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 acetonitrile (CH3CN, 99%) (LabScan; Dublin, Ireland)

col (Spínola et al., 2014): 50 ␮L aliquots (5 mg of dry extract, DE,

and ultrapure water (Milli-Q Waters purification system; Millipore;

per mL of methanol) were mixed with 1.25 mL of FCR (diluted 1:10

Milford, MA, USA) were used for the HPLC–MS analyses.

with water) and 1 mL of 7.5% Na2CO3 aqueous solution. After 30 min

in the dark at room temperature, the absorbance was measured

2.2. Sample preparation and extraction of phenolic compounds

at 765 nm in a Perkin Elmer UV–vis Lambda 2 spectrophotome-

ter (Oberlingen, Germany). The amounts of total phenolics were

Samples of U. europaeus were collected in the wild at two differ-

expressed as mg of gallic acid equivalents (GAE)/100 g of DE.

ent locations of Madeira Island (Funchal and Machico) in January

2015. For analysis, leaves were separated from flowers, lyophilized

2.5.2. Total flavonoid content (TFC)

to dryness (Alpha 1-2 LD plus freeze dryer, CHRIST), ground to

◦ The total flavonoid content was determined as follows (Spínola

−

powder in a mechanic grinder, and stored at 20 C. Then, 1 g of −1

et al., 2014): 0.5 mL of methanolic solutions (2.5 mg mL ) was

dried leaves was extracted with 25 mL of methanol using a soni-

mixed with 1.5 mL of methanol, 2.8 mL of distilled water, 0.1 mL

cator Bandelin Sonorex (Germany) at 35 Hz and 200 W for 60 min −1

of CH3COOK (1 mol L ), and 0.1 mL of AlCl3·6H2O (10% in MeOH).

(room temperature). Chlorophylls were removed by adsorption on

The absorbance was measured at 415 nm after 30 min of reaction

activated charcoal, and extracts were filtered and concentrated

(room temperature, in the dark). The final results were expressed

to dryness under reduced pressure in a rotary evaporator (Buchi

◦ as mg of rutin equivalent (RUE)/100 g DE.

Rotavapor R-114; USA) at 40 C. The resulting extracts were stored

◦

at 4 C until analysis.

2.5.3. ABTS radical scavenging activity

Determination of antioxidant activity was based on a previ-

2.3. Chromatographic conditions

ous procedure (Spínola et al., 2014): 40 ␮L of methanolic solution

−1 •+

(5 mg mL ) was added to 1.96 mL of ABTS solution (diluted in

The HPLC analysis was performed on a Dionex ultimate 3000

PBS pH 7.4 until the absorbance is 0.700 ± 0.021). The reduction of

series instrument (Thermo Scientific Inc.) coupled to a binary pump,

◦

absorbance at 734 nm was measured during 6 min, and the results

an autosampler and a column compartment (kept at 20 C). Sepa-

were expressed as ␮mol of Trolox equivalent (TE)/100 g DE.

ration was carried out in a Phenomenex Gemini C18 column (5 ␮m,

V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 11

2.5.4. DPPH radical scavenging activity was applied to the autoscaled concentrations of the determined

The DPPH assay followed a previously reported method (Spínola polyphenols in Ulex samples from different locations, using an R

−1

et al., 2014): 100 ␮L of methanolic solution (5 mg mL ) was added statistical software package.

−1

to 3.5 mL of DPPH radical solution (0.06 mol L ). Absorbance was

measured at 516 nm, after 30 min of reaction in the dark (room

3. Results and discussion

temperature) andresults were expressed as ␮mol TE/100 g DE.

The analysis of the phenolic composition of U. europaeus leaves

2.5.5. Nitric oxide (NO) scavenging activity n

and flowers by HPLC-ESI–MS was carried out in positive and nega-

The antiradical activity was determined spectrophotometri-

3 tive ionization modes. Two independent assays were performed for

cally using a model Victor microtiter reader (Perkin-Elmer,

each sample, and no relevant variations were observed concerning

Ueberlingen, Germany), with slight modifications from a previous

the nature and relative intensities of the detected fragments. The

procedure (Sousa et al., 2008). Briefly, 50 ␮L of 20 mM sodium nitro-

−1 base peak chromatograms of the methanolic extracts are shown in

prusside was mixed with 50 ␮L of sample (5 mg mL ) for 60 min,

Fig. 1 (only the most abundant compounds are numbered for the

at room temperature, under light. All solutions were prepared in

sake of clarity).

0.1 M phosphate buffer (pH 7.4). After incubation, 50 ␮L of Griess

An essential step was to determine the molecular ion of each

reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihy- − 1

compound. In the negative ionization mode (ESI ) MS spectrum,

drochloride in 2% phosphoric acid), was added to each well. Then,

the most intense peak usually corresponded to the deprotonated

the absorbance was read at 550 nm and the results were expressed −

−

molecular ion [M H] , although sometimes formic adducts were

as ␮mol TE/100 g DE. −

observed ([M−H + HCOOH] ). The mass spectra of the conjugated

phenolic compounds showed the aglycone ion as result of the loss

2.5.6. Superoxide radical (SO) scavenging activity

of sugar moieties like hexosyl or pentosyl (−162, −132 Da, respec-

Superoxide radicals were generated by the NADH/PMS system +

tively). Mass spectra data from the positive ionization (ESI ) mode

according to a described procedure (Ewing and Janero, 1995): 25 ␮L

−1 was only used for confirmation purposes. Compounds were num-

of sample (5 mg mL ) was mixed with 200 ␮L of a solution com-

−1 −1 −1 bered by their order of elution. The structures of the most relevant

posed by 0.1 mmol L EDTA, 62 ␮mol L NBT and 98 ␮mol L

compounds are shown in Fig. 2.

NADH. The reaction was initiated by the addition of 25 ␮L of

−1

33 ␮mol L PMS (containing 0.1 mM EDTA) to each well. All solu-

tions were prepared in 0.1 M phosphate buffer (pH 7.4). The 3.1. Phenolic acids

3

absorbance was read at 550 nm (Victor microtiter reader; Perkin-

− 2

−

Elmer, Ueberlingen, Germany) and the results were expressed as Compound 6 with the [M H] ion at m/z 461 and MS base

␮mol TE/100 g DE. peak at m/z 167 was tentatively characterized as vanillic acid 4-

O-pentosylhexoside (Maier et al., 2015).

−

−

2.6. In vitro inhibition of digestive enzymes Compounds 10, 20 and 32 exhibited the [M H] ions at m/z 341,

2

and suffered the neutral loss of 162 Da (hexoside) yielding an MS

3

→

2.6.1. ˛-Glucosidase inhibition assay base peak at m/z 179. The MS [341 179] fragmentation produced

In a 96-well plate, 50 ␮L of extract solution was combined with an ion at m/z 135, indicating that the ion at m/z 179 corresponded

50 ␮L of enzyme solution and incubated for 20 min in the dark at to caffeic acid (compared with a commercial standard). Therefore,

room temperature (Podsedek˛ et al., 2014). A gradient of concentra- they were tentatively identified as caffeic acid-O-hexoside. Com-

−

−

tions was prepared via serial dilutions of the extracts in distilled pound 7, with [M H] at m/z 503, displayed a fragment ion at

−1 3

water. The reaction was initiated by adding 50 ␮L of 5 mmol L m/z 341 (loss of 162 Da), and its MS fragmentation was similar

−1

p-NPG solution in 0.1 mol L phosphate buffer (pH 7.0). The mix- to compound 10, so this compound was tentatively identified as

◦

ture was incubated at 37 C, in the dark, for 20 min. Finally, 100 ␮L caffeic acid-O-dihexoside. Compounds 63 and 113 also displayed

−1 n

of 0.1 mol L Na2CO3 solution was added and the absorbance MS fragment ions at m/z 179 and 135 and were tentatively char-

was read at 405 nm. Acarbose was used as positive control. The acterized as caffeic acid derivatives.

−1 −

−

inhibitory activity was expressed as the IC50 value (mg mL of Compound 23, with [M H] at m/z 651, suffered the neutral loss

DE), determined from the least-squares regression line of the log- of 326 Da, yielding a fragment ion at m/z 325. The ion at m/z 325

arithmic concentrations plotted against percentage inhibition. exhibited the typical fragmentation of p-coumaric acid-O-hexoside,

3

with MS [651 → 325] fragment ions at m/z 163 and 119 (Sánchez-

2.6.2. ˛-Amylase inhibition assay Rabaneda et al., 2003). It was tentatively characterized as a dimer of

−1

Twenty microliters of extract solution and 40 ␮L of 2 g L starch p-coumaric acid hexoside. Compound 11 was tentatively identified

solution were mixed with 20 ␮L of ␣-amylase (in phosphate buffer as coumaric acid-O-hexoside (formate adduct). Compounds 46 and

7.0) (Podsedek˛ et al., 2014). A gradient of concentrations was pre- 124 also displayed the typical fragmentation pattern of coumaric

pared via serial dilutions of the extracts in distilled water. After acid, so they were tentatively characterized as derivatives.

