Dereplication of Phenolic Derivatives of Qualea grandiflora and Qualea cordata ( Vochysiaceae ) using Liquid Chromatography coupled with ESI-QToF-MS / MS

Um método racional e seletivo de cromatografia de alta eficiência acoplado à espectrometria de massas (CLAE-EM) com ionização por electrospray e analisador do tipo quadrupolo em tempo-de-voo em sequência (ESI-QToF-MS/MS) foi desenvolvido para a desreplicação de derivados fenólicos presentes em Qualea grandiflora e Qualea cordata. Os extratos para a análise foram selecionados por meio dos resultados de atividade antioxidante revelados no ensaio in vitro com DPPH. A análise por HPLC-ESI-QToF-MS/MS foi realizada por detecção contínua das relações massa/carga em alta resolução e pela técnica MS/MS de colisão induzida para fragmentação dos íons moleculares selecionados. A desreplicação da fração AcOEt do extrato hidroalcoólico dos caules de Q. grandiflora permitiu a detecção dos flavonoides: 3’,4’,5’,5,6,7-hexahidroxi-8-metilflavanona, 8-metil-naringenina e 3’,7-dimetoxi-8-metil-4’,5,7-trihidroxiflavanona, assim como os derivativos da benzofenona (bis(4,6-dimetoxi-2-hidroxi-3-metilfenil)-metanona, 3’,4’-dimetoxi-8-metil5,6,7-trihidroxiflavanona, 7-metoxi-6-metil-3’,4’,5-trihidroxiflavanona, 6,8-dimetil-3’-metoxi4’,5,7-trihidroxiflavanona e 3’,5’-dimetoxi-6,8-dimetil-4’,5,7-trihidroxiflavanona) foram detectados na fração AcOEt do extrato hidroalcoólico das folhas de Q. cordata.


Introduction
][7][8] Previous phytochemical analysis of some species of this family led to the identification of lupane, oleanane and ursane triterpenes, steroids, flavonoids as well as ellagic acid derivatives (EAD). 9ol.24, No. 5, 2013   P h a r m a c o l o g i c a l s t u d i e s f o c u s e d o n t h e gastroprotective and anti-inflammatory actions of some Qualea species indicated a relationship between the pharmacological effects and the presence of EAD and polyphenolic compounds. 10ince the therapeutic effects reported for Qualea are a result of their use as complex herbal preparations, avant garde analytical techniques are necessary to identify and detect the bioactive constituents of the crude mixture without performing any time consuming isolation.Dereplication therefore may be applied for that purpose since allows a fast in situ detection of compounds from complex matrixes, such as crude herbal extracts, in low-amounts, by the use of chromatographic and/or spectroscopic techniques. 11n this work, Qualea grandiflora and Qualea cordata, two Brazilian Vochysiaceae species, were analyzed by means of HPLC-ESI-QToF-MS/MS guided by the in vitro DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging capacity aiming at the detection of bioactive polyphenolic molecules.

Plant material
Leaves and stem bark of Q. grandiflora and Q. cordata were collected in 2006 in Assis City (São Paulo State, Brazil), and identified by Prof. Giselda Durigan.Voucher numbers (GD-2033 for Q. grandiflora and GD-2125 for Q. cordata) were deposited in the Herbarium of the Botanic Garden in São Paulo, Brazil.

Extraction
The leaves and stem bark of Q. grandiflora and Q. cordata were dried at 40 ºC under forced ventilation, powdered in an analytical mill and extracted by maceration using an ultrasonic bath for 30 min with EtOH/H 2 O (8:2, v/v).After performing the extract concentration under vacuum in a rotatory evaporator (Büchi, Falwil, Switzerland), they were fractionated by liquid-liquid extraction with hexane and EtOAc.All solvents used were of analytical grade (Mallinckrodt, Paris, Kentucky, USA).

Determination of the radical scavenging activity
DPPH was used as a stable radical in a methanol solution (200 μmol, 2 mL).Rutin was used as the standard compound at 100, 80, 40, 20, 10 and 5 μmol L -1 .Concentrations of 1.7, 2.7, 3.3, 6.7, 10.0, 33.3 and 66.7 μg mL -1 were prepared using crude extracts and fractions from both species (leaves and stem bark).A DPPH solution (2 mL) in methanol (1 mL) was set as the negative control.The samples and the standard (100 μL), previously dissolved in methanol, were added to 200 μL of the fresh prepared reagent.Each mixture was shaken and held for 30 min at room temperature, in the dark.The evaluation of the reduced form of DPPH, generated in situ, was performed measuring its absorbance at 517 nm in a spectrophotometer Synergy HT (Bio-Tek instruments, USA).The radical scavenging activity of the samples was expressed in terms of IC 50 (concentrations that induce 50% inhibition of cell growth).

