Journal of Food Processing and Preservation ISSN 1745-4549

COMPARISON OF ULTRASOUND AND MACERATION

TECHNIQUES FOR THE EXTRACTION OF POLYPHENOLS
FROM THE MANGO PEEL
MUHAMMAD NAEEM SAFDAR1,2,3, TUSNEEM KAUSAR1 and MUHAMMAD NADEEM1
1
Institute of Food Science and Nutrition, University of Sargodha, Sargodha, Pakistan
2
Food Science and Product Development Institute, National Agricultural Research Centre, Islamabad, Pakistan

3
Corresponding author. ABSTRACT
TEL: 1 92-331-5092088;
FAX: 192-51-9255034; An investigation was carried out to extract the polyphenols from mango peels by
EMAIL: naeemsafdar03@yahoo.com ultrasound-assisted extraction (UAE) and maceration techniques and to assess
the antioxidant potential and quantification of phenolic compounds by high per-
Received for Publication February 10, 2016
formance chromatography. Results showed that highest extraction yield obtained
Accepted for Publication March 14, 2016
through solvent methanol at 80% concentration level while ultrasound-assisted
doi:10.1111/jfpp.13028 extraction was more efficient technique and led to comparatively higher polyphe-
nols content than maceration. Highest polyphenols were extracted with 80% etha-
nol (67.58 mg GAE/g of extract) by employing UAE whereas least polyphenols
(18.66 mg GAE/g of extract) were obtained with 100% ethyl acetate through mac-
eration technique. The high antioxidant activity of mango peel extracts was exhib-
ited in three antioxidant assays with 80% ethanolic extracts had the highest
antioxidant activity 31.51 1 0.79 mM/100 g for ferric reducing antioxidant power,
highest scavenging activity 83.19 1 0.96% and 67.23 1 0.85% for 2,2-Diphenyl-1-
picrylhydrazyl and superoxide anion radical assays, respectively. Strong correla-
tions between total polyphenols and antioxidant activity were observed.

PRACTICAL APPLICATIONS
Polyphenols are the natural antioxidants in plants having a significant role in
human health by prevention of certain oxidative stress-related diseases. Mango
peels are the major agro-industrial wastes in fruit juice processing which are not
further utilized despite being an abundant source of phenolic compounds even
more than the mango pulp. The study revealed that mango peels as a potential
source of phenolic compounds which may be utilized as an ingredient for the
preparation of functional foods.

INTRODUCTION enzyme cofactors, providing protection to dietary antioxi-

Oxidative stress is the condition due to over-production of
free radicals and reactive oxygen species in the body and
considered as the root cause in the pathogenesis of certain
chronic diseases like atherosclerosis, cardiovascular disease,
diabetes mellitus, cancer, hypertension, aging, rheumatoid
arthritis, nerve cell damage like Parkinson’s or Alzheimer’s
disease, obesity, reduced immunity, etc. (Jayakumar et al.
2009; Dai and Mumper 2010; Zujko and Witkowska 2011).
Polyphenols are the natural antioxidants in plants, especially
in fruits and vegetables having a significant role in human
health owing to their scavenging of free radicals, antioxidant
dants like vitamin E and C as well as chelation of pro-
oxidant metal ions in the body (Wojdyło et al. 2007; Osman
et al. 2009; Aadil et al. 2015a; Jabbar et al. 2015a). Various
epidemiological studies have reported a positive correlation
between fruits and vegetable intake with a decline in the rate
of cardiovascular disease, aging, certain cancers and other
degenerative diseases attributed to the antioxidant activity
of phenolic compounds in fruits and vegetables (Dilas et al.
2009; Huang et al. 2011; Sesso et al. 2012).
Fruit wastes, especially peels, have a comparatively higher
concentration of phenolic compounds such as phenolic