◦

incubation at 37 C for 20 min in the dark, the reaction was stopped Several derivatives of ferulic acid were observed in the analyzed

−1

by the addition of 80 ␮L of HCl followed by 100 ␮L of 5 mmol L samples. Compound 16 was tentatively identified as a dimer of

−1 −

−

I2 (in 5 mmol L KI), and the absorbance was read at 620 nm. ferulic acid-O-hexoside. Compound 26, with [M H] at m/z 487,

Acarbose was used as positive control. The inhibitory activity was suffered neutral losses of 132 Da (pentoside) and 132 + 162 Da (pen-

expressed as the IC50 value, as described previously. toside + hexoside), yielding fragments at m/z 355 and 193; it was

tentatively characterized as ferulic acid-O-pentosylhexoside. Com-

2.7. Statistical analysis pounds 31, 37, and 42 displayed the deprotonated molecular ion

at m/z 355. Compound 31 suffered the neutral loss of 162 Da (hex-

2

All samples were assayed in triplicate (n = 3) and results are oside) to yield the MS base peak at m/z 193, which displayed the

given as the means ± standard deviations. Data were analyzed by fragment ions characteristic of ferulic acid at m/z 178, 149 and

means of a one-way ANOVA using SPSS for Windows, IBM SPSS 134. Hence, this compound was tentatively characterized as fer-

Statistics 20 (SPSS, Inc., USA). A value of p < 0.05 was consid- ulic acid-O-hexoside (Fang et al., 2002). Compound 37 showed a

−

ered statistically significant. Principal component analysis (PCA) fragment ion at m/z 337 [M−H−18] , indicative of a 6-C glycoside

12 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27

n

Fig. 1. HPLC-ESI/MS base peak chromatograms (BPC) of the methanolic extracts from Ulex europaeus leaves and flowers collected in Funchal.

(Waridel et al., 2001), whereas the absence of the loss of water in 71, 89, and 96 were tentatively identified as quercetin-O-hexoside

the spectrum of 42 indicated a 8-C glycoside. Hence, compounds 37 isomers.

−

and 42 were tentatively identified as ferulic acid-6-C-hexoside and Compounds 17 and 22, with [M−H] at m/z 609, suffered the

ferulic acid-8-C-hexoside, respectively. Following a similar frag- sequential losses of two hexosides, to produce fragment ions at

mentation pattern, compound 45 was tentatively characterized as m/z 447 and 285, and were tentatively identified as kaempferol-O-

−

a ferulic acid-C-hexoside derivative (loss of 60, 90, 120, and 162 Da). dihexoside (Ye et al., 2005). Compound 33 displayed the [M−H]

Compounds 28, 41, and 126 could not be fully identified and were ion at m/z 449 and, after the neutral loss of 162 Da (hexoside),

tentatively characterized as ferulic acid derivatives. yielded a fragment ion at m/z 287, which was tentatively iden-

− 2

Compound 39, with [M−H] ion at m/z 385, exhibited MS tified as dihydrokaempferol, based on the 287 → 259 transition.

− −

fragment ions at m/z 265 [M−H−120] , 295 [M−H−90] and Hence, 33 was tentatively characterized as dihydrokaempferol-O-

−

325 [M−H−60] , characteristic of C-glycosides. The aglycone was hexoside (Llorent-Martinez et al., 2015).

observed at m/z 223. This fragmentation is consistent with the bibli- Compound 18 exhibited the deprotonated molecular ion at m/z

ographic data for sinapic acid-C-hexoside (Vallverdú-Queralt et al., 465, and suffered the neutral loss of 162 Da to yield the aglycone

3

2014). at m/z 303. The MS [465 → 303] spectrum displayed fragment

Compound 84 exhibited the deprotonated molecular ion at m/z ions at m/z 177 and 125. This fragmentation pattern is consistent

515 and was tentatively identified as 3,5-O-dicaffeoylquinic acid by with taxifolin-O-hexoside (Hashim et al., 2013). In addition, the

4

comparison of its fragmentation with bibliographic data (Gouveia MS spectrum provided more fragment ions described for taxifolin

and Castilho, 2009). (dihydroquercetin) (Ye et al., 2012), confirming the identification

of the aglycone.

−

Compound 21, with [M−H + HCOOH] at m/z 639, produced

3.2. Flavonoids − −

fragment ions at m/z 473 [M−H−120] , 431 [M−H−162] , and

−

− 269 [M−H−162−162] , yielding the aglycone at m/z 269. This

Compound 8, [M−H + HCOOH] at m/z 653, was tentatively

fragmentation pattern has been previously described for apigenin-

identified as acacetin-O-dihexoside (formate adduct), since it suf-

C-hexoside-O-hexoside (Yang et al., 2011). Compound 51 exhibited

fered the neutral loss of 208 Da (hexoside + formic acid), yielding a

the deprotonated molecular ion at m/z 431, and suffered the

fragment ion at m/z 445, followed by the loss of another hexoside

neutral loss of 162 Da to yield apigenin, so it was tentatively iden-

residue (445 → 283). The aglycone was characterized as acacetin

tified as apigenin-O-hexoside (Qiao et al., 2011). Compound 73,

due to the fragment ion at m/z 268 (Parejo et al., 2004). The pos- −

with [M−H + HCOOH] at m/z 477, displayed similar fragmentation

itive mode confirmed this identification, showing the 285 → 270

pattern than 51 and was also tentatively characterized as apigenin-

transition, typical from acacetin (Shi et al., 2011). n

− O-hexoside. Compounds 80 and 90 presented MS fragment ions

Compound 9, with [M−H + HCOOH] at m/z 623, suffered the

at m/z 431, 311, and 283, which are consistent with apigenin-C-

sequential losses of hexoside + formic (623 → 415) and hexoside

hexoside (Qiao et al., 2011), so they were tentatively characterized

(415 → 253), yielding the aglycone daidzein at m/z 253 (Kang et al.,

as derivatives. Compound 123 was tentatively identified as the

2007), so it was tentatively identified as daidzein-O-dihexoside.

− aglycone apigenin.

Compounds 13 and 44 exhibited [M−H] ions at m/z 625, and −

n Compound 24 showed [M−H + HCOOH] at m/z 507 and

suffered consecutive losses of 162 Da to yield MS fragment ions

suffered a neutral loss of 208 Da (162 + 46 Da). Sequential fragmen-

at m/z 463 and 301. The ion at m/z 301 displayed the characteristic −

tations produced fragment ions at m/z 299 [M−H−162] and 284

fragmentation pattern of quercetin (comparison with a commercial −

[M−H−162−15] . Based on literature (Ma et al., 2014), 24 was

standard). Hence, these compounds were tentatively identified as  

tentatively identified as 3 5,7-trihydroxyisoflavone-4 -methoxy-

quercetin-O-dihexoside isomers. In a similar way, compounds 56,

V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 13

R1 OH R2 HO O HO 8 O R 7 9 2 1 R3 3 6 4 R 5 4 R2 O OH O

Acacetin R =H ; R =OCH ; R =H ; R =H

1 2 3 3 4 Eriodictyol R =OH ; R =OH

1 2

Apigenin R =H ; R =OH ; R =H ; R =H

1 2 3 4 Naringenin R =H ; R =OH

1 2

Kaempferol R =H ; R =OH ; R =H ; R =OH

1 2 3 4 Liquiritigenin R =H ; R =H

1 2

Luteolin R =OH ; R =OH ; R =H ; R =H

1 2 3 4

Rutin R =H ; R =OH ; R =OH ; R =O-Rutinose

1 2 3 4

Quercetin R =OH ; R =OH ; R =H ; R =OH 1 2 3 4 O OH

R4 O HO OH

R3 Isoliquiritigenin

R2 O R1

O

Daidzein R =OH ; R =H ; R =H ; R =OH 1 2 3 4

Formononetin R =OCH ; R =H; R =H; R =OH

1 3 2 3 4 OH

Genistei n R =OH; R =OH; R =H; R =OH 1 2 3 4

Glycitein R =OH ; R = H ; R =OCH ; R =OH HO

1 2 3 3; 4

Prunetin R =OH; R =OH ; R =H; R =OCH R 1 2 3 4 3

Caffeic ac ic R=OH

Coumaric acid R=H

Feruli c acid R=OCH

3

Fig. 2. Chemical structures of the main compounds identified.



3 -O-␤-glucopyranoside. With an extra sugar residue, compound as daidzein-O-hexoside (formate adduct) and compound 77, with

  −

27 was tentatively characterized as 3 5,7-trihydroxyisoflavone-4 - [M−H] at m/z 253, as daidzein.



methoxy-3 -O-␤-diglucopyranoside. Five naringenin derivatives were identified in the extracts. In all

Compounds 29, 40, and 66 were tentatively characterized cases, the aglycone was observed at m/z 271, with typical fragment

as quercetin-O-hexoside-O-(malonyl)hexoside considering bibli- ions at m/z 151 and 107, indicating naringenin (Chanforan et al.,

ographic data (Gouveia and Castilho, 2009). Compound 64 was 2012). Compound 36 displayed the 433 → 271 transition and was

identified as quercetin-O-rutinoside (rutin) by comparison of its tentatively characterized as a derivative of naringenin-O-hexoside.

−

fragmentation pattern with a commercial standard. Compound 53, with [M−H] at m/z 595, suffered consecutive

− 2 3

Compound 34, with [M−H] at m/z 611, suffered consecu- losses of two hexosides, yielding MS and MS fragment ions at

tive losses of two hexosides, to yield the aglycone at m/z 287. m/z 433 and 271, respectively, so it was tentatively characterized

−

This aglycone was characterized as eriodictyol considering its as naringenin-O-dihexoside. Compound 60, with [M−H] at m/z

3

main fragment ion at m/z 151 (Hvattum, 2002), so 34 was 681, presented a neutral loss of 204 Da (acetylhexoside) in MS

tentatively identified as eriodictyol-O-dihexoside. Compound 62, [681 → 475] to yield naringenin, and was tentatively characterized

−

with [M−H] ion at m/z 449, was tentatively characterized as as a derivative of naringenin-O-acetylhexoside. Compounds 83 and

−

eriodictyol-O-hexoside. 104, with [M−H] at m/z 433, suffered the neutral loss of a hexoside

−

Compounds 35 displayed [M−H] ion at m/z 415 and, after moiety, and were tentatively identified as naringenin-O-hexoside.

a neutral loss of 162 Da, yielded the aglycone daidzein at m/z The aglycone naringenin was attributed to compound 121.

