Ulex Europaeus: from Noxious Weed to Source of Valuable Isoflavones And

Industrial Crops and Products 90 (2016) 9–27 Contents lists available at ScienceDirect Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop Ulex europaeus: from noxious weed to source of valuable isoflavones and flavanones a b c Vítor Spínola , Eulogio J. Llorent-Martínez , Sandra Gouveia-Figueira , a,∗ Paula C. Castilho a CQM—Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9020-105 Funchal, Portugal b University of Castilla-La Mancha, Regional Institute for Applied Chemistry Research (IRICA), Ciudad Real 13071, Spain c Department of Chemistry, Umeå University, 901 87 Umeå, Sweden a r t i c l e i n f o a b s t r a c t Article history: The screening and quantification of the main phenolic compounds in leaves and flowers of Ulex europaeus Received 24 February 2016 (gorse) was carried out by high-performance liquid chromatography with electrospray ionization mass Received in revised form 27 May 2016 n spectrometric detection (HPLC-ESI–MS ) after ultrasound-assisted extraction with methanol. About 98% Accepted 3 June 2016 of compounds corresponded to flavonoids, distributed as flavonols, flavones, isoflavones and flavanones. Flavonols were mainly quercetin glucosides; most of the found flavones were apigenin derivatives and Keywords: the isoflavone group was dominated by glycitin. The flavanone group was composed mainly of liquir- Ulex europaeus itigenin derivatives, substances usually found in liquorice (Glycyrrhiza ssp) and associated with high Isoflavones Liquiritigenin pharmacological relevance; in Ulex they represent about 25% of total polyphenols content. Phenolic acids HPLC-ESI/MSn analysis and saponins were also detected, as minor components. In vitro antioxidant activity (nitric oxide, super- Antioxidant activity oxide assays, ABTS and DPPH assays) of leaves and flowers, and their inhibitory effects towards digestive ␣ ␣ Digestive enzymes inhibition enzymes related to carbohydrate metabolism ( -glucosidase and -amylase) were also studied. © 2016 Elsevier B.V. All rights reserved. 1. Introduction are a rich source of isoflavonoids (in particular isoflavones and pterocarpans) with relevant antifungal activity, insecticide or cyto- The genus Ulex L. (Fabaceae) is well represented in Portugal, toxic effects (Máximo et al., 2002a, 2002b, 2000; Veitch, 2007). where ten species are recognized and some of them are endemic Isoflavones are considered to belong to the “phytoestrogen” class, (Máximo et al., 2002a, 2002b). Ulex europaeus L. (gorse, furze or due to their similar effects to mammalian estrogens (Veitch, 2013). whin) is native mainly to Western Europe and was introduced in Genistein, daidzein and glycitein are considered the most impor- the early 19th century in Madeira Island (Portugal), where it rapidly tant isoflavones, due to their varied biological actions, dependent became invasive. It blooms all year, but mainly between January on their aglycone and conjugated forms (Vitale et al., 2013). These and June (Press and Short, 1994). The flowers have been used in isoflavones are abundant in soybeans and related products and in folk medicine as infusions with sugar cane liquor as antirheumatic, other edible Fabaceae, such as lupin and fava beans. Flavanones and also for the treatment of liver diseases, diabetes, asthma, are intermediates in the biosynthesis of other flavonoids and are and hypertension (Rivera and Obón, 1995), whereas leaves were present in most plants, but accumulate particularly in Asteraceae used as forage shrub considering their high crude protein con- and Fabaceae. Flavanones and their isomeric chalcones intercon- tent (Tolera et al., 1997). Previous reports stated that Ulex species vert enzymatically in most of these species, so it is common to find both types of structures. Naringenin and liquiritigenin are the flavanones that present the most interesting biological activities. In particular, liquiritigenin n Abbreviations: DE, dry extract; HPLC-ESI–MS , high-performance liquid chro- and its conjugated forms display antioxidant, anti-inflammatory ␤ matography with electrospray ionization mass spectrometric detection; NADH , and antitumor activities and neuroprotective effects (Peng et al., nicotinamide adenine dinucleotide reduced; NBT, nitroblue tetrazolium chloride; 2015). Licorice root is the main commercial source of liquiritigenin NO, nitric oxide; PCA, Principal component analysis; PMS, phenazinemethosulfate; derivatives (Tian et al., 2009) and most publications describing pNPG, pnitrophenyl-␣-d-glucopyranoside; SO, superoxide; TFC, total flavonoid con- the bioactivity of these compounds refer to isolates from liquorice tent; TIPC, total individual phenolic content; TPC, total phenolic content. ∗ Corresponding author. extracts. Harborne (Harborne, 1972) reported the presence of E-mail addresses: [email protected], [email protected] (P.C. Castilho). http://dx.doi.org/10.1016/j.indcrop.2016.06.007 0926-6690/© 2016 Elsevier B.V. All rights reserved. 