Application of an Ionic Liquid in the Microwave Assisted Extraction of Cytotoxic Metabolites from Fruits of Schinus terebinthifolius Raddi ( Anacardiaceae )

This work reports the application of an ionic liquid (1-butyl-3-methylimidazolium bromide, BMImBr) in the microwave assisted extraction (MAE) of metabolites from fruits of Schinus terebinthifolius. Dried fruits were individually extracted using BMImBr:H2O 1:1, v/v (experiment 1) and pure H2O (experiment 2) by MAE (10 min at 60 °C). After partition using EtOAc, the yield to experiment 1 was about 23% while to experiment 2 was 0.1%. The EtOAc fraction obtained from experiment 1 was purified by chromatographic methods to afford 3-oxotirucalla-7,24Z-dien27-oic acid, 3a-hydroxytirucalla-7,24Z-dien-27-oic acid, 3a-acetoxytirucalla-7,24Z-dien-27-oic acid, gallic acid, and ethyl gallate, being the first occurrence of the third compound as natural product. Cytotoxic activity was evaluated in vitro against cancer cell lines (A2058, HeLa, SiHa, HCT, SKBR-3, U87, and B16F2Nex2), being 3a-acetoxytirucalla-7,24Z-dien-27-oic acid the more active metabolite with IC50 ranging from 10.9 ± 1.3 to 17.3 ± 1.4 μg mL, lower than that determined to positive control cisplatin.

As reported in previous papers, [4][5][6][7][8][9][10][11][12] the extraction procedures of bioactive compounds involve the use of toxic organic solvents such as MeOH, acetone, CHCl 3 and hexane in large quantities, leading to a significant environmental impact.The growing interest in adopting more environmentally compatible technologies make these processes inadequate.][15][16] Moreover, ionic liquids have modular properties, since there are numerous possible combinations of cations, anions and substituents, which originate salts with different densities, viscosities, polarities and hydrophilicities. 16ecently, the use of ionic liquids in the natural products chemistry has gained importance. 17 Among the various applications, the use of ionic liquids as extracting agents of compounds with different polarities from aqueous extracts such as organic acids, amino acids, phenols and alkaloids emerged as an alternative process of the high efficiency. 17][20][21][22][23][24][25][26][27][28][29] Due to the high viscosity of the ionic liquids, usually they are used dissolved in water, which lowers the cost of the process.Finally, it is noteworthy that ionic liquids are compounds that absorb microwave very efficiently, making them excellent solvents when this technology is required. 18,30Microwave heating is a very efficient process due to the microwaves couple directly with the molecules that are present in the reaction/extraction mixture, leading to a more efficient procedure.The two fundamental mechanisms for transferring energy from microwaves to the substance are dipole rotation and ionic conduction.Due to the ionic character, ionic liquids absorb microwave irradiation extremely well and transfer energy quickly by ionic conduction. 31It is worth to mention that the use of microwave is considered a leading technology and sustainable, as it decreases the extraction time, minimizes energy expenditure, increases process efficiency and hence promotes cost reduction of the final product.
In order to minimize the problems associated to the use of conventional methods in the extraction of bioactive compounds of interesting plant species, this work has two main objectives: report the application of an ionic liquid (1-butyl-3-methylimidazolium bromide, BMImBr) in the microwave-assisted extraction process of metabolites from fruits of S. terebinthifolius and evaluate the cytotoxic potential of obtained EtOAc fraction, as well as purified metabolites, against several tumor cell lines.

General experimental procedures
Microwave assisted extraction (MAE) experiments were performed with a MAS-I microwave oven (2450 MHz, Sineo Microwave Chemistry Technology Company) with a maximum delivered power of 1000 W. The temperature was monitored by an infrared probe inside the microwave oven.Sephadex LH-20 (Sigma-Aldrich) was used for the column chromatographic separation, while silica gel 60 PF 254 (Merck) and silica gel 60 RP-18 F 254 (Merck) were used for analytical and preparative thin-layer chromatography (TLC), respectively.High performance liquid chromatography (HPLC) analysis were performed in a Dionex Ultimate 3000 chromatograph, using a Luna Phenomenex RP-18 column (3 µm, 150 × 5 mm) and an UV-diode array detector (DAD). 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded at 300 and 75 MHz, respectively, in a Bruker Ultrashield 300 Avance III spectrometer using CDCl 3 (Tedia Brazil) or CD 3 OD (Aldrich) as solvents.

