Food Chemistry 129 (2011) 454–462

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Food Chemistry

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In vitro studies to assess the antidiabetic, anti-cholinesterase and antioxidantpotential of Spergularia rubra

Juliana Vinholes a, Clara Grosso a, Paula B. Andrade a,⇑, Angel Gil-Izquierdo b, Patrícia Valentão a,Paula Guedes de Pinho c, Federico Ferreres b,⇑a REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, R. Aníbal Cunha, 164, 4050-047 Porto, Portugalb 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, Spainc REQUIMTE/Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, R. Aníbal Cunha, 164, 4050-047 Porto, Portugal

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 January 2011Received in revised form 9 March 2011Accepted 28 April 2011Available online 6 May 2011

Keywords:Spergularia rubraMetabolic profileAntioxidant activitya-Glucosidase inhibitionCholinesterases inhibition

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.04.098

⇑ Corresponding authors. Tel.: +351 222078934;Andrade).

E-mail addresses: pandrade@ff.up.pt (P.B. AndradFerreres).

Spergularia rubra is distributed all over the world, being its infusion used as diuretic. In spite of its largeuse, the antidiabetic, anti-cholinesterase and antioxidant activities of this species have not been assessedand its chemical composition is scarcely known. In the work herein a hydromethanolic extract was stud-ied.

Thirty-six phenolic compounds were determined by HPLC-DAD, comprising non-acylated C-glycosylflavones (38%), C-glycosyl flavones acylated with aromatic acids (36%), C-glycosyl flavones acylated withaliphatic acids (13%) and 10% corresponded to C-glycosyl flavones with a mixed acylation. Organic acids(oxalic, citric, malic, quinic and fumaric acids) and fatty acids (azelaic, myristic, palmitic, linoleic, linole-nic and stearic acids) are described for the first time. Their determination by HPLC-UV and GC-IT–MSallowed finding concentrations of 192.15 and 34.87 g/kg, respectively.

The extract showed a dose-dependent response against DPPH, superoxide and nitric oxide radicals. Thesame effect was observed in the a-glucosidase inhibitory assay and against acetylcholinesterase andbutyrylcholinesterases. The bioactivities observed may be due, at least partially, to the presence of thedifferent metabolites determined in the present study. The results suggest that the dried extract of S.rubra may be interesting for incorporation in pharmaceutical preparations for human health, since itcan suppress hyperglycaemia and inhibit cholinesterases, and or as food additive due to its antiradicalactivity.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Spergularia (Caryophyllaceae) is a cosmopolitan genus distrib-uted from temperate to subtropical climatic zones of all continents,excepting Antarctica (Hartman & Rabeler, 2005). Several specieshave been studied from the chemical and pharmacological pointsof view (Jouad, Eddouks, Lacaille-Dubois, & Lyoussi, 2000; Jouad,Lacaille-Dubois, & Eddouks, 2001; Jouad, Lemhadri, Maghrani,Zeggwagh, & Eddouks, 2003) revealing to be active as hypoglycae-mic (Jouad et al., 2000), diuretic (Jouad et al., 2001), antihyperten-sive and cholesterol-lowering agents (Jouad et al., 2003).

Spergularia rubra aerial parts are widely used for the prepara-tion of an infusion, consumed for its diuretic properties. However,literature concerning the chemical composition of this species is

ll rights reserved.

fax: +351 222003977 (P.B.

e), federico@cebas.csic.es (F.

scarce and despite the identification of some compounds no quan-tification data is reported. For instance, the occurrence of phytoec-dysteroids reported in S. rubra by Zibareva et al. (2003). In addition,some di-C-glycosyl-flavones were isolated from this species andtheir structures elucidated by spectroscopic means (Bouillantet al., 1979, 1984) and more recently the identification of 36 C-gly-cosyl-flavones, including acylated derivatives, was reported by ourgroup (Ferreres et al., 2011). This kind of compound shows a broadspectrum of activities, such as antibiotic, pancreatic lipase inhibi-tors (Lee et al., 2010), radical scavenging (Aquino et al., 2001;Lin, Chen, Lee, & Lin, 2002; Rauter et al., 2009), cholinesterase inhi-bition and anti-hyperglycaemic (Rauter et al., 2009).

Diabetes mellitus is an emerging health problem in westernsocieties. The scientific community is searching for new naturalcompounds with antidiabetic properties to overcome any resis-tance developed by patients to the currently used drugs. a-Gluco-sidase inhibitors act by reversible inhibition of a-glucosidase, anenzyme responsible for the breakdown of a-glycosidic bonds incomplex carbohydrates to release absorbable monosaccharides

J. Vinholes et al. / Food Chemistry 129 (2011) 454–462 455

(Li, Quia, & Li, 2010). Until now, there is only a brief reference tothe hypoglycaemic activity of S. rubra in a conference proceeding(Jouad & Lyoussi, 1998) without mention to the involvedmechanism.

