You are on page 1of 4

Arabian Journal of Chemistry (2011) 4, 321–324

King Saud University

Arabian Journal of Chemistry


Antioxidant activity, phytochemical screening, and total phenolic content of extracts from three genders of carob tree barks growing in Morocco
Hanane El Hajaji a, Nadya Lachkar b, Katim Alaoui b, Yahya Cherrah b, Abdellah Farah c, Abdesslam Ennabili c, Brahim El Bali d, Mohammed Lachkar

a Laboratoire d’Inge´nierie des Mate´riaux Organome´talliques et Mole´culaires, Unite´ associe´e au CNRST (URAC 19), Faculte´ des Sciences, Universite´ Sidi Mohamed Ben Abdellah, BP 1796 (Atlas), 30000 Fe`s, Morocco b Laboratoire de Pharmacologie et Toxicologie, Faculte´ de me´decine et de Pharmacie, Universite´ Mohamed V Rabat-Souissi, Rabat, Morocco c National Institute of Medicinal and Aromatic Plants, University of Sidi Mohamed Ben Abdellah, BP 8862, 30100 Fe`s, Morocco d Laboratory of Mineral Solid and Analytical Chemistry (LMSAC), Department of Chemistry, Faculty of Sciences, University Mohamed I, P.O. Box 717, 60000 Oujda, Morocco

Received 15 May 2010; accepted 28 June 2010 Available online 3 July 2010

KEYWORDS Antioxidant activity; Phenolic content; Ceratonia siliqua L.; Gender; Female grafted; Spontaneous female; Spontaneous male

Abstract We evaluated the in vitro antioxidant property and phytochemical constituents of the crude ethyl acetate and methanol extract of the three genders of carob tree barks (spontaneous male, spontaneous female, and grafted female). The scavenging activity on DPPH (1,1-diphenyl-2-picrylhydrazyl) was determined, as well as the phenolic contents (Folin–Ciocalteu method) of both the extracts. The highest antioxidant activity and the higher amounts of total phenols were shown in methanol crude bark extract for the three genders. Variety significantly affected the phenol content and the antioxidant activity, with the spontaneous male variety globally showed a higher polyphenol concentration and antioxidant activity than the grafted female and spontaneous female.
ª 2010 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +212 671 556 742; fax: +212 535 733 171. E-mail address: (M. Lachkar). 1878-5352 ª 2010 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. doi:10.1016/j.arabjc.2010.06.053

1. Introduction Epidemiological and experimental studies reveal a negative correlation between the consumption of diets rich in fruits and vegetables and the risks for chronic angiogenic diseases, such as cardiovascular diseases, arthritis, chronic inflammation, and cancers (Chen et al., 2005; Middleton et al., 2000; Prior, 2003; Saleem et al., 2002; Zhang et al., 2005). These physiological functions of fruits and vegetables may be partly attributed to their abundance of phenolics. Phenolic

Production and hosting by Elsevier

000 tones per year and the principal producing countries are Spain.322 compounds are a complex group of substances that have attracted considerable attention due to their roles in providing flavor and color characteristics of food and in human health (Visioli and Galli. Dried coarse powder of the bark was placed into the extractor of a Soxhlet. 2005). The tests were carried out in triplicate. 2004). 2004) and other studied the variation and the composition of phenolic compounds of carob pods grown in different regions of Morocco (Rakib et al. Briefly. ethyl acetate. For these reasons. 2. 1993. Due to its interesting morphophysiological characteristics.). All tests were carried out in triplicate and the results are given as gallic acid equivalents (GAE).4. Mohamed Ben Abdellah University. sugar. flavonoids.1. World carob pod production is estimated about 310. there are no such phytochemical reports concerning Moroccan cultivars. 3. 1977) and the early work of Singleton and Rossi (Singleton and Rossi. Experimental 2. Tous.. 1997). The morphological and physiological traits are traditionally used for the identification and the management of carob cultivars (Tous and Batlle. They are more important in food industry and are a source of many products such as gum. the absorbance of the resulting solution was measured at 764 nm. 1971. and methanol. The absorbance of the mixture was measured at 517 nm by using a double-beam UV–vis Camspec M550 spectrophotometer.2. the total phenolic content. 1990. Gharnit et al. so the present investigation was designed to determine the phytochemical screening of the main secondary metabolites classes. and social reasons (Battle and Tous. aging.. Morocco. The total phenolic contents was determined spectrophotometrically using the Folin–Ciocalteu method. and alcohol (Carlson.. adaptation to poor soils (Battle and Tous.. 2. 1958Þ where AB is the absorbance of the control reaction (containing all reagents except the test compound). To our knowledge.0. Italy. Many of the benefits associated with consumption of phenolic-rich foods are associated with their antioxidant activities (Vinson et al.0007 gallic acid (lg) À 0. The scavenging activity on the DPPH radical was expressed as inhibition percentage using the following equation: %Inhibition ¼ ½ðAB À AS Þ=AB Š  100 ðBlois. 100 ll of various concentrations of the extract in methanol was added to 10 ml of a methanol solution of DPPH (1. Fe 2. After 2 h. 1997). 1992. 0. economic. Sawa et al. 2. At the end of the extraction the respective solvents were concentrated by evaporation. Carob (Ceratonia siliqua L.25 mg/L). and their seeds (Brito de Carvalho. pods (beans).. Plant materials were collected in 2007 from the Province of Chafchaouen (NW of Morocco) and identified by Professor A.0. Antioxidant activities of plant polyphenols have been claimed to have beneficial health functions for retarding. a plant rich in phenols and polyphenols. Papagiannopoulos et al. Authenticated voucher specimens were deposited in the Herbarium of The National Institute of Medicinal and Aromatic Plants. 1997). Greece. 1. 2.. and preventing cancer and cardiovascular diseases (Scalbert et al. Preparation of the extracts H. 1980).0. female. The extract concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage plotted against extract concentration (4. and Cyprus (Battle and Tous. Butylatedhydroxytoluene (BHT) was used as positive control. A mixture of 100 ll of methanol and 10 ml of DPPH solution was used as the control. 1993. Recently. Ennabili. Carob tree requires little maintenance (Fletcher et al. and AS is the absorbance of the test compound. Many investigators have studied the carob trees. and grafted female carob trees growing in Morocco. 1997). Ingram et al. This reagent is based on the Slinkard and Singleton method (Slinkard and Singleton. 2003. 1998..1-diphenyl-2-picrylhydrazyl (DPPH) free radical. Results and discussion 3.5 and 0. Phytochemical screening The ethyl acetate and methanol extracts were screened for phytochemical constituents (alkaloids. 1965) is a colorimetric oxidation/reduction method for phenolic compounds. Morocco. 1996).. 1992). 1986. the natural antioxidants have recently become a major area of research. 2010). Hajaji et al. Plant materials This work was conducted on Dkar (spontaneous female (F)). The fruits are used for diverse purposes as fodder. 1987. Determination of free radical scavenging activity by DPPH method The antioxidant potential of the ethyl acetate extract and the methanolic extract was determined on the basis of their scavenging activity of the stable 1. Briefly. it has been introduced to the Mediterranean basin. and hermaphrodite flowers are generally borne on different trees. 1966. Portugal.. Charalambous and Papaconstantinou. The mixture was vigorously shaken and then allowed to stand at room temperature for 30 min in the dark. The extraction was carried out by using solvents of increasing polarity starting from hexane. 1997) and it is considered to be an important component of vegetation for environmental. Spanos and Wrolstad. thus male.E. unproductive Dkar (spontaneous male (M)).1. The concentrations of phenolic compounds were calculated according to the following equation that was obtained from the standard as gallic acid graph: Absorbance = 0. Spontaneous female. The carob is a dioecious species with some hermaphroditic forms. Sidi ` s. such as resistance to drought and salinity. Marakis et al. and antioxidant activity of barks of spontaneous male.Thompson.. extracts were reacted with Folin–Ciocalteu reagent and then neutralized with sodium carbonate solution (25%).3.0642 (R2: 0. 1999).9964). investigators isolated and identified the major polyphenols in carob fibers (Owen et al. is a slow-growth evergreen tree. Phytochemical screening Phytochemical screening of the crude ethyl acetate and methanolic extract of the three categories of carob tree barks .01 · 10À2 M). and Lanta (grafted female (L)) of carob tree barks. dichloromethane. saponins and tannins) using simple qualitative methods of Paris and Nothis (Paris and Nothis.

and total phenolic content of extracts from three genders revealed the presence of flavonoids and tannins. the methanolic extracts showed a very good scavenging effect compared with ethyl acetate extracts. It also showed that Figure 1 Antioxidant activity of ethyl acetate extract. The result of the DPPH scavenging assay is shown in Figs.54 0.6 g/l) followed by spontaneous male (IC50 = 2. 1 and 2. The result of total phenol contents is shown in Table 2. Determination of free radical scavenging activity by DPPH method Table 2 barks.6 1 0. the result of phytochemical test has been summarized in Table 1. . the results of DPPH analyses for the methanol extract demonstrated that the most active radical scavengers were found in the spontaneous male (IC50 = 0.5 0. IC50 (g/l) values of fractions of three genders of carob Sample EtOAc MeOH EtOAc MeOH EtOAc MeOH BHT IC50 (g/l) 2. and spontaneous female (IC50 = 1.52 0.6 g/l).8 g/l). phytochemical screening. barks varied from 0. the sequence for DPPH radical scavenging ability was grafted female (IC50 = 1.58 0.Antioxidant activity.2 Spontaneous female Spontaneous male Grafted female – 3. Varieties Phytochemicals Ethyl acetate Methanolic extracts extracts À + + À À + + À À + + À À + + À À + + À À + + À Figure 2 Table 3 barks. barks categories tested. In the ethyl acetate extracts. 4. Flavonoids and tannins are phenolic compounds and plant phenolics are a major group of compounds that act as primary antioxidants or free radical scavengers (Polterai. barks were investigated for their antioxidant activity. respectively by grafted female (IC50 = 1 g/l). Categories Spontaneous female Spontaneous male Grafted female Total polyphenolic contents of three genders of carob Sample EtOAc MeOH EtOAc MeOH EtOAc MeOH Total phenolic content (g/l GAE) 0. Spontaneous female Alkaloids Flavonoids Tannins Saponins Spontaneous male Alkaloids Flavonoids Tannins Saponins Grafted female Alkaloids Flavonoids Tannins Saponins Key: + = present. and spontaneous female (IC50 = 2.7 g/l) followed.46 to 0. The total phenolic contents from the ethyl acetate and methanolic extracts of the three varieties of Ceratonia siliqua L.7 1.8 g/l). The presence of flavonoids and tannins in the plants is likely to be responsible for the free radical scavenging effects observed. It is found that antioxidant compounds in ethyl acetate and methanol extracts have the free radical scavenging ability. As shown in Table 3.46 0. 323 Table 1 Phytochemicals detected in extracts of three genders of carob barks. Conclusion This study confirms that the barks of the carob tree contain high amounts of polyphenol compounds. among the three Ceratonia siliqua L. Alkaloids and saponins were not detected.76 (g/l GAE). Thus. À = absent. the methanolic extract has the highest phenolic content than ethyl acetate extract.8 2.2. 1997). In this research.62 The antioxidant activity of each extract is tested with DPPH scavenging assay. Categories Antioxidant activity of methanol extract. These phenolic compounds and most other reported bioactive compounds are generally more soluble in polar solvents.8 1.76 0. Six samples from the three categories of Ceratonia siliqua L.

