Phenolic Compounds in Chilean Mistletoe (Quintral, Tristerix tetrandus) Analyzed by UHPLC–Q/Orbitrap/MS/MS and Its Antioxidant Properties

Mass spectrometry has become a method of choice to characterize bioactive compounds in biological samples because of its sensitivity and selectivity. Hybrid ultra-HPLC hyphenated with Orbitrap mass analyzer is an innovative state of the art technology that allows fast and accurate metabolomic analyses. In this work the metabolites of a Chilean mistletoe endemic to the VIII region of Chile were investigated for the first time using UHPLC mass analysis (UHPLC-PDA-HESI-Orbitrap MSn). The anthocyanins, together with the non-pigmented phenolics were fingerprinted and correlated with the antioxidant capacities measured by the bleaching of the DPPH radical, the ferric reducing antioxidant power (FRAP), the superoxide anion scavenging activity assay (SA), and total content of phenolics, flavonoids and anthocyanins measured by spectroscopic methods. Six anthocyanins were identified, and among them, the 3-O-glycosides of delphinidin and cyanidin were the major ones. In addition, several phenolic acids (including feruloylquinic acid, feruloyl glucose, chlorogenic acid) and several flavonols (luteolin, quercetin, apigenin, isorhamnetin and glycoside derivatives) were also identified. The mistletoe leaves showed the highest antioxidant activity as measured by the DPPH radical bleaching, ferric reducing antioxidant power and superoxide anion scavenging activity tests (13.38 ± 0.47 µg/mL, 125.32 ± 5.96 µmolTE/g DW and 84.06 ± 4.59 at 100 µg/mL, respectively).


