Characterization of Proanthocyanidins from Parkia biglobosa (Jacq.) G. Don. (Fabaceae) by Flow Injection Analysis — Electrospray Ionization Ion Trap Tandem Mass Spectrometry and Liquid Chromatography/Electrospray Ionization Mass Spectrometry

The present study investigates the chemical composition of the African plant Parkia biglobosa (Fabaceae) roots and barks by Liquid Chromatography - Electrospray Ionization and Direct Injection Tandem Mass Spectrometry analysis. Mass spectral data indicated that B-type oligomers are present, namely procyanidins and prodelphinidins, with their gallate and glucuronide derivatives, some of them in different isomeric forms. The analysis evidenced the presence of up to 40 proanthocyanidins, some of which are reported for the first time. In this study, the antiradical activity of extracts of roots and barks from Parkia biglobosa was evaluated using DPPH method and they showed satisfactory activities.

characteristics responsible for the antioxidant activity of a plant extract is its ability to scavenge free radicals, which can play a part in the protection against the harmful action of ROS. Thus, the aim of the present study was to characterize the proanthocyanidins profile as well as to evaluate the antiradical activity of the root and bark extracts of P. biglobosa.

FIA-ESI-IT-MS n Analysis
Direct flow injection -electrospray ionization -mass spectrometry analysis can be used to establish the polyphenol profile of complex extracts [35], so technique was employed to obtain a preliminary qualitative metabolic fingerprint of the bark and root extracts of P. biglobosa, after a clean-up using LLE and SPE. The collected eluates were analyzed by ESI-IT-MS n both in positive and negative ionization mode. The acidic nature of all the compounds present in extract of P. biglobosa makes negative ionization a good choice for obtaining high sensitivity [36]. Both extracts gave similar results (data not shown). Additional structural information was obtained by CID-MS-MS experiments. For identification and characterization of PAs, four main fragmentation mechanisms were observed: the retro-Diels-Alder (RDA), quinone methide (QM), heterocyclic ring fission (HFR) and the benzofuranforming (BFF) mechanisms [22,37,38].

HPLC/ESI-IT-MS Analysis
It has been shown that catechins, which are monomeric units in PAs, are found in plant extracts and almost always identified as catechin and/or (epi)catechin, probably due to stereoselectivity of the enzymes involved in the biosynthesis of these substances [43]. An analytical strategy based on the online HPLC/ESI-IT-MS approach was applied to investigate the presence of isomers of catechins derivatives in P. biglobosa extracts since FIA-ESI-IT-MS n cannot distinguish between stereoisomers [37]. The results obtained revealed a different number of PAs isomers in these extracts and the presence of other B-type PAs which have also been detected in the roots and barks of the species ( Figure 5). The UV spectra of all peaks exhibited absorption maxima bands at 205-215 nm and 270-285 nm, typical of proanthocyanidins [19], confirming the preliminary phytochemical study in both extracts.  Table 1.
Finally, tetramers were the highest m/z values that could be detected by electrospray in these fractions of roots and barks of P. biglobosa. Thus, proanthocyanidins present in this plant are constituted, of procyanidins, with mixed procyanidin-prodelphinidin structures and (epi)gallocatechin oligomers. In order to identify higher polymers, these were concentrated in the aqueous fraction [45] (as described in subsection 3.3 of the Experimental), one analytical alternative for the characterization of polymers includes the matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry, that permits the identification of tannins with higher degree of polymerization [46].
The antiradical activity of the CH 2 Cl 2 /MeOH (1:1, v/v) extracts and the ethyl acetate and aqueous fractions of the barks and roots was evaluated using a DPPH assay, with gallic acid and quercetin as antiradical reference compounds.
The remarkable antioxidant activities exhibited by all the P. biglobosa extracts and fractions tested could be associated with the presence of (epi)catechin derivatives, particularly (epi)gallocatechin-Ogallate [47] and condensed tannins, well known as being very powerful antioxidants [25]. Other polyphenols, such as flavonoids and phenolic acids, are strong antioxidants too. These chemical classes are responsible for the high antiradical activity of extracts obtained from brewery waste streams [48].

Plant Material
Barks and roots of P. biglobosa were collected in October 2008 at Ngaoundere-Cameroon, Africa, and authenticated in loco by Professor Dr. Pierre Marie Mapongmetsem and confirmed in the National Herbarium through the voucher sample (n° 58980 HNC) collected by Dr. Francois Villiers from National Museum of Natural History of Paris.

