Industrial Crops & Products 107 (2017) 232–243

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Industrial Crops & Products

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Profiling of phenolic compounds and antioxidant activity of Melia azedarach MARK

L. leaves and fruits at two stages of maturity
⁎
Yassine M’rabeta, , Nesrine Rokbenia, Stéphanie Cluzetb, Abdennacer Boulilaa, Tristan Richardb,
Stéphanie Krisab, Lamjed Marzoukic, Herve Casabiancad, Karim Hosnia
a
Laboratoire des Substances Naturelles, Institut National de Recherche et d’Analyse Physico-chimique INRAP, Technopole Sidi-Thabet, 2020 Ariana, Tunisia
b
Université Bordeaux, Institut des Sciences de la Vigne et du Vin, Groupe d'Etude des Substances Végétales à Activité Biologique, EA 3675, 210 Chemin de Leysotte, 33882
Villenave d'Ornon, France
c
Laboratoire de Physiologie Fonctionnelle et Valorisation des Bio-Ressources, Institut Supérieur de Biotechnologie de Béja, Avenue Habib Bourguiba, B.P., 382-9000 Béja,
Tunisia
d
Institut des Sciences Analytiques, Laboratoire de Produits Naturels Biosourcés, 5 Rue de la Doua, Villeurbanne, Lyon, France

A R T I C L E I N F O A B S T R A C T

Keywords: The present work aims to investigate the maturation-related changes in phenolic profile of Melia azedarach (L.)
Melia azedarach l leaves (young and old) and fruits (pulp and kernel) at two developmental stages. A total of 26 phenolic com-
Maturation pounds were identified by high-performance liquid chromatography-diode-array detector-hyphenated with
Phenolic profile tandem mass spectrometry (HPLC-DAD-ESI–MS/MS). They include phenolic acids, flavonols and flavanols which
HPLC-DAD-ESI–MS/MS
were found mostly in glycosylated forms. The main phenolic compounds were found to be rutin (5.86–21.33%,
Antioxidant activity
of total integrated peak area at 280 nm), quercetin-3-O-neohesperidoside (3.95–8.76%), kaempferol-3-O-ruti-
noside (2.73–11.23%), feruloylglucaric acid (0.85–11.84%) and feruloylquinic acid derivative (1.94–11.07%).
The pattern distribution of phenolic compounds was organ-specific, and somewhat variable depending on the
stage of maturation. Additionally, the total phenol content was determined as well as the antioxidant capacity
using different in vitro assays. The extracts of young leaves demonstrated the highest total phenol content
(85.4 mg GAE/g extract), and exhibited the strongest scavenging activity against 2,2-diphenyl-1-picrylhydrazyl
DPPH and the highest oxygen radical absorbance capacity ORAC (235 and 328 mg Trolox equivalents/g extract,
respectively) compared to old leaves and immature/mature fruit parts. In contrast, the highest metal chelating
activity (MCA) was observed in immature pulp extracts (22.85 mg EDTA equivalents/g extract). Pearson’s
correlation and principal component analysis (PCA) showed that quercetin-3-O-neohesperidoside, rutin and
kaempferol-3-O-rutinoside were the main contributors on the antioxidant capacity. On the basis of these results,
M. azedarach could serve as an excellent source of natural antioxidants which could be used for pharmaceutical
and cosmeceutical applications.

1. Introduction
Melia azedarach Linn. (Meliaceae), known as “Chinaberry, bead tree,
and Persian lilac”, is a deciduous tree native to Southeast Asia and
Australia. Due to its high tolerance to extreme environments and great
potential to compete for nutritive resources as well as its high growth
rate and prolific seed production, it has been successfully introduced
and naturalized worldwide as ornamental species (Ma et al., 2015).
Various parts of M. azedarach (stem barks, leaves, fruits, and seeds)
have been used since ancient times as folk remedies for a variety of
ailments including stomach irritation, vomiting and bloody diarrhoea
(Suresh et al., 2008), leprosy, vitiligo, kidney stones and intestinal
obstructions, among others (Jafari et al., 2013). The antioxidant, anti-
acetylcholinesterase, anti-tyrosinase (Orhan et al., 2012), antimicrobial
(Pokhrel et al., 2012), cytotoxic (Ntalli et al., 2010), wound healing
(Vijaya et al., 2012), antiulcer (Bahuguna et al., 2009), antiangiogenic
(Kumazawa et al., 2013), antidiabetic (Khan et al., 2014), anti-
hyperlipidemic (Kumar et al., 2013), anti-urolithiatic (Dharmalingam
et al., 2014), anthelmintic (Cala et al., 2012), antiprotozoal (Lee et al.,
2007), antifeedant (Bullangpoti et al., 2012), insecticidal, and nemati-
cidal properties (Aoudia et al., 2012) have also been reported. Most of
these biological activities were attributed to a wide array of bioactive
components including triterpenoids, limonoids, fatty acids and phe-
nolics (Kumazawa et al., 2013). The latter components, recognized for

⁎
Corresponding author.
E-mail address: yassine.mrabet@gmail.com (Y. M’rabet).