On the other hand, Alzheimer’s disease (AD), which is growingall over the world, is a chronic, progressive, neurodegenerative dis-order of the brain leading to changes in behaviour and cognition.The treatment of this disease is based on the cholinergic hypothe-sis that states that the cognitive decline in AD results from the lossof acetylcholine. One possible strategy for enhancing cholinergicactivity in the brain is by inhibition of the enzymes responsiblefor the cleavage of the neurotransmitter, which are acetylcholines-terase (AChE) and butyrylcholinesterase (BuChE). In this way, ace-tylcholine (ACh) is not converted to acetyl-CoA and choline,thereby increasing the availability of ACh to stimulate nicotinicand muscarinic receptors within the brain (Houghton, Ren, &Howes, 2006). To the best of our knowledge, no previous workabout the potential of S. rubra as AChE and BuChE inhibitor hasbeen published.

Moreover, it is well known that a series of human illnesses, suchas cancer, atherosclerosis, cardio- and cerebrovascular diseases,diabetes, immune system impairment, and neurodegenerative dis-eases, is related to the deleterious action of reactive free radicals.Nevertheless, as far as we know the antiradical properties of thisspecies has not been assessed yet.

Therefore, the aim of this work was to increase the knowledgeon the metabolic profile of S. rubra aerial parts, concerning fattyacids, phenolic compounds, and organic acid, and to assess theirantioxidant properties and their potential as a-glucosidase andacetyl and butyrylcholinesterase inhibitor as well, in order to pro-pose plant’s extract as food supplement or additive to be used byfood and pharmaceutical industries.

2. Material and methods

2.1. Plant material

The dried aerial parts of S. rubra (L.) (Caryophyllaceae), corre-sponding to a mixture of different individuals, were purchased inthe local market from a medicinal plants distributor (Morais eCosta & C.ª Lda, Portugal). The identity was confirmed by theauthors following the characteristics described in Floras (FloraIberica, 2003; Hartman & Rabeler, 2005). The acquired samplewas also compared with an individual occurring in nature. Theplant material was powdered (mean particle size lower than910 lm). Voucher specimen was deposited at Laboratório deFarmacognosia, Faculdade de Farmácia, Universidade do Porto(SR-AP-032010).

2.2. Standards and reagents

Reference compounds were purchased from various suppliers:30,7-di-O-glucosyl luteolin, 7-O-glucosyl isovitexin, and chrysoeriolwere from Extrasynthèse (Genay, France), oxalic, citric, malic,quinic and fumaric acid from Sigma (St. Louis, MO, USA), fatty acidmethyl esters kit was purchased from Supelco (Bellafonte, PA, USA)and methyl jasmonate (internal standard) from SAFC (St. Louis,USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH�), b-nicotinamide ade-nine dinucleotide (NADH), phenazine methosulfate (PMS), nitrotet-razolium blue chloride (NBT), 5,50-dithio-bis(2-nitrobenzoic acid)(DTNB), sulphanilamide, acetylcholinesterase (AChE) from electriceel (type VI-s, lyophilised powder), acetylthiocholine iodide (ATCI),butyrylcholinesterase (BuChE) from equine serum (lyophilisedpowder), S-butyrylthiocholine chloride, a-glucosidase (type I frombaker’s yeast) and 4-nitrophenyl a-D-glucopyranoside (PNP-G)

were purchased from Sigma (St. Louis, MO, USA). Acarbose wasfrom Bluepharma� Genéricos (Coimbra, Portugal). N-(1-Naph-thyl)ethylenediamine dihydrochloride, ethanol, potassium dihy-drogen phosphate, sulphuric and formic acids were obtained fromMerck (Darmstadt, Germany). Sodium nitroprussiate dehydrate(SNP) was purchased from Sigma–Aldrich (Steinheim, Germany).Sodium sulphate anhydrous and isooctane were purchased fromPanreac Química SA (Barcelona, Spain). Potassium hydroxide wasfrom Pronalab (Lisboa, Portugal) and boron trifluoride (BF3) 10%methanolic solution from Supelco (Bellafonte, PA, USA).

2.3. Extraction

Aerial parts of S. rubra were extracted with methanol:water(1:1) mixture (1 g/100 ml), as follows: 0.5 h of sonication, 2 h ofstirring (200 rpm) maceration at room temperature, plus 0.5 h ofsonication, filtration and evaporation under reduced pressure.Extractions were carried out in triplicates. The yield of S. rubrahydromethanolic extracts was 0.25 g (±0.02 g) from the startingdry material.

