Antioxidant Capacity, Polyphenolic Content and Tandem HPLC–DAD–ESI/MS Proﬁling of Phenolic Compounds from the South American Berries Luma Apiculata and L

Food Chemistry 139 (2013) 289–299

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Food Chemistry

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Analytical Methods Antioxidant capacity, polyphenolic content and tandem HPLC–DAD–ESI/MS proﬁling of phenolic compounds from the South American berries Luma apiculata and L. chequén ⇑ Mario J. Simirgiotis a, , Jorge Bórquez a, Guillermo Schmeda-Hirschmann b a Laboratorio de Productos Naturales, Departamento de Química, Facultad de Ciencias Básicas, Universidad de Antofagasta, Casilla 170, Antofagasta, Chile b Laboratorio de Química de Productos Naturales, Instituto de Química de Recursos Naturales, Universidad de Talca, Casilla 747, Talca, Chile article info abstract

Article history: Native Myrtaceae fruits were gathered by South American Amerindians as a food source. At present, there Received 16 January 2012 is still some regional consume of the small berries from trees belonging to genus Luma that occurs in Received in revised form 26 December 2012 southern Chile and Argentina. The aerial parts and berries from Luma apiculata and Luma chequen were Accepted 28 January 2013 investigated for phenolic constituents and antioxidant capacity. A high performance electrospray ionisa- Available online 13 February 2013 tion mass spectrometry method was developed for the rapid identification of phenolics in polar extracts from both species. Thirty-one phenolic compounds were detected and 27 were identified or tentatively Keywords: characterised based on photodiode array UV–vis spectra (DAD), ESI–MS–MS spectrometric data and spik- Luma apiculata ing experiments with authentic standards. Twelve phenolic compounds were detected in L. apiculata Luma chequén Myrtaceae fruits and 12 in the aerial parts while L. chequen yielded 10 compounds in fruits and 16 in aerial parts, Arrayán respectively. From the compounds occurring in both Luma species, seven were identified as tannins or South American berries their monomers, 15 were flavonol derivatives and five were anthocyanins. The whole berry and aerial

HPLC–DAD–MS parts extracts presented high antioxidant capacity in the DPPH assay (IC50 of 10.41 ± 0.02 and Phenolics 2.44 ± 0.03 lg/mL for L. apiculata, 12.89 ± 0.05 and 3.22 ± 0.05 for L. chequen, respectively), which can Antioxidants be related to the diverse range of phenolics detected. The antioxidant capacity together with the high polyphenolic contents and compounds identified can support at least in part, their use as botanical drugs. From the compounds identified in both species, 3-O-(600-O-galloyl)-hexose derivatives of myricetin, quercetin, laricitrin and isorhamnetin are reported for the first time for the genus Luma. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction 1995; Muñoz, Barrera, & Meza, 1981). Despite the well known uses and health beneﬁts (Murillo, 1889) of these berries, especially L. The consumption of fruits belonging to the Myrtaceae family is apiculata, their polyphenolic composition and antioxidant activity a common and ancient practice in South America. The Amerindian have not been reported. populations gathered the fruits and the largest in size with the best The medicinal properties of ‘‘Arrayan’’ leaves (L. apiculata, syn. taste were incorporated into South American culinary traditions all Eugenia apiculata DC. or Myrceugenella apiculata (DC.) Kausel, over the continent. Several edible Myrtaceae fruits including the Hoffmann, 1995), include aromatic, astringent, balsamic and Chilean berry ‘‘murtilla’’ (Ugni molinae Turczaninov) and the anti-inﬂammatory activity (Murillo, 1889). In addition, inhibitory murtilla-like berry Myrteola nummularia (Poiret) Berg. have been activity of the xanthine oxidase enzyme has been reported for shown to be a good source of polyphenolic antioxidants these and other Chilean Myrtaceae with similar medicinal uses, (Arancibia-Avila et al., 2011; Reynertson, Yang, Jiang, Basile, & including the treatment of gout, in Chile and Paraguay (Theoduloz, Kennelly, 2008). Franco, Ferro, & Schmeda Hirschmann, 1988; Theoduloz, Pacheco, The Myrtaceae Luma apiculata (DC.) Burret and Luma chequén & Schmeda Hirschmann, 1991). Leaves from the related species L. (Molina) A. Gray are trees with edible black-coloured berries chequén A. Gray (syn: Myrceugenella chequen (Mol.) Kaus have been occurring in southern Chile and Argentina. The berries from both used as an astringent (de Mösbach, 1991). are half the size of commercial blueberries with a more intense col- In the last few years, several biological samples such as alcoholic our but similar aspect and consistence, and have been employed to plant and fruit extracts containing complex mixtures of small and prepare ‘‘chicha’’, a Mapuche fermented beverage (Hoffmann, medium size phenolic and other molecules including very polar and thermally labile constituents have been analysed with the

⇑ Corresponding author. Tel.: +56 55 637229; fax: +56 55 637457. development of reliable LC–MS/MS equipment (Steinmann & Ganz- E-mail address: [email protected] (M.J. Simirgiotis). era, 2011; Wright, 2011). Indeed, the use of liquid chromatography