http://dx.doi.org/10.1016/j.indcrop.2017.05.048
Received 10 February 2017; Received in revised form 25 May 2017; Accepted 26 May 2017
0926-6690/ © 2017 Elsevier B.V. All rights reserved.
Y. M’rabet et al.
their antioxidant potential and their biological activities have been the
subject of few investigations in M. azedarach and different composi-
tional patterns depending on plant part, origin, extraction, and analy-
tical procedures have been published (Aoudia et al., 2012; Orhan et al.,
2012; Kumazawa et al., 2013). In this context, earlier phytochemical
studies showed that the fruits from the Chilean specimen were char-
acterized by the abundance of catechin and kaempferol derivatives
(Chiffelle et al., 2009). Three years later, Aoudia et al. (2012) have
compared the phenolic profiles of pulp water extract of M. azedarach
fruits from Italian and Algerian origins and found that phenolic acids
such as p-coumaric, caffeic and ferulic acids, as well as the flavonols
rutin and isoquercetin were the main compounds. In another report
from Turkey, syringic, chlorogenic, caffeic, and p-coumaric acids were
found as the basic phenolic constituents of leaves and fruits of M.
azedarach (Orhan et al., 2012). Despite these efforts, very little and
fragmentary information is available with respect to maturational ef-
fects on phenolic profiles and their biological activities (Chiffelle et al.,
2009).
With regard to this topic, the present work was intended to com-
prehensively investigate the developmental (in young or newly formed
leaves, and old or completely expanded leaves) and maturational (in
fruits including both kernels and pulps) evolution of phenolic compo-
sition in M. azedarach leaves and fruits, respectively. The evaluation of
the antioxidant activities of different extracts using three in vitro com-
plementary assays: oxygen radical absorbance capacity (ORAC), 1,1-
diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity and
ferrous metal ions chelating capacity (MCA) was also achieved. The
assessment of such an aspect will provides useful insights on the phy-
tochemistry of this species, and consequently promoting its efficient use
as a possible source of natural antioxidants.
2. Material and methods
2.1. Chemicals and reagents
All solvents used for analytical chromatography were of HPLC
grade. Those used for extraction were of a purity > 99%. Methanol and
acetonitrile were obtained from Scharlab S.L. (Sentmenat, Spain).
Formic acid (> 99%) was obtained from Merck (Merck Eurolab,
France). Folin-Ciocalteu reagent, Ferrozine iron reagent, sodium
fluorescein, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH),
2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium carbonate (Na2CO3),
Iron(II) sulphate (FeSO4), Trolox, Ethylenediaminetetraacetic acid
(EDTA), gallic acid, quercetin-3-O-rutinoside, quercetin 3-β-D-gluco-
side, kaempferol 3-O-β −rutinoside and kaempferol 3-β-D-glucoside
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water was
purified using an Elga (Bucks, UK) water purification system with a
resistivity of no less than 18 MΩ/cm−1.
2.2. Plant materials
Young (newly formed) and old leaves (completely expanded) as well
as immature (green) and mature (yellow) fruits were randomly col-
lected from M. azedarach trees growing in the region of Sidi-Thabet
(Northern Tunisia; latitude 36°55'13″(N); longitude 10°5'10″(E); alti-
tude 14 m; average annual rainfall and temperature: 449 mm and 18 °C,
respectively). Fresh fruits are immediately hand-peeled to separate the
outermost pulp (pericarp) from the inner kernel (endocarp, containing
a woody shell enclosing seeds). All materials were dried at 40 ± 2 °C,
in a hot air oven for three days (MMM Venticell, Einrichtungen,
Germany), and grounded into fine powder to pass through a 0.5 mm
sieve.
2.3. Extraction procedure
Twenty grams of powdered raw materials (leaves, fruit pulps and
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Industrial Crops & Products 107 (2017) 232–243
fruit kernels) were first defatted with n-hexane in a Soxhlet apparatus
during 48 h. The defatting process was applied to remove lipophilic
components and pigments in order to simplify the phenolic extraction.
Each dried defatted sample was subsequently extracted twice with 80%
aqueous methanol (1:10, w:v) for 24 h, under constant shaking at
100 rpm, to provide the hydro-methanolic extract. Afterward, the
combined resulting filtrates were concentrated under reduced pressure
at 35 °C, lyophilized and then kept at −20 °C until analysis.
2.4. Determination of total phenolics
The total phenolic content (TPC) in the extracts of M. azedarach was
determined according to the Folin-Ciocalteu method (Singleton et al.,
1999). Briefly, 20 μL of extract (1 mg/mL) was added into 96-well
microplate followed by 100 μL of the Folin-Ciocalteu reagent. After
5 min, 80 μL of 7.5% Na2CO3 were added. After 30 min incubation in
the dark, the absorbance of the solution was measured at 760 nm using
a Fluostar Optima automated apparatus (BMG Labtech., Durham, NC,
USA). Gallic acid was used as the standard, and the results were ex-
pressed as mg of gallic acid equivalents/g of extract (mg GAE/g ex-
tract).
2.5. Characterization of phenolic compounds by HPLC-DAD-ESI–MS/MS
Liquid chromatography analysis was performed on an Agilent 1200
series LC system (Agilent Technologies, Santa Clara, CA) equipped with
a binary pump, a diode-array detector (DAD), and an auto-sampler.
Chromatographic separation was achieved with a Zorbax SB-C18
column 100 mm x 2.1 mm (i.d.), 1.8 μm film thickness (Agilent
Technologies, Palo Alto, CA). Gradient elution of analytes was carried
out with water/0.1% formic acid (solvent A) and acetonitrile/0.1%
formic acid (solvent B) at a flow rate of 0.4 mL/ min, and the injection
volume was 10 μL. The gradient was programmed as follows: 0 min, 1%
B; 2 min, 10% B; 6 min, 35% B; 7 min, 50% B; 8.8 min, 70%B; 10.8 min,
92% B; 11 min, 100% B; and finally, the initial conditions were held for
1 min as re-equilibration step. The mass spectra were acquired with an
Esquire 3000+ ion trap mass spectrometer equipped with an electro-
spray ionisation (ESI) source from Bruker Daltonics (Billerica, MA,
USA). The ESI–MS conditions were as follows: negative ion mode, mass
range from m/z 110–1100 amu, high-voltage capillary ± 3.7 kV; drying
gas (N2) temperature, 365 °C; drying gas flow, 10 L/min; nebuliser gas
pressure, 40 psi; capillary exit 111.2 V; skimmer voltage 40 V; trap
drive 41.5 V. The MS data were handled by Data Analysis 4.0 (Bruker
Daltonics, Bremen, Germany) and mzMine 2.15 (MZmine Development
Team). Tentative identification of phenolic compounds was based on
comparison of their retention time, UV and mass spectra with those of
the authentic standards if available. Otherwise, the structure was pro-
posed based on the UV and mass spectra and literature data (Harbaum
et al., 2007; Plazonić et al., 2009; Simirgiotis et al., 2009; 2015;
Vallverdú-Queralt et al., 2010; Fischer et al., 2011; Gouveia and
Castilho, 2011; Orhan et al., 2012; Chen et al., 2015; Schmeda-
Hirschmann et al., 2015; Llorent-Martínez et al., 2016).
Since we are interested in comparing the relative amounts of phe-
nolic compounds in the different samples, the qualitative analysis was
carried out according to Li et al. (2015) and Pérez-Ràfols and Saurina
(2015). The area of each identified peak was integrated at 280 nm from
samples chromatograms and subjected to several treatments (align-
ment, baseline correction, normalization) using ACD/Spectrus Pro-
cessor package (Advanced Chemistry Development, Inc., Toronto, ON,
Canada). The percent area was calculated accordingly with respect to
total peak areas from chromatograms in triplicate for each sample.
2.6. Determination of antioxidant activities
2.6.1. Free radical scavenging with DPPH% radical
The DPPH radical scavenging activity of M. azedarach extracts was
Y. M’rabet et al.
measured using the method of Brand-Williams et al. (1995), slightly
modified by Miliauskas et al. (2004). Briefly, 50 μL of appropriately
diluted extract sample in 80% methanol was added to 150 μL of freshly
prepared DPPH methanolic solution (300 μM) into 96-well microplates,
and kept in the dark at 37 °C for 20 min. The decrease of absorbance
was monitored at 520 nm (Fluostar Optima plate reader). The DPPH
radical scavenging activities were determined using a regression
equation for a linear range of 50–300 μM of Trolox standards, and the
results were expressed as mg Trolox equivalents/g of extract (mg TE/g
extract).
2.6.2. Oxygen radical absorbance capacity (ORAC) assay
The ORAC activity of different extracts was measured according to
Dávalos et al. (2004). Briefly, 30 μL of appropriately diluted (in phos-
phate buffers pH 7.4) extract sample were put into black 96-well plates,
and 180 μLof fluorescein solution (70 nM) was added. After 5 min pre-
incubation at 37 °C, 90 μL of AAPH solution (12 mM) was added, and
fluorescence was monitored using 485 nm excitation and 530 nm
emission wavelengths at 1 min intervals for 60 min (Fluostar Optima
plate reader) against a blank sample (30 μL phosphate buffer). The area
under the curve of the fluorescence decay (AUC) was calculated for
each sample by integrating the relative fluorescence curve. The net AUC
of the sample was calculated by subtracting the AUC of the blank. The
regression equation between net AUC and Trolox concentration was
