Flavonoids and Methoxy-galloylquinic Acid ... - ACS Publications

Jul 21, 2015 - and Jairo K. Bastos*,†. †. Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University ...
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Flavonoids and Methoxy-galloylquinic Acid Derivatives from the Leaf Extract of Copaifera langsdorffii Desf. Mauro S. Nogueira,†,§ Ricardo A. Furtado,† and Jairo K. Bastos*,† †

Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café s/n, 14040-903 Ribeirão Preto, SP, Brazil § Institute of Pharmaceutical Biology and Phytochemistry (IPBP), University of Münster, Correnstrasse 48, 48159 Münster, Germany S Supporting Information *

ABSTRACT: Despite reports on the pharmacological potential of Copaifera langsdorffii Desf. (Leguminosae-Caesalpinioideae) leaf extract, little is known about its chemical composition. In this work, a phytochemical study from the C. langsdorf f ii ethanol/ H2O 7:3 (v/v) extract was undertaken. Separation was performed by high-speed counter-current (HSCCC) and Sephadex LH20 column chromatographies, followed by preparative HPLC. The EtOAc- and H2O-soluble fractions of the extract furnished the flavonoids quercitrin (1) and afzelin (2) and 3-O-(3-O-methyl-galloyl)quinic acid (3), respectively. The H2O-soluble fraction furnished 3,4-di-O-(3-O-methyl-galloyl)quinic acid (4), 3,5-di-O-(galloyl)-4-O-(3-O-methyl-galloyl)quinic acid (5), and 3,5-di-O(3-O-methyl-galloyl)-4-O-(galloyl)quinic acid (6). Their chemical structures were elucidated by NMR means. KEYWORDS: Copaifera langsdorffii, 3-O-methyl-galloylquinic acid, quercitrin, afzelin, HSCCC

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Counter-current chromatography was carried out using high-speed counter-current chromatography (HSCCC) Quatro CCCTM MK 5 and MK 6 (AECS-QuikPrep Ltd., London, UK) equipped with two coils holding two PTFE pipe columns each (coil 1, column A, 208 mL, i.d. = 3.2 mm, and column B, 29 mL, i.d. = 1.0 mm; coil 2, column C, 102 mL, i.d. = 2.0 mm, and column D, 105 mL, i.d. = 2.0 mm) and a constant-flow gradient HPLC pump. Separations by HSCCC were conducted by connecting C and D in the tail-to-head mode (normal phase elution mode) at 35 °C adjusted temperature, 850 rpm coil rotation speed, and flow rate of 2 mL/min; 10 mL fractions were collected. Analytic HPLC was performed on a Shimadzu SCL-10Avp (Kyoto, Japan) multisolvent delivery system equipped with a Shimadzu SPD-M10Avp photodiode array detector. Analyses were performed using analytical reverse phase column C18 CLC-ODS (M) 25 cm × 4.6 mm (Shimadzu) with a particle diameter of 5 μm; the mobile phase consisted of acidified water (A) (0.01% trifluoroacetic acid (TFA)) and MeOH (B) in gradient conditions as follows: 15− 50% of B (45 min), 50−90% of B (45−65 min) and 90−15% of B up to 70 min; flow rate, 1 mL/min. Preparative HPLC was undertaken in a Shimadzu PREP-HPLC Proeminence model equipped with controller CBM-20 A, UV detector model SPD-20 A, two pumps LC-6 AD, degasser DGU-20 A5, and autosampler collector FCR-10 A. Separations were performed using preparative reverse phase column C18 PREP-ODS (H) (25 cm × 20 mm, Shimadzu). The UV spectra of the isolated compounds were extracted from their chromatogram data performed with the analytic HPLC method. Analytical TLC were carried out on silica gel plates 60F254 (Merck), sprayed with reagent solution of NP in ethanol (1 wt %/v of diphenylboric-β-ethylamino ester) and observed in UV light at a wavelength of 360 nm. 1H NMR, 13 C NMR, and two-dimensional spectroscopic techniques such as HSQC, HMBC, and 1H−1H COSY were recorded on a BrukerAvance DRX500 spectrometer operating at a frequency of 500 MHz (1H NMR) and 125 (13C NMR), and exceptionally for compounds 3