(HPLC, UPLC) coupled to diverse mass spectrometers such as hybrid Freezone 4.5 L, Kansas, MO, USA). One gram of lyophilised material quadrupole time of flight (Q-TOF) or electrospray ionization-ion was finally pulverised in a mortar and extracted thrice with 25 mL trap (Q-ESI) analyzers with complementary properties have been of 0.1% HCl in MeOH in the dark for 1 h each time. The extracts used in the last years for metabolic profiling and biological analysis were combined, filtered and evaporated in vacuo (40 °C). The ex- (Aliferis & Chrysayi-Tokousbalides, 2011; Kang et al., 2011; Mattoli tracts were suspended in 10 mL ultrapure water and loaded onto et al., 2011). a reverse phase solid phase extraction cartridge (SPE, Varian Bond The LC–MS methods proved to be superior to GC–MS since no Elut C-18, 500 mg/6 mL). The cartridge was rinsed with water prior derivatisation of polar samples (bearing hydroxyl and car- (10 mL) and phenolic compounds were eluted with 10 mL MeOH boxyl groups) is required (Hao, Zhao, & Yang, 2007). Quality con- acidified with 0.1% HCl. The solutions were evaporated to dryness trol of herbal drugs and medicinal plants is currently performed under reduced pressure to give 73.60 mg of L. apiculata fruits, with ESI–MS (Steinmann & Ganzera, 2011). HPLC–ESI–MS was 93.60 mg of L. apiculata aerial parts, 67.4 mg of L. chequén fruits used to analyse carotenoids (Maoka, 2009), anthocyanins (Barnes, and 40.6 mg of L. chequén aerial parts, respectively (for extraction Nguyen, Shen, & Schug, 2009), phenolic acids (Fischer, Carle, & yields see Table 1). The extracts were then dissolved in 2 mL 0.1% Kammerer, 2011) and alkaloids (He et al., 2011) in edible fruits. HCl in MeOH, filtered through a 0.45 lm micropore membrane Chilean native berries such as calafate (Berberis spp.) (Ruiz et al., (PTFE, Waters) before use and 10 ll were injected into the HPLC 2010) maqui (Aristotelia chilensis) and murta (U. molinae)(Rubilar instrument for analysis. et al., 2006) were also analysed using this precise technique. Despite the traditional use of L. apiculata and L. chequen, we were 2.3. HPLC analysis not able to find studies about phenolics constituents or antioxidant capacity of edible fruits from either species. The main goals and nov- A Merck-Hitachi (LaChrom, Tokyo, Japan) instrument equipped elty of this work is the profiling of phenolics as well as the measure- with an L-7100 pump, an L-7455 UV diode array detector, a D-7000 ment of antioxidant capacity and polyphenolic content of extracts chromato-integrator and a column compartment was used for from the berry fruits and leaves of the native Chilean L. apiculata analyses. The sample was separated on a Purospher star-C18 col- and L. chequen, which is a continuation of our studies on South umn (250 mm 5 mm, 4.6 mm i.d., Merck, Germany). The mobile American food plants (Simirgiotis & Schmeda-Hirschmann, 2010a). phase consisted of 10% formic acid in water (A) and acetonitrile (B). A gradient program was used for HPLC–DAD and ESI-MS as fol- 2. Materials and methods lows: 90% A in the first 4 min, linear gradient to 75% A over 25 min, then linear gradient back to initial conditions for other 2.1. Chemicals and plant material 15 min. The mobile phase flow rate was 1 mL/min. The column temperature was set at 25 °C; the detector was monitored at

Folin–Ciocalteu phenol reagent (2 N), Na2CO3, AlCl3, FeCl3, 520 nm for anthocyanins and 320–280 nm for other compounds NaNO2, NaOH, D(+) glucose, D(+) galactose, L(+) rhamnose, D() ri- while UV spectra from 200 to 600 nm were recorded for peak bose, quercetin, sodium acetate, HPLC-grade water, HPLC-grade characterisation. acetonitrile, thin layer chromatography (TLC, Kieselgel F254) plates, reagent grade MeOH and formic acid were obtained from Merck 2.4. Mass spectrometric conditions (Darmstadt, Germany). Myricetin 3-O-rhamnoside (myricitrin), quercetin 3-O-rhamnoside (quercitrin), cyanidin, myricetin, isorh- An Esquire 4000 Ion Trap mass spectrometer (Bruker Daltoniks, amnetin, syringetin, petunidin, pelargonidin, peonidin, malvidin Germany) was connected to an Agilent 1100 HPLC instrument via and their 3-O-glucosides (all standards with purity higher than ESI interface for HPLC–ESI-MS analysis. Full scan mass spectra 95% by HPLC) were purchased either from ChromaDex (Santa were measured between m/z 150 and 2000 u in positive ion mode Ana, CA, USA) or Extrasynthèse (Genay, France). Gallic acid, TPTZ for anthocyanins and negative ion mode for other compounds. (2,4,6-tri(2-pyridyl)1,3,5-triazine), Trolox and DPPH (1,1-diphe- High purity nitrogen was used as nebuliser gas at 27.5 psi, 350 °C nyl-2-picrylhydrazyl radical) were purchased from Sigma–Aldrich and at a ﬂow rate of 8 l/min. The mass spectrometric conditions Chemical Co. (USA). for negative ion mode were: electrospray needle, 4000 V; end plate Aerial parts and ripe fruits of L. apiculata (DC.) Burret (local name: offset, 500 V; skimmer 1, 56.0 V; skimmer 2, 6.0 V; capillary Arrayán), and L. chequén (Molina) A. Gray (local name: Chequén), exit offset, 84.6 V; and the operating conditions for positive ion were collected by Luis Bermedo Guzmán and Mario J. Simirgiotis mode were: electrospray needle, 4000 V; end plate offset, in Re-Re, Región del Bio-Bio, Chile in May 2011. Voucher herbarium 500 V; skimmer 1, 56.0 V; skimmer 2, 6.0 V; capillary exit offset, specimens and fruit samples were deposited at the Laboratorio de 84.6 V; capillary exit, 140.6 V. Collisionally induced dissociation Productos Naturales, Universidad de Antofagasta, Antofagasta, Chile, (CID) spectra were obtained with a fragmentation amplitude of with the numbers La-111505-1 and Lc-111505-2, respectively. 1.00 V (MS/MS) using ultrahigh pure helium as the collision gas.