Plant material
Fruits of S. terebinthifolius were randomly collected from an individual tree at Mogi-Guaçu region (São Paulo State, Brazil) on February 2010 by Dr. Maria Claudia Marx Young from Instituto de Botânica (São Paulo, Brazil), where a reference specimen (SP272591) was deposited.
Preparation of BMImBr and the extraction using this ionic liquid 1-Butyl-3-methylimidazolium bromide (BMImBr) was prepared as previously described in the literature, with minor modifications. 32Succinctly, the molar ratio was 1:1.2 of 1-methylimidazole (99%, Aldrich) and the 1-bromobutane (99.9%,Aldrich), respectively.Residual 1-bromobutane was removed by distillation under reduced pressure.Before extraction, the fruits of S. terebinthifolius were dried at 40 °C during 48 h.After grinding, the extractions were conducted in triplicate.To each procedure, the plant material (approximately 2.0 g) was extracted by microwave-assisted extraction (MAE, Figure 1) with 20 mL of mixture containing H 2 O and BMImBr 1:1 (v/v) (experiment 1).Additionally, the same procedure was conducted using distillated H 2 O as solvent (experiment 2).To all MAE experiments, the extraction time was 10 min and extraction temperature was 60 °C.
The obtained aqueous extracts were filtered and then diluted to 5 mL with deionized H 2 O. Sequentially, each aqueous solution was extracted using EtOAc (3 × 25 mL).After dried over Na 2 SO 4 and distillation of the solvent under reduced pressure, were obtained 460 ± 12 mg (experiment 1) and 2.0 ± 0.3 mg (experiment 2) of each EtOAc fractions.

In vitro cytotoxic activity
Compounds 1-5 as well as the EtOAc fraction from fruits of S. terebinthifolius (experiment 1) were suspended in DMSO at final concentration of 10 mg mL -1 and then added to complete RPMI medium supplemented with 10% fetal calf serum.Different concentrations of the compounds 1-5 and EtOAc extract, ranging from 100 to 0 µg mL -1 , were incubated with 1 × 10 4 cells in a 96-well plate at 37 °C and 5% CO 2 .After 24 h of incubation, cell viability was assessed using the Cell Proliferation Kit I (MTT) (Sigma), an MTT-based colorimetric assay as previously described. 33,34Readings were made in a plate reader at 570 nm with a reference of 650 nm.All experiments were performed in triplicates using cisplatin (Sigma) and DMSO 1% as positive and negative controls, respectively.

