Vo l. 9(14), p p . 479-485, 10 Ap ril, 2015
DO I: 10.5897/ JMPR2011.685
Artic le Numb e r: 056640E52580
ISSN 1996-0875
Co p yrig ht © 2015
Autho r(s) re ta in the c o p yrig ht o f this a rtic le
http :/ / www.a c a d e mic jo urna ls.o rg / JMPR
Journal of Medicinal Plants Research
Full Length Research Paper
Identification of phenolic compounds by high
performance liquid chromatography/mass spectrometry
(HPLC/MS) and in vitro evaluation of the antioxidant
and antimicrobial activities of Ceratonia siliqua leaves
extracts
Anis Ben Hsouna1, Mohamed Trigui1, Raoudha Mezghani Jarraya2, Mohamed Damak2 and
Samir Jaoua3*
1
Biopesticides Team (LPIP), Center of Biotechnology of Sfax, P.B. 1177, 3018 Sfax, Tunisia.
Laboratory of Natural Substances Chemistry, Faculty of Science of Sfax, B.P.1171, 3038 Sfax, Tunisia.
3
College of Arts and Sciences, Qatar University, P.O. Box. 2713. Doha, Qatar.
2
Received 26 May, 2011; Accepted 11 July, 2013
Ceratonia siliqua is a typical plant of the Mediterranean area, which is mainly used as animal and human
food and in folk medicine for treating some diseases such as antidiarrheal and diuretic. The present
study was planned to evaluate the potential of antimicrobial and antioxidant activities of C. siliqua
leaves extract and the identification of bioactive compounds by high performance liquid
chromatography/mass spectrometry (HPLC/MS) in the active extract. The antioxidant activities of the
different organic extracts of C. siliqua were assayed by 2,2-diphenyl-l-picrylhydrazyl (DPPH) and βcarotene tests. Among the tested extracts, results showed that the ethyl acetate extract displayed the
greatest DPPH scavenging ability with an IC50 of 1.8 µg/ml and a strong β-carotene bleaching inhibition
after 120 min of incubation with an IC50 of 24.01 µg/ml. The investigation of the phenolic and flavonids
content showed that the ethyl acetate extract of C. siliqua revealed the highest phenolic contents. Only
the ethyl acetate extract of C. siliqua (EACs) showed antimicrobial activity with a broad-spectrum
microbiocide with diameter inhibition zones ranging from 12 to 24 mm and MIC values of 0.312 to 1.25
mg/ml. The HPLC finger print of EACs active extract showed the presence of six phenolic compounds.
They included (1) 1,6-Di-galloyl-glucose, (2) 1,2,6-Tri-galloyl-glucose, (3) Myricetin glucoside, (4) 1,2,3,6Tetra-galloyl-glucose, (5) Myricetin rhamnoside and (6) Syringic acid. These results are a good
agreement of the popular use and experimentally observed effects of C. siliqua and would promote the
reasonable usage and exploitation of the biomolecules of this important plant.
Key words: Ceratonia siliqua, Chemical composition, antimicrobial activity, antioxidant activity, phenolic
compounds.
INTRODUCTION
In recent years, there has been considerable interest in
the finding of antioxidants and antimicrobial compounds
from natural sources to control human and plant diseases
(Tepe et al., 2005). Natural antioxidant inhibited oxidative
480
J. Med. Plants Res.
damage of food products and may prevent inflammatory
conditions (Khanna et al., 2007), ageing and
neurodegenerative disease (Fusco et al., 2007).
The market constantly addresses its attention to
secondary metabolites produced by plants to check their
properties and to evaluate their possible use in the
industry. Also, the scientific interest in these metabolites
has been increased today with the search of new
antimicrobial agents, due to the increasing development
of the resistance pattern of microorganisms to most
currently used antimicrobial drugs. A number of natural
products such as phenolics, flavonoids, coumarins,
curcuminoids or terpenes have been characterised by
these metabolites which allows easy transport across cell
membranes to induce different biological activities,
including
antioxidant,
anti-inflammatory
and
anticholinesterase effects (Loizzo et al., 2007).