Introduction
The genus Tristerix comprises 11 species growing only in South America in places near the Andes Mountains from Chile-Argentina to Colombia and Ecuador. Tristerix tetrandus Mart. (Loranthaceae, local name quintral or quintral del álamo) is a medicinal mistletoe species native to southern Argentina, and central and southern Chile. It is a parasite plant of aspen (Populus sp.) colliguay (Colliguaya odorifera) Maqui (Aristotelia chilensis) willow (Salix sp.) and other native Chilean species. The mistletoe plants are often gathered by local collectors, dried and sold in local markets. The study of this plant is important because this Chilean mistletoe has traditionally been used in alternative medicine as an anti-inflammatory, digestive [1], hemostatic and hypocholesterolemic [2] remedy and as an anxiolytic agent [3]. The related mistletoe Tristerix corimbosus is used also as an astringent [3]. Anthocyanins, which belong to the flavonoids subclass, are well known pigmented bioactive compounds [4]. They are widely distributed in fruits and vegetables, such as blueberries, blackberries, raspberries, strawberries, blackcurrants, elderberries, grapes, cranberries, red cabbage, red radishes, and spinach [5]. They are very stable in acidic conditions (pH 2) in which they exist as red-colored flavylium (2-phenylbenzopyrilium) cations [5,6]. These compounds, including their associated flavonoids and phenolic acids, have demonstrated ability to protect against a myriad of human diseases, and present several beneficial effects such as antioxidant, anti-allergic, antimicrobial, anti-inflammatory, anti-hyperglycemic and anticancer activities [5,[7][8][9][10][11][12], among others. The separation and characterization of phenolics in native plants is important for further research since they can be important for the preparation of nutraceuticals with some of the mentioned activities. The use of liquid chromatography (HPLC, UPLC, UHPLC) coupled to several mass spectrometers such as time of flight (TOF or Q-TOF), quadrupole-Orbitrap (Q or Q-OT), triple quadrupole (TQ) or quadrupole-electrospray ionization (Q-ESI) for metabolic profiling and biological analysis in dietary supplements, plants, fruits and vegetables has increased in the last years [13][14][15][16][17][18]. The hyphenated liquid chromatography-mass spectrometry (LC-MS) methods are superior to gas chromatography-mass spectrometry (GC-MS) methods since no prior derivatization of polar samples (bearing hydroxyl and carboxyl groups) is required [19]. Quality control of herbal drugs and medicinal plants is also performed with LC-MS [13,15]. Indeed, LC-MS was used for the analysis of carotenoids [20], anthocyanins [21], phenolic acids [22] and alkaloids [23] in edible fruits and flowers, among other constituents. Since we were not able to find LC-MS analyses nor reports on phytochemical compounds from this mistletoe species or related ones, and in continuation of our search for interesting polyphenols and other bioactive compounds in native Chilean plants [24][25][26][27][28], in the present work the polyphenolic fingerprints and phenolic content of the leaves and flowers of this species (Figure 1) from the VIII region of Chile were correlated with the antioxidant capacities measured by the bleaching of the DPPH radical, the ferric reducing antioxidant power (FRAP), the superoxide anion scavenging activity assays (SA). The compounds were identified for the first time with the help of PDA analysis and high resolution Orbitrap mass spectrometry (HPLC-ESI-OT-MS) plus comparison with authentic standards. which belong to the flavonoids subclass, are well known pigmented bioactive compounds [4]. They are widely distributed in fruits and vegetables, such as blueberries, blackberries, raspberries, strawberries, blackcurrants, elderberries, grapes, cranberries, red cabbage, red radishes, and spinach [5]. They are very stable in acidic conditions (pH 2) in which they exist as red-colored flavylium (2-phenylbenzopyrilium) cations [5,6]. These compounds, including their associated flavonoids and phenolic acids, have demonstrated ability to protect against a myriad of human diseases, and present several beneficial effects such as antioxidant, anti-allergic, antimicrobial, anti-inflammatory, anti-hyperglycemic and anticancer activities [5,[7][8][9][10][11][12], among others. The separation and characterization of phenolics in native plants is important for further research since they can be important for the preparation of nutraceuticals with some of the mentioned activities. The use of liquid chromatography (HPLC, UPLC, UHPLC) coupled to several mass spectrometers such as time of flight (TOF or Q-TOF), quadrupole-Orbitrap (Q or Q-OT), triple quadrupole (TQ) or quadrupole-electrospray ionization (Q-ESI) for metabolic profiling and biological analysis in dietary supplements, plants, fruits and vegetables has increased in the last years [13][14][15][16][17][18]. The hyphenated liquid chromatography-mass spectrometry (LC-MS) methods are superior to gas chromatography-mass spectrometry (GC-MS) methods since no prior derivatization of polar samples (bearing hydroxyl and carboxyl groups) is required [19]. Quality control of herbal drugs and medicinal plants is also performed with LC-MS [13,15]. Indeed, LC-MS was used for the analysis of carotenoids [20], anthocyanins [21], phenolic acids [22] and alkaloids [23] in edible fruits and flowers, among other constituents. Since we were not able to find LC-MS analyses nor reports on phytochemical compounds from this mistletoe species or related ones, and in continuation of our search for interesting polyphenols and other bioactive compounds in native Chilean plants [24][25][26][27][28], in the present work the polyphenolic fingerprints and phenolic content of the leaves and flowers of this species (Figure 1) from the VIII region of Chile were correlated with the antioxidant capacities measured by the bleaching of the DPPH radical, the ferric reducing antioxidant power (FRAP), the superoxide anion scavenging activity assays (SA). The compounds were identified for the first time with the help of PDA analysis and high resolution Orbitrap mass spectrometry (HPLC-ESI-OT-MS) plus comparison with authentic standards.