INTRODUCTION Copaifera langsdorffii Desf. (Leguminosae-Caesalpinioideae), popularly known as copaiba, grows widely in various states of Brazil, mainly in Pará and Ceará.1 Copaifera species are known worldwide for the oil resin obtained from their trunks, which has been extensively used in folk medicine in Brazil and presents an important commercial value. Several studies have shown the pharmacological potential of its oleoresin, for example, preventing gastric ulceration,1 promoting wound healing,2 gastrointestinal protection in colitis,3 and also as anti-inflamatory, antioxidant,4 and antinociceptive agents.5 Unsurprisingly, most chemical investigations of copaiba are related to the oil resin and its volatile fractions content, which is well-known to contain a variety of sesquiterpenes and diterpene acids, for example, (−)-copalic acid.6−9 Investigations on the nonvolatile content of copaifera seeds showed that coumarins and xyloglucans are their major components and highlighted their potential use in the cosmetic or pharmaceutical industries.10 More recently our research group has promoted the biological potential of C. langsdorf f ii leaves, reporting the positive effect of the hydroalcoholic extract from aerial parts of C. langsdorf fii against induced urolithiasis in rats,11 and the ability of the leaves extract to promote CaOx crystal dissolution and avoid its formation in vitro and in vivo.12 These results have indeed supported ethnopharmacological reports that indicate the use of C. langsdorf f ii leaves to prevent kidney stone formation in urolithiasic patients. On the basis of the reported pharmacological potential of the C. langsdorf f ii leaves,11−16 it was decided to undertake the phytochemical investigation of the C. langsdorf fii leaf extract.

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Received: March 30, 2015 Revised: July 16, 2015 Accepted: July 21, 2015

MATERIALS AND METHODS

Apparatus and Reagents. Column chromatography was performed on Sephadex LH-20 (GE Healthcare Bio-Sciences AB). © XXXX American Chemical Society

A

DOI: 10.1021/acs.jafc.5b01588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. 1H NMR and 13C NMR Spectroscopic Data for 3−6 3 δ 13C

δ 1H

quinic acid moiety 1 73.2 2a 35.4 2b 3 71.5 4 72.1 5 66.9 6ax 39.9 6eq 7 (COOH) 176.3 galloyl moieties 1′ 120.6 2′ 105.3 3′ 147.7 4′ 139.1 5′ 145.1 6′ 110.1 7′(OCH3) 56.0 COO−′ 165.4 1″ 2″ 3″ 4″ 5″ 6″ 7″ (OCH3) COO−″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ (OCH3) COO−‴ a

1.95 2.05 5.27 3.55 3.92 1.81 1.92

dd dd dt dd td dd dd

4 J (Hz)

13.8, 3.9 13.8, 3.6 6.7, 3.9, 3.6 7.5, 3.6 7.5, 7.5, 4 13.5, 7.5 13.5, 4

δ 13C 73.6 37.3 68.2 73.9 66.4 37.6

δ 1H

2.06 2.25 5.49 5.07 4.26 1.91 2.22

bd m*b m dd m dd m*

5 J (Hz)

δ 13C

13, 3

73.1 36.0

7.8, 2.6 13.5, 7

174.7

7.12 d

1.5

7.15 d 3.79 s

1.5

119.1 104.8 147.8 139.5 145.2 105.0 55.8 164.8 119.3 110.6 147.7 139.5 145.2 110.8 55.8 165.3