10 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 isoliquiritigenin glucosides in the flowers of Ulex as early as 1962. 250 × 3.0 mm i.d.) using a mobile phase composed by CH3CN (A) −1 However, the screening and quantitative relevance of these com- and water/formic acid (0.1%, v/v) at a flow rate of 0.4 mL min . ponents of Ulex is not described. The phytochemical composition of The following gradient program was used: 20% A (0 min), 25% A n Ulex europaeus L. is here determined by means of an HPLC-ESI–MS (10 min), 25% A (20 min), 50% A (40 min), 100% A (42–47 min) and −1 method. The quantification of the main polyphenols from leaves 20% A (49–55 min). Sample solutions (5 mg mL ) were prepared and flowers was also carried out, as well as their in vitro antiox- by dissolving the dried extract in the initial HPLC mobile phase. idant activities (ABTS, DPPH, nitric oxide and superoxide assays) After filtration through 0.45 ␮m PTFE membrane filters, 5 ␮L was and inhibitory effects towards digestive enzymes related to carbo- injected. n hydrate metabolism (␣-glucosidase and ␣-amylase). For HPLC-ESI–MS analysis, a Bruker Esquire model 6000 ion trap mass spectrometer (Bremen, Germany) with an ESI source was n 2. Experimental used. MS analysis was performed in negative and positive modes and scan range was set at m/z 100–1000 with a speed of 13,000 Da/s. 2.1. Chemicals and reagents The ESI conditions were as follows: drying and nebulizer gas (N2) −1 flow rate and pressure, 10 mL min and 50 psi; capillary temper- ◦ All reagents and standards were of analytical reagent ature, 325 C; capillary voltage, 4.5 keV; collision gas (He) pressure −5 n × grade unless stated otherwise. Folin-Ciocalteu’s phenol reagent and energy, 1 10 mbar and 40 eV. The acquisition of MS data n (FCR), sodium chloride, potassium chloride, gallic acid (>98%), was made in auto MS mode, with an isolation width of 4.0 m/z, and n 4 quercetin hydrated (>99%) and potassium acetate (>99.5%) were a fragmentation amplitude of 1.0 V (MS up to MS ). Esquire con- obtained from Panreac (Barcelona, Spain). 6-hydroxy-2,5,7,8- trol software was used for the data acquisition and Data Analysis tetramethylchroman-2-carboxylic acid (Trolox), 2,2 azinobis-(3- for processing. ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1- picrylhydrazyl (DPPH) and methanol (99.9%) were obtained from 2.4. Quantification of phenolic compounds Fluka (Lisbon, Portugal). Kaempferol ( > 99%) was purchased from Acros Organics (Geel, Belgium). Apigenin (≥99%) was obtained from For this quantitative analysis, one polyphenol was selected Extrasynthese (Genay, France). N-(1-naphthyl)ethylene-diamine as the standard for each group, being used to calculate individ- dihydrochloride (≥98%), phenazinemethosulfate (PMS, ≥90%), sul- ual concentrations by HPLC-DAD. Caffeic and gallic acids were fanilamide (≥99%), ␤-nicotinamide adenine dinucleotide reduced used for hydroxycinnamic and hydroxybenzoic acids, respectively. (NADH, ≥94%), caffeic acid (≥98%), protocatechuic acid (98%, Quercetin, apigenin, and liquiritin were the standards used for the HPLC), rutin (≥95%), potassium persulfate (99%), sodium carbon- flavonols, flavones, and flavanones, respectively. Stock standard − ate (100%), ␣-glucosidase from Saccharomyces cerevisiae (type I), 1 solutions (1000 mg L each) were prepared in methanol, and cal- ␣-amylase from porcine pancreas (type VI-B), p-nitrophenyl-␣-d- ibration curves were prepared by diluting the stock solutions with − glucopyranoside (pNPG) and formic acid (98%) were purchased 1 the initial mobile phase. Six concentrations (5–100 mg L ) were from Sigma-Aldrich (St. Louis, MO, USA). Nitroblue tetrazolium used for the calibration, plotting peak area versus concentration chloride (NBT, 90%) was obtained from Acros Organics and o- 2 (R ≥ 0.967 in all cases). Total individual phenolic contents (TIPC) phosphoric acid (85%) from BDH AnalaR. Hydrochloric acid (37%) were defined as the sum of the quantified phenolic compounds. was purchased from Fischer Chemicals (Leicestershire, UK). Liquir- itin (≥ 98%) was obtained from Biopurify phytochemicals LTD 2.5. Total phenolic and flavonoid contents and in vitro (Chengdu, China). Potato starch (p.a.), potassium iodate (99.5%), antioxidant assays sodium nitroprusside (99%), and ethylenediaminetetraacetic acid (EDTA; >99%) were obtained from Merck (Darmstadt, Germany). 2.5.1. Total phenolic content (TPC) ® Acarbose (Acarbose Generis ) was purchased from a drug store. The TPC was determined following a previous described proto- LC–MS grade

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