2.2. Sample preparation 2.5. Alkaline and acid hydrolysis of MeOH extracts

Fresh Luma fruits and aerial parts (leaves and stems) were sep- To verify acylation of the ﬂavonol glycoside derivatives 18, 23, arately homogenised in a blender and freeze-dried (Labconco 24 and 28 from the HPLC ﬁngerprint, the SPE MeOH extracts

Table 1 Total phenolic content (TPC), total ﬂavonoid content (TFC), total anthocyanin content (TAC), ferric reducing antioxidant power (FRAP), scavenging of the free radical DPPH and percent w/w extraction yield of Luma methanolic extracts on the basis of freeze-dried starting material.

a Species and plant part TPC (mg/g) TFC (mg/g) TAC (mg/g) FRAP (lmol/g) DPPH (IC50, lg/mL) w/w extraction yield (%) L. apiculata fruits 29.44 ± 0.10 13.31 ± 0.01 21.03 ± 2.14 93.4 ± 0.0 10.41 ± 0.02 7.36 L. chequén fruits 5.15 ± 0.00 1.51 ± 0.00 1.57 ± 0.00 76.2 ± 0.0 12.89 ± 0.05 9.36 L. apiculata aerial parts 179.83 ± 0.38 139.70 ± 1.48 - 170.5 ± 0.1 2.44 ± 0.03 6.74 L. chequen aerial parts 327.09 ± 0.80 126.54 ± 1.15 - 135.6 ± 0.3 3.22 ± 0.05 4.06

a Measurements are expressed as mean ± SD of three parallel determinations (All values are significantly different at p < 0.05). M.J. Simirgiotis et al. / Food Chemistry 139 (2013) 289–299 291 obtained as explained above (2 mL) was hydrolysed with 2 mL of 2.7. Polyphenol, flavonoid and anthocyanin contents 2 mol/L sodium hydroxide as previously reported (Simirgiotis, Cal- igari, & Schmeda-Hirschmann, 2009). The mixture was kept for ’The total polyphenolic contents (TPC) of Luma fruits and leaves 16 h at room temperature, neutralised with concentrated hydro- were determined by the Folin–Ciocalteau method (Simirgiotis, chloric acid, filtered (0.45 lm, PTFE Waters) and directly analysed Caligari, et al., 2009; Simirgiotis, Theoduloz, Caligari, and Schme- (10 ll) by HPLC–DAD and ESI–MS–MS. Another portion of pro- da-Hirschmann, 2009) with some modifications. An aliquot of each cessed SPE MeOH extracts (2 mL) was dissolved in 4 mol/L HCl processed SPE extract (200 ll), was added to the Folin–Ciocalteau (2 mL) in order to further confirm the identity of flavonol aglycones reagent (2 mL, 1:10 v/v in purified water) and after 5 min of reac- by HPLC. After stirring the solution at 90 °C for 60 min, water was tion at room temperature (25 °C), 2 mL of a 100 g/L solution of Na2- added (10 mL), the solvent and hydrochloric acid were removed CO3 was added. Sixty minutes later the absorbance was measured under reduced pressure and the remaining aqueous solution at 710 nm. A calibration curve was performed with the standard (10 mL) was again submitted to SPE to wash the remaining acid gallic acid (concentrations ranging from 16 to 500 lg/mL, and sugars. The identification of the sugars in the aqueous solution R2 = 0.999) and the results expressed as mg gallic acid equiva- was performed by TLC using different sugars as standards as re- lents/g dry mass. ported by Figueirinha, Paranhos, Pérez-Alonso, Santos-Buelga, Determination of total flavonoid content (TFC) of the methano- and Batista (2008). After SPE, the resulting methanolic solution lic extracts was performed as reported previously (Simirgiotis

(2 mL) was filtered (0.45 lm, PTFE Waters) and directly analysed et al., 2008) using the AlCl3 colorimetric method. Quantification (10 ll) by HPLC–DAD–ESI/MS. In order to measure the recovery was expressed by reporting the absorbance in the calibration graph of phenolics for the total extraction procedure, a standard anthocy- of quercetin, which was used as the flavonoid standard (from 0.1 to anin (pelargonidin, 0.5 mg/mL) a standard flavonoid (quercetin, 65.0 lg/mL, R2 = 0.994). Results are expressed as mg quercetin 0.5 mg/mL) and a standard phenolic acid (gallic acid, 0.5 mg/mL) equivalents/g dry mass. The assessment of total anthocyanin con- were added to a fresh sample (one gram) of freeze-dried L. apicula- tent (TAC) was carried out as described by (Lee, Durst, & Wrolstad, ta fruits (three times) used as matrix, extracted, SPE processed as 2005). Absorbance was measured at 510 and 700 nm in buffers at above and recovery was calculated using HPLC–DAD. pH 1.0 and 4.5. Pigment concentration is expressed as mg cyanidin 3-glucoside equivalents/g dry mass and calculated using the 2.6. Antioxidant assessment formula:

A MW DF 103 2.6.1. Free radical scavenging activity TA ðmg=gÞ¼ The free radical scavenging activity of the extracts was deter- e 1 mined by the DPPH assay as previously described (Simirgiotis & where A =(A510nm A700nm) pH 1.0 (A510nm A700nm) pH 4.5; Schmeda-Hirschmann, 2010a), with some modifications. DPPH MW (molecular weight) = 449.2 g/mol; DF = dilution factor; radical absorbs at 517 nm, but upon reduction by an antioxidant 1 = cuvette pathlength in cm; e = 26,900 L/mol cm, molar extinc- 3 compound its absorption decreases. Briefly, 50 lL of processed tion coefficient for cyanidin 3-O-b-D-glucoside. 10 : factor to con- SPE MeOH extract or pure compound prepared at different concen- vert g to mg. All spectrometric measurements were performed trations was added to 2 mL of fresh 0.1 mM solution of DPPH in using a Unico 2800 UV–vis spectrophotometer (Shangai, Unico methanol and allowed to react at 37 °C in the dark. After 30 min instruments, Co., Ltd.). the absorbance was measured at 517 nm. The DPPH scavenging ability as percentage was calculated as: DPPH scavenging abil- 2.8. Statistical analysis ity = (Acontrol Asample/Acontrol) 100. Afterwards, a curve of % DPPH bleaching activity versus concentration was plotted and IC values 50 The statistical analysis was carried out using the originPro 9.0 were calculated. IC denotes the concentration of sample required 50 software packages (Originlab Corporation, Northampton, MA, to scavenge 50% of DPPH free radicals. The lower the IC value the 50 USA). The determination was repeated at least three times for each more powerful the antioxidant capacity. If IC 6 50 lg/mL the 50 sample solution. Analysis of variance was performed using ANOVA. sample has high antioxidant capacity, if 50 lg/mL < IC 6 100 lg/ 50 Significant differences between means were determined by stu- mL the sample has moderate antioxidant capacity and if IC > 200 - 50 dent’s t-test (p values < 0.05 were regarded as significant). lg/mL the sample has no relevant antioxidant capacity. Gallic acid (from 1.0 to 125.0 lg/mL, R2 = 0.991) and quercetin (from 1.0 to 125.0 lg/mL, R2 = 0.993) were used as standard antioxidant com- 3. Results and discussion pounds, and were determined to have IC50 values of 1.1 lg/ml (6.8 lmol/L) and 7.5 lg/ml (24.8 lmol/L), respectively. In the present study, we assessed the polyphenolic profile of aerial parts and fruits of L. apiculata and L. chequen collected in 2.6.2. Ferric reducing antioxidant power the Bio-Bio Region, Chile, and evaluated its antioxidant capacity The determination of ferric reducing antioxidant power or ferric as well as the total phenolic, total flavonoid and total anthocyanin reducing ability (FRAP assay) of the extracts was performed as de- content by spectrophotometric methods. The fresh fruits and aerial scribed by (Benzie & Strain, 1996) with some modifications. The parts were extracted with methanol and the resulting extracts stock solutions prepared were 300 mM acetate buffer pH 3.6, were processed by solid phase extraction. The weight/weight 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) solution in 40 mM extraction yields of the extracts were 7.36%, 9.36%, 6.74% and

