
3
, ethyl acetate (EtOAc), and water fractions were measured at different stages of
peel.
Preparation of Test and Standard Solution
The extract and standard antioxidant ascorbic acid were dissolved in freshly distilled
DMSO (Dimethyl Sulfoxide) separately and used for in vitro antioxidant activity using
different method. For total phenol content a solution of extract in methanol was used.
Gallic acid was dissolved in distilled water and used as a standard for total phenol
estimation [10].
Determination of Total Phenolic Content
The amount of total soluble phenolics was determined according to the FolinCiocalteu's
method Anagnostopoulou et al. with slight modification [11]. The reaction mixture consisted
of 0.5 ml of the extract (10 mg/ml), 5 ml of distilled water, and 0.5 ml of the Folin
Ciocalteu's reagent. After a period of 3 min, 1 ml of saturated 5% sodium carbonate solution
was added. The 10-ml volumetric flasks were shaken and allowed to stand for 1 h. The
absorbance was measured at 725 nm (each measurement repeated three times) in a UV-visible
spectrophotometer (the same equipment was used in the DPPH test). The total phenolic
content was expressed as milligrams gallic acid per gram dry extract, milligram gallic acid/
100 g dry fruit peel. Total content of phenolic compounds of extracts in gallic acid
equivalents (GAE) was calculated by the following formula [12].
1194 Appl Biochem Biotechnol (2011) 164:11921206
C¼c:V=m
Where
CTotal content of phenolic compounds, milligrams per gram plant extract, in GAE
cThe concentration of gallic acid established from the calibration curve, milligrams per
milliliter
VThe volume of extract, milliliters
MThe weight of pure plant methanolic extract (grams).
Determination of Antioxidant Activity
Scavenging Activity on 1, 1-Diphenyl-2-picrylhydrazyl Radicals
Inhibiting action of dry matter of banana peel tissue against stable free radical was
measured by the following method: the radical scavenging activities of the plant extracts
against 2, 2-diphenyl-1-picryl hydroxyl radical (Sigma-Aldrich) were determined by UV
spectrophotometer at 517 nm. Radical scavenging activity was measured by a slightly
modified method previously described Ayoola et al. [13]; Leong and Shui [14]. The
following concentrations of the extracts were prepared 10, 50, 100 mg/ml. (Analar grade).
Vitamin C was used as the antioxidant standard at a stock concentration of 10 mg/ml. One
milligram of the extract was placed in a test tube and 3 ml of methanol was added followed
by 0.5 ml of 1 mM DPPH in methanol. A blank solution was prepared containing the same
amount of methanol and DPPH. The mixture was shaken immediately after adding DPPH
and allowed to stand at room temperature in dark and the decrease in absorbance at 517 nm
was measured after 30 min until the reaction reached a plateau. These experiments were run
in duplicate. The radical scavenging activity was calculated using the following formula:
% inhibition ¼AoA½=Ao
fg
100
Where Ao is the absorbance of DPPH without sample (control) at 517 wavelengths; Ais the
A
517
of sample and DPPH (test sample).
Scavenging Effect on Superoxide AnionsNBT Reduction System
SOD activity was assayed using the NBT method [15]. SOD activity of banana peel tissue
extract in 70% acetone, water, ethyl acetate, chloroform, and hexane were determined by
photochemical reduction of NBT, according to Giannopolitis and Ries [16] with slight
modifications, using the assay system consisting of methionine, riboflavin, and NBT. NBT
was reduced to blue formazan by O
2
, which has a strong absorbance at 560 nm. However, the
presence of SOD inhibits this reaction. Two sets of each sample were used. The reaction mixture
consists of 1.3μM riboflavin, 13 mm methionine, 63 μM NBT, 0.05 M sodium carbonate
(pH 10.2) and the appropriate volume of extract. Distilled H
2
O was added to bring to the final
volume of 3 ml. The test tubes were inverted twice. One set of the reaction tube was covered
with a black cloth as control. The other set was placed approximately 30 cm below a blank of
two 15-W fluorescent lamps. The reaction was initiated by turning the light on for 10 min.
Following light exposure, the tubes were covered with a black cloth to prevent further reaction.
