View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Arabian Journal of Chemistry (2015) xxx, xxx–xxx King Saud University Arabian Journal of Chemistry www.ksu.edu.sa www.sciencedirect.com ORIGINAL ARTICLE The chemical composition on fingerprint of Glandora diffusa and its biological properties Fa´tima Fernandes a, Paula B. Andrade a, Federico Ferreres b, Angel Gil-Izquierdo b, Isabel Sousa-Pinto c,d, Patrı´cia Valenta˜ o a,* a REQUIMTE/LAQV, Laborato´rio de Farmacognosia, Departamento de Quı´mica, Faculdade de Farma´cia, Universidade do Porto, R. Jorge Viterbo Ferreira, n° 228, 4050-313 Porto, Portugal b Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus University Espinardo, Murcia, Spain c Interdisciplinary Centre for Marine and Environmental Research (CIIMAR/CIMAR), Rua dos Bragas n° 289, 4050-123 Porto, Portugal d Faculty of Sciences, University of Porto (FCUP), Rua do Campo Alegre s/n, 4169-007 Porto, Portugal Received 26 October 2014; accepted 25 January 2015 KEYWORDS Abstract Glandora diffusa (Lag.) D.C. Thomas is a medicinal species widely consumed as herbal Glandora diffusa (Lag.) D.C. tea. Despite being commercialized by several herbs distributors, the genuineness of the marketed Thomas; product is unknown. Among secondary metabolites with proven taxonomic interest, the phenolics Metabolic profile; profile was herein used as ‘‘fingerprint’’ of three commercial G. diffusa samples. Furthermore, the Cholinesterase; knowledge on the composition of this species was extended and its amino acids, fatty acids, sterols Cytotoxicity and triterpenes profiles were studied for the first time. The phenolics profile was characterized by HPLC-DAD. All other metabolites were determined by GC–MS. Despite similar qualitative pro- files, significant quantitative differences were observed among the three samples. Their potential as antioxidant and anti-Alzheimer and cytotoxicity was evaluated and relationship between chemi- cal composition and activities was considered. Ethanolic extracts showed a potent dose-dependent response against DPPHÅ, a mild inhibitory effect on both acetylcholinesterase (AChE) and butyryl- cholinesterase (BuChE) and to be toxic to both human colorectal (Caco-2) and gastric adenocarci- noma (AGS) cells only for high concentrations. This work covers interdisciplinary aspects in the pharmaceutical and biomedical sciences, by focusing on metabolic profiling and quality assurance of a medicinal species used in folk medicine. The results improve the knowledge on G. diffusa and * Corresponding author. Tel.: +351 220428653; fax: +351 226093390. E-mail address: [email protected] (P. Valenta˜ o). Peer review under responsibility of King Saud University. Production and hosting by Elsevier http://dx.doi.org/10.1016/j.arabjc.2015.01.012 1878-5352 ª 2015 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/4.0/). Please cite this article in press as: Fernandes, F. et al., The chemical composition on fingerprint of Glandora diffusa and its biological properties. Arabian Journal of Chemistry (2015), http://dx.doi.org/10.1016/j.arabjc.2015.01.012 2 F. Fernandes et al. encourage its use, not only as herbal tea, as it is traditionally consumed, but also in pharmaceutical preparations, ethanol being a cheap and feasible solvent to recover its bioactive components. ª 2015 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/4.0/). 1. Introduction As far as we are aware, the only study available is related to the characterization of its phenolics and was performed by Medicinal plants have been used for centuries throughout the our group (Ferreres et al., 2013). Thus, the aim of this work world and many people still rely on indigenous medicinal was to use phenolics profile as ‘‘fingerprint’’ for authenticity plants for their safe or primary health care needs (Daur, control of commercialized G. diffusa material and to improve 2013). The reasons for the application of specific medicinal the knowledge on its metabolic composition. For this purpose, plants in the treatment of certain diseases are being discovered three distinct commercial samples of G. diffusa were analysed and the use of those species gradually abandons the empiric regarding primary and secondary metabolites. Since the chemi- framework and becomes reasoned on explicatory facts cal profile can greatly influence the biological effects (Milella (Petrovska, 2012). Nowadays, the potential of a panoply of et al., 2011), the samples were also tested as inhibitor of acetyl- cholinesterase (AChE) and butyrylcholinesterase (BuChE), phytochemicals, such