Biochemical Systematics and Ecology 104 (2022) 104486

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Biochemical Systematics and Ecology

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Variability of phenolic compounds and antioxidant activities of ten

Ceratonia siliqua L. provenances
Amira Richane a, c, *, Ben Mansour Rim b, Megdiche wided b, Ksouri Riadh b, Attia Khaoula c,
Moujahed Nizar c, Ben Ismail Hanen a, d
a
Department of Agri-Food Industries, National Agronomic Institute of Tunisia (INAT), UR17AGR01, University of Carthage, 1082, Tunis, Tunisia
b
Laboratory of Medicinal and Aromatics Plant, Biotechnology Centre of Borj-Cedria, BP 901 2050, Hammam-Lif, Tunisia
c
Ecosystems and Aquatic Resource Unit, National Agronomic Institute of Tunisia (INAT), University of Carthage, UR03AGRO1, 1082, Tunis, Tunisia
d
Biodiversity, Biotechnology and Climate Change Laboratory, University of Tunis El Manar, LR11ES09, 2092, Tunis, Tunisia

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

Keywords: In the present study, Tunisian carob (Ceratonia siliqua L.) pulp samples harvested from ten different geographic
Ceratonia siliqua L. regions (Bizerte, Beja, Ariana, Ben Arous, Jendouba, Siliana, Zeghouan, Nabeul, Sousse, Kairouan) were analyzed
Geographical origin for their bioactive phytochemical content and antioxidant activities. Total phenolics (TPC), flavonoids (TFC),
RP-HPLC analysis
condensed tannins (CT) were determined using Folin-Ciocalteau, aluminium trichloride (AlCl3) and vanillin
Phenolic profiles
assays, respectively. Significant variations (p < 0.05) in total polyphenols (TPC), flavonoids (TFC) and condensed
tannins (CT) contents were observed between the analyzed provenances. The highest levels of TPC (23.11 mg
GAE/g DR) and TFC (10.58 mg CE/g DR) were detected in Kairouan site, whereas Beja sample exhibited the
higher amount of CT (3.75 mg CE/g DR). Reversed phase RP-HPLC analysis revealed that gallic acid
(501.26–1628.46 mg/kg), ellagic acid (398.85–940.53 mg/kg) and isoquercetin (264.79–817.61 mg/kg) were
the major phenolics in all studied extracts, with concentrations varying significantly according to the
geographical origin. Using total antioxidant capacity, DPPH, ABTS and FRAP assays, carob pulp extracts were
found to have substantial antioxidant activities, which were correlated to their phenolic contents. Multivariate
statistical analysis (PCA) performed on phenolic profiles and antioxidant activities revealed three distinctive
groups of samples. It was concluded that Ceratonia siliqua L.. pulp from lower semi-arid and upper arid regions of
Tunisia presented appreciable amount of phenolic compounds and high antioxidant capacities. This resource
deserves further attention mainly for its high potential in culinar, pharmaceutical and cosmetic utilisations.

1. Introduction more regressing, and replaced with more profitable species such as Olea
europea, Ficus carica and Vitis vinifera and for charcoal production (Afif
Ceratonia siliqua L. is an evergreen tree from the Fabaceae family. It et al., 2008). According to the Food and Agriculture Organisation of the
has been cultivated since ancient time throughout the Mediterranean United Nations data (FAO), Tunisia is among the leading carob fruits
region for restoration of degraded arid areas and for human and animal producing countries in the world, with a mean annual production of 912
consumption (Afif et al., 2008; Tous et al., 2013). Moreover, its aerial tons/year (FAO, 2017).
parts, i.e. fruits, flowers and leaves, are traditionally used in folk med­ Currently, only the carob seeds (10% of the whole pod) are applied
icine of many countries, including, Greece, Italy, Spain, Morocco, industrially for producing a natural additive (E410) added as flavoring,
Turkey, Algeria, Syria and Tunisia, to cure gastro-intestinal diseases stabiliser and thickener in food products (Bouzouita et al., 2007; Goulas
such as diarrhea and chronic gastric ulcers (Rtibi et al., 2017). In et al., 2016); while the pulp, representing 90% of the pod, could be
Tunisia, carob populations grew spontaneously in association with Pis­ considered as by-product susceptible of valorization (Roseiro et al.,
tacia lentiscus and/or Olea europea in arid and semi-arid regions of the 2013). Carob pulp contains high amounts of carbohydrates (40–60
North and the Centre of the country, as well as in the Northern coast mg/100g DM), dietary fibers (around 45 mg/100g DM), minerals (cal­
(Afif et al., 2008). However, most of these populations are more and cium, phosphorus and potassium) and low amounts of fat (0.5–1

* Corresponding author. National Agronomic Institute of Tunisia (INAT), 1082, Tunis, Tunisia.
E-mail address: [email protected] (A. Richane).