Tunisian flora is remarkable for its diversity of medicinal
plants. Ceratonia siliqua commonly known as Carob,
belongs to the family of Leguminosae. The tree attains a
mature height and spread of 6 to 12 m, and sometimes
more than 20 m, with branches extended to ground level
(Shigenorik et al., 2002). The leaves and fruits of this
plant are used to treat a variety of diseases. Carob pods
have traditionally been used as animal and human food
and currently the main use is the seed for gum extraction.
Bark and leaves have been used in Tunisian folk
medicine as laxative, diuretic, antidiarrheal and for the
treatment of gastroenteritis of lactating babies (Kivçak
and Mert, 2002). Furthermore, recent studies have
confirmed
the
presence
of
antioxidant,
hypocholesterolemic activities in pods of C. siliqua
(Dimitris and Makris, 2004) and antiproliferative effects
on T1 cell line and substances acting on peripheral
benzodiazepine receptor in its leaves (Avallone et al.,
2002; Corsi et al., 2002).
The aims of this study were to screen the different
fraction of C. siliqua for its antioxidant, antimicrobial
activities and to identify its major phenolic compounds for
the first time. The antimicrobial activity was evaluated
against different microorganisms, including Gram-positive
and negative bacteria. The antioxidant potential was
studied by two distinct assays: scavenging of 2,2diphenyl-l-picrylhydrazyl (DPPH) radicals and β-carotene
bleaching assay. Moreover, this study characterized the
active
extract
by
high
performance
liquid
chromatography/mass spectrometry (HPLC/MS) and
discussed their possible health benefits.
MATERIALS AND METHODS
Chemicals
DPPH and butylated hydroxytoluene (BHT) were purchased from
Sigma Chemical France. Folin-Ciocalteu reagent, sodium carbonate
(Na2CO3) and other solvents were of analytical grade and were
freshly prepared in distilled water.
Preparation of C. siliqua leaf extracts
Fresh leaves of C. siliqua were collected from Chebba (Mahdia,
Tunisia, latitude 35.23° and longitude 11.11°) and a voucher
specimen (LBPes C.S. 15.01) was deposited in the laboratory of
Biopesticides of the Centre of Biotechnology of Sfax. The dried
leaves were ground to fine powder using a grinder and the resulted
material (100 g of powder) was extracted by maceration in ethanolwater 80% with shaking, at room temperature. 3.5 g of the dried
hydroethanolic crude extract was suspended in 100 ml distilled
water and was sequentially partitioned with n-hexane (3 × 250 ml),
dichloromethane (3 × 250 ml) and ethyl acetate (3 × 250 ml). The
resulting three fractions were evaporated under vacuum to dryness
to give the hexane (m = 20 mg), the dichloromethane (m = 15 mg)
and the ethyl acetate (m = 1.5 g) fractions. The remaining aqueous
layer was lyophilised to give water fraction (m = 150 mg). The stock
solutions were kept at 4°C in the dark until further analysis.
Total phenolic content
Total phenolic content (TPC) was determined using the FolinCiocalteu method (Waterman and Mole, 1994) adapted to a
microscale. Briefly, 10 µl of diluted sample solution were shaken for
5 min with 50 µl of Folin-Ciocalteu reagent. Then 150 µl of 20%
Na2CO3 were added and the mixture was shaken once again for 1
min. Finally, the solution was brought up to 790 µl by the addition of
distilled water. After 90 min, the absorbance at 760 nm was
evaluated using a spectrophotometer SmartSpecTm3000 (Bio-Rad;
Hercules, CA, USA). Gallic acid was used as an internal standard
for the calibration curve. The phenolic content was expressed as
mg of gallic acid equivalent per gram of dry sample (mg GAE/g)
using the linear equation based on the calibration curve.
Determination of total flavonoids content
The
flavonoids
content
in
extracts
was
determined
spectrophotometrically according to Quettier-Deleu et al. (2000),
using a method based on the formation of a complex flavonoid–
aluminium, having the maximum absorption at 430 nm. The
flavonoids content was expressed in mg of quercetin equivalent per
gram of dry plant extract (mg QE/g).
Antioxidant testing assays
DPPH radical scavenging activity
Radical scavenging activity of the different fractions was
determined using DPPH radical as a reagent according to the
method of Kirby and Schmidt (1997) with some modifications.