−

253 (Kang et al., 2007), so it was tentatively characterized as Compounds 43, 50, and 57 displayed [M−H + HCOOH] at m/z

daidzein-O-hexoside. Compound 48, followed a similar fragmen- 491 and suffered the neutral loss of 208 Da (hexoside + formate) to

tation pattern after loss of 46 Da;it was also tentatively identified yield the base peak at m/z 283. This fragment might correspond

to glycitein or biochanin A. However, the absence of a product ion

14 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27

at m/z 224 is corroborative with glycitein aglycone (Kang et al., of 162 Da (429 → 267). The aglycone at m/z 267 was attributed

2007). Thus, these compounds were tentatively characterized as to formononetin (Kang et al., 2007), hence, 98 was tentatively

isomers of glycitein-O-hexoside. Compounds 69 and 101 exhibited characterized as formononetin-O-hexoside. Compound 115 was

the deprotonated molecular ions at m/z 283 and were tentatively tentatively identified as formononetin.

characterized as glycitein isomers. Prunetin-O-hexoside and prunetin-O-acetylhexoside were

− −

Compounds 49, 70, and 91, with [M−H] at m/z 447, lost 162 Da attributed to compounds 110 and 120, with [M−H + HCOOH]

2 3 −

in MS , yielding the base peak at m/z 285 (aglycone). The MS and [M−H] at m/z 491 and 487, respectively, since the aglycone

[447 → 285] fragmentation displayed a fragment ion at m/z 241, was observed at m/z 283 and its 283 → 255 transition matches

characteristic from luteolin. Hence, they were tentatively identi- the pattern previously described in literature for prunetin (Aisyah

−

fied as luteolin-O-hexoside isomers. Compound 61, with [M−H] et al., 2013).

n −

at m/z 767, displayed MS fragment ions at m/z 447, 429, 327, 309, Compound 119 exhibited [M−H] ion at m/z 313 with frag-

− −

and 299, which are typical from luteolin-6-C-hexoside (isoorientin) ment ions at m/z 298 [M−H CH3] , 283 [M−H−2CH3] , 269

− −

(Llorent-Martinez et al., 2015), so it was tentatively character- [M−H CO2] and 255 [M−H CO2 CH3] . Thus, 119 was ten-



ized as a derivative. Compound 68 was tentatively characterized tatively characterized as 5,4 -dihydroxy-3,7-dimethoxyflavone

as luteolin-8-C-(3-hydroxy-3-methylglutaryl)hexoside due to the (Wang et al., 2008).

−

absence of an ion at m/z 429 [M−H−18] , characteristic from the

6-C hexoside. The loss of 144 Da corresponds to a 3-hydroxy-3- 3.3. Other compounds

methylglutaryl moiety (Montoro et al., 2013).

Many liquiritigenin derivatives were found in the methano- Compounds 1 and 2 were tentatively characterized as oligosac-

−

lic extracts of leaves and flowers. Compound 54, with [M−H] charides considering bibliographic data (Llorent-Martinez et al.,

at m/z 579, suffered the neutral losses of two hexosides, yielding 2015).

liquiritigenin at m/z 255 (Wang et al., 2008), so it was tenta- Compound 5 was tentatively identified as citric acid, considering

−

−

tively characterized as liquiritigenin-O-dihexoside. Compounds its [M H] ion at m/z 191, and the characteristic base peak at m/z

59, 65, 76, 103, and 105 displayed the deprotonated molec- 111 (Spínola et al., 2014).

−

−

ular ion at m/z 417, and suffered the neutral loss of 162 Da, Compound 30, with [M H + HCOOH] at m/z 431, suffered the

yielding liquiritigenin. Compound 65 was identified as liquiritin neutral loss of 46 Da (formate) to yield an ion at m/z 385, which



(liquiritigenin-4 -O-glucoside) by comparison with a commer- was tentatively identified as roseoside considering bibliographic

cial standard. Compounds 59, 76, 103, and 105 were tentatively data (Spínola et al., 2014).

characterized as liquiritin isomers. Compound 78 displayed the Based on previous data (Ye et al., 2005), compound 58 was

deprotonated molecular ion at m/z 621 and suffered neutral tentatively identified as a derivative of 4-methyl-3-methoxy-9␣-

losses of 162 Da and 204 Da, so it was tentatively characterized hydroxyligballinol.

−

as liquiritigenin-O-hexoside-O-acetylhexoside. Compounds 47, 67, Compound 72 displayed [M−H] at m/z 519, and suffered a hex-

2

79, 88, 93, and 99 also presented liquiritigenin fragment ions in oside loss to yield the MS base peak at m/z 357. The ion at m/z

their fragmentation patterns, and were tentatively characterized 357 matched pinoresinol considering bibliographic data (Eklund

as derivatives. It is important to mention that liquiritigenin and et al., 2008), so 72 was tentatively characterized as pinoresinol-O-

isoliquiritigenin present the same fragmentation patterns (Wang hexoside.

−

et al., 2008); therefore, some of the compounds may be liquiriti- Compounds 92 and 94, with [M−H] at m/z 621 and 591,

genin or isoliquiritigenin derivatives. Compounds 86 and 117, with respectively, (Table 1) suffered the neutral loss of 204 Da, yielding

−

[M−H] ion at m/z 459, suffered the neutral loss of 204 Da, yielding fragment ions at m/z 417 and 387, respectively. The fragment ion at

the aglycone liquiritigenin, so they were tentatively characterized m/z 417 was identified as syringaresinol, whereas the ion at m/z 387

as liquiritigenin-O-acetylhexoside. Compound 127 displayed the corresponded to medioresinol (Eklund et al., 2008). Thus, 92 and 94

−

[M−H] ion at m/z 255 and was tentatively characterized as liquir- were tentatively characterized as syringaresinol-O-acetylhexoside itigenin. and medioresinol-O-acetylhexoside, respectively.

−

Compound 75 exhibited [M−H + HCOOH] ion at m/z 507 and, Compounds 116, 118, 125, 128, and 129 were tentatively char-

after losing 46 Da (formate), displayed a fragmentation pattern pre- acterized as saponins comparing the experimental fragmentation

viously described for trihidroxy-methoxyl flavanone-O-hexoside patterns observed with those reported in bibliography for other

(Qiao et al., 2011). saponins (Pollier et al., 2011).

−

Compounds 81 and 106, with [M−H] at m/z 505, suffered the

neutral loss of 204 Da (acetylhexoside) to yield quercetin and were 3.4. Quantification of individual polyphenols

tentatively characterized as quercetin-O-acetylhexoside isomers.

Compound 82 displayed the deprotonated molecular ion at m/z In total, 61 main polyphenols present in U. europaeus were

447 and suffered the neutral loss of 162 Da, yielding the agly- quantified by an HPLC-DAD method (Table 2). It was not pos-

cone kaempferol at m/z 285, so it was tentatively identified as sible to quantify all the identified compounds due to their low

−

kaempferol-O-hexoside. Compound 109 exhibited [M−H] at m/z UV-absorption and/or low concentrations.

489 and, after the loss of 204 Da, yielded kaempferol. Compound The phenolic composition of different morphological parts var-

−

112 was identified as kaempferol, with [M−H] at m/z 285 and ied qualitatively and quantitatively, being the flowers a richer

typical fragment ions at m/z 257, 229 and 151 (comparison with an source of these phytochemicals. Significant differences (p < 0.05)

analytical standard). were observed in TIPC between morphological parts.

Compound 85 presented the deprotonated molecular ion at m/z The results indicated that flavones, flavanones, and flavonols

431 and, after the loss of a hexoside moiety, yielded the aglycone were the dominant class of compounds in the leaves and flowers

at m/z 269, which was identified as genistein (Kang et al., 2007). of Ulex, which is corroborant with the LC–MS screening. Moreover,

Hence, 85 was tentatively characterized as genistin (genistein-O- leaves and flowers collected in Funchal had higher concentrations

hexoside). of polyphenols than Machico counterparts. However, no significant

−

Compound 98 displayed the [M + HCOOH H] ion at m/z 475, differences (p < 0.05) were found among flowers counterparts.

− 2

and the [M−H] ion was observed in MS at m/z 429. The frag- Hydroxycinnamic acids were residual components in the

mentation pattern of this compound exhibited the neutral loss methanol extracts, more abundant in flowers than leaves but

V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 15 (2015)

(2009) (2009)

al.

et

(2014) Castilho Castilho

al.

and and

et

Ref. – Llorent-Martinez – – Spínola – – – – Gouveia – – Gouveia

M

part

F F F F

FFlowers F F

M, M M, M, M,

M, M, M,

Flowers All All Leaves All Morphological All Flowers Leaves All Flowers Flowers Leaves Flowers

-dihexoside -hexoside O O -dihexoside -dihexoside -dihexoside O dihexose)

acid-O-hexoside

O

identity O adduct) adduct) adduct) +

acid polymer

acid- acid-

acid

Machico).

