Isolation, Structural Characterization, and Valorization of Pectic Substances from Algerian Argan Tree Leaves (Argania spinosa (L.) Skeels)

Pectic polysaccharides were solubilized from Algerian argan tree leaves by sequential extraction with water at 100C (water-soluble pectin; AL-WSP) and EDTA solution at 80C (chelating-soluble pectin; AL-CSP). Both AL-WSP andAL-CSPwere rich in arabinose (28% and 74.5%, resp.) and had a high content of uronic acid (38.5% and 21.5%, resp.). Pectic substances were deesterified and fractionated by anion exchange chromatography, giving five fractions for each extract. Most of the fractions were characterized by methylation analysis and then analyzed by Cnuclearmagnetic resonance spectroscopy.The results showed that AL-WSP consisted of rhamnogalacturonan type I, with arabinan and galactan branching at the O-4 position of the main rhamnose chain, while ALCSP consisted of rhamnogalacturonan type I and a block of homogalacturonan. Antioxidant activities of AL-WSP andAL-CSPwere evaluated by electronic spin resonance. The results showed that the antioxidant potential of AL-WSP (8.1%) and AL-CSP (−1.2%) was significantly lower than that of vitamin E.


Introduction
Argan tree (Argania spinosa (L.) Skeels), also known as "iron tree, " is a woody species belonging to the Sapotaceae family and is endemic to Algeria and Morocco [1,2].Its geographic distribution covers a relatively large area of southwestern Algeria in northern Tindouf, where it is the second most common tree after Acacia raddiana [3].The argan tree is important both for the local economy and for ecological sustainability.The woodlands protect against soil erosion and desertification owing to their deep-growing roots; they shade different types of crops and help maintain soil fertility in arid zones [4].Plant cell walls are known to be potential sources of pharmacologically active polysaccharides [5,6].Recently, pectins, a group of anionic polysaccharides that are used in traditional pharmaceutical applications, have attracted a lot of attention and have been subjected to extensive structural study [7].Pectins are polydisperse macromolecules having high heterogeneity in terms of molecular mass and chemical structure.Their composition is affected by their origin, localization within the plant, and the extraction method used to obtain them.Pectins have a complex structure, which in its majority contains blocks of homogalacturonan (known as "smooth regions") and rhamnogalacturonan (known as "smooth regions").
Studies on the analysis of polysaccharides isolated from argan tree leaves are limited.Ray et al. conducted a study on hemicelluloses isolated from leaves of Moroccan argan tree [8].The objectives of this study were to (1) study pectic substances extracted from leaves of the Algerian argan tree and (2) evaluate the antioxidant activity of these substances against the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical by using electronic spin resonance (ESR).To the best of our knowledge, this is the first report on pectic substances isolated from the leaves of the Algerian argan tree.Algeria.After collection, leaves were dried in a ventilated oven (40 ∘ C), ground (particle size < 200 m), and stored in desiccators at room temperature.

Cell Wall Preparation.
Fifty grams of milled powder was subjected to two successive extractions with 50 : 50 ethanol-toluene solution at room temperature for 14 h.The supernatant was discarded to remove debris, cytoplasmic organelles, and starch granules.The residue was filtered through a blotting cloth and mixed with ethanol 80% by continuous stirring for 2 h to remove any traces of toluene.Then, the residue was washed three times with distilled water and acetone solution, dried in a ventilated oven at 60 ∘ C, and weighed [9].Fractions were eluted at a constant flow of 30 mL⋅h −1 with 300 mL of 0.05 M phosphate buffer and then eluted three more times with 300 mL of 0.05 M phosphate buffer containing 0.25 M, 0.5 M, and 1 M sodium chloride, respectively.Five fractions were collected for each extract, dialyzed against distilled water, and lyophilized.DEAE-Trisacryl M column was regenerated with 0.5 M sodium hydroxide solution (Figure 2).