Briefly, 1 ml of a 4% (w/v) solution of DPPH radical in ethanol was
mixed with 500 µl of sample solutions in ethanol (different
concentrations). The mixture was incubated for 20 min in the dark
at room temperature. Scavenging capacity was read spectrophotometrically by monitoring the decrease of the absorbance at
517 nm. Lower absorbance of the reaction mixture indicates higher
free radical scavenging activity. Ascorbic acid was used as
*C o rre sp o nd ing a utho r. E-ma il: sa mirja o ua @ q u.e d u.q a . Te l: (+974)44034563. Fa x: (+974)44034531.
Autho r(s) a g re e tha t this a rtic le re ma in p e rma ne ntly o p e n a c c e ss und e r the te rms o f the C re a tive C o mmo ns Attrib utio n
Lic e nse 4.0 Inte rna tio na l Lic e nse
Hsouna et al.
481
standard. The percent DPPH scavenging effect was calculated
using the following equation: DPPH scavenging effect (%) = (Acontrol
- Asample / Acontrol) × 100. Where Acontrol is the absorbance of the
control reaction where the sample is replaced by 500 µl ethanol.
Tests were carried out in triplicate.
evaluated by measuring the diameter of circular inhibition zones
around the well. Tests were performed in triplicate.
β-Carotene bleaching assay
MIC of extract of C. siliqua were determined according to Gulluce et
al. (2007) with minor modifications. The test was performed in
sterile 96-well microplates with a final volume in each microplate
well of 100 µl. For susceptibility testing, 100 μl of Mueller-Hinton
broth or potatoes dextrose broth was distributed from the second to
the twelfth test wells. A stock solution of the extract was prepared
by dissolving 100 µl of the extract in dimethyl sulfoxide and then
adjusted to a final concentration of 50 mg/ml by Mueller-Hinton
broth. The first well of the microplate was prepared by dispensing
160 µl of the growth medium and 40 µl of the tested extract to reach
a final concentration of 10 mg/ml and then 100 μl of scalar dilutions
were transferred from the second to the ninth well. Thereafter and
from each well, 10 μl of the suspension were removed and replaced
by the bacterial suspensions to final inoculum concentrations of 106
CFU/ml. The final extracts concentrations adopted to evaluate the
antimicrobial activity were 0.039 to 10 mg/ml.
The tenth well was considered as positive growth control
containing Mueller-Hinton media for bacterial strains, since no
extract solution was added. Another well containing 10%
dimethylsulfoxide (v/v), without extract, was used as negative
control. The plates were then covered with the sterile plate covers
and incubated at 37°C for 24 h. The MIC was defined as the lowest
concentration of the total extract tested at which the microorganism
does not demonstrate visible growth after incubation. As an
indicator of microorganism growth, 25 µl of 3-(4,5-Dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (0.5 mg/ml)
dissolved in sterile water were added to the wells and incubated at
37°C for 30 min (Eloff, 1998). Where microbial growth was
inhibited, the solution in the well remained clear after incubation
with MTT. For the determination of MBC, a portion of liquid (5 µl)
from each well that showed no change in colour was plated on
nutrient agar and incubated at 37°C for 24 h. The lowest
concentration that yielded no growth after this sub-culturing was
taken as the MBC, indicating that >99.9% of the original inoculum
was killed. The determinations of MIC and MBC values were done
in triplicate.
The antioxidant activity was determined according to the -carotene
bleaching method described by Pratt (1980). A stock solution of carotene/linoleic acid mixture was prepared as follows: 0.5 mg of carotene was dissolved in 1 ml of chloroform with 25 µl of linoleic
acid and 200 mg of Tween-20. Chloroform was completely
evaporated, using a vacuum evaporator. Then, 100 ml of distilled
water, saturated with oxygen (30 min), were added and the
obtained solution was vigorously shaken. 4 ml of this reaction
mixture were dispensed into test tubes and 200 µl of each sample,
prepared at different concentrations, were added. The emulsion
system was incubated for 2 h at 50°C. The same procedure was
repeated with BHT as positive control, and a blank as a negative
control. After this incubation period, the absorbance of each mixture
was measured at 490 nm. Antioxidant activity in -carotene
bleaching model in percentage (A %) was calculated with the
following equation: A% = 1- (A0 –At/A’0-A’t) × 100, where A0 and A’0
are absorbances of the sample and the blank, respectively,
measured at zero time, and A t and A’t are absorbances of the
sample and the blank, respectively, measured after 2 h. All tests
were carried out in triplicate.