Antioxidant Capacity and Total Phenolics, Anthocyanin and Flavonoids Contents
Three antioxidant assays were employed for this study: the DPPH antiradical activity assay, the superoxide anion inhibition assay, and the ferric reducing activity measured as micromoles of the standard Trolox (Table 1). The antioxidant capacities were supported by the measurement of total anthocyanins in the flowers (TAC), as well as the phenolic (TPC) and flavonoid (TFC) contents in flowers and leaves. For the TPC assay, it has to be stressed that the Folin-Ciocalteu reagent employed reacts with all the oxidants present in the plant extract. Consequently, depending on the extract, this method could overestimate the real phenolic level in the extract. Therefore, data for TPC obtained with the Folin-Ciocalteu's method could be artefactual and the interpretation of the results erroneous [29], for this reason we have supported the results of this assay with the other complementary ones. The leaves showed more antiradical DPPH quenching activity than the flowers, possibly due to the quantity of phenolics (Table 1) found in the leaves. The DPPH value of T. tetrandus leaves was close to that of standard cyanidin-3-glucoside (Table 1) and the synthetic antioxidant butylated hydroxytoluene (BHT, 61.13 µM) [30]. The European mistletoe Viscum album has been extensively studied and the antioxidant activity already reported [31][32][33][34], indeed, several studies showed that V. album possess remarkable cholinesterase and tyrosinase inhibitory and antioxidant properties [32]. The TPCs of T. tetrandus leaves were close to that exhibited by V. album L. ssp. album hosting Cerasus vulgaris Miller (Sourcherry), Pinus nigra Arn., (Pine) and Crataegus sp. (around 31, 33 and 37 mg GAE per g extract respectively) [32]. It was also close to that reported from Nolana aplocarioides from Northern Chile (around 30 mg per g) [35]. The TPC of the leaves was also close to that reported for blackberry (Rubus ulmifolium) bud preparations (350˘8 mg/100 g fresh weight, considering 90 percent of water loss) [36] and was higher to that reported for the superfruit goji (281.91 mg/100 g fresh weight) [37]. The TFC was also close to the value reported for Nolana aplocarioides (around 22 mg quercetin per g dry weight) [35]. The TPC and FRAP activity of the flowers was similar to that reported for flowers of Helianthus annus [38]. The TAC of the flowers was close to that of standard cyanidin-3-glucoside (Table 1), and was also close to that reported from the Chilean berries Luma apiculata (15.24˘1.29 mg cyanidin 3-O-glucoside/g dry weight) [39]. The TAC of the flowers was also double to that reported for the Black Diamond blackberry (Rubus fruticosus) variety (119.3˘1.2 mg/100 g fresh weight, considering 90 percent of water loss) [40] and similar to black currant (Ribes nigrus) var. Black Down (170.0˘1.7 mg/100 g fresh weight) [41]. The SAA scavenging of the leaves was somehow lower to that reported for leaves of strawberries (67.60%˘1.01% inhibition) [42].

MS-PDA Identification of Phenolic Acids in Chilean Mistletoe (Lorantaceae) From Southern Chile
The hybrid machine used in this study combines the rapid separation of the ultra-HPLC technique with photodiode (PDA) detection with flow rates up to 2 mL per minute, zero dead volume, the effective ionization of the heated electrospray probe (HESI II), the high resolving power performance of the orbital trap (Orbitrap, OT), and selectivity of a quadrupole, (reaching resolutions of up to 70,000 FWHM at m/z 200), and the outstanding diagnostic power of a high resolution collision (HCD) cell. Qualitative data regarding the phenolic compounds of mistletoe extracts are shown in Table 2. We have identified 28 compounds in the leaves and six in the flowers.The compounds in the flowers and leaves were detected and identified using UHPLC with total ion current (TIC) in positive mode for anthocyanins and negative mode for the other phenolic compounds using OT-HESI-MS (Table 2) and UV-visible data (PDA, Figure 2, Table 2).    Table 2.
The optimal conditions for the separation of the phenolics were obtained with a fast linear gradient solvent system of 0.1% aqueous formic acid (solvent A) and acetonitrile 0.1% formic acid (solvent B) with a flow rate of 1.0 mL/min −1 using an UHPLC C18 column as a stationary phase. Several common compounds were in the present study identified accurately using the HCD cell including proanthocyanidins, phenolic acids and flavonoids. Peaks 20, 22, 25, 27, 29 and 34 were detected in the flowers and the other peaks in the leaves. Below is the detailed explanation of the characterization. Figure 3 shows full MS spectra and structures of several compounds detected.   Table 2.
The optimal conditions for the separation of the phenolics were obtained with a fast linear gradient solvent system of 0.1% aqueous formic acid (solvent A) and acetonitrile 0.1% formic acid (solvent B) with a flow rate of 1.0 mL/min´1 using an UHPLC C 18 column as a stationary phase. Several common compounds were in the present study identified accurately using the HCD cell including proanthocyanidins, phenolic acids and flavonoids. Peaks 20,22,25,27,29 and 34 were detected in the flowers and the other peaks in the leaves. Below is the detailed explanation of the characterization. Figure 3 shows full MS spectra and structures of several compounds detected.  Table 2.
The optimal conditions for the separation of the phenolics were obtained with a fast linear gradient solvent system of 0.1% aqueous formic acid (solvent A) and acetonitrile 0.1% formic acid (solvent B) with a flow rate of 1.0 mL/min −1 using an UHPLC C18 column as a stationary phase. Several common compounds were in the present study identified accurately using the HCD cell including proanthocyanidins, phenolic acids and flavonoids. Peaks 20, 22, 25, 27, 29 and 34 were detected in the flowers and the other peaks in the leaves. Below is the detailed explanation of the characterization. Figure 3 shows full MS spectra and structures of several compounds detected.      Table 2.

