Determination of Polyphenols Content in Carob Pulp from Wild and Domesticated Moroccan Trees

Carob (Ceratonia siliqua L.) fruit pulp from 12 wild and 8 domesticated trees from 15 Moroccan localities were investigated for their polyphenols contents: total polyphenols content (53.22 118.04 mg and 57.46 183.31 mg), total flavonoids (1.41 4.83 mg and 1.62 7.46 mg) and condensed tannins (1.47 7.36 mg and 1.85 6.66 mg) in one carob fresh pulp for wild and domesticated trees, respectively. Fruit pulp from trees in the same region shows variable contents in polyphenols, flavonoids and condensed tannins.


Introduction
Ceratonia siliqua L. (carob tree) is a perennial Fabaceous tree which is flourishing in the Mediterranean area since antiquity [1] [2].It grows usually in mild and dry places with marginal and prevailing calcareous soils [1].The tree attains a mature height and spread of 6 -12 m, and sometimes more than 20 m, with branches extended to ground level [3].
Carob pod is the fruit of carob tree and is mostly used in the food industry for locust bean gum: polysaccharides (galactomannans) contained in the endosperm of the seeds.
Due to their chemical composition, carobs are used in industry, in food and in medicine [12].Industrial use lies in gum production, taken from seeds after shuttering and separation, used as a thickening agent in many commercial food products as well as in textile industry [13].Moreover, different human foods can be derived from the pulp such as sugar syrups or molasses, unroasted and roasted carob powder used as cocoa substitutes, or especially tannin rich preparations as anti-diarrheic products [7] [14].Furthermore, carob pods have demonstrated interesting anti-cardiovascular and antioxidant properties, apparently related to their phenolic compounds content [3] [12] [15].
As Morocco is the fifth carob producer in the world after Spain, Italy, Portugal and Greece [16], plus the biological importance of some chemical compounds and their strong association with health benefits, an effort has been made to quantify total polyphenols, flavonoids and tannins in carob pulps of 12 wild and 8 domesticated trees from 15 different localities in Morocco, in order to evaluate differences between these two tree types.

Plant Material
Carob mature fruits have been collected from 12 wild trees and 8 domesticated ones from 15 different localities in Morocco.Collection has been carried out between July and September 2012.Fruits were stored in the dark at room temperature (25˚C ± 2˚C).
Origins, localities, numbers, latitudes, longitudes and altitudes may be seen in Table 1.

Polyphenols Extraction
20 g of powder was macerated in 200 mL distillated water 6 h at 4˚C.Then, this mixture was centrifuged 15 min at 5000 rpm.The fresh residue was extracted with 70% acetone (ratio of extraction 1:5; w/v) with a magnetic stirrer (30 min; 350 rpm), then centrifuged 10 min at 5000 rpm.This extraction was repeated two times.The three supernatants were pooled and incubated at 4˚C until analyses.

Total Polyphenols Content
The total polyphenols content was determined by the Folin-Ciocalteu colorimetric method [17] [18], with some modifications.Carob pulp extract was diluted at 1:10 in distilled water and 0.6 mL was mixed with 5 mL distillated water and 3 mL Folin-Ciocalteu reagent 1N diluted at 1:10.After 3 min waiting, 1 mL of sodium carbonate solution (10% Na 2 CO 3 in distillated water) was added.Finally, absorbance was measured by spectrophotometer UV-Vis (UV-1800; Shimadzu, Kyoto, Japan) at 725 nm, after 2 h incubation at obscurity and 20˚C.Results were expressed as mg gallic acid equivalents by g of dry carob pulp powder (mg GAE/g).

Total Flavonoids Content
Flavonoids content was determined by the method described by Djeridane et al. [19] [20].Carob pulp extract was diluted at 1:5 and 4.5 mL were mixed with 4.5 mL of aluminum chloride (2%).Absorbance was measured by Spectrophotometer UV-Vis at 430 nm, after 45 min incubation at obscurity and 20˚C.Results were expressed as mg quercetin equivalents by g of dry carob pulp powder (mg QE/g).