https://doi.org/10.1016/j.bse.2022.104486
Received 12 March 2022; Received in revised form 8 August 2022; Accepted 20 August 2022
Available online 2 September 2022
0305-1978/© 2022 Elsevier Ltd. All rights reserved.
A. Richane et al. Biochemical Systematics and Ecology 104 (2022) 104486

mg/100g DM) and protein (3–4 mg/100g DM) (Tous et al., 2013; Goulas further analysis.
et al., 2016). Moreover, the pulp is a rich source of polyphenolic com­
pounds that illustrate a wide range of in vitro biological activities
including antioxidant, antibacterial, anti-cancer and anti-inflammatory 2.2. Extraction procedure
actions (Stavrou et al., 2018; Liu et al., 2019; Sun et al., 2019).
Despite its nutritional, health and economic importance, carob pulp Carob pulp sample (2.5g) was homogenized with 25 mL of aqueous
remains, so far, underexploited and it is generally intended for animal methanol (80%, v/v) under magnetic stirring during 30 min. The ho­
nutrition or is used for production of traditional specialities such as mogenates were stored overnight at 4 ◦ C and then filtrated through No.4
molasses or syrup (El Batal et al., 2016; Ben Othmen et al., 2021). There Whatman filter paper. Filtrates were concentrated under vacuum at
are few data about the phytochemical profiles of carob pulp and results 45 ◦ C to eliminate the extraction solvent and the dry extracts were re-
are quite different, possibly due to the different carob cultivars, origins dissolved in methanol and preserved in hermetic and dark glass bot­
and storage conditions, and also the use of different analytical methods tles at − 20 ◦ C until further analysis.
(Papagiannopoulos et al., 2004; Custódio et al., 2007; Roseiro et al.,
2013). This may lead to encouraging further researches to improve
literature data on this species. 2.3. Determination of total phenolic contents
Recent studies on Ceratonia siliqua L. pods revealed the influence of
several agronomic and technological factors on their phenol com­ Total polyphenols were assayed by the Folin-Ciocalteu reagent and
pounds, such as developmental stage (Vekiari et al., 2011; Benchikh gallic acid as standard according to Ben Mansour et al. (2020). Absor­
et al., 2014; Ydjedd et al., 2017), tree gender (Custódio et al., 2011; bance was checked at 760 nm. Contents were expressed as mg gallic acid
Hajaji et al., 2011) and the extraction methodologies (Roseiro et al., equivalent per gram of dry residue (mg GAE/g DR) through the cali­
2013; Brahim, 2017; Quiles-Carrillo et al., 2019). Geographic origin has bration curve with gallic acid, ranging from 0 to 500 mg/mL. All sam­
also a preponderant effect on phenolic composition of plant tissues ex­ ples were analyzed in triplicate.
tracts (Jaouadi et al., 2019). In the present study, we investigated, for
the first time, the effect the geographic origin on the phenolic profiles
and In Vitro antioxidant activities of Tunisian carob (Ceratonia siliqua L.) 2.4. Determination of total flavonoid contents
pulp extracts.
Total flavonoid content (TFC) of each carob methanolic extract, was
2. Materials and methods estimated using a colorimetric assay developed by Falleh et al. (2012).
Flavonoids was first reacted with sodium nitrite (NaNO2), followed by
2.1. Plant material the formation of a coloured flavonoid-aluminum complex which can be
monitored spectrophotometrically at wavelength of 510 nm (Wanyo
Mature fruits of carob (Ceratonia siliqua L.) were harvested in et al., 2011). An aliquot (250 mL) of the samples was mixed with 75 mL
September to October 2020 from ten different geographic regions of the of NaNO2 (5%; w/v) for 6 min, before adding 150 mL AlCl36H2O (10%;
North and the Center of Tunisia belonging to five different bioclimatic w/v). After 5 min at room temperature (RT), 500 mL of NaOH (1 M) was
zones, i.e. sub humid (Sh), upper semi-arid (Usa), mean semi-arid (Msa), added. The final volume was adjusted to 2.5 mL with H2O and scrupu­
lower semi-arid (Lsa), and upper arid (Ua) bioclimates according to lously mixed. Absorbance of the mixture was determined at 510 nm and
Emberger’s (1966) pluviothermic coefficient. The detailed data about converted to flavonoid concentration from a catechin standard curve
the collection sites such as locations, temperature, altitude, and rainfall and expressed as mg catechin equivalents/g of dry residue (mg CE/g
are shown in Table 1. For each region, 15 to 30 trees were randomly DR).
chosen depending on the population size with a minimum distance of
100 m, and healthy pods at full maturity were sampled. Identification of
the collected plants was conducted by Pr. MOUJAHED Nizar, and 2.5. Determination of condensed tannins contents
voucher specimens (C.s. INAT, 20) have been deposited at the National
Agronomic Institute of Tunisia. In the laboratory, seeds were manually The condensed tannins content was determined according to the
removed. The remaining biomass was oven dried at 40 ◦ C and then method of Sun et al. (1998). 50 mL of extract (1 mg/mL) were mixed
ground to obtain a fine powder, with particle size less than 1 mm of with 3 mL vanillin (4%; w/v). Then, 1.5 mL 2M hydrochloric acid was
diameter. Carob pulp powder were then deposited in labeled poly­ added to the mixture. After incubation for 15min at RT, absorbance was
ethylene bags (date of collection and locality) and stored at 4 ◦ C for measured at 500 nm. Results were expressed as mg catechin equiv­
alents/g of dry residue (mg CE/g DR).

