View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositori d'Objectes Digitals per a l'Ensenyament la Recerca i la Cultura Int. Agrophys., 2015, 29, 299-306 doi: 10.1515/intag-2015-0035 Water-soluble polysaccharides from Opuntia stricta Haw. fruit peels: recovery, identification and evaluation of their antioxidant activities** Mohamed Koubaa1*, Ameni Ktata2, Francisco J. Barba3*, Nabil Grimi1, Houcine Mhemdi1, Fatma Bouaziz2, Dorra Driss2, and Semia Ellouz Chaabouni2,4 1Sorbonne Universités, Université de Technologie de Compiègne, Laboratoire Transformations Intégrées de la Matière Renouvelable (UTC/ESCOM, EA 4297 TIMR), Centre de Recherche de Royallieu, B.P. 20529, 60205 Compiègne Cedex, France 2Enzyme Bioconversion Unit (UR13ES74), National School of Engineering, P.O. Box 1173-3038, Sfax University, Tunisia 3Faculty of Pharmacy, Nutrition and Food Science Area, Universitat de València, Avda. Vicent Andrés Estellés, s/n. 46100 Burjassot, Spain 4Common Service Unit of Bioreactor coupled with an ultrafilter, National School of Engineering, P.O. Box 1173-3038, Sfax University, Tunisia Received March 11, 2015; accepted June 1, 2015 A b s t r a c t. Opuntia stricta Haw. is considered as one of ranging from phytotherapy to the modern food industries the most common cactus plant growing in Tunisia. Extracting (Belton et al., 2003). The extraction of antioxidant mo- valuable compounds from its fruit peel, considered as by-product, lecules represents an important way of agricultural waste is drawing more and more attention, making it on the verge of commercialization. Water-soluble polysaccharides were extracted valorisation, especially due to the technological advances from Opuntia stricta Haw. peels, and their chemical composi- in molecular separation and identification (Roselló-Soto et tion assessed using thin layer chromatography. The antioxidant al., 2015; Wijngaard et al., 2012), as well as due to the uses activities of the extracted polysaccharides were assessed using of emerging technologies replacing thus the conventional 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity, ones (Galanakis, 2012, 2013). Numerous antioxidant mol- total antioxidant activity and reducing power capacity. The extrac- ecules derived from plant extracts have been widely used tion yield of water-soluble polysaccharides was 7.53±0.86%. The chemical composition revealed the presence of rhamnose, ara- as additives in food formulation (Sinha et al., 2008), in part binose, glucose, mannose, galactose and galacturonic acid. The due to their biological activities (Roselló-Soto et al., 2015). infra-red spectroscopic analysis showed a similar structure to that To address the problems of oxidation and contamination of Opuntia ficus-indica polysaccharide peels. Additionally, the of foodstuffs, many synthetic antioxidant molecules are extracted polysaccharides exhibited high antioxidant activities. often added. However, regarding their potential toxicity In fact, the free radical scavenging activity (half inhibition con- and carcinogenicity, they have been restricted by legisla- centration = 6.5 mg ml-1 with 94.9% inhibition at 50 mg ml-1), the total antioxidant activity (100 μg ascorbic acid equivalent at tion in many countries (Madhavi et al., 1996). Hence, their 50 mg polysaccharides) and the reducing power capacity (absor- replacement by natural molecules has been widely studied. bance 700 nm = 0.7 at 50 mg ml-1), appeared to be interesting The antioxidant potential of different polysaccharides has compared to natural and synthetic antioxidants. Therefore, water- been demonstrated. Moreover, they are harmless and do not soluble polysaccharides from Opuntia stricta Haw. fruit peels cause side effects (Warrand, 2006). In addition, these high could be a natural alternative to replace synthetic antioxidants. molecular weight polymers may exhibit therapeutic pro- K e y w o r d s: Opuntia stricta Haw. peels, polysaccharide extraction, water-soluble polysaccharides, antioxidant activity perties eg anti-tumor, anti-inflammatory, and anti-micro- bial activities (Caili et al., 2007; Chen et al., 2008; Krichen INTRODUCTION et al., 2015; Leung et al., 2006; Mokni Ghribi et al., 2015). All of these activities and others are closely related to Plant extracts constitute natural sources of antioxidant the physicochemical properties of each polysaccharide compounds (Dent et al., 2013; Polya, 2003; Šic Žlabur et molecule such as the type of the sugar residues, the chemi- al., 2015). They are hence used in numerous applications cal composition, the molecular weight and the degree of *Corresponding author e-mail: [email protected], branching. Numerous research groups have been interested [email protected] **This work was funded by the Ministry of Higher Education and © 2015 Institute of Agrophysics, Polish Academy of Sciences Scientific Research of Tunisia. 