G Model
ARTICLE IN PRESS
BIOMAC 5148 1–8
International Journal of Biological Macromolecules xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Depolymerization of polysaccharides from Opuntia ficus indica: Antioxidant and antiglycated activities
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Mohamed Aymen Chaouch a , Jawhar Hafsa b , Christophe Rihouey c , Didier Le Cerf c,∗ , Hatem Majdoub a,∗ a Laboratoire des Interfaces et des Matériaux Avancés (LIMA), Faculté des Sciences de Monastir, Université de Monastir, Bd. de l’environnement, 5019 Monastir, Tunisia b Laboratoire de Biochimie, Faculté de Médecine, Université de Sousse, 4002 Sousse, Tunisia c Normandie Université, Laboratoire Polymères Biopolymères Surfaces (PBS), UMR 6270 & FR 3038 CNRS, Université de Rouen, 76821 Mont Saint Aignan, France
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Article history: Received 25 March 2015 Received in revised form 1 June 2015 Accepted 2 June 2015 Available online xxx
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Keywords: Opuntia ficus indica Polysaccharide Free radical depolymerization Antioxidant activity Antiglycation activity
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1. Introduction
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The extraction, purification and degradation of polysaccharides from Opuntia ficus indica cladodes, as well as the evaluation of their antioxidant and antiglycated activities in vitro were investigated. The optimization of the extraction showed that extraction by ultrasound at 40 ◦ C presented the best carbohydrates yield. The degradation of the extracted polysaccharides was achieved by free radical depolymerization with H2 O2 in the presence of copper(II) acetate for various reaction times. Sugar contents were determined by colorimetric assays. The macromolecular characteristics of the different isolated and degraded carbohydrates were carried by size exclusion chromatography (SEC/MALS/VD/DRI). These experiments showed that all samples are polysaccharides, which are probably pectins and that molecular weight (Mw ) has decreased from 6,800,000 to 14,000 g/mol after 3 h of depolymerization without changing the structure. Preliminary antioxidant and antiglycated tests indicated that degraded polysaccharides for 2 and 3 h showed even better antioxidant and antiglycated activities. © 2015 Published by Elsevier B.V.
The antioxidants exhibit significant efficiency to protect the human body against damage caused by reactive oxygen species which attack biological molecules, such as lipids, proteins, enzymes, DNA and RNA, resulting in cells or tissues lesions associated with aging, atherosclerosis, and carcinogenesis. Nowadays great interest is awarded to natural antioxidants due to problems related to safety and toxicity of synthetic antioxidants. Many natural antioxidants have already been isolated from different kind of plants such as oil seeds, cereals, vegetables, leaves, roots, herbs and spices [1]. Over the past few years, the contribution of advanced glycation endproducts for diabetes and aging has received considerable attention. The modification of proteins by glucose through the glycation process leads to the formation of advanced glycation endproducts. In fact, glycation is a spontaneous non-enzymatic amino-carbonyl reaction between reducing sugars and proteins [2].
∗ Corresponding author. Tel.: +216 98355740. E-mail address: [email protected] (H. Majdoub).
As part of the research of new sources of natural antioxidation and antiglycation agents, we were interested in a plant which is resistant to water stress and tolerant to poor soils, Opuntia ficus indica (OFI). This plant is native to Mexico and South America, but is also widely distributed in the Mediterranean, Africa and Australia [3]. The physicochemical analysis of OFI young cladodes reveals similarities in the composition compared to wide used vegetables such as spinach, tomato and lettuce. Polysaccharide extracts from plants, like those from the Cactaceae family, represent an important source of additives for several industries, especially in food and drug industry [4]. The major components in OFI cladodes are pectins which are heteropolysaccharides characterized by a high content of galacturonic acid (monomers linked by ␣-(1-4) links and partially acetylated or esterified with methyl groups) [5]. Pectins have specific physico-chemical properties due to their polyelectrolyte character. Forni et al. proposed a method of pectin extraction from prickly pear peels in two stages: an extraction of alcohol insoluble substances (AIS) followed by pectins extraction from AIS under hot acid conditions [6]. Pectin’s extraction procedure from OFI cladodes in cold water with extensive ultrafiltration was performed by Majdoub et al. [7].
http://dx.doi.org/10.1016/j.ijbiomac.2015.06.003 0141-8130/© 2015 Published by Elsevier B.V.