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THE POTENTIAL OF ULTRASOUND AGAINST MACERATION
acids, flavonoids, anthocyanins, carotenoids and thus has
more antioxidant potential than in most of the fruit pulp
(Deng et al. 2012; Goulas and Manganaris 2012; Jabbar et al.
2015b). Mango peel is the major by-product during mango
processing which constitutes approximately 15–20% of the
mango fruit weight. Mango peel is not further utilized and
discarded as waste despite being an abundant source of phyto-
chemicals such as polyphenols, carotenoids, vitamin E, ascor-
bic acid and dietary fiber. It contains more phenolic
compounds than mango pulp (Ajila et al. 2007). Magniferin,
quercetin, kaempferol, ellagic acid and rhamnetin are some of
the phenolic compounds in mango peel with potential health
benefits like antioxidative, anti-atherosclerosis, anti-muta-
genic and anti-carcinogenic activities (Masibo and He 2008).
Phenolic compounds are generally extracted by using
organic solvents such as ethanol, methanol, ethyl acetate
and acetone (Ross et al. 2009). Maceration is the conven-
tional solid-liquid extraction technique in which the sample
is kept in contact with the solvent at ambient or high tem-
perature for a longer time period with or without agitation
until the bioactive compounds present in sample are com-
pletely solubilized in the solvent (Garcia-Salas et al. 2010).
Ultrasound-assisted extraction of polyphenols involves mix-
ing the sample with organic solvent in a flask or beaker and
placing it in an ultrasonic bath with pre-set time and tem-
perature. Sound waves are produced during the process
which ruptures sample cell walls, thus lead to extraction of
phenolic compounds. Generally, ultrasound-assisted extrac-
tion time is less than 1 h, but the extraction yield is 6–35%
more than traditional extraction techniques with longer
extraction time of 12 or more hours (Vilkhu et al. 2008; Jab-
bar et al. 2014; Aadil et al. 2015b). During a study on orange
peel polyphenols extraction through ultrasound-assisted
extraction, Khan et al. (2010) observed high extraction yield
of phenolic compounds at ultrasound frequency 25 kHz.
Similarly, Pan et al. (2011) studied pomegranate peel poly-
phenols and reported that for the extraction of bioactive
compounds, 20–100 kHz ultrasonic radiations was effective
and could be efficiently utilized due to high reproducibility,
low energy and solvent consumption, low temperature
employed and thus lower loss of phenolic compounds.
Antioxidant activity assessment of phenolic compounds
in vitro involves reagents and chemicals to generate free rad-
icals and the radical scavenging ability of antioxidant to be
tested may be measured. Various assays are currently
employed for antioxidant activity determination such as fer-
ric reducing antioxidant power (FRAP), 2,2-Diphenyl-1-
picrylhydrazyl (DPPH), superoxide radical scavenging assay,
oxygen radical absorbance capacity (ORAC), trolox equiva-
lent antioxidant capacity (TEAC), etc. Among all, DPPH
radical scavenging assay is the most widely utilized tech-
nique to measure antioxidant capacity (Bendini et al. 2006;
Perez-Jimenez et al. 2009).
2 Journal of Food Processing and Preservation 00 (2016) 00–00 V
M.N. SAFDAR, T. KAUSAR and M. NADEEM
For the quantification of phenolic compounds, high per-
formance liquid chromatography (HPLC) is the generally
employed technique. Phenolic compounds are identified by
UV-visible or photo diode array detectors at 190–380 nm
wavelength with methanol and acetonitrile as the commonly
utilized mobile phase utilized for quantification of polyphe-
nols (Cicchetti and Chaintreau 2009; Zarena and Sankar
2012). Keeping in view the above-mentioned facts, a
research study was designed to optimize extraction condi-
tions for polyphnols from mango peels, determination of
antioxidant activity and quantification of phenolic com-
pounds in mango peels.
MATERIALS AND METHODS
Plant Material
Mango (Mangifera indica L.) of Chaunsa variety were pro-
cured from the fruit market Islamabad and taken to Food Sci-
ence and Product Development Institute (FSPDI) research
laboratory, National Agricultural Research Center (NARC).
Fruits were thoroughly washed under tap water to remove
dirt, dust, micro flora and pesticide residue on the surface.
Peeling of mangoes was carried out and mango peels were
further cut into small pieces. The adhering pulp was thor-
oughly removed from the peels by stainless steel knife and
then oven-dried at 50C for 48 h in hot air oven till moisture
content below 10%. Dried peels were grinded to fine powder
through the cyclotec sample mill with sieve size 0.5 mm. The
mango peel powder was packed in air-tight polyethylene zip
bags and stored at refrigeration temperature (6 6 1C).
Proximate Analysis of Peel Powder
The mango peel powder was analyzed for moisture, ash,
crude protein, crude protein, crude fat and crude fiber
according to standard methods of AOAC (2012). Available
carbohydrate in peel powder was estimated by difference
[100 2 (% moisture 1 % ash 1 % crude protein 1 % crude
fat 1 % crude fiber)].
Extraction of Polyphenols
Maceration and ultrasound-assisted extraction procedures
were employed for polyphenols extraction from mango peel
powders.
Maceration
Mango peel powders were subjected to extraction through