Unknown Caffeic Coumaric Unknown Hexose Oligosaccharide (pentose Quercetin- Vanillic Caffeic Daidzein- (formate 4-pentosylhexoside Assigned Citric Unknown (formate Acacetin- (formate M:

Funchal;

(F: 240 255 175

(68), 113 119

(22), (16) (12), (47),

(100), 478

(12), (95) 163

Island (100), (28), (34), (100), 131 131 467 299

158

(34) (19), 267 208

(26), 271 303 119 143

(17), (37), (10) (31) (83) (52),

(100) (100), (32), 131 497

179 Madeira

(90), (20), (100), (11), (100), (32), 143 113 108 268 108 300

(55)

252 200 479 of

(28), (44), (18) (47)

(36), 143 149 268 209 272 321

151 (18), (19), (93), (38), (75),

(35)

peak) (59), (98), (34), (100), 179 539 152 301

207

(55), (23), (12), (35), (24), (93), (100) (100) (100) 161 161 123 282 123

(58), 175

(100)

202 480 253 301

locations

base (100)

(22) (16), (48)

(100), (100), (100) (100), (100) (100) (100) (32), 161 161 96 520 109 108 269 225 299 135

(% 179

(23), z

341 (100), (100), (23), (34), (100), (100), (11), (100) (100) (100), (100) (17), 135 462 179

/

341 625 111 167 445 415 163 271

m 179]: 179]: 158]: 479]: 123]: 152]: 283]: 253]: 301]: 179]: 303 (17),

n

179 179 218 497 152 283 295 135 119 124 302 179 different

→ → → → → → → → → →

(16), (12.0), (100) (22), (12), (88), (16), (18), (100), (19), (40), (100), (100),

227

(25), (100) (25) (18) two

(52) 341]: 341 341]: 341 262]: 262 625]: 625 167]: 167 167 445]: 445 415]: 415 179]: 163]: 152]: 463]: 463 341]: 341

427 342 262 626 173 329 446 416 179 325 315 463 341 at

→ → → → → → → → → → → → → → → → → → → → → → 101 101 96 321 152 (11), (24)

(100) (100)

89 303 254 89 [473]: [473 [473 [683]: [683 [683 [525]: [525 [525 [643]: [643 [643 [191]: [461]: [461 [461 [461 [503]: [503 [503 [653]: [653 [653 [623]: [623 [623 [341]: [341 [371]: [371 [447]: [447 [625]: [625 [625

(15), (13), (39), (36), (46), (12) collect

2 3 4 2 3 4 2 3 4 2 3 4 2 2 3 4 4 2 3 4 2 3 4 2 3 4 2 3 2 3 2 3 2 3 4 MS MS MS MS MS HPLC-DAD-ESI/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS 271 MS 113 119 MS MS (20), (22), MS MS 437 MS MS MS (34), (33), MS (42) (11) MS MS MS MS 142 161 europaeus

) z Ulex

/ of m

( − H]

− flowers

[M 473 683 525 643 191 461 503 653 623 341 371 447 625 and

leaves

in

(min) 3.0 3.3 3.8 4.0 4.3 4.6 4.8 5.2 5.5 5.8 6.0 6.2 6.3 R t found

1

◦

N 1 3 6 11 2 4 5 7 8 9 10 12 13 Compounds Table

16 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 (2003)

al. (2009)

et

(2013)

Castilho

(2011) al. (2002)

(2014) and

(2005) (2005) et

al.

al.

al.

al. al. et et

et

et et

Ref. – – Fang Ye Hashim – Gouveia Yang Ye Sánchez-Rabaneda Ma – –

M

part

F F F F

F F F FFlowers

M, M, M, M, M

M, M, M, M, F

Leaves All Flowers Morphological Leaves Leaves Flowers Flowers Leaves All Flowers Leaves Flowers

- 

dimer

- O

(formate -hexoside

- O ␤ -dihexoside All - -hexoside -hexoside O O O O - -hexoside -hexoside-  acid- C

O

identity adduct)

acid- acid-

-pentosylhexoside O 5,7-trihydroxyisoflavone-4  Taxifolin- Unknown Coumaric Caffeic Kaempferol-O-dihexoside 3 Unknown glucopyranoside dimer Ferulic acid- Unknown Kaempferol- Unknown hexoside (formate Apigenin- Assigned methoxy-3 adduct) Ferulic 230 (19)

(39), 238 134 199 224

(24), (18) (83),

(33), (10), 179

149

(10), 309

(39), (77), (84), (59), 163 125 125

283 131

(13), 255

(12), (100), 239 149 241 239

(36), (16), (21),

(100) (199) (44), (18),

191

(11) 349 193 (64)

(43), (26), (16), (49), (100), 165 177 167

431 310 134 207

58

(60), (54) 185

(31), (100) (18), 253 150 240 242 257

(26)

(21), peak) (27), (100), (63), (91), (100), (100), (100) (100)

203 285

(73)

393 115 217

(24)

(59), (100) (95), (32), (100) (100), (14), (13), (100)

119

85

(100)

432 311 203 243 149 285 268 285 85(100), 249

base

(22) (52) (37) (34.6)

196 (25), (100) (100), (100) (100), (40), (26), 267 231 178 97 267 119 257 243 283 284

(% (2),

(37), z

(69), (100) (51), (19), (100) (50), (100), (75), (100) (16), (12), (100) (42), (18), 135 285 285 299 /

411 355 329 473 447 143 337

95 (19), m

473

282]: 249]: 193]: 125]: 268]: 163]: 285]: 285]: 299]: 285]: (12)

n

445 250 193 269 333 135 269 163 178 285 286 286 299 115

→ → → → → → → → → →

201 (20), (27), (16), (48), (100), (3), (100) (100), (100), (19), (100), (26), (16),

(54) 217

(100),

473]: 473 267]: 267 355]: 355 303]: 303 329]: 369]: 329 179]: 431]: 431 325]: 325 193]: 447]: 447 447]: 447 461]: 461 115]:

474 555 625 303 369 179 593 325 447 448 461 159 355

249 (23),

→ → → → → → → → → → → → → → → → → → → → → → → → 113 (100) (68) (21),

212

175 125 229 [593]: [593 [593 [681]: [681 [681 [711]: [711 [711 [609]: [609 [609 [465]: [465 [465 [431]: [431 [431 [431 [341]: [341 [639]: [639 [639 [609]: [609 [609 [651]: [651 [651 [507]: [507 [507 [205]: [205 [487 [487]:

(100) (100), (16), (43) (21)

2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 2 3 3 4 2 3 2 3 4 2 3 4 2 3 4 2 3 4 2 3 2 3 MS MS MS MS MS MS MS HPLC-DAD-ESI/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS (100) MS MS 267 MS 282 MS MS MS MS MS MS 107 MS 129 (12), MS MS MS 125 MS MS MS (22), (100) (14), (100),

) z / m ( − H]

− [M 711 465 431 341 639 487

)

(min) 6.5 593 6.6 681 6.9 7.17.0 609 7.3 7.5 7.7 7.98.0 609 8.1 651 8.3 507 8.4 205 R t Continued (

1

◦ N 14 17 18 19 21 22 23 24 25 15 16 20 26 Table

V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 17 (2014)

(2015)

(2009) (2009) (2009) al.

al.

et

et

(2014) Castilho Castilho Castilho

(2002) al.

and and and

et al.

et

Ref. – – Gouveia Spínola Fang Gouveia Llorent-Martinez – – – – – Vallverdú-Queralt Gouveia – – –

F M

M,

part

F F F F F F F

F FFlowers F F

Flowers M, M, M, M, M M, M, M,

M, M, F M, F M, F

Flowers Flowers Flowers All Flowers Leaves All Morphological Leaves Flowers Flowers Flowers Flowers Leaves Leaves Leaves Leaves Leaves - 

- -

O O

-

O (formate

- -hexoside -hexoside ␤ C C -hexoside - -hexoside -hexoside -hexoside -dihexoside C O O O -hexoside- -hexoside- O derivative derivative O -

-hexoside -hexoside  O O O

O identity adduct) adduct)

acid- acid acid- acid-6- acid acid-8- acid-

5,7-trihydroxyisoflavone-4  malonyl(hexoside) derivative diglucopyranoside malonyl(hexoside) hexoside Quercetin- 3 Roseoside (formate Ferulic Ferulic Ferulic Naringenin- Unknown Glycitein- (formate Daidzein- Sinapic Ferulic Caffeic Quercetin- Dihydrokaempferol- Ferulic adduct) Eriodictyol- Assigned methoxy-3 107 163

(37) (21) 149 300

(10) (26), (68)

269 (56.4), (14), (100),

(49) (21), 193 259

(100), (100) (12), (19), 149 462 301

269 154 224

(57) (11) (25) (32) 134 165

(16.7), (63),

151 134 151 301

(100), (77) (22), (17) (15) (13) (23), (63),

(60), (24), 175 153 223 193

283 (10.3), (33) 235

(19), (78), (17), (100), (12), (100) (100), 287 134 164 140 243 134 463 463 (100)

225 161

(16), (57) (82), (77), (13) 270 299

(100) (100), (100) (100) 152 149 281 149 240 323 208

461

(12), (100),

(49.4), (24) (10), (44),

peak) (100) (100), (44), (100), (100) (100) (100), (55), (100), (100), 223 252 265 235 287

327 193 283 193

(100), (100) (15), (71), (89), (72), (14), (15), (63), (100), 288 265 297 151 149 504

134 205 149 252 312 223 134 268 505 259

base (21.3),

(22) (24)

(60), (100), (100), (23), (100), (18), (100), (16), (100) (100), 284 151 177 178 283 209 178 241 344 301

(% (12), (78),

z 462 (100), (17), (48), (39), (100) (100), (42), (100) (100), (26), (22), (41), (100) (16), (15), (17), (100), 135 271

/

235 385 253 328 295 235 265 284 667 449

m 327 295

299]: 505]: 287]: 271]: 193]: 312]: 223]: 505]: 193]: 268]:

n 298 149 506 223 178 135 260 287 288 271 193 313 224 505 149 193 269

→ → → → → → → → → →

(18.8), (18), (30), (18), (100) (100), (9), (13), (100), (8), (19), (65), (100) (12), (62), (18), (20),

(77)

461]: 461 193]: 667]: 667 385]: 193]: 179]: 287]: 449]: 449 253]: 433]: 433 265]: 265 327]: 327 295]: 295 667]: 667 193]: 265]: 265 283]: 283

623 295 668 386 193 179 421 450 254 433 337 489 325 667 265 295 445

(27)

→ → → → → → → → → → → → → → → → → → → → → → → → → → → 208 (100)

271

149 [669]: [669 [669 [531]: [531 [711]: [711 [711 [431]: [431 [355]: [355 [341]: [341 [449]: [449 [611]: [611 [611 [415]: [415 [631]: [631 [631 [355]: [355 [355 [537]: [537 [537 [385]: [385 [385 [711]: [711 [711 [459]: [459 [355]: [355 [355 [491]: [491 [491