69.2 72.0 68.3 38.0

δ 1H

2.02 dd 2.37 dd 5.58 ddd 5.32 dd 5.72 td 2.02−2.17 m 2.02−2.17 m

6 J (Hz)

13.5, 4 13.5, 3.9 4, 3.9, 3.5 9, 3.5 9, 9, 3.5

176.2

6.97 d

1.7

7.03 d 3.77 s

1.7

7.04 d

1.7

7.13 d 3.75 s

1.7

119.8 109.1 145.5 138.6 145.5 109.1 165.6 119.0 104.8 145.3 139.7 147.7 110.7 55.8 165.0 119.2 108.8 145.6 138.8 108.8 145.6 165.2

δ 13C NMa 35.6 68.6 70.5 68.9 37.3

δ 1H

1.96 2.39 5.54 5.28 5.72 2.15 2.32

dd dd ddd dd ddd dd dd

J (Hz)

13.5, 3.5 13.5, 3 3.5, 3, 3 8, 3 8, 8, 3 12.5, 8 12.5, 3

175.2

7.02 s

7.02 s

6.85 d

1.8

6.97 d 3.77 s

1.8

6.88 s

6.88 s 6.88 s

120.0 110.8 147.6 139.4 145.3 105.4 55.6 165.3 118.8 108.6 145.5 138.8 145.5 108.6 165.2 119.0 110.4 147.7 139.6 145.3 104.9 55.8 164.9

7.20 bs

7.01 d 3.7 s

1.8

6.8 bs

6.8 bs

7.20 bs

6.97 d 3.78 s

1.8

b

NM, not measured. An asterisk (*) indicates overlapped signals. Extraction and Isolation. Dried leaves of C. langsdorf f ii (200 g) were ground and exhaustively extracted with ethanol/H2O (7:3) (1.2 L, three times) by maceration at room temperature. The lyophilized extract (50 g) was suspended in MeOH/H2O (9:1, 300 mL) and partitioned first with hexanes (300 mL, three times). The MeOH/H2O phase was evaporated until elimination of the methanol. The remaining fraction was added of water and successively partitioned with dichloromethane (CH2Cl2, 300 mL, three times) and ethyl acetate (EtOAc, 300 mL, three times). After evaporation of the solvents, the hexanes-soluble fraction (9.7 g), CH2Cl2-solube fraction (2.6 g), EtOAc-soluble fraction (13.8 g), and the H2O-soluble fraction (21 g) were obtained. The EtOAc-soluble fraction (3 g) was subjected to HSCCC using interconnected 2 mm diameter columns C (102 mL) and D (105 mL). Solvent system was optimized on the basis of the partition coefficients Kd of compounds 1 and 2 after dissolution of the EtOAc-soluble fraction in the upper and lower phases. Values of (Kd) were assessed by the relation of their peak areas from the analytical chromatograms of both upper and lower solutions. The final solvent system consisted of hexanes/n-BuOH (1:1) (upper phase) and MeOH/H2O (0.4:1) (lower phase), which exhibited a settling time of 25 s, and optimized Kd values for 1 (Kd = 0.6) and 2 (Kd = 0.95). The coil was filled with the stationary phase (lower phase) in a tail-tohead mode, and after a starting rotation program, the mobile phase was pumped into the coil until equilibrium, that is, when only mobile

and 5 on a Bruker-Avance DRX800 spectrometer operating at frequency of 200 MHz (13C NMR). Samples were prepared in Aldrich deuterated dimethyl sulfoxide (DMSO-d6). The mass spectrometry analyses were conducted in two different spectrometers, both equipped with electrospray ionization source (ESI) in negative mode over a mass range of m/z 50−1500. Mass spectra obtained for compound 3 were recorded on a Bruker Daltonics time-of-flight mass spectrometer (a) and those for compounds 4, 5, and 6 on a Thermo Scientific LTQ-FT-Ultra mass spectrometer (b). The following instrument settings were used: (a) nebulizer gas nitrogen (0.4 bar), dry gas nitrogen (4 L/min), 220 °C, capillary voltage of 4500 V, end plate offset −500 V, sample (0.5 g mL−1) dissolved in MeOH/H2O 1:1 and introduced in the electrospray source with a direct infusion pump operating at a flow of 5 μL/min; (b) nebulizer gas nitrogen (0.4 bar), dry gas nitrogen (9 L/min), 270 °C, capillary voltage of 47.96 V, source voltage of 40 V, samples dissolved in MeOH/H2O 8:2 and introduced in the electrospray source with a direct infusion pump operating at a flow of 5 μL/min. Melting points were performed using Fisatom device (model 431, Brazil, series 1237344). Plant Material. Leaves of C. langsdorf f ii were collected in the Campus of the University of São Paulo, Ribeirão Preto, Brazil. The plant material was identified by Dr. Milton Gropo from the herbarium of the Faculdade de Filosofia, Ciências e Letras, Ribeirão Preto, Brazil, where a voucher specimen (SPFR 10120) was deposited. B