as phenolic compounds, fatty acids, enzymes associated with the aetiology of Alzheimer’s disease sterols, triterpenes and glucosinolates, in the prevention or (AD) (Lle´o, 2007), for antiradical capacity, since reactive treatment of several pathologies, such as cardiovascular species are in the origin of several pathologies, and for diseases, diabetes, neurodegenerative disorders and cancer, is cytotoxicity on gastric and intestinal carcinoma cells lines, as known (Burda and Oleszek, 2001; Fan et al., 2010; Ferreres gastrointestinal cancers rank second in overall cancer-related et al., 2014; Hooper and Cassidy, 2006; Lle´o, 2007; deaths (Surh, 2003). Ramadan and El-Shamy, 2013; Surh, 2003). In fact, the plant The comparison of the samples is interesting because the kingdom is an inexhaustible source of health-promoters differences observed will be representative of the procedure metabolites (El-Chaghaby et al., 2014). followed by each supplier, from production to selection, In the recent years, the quest for natural food additives has transformation and conservation of the vegetal materials. This become an increasing concern. Consumers’ demand for health- information may be important for consumers’ choice. ier foods has been the initiative for many researchers seeking for natural alternatives (El-Chaghaby et al., 2014). Several herbs with recognized therapeutic applications and used as 2. Materials and methods medicinal plants are often undervalued by unawareness of their metabolic profile, as they are often important sources 2.1. Standards and reagents of bioactive compounds. Glandora diffusa (Lag.) D.C. Thomas (Boraginaceae) Quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, isorham- (synonyms: Lithospermum diffusum Lag. and Lithodora diffusa netin-3-O-glucoside and rosmarinic acid were from Extrasyn- (Lag.) I.M. Johnst.) is one of the six species of the genus these (Genay, France). Caffeic and p-coumaric acids, alanine, Glandora, being commonly known as ‘‘scrambling-gromwell’’. glycine, valine, isoleucine, serine, threonine, trans-4-hydrox- This herbal species is a medicinal plant spontaneously found in yproline, norvaline, pelargonic acid, capric acid, lauric the Mediterranean area (Ferreres et al., 2013; Thomas et al., acid, myristic acid, palmitic acid, margaric acid, linoleic acid, 2008). Its pollen is present in some honeys and the herbal a-linolenic acid, oleic acid, stearic acid, docosahexaenoic acid tea, as it is traditionally consumed, is used as diuretic, (DHA), methyl linolelaidate, as well as lanosterol, cholesterol, depurative and anti-hypertensive (Sa´-Otero et al., 2006). These campesterol, betulin, stigmasterol, b-sitosterol, lupeol, lupeol properties sparked the attention of researchers and pharmaco- acetate, desmosterol, N-methyl-N-(trimethylsilyl) trifluoroac- logical studies have been conducted in order to explore other etamide (MSTFA), 1,1-diphenyl-2-picrylhydrazyl radical potentialities of G. diffusa. In a recent work, Ferreres and (DPPHÅ), b-nicotinamide adenine dinucleotide reduced form collaborators reported the presence of high amounts of several (NADH), sodium pyruvate, AChE from electric eel (type kinds of phenolics in the aqueous extract, its good antiradical VI-s, lyophilized powder), acetylthiocholine iodide (ATCI), activity and strong ability to inhibit a-glucosidase, an BuChE from equine serum (lyophilized powder), S-butyrylth- enzyme related to diabetes mellitus (Ferreres et al., 2013). iocholine chloride (BTCC), 5,50-dithiobis(2-nitrobenzoic Nevertheless, the interest in these metabolites can go further. acid) (DTNB), dimethyl sulfoxide (DMSO), triton X-100 and As secondary metabolism products, phenolic compounds are (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide specific of certain families, constituting important chemo- (MTT) were obtained from Sigma–Aldrich (St. Louis, MO, taxonomic markers. According to this, phenolic profiles can USA). The n-alkane series (C8–C40) was purchased from be successfully used in the determination of authenticity of Supelco (Bellefonte, PA, USA). Methanol, potassium dihydro- different products from vegetal origin (Ferreres et al., 2014). gen phosphate, acetonitrile and phosphoric, formic and acetic In Portugal, G. diffusa aerial parts are widely commercial- acids were obtained from Merck (Darmstadt, Germany) and ized by several plants distributors, being sold in herbal shops. sulphuric acid from Pronalab (Lisboa, Portugal). Reagents The marketed product (the one that is consumed) corresponds for cell culture were obtained