Antimicrobial activity
Microorganisms and growth conditions: Authentic pure cultures
of bacteria were obtained from international culture collections
(ATCC) and the local culture collection of the Center of
Biotechnology of Sfax, Tunisia. They included Gram-positive
bacteria: Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 14579,
Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis
ATCC 12228, Enterococcus faecalis ATCC 29212, Micrococcus
luteus ATCC 1880 and Gram-negative bacteria:
Klebsiella
pneumoniae ATCC 10031, Escherichia coli ATCC 8739 and
Pseudomonas aeruginosa ATCC 9027.
The bacterial strains were cultivated in Muller-Hinton agar (MH)
(Oxoid Ltd, UK) at 37°C except for Bacillus species which were
incubated at 30°C. Working cultures were prepared by inoculating a
loopful of each test bacteria in 3 ml of Muller-Hinton broth (MH)
(Oxoid Ltd, UK) and were incubated at 37°C for 12 h. For the test,
final inoculum concentrations of 106 CFU/ml bacteria were used.
Agar diffusion method: Antimicrobial activities of the C. siliqua
were evaluated by means of agar-well diffusion assay according to
Güven et al. (2006) with some modifications. Fifteen milliliters of the
molten agar (45°C) were poured into sterile petri dishes (Ø 90 mm).
Working cell suspensions were prepared and 100 µl was evenly
spread onto the surface of the agar plates of Mueller-Hinton agar
(Oxoid Ltd, UK) for bacteria. Once the plates had been aseptically
dried, 06 mm wells were punched into the agar with a sterile
Pasteur pipette.
The extract was dissolved in dimethylsulfoxide/water (1/9; v/v) to
a final concentration of 50 mg/ml. Thus, 80 μl were placed into the
wells and the plates were incubated at 37°C for 24 h for bacterial
strains. Gentamicin (10 μg/wells) was used as a positive control.
Negative control consisted of 10% dimethyl sulfoxide (DMSO)
which is used to dissolve the extract. Antimicrobial activity was
Determination of minimal inhibitory concentration (MIC) and
minimal biocide concentration (MBC)
HPLC-MS analysis of phenolic compounds
Reverse phase high performance liquid chromatography was used
to analyse phenolic compounds present in the active fraction of C.
siliqua (EACs), using the separation module (Knawer Analogy)
equipped with a C18 column (Zorbax, 2.6 × 250 mm, 3.5 µm
particle size) and a diode array detector (DAD). The samples were
eluted with a gradient system consisting of solvent A (water, 0.1%
formic acid) and solvent B (Acetonitrile, 0.1% formic acid), used as
the mobile phase, with a flow rate of 500 µl/min. The temperature of
the column was maintained at 25°C and the injection volume was
10 µl. The gradient system started from 90% A at 1 min, to 20% B
at 4 min, 80% B at 30 min, 100% B at 32 min, 100% B at 36 min
and 20% B at 38 min.
The peaks of the phenolic compounds were monitored at 280
nm. Electrospray ionisation mass spectroscopic (ESI–MS) analysis
of phenolic compounds in ethyl acetate fraction was performed
using an Applied Biosystems (LC/MSD TRAP × CT). Mass spectra
were achieved by electrospray ionisation in both positive and
negative modes. The capillaries 4500 (negative) and 3500 V
(positive) were used in this study. The electrospray probe-flow was
adjusted to 8 ml/min. Continuous mass spectra were obtained by
482
J. Med. Plants Res.
Table 1. Amounts of total flavonoid, total phenolic compounds and evaluation of the IC50 values of the DPPH free radical
scavenging assay of the different fractions of C. siliqua and Ascorbic acid was used as standard. Each value represents
the mean ± S.D. of three experiments.
Extract/Fraction
Hexane
Dichloromethane
Ethyl acetate
Water
Ascorbic acid
Phenolic content (mg GAE/g)
91.20 ± 3.94
139.58 ± 6.55
680 ± 8.33
130 ± 5.62
-
Flavonoid content (mg QE/g)
ND
75.94 ± 7.68
193.30 ± 3.07
21.71 ± 8.71
-
DPPH IC50 (µg/ml)
ND
41.01
1.80
8.65
3.50
mg GAE /g): mg of gallic acid equivalent per g of dry plant extract; mg QE/g: mg of quercetin equivalent per g of dry plant extract.