DOI: 10.1021/acs.jafc.5b01588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Chromatographic profile of the EtOAc- and H2O-soluble fractions of the leaf hydroalcoholic extract of C. langsdorf f ii acquired by HPLCDAD detected at the typical relative absorption maxima of flavonols (360 nm) and galloyquinic acids (280 nm). Peaks: 1, quercetrin; 2, afzelin; 3−9, methoxy-galloylquinic acid derivatives. C23H24O14 524.1166; 357 [M − 166(MeG) − H]−, 339 [M − 184(MeG acid) − H]−; UV λmax 219 and 278 nm (MeOH/H2O 0.01% TFA); 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz), see Table 1. Data for 3,5-Di-O-(galloyl)- 4-O-(3-O-methyl-galloyl)quinic Acid (5). (1S,3R,4S,5R)-1-Hydroxy-4-(3-methoxy-4,5dihydroxybenzoyl)oxy)-3,5-bis((3,4,5-trihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: dark yellow amorphous solid; [α]24D −136.6 (c 0.08, methanol); melting point 90−94 °C; ESI-LQT-FTMS m/z 661.1046 [M − H]−, calculated for C29H26O18 662.1119, 509 [M − 152(G) − H]−, 491 [M − 170(G acid) − H]−, 357 [M − 152(G) − 152(G) − H]−; UV λmax 220 and 275 nm (MeOH/H2O 0.01% TFA); 1 H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 200 MHz), see Table 1. Data for 3,5-Di-O-(3-O-methyl-galloyl)-4-O-(galloyl)quinic Acid (6). (1S,3R,4S,5R)-1-Hydroxy-4-(3,4,5-trihydroxybenzoyl)oxy)3,5-bis((3-methoxy-4,5-dihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: dark yellow amorphous solid; [α]23D −202.7 (c 0.12, methanol); melting point 100−107 °C; ESI-LQT-FTMS MS m/z 675.1205 [M − H]−, calculated for C30H28O18 676.1275; 523 [M − 152(G) − H]−, 509 [M − 166(MeG) − H]−, 491 [M − 184(MeG acid) − H]−, 339 [M − 152 − 184 − H]−/[M − 166 − 170 − H]−; UV λmax 217 and 275 nm (MeOH/H2O 0.01% TFA); 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz), see Table 1.