HCl, and 20 mM FeCl36H2O solution. Plant extracts or standard 4.06% for L. apiculata fruits, L. chequén fruits, L. apiculata aerial parts methanolic Trolox solutions (150 lL) were incubated at 37 °C with and L. chequen aerial parts, respectively. The identity of phenolic 2 mL of the FRAP solution (prepared by mixing 25 mL acetate buf- compounds from the extracts was investigated by high-perfor- fer, 5 mL TPTZ solution, and 10 mL FeCl36H2O solution) for 30 min mance liquid chromatography paired with UV photodiode array in the dark. Absorbance of the blue ferrous tripyridyltriazine (HPLC–DAD) and triple quadrupole ion trap-electrospray ionisa- complex formed was then read at 593 nm. Quantification was per- tion tandem mass spectrometry (HPLC–ESI/MS). Anthocyanins formed using a standard calibration curve of antioxidant Trolox were monitored in ESI positive mode while other compounds were (from 0.2 to 2.5 lmol/mL, R2: 0.995). Samples were analysed in measured in negative mode. While L. chequén is reported to pro- triplicate and results are expressed in lmol TE/gram dry mass. duce several flavanones (Labbe et al., 1992), these compounds 292 M.J. Simirgiotis et al. / Food Chemistry 139 (2013) 289–299 were not identified in our Luma samples. The pattern of methoxy- the different antioxidant capacity (10.41 ± 0.02/12.89 ± 0.05 lg/mL lated flavonoids glycosides (laricitrin, myricetin, isorhamnetin) de- in the DPPH assay and 93.4 ± 0.0/76.2 ± 0.0 lmol Trolox/g in the tected in Luma resembles that reported in Vitis vinifera cv. Petit FRAP assay, respectively, Table 1). The TAC value for L. apiculata Verdot grapes (Castillo-Muñoz et al., 2009). fruits (21.03 ± 2.14 mg/g) was almost three times of that reported for the blueberries Vaccinium uliginosum (9.01 ± 0.06 mg/g of 3.1. Total phenolic, anthocyanin and flavonoid contents and freeze-dried powder) (Li et al., 2011). The small Korean fruits (Lir- antioxidant capacity of Luma extracts iope platyphylla) of a comparable shape, size and colour to Luma fruits (Fig. 2) produced similar HPLC–DAD anthocyanin fingerprint In this study, the phenolic profiles of methanolic extracts from (Lee & Choung, 2011). 2 Luma fruits and aerial parts were compared by HPLC–DAD The phenolic content correlated with antioxidant capacity (R : (Fig. 1). Antioxidant capacity of the extracts was measured and cor- 0.778 for TP/DPPH assay) while the antioxidant assays correlated 2 related with the total phenolic, flavonoid and anthocyanin con- with one another (R : 0.922). The phenolic content of L. apiculata tents. The total phenolic, anthocyanin and flavonoid contents as fruits was even higher than those reported for 17 berry cultivars well as the extraction yield and antioxidant capacity measured including raspberries (Rubus idaeus), blackberries (Rubus fructico- by the bleaching of DPPH radical and ferric reducing antioxidant sus), red currant (Ribes sativum), gooseberry (Ribes glossularia) power are given in Table 1. The flavonoid content of the L. apiculata and cornelian cherry (Cormus mas)(Pantelidis, Vasilakakis, Man- fruit extract forms about 45% of its total phenolic content, while for ganaris, & Diamantidis, 2007). The L. apiculata aerial parts showed L. chequen is only 29% (Table 1). The aerial parts of Luma had higher a FRAP value (170.5 ± 0.1 lmol TE/g) close to those reported for 50 total phenolic content (179.83 ± 0.38 and 327.09 ± 0.80 mg/g gallic red and 18 green pepper collections (FRAP mean values: 185 and acid equivalents for L apiculata and L. chequén, respectively) than 157 lmol TE/g dry weight, respectively), while L. chequén FRAP val- the fruits (Table 1) with total phenolic content values similar to ues for aerial parts and fruits (135.6 ± 0.3 and 76.2 ± 0.0 lmol TE/g, that reported for green tea (213 ± 5.9 mg/g gallic acid equivalents) respectively) were higher than common foods like beet and two times the values reported for mate leaves (112.1 ± 4.1 mg/ (86 ± 29 lmol TE/g), cauliflower (51 ± 12 lmol TE/g), spinach g gallic acid equivalents) (Piccinelli, De Simone, Passi, & Rastrelli, (64 ± 13 lmol TE/g), broccoli (41 ± 11 lmol TE/g), tomato 2004). Anthocyanins identified were present in both Luma fruits, (56 ± 8 lmol TE/g) and white cabbage (39 ± 17 lmol TE/g), among but concentration of these pigments were different, (Total antho- others (Ou, Huang, Hampsch-Woodill, Flanagan, & Deemer, 2002). cyanin content: 21.03 ± 2.14 mg/g for L. apiculata and Phenolic and flavonoid content of both Luma leaves were higher 1.57 ± 0.00 mg/g for L. chequen fruits) which is in accordance with than those of the fruits (Table 1). The values were also higher than

Fig. 1. HPLC–DAD chromatograms of Luma extracts. (a) Chromatograms at 280 nm. (A) L. apiculata fruits, (B) L. chequén fruits, (C) L. apiculata leaves, (D) L. chequén leaves, and (b) chromatograms at 520 nm. (E) L. apiculata fruits, (F) L. chequén fruits. M.J. Simirgiotis et al. / Food Chemistry 139 (2013) 289–299 293

Fig. 1. (continued) those reported for seven black and fresh tea leaves from Asia where was a powerful tool for their characterisation. The 31 compounds the highest value reported was for the brand Ouvagalia tea with detected and 27 identified or tentatively identified are listed in Ta- 110 ± 10 mg/g GAE and 80 ± 7 mg/g quercetin equivalents (Luxi- ble 2, along with UV–vis and MS data. Fig. 2 shows structures of mon-Ramma et al., 2005). L. apiculata aerial parts showed a pheno- several compounds identified while Fig. 3 shows structures and full lic content close to that reported for Rosa chinensis methanol MS and MS–MS spectra of some representative compounds. Peaks extract (189 mg ± 13 GAE/g dry weight) (Cai, Xing, Sun, Zhan, & 1–8 and 10–12 were tentatively identified as tannins (hydrolysable Corke, 2005). or proanthocyanins) or their monomers, peaks 30 and 31 were simple flavonols while peaks 15, 16, 18, 20–29 were glycosyl flavo- 3.2. Identification of phenolic constituents nol derivatives, and among those, peaks 15, 20, 21 and 25 were flavonols acylated with gallic acid. Peaks 9, 13, 14, 17 and 19 were Phenolics occurring in Luma fruits and aerial parts extracts were anthocyanins, and peaks 2, 8, 10 and 12 remain as unidentified separated by HPLC and UV–vis spectra were obtained using a phenolics. The identification of peaks is listed below. diode-array detector. HPLC fingerprints were generated (Fig. 1) and phenolic compounds subsequently analysed by ESI–MS–MS. A preliminary analysis of DAD spectrum obtained for the peaks 3.3. Tannins and flavanol derivatives gave a first indication of the family of phenolic compounds (Simir- giotis, Caligari, et al., 2009; Simirgiotis, Theoduloz, et al., 2009). Peaks 1–8 and 10–12 were tentatively identified as hydrolysa- Some compounds were identified by co-elution with standard ble tannins, epimeric procyanidins or flavanol derivatives. Peak 1 phenolics. For those compounds not commercially available, full was identified as the hydrolysable tannin hexahydroxydiphenoyl- scan mode followed by ESI–MS–MS experiments in negative mode glucose (HHDP-glucose, (Fig. 2) with a MW 482, a [MH] ion at 294 M.J. Simirgiotis et al. / Food Chemistry 139 (2013) 289–299

