Progress in Nutrition 2019; Vol. 21, Supplement 1: 458-470 DOI: 10.23751/pn.v21i1-S.5672 © Mattioli 1885 Original article Bioactivity-guided isolation of compounds with antiproliferative activity from Teucrium chamaedrys L. subsp. sinuatum (Celak.) Rech. f. Ibrahim Halil Gecibesler1, Ibrahim Demirtas2, Serkan Koldas2, Lutfi Behcet3, Fatih Gul2, Muhammed Altun2 1Department of Occupational Health and Safety, Laboratory of Natural Product Research, Faculty of Health Sciences, Bingol Uni- versity, 12000, Bingol, Turkey - Email: [email protected]; 2Department of Chemistry, Faculty of Science, Cankiri Karatekin University, 18200 Cankiri, Turkey; 3Department of Biology, Faculty of Science and Art, Bingol University, 12000 Bingol, Turkey Summary Purpose: To reach active compounds from aerial parts of Teucrium chamaedrys L. subsp. sinuatum (TCS) under the guidance of anticancer assay. Methods: The active compounds of T. chamaedrys were isolated using chromatographic techniques including column and preparative thin layer chromatography. Isolated com- pounds were characterized using 1D and 2D NMR techniques and high-resolution HPLC–TOF/MS. In the present study, we evaluated in vitro antiproliferative activities of isolated compounds from TCS using xCEL- Ligence Real Time Cell Analysis system (RTCA). Findings: Three phenolic compounds, namely luteolin-7-O- glucoside (cinaroside, compound 1), 5,6,3’-trihydroxy-7,4’ dimethoxyflavone (nuchensin, compound 2), and (E)-p-coumaroyl-O-β-D-glucoside (compound 3) were isolated for the first time from Teucrium chamaedrys subsp. sinuatum (TCS). The phenolic compounds have antiproliferative effect against the HeLa (Human uter- ine cancer), C6 (Rat brain tumor) and PC3 (human prostate carcinoma) cells. Conclusions: The results demon- strated that T. chamaedrys subsp. sinuatum and its active components may be suggested as a promising natural antiproliferative agent against HeLa, C6 and PC3 cells. Key words: phytochemical content, antiproliferative activity, HeLa, C6, PC3, Teucrium chamaedrys L. subsp. sinuatum Introduction Some species has been used to support medicinal treat- ments of certain diseases for a long time (2-4). Teucrium Teucrium L. (Lamiaceae) is a cosmopolitan genus chamaedrys was used in the treatment of diseases such that differs from other related genera. It comprises more as abscesses, gout, digestive and respiratory tract infec- than 300 species and almost 50 of these are known in tions (5). Among all disease groups the most serious Europe and distributed mainly in the Mediterranean one is cancer, therefore, there is excessive scientific and basin. It is a perennial herbaceous plant with half-lig- commercial interest in the continuing discovery of new neous and shrub-like low stem being up to 50 cm high. anti-cancer products from natural sources (6). In can- T. chamaedrys subsp. sinuatum has a branched stem with cer chemotherapy, plant-derived drugs became consid- oval serrated leaves and tiny blooms on the branch-tops. erably important products due to the generally higher The plant inhabits rocky limestone areas, dry mountain activities, therefore, many new drugs have been applied meadows and pastures, edges of the sparse oak and pine to treatment/prevention of cancer derived from second- forests up to 2600 m above the sea level in Central Eu- ary metabolites of the plants (7,8). Some drugs used to rope, in the Mediterranean region and western Asia (1). treat cancer have been isolated from natural products Bioactivity-guided isolation of compounds with antiproliferative activity from Teucrium chamaedrys L. subsp. sinuatum (Celak.) Rech. f. 459 (9). In addition, many studies exhibited the individual for a variety of cell-based assay applications, such as polyphenols or crude extracts of plants have potential compound-mediated cytotoxicity (26), cell invasion and anti-cancer effects, either antiproliferation, cancer cell migration (23), quality control of cells (27), and cell pro- cycle arrest or apoptosis (10,11). Many plant-derived liferation and differentiation (28,29). In this study, we new drugs have been applied for the treatment or pre- reported the evaluation of the antiproliferative activities vention of various diseases due to their attractive bio- of crude extracts of TCS, the isolation and characteriza- logical activities (12). The recent study on antiprolifera- tion of flavonoids and flavonoid glycosides from polar tive effect demonstrated that the isolated flavonoids and extracts. To the best of our knowledge, there is no report fatty acids were effective from 100 to 1000 μg/ml (13). on the isolation of flavonoids and flavonoid glycosides The interest towards Teucrium species has increased due and their antiproliferative activities against HeLa, PC3 to an anticancer activity evidenced for plant extracts and and C6 cell lines from TCS. Thus, the present research isolated compounds of these plants (14). Teucrium is reports the first bioassay-guided isolation of these com- one of the richest sources of diterpenes and more than pounds from TCS with antiproliferative activities. 