Results and Discussion
][6][7][8][9][10][11][12] Aiming the extraction of cytotoxic derivatives from fruits of this species, in this work was employed an alternative extraction method based in the use of ionic liquid extraction assisted by microwave.Using this approach, extracting plant material with BMImBr aqueous solution at 3 mol L -1 (experiment 1), followed by partition using EtOAc, were obtained 460 ± 12 mg (yield 23%) of EtOAc fraction.The same procedure was conducted using pure distillated H 2 O (experiment 2) to afford 2.0 ± 0.3 mg (yield 0.1%) of EtOAc fraction, at the same MAE conditions.Each experiment was conducted in triplicate.This result showed that the use this ionic liquid affected the extraction of S. terebinthifolius metabolites, which could be explained by the dissolution of BMImBr in H 2 O to form ionic liquid clusters due to its strong interactions, such as ionic interaction and hydrogen bonding.Therefore, it is possible to assume that these clusters could accommodate the metabolites and establish interactions with the ions, optimizing the extraction process.In addition, based in the fact that ionic liquids are microwave absorber, this heating method showed to be more efficient than that using exclusively H 2 O.Moreover, the efficiency of ionic liquids containing bromide anion in dissolving organic compounds have been indicated by the previously reported results. 18,30,35Thus, using reduced amount of BMImBr in H 2 O was observed an efficacy in extraction of metabolites from fruits of S. terebinthifolius.
According to the colorimetric assay of MTT and light microscopy, all isolated compounds killed 100% of cytotoxic cells at the highest tested concentration, resulting in IC 50 values in the range of 10.9 ± 1.3 to 94.0 ± 1.5 µg mL -1 .Comparison of the IC 50 values to related tirucallane derivatives 1-3 demonstrated that these three derivatives displayed different cytotoxic potentials, suggesting an important effect caused by the substituent in C-3.Compound 1, which displayed a carbonyl group, showed higher IC 50 values, ranging from 25.0 ± 1.3 (U87) to 53.0 ± 1.2 µg mL -1 (HCT), in comparison to the other tirucallane derivatives 2 and 3.As could be seen in Table 1, compound 2, which displayed a hydroxyl group at C-3, was more active than 1, except to U87, A2058 and B16F10 Nex 2 cell lines, suggesting that the reduction of carbonyl group at C-3 could cause an important effect in the cytotoxicity.Otherwise, the IC 50 values determined to compound 3 indicated an expressive increase in the cytotoxicity since IC 50 values were lower than 20 µg mL -1 to all tested cell lines, indicating that the presence of acetoxyl group at C-3 play an important role in the cytotoxic effects of tirucallane derivatives.Additionally, it is important to mention that compound 3 displayed higher potential than positive control cisplatin in five cell lines, suggesting that this compound could be used as scaffold to the development of new compounds with promising cytotoxic activity based in natural products.
Compounds 4 and 5 have previously been described as cytotoxic against cell lines HeLa and A2058, 12 although in this study have been tested against other cell lines.Thus, these compounds showed cytotoxic activity to all tested cell lines with IC 50 values ranging from 12.3 ± 0.5 to 94.0 ± 1.5 µg mL -1 .As could be seen in Table 1, compound 4 was the more active derivative, especially to B16F10Nex2 (15.2 ± 1.6 µg mL -1 ), HeLa (13.3 ± 0.8 µg mL -1 ), HCT (12.3 ± 0.5 µg mL -1 ), SKBR-3 (23.9 ± 2.3 µg mL -1 ) and U87 (21.6 ± 0.7 µg mL -1 ) cells.Similarly to the previous results to antiparasitic activity, 10 the obtained data indicated that the presence of free carboxyl group in the gallic acid is crucial to the cytotoxic potential.However, the use of galloyl derivatives as bioactives must be carefully employed since these compounds could be considered as PAINS (Pan Assay Interference Compounds) since they displayed a catechol unit which can interfere in bioassays via different mechanisms. 36espite the isolated compounds 1, 2, 4 and 5 have already been described for the leaves of S. terebinthifolius, [10][11][12] this study demonstrate, at first time, that it is possible to extract these bioactive compounds using a more sustainably method based in the microwave assisted by ionic liquid.Additionally, this work report the cytotoxic effects of compounds 1-5, highlighting the activity observed for compound 3 which displayed lower IC 50 values of those observed to positive control (cisplatin) to five cell lines.

Conclusion
Ionic liquids have attracted considerable attention as solvents in the extraction and separation of bioactive compounds from plants owing to their excellent properties.In this work, the application of microwave-assisted extraction (MAE) using an ionic liquid aqueous solution (1-butyl-3-methylimidazolium bromide, BMImBr) showed to be an efficient procedure to the extraction of cytotoxic metabolites from fruits of S. terebinthifolius.After partition with EtOAc, the extraction yield was approximately 23% in one single step and short time (10 minutes, experiment 1).Comparatively, the use of pure H 2 O as solvent extractor (experiment 2) afforded a yield of 0.1%, showing that the presence of BMImBr dissolved in H 2 O is fundamental for the success of the extraction.The ionic liquid changes the dissipation factor of solution and the transfer of energy from microwaves to sample, which affected extraction efficiency.The proposed technique was green, simple and rapid.It is strongly believed that an ionic liquid aqueous solution as solvents in the MAE of useful substances in natural sources is a new and interesting approach.Additionally, the EtOAc fraction (experiment 1) displayed cytotoxic activity and was subjected to several chromatographic steps to afford five bioactive compounds, including three tirucallane triterpenoids (1-3) and two gallate derivatives (4-5).Among the isolated compounds, 3a-acetoxytirucalla-7,24Z-dien-27-oic acid (3) displayed higher cytotoxic potential against five cell lines, suggesting that this triterpene could be considered as a lead anti-leukemic agent.

Figure 1 .
Figure 1.MAE apparatus used for the extraction of fruits of S. terebinthifolius.

Table 1 .
Cytotoxic activity of crude EtOAc fraction (experiment 1) and compounds 1-5 isolated from fruits of S. terebinthifolius after extraction using MAE/BMImBr as well as positive control cisplatin against different tumor cell lines