maceration technique according to the procedure described
by Elfalleh et al. (2012) with slight modifications.
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M.N. SAFDAR, T. KAUSAR and M. NADEEM
Preliminary studies were performed to evaluate an optimal
sample to solvent ratio (1:10, 1:15 and 1:20) and extraction
temperature (30 and 40C). After preliminary studies,
extraction was carried out by different solvents, i.e., etha-
nol, methanol, acetone and ethyl acetate at three solvent
concentrations (50, 80 and 100%) with sample to solvent
ratio 1:15 and extraction temperature 40C. Briefly, 5 g of
mango peel powder samples were extracted by specific sol-
vent, concentration level, extraction temperature and sam-
ple to solvent ratio into 250 mL conical flasks and placed
in a shaking water bath (Tecator 1024, FOSS Analytical
AB, Pal Anders vag, Hoganas, Sweden) for 20 h. The
extracts were filtered through Whatman filter paper 1 and
centrifuged (Beckman J2-21, Brea, CA) at 5,000 rpm for
10 min. Supernatant was collected and the solvent was
evaporated by rotary evaporator (Buchi Labortechnik,
Rotavapor R-300, Meierseggstrasse, Flawil, Switzerland)
under vacuum at 45C to obtain the extract which was
further filtered through 0.45 mm membrane filter, collected
in amber glass bottles and stored at refrigeration
temperature.
Ultrasound-Assisted Extraction
Extraction of polyphenols from mango peel powder was
conducted by ultrasound-assisted extraction technique as
described by Bimakr et al. (2013) with slight modifications.
Preliminary studies were carried out to determine optimal
sample to solvent ratio (1:10, 1:15 and 1:20), extraction
temperature (35, 45 and 55C) and extraction time 40, 50,
60 and 70 min. After preliminary studies, 5 g mango peel
powder samples were extracted by solvents ethanol and
methanol at 50, 80 and 100% concentration level at opti-
mal extraction conditions like sample to solvent ratio 1:20,
extraction temperature 45C and extraction time 60 min
into 125 mL (diameter: 57 mm/height 105 mm) reagent
bottles placed in a sonicator (Bandelin RK 510 H Sonorex,
Heinrichstrabe, Berlin, Germany) at 35 kHz frequency
with 100% amplitude level. The observed rise in tempera-
ture of the samples due to sonication was 2–5C. The tem-
perature was controlled by an automatic control system.
Extracts were subjected to filtration, centrifugation, solvent
vacuum evaporation, microfiltration, collection in amber
glass bottles and refrigerated storage similarly as macera-
tion extracts.
Yield (%) of Peel Extracts
The percent yield of mango peel extracts through macera-
tion and ultrasound-assisted extraction was assessed by
dividing the weight of the extract with the sample weight
and multiplying by 100.
Journal of Food Processing and Preservation 00 (2016) 00–00 V
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THE POTENTIAL OF ULTRASOUND AGAINST MACERATION
Total Polyphenols Determination
The total polyphenol content of mango peel extracts was
measured by the Folin–Ciocalteau method as described by
Singleton et al. (1999). Methanolic solution of mango peel
extracts 10 mg/mL were prepared for the analysis. Briefly,
0.5 mL methanolic extract solution was mixed with 2.5 mL
of 10% Folin–Ciocalteu’s reagent dissolved in distilled water
and 2.5 mL 7.5% sodium carbonate. Blank contained 0.5 mL
methanol, 2.5 mL Folin–Ciocalteu reagent (10 times diluted)
and 2.5 mL of 7.5% sodium carbonate. Then the samples
were incubated at 25C for 30 min for the development of a
blue colour. The absorbance was measured at 765 nm with
UV-vis Spectrophotometer (Agilent 8453, Santa Clara, CA).
A similar procedure was carried out in gallic acid standard
solution and the calibration curve was prepared from vari-
ous concentrations of gallic acid. Total polyphenol content
was expressed as milligram gallic acid equivalent (GAE)/g of
extract.
HPLC Analysis of Phenolic Compounds
Identification and quantification of phenolic acids and fla-
vonoids in the extracts were determined with high perform-
ance liquid chromatography according to the method
described by Salvador et al. (2006) with slight modifications.
Mango peel extract samples filtered through 0.45 mm mem-
brane filter were injected into the HPLC system consisted of
Perkin Elmer HPLC equipped with Binary LC pump 250,
LC 295 UV/VIS detector, reversed phase C18-WP.100 col-
umn (CNW Technology, Kolner Landstrasse, Dusseldorf,
Germany) with internal dimensions 4.6 mm 3 250 mm,
5 mm. The mobile phase consisted of linear gradient with a
combination of solvent A (acetonitrile) and solvent B (dis-
tilled water/acetic acid, 99:1, v/v, pH 2.30 6 0.1). Following
gradient program was used for the separation of flavonoids
and phenolic acids: 20% A (5 min), 80% A (10 min) and
20% A (5 min). The analyses were conducted at a flow rate
of 1 mL/min with the UV detector set at 280 nm for phenolic
acids and 370 nm for flavonoids and sample injection vol-
ume 20 mL. The analytes were identified by comparing the
retention times and spike samples with polyphenol stand-
ards and subsequent quantification of phenolic compounds
were determined.
Antioxidant Activity Evaluation
Ferric Reducing Antioxidant Power Assay. The
FRAP assay was carried out by the procedure described by
Benzie and Strains (1996) with some modifications. The
FRAP reagent was prepared by mixing 25 mL of 0.3 M acetate