(15.9) (100) (31), (39)

2 3 4 2 3 2 3 4 2 3 2 3 2 3 2 3 2 3 4 2 3 2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 2 3 2 3 4 2 3 4 HPLC-DAD-ESI/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS (100), MS MS 153 223 MS MS MS MS 301 MS (14) MS MS MS MS MS MS MS MS MS MS MS (42) MS MS 176 MS MS MS (22), (14.7) MS

) z / m ( − H]

− [M 669 531 711 431 355 341 449 611 415 631 355 537 385 711 459 355 491

)

(min) 8.6 8.6 8.9 9.0 9.0 9.5 9.6 9.8 R 10.0 10.2 10.3 10.5 10.5 10.8 t 11.1 11.5 11.8 Continued (

1

◦ N 27 30 31 32 33 34 40 41 28 29 35 36 37 38 39 42 43 Table

18 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 (2012)

al.

et (2011) (2011)

(2005)

al. al.

al. et et

et

Ref. – – – – Qiao – Qiao – Chanforan – – – – Ye

F F M

M,

part

Flowers

F FFlowers F F

Flowers M M

F– M, M, M, F M,

Leaves All Leaves All Leaves Leaves Morphological All All All Leaves Flowers All Leaves Flowers

-

␣

-dihexoside derivative O

derivative All -hexoside

-dihexoside C -dihexoside -hexoside O -hexoside -hexoside -hexoside -hexoside O O -hexoside acid O O O

O

identity adduct) adduct) adduct) adduct) O

acid-

Glycitein- (formate Daidzein- (formate Unknown Naringenin- Quercetin- Glycitein- (formate Coumaric Luteolin- Apigenin- (formate Quercetin- derivative Liquiritigenin- 4-Methyl-3-methoxy-9 hydroxyligballinol derivative Ferulic Assigned Liquiritigenin Unknown 312 101 211

151 179

(93), (18), (28),

(26),

(35) (9) (10),

(100), (100), (10), (31), 197 229 223

179

(27) (47) 119 134 212

(100) (100) 103 313 165 229

(59) (67) (82), (31), (9) (12)

(100), (11), 151 327

(96), (48), 253 389

(56), (100) (39), (100), (100) (10), (12), (100) 134 298 240 309 211 241

224 211

(28) (15) (29) (15), (11), 149 239

(50), (16), (100) (100) (100) 119 135 327 135 298 240 243 177

(11), (80), (10), (26) (53),

peak) (100) (100) (100) (100), (100), (100), (64), (100) (42), (100), 301 255 163 283 328

431 254 283 283 357

(100), (41), (15), (66), (10), (34), (13), (28), (25), (100), (100) 149 224 243 151 313

163 255 268 342 268 324 301 239 255 241

base

(13)

(100), (37), (100) (39), (100) (20), (100) (100), (100) (14), (100), (30), (100), (100), 178 145 153 240 328 227 153 299 241 309 273

(%

z (100), (17), (15), (100), (100), (16), (22), (100), (10), (100), (16), (18), (18), (76), (12), 271 /

463 461 417 415 285 284 268 417 341 301 284 429 358 339

m 301]: 193]: 163]: 255]: 268]: 342]: 271]: 255]: 326]: 268]: 324]: (56)

n

302 193 265 256 253 257 269 240 343 271 256 326 269 325 257

→ → → → → → → → → → →

(24), (88), (31), (11), (16), (23), (58), (100), (17), (17), (18), (29), (18), (17), (19),

149

(100) (59)

463]: 463 389]: 389 429]: 429 417]: 417 253]: 285]: 283]: 283 268]: 357]: 357 433]: 433 417]: 417 341]: 341 301]: 283]: 283 339]: 339

464 491 430 459 416 286 445 269 405 433 418 342 343 445 340

(28) (27)

→ → → → → → → → → → → → → → → → → → → → → → → → → → (30) 151 135 (43),

(24)

151 107

89 196 150 [625]: [625 [625 [551]: [551 [551 [475]: [475 [475 [665]: [665 [665 [461]: [461 [447]: [447 [491]: [491 [491 [431]: [431 [567]: [567 [567 [595]: [595 [595 [579]: [579 [579 [461]: [461 [461 [463]: [463 [491]: [491 [491 [565]: [565 [565

(16) (24) (15), (89),

2 3 4 2 3 4 2 3 4 2 3 4 2 3 2 3 3 3 4 2 3 2 3 4 2 3 4 2 3 4 2 3 4 2 3 2 3 4 2 3 4 MS MS HPLC-DAD-ESI/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS (100), MS MS MS MS MS (59), 180 217 (53), MS MS MS MS 169 212 MS MS MS MS MS MS MS MS MS (15), MS MS MS (100),

) z / m ( − H]

− [M 625 461 447 491 431 567 595 579 461 463 491 565

) (min) R t 12.6 12.712.7 551 12.8 475 13.0 665 13.5 13.8 14.0 14.1 14.2 14.5 15.0 15.0 15.3 15.5 Continued (

1

◦ N 44 50 52 54 46 45 47 48 49 51 53 55 56 57 58 Table

V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 19 (2009)

Castilho

(2008) (2008)

(2007) (2007) (2011) (2011) (2011)

(2002) al. al.

and

(2005)

al. al.

al. al. al.

et et

et et al. et et et

et

Ref. Wang – – Hvattum – standard standard Gouveia – – Kang Qiao – Ye Qiao – Qiao Wang Kang

part

F F F

F F F F

M, M M M M, M M M,

M, M, M F M, M,

All Flowers Flowers Leaves Flowers All Leaves Morphological Flowers Flowers Flowers Flowers All Leaves Leaves All Leaves Leaves Flowers All -

O

-

O

- O -hexoside -acetylhexoside -hexoside derivative -(hydroxy-3- -hexoside

-hexoside -hexoside- O O derivative O

-hexoside C C O O -hexoside O isomer isomer

isomer identity adduct)

O

acid

hexoside derivative malonyl(hexoside) Unknown Luteolin- Luteolin-8- methylglutaryl)hexoside Trihidroxy- methoxylflavanone- Quercetin- Luteolin-6- (isoorientin) Liquiritin Naringenin- Eriodictyol- Apigenin- (formate Caffeic Glycitein Assigned Daidzein Pinoresinol- Liquiritin Liquiritin Quercetin- Liquiritigenin- acetylhexoside derivative Rutin 213 241 161 223 184 (56)

(35),

(66) (23) (25), (24) (10)

(100), 221

327

(32), (32) (70), (96), (20) 429

(100), (100), 285 151 297 119 309

(59)

179

(13), (90) 232 119 195 255 224

(46), (100), 253 179

125 (14) (40), (71) (68), (24) (29), (36) (36)

(100), (100), (14), 222 299

(23) (19) (77), 447 357

(100) (100), (100), (83), (21), (100), (100), 119 143 113 301 298 119 284 151

(18),

297 135 193

(12) (37) (13) (59) (14), (21) 107 179 239

(100), (43), (18), 151 255 203 135 255 196 283

131 (11) (18) (43), (64), (75),

peak) (100), (50), (16), (100) (100) (100), (100), (100), (100), (10), 249 269 300 463 135 300

(100) 325 489 369

(23), (29), (22), (48), (85), (100), (100) (100), (64), (100), (34), 135 179 227 161 299

(59),

135 153 298 151 268 463 135 299 179 255

base

(17) (16) (41), (44), 240

(100), (100) (100) (23), (10), (100), (100), (100), (47), 177 297 233 301 153 269 136 240 161 212 284

(% 143

z (29), (100) (100), (100), (13), (100) (35), (100), (28), (83), (100), (10), (92), (10), (100), (23), (29), 433 323 505 209 /

(42), 490 268 285 357 365 301 431 461 393

m 271]: 299]: 323]: 463]: 255]: 297]: 240]: 151]: 268]: 221]: 446]: (20) (35),

n

153 271 300 151 323 154 505 255 339 24 241 136 269 221 153 271 446 271

→ → → → → → → → → → →

(100) (100), (26), (100) (100), (100) (100), (100) (33), (14), (16), (20), (11), (100) (100), (15), (100) (27), (33),

135 149

(86) (6) (100)

255]: 475]: 475 327]: 327 287]: 517]: 517 255]: 667]: 667 459]: 459 357]: 357 268]: 268 285]: 301]: 357]: 357 431]: 431 325]: 325 255]: 301]: 461]: 461

255 475 539 287 517 255 667 459 269 286 301 358 432 383 255 224 343 462 447

→ → → → → → → → → → → → → → → → → → → → → → → → → → → → → (44), 151 (84), 269 327 (41)

149 161 225 [417]: [417 [681]: [681 [681 [767]: [767 [767 [449]: [449 [679]: [679 [679 [609]: [609 [417]: [417 [711]: [711 [711 [751]: [751 [751 [591 [591 [283]: [283 [283 [447]: [447 [463]: [463 [519]: [519 [519 [477]: [477 [477 [427]: [427 [427 [507]: [507 [507 [417]: [417 [253]: [591]:

(20), (15), (21),

2 3 2 3 4 2 3 4 2 3 2 3 4 2 3 2 3 2 3 4 2 3 4 2 3 4 2 3 4 2 3 2 3 2 3 4 2 3 4 2 3 4 2 3 4 2 3 2 MS MS MS MS MS HPLC-DAD-ESI/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS 357 MS MS MS 152 MS 283 MS MS MS MS (15) (30), MS MS MS (36), MS (38), MS (59) MS MS MS MS MS MS (27) MS MS MS

) z / m ( − H]

− [M 417 681 767 449 679 609 417 711 751 591 283 447 463 519 477 427 507 417 253

) (min) R 20.0 20.0 20.7 t 15.8 16.1 16.3 16.3 16.6 17.2 17.7 17.8 18.2 18.9 19.4 19.5 21.5 21.7 23.2 23.5 Continued (

