Integrative Analysis Based on HPLC-DAD-MS / MS and NMR of Bertholletia excelsa Bark Biomass Residues : Determination of Ellagic Acid Derivatives

Bertholletia excelsa Bonpl. (Lecythidaceae) is a South American tree worldwide known for providing the Brazil nuts. In the Amazon Region, B. excelsa is found in monocultures, integrating agroforestries and providing raw materials for food and timber industries. Through the application of an integrative analysis based on high-performance liquid chromatography coupled with diode array detector and tandem mass spectrometry (HPLC-DAD-MS/MS) and nuclear magnetic resonance (NMR) techniques, the present study showed that B. excelsa bark biomass residues contain large quantities of ellagic acid (EA) and its derivatives. Qualitatively, five compounds were characterized for the first time in this species. Quantitations were carried out to determine the total amount of these compounds in outer and inner bark tissues. A total of 4.96 and 44.09 g of EA derivatives per kg of dry residues was determined for the outer and inner barks, respectively. Among the EA derivatives, eschweilenol C, ellagic acid and valoneic acid dilactone were the main compounds. These results pointed B. excelsa barks as a valuable biomass residue with potential to be source of health-promoting compounds. Therefore, a potential raw material as source of valuable bioactive phenolic compounds is described herein.


Introduction
The Brazil nut (Bertholletia excelsa, Bonpl.) is a South American tree and is also the name of its commercially harvested edible seeds. 1,24][5] The economic interest for exportation relies basically on the edible seeds that are largely applied in food and cosmetic industries, generating $ 30 million annually in Brazil. 6This species also produces high-quality timber, whose extraction is allowed only for planted trees by the Brazilian law. 5,7ery year, farms and furniture industries in the Amazonas state of Brazil generate tons of cake and woods residues.So far, only waste material from pressed nuts were proposed as addictive ingredients for the enrichment of animal food and nutritional bars due to its polyphenol content. 8This material has been proved to be an interesting source of hydrophilic bioactive compounds such as phenolic acids and flavonoids. 8bout the timber industry, the destination of trunk barks and sawdust are simply their discharge. 7,91][12][13] For that reason, the number of investigations concerning the extraction of these compounds from biomass wastes increased. 85][16] Although less sensitive than mass spectrometry (MS), nuclear magnetic resonance (NMR) likewise provides a powerful complementary technique for the confirmation and quantification of metabolites directly in plant extracts. 17,18In an integrative way, these techniques enable numberless analytical approaches, whether in the prior recognition of complex matrices, or seeking pharmacologically active substances. 19,20hus, the aim of the present work was the qualitative and quantitative characterization of phenolic compounds in the barks waste of B. excelsa from a regularized Brazil nut crop.In order to achieve this goal, an integrative strategy consisting of HPLC-DAD-MS/MS and NMR was applied.Moreover, a simplified protocol for selective extraction and quantification of phenolic compounds was proposed.

Biomass residue sample
A piece of log wood (ca. 10 kg) from B. excelsa was provided by the agroindustry Agropecuária Aruanã S. A. during September of 2016 (Itacoatiara, Amazonas State, Brazil, 58°49'48.0"W03°00'17.0"Swhich constituted the largest monoculture of B. excelsa of the state.The sample was obtained directly from a living tree.The access to genetic heritage was registered at Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen) under the code No. A442001.

Biomass residue extraction and fractionation
The plant material was cleaned and dried at ambient temperature (ca.20 °C) for 20 days.Then, the barks were manually separated from the wood, and split off in outer and inner barks.An aliquot of each material (ca.15 g) was powdered using an analytical knife micro-mill (Q298A21, Quimis, Diadema, SP, Brazil).An amount of 1 g of pulverized material was extracted with distilled water (50, 100 or 150 mL) under agitation for 24 h at 20 °C.All extracts were filtered through Whatman 43 filter paper (Sigma-Aldrich, St. Louis, MO, USA) and then freeze-dried in a ModulyoD-230 apparatus (Thermo Fisher, New York, NY, USA).In order to evaluate the clean-up efficiency of the solid phase extraction (SPE), the inner bark aqueous extract prepared by the 100 mL-protocol was passed through a cartridge containing 10 g of KP-C18-HS phase (Biotage, VA, USA) previously activated with methanol HPLC (20 mL) and conditioned with water (60 mL).The column was washed with water (60 mL) and further eluted with methanol HPLC (60 mL).The methanol fraction was dried under a nitrogen gas stream, while the aqueous fraction was freeze-dried.All the experiments were performed in triplicate and the results were expressed as mean ± standard deviation (SD).