2.4. Phenolic compounds HPLC-DAD analysis

For phenolic compounds determination, 20 ll of the redissolvedhydromethanolic (30.4 mg/ml MeOH/H2O) were analysed on ananalytical HPLC unit (Gilson), using a ACE 3 C-18-AR column(150 � 4.6 mm, 3 lm particle size, Advanced ChromatographyTechnologies, Aberdeen, Scotland). The mobile phase consisted oftwo solvents: water (1% acetic acid) (A) and methanol (B), startingwith 20% B and using a gradient to obtain 50% at 30 min, 60% at55 min, 90% at 57 min., at a solvent flow rate of 0.5 ml/min. Detec-tion was achieved with a Gilson diode array detector. Spectral datafrom all peaks were collected in the range of 200–400 nm, andchromatograms were recorded at 350 nm. The data were processedon an Unipoint System software (Gilson Medical Electronics,Villiers le Bel, France). The compounds were identified by compar-ing their elution order and UV–Vis spectra in the 200–400 nmrange with those of flavonoids already reported by us for hydrome-thanolic extract of S. rubra (Ferreres et al., 2011).

Peak purity was checked by the software contrast facilities.Phenolic compounds quantification was achieved by the absor-bance recorded in the chromatograms relative to external stan-dards by using the following equation:

CðcÞ ¼ AðcÞAðstÞ � CðstÞ

where C(c) is the concentration of the compound in the sample, A(c)is the peak area of the compound in the sample chromatogram, C(st)is the concentration of the standard in the reference solution andA(st) is the area of the peak for the standard in the referencechromatogram.

Since there is no standard of the C-glucosyl flavonescommercially available, luteolin derivatives were quantified as30,7-di-O-glucosyl luteolin, apigenin derivatives as 7-O-glucosylisovitexin and chrysoeriol derivatives as chrysoeriol. In the cases ofco-elution, the UV spectra allowed to notice which was the mostabundant derivative. Thus, 6-C-glucosyl-8-C-arabinosyl apigeninand 6-C-glucosyl-8-C-(40 0 0-malonyl)glucosyl luteolin (compounds8 + 9) were quantified together as 30,7-di-O-glucosyl luteolin, whilethe pairs 20 0-O-glucosyl-6-C-arabinosyl apigenin plus 6-C-glucosyl-8-C-(20 0 0-p-coumaroyl)glucosyl luteolin (compounds 22 + 23),6-C-glucosyl-8-C-(20 0 0-feruloyl)glucosyl chrysoeriol plus 6-C-gluco-syl-8-C-(20 0 0-dihydroferuloyl)glucosyl apigenin (compounds30 + 31), and 6,8-diC-(60 0malonyl, feruloyl)glucosyl chrysoeriol plus

0.00

0.50

0 20 40 60

26

27

2829

30+31

3233

34

35+36

25

1

2

3

4

5

6

7

8+9

10

11

12

13

14

1516

21

22+23

24

17 18

1920

Arb

itra

ryun

its

Time (min.)

Fig. 1. HPLC-DAD chromatogram of phenolic compounds from Spergularia rubra MeOH/H2O extract. Identity of compounds as in Table 1.

Table 1Flavonoids composition of S. rubra (g/kg of plant hydromethanolic extract).a

