Journal of Food Measurement and Characterization (2019) 13:3242–3249

https://doi.org/10.1007/s11694-019-00246-w

ORIGINAL PAPER

Characterization of phenolic compounds in sweet lime (Citrus limetta)

peel and freshly squeezed juices by LC‑DAD‑ESI‑MS/MS and their
antioxidant activity
Ozlem Kilic Buyukkurt1 · Gamze Guclu2 · Hasim Kelebek3 · Serkan Selli2

Received: 15 May 2019 / Accepted: 5 August 2019 / Published online: 10 August 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Sweet lime (Citrus limetta) is one of the citrus species and it is generally consumed fresh or processed to fruit juice. The
squeezing method of fruit is an important factor affecting the quantity and composition of phenolic compounds of the
juice. The aim of this study was to identify and quantify the phenolic compounds and determine the antioxidant activities
of sweet lime peel and juices. For this purpose, sweet lime juices were prepared using two different methods. Firstly, sweet
lime fruits were hand-squeezed without peeling. Secondly, fruits were hand-squeezed after the manual removal of the peel.
The phenolic compounds of the peel and the two types of juices were analyzed by LC-DAD-ESI-MS/MS. Twenty phenolic
compounds were determined in all extracts, 18 of which were flavonoids and two were phenolic acids. The major compound
was hesperidin both in the peel and juices. The procyanidin B dimer and luteolin were detected for the first time in sweet
lime peel and juices in the present study. The amount of phenolic compounds of the peel was significantly higher than those
of the juices. The total phenolic contents of the sweet lime juice increased about 13% from 444.55 to 502.54 mg/L due to
the effect of the peel. The antioxidant capacity of the peel was found to be higher than those of the juices resulting from its
high phenolic content.

Keywords Sweet lime · Citrus limetta · Peel · Juice · Phenolic compounds · Antioxidant

Introduction recognized as sweet lime. It has a flattened vertex, a notice-

able nipple, a light yellow and thin peel [4–6]. Sweet lime is
The fruit of citrus which belongs to the family Rutaceae, generally consumed fresh or processed to fruit juice.
is one of the most popular fruits in the world because of Citrus fruits have many health benefits thanks to their
its nutritional value, flavor, aroma and color properties [1, bioactive compounds mainly phenolics [1]. The phenolic
2]. Citrus fruits are produced in approximately 100 million compounds consist of phenolic acids and flavonoids such as
tons in the world while Turkey has a share of about 3.5 mil- flavones, flavanones, and flavonols. Flavonoid compounds
lion tons [3]. The most important citrus fruits are oranges exist in the aglycone or glycoside forms in citrus fruits [7].
followed by mandarins, lemons, grapefruits, bergamots, Polymethoxylated flavones (tangeretin, sinensetin, nobiletin,
and sweet limes. Citrus limetta is one of the citrus species etc.) and flavanone glycosides (hesperidin, neohesperidin,
narirutin, naringin, etc.) are found only in citrus fruits [8,
* Serkan Selli 9]. Hesperidin, neohesperidin, eriocitrin, quercetin, diosmin,
sselli@cu.edu.tr and kaempferol are recognized as basic phenolic compounds
1
while rutin and narirutin are known as minor compounds in
Department of Food Technology, Kadirli Applied Sciences sweet lime. Caffeic, p-coumaric, sinapic, ferulic and chloro-
School, Osmaniye Korkut Ata University, 80760 Osmaniye,
Turkey genic acid are the most common phenolic acids in sweet lime
2 [2, 4, 6, 10, 11]. These phenolic compounds are the second-
Department of Food Engineering, Faculty of Agriculture,
Cukurova University, 01330 Adana, Turkey ary metabolites and have antioxidant properties. Moreover,
3 these compounds have antiviral, antitumoral, antimicrobial,
Department of Food Engineering, Faculty of Engineering,
Adana Alparslan Turkes Science and Technology University, anti-allergenic and anti-inflammatory effects [12, 13].
01250 Adana, Turkey

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Characterization of phenolic compounds in sweet lime (Citrus limetta) peel and freshly… 3243

