Evaluation of the Antiproliferative Activity of the Leaves from Arctium lappa by a Bioassay-Guided Fractionation

Arctium lappa L. (Asteraceae) is used in folk medicine around the World, and shows several kinds of biological activity, particularly in vitro antitumor activity in different cell lines. This study evaluated the antiproliferative activity of the crude extract, semipurified fractions, and isolated compounds from the leaves of A. lappa, through bioassay-guided testing in Caco-2 cells. The crude extract was obtained with a 50% hydroethanolic extract and then partitioned with hexane, ethyl acetate, and n-butanol. The ethyl-acetate fraction (EAF) showed antiproliferative activity. This fraction was subjected to sequential column chromatography over silica gel to afford onopordopicrin (1), mixtures of 1 with dehydromelitensin-8-(4'-hydroxymethacrylate) (2), a mixture of 2 with dehydromelitensin (3), mixture of 1 with melitensin (4), dehydrovomifoliol (5), and loliolide (6). The compounds were identified by spectroscopic methods (NMR, MS) and comparison with literature data. This is the first description of compounds 2–5 from this species. The compounds tested in Caco-2 cells showed the following CC50 (µg/mL) values: 1: 19.7 ± 3.4, 1 with 2: 24.6 ± 1.5, 2 with 3: 27 ± 11.7, 1 with 4: 42 ± 13.1, 6 30 ± 6.2; compound 5 showed no activity.


Introduction
Greater burdock, Arctium lappa L. (Asteraceae), was introduced from Japan to Brazil, where it is widely used in folk medicine [1]. The first report on its antitumor activity was provided by Foldeak and Dombradi [2]. Testing in prostate-cancer cells revealed that the methanol extract of burdock seeds showed activity against the LNCaP cell line. Bioassay-guided cytotoxicity fractionation isolated the compounds lappaol A, C, and F, arctignan E, and arctiin. Lappaols A, C, and F showed activity, with IC 50 values of 8, 16, and 40 g/mL, respectively [3]. The roots contain inulin [4] and flavonoids [5]; and the fruits contain lignan [6]. Sesquiterpene lactones in Asteraceae characteristically show cytotoxic, antitumor, and other activities [7]. Onopordopicrin (a sesquiterpene lactone) isolated from the leaves of A. lappa [8] showed an IC 50 of 15 mol/L by MTT and PTP assays against a cell line of promyelocytic leukemia (HL60) [9]; in another experiment, onopordopicrin inhibited the tumor necrosis factor [10]. This study used a bio-guided assay to evaluate the antiproliferative activity of the crude extract, semipurified fraction, and isolated compounds from leaves of A. lappa, against Caco-2 cells.

Antiproliferative Activity
In the search for new anti-cancer drugs, numerous plant derivatives and phytochemicals have been evaluated for the prevention of this disease [11]. In the present study, active compounds were isolated by means of bioassay-guided methods in Caco-2 tumor cells. The CE showed CC 50 (µg/mL) of 347.5, NHF > 500, NBF > 500, and EAF 7.24; of the 22 subfractions obtained from EAF#3#2, only three were active against Caco-2 cells (CC 50 µg/mL): Subfractions 12 (23.8), 13 (25.6), and 14 (25.0) ( Table 1). The compound onopordopicrin (1) shows promise against some tumor-cell lines, including those tested in this experiment. This compound, obtained from subfractions 13 and 14, showed CC 50 of 19.7 ± 3.4 µg/mL against Caco-2 cells. Onopordopicrin was previously reported as cytotoxic in vitro against KB cell lines (human nasopharyngeal squamous cell carcinoma) [12]. This sesquiterpene lactone was the major component of the fractions, and was isolated in all the most active fractions and in considerable quantity, especially in fraction 13 (95.6 mg). This suggests that the compound responsible for the antiproliferative activity of the extract on the Caco-2 cell line is onopordopicrin (1).
Savina et al. [13] determined that the onopordopicrin content in the ethyl-acetate fraction from the juice of leaves of A. lappa is in the range of 0.035-0.005%, with a total sesquiterpene content of approximately 0.01%. In this study, the isolated compound comprised 0.02% (118.4 mg) of CE (575 g). Therefore, compound 1 is appropriate for use in studies to validate the analytical methodology, to determine its mechanism of action, and to evaluate its toxicity, and can also be used as a biological marker. Further, detailed evaluation is needed; Rocha [14] noted reservations regarding the oral administration of sesquiterpene lactones for therapeutic purposes, because of their toxicity. Subfraction 12, with a mixture of onopordopicrin (1) and dehydromelitensin-8-(4'-hydroxymethacrylate) (2), showed a CC 50 of 24.6 ± 1.5 µg/mL. This activity is probably related to the presence of onopordopicrin (1), as the samples containing onopordopicrin showed similar results to the EAF.

General
NMR spectra were recorded in CDCl 3 and/or CD 3 OD/CDCl 3 at ambient temperature, with TMS as the internal standard, in a Varian Mercury Plus 300 BB, 300 MHz. The MS spectra were obtained by HR-ESI-TOF of MicroTof (Bruker-Daltonics, Bremen, Germany) at the Organic Chemistry Institute of Münster University, Germany.

Antiproliferative Activity Assay
The antiproliferative activity was assessed by sulforhodamine colorimetric assay according to Skehan et al. [21]. A suspension of 5 × 10 5 cells/mL of Caco-2 cells in DMEM medium supplemented with 10% FBS and 50 µg/mL of gentamicin was added to each well in a 96-well microplate (TPP ® ). The plates were incubated in a 5% CO 2 -air mixture at 37 °C to obtain confluent growth of the cells. After 24 h, the medium was removed, and one of several concentrations of the crude extracts, fractions, or isolated compounds (1,5,10,25,50,100,200, and 500 µg/mL) was added to each well containing the cells, and the plates were incubated for 48 h. The nonadherent cells were removed by washing with DMEM medium, and the adhered cells were fixed with 50 µL/well of 10% trichloroacetic acid (Synth ® ) at 4 °C for 1 h; after that, they were washed with water, and 50 µL/well of sulforhodamine B (0.4% w/v) was added; the microplate was then maintained at 4 °C for 30 min. Next, the sulforhodamine B was removed, and the microplate was washed four times with 1% acetic acid; then, 150 µL/well of 10 mmol/L unbuffered Tris-base solution (Sigma Chemical Co., St Louis, MO, USA) was added. Next, the absorbance of each individual well was read in a 96-well plate reader (BIOTEK Power Wave XS) at 530 nm. Each experiment was performed in triplicate on three different occasions. The antiproliferative activity was determined according to the following formula: % cell destruction = 1 − (ODtc/ODcc), where: ODtc = optical density of the treated cells and ODcc = optical density of the control cells.

Statistical Analysis
The statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). The results were expressed as mean ± standard deviation and were analyzed using one-way