Extraction and Sample Preparation
Dried and powdered barks (1 g) and roots (1 g) of P. biglobosa were macerated separately for a week, at room temperature, with 100 mL of a 1:1 (v/v) mixture of CH 2 Cl 2 /MeOH. The solutions were evaporated to dryness under vacuum to give 100 mg (10%) of crude bark and root extracts. In order to minimize the interference of very high order polymeric compounds, a combination of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) was employed [45]. In the first step, 100 mg of each extract were, separately, submitted to LLE between EtOAc and H 2 O (50 mL of each solvent). After drying, the EtOAc fractions afforded 30 mg of each sample. An aliquot (10 mg) of the EtOAc fraction of each extract were submitted to the SPE using RP18 cartridge, eluted with H 2 O/MeOH 8:2 (v/v) (5 mL). The eluate was filtered through the nylon membrane and directly analyzed by ESI-IT-MS n as well as by HPLC/ESI-MS.

FIA-ESI-IT-MS n
Flow injection analysis (FIA) was performed using a ThermoFisher Scientific ion trap mass spectrometer (San Jose, CA, USA) equipped with an electrospray ionization source. The MS and MS/MS analysis in negative ion mode were selected after calibration infusing a standard solution of (+)-catechin (1 µg·mL −1 in methanol) at a flow rate of 5 µL·min −1 and working under the following conditions: capillary voltage −31 V, spray voltage 5 kV, tube lens offset 75 V, capillary temperature 300 °C, sheath gas (N 2 ) flow rate 8 (arbitrary units). Negative ion mass spectra were recorded in the range m/z 100-2000 Da. The first event was a full -scan mass spectrum to acquire data on ions in the m/z range. The second scan event was an MS/MS experiment performed by using data-dependent scan that was carried out on deprotonated molecules from the compounds at collision energy of 30% and activation time of 30 ms. Data acquisition and processing were performed using the Xcalibur software.

HPLC/ESI-IT-MS
Analysis were performed by HPLC/ESI-MS using a SURVEYOR MS micro HPLC system coupled on-line with an LCQ Fleet ion trap mass spectrometer (Thermo Fisher Scientific). HPLC separation was conducted on a Synergi Hydro RP-18 column (250 × 4.6 mm, L × i.d.; 4 µm, Phenomenex), at a flow rate of 800 µL·min −1 . Gradient elution was performed by using H 2 O (A) and MeOH (B), both added of 0.1% acetic acid, as mobile phases. After a 5 min hold at 5% B, elution was performed according to the following conditions: from 5% B to 25% B in 50 min; 25% to 100% B in 60 min. The column effluent was split into two by means of a "T junction" placed after the chromatographic column and analyzed "on-line" both by ESI/MS and UV-DAD; 80% of the effluent sent to the UV-DAD detector; 20% of the effluent was analyzed by ESI/MS in negative ion mode. The mass spectra were acquired and processed using the Xcalibur software (version 1.3) provided by the manufacturer. Capillary voltage 20 V, capillary temperature 275 °C, nitrogen as drying gas at a flow rate 4 (arbitrary units) and as nebulizing gas, ion spray voltage 5 kV, tube lens offset 80 V, sweep gas at a flow rate 15 (arbitrary units). Data were acquired in MS 1 scanning modes in the range of m/z 100-2,000 Da. The ESI interface and MS parameters were optimized to obtain maximum sensitivity. PAs were identified based on observation of the m/z values of their deprotonated molecules.

Antiradical Activity Determination
DPPH assays were used to measure the free radical scavenging potential of the extracts [49]. The After 30 min of reaction, absorbance was immediately recorded at 517 nm. Quercetin and gallic acid (Sigma Aldrich ® , St Louis, MO, USA) standard solutions were prepared and analyzed under the same conditions. The results were expressed as 50% inhibitory concentration (IC 50 in μg·mL −1 ). All analyses were the mean of triplicate measurements ± standard deviation. Lower IC 50 value indicates higher antioxidant activity.

Conclusions
To the best of our knowledge no study had previously described the tannins profile of Parkia biglobosa. Thereby, with the aim of providing further scientific contributions to support and improve African economy, we decided to carry out an analytical study to define the metabolic fingerprinting of this plant.
Effective, rapid and sensitive HPLC/ESI-IT-MS and FIA-ESI-IT-MS n methods were developed for characterizing the oligomeric constituents in the plants. The work established the profile of proanthocyanidins in the extract of roots and barks of P. biglobosa. Mass spectral data indicated that B-type oligomers are present, namely procyanidins and prodelphinidins, with their gallate and glucuronide derivatives, some of them in different isomeric form. With this method, some proanthocyanidins with novel structures were determined for the first time. Tandem Mass Spectrometry (MS n ) was demonstrated to be a useful analytical tool of choice for characterizing PAs, since more information about the structural details of the different molecules can be elucidated from the fragmentation of the precursor ions. The occurrence of proanthocyanidins is in accordance with chemical constituents of this genus, since tannins were previously identified in P. clappertoniana [14].
The antiradical activity of the extracts and the fractions was evaluated using the DPPH assay, with gallic acid and quercetin as antiradical reference compounds. The study suggests that the high antiradical activity of extracts and fractions from P. biglobosa can be attributed to the proanthocyanidin derivatives present in this plant.