Characterization of Flavonoids and Phenolic Acids in Myrcia bella Cambess. Using FIA-ESI-IT-MSn and HPLC-PAD-ESI-IT-MS Combined with NMR

The leaves of Myrcia DC. ex Guill species are used in traditional medicine and are also exploited commercially as herbal drugs for the treatment of diabetes mellitus. The present work aimed to assess the qualitative and quantitative profiles of M. bella hydroalcoholic extract, due to these uses, since the existing legislation in Brazil determines that a standard method must be developed in order to be used for quality control of raw plant materials. The current study identified eleven known flavonoid-O-glycosides and six acylated flavonoid derivatives of myricetin and quercetin, together with two kaempferol glycosides and phenolic acids such as caffeic acid, ethil galate, gallic acid and quinic acid. In total, 24 constituents were characterized, by means of extensive preparative chromatographic analyses, along with MS and NMR techniques. An HPLC-PAD-ESI-IT-MS and FIA-ESI-IT-MSn method were developed for rapid identification of acylated flavonoids, flavonoid-O-glycosides derivatives of myricetin and quercetin and phenolic acids in the hydroalcoholic M. bella leaves extract. The FIA-ESI-IT-MS techinique is a powerful tool for direct and rapid identification of the constituents after isolation and NMR characterization. Thus, it could be used as an initial method for identification of authentic samples concerning quality control of Myrcia spp extracts.


Introduction
Nowadays, the main concerns about natural medicinal products are effectiveness, safety and the quality of the herbal drugs [1,2]. Consequently, it is essential to identify and measure all the bioactive constituents of medicinal plants in order to ensure the biological research reliability and repeatability as well as to ensure enhancing the quality control over the pharmacological benefits and/or hazardous. HPLC-MS plays a prominent role as an analytical tool for detecting and identifying pharmacologically active metabolites and/or reactive metabolites [3,4]. When compared to other detection methods, MS not just allows determining natural compounds chemical structure with known and unknown structures, but also offers excellent sensitivity to low amount of samples within relatively short analysis time as well as plays an important role in screening flavonoids and other phenolics [5][6][7][8].
The Brazilian savanna, called Cerrado, is included in the list of global hotspots for conservation due to its high concentrations of endemic species that had suffered heavy habitat losses [9]. Despite its relevance, the Brazilian savanna has been continuously destroyed in order to create pastures and crop fields. Nowadays, in the state of Sao Paulo, southeastern of Brazil, remnants are very reduced and fragmented [10]. The Myrtaceae is an important family in the Brazilian savanna, with more than 1,000 species countrywide and Myrcia, with 350 species, is one of the richest genus [11]. Myrcia bella is a common and important species in many savanna fragments, distributed in the state of Sao Paulo [12,13]. Indigenous and traditional communities in Brazil have used some species of this genus as astringent, against diabetes, diarrhea, diuretic, to stop bleeding, against hypertension and ulcers [14,15]. Most phytochemical studies on the Myrcia species are related to essential oils [16][17][18][19][20]; however Yoshikawa et al. [21] and Matsuda et al. [22] described the features of flavanone glucosides, acylated flavanone glucosides, acetophenones, flavonols and gallic acid in the leaves of Myrcia multiflora (Lam.) DC.
The present work aimed to assess the qualitative and quantitative profiles of M. bella hydroalcoholic extract, due to the potential use of the Myrcia species in tradittional medicine and also due to its comercial exploration as an herbal drug to be used in the treatment of diabetes mellitus [15,23,24] once the existing legislation in Brazil determines that a standard method must be developed in order to be used for quality controlling raw plant materials [25].