Please cite this article in press as: M.A. Chaouch, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.06.003
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Several researches have indicated that high molecular weight and high viscosity of polysaccharides restricted their pharmaceutical application. Generally, several structural parameters influence the biological activities of carbohydrates, such as the substitution degree, the functional groups and the molecular weight. Recently, it has been reported that the polysaccharide’s activity is strongly influenced by the molecular weight distribution [8]. Chemical modification of carbohydrates provides the possibility to obtain new pharmaceutical agents [9]. Other research has shown that pectin interacts directly with oxidants and free radicals. In addition to that, it has been suggested that a relatively low molecular weight and a high galacturonic acid content in polysaccharides appeared to increase the antioxidant activity [10]. The degradation of polysaccharides was achieved by enzymatic (pectinase [11], pectin methyl esterase, polygalacturonase [12]), chemical (acid hydrolysis [13], ozonolysis [14], free radical depolymerization [15]), physical (sonochemistry [16], microwave, irradiation [17], etc.) and thermal processes [17]. These methods are generally dependent on the structure, the conformation, the applicability of polysaccharides and the reactive agents [18]. Freeradical depolymerization is an interesting chemical process for polysaccharide’s degradation because it provides constant composition, reproducibility and the retention of primary structure after depolymerization. In addition, the reaction conditions are mild and the reagents are cheap and suitable for use on a large scale [19]. In this work we are interested in the optimization of the extraction and the degradation of pectic polysaccharides from OFI cladodes and the study of the influence of their molecular weight on the antioxidant and antiglycated activities. 2. Materials and methods 2.1. Materials Prickly pear cladodes were harvested in the area of Monastir (Tunisian Sahel) at the end of spring (flowering period of the plant). The cladodes specimens were washed and cut into small pieces. 2.2. Optimization of polysaccharides extraction At industrial level, pectin is extracted in hot water (60–100 ◦ C) acidified with a mineral acid (sulfuric, nitric, hydrochloric or citric acid) during 0.5–6 h at a pH ranging from 1.5 to 3. These methods are time consuming and lead to pectin degradation. So, in order to produce pectin with high yield and quality, it is of great importance to introduce novel processes [20]. We chose to vary the physical treatment: for this, 1000 g of fresh cladodes were crushed with distilled water and split into 4 fractions: The first fraction was subjected to 10 min of ultrasound (100 W) [21] (PCU), the second was treated by microwave for 150 s (PCM) [21], the third was frozen overnight and then thawed at room temperature (PCF) [22], whereas the fourth did not undergo any treatment (PCW). The different fractions were kept under mechanical stirring in water for 2 h at a rate of 300 rpm at room temperature (solid–liquid ratio 1:30 (w/v)) and then filtered through a G0 sintered glass funnel. The filtrates were centrifuged for 20 min at 3000 × g and then freeze dried (Fig. 1). In order to purify the extracted products, two more steps were carried out: a precipitation with 80% ethanol followed by filtration on a G2 funnel [23]. The precipitate was then dialyzed against deionized water using dialysis tubing with molecular weight cut off 30 kDa in order to eliminate salts and compounds of low molecular weight, until conductivity equaled that of the deionized water. Finally, the dialysate was freeze dried.
2.3. Free radical depolymerization In order to degrade high molecular weight polysaccharides extracted from prickly pear cladodes [24,25], we proceeded to the free radical depolymerization by Fenton reaction according to the method described by Wu et al. [19] We began with the preparation of 25 mL of a polysaccharidic solution (40 mg/mL). Then, the solution was introduced into an electrochemical cell and the temperature was set at 60 ◦ C using a thermostated bath. Subsequently, 0.25 g of copper acetate was added and the initial pH was adjusted to 6.2 with NaOH solution (8 M). The depolymerization reaction is started with the addition of a H2 O2 solution (12%) at a rate of 5 mL/h which was controlled by a peristaltic pump. The pH of the solution was fixed at 6.2 using a pH-stat enabling the addition of the NaOH solution (8 M) when the pH drops below 6.2. The degradation was carried out for periods of 1, 2 and 3 h, leading respectively to the products PCU1 , PCU2 , and PCU3 . For each sample, 20 mL was taken in a beaker to which was added 7.5 mL of glacial acetic acid and then 12.5 mL of resin (Chelex 100) to remove copper. The resin Chelated cupric and cuprous ions. The resin was removed and replaced as many times as needed until it becomes colorless. The treatment was complete when the resin became colorless. Finally, the solution was dialyzed for 24 h against deionized water using dialysis tubing with molecular weight cut off 7000 Da and then freeze-dried.
2.4. Size exclusion chromatography Analysis of various samples was performed using size exclusion chromatography (SEC) equipped with a triple detection: multiangle light scattering (MALS) (Down HELEOS II, Wyatt Technology, CA, USA), viscometer detector (VD) (Viscostar II, Wyatt Technology, CA, USA) and differential refractive index (DRI) (RID 10 A Shimadzu, Japan). The SEC system consists of a pump (LC10 Ai Shimadzu, Japan) at a flow rate 0.5 mL/min of LiNO3 0.1 mol/L and two columns 8.0 mm × 300 mm (internal diameter × length) OHPAK SB 804 and 806 HQ (Shodex) in series. The paking material is polyhydroxymethacrylate. The samples were dissolved in the eluent (LiNO3 0.1 mol/L) at 2 g/L. The dissolution was carried out by stirring at 380 rpm for 24 h at room temperature. 3 mL solutions were filtered through membrane 0.45 microns (regenerated cellulose) before injection. The analyzes from MALS were performed by a data processing Zimm “order 1” using angles from 34.8◦ to 142.8◦ [26]. The corresponding value of dn/dc is 0.15 mL/g, as published in a previous paper [7]. The Astra 6.0.1.7 software package is set to collect and extrapolate data with the aim to obtain for each elution volume the molecular weight and the gyration radius (if Rg > 10 nm) [27]. With an integration of the peak, we calculated the number (Mn ) and weight (Mw ) average molecular weight and the z-average gyration radius. The differential viscosimeter detector permits to obtain for each elution fraction the intrinsic viscosity. An integration of the peak gives the average intrinsic viscosity, which allowed us to obtain the average hydrodynamic volume (Vh ) using the Einstein–Simha Q3 equation: Vh =
[]M NA
where NA is Avogadro’s number, M is the molecular weight, [] is the intrinsic viscosity (mL/g), and is a conformational parameter that takes the value of 2.5 in the case of a spherical conformation.