The phenolic compounds of sweet lime are affected by from sweet limes cut into two parts. Two juice samples were
genotype, environmental conditions (soil type, climate, prepared using two different methods. Firstly, sweet lime
and fertilizers), cultivar, fruit ripeness, postharvest han- fruits were hand-squeezed without peeling. Secondly, fruits
dlings, and storage conditions. In addition, the extraction were hand-squeezed after the manual removal of the peel.
method, heat treatment and fermentation also affect the phe- Sweet lime peels were also used for phenolic analysis. A
nolic compounds in fruit juices [6, 9, 14]. Jeong et al. [15] total of about two liters from each juice were prepared and
reported that heat treatment, far-infrared radiation and fer- stored at − 20 °C until the analysis.
mentation result in release of phenolic compounds in citrus
peel. Besides, the squeezing method of fruit is an important
factor affecting the quantity and composition of the phenolic Chemicals
compounds. Hand-squeezed sweet lime juice contains great
quantity of phenolics because of the compounds arising from Distilled water used during the analysis was purified through
the peel [16]. Non-edible parts of the citrus fruit for example a Millipore-Q system (resistivity over 18 MΩ cm; Mil-
peel is rich in terms of phenolic compounds as well as edible lipore Corp., Saint-Quentin, France) in the present study.
parts of the fruit such as juice and pulp [11, 17, 18]. The standard of phenolic compounds (Vicenin 2/23,666-
Most of the extant studies have generally focused on the 13-9, Eriocitrin/13,463-28-0, Orientin/28,608-75-5,
antioxidant activity and total phenolic contents of the juice Naringin/10,236-47-2, Rhoifolin/17,306-46-6, Dios-
and/or peel of sweet lime. A limited number of studies elu- min/520-27-4, Hesperidin/520-26-3, Procyanidin B/29,106-
cidated the individual phenolic compounds in sweet lime 49-8, Ferulic acid/1135-24-6, Luteolin/491-70-3, Iso-
juices. Barreca et al. [4] investigated the flavonoid compo- phorone/78-59-1 ( ± )-abscisic acid (ABA)/14,375-45-2)
sition of crude sweet lime juice by LC-DAD-ESI-MS/MS. and DPPH were procured by Sigma-Aldrich (Steinheim,
Durand-Hulak et al. [8] identified 64 phenolic compounds in Germany, St. Louis, Missouri, USA).
some of Citrus leaves and fruits (orange, mandarin, grape-
fruit) using a UPLC-MS. Mcharek and Hanchi [13] analyzed
the phenolic constituents in lemon (Citrus limon) peels by General analysis
LC-MS/MS. Nogata et al. [16] determined the concentra-
tions of polymethoxylated flavones (tangeretin, nobiletin, The pH and total acidity analyses of sweet lime juices were
and sinensetin) in a mandarin-type citrus fruit, ponkan (Cit- performed according to the method of Selli et al. [19]. The
rus reticulata) juices using LC-DAD system. Rodríguez- color of the sweet lime juices were measured by a Hunter
Rivera et al. [18] analyzed the phenolic compounds in peels colorimeter (HunterLab, Color QuestXE, USA). A CIE Lab
of Citrus limetta by the combination of preparative high- color profile (L*, a*, b*) was recorded. Juice samples were
speed countercurrent chromatography and LC-ESI-MS/MS. placed in a sample cell (6 cm diameter, 2 cm deep) and the
However, there has been no data in the literature on the use data was collected [20].
of LC-DAD-ESI-MS/MS for the determination of phenolic
compounds both in peel and juice of sweet lime. Therefore,
the aims of the present study were: (i) to determine the phe- Phenolic compounds analysis
nolic compounds of sweet lime peels, (ii) to investigate the
effect of phenolic compounds passing from peel to juice by Extraction of phenolic compounds
examining two juice samples one squeezed without peel-
ing and another squeezed after peels were removed, (iii) to The phenolic extraction of sweet lime juices was modified
evaluate the antioxidant activities of all sweet lime extracts according to the method of Rodríguez-Rivera et al. [18]. The
of the peel and the juices. juice was diluted with 80% methanol (ratio 1:1 v/v) and the
mixture was centrifuged at 12,000 rpm for 5 min and 6 °C.
The supernatant was filtered with a 0.45 µm membrane filter
Materials and methods and the extracts were stored at − 20 °C until the analysis. The
extracts were also used to define the antioxidant capacity.
Materials The phenolic extraction of the peel was modified accord-
ing to the method of Moraes Barros et al. [14]. Firstly the
Sweet lime (Citrus limetta) fruits were harvested from the peels were frozen and powdered by a grinder. 0.5 g pow-
city of Karatas, Adana, Turkey in September 2017. The dered peel was mixed with 20 ml 75% methanol and agitated
mean maturity index values of sweet limes were 19.35. The for one hour at 4 °C under ­N2. The mixture was centrifuged
fruits were washed using tap water before preparing the sam- at 5500 rpm for 15 min. After the filtration, the extracts were
ples. A kitchen-type manual juicer was used to obtain juice stored at − 20 °C until the analysis.