Identification of Constituents by Combination of NMR and FIA-ESI-IT-MS/MS n
In total, 24 constituents ( Figure 1) were identified in the 70% EtOH extract. Eighteen of them were isolated and characterized by UV, MS and NMR spectral data and six were tentatively identified considering retention time values, co-chromatography with authentic samples, UV and MS spectral data. In Figure 2, the HPLC-PAD chromatogram of the M. bella 70% EtOH leaves extract is presented. Data concerning identification of the peaks are shown in Table 1, where the retention time, UV-vis absorptions and electrospray ionization mass spectrometry in negative ion mode of all the compounds detected are reported. Once this is the first experiment on chemical characterization of constituents in M. bella, it was necessary to completely characterize them; therefore purification and consecutive identification by 1D and 2D NMR spectroscopy was carried out.      The loss of 152 Da in MS spectra of compound 18 suggested the presence of a galloyl unit. 13 C-NMR signals were in agreement to MS data and confirm the presence of galloyl unit [29].
In addition to the experiments which led to the isolation and identification of compounds, an analysis was also carried out to confirm the chemical composition of the 70% EtOH extract employing the combination of multi-stage analysis of selected ions by ESI-IT-MS/MS n and HPLC-PAD-ESI-MS. The main objective of this analysis was to evaluate the ability of this technique to produce spectral information on the chemical constitution sample analyses quickly and directly, without the need for pretreatment steps and/or chromatographic separations. Six constituents (compounds 3-4 and 21-24) were tentatively identified. Within the exception of the isolated compounds 1, 2, 5, 6 and 12, all other compounds were simultaneously identified by HPLC-PAD-ESI-MS. The typical precursor ions spectrum in negative ion mode of 70% EtOH leaves extract of M. bella is presented in Figure 3.   Finally, the multi-stage analysis of selected ions by FIA-ESI-IT-MS/MS n provided rich information of the compounds of the 70% EtOH extract providing the identification of minor compounds not isolated without purification or pre-treatment.
The repeatability, based on three samples with known concentration, was analyzed by HPLC and the relative standard deviation (% RSD) of the standards was calculated. RSD values ranged between 0.32 and 3%. The overall intraday time variations of the standards were less than 0.32-2.40% for gallic acid and 0.39-3.0% for quercetin and interday time variations were less than 0.52-2.32% for gallic acid and 0.47-1.47% for quercetin. Precision data are displayed in Table 2.

Quantitative Analysis
In a first look at the chromatogram it became evident that the main constituents were quercetin glycosides derivatives containing one sugar unit. In order to make the most pratical method, we used two reference standards available on the market. Quercetin glycosides derivatives were expressed as quercetin, whereas phenolic acids derivatives were expressed as gallic acid. The quantitative analysis results are reported in Table 3.

Solvents and Chemicals
The solvents used on HPLC analysis were HPLC grade; MeOH and formic acid (85% v/v) for HPLC were purchased from Merck (Sao Paulo, Brazil). Water was purified by a Milli-Q plus system from Millipore ® . PTFE membrane filter (0.45 mm) was also purchased from Merck. All laboratory chemicals used in the current study in the isolation protocol were of reagent grade.

Plant Material
Samples of M. bella leaves were collected in November 2010 at the Botanical Garden of Bauru (22°20'30" S e 49°00'30" W)-SP, Brazil. Voucher specimens were prepared and identified by A. L. Dokkedal and stored at the Herbarium of the UNESP-Univ Estadual Paulista "Júlio de Mesquita Filho"-UNBA (Bauru-SP, Brazil) under code number 5508.

Standards
The following standards were used for quantitative analysis: gallic acid and quercetin, which were purchase from Sigma-Aldrich (Sao Paulo, Brazil).

Isolation of the Characteristic Constituents
Fresh leaves were dried at 40 °C for 48 h. The separated powdered leaves (1.3 kg) were extracted with EtOH-H 2 O (7:3) by percolation at room temperature for 2 months [35]. The ethanolic solution was filtered and concentrated to dryness under reduced pressure at 40 °C furnishing 364 g of the hydroalcaholic extract (70% EtOH); a portion (15 g) of this extract was redissolved in MeOH-H 2 O (1:4) and partitioned with equal volumes of n-hexane, dichloromethane, n-butanol and water (Fr 1 -Fr 4 ) to obtain four major fractions. HPLC-PAD analysis showed that fraction Fr 3 was richer in flavonoid glycosides. Fraction Fr 3 (3 g) was dissolved in 15 mL of MeOH and subjected to size exclusion chromatography using a Sephadex LH-20 column (85 × 2.5 cm; H × i.d.) with peristaltic pump and automatic collector Redifrac using MeOH as mobile phase yielding 366 fractions (10 mL each) (Mb 1 -Mb 366 ). Fractions Mb 77 -Mb 366 were combined by similarity and were subjected to purification by semipreparative HPLC-RI (Knauer ® 2300) using a C 18

HPLC-PAD Analysis Instrumentation
The HPLC system consisted of a PU-2089S Plus (Jasco ® ) pump equipped with a MD-2015 Plus Photodiode Array Detector (PAD, Jasco ® ) and AS-2055 automatic injector (Jasco ® ). The column was a Luna C 18 column (250 × 4.6 mm, i.d.) with a particle size of 5 mm (Phenomenex ® ) maintained at 35 °C, and managed by the Jasco ChromPass software. The eluents were: A (MeOH + 0.1% formic acid) and B (H 2 O + 0.1% formic acid.). The gradient condition was: 5-45% of A in B in 190 min. Injected volume of the samples was 20 μL solution. The UV-vis spectra were recorded between 200 and 600 nm, and the chromatographic profiles were registered at 254, 280 and 360 nm.