determined, and ORAC values were expressed as mg of Trolox
equivalents/g of extract dry weight (mg TE/g extract).
2.6.3. Metal chelating activity (MCA)
The chelation of Fe2+ ions by M. azedarach extracts was estimated
following the method of Dinis et al. (1994). Briefly, 60 μL of FeSO4
(0.3 mM) was mixed with 40 μL of appropriately diluted extract sample
in 80% methanol in 96-well microplates. The reaction was initiated by
the addition of 80 μL of ferrozine solution (2 mM), and the mixture was
vigorously shaken and left to stand at room temperature for 10 min.
The decrease of purple ferrozine-Fe2+ complex was monitored at
550 nm using MRX II plate reader (Dynex Technologies Inc., Chantilly,
VA). The EDTA was used as the chelating agent standard (10–75 μg/
mL) and the results were expressed as mg EDTA equivalents/g of ex-
tract dry weight (mg EDTAE/g extract).
2.7. Statistical analysis
Data are expressed as means ± standard deviations (SD). Means
comparison was achieved using one-way analysis of variance (ANOVA)
followed by Tukey’s honestly significant difference (HSD) post-hoc test
at the significance level of 5%. Pearson’s correlation analysis and
principal component analysis (PCA) were performed in order to identify
the relationship between TPC, proportions of phenolic components and
parameters of antioxidant activities. The input dataset was arranged in
a XIJ matrix, where I corresponded to rows (6 samples × 3 replicates)
and J corresponded to columns (33 variables). The variables comprised:
(i) the pre-processed areas obtained by integration of identified peaks at
280 nm, (ii) phenolic compounds groups (phenolic acids, flavonols, and
flavanols) proportions obtained by summing the corresponding peaks
area from (i), (iii) corresponding TPC, and (iv) antioxidant activities
(ORAC, DPPH and MCA). Zero-centring and scaling were applied to
data before PCA.
The statistical software RStudio version 1.0.44 (RStudio Inc.,
Boston, MA) was used for all analyses.
3. Results and discussion
3.1. Total phenols content (TPC)
As polyphenols are one of the major bioactive compounds acting as
primary antioxidants, metal chelators or free radical scavengers, the
234
Industrial Crops & Products 107 (2017) 232–243
Fig. 1. Total phenol contents (TPC) in leaves (YL: young leaves; OL: old leaves) and fruits
(IP: immature pulps; MP: mature pulps; IK: immature kernels, MK: mature kernels) of
Melia azedarach at two developmental and maturational stages. Different superscripts
indicate statistically significant differences (p < 0.05).
determination of their total content in different extracts is of utmost
importance. In the present study, TPC varied significantly (p < 0.05)
between the plant parts and growth stages (Fig. 1).
The highest TPC was found in the extracts of young leaves (85.4 mg
GAE/g extract), followed by those of old leaves (59.1 mg GAE/g ex-
tract) and immature kernels (45.9 mg GAE/g extract). In contrast, the
lowest values were observed in the extracts from mature pulps and
kernels (11.5 and 20.9 mg GAE/g extract, respectively). In general, it
appears that development and maturation of M. azedarach leaves and
fruit parts (kernels and pulps) were associated with a concomitant de-
crease in the TPC. This developmental regulation can be explained by
the highest expression levels of the enzymes responsible for poly-
phenols synthesis during the early stages of leaves development and
fruits maturation. In contrast, an extensive degradation of polyphenols
during the course of maturation could be the reason of the drastic de-
crease in TPC as observed in lotus seeds (Liu et al., 2015). From bio-
logical stand point, the active biosynthesis and accumulation of poly-
phenols might have a defensive role against foraging insect during the
early development/maturation stages of leaves and fruits, which are
normally heavily defended. Support to this assumption is provided by
Bentes and Mercadante 2014, who showed that the accumulation of
defensive phenolic compounds during the early stages of maturation of
genipap (Genipa americana L.) fruits was associated with the high
availability of low molecular mass precursors. As displayed in this
study, the accumulation of TPC during the early stages of maturation is
consistent with that observed in other species such as pistachio (Kilic
et al., 2016), strawberry (Mandave et al., 2014), and lotus (Liu et al.,
2015).
When compared with the few available publications regarding M.
azedarach, our TPC values are nearly similar to those observed for the
Algerian (Aoudia et al., 2013) and Indian (Pokhrel et al., 2015) speci-
mens with an average TPC values ranging from 89 to 92 mg GAE/g in
leaf samples. In general, the present results indicate that irrespective to
the stage of maturation, leaves and fruits of M. azedarach could re-
present a potential source of phenolic compounds. To get more insight
into the identity of phenolic compounds in the methanol extracts of
leaves and fruits, HPLC-ESI–MS/MS analyses were performed.
3.2. Characterization of phenolic compounds
The fingerprinting of methanol extracts of M. azedarach leaves and
fruits at two stages of maturity led to the identification of 26 phenolic
compounds (Fig. 2).
They include 12 phenolic acids, 11 flavonols and 3 flavanols, which
were tentatively identified based on their HPLC retention time, UV
spectra, deprotonated molecule, and mostly on their MS2 fragmentation
Y. M’rabet et al.
Fig. 2. HPLC-DAD chromatograms (recorded at 280 nm) of the methanol extract of leaves (YL: young leaves; OL: old leaves) and fruits (IP: immature pulps; MP: mature pulps; IK:
immature kernels, MK: mature kernels) of Melia azedarach at two developmental and maturational stages. Peak assignments are depicted in Table 1, * indicates the identified compounds.
behaviour previously reported in the literature due to the lack of re-
ference standards for most components (Table 1). This study was car-
ried out using an ion-trap machine, where both positive and negative
electrospray ionisation modes (ESI) were applied. Although the two
modes analysis were complementary, visualization of both chromato-
grams revealed more intense and well-resolved chromatographic peaks
in the negative compared to the positive ion mode. Therefore, negative
ESI mode was adopted for the identification.
3.2.1. Phenolic acids
Phenolic acids, eluted between 1.85 and 4.41 min were easily dis-
tinguished based on their UV absorption maxima at 230–240 nm and
320–330 nm and a shoulder around 290–300 nm.
Two caffeic acid hexosides derivatives (peaks 1 and 12) were de-
tected in M. azedarach. Peaks 1 eluted at 1.85 min, exhibited a depro-
tonated molecular ion [M-H]− at m/z 387, releasing two MS2 fragments
ions. The fragment ion [M-H-46]− at m/z 341 (loss of formic acid) and
the second fragment ion [M-H-46-162]− (loss of hexose moiety) at m/z
179 [caffeic acid-H]−, suggested that peak 1 is a caffeic acid hexoside
derivative (Vallverdú-Queralt et al., 2010). This component (caffeic
hexoside derivative I) was detected in both leaves and fruits (pulps end
kernels) at immature and mature stages with the highest proportion
(4%, relative area at 280 nm) being recorded in mature pulps (Table 2).
Peak 12 eluted at 4.41 min, showed a quasi-molecular ion [M-H]−
at m/z 431, which yielded MS2 fragments at m/z 385 ([M-H-46]−, loss
of formic acid), at m/z 341 ([M-H-90]−, loss of C3H6O3) and a third
235
Industrial Crops & Products 107 (2017) 232–243
peak at m/z 179 ([M-H-90-162]−, loss of hexose moiety) indicative to
the aglycone caffeic acid. The fragmentation scheme was similar to that
obtained for caffeic acid hexose derivatives (Vallverdú-Queralt et al.,
2010). This component (caffeic acid hexoside derivative II) was omni-
present in leaves and fruits with preferential accumulation in mature
pulps (3.8%). Peak 2 eluted at 2.31 min, showed a quasi-molecular ion
[M-H]− at m/z 371 and its MS2 fragmentation gave a fragment ion [M-
H-162]− at m/z 209 (loss of hexose residue) and a “dehydrated” frag-
ment ion [M-H-162-18]− at m/z 191, characteristic of ferulic acid
(Simirgiotis et al., 2009; Schmeda-Hirschmann et al., 2015). Conse-
quently, peak 2, which was only detected in young and old leaves with
a percentage ranging from 0.1 to 0.8% in young and old leaves, re-
spectively (Table 2). Irrespective to the stage of maturity, peak 3 eluted
at 2.89 min, with a characteristic UV absorbance of hydroxycinnamic
acids (UV around 310 nm), was present in both leaves and fruits
(0.2–2.5%) and exhibited a deprotonated molecular ion [M-H]− at m/z
315, yielding in MS2 an intense base peak [M-H-162]− at m/z 153 (loss
of hexose moiety), indicative to the aglycone protocatechuic acid. This
component was tentatively identified as protocatechuic acid hexose and
has already been found in leaves and fruits of M. azedarach (Orhan
et al., 2012). Peak 4 eluted at 3.01 min, showed a deprotonated mo-
lecular ion [M-H]− at m/z 329 and a fragment ion [M-H-162]− at m/z
167 due to the loss of hexose moiety, revealing the compound to be
vanillic acid hexoside (Fischer et al., 2011). This component was om-
nipresent in both analysed plant parts with the highest proportion (3%)
being observed in mature pulps. Peak 5, eluted at 3.64 min was
 Table 1
Retention time, UV, MS2, tentative identification and occurrence of phenolic compounds in Melia azedarach leaves and fruits