Qualitative analysis of the phenolic compounds
Aqueous extracts and SPE fractions were solubilized in methanol at 1 mg mL -1 and injected (2 μL) into a Phenomenex Luna C18 column (5 μm, 150 × 4.6 mm i.d.) (Torrance, CA, USA).Separations were performed by an Accela HPLC system (Thermo Scientific, Waltham, MA, USA).The mobile phase consisted of 1% formic acid aqueous solution (A) and methanol (B) at a flow rate of 1 mL min -1 .Elution was performed in gradient mode consisting of 20% B to 100% B over 20 min, followed by 10 min at isocratic mode with 100% B. Absorptions were registered from 240 to 400 nm.The outlet from the diode array detector (DAD) was connected through a split valve (flow rate of 0.3 mL min -1 ) to a triple quadrupole mass spectrometer model TSQ Quantum Access (Thermo Scientific, Waltham, MA, USA), equipped with an electrospray ionization (ESI) interface and running in the negative ion mode.The ionization settings were as follows: spray voltage, 3000 V; sheath gas pressure, 35 (arb); ion sweep gas pressure, 0.0 (arb); aux gas pressure, 15 (arb); capillary temperature, 270 °C; capillary offset, −35 V; skimmer offset, 0 V; mass range, m/z 150 to 950; collision energy, 30 eV.

NMR analysis
The methanol fraction obtained from the SPE fractionation (30 mg) was solubilized in 600 μL of DMSO-d 6 and subjected to one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy on the AVANCE III HD spectrometer (Bruker, Karlsruhe, Germany) operating at 11.75 T, observing 1 H and 13 C at 500.13 and 125.76 MHz, respectively.Chemical shifts (d) were presented in ppm relative to the tetramethylsilane signal at 0.00 ppm as internal reference and the coupling constants (J) were given in hertz.

Quantitative analysis
The quantification experiments proceeded by HPLC-DAD using the same chromatographic system and elution conditions of qualitative analysis.The content of EA and its derivatives was established with reference to a calibration curve built with EA at 254 nm. 21,22A stock solution of EA (1 mg mL -1 ) was prepared in DMSO and dilutions were done at 5 different levels for calibration curve (0.3, 5, 15, 25 and 50 μg mL -1 ) (y = 1 × 10 -5x − 0.204, correlation coefficient, R 2 = 0.999).This solvent was chosen to overcome the solubility issues previously reported to EA. 23 Outer and inner bark aqueous extracts, and methanol SPE fraction were diluted in DMSO at 1 mg mL -1 .All injections were performed in triplicate and the results were expressed as mean ± standard deviation (SD).

Statistical analysis
Results were expressed by means of values ± standard error of three separate determinations.Comparison of means was performed by one-way analysis of variance (ANOVA) (p < 0.05) followed by post hoc Tukey honestly significant difference (HSD) test (p < 0.01).