phase came out from the coil. Nearly 85% retention of the stationary phase inside the coils was obtained before sample injection. Fractions were combined after TLC analysis furnishing F8−14 (320 mg), F15− 23 (215 mg), F24−26 (65 mg), F27−33 (93 mg), F34−41 (267 mg), and F42−51 (570 mg). Fractions F15−23 (90% of 1) and F34−41 (90% of 2) were subjected to preparative HPLC, performed with an elution program of 35−80% MeOH/H2O in 20 min to yield 1 (108 mg) and 2 (94 mg). The H2O-soluble fraction (3.5 g) was subjected to counter-current chromatography with an optimized solvent system consisting of n-BuOH/EtOAc/H2O (0.1% formic acid) (1:1:1) and a settling time of 15 s, suitable for the distribution of four compounds in both upper and lower phases. The Kd values obtained for those compounds were 0.37, 0.46, 0.60, and 1.05. The coil was filled with the stationary phase (lower phase) in a tail-to-head mode. Retention of the stationary phase before sample injection of 84% was obtained. Fractionation furnished the subfractions F15−18 (134 mg), F21−25 (132 mg), F26−29 (75 mg), and F30−38, which were later purified by preparative HPLC to yield compounds 3, 7, 8, and 9. After purification and storage, three of them underwent chemical degradation. Therefore, only compound 3 (31 mg), obtained from F15−18, was fully elucidated. The H2O-soluble fraction (3.5 g) was subjected to chromatography on a Sephadex LH-20 column, eluted with MeOH to afford several subfractions. Those subfractions were purified in preparative HPLC to yield 4 (28 mg), 5 (33 mg), and 6 (25 mg). The mobile phase optimized for the PREP-HPLC consisted of water (A) (0.01% TFA) and MeOH (B) in gradient condition as follows: 5− 15% of B (20 min), 15−34% of B (20−40 min), and 34−80% of B (40−85 min); flow rate = 9 mL/min. Compounds 4−6 had their chemical structures elucidated. Data for 3-O-(3-O-Methyl-galloyl)quinic Acid (3). (1S,3R,4S,5R)-1,4,5-Hydroxy-3-((3-methoxy-4,5-dihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: white amorphous solid; [α]22D −36.5 (c 0.1, methanol); melting point 115−120 °C; ESI-qTOF-MS m/z 357.0904 [M − H]−, calculated for C15H18O10 358.0900, 191 [M − 166(MeG) − H]−; UV λmax 220 and 275 nm (MeOH/H2O 0.01% TFA); 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 200 MHz), see Table 1. Data for 3,4-Di-O-(3-O-methyl-galloyl)quinic Acid (4). (1S,3R,4S,5R)-1,5-Hydroxy-3,4-bis((3-methoxy-4,5dihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: light brown amorphous solid; [α]22D −38.7 (c 0.02, methanol); melting point 83− 84 °C; ESI-LQT-FTMS m/z 523.1093 [M − H]−, calculated for

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RESULTS AND DISCUSSION C. langsdorf f ii leaf hydroalcoholic extract was dissolved in MeOH/H2O and partitioned with hexanes, dichloromethane, and ethyl acetate (EtOAc) to furnish fractions soluble in these solvents and a water-soluble fraction. A high content of phenolic compounds was found in the EtOAc- and H2Osoluble fractions (Figure 1), including the known flavonoids quercetin-3-O-α-L-rhamnopyranoside (quercetrin, 1) and kaempferol-3-O-α-L-rhamnopyranoside (afzelin, 2), which were previously reported as validated markers for flavonoid quantification in C. langsdorf f ii leaf extracts.14 As the reported pharmacological potential of the extract was primarily suggested to be due to its phenolics content, phytochemical investigations were oriented toward the EtOAc- and H2O-soluble fractions. C

DOI: 10.1021/acs.jafc.5b01588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry The method developed in HSCCC was selective for separation of 1 and 2 from the EtOAc-soluble fraction to yield subfractions containing >90% of these compounds. They were identified by comparison of their UV and 1H NMR spectra (Supporting Information) with the literature.13,17 The separation of those compounds by liquid−liquid chromatography brought several advantages compared to separations previously performed in silica gel or Sephadex column chromatography, as follows: (a) no content loss due to adsorption and suitability for polar fractions; (b) higher purity for compounds of similar molecular size as the separation is based on their Kd; (c) reproducibility; and (d) lower solvent consumption for preparative separations. Column chromatography performed on Sephadex LH-20 and HSCCC, followed by preparative HPLC purification, led to the isolation of three galloylquinic acid derivatives (4−6) from the H2O-soluble fraction, which accounts for roughly 45% (w/w) of C. langsforf f ii leaf extract (Figure 1). Method of separation in HSCCC was also performed for the H2O-soluble fraction. Four semipurified subfractions yielded compounds 3, 7, 8, and 9 after purification in preparative HPLC. Compounds 7−9 underwent chemical degradation most probably by hydrolysis of ester bonds due to remaining acid from the mobile phase on the fractions. The UV absorption (typical λmax at 220 and 275 nm, Figure 1), along with 1H NMR spectra of the isolated compounds (Supporting Information) indicated that the fraction is composed of a series of galloylquinic acid derivatives. The isolated compounds peculiarly bear a methyl group in at least one of their galloyl moieties. Leaf extraction with aqueous ethanol, followed by direct HPLC analysis, was undertaken, confirming that these methylated compounds are truly present in the plant biomass (data not shown). The methoxy-galloylquinic acids (3−6) (Figure 2) are, to the best of our knowledge, first reported. Their structural