Peak No.* Tentative identification RT (min) UVmax (nm) [M-H]−(m/ MS2 m/z (relative abundance%) Occurrence in M. azedarach parts
Y. M’rabet et al.

z)
Leaves Fruits

Kernels Pulps

YL OL IK MK IP MP

Phenolic acids
1 Caffeic acid hexoside derivative 1.85 290, 325 387 341 (100), 179 (25) + + + + + +
I
2 Hydroxyferulic acid hexoside 2.31 – 371 353 (91), 335 (11), 334 (30), 209 (100), 201 (5), 191 (31), 129 (16) + + − − − −
3 Protocatechuic acid hexoside 2.89 234, 292, 314 315 153 (100), 151 (70), 109 (83) + + + + + +
4 Vanillic acid hexoside 3.01 296, 328 329 167 (100), 151.6 (75) + + + + + +
5 Coumaroyl-quinic acid 3.64 280, 312 355 337 (45), 191 (100), 147 (36) + + − + − −
6 Ferulic acid hexoside I 3.72 298, 326 355 193 (100), 149 (83), 134 (78) + + − − + +
7 Feruloylquinic acid derivative 3.87 299, 324 385 367 (20), 191 (100),185 (10), 119 (8) + + − − + +
8 Ferulic acid dihexoside 3.89 – 517 337 (44), 193 (100), 175 (71) − − − − + +
9 Ferulic acid hexoside II 4.13 298, 326 355 193 (100), 149 (1), 134 (18) + + + + + +
10 Feruloylglucaric acid 4.26 244, 323 385 191 (100), 173 (2) + + + + + +
11 Feruloylquinic acid 4.38 229, 320 367 367 (13), 191 (100), 185 (11), 119 (9) + + + + + +
12 Caffeic acid hexoside derivative 4.41 290, 325 431 385 (17), 341 (10), 179 (100), 125 (35) + + + + + +
II
Flavonols
13 Myricetin-O-rutinoside 4.65 286, 356 625 625 (3), 479 (15), 317 (100), 301 (5) + + − − − −
14 Myricetin hexoside 4.73 264, 356 479 332 (66), 317 (100), 277 (34) − − − − + +
15 Quercetin-3-O- 4.98 256, 352 609 591 (3), 463 (6), 301 (100), 300 (18), 179 (3) + + + + + +

236
neohesperidoside
16 Quercetin-3-O-rutinoside 5.05 256, 354 609 507 (45), 463 (2), 343 (10), 302 (22), 301 (100), 300 (30), 185 (38) + + + + + +
(Rutin)
17 Quercetin-3-O-glucoside 5.19 256, 352 463 301 (100), 300 (13), 151 (2) + + − − + +
18 Kaempferol-O-dihexoside 5.22 256, 348 609 447 (14), 285 (100), 284 (61), 241 (2) + + − − − −
19 Kaempferol-3-O-rutinoside 5.35 266, 351 593 327 (4), 285 (100), 284 (8), 199 (2) + + − − + −
20 Isorhamnetin-3-O-rutinoside 5.45 256, 356 623 461 (5), 315 (90), 301 (8), 300 (100), 271 (20) − + − − + +
21 Quercetin pentoside 5.49 256, 352 433 365(7), 343(9), 301 (100), 300 (48) + + − − − −
22 Kaempferol-3-O-glucoside 5.57 262, 343 447 447 (3), 329 (2), 285 (100), 284 (77) + + − − − −
23 Kaempferol-3-O-rhamnoside 5.88 289,340 431 429 (100), 428 (51),285 (46) + + − − − −
Flavanols
24 Catechin hexoside 3.49 293, 334 451 289 (96), 244 (15), 205 (9), 179 (6), 171 (18), 170 (26), 163 (8), 160 (6),152.5 (7), 136 (15), 128 + + + + − −
(49)
25 (Epi)catechin methyl gallate 5.66 278 455 289 (100) + + − − − −
26 Procyanidin dimer B 6.54 211, 247, 317 577 451 (4), 407 (32), 359 (100), 289 (48) + + + + + +
Others
27 Toosendanin 7.76 220 573 531 (100), 453 (1.5), 451 (0.07), 425 (3) + + + + + +
Non identified
28 NI 5.19 230, 280, 310 563 562 (100), 542 (65), 487 (34), 412.8 (42) − − + + − −
29 NI 5.29 214, 263, 298 375 356.9 (10), 345 (6), 328 (2), 327 (100), 315 (6), 313 (6), 312 (3), 294 (1.5), 195 (64), 180 (3), 179 − − + + − −
(31), 165 (36), 161 (5), 150 (14), 146 (9),
30 NI 6.43 214, 222, 272 619 362.7 (10), 361 (6), 348.6 (3), 345 (100), 335 (13), 321 (9), 316 (2),295 (4), 279 (23), 275 (2), 269 − − + + − −
(10), 269 (4), 257 (17),251 (4), 169 (2), 145 (2)
31 NI 7.14 231, 278, 325 427 383 (9), 369 (38), 343 (34), 333 (20), 321 (15), 318 (7), 307 (100), 305 (8), 268 (16), 267(36), 265 − − + + − −
(4), 265(10), 249 (4), 238.6 (7), 212.8 (7), 174.5 (14), 169 (17)
32 NI 8.44 220, 293, 330 857 825 (100), 813 (6), 811 (8), 798 (1), 797 (37), 763 (3), 757 (2), 753(9), 709 (1), 695 (3), 667 (9), 649 − + + + + +
(10.5), 635 (2)
33 NI 9.08 208, 320 629 471 (5), 470 (2.5), 465 (1), 464 (3), 461 (3), 283 (100), 281 (3), 219 (3), 198 (39), 197 (35), 184 (5), + − + + + +
183 (4), 147 (5)
(continued on next page)
Industrial Crops & Products 107 (2017) 232–243
Y. M’rabet et al. Industrial Crops & Products 107 (2017) 232–243

Numbered according to Fig. 2; RT: retention time; NI: non-identified; YL: young leaves, OL: old leaves, IK: immature kernel, MK: mature kernel, IP: immature pulp, MP: mature pulp; +: detected/-: not detected based on the presence/absence of
detected in leaves (0.4-1.2%) and mature kernels (2%). It displayed a

MP
quasi-molecular ion [M-H]− at m/z 355 and gave an MS2 fragment ion

+
+
+
[M-H-18]− at m/z 337 due to the loss of H2O molecule, and an intense
Pulps fragment ion[M-H-18-146]−at m/z191 (quinic acid), due to the loss of

+
+
+
IP
Occurrence in M. azedarach parts

coumaroyl residue. Consequently, peak 5 was tentatively identified as

MK coumaroyl-quinic acid (Harbaum et al., 2007; Plazonić et al., 2009).
+
+
+
Kernels