Flavonoid MeOH/H2O

1 7-O-glucosyl-6,8-diC-glucosyl luteolin 0.73 ±0.082 6,8-diC-glucosyl luteolin 5.21 ±0.163 6,8-diC-glucosyl apigenin 14.46 ±0.964 6,8-diC-arabinosyl luteolin 1.04 ±0.055 6,8-diC-glucosyl chrysoeriol 4.27 ±0.196 6-C-(60 0-acetyl)glucosyl-8-C luteolin 1.38 ±0.047 6-C-(60 0-malonyl)glucosyl-8-C luteolin 1.38 ±0.088 + 9 6-C-glucosyl-8-C-arabinosyl apigenin + 6-C-glucosyl-8-C-(4’’’-malonyl)glucosyl luteolin 4.79 ±0.2610 6-C-arabinosyl-8-C-glucosyl chrysoeriol 0.93 ±0.0711 6-C-(20 0-feruloyl)glucosyl-8-C luteolin 0.70 ±0.0512 6-C-glucosyl-8-C-arabinosyl apigenin 1.36 ±0.0913 6-C-(40 0-malonyl)glucosyl-8-C apigenin 4.69 ±0.4314 7,20 0-diO-glucosyl-6-C-arabinosyl apigenin 21.87 ±1.7515 6-C-(40 0-malonyl)glucosyl-8-C chrysoeriol 0.95 ±0.0616 7-O-glucosyl-6-C-arabinosyl-8-C-(6’’’-malonyl)arabinosyl chrysoeriol 6.08 ±0.2417 6-C-(20 0-feruloyl)glucosyl-8-C apigenin 3.92 ±0.2518 7-O-glucosyl-6-C-(20 0-malonyl)arabinosyl-8-C-arabinosyl chrysoeriol 0.92 ±0.1519 7-O-glucosyl-6-C-glucosyl-8-C-(20 0 0-sinapoyl)glucosyl luteolin 1.04 ±0.0520 7-O-glucosyl-6-C-glucosyl-8-C-(20 0 0-feruloyl)glucosyl luteolin 0.77 ±0.0721 6-C-glucosyl-8-C-(20 0 0-sinapoyl)glucosyl luteolin 2.28 ±0.1222 + 23 20 0-O-glucosyl-6-C-arabinosyl apigenin + 6-C-glucosyl-8-C-(2’’’-p-coumaroyl)glucosyl luteolin 2.78 ±0.2324 6-C-glucosyl-8-C-(20 0 0-feruloyl)glucosyl luteolin 4.62 ±0.1825 6-C-glucosyl-8-C-(20 0 0-dihydroferuloyl)glucosyl luteolin 2.18 ±0.1226 6-C-glucosyl-8-C-(20 0 0-sinapoyl)glucosyl apigenin 7.33 ±0.6627 6-C-glucosyl-8-C-(20 0 0-sinapoyl)glucosyl chrysoeriol 1.72 ±0.1228 6,8-diC-(2’’’malonyl, feruloyl)glucosyl luteolin 1.26 ±0.1029 6-C-glucosyl-8-C-(20 0 0-feruloyl)glucosyl apigenin 10.30 ±0.2930 + 31 6-C-glucosyl-8-C-(20 0 0-feruloyl)glucosyl chrysoeriol + 6-C-glucosyl-8-C-(2’’’-dihydroferuloyl)glucosyl apigenin 10.92 ±0.7432 6-C-glucosyl-8-C-(20 0 0-dihydroferuloyl)glucosyl chrysoeriol 1.63 ±0.1233 6,8-diC-(60 0 0malonyl, sinapoyl?)glucosyl chrysoeriol 0.83 ±0.0434 6,8-diC-(60 0 0malonyl, sinapoyl?)glucosyl apigenin 2.40 ±0.1735 + 36 6,8-diC-(60 0malonyl, feruloyl?)glucosyl chrysoeriol + 6,8-diC-(6’’’malonyl, feruloyl)glucosyl apigenin 7.35 ±0.26

Total 132.07

a Results are expressed as mean ± standard deviation of three assays.

456 J. Vinholes et al. / Food Chemistry 129 (2011) 454–462

Fig. 2. HPLC-UV chromatogram of organic acids from Spergularia rubra MeOH/H2O extract (A) and standards mixture (B). 1- Oxalic acid; 2- Citric acid; 3- Malic acid; 4- Quinicacid; 5- Fumaric acid and MP- mobile phase.

J. Vinholes et al. / Food Chemistry 129 (2011) 454–462 457

6,8-diC-(60 0 0malonyl, feruloyl)glucosyl apigenin (compounds35 + 36) were quantified as 7-O-glucosyl isovitexin (Fig. 1, Table 1).

2.5. Organic acids

2.5.1. ExtractionOrganic acids profile was determined by redissolving 42.14 mg

of the dried hydromethanolic extract in 1 ml of H2SO4 (0.01 N), fol-lowed by filtration and analysis by HPLC/UV (20 ll).

2.5.2. HPLC-UV analysisOrganic acids were analysed on an analytical HPLC unit (Gilson),

using an ion exclusion Nucleogel Ion 300 OA column(300 � 7.7 mm) (Germany), in conjunction with a column heatingdevice set at 30 �C. Elution (70 min) was carried out at a solventflow rate of 0.2 ml/min, isocratically, with H2SO4 0.01 N as mobilephase. Detection was performed with a Gilson UV–Vis detector at214 nm. The organic acids quantification was achieved by theabsorbance recorded in the chromatograms relative to external

Table 2Organic acid composition of S.rubra (g/kg of plant hydrome-thanolic extract)a.

Organic acid Content

1 Oxalic 71.23 ±5.992 Citric 12.32 ±2.643 Malic 36.57 ±5.504 Quinic 63.24 ±0.175 Fumaric 8.80 ±0.91

Total 192.15

a Results are expressed as mean ± standard deviation ofthree assays.

Table 3Free fatty acids composition (as methyl esters) of Spergularia rubra(g/kg of hydromethanolic extract)a.

Fatty acids Content

1 Azelaic acidb 1.43 ±0.132 Myristic acidc 0.86 ±0.103 Palmitic acidc 13.25 ±0.604 Linoleic acidc 1.62 ±0.155 Linolenic acidc 14.77 ±1.076 Stearic acidc 2.94 ±0.22

Total 34.87

a Results are expressed as mean ± standard deviation of threeassays.

b Expressed in equivalents of caprylic acid (C8:0). Tentativelyidentified by NIST 05 database, with fit/retrofit values of 85/90,respectively.

c Compound quantified with correspondent standard.

458 J. Vinholes et al. / Food Chemistry 129 (2011) 454–462

standards, using the equation indicated above for phenolic com-pounds quantification.

2.6. Fatty acids

2.6.1. DerivatizationThe extraction and derivatization procedure was performed

according to Ribeiro, Guedes de Pinho, Andrade, Baptista, and Va-lentão (2009) with some modifications. Briefly, 20 ll of methyljasmonate (1 g/l) (internal standard) was added to 200 ll of redis-solved plant hydromethanolic extract in MeOH/H2O (1:1). Themixture was dried under reduced pressure (40 �C) and redissolvedin 2 ml of methanol, followed by simultaneous hydrolysis andderivatization with 1 ml of KOH methanolic solution (11 g/l), at90 �C for 10 min. Derivatization was assured by treatment with1 ml of BF3 methanolic solution (10%), at 90 �C, for 10 min, result-ing in methyl ester forms. The fatty acid methyl esters (FAME)were purified with 2 � 6 ml of isooctane and anhydrous sodiumsulphate was added to assure the total absence of water. The ex-tract was then evaporated under a stream of nitrogen and redis-solved in 200 ll of isooctane.

2.6.2. Gas chromatography-ion trap-mass spectrometry (GC–IT-MS)analysis

The FAME quantification was performed by external calibrationcurves obtained by GC-IT-MS for the respective methyl esters ofcaprylic, myristic, palmitic, stearic, linoleic and linolenic acids, ina concentration series of 4.00, 2.00, 1.00, 0.50, and 0.25 mg/l pre-pared in isooctane.

The standard mixture/sample extract were analysed using aVarian CP-3800 gas chromatograph (USA) equipped with a VARIANSaturn 4000 mass selective detector (USA) and a Saturn GC/MSworkstation software version 6.8. A VF-5 ms (30 m � 0.25 mm �0.25 lm) column (VARIAN) was used. The injector port was heatedto 250 �C. Injections were performed in split mode, with a ratio of1/40. The carrier gas was helium C-60 (Gasin, Portugal), at a con-stant flow of 1 ml/min. The oven temperature was set at 40 �Cfor 1 min, then increasing 5 �C/min to 250 �C, 3 �C/min to 300 �Cand held for 15 min. All mass spectra were acquired in electron im-pact (EI) mode. Ionisation was maintained off during the first4 min, to avoid solvent overloading. The Ion Trap detector wasset as follows: transfer line, manifold and trap temperatures were280, 50, and 180 �C, respectively. The mass ranged from 50 to600 m/z, with a scan rate of 6 scan/s. The emission current was50 lA, and the electron multiplier was set in relative mode to autotune procedure. The maximum ionisation time was 25.000 ls, withan ionisation storage level of 35 m/z. The injection volume for li-quid extracts was 1 ll and the analysis was performed in Full Scanmode. Identification of compounds was achieved by comparison oftheir mass spectra with those from pure standards analysed underthe same conditions, and from NIST 05 MS Library Database. Theextracts were analysed and the amounts of fatty acids methyl es-ters present in samples was achieved from the calibration curveof the respective FAME standards. The FAMEs values were thenconverted into their respective fatty acid contents.

2.7. Biological potential

2.7.1. GeneralFor the evaluation of the biological potential of S. rubra

hydromethanolic extract in vitro microassays were performed in96-well plates, applying spectrophotometric methods. Absorbancewas measured in a Multiskan Ascent plate reader (Thermo ElectronCorporation). The EC50 values were calculated for three indepen-dent assays performed in triplicate.

2.7.2. In vitro antioxidant activity2.7.2.1. DPPH� assay. The free radical-scavenging activity of the ex-tract (0.03–0.92 mg/ml) was determined by monitoring the de-crease of absorbance of DPPH� at 515 nm, according to Oliveiraet al. (2010).

2.7.2.2. Superoxide anion O��2 assay. O��2 was generated by theNADH/PMS system, as referred in Oliveira et al. (2010). The extractwas tested in a concentration range between 0.40 and 7.05 mg/ml.The absorbance of the reaction mixture was determined at 560 nmat room temperature, for 2 min.

2.7.2.3. Nitric oxide (�NO) assay. The antiradical activity was deter-mined using a previously described procedure (Oliveira et al.,2010), with different extract concentrations (0.10–1.58 mg/ml).The absorbance was measured at 562 nm.

2.7.3. a-Glucosidase inhibitory activitya-Glucosidase inhibitory activity was assessed by modification

of a previously reported procedure (Chan, Sun, Reddy, & Wu,2010). Briefly, each well contained 100 ll of 2 mM 4-nitrophenyla-D-glucopyranoside (PNP-G) in 100 mM potassium phosphate buf-fer (pH 7.0) and 20 ll of the extract (0.27–4.92 mg/ml) or acarbose(positive control) also in buffer. The reaction was initiated by theaddition of 100 ll of the enzyme solution (56.6 mU/ml). The plateswere incubated at 37� C for 10 min. The absorbance of 4-nitrophe-nol released from PNP-G at 400 nm was measured. The increase inabsorbance (DA) was compared with that of the control (buffer in-stead of sample solution) to calculate the inhibitory activity.

