Phytochemical Study of Prickly Pear from Southern Morocco

Journal of the Saudi Society of Agricultural Sciences (2014) xxx, xxx–xxx

King Saud University Journal of the Saudi Society of Agricultural Sciences

www.ksu.edu.sa www.sciencedirect.com

FULL LENGTH ARTICLE Phytochemical study of prickly pear from southern Morocco

Z. Bouzoubaaˆa, Y. Essoukrati b, S. Tahrouch b, A. Hatimi b, S. Gharby c, H. Harhar c,* a Laboratoires d’Agrophysiologie et Physiologie de Poste re´colte, UR Ressources Naturelles et Produits de Terroirs, INRA-CRRA-Agadir-Morocco, B.P. 124, Inezgane, Morocco b Laboratoires de Biotechnologie Ve´ge´tale, Universite´ Ibn-Zohr, Faculte´ des Sciences, Agadir, Morocco c Laboratoire de Chimie des Plantes et de Synthe`se Organique et Bioorganique, Faculte´ des Sciences, Universite´ Mohammed V-Agdal, BP 1014, Rabat, Morocco

Received 29 April 2014; revised 14 September 2014; accepted 22 September 2014

KEYWORDS Abstract This work concerns the phytochemical study of the prickly pear pulp’s fruits of two Opuntia ﬁcus indica; opuntia cultivars; Achefri and Amouslem widely present in two regions of southern Morocco; Phytochemical characteriza- Arbaa Sahel and Asgherkis that are different in their altitude and annual rainfall. The results of tion; the phytochemical study show that the levels of antioxidants have a non-signiﬁcant difference Southern Morocco; between the fruits of the two sites (comparing Amouslem and Achefri in the same site, on the Antioxidants; one hand, for the differences due to the variety or cultivar, on the other hand between Amouslem Polyphenols and Achefri from the two sites to show the site effect). ª 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction Spanish conquerors in the 16th and 17th century. The culture of Opuntia covers about 200,000 ha in North Africa 30–40% Opuntia cactus is a native species of Mexico (Mulas et al., in Tunisia (Le Houe´rou, 1985). Opuntia cactus was introduced 1992). It was one of the food bases for the indigenous popula- in Morocco in the early 17th century from Spain (Schielief tions (Hoffman, 1995). It was introduced in southern Spain et al., 2000). With the exception of the Sahara and mountain- after Columbus’s ﬁrst expedition to the New World. Thereaf- ous areas, Opuntia are widely represented in the Moroccan ter, it was spread throughout the Mediterranean Basin by the rural landscape, into plantations more or less regular, around villages or plant closing limiting parcels of crops or orchards (Arba, 1983; Arba et al., 2000; Bouzoubaaˆet al., 2014). * Corresponding author. Tel.: +212 6 68 79 99 42. The culture of Opuntia exists almost in all regions of the E-mail address: [email protected] (H. Harhar). country with relatively variable area; it occupies an estimated Peer review under responsibility of King Saud University. area with about 54,530 ha, representing 11.07% of the total area of the fruit trees (Boujghagh and Chajia, 2001). Its geo- graphical distribution is quite large since it found both in coastal areas from Sidi Ifni in the South to Tangier in the Production and hosting by Elsevier http://dx.doi.org/10.1016/j.jssas.2014.09.002 1658-077X ª 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