ND: not detected.
scanning from 50 to 900 m/z. Identification of the phenolic
compounds of each fraction was achieved by comparison with
ESI-MS spectra comparisons with literature reports.
Statistical analysis
Experimental results concerning this study were expressed as
means ± standard deviation of three parallel measurements. The
significance of difference was calculated by Student’s t test, and
values p<0.05 and p<0.001 were considered to be significant and
highly significant, respectively.
RESULTS AND DISCUSSION
Total phenolics (TPC) and flavonoids
C. siliqua leaves fractions were investigated for their TPC
by the Folin–Ciocalteu assay and for their flavonoids by
AlCl3 reagent. As shown in Table 1, the TPC values
expressed as mg gallic acid equivalents/g of dry extract
(mg GAE/g) of the successive C. siliqua leaves extracts
ranged between 91.2 to 680 mg GAE/g and were in the
following order: ethyl acetate fraction > dichloromethane
fraction > water fraction > hexane fraction.
Ethyl acetate fraction of C. siliqua leaves (EACS
fraction) have a total flavonoid content of 193.3 mg of
quercetin equivalents/g of dried extract, while the water
fraction have the lowest concentration. It was observed
that the amount of flavonoids in the analyzed plant
extracts showed a high correlation with the total amount
of phenolics.
Total antioxidant capacity
DPPH test
The DPPH, a stable free radical, has been widely used to
evaluate the free radicals scavenging ability of various
natural products (Porto et al., 2000). The effect of the
different C. siliqua fractions on DPPH radical scavenging
showed a dose-dependent activity that can be evaluated
by the determination of the IC50 values corresponding to
the amount of the fraction required to scavenge 50% of
DPPH radicals present in the reaction mixture. High IC50
values indicate low antioxidant activity. As shown in
Table 1, the most potent radical scavenger extract was
EACS fraction (IC50 = 1.8 µg/ml), followed by water
fraction (IC50 = 8.65 µg/ml) and dichloromethane fraction
(IC50 = 41.01 µg/ml). Therefore, it can be concluded that
these extracts were able to reduce the stable free radical
DPPH to the yellow-colored diphenylpicrylhydrazine.
Additionally, the IC50 of this fraction was better than the
reported IC50 values obtained for gallic acid (IC50 = 64
µg/ml) and BHA (IC50 = 114 µg/ml) (Ozsoy et al., 2009).
The antioxidant activity of the EACS fraction could be
attributed to its high total phenolic content. The key role
of these compounds as scavengers of free radicals was
reported in several studies (Komali et al., 1999; Moller et
al., 1999).
Based on these results, EACS fraction have been
chosen rich in phenolic compounds, to identify its
chemical composition and investigate its antioxidant and
antimicrobial activities.
β-carotene bleaching method
The inhibitory effect of the ethyl acetate fraction of C.
siliqua on lipid peroxidation was determined by the carotene/linoleic acid bleaching test. The antioxidant
activity of C. siliqua was evaluated using different
concentrations of extracts and was compared with BHT
used as reference. Results are presented in Table 2. The
addition of the EACS fraction and the BHT at a
concentration of 5 and 20 µg/ml prevented the bleaching
of β-carotene with different degrees. Antioxidant activities
of 38.95 and 75.79% were obtained using 20 µg/ml of the
EACS fraction and BHT, respectively. The C. siliqua
fraction was found to hinder the extent of β-carotene
bleaching by quenching peroxide radicals to terminate
the peroxidation chain reaction. This fraction possessed
better antioxidant activity than other extracts such as
water extract of chestnut fruit as described by Barreira et
Hsouna et al.
483
Table 2. Inhibition of lipid peroxidation obtained by ethyl acetate fraction from C. siliqua leaves
and BHT as assessed by the coupled oxidation of β-carotene and linoleic acid over 120 min. Data
represent the means ± SD (n=3).
Inhibition peroxidation lipidique
Concentration (µg/ml)
1
2.5
5
20
IC50
Ethyl acetate extract
6.26 ± 1.03
13.78 ± 0.49
18.63 ± 1.80
38.95 ± 3.50
24.01 ± 2.24
BHT
52.71 ± 0.46
58 ± 1.25
62.98 ± 1.30
75.79 ± 1.50
5.01 ± 1.08
Table 3. Antimicrobial activities of ethyl acetate extract of C. siliqua against food borne and spoiling bacteria and the
determination of the minimal inhibition concentration (MIC) and the minimal biocidal concentration (MBC) in µg/ml.