2.7.4. AChE and BuChE inhibitory activityThe inhibition of AChE activity was determined based on

Ellman’s method, as previously reported (Pereira et al., 2010).The absorbance was measured at 405 nm and the rates of reactionswere calculated by Ascent Software version 2.6 (Thermo

Fig. 3. GC-IT-MS chromatogram of S. rubra MeOH/H2O extract methyl esters of free fatty acids. 1- Azelaic acid; 2- Myristic acid; 3- Palmitic acid; 4- Linoleic acid; 5- Linolenicacid; 6- Stearic acid and IS-Methyl jasmonate.

J. Vinholes et al. / Food Chemistry 129 (2011) 454–462 459

Labsystems Oy). The BuChE inhibition assay was performed in asimilar way, using 25 ll of substrate (15 mM BTCCI) and 25 ll ofenzyme (0.1 U/ml of BuChE). Extracts were evaluated at differentconcentrations (0.61–9.88 mg/ml).

3. Results and discussion

3.1. Metabolic profile

3.1.1. Phenolic compoundsPhenolic compounds in the aerial parts of S. rubra were recently

identified by our group (Ferreres et al., 2011). These included non-acylated flavones, C-glycosyl flavones acylated with aromatic oraliphatic acids and others with mixed acylation, that is, with botharomatic and aliphatic acids. The analysis by HPLC–DAD (Fig. 1)

performed in the present work allowed their quantification forthe first time. Concentrations ranging from 0.70 to 21.87 g/kg werefound in the extract (Table 1). Phenolic compounds corresponded to13.21% of S. rubra hydromethanolic extract and were mainlyrepresented by apigenin derivatives (64%), followed by luteolin(23%) and chrysoeriol derivatives (13%). Excluding the co-elutingcompounds, the non-acylated C-glycosyl-flavones and those acyl-ated with aromatic acids were clearly responsible for the majorityof phenolics in the extract, accounting for 38% and 36% of theirtotal, respectively. The most representative compound in eachgroup was 7,20 0-diO-glucosyl-6-C-arabinosyl apigenin (14, 17%), 6-C-glucosyl-8-C-(20 0 0-feruloyl)glucosyl apigenin (29, 8%), 7-O-gluco-syl-6-C-arabinosyl-8-C-(60 0 0-malonyl)arabinosyl chrysoeriol (16,4%), and the pair 6,8-diC-(60 0malonyl, feruloyl)glucosyl chrysoeriolplus 6,8-diC-(60 0 0malonyl, feruloyl)glucosyl apigenin (35 + 36, 6%)

A

B

C

0.01 0.1 1

0

20

40

60

80

100

Concentration in Log 10 (mg/mL)

DPP

H. S

cave

ngin

g (%

)

0.1 1 10

0

20

40

60

80

100

Concentration in Log 10 (mg/mL)

. NO

sca

veng

ing

(%)

0.1 1 10

0

20

40

60

80

100

Concentration in Log 10 (mg/mL)

O2• -

scav

engi

ng (%

)

Fig. 4. Scavenging activity of S. rubra MeOH/H2O extract against (A) DPPH�, (B)nitric oxide radical (�NO), and (C) superoxide radical (O��2 ). Values show mean ± SEMof three experiments performed in triplicate.

460 J. Vinholes et al. / Food Chemistry 129 (2011) 454–462

(Table 1). As far as we know, no phenolics content was determinedbefore in S. rubra hydromethanolic extract or in its vegetal material.

3.1.2. Organic acidsConcerning the organic acid composition, five compounds,

namely oxalic, citric, malic, quinic, and fumaric acids were foundin S. rubra (Fig. 2), in concentrations ranging from 8.80 to71.23 g/kg of dried extract (Table 2). To our knowledge, the organicacids composition of S. rubra is reported for the first time.

Oxalic and quinic acids were responsible for 70% of the total or-ganic acids content, corresponding to 37% and 33%, respectively(Table 2). The high content of oxalic acid in S. rubra was expectedsince Caryophyllaceae family is one of the most extreme oxalateaccumulators (more than 5% oxalate by dry weight) (Libert &Franceschi, 1987). However, due to its high oxalic acid content,some care must be taken, and the consumption of high dosesshould be avoided because oxalic acid has a predicted LD50 valueof 375 mg/kg body weight (Ferreres et al., 2006). The toxicity ofoxalic acid is related with its capacity to form insoluble calciumsalts, which cause a disturbance in the calcium concentrationsand affect the blood coagulation mechanism (DerMarderosian &Beutler, 2002). Additionally, the ingestion of oxalates in high dosesmay result in gastrointestinal symptoms, and the systemic absorp-tion may lead to kidney damage (Newall, Anderson, & Phillipson,1996).