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3244 O. K. Buyukkurt et al.

LC‑DAD‑ESI‑MS/MS analysis of phenolic compounds Statistical analysis

The extracts were filtered through a membrane filter (What- The data of the present study were exposed to one way anal-
man Inc., Clinton, NJ, USA) with a pore size of 0.45 micron ysis of variance (ANOVA) using SPSS 20.0 (SPSS Inc., Chi-
before injection to LC-MS/MS (Agilent 1260 HPLC sys- cago, IL), and Duncan’s multiple comparison test was used
tem; Agilent Technologies, Palo Alto, California, USA). to find significant differences. Principal component analysis
The HPLC equipment was composed of an autosampler (PCA) was also applied using the XLSTAT statistical pro-
(G1367 E, 1260 HIP ALS), a binary pump (G1312 B, 1260 gram (2015) for Windows (Addinsoft, New York, NY).
Bin pump), a degasser (G1322 A, 1260 Degasser) and a
diode array detector (G1351D 1260 DAD VL). The column
used was a Phenomenex Luna reversed-phase C-18 column Results and discussion
(4.6 mm × 250 mm, 5 μm) (Torrance, California, USA). The
mobile phase consisted of two solvents: Solvent A, water/ General composition of sweet lime juices
formic acid (99:1; v/v) and Solvent B, acetonitrile/solvent
A (60:40; v/v). Phenolic compounds were eluted according The general composition of sweet limes juices is given in
to the method described by Kelebek et al. [21]. The stand- Table 1. No significant differences were found between the
ard curves were acquired utilizing the commercial stand- pH, total acidity and color of the juice with hand-squeezed
ards at concentrations normally exist in extracts (nearly and the juice with hand-squeezed after peeling. The results
1–100 mg/L) and getting regression coefficients (­ r2) above of pH and total acidity were similar to previous studies. In
0.995 in all cases. In the absence of reference compounds, a study by Moraes Barros et al. [14], pH was determined as
the calibration of structurally related substances was used, 5.52 in sweet lime juice. Another study by Cano-Lamadrid
taking into account the molecular weight correction factor. et al. [23], total acidity was determined as 0.66% and the val-
The limits of detection (LOD) and limit of quantification ues of L*, a*, b* were determined as 46.09, − 1.33, − 1.77,
(LOQ) were calculated at signal-to-noise ratios (S/N) of 3 respectively in C. limetta juice. The color of the juices was
and 10, respectively. brighter and more yellow compared to the previous study.