Condensed Tannins Content
Condensed tannins content was determined by Butanol-HCl method [21] [22].0.5 mL of the extract was mixed with 3 mL of Butanol-HCl solution (95:5 v/v) and 0.1 mL of ammonium iron (II) sulfate (2% in HCl 2N).Absorbance was measured by Spectrophotometer UV-Vis at 550 nm, after 1 h of incubation in boiling water bath protected from light.Results were expressed as mg proanthocyanidin equivalents by g of dry carob pulp powder (mg PE/g).

Statistical Analysis
All measurements were run in triplicates (n = 3), and the values were averaged and given along with standard error (±SE).Analyses were performed with Statistical 6; averages were compared by Duncan test and values beyond p ≤ 0.05 were considered to be significant.

Total Polyphenols Content
For wild trees, TPC F ranged from 7.00 to 19.36 mg GAE/g, TPC D from 7.80 to 22.75 mg GAE/g and TPC P from 53.22 to 118.04 mg GAE/one fresh pulp.Ghorghez powder showed the higher TPC  2.

Total Flavonoids Content
For wild trees, TFC F ranged from 0.  For domesticated trees, TFC F ranged from 0.19 to 0.65 mg QE/g, TFC D from 0.21 to 0.73 mg QE/g and TFC P from 1.62 to 7.46 mg QE/g.Fresh and dry powders from Zoumi 1 tree were the richest in flavonoids (0.65 and 0.73 mg QE/g, respectively), followed by M'hannech II tree (0.48 and 0.57) and Kitane tree (0.45 and 0.48).Fresh and dry powders of Moulay Idriss Zerhoun 2 tree were the poorest in flavonoids with a TFC F and a TFC D of 0.19 and 0.21 mg QE/g.Pulp from Zoumi 1 tree showed the highest TFC P (7.46 mg QE/one fresh pulp), followed by Ain Beida 1(4.65) and Kitane (4.35) trees.The poorest pulp in flavonoids was from Moulay Idriss Zerhoun 2 tree with a TFC P of 1.62 mg QE/one fresh pulp Table 3.

Condensed Tannins Content
For wild trees, CTC F ranged from 0.30 to 0.85 mg PE/g, CTC D from 0.35 to 1.02 mg PE/g and CTC P from 1.47 to 7.36 mg PE/one fresh pulp.The highest CTC F and CTC D corresponded to the powder of Isslan tree (0.85 and 1.02 mg PE/g, respectively), followed by the powders from Ghorghez (0.72 and 0.84) and Midar (0.68 and 0.80) trees.The lowest CTC F and CTC D corresponded to the powder of Ain Koub (0.30 and 0.35 mg PE/g).The richest pulp in condensed tannins belonged to Ain Kalaa tree (7.36 mg PE/one fresh pulp), followed by Ain Beida 2 (6.61) and Midar (5.35) trees.The poorest pulp in condensed tannins belonged to Guelmouss tree with 1.47 mg PE/one fresh pulp Table 4.
For domesticated trees, CTC F ranged from 0.22 to 0.59 mg PE/g, CTC D from 0.24 to 0.68 mg PE/g and CTC P from 1.85 to 6.66 mg PE/one fresh pulp.Powder from Moulay Idriss Zerhoun 3 tree showed the highest CTC F and CTC D (0.59 and 0.68 mg PE/g, respectively), followed by the powders from Zoumi 1 (0.48 and 0.54) and Kitane (0.47 and 0.51) trees.Powder from Oued Laou tree showed the lowest CTC F and CTC D (0.22 and 0.24 mg PE/g).The richest pulp in condensed tannins was from Moulay Idriss Zerhoun 3 tree with 6.66 mg PE/one fresh pulp, followed by Ain Beida 1 (6.37) and Zoumi 1 (5.47) trees.The poorest pulp in condensed tannins was from Oued Laou tree with 1.85 mg PE/one fresh pulp Table 4.

Discussion
The present study highlights differences in polyphenols contents among powders and pulps from wild and domesticated trees of Ceratonia siliqua L.
Thereby, carob dry powders from wild trees show total polyphenols, flavonoids and condensed tannins contents reaching 22.75 mg GAE/g, 1.02 mg QE/g and 1.02 mg PE/g, respectively.Also, pulps from wild trees show total polyphenols, total flavonoids and condensed tannins contents by one fresh carob pulp reaching 118.04 mg GAE, 4.83 mg QE and 7.36 mg PE, respectively, while domesticated trees show lower contents for powders.Thus, total polyphenols, flavonoids and condensed tannins contents reach 18.06 mg GAE/g, 0.73 mg QE/g and 0.68 mg PE/g, respectively.For one fresh carob pulp, they reach 183.31 mg GAE, 7.46 mg QE and 6.66 mg PE, respectively.Indeed, Marakis et al. [23] found that wild carob populations are richer in tannins than domesticated ones.Our results are in the interval of results of other studies on carob tree.Difference depends on genetic, geographical, environmental, physiological and cultural conditions, sample preparations, extraction protocol and method of analysis.
It was also found that the high growth activity in May is expressed both by higher photosynthetic rates and an increase in polyphenols, compared to June and October [25].
Benchikh et al. [26] have also compared pulps from one carob wild tree and two cultivated ones, in unripe stage, mid-ripe stage and ripe stage and observed that the total phenolic content and total flavonoids decreased with ripening.In addition, Ouzounidou et al. [25] observed that total polyphenols content gradually decreased with ripening.
Small pieces of pods (0.5 -1.0 cm) eluted with water (1:4) using peristaltic pump, carob pods reduced to fine powder and mixed with water at a ratio (1:4) during 2 h at 45˚C and particle size of 0.5 -1.0 cm prepared as the previous powder were compared; the first resulted eluant and the two latter extracts were centrifuged at 3000 rpm and 0˚C for 30 min; resulted extracts contained 0.06, 0.11 and 0.07 g TAE/100 mL of extract [27].Likewise, two types of carob juice were tested, 0.5 -1.0 cm and fine particle size; both particles were suspended in water and stirred at 43°C for 160 min.Low differences were observed for total polyphenols content (19.8 ± 1.2 mg vs.20.3 ± 1.4 mg GAE/g of dry matter), total tannins content (4.3 ± 0.2 vs. 4.5 ± 0.3 mg CE/g of dried extract) and total flavonoids content (1.2 ± 0.1 vs. 1.5 ± 0.1 mg CE/g of dried extract) [28].
Optimization of yield can be accomplished by applying and controlling parameters of some processes like roasting.Kibbled carob was roasted at 135˚C, 150˚C and 165˚C during 0, 5, 10, 15, 20, 30, 45, 60, 75 and 90 min.Roasted samples were then ground, sieved and stored at 4˚C till extraction and analysis.Total phenolic content of nonroasted kibbled carob was 0.570 g/100 g of dry matter and increased up to 0.865, 0.986 and 1.131 g/100 g during the roasting process at 135˚C, 150˚C and 165˚C, respectively.
Total polyphenols content was almost stable in the initial stage and gradually increased to a maximum level up to 75 min [29].It was also found that the most acceptable roasted carob powder was obtained by roasting kibbled carob at 150˚C for 60 min [30].
Different methods for polyphenols extraction are reported in literature including conventional methods using solvents, ultrasound extraction [35] and supercritical extraction [35] [36].
Kumazawa et al. [3] followed by [20] extracted carob pods, crushed and dried, during 12 h in cold water (3˚C).This operation, repeated two times, eliminated monosaccharides.Also, polyphenols extraction from carob pods has been performed with water at room temperature [35] [37] [38].Also, it was demonstrated that extraction time (0.5, 1, 3, 5, 7 and 9 h) had no effect on the quantity of extracted phenolic compounds at a ratio of 1:10 (w/v); but increasing the ratios carob pulp/water produced extracts richer in soluble polyphenolic compounds [38].Phenol compounds have often been extracted from carob pod with boiling water [3] [39].In a study applied in the industrial scale, 3 kg of carob pods, from trees in Turkey, was mixed with 17 L of water in a tank of 25 L, equipped with heating/cooling system with circulation water by pumping.Three temperatures were tested: 20˚C, 50˚C and 85˚C.It was found that increasing the extraction temperature resulted in higher amounts of total polyphenols content [40].
Benchikh and Louailèche [45] have first extracted fresh carob pods powder with water, 60% ethanol, 60% methanol and 60% acetone, using bath shaker.In another study, water, ethanol, acetone and ethyl acetate were evaluated.Maximum quantities of polyphenols were obtained by 10% aqueous ethanol and maximum quantities of proanthocyanidins were obtained with 70% aqueous acetone [46].
A series of non toxic solvent systems, composed of water/ethanol, acidified and non acidified with either acetic or citric acid, were tested for their efficiency in extracting polyphenols from dried and ground carob kibbles.It was found that 30% ethanol was the best solvent system, providing extracts with high total polyphenolic content (9.32 ± 0.78 mg GAE/g of dry powder) and high antioxidant activity.The highest yield of total polyphenols was achieved at 60˚C [47].
In addition, carob kibbles (by-product of carob been gum production) from Portugal were submitted to an aqueous extraction to extract sugars, and supercritical fluid extraction was applied to the solid residue of that aqueous extraction, by using compressed carbon dioxide as the solvent and 80% ethanol as a co-solvent.Pressure and temperature were studied in the ranges 15 -22 MPa and 40˚C -70˚C, respectively.Par-ticle diameter and co-solvent percentage are in ranges of 0.27 -1.07 mm and 0% -12.4%, respectively, as well as the flow rate of supercritical CO 2 between 0.28 and 0.85 kg/h.The best results were found at 22 MPa, 40˚C, 0.27 mm particle size, about 12.4% of co-solvent and a flow rate of 0.29 kg/h [36].
In the present study water was used as solvent system for polyphenols extraction, followed by acetone.It was proved that it attacks bonds established between polyphenolic structures and other macromolecules like proteins and lipids.Furthermore, reports about carob pod tannins have shown that they had a low solubility in solvents like methanol and ethanol.Actually, carob pod tannins are strongly polymerized with molecular weight attending 32,000 g/mol and have non-porous granular form [5] [7].
Methods of analysis can also explain the variation of contents.In this work, the condensed tannins content have been determined the proanthocyanidin assay (butanol-HCl method).There is also the vanillin assay (vanillin-H 2 SO 4 method) that provides the quantity of flavanols including catechins and proanthocyanidins [38].The proanthocyanidin assay provides the degree of polymerization of proanthocyanidins, with a very specific reaction based on conversion to anthocyanidins by means of auto-oxidation following acid-catalyzed cleavage of the interflavonoid bonds [22].Therefore, the ratio obtained by the vanillin assay divided by that obtained by the proanthocyanidin assay provides a rough estimate of the degree of polymerization of flavanols.Thereby, in the study carried out by Kumazawa et al. [3], the values determined by the vanillin assay and the proanthocyanidin assay were 4.37 ± 0.1 and 1.36 ± 0.01 g/100 g of carob pod crude polyphenols, respectively, thus indicating that the degree of polymerization of flavanols was 31.1% (1.36/4.37).Tannins content can also be determined with the gravimetric method using cupper acetate as an agent of association with phenol compounds [39].Moreover, contrary to our results, Gaouar-Borsali [20] obtained more flavonoids than tannins.