Fig. 2. Structures of compounds identified in Chilean Luma berries. (a) Tannins, peaks 1, 5, 6 and 7 and corresponding fragmentation pattern. (b) Anthocyanins: peaks 9, 13, 14, 17 and 19. (c) Flavonol/glycoside derivatives: peaks 15, 16, 18, 20–31. m/z 481 yielding diagnostic fragments at m/z 301, 283, 257 and MS–MS fragments as for compounds 3 and 5. This compound 229) as reported (Salminena, Ossipova, & Pihlajaa, 2002; Simirgio- was characterised as the ellagitannin furosinin (Takuo, 2005). Peak tis & Schmeda-Hirschmann, 2010a). Peak 3 was also a hydrolysable 7 showed a [MH] ion at m/z 577 (Fig. 3a) and MSn ions at m/z tannin with a molecular ion at m/z 783, an MS2 ion at m/z 481, pro- 425 (RDA rearrangement from one heterocicle of the dimer), m/z ducing a daughter MS3 ion at m/z 301 (with MS4 ions at m/z 283, 407 (loss of water from fragment at m/z 425) and m/z 289 (epicat- 257 and 229 assigned to one HHDP unit or ellagic acid) identified echin, diagnostic fragments at m/z 245, 205 and 179 (Stoggl, Huck, as a bis-HHDP-glucose/hexose as previously reported (Fischer & Bonn, 2004) and was identified as procyanidin B1 by comparison et al., 2011; Simirgiotis & Schmeda-Hirschmann, 2010b). Peak 4 with literature (Sun, Liang, Bin, Li, & Duan, 2007) and spiking was assigned as the ellagitannin castalagin or its isomer vescalagin experiments with authentic compound. MS/MS analysis of peak both with a [MH] at m/z 933 (Figs. 2 and 3a) and MS ions at m/z 11 with a molecular ion at m/z 457 showed MS2 ions at m/z 331, 631 (Fig 2, loss of HHDP unit), 481 (loss of gallic acid moiety) and 169 (gallic acid moiety), and 305 (deprotonated epigallocatechin). 301 (loss of galloyl-glucosyl moiety from the parent MS2 ion at m/z This compound was identified as epigallocatechin gallate (Mark- 631 (Simirgiotis & Schmeda-Hirschmann, 2010b). Peak 5 was iden- owicz Bastos et al., 2007) by comparison with an authentic sample. tified as an ellagic acid hexoside (pedunculagin I) showing an [MH] ion at m/z 633 and fragment ions at m/z 615, 481 and 3.4. Anthocyanins 301 (Fischer et al., 2011). Furthermore, peak 6 was identified as another bis-HHDP-hexose derivative with a mass difference of 32 U Five known anthocyanins were identified in the fruits, (peaks 9, ([MH] ion at m/z 815, C34H23O24) and the same UV data and 13, 14, 17 and 19, Fig. 3b) with molecular ions in positive mode at M.J. Simirgiotis et al. / Food Chemistry 139 (2013) 289–299 295

Fig. 2. (continued)

m/z 465, 449, 479, 463 and 493 and showing characteristic MS2 UV spectra and [MH] ions at m/z 449, 479 and 463 respectively ions at m/z 303 (MS3 ion at m/z 257), 287 (MS3 ions at m/z 213, all yielding a MS2 daugther ion at m/z 317 (myricetin) and were 147), 317 (MS3 ion at m/z 302), 301 (MS3 ion at m/z 286) and tentatively identified as myricetin 3-O-pentose, 3-O-hexose and 331 (MS3 ion at m/z 299, 179) respectively, corresponding to 3-O-rhamnose (Michodjehoun-Mestres et al., 2009). Peak 24 with delphinidin 3-O-glucoside (kmax: 275-341sh-512), cyanidin-3-O- a MW 494 (full ESI–MS main peak: 493 U, Fig. 3c) was tentatively glucoside, (kmax: 278-503), petunidin-3-O-glucoside (kmax: 275- identified as a methyl-myricetin hexoside derivative (laricitrin 343sh-512), peonidin-3-O-glucoside, (kmax: 268-357sh-503), and derivative). A flavonol derivative with a [MH] ion of 493 U malvidin-3-O-glucoside (kmax: 275-343sh-512), respectively. The was identified as quercetin 3-methoxy-hexoside in blueberries identity was corroborated by co-elution with standard anthocya- (Cho, Howard, Prior, & Clark, 2005). However, MS–MS data of com- nins and literature data. After extraction and SPE method the pound 27 is in agreement with laricitrin 3-O-hexose (Castillo- recovery of an external standard compound (pelargonidin) was Muñoz et al., 2009) or myricetin 30 methyl ether-3-O-hexose 97 ± 7% by HPLC. (Min et al., 2010). Further fragmentation of the ion at m/z 493 yielded an ion at m/z 331 (laricitrin, or myricetin 50 methyl ether), 3.5. Flavonol derivatives which in turn, yielded an ion at m/z 316 (myricetin-2 H). In the same manner, compound 21 with UV spectral data of 255, 293, Peaks 15, 16, 18, 20–31 were identified as flavonol derivatives 358 nm and a [MH] ion at 645 (Fig. 3c, MSn ions at 493 and since the shape of the UV spectra were similar to those reported 331 U) was tentatively identified as myricetin 50 methyl ether-(600 (Mabry, Markham, & Thomas, 1970; Simirgiotis, Caligari, et al., galloyl) 3-O-hexose (Min et al., 2010). 2009; Simirgiotis, Theoduloz, et al., 2009; Sun et al., 2007). The Full scan MS main peak for compound 28 (m/z 477 [MH]) linkages of gallic acid moieties for the 3-O-acylated flavonols were was consistent with the molecular formula C22H22O12. MS–MS located in position 600 of the sugar hydroxyl by characteristic MS fragmentation pattern (Fig 3c, MS2 315 U and MS3 300 U) was coin- fragmentation (Ferreres et al., 2008). MS–MS analysis of all of those cident with that reported for isorhamnetin-3-O-glucoside (Gutzeit, compounds showed characteristic ions at m/z 179 and 151 (Fig. 3c) Wray, Winterhalter, & Jerz, 2007). In the same manner, peak 25 which were confirmed to be produced by RDA rearrangement of was characterised as isorhamnetin-3-O-(600-O-galloyl)-glucose 5,7-dihydroxy-flavon-3-ols such quercetin, isorhamnetin and (Fig. 3c) and peak 31 was characterised as the aglycon isorhamne- myricetin using deuterium labelling experiments (McNab, Ferreira, tin since it showed the characteristic UV spectra and has an Hulme, & Quye, 2009). Peaks 16, 18 (Fig 3c) and 22 showed similar [MH] ion at m/z 315 consistent with a molecular anion of 296 M.J. Simirgiotis et al. / Food Chemistry 139 (2013) 289–299