Sample preparation
Prior to the reverse phase LC analyses, approximately 20.0 mg of the selected extracts were dissolved in 1.0 mL of methanol/H 2 O (2:8, v/v) doped with 2% of acetic acid (pH 4).For removal of non-polar molecules, each solution was purified by solid phase extraction (SPE), using Strata C 18 cartridges (Phenomenex, Torrance, CA, USA), previously activated with 5.0 mL of methanol and equilibrated with 5.0 mL of methanol/H 2 O (1:1, v/v).The resulted samples were dried under nitrogen and set to a final volume of 5.0 mL of methanol/H 2 O (1:1, v/v), filtered through a 0.45 μm GHP filter, and aliquots of 20.0 μL were injected directly into the HPLC.
The analyses were held using the following elution gradient: solvent A = aqueous acetic acid, 0.1% (v/v); solvent B = MeOH, acetic acid, 0.1% (v/v); elution profile = 0-30 min, 5-95% B (linear gradient), 30-35 min, 100% B (isocratic), 35-40 min, 95-5% B (return to initial conditions) and using a flow rate of 3.0 mL min −1 .ESI mass spectra, precursor as well as product ions were acquired in positive ion mode and recorded between m/z 50-1000.The mass spectrometer parameters were maintained constant for all analyses: 1000 scans per s; spectrum interval, 2 s; drying gas flow, 5.0 L min −1 ; drying gas temperature, 180 °C; nebulizer gas pressure, 4 bar.For on-line MS/MS, m/z of the ion to be fragmented in the collision cell was used as the input for the mass spectrometer software.Collision induced dissociation (CID) fragmentation was performed using N 2 (collision gas) on the isolated protonated molecules using collision energies between 10 and 40 eV.

Dereplication strategy
For dereplication of the plant extracts, a database was built containing all molecules and spectroscopic data reported for the Vochysiaceae. 9Thus, from the comparative analysis, the presence of the substances previously described 11 was detected.The scientific reports led to the creation of a database containing 92 molecules including 38 pentacyclic triterpenes, 10 ellagic acid derivatives, 36 flavonoids from the structural classes of flavanones, flavones, dihydroflavonols, flavanes and pyrrolidinoflavanes, 2 benzophenones, among others. 9or the on-line molecular identification, the high-resolution m/z ratio observed for the major chromatographic peaks were compared with the database in order to obtain the molecular formulas.From each formula, it was determined the relative error in ppm.Only molecular formulas bearing less than 10 ppm of error were considered for further MS/MS studies.Final molecules were proposed using collision induced MS/MS through the establishment of rational fragmentation patterns.

Determination of radical scavenging chromatographic profile
In order to evaluate the radical scavenging activity of the metabolites dereplicated by HPLC-ESI-QToF-MS/MS, the EtOAc fraction from the stem bark of Q. grandiflora and the EtOAc fraction from the leaves of Q. cordata were analyzed on a Shimadzu LC-20A HPLC apparatus controlled by the software CLASS-VP 6.14 coupled with a Gilson F203 fraction collector (Gilson, Middleton, WI, USA).The samples were injected (20 μL) at the same chromatographic conditions used on LC-MS experiments and the on-flow elution were collected in 96 well plates (2 mL well volume).For the scavenging activity evaluation, 100 μL of each collected well were transferred to 96 well microplates (350 μL well volume) and added 200 μL of 100 μmol L -1 DPPH, prepared according to previous description.After gentle mixing and 30 min standing at room temperature, in the dark, the level of oxidation was determined using UV spectroscopy.Methanol (20 μL) was injected on LC, collected and tested as negative control.

Results and Discussion
Polyphenolic compounds usually show antioxidant properties associated with the capacity of delay or prevent free radical mediated oxidation by forming a stable free radical. 12Since polyphenolic compounds are associated with anti-inflammatory and anti-ulcer activity in some Qualea species, 10 a bio-guided dereplication of Q. grandiflora and Q. cordata was devised based on in vitro radical scavenging assay.The results of the antioxidant activity showed a strong scavenging capacity for the hydro-alcohol extracts and fractions of Q. grandiflora and Q. cordata, especially for the EtOAc fraction from the stem bark of Q. grandiflora (IC 50 : 10.0 μg mL -1 ), final aqueous fraction from the stem bark (IC 50 : 15.0 μg mL -1 ) and crude extract from the leaves (IC 50 : 22.0 μg mL -1 ).EtOAc fraction from the leaves of Q.cordata also showed a strong scavenging activity (IC 50 : 10.0 μg mL -1 ), when compared with the positive control rutin (IC 50 : 5.0 μg mL -1 ).
In view of the above results, the EtOAc fraction from the stem bark of Q. grandiflora and the EtOAc fraction from the leaves of Q. cordata were selected for dereplication analyses as described in the Experimental section in order to detect and elucidate molecular metabolites that may be responsible for the observed scavenging activity.