Methylation Analysis.
Polysaccharides were methylated using sodium hydroxide and methyl iodide in dry dimethyl sulfoxide, according to the method described by Hakomori [14].Permethylated polysaccharides were hydrolyzed with 2 N trifluoroacetic acid at 100 ∘ C for 3 h.Subsequently, they were converted into their corresponding alditol acetates by successive treatments with sodium borohydride and were acetylated with acetic anhydride in the presence of pyridine that acts as a catalyst.
2.4.5.Mass Spectrometry.Permethylated polysaccharides were characterized by a gas chromatograph (HP-Agilent 6850) using a 530 m capillary column (SP 2380).The injector temperature is 260 ∘ C with a constant flow of 4 mL⋅min −1 nitrogen carrier gas.Column temperature was held at 165 ∘ C for 4 min and ramped at 2.5 ∘ C⋅min to 225 ∘ C and held for 3 min.After separation in capillary column, methylated derivatives were analyzed by a mass spectrometer (Agilent 5975C, Agilent technologies Inc., Santa Clara, CA, USA).

Nuclear Magnetic Resonance (NMR) Spectroscopy.
Samples of 15 mg were dissolved in 0.5 mL deuterated water (NMR 13 C: 15 mg in 0.5 mL solvent) and NMR spectra were recorded on a Bruker Avance 400 spectrometer equipped with a quadruple nucleus probe at room temperature or at 60 ∘ C and a frequency of 100.62 MHz for 13 C.Chemical shifts  were expressed in ppm by frequency.The detected frequency for 13 C was referenced against tetramethylsilane.
where ref and compound are the values of the double integrals of ESR spectra of the reference (DPPH + water) and the tested solution (DPPH + pectin extracts in water), respectively; bg represents the background signal (water).Obtained data were the average of three independent measurements.Inhibition of 50% DPPH scavenging was calculated from inhibition versus concentration curves.The presence of arabinose, galactose, and rhamnose suggested the lack of homogalacturonans in most of the polysaccharide fractions.Thus, the high content of arabinose relative to galactose suggested the presence of side chains of arabinan and/or arabinogalactan.Homogalacturonans ratio ranged from 0.82 to 1 for AL-CSP fractions, which, according to Voragen and Schols [16,17], reveals a predominance of rhamnogalacturonan type I.These results do not exclude the possibility of the presence of rhamnogalacturonan type II as well.2. These results showed that AL-WSP and AL-CSP fractions appear as mixtures and subfractions contain acidic polysaccharides.However, each of them was characterized by the dominance of a single subfraction.Most of the subfractions were eluted with 0.25 M NaCl with total percentage of AL-WSP(3) and AL-CSP(3) to be 23% and 16.5%, respectively.A significant amount (17.5%) of AL-WSP(1) fraction, apparently neutral, was recovered with phosphate buffer that did not contain NaCl.Table 3. AL-WSP(3) and AL-CSP(3) were acidic and contained approximately 14% and 25.5% uronic acid, respectively.They also had significant amounts of arabinose (65% in AL-WSP(3) and 68% in AL-CSP( 3)).The presence of rhamnose and galactose was, on average, 15% in AL-WSP(3) and 12% in AL-CSP(3), suggesting that the chains of arabinan and galactan on the rhamnogalacturonan skeleton are connected.