220 diterpenes were determined and they demonstrated insect-repellent and medicinal activities (15-17). Cell-based in vitro screening of potential antican- Material and Methods cer compounds is one of the most essential processes in new drug development. Conventional end point General experimental procedures cytotoxicity assays, such as 3-(4, 5-dimethylthiazol- 2-ly)-2,5-diphenyltetrazolium bromide (MTT) assay NMR spectra were recorded on Bruker Avance (18,19), fluorescence microscopy (20), and the lactate III Spectrometer at 400 MHz. Mass spectra was run dehydrogenase (LDH) assay (21), play an important on Agilent LC-TOF/MS spectrometer. IR spectra role in the screening of anticancer activities of com- were recorded on KBr plates on Jasco 430 FT/IR pounds isolated from natural sources. Although useful Spectrometers. UV spectra were recorded in MeOH and widespread, these conventional methods are la- on JASCO V 530. Silica gel 60 F254 (Merck) was used bor-intensive and require a sequence of manipulation for TLC analyses. Spots on TLC were visualized by steps which may lead to deviation of the results (19). UV irradiation (254 and 366 nm), and by staining Moreover, they can hardly meet the demands for real- with Ce(SO4)2 (1% in 10% aqueous H2SO4) followed time and dynamic monitoring of drug-cell interaction by heating (105 ºC, 5 min). process. Therefore, it is imperative to develop a new method which can overcome these drawbacks. Plant material The xCELLigence RTCA system (ACEABIO, USA) is an impedance-based, label-free biosensor tech- Aerial parts of TCS were collected at vegetative nology which has a demonstrated applicability to pro- state at an altitude of 1550 m in July 2011 in Bingol, vide continuous monitoring of cellular status, including Turkey (N:39°10’3125’ E:40°22’1648’). The plant was cell growth, morphological changes and cell death in identified by one of the co-authors, Prof. Dr. L. Behçet, a real-time manner (22-24). As the cells interact with and a voucher specimen (No: 782) was deposited in the the microelectrodes integrated onto the bottom of the Herbarium of Biology Department, Bingol University, standard E-plate, the instrument measures the corre- Turkey. The plant aerial parts were air-dried, protected sponding changes of electrical impedance automatically from direct sunlight and powdered. The powder was when changes occur in the biological status of the cells. kept in a closed container at 10 ºC. The cellular responses are monitored and expressed as cell index (CI) (25). Thus, a label-free, non-invasive, Extraction and isolation of constituents real-time, automated detection can be performed us- ing the xCELLigence system. Furthermore, a number Part of the air-dried and powdered plant mate- of studies have proved that the new system is applicable rial of TCS (whole plant; 126 g out of 960 g) was first 460 I. H. Gecibesler, I Demirtas, S. Koldas, et al. extracted with MeOH:CH2Cl2 (1:1; 3x1.5 L). After 5 μL. Solvent program was as follow: 0-1 min 10% B; filtration, the solvent was removed by rotary evapora- 1-12 min 40% B; 12-14 min 90% B; 14-17 min 90% tion to give a crude extract (16.98 g). An aliquot of B; 17-18 min 10% B; 18-25 min 10% B. Analyses were the crude extract (12 g) was subjected to open column triplicated (30). chromatography by using silica gel (250 g), eluted initially with hexane followed by hexane:CH2Cl2 Real-Time cell proliferation assays (HD1 and HD5); CH2Cl2:EtOAc (DE1 and DE5); EtOAc:MeOH (ME1 and ME7) along a linear gradi- Antiproliferative tests were performed against ent and finally with 100% MeOH. ME-1U and ME- HeLa, C6 and PC3 cells by using xCELLigence Real 2U and ME-1A and ME-2A were obtained from Time Cell Analyzer (RTCA) in an incubator (5% CO2 the ME-1 and ME-2 fractions after purification by and humidity 95%, 37 °C). Dulbecco’s Modified Eagle preparative thin layer chromatography (P-TLC) with Medium-High Glucose (DMEM) with 10% fetal bo- MeOH:CH2Cl2 (1:4) as the solvent system. ME-1U vine serum and 2% penicillin-streptomycin was used as and ME-2U parts exhibited the highest anticancer cell culture medium during assessments. Firstly, 50 μL activity against HeLa and C6 cells. To increase the of medium was added to each well and the plate was amount of ME-1U and ME-2U parts, aerial parts of left in the hood for 15 min and in the incubator for 15 TCS (834 g) was selectively extracted with n-hexane min to let both the E-plate’s golden electrode well bot- (5x11 L), CH2Cl2 (5.5 x 9 L), EtOAc (5 x 8 L), and toms and medium reach a thermal equilibrium. Then, MeOH (5.5 x 12 L) to yield 10.47 g, 16.18 g, 40.09 g the E-plate 96 was inserted into the RTCA SP station and 22.6 g of extracts, respectively.