Biomass residue extraction
Since chemical differences between inner and outer barks were previously established 24 and water has been preferentially used to prepare folk medicines B. excelsa, 25 we choose to study the phenolic composition of aqueous extracts from the biomass waste of Brazil nut timber industry.
The extraction with water followed by lyophilization yielded a powder material.This process was tested with different amounts of distilled water for each 1 g of biomass powdered.A difference of yield (%mg of extract per g of dry plant material) was observed among the use of 50 and 100 mL of distilled water, increasing from 94.6 ± 2.1 (9.5%, m/m) to 103.4 ± 2.8 mg (10.3%, m/m) for the outer bark, and from 443.6 ± 4.2 (44.4%, m/m) to 473.7 ± 5.5 mg (47.3%, m/m) for the inner bark.No significant differences were observed between the use of an amount of 100 and 150 mL of water for extractions (Table 1).Furthermore, post hoc Tukey HSD test confirmed the raw observation of the extract yields, in which the lack of significance (p > 0.01) among 100 and 150 mL volumes highlighted the limit of extract recovery to a given solvent amount.These observations enabled the choice of 100 mL as solvent volume and the inner bark extract as the raw material to be investigated concerning to the clean-up efficiency by the SPE.This procedure yielded 320.7 ± 6.8 mg (32.1%, m/m) of methanol fraction and 151.4 ± 4.4 mg (15.1%, m/m) of aqueous fraction, providing a yield for total fractions of 47.2%.

Characterization of phenolic compounds
The analysis of the crude extracts by HPLC-DAD-MS/MS displayed that both parts of the timber waste, outer and inner barks, had similar chemical profiles (Figures 1a and  1b).A discrete difference regarding the relative abundance of individual compounds was noticed.The main peaks marked as compounds 1 to 6 displayed UV spectrum profiles with absorbances between 200-400 nm.Two characteristic absorption bands at 254-255 and 361-368 nm were compatible with phenolic compounds (Table 2). 21,22hrough the MS spectra, the phenolic compounds were identified based on their fragmentation under collision-induced dissociation (CID).Compound 1 (Rt 7.0 min) displayed a deprotonated peak at m/z 469, and three main fragments at m/z 299, 300 and 301, consistent with the structure of valoneic acid dilactone (Figure 2). 26Compounds 2 (Rt 7.9 min), 3 (Rt 9.7 min) and 4 (Rt 10.1 min) displayed the deprotonated m/z at 463, 433 and 447, respectively.After fragmentation, all compounds displayed a single fragment at m/z 301, indicative of EA glycosides.Comparison with compounds previously isolated in Brazil nut pointed to ellagic acid hexoside (2), ellagic acid pentoside (3) and ellagic acid desoxyhexoside (4). 21,22Compound 5 (Rt 10.6 min) was identified as EA by means of its deprotonated mass (m/z 301) and fragments (m/z 185 and 229) in comparison with an original standard (Figure 3).This reinforced our structural proposals about the phenolic content in wasted barks of Brazil nut timber.The presence of EA ( 5) in the bark of B. excelsa was early cogitated, 27 however, its confirmation through analytical instrumentation was only performed in the nuts. 28According to this previous report, a similar compound related to 4 (m/z 447 → 301) was detected in Brazil nut brown skin, but its content was not determined.In addition, compound 6 (Rt 11.4 min) was also an EA derivative.Its deprotonated ion at m/z 461 displayed as main fragments the m/z 300 and 315.Thus, this phenolic was identified as methyl ellagic acid deoxyhexoside (6). 21,22o obtain more structural informations regarding to the main compound 4, the SPE methanol EA derivativesenriched fraction (Figure 1d) was directly subject to one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy.Several diagnostic signals were observed in the 1 2. for deshielded proton at d H 7.74 with carbons at d C 107.3, 114.5, 141.3, 146.2 and 158.9 (Figure 4a), consistent for an EA unit. 29Additionally, the anomeric hydrogen displayed HMBC correlations with the carbon atoms at d C 69.9 and 146.2 (Figure 4b), which is characteristic of a rhamnose moiety.This structural evidence was confirmed upon the correlations between the methyl group and the carbon at d C 69.9 (Figure 4c).Through the observation of a nuclear Overhauser effect (NOE) between H-5 (d H 7.74) and H-1" (d H 5.46) in the nuclear Overhauser enhancement spectroscopy (NOESY) spectrum (Figure 4d), the rhamnose moiety was confirmed to be linked to C-4.The anomeric configuration was attributed as α through the characteristic chemical shift and  small coupling constant (J 1.5 Hz) expected for α anomeric hydrogens. 30Therefore, compound 4 was identified as 4-(α-rhamnopyranosyl) ellagic acid (eschweilenol C).This compound has been previously reported in the bark of Eschweilera coriacea (Lecythidaceae). 313]32 As important natural polyphenols, EA compounds are widely contained in various mushy fruits.Due to their strong antioxidant activities, some compounds have been used to scavenge cancer-causing toxins from the human body and improve immunity. 32