Table 1
Locations, bioclimatic and soil characteristics of the collection sites.
Provenance Geographic region P (mm) T (◦ C) Latitude (N) Longitude (E) Altitude (m) Soil type

Bizerte Sh 706 18.1 36◦ 92’ 09◦ 38′ 67 Calcareous

Beja Sh 850 17.5 37◦ 10′ 09◦ 08′ 385 Clay-sandstone
Ariana Usa 450 18.6 36◦ 56′ 10◦ 08′ 197 Sandstone
Ben Arous Usa 400 18.7 36◦ 05′ 09◦ 30′ 890 Sandstone
Jendouba Usa 660 16.9 36◦ 25′ 08◦ 44′ 150 Clay-sandstone
Siliana Msa 429 18.4 35◦ 48′ 09◦ 21′ 450 Calcareous
Zeghouan Msa 515 18.3 36◦ 23′ 10◦ 07′ 325 Calcareous
Nabeul Msa 401 18.6 36◦ 41′ 10◦ 35′ 437 Clay-sandstone
Sousse Lsa 448 18.1 35◦ 30′ 10◦ 50′ 230 Calcareous clay
Kairouan Ua 335 19.6 35◦ 09′ 10◦ 08′ 665 Calcareous

P: average annual precipitation (mm), T: average annual temperature (◦ C). Bioclimatic zones were defined according to Emberger’s (1966) pluviothermic coefficient
(Q2). Q2 = 2000 P/(M2− m2) where P is the mean annual rainfall (mm). M is the average maximum temperature (K) in the warmest month (June) and m (K) is the mean
minimum temperature in the coldest month (February). Sh, Usa, Msa, Lsa and Ua are: sub humid, upper semi-arid, mean semi-arid, lower semi-arid and upper arid
climates, respecively.

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A. Richane et al. Biochemical Systematics and Ecology 104 (2022) 104486

2.6. RP-HPLC evaluation of phenolic compounds diluted radical solution, and absorbance at 734 nm was recorded after 6
min of incubation at room temperature. The inhibition percentage of
The characterization of phenolic compounds in metanolic extracts of ABTS⋅þ radical (IP %) was calculated with the following formula:
Ceratonia siliqua L. pulp was performed using a High-Performance Liquid
Chromatography system (HPLC) (Agilent technologies 1260, Germany, IP% = (A0 - A1) / A0 * 100
Japan) equipped with reverse-phase C18 column, (4.6×100 mm and 3.5 Where, A0 and A1 are the absorbance values of control and test extract,
mm particle size), (Zorbax Eclipse XDB C18). The diode array detector respectively. As for the antiradical activity, ABTS.þ scavenging ability
was set to a scanning range of 200–400 nm. 3 μL of sample was injected was expressed as IC50 values (e.g. the concentration of extract necessary
to HPLC system under flow rate of 0.4 mL/min. The mobile phase is to reduce 50% of the initial ABTS⋅þ radical concentration), calculated
composed of two solvents: methanol and Milli-Q water (0.1% formic from the curve of inhibition percentage (IP %) versus extract concen­
acid). The gradient elution was illustrated as follows: 0–5 min, 10–20% tration. BHT was used as reference antioxidant.
A; 5–10 min, 20–30% A; 10–15 min, 30–50% A; 15–20 min, 50–70% A;
20–25 min, 70–90% A; 25–30 min, 90–50% A; 30–35 min, return to 2.7.4. Ferric reducing power assay (FRAP)
initial conditions. All chromatographic conditions were performed ac­ This antioxidant activity was focused on the reduction of the triva­
cording to the method described previously by Mkadmini Hammi et al. lent iron produced by the FeCl3 (Ksouri et al., 2009). The intensity of the
(2017). Peaks were monitored at 280 nm, and the identification of blue-green color was measured at 700 nm. Results were expressed as
phenolic compounds was done by comparison of their retention time EC50 value (mg/ml) which is the effective concentration giving an
and UV spectra with those of pure standards. For the quantitative absorbance of 0.5 and was obtained from linear regression analysis.
analysis, a calibration curve was performed for each identified phenolic
compound using the available standards at 280 nm.
2.8. Statistical analysis

2.7. Evaluation of antioxidant activities All data are expressed as mean ± standard deviation (mean ± SD) of
three determinations. Quantitative differences were assessed using
2.7.1. Total antioxidant capacity (TAC) analysis of variance (one-way ANOVA) followed by Duncan’s multiple
Total antioxidant capacity of carob pulp extracts was based on the range tests at p < 0.05. The linear correlation coefficients between the
reduction of Mo (VI) to Mo (V) and subsequent formation of a green phenolic compounds and the antioxidant activities (by TAC, DPPH,
phosphate/Mo5+ complex at acid pH according a protocol described ABTS and FRAP assays) were calculated using Pearson’s correlation
previously (Boulaaba et al., 2019). An aliquot (100 mL) of diluted ex­ analysis. Classification of samples harvested from different geographical
tracts was combined with 1 mL of reagent solution (0.6N sulfuric acid, origins according to their phenolic compounds and antioxidant activities
28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes was performed by the Principal Component Analysis (PCA). The SPSS
were incubated in a thermal block at 95 ◦ C for 90 min. Then, the mix­ program (IBM SPSS Statistics version 21.) was used for all statistical
tures were cooled to room temperature and the absorbance of each so­ analyses.
lution was measured at 695 nm against blank in a UV–vis
spectrophotometer. Antioxidant capacity was expressed as mg gallic 3. Results and discussion
acid equivalent per gram dry residue (mg GAE/g DR).
3.1. Total phenolic, flavonoid and condensed tannins contents
2.7.2. DPPH free radical scavenging activity
The radical scavenging activity of the analyzed carob pulp extracts This work was carried out on different geographic regions of Tunisia.
against DPPH. radical was determined using the method described by Each provenance is here defined as a region characterized by similar
Hanato et al. (1988). An aliquot (1 ml) of each extract at various con­ topographic and climatic conditions with a homogeneous flora.
centrations was mixed with 250 μl of freshly prepared methanolic so­ Geographic characteristics such as altitude, central latitude and longi­
lution of 0.2 mM DPPH. The mixtures were shaken vigorously and tude as well as the mean precipitation and temperature of these prove­
allowed to react in the dark for 20 min at room temperature, and then nances are summarized in Table 1. The total phenolic, flavonoid and
absorbance was read at 517 nm using a UV–vis spectrophotometer. The condensed tannins contents from the different provenances of C. siliqua
inhibition percentage (IP %) of DPPH. radical was calculated using the L. pulp are presented in Fig. 1. Values are ranged from 13.19 to 23.11 mg
following equation: GAE/g DR for TPC, from 5.45 to 10.58 mg CE/g DR for TFC and from
IP% = (A0 - A1) / A0 * 100 0.54 to 3.75 mg CE/g DR for CT. The highest levels of TPC (23.11 mg
GAE/g DR) and TFC (10.58 mg CE/g DR) were found for Kairouan
Where A0 is the absorbance of the control and A1 is the absorbance of the sample belonging to the upper arid climate, while the lowest ones (13.19
carob pulp extract. Antiradical activity was expressed as IC50, defined as GAE/g DR and 5.45 mg CE/g DR, respectively) were recorded in Bizerte
the concentration of extract necessary to reduce 50% of the initial DPPH sample (sub-humid climate). For CT, the highest level was observed in
radical concentration, and graphicaly calculated from the curve of in­ Beja sample (3.75 mg CE/g DR) followed by Bizerte (3.69 mg CE/g DR)
hibition percentage (IP %) versus extract concentration. The lower value and Sousse (2.93 mg CE/g DR) ones. The lowest content of CT (0.54 CE/
of IC50 corresponded to the strongest antiradical activity of carob pulp g DR) was found in the extract of Ariana region. The amount of TPC, TFC
extract. BHT was used as reference antioxidant. and CT was significantly (p < 0.05) influenced by geographical origin.
The quantification of total phenolic compound contents is the first step
2.7.3. ABTS radical assay used when assessing the antioxidant activity of plant and food extracts
The ABTS assay was done according to the method described by Re because of high interest of these compounds as alternative for synthetic
et al. (1999). The stock solution of ABTS.+ was freshly prepared by antioxidant to prevent lipid peroxidation in food systems (Benchikh
mixing equal volumes (5 ml) of 14 mM ABTS⋅+ solution and 4.9 mM et al., 2014). Several reports (Makris and Kefalas, 2004; Custódio et al.,
potassium persulfate solution and then incubating the resultant mixture 2011; Sebai et al., 2013; Benchikh et al., 2014) discuss phenolic contents
in the darkness for 12–16 h for radical stabilization. Before analysis, this in C. siliqua.L pulp extracts in the literature. Values of TPC in the present
stock solution was diluted with absolute ethanol until reaching an study are markedly lower than those reported by Ydjedd et al. (2017)
absorbance of 0.700 ± 0.02 at 734 nm. Then, an aliquot (50 μl) of each who found TPC amount of about 44 mg GAE/g DR for an extract of carob
test extract at different concentrations was mixed with 950 μl of the pulp growing wild in Algeria. However, Makris and Kefalas (2004)

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A. Richane et al. Biochemical Systematics and Ecology 104 (2022) 104486

Fig. 1. Total polyphenols (TPC), flavonoids (TFC) and condensed tannins (CT) contents of methanolic extracts of C. siliqua L. pulp from different locations in Tunisia.
For each phenolic class, bars with different letters are significantly different (p < 0.05). Sh, Usa, Msa, Lsa and Ua are: sub humid, upper semi-arid, mean semi-arid,
lower semi-arid and upper arid climates, respecively.