Conclusions
In the present study, pods from Moroccan carob were found to have obvious variability of polyphenols content.Indeed, the twenty studied trees showed great variability in total polyphenols, flavonoids and condensed tannins contents.
Generally, it was found that pods powders from wild trees are richer in polyphenols than those from domesticated trees.Nevertheless, the second one has sometimes a tendency to reach the content of the first one.In addition, polyphenols content in the fresh pulp depends on its size and weight.
Also, pods from trees in the same region showed variable contents in polyphenols, flavonoids and condensed tannins.
This report is the one among others researches which proves that carob pulp (kibbled pod), especially from Morocco, is a rich source of polyphenols, shown to be natural bioactive compounds with several benefits.Also, it offers a tool for socio-economic development of local populations as an implement to some government institutions interested by protecting, valorizing genetic resources and developing the regional infor-mation system for the management of biodiversity.Moreover, the high pulp content of polyphenols found may encourage people to consume carob deseeded fruits.
F and TPC D(19.36 and 22.75 mg GAE/g), followed by Guelmouss (18.61 and 21.21) and Ain Asserdoune(17.25 and 19.41).Kassita powder had the lowest TPC F and TPC D (7.00 and 7.80 mg GAE/g).The richest pulp in polyphenols belonged to the tree of Ain Beida 2 (118.04 mg GAE/one fresh pulp) followed by Zinat (109.36) and Ghorghez (107.83).The poorest pulp in polyphenols was from Guelmouss (53.22 mg GAE/one fresh pulp) Table TFC F : Total Flavonoids Content in Fresh powder, TFC D : Total Flavonoids Content in Dry powder, TFC P : Total Flavonoids Content in one fresh Pulp.The data represent Mean ± SE of replicates (n = 3).Values in the same rows carrying different letters are significantly different between treatments and control by Duncan's multiple range tests at p ≤ 0.05.