V. R. J. Morocco) for assistance during sampling of plant material. I. Chron.W. Hao.. Visioli. Kolybaba.. Brito de Carvalho.. Singleton. J. p. 1987. Cale Polterai.. 1999. Ingram. H.. Vinson.. Chim.324 the amounts of those compounds were significantly affected by the variety of the tree. Olive oil phenols and their potential effects on human health. Composition and Uses of Carob Bean.) and derived products by HPLC-UV-ESI/MS.-K.. 52. Saltmarsh. Haubne. Alkylperoxyl radical-scavenging activity of various flavonoids and other phenolic compounds: implications for the anti-tumor – promoter effect of vegetables. 1990. The Australian New Crops Newsletter. Abouricha. R. Chem. M. The carob: evaluation of trees. 1980. 257–258. Su. 46... Nair. Y. 91...K. C. Morocco) and the Sidi Mohamed Ben Abdellah University. J. H. Sanders. 22. Chem. Saleem. Vitic.. 1992. Int. 41–51. Polyphenols: antioxidants and beyond. Lambrakis. Rabat.. P. Haber.. 7.. Yang.. Hull. 415–440. Mulet. In: El Algarrobo (Ed).. Revs. 78. The phenolic compounds are commonly found in the plant kingdom. 570S–578S.W. 215–217.. 46. S.. Sawa. 1. F.C.. 2004. Charalambous. The Lancet 350. 2003. Phenol antioxidant quantity and quality in foods: vegetables.) from the province of Chefchaouen (NW of Morocco). Zubik. pear... 1993. K. 25. Ethnopharmaco... 3784–3791. Food Chem.. 2002. G. Moroccan J. 41. Identification and quantification of polyphenols in carob fruits (Ceratonia siliqua L. Carob Agroforestry in Portugal and Spain. J. Papaconstantinou. p.. C. 274–287.L. and white grape juices and their changes with processing and storage: a review. I. Madrid. Alaoui.). K. p.. The effects of plant flavonoids on mammalian cells: implications for . Akaike. Pihlaja.. Liu. Tree Crops J.. Agric. I. Sci. Toxicol. Chicha. L. 1996.L. A. Kandaswami. 397– 402. Wang.. A. Agric. S. J.... Fletcher.A. pods and kernels. Scalbert.. and cancer. Food. E. T. 1993.. Fruits and vegetables in the prevention of cellular oxidative damage. A. Hsu. (Ed. 673– 751. International Plant Genetic Resources Institute. 3630–3634. 27. Floral characterization of carob tree (Ceratonia siliqua L. Batlle. R.R..L. Chen. Human tumor cell growth inhibition by non-toxic anthocyanidins. Current Org. Am. Case-control study of phyto-oestrogens and breast cancer. Isozyme polymorphism in carob cultivars. the pigments in fruits and vegetables. 52.. Galli. Tous. (Eds.E. K. Isolation and structure elucidation of the major individual polyphenols in carob fiber. 76. Erben. 213– 224. Owen. W.E. M. Vitic. 28. Am.. W.... Ono. Proceedings of the II International Carob Symposium. New Fungal Strains for Microbial Protein Production from Carob Beans.. Galensa. J. R. In: Fito. 174. which we gratefully acknowledge. Nakao. Food.). S.. Husheem. 81. Biol. Enol. T. 4292–4296. A.. 2004..-C. Tous... J.. Food. Owen.A. 1727. E. California Rare Fruit Growers Yearbook III. Wrolstad.S... Spain.. M. 81. 1997. Tous.. Pharmacol... Criteria for evaluation of carob varieties. 1465–1472..). Bartsch. C. K. 144–158. S. T. Am.D. Maeda. inflammation. Y. C.. Life Sci. 3. J. O.. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. 2005. Inhibition of cancer cell growth by crude extract and the phenolics of Terminalia chebula retz. Huang. Middleton Jr.. Rossi. E.. El Mtili. F. M.H. Determination of phenolic composition of carob pods grown in different regions of Morocco. P. Gharnit. Mundi-Prensa. M.. S. 1965. 3. Marakis.. Marakis. A. Lopez. B. B. Italy. Food Chem. N. J. M.-J. Chem. Tannin chemistry of nine Cretan carob varieties. 47. 40. Rome.. H.-Y.. Blois. N. Antioxidant determinations by the use of a stable free radical. V. L. 1986. Spiegelhalder. P. M.. X. Paris. Hajaji et al. R. M.S.-P. 601–607.. Bouli.. Agric. 2005. J. 1966. Chem. Sur quelques plantes de nouvelle ´ donie. D. Food. Plantes me ´ dicinales et Phytothe ´ rapie 4. Carlson. J. 1958. Diamantoglou.. Prod.. Fruit J. A. 1997. Enol.. 1992. Nutr. 990– 994. Clin. Zhang. Food. J.. Singleton. Vareed. Harkonen. 281–290. J.G... A. The Carob in California.. Chem. Ph.. and they have been reported to have multiple biological effects including antioxidant activity. Nat. 2010. J.. 49– 55. Antioxidants and free-radical scavengers of Natural Origin. Thompson. Via delle Sette Chiese 142 00145. Nothis. 1977. 1478–1487. Sayah.. Clin.R...-D. 2003. J. Thesis. Nutr. B. Cyprus Agricultural Research Institute Ministry of Agriculture and Natural Resources Nicosia. 16. J. In: Charalambous.. R. H. Spanos. 2005. 2000. H. Papagiannopoulos. Prior.J. Hort.. The authors also thank the ADEMN Association (Chefchaouen. 1. 1997. 327–336. R. Rakib. Greece. Carob Tree (Ceratonia siliqua L. Agric. Theoharides. A. D.. N. Acknowledgments This work was financially supported by the CNRST (Centre National de la Recherche Scientifique et Technique. Hansali. A. 134–140. Chem. University of Athens. Ennabili. Agric. J. Nature 181. M. M. Wollseifen... 1199–1200. Total phenol analysis: automation and comparison with manual methods. J. Phenolics of apple. Johnson. Cyprus.T. 1971. 61. Am. Current results on the chemical composition of the carob bean. Mulberry extract inhibits the development of atherosclerosis in cholesterol-fed rabbits. Haber.G. Mellenthin. Issue No. heart disease. 1998. Slinkard. 1997.. Valencia.. H. M. References Battle. J.