Mass spectra of peaks 6 and 9 (eluted at 3.72 and 4.13 min, respec-
Fruits

tively) displayed a quasi-molecular ion [M-H]− at m/z 355 and two

+
+
+
IK

fragment ions with one at [M-H-162]− at m/z 193 for ferulic acid
through the loss of hexose moiety, and the other fragment ion [ferulic
OL

−
+
−

acid-CO2-H]− at m/z 149 for decarboxylated ferulic acid after elim-

Leaves

ination of hexose and CO2. Conclusively, peaks 6 and 9 were tentatively

YL

−
+
−

identified as ferulic acid hexoside I and II. Both components were

preferentially biosynthesized/accumulated in leaves and pulps. Peaks 7
611 (69), 610 (6), 593 (12), 587 (35), 569 (100), 551 (8), 549 (2), 547 (26), 545 (8), 541 (8), 527

eluted at 3.72 min, showed a deprotonated molecular ion [M-H]− at m/

z 385 and gave an MS2 fragment ion [M-H-18]− at m/z 367 (fer-
uloylquinic acid), due to the loss of H2O molecule and a fragment ion at
m/z 191 consistent with quinic acid adduct. Peak 7 was therefore
tentatively identified as feruloylquinic acid derivative (Simirgiotis
et al., 2015). This phenolic acid showed the same pattern of distribution
observed for ferulic acid hexoside I and II. Peak 11 eluted at 4.38 min
showed similar fragmentation pattern as peak 7, consequently peak 11
was tentatively identified as feruloylquinic acid (Simirgiotis et al.,
2015; Gouveia and Castilho, 2011). However, it distribution was lim-
ited to leaves and pulps. Peak 8 eluted at 3.89 min exhibited a depro-
tonated molecular ion [M-H]− at m/z 517 and a fragment ion [M-H-
324]− at m/z 193 (ferulic acid) through the loss of two hexosyl moi-
eties (162 + 162 amu). Accordingly, peak 8 was probably a ferulic acid
dihexoside (Chahdoura et al., 2015; Bystrom et al., 2008) and was
found in immature and mature pulps. The fragmentation pattern of
peak 10 (eluted at 4.26 min; [M-H]− at m/z 385) was matching the
previously reported for feruloylglucaric acid (Spínola et al., 2015;
MS2 m/z (relative abundance%)

Goulas and Manganaris, 2012). This component was omnipresent in

689 (4), 673.5 (2), 601 (100)

both plant parts and in all development and maturation stages with
(11), 517 (5), 511 (10)

preferential accumulation in immature (11%) and mature pulps (6.8%).

542 (4), 541 (100)

3.2.2. Flavonols
According to their UV spectra (absorption maxima around
350–360 nm), flavonols were also found in the studied samples, most of
them associated to myricetin (2 compounds), quercetin (4 compounds),
kaempferol (4 compounds) and isorhamnetin derivatives (1 com-
pound).
[M-H]−(m/

Peak 13 eluted at 4.65 min, showed a quasi-molecular ion [M-H]−

629/297

at m/z 625 and its MS2 spectrum produced fragment ion at m/z 479 [M-
643
583

H-146]− due to the loss of rhamnose moiety, and an intense fragment

z)

ion at m/z 317 [M-H-308]− (loss of rutinoside moiety) that correspond

UVmax (nm)

to myricetin as aglycone. With these information, compound 13 can be

208, 323
282, 324
208, 326

assigned to myricetin-O-rutinoside (Llorent-Martínez et al., 2016). This

component was only found in young (0.23%) and old leaves (1.14%).
Peak 14 eluted at 4.73 min exhibited a deprotonated molecular ion [M-
RT (min)

H]− at m/z 479 and MS2 ion [M-H-162]− at m/z 317 through the loss
9.29
9.37
9.42

of hexose residue was tentatively identified as myricetin-hexoside

(Saldanha et al., 2013). The distribution of this flavonol was limited to
pulps (1.22 and 2.67 for immature and mature pulps, respectively).
Peaks 15 and 16 eluted respectively at 4.98 and 5.05 min, showed a
quasi-molecular ion [M-H]− at m/z 609, releasing in MS2 an intense
characterized mass fragment [M-H]−.
Tentative identification

fragment ion [M-H-308]− at m/z 301 indicative to the quercetin

aglycone. The loss of 308 amu is indicative to rutinose or neohesper-
idose moieties linked through an O-glycosidic bond (Brito et al., 2014).
Considering their order of elution, peak 15 was tentatively identified as
Table 1 (continued)

quercetin-O-neohesperidoside, whereas peak 16 was unambiguously

NI
NI
NI

identified as quercetin-3-O-rutinoside (Chen et al., 2015). The identity

of peak 16 was further confirmed by co-injection of the standard rutin
Peak No.*

(RT = 5.1 min). Both quercetin-O-neohesperidoside and rutin were

34
35
36

omnipresent in all parts analysed with the highest proportions being

recorded in young leaves (Table 2). Peak 17 eluted at 5.19 min showed
a

237
Y. M’rabet et al. Industrial Crops & Products 107 (2017) 232–243

Table 2
Relative percentage composition (% peak area calculated at 280 nm) of different phenolic compounds of Melia azedarach leaves and fruits.

Compounds Leaves Fruits F-value Significance

Pulps Kernels
YL OL IP MP IK MK

a a a a a
1 1.18 ± 0.28 1.34 ± 0.46 3.88 ± 1.13 4.35 ± 0.16 2.53 ± 0.66 3.17 ± 2.47a 3.768 *
2 0.12 ± 0.04 0.77 ± 0.58 nd nd nd nd – –
3 0.26 ± 0.05b 0.47 ± 0.07b 1.63 ± 0.65ab 2.53 ± 0.7a 1.19 ± 0.83ab 0.52 ± 0.29b 8.088 **
4 0.33 ± 0.03b 0.76 ± 0.11b 1.87 ± 0.47ab 3.21 ± 0.84a 1.4 ± 0.91b 0.68 ± 0.41b 10.38 ***
5 0.39 ± 0.07b 1.18 ± 0.05ab nd nd nd 2.08 ± 0.31a 88.78 ***
6 0.54 ± 0.07d 1.22 ± 0.04c 3.18 ± 0.21b 5.4 ± 0.18a nd nd 337.4 ***
7 1.94 ± 0.15c 4.34 ± 0.1bc 11.07 ± 1.37a 6.73 ± 0.49b nd nd 33.84 ***
8 nd nd OL (7) OL (7) nd nd – –
9 1.14 ± 0.16d 1.93 ± 0.12cd 9.18 ± 0.66a 5.88 ± 0.46b 2.01 ± 0.6cd 2.48 ± 0.22c 159.6 ***
10 0.85 ± 0.18d 3.02 ± 0.06c 11.48 ± 0.68a 6.84 ± 0.67b 3.77 ± 0.96c 3.49 ± 0.22c 131.6 ***
11 0.28 ± 0.21c 2.34 ± 0.03b 4.45 ± 0.64a 4.81 ± 0.08a 2.59 ± 0.32b 2.99 ± 0.12b 82.13 ***
12 0.25 ± 0.22c 2.07 ± 0.35b 2.19 ± 0.51b 3.84 ± 0.11a 2.89 ± 0.33ab 2.86 ± 0.46b 34.37 ***
13 0.23 ± 0.06 1.14 ± 0.09 nd nd nd nd – –
14 nd nd 1.22 ± 0.6 2.67 ± 0.37 nd nd – –
15 8.76 ± 0.01a 7.94 ± 0.01a 4.88 ± 0.52b 5.27 ± 0.42b 6.5 ± 5.02b 3.95 ± 0.61b 26.77 **
16 21.33 ± 0.13a 14.8 ± 0.01b 5.86 ± 0.5d 6.04 ± 0.64d 11.48 ± 0.33c 11.4 ± 0.18c 104.1 ***
17 OL (18) OL (18) 2.56 ± 0.92 2.84 ± 0.18 nd nd – –
18 4.12 ± 0.07 4.66 ± 0.09 nd nd nd nd – –
19 11.23 ± 0.14a 6.97 ± 2.51b 2.73 ± 1.19c nd nd nd 22.66 ***
20 nd 5.73 ± 0.12a 2.28 ± 0.89b 1.73 ± 0.1b nd nd 24.94 ***
21 0.07 ± 0.01 0.85 ± 0.03 nd nd nd nd – –
22 0.94 ± 0.02 1.54 ± 0.04 nd nd nd nd – –
23 0.57 ± 0.09 0.51 ± 0.01 nd nd nd nd – –
24 0.35 ± 0.08b 2.49 ± 0.08a nd nd 2.75 ± 1.44a 2.1 ± 0.51a 22.64 ***
25 0.94 ± 0.04 1.49 ± 0.13 nd nd nd nd – –
26 0.54 ± 0.05c 0.25 ± 0.15c 2.65 ± 1bc 2.64 ± 0.65bc 11.5 ± 5.09a 8.16 ± 1.41ab 12.62 ***