Dereplication and detection of bioactive compounds
The active compounds of the EtOAc fraction from the stem bark of Q. grandiflora and the EtOAc fraction from the leaves of Q. cordata were analyzed using a DPPH bioassay coupled with HPLC-ESI-QToF-MS/MS towards the dereplication process.Figures 1 and 2 show the total ion current chromatograms (TIC, positive ion mode) as well as the percentage of DPPH inhibition (superimposed).Retention time, observed m/z and calculated m/z, relative errors, molecular formulae and MS/MS fragments are outlined in Tables 1 and 2.
To validate the HPLC-MS results, rational mechanistic fragmentation proposals were established for all detected molecules using CID tandem mass spectrometry.
Fragmentation patterns for all detected flavonoids are depicted in Figure 4 following the proposed pathway: all compounds showed abundant [M + H] + ions consistent with a retro-Diels-Alder (RDA) cleavage followed by elimination of carbon monoxide and/or neutral losses.Additionally, no dehydration previous to RDA was observed, suggesting that C-3 is not oxidized. 13or the compound 1, a trihydroxylation on B ring due to RDA fragmentation (m/z 153.05), followed by a loss of water on the ion containing the B ring (m/z 135.04), was proposed.
For 8-methyl-naringenine (2), a second RDA reaction to explain the ion m/z 121.06 and subsequent allene formation for m/z 95.05 were proposed.
For O-methyl flavanones, the substituent positions were proposed by RDA rearrangements, radical fragmentations and neutral eliminations. 13,14ccording to literature, C-5 and C-8 O-methyl flavanones lead to prominent loss of methyl radical (m/z 15), not observed on compounds 3 and 6, suggesting they are 7-O-methyl flavanones, already reported for Vochysiaceae species. 9,14or the substances 3 and 7, MS data suggested an O-methyl substituent at B or C rings due to an observed RDA fragment.Furthermore, the absence of characteristic O-methyl fragmentations ([M -15] + and [M -16] + ) indicated the substituent position at C-3'. 13,14 For the substance 5, a neutral loss of methane indicated the presence of an O-methyl group at C-3' and C-4',     in which the fragmentation can be rationalized by the formation of a C-3'/C-4' methylenedioxy intermediate.In the case of 3,3'-O-methyl substituents, methane should be expected to be observed. 13olecule 8 showed the absence of [M -15] + and [M -16] + indicating that the O-methyl groups may be at C-3' and C-5' (meta positions), restricting the formation of the methylenedioxy intermediate (3',4'-O-dimethyl) or C-2'/C-3 furan ring (3-O-methyl). 13,14he detected benzophenone was proposed as bis(2-hydroxy-4,6-dimethoxy-3-methylphenyl)-metanone (substance 4, Figure 5), justified by the cleavage of the carbonyl bond (m/z 195.07) followed by a loss of carbon monoxide (m/z 167.07).The observed ions are in agreement with MS data previously reported from the isolated benzophenone. 15he percentage of DPPH inhibition of the detected peaks showed the high radical scavenging activity, supporting the preliminary antioxidant results obtained for the fractions.This result suggests that the activity may be related to the dereplicated molecules since the observed phenolic compounds bear substituent arrangements that allow the delocalization of radicals.
The substitution pattern of the methyl groups proposed for carbons C-6 and C-8 is unusual in plants but already described in some Vochysiaceae species. 9All MS and MS/MS experiments carried out were unable to confirm the exact position of these substituents since there was no observed fragmentation capable to provide such information.Other analytical strategies such as derivatization followed by MS analysis or the use of nuclear magnetic resonance (NMR) may shed further information in order to conclude the C-methyl pattern for those flavanones.

Conclusions
The dereplication of Qualea species using HPLC-ESI-QToF-MS/MS was applied for the first time.LC-MS technique coupled with in vitro DPPH assay has proven to be rapid and sensitive, providing a great deal of preliminary information about the content and nature of radical scavenging properties from detected molecules without the need of isolation.In this study, seven flavanones and a benzophenone were detected by retention times, accurate ESI mass spectra and further CID tandem experiments that allowed the proposal of rational fragmentation pathways for each compound.

Figure 1 .
Figure 1.Total ion current (TIC) and percentage of DPPH inhibition (grey bars) of EtOAc fraction from the stem bark of Q. grandiflora.

Figure 2 .
Figure 2. Total ion current (TIC) and percentage of DPPH inhibition (grey bars) of EtOAc fraction from the leaves of Q. cordata.

Figure 4 .
Figure 4. Proposed pathway fragmentation of the protonated flavonoids from Qualea species.

Table 2 .
HPLC-ESI-QToF-MS/MS data for the ethyl acetate fraction from the leaves of Q. cordata

Table 1 .
HPLC-ESI-QToF-MS/MS data for ethyl acetate fraction from the stem bark of Q. grandiflora