1

◦ N 59 60 63 67 70 71 73 74 76 77 61 62 64 65 66 68 69 72 75 Table

20 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 (2003)

al.

et

(2012)

al. (2014)

(2008)

et al. (2011)

al.

et al.

et

et

Ref. – – – – Spínola Chanforan Sánchez-Rabaneda – – – – – Qiao – Wang

F F F

M, M, M,

part

F F F

F F F F

Flowers M, M M, M, F

F M, M MFlowers M, F,Flowers M, M,

Leaves Leaves Leaves Leaves Leaves Morphological All Flowers Flowers Leaves All Leaves Flowers Leaves Flowers Flowers -

O

acid

- - -hexoside- O O O derivative derivative derivative -hexoside

-hexoside All O -acetylhexoside -hexoside O -hexoside O O -hexoside C

identity O

-Dicaffeoylquinic O Quercetin- Genistin Quercetin- Luteolin- Syringaresinol- acetylhexoside Unknown derivative Apigenin- Liquiritigenin Liquiritigenin- acetylhexoside Kaempferol- Apigenin-C-hexoside derivative 3,5- Liquiritigenin- acetylhexoside Assigned Liquiritigenin Naringenin- Liquiritigenin 65 91 (42) (14)

93 (32), 153 222

(27), (8), (66), (49), (90), (47),

(100), 341

417 271

(20), (40) 413

(50), (62), (26), (100),

229 121 166 151 223 119

151

229 119

111 (100), (54), (100), (100), 107 155 251

(11) (16), (99),

(100) (100), (35), (100), (100), (100),

(24) 431 431 301 459

(17), (17)

(100), (100) (36), (58), (41), (100) (100),

135 239 224

293 151 169 181 179 135

(27) (78) 119

112 118 (16), (100) (50), (100) (10), (21), 135 283 122 171 265 135 135

(46), (15) (14)

peak) (100), (23), (100) (20), (100) (10), (86), (12), (43), (100), 255 255

432 268 473 369 343 460

(16),

(100), (33), (16), (52), (86), (33), (100) (11), (48),

107 179

(100), 133

255 294 283 241 255 136 179 182 255 239

base

(32), (40),

164 (18), (55), (11), (20), (100) (58), (15), 135 153 284 169 173 293 166 153 153

(% 127

(10), z

(100) (100) (100) (14), (100), (100), (50), (29), (15), (25), (18), (100) (17), (60), (21), (52), 417 417 /

473 269 370 515 301 621 445

(79), m

255]: 255]: 311]: 151]: 191]: 293]: 181]: 255]: 255]: 163 (39),

n

255 255 311 267 151 191 240 153 351 284 257 402 417 255 257 271

(26) → → → → → → → → →

173 (100), (32), (100) (100) (100) (11), (100) (14), (23), (100) (100) (100), (100) (17), (14), (33),

(32) 137

(24), 71

459]: 459 459]: 459 431]: 431 284]: 271]: 271 353]: 353 268]: 255]: 369]: 369 301]: 311]: 285]: 417]: 417 673]: 673 301]: 459]: 459

459 513 284 271 353 271 255 455 302 719 285 417 673 459 622 463

283

→ → → → → → → → → → → → → → → → → → → → → → → → → (55) 164 (21), (100),

(79),

311 145 85 175 [621]: [621 [621 [665 [665 [575]: [575 [575 [505 [447]: [447 [433]: [433 [433 [515]: [515 [515 [431]: [431 [459]: [459 [577]: [577 [577 [503]: [503 [503 [463]: [463 [845]: [845 [447]: [447 [621]: [621 [621 [835]: [835 [835 [505]: [665]:

(12) (16), (23) (100), (27) (5) (15)

2 3 4 2 3 4 2 3 4 2 3 2 3 2 3 4 2 3 4 2 3 2 3 2 3 4 2 3 4 2 3 2 3 2 3 2 3 4 2 3 4 (10)

MS MS MS MS HPLC-DAD-ESI/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS (16), (28), MS MS MS MS 311 MS MS MS MS MS MS 107 MS MS 196 MS MS MS (60) MS 121 91 MS (58), (16) 223 107 MS MS (66), 151

) z / m ( − H]

− [M 621 575 505 447 515 431 459 577 503 463 845 447 621 835

)

(min) R t 23.5 24.425.3 665 25.4 25.4 26.026.4 433 26.7 27.4 27.7 27.8 28 28.3 28.4 28.5 28.6 Continued (

1

◦ N 78 83 84 85 92 86 79 80 81 82 87 88 89 90 91 93 Table

V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 21 (2012)

al. (2008)

(2013)

(2008)

(2007) et

al., al.

al.

al.

et et

et

et

Wang Ref. – – – – – – Kang – ( Chanforan Wang – – – – Aisyah – standard –

part

F F F F F F

F F F F F F

M, M, M M, M, M, M, M

M, M, M, F M, M, M,

All Leaves Leaves All Morphological Flowers Flowers Leaves All Leaves Leaves All Flowers All Flowers Leaves Flowers Flowers Leaves Flowers Flowers

(formate

-hexoside

- - O O O -acetylhexoside -hexoside O derivative -hexoside -acetylhexoside O derivative

-hexoside O O isomer isomer

O isomer identity adduct)

acid acid

Naringenin- Medioresinol- Unknown Glycitein Liquiritigenin- acetylhexoside derivative Liquiritin Prunetin- Unknown Unknown Formononetin- (formate Kaempferol- adduct) Quercetin- Kaempferol Unknown Unknown Caffeic Assigned Liquiritin Sinapic Unknown acetylhexoside Quercetin- 239 208

(80), 161

(84), (11), (18) (58) (12) (11)

(44), 197 135

(73), (36), 473

(37), 151 213 281 151 107 107

257

(57), (56), (20) 283 209

(11), (100) 163

(29), (12), (17), (22) (42)

(23), (100), (100) (100), (100) (100), 201 153 180

(86), 475 393

(100), (100) (28), (100) 166 349 213 119 149

283 282 151 151 229 151

(30), (32), (56), (14) 223

(17), (20), (100) 333 135 238 184

(100)

peak) (100), (100), (100) (86), (100) (100) (28), (10), (100), (99), (100) (100), (100), (47), (96), 214 205 199 135

494 394 283

(100) (100) (61), (100), (24), (66), (12), (100) 267

181 377 252 240 351 151 135 283 169 255 230 179 135 164 179

base

(12) (69); (44) (20)

(27), (12), (25), (100), (100), (100), 166 132 349 224 153 107 253 196

(% (17),

(100) z

(31), (100) (13), (17), (100) (100) (19), (54), (30), (22), (44), (14), (17), (27), (67), (100) (13), (36), (10), 255 223 240 151 /

515 411 208 284 223 179

m 268

181]: 187]: 377]: 252]: 255]: 240]: 151]: 282]: 133

n

372 187 378 253 255 197 241 365 153 177 153 335 257 268 208 239 135 193 257 (43)

→ → → → → → → →

(100) (100) (22), (2), (100), (100), (100), (11), (100) (100) (100) (100) (73), (100) (32), (41), (100), (100) (22), (100)

91

(90),

387]: 387 231]: 231 301]: 395]: 395 267]: 267 459]: 459 241]: 268]: 268 393]: 255]: 271]: 271 255]: 353]: 353 285]: 283]: 223]: 257]: 179]: 301]:

387 231 301 557 429 459 241 268 531 255 271 255 353 224 285 445 385 257 205 301

→ → → → → → → → → → → → → → → → → → → → → → → → → → → (90) (49) 215 (30), (14)

215 196 133 132 [591]: [591 [591 [439]: [439 [439 [463]: [463 [617]: [617 [617 [475]: [475 [475 [751]: [751 [751 [269]: [269 [283]: [283 [283 [675]: [675 [417]: [417 [433]: [433 [433 [417]: [417 [505]: [505 [515]: [515 [515 [253]: [489]: [489 [491]: [491 [591]: [591 [285]: [285 [367]: [367

(25) (100) (17) (29),

2 3 4 2 3 4 2 3 2 3 4 2 3 4 2 3 4 2 3 2 3 4 2 3 2 3 2 3 4 2 3 2 3 2 3 4 2 2 3 2 3 2 3 2 3 2 3 MS MS HPLC-DAD-ESI/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS 239 MS MS MS MS (63), MS MS 136 161 MS (24), MS MS MS MS MS MS MS MS MS (16), MS (80), MS MS MS 395 (100)

) z / m ( − H]

− [M 591 439 463 617 475 751 269 283 675 417 433 417 505 515 253 489 491 591 285 367

(min) ) R 30.0 30.7 t 29.0 29.9 29.9 31.2 31.3 31.6 31.8 31.9 32.3 32.5 32.7 32.8 33.5 33.7 34.2 34.3 34.9 35.1 Continued (

1

◦ N 94 102 104 105 106 111 99 95 96 97 98 100 101 103 107 108 109 110 112 113 Table

22 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 (2012)

al.