reported lower TPC (9.25 mg GAE/g DR) amount in Greek carob pulp. fact, the highest levels of gallic acid (1628.46 mg/kg), syringic acid
Literature data regarding flavonoid content of carob are limited; Sebai (198.57 mg/kg), kaempferol (183.15 mg/kg) and apigenin (696.12
et al. (2013) reported a flavonoid content of 4.14 mg CE/g DR, which mg/kg) were found in Kairouan sample. Extract of Bizerte had the
was lower than the values recorded for the provenances assayed in the highest amounts of naringenin (362.48 mg/kg), p-coumaric acid (79.34
present study. These differences between our results and those reported mg/kg) and resorcinol (98.34 mg/kg). In turn, carob pulp from Nabeul
previously could be attributed to various factors such as cultivar, region showed the highest content of caffeic acid (139.37 mg/kg) and
maturity, geographic origin, storage conditions and use of different epicatechin-3-O-gallate (79.31 mg/kg), whereas Zaghouan sample
solvants of extraction and analytical methods (Fan and Beta. 2017). exhibited the highest amount of ellagic acid (940.53 mg/kg), catechin
On the other side and in comparison with other fruits, our results (269.30 mg/kg) and isoquercetin. Finally, luteolin was detected at the
showed that the C. siliqua.L pulp might be considered as a rich source of highest concentration in the sample harvested in Sousse region (lower
polyphenols content. In fact, it presented a greater TPC content semi-arid climate). The minor phenolic compounds identified in this
compared with other fruits, such as peach (4.85 mg GAE/g DW), orange study were ferulic acid, trans-cinnamic acid, sinapic acid, catechol,
(6.22 mg GAE/g DW), dates (5.77 mg of GAE/g DW), apricot (4.31 mg of kaepmferol-3-O-rutinoside, crismaritin and luteolin-7-O-glucoside, wich
GAE/g DW) and banana (4.55 mg GAE/g DW) (Stavrou et al., 2018). are detected at lower concentrations only in a few of the localities. For
example, ‘5-caffeoylquinic acid (currently known as 5-caffeoylquinic
3.2. RP-HPLC-DAD analysis of phenolic compounds acid (5-CQA) as per IUPAC numbering system (1976)) (10.84–12.23
mg/kg), epigallocatechin (18.04–19.25 mg/kg), catechol (23.21–27.36
The phenolic composition of carob pulp is quite different among the mg/kg) and kaempferol-3-O-rutinoside (12.19–13.66 mg/kg) were
literature, depending not only on the carob provenance, weather con­ detected only in the samples collected from Sousse and Kairouan areas.
ditions, geographical origin, harvesting and storage, but also on tech­ Ferulic acid (19.56–20.36 mg/kg) and trans-cinnamic acid (8.98–10.37
nological factors such as the extraction methodologies (Ydjedd et al., mg/kg) were detected in samples of Bizerte and Beja regions. Sinapic
2017). Reversed phase HPLC-DAD analysis of the aqueous methanol acid (10.71–20.30 mg/kg), crismaritin (23.96–30.81 mg/kg) and
extracts of Ceratonia siliqua.L pulp revealed a total of 22 phenolic luteolin-7-O-glucoside (20.87–36.20 mg/kg) were found only in samples
components, including 9 phenolic acids and 13 flavonoids. Retention from the upper and mean semi-arid zones. In line with our results, a
times (RT) and the average concentration of the identified compounds large variability in individual phenolic compounds (phenolic acids and
are presented in Table 2. A typical chromatogram of carob pulp extract flavonoids) contents according to growing site have been observed for
(Kairouan sample) is presented in Fig. 2. Gallic acid (501.26–1628.46 other fruits like strawberries (Kim and Shin. 2015) and apples (Yuri
mg/kg), ellagic acid (398.85–940.53 mg/kg) and isoquercetin et al., 2009). This variation could be due to diverse factors such as ge­
(264.79–817.61 mg/kg) were the major components in all tested ex­ netic background, environmental conditions and edaphic factors
tracts, followed by apigenin (194.66–696.12 mg/kg) and naringenin (Jaouadi et al., 2019; Li et al., 2020).
(96.53–362.48 mg/kg). Those natural antioxidants have been reported
to possess a wide range of in vitro biological properties, such as antiox­ 3.3. Antioxidant activities
idant, anti-inflammatory and anticancer effects (Esfanjani et al., 2018),
which give carob pulp a high pharmaceutical value and encourage their By the way, no standardized methods available to evaluate the
use in food formulations, nutraceutical, and pharmaceutical industries. antioxidant capacity of plant extract. Numerous methods have been
Furthermore, as Table 2 shows, syringic acid, catechin, kaempferol, employed to estimate the antioxidant potential (Jallali et al., 2014). In
caffeic acid, resorcinol, epicatechin-3-O-gallate, p-coumaric acid and the current study, Total antioxidant capacity (TAC), DPPH and ABTS
luteolin were also identified in all provenances. A comparison with other radicals scavenging effect and ferric reducing power (FRAP) assays were
studies on the individual phenolic compunds of Ceratonia siliqua.L pulp used to assess the antioxidant activity of carob pulp provenances. Re­
is difficult since few papers were reported on the subject and they used sults in Table 3 demonstrated that methanol extracts of carob pulp
different extraction processes and analytical techniques leading to exhibited higher antioxidant activity, evaluated by TAC (57.19–88.92
different phenolic profiles (Papagiannopoulos et al., 2004; Roseiro et al., mg GAE/g DR), DPPH (IC50 = 98.08–192.52 μg/ml), ABTS (IC50 =
2013). Statistical analysis showed significant differences (p < 0.05) 19.16–54.92 μg/ml) and FRAP (EC50 = 6.30–23.55 μg/ml), compared to
among the ten studied extracts for all identified phenolic compounds. In butylated hydroxyanoside (BHA) and ascorbic acid as reference

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 A. Richane et al.
Table 2
Individual phenolic compounds (mg/kg of DR) identified in the aqeuous methanolic extracts of Ceratonia siliqua L. pulp from different locations in Tunisia.
Compounds RT Sh Usa Msa Lsa Ua