*Peak assignments are depicted in Table 1.

Values (mean ± SD, n = 3) represent the contribution of each metabolite to the total area integrated at 280 nm, for identified compounds only (cf. Table 1).
YL: young leaves, OL: old leaves, IK: immature kernel, MK: mature kernel, IP: immature pulp, MP: mature pulp.
OL(x): area overlap with the compound (x).
nd: Non-detected or not confirmed with mass spectrometry analysis.
–: not determined.
The differences between means with different superscript letters are statistically significant (p < 0.05).

a quasi-molecular ion [M-H]− at m/z 463, and a fragment ion [M-H-
162]− at m/z 301 (quercetin), owing to the loss of hexose residue, was
tentatively identified as quercetin-O- hexoside (Carocho et al., 2014).
Quercetin 3-β-D-glucoside standard was co-injected, and had a similar
retention time (RT = 5.2 min). Hence, we can suggest that the hexose
moiety was bound to position C3 of quercetin which was previously
confirmed by NMR in leaves of M. azedarach (Jafari et al., 2013). In this
study, this quercetin derivative was found in leaves (young and old) and
pulps (immature and mature). Peak 18 eluted at 5.22 min exhibited a
deprotonated molecular ion [M-H]− at m/z 609 and the MS2 spectrum
of the deprotonated molecule gave a base peak [M-H-324]− at m/z 285
(kaempferol aglycone), owing to the loss of two hexosyl residues (162
+162 amu). The fact that the two residues were lost simultaneously
suggested that they might constitute a disaccharide O-linked to the
aglycone. Accordingly, peak 18 was tentatively identified as kaemp-
ferol-O-dihexoside (Bresciani et al., 2015). Peak 19 eluted at 5.35 min,
with a deprotonated molecular ion [M-H]− at m/z 593, was tentatively
identified as kaempferol-3-O-rutinoside (Guimarães et al., 2013), owing
to the loss of rutinosyl moiety (308 amu) to yield a fragment ion [M-H-
308]−at m/z 285 (kaempferol aglycone). The identification of this
component was further supported based on the co-injection of reference
compound (RT = 5.4 min) and has already been found in leaves of M.
azedzrach (Jafari et al., 2013; Kumazawa et al., 2013). Peak 20 eluted at
5.45 min, showed a quasi-molecular ion [M-H]− at m/z 623, and it MS2
gave an intense fragment ion [M-H-308]− at m/z315 (isorhamnetin
aglycone) through the loss of rutinosyl moiety (308 amu). Accordingly,
peak 20 was tentatively identified as isorhamnetin-3-O-rutinoside
(Brito et al., 2014). This flavonol was found in young leaves (5.73%)
and both immature (2.28%) and mature (1.73%) pulps. Peak 21 eluted
at 5.49 min had a deprotonated molecular ion [M-H]− at m/z 433
andMS2 fragmentation ion [M-H-132]−at m/z 301 (quercetin aglycone)
through the loss of pentosyl residue (132 amu). This peak was therefore
tentatively assigned to quercetin pentoside (Chen et al., 2011) and was
found in young (0.07%) and old leaves (0.85%), but it lacked in fruits
(pulps and kernels). Peaks 22 (eluted at 5.57 min) and 23 (eluted at
5.88 min) exhibited quasi-molecular ions [M-H]− at m/z 447 and 431,
respectively, that yielded MS2 fragments at m/z 285 (kaempferol
aglycone) from the loss of hexoside (162 amu) and rhamnoside
(146 amu), respectively. Consequently, peaks 22 and 23 were tenta-
tively identified as kaempferol-O-hexoside and kaempferol-3-O-rham-
noside, respectively (Schmeda-Hirschmann et al., 2015). Peak 22 could
be further assigned to kaempferol-3-O-glucoside based on the retention
time matching with the standard (RT = 5.6 min). Both kaempferol
derivatives were detected only in leaves. It is worthy to note that the
biosynthesis/accumulation of kaempferol derivatives including
kaempferol-O-dihexoside, kaempferol-3-O-rutinoside, kaempferol-3-O-
glucoside and kaempferol-3-O-rhamnoside was mainly achieved in
leaves (Tables 1 and 2).
3.2.3. Flavanols
Flavanols were easily distinguished from the other phenolic groups
by their UV maxima absorbance at 280 nm. In the current study, three
flavanols were identified. Peak 24 eluted at 3.49 min showed a de-
protonated molecular ion [M-H]− at m/z 451 and an MS2 fragment ion
[M-H-162]− at m/z 289 (catechin aglycone) through the loss of hexose
residue. Accordingly, peak 24 was tentatively identified as catechin
hexoside (Ojwang et al., 2013). The distribution of this flavanol was
limited leaves (young and old) and kernels (immature and mature).
Peak 25 eluted at 5.66 with a quasi-molecular ion [M-H]− at m/z 455
showed the loss of methyl gallate (166 amu) originating a fragment ion