Correlations between the quinic acid protons were established via HMBC spectra, along with 1H−1H COSY cross-peaks H-2/H-6, H-2/H-3, H-3/H-4, H-4/H-5, and H-5/ H-6 (Figure 3). Inference of the quinic acid chair conformation and assignment of its proton signals was reasoned primarily on the large vicinal coupling constants between the signals of H-4 and H-5 and between H-5 and H-6ax. The presence of one galloyl moiety bearing a methoxyl group (MeG) in compound 3 was indicated by loss of 166 units in the negative ESI-MS spectrum. This was confirmed by the signals of two aromatic protons meta coupled at δ 7.12 and 7.15 in the 1 H NMR spectrum, one methoxyl group (δ H, 3.79; and δ C, 56.0), and the signal of the aromatic hydroxyl carbon C-3′ (δ 147.7) shifted downfield in comparison to C-6′ (Table 1). The long-range correlation in the HMBC spectrum between C-3′ and the methylic protons (δ 3.79) confirmed the substitution at position C-3′ (Figure 3). The quinic acid unit was indicated by the 1H NMR chemical shifts of four sp3 methylene protons at δ 1.81, 1.92, 1.95, and 2.05, along with three oxymethine protons at δ 3.55, 3.92, and 5.27. Those resonances were in agreement with two sp3 methylene and three oxymethine carbons in the 13 C NMR spectrum (Table 1). The 1H−13C long-range correlation between the proton H-3 (δ 5.27) and the carbonyl carbon signal at δ 165.4 (COO−′) indicated that the MeG moiety was linked to the quinic acid by an ester bond at C-3. In the 1H NMR spectrum, the signal of a dd (J = 7.5 and 3.2 Hz) was assigned to the proton H-4, indicating the axial position of H-4 vicinal to an equatorial proton at δ 5.27 (H-3) and an axial proton at δ 3.92 (H-5). The signal for H-4 in quinic acid derivatives is usually well-defined, even in 1H NMR spectra from low-field instruments,28 by its 3JH,H magnitudes (Table 1) and the 1H−1H COSY cross-correlation with both protons H-3 and H-5 (Figure 3). The proton H-6ax (δ 1.81, dd) was distinguished from another three methylene protons by the greater J H,H magnitudes (2J6ax,6eq = 13.5 Hz and 3J6ax,5ax = 7.5 Hz). The signal at δ 1.92 (m) was associated with C-6 in the HSQC spectrum and, therefore, it was assigned to 6-Heq. As the equatorial−equatorial and axial−equatorial coupling constants between the protons at C-2 and the proton H-3 assumed similar magnitudes, roughly 4 Hz, the relative position of the pair of methylene protons at C-2 (δ 1.95 and 2.05) was not defined. Therefore, they were rather assigned as H-2a and H-2b. The assignment of H-2ax and H-2eq has been previously described by using the signal multiplicity pattern, dd for H-2ax and ddd for H-2eq, in the 1H NMR spectra in the analysis of dicaffeoylquinic and divanilloylquinc acids.29,30 Particularly for H-2eq and H-6eq a pattern of ddd has been observed in these derivatives, to which the smallest coupling constant value (nearly 1.5 Hz) is attributed an 4J2eq,6eq W-coupling.23,30,31 Negative ESI-MS data for compound 4 suggested the presence of two MeG moieties. They were confirmed by signals of two sets of aromatic protons meta coupled in the 1H NMR spectrum (Table 1) and two methoxyl signals (δ H, 3.77/δ C, 55.8; and δ H, 3.75/δ C, 55.8). The substitution position was suggested by the aromatic hydroxyl carbons C-3′ (δ 147.8) and C-3″(δ 147.7) shifted downfield in comparison to the carbons at δ 145.25 (C-5′ and C-5″) and confirmed by the long-range correlations in the HMBC spectrum (Figure 3). The signal of the oxygenated methine proton at δ 5.07 (dd, J = 7.8 and 2.6 Hz) was assigned to proton H-4. Among four methylene proton signals, that at δ 1.91 was assigned to H-6ax due to a greater axial−axial coupling constant (3J = 7 Hz). The