Bizerte Beja Ariana Ben Arous Jendouba Siliana Zaghouan Nabeul Sousse Kairouan

Phenolic acids
Gallic acid 8,2 501.26 j±1.23 527.34i±0.56 908.26e±0.23 969.21d ± 0.12 994.52c±0.21 856.35g ± 0.45 898.96f±0.19 814.67h ± 0.71 1310.34b ± 1.2 1628.46a±0.4
Caffeic acid 17,42 51.39j±0.2 56.39i±1.09 109.12f±0.36 116.57d ± 0.33 112.52e±0.39 132.83b ± 0.28 120.76c±0.09 139.37a±0.13 89.13g ± 0.23 71.42h ± 0.16
5-caffeoylquinic acid 15,75 nd nd nd nd nd nd nd nd 10.84a±1.11 12.23b ± 0.56
Syringic acid 17,8 33.43j±0.07 43.37i±0.12 92.81h ± 0.11 95.74g ± 0.36c 99.87f±0.85 111.71d ± 0.64 107.48e ±0.91 123.09c±0.24 164.54b ± 0.34 198.57a±1.02
Ellagic acid 22,28 398.85j±1.02 413.96i±1.03 600.38g ± 0.36 763.54d ± 0.14 684.56f±0.12 892.5b ± 1.31 940.53a±1.12 808.36c±2.03 695.53e±0.96 543.72h ± 0.9
Ferulic acid 20,23 20.36a±0.04 19.56b ± 0.04 nd nd nd nd nd nd nd nd
Sinapic acid 19,98 nd nd 12.52d ± 0.62 19.91b ± 0.75 10.71f±1.04 14.96c±0.21 11.18e±0.04 20.3a±0.14 nd nd
Trans-cinnamic acid 24,36 10.37 a ±0.04 8.96 b ± 0.04 nd nd nd nd nd nd nd nd
p-Coumaric acid 19,69 79.34a±0.14 78.38b ± 0.21 21.36h ± 0.19 19.28i±0.35 15.29j±0.58 29.13f±0.64 32.37c±0.74 38.41g ± 0.51 42.4e±0.88 53.19d ± 0.14
Flavonoids
5

Apigenin 25,9 194.66j±1.02 268.79i±0.02 452.45g ± 1.23 460.69f±0.36 444.31h ± 0.78 599.81c±0.12 577.84e±0.09 587.94d ± 0.36 641.9b ± 0.96 696.12a±1.23
Epigallocatechin 14,7 nd nd nd nd nd nd nd nd 19.25a±0.36 18.04b ± 0.98
Epicatechin-3-O-gallate 18,27 54.3 h ± 1.09 55.17 g ± 0.98 68.33 d ± 0.29 65.53 e±1.23 64.35 f ±0.03 77.62 b ± 0.98 75.67 c ±0.28 79.31 a±0.21 41.94 i±1.36 29.63 j±0.25
Catechin 14,31 36.55j±1.23 45.18i±0.07 171.05b ± 0.09 139.48d ± 0.63 98.51f±0.78 121.35e±1.45 269.30a ±1.96 148.26c±1.23 72.49h ± 2.69 83.38g ± 0.36
Luteolin 24,79 7.89j±1.23 9.71i±1.23 17.23e±1.23 19.49d ± 0.36 20.52c±0.21 12.33h ± 0.36 14.82f±1.23 13.28g ± 1.23 44.14a±0.78 32.32b ± 0.45
Crismaritin 26,98 nd nd 30.18d ± 1.96 30.63b ± 0.12 27.61e±0.02 30.81a±0.98 30.31c±0.98 23.96f±0.98 nd nd
Catechol 12,03 nd nd nd nd nd nd nd nd 23.21b ± 1.78 27.36a±0.23
Luteolin-7-O-glucoside 20,94 nd nd 27.93b ± 0.96 36.2a±1.23 24.55d ± 0.36 26.91c±0.23 23.74e±0.47 20.87f±0.45 nd nd
Isoquercetrin 21,48 398.61i±0.29 264.79j±0.06 534.1g ± 1.89 578.78e±1.36 633.22d ± 1.89 797.41b ± 0.09 817.61a±0.26 701.4c±0.03 429.38h ± 0.78 544.76f±0.12
Naringenin 24,32 362.48a±1.23 340.57b ± 1.23 96.53 j±0.12 134.53h ± 0.96 104.71i±1.25 140.26f±1.25 168.55e±0.12 139.43g ± 0.98 251.68d ± 0.23 262.88c±0.42
Kaempferol 25,46 98.37j±0.96 109.72h ± 1.15 121.84f±0.22 114.87g ± 0.38 105.78i±0.56 139.63e±0.98 141.7d ± 0.78 158.71c±0.98 162.25b ± 1.14 183.15a±0.58
Kaempferol-3-O-rutinoside 22,54 nd nd nd nd nd nd nd nd 12.19b ± 0.58 13.66a±0.98

Biochemical Systematics and Ecology 104 (2022) 104486

Resorcinol 10,31 98.34a±1.11 89.4b ± 1.09 57.31e±0.36 50.41g ± 0.22 56.26f±0.45 32.42j±0.98 39.2i±0.23 45.37 h ± 0.13 63.41d ± 0.66 71.66c±0.63

Results were given as means ± SD. Different letters for the same line indicate significant differences at p < 0.05.
nd: not detected. Sh, Usa, Msa, Lsa and Ua are sub humid, upper semi-arid, mean semi-arid, lower semi-arid and upper arid climates, respecively.
A. Richane et al. Biochemical Systematics and Ecology 104 (2022) 104486

Fig. 2. Typical RP-HPLC chromatogram of Ceratonia

siliqua L. pulp extract monitored at 280 nm.
Peak numbers corresponding to: 1: gallic acid; 2:
resorcinol, 3: catechol, 4: catechin, 5: epi­
gallocatechin, 6: 5-caffeoylquinic acid, 7: caffeic acid,
8: syringic acid, 9: epicatechin-3-o-gallate, 10: p-
coumaric acid, 11: isoquercetin, 12: ellagic acid, 13:
kaempferol-7-o-glucoside, 14: naringenin, 15: luteo­
lin, 16: kaempferol, 17: apigenin.

observed between extracts of the investigated geographical origins.