Sample Preparation
Flowers and leaves (three individuals of each) were separately collected and extracted with acidified methanol and the resulting extracts were processed by solid phase extraction. Fresh flowers were carefully washed, separately homogenized in a blender and freeze-dried (Freezone Freeze dry system plus 2.5 L, Labconco Corporation, Kansas City, MO, USA). Ten grams of the lyophilized flowers and leaves were finally pulverized in a mortar, (separately) defatted thrice with 100 mL of n-hexane and then extracted with 100 mL of 0.1% HCl in MeOH in the dark in an ultrasonic bath for one hour each time. The extracts from each sample were combined, filtered and evaporated in vacuo in the dark (40˝C). The extracts were suspended in 20 mL ultrapure water and loaded onto an XAD-7 (100 g) column. The column was rinsed with water (100 mL) and phenolic compounds were eluted with 100 mL of MeOH acidified with 0.1% HCl. The solutions were combined and evaporated to dryness under reduced pressure (40˝C) to give 567.23 mg and 895.3 mg of extract from T. tetrandus leaves and flowers, respectively.

Instrumentation
A Thermo Scientific Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany) equipped with a quaternary Series RS pump and a Thermo Scientific Dionex Ultimate 3000 Series TCC-3000RS column compartments with a Thermo Fisher Scientific Ultimate 3000 Series WPS-3000RS autosampler (Thermo Fisher Scientific) and a rapid separations PDA detector controlled by Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, USA and Dionex Softron GmbH division of Thermo Fisher Scientific) hyphenated with a Thermo high resolution Q Exactive focus mass spectrometer (Thermo Fisher Scientific) were used for analysis. The chromatographic system was coupled to the MS with a Heated Electrospray Ionization Source II (HESI II). Nitrogen (purity > 99.999%) obtained from a Genius NM32LA nitrogen generator (Peak Scientific, Billerica, MA, USA) was employed as both the collision and damping gas. Mass calibration for the Orbitrap was performed once a week, in both negative and positive modes, to ensure a working mass accuracy lowers than or equal to 5 ppm. Caffeine, N-butylamine (Sigma-Aldrich) were the calibration standards for positive ions and buspirone hydrochloride, sodium dodecyl sulfate, and taurocholic acid sodium salt (Sigma-Aldrich) were used to calibrate the mass spectrometer. These compounds were dissolved in a mixture of acetic acid, acetonitrile, water and methanol (Merck) and were infused using a Chemyx Fusion 100 syringe pump (Thermo Fisher Scientific). Q Exactive 2.0 SP 2, XCalibur 2.3 and Trace Finder 3.2 softwares (Thermo Fisher Scientific and Dionex Softron GmbH Part of Thermo Fisher Scientific) were used for UHPLC-mass spectrometer control and data processing, respectively.

MS Parameters
The HESI parameters were optimized as follows: sheath gas flow rate 75 units; aux. gas unit flow rate 20; capillary temperature 400˝C; aux gas heater temperature 500˝C; spray voltage 2500 V (for ESI´); and S lens RF level 30. Full scan data in both positive and negative was acquired at a resolving power of 70,000 full width half maximum (FWHM) at m/z 200. For the compounds of interest, a scan range of m/z 100-1000 was chosen; the automatic gain control (AGC) was set at 3ˆ10 6 and the injection time was set to 200 ms. Scan-rate was set at 2 scans¨s´1. External calibration was performed using a calibration solution in positive and negative modes before each sample series. In addition to the full scan acquisition method, for confirmations purposes, a targeted MS/MS analysis was performed using the mass inclusion list and expected retention times of the target analytes, with a 30 s time window, with the Orbitrap spectrometer operating both in positive and negative mode at 17,500 FWHM (m/z 200). The AGC target was set to 2ˆ10 5 , with the maximum injection time of 20 ms. The precursor ions are filtered by the quadrupole which operates at an isolation window of m/z 2. The fore vacuum, high vacuum and ultrahigh vacuum were maintained at approximately 2 mbar, from 105 to below 1010 mbar, respectively. Collision energy (HCD cell) was operated at 30 kv. Detection was based on calculated exact mass and on retention time of target compounds, presented in Table 2. The mass tolerance window was set to 5 ppm for the two analysis modes.