(2011) (2011) (2011) (2011) (2011)

(2008) (2008)

(2007) et (2011)

al. al. al. al. al.

al. al.

al. al.

et et et et et

et et

et et

Ref. – Kang Pollier – Pollier Wang – Chanforan – Qiao – Pollier – Wang Pollier Pollier

M

part

F F F F

F F F F F

M, M, M, M,

M, M FFlowers M, M, M, M,

All Leaves Morphological Leaves Flowers All All Leaves Flowers Leaves Flowers Leaves All Leaves Flowers All Leaves

- derivative O

derivative

-acetylhexoside

acid

O identity

acid

-dihydroxy-3,7-  Liquiritigenin- acetylhexoside 5,4 Saponin-5 Ferulic Saponin-1 Saponin-4 Apigenin Coumaric Formononetin Prunetin- Unknown Saponin-3 Saponin-2 Liquiritigenin Naringenin Unknown dimethoxyflavone 541 212

(36), (36), (37), 545 409 (74),

(17) (33), (59), (37), (25.8), (23),

242

(5), (100) (100), (100),

201 (32)

(56), 603 749 733 (100),

395

(100) (14), 133 119 489

765 242 283

(100),

613 525 457 557

489

(25) (6), (7), 213 (17.8) 197

(20.4),

(100), (15),

269 570 (5), (40),

(61) (46), (44), 439 (10)

(24),

(30), (36), (32), (76), (100), 457 767 751 (73),

131

(12.5),

255

(63),

225 629

783 455

(82), (78), (100) (100) (6), 134 195 490

(47.5), 453

333

629 749 597 497 135 557

253

(22) (56), (2) (13), (15) (22), 227

(23), 321 585 161 455 489 (47),

200 (37.4), (100),

(100), (60), (37)

(69),

(13), (25) (23),

peak) Assigned (48), (10), (18), (100), (26), (68), (100) (14) (12) 809 119 525 811 115 795

(31),

227

161 269 (38), (25), (15.9), (100), (11), (32), (80), 647 208 559 254 91 83

473 457

335

765 136 895 149 733 553 479 603

base (47.2),

(28), (30), (10), (30),

(18)

(41),515 (100) (100) (100), (100), (37), 334 507 597 255 241 317 499

(% (20),

(59), (62), z

201 (22.0), (100), (51), (62), (100), (40.8), (100), (100) (100) (38), (57), (100), (24), (48), (100), (32), 911 895 134 879 / (26),

(100),

119

537 283 151 135 615 233

m 648

349]: 557]: 615]: 269]: 255]: 479]: 541]: (44) 541 525 (68)

n

200 223 911 153 615 283 255 107 480 181 119 921 178 153 879 571 629

349

→ → → → → → →

(36), (100) (100), (100) (9), (100) (14), (24), (100) (40), (100), (100), (39), (100), (25), (24.1), (100),

151 157

(56) (49) (26), (28),

(43), 377 252]: 956]: 955 255]: 647]: 647 298]: 298 283]: 283 151]: 641]: 641 225]: 163]: 939]: 939 193]: 135]: 923]: 615]:

377]:

597 252 955 255 665 298 284 177 641 253 939 193 153 923 633 (34.2), 227

(53) (8)

→ → → → → → → → → → → → → → → → → → → → → → 215 489 (57), 613 597 (48),

→

647

241 457 473

209 197 [659]: [659 [267]: [267 [974]: [974 [974 [459]: [459 [828]: [828 [828 [313]: [313 [313 [487]: [487 [487 [271]: [271 [687]: [687 [687 [269]: [269 [351]:163 [351 [957]: [957 [957 [381]: [381 [255]: [255 [942]: [942 [795]: [795

(100) (16), (2), (17) (31), (44) (47.6), (18.5) (32), (26), (27) [659

2 3 4 2 3 2 3 4 2 3 2 3 4 2 3 4 2 3 4 2 3 2 3 4 2 3 2 3 2 3 4 2 3 2 3 2 3 2 3 (12)

MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS 161 241 MS (100), (100), MS MS MS MS MS MS 377 MS MS MS MS (51) 451 631 615 489 MS 557 441 MS MS MS 239 (49.2), (23), 91 MS (76), 665 ) HPLC-DAD-ESI/MS z / m ( −

H]

− 659 267 973.5 827.5 487 271 687 957.5 381 255 795.5

(min) [M ) R 40.4 40.8 t 35.2 35.4 36.0 36.436.5 459 37.7 313 38.1 39.2 39.4 39.639.9 269 351 43.5 44.746.1 941.5 Continued (

1

◦ N 114 116 117 119 120 122 124 126 128 129 115 118 121 123 125 127 Table

V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 23

Table 2

Quantification of the main polyphenolic compounds present in leaves and flowers of Ulex europaeus (mg per 100 g DE).

Leaves Flowers

◦

N MW Hydroxycinnamic acids Machico Funchal Machico Funchal

7 503 Caffeic – – 17.03 ± 0.53 19.43 ± 0.37

acid-O-dihexoside

20 341 Caffeic 27.38 ± 1.76 31.89 ± 1.80 64.17 ± 2.13 90.68 ± 3.06

acid-O-hexoside

32 341 Caffeic – – 84.89 ± 3.79 92.52 ± 4.13

acid-O-hexoside

84 515 3,5-O-Dicaffeyolquinic 42.57 ± 2.78 – – –

acid

± ±

28 531 Ferulic acid derivative 1.88 0.11 6.77 0.22 – –

37 355 Ferulic – 23.18 ± 1.07 – –

acid-6-C-hexoside

41 459 Ferulic acid derivative – 2.68 ± 0.17 – –

45 551 Ferulic acid-C-hexoside – 3.34 ± 0.21 – –

derivative

11 371 Coumaric 8.21 ± 0.34 10.33 ± 0.41 – –

acid-O-hexoside

23 651 Coumaric – – 47.45 ± 3.23 139.36 ± 9.14

acid-O-hexoside dimer

46 475 Coumaric acid 38.86 ± 2.16 43.64 ± 2.77 – –

derivative

124 351 Coumaric acid 14.93 ± 0.94 – 16.71 ± 1.11 –

derivative

111 591 Sinapic acid derivative 3.58 ± 0.09 9.22 ± 1.11 – –

± ±

±

± Total 137.39 8.18 151.05 7.79 280.36 8.07 342.00 51.34

Hydroxybenzoicacids

6 461 Vanillic acid 25.37 ± 1.09 34.55 ± 1.20 42.12 ± 1.54 47.09 ± 1.38

4-hexosylpentoside

Total 25.37 ± 1.09 34.55 ± 1.20 42.12 ± 1.54 47.09 ± 1.38

Flavonols

13 625 Quercetin-O- – – 864.25 ± 11.75 887.77 ± 14.76

dihexoside

29 711 Quercetin-O-hexoside- – – 92.34 ± 3.36 107.37 ± 4.45

O-malonyl(hexoside)

40 711 Quercetin-O-hexoside- 25.00 ± 1.36 35.20 ± 2.05

O-malonyl(hexoside)

44 625 Quercetin-O- 311.21 ± 8.64 333.47 ± 9.03

dihexoside

56 463 Quercetin-O-hexoside 21.00 ± 0.63 24.45 ± 0.94 594.45 ± 5.64 627.23 ± 4.73

81 505 Quercetin-O- – – 17.8 ± 0.75 21.48 ± 0.71

acetylhexoside

96 463 Quercetin-O-hexoside – – 986.65 ± 43.01 1023.57 ± 48.32

isomer

106 505 Quercetin-O- – – 48.34 ± 1.33 63.08 ± 1.89 acetylhexoside

isomer

17 609 Kaempferol-O- 241.85 ± 13.78 281.23 ± 12.90 426.29 ± 14.55 456.59 ± 11.39

dihexoside

33 449 Dihydrokaempferol-O- 242.72 ± 9.45 258.93 ± 8.99 301.60 ± 10.11 325.01 ± 9.76

hexoside

82 447 Kaempferol-O- 55.13 ± 1.97 57.34 ± 2.04 55.17 ± 3.35 64.64 ± 3.12

hexoside

Total 560.71 ± 25.83 621.96 ± 26.12 3723.10 ± 198.41 3989.40 ± 254.66

Flavones

8 653 Acacetin-O-diglucoside 107.12 ± 3.32 115.16 ± 3.65 126.71 ± 4.35 135.36 ± 4.77

21 639 Apigenin-C-hexoside- 521.89 ± 4.56 562.50 ± 5.21 1396.32 ± 43.74 1854.62 ± 54.23

O-hexoside

49 447 Luteolin-O-hexoside 55.32 ± 3.65 67.82 ± 2.82 267.26 ± 10.22 297.90 ± 9.75

51 431 Apigenin-O-hexoside 77.84 ± 2.55 107.14 ± 3.44 170.34 ± 9.41 200.58 ± 8.56

73 477 Apigenin-O-hexoside 232.07 ± 7.53 244.49 ± 7.01 654.72 ± 10.35 770.33 ± 12.49

80 575 Apigenin-C-hexoside 21.92 ± 1.62 38.69 ± 1.88 – –

derivative

90 845 Apigenin-C-hexoside 40.17 ± 2.33 42.83 ± 2.99 – –

derivative

123 269 Apigenin – – 16.77 ± 1.42 17.94 ± 2.15

Total 1056.83 ± 77.43 1178.63 ± 79.33 2632.12 ± 186.73 3276.73 ± 235.11

Isoflavones

9 623 Daidzin-O-dilhexoside 99.87 ± 2.9 116.37 ± 2.1 214.70 ± 5.70 250.16 ± 5.63



24 507 3 5,7- – – 159.21 ± 3.12 166.37 ± 3.24 trihydroxyisoflavone- 4-methoxy-3-O-␤- glucopyranoside

24 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27

Table 2 (Continued)

Leaves Flowers

◦

N MW Hydroxycinnamic acids Machico Funchal Machico Funchal

43 491 Glycitin isomer 1027.11 ± 55.1 1234.53 ± 53.88 1596.06 ± 53.78 1654.33 ± 50.01

48 461 Daidzein-O-hexoside 179.59 ± 4.11 219.35 ± 3.77 569.84 ± 27.41 695.99 ± 26.44