buffer (pH 3.6) with 2.5 mL TPTZ solution (0.01 M) and
2.5 mL of FeCl36H2O (0.02M). A 200 mL diluted sample was
added to 1.5 mL FRAP reagent and warmed at 37C for
3
THE POTENTIAL OF ULTRASOUND AGAINST MACERATION
TABLE 1. PROXIMATE COMPOSITION OF MANGO PEEL POWDER
Mango peel powder Moisture (%) Ash (%) Crude fat (%)
8.13 6 0.10 5.10 6 0.06 1.75 6 0.07
All values are mean of three replications.
10 min. The absorbance was measured at 593 nm and antioxi-
dant activity of the sample was expressed as mmol/100g of
extract.
DPPH Radical Scavenging Assay. The antioxidant
activity of mango peel extracts was carried out by DPPH
(1,1-diphenyl-2-picryl-hydrazyl) assay according to the
method of Brand-William et al. (1995) with slight modifica-
tions. Briefly, 24 mg DPPH was dissolved in 100 mL metha-
nol to prepare a stock solution. The working standards were
prepared by diluting DPPH stock solution with methanol to
obtain about 0.98 (60.02) absorbance at 517 nm. A 3 mL of
the solution was mixed with 100 mL of samples at different
concentrations (25–400 mg/mL), well shaken, incubated in
the dark at room temperature for 15 min and absorbance
was measured at 517 nm. The scavenging activity was calcu-
lated based on the DPPH radical percentage scavenged.
% Inhibition of DPPH radical 5 Ac 2As 3100;
Superoxide Radical Scavenging Power Assay. The
antioxidant activity of mango peel extracts was determined
by superoxide radical scavenging assay in accordance with
the procedure described by Vaidya et al. (2008). Initially,
1 mL mango peel extract at different concentration (25–
400 mg/mL) was added to 1 mL sodium carbonate (5%),
0.3 mL EDTA (0.5%) and 0.4 mL nitroblue tetrazolium
(150 mm). After mixing all the reagents, absorbance was
measured immediately at 560 nm. The reaction was initiated
by the addition of 0.4 mL hydroxlylamine hydrochloride
and incubated at 25C for 5 min. The NBT reduction was
determined with a spectrophotometer at 560 nm. A parallel
control (without extract) and standard ascorbic acid were
also analyzed in the similar manner. The % scavenging
activity was calculated as:
% Inhibition of superoxide radical 5 ½12A1 =A0  3100;
where A1 is the absorbance of extract sample and A0 is the
absorbance of control.
Statistical Analysis
Data was statistically analyzed by applying analysis of var-
iance (ANOVA) technique to determine significance level.
The least square design test was used to calculate least signif-
icant difference between means. Minitab software was used
for conducting statistical analysis of data.
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M.N. SAFDAR, T. KAUSAR and M. NADEEM
Crude protein (%) Crude fiber (%) Carbohydrates (%)
3.88 6 0.05 13.51 6 0.28 67.63 6 0.31
RESULTS AND DISCUSSION
Proximate Composition
Proximate composition estimation is significant to assess the
raw material quality. Data regarding proximate composition
of mango peel powder are presented in Table 1. The mango
peel powder had the optimum moisture content (8.13%), ash
(5.10%), crude protein (3.88%), crude fat (1.75%), crude
fiber (13.51%) and available carbohydrates (67.63%). Earlier,
Ashoush and Gadallah (2011) observed the crude protein
3.60%, crude fat 1.23%, crude fiber 9.33% and ash 3.88% in
mango peel powder. Similarly, Abdeldaiem and Hoda (2012)
reported the chemical composition of mango peel powder as
moisture 10.37%, crude fat 2.28%, crude protein 3.61%, ash
3.35%, carbohydrates 29.15% and dietary fiber 51.24%. The
variation in proximate composition of mango peel powder
among different studies may be due to varietal differences,
agronomic practices, climatic conditions as well as topo-
graphic locations (Palafox-Carlos et al. 2011).
Extraction Yield
The percent yield of mango peel extracts through maceration
and ultrasound-assisted extraction at different solvent con-
centration levels shows that highest extraction yield was
obtained with solvent methanol either through maceration
or ultrasound-assisted extraction (Fig. 1). As regards macer-
ation technique, extraction with 80% methanol lead to maxi-
mum yield (25.25%) followed by 80% ethanol extraction
(24.51%) while lowest extraction yield (6.15%) was observed
in samples extracted with 50% ethyl acetate. In case of
ultrasound-assisted extraction (UAE), mango peel powders
extracted with 80% methanol had a comparatively higher
yield (25.21%) than samples extracted with solvent ethanol.
Statistically, solvent concentration levels were significantly
different from each other for solvent methanol and ethanol
employed during UAE method, while there were nonsignifi-
cant difference between concentration levels 50 and 80% for
solvent ethanol as well as 80 and 100% for ethyl acetate dur-
ing maceration technique. However, the solvent concentra-
tion level 80% was more effective than 50 or 100% solvent
concentration of all solvents employed during maceration
and UAE. Overall, maceration technique had a compara-
tively more extraction yield at all solvent concentration levels
than ultrasound-assisted extraction. Variations in extraction
yield among various solvents employed may be due to differ-
ent polarities of solvents. Palmeira et al. (2012) reported
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M.N. SAFDAR, T. KAUSAR and M. NADEEM THE POTENTIAL OF ULTRASOUND AGAINST MACERATION

