INTRODUCTION

Heliocarpus terebinthinaceus (Tiliaceae) is commonly known as cuetla and is native from southern Mexico and Central America. According to the local medicinal knowledge, the aerial parts of this plant possess properties against acne and gastrointestinal disorders. Moreover, it is well known the therapeutic use of the flowers of the Tilia (Tiliaceae) genus to control cough and bronchitis (Gruenwald et al., 2000) and for their anti-inflammatory, antinociceptive (Toker et al., 2004a) and anxiolytic activity (Herrera-Ruiz et al., 2008).

Since plants of Tilia (Tiliaceae) genus have shown both antioxidant (Dimitrios, 2006) and antimicrobial (Nohynek et al., 2006) activities, it is possible that such functional properties may be associated with polyphenolic compounds, including flavonoids. It has been reported that the aerial parts of plants belonging to the Tiliaceae family contain secondary metabolites with a flavonoid structure. For example, kaempferol-3, 7-O-α-L-dirhamnoside and quercetin-3, 7-O-α-L-dirhamnoside have been isolated from Tilia argentea (Toker et al., 2004b).

Consequently, the study of natural products of Tiliaceae plants is very relevant to relate the antioxidant activity to the specific flavonoids present in these plants. This study describes the quantification of total polyphenols (TPP) and flavonoids (TF), free radical-scavenging capacity, as well as the isolation of tiliroside from H. terebinthinaceus seeds. This is the first phytochemicals study of H. terebinthinaceus.

Experimental
Materials and equipment: Folin-Ciocalteu reagent, gallic acid, rutin, 2,6-di-t-butyl-4-methylphenol (BHT), 2,2-diphenyl-1-picrylhidracyl (DPPH), 2-aminoethyl diphenylborinate, KBr, HCO₂H, Et₂O, DMSO, DMSO-d₆ and D₂O were purchased from Sigma-Aldrich Inc. (USA) Na₂CO₃, hexane, CHCl₃, EtOAc, acetone and MeOH, were purchased from J.T. Baker (USA) and EtOH from Reasol (Mexico).

Absorbance readings were obtained with a microplate reader ELx808 (BioTek Instruments Inc., USA). Solvent degassing was performed in an 8510 sonicator (Branson Ultrasonics Co., USA). Solvent evaporation was carried out in a LABOROTA 4000-efficient rotary evaporator (Heidolph, Germany), equipped with a MZ2C vacuum pump (Vacuubrand, Germany) and an ECO-SEV-10 cold-water recirculating bath (SEV, Mexico).

Table 1:	TPP, TF and free radical-scavenging capacity in seeds of H. terebinthinaceus extracted by Soxhlet
aGAE: Gallic acid equivalents. bRE: Rutin equivalents. †BHT: Positive control. Each value is the Mean±S.D of at least three replicates, SC50 is the final concentration of extract in the well required to decrease the initial DPPH concentration by 50%. DM: Dry matter. Calibration equation for TPP, y = 0.0029x-0.0278 (R2 = 0.999). Calibration equation for TF, y = 0.0189x-0.0721 (R2 = 0.995)

Chromatographic purification was performed using flash column chromatography with silica gel packing (230-400 mesh, 60 Å) followed by preparative thin layer chromatography (TLC) employing Techware Analtech plates of 20x20 cm and 500 μm of thickness. The tiliroside IR spectrum was obtained using a Spectrum GX instrument (Perkin Elmer, USA) with KBr plates. The high resolution mass spectrum (HRMS) was obtained using an LC/MSD TOF instrument (Agilent Technologies, USA) equipped with an APCI source. TLC analyses were performed with silica gel UVG-54 plates (Merck, USA). TLC spots were visualized at 254 nm. The melting point was measured with a Fisher-Johns apparatus and is uncorrected. NMR data were recorded on a GSX-DELTA ECLIPSE+400 spectrometer (Jeol, Japan), operating at 399.78 and 100.53 MHz for ¹H and ¹³C, respectively. Chemical shifts were referenced to tetramethyl silane (TMS). The tiliroside structure was assigned by analyzing the 1D, 2D (HETCOR and COSY) data using ¹H and ¹³C NMR experiments.

Plant material: Seeds of Heliocarpus terebinthinaceus were collected at the Universidad Technologica de la Mixeca Campus (1751 m of altitude, 17°49'32'' N, -97°48'4'' W) in Huajuapan de León, Oaxaca, Mixteca. The voucher specimen was deposited in the SERBO Herbarium, San Sebastián Tutla, Oaxaca, Mexico (http://serboax.org/ herbario/). The seeds were selected, dried and crushed in a domestic worm mill and stored in amber vials at -20°C previously to sample pretreatment.

Preparation of extracts: Crushed seeds of H. terebinthinaceus (81 g) were successively extracted in a Soxhlet apparatus during 5 h using hexane, Et₂O, CHCl₃, EtOAc, acetone and MeOH (180 mL of each solvent). The corresponding extracts were obtained by evaporation under vacuum in a rotary evaporator and stored at -20°C prior to analysis. Extracts yields are given in Table 1.

Total polyphenols assay: The assay method reported by Santos-Sanchez et al. (2012) was used. The TPP content was expressed as mg of gallic acid equivalents (GAE) per 100 g of dry matter (DM).

Total flavonoids assay: The spectrophotometer assay for the quantitative determination of flavonoid content was adapted to a microplate reader instrument from the work of El Hariri et al. (1991). Briefly, 100 mu: μL of extract or standard were mixed with 50 mu: μL of 2-aminoethyl diphenylborinate (1%in MeOH). After 30 min settling, the sample absorbance was determined at 405 nm. The TF content was expressed as mg of rutin equivalents (RE) per 100 g DM. All measurements were performed in triplicate.

Free radical-scavenging capacity assay: The assay reported by Julian-Loaeza et al. (2011) was adapted to a microplate reader instrument. Briefly, 10 μL of extract were mixed with 100 μL of DPPH• solution (125 mM in DMSO). The reaction mixture was covered and kept in the dark at room temperature for 30 min, followed by absorbance measuring at 545 nm. BHT was used as control. The DPPH• scavenging was calculated using the equation: S (%) = 100 x (A_DPPH - A_s)/A_DPPH, where A_DPPH is the absorbance value of the DPPH^● blank and A_s is the absorbance value of the tested sample or control. Sample SC₅₀ data were obtained by plotting the decrease in DPPH• absorbance, S(%), as a function of extract concentration. The SC₅₀ value indicates the concentration of the extract that causes 50% of radical scavenging. All measurements were performed in triplicate.

Purification: The EtOAc extract (700 mg) was redissolved in acetone and was introduced into a flash column packed with silica gel. A solvent gradient was performed as follows: hexane, the initial eluent, was swapped to CHCl₃ by employing CHCl₃ 20% composition changes until the eluent was CHCl₃ 100%. Then CHCl₃:EtOAc (1:1) was used as eluent and was swapped to EtOAc using composition changes with EtOAc 20%. The tiliroside was obtained in the CHCl₃:EtOAc (3:7) fraction, which was further purified by preparative TLC using acetone:CHCl₃:formic acid (HCO₂H) (3:2:0.1) as eluent. Isolation was achieved by crystallization with a mixture of acetone:hexane at 4°C to obtain 2.7 mg of tiliroside, which accounted for 0.39% of the EtOAc extract.

Structure elucidation of kaempferol-3-O-(6’=-O-E-p-coumaroyl)-β-D-gluco-pyranoside (tiliroside): Light yellow solid, mp 255-258°C (lit. 257-260°C, Bajaj et al., 1986), R_f (AcOEt) 0.27. FT-IR (KBr, _max, cm^-1) 3251 (OH), 1684 (C = O), 1608 (C = C), 1183 (C-O). HRMS⁺ m/z 595.1430 [M + H]⁺ (calcd for C₃₀H₂₇O₁₃, 595.1452), 617.1242 [M + Na]⁺ (calcd for C₃₀H₂₆O₁₃Na, 617.1271). ¹H NMR (DMSO-d₆, δ, ppm, J/Hz): 10.2 (1H, s, OH), 7.99 (2H, d, J = 8.6, H-2’, H-6’), 7.36 (2H, d, J = 8.6, H-2’’’, H-6’’’), 7.34 (1H, d, J = 15.9, H-7’’’), 6.86 (2H, d, J = 8.6, H-3’, H-5’), 6.79 (2H, d, J = 8.6, H-3’’’, H-5’’’), 6.38 (1H, d, J = 1.7, H-8), 6.15 (1H, d, J = 1.7, H-6), 6.11 (1H, d, J = 15.9, H-8’’’), 5.45 (1H, d, J = 7.3, H-1”), 5.20 (1H, br.s, OH), 5.15 (1H, br.s, OH), 4.28 (1H, dd, J = 11.8, 1.4, H-6”A), 4.03 (1H, dd, J = 11.8, 6.5, H-6”B), 3.38 (1H, ddd, J = 9.1, 6.5, 1.4, H-5”), 3.25 (1H, t, J = 8.6, H-3”), 3.21 (1H, dd, J = 8.6, 7.3, H-2”), 3.16 (1H, dd, J = 9.1, 8.6, H-4”). ¹³C NMR (DMSO-d₆, δ, ppm): 177.9 (s, C-4), 166.7 (s, C-9’’’), 164.8 (s, C-7), 161.7 (s, C-5), 160.5 (s, C-4’), 160.3 (s, C-4’’’), 157.0 (s, C-8a), 156.9 (s, C-2), 145.1 (d, C-7’’’), 133.7 (s, C-3), 131.4 (d, C-2’, C-6’), 130.7 (d, C-2’’’, C-6’’’), 125.5 (s, C-1’), 121.4 (s, C-1’’’), 116.3 (d, C-3’, C-5’), 115.6 (d, C-3’’’, C-5’’’), 114.2 (d, C-8’’’), 104.4 (s, C-4a), 101.6 (d, C-1”), 99.3 (d, C-6), 94.2 (d, C-8), 76.8 (d, C-3”), 74.8 (d, C-5”), 74.7 (d, C-2”), 70.6 (d, C-4”), 63.5 (t, C-6”).

Statistical analysis: Data obtained from experiments were expressed as mean±standard deviation.