Dakiche et al Tropical Journal of Pharmaceutical Research December 2016; 15 (12): 2563-2570 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v15i12.5 Original Research Article Biological activities and phenolic contents of Argania spinosa L (Sapotaceae) leaf extract Hadjira Dakiche1,2*, Mustapha Khali1, Ahmed Khaled Abu-el-Haija3, Ammar Al- Maaytah3 and Qosay Ali Al-Balas3 1Laboratoire de Protection et de Valorisation des Ressources Agro-Biologiques, Faculté de Sciences de la Nature et de Vie, Université Saâd Dahlab Blida, BP 270, Route de Soumaâ, Blida 09000, 2Centre de Recherche Scientifique et Technique en Analyses Physico-chimiques, BP 248, Alger RP, 16004 Alger, Algeria, 3Faculty of Pharmacy, Jordan University of Science and Technology, PO Box 3030, Irbid 22110, Jordan *For correspondence: Email: [email protected]; Tel: +213 555 497 211 Received: 20 June 2016 Revised accepted: 16 November 2016 Abstract Purpose: To investigate the phenolic profile and biological activities of Argania spinosa L. leaves. Methods: The crude methanol extract of leaves of A. spinosa L. Skeels was obtained by ultrasonic extraction, and the total polyphenolic and flavonoid contents were determined by ultra-high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (UPLC-ESI- QTOF-MS). In vitro antioxidant activity was determined by 2,2-di-phenyl-1-picryl-hydrazil (DPPH) radical assay and antimicrobial activity evaluated using agar disk diffusion method against reference pathogenic strains (Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 14579, Yersinia enterocolitica ATCC 23715, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922). Cytotoxic activity was evaluated by methyl-thiazolyldiphenyl-tetrazolium bromide (MTT) assay. Results: The results revealed abundant polyphenols and flavonoids (221.39 ± 5.70 µg GAEq/1 g and 66.86 ± 3.36 µg CAEq/1 g, respectively) in the leaf extract. UPLC-DAD-ESI-QTOF-MS profiling showed the presence of myrecitin-3-galactoside, myrecitin-3-gluctoside, myrecitin-3-xyloside, quercetin-3- galactoside, quercetin-3-glucoside, quercetin-3-arabinofuranoside, and quercetin-3-rhamnoside. DPPH assay of the leaf extract yielded a half-maximal effective concentration (EC50) value of 125.60 ± 1.87 µg. A. spinosa L. leaves exhibited antibacterial activity against Gram (+) and Gram (-) bacteria, with particularly marked activity against B. subtilis (inhibition zone, 16 mm). Cytotoxicity data showed that the extract inhibited the proliferation of PC3 cells (IC50 ~ 600 µg). Conclusion: A. spinosa L. leaf extract is rich in valuable biologically active compounds and could represent a new resource for natural and preventive therapies. Keywords: Argania spinosa, Polyphenols, Flavonoids, Antioxidant, Antibacterial, Cytotoxic Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION [1]. This species is the most original essence of North Africa and is endemic to Algeria, growing Algeria has a diverse collection of flora, the in the southwest of Tindouf Province, from the majority of which may have therapeutic potential. Jebel Ouarkziz to Hamada, and in south-western Argania spinosa L., also known as the Argan Morocco [2]. This tree has been successfully tree, belongs to the Sapotaceae family and cultivated in some regions of northern Algeria, represents the only species of the genus Argania Trop J Pharm Res, December 2016; 15(12): 2563 Dakiche et al such as Stidia (Mostaganem Province), Chlef, method [6]. Gallic acid was used as the standard and Mascara. to generate a calibration curve, and measurements were carried out at 725 nm using Due to the presence of Argan oil, which has, for a UV-vis spectrophotometer (Shimadzu UV centuries, been used in the traditional treatment 1650, Italy). The results were expressed as of rheumatism, constipation, diabetes, and milligrams of gallic acid-equivalent per gram dry respiratory difficulties, as well as wounds and dry mass (mg GAE/g dry mass). skin, the seed kernel is the most valuable part of A. spinosa L. [3]. To date, the use of by-products Total flavonoids were estimated according to the or parts of Argan trees other than the kernel has method of Grand et al [7]. Quercetin was used as been limited (e.g., Argan leaves are used only in the standard to generate the calibration curve, livestock feed). This represents an economic loss and measurements were carried out at 510 nm due to its content of bioactive compounds. using a UV-vis spectrophotometer (Shimadzu UV Several phenolic compounds have been 1650, Italy). Results were expressed as detected in A. spinosa L. leaves from Morocco; milligrams of quercetin-equivalent per gram dry the main constituents are quercetin, hyperoside, mass (mg QAE/g dry mass). and myricetin [4], which have antioxidant, antimicrobial, and cytotoxic activities [5]. Polyphenol characterization by UPLC–ESI– QTOF–MS The present study was conducted to investigate and explore the potential of Argan leaves as a Polyphenolic compounds were identified using by-product, analyze phenolic compounds using an Acquity Ultra-Performance LC system (UPLC) UPLC-DAD-ESI-QTOF-MS and assess the coupled to a XEVO-G2-QTOF (quadrupole time- biological activities and pharmaco-toxicological of-flight) mass spectrometer (Waters properties (e.g., antioxidant, antibacterial, and Corporation, Manchester, United Kingdom), cytotoxic effects) of leaves of the Argan tree. equipped with an electrospray ionization (ESI) source operating in positive and negative modes, EXPERIMENTAL using the binary solvent manager software (Waters Corporation, UK). Plant materials Chromatographic separation was carried out on A. spinosa L. leaves were collected in the Stidia a UPLC HSS T3, C18 column (1.8 μM, 2.1 × 100 o region, Mostaghanem, north-western Algeria mM, Waters Corporation) at 30 C. The mobile (latitude 35°48’N, longitude 0°03’E; altitude 35 phase comprised Solvent (A) (0.1 % formic acid, m) from June to July 2014. The authentication v/v) and Solvent (B) (100 % methanol). The was performed by Professor H. Abdelkrim gradient used was 0–5 min, 0–10 % B; 5–10 min, (National Hight School of Agronomy, Algiers, 10–15 % B; 10–15 min, 15-20 % B; 15–20 min, Algeria). The voucher specimen was deposited in 20–30 % B; 20–30 min, 30-40 % B; and 30–35 the herbarium of Laboratory of Protection and min, 40-0 % B. The solvent flow rate was 0.4 Valorization of Agro-biological Resources, mL/min. The injection volume was 2 μL. University Saad Dahleb Blida, Algeria. The leaves were air-dried, powdered, and conserved Analysis was performed at a wavelength of 280 until used. nm, data depending MS scanning from 50 to 1500 m/z. Analysis was performed in positive Preparation of extract and negative ion modes using an electrospray source with a source temperature of 120 °C and Powdered A. spinosa L. leaves (20 g) were a desolvation temperature of 500 °C, a capillary defatted using petroleum ether (60 – 80 °C) and voltage of 1 kV, and a sampling cone voltage of then extracted with 200 ml of methanol/water (70/30, v/v) in an ultrasonic bath for 30 min. The 30 eV. Highly pure N2 (˃ 99.9995 %) was used solvent was then evaporated, and the extract as the desolvation gas (flow rate, 10 L/h) and was obtained with a yield of 13 %. The extract cone gas (flow rate, 1000 L/H). was stored in the dark at -20 °C until use. Chemical analysis Data were acquired using the MassLynx software (version 4.1, Waters). The exact masses and MS Total polyphenol and flavonoid determination fragmentation data were compared with those in the respect for phytochemicals and Mass Bank The total phenolics in the methanolic extract metabolomics data banks and in previous reports were determined by the Folin and Ciocalteu to identify phenolic compounds. Trop J Pharm Res, December 2016; 15(12): 2564 Dakiche et al Antioxidant assay Cytotoxicity assay DPPH assay of the methanolic extract of leaves The PC-3 human prostate cancer cell line was of A. spinosa L was carried out as described by used (ATCC). Cells were plated in RPMI 1640 Blois [8]. Dried extract was weighed and medium supplemented with 10 % FBS and 1 % reconstituted to known concentrations. The penicillin-streptomycin. All cultures were extract (0.3 mL) and a methanolic solution of incubated at 37 °C in a humidified atmosphere containing 5 % CO2. Cells were plated at 5.0 × DPPH (2.7 mL; 5 mg in 100 mL methanol) were 3 mixed and incubated at room temperature for 1 h 10 /well in 96-well plates and allowed to attach in the dark. The absorbance at 515 nm was then for 24 h. Cells were then treated for 24 h under measured (Shimadzu UV 1650, Italy). A control the conditions described above in the presence of 50–1000 μg/mL plant extract dissolved in solution was prepared with 0.3 mL methanol and DMSO followed by filtration through a 0.22-μm 2.7 mL methanolic solution of DPPH. Ascorbic syringe filter. Stock solutions were further diluted acid and Trolox were used as reference in culture medium to the appropriate compounds. Radical scavenging activity is concentrations. DMSO (1 %) functioned as a expressed as the percentage inhibition of DPPH vehicle control and had no effect on cell growth (D) as in Eq 1. [10]. D (%) = {(Ac – At)/Ac}100 …………… (1) The methyl-thiazolyldiphenyl-tetrazolium bromide (MTT) assay, which is based on conversion of where Ac and As are the absorbance of control the yellow tetrazolium salt-MTT to purple and test samples, respectively.