3.1.3. Fatty acidsSix fatty acids (FA) were determined as their methyl esters deriv-

atives, with five of them being fully identified by GC–MS inhydromethanolic extract of S. rubra. FA compounds with monocar-boxylic structures comprising C14:0 to C18:2 were found (Table 3,Fig. 3), plus one dicarboxylic acid. As far as is known this is the firstreport on S. rubra free fatty acids composition. Linolenic (9,12,15-octadecatrienoic acid, C18:3) and palmitic acid (hexadecanoic acid,C16:0) represented equally almost 80% of the total determined fattyacids. Apart from azelaic acid (nonadioic acid), the FAs found consti-tute structural lipids in plant cell membranes (Taiz & Zeiger, 1998).

3.2. Biological potential

3.2.1. Antioxidant activityThe evaluation of hydromethanolic extract of S. rubra as radical

scavenger was performed against DPPH�, O��2 , and �NO radicals. Adose-dependent response was observed in all assays (Fig. 4A–C),and EC50 values of 0.38, 1.08, and 0.51 mg/ml, were found, respec-tively. To the best of our knowledge this is the first report on thein vitro antioxidant activity of Spergularia genus. The inhibitoryactivity over O��2 and �NO radicals is seen of great importance sinceit prevents the formation of other deleterious radicals, like perox-ynitrite, which plays an important role in cardiovascular, neurolog-ical and airway diseases and is also able to nitrosylate biomolecules,for instance, proteins (Pacher, Beckman, & Liaudet, 2007).

The results obtained may be attributed, at least partially, to theC-glycosyl flavones. This fact is corroborated by the works ofRauter et al. (2009) and Aquino et al. (2001) who reported DPPH�

scavenging by rich C-glycosyl flavones fractions of Genista teneraand Anthurium versicolor, respectively. Furthermore, the secondmajor phenolic compound in S. rubra, 6,8-diC-glucosyl apigeninhad already shown a strong anti-radical activity (Velozo et al.,2009). Furthermore, Lin et al. (2002) confirmed that 6-C-glucosylapigenin was active against lipid peroxidation being this com-pound less toxic when compared with kampferol and quercetin,two well known antioxidants (Lin et al., 2002). In addition, theantioxidant activity of phenolic compounds is related with thepresence of a double bond between C-2 and C-3 and a free hydro-xyl group at C-40, which were present in all flavone derivatives

found in S. rubra, a o-di-OH B-ring, present only in luteolin deriva-tives, besides a free hydroxyl at C-3, absent in the determined com-pounds (Bors, Heller, Michel, & Saran, 1990).

On the other hand, the high content of oxalic acid in S. rubramay give an important contribution to the antiradical activity sinceoxalate was reported as a very efficient lipid peroxidation inhibitor(TBAR’s) (Kayashima & Katayama, 2002). Also quinic acid, found insimilar content, may be important, as it has proved to be a goodantioxidant in clinical trials (Pero, Lund, & Leanderson, 2009).

3.2.2. In vitro a-glucosidase inhibitory activityThe goal in diabetes mellitus treatment is to achieve blood

glucose levels as close to normal as possible. a-Glucosidase is anenzyme that catalyses the final step in the digestive process of car-bohydrates, and hence a-glucosidase inhibitors (e.g. acarbose,

0.1 1 10 100

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Fig. 6. Acetyl (A) and butyrylcholinesterase (B) inhibition of Spergularia rubraMeOH/H2O extract. Results show mean ± SEM of three experiments performed intriplicate.

J. Vinholes et al. / Food Chemistry 129 (2011) 454–462 461

miglitol, and voglibose) could retard the use of dietary carbohy-drates to suppress postprandial hyperglycemia (Li et al., 2010).

The hydromethanolic extract of S. rubra was able to inhibit thea-glucosidase in a dose-dependent manner, with an IC50 value of2.55 mg/ml (Fig. 5). If, by one hand, this value was higher than thatfound for the positive control acarbose (IC50 = 0.30 mg/ml), the S.rubra extract inhibition is considerably better than that reportedfor some plants used in the treatment of diabetes, like Viscumalbum (IC50 = 11.7 mg/ml) (Seçil, Timur, Okutucu, & Zihnioglu,2005) and Glycyrrhiza uralensis (IC50 = 20.1 mg/ml) (Li et al.,2010). According to the results found, the inhibition of a-glucosi-dase may be one of the mechanisms involved in the hypoglycaemiceffect of S. rubra.