Please cite this article in press as: M.A. Chaouch, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.06.003
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Fig. 1. Polysaccharide’s extraction from OFI cladodes by varying the physical treatment.
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2.5. Colorimetric assays The content of polysaccharides was determined for all the extracted polysaccharides by the phenol–H2 SO4 method using galactose as standard [28]. The content of galacturonic acid in the extracts containing pectin is an important parameter in the quantitative and structural analysis of this polysaccharide. The micro-titer plate method was used for the determination of galacturonic acid amount as described earlier by Casaretti et al. [29]. A serial dilution of standard (glucuronic acid) and samples (50 L, 1 mg/mL) was placed in a 96 well plate. Subsequently, 200 L of 25 mM sodium tetraborate in sulphuric acid solution was added. Then the plate was heated at 100 ◦ C for 10 min in an oven. After cooling at room temperature for 15 min, 50 L of 0.125% carbazole in absolute ethanol was carefully added. After heating at 100 ◦ C for 10 min in an oven and cooling at room temperature for 10 min, the plate was read at 550 nm.
2.6. ATR-FTIR The chemical composition of extracted and degraded samples was obtained using PerkinElmer Spectrum Two ATR-FTIR, over the wave number range between 4000 and 400 cm−1 .
2.7. Antioxidant activity 2.7.1. DPPH radical scavenging assay The free radical scavenging effect of extracted and degraded samples was estimated according to the method of Espin et al. [30]. Briefly, 1 mL of each sample (0.5, 1, 1.5 and 2 mg/mL) was mixed with 4 mL of methanolic solution of DPPH (100 M). The reaction mixture was vortexed and incubated for 30 min in room temperature. The absorbance of the solution was measured at 517 nm. Ascorbic acid (AA) was used as standard. The
inhibitory percentage of DPPH was calculated using to the following equation:
DPPH scavenging effect (%) = 1 −
Abssample
Abscontrol
× 100
2.7.2. Nitric oxide radical scavenging assay The procedure is based on the principle that sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of NO• compete with oxygen, leading to reduced production of nitrite ions. 2 mL of different solutions (0.5, 1, 1.5 and 2 mg/mL) were mixed with sodium nitroprusside (4 mL of 10 mM, in PBS pH 7.4), and incubated at room temperature for 150 min. After the incubation period, 0.5 mL of Griess reagent was added [31]. The absorbance of the chromophore formed was measured at 546 nm. Ascorbic acid (AA) was used as standard. The NO• scavenging activity was calculated using the following equation:
NO scavenging activity (%) = 1 −
Abssample Abscontrol
212 213
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215 216 217 218 219 220 221 222 223 224 225 226 227
× 100
2.7.3. Linoleic acid peroxidation with TBARS assay In this assay antioxidant capacity is determined by measuring the thiobarbituric acid-reacting substances (TBARS) arising from linoleic acid peroxidation [32]. The reaction mixture contained 500 L linoleic acid (20 mM), 500 L Tris HCl (100 mM, pH 7.5), 100 L FeSO4 .7H2 O (4 mM) and a varying concentration of each sample (0.5, 1, 1.5 and 2 mg/mL). Linoleic acid peroxidation was initiated by the addition of 100 L of ascorbic acid (2 mM), incubated for 30 min at 37 ◦ C and achieved by the addition of 2 mL trichloroacetic acid (10%). Therefore 1 mL of the mixture was added with 1 mL of thiobarbituric acid (1%), followed by heating for 10 min at 95 ◦ C in water bath. The mixtures were centrifuged at 3500 × g for 10 min and the absorbance of TBARS in the
Please cite this article in press as: M.A. Chaouch, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.06.003
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Table 1 Polysaccharide extraction yields for different methods at room temperature.
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Samples
PCU
PCM
PCF
PCW
Extraction process Yield relative to fresh matter (%) Yield relative to dry matter (%)
Ultrasound 3.2 45.7
Microwave 2.7 39.0
Freezing 2.9 41.7
Without treatment 3.0 43.6
supernatant was measured at 532 nm. Ascorbic acid (AA) was used as standard. The percentage of antioxidant activity is determined using the following equation: Linoleic acid peroxidation inhibition (%) =
A −A c s Ac − An
× 100
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Ac = absorbance of control (without extract); As = absorbance of extract; An = absorbance of blank (without extract and FeSO4 ·7H2 O).
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2.8. In vitro glycation of protein
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The inhibition of protein glycation was determined by the method of Yang et al. [33] with slight modifications. Bovine serum albumin (20 mg/mL, in 20 mM PBS, pH 7.4) containing 0.1% (w/w) sodium azide (in PBS pH 7.4) was preincubated with polysaccharide (0.5, 1, 1.5 and 2 mg/mL) or aminosalicylic acid (ASA) for 10 min at room temperature (20 ◦ C). 1 M galactose solution was added to the reaction mixture and bacteria were eliminated by membrane filtration with a pore size of 0.2 m (Denville® syringe filter, PES, 0.22 m, 25 mm diameter). The solutions were incubated in dark at 60 ◦ C for 72 h. The reaction mixture without d-galactose was used as a blank solution and aminosalicylic acid was used as standard [34,35]. Fluorescence intensity was measured using a fluorometer (VersaFluor spectrofluorometer, Bio Rad), with an emission wavelength of 450 nm and an excitation wavelength of 370 nm. The percentage of antiglycated activity was calculated using the following equation:
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Inhibition % =
1 − FBSA+galactose+sample
FBSA+galactose
× 100
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where F is fluorescence intensity. Measurements were performed in triplicate and results are expressed as percentage inhibition of formation of the glycated protein.
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3. Results and discussion
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3.1. Extraction and depolymerization
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3.1.1. Extraction yields Table 1 shows the average yield for the different extraction methods. From the extraction data, it appears that the extraction by ultrasound presented the higher carbohydrates amount (3.2%). The mechanism of the extraction by ultrasound involves two types of physical processes: diffusion through the cell walls and washing out (rinsing) the cell contents once the walls are broken [36]. The increase in ultrasound treatment time may enhance both activities resulting in a high extraction yield, which explains the
observed increase in our extraction yield compared to other methods. The use of high ultrasonic power (>100 W) causes degradation effects [37,38], which explains the choice of the value 100 W. Furthermore, we tried to study the effect of the temperature on the extraction yield. We chose to vary the temperature from 25 to 100 ◦ C. The extraction at 40 ◦ C had the best yield (3.5% relative to fresh matter). While yields were 3.2%, 3.1% and 3.0% at 25 ◦ C, 70 ◦ C and 100 ◦ C, respectively. PCU sample was chosen for the depolymerization study. 3.1.2. Depolymerization After optimization of the extraction, we proceeded to the free radical depolymerization by Fenton reaction at three times (1, 2 and 3 h) leading respectively to the products PCU1 , PCU2 , and PCU3 . The yields of different depolymerization reactions were 14.0%, 9.7% and 9.2% respectively for PCU1 , PCU2 and PCU3 . 3.2. Identification
PCU PCU1 PCU2 PCU3 *
Mn (g/mol) 4,000,000 50,000 23,000 11,000
Table 2 shows the neutral sugars and the uronic acids rates for the extracted and degraded samples. From these data, we notice that all extracted and degraded samples are polysaccharides which are probably pectin since they contain high amounts of galacturonic acid (31–44%). These results are consistent with those found by gas chromatography according to Majdoub, Forni and Cárdenas [6,7,39]. On the other hand, the Gal acid content increases with free radical depolymerization from 31.2% for PCU to 43.8% for PCU3 unlike neutral sugars content which remarkably decreases from 54.2% to 30.3%. These data allow us to conclude that the cutoff was carried out at the junctions of neutral sugars. These results are consistent with those found by Garna et al. and Emega et al. who studied the variation of the galacturonic acid rate during acid and enzymatic hydrolysis of pectin [40,41]. The FT-IR spectra of native PCU, PCU1 , PCU2 and PCU3 are shown in Fig. 2. The four samples displayed a broad stretching intense characteristic peak at around 3300 and 3315 cm−1 for the stretching vibration of hydroxyl group. Each particular polysaccharide has a specific band in the 1200–1000 cm−1 region. This region is dominated by ring vibrations overlapped with stretching vibrations of (C OH) side groups and the (C O C) glycosidic bond vibration [42]. The stretching signals at 1031 cm−1 suggested the presence of C O bonds. In addition, signals at 1635 cm−1 were due to the asymmetric stretch vibration of C O of galacturonic acid. The signals at 2923 cm−1 and 1419 cm−1 are attributed respectively to the stretch vibration of C H and the symmetric stretch vibration of COO− of galacturonic acid and the stretch vibration of C O within COOH [2]. These results show that the extracted and degraded samples have the same structures of the main chain. These data suggest
Mw (g/mol)
Ð (Mw /Mn )
Rg (nm)
[] (mL/g)
Rh (nm)
Neutral sugars (%)
Gal acid (%)
6,800,000 117,000 49,000 14,000
1.7 2.4 2.1 1.2
185
385 11.8 8.5 6.4
72 5.5 3.7 2.4
54.2 53.6 32.6 30.3
31.2 35.9 41.4 43.8
* * *
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290 291 292 293 294 295
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Table 2 Results of size exclusion chromatography analyzes and sugar determination. Samples
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ARTICLE IN PRESS
BIOMAC 5148 1–8
International Journal of Biological Macromolecules xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Depolymerization of polysaccharides from Opuntia ficus indica: Antioxidant and antiglycated activities
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Mohamed Aymen Chaouch a , Jawhar Hafsa b , Christophe Rihouey c , Didier Le Cerf c,∗ , Hatem Majdoub a,∗ a Laboratoire des Interfaces et des Matériaux Avancés (LIMA), Faculté des Sciences de Monastir, Université de Monastir, Bd. de l’environnement, 5019 Monastir, Tunisia b Laboratoire de Biochimie, Faculté de Médecine, Université de Sousse, 4002 Sousse, Tunisia c Normandie Université, Laboratoire Polymères Biopolymères Surfaces (PBS), UMR 6270 & FR 3038 CNRS, Université de Rouen, 76821 Mont Saint Aignan, France
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Article history: Received 25 March 2015 Received in revised form 1 June 2015 Accepted 2 June 2015 Available online xxx
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Keywords: Opuntia ficus indica Polysaccharide Free radical depolymerization Antioxidant activity Antiglycation activity
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1. Introduction
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The extraction, purification and degradation of polysaccharides from Opuntia ficus indica cladodes, as well as the evaluation of their antioxidant and antiglycated activities in vitro were investigated. The optimization of the extraction showed that extraction by ultrasound at 40 ◦ C presented the best carbohydrates yield. The degradation of the extracted polysaccharides was achieved by free radical depolymerization with H2 O2 in the presence of copper(II) acetate for various reaction times. Sugar contents were determined by colorimetric assays. The macromolecular characteristics of the different isolated and degraded carbohydrates were carried by size exclusion chromatography (SEC/MALS/VD/DRI). These experiments showed that all samples are polysaccharides, which are probably pectins and that molecular weight (Mw ) has decreased from 6,800,000 to 14,000 g/mol after 3 h of depolymerization without changing the structure. Preliminary antioxidant and antiglycated tests indicated that degraded polysaccharides for 2 and 3 h showed even better antioxidant and antiglycated activities. © 2015 Published by Elsevier B.V.
The antioxidants exhibit significant efficiency to protect the human body against damage caused by reactive oxygen species which attack biological molecules, such as lipids, proteins, enzymes, DNA and RNA, resulting in cells or tissues lesions associated with aging, atherosclerosis, and carcinogenesis. Nowadays great interest is awarded to natural antioxidants due to problems related to safety and toxicity of synthetic antioxidants. Many natural antioxidants have already been isolated from different kind of plants such as oil seeds, cereals, vegetables, leaves, roots, herbs and spices [1]. Over the past few years, the contribution of advanced glycation endproducts for diabetes and aging has received considerable attention. The modification of proteins by glucose through the glycation process leads to the formation of advanced glycation endproducts. In fact, glycation is a spontaneous non-enzymatic amino-carbonyl reaction between reducing sugars and proteins [2].
∗ Corresponding author. Tel.: +216 98355740. E-mail address: [email protected] (H. Majdoub).
As part of the research of new sources of natural antioxidation and antiglycation agents, we were interested in a plant which is resistant to water stress and tolerant to poor soils, Opuntia ficus indica (OFI). This plant is native to Mexico and South America, but is also widely distributed in the Mediterranean, Africa and Australia [3]. The physicochemical analysis of OFI young cladodes reveals similarities in the composition compared to wide used vegetables such as spinach, tomato and lettuce. Polysaccharide extracts from plants, like those from the Cactaceae family, represent an important source of additives for several industries, especially in food and drug industry [4]. The major components in OFI cladodes are pectins which are heteropolysaccharides characterized by a high content of galacturonic acid (monomers linked by ␣-(1-4) links and partially acetylated or esterified with methyl groups) [5]. Pectins have specific physico-chemical properties due to their polyelectrolyte character. Forni et al. proposed a method of pectin extraction from prickly pear peels in two stages: an extraction of alcohol insoluble substances (AIS) followed by pectins extraction from AIS under hot acid conditions [6]. Pectin’s extraction procedure from OFI cladodes in cold water with extensive ultrafiltration was performed by Majdoub et al. [7].
http://dx.doi.org/10.1016/j.ijbiomac.2015.06.003 0141-8130/© 2015 Published by Elsevier B.V.