FIG. 1. YIELD (%) OF MANGO PEEL

EXTRACTS BY MACERATION AND
ULTRASOUND-ASSISTED EXTRACTION
Values are presented as mean 6
standard error of triplicate analyses.
Same alphabetical letters denote a
nonsignificant difference at P < 0.05.

maximum polyphenols extraction yield 33.7% was obtained
from mango peels of “Espada” variety with 70% ethanol sol-
vent. Similarly, Wang et al. (2008) observed that the yield of
phenolic compounds from plants was associated with certain
extraction parameters like solvent concentration, extraction
temperature and time.
Total Polyphenols Content
Maceration and UAE methods were applied for the extrac-
tion of polyphenols which were then determined by Folin–
Ciocalteau reagent assay (Figs. 2 and 3). It is evident from
the data that ultrasound-assisted extraction was more effi-
cient technique and led to comparatively higher polyphe-
nols content than maceration. In case of maceration
extraction, ethanol was the most effective solvent followed
by methanol while ethyl acetate had the lowest polyphenols
extraction. Highest total polyphenols (59.74 mg GAE/g of
extract) were extracted with solvent ethanol at 80% concen-
tration level, whereas least polyphenols (18.66 mg GAE/g of
extract) were obtained with 100% ethyl acetate.
As regards ultrasound-assisted extraction, maximum pol-
yphenols were extracted with 80% ethanol (67.58 mg GAE/g
of extract) while 100% methanol extracted minimum

FIG. 2. TOTAL POLYPHENOL

CONTENT (mg GAE/g OF EXTRACT)
OF MANGO PEEL EXTRACTS BY
MACERATION TECHNIQUE
Values are presented as
mean 6 standard error of triplicate
analyses. Different alphabetical
letters denote a significant
difference at P < 0.05.

Journal of Food Processing and Preservation 00 (2016) 00–00 V

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THE POTENTIAL OF ULTRASOUND AGAINST MACERATION
phenolics (49.13 mg GAE/g of extract). Least significant dif-
ference (LSD) test reveals that solvent concentration levels
had a significant effect on phenolic extraction and were sig-
nificantly different from each other at all concentration lev-
els for each solvent as well as for extraction method. The
total polyphenols content of mango peel extracts obtained
by UAE was 13.12% more than polyphenols extracted
through maceration technique at 80% ethanol solvent con-
centration. Petigny et al. (2013) employed maceration and
UAE methods for the extraction of polyphenols from boldo
leaves and observed 20% more polyphenolic content
extracted through UAE than maceration. Due to ultrasonic
cavitation phenomenon of UAE, the cavitation generates
currents in the solvent which leads to elevate the mass trans-
fer rate between the sample material and the solvent
medium (Da Porto and Decorti 2009) causing mechanical
effects on samples cell walls resulting in cell disruption and
particle breakdown (Paniwnyk et al. 2009). The advantage
of the UAE over maceration technique is the comparatively
high extraction of polyphenols in shorter time, thus saving
energy inputs (Veggi et al. 2013). Furthermore, phenolic
compounds being thermo sensitive remain stable during
UAE as compared to soxhlet and other conventional techni-
ques where elevated temperature are employed (Zhang et al.
2008).
Extraction of polyphenols also depends on the type of sol-
vent employed. Ajila et al. (2007) compared various solvents
such as ethanol, acetone and water for extraction of polyphe-
nols from mango peel and concluded that ethanol and ace-
tone were more efficient solvent than water with polyphenol
content 92.62, 90.02 and 55.05 mg GAE/g, respectively.
Total polyphenols of each solvent at absolute concentra-
tion level were lowest for both extraction methods which
6 Journal of Food Processing and Preservation 00 (2016) 00–00 V
M.N. SAFDAR, T. KAUSAR and M. NADEEM
FIG. 3. TOTAL POLYPHENOL CONTENT
OF MANGO PEEL EXTRACTS BY
ULTRASOUND-ASSISTED EXTRACTION
Values are presented as mean 6
standard error of triplicate analyses.
Different alphabetical letters denote a
significant difference at P < 0.05.
established the findings of Chan et al. (2009) that absolute
solvent could not ensure fair extraction of polyphenols than
aqueous solvents. Selection of proper extraction solvent is
vital for complex food matrices as it will estimate the type
and quantity of polyphenols being extracted. Variations in
extracted polyphenol content depend on the polarities of
the solvents used as well as their concentration level either
aqueous or absolute. Generally, aqueous alcohols such as
methanol and ethanol are employed in phenolic compounds
extraction from plant materials (Hayouni et al. 2007). Sol-
vent ethanol categorized under GRAS (generally recognized
as safe) is preferred due to its application in the food system.
Ethanol enhances the solubility of a solute while water accel-
erates it’s desorption from sample matrix (Mustafa and
Turner 2011). The low solubility of phenolic compounds in
absolute solvents may be due to the strong hydrogen bond-
ing between protein and polyphenols. However, the solubil-
ity increases upon addition of water to organic solvents that
weakens the hydrogen bonds (Wissam et al. 2012). In a
related study, Nepote et al. (2005) investigated the phenolic
content of peanut skins with different concentrations of
ethanol and reported that 50% ethanol, lead to highest poly-
phenol content which decreased with the increase in ethanol
concentration above 70%.
Ferric Reducing Antioxidant Power
(FRAP) Assay
The antioxidant power of a sample extract corresponds to
reducing ability which depends on its ability to transfer elec-
tron towards FRAP reagent. The FRAP data (Fig. 4) indicates
that mango peels polyphenols had high antioxidant activity
extracted with ethanol as well as methanol solvents. However,
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M.N. SAFDAR, T. KAUSAR and M. NADEEM THE POTENTIAL OF ULTRASOUND AGAINST MACERATION

