Pectic polysaccharides were solubilized from Algerian argan tree leaves by sequential extraction with water at 100°C (water-soluble pectin; AL-WSP) and EDTA solution at 80°C (chelating-soluble pectin; AL-CSP). Both AL-WSP and AL-CSP were 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 13C nuclear magnetic 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 AL-CSP consisted of rhamnogalacturonan type I and a block of homogalacturonan. Antioxidant activities of AL-WSP and AL-CSP were 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.
Argan tree (
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 [
Argan tree leaves used in this study were collected in June 2007 from Tindouf Province in southwest Algeria. After collection, leaves were dried in a ventilated oven (40°C), ground (particle size < 200
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 [
Each dried residue was subjected to extraction with ethanol 80% at 90°C for 20 min. The residue was dissolved in distilled water, and the supernatant was recovered by centrifugation. The insoluble material was washed twice with distilled water at 100°C for 2 h to obtain water-soluble pectin (AL-WSP). The remaining residue was treated with 1% ethylenediaminetetraacetic acid (EDTA) solution at 80°C for 6 h to obtain chelating soluble pectin (AL-CSP). All extracts were filtered through a porous glass frit (Porosity 3) and transferred to presoaked dialysis tubing (Spectra/Por; molecular weight cutoff 6,000–8,000 Da). Then it was precipitated with ethanol solution (3 volumes), centrifuged, and finally lyophilized (Figure
Schematic representation of water-soluble (AL-WSP) and chelating soluble (AL-CSP) pectins.
The composition of neutral monosaccharides was determined from their alditol acetates on a Hewlett-Packard 5890A gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA), equipped with a polar-fused silica capillary column (30 m × 0.53 mm) and a flame ionization detector (FID) coupled to a Hewlett-Packard 3395 integrator. Column temperature was held at 195°C for 4 min and ramped at 2.5°C·min−1 to 225°C and held for 3 min with a constant flow of 4 mL·min−1 nitrogen carrier gas. The injector and detector temperature were 260°C and 280°C, respectively. Quantification of monosaccharides was conducted using myoinositol as an internal standard against a mixed standard solution of monosaccharides (rhamnose, fucose, xylose, arabinose, mannose, galactose, and glucose) [
The content of uronic acid was estimated using the assay of Blumenkrantz and Hansen [
Fractionation of pectin was performed by ion exchange chromatography. A 400 mg pectin sample was deesterified using 0.1 M sodium hydroxide solution overnight at 4°C under nitrogen. The solution was neutralized with 1 M (pH 5.0) hydrochloric acid to obtain the acid form of pectin. Extract was dissolved in approximately 100 mL of 0.05 M phosphate buffer (pH 6.3) and applied to a DEAE-Trisacryl M column (2 × 20 cm). 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
Ion exchange chromatography fractionation of water-soluble (AL-WSP) and chelating-soluble (AL-WSP) pectins.
Polysaccharides were methylated using sodium hydroxide and methyl iodide in dry dimethyl sulfoxide, according to the method described by Hakomori [
Permethylated polysaccharides were characterized by a gas chromatograph (HP-Agilent 6850) using a 530
Samples of 15 mg were dissolved in 0.5 mL deuterated water (NMR 13C: 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 13C. Chemical shifts
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 [
Monosaccharide extraction yield and composition of different polysaccharide fractions, determined by GC as alditol acetates, are presented in Table
Monosaccharide yield and composition of water-soluble AL-WSP and chelating-soluble AL-CSP pectins obtained by gas chromatography (GC).
Fraction | Yielda | UAb | Sugar compositionc | ||||||
---|---|---|---|---|---|---|---|---|---|
Rha | Fuc | Ara | Xyl | Man | Gal | Glc | |||
AL-WSP | 6.5 | 38.5 | 9 | — | 28 | 1 | 0.5 | 11 | 50.5 |
AL-CSP | 7 | 21.5 | 6 | 4.5 | 74.5 | 1.5 | — | 6.5 | 7 |
aExpressed as % of 15 g starting leaf powder (dry weight); bexpressed as relative weight percentages; cpercentage 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: chelating-soluble pectin; WSP: water-soluble pectin; AL: argan tree leaves.
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 [
The percentages of AL-WSP and AL-CSP fractions obtained with different eluting solutions are summarized in Table
Mass yield of water-soluble AL-WSP and chelating-soluble AL-CSP pectins obtained by ion exchange chromatography.
Elution solution | Fractiona | |
---|---|---|
AL-WSP | AL-CSP | |
(1) Buffer | 17.5 | 7.5 |
(2) Buffer + 0.125 M NaCl | 5 | 6.5 |
(3) Buffer + 0.25 M NaCl | 23 | 16.5 |
(4) Buffer + 0.5 M NaCl | 10.5 | 13 |
(5) Buffer + 1 M NaCl | 3.5 | 6 |
Total |
|
|
Buffer: phosphate buffer (0.05 M, pH 6.3); aexpressed as % of 400 mg AL-WSP and AL-CSP; CSP: chelating-soluble pectin; WSP: water-soluble pectin; AL: argan tree leaves.
Monosaccharide compositions of AL-WSP(3) and AL-CSP(3) are presented in Table
Monosaccharide composition of water-soluble AL-WSP(3) and chelating-soluble AL-CSP(3) pectins.
Fraction | Yielda | UAb | Sugar compositionc | ||||||
---|---|---|---|---|---|---|---|---|---|
Rha | Fuc | Ara | Xyl | Man | Gal | Glc | |||
AL-WSP(3) | 23 | 14 | 15.5 | 0.5 | 65 | 2.5 | — | 13.5 | 3 |
AL-CSP(3) | 16.5 | 25.5 | 14.5 | 0.5 | 68 | 2.5 | — | 11.5 | 3 |
aExpressed as % of 400 mg AL-WSP(3) and AL-CSP(3) eluted with 0.25 M NaCl; bexpressed as relative weight percentage; cpercentage 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: chelating-soluble pectin; WSP: water-soluble pectin; AL: argan tree leaves.
The results of methylation analysis are summarized in Table
Partially methylated alditol acetates ofwater-soluble AL-WSP(3) and chelating-soluble AL-CSP(3) pectins obtained by gas chromatography-mass spectrometer (GC-MS).
Partially methylated |
AL-WSP(3)a | AL-CSP(3)a |
---|---|---|
3,4-Di-methyl-rhamnose | 11.6 | 4.6 |
3-Methyl-rhamnose | 7.1 | 4.5 |
Total |
|
|
|
||
2,3,5-Tri-methyl-arabinose | 23.7 | 28 |
2,5-Di-methyl-arabinose | 3.6 | 3.3 |
2,3-Di-methyl-arabinose | 26.7 | 27.4 |
2-Methyl-arabinose | 12.7 | 24 |
3-Methyl-arabinose | 4.8 | 3.7 |
Total |
|
|
|
||
2,3,4,6-Tetra-methyl-galactose | 4.8 | 2.3 |
2,3,6-Tri-methyl-galactose | 4.8 | 2.1 |
Total |
|
|
aPercentage of peak area of
AL-CSP(3) and AL-WSP(3) have the same characteristics, but the proportion of (1 → 2) linked and O-4 branched rhamnose (3-methyl-rhamnose) is higher in AL-CSP than in AL-WSP. Methylation analysis revealed that the proportion of uronic acid (25.5%) in AL-CSP was higher than that of rhamnose (14.5%). These results suggest that AL-CSP(3) has a skeleton that contains both homogalacturonan [→ 4)-galactose-A-(1 →] blocks and rhamnogalacturonans [→ 4)-galactose-A-(1 → 2)-rhamnose-(1 →] blocks [
Figure
13C spectra of water-soluble (AL-WSP3) and chelating-soluble (AL-WSP3) pectins.
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 [
DPPH scavenging activities of AL-WSP(3) and AL-CSP(3) are shown in Figure
Antioxidant activity of water-soluble (AL-WSP3) and chelating-soluble (AL-WSP3) pectins.
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.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank Professor Vincent Gloaguen, Assistant Director of Laboratory of Chemistry of Natural Substances, EA1069, University of Limoges, France, for his help in ESR analysis and Professor Redouane Boursali, Director of the Research Center on Plant Macromolecules (UPR5301, France), for his help in methylation and NMR analysis. This research was funded by Dr. Moulay Tahar, University of Saida, Algeria.