Please cite this article in press as: M.A. Chaouch, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.06.003
42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
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Several researches have indicated that high molecular weight and high viscosity of polysaccharides restricted their pharmaceutical application. Generally, several structural parameters influence the biological activities of carbohydrates, such as the substitution degree, the functional groups and the molecular weight. Recently, it has been reported that the polysaccharide’s activity is strongly influenced by the molecular weight distribution [8]. Chemical modification of carbohydrates provides the possibility to obtain new pharmaceutical agents [9]. Other research has shown that pectin interacts directly with oxidants and free radicals. In addition to that, it has been suggested that a relatively low molecular weight and a high galacturonic acid content in polysaccharides appeared to increase the antioxidant activity [10]. The degradation of polysaccharides was achieved by enzymatic (pectinase [11], pectin methyl esterase, polygalacturonase [12]), chemical (acid hydrolysis [13], ozonolysis [14], free radical depolymerization [15]), physical (sonochemistry [16], microwave, irradiation [17], etc.) and thermal processes [17]. These methods are generally dependent on the structure, the conformation, the applicability of polysaccharides and the reactive agents [18]. Freeradical depolymerization is an interesting chemical process for polysaccharide’s degradation because it provides constant composition, reproducibility and the retention of primary structure after depolymerization. In addition, the reaction conditions are mild and the reagents are cheap and suitable for use on a large scale [19]. In this work we are interested in the optimization of the extraction and the degradation of pectic polysaccharides from OFI cladodes and the study of the influence of their molecular weight on the antioxidant and antiglycated activities. 2. Materials and methods 2.1. Materials Prickly pear cladodes were harvested in the area of Monastir (Tunisian Sahel) at the end of spring (flowering period of the plant). The cladodes specimens were washed and cut into small pieces. 2.2. Optimization of polysaccharides extraction At industrial level, pectin is extracted in hot water (60–100 ◦ C) acidified with a mineral acid (sulfuric, nitric, hydrochloric or citric acid) during 0.5–6 h at a pH ranging from 1.5 to 3. These methods are time consuming and lead to pectin degradation. So, in order to produce pectin with high yield and quality, it is of great importance to introduce novel processes [20]. We chose to vary the physical treatment: for this, 1000 g of fresh cladodes were crushed with distilled water and split into 4 fractions: The first fraction was subjected to 10 min of ultrasound (100 W) [21] (PCU), the second was treated by microwave for 150 s (PCM) [21], the third was frozen overnight and then thawed at room temperature (PCF) [22], whereas the fourth did not undergo any treatment (PCW). The different fractions were kept under mechanical stirring in water for 2 h at a rate of 300 rpm at room temperature (solid–liquid ratio 1:30 (w/v)) and then filtered through a G0 sintered glass funnel. The filtrates were centrifuged for 20 min at 3000 × g and then freeze dried (Fig. 1). In order to purify the extracted products, two more steps were carried out: a precipitation with 80% ethanol followed by filtration on a G2 funnel [23]. The precipitate was then dialyzed against deionized water using dialysis tubing with molecular weight cut off 30 kDa in order to eliminate salts and compounds of low molecular weight, until conductivity equaled that of the deionized water. Finally, the dialysate was freeze dried.
2.3. Free radical depolymerization In order to degrade high molecular weight polysaccharides extracted from prickly pear cladodes [24,25], we proceeded to the free radical depolymerization by Fenton reaction according to the method described by Wu et al. [19] We began with the preparation of 25 mL of a polysaccharidic solution (40 mg/mL). Then, the solution was introduced into an electrochemical cell and the temperature was set at 60 ◦ C using a thermostated bath. Subsequently, 0.25 g of copper acetate was added and the initial pH was adjusted to 6.2 with NaOH solution (8 M). The depolymerization reaction is started with the addition of a H2 O2 solution (12%) at a rate of 5 mL/h which was controlled by a peristaltic pump. The pH of the solution was fixed at 6.2 using a pH-stat enabling the addition of the NaOH solution (8 M) when the pH drops below 6.2. The degradation was carried out for periods of 1, 2 and 3 h, leading respectively to the products PCU1 , PCU2 , and PCU3 . For each sample, 20 mL was taken in a beaker to which was added 7.5 mL of glacial acetic acid and then 12.5 mL of resin (Chelex 100) to remove copper. The resin Chelated cupric and cuprous ions. The resin was removed and replaced as many times as needed until it becomes colorless. The treatment was complete when the resin became colorless. Finally, the solution was dialyzed for 24 h against deionized water using dialysis tubing with molecular weight cut off 7000 Da and then freeze-dried.
2.4. Size exclusion chromatography Analysis of various samples was performed using size exclusion chromatography (SEC) equipped with a triple detection: multiangle light scattering (MALS) (Down HELEOS II, Wyatt Technology, CA, USA), viscometer detector (VD) (Viscostar II, Wyatt Technology, CA, USA) and differential refractive index (DRI) (RID 10 A Shimadzu, Japan). The SEC system consists of a pump (LC10 Ai Shimadzu, Japan) at a flow rate 0.5 mL/min of LiNO3 0.1 mol/L and two columns 8.0 mm × 300 mm (internal diameter × length) OHPAK SB 804 and 806 HQ (Shodex) in series. The paking material is polyhydroxymethacrylate. The samples were dissolved in the eluent (LiNO3 0.1 mol/L) at 2 g/L. The dissolution was carried out by stirring at 380 rpm for 24 h at room temperature. 3 mL solutions were filtered through membrane 0.45 microns (regenerated cellulose) before injection. The analyzes from MALS were performed by a data processing Zimm “order 1” using angles from 34.8◦ to 142.8◦ [26]. The corresponding value of dn/dc is 0.15 mL/g, as published in a previous paper [7]. The Astra 6.0.1.7 software package is set to collect and extrapolate data with the aim to obtain for each elution volume the molecular weight and the gyration radius (if Rg > 10 nm) [27]. With an integration of the peak, we calculated the number (Mn ) and weight (Mw ) average molecular weight and the z-average gyration radius. The differential viscosimeter detector permits to obtain for each elution fraction the intrinsic viscosity. An integration of the peak gives the average intrinsic viscosity, which allowed us to obtain the average hydrodynamic volume (Vh ) using the Einstein–Simha Q3 equation: Vh =
[]M NA
where NA is Avogadro’s number, M is the molecular weight, [] is the intrinsic viscosity (mL/g), and is a conformational parameter that takes the value of 2.5 in the case of a spherical conformation.