Table 3
These can be explained by the observed variation in their phytochemi­
Antioxidant activities of aqeuous methanolic extracts of C. siliqua.L pulp from
cals contents (Mena et al., 2011). Plants antioxidant activity might be
different locations in Tunisia.
attributed to the phenolic compounds content as well as their chemical
Sample DPPH Test ABTS Test FRAP (EC50 TAC (mg structure (Barku et al., 2013). To verify this finding, correlation analysis
(IC50 μg/ml) (IC50 μg/ml) μg/ml) GAE/g DR)
between antioxidant activities and phenolic compounds content (total
Bizerte 192.52a ± 2.15 54.92a ±0.23 23,55a ±0.96 57.19j±0.89 and individual phenolics) was carried out. As shown in Table 4, signif­
(Sh)
icant correlation was noticed between the antioxidant activity by TAC,
Beja (Sh) 186,88b ± 48,15b ± 1.45 19,29b ± 60.25h ±
3.96 1.23 1.23 DPPH, ABTS and FRAP assays and the amount of TPC (r = 0,91, − 0.76,
Ariana 174,14c ±0.16 29,31f ±0.63 17,43c±1.45 59,46i±2.36 − 0.75 and − 0.85, respectively) and TFC (r = 0.90, − 0.75, − 0.78 and
(Usa) − 0.87, respectively) (Table 4). These results agree with previous in­
Ben Arous 102,83h ± 31,25e±0.23 13,21d ± 63,91g ± 3.09 vestigations that underlined high relationship between total phenolics
(Usa) 0.14 2.23
Jendouba 122,09g ± 0.41 26,1g ± 1.25 9,48f ±0.47 65,19f ±1.23
content and antioxidant activity of many plant extracts, due to the
(Usa) effectiveness of these compounds as radical quenchers, transition metal
Siliana 164,11d ± 23,36h ± 2.45 10,59e±2.36 68,93e±2.36 chelators and reducing agents, etc (Djeridane et al., 2006; Ghasemi
(Msa) 0.32 et al., 2009; Ebrahimzadeh et al., 2014). Regarding the individual
Zaghouan 152,64e ±0.92 39.39d ± 0.86 8,76g ± 1.56 77,14c ±0.98
phenolic compounds detected, gallic acid, syringic acid, luteolin,
(Msa)
Nabeul 137,17f ±0.08 45.21c ±1.23 8.18h ± 0.36 72,28d ± kaempferol and apigenin showed significant correlation with in vitro
(Msa) 0.14 antioxidant properties, which implied that these compounds are the
Sousse 100.65i±0.66 22.07i±0.12 7,28i ±0.36 85.76b ± major contributors to the antioxidant activity of C. siliqua L. pulp extract.
(Lsa) 0.23 In line with our results, some authors have reported the impact of gallic
Kairouan 98,08j ±1.13 19,16j ±0.08 6,3j ±1.45 88,92a ±1.45
(Ua)
acid, isoquercetin and apigenin in the free radical scavenging activity
Standard antioxidants due to their structure (presence of several hydroxyl substituents) that
BHT 269.236 ± 67.56 ± 1.14 – – allow them to donate hydrogen to free radicals (Kabtni et al., 2020). In
2.13 addition, it is noticeable that the chemical interactions (synergism or
Ascorbic – – 30.36 ± 0.21 51.09 ± 0.21
acid
Table 4
Results were given as means ± SD. Different letters for the same line indicate
Correlations between the antioxidant activities and phenolic compounds content
significant differences at p < 0.05. Sh, Usa, Msa, Lsa and Ua are sub humid,
of Ceratonia siliqua L. pulp.
upper semi-arid, mean semi-arid, lower semi-arid and upper arid climates,
respecively. Antioxidant activities

Compounds TAC DPPH ABTS FRAP

antioxidants. The extract from Kairouan region showed the stronger TPC 0.91** − 0.76a − 0.75a − 0.85**
TFC 0.90** − 0.75a − 0.78a − 0.87**
antioxidant power mesured by all tests: TAC (88.92 mg GAE/g DR),
CT 0.06 0.06 0.16 0.21
DPPH (IC50 = 98.08 μg/ml), ABTS (IC50 = 19.16 μg/ml) and FRAP (EC50 Gallic acid 0.84** − 0.76** − 0.79** − 0.69a
= 6.30 μg/ml) (Table 3). In contrast, the lowest TAC (57.19 mg GAE/g Caffeic acid 0.04 − 0.27 − 0.22 − 0.49
DR), DPPH (IC50 = 192.52 μg/ml), ABTS (IC50 = 54.92 μg/ml) and FRAP Syringic acid 0.71a − 0.78** − 0.76** − 0.71a
Ellagic acid 0.45 − 0.46 − 0.48 − 0.72
(EC50 = 23.55 μg/ml) values were found in Bizerte sample. These results
Apigenin 0.85** − 0.70a − 0.72a − 0.87**
suggest that carob pulp might constitute a good alternative to replace Isoquercetin 0.25 − 0.23 − 0.31 − 0.54
cytotoxic synthetic antioxidants like BHA and BHT, which are widely Resorcinol − 0.26 0.35 0.40 0.40
used in food, pharmaceuticals and cosmetic industries (Gordobil et al., Catechin 0.17 − 0.25 − 0.22 − 0.25
2018). Additionally, using DPPH and FRAP assays, the antioxidant ac­ Naringenin 0.03 0.26 0.24 0.35
Kaempferol 0.91** − 0.78a − 0.84** − 0.74a
tivity of carob pulp extracts was higher than that of other fruits (Meyers
p-coummaric acid 0.08 − 0.42 − 0.40 − 0.43
et al., 2003; Wojdyło et al., 2008; Lachman et al., 2009), vegetables Luteolin 0.77** − 0.75a − 0.69a − 0.70a
(Ismail et al., 2004), and medicinal plants (Rašković et al., 2014). Epicatechin-3-O-gallate − 0.47 0.34 0.34 0.07
Significant differences (p < 0.05) in antioxidant activities were a
, ** = significant at p < 0.05 and p < 0.01, respectively.

