Extraction and Characterization of Phenolic Compounds from Rose Hip (Rosa canina L.) Using Liquid Chromatography Coupled with Electrospray Ionization-Mass Spectrometry

Wild berry are a rich of natural compounds which provide them high antioxidant potential. The compounds which provide them these proprieties are known to be vitamins, flavonoids, anthocyanins and phenolic acids. The aim of this study was to extract, identify and characterize bioactive compounds from rose hip (Rosa canina L.) currently found in Romania. A qualitative high-performance liquid chromatography coupled with electrospray ionization mass spectrometric (ESI-MS) detection in positive ion mode has been used to identify phenolic compounds from rose hip crude extract. The chromatograms revealed the presence of a large number of compounds (19), identified and grouped as phenolic acids and flavones/ols, flavan3-ols and also anthocyanins. Based on obtained results these berries can be highly recommended as part of our diet. Also this finding represents a contribution to the chemical characterization of phenolic profile of rose hip.


Introduction
The genus Rosa L. (Rosaceae) covers more than 100 wild species and thousands of cultivars, which are widespread and grow in the north part of Europe, Asia, Middle East and North America (Rehder, 1940;Gu et al., 2003). Rosa canina L. (rose hip) is an important berry for food industry due to its rich chemical composition. The small berries are an important source of colour, flavours and bioactive compounds with potential health benefits (Hvattum, 2002;Wu et. al., 2005;He et al., 2010;Jodee et al., 2011;Rugină et al., 2012;Tayefi-Nasrabadi et al., 2012;Widen et al., 2012;Dulf et al., 2013;Yang et al., 2015). Rose hip contains compounds with antioxidant potential such as ascorbate, ß-carotene, glutathione, a-tocopherol, anthocyanins and other phenolics (Ercisli, 2007;Nojavan et al., 2008;Tozzi et al., 2008;Tumbas et al., 2012;Czyzowska et al., 2015). So far, fruits of Rosa canina L. (Rosaceae), rose hips, are known for their high content of vitamin C and for their great flavour. Rosa canina L. grows naturally in Romania and, as reported previously, fruit and flavoured teas are very popular in many European countries (Pękal et al., 2011). Rose hip can be consumed in both raw and dried state, being used in the herbal tea for flavour and a natural dose of vitamin C. The chemical composition of rose hip has been very rarely evaluated in comparing with other berries containing similar compounds. The polyphenols contained by rose hip are a large group of secondary metabolites. They are widely distributed in vegetables and berries ranging from simple molecules, such as phenolic acids, to complex molecules with numerous phenolic groups, e.g. acylated flavonoid glycosides, proanthocyanidins or tannins. Polyphenols occur primarily in conjugated form, linked to sugars moieties, and to other compounds, such as carboxylic and organic acids, amines, lipids and even to other polyphenols (Bravo, 1998;Guimarães et al., 2013).
Rose hip consumption has been associated with preventive and therapeutic proprieties against a wide range of degenerative diseases, including the inflammatory arthritis disorder, rheumatism, gastrointestinal disorders or cancer (Larsen et al., 2003;Rein et al., 2004;Christensen et al., 2008;Fujii et al., 2009;Andersson et al., 2012). In a recent study Widen et al. (2012) has evaluated erythrocyte antioxidant protection of rose hips. They have investigated the degree of amelioration of oxidative damage in an erythrocyte in vitro bioassay by comparing the e ects of a reducing agent on erythrocytes alone with the effect on erythrocytes pretreated with berry extracts. The obtained results revealed that the maximum protection against oxidative stress, 59.44%, was achieved when incubating the cells with the first eluted meta-phosphoric extract. Removal of ascorbic acid from this extract increased the protection against oxidative stress to 67.9%. These results clearly indicate that rose hips contain a were colourless. The acidic condition was created in order to prevent anthocyanins from degradation. The obtained extracts were filtered through multiple layers of cotton and concentrated at 35 °C under reduced pressure (Rotavapor R-124, Buchi, Switzerland). Thereafter, it was dissolved in a known amount of acidified water, filtered through 0.45 µm Millipore filter and analyzed by liquid chromatography.

HPLC-DAD/-ESI-MS identification of anthocyanins, flavonol glycosides and phenolic acids
Samples were analyzed on an Agilent 1200 system equipped with a binary pump delivery system LC-20 AT (Prominence), a degasser DGU-20 A3 (Prominence), a diode array SPD-M20 A and an UV-VIS detector (DAD). Volumes of 10 µl were injected on an Eclipse XDB-C18 (5µm, 150x4.6 mm) column. The mobile phase consisted in: solvent A -bidistilled water and 0.1% formic, B -acetonitrile. The gradient elution system started with 10% B for 9 min. The percent of B increased to 12% at 17 min and continued up to 25% B at 30 min, between minute 30 and 50 the percentage of B being 90%. DAD recorded full spectra. In-line MS data were recorded by directing the LC flow to a Quadrupole 6110 mass spectrometer (Agilent Technologies, Chelmsford, MA) equipped with an ESI probe. Flow rate was 0.5 ml/min and column temperature was maintained at 25 °C. The chromatograms were monitored at 280, 340 and 520 nm, respectively. The compounds identification and peak assignments were done based on their retention times, UV-VIS spectra and also compared with standards and published data. As a confirmation the samples were analyzed by HPLC-ESI-MS. The measurements were performed in the positive mode with an ion spray voltage of 3000 V, and a capillary temperature of 350 °C. Data were collected in full scan mode within the range 280 to 1000 m/z. Identification of anthocyanins, flavonol glycosides and phenolic acids was carried out based on molecular mass determination, masses and occurrence of fragments, elution order and literature data reported previously (Koponen et al., 2007;Adam et al., 2013;Guimarães et al., 2013).