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Fig. 1. Polysaccharide’s extraction from OFI cladodes by varying the physical treatment.
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2.5. Colorimetric assays The content of polysaccharides was determined for all the extracted polysaccharides by the phenol–H2 SO4 method using galactose as standard [28]. The content of galacturonic acid in the extracts containing pectin is an important parameter in the quantitative and structural analysis of this polysaccharide. The micro-titer plate method was used for the determination of galacturonic acid amount as described earlier by Casaretti et al. [29]. A serial dilution of standard (glucuronic acid) and samples (50 L, 1 mg/mL) was placed in a 96 well plate. Subsequently, 200 L of 25 mM sodium tetraborate in sulphuric acid solution was added. Then the plate was heated at 100 ◦ C for 10 min in an oven. After cooling at room temperature for 15 min, 50 L of 0.125% carbazole in absolute ethanol was carefully added. After heating at 100 ◦ C for 10 min in an oven and cooling at room temperature for 10 min, the plate was read at 550 nm.
2.6. ATR-FTIR The chemical composition of extracted and degraded samples was obtained using PerkinElmer Spectrum Two ATR-FTIR, over the wave number range between 4000 and 400 cm−1 .
2.7. Antioxidant activity 2.7.1. DPPH radical scavenging assay The free radical scavenging effect of extracted and degraded samples was estimated according to the method of Espin et al. [30]. Briefly, 1 mL of each sample (0.5, 1, 1.5 and 2 mg/mL) was mixed with 4 mL of methanolic solution of DPPH (100 M). The reaction mixture was vortexed and incubated for 30 min in room temperature. The absorbance of the solution was measured at 517 nm. Ascorbic acid (AA) was used as standard. The
inhibitory percentage of DPPH was calculated using to the following equation:
DPPH scavenging effect (%) = 1 −
Abssample
Abscontrol
× 100
2.7.2. Nitric oxide radical scavenging assay The procedure is based on the principle that sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of NO• compete with oxygen, leading to reduced production of nitrite ions. 2 mL of different solutions (0.5, 1, 1.5 and 2 mg/mL) were mixed with sodium nitroprusside (4 mL of 10 mM, in PBS pH 7.4), and incubated at room temperature for 150 min. After the incubation period, 0.5 mL of Griess reagent was added [31]. The absorbance of the chromophore formed was measured at 546 nm. Ascorbic acid (AA) was used as standard. The NO• scavenging activity was calculated using the following equation:
NO scavenging activity (%) = 1 −
Abssample Abscontrol
212 213
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× 100
2.7.3. Linoleic acid peroxidation with TBARS assay In this assay antioxidant capacity is determined by measuring the thiobarbituric acid-reacting substances (TBARS) arising from linoleic acid peroxidation [32]. The reaction mixture contained 500 L linoleic acid (20 mM), 500 L Tris HCl (100 mM, pH 7.5), 100 L FeSO4 .7H2 O (4 mM) and a varying concentration of each sample (0.5, 1, 1.5 and 2 mg/mL). Linoleic acid peroxidation was initiated by the addition of 100 L of ascorbic acid (2 mM), incubated for 30 min at 37 ◦ C and achieved by the addition of 2 mL trichloroacetic acid (10%). Therefore 1 mL of the mixture was added with 1 mL of thiobarbituric acid (1%), followed by heating for 10 min at 95 ◦ C in water bath. The mixtures were centrifuged at 3500 × g for 10 min and the absorbance of TBARS in the
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Table 1 Polysaccharide extraction yields for different methods at room temperature.
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Samples
PCU
PCM
PCF
PCW
Extraction process Yield relative to fresh matter (%) Yield relative to dry matter (%)
Ultrasound 3.2 45.7
Microwave 2.7 39.0
Freezing 2.9 41.7
Without treatment 3.0 43.6
supernatant was measured at 532 nm. Ascorbic acid (AA) was used as standard. The percentage of antioxidant activity is determined using the following equation: Linoleic acid peroxidation inhibition (%) =
A −A c s Ac − An
× 100
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Ac = absorbance of control (without extract); As = absorbance of extract; An = absorbance of blank (without extract and FeSO4 ·7H2 O).