North, as in several continental regions (Boujghagh and located at 15 km from the sea and 17 km southwest of the Tiz- Chajia, 2001). The best plantations are located in coastal areas nit city and Asgherkis a continental site located at 709 m of and more specifically in the coastal strip of more than 10 km altitude, and 57 km in the south of the Ait Baha city (70 km wide undergoing maritime influence, the plant benefits the from Agadir) (Fig. 1). night and morning fog, very frequently in this area throughout year (Boujghagh and Chajia, 2001; Bouzoubaa et al., 2014). In 2.2. Plant material Morocco, at the Tiznit province, the culture of Opuntia occupies the largest area with about 17,600 ha mainly in the regions The studied fruits were harvested between July and September of Sidi Ifni and Ait Baamrane accounting for 65% of it (Dehbi 2010. Only ripe and healthy fruits were selected, they were and Radwan, 2000). Tensift and the Central regions are among immediately stored in a cold room at a temperature of 4 C. the areas where regular plantations have been installed as part The present plant material in the study sites is composed of of national operation against erosion. In the region of Moulay two populations: Idriss, near Meknes, a cactus plantation for the production of fruits has been a steady expansion. In Beni Smir, near Oued a- A population consists of individuals with thorny rackets Zem, a cactus collection was established in 1944 on an area and small oval fruits, with yellow or red color, and yel- of 500 ha (Habibi, 2004). low–orange pulp with a thinner crust. This population is The cactus has been largely ignored by scientists until the locally known as Achefri. beginning of 1980; this renewed interest is partly attributed b- A population consists of individuals with spineless rack- to the multifunctionality of prickly pear fruit. Recent studies ets and oval fruits with large caliber, yellow or red color, have revealed their high levels of certain chemical compounds, and orange pulp with a thicker crust. This population is which can make this fruit an added nutritional value, such as locally known as Amouslem Fig. 2. taurine, calcium, magnesium, phenolic compounds and betalains (Piga, 2004; Zine et al., 2013). Cactus also knows a renewed interest in several countries 2.3. Physico-chemical analyses due to its ecological role, environmental and socio-economic: the fight against erosion and desertification, and fruit production fodder (Bouzoubaa et al., 2014). The prickly pear is also From the sample kept cold, 30 fruits per cultivar were ran- the origin of products and by-products to various industrial domly selected with a view to determine the various physico- uses, medicinal, pharmaceutical and cosmetics (Barbera, 1995). chemical parameters. The determination of the color of the In Morocco, the new strategy of agricultural development bark and the pulp was carried out using a chromameter concretized by the Green Morocco Plan (PMV) is a real oppor- (KONICA MINOLTA CR-400). tunity for the development of different sectors related to cac- The sugar content of the juice was determined using a tus, knowing that the PMV provides the consecration of a refractometer (DIGIT 032), the results are expressed as Brix. million hectares fruit species requiring little water, such as The free acidity, expressed as a percentage of citric acid is olive, carob or cactus (Bouzoubaa et al., 2014). The area determined by titration; 10 ml of the juice of prickly pear reserved for the latter in each region will experience a remark- was placed in a 100 ml beaker equipped with a magnetic stir- able increase, especially in the Guelmim-Smara and Rhamna rer, 20 ml of distilled water was added and the homogenized regions with additional 70,000 and 50,000 ha respectively. mixture is titrated with NaOH (0.1 N) to pH = 8.1 (Medina Moroccan cactus has a very high genetic variability, several et al., 2007). cultivars exist (Bouzoubaa et al., 2014), and are distinguished For the determination of dry matter, fruits were washed and by the flowering period (early, late), the flower color (yellow, peeled, 30 g of the pulp obtained was precisely weighed and orange and pink), fruit color and pulp (green, yellow, orange, placed in clean glass Petri dishes and pre-weighed. Then they red and purple), fruit shape (oval, round or oblong), the orga- were placed in an oven at 100 C. After 24 h, periodical weigh- noleptic characteristics of fruit (Boujghagh Chajia, 2001), and ing was continued at intervals of 2 h until a constant weight. the antioxidant content (Kuti, 2004; Tesoriere et al., 2005). The fruits of the prickly pear exhibit both intra-site and 2.4. Total polyphenols inter-site variability in the shape, color, weight, sugar, acids, antioxidants, etc. These parameters vary from one cultivar to The polyphenol content was determined using the Folin– another and are strongly influenced by the environment Ciocalteu spectrophotometrically according to the Singleton (Parish and Felker, 1997). In this context, we are interested method (Singleton et al., 1999) using caffeic acid as standard. in the study of antioxidants (polyphenols, flavonoids and In an alkaline medium, polyphenols reduce phosphomolybdic betalains) of the pulp’s fruit of both cultivars in the regions acid of Folin–Ciocalteu reagent (Catalano et al., 1999); this of Tiznit and Ait Baha. These cultivars are locally named reduction is reflected by the appearance of a dark blue color. Achefri and Amouslem. The color produced, (whose maximum absorption is between 725 and 765 nm) is proportional to the amount of polyphenols 2. Materials and methods present in plant extracts (Ghazi and Sahraoui, 2005).

2.1. Sampling sites 2.5. Total ﬂavonoı¨des

The fruit samples were collected from two different sites in The ﬂavonoid in the raw juice has been extracted by the meth- their relief and climate; Arbaa Sahel had a maritime inﬂuence, anol–water mixture (80:20, v/v). After stirring, sonication and

Altitude :709m ; Annual rainfall : 224mm/year. Latitude : N 29°49’00’’ ; Longitude : W 9° 12’00’’

Altitude :327m ; Annual rainfall : 143mm/year. Latitude : N ’ 29°38’00’’ ; Longitude : W 9° 52’00’’

Figure 1 Sampling sites: Asgherkis and Arbaa¨ Sahel localization.

cv. Achefri (Opuntia megacantha) (Asgherkis. cv. Amouslem (Opuntia ficus indica) Ait Baha) thorny bark (Asgherkis. Ait Baha), smoothy bark

cv. Achefri (Opuntia megacantha) (Arbaa Sahel, cv. Amouslem (Opuntia ficus indica) (Arbaâ Tiznit) thorny bark Sahel, Tiznit), smoothy bark

Figure 2 Cross section of Achefri and Amouslem cultivars from the two studied sites Tiznit and Ait Baha.

3.1. Color and weight 3.4. Volatile and dry matter content

In the site of Arbaa Sahel, fruits of the cultivar Achefri have The fruits of four cultivars studied contain 80% of water. This yellow pulp with an average weight of 50.58 ± 10.11 g, while water content is high, but it is still lower than the results of the the Amouslem fruits have orange pulp with an average weight study. Regarding the remaining part, the dry matter content of of 64.55 ± 11.94 g. On the other hand, Asgherkis fruits have 19% is higher than the theoretical value (16%) identified by orange pulp with an average weight of 37.92 ± 9.26 g for the the literature (Askar and El Samahy, 1981). This very high Achefri population and 43.25 ± 13.66 g for the Amouslem water content in prickly pears is a parameter that reflects the population (Table 1). Statistical analyses showed a significant high perishability of this type of fruit and limits its ability to difference between these middleweights, in fact cultivars of site storage at room temperature. ArbaaˆSahel have pulps with weigh more than that of Asgher- kis (Table 1)(Bouzoubaaêt al., 2014). 3.5. Total polyphenols

3.2. Sugar content Phenolic compounds, also called polyphenols, are metabolic products widely distributed in plant foods; they have many Asgherkis fruits have an average sugar content of 14.91 Brix biological and pharmacological properties that could provide for the Achefri cultivar and 15.87 Brix for the Amouslem cul- protection against chronic disease. These compounds have tivar. Indeed, these fruits are signiﬁcantly sweeter than those of more antioxidant effect than vitamins, they are able to neutral- the ArbaaˆSahel region (Table 1), it could be explained by the ize the effects of oxidative free radicals (Shahidi et al., 1992).

Table 1 Comparison of physico-chemical parameters of the studied pulp fruit. Parameters ArbaaˆSahel Asgherkis Achefri Amouslem Achefri Amouslem Pulp color Yellow Orange Orange Orange Weight pulp (g) 50.58 ± 10.11b 64.55 ± 11.94a 37.92 ± 9.26c 43.25 ± 13.66c Sugar content 13.50 ± 0.95c 13.15 ± 0.88c 14.91 ± 2.06b 15.87 ± 1.64a Acidity (%) 0.054 ± 0.008a 0.058 ± 0.007a 0.055 ± 0.008a 0.058 ± 0.011a Volatile matter (%) 80.03 ± 1.52a 80.33 ± 1.17a 80.03 ± 1.24a 80.37 ± 1.80a Dry matter (%) 19.96 ± 1.52a 19.66 ± 1.17a 19.96 ± 1.24a 19.62 ± 1.80a The values in the same line followed by the same letter are not signiﬁcantly different with IC = 95.

ArbaaˆSahel fruits have an average content of polyphenols of Asgherkis fruits have a slightly higher levels compared to those 289.39 ± 59.10 lg/g and 337.57 ± 67.53 lg/g for Achefri and of ArbaaˆSahel (Table 1). The average rate of total flavonoids Amouslem cultivars respectively. While in the Asgherkis site, is between 50.24 and 69.95 lg/g, these results are consistent this content is in the range of 404.84 ± 16.72 lg/g and with the results reported by Kuti (2004) who presented the val- 447.27 ± 76.77 lg/g for Achefri and Amouslem cultivars, ues from 9.8 to 93.5 lg/g, but lower than the results provided respectively (Table 2). In both sites, inter-cultivar comparison by Chavez-Santoscoy (2009) ranging from 95.8 to 373.3 lg/g. shows that the fruits of the Amouslem population contain higher Indeed, prickly pears contain flavonoid contents similar to polyphenol content than the fruits of the Achefri population. other fruits and vegetables (Peterson and Dwyer, 1998). On the other hand, the inter-site comparison shows that phenols are higher in Asgherkis fruits than in the ArbaaˆSahel 3.7. Betalains one. This difference between the two sites is the result of the effect of a number of factors, the main ones being genetic, pre- The coloration of prickly pears is due to the combination of cipitation, light, topography, soil type and maturity (Harris pigments yellow (betaxanthins) and red (betacyanins) (Forni and Karmas, 1977). The mean content of polyphenols com- et al., 1992); these azo pigments replace anthocyanins in most prised between 289.39 and 447.27 lg/g is higher than those families of Caryophyllales including cactacea (Zryd and indicated by Chavez-Santoscoy (2009) who determined the val- Christinet, 2004). Their identification in the fruits of the ues of 55.4–226.3 lg/g of nine varieties of Opuntia. Our results prickly pear has recently undergone a resurgence of interest are identical to those found by Gil et al. (2002) on the peach, (Stintzing et al., 2005). In addition to color, these pigments plum and nectarine ranging from 91 to 1042 lg/g. have higher antioxidant properties than the ascorbic acid (Stintzing et al., 2005). 3.6. Total flavonoids Prickly pears are a high source of betalains (Tesoriere et al., 2005), their concentration varies between 66 and 1140 lg/g of The class of flavonoids accounted for the largest part of the pulp (Stintzing et al., 2005). The ratio betacyanin:betaxanthin polyphenols (2/3), the rest (1/3) being represented by the phe- varies from 0 to 11.7 according to the color of the pulp nolic acids such as gallic acid and caffeic acid (Harborne and (Stintzing et al., 2005). According to the literature, the betanin, Williams, 2000). Flavonoids are divided into several groups; phyllocactin and the neobetanin are betacyanins found in the the most important are anthocyanins, flavonols, flavones and Opuntia fruits, with a predominance of betanin (95%), while flavanones (Ross and Kasum, 2002). indicaxanthin, miraxanthine II, vulgaxanthine I, vulgaxan- The pulp of cactus fruit contains a high content of polyphe- thine II and vulgaxanthine V are the betaxanthins present in nols and an antioxidant cactus fruit effect is mainly due to the Opuntia (Stintzing et al., 2000). The indicaxanthin was the first presence of quercetin, kaempferol, isorhamnetin and of their betaxanthin identified in the O. ficus-indica fruits (Piattelli derivatives, quercetin remains the predominant flavonoid in et al., 1964), while betanin has been identified in the fruits of Opuntia (Kuti et al., 2004). Several types of flavonoids have Opuntia dillenii (Zhang and Liu, 1992). been reported in the cactus Opuntia, their types and concentra- Betalains are powerful antioxidants, because of their oxido- tions vary depending on the variety and stage of maturation reduction properties; these compounds inhibit the oxidation of (Wallace et al., 1986). microsomal membranes and LDL oxidation in vitro (Tesoriere Kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside et al., 2003). In our study, in the Asgherkis site, the fruits of the and isorhamnetin-3-O-glucoside were identified in extracted Achefri cultivar have average contents of betalains of juice of Opuntia ficus-indica (Galati et al., 2003). Some authors 91.01 ± 8.11 lg/g, betaxanthins of 84.26 ± 7.44 lg/g and have indicated that plants of the family Cactaceae produce betacyanins of 6.74 ± 0.91 lg/g, while those of the Amouslem essentially the flavonol 3-O-glucosides (quercetin, kaempferol cultivar present contents of about 94.59 ± 18.13 lg/g, and isorhamnetin), dihydroflavonols, flavanones and flavano- 87.70 ± 17.95 lg/g and 6.89 ± 0.31 lg/g of betalains, betax- nols (Burret et al., 1982; Miller and Bohmt, 1982). At the anthins and betacyanins, respectively (Table 2). Indeed, these ArbaaˆSahel site the fruits have an average level of flavonoids contents are almost 3 times higher than the fruits of Arbaaˆ of 50.24 ± 3.85 lg/g and 52.58 ± 4.48 lg/g for the Achefri Sahel site. However, analysis of variance of the three parame- and Amouslem cultivars, respectively. While in the Asgherkis ters showed a non-significant difference. site the fruits of the population Achefri have an average grade The results of the quantitative study of betaxanthins and of 67.02 ± 7.20 lg/g and those of the population Amouslem betacyanins show that the studied fruits have a high concentra- have a concentration of 69.95 ± 3.94 lg/g (Table 2). Indeed, tion of yellow pigments compared to that of red pigments,

Table 2 Comparison of phytochemical parameters of the studied fruits. Parameters ArbaaˆSahel Asgherkis Newman and Keuls test Achefri Amouslem Achefri Amouslem Polyphenols totaux (lg AC/g) 289.39 ± 59.10 337.57 ± 67.53 404.84 ± 16.72 447.27 ± 76.77 NS Flavonoı¨ des (lg Q/g) 50.24 ± 3.85 52.58 ± 4.48 67.02 ± 7.20 69.95 ± 3.94 NS Betalaines (lg/g) 29.90 ± 5.29 37.25 ± 7.10 91.01 ± 8.11 94.59 ± 18.13 NS Betaxanthines (lg Ind/g) 28.08 ± 4.71 35.36 ± 6.61 84.26 ± 7.44 87.70 ± 17.95 NS Betacyanins (lg Bet/g) 1.81 ± 0.71 1.88 ± 0.67 6.74 ± 0.91 6.89 ± 0.31 NS AC: caffeic acid, Q: Quercetin, Ind: indicaxanthin, Bet: betanin.

Please cite this article in press as: Bouzoubaaˆ, Z. et al., Phytochemical study of prickly pear from southern Morocco. Journal of the Saudi Society of Agricultural Sciences (2014), http://dx.doi.org/10.1016/j.jssas.2014.09.002 6 Z. Bouzoubaaêt al. which explains the yellow or orange color of fruit pulp ana- nelle acque di vegetazione dei frantoi oleari e negli effluenti dei loro lyzed. On the other hand, our data also show that fruits that impianti di depurazione. Un confronto fra il metodo di Folin- represent high contents of betalains are the richest in polyphe- Ciocalteau e l’HPLC. Agrochimica (Italy). nols (Stintzing et al., 2005). Chavez-Santoscoy, R., Gutierrez-Uribe, J., Serna-Saldı´var, S., 2009. Phenolic composition, antioxidant capacity and in vitro cancer cell We analyzed that the average concentrations of betalains cytotoxicity of nine prickly pear (Opuntia spp.) juices. Plant Foods varied between 29.90 and 94.59 lg/g in fresh fruits (Table 2), Hum. Nutr. (Formerly Qualitas Plantarum) 64 (2), 146–152. these values are much lower than those reported by Stintzing Dehbi, Z., Radouane, A., 2000. Le figuier de barbarie a` Aı¨ t Baaˆmrane, et al. (2005) who determined values in the range of 65.9– Atouts et perspectives de de´veloppement. IIe` me Journeé Nat. Cult. 1140.4 lg/g in seven varieties of Opuntia, and consistent with Cact. DPA El Kelaaˆ, 2–5. those reported by Chavez-Santoscoy (2009) which vary El mehrach, K., 2003. Effet du sol de´sinfecteét du sol non de´sinfecte´ between 6.3 and 490.4 lg/g. sur la croissance et le profil pheńolique de deux le´gumineuses arborescentes du Souss-Massa: Acacia gummifera et Ceratonia 4. Conclusion siliqua. Me´moire DESA, Faculte´des sciences, Universiteíbn zohr, Agadir, Maroc, p. 35. Forni, E., Polesello, A., Montefiori, D., Maestrelli, A., 1992. High- The physico-chemical study showed that the titratable acidity performance liquid chromatographic analysis of the pigments of and water content did not differ significantly between the cul- blood-red prickly pear (Opuntia ficus indica). J. Chromatogr. A 593 tivars of the two studied localities. Our results also showed (1–2), 177–183. that the fruits of the Amouslem cultivar of the ArbaaˆSahel Galati, E., Mondello, M., Giuffrida, D., Dugo, G., Miceli, N., region are characterized by a high sugar content. This is felt Pergolizzi, S., Taviano, M., 2003. Chemical characterization and in the commercial point of view since this prickly pear is the biological effects of Sicilian Opuntia ficus indica (L.) Mill. fruit juice: antioxidant and antiulcerogenic activity. J. Agric. Food most searched and sold at the local market. Chem. 51 (17), 4903–4908. The spectrophotometric determination of total polyphenols Ghazi, F., Sahraoui, S., 2005. Evolution des compose´s pheńoliques et shows values between 289.39 and 447.27 lg/g. These results des caroteńoı¨ des totaux au cours de la maturation de deux varie´te´s indicate that the prickly pears contain significant levels of de datte communes Tantboucht et Hamraia. me´moire d’ingeńiorat polyphenols. Quantitative analysis of betalain shows that ana- en agronomie, El Harrach, Tunisie. lyzed fruits have high levels of betaxanthins, ranging from Gil, M., Toma´s-Barberań, F., Hess-Pierce, B., Kader, A., 2002. 28.08 to 87.70 lg/g. Indeed the fruit pulp can be considered Antioxidant capacities, phenolic compounds, carotenoids, and as a potential source of yellow and purple pigments widely vitamin C contents of nectarine, peach, and plum cultivars from used in the food industry. In general, we can conclude that California. J. Agric. Food Chem. 50 (17), 4976–4982. the fruits of the prickly pear are a potential source of polyphe- Habibi, Y., 2004. 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