Quantification of phenolic compounds
The total amounts of EA and its derivatives in outer and inner bark residues found by HPLC analyses tissues were 4.96 and 44.09 g of EA derivatives per kg of dry residues, respectively (Table 3).Individually, the main compounds in the outer and inner bark wastes were eschweilenol C (1.36 ± 0.09 and 14.97 ± 0.57 g kg -1 ), ellagic acid (1.85 ± 0.19 and 13.25 ± 0.62 g kg -1 ), valoneic acid dilactone (1.27 ± 0.11 and 10.10 ± 0.48 g kg -1 ) and ellagic acid pentoside (0.26 ± 0.02 and 4.45 ± 0.37 g kg -1 ).Alternatively, the SPE methanol fraction for the inner bark presented an amount of 43.56 g of EA derivatives per kg of dry residues, providing to be a simple protocol for selective extraction of EA derivatives.This high concentration of phenolic compounds was close to that reported to Longan seeds (Dimocarpus longan). 33Nowadays, the extracts or products of EA have been put into use in food industry, disease prevention and treatment, and cosmetic production in several countries. 32,33Therefore, the barks waste from the timber obtaining of Brazil nut are an abundant source of EA derivatives.

Conclusions
The integrative approach proposed to directly analyze the crude extracts of Bertholletia excelsa bark residues from  the Amazon timber industry showed to be a useful strategy for characterization and quantification of ellagic acid (EA) and its derivatives.Besides, the SPE protocol proved to be a simple way to selectively extract of phenolic compounds in this matrix.The results of this study highlighted for the first time the B. excelsa bark residues as a promising source of health-promoting compounds, therefore a potential raw material for food, pharmaceutical and chemical industries.The findings of this work may guide future uses of rich biomass that actually is simply dumped into the environment.
H NMR spectrum, among them, aromatic protons at d H 7.74-7.46,an anomeric hydrogen at d H 5.46 (1H, d, 1.5 Hz) and a methyl group at d H 1.14 (3H, d, 6.4 Hz) might be highlighted.The heteronuclear multiple bond correlation (HMBC) experiment evidenced correlations

Figure 1 .
Figure 1.HPLC chromatograms of the inner (a) and outer bark (b) aqueous extracts; and aqueous (c) and methanol SPE fraction (d) from inner bark at 254 nm.The peaks labeled 1 to 6 correspond to the compounds listed in Table2.

Figure 3 .
Figure 3.Chemical structures of the phenolic compounds identified in Bertholletia excelsa residues.

Figure 4 .
Figure 4. Main correlations observed through HMBC (a, b and c) and NOESY (d) experiments for methanol fraction (DMSO-d 6 ) and eschweilenol C tentatively identified.

Table 1 .
Yields of extracts and fractions of Bertholletia excelsa biomass residues a Aqueous extract; b SPE: solid phase extraction, methanol fraction; c extraction performed with inner bark; d SPE: solid phase extraction, aqueous fraction; e mean ± standard deviations (SD); f distribution analysis by one-way analysis of variance (ANOVA) (p < 0.05) and significance analysis by post hoc Tukey HSD test (p < 0.01).

Table 2 .
Ellagic acid and derivatives identified in Bertholletia excelsa biomass residues using HPLC-DAD-MS/MS analyses a Main observed fragments.

Table 3 .
Concentration of ellagic acid and derivatives in Bertholletia excelsa biomass residues according to HPLC-UV at 254 nm a Aqueous extract prepared by the 100 mL-protocol; b SPE: solid phase extraction, methanol fraction; c extraction performed with inner bark; d mean ± standard deviations (SD).