FIA-ESI-IT-MS/MS n and HPLC-ESI-IT-MS Analysis Instrumentation
The chromatographic profile of the crude extract of M. bella was performed using Accela High Speed LC (Thermo Scientific ® , San Jose, CA, USA), Phenomenex ® Luna C 18  Using this method, we determined the most intense parent ion for each peak in the chromatogram. A second event, FIA-ESI-IT-MS in the negative ion mode, was performed using the same equipment described above, equipped with Xcalibur software. The 70% EtOH extract was dissolved in MeOH-H 2 O (8:2) and infused in the ESI source by flow injection analysis (FIA) using a syringe pump; the flow rate was 33 μL·min −1 . The capillary voltage was −20 V, the spray voltage was 4 kV, and the tube lens offset was −55 V. The capillary temperature was 275 °C. Nitrogen was used both as drying gas at a flow rate of 60 (arbitrary units) and as nebulising gas. The nebulizer temperature was set at 280 °C, and a potential of −4 V was used on the capillary. Negative ion mass spectra were recorded in the range m/z 100-1,550. Two scan events were prescribed to run in the LCQ mass spectrometry. The first event was a full-scan spectrum to acquire data on the deprotonated compounds within the scan range. The second scan event was a MS/MS experiment performed using a datadependent scan on deprotonated molecule [M − H] − . The collision energy for MS/MS was adjusted to 10-25%.

Identification of Peaks and Peak Purity
Identification of all constituents was performed by HPLC-PAD and MS analysis by comparing the retention time, the UV and MS spectra of the peaks in the samples with those of authentic reference samples or isolated compounds. The purity of peaks was checked by a PAD coupled to the HPLC system, comparing the UV spectra of each peak with those of authentic references samples and/or by examination of the MS spectra.

Linearity Limitation of Detection, Limitation of Quantification and Precision
The optimized HPLC-PAD method was validated for the simultaneous analysis of gallic acid and quercetin in terms of linearity, limit of detection, limit of quantification, precision and accuracy. The calibration curves were obtained by the external standard method on eight levels of concentration of standard mixtures, with three injections per level. Chromatogram peak areas at 280 nm for gallic acid and at 360 nm for quercetin were plotted against the known concentrations of the standard solutions to establish the calibration equations. A linear regression equation was calculated by the least squares method. The detection limit (LOD) and limit of quantification (LOQ) were calculated from the residual standard deviation of the regression (σ) line and the slope (S) as follows: LOD = 3.3σ/S; LOQ = 10σ/S. Three different concentrations of standard mixtures (0.05; 0.1 and 0.2 mg·mL −1 ) were used for intra-and interday precision testing. The areas under curves and retention times of the three consecutive injections, performed at each concentration on three different days, were used to calculate % RSD (relative standard deviation) interday precision. Intraday precision data for peak areas and retention times were calculated from six non-consecutive injections, performed at each concentration on the same day.

Quantitative Determination of Constituents
The method of external standard was applied to quantify each compound. Quantification of individual constituents was performed using a regression curve, each point in triplicate. Measurements were performed at 360 nm, which is the maximum absorbance for flavonols and 280 nm for phenolic acids.

NMR Analysis
The 1 H-NMR and 13 C-NMR 1D and 1 H-NMR 2D-NMR 13 C g-HMBC experiments were performed on a Bruker ® 300 MHz (7.0 T) nuclear magnetic resonance spectrometer and/or on a Varian Inova ® 500 MHz (11.7 T) nuclear magnetic resonance spectrometer (for sample preparation for the NMR experiments pyridine-d 5 and dimethyl sulfoxide (DMSO-d 6 , Cambridge Isotope Laboratories, Inc., Andover, MA, USA) were used.

Conclusions
The qualitative and quantitative profiles of the 70% EtOH extract from the leaves of M. bella were analyzed. The combined NMR, FIA-ESI-IT-MS/MS n and HPLC-PAD-ESI-MS techniques allowed the unambiguously identification of 24 compounds. Eighteen constituents were isolated and confirmed by NMR and/or MS analysis and six were tentatively identified. Main secondary metabolites were flavonoid glycosides, mainly derivatives of quercetin and myricetin. The HPLC-PAD analysis also revealed the presence of peaks with typical UV spectra of phenolic acids derivatives in the chromatogram. The genus is reported to contain flavonol-O-glycosides of myricetin and quercetin as well as flavanones glucosides and acetophenone glucosides in polar extracts [20][21][22]. The FIA-ESI-IT-MS techinique is a powerful tool for direct and rapid identification of the constituents after isolation and NMR characterization. Thus, it could be used as a starting method for identification of authentic samples for the purposes of quality control of Myrcia spp extracts.