Figure 2. Chemical structures of compounds 3−6.

elucidation is discussed in detail in this work. Numeration of the quinic acid is rather controversial in the literature for galloylquinic acids.18−22 Therefore, to avoid misinterpretation when accessing some references cited throughout this work, the assignments to C-2, C-3, C-5, and C-6 positions might be inverted to C-6, C-5, C-3, and C-2, respectively. The numbering form we adopted has been also described in recent papers related to quinic acid derivatives.23−27 D

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Figure 3. 1H−1H COSY (bold) and key HMBC correlations of compounds 3−6.

signal at δ 4.26, which was cross-correlated in the 1H−1H COSY (Figure 3) with the signal of H-6ax and H-4 (δ 5.07), was assigned to proton H-5. The lasting oxygenated methine signal at δ 5.49 was assigned to H-3. The 1H−13C long-range correlation of the H-4 proton at δ 5.07 with the carbonyl carbon at δ 164.8 (COO−″) indicated one MeG moiety was linked to the quinic acid at the C-4 position. Due to a lack of correlation between quinic acid protons and the carbonyl carbon at δ 165.3 in the HMBC spectrum, the linkage position of the second MeG group was solved by the chemical shift of the proton signals. Acylation of an hydroxyl group produces a paramagnetic shift from 1.3 to 1.8 Hz on the oxymethine proton.23,28,29,31 Therefore, the MeG moiety should be attached to the hydroxyl group at C-3, as the signal of H-3 (δ 5.49) was shifted downfield in comparison to H-5 (δ 4.26). As protons H-3 and H-5 appeared as broad singlets or coalesced signals in the 1H NMR spectrum (Figure 9 in the Supporting Information), they could not be unambiguously assigned by differences in 3JH,H magnitudes. Nevertheless, they should be alternatively differentiated by their signal half-width values (Jhw/2). Assuming the vicinal proton−proton angular dependence of the coupling constants, as the protons H-3, H-4, and H-5 are in equatorial, axial, and axial positions, respectively, a smaller half-width is expected for H-3, which couples with protons H-2 and H-4 at smaller values. For H-5, which couples at greater values with H-6ax and H-4, a larger half-width is expected. However, their values in compound 4 were similar (Jhw/2 = 8 and 10.5) and lower than usually described in the literature (Jhw/2 = 8−10 Hz, H-3; Jhw/2 = 20−28 Hz, H-5).22 This may be explained by the presence of two main relative conformers of the quinic acid in a ratio of nearly 1:1 (H-5eq:H-