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2.8. In vitro glycation of protein
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The inhibition of protein glycation was determined by the method of Yang et al. [33] with slight modifications. Bovine serum albumin (20 mg/mL, in 20 mM PBS, pH 7.4) containing 0.1% (w/w) sodium azide (in PBS pH 7.4) was preincubated with polysaccharide (0.5, 1, 1.5 and 2 mg/mL) or aminosalicylic acid (ASA) for 10 min at room temperature (20 ◦ C). 1 M galactose solution was added to the reaction mixture and bacteria were eliminated by membrane filtration with a pore size of 0.2 m (Denville® syringe filter, PES, 0.22 m, 25 mm diameter). The solutions were incubated in dark at 60 ◦ C for 72 h. The reaction mixture without d-galactose was used as a blank solution and aminosalicylic acid was used as standard [34,35]. Fluorescence intensity was measured using a fluorometer (VersaFluor spectrofluorometer, Bio Rad), with an emission wavelength of 450 nm and an excitation wavelength of 370 nm. The percentage of antiglycated activity was calculated using the following equation:
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Inhibition % =
1 − FBSA+galactose+sample
FBSA+galactose
× 100
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where F is fluorescence intensity. Measurements were performed in triplicate and results are expressed as percentage inhibition of formation of the glycated protein.
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3. Results and discussion
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3.1. Extraction and depolymerization
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3.1.1. Extraction yields Table 1 shows the average yield for the different extraction methods. From the extraction data, it appears that the extraction by ultrasound presented the higher carbohydrates amount (3.2%). The mechanism of the extraction by ultrasound involves two types of physical processes: diffusion through the cell walls and washing out (rinsing) the cell contents once the walls are broken [36]. The increase in ultrasound treatment time may enhance both activities resulting in a high extraction yield, which explains the
observed increase in our extraction yield compared to other methods. The use of high ultrasonic power (>100 W) causes degradation effects [37,38], which explains the choice of the value 100 W. Furthermore, we tried to study the effect of the temperature on the extraction yield. We chose to vary the temperature from 25 to 100 ◦ C. The extraction at 40 ◦ C had the best yield (3.5% relative to fresh matter). While yields were 3.2%, 3.1% and 3.0% at 25 ◦ C, 70 ◦ C and 100 ◦ C, respectively. PCU sample was chosen for the depolymerization study. 3.1.2. Depolymerization After optimization of the extraction, we proceeded to the free radical depolymerization by Fenton reaction at three times (1, 2 and 3 h) leading respectively to the products PCU1 , PCU2 , and PCU3 . The yields of different depolymerization reactions were 14.0%, 9.7% and 9.2% respectively for PCU1 , PCU2 and PCU3 . 3.2. Identification
PCU PCU1 PCU2 PCU3 *
Mn (g/mol) 4,000,000 50,000 23,000 11,000
Table 2 shows the neutral sugars and the uronic acids rates for the extracted and degraded samples. From these data, we notice that all extracted and degraded samples are polysaccharides which are probably pectin since they contain high amounts of galacturonic acid (31–44%). These results are consistent with those found by gas chromatography according to Majdoub, Forni and Cárdenas [6,7,39]. On the other hand, the Gal acid content increases with free radical depolymerization from 31.2% for PCU to 43.8% for PCU3 unlike neutral sugars content which remarkably decreases from 54.2% to 30.3%. These data allow us to conclude that the cutoff was carried out at the junctions of neutral sugars. These results are consistent with those found by Garna et al. and Emega et al. who studied the variation of the galacturonic acid rate during acid and enzymatic hydrolysis of pectin [40,41]. The FT-IR spectra of native PCU, PCU1 , PCU2 and PCU3 are shown in Fig. 2. The four samples displayed a broad stretching intense characteristic peak at around 3300 and 3315 cm−1 for the stretching vibration of hydroxyl group. Each particular polysaccharide has a specific band in the 1200–1000 cm−1 region. This region is dominated by ring vibrations overlapped with stretching vibrations of (C OH) side groups and the (C O C) glycosidic bond vibration [42]. The stretching signals at 1031 cm−1 suggested the presence of C O bonds. In addition, signals at 1635 cm−1 were due to the asymmetric stretch vibration of C O of galacturonic acid. The signals at 2923 cm−1 and 1419 cm−1 are attributed respectively to the stretch vibration of C H and the symmetric stretch vibration of COO− of galacturonic acid and the stretch vibration of C O within COOH [2]. These results show that the extracted and degraded samples have the same structures of the main chain. These data suggest
Mw (g/mol)
Ð (Mw /Mn )
Rg (nm)
[] (mL/g)
Rh (nm)
Neutral sugars (%)
Gal acid (%)
6,800,000 117,000 49,000 14,000
1.7 2.4 2.1 1.2
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385 11.8 8.5 6.4
72 5.5 3.7 2.4
54.2 53.6 32.6 30.3
31.2 35.9 41.4 43.8
* * *
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290 291 292 293 294 295
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Table 2 Results of size exclusion chromatography analyzes and sugar determination. Samples
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