238
Y. M’rabet et al.
at m/z 289 and being identified as (epi)catechin methyl gallate (De la
Luz Cádiz-Gurrea et al., 2014). It was only detected in young (0.94%)
and old leaves (1.49%). Peak 26 eluted at 6.54 min was only found in
mature pulp (2.64%). It showed a quasi-molecular ion [M-H]− at m/z
577, yielding an intense fragment ion [M-H-170]− at m/z 407, and [M-
H-288]− at m/z 289. This fragmentation pattern (loss of 170 and
288 amu) was consistent with previous descriptions (Tala et al., 2013;
Rockenbach et al., 2012), and allowed the identification of peak 26 as
procyanidin dimer B.
When compared with earlier reports, the few compositional data are
conflicting and greatly differed from the present results, owing to
origin, plant part analysed, extraction and analytical procedure. For
example, by using UV and RMN spectroscopy, Salib et al. (2008) have
reported that flavonols glycosides namely kaempferol-3-O-rutinoside,
kaempferol-3-O-rhamnoside, quercetin 3-O-rhamnoside, rutin, kaemp-
ferol and quercetin were the most plentiful phenolic compounds in the
leaf ethanol extract of M. azedarach from Egypt. The HPLC-DAD ana-
lysis of the water extracts from immature and mature fruits from Chi-
lean specimen revealed that catechin and kaempferol were the main
flavonoids (Chiffelle et al., 2009). Three years later, Orhan et al. (2012)
compared the phenolic profiles of leaves and fruits from Turkish spe-
cimen and found that phenolic acids (i.e. protocatechuic, p-coumaric,
vanillic, gallic, caffeic, syringic, and chlorogenic acids) were the main
components in methanol, ethanol, and ethyl acetate extracts. The same
phenolic profile has also been described in the aqueous extracts of pulps
and leaves from M. azedarach collected from Italy and Algeria (Aoudia
et al., 2012; 2013). Rutin, kaempferol-3-O-robinobioside and kaemp-
ferol-3-O-rutinoside were detected as the main components in the
subcritical water extract of M. azedarach leaves from Japan (Kumazawa
et al., 2013). In general, the present results are in partial agreement
with those reported by Salib et al. (2008) and Kumazawa et al. (2013).
The presence of conjugated phenolic acids has not been reported in
none of these studies. To shed more light on the differential distribution
of phenolic components between different tissues (leaves, kernels and
pulps) and among stages of maturation, a set of marker components was
established.
3.2.4. Marker components
To be considered as marker, the presence/absence of a given com-
ponent should be confirmed in all samples from the same origin (Hosni
et al., 2013). Table 3 lists the marker components selected based on the
above-mentioned criterion.
As shown, the occurrence of hydroxyferulic acid hexoside (2),
myrcetin-O-rhamnoside (13), kaempferol-O-dihexoside (18), quercetin
pentoside (21), kaempferol-3-O-glucoside (22), kaempferol-3-O-rham-
noside (23) and (epi)catechin methyl gallate (25) could be considered
as specific to leaves irrespective to their stage of maturity. The
Table 3
Typical phenolic compounds of leaves and fruits of Melia azedarach at two stages of maturation.
Organ/Parts Development/ Typical components
Maturation
Presence of
Leaves Young Hydroxyferulic acid hexoside (2), myricetin-O-rutinoside
(13), kaempferol-O-dihexoside (18), quercetin pentoside
Old (21), kaempferol-3-O-glucoside (22), kaempferol-3-O-
rhamnoside (23), (Epi)catechin methyl gallate (25)
Fruit pulp Immature Ferulic acid dihexoside (8), myricetin hexoside (14)
Mature
Fruit kernel Immature
Mature
239
Industrial Crops & Products 107 (2017) 232–243
difference between the two stages of leaves maturity was only noticed
in the presence of isorhamnetin-3-O-rutinoside (20) which was typical
to old leaves. Kernel extracts can be distinguished from leaf and pulp
extracts by the absence of Ferulic acid hexoside I (6), feruloylquinic
acid derivative (7) and quercetin-O- hexoside (17) and the presence of
coumaroyl-quinic acid (5) only in mature stage. Ferulic acid dihexoside
(8) and myricetin hexoside (14) arise only in pulps extracts with cou-
maroyl-quinic acid (5) highlighting the mature stage. From practical
stand point, the established set of marker components could be useful to
discriminate between organs and the stage of maturity.
3.2.5. Unidentified components
Two components with UV maxima absorbance around 230 and
290 nm (peaks 29 and 30) belonging presumably to flavanones or di-
hydrochalcones (Portet et al., 2008), and 7 compounds (peaks 28, 31-
36) with UV maxima absorbance around 300 and 340 nm consistent
with the classes of flavones or chalcones (Portet et al., 2008) could not
be identified based on their fragmentation patterns.
3.3. Antioxidant activity
The antioxidant activity of methanol extracts from leaves, pulps and
kernels at two stages of maturity was evaluated using three com-
plementary in vitro tests.
3.3.1. The 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
Data depicted in Table 4, showed that among the six extracts, those
issued from young leaves were found to be the most DPPH radical
scavengers with an average value of 235 mg Trolox equivalents (TE)/g
extract, while those issued from mature pulps were the least DPPH
radical scavengers (20.5 mg TE/g extract). The hydrogen/electron do-
nating ability of tested extracts showed the following rend: young
leaves > old leaves > immature kernels > immature pulps > mature
pulps > mature kernels. The highest DPPH radical scavenging activity
of young leaves could be attributed to its high TPC content. These re-
sults are in good agreements with those of Orhan et al. (2012) who
reported that leaf extracts of M. azedarach were more active against the
DPPH radicals than fruit extracts.
3.3.2. Oxygen radical absorbance capacity (ORAC) assay
In accordance with data obtained in DPPH assay, extracts from
young leaves exhibited the highest ORAC activity with an average value
of 328 mg TE/g extract, while the lowest activity was found in extracts
from immature pulps (20.68 mg TE/g extract) (Table 4). The ranking of
ORAC activity was found to be roughly the same with the ranking of
TPC values, suggesting that the ability of different extracts to transfer a
hydrogen atom was tightly associated with the TPC. This is consistent
Absence of
Isorhamnetin-3-O-
rutinoside (20)
Isorhamnetin-3-O-
rutinoside (20)
Kaempferol-3-O- Catechin hexoside (24)
rutinoside (19)
Kaempferol-3-O-
rutinoside (19)
Ferulic acid hexoside I (6), Coumaroyl-quinic
acid (5)
Coumaroyl-quinic feruloylquinic acid derivative
acid (5) (7), quercetin-O- hexoside
(17)
Y. M’rabet et al.
Table 4
Estimation of antioxidant activity of Melia azedarach parts at two different stages of development.
Leaves
YL OL
ORAC (mg TE/g Extract) 328.18 ± 15.65a* 217.18 ± 15.05b
DPPH (mg TE/g Extract) 234.81 ± 6.46a 175.93 ± 5.65b
MCA (mg EDTA/g Extract) 15.97 ± 0.71b 11.93 ± 0.84c
*Different letters beside the values indicate significant differences between means as detected by Tukey HSD post-hoc test (p < 0.05). YL: young leaves, OL: old leaves, IK: immature
kernel, MK: mature kernel, IP: immature pulp, MP: mature pulp.
with the results of Aoudia et al. (2013) who showed that the highest
ORAC activity in M. azedarach leaves in comparison to seeds and al-
monds was attributed to their high TPC.
3.3.3. Metal chelating activity (MCA)
In contrast to the DPPH and ORAC assay where extract from young
leaves was found as the most potent radical scavenger, the MCA of
immature kernel extracts showed the highest capacity (22.85 mg EDTA
equivalents/g extract), while the lowest capacity was observed with old
leaves extracts (11.93 mg EDTA equivalents/g extract) (Table 4). The
ability of different extracts to act as metal chelators was as follow:
immature kernels > mature pulps > young leaves > mature kernels >
immature pulps > old leaves. These results were in good agreement
with those reported by Orhan et al. (2012) who revealed that fruits and
leaves of M. azedarach exhibited MCA higher than the standard EDTA.
It is interesting to note that contrarily to DPPH and ORAC assays,
the MCA activity in M. azedarach seems to be not correlated with the
TPC. These results suggest that the radical scavenging activity of M.
azedarach against DPPH and oxygen radicals was mediated through the
same phenolic components, on one hand, and that both assays (DPPH
and ORAC) rely on the same reaction mechanism (hydrogen donating
mechanism), on the other hand (Rahman et al., 2015; Wong et al.,
2006). They also suggest that the active components against DPPH and
oxygen free radicals were not effective as metal chelators, and the
presence of other sequesters components like organic acids, amino acids
and sugars could explain the observed results. To confirm such as-
sumptions, correlation and principal component analyses were per-
formed for TPC, phenolic components and parameters of antioxidant
activities of all extracts.
3.4. Pearson’s correlations and principal component analysis (PCA)
As depicted in Fig. 3, different degrees of correlations were found
between TPC, DPPH, ORAC, MCA and the identified phenolic com-
pounds in the methanol extract of M. azedarach.
The TPC showed significant and strong positive correlation with
DPPH (r = 0.98; p < 0.001) and ORAC (r = 0.96; p < 0.01) values
(Table 5).
In contrast, a non-significant and negative correlation (r = −0.3;
p > 0.05) was observed between TPC and MCA. Correlation analyses
among the three in vitro assays revealed significant and strong positive
relationship between DPPH and ORAC (r = 0.92; p < 0.01), while, a
negative and insignificant correlations were found between DPPH
versus MCA (r = −0.41; p > 0.05) and ORAC versus MCA
(r = −0.28; p > 0.05). Confirming our previous deductions, these
results clearly demonstrate that radical scavenging activities in both
DPPH and ORAC assays were mediated through the same phenolic
components with flavonols being the main contributors (r = 0.96;
p < 0.01 for flavonols versus ORAC and r = 0.85; p < 0.05 for fla-
vonols versus DPPH) in the observed activities. Among the identified
flavonols, quercetin-3-O-neohesperidoside (15), rutin (16), kaempferol-
3-O-rutinoside (19) and kaempferol-O-dihexoside (18) were found as
the main radical scavengers (Table 5). These results were consistent
with those of Matsuda et al. (2003) who showed that rutin and
240
Industrial Crops & Products 107 (2017) 232–243
Fruits
IK MK IP MP
20.68 ± 5.5d 23.7 ± 3.78d 67.37 ± 12c 28.45 ± 2.52d
30.14 ± 8.29d 20.5 ± 3.55d 141.66 ± 37.71c 17.56 ± 4.15d
22.85 ± 0.6a 13.16 ± 1.4c 12.25 ± 0.79c 17.49 ± 2.84b
quercetin-3-O-neohesperidoside have a potent DPPH radical scavenging
activity with IC50 values of 4.3 and 5.7 μM, respectively. More recently,
Zhang et al. (2016) reported that quercetin derivatives (i.e. rutin) and
kaempferol derivatives were potent radical scavengers. In a compara-
tive study regarding the ORAC radical scavenging activity of seven
phenolic compounds including 3 flavonols and 4 flavanols, Su et al.
(2014) showed that rutin and quercetin-3-rutinoside-7-rhamnoside ex-
hibited the highest ORAC activity, owing to their chemical structures
(position of glycosylation, type of esterified sugar, 2,3-double bond in
conjugation with 4-oxo function in the C-ring, and the twist angles of
the B-ring compared to the A- and C-rings introduced by glycosylation)
which determine their ability to scavenge free radicals. However, the
inability of these components to chelate metal ions could be attributed
to the absence of suitable functional groups that could chelate metal on
one hand, and the glycosylation which hindered metal bending on the
other hand (Wong et al., 2006). In their structural-activity relation-
ships, Badmus et al. (2016) concluded that kaempferol-3-O-rutinoside
exhibited strong antioxidant activity.
It is worth mentioning that the low contribution of phenolic acids on
the antioxidant activity of M. azedarach (Table 5; Fig. 4) could be at-
tributable to the presence of glycosyl moieties in their structures,
masking hence their functional groups namely hydroxyl (eOH) and
methoxy group (eOCH3), which ultimately reduced their hydrogen-
donating ability as previously reported by Cai et al. (2006).
Along with the correlation analyses, principal component analysis
(PCA) using the same set of variables was performed (Fig. 4). At first
glance, PCA analyses corroborate our previous conclusions about the
differential repartition of the identified components and their con-
tribution on the antioxidant activity among various plant parts. The
PCA revealed the two principal axes represented 75% of the total in-
formation (47.8 and 27.3% for PC 1 and PC 2, respectively). Both young
and old leaves were clearly separated from kernels and pulps (immature
and mature) in the PCA biplot, due to their high flavonol contents and
their strongest anti-radical activity against DPPH and ORAC. The pre-
ferential accumulation of phenolic acids in immature and mature pulps
allowed the separation of pulps from the remaining plant parts (leaves
and kernels). They were also slightly correlated with their ability to
chelate metals. The extracts from immature and mature kernels were
easily distinguished from those of pulps and leaves by their high fla-
vanol contents and low ORAC and DPPH radical scavenging activity.
4. Conclusions
Altogether, these results clearly supported the plant-organ specific
biosynthesis/accumulation of phenolic compounds which were tightly
dependent on the development and maturation stages of leaves and
fruits. The evaluation of the antioxidant activity revealed that young
leaves exhibited the highest radical scavenging activities against the
DPPH and ORAC radicals owing to their TPC and flavonol contents. In
contrast, the strongest metal chelating activity was observed in im-
mature kernels. The results also substantiate the traditional uses of M.
azedarach in treating various oxidant-related disorders, and increase the
interest and potential use of this under-estimated species as con-
solidated source of pharmaceutical agents. However, they suggest the
Y. M’rabet et al. Industrial Crops & Products 107 (2017) 232–243