peel samples extracted with ethanol had higher antioxidant

activity than samples extracted with methanol, but the differ-
ence between the two solvents was non-significant. As regards
the solvent concentration level, 80% ethanol had the highest
antioxidant activity (31.51 6 0.79 mM/100 g) followed by
80% methanol (30.22 6 0.88 mM/100 g) while polyphenols
extracted with 100% methanol had the least activity
(24.76 6 0.70 mM/100 g). The LSD test shows that there were
nonsignificant difference between 50 and 80% concentration,
but were significantly different from 100% concentration for
both solvents and extraction techniques. The antioxidant
activity of mango peel extracts may be attributed to phenolic
compounds present in mango peels. Berardini et al. (2005)
assessed the FRAP activity of mango peel ethanolic and water
extracts. They observed higher antioxidant activity in etha-
nolic extract 436 mmol Trolox/100 g as compared to water
extraction 361 mmol Trolox/100 g. Similarly, Zulkifli et al.
(2012) reported FRAP value for mango peel as
37.51 6 0.62 mM/100 g using water extraction, whereas
Imran et al. (2013) investigated the antioxidant potential of FIG. 4. FRAP ACTIVITY (mM/100 g) OF MANGO PEEL EXTRACTS
peels of different mango varieties and observed FRAP values Values are presented as mean 6 standard error of triplicate analyses.
Means followed by same letters do not differ significantly (P < 0.05).
in the range of 6.25 6 0.11 to 8.96 6 0.42 mmol/100 g of
mango peels extracted with ethanol. The difference in FRAP
activity of mango peel among different studies may be influ- DPPH scavenging activity in ripened and unripened mango
enced by type of mango variety, solvent as well as solvent peel extracts. Among solvents, ethanol extracted samples
concentration. exhibited more scavenging activity than samples extracted
with methanol. The extracting solvent effect on DPPH radi-
cal scavenging activity was earlier reported by Turkmen
DPPH Radical Scavenging Activity et al. (2007). Similarly, Imran et al. (2013) observed that the
DPPH assay has been employed widely to evaluate the free antioxidant activity of mango peel extracts is influenced by
radical scavenging activity of different plant extracts. It is a solvent type and concluded that ethanolic extracts of mango
stable free radical dissolves in either ethanol or methanol, peels had higher DPPH inhibitory activity in comparison
which DPPH free radical reduction is determined by the with water and acetone extracts.
decrease in its absorption at 517 nm and the color of the
DPPH assay solution changes from purple to light yellow. Superoxide Radical Scavenging Power Assay
The scavenging potential of plant extracts antioxidants cor-
responds to the degree of discolouration (Kalpna et al. Superoxide anion radical is produced through different bio-
2011). chemical process, though itself a weak oxidant but may leads
The effect of different solvents and their concentration to the generation of dangerous and powerful hydroxyl radi-
levels on DPPH radical scavenging activity of mango peel cals and singlet oxygen responsible for oxidative stress-
extracts (Figs. 5 and 6) reveals high antioxidant activity of related disorders. The antioxidants scavenge the superoxide
all sample extracts. However, highest scavenging activity anion and the percentage scavenging of superoxide anion
(83.19 6 0.96) was exhibited by samples extracted with sol- radical increases with higher concentration of antioxidants
vent ethanol at 80% concentration level, whereas samples (Jayakumar et al. 2009).
extracted with 100% methanol had the lowest scavenging The effect of different solvents and their concentration lev-
activity (59.17 6 0.81). Aqueous solvent extracts had higher els on superoxide anion radical scavenging activity of mango
inhibitory activity against DPPH radical as compared to peel extracts (Figs. 7 and 8) reveals high antioxidant activity
corresponding absolute solvents which may be attributed to of all sample extracts. However, mango peel ethanolic extracts
higher polyphenol content in these extracts. When com- at 80% concentration level exhibited highest activity to scav-
pared to standard ascorbic acid, DPPH radical scavenging enge superoxide anion radical (67.23 6 0.85) followed by
activity of mango peel extracts was relatively less than stand- 50% ethanolic extract (61.35 6 1.17) whereas 100% metha-
ard ascorbic acid (95.83 6 0.75). Kim et al. (2010) reported nolic extract had least scavenging activity (51.96 6 1.02).
a linear association between polyphenol concentration and Absolute solvent concentration demonstrated lowest