Table 2 Identiﬁcation of phenolic compounds in Luma fruits and aerial parts by LC–DAD, LC–MS and MS/MS data.

Peak Rt (min) HPLC–DAD kmax (nm) ESI mode [MH] [2MH] MS–MS ions (m/z) Tentative identiﬁcation Species/plant (m/z) (m/z) part 1 2.0 275 – 481 301, 257, 229 HHDP-glucose Cf 2 2.3 290 – 533 191 Unknown quinic acid derivative Ap, Cp 3 2.9 270 – 783 633, 481, 301 Bis-HHDP hexose Cf 4 2,9 262 – 933 631, 451, 301 Castalagin or Vescalagin Af 5 3.0 275 – 633 615, 481, 301 Galloyl HHDP hexose (pedunculagin I) Ap, Cp 6 3.6 270 – 815 481, 301, 257, 229 Furosinin Cf 7 4.1 280 – 577 407, 289 Procyanidin B1 Cp 8 4.7 275 – 357 715 169, 125 Unknown gallotannin Cf 9 6.8 275, 341sh, 512 + 465 303, 257 Delphinidin 3-O-hexose Af, Cf 10 10.0 275 – 509 357, 169 Unknown Gallotannin Cf 11 9.7 275 – 457 331, 169 Epigallocatechin gallate Af, Ap 12 10.0 275 – 509 357, 169 Unknown Gallotannin Cp 13 10.1 278, 503 + 449 287, 213, 147 Cyanidin-3-O-glucose Af, Cf 14 11.3 275, 343sh, 512 + 479 317, 302 Petunidin 3-O-glucose Af,Cf 15 12.1 257, 292sh-361 – 631 479, 317 My-3-O-(600-O-galloyl)-hexose Af, Cp, Ap 16 13.5 257, 362 – 449 317, 179 My-3-O-ribose Ap 17 14.1 268, 357sh, 503 + 463 301, 286 Peonidin 3-O-glucose Af,Cf 18 14.4 254–362 – 479 959 317, 179 My-3-O-galactose (myricitrin) Af,Cf, Cp 19 15.0 275, 343sh, 512 + 493 331, 299, 179 Malvidin 3-O-glucose Af, Cf 20 15.9 255, 290sh, 356 – 615 1231 463, 301 Q-3-O-(600-O-galloyl)-hexose Af, Cp, Ap

21 16.7 255, 293sh, 358 – 645 493, 331, 315, 179, 151 L-(600-O-galloyl)3-O-hexose Cp 22 17.7 254, 360 – 463 927 317, 179, 151 My 3-O-rhamnose Af, Cp, Ap 23 18.8 254, 354 – 463 927 301, 257, 179, 151 Q-3-O-glucose (isoquercitrin) Af, Cp, Ap

24 19.4 266, 361 – 493 331-316, 179 L3-O-hexose Cp 25 20.3 264, 292sh, 357 – 629 477, 315, 300 IRh-3-O-(600-O-galloyl)-hexose Ap, Cp 26 21.3 255, 355 – 433 301, 179, 151 Q-3-O-ribose Ap 27 22.8 254, 355 – 447 895 301, 179, 151 Q-3-O-rhamnose (quercitrin) Ap 28 23.0 265, 354 477 955 315, 300 IRh-3-O-glucose Cp 29 23.3 265, 357 – 507 1015 344, 345, 315 Syringetin-3-O-glucose Cp 30 25.1 265, 357 – 345 330, 315 Syringetin Ap, Cp 31 25.5 265, 354 315 300, 179, 151 IRh Cp

Abbreviations. Compounds: Q: quercetin; My: myricetin; IRh: isorhamnetin; L: Laricitrin; Species and plant parts: L. apiculata: (Ap) aerial parts (Af) fruits; L. chequén: (Cp) aerial parts (Cf) fruits.