Strains
IZa (mm)
Ethyl acetate fraction
MICb (mg/ml)
MBCc (mg/ml)
Gentamicind IZ (mm)
Gram positive bacteria
Bacillus cereus
Bacillus subtilis
Staphylococcus aureus
Enterococcus feacalis
Staphylococcus epidermidis
Micrococcus luteus
20
22
24
17
20
23
1.25
1.25
1.25
0.625
1.25
0.312
5
5
5
5
5
0.625
23
20
19
23
20
20
Gram negative bacteria
Escherichia coli
Pseudomonas aeruginosa
Klebsiella pneumoniae
12
18
15
1.25
0.312
1.250
1.25
10
10
18
14
18
Data are reported as the mean ± SD from three different experiments. Gentamicin was used as a standard antibiotic at a
a
b
concentration of 10 µg/well. Inhibition zones. Minimal inhibitory concentration measured by the broth microdilution using 96-well
c
d
microplates method. Minimal bactericidal concentration by subculture on nutrient agar at 37°C. Gentamicin (10 µg/well).
al. (2008). The inhibition of lipid peroxidation by addition
of EACS leaves fraction can be used to improve the
quality and stability of food products. This antioxidant
activity could be attributed to its high phenolic content.
The significant correlation between the phenolic content
and the antioxidant activity of various vegetable extracts
has been previously observed (Velioglu et al., 1998).
Antibacterial activity
The antibacterial activity of the different fractions of C.
siliqua was quantitatively assessed by measuring the
diameter of the inhibition zone around the well and the
determination of the MIC and MBC. As shown in Table 3,
among the tested extracts, only the EACS fraction
showed antimicrobial activities. This fraction inhibited the
growth of various species belonging to both Grampositive and Gram-negative bacteria. The diameter of the
inhibition zones were in the range of 12 to 24 mm. The
largest growth inhibition "halo" was observed from S.
aureus (24 mm) and M. luteus (23 mm). The inhibition
zone diameter of the EACS fraction was comparable with
that of gentamicin used as standard antibiotic and
positive control. The observed differences in the inhibition
zones within pathogenic bacteria could be probably due
to cell membrane permeability or other genetic factors.
Earlier reports have shown that Gram positive-bacteria
are more sensitive than Gram negative form (El-Astal et
al., 2005).
The MIC values of the EACS fraction ranged from
0.312 to 1.25 mg/ml and the MBC were from 0.625 to 10
mg/ml (Table 3). Furthermore, EACS fraction showed the
most potent inhibition for P. aeruginosa and M. luteus
(MIC = 0.312 mg/ml). Infections caused by P. aeruginosa,
especially those with multi-drugs resistance, are among
the most difficult to treat with conventional antibiotics. In
this study, the growth of P. aeruginosa was remarkably
484
J. Med. Plants Res.
Table 4. LC–ESI–MS data for phenolic compounds in ethyl acetate fraction of C. siliqua leaves.
Peak
1
2
3
4
5
6
HPLC retention time (min)
10
12.4
14.9
15.1
16.2
17.4
Molecular mass (M)
484
636
480
788
464
198
inhibited by the leaves of EACS fraction (MIC = 0.312
mg/ml). These results show that C. siliqua fraction can be
used to minimize problems of drug resistance and protect
foods against multiple pathogenic bacteria.
To the best of our knowledge, this is the first study that
demonstrates that EACS fraction contains antimicrobial
substances or to the high level of phenolic components in
leaves used by plants as defence mechanisms against
pathogenic microorganisms (Cowan, 1999).
Identification of phenolic compounds by HPLC-MS
The EACS fraction was analyzed by HPLC-MS in order to
identify its chemical composition. The identification of the
different peaks was based on their retention times in
HPLC, their characteristic UV/Visible and mass spectra
with positive and negative ionization at different
fragmentation voltages, in comparison to authentic
standards. Close examination of the MS and MS2 spectra
obtained from the LC-UV profile of the EACS fraction
confirmed the presence of individual components from
several classes: simple phenol, free flavonol,
glycosylated flavonol and isoflavone (Table 4). By
comparing mass spectra with those of literature data
given by Owen et al. (2003), Peak 1 (tR= 10 min) was
identified as 1,6-Di-galloyl-glucose and the [M-H]+ peak
was at m/z 484.