Study by NMR Spectroscopy.
Figure 3 shows NMR spectra ( 13 C) of AL-WSP(3) and AL-CSP(3).Chemical shift assignment was compared with already published spectral data on the structural characterization of pectins [25,29,30].The detected signals were characteristic of -1,5 arabinan (Ara), -1,4 galactan (Gal), -1,4 galacturonan (AGal), and -1,2 rhamnose (Rha).Between 108.17 ppm and 107.76 ppm in the 13 C spectrum of AL-WSP(3), several characteristic signals of (1 → 5) linked arabinose residues were detected.C1 of galacturonic acid and C6 of galactose were identified by 99.6 ppm and 61.7 ppm signals, respectively.The signal at 176.1 ppm was characteristic of carboxyl functional groups of (C6) galacturonic acid units, while the signal at 17.2 ppm was characteristic of (C6) methyl groups of rhamnose residues.These results were in agreement with those obtained by methylation analysis and confirmed that AL-WSP(3) consisted of a main rhamnogalacturonan skeleton.The O-4 position side chains of (1 → 4)-linked galactan, or (1 → 5)linked arabinan, are branched at O-2 and/or O-3 positions.Spectrum speed of AL-CSP(3) and AL-WSP(3) was similar.The signals at 99.12 ppm and 98.62 ppm were characteristic of the anomeric carbons of (2 → 1)-linked rhamnose residues and galacturonic acid bound by a (1 → 4) link, respectively.The signals at 17.55 ppm and 175.01 ppm were characteristic of methyl rhamnose and the carboxylic  acid functions of galacturonic residues, respectively.However, multiple signals at 99.60 ppm, 78.51 ppm, 71.94 ppm, 68.73 ppm, and 67.53 ppm were characteristic of (1 → 4)linked galacturonic acid units that form blocks of homogalacturonan.These results confirmed that AL-CSP(3) consisted of a main skeleton that has homogalacturonan and rhamnogalacturonan blocks substituted by a galactan and/or arabinan side chain.AL-WSP(3) and AL-CSP(3) can be considered as models of pectin structure synthesized in the cell wall of argan tree leaves.Both fractions have rhamnogalacturonan type I structures, while AL-CSP(3) also has homogalacturonan type structures.The presence of arabinose and galactose in AL-WSP(3) and AL-CSP(3) that was detected by NMR and methylation analysis revealed two lateral arabinan and galactan branches that were separate and differed in the length of strings (arabinose stings were longer than those of galactose).The structure of rhamnogalacturonan type I is the same in AL-WSP(3) and AL-CSP(3) with minor variations.These results are in agreement with previous studies in sugar beet pulp [31] and prickly pear peel [27] but differ from those in potato peel [32], potato pulp [33], and soybeans [34] in which rhamnogalacturonan type I structure is variable.and AL-CSP(3) showed an antioxidant potential of 8.1% and −1.2%, respectively.These percentages were lower than those of vitamin E (100%).These results were in agreement with previous reports in which 4-O-methyl-glucuronoxylan from Castanea sativa hardwood had insignificant antioxidant activity [35].It has been shown that the extraction method modifies the structure of polysaccharides by removing acetyl groups or creating new functions and therefore influences their biological properties [36,37].

Conclusion
From the foregoing results it can be concluded that AL-WSP(3) and AL-CSP(3) can be considered as models of the pectin structure synthesized in the cell wall of Algerian argan tree leaves.These fractions have similar rhamnogalacturonan type I structures with a block in addition to the homogalacturonan type AL-CSP(3) fraction, and with side chains of either arabinan or galactooligosaccharides attached to O-4 of the backbone rhamnose units.The pectic substances AL-WSP(3) and AL-CSP(3) showed an antioxidant potential lower than those of vitamin E, universally known as a reference of antioxidant compound.The results presented in this primary study need to be conducted by using green chemistry processes, given that the polysaccharide derivatives are considered as potential candidates for natural antioxidant additives in the food and cosmetic industries, because of their environmentally friendly and economic extraction process.

Table 1 :
Monosaccharide yield and composition of water-soluble AL-WSP and chelating-soluble AL-CSP pectins obtained by gas chromatography (GC).
[15]pressed as % of 15 g starting leaf powder (dry weight); b expressed as relative weight percentages; c percentage of peak area relative to total peak areas, determined by GC; UA: uronic acid; Rha: rhamnose; Ara: arabinose; Xyl: xylose; Glc: glucose; Gal: galactose; Man: mannose; Fuc: fucose; CSP: chelatingsoluble pectin; WSP: water-soluble pectin; AL: argan tree leaves.2.4.7.Antioxidant Activity.Antioxidant activity was determined by electron spin resonance (ESR) on a Bruker ESP 300E spectrometer.Different pectin extracts were evaluated for their DPPH radical scavenging activity[15].Tested compounds were dissolved in water at different concentrations and scattered for 10 min in an ultrasonic bath.Radical scavenging activity was evaluated after mixing 50 L of 5 × 10 −4 M DPPH in ethanol with 50 L of pectin extract solution.ESR spectra were recorded 5 min after mixing under the following conditions: 100 kHz modulation frequency, 9.78 GHz microwave frequency, 4 mW microwave power, 1.97 modulation amplitude, and 10.24 ms time constant.Inhibition was calculated as follows:

Table 4
a Percentage of peak area of O-methylalditol acetates relative to total area, determined by GC-MS.