Fig. 3. Pearson’s correlation coefficients between

total phenols content (TPC), identified phenolic
components percentage areas, phenolic groups pro-
portions (phenolic acids, flavonols and flavanols)
and antioxidant parameters (DPPH, ORAC and
MCA). Colour saturation represents the correlation
strength according to the scale below the figure.

importance of considering suitable stage of maturation and plant part Acknowledgments

used.
The authors are thankful to the “Direction Générale de la Recherche
Scientifique” (DGRS, Tunisia) and the “Centre National de la Recherche
Conflict of interest statement Scientifique” (CNRS, France) for financial support, Project “Laboratoire
International de Recherche Analytique” (LIRA-Tunisia).
The authors declare that there was no conflict of interest

Table 5
Pearson’s correlation coefficients (r) between TPC, antioxidant activities (ORAC, DPPH and MCA), phenolic acids, flavonols, flavanols (% determined by the integration of identified
peaks area) and the main contributors (% peak area of compounds 15, 16, 18 and 19).

Phenolic Acids Flavonols Flavanols TPC ORAC DPPH MCA 15 16 18

Flavonols −0.35
Flavanols −0.54 −0.49
TPC −0.66 0.87* −0.13
ORAC −0.53 0.96** −0.37 0.96**
DPPH −0.64 0.85* −0.09 0.98*** 0.92**
MCA 0.5 −0.28 −0.29 −0.3 −0.28 −0.43
15 −0.47 0.91* −0.28 0.93** 0.93** 0.97** −0.46
16 −0.78 0.82* −0.07 0.94** 0.93** 0.89* −0.3 0.81
18 −0.38 0.99*** −0.44 0.84* 0.93** 0.83* −0.33 0.87* 0.8
19 −0.3 0.95** −0.57 0.87* 0.95** 0.82* −0.02 0.85* 0.82* 0.91*

*, ** and ***: correlation is significant at the level 0.05, 0.01 and 0.001, respectively. Peak assignments are depicted in Table 1.

241
Y. M’rabet et al.
Fig. 4. Scatter plot of principal components analysis (PCA) PC1 versus PC2. A) variables ordination map corresponding to peak areas of identified phenolic components (sym-
bols = “circle”), the total proportions of phenolic groups (“stars”) TPC (“square”), and antioxidant activities scores for DPPH, ORAC and MCA (“triangle”). B) Melia azedarach samples
ordination map: YL: young leaves; OL, old leaves; IP, immature pulps; MP, mature pulps; IK: immature kernels, MK, mature kernels.
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