The second peak (tR = 12.4 min and [M-H]- of 635), was
identified as 1,2,6-Tri-galloyl-glucose. Peak 3 (tR = 14.9
min) showed [M-H]- of 480 and was identified as
myricetin glucoside. The value of the characteristic
fragment ion of the latter peak (m/z =316), was also
identical with the reported in the literature. The fourth
peak (tR= 15.1 min, [M-H]_ =787 and a fragment ion value
of 617) was identified as 1,2,3,6-Tetra-galloyl-glucose
which is the main polyphenol. The fifth peak (tR =
16.2min, [M-H]- of 463) was identified as myricetin
rhamnoside. The value of its fragment ion, found to be of
316, was similar to that reported in carob fibre. The last
peak (tR = 17.4min, [M-H]- of 197) was identified as the
synergic acid. All these results are in agreement with
those reported in the literature by Owen et al. (2003) in
carob fibre.
This is the first report on detection of phenolic
Major fragment ions m/z
331
465
316
617.1
316
177
Compounds
1,6-Di-O-galloyl-glucose
1,2,6-Tri-O-galloyl-glucose
Myricetin glucoside
1,2,3,6-Tetra-O-galloyl-glucose
Myricetin rhamnoside
Syringic acid
compounds in C. siliqua leaves to the best of our
knowledge. Our data showed that in comparison to the
carob pod itself, leaves of C. siliqua contains higher
concentrations of phenolic compounds than detected in
earlier report (Corsi et al., 2002; Porto et al., 2000). Corsi
et al. (2002) have demonstrated that the phenolic fraction
of carob pod by infusion was dominated by gallic acid
with minor contributions of catechins, epigallocatechin
and epicatechin gallate. As part of our investigation on
the constituents of C. siliqua leaves, we have
demonstrated that the actively ethyl acetate fraction
contained mainly syringic acid, myricetin glycosides and
gallic acids derivatives.
Further screening of the reported phenolics failed to
identify catechin, epicatechin, quercitin and kaempferol.
Therefore, the proximate analysis of Tunisian C. siliqua
constituents, compared to those obtained in previous
work, showed some variation in the composition. This
variation in composition can be attributed to the diversity
of geographical environments (soil, sunlight, temperature,
precipitation, etc). Custódio et al. (2009) have reported
previously that gender significantly affected the phenolic
profile with the hermaphrodites being generally richer in
phenols
Conclusion
Conclusively, this study is the first report dealing with the
in vitro biological activities of the ethyl acetate fraction of
C. siliqua leaves. It exhibited a high content of total
phenolic compounds and the strongest antioxidant
activity. In addition, this fraction inhibited the peroxidation
of linoleic acid, and acted strong hydrogen-donating
agents in the DPPH assay. The antioxidant ability of this
fraction was strongly correlated with the phenolic and
flavonoid content. Furthermore, six compounds were
identified from C. siliqua leaves, namely 1,6-di-galloylglucose, 1,2,6-tri-galloyl-glucose, myricetin glucoside,
1,2,3,6-tetra-O-galloyl-glucose, myricetin rhamnoside and
syringic acid.
The results of this study demonstrated that C. siliqua
leaves might be a good candidate for employment as
antimicrobial activities and an excellent food preservative
against bacteria growth. These two properties of plant
Hsouna et al.
fraction are of great interest for food industry, since their
possible use as natural additives emerged from a
growing tendency to replace synthetic antioxidants by
natural ones. Further investigation is required to examine
the cytotoxicity effects of leaves of EACS fraction.
ACKNOWLEDGEMENTS
This work was supported by grants from the Tunisian
“Ministry of Higher Education, Scientific Research and
Technology”. The authors wish to thank Pr. Adnane
Hammami head of the Research Laboratory 'Microorganismes et Pathologie Humaine', EPS Habib
Bourguiba, Sfax, Tunisia for providing some of the
pathogenic bacterial strains and technical assistance.
Conflicts of interest
The authors declare that they have no conflicts of
interest.
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