Results and Discussion
Rose hip (Rosa canina L.) varieties were not extensively studied, due to this fact the present study relieved that they are a rich source of anthocyanins, flavonol glycosides and phenolic acids. The identification of phenolic compounds present in rose hip crude extract was performed by comparing their UV-Vis spectra with published data and available standards, as well, trough HPLC-DAD-ESI/MS. A total of 19 different types of phenolic compounds were identified in crude extract. The main classes of phenolic compounds identified were grouped in phenolic acids and flavones/ols, flavan-3-ols and also anthocyanins. HPLC profile of polyphenols was recorded at three wavelengths: 280 nm for phenolic acids, 340 nm for flavan-3-ols and 520 nm for anthocyanins. The obtained chromatograms with each identified peak are showed and discussed below.

Phenolic acids and flavones/ols
In crude extract of rose hip, hydroxycinnamic acids were the major class of phenolic acids which is in agreement with literature (Hvattum, 2002;Guimarães et al., 2013). Peak assignments of the different identified compounds are presented promising level of clinically relevant antioxidant protection (Widen et al., 2012). Another published study reports that the rose hip fractions rich in flavonoid inhibit cell proliferation in HeLa, MCF7 and HT-29 cell lines (Tumbas et al., 2012). The potential health benefits of rose hips are due to the polyphenolic compounds found in these berries that are mainly flavonoid, phenolic acids and anthocyanins.
Since these classes of compounds are normally found as complex molecules in natural sources, several techniques have been developed for their separation and identification: mass spectrometry (MS) is the most used with several techniques, such as electrospray ionization mass spectrometry (ESI-MS), liquid chromatography coupled with mass spectrometry (LC/MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with mass spectrometric (MS/MS), high performance thin layer chromatography (HPTLC) and also nuclear magnetic resonance NMR (Nawwar et al., 1994;Wang et al., 1999;Reed et al., 2005;Mandal et al., 2008;Ferrari et al., 2011;Savage et al., 2011;Hingse et al., 2014;Ali et al., 2015;Jin et al., 2015). Being one of the most common fruit used in our country as herbal tea, the proposal of this work was to characterize their chemical profile. Therefore, the objective of the present study was to identify individual phenolic compounds in rosehip using the HPLC-ESI-MS.

Plant material
Fully ripened rose hips (Rosa canina L.) from spontaneous flora of Transylvania (Cluj, Feleac) were harvested during the months of September and October 2011. Samples were randomly collected from several plants and the form and variety of biotype to be analysed was taxonomically classified after a preliminary botanical identification. The identified sample was Rosa canina var. lutetiana f. flexibilis. Rose hips without calyxes were washed with water and kept in a freezer at ˗20 °C prior to the analysis.

Extraction of polyphenolic compounds from rose hip
The method used to obtain crude extract rich in phenolic compounds was done according to previous studies with minor modifications (Cuevas-Rodriguez et al., 2010;Rugină et al., 2012). Berries samples, 1 g each, were grounded using an ultraturax (Miccra D-9 KT Digitronic, Germany) and weighed, followed by addition of 10 ml of acidified methanol (0.3% HCl (v/v)). The extraction process was repeated until the samples 351 in Table 1. The analysed crude extract was characterized by the presence of a large number of phenolic acids and flavones/ols, more specifically 14 individual compounds (Fig. 1).
Quercetin derivates which have λmax around 355 and an MS fragment at m/z 301 were mainly identified in rose hip crude extract (peak 1, 3, 4, 5, 6, 14). Structure, fragmentation, full ESI-MS and spectra of quercetin is showed in Fig. 1.
Detected quercetin derivates were assigned to quercetin pentoside ([M-H] at m/z 435); peaks 3-6 ([M-H] at m/z 479) were tentatively identified as quercetin hexuronide, quercetin 3-O-glucoside, quercetin glucuronide and quercetin hexuronide. This identification of quercetin glycosides was done based on process of releasing a 301 (m/z) ion fragment in the MS/MS spectrum.
The obtained results are in agreement to available literature (Jin et al., 2015). However, to the best of our knowledge, this is the first report that relieved the presence of resveratrol in the rose hip crude extract ( Fig. 1 at 280 nm).

Flavan-3-ols
Catechin (flavan-3-ols) is the most frequent form of flavanols that occurs in plants (Vagiri et al., 2012). The identified flavan-3-ols in rose hip crude extract are catechin and catechin hexoside, their galloyl derivates, according to their comparison with standards, published data and also confirmed by ESI-MS analysis.
The presence of catechin in rose hip has been previously reported (Hvattum, 2002;Guimarães et al., 2013). We  Table 2.  Recent performed studies relieved that rose hip contains significant amounts of phenolic acids with antioxidant activity (Roman et al., 2013), the major compounds being catechin, a proanthocyanidin monomer, a proanthocyanidin dimmer, rutin, quercetin galactoside, quercetin glucoside and cyanidin-glucoside 352 (Widen et al., 2012). The identified compounds in this study are in agreement with published data related to the phenolic compounds in rose hip (Hvattum, 2002;Widen et al., 2012;Guimarães et al., 2013).