6
A. Richane et al. Biochemical Systematics and Ecology 104 (2022) 104486

antagonism) among various major and minor phenolic constituents may

also influence the antioxidant activity of plant extracts (Dai and
Mumper. 2010).

3.4. Multivariate analysis (PCA)

Principal component analysis (PCA) was carried out to discriminate

and to cluster the ten investigated samples according to their phenolic
composition and antioxidant activities. The two first principal compo­
nents (PCs) expalined 86.83% of the total variability. PC1 explained the
great part of the total variability (54.81%) and was positively correlated
with TPC, TFC, gallic acid, syringic acid, apigenin, kaempferol, luteolin
and total antioxidant capacity (TAC), while it was negatively correlated
with IC50 (DPPH and ABTS) and EC50 (FRAP) values. PC2 accounted for
32.02% of the total variance and was highly correlated with caffeic acid,
ellagic acid, isoquercetin, catechin, epicatechin-3-O-gallate, resorcinol,
naringenin and p-coumaric acid (Fig. 3A). Scatter plot of PCA defined by
the two first axes (PC1 and PC2) allowed a separation of samples into
three distinctive groups (I-III) (Fig. 3B). The first group includes the
Kairouan and Sousse samples characterized by the highest concentra­
tions of TPC, TFC, gallic acid, apigenin, syringic acid, luteolin and
kaempferol and the highest antioxidant capacities. The upper semi-arid
(Ariana, Ben Arous and Jendouba) and the mean semi-arid (Siliana,
Zaghouan and Nabeul) samples constitute the second group situated at
the positive side of the axis 2. This group was mainly distinguished by
high contents of ellagic acid, isoquercetin, caffeic acid, catechin and
epicatechin-3-O-gallate. Finally, the third group, integrated the negative
side of axis 1 and 2, consists of samples from Bizerte and Beja wich are
Fig. 3. Principal component analysis (PCA) of Ceratonia siliqua L. pulp samples
distinguished by the highest levels of CT, naringenin, resorcinol and p-
from different locations in Tunisia based on their phenolic composition and
coumaric acid, the lowest amounts of all the remaining individual antioxidant capacities. (A) variables. (B) loadings.
compounds and the lowest antioxidant capacities. Findings from PCA
confirmed the variability in the phenolic composition of Tunisian carob
promising populations considering their strong phenol content and their
pulp according to geographical origins. It is well know that phyto­
high antioxidant capacity. Additional biological testing will be neces­
chemical composition of plants and consequently, their biological ac­
sary to prove the highly beneficial properties of these provenances. In
tivities are influenced by diverse factors such as genetic background,
vivo assays are essential and they should be carried out to further
environmental conditions and edaphic factors (Jaouadi et al., 2019). In
confirm their uses.
the present study, the multivariate analysis (PCA) showed a clustering of
samples collected from sites that differed for climatic condition, soil
type, and elevation above sea level, such as the group formed by Kair­ Author contributions
ouan (Upper arid climate; 665 m of altitude) and Sousse (lower semi arid
climate; 230 m of altitude). Also, as shown in Fig. 3B, samples of Ariana, All authors contributed to the study conception and design. Richane
Ben Arous, Jendouba, Siliana, Zaghouan and Nabeul belonging to Amira: Conceptualization, Methodology, Investigation, Data curation,
different climatic zones and growing at altitudes that ranged from 150 to Formal analysis, Writing - original draft, Visualization. Ben Mansour
890 m are very close and clustered togherther, suggesting similarities in Rim, Megdiche Wided and Ksouri Riadh: Investigation, Methodology,
their phenolic composition and antioxidant activities. Hence, the evi­ Validation, Writing - review & editing. Attia Khaoula: Investigation,
denced variability between examined samples might be mostly due to Visualization. Nizar Moujahed, Ben Ismail Hanen: Supervision, Re­
genetic factors rather than to environmental and ecogeographic condi­ view and Editing.
tions. Studies conducted by Afif et al. (2008), based on Randomly
Amplified Polymorphic DNA (RADP) and isozymic markers, have shown Declaration of competing interest
that Tunisian Ceratonia siliqua.L populations (other than those reported
in this study) presented high genetic differentiation and different levels The authors declare that they have no conflict of interest.
of polymorphism within populations. Our results are in agreement with
those of previous studies that evaluated the phenolic variability in other
Acknowledgements
plant species, highlighting the high impact of the genetic fector on
phenolic profiles and antioxidant capacities (Crespo et al., 2010; Zaouali
The authors would like to thank Prof. Riadh Ksouri, Director of the «
et al., 2010).
Laboratory of Medicinal and Aromatics Plant, Biotechnology Centre of
Borj-Cedria » for their help and suggestions.
4. Conclusion

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