5ax) at 20 °C, as shown in a molecular model performed in molecular operations environment (MOE; 2010.09) (Figure 28 in the Supporting Information). It is noteworthy that in case H6ax is not assigned, unambiguous assignment of H-3 and H-5 in compounds alike may be possible only by optimizing the NMR experiments to favor the equilibrium toward one or the other conformer, that is, by performing analysis in different temperatures. Detailed experimental proof of the quantitative conformation equilibrium in quinic acid derivatives has been described by Pauli and et al.31 In compound 5, the presence of two G moieties was confirmed by the 1H NMR signals of two pairs of symmetric aromatic protons at δ 7.02 (H-2′/H-6′) and δ 6.88 (H-2‴/H6‴). The MeG unity was confirmed by the signals of asymmetric aromatic protons at δ 6.85 (H-2″) and δ 6.97 (H-6″). Methylation on the hydroxyl at C-3″ position was confirmed by the long-range correlation between C-3″ and the methylic protons (δ 3.53) (Figure 3). Cross-correlation of the proton H-5 (δ 5.72) with two methylene protons at δ 2.11−2.22 in the 1H−1H COSY spectrum indicated their position at C-6. Thus, the second pair of methylene protons (δ 2.02 and 2.37) should be attached at the C-2 position, which was confirmed by their crosscorrelation with H-3 (δ 5.58) in the 1H−1H COSY spectrum. 1 H−13C long-range correlation between the proton H-4 (δ 5.32) and the carbonyl carbon at δ 165.0 (COO−″) indicated the linkage of the MeG moiety at the C-4 position. No 1H−13C long-range correlations were observed between the oxymethine protons H-3 and H-5 with the carbonyl carbons at δ 165.2 and 165.6 in the HMBC spectrum. However, the paramagnetic shifts of those proton signals in the 1H NMR spectrum E

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Journal of Agricultural and Food Chemistry

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suggested that the two G moieties should be linked to quinic acid at the C-3 and C-5 positions. In the 1H NMR spectrum of compound 6, signals of two pairs of asymmetric aromatic protons at δ 7.20 (H-2′)/δ 7.01 (H-6′) and δ 7.20 (H-2‴)/δ 6.97 (H-6‴) and two aromatic protons meta coupled at δ 6.8 (H-2″/H6″) (Table 1) confirmed the presence of two MeG and one G, respectively. The methylated position was confirmed by the long-range correlations in the HMBC spectrum between C-3′ and the methylic protons at δ 3.7 and between C-3‴ and the methylic protons at δ 3.78 (Figure 3). The signal at δ 2.15 was assigned to H-6ax (2J6ax,6eq = 12.5 Hz, 3 J6ax,5 = 8 Hz). Proton H-3 (δ 5.54) was easily assigned by its signal multiplicity (ddd) and coupling constant magnitudes (3J3,4 = 3.5 Hz, 3J2ax,3 = 3 Hz, 3J2eq,3 = 3 Hz). In the 1H−1H COSY spectrum, the signal of H-3 was cross-correlated with protons at δ 1.96 and 2.39, which were assigned to the pair of methylene protons at the C-2 position. The presence of a 1 H−13C long-range correlation between H-4 (δ 5.28) and the carbonyl carbon at δ 165.2 and between H-3 (δ 5.54) and the carbonyl carbon at δ 165.3 indicated the linkage of G and MeG at the C-4 and C-3 positions, respectively. MeG, with the carbonyl carbon at δ 164.9, was linked therefore into the C-5 position. This study will proceed to further pharmacological investigation of the isolated compounds.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01588. HPLC-UV-DAD chromatograms, 1D/2D NMR, MS, and UV spectra, and calculated dihedral angles (H−C− C−H) for the quinic acid in all conformers of compound 4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(J.K.B.) Phone: +55 16 3315-4162. Fax: +55 16 3315-4879. Email: [email protected]. Funding

We thank the State of São Paulo Research Foundation (FAPESP) for financial support, Grants 2009/10975-3 and 2011/13630-7. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Dr. Thomas J. Schmidt for the use of MOE. We are also thankful to Prof. Milton Groppo for identifying the plant material and to Dr. Antonio Gilberto for helping with optimized NMR measurements.

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ABBREVIATIONS USED HSCCC, high-speed counter-current chromatography; MOE, molecular operations environment; Kd, Partitioning coefficient; MeG, 3-O-methyl-galloyl; G, galloyl

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REFERENCES

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DOI: 10.1021/acs.jafc.5b01588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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G

DOI: 10.1021/acs.jafc.5b01588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX