Galloylquinic acid derivatives from Copaifera langsdorffii leaves display gastroprotective activity

Accepted Manuscript Galloylquinic acid derivatives from Copaifera langsdorffii leaves display gastroprotective activity Erick V.S. Motta, Marivane Lemos, Juliana C. Costa, Vilmar Cláudio Banderó-Filho, Astrid Sasse, Helen Sheridan, Jairo K. Bastos PII:

S0009-2797(16)30649-4

DOI:

10.1016/j.cbi.2016.11.028

Reference:

CBI 7871

To appear in:

Chemico-Biological Interactions

Received Date: 6 July 2016 Revised Date:

11 November 2016

Accepted Date: 24 November 2016

Please cite this article as: E.V.S. Motta, M. Lemos, J.C. Costa, V.C. Banderó-Filho, A. Sasse, H. Sheridan, J.K. Bastos, Galloylquinic acid derivatives from Copaifera langsdorffii leaves display gastroprotective activity, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.11.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Galloylquinic acid derivatives from Copaifera langsdorffii leaves

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display gastroprotective activity

Sasseb, Helen Sheridanb and Jairo K. Bastosa*

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 14040-903,

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Ribeirão Preto, SP, Brazil. b

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Erick V. S. Mottaa, Marivane Lemosa, Juliana C. Costaa, Vilmar Cláudio Banderó-Filhob, Astrid

School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Sciences Institute,

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Trinity College Dublin, University of Dublin, Dublin 2, Ireland.

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ABSTRACT: Nine new methylated galloylquinic acids were isolated from an aqueous fraction of Copaifera langsdorffii (Fabaceae-Caesalpinioideae) leaf hydroalcoholic extract (3–8, 11, 12, and 14), along

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with three known methylated galloylquinic acids (1, 2, and 15) and four galloylquinic acids (9, 10, 13, and 16). These compounds were characterized by nuclear magnetic resonance

spectroscopy and mass spectrometry. They were further tested in a gastroprotection assay

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(Ethanol-HCl induced ulcer model in mice), in which all of them significantly reduced the total lesion area, and increased the cure ratio in comparison with pantoprazole. Also, the tested

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compounds displayed cytotoxicity against gastric adenocarcinoma cells.

Abbreviations:

HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography coupled to

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mass spectrometry; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond correlation.

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Keywords:

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Galloylquinic acid, phenol, copaiba, gastroprotection, adenocarcinoma, ulcer.

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1. Introduction Copaifera langsdorffii Desf. (family Fabaceae, subfamily Caesalpinioideae), popularly known as “copaiba” or “pau d’oleo”, is a large tree widely distributed in Brazil, from the

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Amazon rainforest to savannah vegetation [1]. The hydroalcoholic extract prepared from its leaves has been pharmacologically and chemically studied, displaying positive effect on

urolithiasis induced in rats [2], prophylactic potential to treat kidney stones recurrence in rats [3],

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and remarkable gastroprotective activity in mice [4]. The major secondary metabolites found in C. langsdorffii leaves that could be associated with such a gastroprotective action are

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galloylquinic acid derivatives [5], and two flavonoid heterosides (quercitrin and afzelin) [5,6]. These phenolic compounds are usually obtained in polar fractions, after liquid-liquid partition, which has hindered their isolation and characterization by conventional chromatographic techniques. Some methylated galloylquinic acid derivatives were recently isolated: 3-O-(3-O-

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methyl galloyl) quinic acid (1), 3,4-di-O-(3-O-methyl galloyl) quinic acid (2), and 3,5-di-O(galloyl)-4-O-(3-O-methyl galloyl) quinic acid (15) (Figure 1) [5]. This was possible due to the use of specific chromatographic techniques, such as high speed countercurrent chromatography,

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Sephadex LH-20 column chromatography, and semi-preparative HPLC. After isolating and identifying these compounds, it was possible to predict the presence of many other derivatives

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with very similar polarities in the hydroalcoholic extract, which seem to be responsible for the chemical complexity and pharmacological properties observed for this species leaves. Considering that galloylquinic acid derivatives are the main group of secondary

metabolites present in C. langsdorffii leaves, and that they can be related to the biological properties reported for this species [2–4,7–9], we have isolated and identified other galloylquinic acid derivatives from C. langsdorffii leaves to investigate their pharmacological potential. In this

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study, the isolated galloylquinic acid derivatives displayed promising gastroprotective activity in comparison with pantoprazole in an Ethanol-HCl induced ulcer model in mice, as well as

2. Materials and methods 2.1 Phytochemical studies 2.1.1 General Experimental Procedures

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cytotoxic activity against gastric adenocarcinoma cells (AGS).

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Column chromatography was performed on a Sephadex LH-20 (GE Healthcare Bio-Sciences AB). Analytical HPLC analyses were carried out using a a Synergi Polar-RP analytical column

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(150 × 4.6 mm, 4 µm, Phenomenex) and a Waters 1525 binary solvent delivery system equipped with a Waters 2998 photodiode array detector. Preparative HPLC analyses were carried out using a Synergi Polar-RP column (250 × 10 cm, 4 µm, Phenomenex) and a Shimadzu CBM-20A binary system coupled to a SPD-20A UV-visible detector, two LC-6AD pumps, and a FCR-10A

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auto collector. In both cases, the mobile phase was made up of formic acid-water (0.1:99.9, solvent A), and methanol (solvent B) in gradient conditions as follows: 15-50% of B (45 min), 50-100% of B (45-48 min), 100% B (48-52 min), 100-15% B (52-55 min), 15% de B (55-60

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min); flow rate of 1 mL/min (analytical system) and 4.7 mL/min (preparative system). Chromatograms were recorded at 280 nm. Analytical Thin-Layer Chromatography (TLC) was

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conducted on silica gel 60F254 (Merck) and eluted with ethyl acetate–acetic acid–formic acid– water (100:11:11:27). Spots were visualized by UV light at 254 and 366 nm, and by spraying 5% H2SO4 followed by heating. NMR spectra were acquired on a Bruker Avance DRX-500 spectrometer operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR in CD3OD, or (CD3)2SO; multiplicity determinations (DEPT) and 2D NMR spectra (HMQC and HMBC) were obtained using standard Bruker pulse programs. Residual signals from the solvents were used as

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an internal reference. The chemical shift values (δ) are given in parts per million (ppm), and the coupling constants (J) are in Hz. Mass spectrometry analyses were carried out using a Bruker Daltonics time-of-flight mass spectrometer with electrospray ionization source (ESI) in negative

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mode over a mass range of 50-1500 m/z. The equipment parameters were used as following: nebulizer gas nitrogen (0.4 bar), dry gas nitrogen (4 L/min), 220 ºC, capillary voltage of 4500 V, end plate offset -500 V, samples were dissolved in methanol-water 1:1 and injected into the

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electrospray source with a direct infusion pump operating at a flow rate of 5 µL/min. The

specific rotation [α]D was performed using polarimeter (Jasco P-2000, serial No. A104161232,

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Japan) at 25 °C and at 589 nm. All samples were dissolved in methanol (HPLC grade). Three readings were recorded. Melting points were performed using FisatomTM device (Mod. 431, Brazil, Serie 1237344). 2.1.2 Plant Material

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Copaifera langsdorffii leaves were collected in Ribeirão Preto, São Paulo, Brazil (21º10’S, 47º50’W). The plant material was identified by Professor Milton Groppo Júnior, and a voucher specimen (SPFR 10120) was deposited in the herbarium of the University of São Paulo, Campus

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Ribeirão Preto.

2.1.3 Extraction and Isolation

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Leaves (2.0 kg) were dried at 40 ºC in an oven with air circulation and ground using a knife mill. The resultant powder was submitted to maceration three times sequentially using ethanol-water 7:3 for 72 h, followed by filtration using filter paper. The extracts obtained were concentrated under vacuum, and lyophilized to furnish the total crude extract (754.9 g). Part of the crude extract (100.1 g) was dissolved in methanol-water 6:4, and partitioned with the organic solvents dichloromethane, ethyl acetate, and n-butanol in sequence, furnishing after concentration and

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lyophilisation, the following yields: 5.2 g, 28.7 g, and 16.1 g, respectively. The remaining aqueous fraction was lyophilized, yielding 44.8 g, from which 6.06 g were loaded on a 300 g Sephadex LH 20 glass column (70 cm × 5 cm i.d.) and eluted with an increasing amount of

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methanol in water (1:9 to 9:1). Two hundred and fifty fractions of 20 mL each were collected and combined on the basis of their TLC profiles into 13 fractions. The richest galloylquinic acids fractions were further purified by preparative HPLC (Table 1).

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Data for 3-O-(galloyl)-4-O-(3-O-methyl galloyl) quinic acid (3). (1S,3R,4S,5R)-1,5-Hydroxy-3 ((3,4,5-trihydroxybenzoyl)oxy)-4((3-methoxy-4,5-dihydroxybenzoyl)oxy)cyclohexane

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carboxylic acid rel: light brown amorphous powder; mp 184-190°C; [α]D25 -53.9 (c 0.23; CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 226.0, 274.6; ESI-qTOF-MS m/z 509.0929 [M – H]–.

Data for 3,5-di-O-(3-O-methyl galloyl) quinic acid (4). (1S,3R,4S,5R)-1,4-Hydroxy-3,5-bis

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((3-methoxy-4,5-dihydroxybenzoyl)oxy)-cyclohexane carboxylic acid rel: light brown amorphous powder; mp 161-163°C; [α]D25 -55.1 (c 1.01; CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 223.7, 276.9; ESI-qTOF-MS m/z 523.1481 [M – H]–.

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Data for 3-O-(galloyl)-5-O-(3-O-methyl galloyl) quinic acid (5). (1S,3R,4S,5R)-1,4-Hydroxy3((3,4,5-trihydroxybenzoyl)oxy)-5((3-methoxy-4,5-dihydroxybenzoyl)oxy)cyclohexane

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carboxylic acid rel: light brown amorphous powder; mp 205-210 °C; [α]D25 -48.7 (c 1.68; CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 224.9, 275.8; ESI-qTOF-MS m/z 509.1165 [M – H]–. Data for 3-O-(3-O-methyl galloyl)-5-O-(galloyl) quinic acid (6). (1S,3R,4S,5R)-1,4-Hydroxy3((3-methoxy-4,5-dihydroxybenzoyl)oxy)-5((3,4,5-dihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: light brown amorphous powder; mp 195-205 °C; [α]D25 -25.3 (c 1.08;

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CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 222.5, 275.8; ESI-qTOF-MS m/z 509.0929 [M – H]–. Data for 4,5-di-O-(3-O-methyl galloyl) quinic acid (7). (1S,3R,4S,5R)-1,3-Hydroxy-4,5-bis((3-

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methoxy-4,5-diihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: light brown amorphous powder; mp 165-170 °C; [α]D25 -85.8 (c 0.58; CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 224.9, 279.3; ESI-qTOF-MS m/z 523.1088 [M – H]–.

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Data for 3-O-(3-O-methyl galloyl)-4-O-(galloyl) quinic acid (8). (1S,3R,4S,5R)-1,5-Hydroxy3((3-methoxy-4,5-dihydroxybenzoyl)oxy)-4((3,4,5-trihydroxybenzoyl)oxy)cyclohexane

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carboxylic acid rel: light brown amorphous powder; mp 170-175 °C; [α]D25 -79.7 (c 0.19; CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 224.9, 274.6; ESI-qTOF-MS m/z 509.0925 [M – H]–.

Data for 4-O-(3-O-methyl galloyl)-5-O-(galloyl) quinic acid (11). (1S,3R,4S,5R)-1,3-Hydroxy-

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4((3-methoxy-4,5-dihydroxybenzoyl)oxy)-5((3,4,5-trihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: light brown amorphous powder; mp 170-172 °C; [α]D25 -113.2 (c 0.15; CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 226.0, 276.9;

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ESI-qTOF-MS m/z 509.0930 [M – H]–.

Data for 4-O-(galloyl)-5-O-(3-O-methyl galloyl) quinic acid (12). (1S,3R,4S,5R)-1,5-Hydroxy-

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3((3-methoxy-4,5-dihydroxybenzoyl)oxy)-4((3,4,5-trihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: light brown amorphous powder; mp 168-169 °C; [α]D25 -60.7 (c 0.64; CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 224.9, 276.9; ESI-qTOF-MS m/z 509.0925 [M – H]–. Data for 3,4,5-tri-O-(3-O-methyl galloyl) quinic acid (14). (1S,3R,4S,5R)-1-Hydroxy3,4,5tri((3-methoxy-4,5-dihydroxybenzoyl)oxy)cyclohexane carboxylic acid rel: light brown

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amorphous powder; mp 180-185°C; [α]D25 -116.6 (c 0.77; CH3OH); UV (online spectrum from HPLC in MeOH-H2O with 0.1% FA) λ nm 224.9, 278.1; ESI-qTOF-MS m/z 689.1346 [M – H]–. For 1H and 13C NMR spectroscopic data of all compounds see Tables 2 and 3, and Supporting

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information. 2.2 Biological Assays 2.2.1 Drugs, Reagents and Solvents

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Cremophor, ethanol, chloridric acid and pantoprazole were purchased from Sigma–Aldrich (St. Louis, MD, USA). Gallic acid and quinic acid were purchased from TCI (Portland, OR 97203

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USA). All the other reagents and solvents used were of analytical grade. 2.2.2 Animals

Balb-C male mice (18–23 g) from the animal facility of the central biotery of the University of São Paulo, Campus Ribeirão Preto, were maintained under standard laboratory conditions (25 ±

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2 ºC at 40–60% relative humidity in a 12 h light-dark cycle), with free access to water and a balanced ration Nuvilab CR-1 from Nuvital Nutrientes S/A (Brazil). 12 h prior to the experiments they were transferred to the experimental laboratory and given only water ad

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libitum. In all the experiments, the animals were kept in cages with wide-mesh, raised flooring, to prevent coprophagy. The experiments were approved by the Research Ethics Committee on

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Animal Use of the University of São Paulo, Ribeirão Preto (Protocol # 12.1.1018.53.5) in accordance with the Federal Government Legislation on animal care (Brazil, Law No. 11,794, 8 October 2008).

2.2.3 Ethanol-HCl induced ulcer model The experiment was performed according to the method described by Mizui and Douteuchi [10], with modifications. After 12 h of fasting, mice were randomly divided into groups of six animals

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each, and the treatments (controls, gallic acid, quinic acid and galloylquinic acids) were administered orally by gavage (for dose details, see Table S1). One hour after treatment, all mice received a 60% ethanol–0.3 M HCl solution, in a dose of 10 mL/kg, to induce gastric ulcer. One

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hour later, the animals were sacrificed by cervical dislocation, and the stomachs were removed and opened along the greater curvature. The stomachs were gently rinsed with saline to remove the gastric contents and blood clots, for subsequent scanning. The images obtained were

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analyzed using specific ImageJ software to measure each lesion point. Both, the total area of lesion and the percentage ratio of lesion area in relation to total stomach area were determined.

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In addition, the inhibition, expressed as percent, was calculated using the following equation: Inhibition (%) = [(percentage average of total area of lesion (%mm2) in treated group / percentage average of total area of lesion (%mm2) in control group) − 1] × 100 2.2.4 Cell culture conditions

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AGS gastric adenocarcinoma cells (ATCC CRL-1739) were cultured (at 37 ºC, 5% CO2, atmospheric O2) in DMEN + Ham’s F12 medium (Sigma-Aldrich, Darmstadt Germany) supplemented with 10% fetal bovine serum (Invitrogen, USA), 2 mM L-glutamine (Invitrogen,

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USA), 100 µg/mL penicillin, 100 µg/mL streptomycin, and 100 µg/mL amphotericin B (Invitrogen, USA). The cell suspension containing 5 × 104 cells/mL was aliquoted into 96 well

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plates (200 µL/well). After 24 h, cells were treated with the tested samples at different concentrations (0.1, 0.3, 1, 3, 10, 30 or 100 µM/well) and positive control compounds, paclitaxel and camptothecin (0.1, 0.5, 1, 5, 10, 50 or 100 nM/well), as well as with control of the solvent (DMSO 2%) and negative control (medium). After treatment, the plates were incubated at 37 ºC, 5% CO2, and atmospheric O2 for 24 h, 72 h and 120 h. The cytotoxic activity was determined by the acid phosphatase test [11]. The plates were washed with 100 µL of PBS, and subsequently

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incubated for 1h at 37 ºC, 5% CO2, and atmospheric O2 with a solution of 4-nitrophenyl phosphate disodium hexahydrate, at a concentration of 2.7 mg/mL to 0.1 M sodium acetate, 0.1% Triton X-100, pH 5.5. After this incubation, it was added 50 µL of 1 M NaOH to stop the

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reaction. The absorbance was determined in a microplate reader at 405 nm using microplate reader (EZ Reader 2000, Biochrom Ltd., Cambridge, UK). Cell viability was determined

according to the following equation: Cell viability (%) = (absorbance average of treatment –

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absorbance average of blank) / absorbance average of control.

After the calculation of the cell viability, the 50% cytotoxic concentration was determined by

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sigmoidal regression data using the average of three wells with the test with three replicates. 2.2.5 Assessment of cell death by propidium iodide (PI) staining and flow cytometry analysis

The cell suspension containing 25 × 104 cells/mL was aliquoted into 24 well plates (600

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µL/well). After 24 h, cells were treated with C. langsdorffii crude extract at different concentrations (10 or 30 µg/well) and positive control compounds, paclitaxel and camptothecin (10 or 50 nM/well), as well as with control of the solvent (DMSO 2%) and negative control

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(medium). After treatment, the plates were incubated at 37 ºC, 5% CO2 for 24 h, 72 h and 120 h. The cells were separated using 2 mL EDTA and trypsin, followed by centrifugation at 2000 g for

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5 min. Cells were resuspended in 1 mL ice-cold 70% ethanol and incubated for at least 1 h at -20 ºC to be fixed, and then were washed twice with 2 mL of PBS and centrifuged. Next, cells were resuspended with 1 mL phosphate-citrate wash buffer (200 mM Na2HPO4, 100 mM citric acid) followed by centrifugation at 2000 g for 1 min. PI dye solution (1 mL, Sigma) was added, followed by treatment with 50 µL of 100 µg/mL RNAase (Fermentas, Germany) to ensure that only DNA was stained. Then, it was incubated in the dark at room temperature for 30 min.

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Finally, dead cells were analyzed using flow cytometry (Accuri C6, BD Biosciences, USA) [12,13]. 2.2.6 Statistical Analysis

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Statistical analysis was performed using the software GraphPad Prism® 5.0 (GraphPad Software, San Diego, CA, USA). When comparing two groups, data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey-Kramer’s pair wise test. Data are expressed as mean ±

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standard deviation. Significance was accepted at p ≤ 0.05. The dose-response curves of the

compounds were fitted by means of the computer program Origin® 9.1 for Windows (Origin

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Lab, Northampton, MA, USA), and IC50 values were calculated. 3. Results and discussion

3.1 Isolation and identification of galloylquinic acid derivatives

The phytochemical study of the hydroalcoholic extract of C. langsdorffii leaves yielded 16

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secondary metabolites (Figure 1). They were identified as galloylquinic acids by comparison of experimental spectroscopic (1H NMR and 13C NMR) and spectrometric (LC-MS) data with literature values [5,14–19]. Multiplicity determinations (DEPT) and 2D NMR spectra (HSQC

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and HMBC) were also used for chemical structural elucidation. Among the obtained compounds, which were numbered according to their order of elution on a Sephadex LH-20 (Table 1), three

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disubstituted (9, 10 and 13) and one trisubstituted (16) galloylquinic acids have been reported by other research groups from other plant sources.16–18,20–22 Three methylated galloylquinic acids (1, 2, and 15) have been previously isolated from C. langsdorffii [5], whereas eight disubstituted (3– 8, 11, and 12), and one trisubstituted (14) methylated galloylquinic acids are, to the best of our knowledge, first reported. Their 1H and 13C NMR data are presented in Tables 2 and 3, respectively. The gradient elution mode with methanol-water used during the chromatographic

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process on a Sephadex LH-20 allowed the separation of some galloylquinic acid derivatives with similar polarity and retention times, but distinct molecular weights, into different fractions that were further purified by preparative HPLC, such as the pairs 3 and 13, 11 and 16, 2 and 12, 4 and

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15 (Figure 2).

A detailed structural elucidation is provided for the new methylated galloylquinic acids (3–8, 11, 12, and 14). Compounds 8 (C22H22O14) and 3 (C22H22O14) were obtained as light brown

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amorphous powders, with molecular formulae supported by ESI-qTOF-MS through [M – H]– ion peak at m/z 509.0925 and 509.0929, respectively. The 1D and 2D NMR spectra of these

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compounds were very similar to that of compound 9 (3,4-di-O-galloylquinic acid, Figures S9a, S9b, and S9c, Supporting information), showing the presence of a quinic acid core substituted at C-3 and C-4 positions by galloyl subunits. Their 13C NMR spectra exhibited five aliphatic signals due to two methylene carbons (C-2 and C-6), and three oxymethine carbons (C-3, C-4

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and C-5), confirmed by DEPT, as well as a quaternary carbon (C-1), and a carboxy signal (C-7). These spectroscopic data agreed with those of quinic acid [17]. Two galloyl subunits were indicated by the carbon signals with chemical shifts between 100-170 ppm (13C NMR), as well

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as by the hydrogen signals with chemical shifts between 6.8-7.4 ppm (1H NMR). The position of the galloyl subunits on the quinic acid was determined from 1H NMR and 2D NMR analyses.

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Chemical shifts, multiplicities and couplings of H-3 equatorial and H-4 axial were in agreement with an esterification of the hydroxy groups linked to C-3 and C-4, respectively, while H-5 axial was linked to C-5 in lower field. Besides, HMBC spectrum showed key correlations of H-3 quinic acid to C-7a galloyl subunit, and H-4 to C-7b, confirming the attachment of the galloyl subunits to C-3 and C-4 positions. Furthermore, their spectra showed signals of methyl groups bonded to oxygen, which were not reported in compound 9, and because of that, the couplings of

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some galloyl protons were observed as doublets in 1H NMR. For compound 8, signals due to hydrogen H-8a (δ 3.77, 3H, s) and carbon C-8a (δ 56.48) in 1H and 13C NMR, respectively, were related to a methyl group bonded to oxygen. The HMBC spectrum demonstrated correlation of

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H-8a to C-3a (δ 148.99), indicating the replacement of the hydrogen by a methyl group. Due to the presence of a methyl group, its galloyl protons were observed as doublets in the 1H NMR, H2a (δ 7.16, d, 1.8 Hz) and H-6a (δ 7.19, d, 1.8 Hz). The key HMBC correlations of H-3 to C-7a,

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H-2a to C-7a, H-2a to C-3a, and H-8a to C-3a were visualized in the HMBC spectrum,

confirming the attachment of the methylated galloyl subunit in C-3 position of quinic acid

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(Figure 3). Thus, compound 8 was identified as 3-O-(3-O-methyl galloyl)-4-O-(galloyl) quinic acid (Figures S8a, S8b, and S8c, Supporting information). For compound 3, the key HMBC correlations of H-3 to C-7b, H-2b to C-7b, H-2b to C-3b, and H-8b to C-3b indicated a methylated galloyl subunit linked to quinic acid at C-4, and it was named as 3-O-(galloyl)-4-O-

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(3-O-methyl galloyl) quinic acid (Figures S3a, S3b, and S3c, Supporting information). Compound 2, named as 3,4-di-O-(3-O-methyl galloyl) quinic acid was also isolated and identified (Figures S2a, S2b, and S2c, Supporting information) [5].

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The other compounds were also isolated as light brown amorphous powders and had their molecular formulae supported by ESI-qTOF-MS through [M – H]– ion peaks, as following: 4

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(C23H24O14, m/z 523.1481), 5 (C22H22O14, m/z 509.1165), 6 (C22H22O14, m/z 509.0929), 7 (C23H24O14, m/z 523.1088), 11 (C22H22O14, m/z 509.0930), 12 (C22H22O14, m/z 509.0925), 14 (C31H30O18, m/z 689.1346), and 15 (C29H26O18, m/z 661.1059). The 1D and 2D NMR spectra of compounds 4, 5 and 6 were very similar to that of compound 10 (3,5-di-O-galloylquinic acid, Figures S10a, S10b, and S10c, Supporting information), showing the presence of a quinic acid core substituted at C-3 and C-5 positions by galloyl subunits.

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Besides, their spectra showed signals of methyl groups bonded to oxygen. Compound 4 was named as 3,5-di-O-(3-O-methyl galloyl) quinic acid (Figures S4a, S4b, and S4c, Supporting information), compound 5 as 3-O-(galloyl)-5-O-(3-O-methyl galloyl) quinic acid (Figures S5a,

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S5b, and S5c, Supporting information), and compound 6 as 3-O-(3-O-methyl galloyl)-5-O(galloyl) quinic acid (Figures S6a, S6b, and S6c, Supporting information).

The 1D and 2D NMR spectra of compounds 7, 11 and 12 were very similar to that of compound

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13 (4,5-di-O-galloylquinic acid, Figures S13a, S13b, and S13c, Supporting information),

showing the presence of a quinic acid core substituted at C-4 and C-5 positions by galloyl

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subunits. Their spectra showed signals of methyl groups bonded to oxygen. Compounds 7, 11, and 12 were respectively named as 4,5-di-O-(3-O-methyl galloyl) quinic acid (Figures S7a, S7b, and S7c, Supporting information), 4-O-(3-O-methyl galloyl)-5-O-(galloyl) quinic acid (Figures S11a, S11b, and S11c, Supporting information), and 4-O-(galloyl)-5-O-(3-O-methyl galloyl)

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quinic acid (Figures S12a, S12b, and S12c, Supporting information). The 1D and 2D spectra of compounds 14 and 15 were very similar to that of compound 16 (3,4,5-tri-O-galloylquinic acid, Figures S16a, S16b, and S16c, Supporting information), showing

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the presence of a quinic acid unit substituted at C-3, C-4 and C-5 positions by galloyl subunits. Their spectra showed signals due to methyl groups bonded to oxygen. Compound 14 possesses

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three methyl groups, one in each galloyl subunit, and it was named as 3,4,5-tri-O-(3-O-methyl galloyl) quinic acid (Figures S14a, S14b, and S14c, Supporting information). Compound 15 bears only one methyl group and was named as 3,5-di-O-(galloyl)-4-O-(3-O-methyl galloyl) quinic acid (Figures S15a, S15b, and S15c, Supporting information). Since the quinic acid has its stereochemistry pre-established from the shikimate pathway and through the coupling constants obtained in the 1H NMR spectra, it was possible to assign the

14

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position of the oxymethine hydrogens H-3 and H-5. Taking into consideration that H-3 is in equatorial position, it is expected two 3Jax-eq (2-6 Hz) and one 3Jeq-eq (2-5 Hz) due to its interaction with H-2 axial, H-4 axial, and H-2 equatorial, respectively. On the other hand, H-5 is

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in axial position, and that is why it exhibits two 3Jax-ax (10-14 Hz) and one 3Jax-eq due to its

interaction with H-6 axial, H-4 axial, and H-6 equatorial. These differences make possible to distinguish between H-3 and H-5 and provide the stereochemistry of the quinic acid for all

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galloylquinic acids (Table 2).

The presence of methyl groups in galloylquinic acids was first reported recently [5]. To exclude

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the possibility of having produced artefact compounds during the isolation procedures, fresh plant material was extracted with aqueous ethanol at room temperature and analyzed by HPLC in comparison with the isolated compounds, confirming that the isolated methylated compounds are natural (Figure 2).

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Non-methylated galloylquinic acids have been detected in other plant leaves [18,21–23], and barks as well [14,16]. Their main sources are Caesalpinia spinosa, which belongs to the same family and subfamily of C. langsdorffii, and Camellia sinensis (Theaceae), whose leaves are

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used for preparing green and black teas [20]. In addition to phytochemical studies, important biological activities have been reported in literature for non-methylated galloylquinic acids,

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which include selective inhibition of HIV reverse transcriptase, and human DNA polymerases, as well as anti-HIV activity. 4,5-di-O-galoylquinic acid and 1,3,4,5-tetra-O-galloylquinic acid have displayed moderate selective cytotoxicity against melanoma cells [24], whereas the 1,3-di-Ocaffeoylquinic acid exhibited anti-ulcer activity [25]. Furthermore, as polyphenols in general, they display antioxidant activity [26,27]. 3.2 Galloylquinic acids display gastroprotective activity

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Lemos et al., 2015 [4] reported that the gastroprotection of C. langsdorffii leaves extract could be related to both the antioxidant components of the extract and the stimulation of mucus production in the gastric mucosa in mice. It is well known that polyphenols in general, including

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flavonoids and galloylquinic acids display antioxidant activity [26]. Therefore, taking into

account the positive association between phenolic compounds and gastroprotection, as well as the promising results found for C. langsdorffii leaf extract, afzelin and quercitrin in a previous

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study using ethanol-HCl induced ulcer model in mice [4], it was undertaken the gastroprotective activity. For that, n-butanol and aqueous fractions obtained from the hydroalcoholic extract of C.

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langsdorffii, which are rich in galloylquinic acids, along with 16 isolated galloylquinic acids were assayed. Groups treated with 30, 100, and 300 mg/kg of n-butanol fraction showed significant reduction in total area (p<0.01) and percentage of lesions (p<0.05), in comparison with control group. The cure ratios for the three groups were 40.91±4.85, 51.57±4.75, and

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64.73±4.89%, respectively. The aqueous fraction significantly reduced the total area (p<0.01) and percentage of lesion (p<0.05) by 32.29±4.55, 33.83±4.07 and 35.54±6.96%, respectively, but it was less effective than n-butanol fraction (Figure 4, Table S1). The isolated galloylquinic

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acids, as well as the commercial standards of gallic acid and quinic acid, which are products of the galloylquinic acid hydrolysis, were evaluated at doses of 30 mg/kg in the ethanol/HCl-

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induced ulcer model. The galloylquinic acids tested displayed gastroprotective activity in comparison with pantoprazole, the positive control, which corroborates the folk uses of this plant (Figure 5, Table S1).

In this gastroprotection assay, ethanol is responsible for decreasing the defence

mechanisms present in the stomach [28], reducing the blood flow and contributing to the development of necrosis and solubilisation of mucus, which increases the flow of sodium and

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potassium ions into the lumen, as well as pepsin secretion, H+ and histamine. Ethanol also promotes oxidative stress, increases the activity of xanthine oxidase and levels of malondialdehyde and reduction of total glutathione levels in gastric cells [29–31]. Studies on

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Byrsonima intermedia leaf extracts led to the suggestion that the main secondary metabolites found in its leaves, such as phenolic acids, flavonoids and galloylquinic acids, might be

responsible for the gastroprotective activity in rodents [30]. Similar gastroprotective activity was

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observed for hydroalcoholic extracts of Camellia sinensis leaves, which contains a huge variety of phenolic compounds, including galloylquinic acids and flavonoids [31].

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The mechanism of action of galloylquinic acid compounds in gastroprotection may be related mainly to the increase of mucus production and decrease of the oxidative stress in the stomach, as reported by Lemos et al., 2015 [4] for the hydroethanolic extract of C. langsdorffii leaves, in which the major class of compounds are galloylquinic acid derivatives.

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The crude extract of C. langsdorffii leaves, its isolated galloylquinic acids, as well as gallic and quinic acids, were further tested in a cytotoxicity assay against gastric adenocarcinoma cells (AGS). All treatments exhibited cytotoxicity in a dose and time-dependent effect in

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comparison with the positive controls paclitaxel and camptothecin (Table 4). Besides, AGS cells treated with crude extract of C. langsdorffi leaves and stained with propidium iodide (PI) showed

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approximately 30% of dead cells after 24 h and 72 h, respectively. Increased cell death occurred only at 120 h, by displaying 63% and 70% of cell death for concentrations of 10 and 30 µg/mL, respectively (Table 5). Cell death resulting from necrosis or apoptosis processes shows typical characteristics, including fragmentation and loss of genetic material, DNA. The use of PI, a fluorochrome able to bind to damaged DNA results in a rapid and accurate information in the percentage of viable cells [32].

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Gastric cancer is a serious global public health, requiring the development of new drugs associated with gastroprotective agents [33]. Natural products, such as phenolic compounds, exhibit a broad spectrum of chemopreventive properties against different cancers in both cell

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culture and animal models. They negatively influence various cell signaling pathways that allow tumor growth, such as proliferation and angiogenesis [34]. Moreover, there are some evidences that flavonoids, condensed tannins, and galloylquinic acids lead to a decrease incidence of

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cancer, especially by reducing reactive oxygen species. [24,35–38].

One important issue is the bioavailability of the natural compounds as reported by

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Kramer et al., 2007 [39], regarding the measurement and modeling of the bioavailability in in vitro assays and the prediction for the in vivo assay systems. In this regard, it is important to point out that the tested compounds might act mainly in the stomach mucous in the gastroprotection, with no further need of systemic absorption and bioavailability. However,

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considering that the tested galloylquinic compounds are esters and may undergo partial hydrolysis in in vivo systems furnishing gallic and quinic acids, these compounds were also tested. Gallic acid has been evaluated in many biological assays and Ahad et al., 2015 [40]

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reported that gallic acid ameliorates renal functions by inhibiting the activation of p38 MAPK in experimentally induced type 2 diabetic rats, as well as Chen et al., 2016 [41], which reported that

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gallic acid abolishes the EGFR/Src/Akt/Erk-mediated expression of matrix metalloproteinase-9 in MCF-7 breast cancer cells. Therefore, the data reported here may contribute to the development of new phytotherapic medicines to treat stomach diseases. However, further studies are necessary to fully investigate the gastroprotection mechanism, as well as to unravel both the molecular mechanism of action of these compounds and their possible selectivity for cancer cells.

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4. Conclusions This paper reports new natural methylated galloylquinic acid derivatives from C. langsdorfii, which are majorly responsible for the gastroprotective activity previously found for the crude

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extract of this plant, corroborating its folk use. Additionally, the isolated galloylquinic acids displayed moderate cytotoxicity against gastric adenocarcinoma cells. Therefore, the reported scientific data shows not only the chemical complexity of C. langsdorffii leaves, but also its

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association with gastroprotection, one of the promising pharmacological properties related to this species.

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Supporting information

The Supporting Information is available online free of charge. Corresponding Author

* Tel: +55 16 3602-4230. Fax: +55 16 3633-2363. Email: [email protected]

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Funding

The authors are grateful to São Paulo Research Foundation (FAPESP), grant # 2011/13630-7, scholarships # 2012/09727-8 and # 2012/01336-0, as well as to Conselho Nacional de

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Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PDSE), grant# BEX 7277/14-8 and Trinity College Dublin

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(TCD award n. 203327.13221), for their financial support. Conflict of interest

The authors declare that there are no conflicts of interest among themselves or with any public or private company in relation to this manuscript. Acknowledgment Authors are thankful to Milton Groppo Junior for identifying the plant material.

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TABLES

Table 1. Fractions and isolated compounds from aqueous fraction of Copaifera langsdorffii leaf

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hydroalcoholic extract.

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Table 2. 1H NMR data of the galloylquinic acids isolated from Copaifera langsdorffii leaves.

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Table 3. 13C NMR data of the galloylquinic acids isolated from Copaifera langsdorffii leaves.

Table 4. The IC50 values of C. langsdorffii crude extract and its galloyl quinic acids in acidphosphatase assay (the data are expressed as mean ± SEM, in triplicate), at 24 h, 72 h and 120 h.

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CLE, Copaifera langsdorffii extract.

Table 5. Percentage of living and dead cells by flow cytometry AGS cell line. CLE, Copaifera

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langsdorffii extract.

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206.3

99–107

210.1

108–116

217.7

117–137 163–185 186–200

99.0 181.6 177.0

Weight (mg) 21.2 78.1 74.2 27.9 32.0 28.6 45.2 28.1 34.1 36.2 38.6 40.4 45.9 18.9 53.9 39.9

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89–98

Compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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Weight (mg) 94.1 147.1 160.8

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Fractions 38–41 60–76 77–88

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Table 1.

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Table 2. δH (integral, multiplicity, J in Hz) Compound 1 Compound 2

Compound 3

Compound 4

2

2.18 (1H, m)a 2.22 (1H, m)a 5.47 (1Heq, m) 3.67 (1Hax, dd, 3.0, 8.8) 4.24 (1Hax, ddd, 3.6, 8.8, 9.4) 1.96 (1Hax, dd, 9.4, 12.4) 2.16 (1Heq, dd, 3.6, 12.4) 7.29, 7.26 (1H, d, 1.7) 3.88 (3H, s) -

2.20 (1Hax, dd, 6.8, 15.0) 2.42 (1Heq, dd, 3.1, 15.0) 5.73 (1Heq, ddd, 3.1, 3.3, 6.8) 5.15 (1Hax, dd, 3.3, 9.3) 4.52 (1Hax, ddd, 4.2, 9.3, 9.7) 2.12 (1Hax, dd, 9.7, 11.1) 2.29 (1Heq, dd, 4.2, 11.1) 7.14 (2H, s) 6.97, 7.15 (1H, d, 1.5) 3.52 (3H, s)

2.35 (1Hax, dd, 6.8, 13.8) 2.28 (1Heq, dd, 3.0, 13.8) 5.50 (1Heq, ddd, 2.9, 3.0, 6.8) 4.08 (1Hax, dd, 3.0, 7.0) 5.59 (1Hax, ddd, 3.2, 7.0, 7.8) 2.23 (1Hax, dd, 7.8, 13.6) 2.40 (1Heq, dd, 3.2, 13.6) 7.21, 7.20 (1H, d, 1.5) 3.91 (3H, s) 7.31, 7.28 (1H, d, 1.6) 3.90 (3H, s)

3 4 5 6 2a, 6a 8a 2b, 6b 8b 2 3 4 5 6 2a, 6a 8a 2b, 6b 8b

Compound 6

Compound 7

2.35 (1Hax, dd, 3.2, 13.4) 2.25 (1Heq, m) 5.48 (1Heq, m) 4.08 (1Hax, dd, 3.1, 7.2) 5.55 (1Hax, ddd, 3.7, 7.2, 7.8) 2.20 (1Hax, dd, 7.8, 13.2) 2.38 (1Heq, dd, 3.7, 13.2) 7.22, 7.20 (1H, d, 1.9) 3.92 (3H, s) 7.16 (2H, s) -

2.15 (1Hax, dd, 5.4, 14.0) 2.36 (1Heq, dd, 2.7, 14.0) 4.47 (1Heq, ddd, 2.7, 3.1, 5.4) 5.24 (1Hax, dd, 3.1, 8.6) 5.75 (1Hax, ddd, 6.1, 8.2, 8.6) 2.302.40 (1Hax, dd, 8.2, 14.5) 2.302.40 (1Heq, dd, 6.1, 14.5) 7.17, 7.20 (1H, d, 1.9) 3.81 (6H, s) 7.08, 7.11 (1H, d, 1.8) -

Compound 8

2.19 (1Hax, dd, 5.3, 14.4) 2.44 (1Heq, dd, 3.5, 14.4) 5.75 (1Heq, ddd, 3.2, 3.5, 5.3) 5.15 (1Hax, dd, 3.2, 8.2) 4.41 (1Hax, ddd, 5.3, 7.5, 8.2) 2.182.24 (2H, m) 7.16, 7.19 (1H, d, 1.8) 3.77 (3H, s) 7.03 (2H, s) -

Compound 9

Compound 10

Compound 11

Compound 12

2.18 (1Hax, dd, 7.0, 14.6) 2.44 (1Heq, dd, 3.3, 14.6) 5.72 (1Heq, ddd, 2.8, 3.3, 7.0) 5.12 (1Hax, dd, 2.8, 8.6) 4.44 (1Hax, ddd, 4.1, 8.6, 8.6) 2.16 (1Hax, dd, 8.6, 11.7) 2.24 (1Heq, dd, 4.1, 11.7) 7.08 (2H, s) 7.02 (2H, s) -

2.33 (1Hax, dd, 7.1, 14.4) 2.25 (1Heq, dd, 3.3, 14.4) 5.47 (1Heq, ddd, 3.1, 3.3, 7.1) 4.06 (1Hax, dd, 3.1, 7.2) 5.54 (1Hax, ddd, 3.6, 7.2, 7.6) 2.20 (1Hax, dd, 7.6, 13.6) 2.38 (1Heq, dd, 3.6, 13.6) 7.08 (2H, s) 7.17 (2H, s) -

2.14 (1Hax, dd, 5.5, 14.2) 2.36 (1Heq, dd, 3.1, 14.2) 4.49 (1Heq, ddd, 2.9, 3.1, 5.3) 5.18 (1Hax, dd, 2.9, 8.9) 5.78 (1Hax, m) 2.29-2.33 (2H, m)

2.13 (1Hax, dd, 5.7, 14.0) 2.302.40 (1Heq, dd, 2.8, 14.0) 4.44 (1Heq, ddd, 2.8, 3.0, 5.7) 5.27 (1Hax, dd, 3.0, 7.8) 5.66 (1Hax, m) 2.30-2.40 (2H, m)

7.16, 7.18 (1H, d, 1.6) 3.80 (3H, s) 6.99 (2H, s) -

7.09 (2H, s) 7.07, 7.10 (1H, d, 1.8) 3.81 (3H, s)

Compound 13

Compound 14

Compound 15

Compound 16

EP

1.71 (1Hax, dd, 5.5, 13.5) 2.28 (1Hax, dd, 5.0, 14.5) 2.28 (1Hax, dd, 4.7, 14.7) 1.81 (1Hax , dd, 1.1, 13.3) 2.13 (1Heq, dd, 3.0, 13.5) 2.56 (1Heq, dd, 3.4, 14.5) 2.53 (1Heq, dd, 3.7, 14.7) 2.34 (1Heq, dd, 0.9, 13.3) 3 4.15 (1Heq, ddd, 2.8, 3.0, 5.5) 5.85 (1Heq, ddd, 3.2, 3.4, 5.0) 5.81 (1Heq, ddd, 3.4, 3.7, 4.7) 5.53 (1Heq, ddd, 0.9, 1.1, 1.7) 4 5.03 (1Hax, dd, 2.8, 10.1) 5.52 (1Hax, dd, 3.2, 8.4) 5.48 (1Hax, dd, 3.4, 8.6) 5.27 (1Hax, dd, 1.7, 9.5) 5 5.61 (1Hax, ddd, 5.2, 10.1, 5.88 (1Hax, m) 5.89 (1Hax, ddd, 6.4, 8.6, 5.74 (1Hax, ddd, 9.5, 9.7, 10.4) 14.0) 12.8) 6 2.03 (1Hax, dd, 10.4, 12.8) 2.42 (2H, m) 2.40 (2H, m) 2.14 (1Hax, dd, 11.2, 12.8) 1.91 (1Heq, dd, 5.2, 12.8) 1.97 (1Heq, dd, 9.7, 11.2) 2a, 6a 6.87 (2H, s) 7.26, 7.27 (1H, br s) 7.16 (2H, s) 6.88 (2H, s) 8a 3.83 (3H, s) 2b, 6b 6.91 (2H, s) 6.95, 7.10 (1H, br s) 6.95, 7.09 (1H, d, 1.7) 6.80 (2H, s) 8b 3.58 (3H, s) 3.57 (3H, s) 2c, 6c 7.10, 7.12 (1H, br s) 7.01 (2H, s) 7.04 (2H, s) 8c 3.84 (3H, s) Compounds 1-12,14 and 15 were recorded in CD3OD at 500 MHz. whereas compounds 13 and 16 were recorded in (CD3)2SO at 500 MHz. a Interchangeable

AC C

2

Compound 5 2.32 (1Hax, dd, 7.4, 14.2) 2.24 (1Heq, dd, 3.2, 14.2) 5.52 (1Heq, ddd, 3.1, 3.2, 7.4) 4.06 (1Hax, dd, 3.1, 7.4) 5.55 (1Hax, ddd, 3.7, 7.1, 7.4) 2.20 (1Hax, dd, 7.1, 14.0) 2.39 (1Heq, dd, 3.7, 14.0) 7.08 (2H, s) 7.33, 7.29 (1H, d, 1.8) 3.91 (3H, s)

SC

2

M AN U

2a, 6a 8a 2b, 6b 8b

TE D

3 4 5 6

2.18 (1Hax, dd, 7.4, 14.8) 2.43 (1Heq, dd, 3.7, 14.8) 5.77 (1Heq, ddd, 3.0, 3.7, 7.4) 5.16 (1Hax, dd, 3.0, 8.9) 4.48 (1Hax, ddd, 4.2, 8.9, 9.5) 2.14 (1Hax, dd, 9.5, 12.3) 2.28 (1Heq, dd, 4.2, 12.3) 7.22, 7.24 (1H, d, 1.8) 3.83 (3H, s) 6.83, 6.99 (1H, d, 1.9) 3.56 (3H, s)

RI PT

Position

26

ACCEPTED MANUSCRIPT

Table 3. δC 1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 1a 2a 3a 4a 5a 6a 7a 8a 1b 2b 3b 4b 5b 6b 7b 8b

75.48 36.82 73.28 75.18 68.25 41.81 178.52 122.15 106.53 148.94 140.38 146.09 112.22 168.10 56.62 -

75.27 37.16 70.41 76.74 66.08 42.01 178.24 121.62 106.42 149.08 140.61 146.27 112.17 167.47 56.56 121.38 106.14 148.85 140.55 146.15 112.09 167.59 56.22

75.29 37.09 70.43 76.97 65.90 42.19 178.14 121.63 110.44 146.02 139.83 146.02 110.44 167.72 121.31 106.05 148.77 140.45 146.45 112.01 167.61 56.13

74.65 37.34 72.51 70.36 72.67 35.84 177.26 121.39 106.33 149.11 140.67 146.18 112.02 167.38 56.72 121.83 106.49 149.03 140.55 146.15 112.20 167.87 56.68

74.84 37.87 72.35 70.71 73.01 36.07 177.81 121.51 110.26 146.43 139.98 146.43 110.26 167.64 121.87 106.54 149.03 140.55 146.15 112.22 167.95 56.68

74.64 37.28 72.67 70.20 72.55 35.83 177.38 121.95 106.34 149.11 140.68 146.18 112.02 167.37 56.77 121.39 110.39 146.38 139.75 146.38 110.39 168.04 -

76.20 38.54 69.32 76.06 69.49 39.32 176.86 121.16 106.37 149.01 140.75 146.21 112.11 167.73 56.59 121.11 106.17 149.01 140.71 146.21 119.93 167.39 56.59

75.03 36.97 70.24 76.01 66.29 41.65 178.03 121.62 106.36 148.99 140.52 146.16 112.12 167.44 56.48 121.37 110.31 146.39 139.87 146.39 110.31 167.81 -

9

10

11

12

13

14

15

16

M AN U

SC

RI PT

Position

AC C

EP

TE D

1 75.12 74.62 76.04 75.82 75.02 74.91 74.89 73.30 2 36.98 37.41 38.44 38.53 36.38 36.89 36.84 36.47 3 70.28 70.45 69.17 69.05 68.88 70.48 70.20 69.91 4 76.45 70.43 76.42 75.28 75.91 73.35 73.56 72.95 5 66.07 72.66 69.02 69.75 68.42 69.72 69.41 68.43 6 41.97 35.84 39.32 38.91 38.04 38.83 38.93 39.69 7 178.06 177.41 176.91 176.84 176.21 177.33 177.43 176.84 1a 121.67 121.45 121.17 121.05 119.36 121.47 121.47 118.95 2a 110.45 110.25 106.36 110.33 108.65 106.47 110.44 108.64 3a 146.36 146.42 148.98 146.41 145.59 149.09 146.46 145.44 4a 139.80 139.98 140.70 139.99 138.72 140.70 139.93 138.53 5a 146.36 146.42 146.15 146.41 145.59 146.30 146.46 145.44 6a 110.45 110.25 112.05 110.33 108.65 112.15 110.44 108.64 7a 167.69 167.54 167.78 167.76 165.49 167.46 167.70 165.17 8a 56.58 56.56 1b 121.35 121.83 121.13 121.12 119.30 120.79 120.79 118.77 2b 110.31 110.39 110.17 106.07 108.79 106.24 106.10 108.69 3b 146.31 146.39 146.42 149.01 145.64 148.94 148.87 145.47 4b 139.85 139.75 139.34 140.69 138.72 140.77 140.67 138.65 5b 146.31 146.39 146.42 146.14 145.64 146.20 146.08 145.47 6b 110.31 110.39 110.17 111.88 108.79 112.00 111.92 108.69 7b 167.86 168.09 167.43 167.38 165.34 167.06 167.13 165.26 8b 56.59 56.27 56.24 1c 121.01 120.99 119.99 2c 106.13 110.19 109.17 3c 149.04 146.39 145.57 4c 140.77 139.98 138.77 5c 146.20 146.39 145.57 6c 111.94 110.19 109.17 7c 167.28 167.43 165.59 8c 56.62 Compounds 1-12,14 and 15 were recorded in CD3OD at 125 MHz. whereas compounds 13 and 16 were recorded in (CD3)2SO at 125 MHz.

27

ACCEPTED MANUSCRIPT

IC50 72 h µM 57.49±1.69

R2 0.994

IC50 120 h µM 47.40±3.72

R2 0.966

Gallic acid

4.68±0.90

0.986

2.95±0.44

0.963

2.33±0.67

0.989

Quinic acid

3.85±0.99

0.987

2.29±0.56

0.950

1.77±0.35

0.916

Compound 1

15.79±1.88

0.952

9.87±2.39

0.989

8.27±2.17

0.989

Compound 2

14.96±3.92

0.979

12.13±2.61

0.902

8.85±2.50

0.982

Compound 3

15.14±2.36

0.919

10.77±2.06

0.917

8.84±2.36

0.988

Compound 4

17.09±2.81

0.989

11.57±1.98

0.934

9.23±2.41

0.989

Compound 5

16.46±2.51

0.917

10.24±2.28

0.904

8.69±2.35

0.988

Compound 6

14.42±3.04

0.985

8.23±1.91

0.912

7.91±2.94

0.980

Compound 7

14.21±3.32

0.986

10.79±2.07

0.929

9.10±2.31

0.900

Compound 8

15.96±2.98

0.987

9.44±2.51

0.907

8.31±1.96

0.986

Compound 9

16.90±3.20

0.986

9.45±2.63

0.986

7.70±2.44

0.968

Compound 10

17.05±2.61

0.917

10.79±2.25

0.984

6.89±2.43

0.989

Compound 11

16.17±3.58

0.983

11.53±2.14

0.930

9.311±2.57

0.987

Compound 12

12.76±4.42

0.979

9.99±2.84

0.985

8.10±2.49

0.986

Compound 13

17.33±2.68

0.908

9.09±3.13

0.988

6.30±1.98

0.980

Compound 14

12.87±4.07

0.980

11.70±2.05

0.924

7.94±2.03

0.907

Compound 15

15.94±3.97

0.981

10.29±2.17

0.910

8.09±2.21

0.988

Compound 16

13.43±6.30

0.965

12.41±2.24

0.903

7.67±2.33

0.986

53.76±15.51

0.952

15.06±4.01

0.911

TE D 6.71±0.83

0.978

1.54±0.42

0.992

AC C

EP

Paclitaxel 67.23±8.40 0.975 (nM) Camptothecin 23.71±2.09 0.989 (nM) a CLE, Copaifera langsdorffii extract

SC

R2 0.994

M AN U

CLE

IC50 24 h µM 100.31±6.31

Treatment

RI PT

Table 4.

28

ACCEPTED MANUSCRIPT

Table 5. 24 h

72 h

120 h

Treatment % Dead cells

% Living cells

% Dead cells

% Living cells

% Dead cells

4.72±2.74

90.52±4.83

15.48±1.26

83.47±5.45

11.70±2.31

Control

82.83±8.28

17.95±6.74

67.18±7.37

29.22±5.95

68.34±4.86

32.62±1.98

DMSO 2%

68.17±16.95

27.96±15.79

73.44±1.98

22.35±1.62

53.07±2.86

39.38±1.83

a

CLE 10 µg/mL

70.59±1.97

25.44±2.39

61.42±16.91

34.73±15.47

31.70±1.84

63.28±1.28

a

CLE 30 µg/mL

62.99±13.86

32.44±14.16

65.04±5.75

31.42±4.99

16.53±4.88

80.15±5.75

Paclitaxicel 10 nM

54.14±29.39

40.97±27.06

25.28±8.70

71.36±8.65

8.30±4.64

85.71±3.70

RI PT

% Living cells 94.13±1.08

Blank

49.25±27.69

47.14±26.75

13.95±3.56

83.60±4.15

6.15±1.69

85.69±4.74

Camptotecina 10 nM

58.57±20.10

38.64±19.03

42.51±2.84

53.47±2.25

9.79±1.95

85.56±1.25

Camptotecina 50 nM

38.51±3.59

58.38±3.82

33.86±5.93

63.25±6.14

6.73±1.48

89.20±3.02

EP

TE D

M AN U

CLE, Copaifera langsdorffii extract

AC C

a

SC

Paclitaxicel 50 nM

29

ACCEPTED MANUSCRIPT

FIGURES Figure 1. Galloylquinic acids isolated from Copaifera langsdorffii leaves.

RI PT

Figure 2. HPLC-UV chromatographic profiles for hydroalcoholic extract and isolated

compounds (1-16) from Copaifera langsdorffii leaves. Stationary phase: Synergi Polar-RP

column (150 x 4.60 mm, 4 µm). Mobile phase: (A) formic acid-water 0.1:99.9 and (B) methanol.

SC

Gradient elution: 15-50% B up to 45 min, 50-100% B up to 48 min, 100% B up to 52 min, 100-

M AN U

15% B up to 55 min, 15% de B up to 60 min. Wavelength: 280 nm. Flow rate: 1 mL/min.

Figure 3. HMBC correlations necessary for the distinction of the galloyl and methylated galloyl subunits in 3-O-(3-O-methyl galloyl)-4-O-(galloyl) quinic acid (8).

TE D

Figure 4. Effects of oral administration of pantoprazole (30 mg/kg), n-butanol and aqueous fractions from Copaifera langsdorffii leaves (both at 30, 100, and 300 mg/kg) on ethanol/HClinduced ulcer model in mice. The results are mean ± S.E.M. for six rats. Statistical comparison

EP

was performed using ANOVA followed by the Tukey-Kramer test. *p<0.05 and **p<0.01 in

AC C

comparison with the control group.

Figure 5. Effects of oral administration of pantoprazole, gallic acid, quinic acid and the 16 galloylquinic acids isolated from Copaifera langsdorffii leaves (all at 30 mg/kg) on ethanol-HCl induced ulcer model in mice. The results are mean ± S.E.M. for six mice. Statistical comparison was performed using ANOVA followed by the Tukey-Kramer’s test. *p<0.05 and **p<0.01 in comparison with the control group.

30

ACCEPTED MANUSCRIPT

Figure 1. OH

O

4

6 5

7

OH

2

HO

1

3

OH

O

O 7a

2a

5a

HO

RI PT

1a 6a a6

3a 4a

8a

O

OH

SC

1

O

OR2

OH 4

6

a7 7b

5

7 1

3

OH

O

OR2

a6 6b

2b c2

O

4b 5b

OH

O 7a

OH 2a

5a

HO

3a

OR1

4

1

O

OH

OR3

EP

5

7

2

3

OH

O

O 6 2 1

OH

O

OR1

HO

OH

OH 2a

5a

3a

4. R1 = R2 = CH3 5. R1 = H, R2 = CH3 6. R1 = CH3, R2 = H 10. R1 = R2 = H

4b

O

6a a6

2a

OR2 3b

a6 6b

1a

OH

1b

3

1a

4a

bb 2 2b

a7 7b

O

7a

5a

HO

7

HO

4 5

7a

6a a6

OH

5b

O

O

5c 6c O O

O

OH

AC C

1

4c

1c 7c

4

6

HO

O

O

O

OH

3c 2c c2

OH

O

OH

7. R1 = R2 = CH3 11. R1 = CH3, R2 = H 12. R1 = H, R2 = CH3 13. R1 = R2 = H

5b

6b O

4a 5a

OH

4b

1b 7b

OR1 3a

a6 6a

OH

3b

1a

3

OR2

2b c2

bb 2 2a

a7 7a

O

2

TE D

2. R1 = R2 = CH3 3. R1 = H, R2 = CH3 8. R1 = CH3, R2 = H 9. R1 = R2 = H

OH O

5

OH

OH

5b

6b O O

O

O 7

4a

HO

4b

1b

7b

6

1a 6a a6

OH

3b

3b

1b

O

2

HO

bb 2 2b

M AN U

O

3a 4a

OR1

OH

14. R1 = R2 = R3 = CH3 15. R1 = R3 = H, R2 = CH3 16. R1 = R2 = R3 = H

31

ACCEPTED MANUSCRIPT

Figure 2.