Furthermore, we notice
variations in polyphenols contents among wild and/or domesticated trees from the same locality.This is the case for Bni Hassane (trees 2 and 3), Mokrissat (wild trees 5, 6 and 7, domesticated trees 16 and 17), Nador (trees 9 and 10) and Moulay Idriss Zerhoun (trees 18, 19 and 20).Within a same location, altitude can change: for example, trees 2 and 3 from Bni Hassane grow at 330 and 876 m.This implies different environmental conditions (climate, soil…), which could influence the content of polyphenols[23].On the other side, trees 18, 19 and 20 have approximate altitudes but different polyphenols contents, which may be explained by the age of tree, its physiological condition and other genetic reasons[23].
Different solvent concentrations(40, 60, 80 and 100%), ratios (15:10, 25:10, 50:10 and 75 mg: 10 mL), extraction times (60, 90 and 120 min) and temperatures (25˚C, 50˚C, 70˚C and 90˚C) were tested.Acetone 60% extract had the highest phenolic and flavonoids contents, followed by methanol 60%, ethanol 60% and water extracts.Phenolic and flavonoids contents increased with the increasing proportion of acetone up to 70%.The lowest phenolic contents were obtained with 100% acetone.The best phenolic and flavonoids contents were obtained with a ratio of 25 mg:10 mL.Extraction time had significant effect on total phenolic and flavonoids content that increase from 60 to 90 min.After 90 min, values decreased significantly.The best extraction of phenolic and flavonoids compounds was obtained at 90˚C.

Table 1 .
Geographic coordinates (latitude and longitude) and altitudes of 20 carob trees in four regions from 15 Moroccan localities.

Table 2 .
Total polyphenols content (TPC) in mg gallic acid equivalents by g of fresh powder, dry powder and by one carob fresh pulp for 12 wild and 8 domesticated carob trees.: Total Polyphenols Content in one fresh Pulp.The data represent Mean ± SE of replicates (n = 3).Values in the same rows carrying different letters are significantly different between treatments and control by Duncan's multiple range tests at p ≤ 0.05.

Table 3 .
19to 0.87 mg QE/g, TFC D from 0.21 to 1.02 mg QE/g and TFC P from 1.41 to 4.83 mg QE/g.Powder from Ghorghez tree showed the highest TFC F and TFC D (0.87 and 1.02 mg QE/g, respectively), followed by powders from Guelmouss tree (0.67 and 0.77), and Zoumi 2 tree (0.49 and 0.55).The lowest TFC F and TFC D were observed in the case of powder from Ain Kalaa tree (0.19 and 0.21 mg QE/g).The richest pulp in flavonoids was from Ghorghez tree with 4.83 mg QE/one fresh pulp, followed by Belyounech (3.96) and Zoumi 2 (3.60) trees.The poorest pulp in flavonoids was from Isslan tree with 1.41 mg QE/one fresh pulp Table3.Total flavonoids content (TFC) in mg quercetin equivalents by g of fresh powder, dry powder and one fresh pulp for 12 wild and 8 domesticated carob trees in Morocco.

Table 4 .
Condensed tannins content (CTC) in mg proanthocyanidin equivalents by g of fresh powder, dry powder and one fresh pulp for 12 wild and 8 domesticated carob trees in Morocco.: Condensed Tannins Content in one fresh Pulp.The data represent Mean ± SE of replicates (n = 3).Values in the same rows carrying different letters are significantly different between treatments and control by Duncan's multiple range tests at p ≤ 0.05.
F : Condensed Tannins Content in Fresh powder, CTC D : Condensed Tannins Content in Dry powder, CTC P