As hypothesised above, the presence of C-glycosyl flavones canbe responsible for the activity of the extract since monoglycosyland diglycosyl flavones were reported to decrease remarkablythe glucose blood levels (Rauter et al., 2009). Although there isno report for the a-glucosidase activity of di-C-glycosyl flavones,major compounds found in S. rubra hydromethanolic extract, somemolecular characteristics responsible for the activity are present inquantified compounds. Therefore, according to the structure activ-ity relationship already described for phenolic compounds asa-glucosidase inhibitors, it can be specified that, as observed forthe antioxidant activity, the presence of unsaturation betweenC-2 and C-3, in addition to the OH-groups in positions C-5 andC-40 for all flavone derivatives found in S. rubra extract, and theOH-group at C-30 for the luteolin derivatives, may be responsiblefor the extract activity (Li et al., 2009; Tadera, Minami, Takamatsu,& Matsuoka, 2006).

Concerning the fatty acids palmitic, linoleic, linolenic and stea-ric, these compounds were able to inhibit a-glucosidase at differ-ent levels (Miyazawa, Yagi, & Taguchi, 2005). According to theseauthors, the inhibitory effect increases, with the increase of doublebonds, thus linolenic acid was the most active, followed by linoleic,palmitic and stearic acids. The effect observed for S. rubrahydromethanolic extract can also be partially attributed to thepresence of palmitic and linolenic acids, and, to a less extent, tostearic acid too.

3.2.3. AChE and BChE inhibitionAlzheimer’s disease is the most common cause of dementia in the

elderly. Nowadays, the search of inhibitors of cholinesterases (acetylor butyryl) is a challenge since these enzymes are associated with theetiology of Alzheimer’s disease (Greig, Lahiri, & Sambamurti, 2002).Under the assay conditions, the hydromethanolic extract of S. rubraexhibited some activity against AChE (EC25 = 3.68 mg/ml) (Fig. 6A)and BuChE (EC25 = 4.29 mg/ml) (Fig. 6B). In fact, concerning AChE

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Fig. 5. a-Glucosidase inhibition of S. rubra MeOH/H2O extract. Results showmean ± SEM of three experiments performed in triplicate.

inhibition the extract revealed to be less active than the positive con-trol physostigmine (EC50 = 1.77 lg/ml) tested before under the sameconditions (Pereira et al., 2010). Nevertheless, taking into accountthe chemical composition of the extract, the presence of C-glycosylflavones may be partially responsible for the activity, as it was alsoverified for other plants (Rauter et al., 2009). In a recent study, itwas shown that the presence of a 4’-OMe group, and the O-glycosylation in position 7 are both important requirements forphenolics activity, however, the length and the interglycosidic link-ages of the sugar chain are also determinant (Fan, Hay, Marston, &Hostettmann, 2008). In contrast, for BChE activity the methoxylgroup at C-40 seems to be not so important (Sawasdee, Sabphon,Sitthiwongwanit, & Kokpol, 2009). The extract activity can beexplained, in part, by the presence of 25% of 7-O-glucosylatedflavones (7-O-glucosyl-6,8-diC-glucosyl luteolin, 7,20 0-diO-gluco-syl-6-C-arabinosyl apigenin, 7-O-glucosyl-6-arabinosyl-8-C-(60 0 0-malonyl)arabinosyl chrysoeriol, 7-O-glucosyl-6-C-(20 0-malonyl)arabinosyl-8-C-arabinosyl chrysoeriol, 7-O-glucosyl-6-C-glucosyl-8-C-(20 0 0-sinapoyl)glucosyl luteolin, and 7-O-glucosyl-6-C-gluco-syl-8-C-(20 0 0-feruloyl)glucosyl luteolin). In addition, palmitic andlinolenic acid, the major FA compounds in S. rubra, were reportedwith moderate AChE inhibitory activity while only linolenic acidwas mild active against BChE (Fang et al., 2010).

4. Conclusion

The study reported herein describes, for the first time, somebiological activities of S. rubra, as well as the organic and fatty acidscomposition and phenolics quantification. The results indicate thatS. rubra aerial parts are a rich source of bioactive compounds show-ing a-glucosidase and cholinesterases (AChE and BuChE) inhibitory

462 J. Vinholes et al. / Food Chemistry 129 (2011) 454–462

properties and radical scavenging capacity. This study adds valueto the species knowledge and due to its complex metabolite con-tent and the cover of different biological activities incites the useof its extract, after drying the solvent, in human health applica-tions, both in pharmaceutics and food industries products.

Acknowledgments

The authors thank ‘‘Consolider Ingenio 2010 Project CSD2007-00063 FUN-C-FOOD’’ and ‘‘Grupo de excelencia de la región deMurcia 04486/GERM/06’’. Clara Grosso thanks the Fundação paraa Ciência e a Tecnologia for the Post-Doc fellowship (SFRH/BPD/63922/2009).

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