Hydroalcoholic extract

AU

0.105 10 1

3 13

0.035

0.070

7

5 9

0.035

8

10 1

3 13

7

5 9

8

226.0

3.00

AU

AU

1.50

1 4 .7 7 4

2 40.00

260.00

280 .0 0

30 0.00 nm

3 20.00

340.00

360 .00

38 0.0 0

4 00.00

42.00

48.00

54.00

3.60

2

220.00

260.00

280.00

300.00 nm

320.00

340.00

360.00

380.00

400.00

2.70 274.6

AU

AU

2.50

AU

2.00

220.00

240.00

42.00

260.00

280.00

300.00 nm

48.00

320.00

340.00

223.7

276.9

AU

2 4.7 37

220.00

30.00

36.00

42.00

260.00

280.00

300.00 nm

48.00

320.00

3.00

AU

2.50

14 .62 6 Extracted

224.9

275.8

AU

2 8 .8 2 3 24.00

30.00

400.00

60.00

0.00 22 0.00

2 40.00

42.00

260.00

280 .0 0

30 0.00 nm

48.00

3 20.00

340.00

360 .00

38 0.0 0

0.00 0.00 3.60

6.00

54.00

4 00.00

60.00

6

3.00

AU

2.50

1.40

AU

220.00

30.00

36.00

0.00 0.00 3.60

222.5

3.00

12.00

18.00

6.00

12.00

18.00

2 5 .5 0 7

1 4 .6 9 8

400.00

224.9

260.00

280 .0 0

30 0.00 nm

3 20.00

340.00

360 .00

38 0.0 0

276.9

24.00

0.50 0.00 220.00

30.00

36.00

240.00

42.00

260.00

280.00

300.00 nm

48.00

320.00

340.00

360.00

380.00

54.00

400.00

60.00

13 10.128 Extracted 3.00

275.8

226.0

2.50 2.00 1.50 1.00 0.50 0.00 220.00

24.00

30.00

36.00

240.00

42.00

260.00

280.00

300.00 nm

48.00

320.00

340.00

360.00

380.00

54.00

400.00

60.00

14 3.00

37.178 Extracted

224.9

278.1

2.50 2.00 1.50 1.00

0.90

2 40.00

60.00

1.00

0.50 0.00

4 00.00

220.00

240.00

260.00

280.00

3.60

7

AU

2.50

300.00 nm

320.00

340.00

360.00

380.00

400.00

1.80

320.00

340.00

360.00

380.00

400.00

15

2.70

25.861 Extracted

224.9

3.00

2.00 AU

AU

1.50

3.00

320.00

340.00

360.00

380.00

400.00

0.00 0.00 3.60

6.00

12.00

18.00

2.70

11.133 Extracted

24.00

30.00

1.00 0.50 0.00 220.00

36.00

30.00 Minutes

36.00

54.00

60.00

17.007 Ex tract ed

275.8

AU

2.00 1.50

220.00

240.00

260.00

48.00

280.00

300.00 nm

320.00

340.00

54.00

360.00

380.00

400.00

60.00

0.00 0.00

6.00

12.00

18.00

24.00

2 7 .2 2 2

0.90

0.00

2 4 .7 1 1

1 5 .6 8 7

1.00

0.50

42.00

300.00 nm

2.50

1.80

AU

24.00

280.00

16

1.00

2 6.8 06

AC C 1 0.21 5

18.00

260.00

48.00

224.9

274.6

2.00

12.00

240.00

42.00

1.50

6.00

1.50

3.00

224.9

AU

300.00 nm

8

2.50

1.80

280.00

3 2 .8 0 1

260.00

1 7 .0 0 4

2.70

240.00

AU

1 1.1 31

3.60

0.90

0.00

220.00

2 5 .2 5 8

EP

0.00

1 7 .3 4 7

2 3 .0 1 3

1.00 0.50

275.8

2.50

1.80

2.00

0.90

22.896 Extracted

226.0

279.3

AU

3.00

2 2 .8 9 2

25 .85 6

2.70

0.00 0.00

380.00

0.00

3.60

0.90

360.00

54.00

1.50

0.00

22 0.00

300.00 nm

2.00

1.50

0.50

280.00

17.888 Extracted

2.50

1.00

0.00

260.00

48.00

1.80

275.8

240.00

42.00

12

2.70

15 .60 5 Extracted

2.00

0.70

340.00

0.00

24.00

AU

2.10

380.00

0.90

0.50

36.00

360.00

TE D

18.00

1 5 .6 0 2

12.00

320.00

276.9

0.50

1.80

1.50 1.00

6.00

400.00

1.50

2.70

2.00

0.90

340.00

54.00

5

1.80

380.00

1.00

AU

2.70

18.00

300.00 nm

2.00

AU

24.00

240.00

280.00

17.325 Extracted

226.0

37.0 86

1 8.36 5 18.00

0.00

1 4 .6 2 4

12.00

12.00

0.90

0.50

260.00

2.50

1.80

1.50 1.00

6.00

6.00

2.70

23.035 Extracted

2.00

0.90

60.00

1 7 .1 3 9

AU

2.50

400.00

0.00 0.00 3.60

240.00

11 3.00

1 1 .20 6

3.00

380.00

54.00

4

1.80

360.00

220.00

1 0.1 2 6

36.00

360.00

0.00

1 7 .8 6 4

0.00

30.00

340.00

0.50

AU

2.70

0.90

0.50

9.5 1 7

27 .8 6 8

24.00

320.00

274.6

1.00

M AN U

18.00

1 1 .5 04

1.00

2 3.3 6 4 12.00

1.80

1.50

2 3.0 30

6.00

400.00

8.484 Extracted

1 7 .3 22

1 0 .5 6 9

3.60

3 10.569 Extr acted

226.0

0.90

380.00

AU

240.00

SC

220.00

1.80

360.00

AU

AU

3 6 .6 8 6

0.00

3.00

340.00

1.50

0.90

0.50

0.00

2.70

320.00

2.50

1.50

0.00 3.60

300.00 nm

2.00

1.00

3 0 .5 1 2

2 7 .5 6 3

0.90

3.00

1.80

2.00

280.00

AU

AU

1.80

260.00

224.9

275.8

2.50

240.00

10

2.70

18.691 Extr acted

224.9

0.00 0.00 2.80

0.00

60.00

20 .3 12

36.00

2 2.9 1 9

30.00

1 4.9 8 5

24.00

8 .4 8 3

18.00

3.00

0.00 0.00 3.60

0.50

AU

12.00

1 8 .6 8 8

6.00

2.70

0.00 0.00 3.60

1.00

0.00

AU

2 .5 8 4 3 .3 8 7

22 0.00

1 8 .4 2 4

0.90

0.50 0.00

46 .0 70

AU

2.00

1.50 1.00

0.00 0.00 3.60

271.0

226.0

2.50

1.80

2.00

2 9 .2 1 3

AU

5.682 Extracted

274.6

2.50

1.80

9

2.70

3.995 Ex tract ed

3.00

RI PT

3.9 9 3

3.60

1

2.70

0.90

14

4 15

11 16 2 12 6

0.000

0.000 3.60

Hydroalcoholic extract

0.105

5 .6 8 1

0.070

0.140

14

4 15

11 16 2 12 6

AU

0.140

0.50 0.00 220 .00

30.00 Minutes

36.00

42.00

240 .00

260 .00

48.00

28 0.0 0

30 0.0 0 nm

32 0.0 0

34 0.00

54.00

36 0.00

38 0.00

4 00.00

60.00

32

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Figure 3.

33

ACCEPTED MANUSCRIPT

Figure 4.

Percentage of lesion (%)

40

**

*

* **

**

10

**

30 0

10 0

Aqueous fraction (mg/kg)

AC C

EP

TE D

M AN U

1-butanol fraction (mg/kg)

30

30 0

10 0

30

zo le

nt op ra

Pa

C

on t ro l

0

SC

*

20

RI PT

30

34

ACCEPTED MANUSCRIPT

Figure 5.

40

RI PT

3-monosubstituted galloylquinic acid

3,5-disubstituted galloylquinic acids 4,5-disubstituted galloylquinic acids 3,4,5-trisubstituted galloylquinic acids

20

**

16

15

**

14

**

12

11

8

7

6

5

4

3

2

1

**

13

**

**

0

Galloylquinic acids (30 mg/kg)

M AN U

Pa nt op ra zo le G al lic ac id Q ui ni c ac id

**

**

**

on tro l

*

*

10

**

10

*

* **

SC

*

9

*

**

**

EP

TE D

(30 mg/kg)

AC C

C

Percentage of lesion (%)

3,4-disubstituted galloylquinic acids

30

35

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

1- New methylated galloylquinic acids were isolated from Copaifera langsdorffii. 2- Galloylquinic acids displayed gastroprotection in comparison with pantoprazole. 3- Galloylquinic acids displayed cytotoxicity against gastric adenocarcinoma cells.