Illuminated mixtures lacking enzyme developed maximum color, while non-illuminated
Appl Biochem Biotechnol (2011) 164:11921206 1195
mixtures did not develop color and were used as control. The absorbances of the illuminated
mixtures were compared to non-illuminated mixtures using a spectrophotometer at 560 nm.
From the graph, the volume of enzyme extract corresponding to 50% inhibition of the reaction
was calculated and considered as one enzyme unit. Protein concentration was determined
according to Bradford using bovine serum albumin as a standard. [17]. SOD activity was
expressed as enzyme units per gram fresh weight units per gram fresh weight of banana peel.
Scavenging Effect on H
2
O
2
-Induced Human Blood Hemolysis (Antihemolytic Activity)
Preparation of Erythrocytes Human blood (510 ml) sample was withdrawn from group of
healthy female (2025 year). Samples were collected into centrifuged tubes coated with
anticoagulant EDTA and the Red Blood Cells were collected by centrifugation of blood at
1,000×gat 4 °C for 20 min. The buffy coat and plasma were removed using a pipette. The
crude RBCs were washed with the same volume of phosphate-buffered saline (PBS) pH 7.4
followed by centrifugation twice. The packed RBCs were then suspended in four volumes
of PBS solution [18].
Assay for Free Radical-Mediated Hemolysis The inhibition of human erythrocyte
hemolysis was done according to Rafat et al. [18] with slight modification. Here, the
erythrocyte hemolysis was performed with H
2
O
2
as free radical initiator, on human
blood. To 500 μl of suspension of erythrocyte in IPB (isotonic phosphate buffer solution
at pH 7.4), 1 ml of dry extract of banana peel extracted in solvent water, chloroform,
70% acetone, ethyl acetate, hexane was added. Concentration of sample 100 mg/ml
prepared in 5% DMSO dissolved in isotonic phosphate buffer pH 7.4 and kept at
4 °C until required. The working solution 10 mg/ml for assay was made by diluting the
stock solution with IPB. The reaction mixture was shaken gently while being incubated
at 37 °C for 3 h. A positive control was prepared by pretreating the erythrocyte
suspension with 1 ml of 10 mg/ml concentration of vitamin C dissolved in IPB. The
inhibitory effect of the extract was compared with standard antioxidant vitamin C. The
non-pretreated erythrocyte suspension was used as the negative control. Oxidative stress
was then induced by adding 1 ml of 10 mM hydrogen peroxide (H
2
O
2
) and incubated at
37 °C for 150 min. After incubation, the volume of all pretreated and non-pretreated
erythrocyte suspensions were adjusted to 9 ml by adding IPB. The released hemoglobin
in the supernatant of the mixtures was measured using spectrophotometer at 540 nm.
Erythrocyte hemolysis in pure water was based on complete erythrocytes hemolysis
(100%) while hemolysis of the pretreated and non-pretreated erythrocytes was
expressed as a percentage of this value.
Percentage inhibition was calculated as described by the equation (1 A
antioxidant
/
A
H2O2
)×100, where A
H2O2
is the absorbance of sample containing no extract and A
antioxidant
is the absorbance of sample containing extract [19].
Statistical Analysis
All measurements were carried out in triplicate and the results are statistically analyzed
using the statistical program to determine the average value. Analysis of variance was
performed using two-way ANOVA and the significant differences (p<0.05) between the
means were performed to determine the effect of solvent polarity and ripening stages on the
content of bioactive compounds and antioxidant capacity of banana peel.
1196 Appl Biochem Biotechnol (2011) 164:11921206
Results and Discussion
Different extracts of banana peel in different stages of ripening possess potent antioxidant
activity, using the DPPH assay and the SOD assay. Fruit ripening has been described as
oxidative phenomenons [20], which require a turnover of active species such as H
2
O
2
and
superoxide anion. It has been reported that tolerance of plant to condition-causing damage
may be associated with their higher ability to remove free radical and active oxygen species
through active oxygen species (AOS) detoxifying enzyme, SOD, and peroxidases implying
that they may play a protecting role from oxidative damage [21].
Banana peel is rich in photochemical compound mainly antioxidant. Our results show
the total phenolic content estimated to be in the range of 1.74±0.09 GAE g/100 g in unripe
stages which is reported by Someya et al. [22] and Nguyen et al. [23].
Table 1shows the results of the extraction of the unripe, ripe, and leaky ripe banana peel
sample with 70% acetone (acetone/water). The highest yield (5.2%) was obtained from
unripe fruit peels while the lowest yield (2.98%) was from leaky ripe fruit peels. The total
phenolic contents measured in unripe to leaky ripe peel was GAE gram per 100 g dry
extract using FolinCiocalteu's assay ranged from 1.74 to 0.844, it is noted that unripe
banana peel demonstrated significantly higher total phenolic content than that of ripe and
leaky ripe peels. Ethyl acetate seems to be the solvent that concentrates best phenolic
substances of intermediate polarity. This is in accordance with the findings by Chung et al.
[24] and Parejo et al. [25].
Kiyoshi and Wahachiro [26] reported that in immature stages 60% polyphenol
compounds with molecular weight (MW) above 2×10
5
and other 40% were those with a
MW below 2 × 10
5
. During ripening polyphenol compounds with MW 2×10
5
and
astringency disappeared. As ripening proceeds only low MW (below 2 × 10
5
) polyphenolic
compounds remain. Our result is in line with this, as the ripening proceeds, polyphenol
compound were degraded.
Choosing the appropriate extraction conditions and solvent is one of the most important
factors in obtaining extracts with a high content of bioactive compounds. The results
obtained coincide with reports that the mixture of acetone/water (70% acetone extract) is an
effective solvent for extracting phenolic compounds (most of the polar and non polar
antioxidants were extracted in this solvent) [2,27]. Our results reinstate the view of earlier
studies that extracting banana peel with 70% was not only very efficient but also produced
extracts with high antioxidant capacity, as confirmed by various model systems. This may
be due to variation in the quality and quantity of phenolic compounds and other bioactive
compounds present in the different extracts, which on fractionation given higher
concentration in ethyl acetate solvent. [5].
Banana peel powders were consecutively fractionated with organic solvents with
increasing polarity. The yields of these extractions and the total phenolic contents are
reported in Table 2. It was found that pooled fraction of extract with polar solvent, gave the
Table 1 Yield of acetone extract (percentage of w/w) and total phenolic content from the banana peel tissue
at the different stage of ripening
Sample (ripening stage) Sample code Percentage of yield (w/w) GAE mg/100 g
Unripe banana peel UP 5.23± 0.585 1743.93± 90.12
Ripe banana peel RP 3.56 ± 0.128 1091.41±47.8
Leaky ripe banana peel LRP 2.63± 0.124 844.61± 10.7
Appl Biochem Biotechnol (2011) 164:11921206 1197
highest yields (1626%) from the 70% acetone extract of banana peel at different stages of
ripening, whereas the yields obtained after fractionation with hexane were the lowest (0.6
0.9%) [27]. The hexane extracts not only gave low yields, but also contained the lowest
levels of phenolic compounds (0.10.3 mg/ml). The extracts of the intermediate polar
solvent, EtOAc contained substantially higher levels of phenolic compounds (ranging from
0.071 to 0.996 GAE g/100 g) than polar water fraction and comparable with those found in
the 70% acetone extracts (Table 1). Similar results were obtained by Mokbel and Fumio
Hashinaga [28] in Cavendish variety of banana peel. Low recovery of phenols in water
fraction could be caused by the oxidation of phenolic compound by polyphenol oxidase.
While, in acetone, ethyl acetate, and chloroform the enzyme is inactivated (Table 3).
Figure 4shows that the antioxidant activity (AEAC) values of the 70% extracts of the
banana peel at the different stages of ripening were between 1.21 + 0.10 g/100 g and 0.485 +
0.07 g/100 g. The antioxidant activity of the extracts was significantly lower than that of
Table 2 Yield and total phenolic content of fractionated dry extracts per 100 g of banana peel tissue at
different stage of ripening
Ripening stage Solvent Sample code Percentage of yield (w/w) GA mg/100 g
Unripe Water WUP 48.76± 1.50 741.5± 4
Chloroform CUP 1.92± 0.223 35.12± 3.0
Ethyl acetate HUP EUP 37.09± 2.77 992.46± 17.9
Hexane HUP 0.41± 0.040 87.63 ±3.65
Ripe Water WRP 59.1± 2.9 371.74 ± 3.82
Chloroform CRP 0.620± 0.055 17.85± 2.45
Ethyl acetate ERP 18.71 ± 1.73 630.14±2.85
Hexane HRP 0.085± 0.007 68.83±3.32
Leaky ripe Water WLP 74.03± 1.45 155.58± 3.20
Chloroform CLP 0.446 ± 0.096 23.81±2.42
Ethyl acetate ELP 15.16± 0.642 583.64± 2.62
Hexane HLP 0.171±0.027 71.26± 3.26
Table 3 Correlation between phenolic content and AOA of 70% acetone extract of fresh banana peel at
different stages of ripening
Variation in ripening stage (70% acetone extract)
Correlation RR
2
(%)
TPC vs. DPPH 0.998 99.6
TPC vs. SOD 0.049 0.24
TPC vs. hemolysis 0.702 49.2
DPPH vs. SOD 0.101 96.4
DPPH vs. hemolysis 0.663 57.4
SOD vs. hemolysis 0.676
a
45.6
Ripening stages in 70% acetone extract of banana peel in the antioxidant activity, scavenging effects on
superoxide anion, DPPH radicals, and H
2
O
2
-induced heamolysis used for correlation analysis were unripe,
ripe, and leaky ripe, respectively
a
Indicate negative correlation
1198 Appl Biochem Biotechnol (2011) 164:11921206
vitamin C at the same concentration (Fig. 1). The extracts of the unripe peels have both
excellent DPPH* radical scavenging (Figs. 2and 3) and superoxide dismuting capacities
(Figs. 6and 7). The hexane fraction possess the lowest antioxidant activity, as determined
by the DPPH assay (AEAC values ranging from 0.097 to 0.008 AEAC g/100 g) and SOD
assay (ranging from 0 to 0.48 unit/g fruit weight from unripe to leaky ripe) which can be
ascribed to their very low polyphenolic contents (Table 2, Figs. 4,5, and 7). From the result
we can conclude that unripe stages of banana has inherent compensatory mechanism
against oxidative stress (tolerance of plant to condition causing damage may be associated
with their ability to remove AOS through AOS detoxifying enzyme, SOD, CAT, etc.
implying that they play a role protecting fruit from oxidative damage) though have less
protein whereas in leaky ripe sample comparatively high protein causes the higher SOD
activity. On fractionation, higher activity reported in ethyl acetate fraction due to the
peculiar behavior of SOD isoenzyme, whereas in water, enzyme activity degrades at water
pH (Figs. 6and 7).
The extracts of the intermediate polar solvents (ethyl acetate) had substantially higher
activity for DPPH* scavenging (ranging from 0.22±0.025 to 0.70 ± 0.018 AEAC g/100 g)
and SOD activity (ranging from 2.19 to 1.67 unit/g fruit weight unripe to leaky ripe) which
is corresponding to their high polyphenolic levels (Table 2).
Similarly, unripe sample shows better SOD activity in water and chloroform fractions.
This is because polyphenol oxidase exhibits lesser activity in immature stages of fruit and
the enzyme extracts in water is not degraded at that particular pH.
Antioxidant potency of the crude extract was further investigated for their capacity to
protect human erythrocytes against damage in vitro (Fig. 8). It is known that polyphenolics
enhance red blood cell resistance to oxidative stress both in vitro and in vivo [29]. Figure 9
shows the inhibitory effect of different concentrations of unripe crude 70% acetone fraction
(550 mg/ml) on an H
2
O
2
-induced hemolysis of human erythrocytes.
Unripe peel extracts showed higher inhibition on erythrocyte hemolysis than that of ripe
peel, while, not much difference in inhibition was noticed between ripe and leaky ripe peel
extract of banana peel. Ajila and Rao [30] reported acetone extract of mango peel in unripe
stages inhibited the H
2
O
2
-induced hemolysis of rat erythrocytes. The results indicated that
0.196
0.122
0.094866667
0
0.05
0.1
0.15
0.2
0.25
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
concentration of standard polyphenol in µg/ml
O.D of Standard
Unripe
Ripe
Leaky ripe
O.D at
725 nm
Fig. 1 Total phenolic content in GAE/10 mg of banana peel extract in 70% acetone at different stages of
ripening. The data are displayed with mean+ standard deviation (bars) of three replications
Appl Biochem Biotechnol (2011) 164:11921206 1199
the differences in hemolysis of peel extracts of unripe and ripe mango fruits might be due to
the synergistic actions of bioactive compounds present, Since, unripe peel extracts were
rich in polyphenols than the corresponding ripe peel extracts, the increased inhibitory effect
of unripe peel extracts on erythrocyte hemolysis might be due to high content of
polyphenols. Furthermore, with the aim of characterizing the compound, we fractionated
the peel extract using solvents of increasing polarity (hexane, ethyl acetate, chloroform, and
water). This was done to investigate the fraction(s) with higher antioxidant potency
0
20
40
60
80
100
120
.2 mg/ml 1.0 mg/ml.8 mg/ml.4 mg/ml
Unripe Ripe Leaky ripe Ascorbic acid
%Discoloration of DPPH
Fig. 3 Antioxidant activity of 70% acetone extract (acetone/water) of unripe ripe, leaky ripe banana peel
extract at different concentration to scavenge free radical of DPPH. The data are displayed with mean +
standard deviation (bars) of three replication
0
10
20
30
40
50
60
70
80
Acetone
extract
Water extract Ethyl acetate
Unripe Ripe Leak
y
ripe
%Discoloration of DPPH
Chloroform
extract
Hexane
Fig. 2 DPPH radical scavenging effect of the unripe, ripe, and leaky ripe banana peels extract in solvent
70% acetone, water,chloroform, ethyl acetate, hexane. The data are displayed with mean+standard deviation
(bars) of three replications
1200 Appl Biochem Biotechnol (2011) 164:11921206
showing best inhibiting activity of erythrocyte hemolysis. The extracts of the polar solvents
had substantially higher inhibitory effect on erythrocyte hemolysis (ranging from 7.3±
0.025% to 34.3±0.018% inhibition) which is corresponding to their high polyphenolic
levels (Tables 2,3). The ethyl acetate fraction of unripe had about three times higher
activity than that of ripe and leaky ripe, respectively. Thus, it is possible that the compound
with antioxidant capacity may have a polar nature.
Correlation Analysis
Calculated coefficients of correlations between antioxidant activity, scavenging effects on
radicals and contents of phenolic compounds of banana peel fractionate in different solvents
are shown in Tables 4and 5. The antioxidant activity of banana peel fractionate was
significantly correlated with their scavenging effects on superoxide anion (P< 0.01),
DPPH* radicals (P<0.05) and H
2
O
2
-induced hemolysis (P<0.01) on varying extractions in
different solvents, whereas no significant difference was observed on varying the ripening
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Unripe banana
peel
Ripe banana
peel
Leaky ripe
banana peel
70% Acetone extract
AEACg/100gm of fruit weight
Fig. 4 Ascorbic acid equivalent
Antioxidant activity of the 70%
acetone extracts from the banana
fruit peels at different stage of
ripening using DPPH assays.
Calibration was done with ascor-
bic acid standard (DPPH assay)
0
100
200
300
400
500
600
700
800
Water extact chloroform
extract
Ethyl acetate
extract
hexane extract
Unripe banana peel
Ripe banana peel
Leaky ripe banana peel
AEACmg/100gm of banana peel extract
Fig. 5 Ascorbic acid equivalent
antioxidant activity milligrams
per 100 g of fractionated extracts
(Table 2) using DPPH assay.
Ascorbic were used as standards
Appl Biochem Biotechnol (2011) 164:11921206 1201
stages. Therefore, the antioxidant activities of banana peel extracts may be due to their
scavenging effects on radicals and inhibiting the human erythrocyte hemolysis induced by
H
2
O
2
.
For scavenging effects on radicals, high correlations (R
2
=91.3931.02) were observed
between various radicals, on varying the extraction in different solvent in order of polarity
in unripe, ripe, and leaky ripe samples. Results indicate that in unripe sample highest
correlation observed between TPC vs. DPPH (91.39%).Whereas in ripe and leaky ripe peel,
good correlation was observed between TPC vs. SOD (92.7% and 78.85%). The
antioxidant activity (P<0.05) and scavenging effects on superoxide anion, percentage of
inhibition of H
2
O
2
-induced hemolysis (P<0.01) of banana peel extracts was also well
correlated with their contents of total phenolic compounds. The same trends were observed
in the correlation of the content of gallic acid and the antioxidant activity and scavenging
effects on superoxide anion. According to recent reports, a highly positive relationship
existed between total phenolics and antioxidant activity in many plant species [31].
Table 5indicates that in each stages, on varying the solvent polarity the lower correlation
between DPPH and SOD observed in leaky ripe. In ripe and leaky ripe peel samples, SOD
activity is not only the cause of inhibition of erythrocyte hemolysis but other phenolic
compounds are also responsible for it. From Table 4, we can conclude that on varying the
ripening stages (variation factor), highest correlation was observed between TPC and DPPH
0
0.5
1
1.5
2
2.5
Acetone chloroform water Etoac hexane
Unri
p
eRipe Leaky ripe
SOD activity (Unit/g fruit wt.)
Fig. 6 Activities of superoxide
dismutase (SOD) in unit per gram
dry extract of banana peel tissue
extracted in different solvent.
Different stage of growth stands
for unripe, ripe, leaky ripe banana
peel sample. The data are
displayed with mean± standard
deviation (bars) of three
replications
0
10
20
30
40
50
60
Acetone chloroform water Etoac hexane
Unri
p
eRi
p
e Leak
y
ri
p
e
% SOD activity
Fig. 7 Percentage of activity of
superoxide dismutase in banana
peel extract at different stage of
growth to scavenge free radical
O
2
generated from NBT reduc-
tion.The data are displayed with
the mean± standard deviation
(bars) of three replications
1202 Appl Biochem Biotechnol (2011) 164:11921206
0
10
20
30
40
50
60
70
80
90
100
5mg/ml 10mg/ml 25mg/ml 50mg/ml
70% acetone extract
of unripe peel
70% acetone extract
of ri
p
e
p
eel
70% acetone extract
of leaky ripe peel
Standard Ascorbic
acid
%inhibition of R.B.C
lysis
Concentation of extracts
Fig. 9 antioxidant activity of banana peel tissue at different concentration to inhibit RBC lyses in respect of
standard ascorbic acid. The data are displayed with mean± standard deviation (bars) of three replications
0
10
20
30
40
50
60
70
80
90
% inhibition 0f R.B.C lysis
Ascorbic acid
10mg/ml
Ascorbic acid 50
mg/ml
unripe banana
peel (10mg/ml)
Ripe banana peel
(10mg/ml)
Leakyripe banana
peel (10mg/ml)
Acetone Water chloroform Ethyl acetate hexane
-
10
Fig. 8 Antioxidant activity of unripe, ripe, leaky ripe banana peel tissue extract in different solvent:
inhibition of human red blood cell (erythrocyte) expressed as percentage value. Ascorbic acid was used as
positive control
Appl Biochem Biotechnol (2011) 164:11921206 1203
(dependent factor) in ethyl acetate and water extract (independent factor). Similarly, highest
correlation was observed between TPC and SOD in water fractions than that of ethyl acetate.
Conclusion
The present study was conducted with a view to exploit banana peels as a source of
valuable components. Antioxidant activities have been detected in peel portion of banana
fruit from unripe stages to leaky ripe stages. Unripe banana peel has the highest antioxidant
activity and antioxidant enzyme in comparison to the other two stages. Fruit maturation and
ripening was accompanied by the decrease in the activities of phenolic compound and
antioxidant enzyme. These events may lead to increased oxidative stress and cause many
metabolic changes associated with ripening and maturation.