- 2 C16H11O7 , which in turn, produced an MS ion at m/z 300, et al., 2006). Compound 26 was identified as a quercetin-3-O- ([M15H]), due to loss of a methyl group and MS3 ions typical pentoside (ribose, loss of 132 U from the molecular anion at m/z of 5,7 dihydroxy-flavonols (Justesen, 2001; McNab et al., 2009; 433) (Simirgiotis, Theoduloz, Caligari, & Schmeda-Hirschmann, Simirgiotis & Schmeda-Hirschmann, 2010a). Peak 29 was identi- 2009). The identity of flavonoids in the extracts was corroborated fied as syringetin-3-O-glucoside with a prominent peak at 507 by comparing DAD (at 280, 320 and 520 nm) chromatograms and [MH] in the full scan mass spectra (Fig. 3c) and MS/MS ions at MS data before and after acid and alkaline hydrolysis. After 345 [MhexoseH] and 315 (Gutzeit et al., 2007), while com- saponification peaks 15, 20, 21 and 25 were not detected, and pound 30 eluting 2 min later was identified as the flavonol aglycon after acid hydrolysis the branched flavonoids described in this myricetin 30,50-di-O-methyl ether (syringetin, ESI–MS–MS data: work gave glucose, ribose, galactose and rhamnose as sugar moi- 315, 300, 179, 151 U). eties (by TLC) and myricetin, isorhamnetin, syringetin, quercetin, The full scan mass spectra of compound 20 showed mainly an cyanidin, petunidin, peonidin, delphinidin and malvidin (by intense ion at m/z 615, which yielded an MS2 ion at m/z 463 (quer- HPLC–DAD and ESI–MS comparison with authentic standards) cetin 3-O-glucoside: isoquercitrin) (Gutzeit et al., 2007) corre- as aglycones. After extraction and SPE method the recovery of sponding to the loss of a gallic acid moiety ([Mgalloyl an external standard flavonoid compound (quercetin) and a phe- moietyH])(Sannomiya et al., 2007) which in turn, fragmented nolic acid (gallic acid) were 88 ± 5%, and 99 ± 7% respectively by to an MS3 ion at m/z 301 (deprotonated quercetin, MS4 ions at m/ HPLC. z 179, 151) by loss of an hexose unit (162 U). UV spectral data of this compound is consistent with the typical UV overlap of a gallic acid moiety (UV shoulder at kmax 290 nm) and a flavonol structure 3.6. Unknown phenolic derivatives (kmax band I: 354 nm, band II: 254 nm)(Djoukeng, Arbona, Argam- asilla, & Gomez-Cadenas, 2008). Thus, this compound was identi- Peak 2 has a [MH] ion at m/z 533 and UV max at 320 nm. A fied as a quercetin-3-O-(600 galloyl) glucoside (Barakat, Souleman, compound with these UV and similar MS characteristics (plus a Hussein, Ibrahiem, & Nawwar, 1999). In the same manner, peak fragment at m/z 179 [caffeic acidH]) was identified as a caffeic 15 (kmax: 257, 292 and 361 nm) was identified as myricetin-3-O- acid derivative occurring in leaves of Helichrysum melaleucum (600 galloyl)glucoside/galactoside (molecular anion at m/z 631 and (Gouveia & Castilho, 2011). Because of its main MS2 fragment at MS–MS ions at 479 and 317 U as reported (Romani, Campo, & m/z 191 (quinic acid, MS3 127), it was partially identified as an un- Pinelli, 2012). known quinic acid derivative. Peak 8 with a [MH] ion at 357 as

Peak 27 was identified as the quercetin 3-O-rhamnoside: well as peaks 10 and 12 both with UV data (kmax 275 nm) and quercitrin by comparison of retention time and MS and UV prop- pseudomolecular ions at m/z 509 and MS-MS ions at m/z 357 erties with authentic sample. This compound is common in Myrt- [M152H] and 169 were tentatively identified as gallotannin aceae plants, and was identified in U. molinae leaves (Rubilar isomers or gallic acid derivatives, since detection of the fragment M.J. Simirgiotis et al. / Food Chemistry 139 (2013) 289–299 297

Fig. 3. Full MS and MS–MS spectra of some selected compounds identiﬁed in Luma berries. (a) Tannins (peaks 3, 4 and 7); (b) anthocyanins (peaks 9, 13, 14 and 17); (c) ﬂavonol derivatives (peaks 18, 21 24, 25, 28 and 29).

MS3 ion at m/z 169 led to the identification of a gallic acid residue the methodology developed is appropriate for rapid analysis and (MW: 170). identification of phenolic substances in extracts from native Luma species and can be potentially used for other edible South Ameri- 4. Conclusions can myrtaceae fruits. The anthocyanin pattern for both Luma species was similar but higher content was found in L. apiculata fruits. The LC–DAD and ESI–MS–MS system used in this work allowed The highest total phenolic content was found in Luma chequen the detection of 31 phenolic compounds and the identification or aerial parts while L. apiculata aerial parts showed the highest total tentative identification of 27 of them in fruits and aerial parts of flavonoid content. The antioxidant properties and high content of L. apiculata and L. chequen. The results obtained pointed out that phenolics and flavonoids found in the aerial parts can explain, at 298 M.J. Simirgiotis et al. / Food Chemistry 139 (2013) 289–299

Fig. 3. (continued) least in part, the traditional use of both native plants and the re- Barakat, H. H., Souleman, A. M., Hussein, S. A. M., Ibrahiem, O. A., & Nawwar, M. A. puted health benefits of the infusions. Further research is needed M. (1999). Flavonoid galloyl glucosides from the pods of Acacia farnesiana. Phytochemistry, 51(1), 139–142. to explore other biological activities of Luma fruits to support their Barnes, J. S., Nguyen, H. P., Shen, S., & Schug, K. A. (2009). General method for potential in the human diet. extraction of blueberry anthocyanins and identification using high performance liquid chromatography–electrospray ionization–ion trap-time of flight-mass spectrometry. Journal of Chromatography A, 1216(23), 4728–4735. Acknowledgment Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘‘Antioxidant Power’’: The FRAP assay. Analytical Biochemistry, 239, 70–76. 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