50 491 Glycitin isomer 33.59 ± 2.23 – 39.28 ± 1.78 41.27 ± 1.84

±

69 283 Glycitein 14.03 0.39 – – –

77 253 Daidzein 45.99 ± 1.45 48.80 ± 0.99 74.31 ± 3.73 77.93 ± 2.99

85 431 Genistin 7.83 ± 0.46 9.97 ± 0.71 19.82 ± 0.77 20.76 ± 1.11

98 475 Formononetin-O- 15.87 ± 0.99 18.14 ± 1.14 18.87 ± 0.79 23.14 ± 0.95

hexoside

110 491 Prunetin-O-hexoside 1.80 ± 0.09 12.52 ± 0.73 – –

± ±

Total 1425.68 95.78 1659.68 99.44 2692.05 ± 167.34 2929.95 ± 188.65

Flavanones

36 631 Narigenin-O-hexoside – – 14.05 ± 1.98 17.51 ± 1.64

derivative

60 681 Narigenin-O- – – 185.45 ± 17.43 – acetylhexoside

derivative

83 433 Narigenin-O-hexoside 115.90 ± 8.14 119.24 ± 6.32 145.45 ± 7.37 159.31 ± 7.01

121 271 Narigenin – – 52.19 ± 3.53 54.55 ± 1.12

±

±

75 507 Trihidroxy- – 153.30 11.75 318.69 10.99 457.26 ± 17.10 methoxylflavanone-O-

glucoside

47 665 Liquiritigenin 43.24 ± 2.56 69.35 ± 3.33 63.71 ± 2.95 72.92 ± 2.37

derivative

59 417 Liquiritin isomer – – 13.28 ± 1.03 17.50 ± 1.17

a

65 417 Liquiritin 12.67 ± 0.97 16.66 ± 0.81 23.20 ± 22.98 25.75 ± 18.99

76 417 Liquiritin isomer – – 496.77 ± 30.39 512.37 ± 35.71

78 621 Liquiritin-O-hexoside- – 60.69 ± 6.32 157.29 ± 8.31 170.75 ± 9.92

O-acetylhexoside

79 665 Liquiritigenin 214.55 ± 16.09 219.05 ± 15.79 249.52 ± 13.73 297.93 ± 10.35

derivative

88 503 Liquiritigenin – – 227.67 ± 14.11 249.62 ± 10.74

derivative

99 751 Liquiritigenin-O- 602.05 ± 34.08 659.81 ± 27.99 1354.62 ± 86.57 1406.06 ± 97.34 acetylhexoside

derivative

103 417 Liquiritin isomer 256.53 ± 17.03 296.79 ± 19.06 543.87 ± 43.65 589.11 ± 39.35

105 417 Liquiritin isomer 83.60 ± 8.49 102.43 ± 7.25 209.24 ± 26.33 294.00 ± 19.55

117 459 Liquiritigenin-O- 9.69 ± 0.27 16.17 ± 0.53 58.81 ± 4.32 68.19 ± 2.01

acetylhexoside

127 255 Liquiritigenin – – 22.05 ± 1.71 36.22 ± 2.09

Total 1338.23 ± 110.34 1713.50 ± 108.92 4135.87 ± 285.37 4428.85 ± 255.32

TIPC 4544.27 ± 334.69 5359.71 ± 313.13 13505.50 ± 1030.42 15013.42 ± 1197.44

a

Confirmed by commercial standard.

Table 3

Data from TPC, TFC, and in vitro antioxidant and enzyme inhibition assays.

TPCa TFCb DPPHc ABTSc NOc SOc ␣-Glucosidased ␣-Amylased

Leaves Funchal 23.48 ± 1.27 10.74 ± 0.51 111.16 ± 4.2 540.77 ± 11.08 22.21 ± 1.33 15.11 ± 1.04 1.97 ± 0.182 3.36 ± 0.247

Flowers Funchal 30.36 ± 1.41 24.96 ± 1.40 134.30 ± 5.3 660.23 ± 10.31 27.08 ± 1.17 17.15 ± 0.98 1.09 ± 0.086 2.57 ± 0.252

Leaves Machico 22.12 ± 0.99 9.81 ± 0.39 107.11 ± 3.7 531.17 ± 9.88 21.07 ± 1.03 14.05 ± 0.86 2.15 ± 0.157 3.43 ± 0.281

Flowers Machico 28.16 ± 1.16 21.16 ± 0.95 129.22 ± 6.6 653.29 ± 10.97 25.86 ± 1.04 16.85 ± 0.77 1.27 ± 0.091 2.79 ± 0.313

Acarbose – – – – – – 1.619 ± 0.023 0.02 ± 0.002

All measurements are expressed as mean ± SD (n = 3).

a

g GAE/100 g DE.

b

g QCE/100 g DE.

c

mmol TE/100 g DE.

d

IC50 value (mg/mL).

with less diversity. Flavonoids were about three times more Since their mass spectra are all very similar, exact identification was

abundant in flowers. As shown in Table 2, there was an even dis- not attempted and they are described in Tables 1 and 2 as liquir-



tribution of flavonoids amongst four classes: isoflavones (mainly itin isomers. A standard of liquiritin (liquiritigenin-4 -O-glucoside)

glycitin) < flavones (mainly apigenin derivatives) < flavonols (domi- was used, showing that in the extracts of Ulex, this was not the most

nated by quercetin glucosides) < flavanones. Significant differences abundant isomer (compound 65).

(p < 0.05) were found in isoflavones, flavonols and flavanones con- Ulex flowers were richer in liquiritigenin derivatives

tents only among leaves counterparts. The flavanone group was (3740 mg/100 g DE) than the roots and rhizomes of Glycyrrhiza

composed largely by derivatives (glucosides) of liquiritigenin; the glabra L. (about 1242 mg/100 g DE) (Martins et al., 2015), where

free aglycone itself was identified in small amounts in flower liquiritigenin apiosides were reported as the main flavonoids,

extracts. All four isomers of liquiritin (liquiritin, isoliquiritin, which curiously were absent in Ulex extracts. Other authors have

neoliquiritin and neoisoliquiritin) were detected and quantified. presented quantitative results in Glycyrrhiza species but expressed

V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27 25

0.5 A 0.4 Leaves M 0.3

0.2

0.1 Flowers M

%) 0

(16

-0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 -0.1 PC2 Flowers F -0.2

-0.3

Leaves F -0.4

-0.5 PC1 (67 %)

1 B 0.8 0.6 0.4 0.2

PC1

0

28 37 41 45 80 110 PC2 -0.2 -0.4 -0.6 -0.8

-1

Fig. 3. (A) PC1 × PC2 of scores scatter plot between different Ulex morphological parts and collection area; (B) PC1 × PC2 of loading plot of the main source of variability

between different Ulex morphological parts and collection area.

differently (mg/100 g dry plant, DP). Tian and co-workers (Tian These results are corroborative with previous screening and

et al., 2009) reported 29 mg/100 DP of liquiritin in commercial quantification of polyphenols and may justify in part the use of

liquorice, while in another study (Montoro et al., 2011), liquiriti- flowers in detriment of leaves in folk medicine (Rivera and Obón,

genin derivatives varied between 1498 and 4049 mg/100 g DP. The 1995). This variation in the distribution of phenolics may be respon-

latter study presented higher concentrations than Ulex flowers sible for the observed biological effects. An explanation for the

(893–1217 mg/100 g DP) analyzed in the present work. noticeably higher concentration of polyphenols in Funchal samples

may be due to the fact that these were collected at approximately

1400 m high (while Machico at about 600 m). At higher altitudes,

3.5. TPC, TFC and in vitro antioxidant assays

plant species are more subject to harsh environmental conditions

such as ray from the sunlight or cold. Stress factors may induce

In the present work, flowers proved to be a better source of phe-

intense synthesis of phenolic compounds as a response to abiotic

nolic compounds than leaves (Table 3). Moreover, samples from

stress in order to prevent oxidative damage of the plant cellular

Funchal presented higher TPC and TFC than Machico counterparts, structures.

which is in agreement with data shown in Table 2. Significant differ-

ences (p < 0.05) were found among flower samples in colorimetric

determinations. 3.6. Inhibition of digestive enzymes

Overall, the results obtained in the four antioxidant assays

revealed Ulex good ability to scavenge free radicals (in particu- Since Ulex was formerly used for the treatment of diabetes and

lar flowers material), which can be partially related to the higher liver diseases (Rivera and Obón, 1995), and liquiritin is described

content of polyphenols (Table 3). Under the assay conditions, as anti-diabetic (Gaur et al., 2014), the effect of methanolic

when comparing collection areas, it is possible to see that Funchal extracts towards key digestive enzymes linked to type II dia-

samples presented a stronger antioxidant capacity than Machico betes was evaluated. Ulex samples were more specific inhibitors

␣ ␣

counterparts. Significant differences (p < 0.05) in the antioxidant of -glucosidase than of -amylase activities (Table 3). In the

␣

activity were observed (except between flower samples in the ABTS -glucosidase assay, the results showed that flowers presented

assay). stronger inhibitory activity than leaves and acarbose. For ␣-

Based on the scavenging capacity observed for both NO and SO amylase, the same trend was observed, but a weaker inhibition

tests, Ulex might also prevent the formation of other biologically effect compared to the commercial drug acarbose was observed.

important oxidative species, like peroxynitrite and hydroxyl radical Statistical differences (p < 0.05) were found between acarbose and

(López-Alarcón and Denicola, 2013; Valko et al., 2007). Ulex samples, between morphological parts in all assays and in

26 V. Spínola et al. / Industrial Crops and Products 90 (2016) 9–27

the ␣-glucosidase assay between flowers samples. The inhibition Martínez acknowledges the financial support from the UCLM

activity could be attributed to the higher content of liquiritigenin Research Plan. This research was supported by FCT with funds from

derivatives in flowers than in leaves (Table 2). Previous studies the Portuguese Government (Project PEst-OE/QUI/UI0674/2013)

(Choi et al., 2010; Gaur et al., 2014) have reported the potent and the Portuguese National Mass Spectrometry Network (Contract

inhibition of liquiritigenin derivatives (neoliquiritin, liquiritin and RNEMREDE/1508/REM/2005).

liquiritigenin) on ␣-glucosidase, higher than acarbose.

Recently, the inhibitory effects of leaves from Senna suratten-

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