Table 4 Correlation between phenolic content and AOA of banana peel fractionate on dry weight bases
Variation in ripening stage
Correlation R
2
(%) Water Ethyl acetate Chloroform Hexane
TPC vs. DPPH 91.0 93.3 74 7.29
a
TPC vs. SOD 97.0 19.8
a
88.36 76.5
a
TPC vs. hemolysis 38.8 65.4 96.8 18.4
DPPH vs. SOD 98.2 3.96
a
96.4 5.47
a
DPPH vs. hemolysis 68.7 39.69 57.4 97.0
a
SOD vs. hemolysis 55.8 78.4
a
74.9 0.43
Ripening stages in banana peel fractionate in the antioxidant activity, scavenging effects on superoxide anion,
DPPH radicals, and H
2
O
2
-induced hemolysis used for correlation analysis were unripe, ripe, and leaky ripe,
respectively
On varying the stage of banana peel, here the correlation in between antioxidant activity on keeping the
extraction in solvent constant (on taken a single stage of extraction)
a
Indicate negative correlation
Table 5 Correlation between phenolic content and AOA of banana peel fractionate on dry wt bases
Variation in solvent polarity (extraction condition)
Correlation R
2
(%) Unripe Ripe Leaky ripe
TPC vs. DPPH 91.39 80.10 31.02
TPC vs. SOD 51.84 92.7 78.85
TPC vs. hemolysis 32.14 96.4 70.22
DPPH vs. SOD 64.80 55.3 9.92
DPPH vs. hemolysis 48.72 92.5 0.102
SOD vs. hemolysis 77.02 80.46 63.2
Solvent to fractionate banana peel extract in the antioxidant activity, scavenging effects on superoxide anion,
DPPH radicals, and H
2
O
2
-induced hemolysis used for correlation analysis were water, acetone, ethyl acetate,
chloroform, and hexane respectively
On varying the extraction process in different solvent, here the correlation in between antioxidant activity on
taken single stages of banana peel
1204 Appl Biochem Biotechnol (2011) 164:11921206
Higher antioxidant potency of ethyl acetate fraction from 70% acetone extract of banana
peel is due to high phenolic content. The chemical activity of polyphenols in terms of their
reducing properties as hydrogen or electron donating agents predicts their potential for action as
free radical scavengers (antioxidants). Polyphenols possess ideal structural chemistry for free
radical scavenging activities and have been shown to be more effective antioxidants in vitro
than vitamins E and C on a molar basis. One of the antioxidant compounds in the banana peel
was determined to be gallocatechin, which was related to the antioxidant activity of the banana
extract show the maximum extraction in non polar solvent. The low recovery of phenolic
compounds obtained with water could be caused by the oxidation of phenolic compounds by
polyphenol oxidase, whereas in 70% acetone, ethyl acetate, chloroform fraction the enzyme is
inactivated. Thus, from the above investigation, it can be concluded that the waste part of
banana peel is a potential candidate for nutraceutical preparation for their antioxidant potency.
Thus, further work can be carried out to isolate the active moiety responsible for the biological
activity, characterize it and commercialize it.
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... The presence of natural components is significantly influenced by genetic factors, the production area, growing conditions, fruit maturity, post-harvest management and processing application (Vu et al., 2018). The phenolic content, flavonoid content and antioxidant capacity of mature and overripe fruit peel are lower than those of green peel (Sundaram et al. 2011). Likewise, as the peel changed from green to yellow, many individual compounds such as naringin, rutin, norepinephrine and dopamine are reduced. ...
... Therefore, the stage of maturation must be considered on the basis of its potential use. In line with a study by Sundaram et al. (2011), due to the high content of polyphenol in unripe banana peel, the inhibitory effect against oxidative hemolysis of human red blood cells is better than the ripe banana peel extract. ...
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In this study we investigated the potential antioxidant properties of blueberry polyphenolics in vitro and vivo, using red blood cell (RBC) resistance to reactive oxygen species (ROS) as the model. In vitro incubation with anthocyanins or hydroxycinnamic acids (HCA) (0.5 and 0.05 mg/ml) was found to enhance significantly RBC resistance to H2O2 (100 μM) induced ROS production. This protection was also observed in vivo following oral supplementation to rats at 100 mg/ml. However, only anthocyanins were found to afford protection at a significant level, this at 6 and 24 h post supplementation. This protection was not consistent with the measured plasma levels of anthocyanins. Indeed, plasma polyphenolic concentrations were highest after 1 h, declining considerably after 6 h and not detected after 24 h. The difference in absorption between anthocyanins and HCA is likely to have contributed to the observed difference in their abilities to afford protection to RBC. This protection represents a positive role following dietary consumption of polyphenolics from blueberries, against ROS formation within RBC in vivo.