Ferric Reducing Antioxidant Power
The determination of ferric reducing antioxidant power or ferric reducing ability (FRAP assay) of the extracts was performed as described by [49] with some modifications. The stock solutions prepared were 300 mM acetate buffer pH 3.6, 10 mM TPTZ (2,4,6-tri (2-pyridyl)-s-triazine) solution in 40 mM HCl, and 20 mM FeCl 3¨6 H 2 O solution. Plant extracts or standard methanolic Trolox solutions (150 µL) were incubated at 37˝C with 2 mL of the FRAP solution (prepared by mixing 25 mL acetate buffer, 5 mL TPTZ solution, and 10 mL FeCl 3 .6H 2 O solution) for 30 min in the dark. Absorbance of the blue ferrous tripyridyltriazine complex formed was then read at 593 nm. Quantification was performed using a standard calibration curve of antioxidant Trolox (from 0.2 to 2.5 µmol/mL, R 2 : 0.995). Samples were analyzed in triplicate and results are expressed in µmol TE/100 grams fresh mass.

Superoxide Anion Scavenging Activity
The enzyme xanthine oxidase is able to generate superoxide anion radical (O 2´) "in vivo" by oxidation of reduced products from intracellular ATP metabolism. The superoxide anion generated in this reaction sequence reduces the nitro blue tetrazolium dye (NBT), leading to a chromophore with a maximum of absorption at 560 nm. Superoxide anion scavengers reduce the speed of generation of the chromophore. The superoxide anion scavenging activities of isolated compounds and fractions were measured spectrophotometrically in a microplate reader as reported previously [24]. All extracts were evaluated at 100 µg/mL. Values are presented as mean˘standard deviation of three determinations.

Polyphenol and Flavonoids Contents
The total polyphenolic contents (TPC) of mistletoe were determined by the Folin-Ciocalteau method [25,26,50] with some modifications. An aliquot of each processed SPE extract (200 µL, approx. 2 mg/mL) was added to the Folin-Ciocalteau reagent (2 mL, 1:10 v/v in purified water) and after 5 min of reaction at room temperature (25˝C), 2 mL of a 100 g/L solution of Na 2 CO 3 was added. Sixty minutes later the absorbance was measured at 710 nm. The calibration curve was performed with gallic acid (concentrations ranging from 16 to 500 µg/mL, R 2 = 0.999) and the results were expressed as mg gallic acid equivalents/100 g fresh mass. Determination of total flavonoid content (TFC) of the methanolic extracts was performed as reported previously [51] using the AlCl 3 colorimetric method. Quantification was expressed by reporting the absorbance in the calibration graph of quercetin, which was used as a standard (from 0.1 to 65.0 µg/mL, R 2 = 0.994). Results are expressed as mg quercetin equivalents/g fresh weight. All spectrometric measurements were performed using a Unico 2800 UV-vis spectrophotometer (Unico Instruments, Co, Ltd., Shanghai, China).

Statistical Analysis
The statistical analysis was carried out using the originPro 9.0 software packages (Originlab Corporation, Northampton, MA, USA). The determination was repeated at least three times for each sample solution. Analysis of variance was performed using ANOVA. Significant differences between means were determined by Tukey comparison test (p values < 0.05 were regarded as significant).

Conclusions
Thirty six compounds including several caffeoyl acids (peaks 2, 3, 4, 9, 16, 17, and 24) three procyanidins (peaks 6, 8 and 10), several flavonols (peaks 11,12,15,21,23,26,28,[31][32][33][34][35][36] two oxylipins (peaks 18 and 19) were detected in the leaves and six anthocyanins (peaks 22, 25, 26, 27, 29 and 34) were detected in the flowers of a native mistletoe from the VIII region of Chile using PDA and Orbitrap-ESI-MS for the first time. However, significant differences in the total phenolic content and antioxidant activity were found between these two plant parts, probably due to the quantity of phenolic compounds detected. The mistletoe leaves showed the highest antioxidant activity measured as the bleaching of the DPPH radical, the ferric reducing antioxidant power and superoxide anion scavenging activity (13.38˘0.47 µg/mL, 125.32˘5.96 µmol TE/g DW and 84.06˘4.59 at 100 µg/mL, respectively). The mistletoe is thus a good candidate for industrial crop production and has also the potential to produce nutraceuticals.