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THE POTENTIAL OF ULTRASOUND AGAINST MACERATION M.N. SAFDAR, T. KAUSAR and M. NADEEM

FIG. 5. EFFECT OF METHANOL

CONCENTRATION LEVELS ON
DPPH SCAVENGING ACTIVITY OF
MANGO PEEL EXTRACTS

FIG. 6. EFFECT OF ETHANOL

CONCENTRATION LEVELS ON DPPH
SCAVENGING ACTIVITY OF MANGO
PEEL EXTRACTS

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M.N. SAFDAR, T. KAUSAR and M. NADEEM THE POTENTIAL OF ULTRASOUND AGAINST MACERATION

FIG. 7. EFFECT OF METHANOL

CONCENTRATION LEVELS ON
SUPEROXIDE ANION RADICAL
SCAVENGING ACTIVITY OF MANGO
PEEL EXTRACTS

inhibitory activity against superoxide anion radical than
aqueous solvent extracts. Overall, superoxide anion radical
scavenging activity of mango peels was comparatively lower
than standard ascorbic acid (87.83 6 0.92). Kalpna et al.
(2011) observed that the acetone extract of mango peels had
highest antioxidant activity than extraction with methanol,
chloroform and hexane. During a related study, Jahan (2011)
investigated the antioxidant activity of different medicinal
plants and reported stronger antioxidant activity of methano-
lic extracts than water extracts which might be due to the
presence of high concentration of hydrophilic and hydropho-
bic phenolic compounds.
Correlation Analysis of Polyphenol Content
and Antioxidant Activity
Coefficient of correlations between the phenolic compounds
content and antioxidant activity of mango peel extracts are

FIG. 8. EFFECT OF ETHANOL

CONCENTRATION LEVELS ON
SUPEROXIDE ANION RADICAL
SCAVENGING ACTIVITY OF MANGO
PEEL EXTRACTS

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THE POTENTIAL OF ULTRASOUND AGAINST MACERATION M.N. SAFDAR, T. KAUSAR and M. NADEEM

TABLE 2. CORRELATION BETWEEN TOTAL POLYPHENOLS AND
ANTIOXIDANT ACTIVITIES (FRAP, DPPH AND SUPEROXIDE RADICAL
SCAVENGING ASSAYS) OF MANGO PEEL EXTRACTS
Superoxide
Assay FRAP DPPH radical
Total polyphenol content 0.63 0.91 0.96
All values were significant at P < 0.05.
presented in Table 2. The scavenging activities of mango
peel extracts on DPPH (r 5 0.9075) and superoxide anion
radical (r 5 0.9576) as well as ferric reducing antioxidant
power (r 5 0.5943) were well correlated with their total pol-
yphenol content. It indicated that the antioxidant activity of
mango peel extracts was due to their polyphenols content.
The similar positive correlation between total polyphenols
and antioxidant activities have been reported in fruit waste
extracts by different researchers (Khammuang and Sarn-
thima 2011; Wong et al. 2014; Zulkifli et al. 2012).
HPLC Analysis of Phenolic Compounds
Identification and quantification of phenolic acids and flavo-
noids in mango peel extracts were determined with high per-
formance liquid chromatography. A total of eleven phenolic
compounds, including four phenolic acids and seven flavo-
noids at wavelength 280 and 370 nm, respectively, were iden-
tified and quantified according to retention time and their
peaks spectral characteristics against those of standards (Table
3). Results revealed that mango peel extracts phenolics varied
considerably as a function of solvent composition and con-
centration level. Methanolic extracts showed comparatively
higher quantity of phenolic compounds than the ethanolic
extracts of mango peels. Methanolic extracts at 50% concen-
tration level had highest phenolic content quantified
(1134.35 mg/g) whereas 100% ethanolic extract had the least
quantified phenolic compounds (591.13 mg/g). Among the
phenolic compounds, coumaric acid was the most abundant
phenolic acid in mango peel extracts. The highest level of
coumaric acid was found in 50% ethanolic extract
(240.54 mg/g). Gallic acid, ferulic acid and epicatechin were
the other phenolic compounds present in high concentration
while myrecetin was the least quantified flavonoid in mango
peel extracts as it was quantified only at 50 and 80% ethanol
concentration level. Means of each phenolic compound
quantified were compared statistically by LSD test to assess
the effect of solvents and their concentration level. Significant
variations were observed in phenolic acids and flavonoids
content with respect to solvent concentrations. Gallic acid
content of 80% ethanolic extract was non-significantly differ-
ent to 50% methanolic extract, but was significantly different
to other concentration levels. Chlorogenic acid and rutin
content of 100% methanolic and 50% ethanolic extracts were
nonsignificant, but were significantly different from other
treatment levels. Phenolic compound magniferin at all con-
centration levels differed significantly while kaempferol com-
pound at 50 and 100% ethanolic extracts differed
nonsignificantly, but were significantly different from other
concentration level methanolic and ethanolic extracts. Over-
all, 50 and 80% methanolic and ethanolic extracts lead to
more phenolic compounds quantified as compared to abso-
lute concentration levels. The antioxidant activity of mango
peel extracts might be due to the phenolic acids and flavo-
noids contribution. Earlier, Chun et al. (2007) claimed that
flavonoids were responsible for the antioxidant activities of
plants. However, Zulkifli et al. (2012) reported that mango
peels contained considerably higher concentrations of gallic

TABLE 3. HPLC QUANTIFICATION OF POLYPHENOLS IN MANGO PEEL EXTRACTS

Phenolic Methanol Ethanol
compounds
(mg/g) 100% 80% 50% 100% 80% 50%
c a b d c
Gallic acid 85.37 6 0.31 142.54 6 1.75 129.82 6 2.04 73.79 6 0.64 91.00 6 0.67 144.18a 6 2.68
Chlorogenic acid 35.29b 6 0.41 21.78e 6 0.34 42.07a 6 0.37 24.52d 6 0.23 28.90c 6 0.44 33.97b 6 0.56
Ferulic acid 99.67e 6 1.79 125.93c 6 2.11 146.64b 6 2.53 80.74f 6 0.63 115.65d 6 2.25 167.21a 6 1.75
Coumaric acid 138.32e 6 1.01 217.86b 6 4.33 194.32c 6 2.90 152.66d 6 2.00 188.97c 6 4.13 240.54a 6 5.32
Catechins 62.34b 6 0.95 83.62a 6 1.36 51.16c 6 0.53 45.73d 6 0.34 67.41b 6 1.28 88.50a 6 2.07
Epicatechins 167.33c 6 1.62 198.40b 6 2.62 242.73a 6 2.19 131.07e 6 0.67 152.13d 6 1.48 125.42e 6 2.71
Magniferin 56.78e 6 0.42 99.75c 6 1.48 124.20a 6 2.46 37.89f 6 1.00 112.18b 6 1.54 65.33d 6 1.09
Quercetin 19.05d 6 0.23 24.41c 6 0.17 35.20a 6 0.35 15.91e 6 0.39 30.46b 6 0.60 16.86de 6 0.46
Myrecetin N.D N.D N.D N.D 11.08a 6 0.42 7.05b 6 0.30
Rutin 41.16d 6 0.98 73.17c 6 1.97 98.43a 6 1.88 N.D 80.24b 6 2.29 37.15d 6 0.65
Kaempferol 84.01a 6 1.80 62.49c 6 0.95 69.78b 6 2.47 28.82e 6 0.26 42.56d 6 0.62 31.73e 6 0.66
Total 789.32e 6 2.41 1049.95b 6 10.00 1134.35a 6 13.30 591.13f 6 2.77 920.58d 6 5.60 957.94c 6 4.25

All values are mean of three replications.

Means followed by same letters do not differ significantly (P < 0.05).
Different superscript letters within same row denote a significant difference (P < 0.05).

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M.N. SAFDAR, T. KAUSAR and M. NADEEM
acid and chlorogenic acid which contributed to their strong
antioxidant activities.
CONCLUSION
Mango peels are a rich source of phenolic compounds with
strong antioxidant activity. Ultrasound-assisted extraction
lead to high polyphenol extraction and more efficient tech-
nique than maceration. Absolute solvent could not ensure
fair extraction of polyphenols than aqueous solvents as well
as lower antioxidant activity in comparison with absolute
solvents. Though methanol and ethanol are efficient sol-
vents for extraction of polyphenols, ethanol categorized
under GRAS is preferred due to its application in the food
system. Strong correlations between total polyphenols and
antioxidant activity were observed. Eleven phenolic com-
pounds, including four phenolic acids and seven flavonoids
were identified and quantified by HPLC. Coumaric acid was
the most abundant phenolic acid in mango peel extracts.
Gallic acid, ferulic acid and epicatechin were the other phe-
nolic compounds present in high concentration while myre-
cetin was the least quantified flavonoid in mango peel
extracts. It was concluded that mango peels being a potential
source of phenolic compounds could be utilized as an ingre-
dient for the preparation of functional foods.
ACKNOWLEDGMENT
This research work was supported by a Research For Agricul-
tural Development Program (RADP), Islamabad, Pakistan.
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