300 M. KOUBAA et al. in extracting polysaccharides from agricultural and indus- um ether as solvent. Total sugar content was determined trial by-products for their valorisation (Galanakis, 2011; according to Dubois et al. (1956). Total ash was determined Galanakis and Schieber, 2014; Sila et al., 2014a). by the combustion of 5 g peel juice in a muffle furnace at Extracting polysaccharides from opuntia fruit peels, 550°C for 4 h. After dry ashing, the mineral composition especially from the ficus-indica species, has been studied (Na, Mg and Ca) was determined by flame atomic absorp- due to its high availability as agro-industrial by-product in tion spectrometry (Analytic Jena ZEEnit 700 spectrometer, arid and semi-arid regions (Habibi et al., 2004; Majdoub et USA) (Jorhem, 2000). al., 2010). In Tunisia, Opuntia stricta Haw. is the second Water-soluble polysaccharides (WSP) were extracted widespread cactus plant growing after O. ficus-indica. from O. stricta Haw. peels as described previously with Numerous studies have been conducted on O. stricta Haw. slight modifications (Ding et al., 2012). 100 g of O. stricta peels, such as the extraction and characterisation of poly- Haw. peel juice was mixed with 500 ml distilled water in phenols, flavonoids, betacyanins (Yeddes et al., 2013) 1 l round bottom flask. The mixture was then boiled with and dyes (Obón et al., 2009), making it on the verge of reflux for 4 h using heating mantle. The mixture was then commercialization. recovered, filtered through Whatman paper using Büchner In this work, chemical composition of Opuntia stricta funnel. The pellet was then extracted a second time under Haw. peels were investigated. Water-soluble polysaccha- the same conditions and the filtrates containing WSP were rides (WSP) were extracted from O. stricta Haw. peels, their pooled and concentrated 20 times using a rotary evaporator structure was investigated using Fourier transform infra- system at 50°C. WSP were recovered overnight by add- red spectroscopy, and their chemical composition was as- ing 100 ml absolute ethanol at -20°C, followed by 15 min sessed using thin layer chromatography. DPPH (2,2-diphe- centrifugation at 5 000 r.p.m. Five washing steps were per- nyl-1-picrylhydrazyl) free radical scavenging activity, total formed in order to remove the colorants from the extracted antioxidant activity, and reducing power capacity of the polysaccharides. Each step consisted of resolubilising the extracted WSP were also studied. WSP in 20 ml distilled water, followed by adding one vo- lume of absolute ethanol, an overnight precipitation and MATERIAL AND METHODS a centrifugation step as described above. A dialysis step was performed on the recovered WSP using bi-distilled water Potassium ferricyanide was purchased from Loba for 3 days, in order to remove the salts. The extracted WSP Chemie (India). Sodium phosphate, sodium tetraborate, were freeze-dried overnight, and visualised using an XL30 sodium dodecyl sulphate, ferric chloride, DPPH, trichlo- ESEM scanning electron microscope. roacetic acid, trifluoroacetic acid, Tris, petroleum ether, Monosaccharide composition of the extracted polysac- glucose, arabinose, mannose, xylose, rhamnose, galactose, charides was determined after acid hydrolysis. 2 mg of galacturonic acid, polygalacturonic acid, cellulose, cit- lyophilized WSP was mixed with 0.5 ml of trifluoroacetic rus peel pectin and beechwood xylan were obtained from acid (2 N) and heated at 120°C for 2, 4, and 6 h. The mix- Sigma-Aldrich (France). Sulphuric acid and ethanol were ture was then completely dried under nitrogen flow, then obtained from Sharlab (Spain). Ammonium molybdate was solubilised in 100 µl distilled water. The obtained hydro- obtained from NenTech Ltd (United Kingdom). lysate (5 µl from each solution) was then analyzed with Opuntia stricta Haw. fruits were collected in the suburb thin layer chromatography using silica gel plate 60 F254 of Sfax city, Tunisia, in February 2014. They were com- (Millipore) as previously described (Ben Jeddou et al., posed of 69% peel, 21% pulp and 10% seeds. The peels 2014). Glucose, arabinose, mannose, xylose, rhamnose, were separated manually from the pulps and the seeds, and galactose and galacturonic acid were used as standards for then blended using a kitchen mixer. The obtained viscous compounds identification (5 µl from each standard solution peel juice was frozen at -20°C until analysis. at 4 mg ml-1). A mixture of chloroform / acetic acid / water Dry matter was determined according to AFNOR stan- (6:7:1 v/v) was used as the mobile phase. After the separa- dards