Phenolic compounds
Analyses of antioxidant capacity
Identified phenolic compounds with their UV absorptions
The phenolic extracts of fruit juices were also used for anti- and ­MS2 fragmentation pattern in negative ion mode are
oxidant capacity. The extraction of sweet lime peels was presented in Table 2. Total of 20 phenolic compounds
modified according to the method of Moraes Barros et al. (vicenin-2, lucenin-2 4′-methyl ether, eriocitrin, scopa-
[14]. Peels were frozen at − 20°C and shaved to reduce their rin, orientin 4′-methyl ether, naringin, rhoifolin, diosmin,
size. 1 g peel powder was extracted with 20 ml 75% metha- hesperidin, procyanidin B dimer, ferulic acid, luteolin,
nol by stirring with a magnetic bar for 90 min, at 4 °C under melitidin, isophrone-O-glc-3-hydroxy-3-methyl-glutaryl
­N2. The mixture was centrifuged for 15 min at 5500 rpm and (HMG), ABA-O-glc-HMG, caffeic acid derivative, dihy-
4 °C. The supernatants were collected for extract and the dro-feruloyl-O-glucosides, dihydro-feruloyl-O-glc-HMG-
extracts were stored at − 20 °C until the analysis. caf, dihydro-feruloyl-O-glc-HMG-caffeoyl-HMG and
The antioxidant capacity of samples was determined by
DPPH assays according to the method of Sonmezdag et al.
[22]. The electron contribution ability of the extract was Table 1  General composition of sweet lime juices
measured by blanching the purple blanched solution of the
Analysis Juice with hand- Juice with hand-
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Each extract squeezed squeezed after
was diluted 1:10 (v/v) with 80% methanol. An aliquot of peeling
0.1 ml of each diluted extract was added to 3.9 ml of DPPH
pH 5.62 ± 0.01a 5.59 ± 0.01a
solution (6 × 10−5 M) in methanol. The mixture was agitated
Total acidity (g/100 ml) 0.48 ± 0.02a 0.57 ± 0.04a
strongly and kept in dark at room temperature for 30 min.
Colour properties
The absorbance was measured at 515 nm after 30 min by a
L* 85.04 ± 0.01a 83.32 ± 0.01a
Cary 60 UV–Vis spectrophotometer (Agilent Technologies,
a* − 1.46 ± 0.01a − 0.75 ± 0.01a
Santa Clara, California, USA). The Trolox calibration curve
b* 17.33 ± 0.01a 19.16 ± 0.01a
was used to calculate the antioxidant activity of samples as
µM Trolox Equivalent. The mean and standard deviation Values expressed are means ± S.D. of three replications
were calculated for two replicates. Means line with different letters are significantly different (p < 0.05)

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Characterization of phenolic compounds in sweet lime (Citrus limetta) peel and freshly… 3245

Table 2  Peak number, retention times (Rt), wavelength (nm) and mass spectral data for analyses of phenolic compounds in sweet lime using LC-
DAD-ESI-MS/MS
Peak No Compounds UV–Vis (nm) Rt (min) MS MS/MS fragments m/z References
[M–H]−
m/z

1 Vicenin-2a 271, 335 38.35 593 575, 512, 473, 383, 353 [18, 27, 29]
2 Lucenin-2 4′-methyl ether 280 (sh), 318 41.45 623 605, 533, 503, 413, 383 [18, 26, 29]
3 Eriocitrina 284, 319 46.99 596 287 [18, 27, 29]
4 Scoparin 256, 268, 347 48.61 461 371, 341 [18]
5 Orientin 4′-methyl ether 253 (sh), 278, 48.91 461 371, 341 [18]
325
6 Naringina 280 49.75 579 459, 271, 151 [18]
7 Rhoifolina 271, 333 50.28 577 463, 431, 311, 269 [29]
8 Diosmina 224 (sh), 345 51.98 607 463, 299 [26]
9 Hesperidina 284, 321 52.29 609 343, 325, 301 [18, 26]
10 Procyanidin B ­dimera 280 40.98 577 425, 289 [26]
11 Ferulic ­acida 238, 290(sh), 322 45.11 193 178, 149, 134 [30–32]
12 Luteolina 276, 326 55.90 285 269, 257, 243, 217 [30–32]
13 Melitidin 285 50.62 723 579, 459 [18, 29]
14 Isophorone-O-glc-HMG 360 58.20 475 137 [18]
15 ABA-O-glc-HMG 360 51.19 585 483, 441, 330, 139 [18]
16 Caffeic acid derivative 327 42.93 421 375, 285, 161 [26]
17 Dihydro-feruloyl-O-glucoside 355 42.35 357 195, 151 [18]
18 Dihydro-feruloyl-O-glc-HMG-caf 360 39.45 663 501, 399, 357, 195 [18]
19 Dihydro-feruloyl-O-glc-HMG-caffeoyl-HMG 360 31.23 807 771, 663, 501, 399, 357, 195 [18]
20 Feruloyl-glucarate 238, 298 (sh), 326 35.02 385 191, 181 [18]
a
Identification confirmed by comparison with standards

feruloyl-gluccarate) were identified and quantified in sweet that the peel had approximately sixfold higher amounts of
lime peel and juice extracts by LC-DAD-ESI-MS/MS. hesperidin than the juices. The presence of the hesperidin
Among the phenolic compounds, 18 were flavonoids and was previously reported in sweet lime juice and peel [4, 18].
two were phenolic acids. In a study by Costa et al. [24], hesperidin was determined as
The results of the quantitative calculation of the phenolic 268.94 ± 22.68 mg/L in C. limetta juices. Gómez-Mejía et al.
compounds of the sweet lime juices and peel are reported [25] also reported that the most abundant flavanone was the
in Table 3. The amount of the total phenolic compounds hesperidin in the orange, lemon, and clementine.
of the peel, the juice obtained by hand-squeezing with Concerning flavone C- and O-glycosides, scoparin
peels and the juice obtained by hand-squeezing after peel- (chrysoeriol 8-C-glucoside) (peak 4, Fig. 1) and orientin
ing were 3127.67 ± 90.38 mg/kg, 502.54 ± 14.52 mg/L and 4′-methyl-ether (diosmetin 8-C-glucoside) (peak 5, Fig. 1)
444.55 ± 12.88 mg/L, respectively. When the amounts of the were identified in sweet lime peel and juices (Table 2). Both
phenolic compounds were compared, it was found that the scoparin and orientin 4′-methyl-ether were found to be in
peel’s compounds had significantly higher values than those significantly higher amounts in the peel as compared to both
of both juices (p < 0.05). On the other hand, when the total juice samples (Table 3).
phenolic contents of the sweet lime juices were evaluated, it Vicenin-2 (apigenin 6,8-di-C-glucoside) and lucenin-2
was seen that the total phenolic content increased by about 4′-methyl ether (diosmetin 6,8-di-C-glucoside) were the
13% from 444.55 to 502.54 mg/L due to peel effect. other typical C-glycoside fragmentation forms found in the
Hesperidin (peak 9, Fig. 1) which belongs to the class sweet lime peel and juice extracts (Table 2). It was reported
of flavanone glycosides was found to be the main phenolic that these compounds had C-linked di-hexoses [26]. Bar-
compound in all sweet lime extracts (Table 2 and Table 3). reca et al. [4] reported that both of vicenin-2 and lucenin-2
The concentration of hesperidin was 316.66 ± 9.22 mg/L 4′-methyl ether were found for the first time in C. limetta
in the juice with hand-squeezed, 290.49 ± 8.46 mg/L juice. Rhoifolin (apigenin 7-O-neohesperidoside) (peak 7,
in the juice with hand-squeezed after peeling and Fig. 1) and diosmin (diosmetin 7-O-rutinoside) (peak 8,
1777.46 ± 51.77 mg/kg in the peel (Table 3). This means Fig. 1) were the other C-glycoside fragmentation forms

13
3246 O. K. Buyukkurt et al.

Table 3  Quantitative calculation of the phenolic compounds of sweet lime extracts

Peak No Compounds Juice with hand- Juice with hand-squeezed Peel (mg/kg)
squeezed (mg/L) after peeling (mg/L)

1 Vicenin-2 20.09 ± 0.59b 17.78 ± 0.52b 171.73 ± 5.00a

2 Lucenin-2 4′-methyl ether 52.12 ± 1.52b 45.05 ± 1.31b 74.84 ± 2.18a
3 Eriocitrin 16.25 ± 0.47b 11.48 ± 0.33b 133.65 ± 3.89a
4 Scoparin 12.70 ± 0.37b 11.39 ± 0.33b 38.03 ± 1.11a
5 Orientin 4′-methyl ether 9.97 ± 0.29b 8.54 ± 0.25b 327.66 ± 9.54a
6 Naringin 2.43 ± 0.02 b 2.39 ± 0.00b 25.66 ± 0.03a
7 Rhoifolin 5.56 ± 0.16b 3.34 ± 0.10b 92.44 ± 2.69a
8 Diosmin 16.79 ± 0.49b 15.04 ± 0.44b 32.37 ± 0.94a
9 Hesperidin 316.66 ± 9.22b 290.49 ± 8.46b 1777.46 ± 51.77a
10 Procyanidin B dimer 9.07 ± 0.26b 5.55 ± 0.16b 46.77 ± 1.36a
11 Ferulic acid 8.58 ± 0.25b 6.99 ± 0.20b 83.2 ± 2.42a
12 Luteolin 1.61 ± 0.02b 1.07 ± 0.03b 232.26 ± 6.76a
13 Melitidin 0.67 ± 0.02b 0.49 ± 0.01b 10.16 ± 0.30a
14 Isophrone-O-glc-HMG 0.16 ± 0.00b 0.19 ± 0.01b 3.06 ± 0.09a
15 ABA-O-glc-HMG 2.08 ± 0.06b 1.56 ± 0.05b 7.52 ± 0.22a
16 Caffeic acid derivative 0.00 ± 0.00b 0.00 ± 0.00b 21.67 ± 0.63a
17 Dihydro-feruloyl-O-glucoside 2.08 ± 0.06a 1.73 ± 0.05b 0 ± 0.0c
18 Dihydro-feruloyl-O-glc-HMG-caf 0.49 ± 0.01b 0.38 ± 0.01b 1.83 ± 0.05a
19 Dihydro-feruloyl-O-glc-HMG-caffeoyl-HMG 10.09 ± 0.29b 7.50 ± 0.22c 12.76 ± 0.37a
20 Feruloyl-glucarate 15.15 ± 0.44b 13.58 ± 0.40b 34.62 ± 1.01a
Total phenolic compounds 502.54 ± 14.52b 444.55 ± 12.88b 3127.67 ± 90.38a
Antioxidant activity (µM Trolox Equivalents) 183.67 ± 3.78b 188.00 ± 0.33b 755.78 ± 0.19a

Values expressed are means ± S.D. of two replications

Data marked with different letters within the same row indicate statistically significant differences (p < 0.05)

identified in the samples. These compounds have saccha- of ferulic acid in the peel was significantly higher as com-
ride units attached through O-glycosidic bonds. In addi- pared to the juice samples. On the other hand, caffeic acid
tion to these, peak 3 and peak 6 were eriocitrin (eriodictyol derivative was not detected in both juice samples while
7-O-rutinoside) and naringin (naringenin 7-O-neohesperi- it was available in the peel extract. Similar to the results
doside), which belong to the flavanone O-glycoside group. in the current study, Costa et. al [24] determined ferulic
These compounds are usually found in Citrus fruits. In a pre- acid in 11.35 ± 0.53 mg/L in C. limetta juices.
vious study, eriocitrin was found to be 516.52 ± 12.84 mg/L Sweet lime also comprises cinnamic acid glucosides
in C. limetta juices [24] which is higher than the values including, dihydro-caffeoyl-, dihydro-feruloyl-gluco-
found in the present study (Table 3). side. The dihydro-feruloyl-O-glucoside was detected in
Peak 10 was identified as B types of procyanidin which very low amounts in both of the juices (2.08 ± 0.09 and
consists of two pieces (epi)catechin unit [26] (Table 2). Also, 1.73 ± 0.07 mg/L), but not in the peel. Peak 18 with a
peak 12 was identified as luteolin which was an aglycon. In [M–H] − ion at m/z 663 and fragment ion m/z 195 was
previous studies; luteolin was determined in Citrus limon recognized as dihydro-feruloyl-O-glc-HMG-caf which
peel and pulp [28, 29]. Procyanidin B and luteolin were had caffeoyl-unit. The fragment ion m/z 195 belonged to
detected for the first time in sweet lime in the present study dihydro-feruloyl moiety [18]. Peak 19 with a [M–H]− ion
even if they were not identified in sweet lime before. It was at m/z 807 was also detected as dihydro-feruloyl-O-glc-
found that the concentrations of procyanidin B and luteolin HMG-caffeoyl-HMG. This compound has caffeoyl-unit
of the peel were significantly higher than those of the juices and two HMG-substitution. Peak 20 with a [M–H]− ion at
(Table 3). m/z 385 presented characteristic UV and MS spectra con-
Ferulic acid (peak 11, Fig. 1) and caffeic acid deriva- sistent with feruloyl-gluccarate (p-feruloyl-glucaric acid
tive (peak 16, Fig. 1) were also identified in the sweet or p-feruloyl-galactaric) in a Rt of 35.02 min.
lime extracts in the present study (Table 3). The amount

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Characterization of phenolic compounds in sweet lime (Citrus limetta) peel and freshly… 3247

13
 3248 O. K. Buyukkurt et al.

◂Fig. 1  LC-DAD and LC-ESI-MS/MS chromatograms of phenolic Moraes Barros et al. [14] reported that the DPPH radical
compounds identified in sweet lime. Peaks correspond to compounds scavenging capacity of the sweet lime peel was higher than
in Table 2. A Peel, B Juice with hand-squeezed after peeling, C Juice
with hand-squeezed
that of the pulp due to high phenolic content of the peel.
Barreca et al. [4] determined the antioxidant activity of
crude C. limetta juice as 261 µM TE and reported that the
PCA results antioxidant activity of the juice was associated with low fla-
vonoid content in the juice. Ali et al. [33] determined the
Principal component analysis (PCA) was performed in order antioxidant activity in different fruit (orange, mango, lemon,
to indicate the relations among the phenolic compounds in papaya etc.) and the antioxidant activity level of sweet lime
the sweet lime peel and juices (Fig. 2). According to the was classified as high compared to other fruits. Guimarães
results of the PCA, two principal components (factors) were et al. [1] reported that the peel extract had higher antioxidant
determined and these two components defined 99.99% of the activity than that of the juice extract. Sir Elkhatim et al. [34]
total variability of the experimental data (F1 was 99.53%, also reported that antioxidant activities of lemon, orange,
and F2 was 0.46%). It was observed that the sweet lime and grapefruit peels were higher than in those of their pulp
extracts were well categorized as peel and juice. However, and seeds.
both of the juices were found to be very close to each other
as can be seen in Fig. 2. It was also found that all phenolic
compounds except hesperidin and total phenolic compounds Conclusion
were positioned very close to each other in the PCA biplot.
Hesperidin was the most abundant compound among the The phenolic compounds of sweet lime peel and juices were
phenolics as can be seen in the PCA graph and Table 3. identified and quantified with the aid of LC-DAD-ESI-MS/
Therefore, hesperidin and the total phenolic compounds MS technique in this study. A total of 20 phenolic com-
were positioned on the positive side of the F1 axis. On the pounds (18 flavonoids and two phenolic acids) were iden-
other hand, the other phenolic compounds were positioned tified in the samples. Two of these phenolic compounds,
on the opposite side of the F1 axis in Fig. 1. procyanidin B and luteolin, were detected for the first time
in sweet lime in the present study. Hesperidin was found
Antioxidant assay in considerable amounts in all sweet lime extracts. The
phenolic compounds in the peel were found to be statisti-
The antioxidant capacity of the sweet lime peel and juice cally higher than those of the juices while the amounts of
extracts were evaluated using DPPH free radical scaveng- the phenolic compounds of the two juice samples were not
ing assays. The results of the antioxidant capacity are pre- statistically different in the study. When the total phenolic
sented in Table 3. It was found that the antioxidant capacity contents of the sweet lime juices were examined, it was seen
of the peel was higher than those of both of the juices. For that the total phenolic content increased by about 13% due
the peel extract, the value of the antioxidant capacity was to peel effect. Similarly, the antioxidant capacity of the peel
755.78 ± 0.19 µM Trolox Equivalent (TE) (Table 3). On was found to be higher than those of the juices due to the
the other hand, no significant difference was found between high total phenolic content of the peel. In conclusion, it is
the antioxidant capacities of the juice with hand-squeezed recommended that the sweet lime juice should be prepared
and the juice with hand-squeezed after peeling. Similarly, by squeezing the fruit with the peel to obtain higher amounts
of phenolics.

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Characterization of phenolic compounds in sweet lime (Citrus limetta) peel and freshly… 3249

Fig. 2  PCA biplot of the phe- Biplot (axes F1 and F2: 99,99%)
nolic compounds of sweet lime
8
peel and juices
Peel

F2 (0,46%)
2
Caffeic acid derivative Rhoifolin
Dihydro-feruloyl-O-glucosides
Orientin 4′-methyl ether Luteolin
Ferulic acid Vicenin-2 Eriocitrin Total phenolic compounds
0 ABA-O-glc-HMG
Procyanidin B dimer Naringin
Diosmin Scoparin Hesperidin
Feruoyl gluccarate Lucenin-2 4′-methyl ether
Dihydro-feruloyl-O-glc-HMG-caf Isophrone-O-glc-HMG
-2 Dihydro-feruloyl -O-glc-HMG-caffeoyl-HMG Melitidin Juice with hand-
squeezed
Juice with hand-
-4 squeezed after peeling
-8 -6 -4 -2 0 2 4 6 8
F1 (99,53%)

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