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And Chemical Composition of Polar Extracts from the Ruderal Species Coleostephus Myconis (L.) Rchb.F

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And Chemical Composition of Polar Extracts from the Ruderal Species Coleostephus Myconis (L.) Rchb.F Journal of Toxicology and Environmental Health, Part A Current Issues ISSN: 1528-7394 (Print) 1087-2620 (Online) Journal homepage: http://www.tandfonline.com/loi/uteh20 Evaluation of the cytotoxicity (HepG2) and chemical composition of polar extracts from the ruderal species Coleostephus myconis (L.) Rchb.f. Sílvia M. F. Bessada, João C. M. Barreira, J. Santos, Carla Costa, Filipa B. Pimentel, Maria João Bessa, João Paulo Teixeira & M.Beatriz P.P. Oliveira To cite this article: Sílvia M. F. Bessada, João C. M. Barreira, J. Santos, Carla Costa, Filipa B. Pimentel, Maria João Bessa, João Paulo Teixeira & M.Beatriz P.P. Oliveira (2017) Evaluation of the cytotoxicity (HepG2) and chemical composition of polar extracts from the ruderal species Coleostephus myconis (L.) Rchb.f., Journal of Toxicology and Environmental Health, Part A, 80:13-15, 641-650, DOI: 10.1080/15287394.2017.1286915 To link to this article: https://doi.org/10.1080/15287394.2017.1286915 Published online: 19 May 2017. Submit your article to this journal Article views: 48 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 2017, VOL. 80, NOS. 13–15, 641–650 https://doi.org/10.1080/15287394.2017.1286915 Evaluation of the cytotoxicity (HepG2) and chemical composition of polar extracts from the ruderal species Coleostephus myconis (L.) Rchb.f. Sílvia M. F. Bessadaa, João C. M. Barreiraa,b, J. Santosa, Carla Costac,d, Filipa B. Pimentela, Maria João Bessac,d, João Paulo Teixeirac,d, and M.Beatriz P.P. Oliveiraa aREQUIMTE/LAQV,Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal; bCIMO-ESA, Instituto Politécnico de Bragança, Bragança, Portugal; cDepartment of Environmental Health, Portuguese National Institute of Health, Porto, Portugal; dEPIUnit – Institute of Public Health, University of Porto, Porto, Portugal ABSTRACT Coleostephus myconis (L.) Rchb.f. (Asteraceae) is a highly disseminated plant species with ruderal and persistent growth. Owing to its advantageous agronomic properties, C. myconis might have industrial applications. However, this species needs to be comprehensively characterized before any potential use. In a previous study, the phenolic composition and antioxidant activity of different C. myconis tissues were characterized. This investigation was extended to examine the cytotoxic potential of selected plant tissues (flowers and green parts) using a HepG2 cell line by utilizing the lysosomal neutral red uptake assay or mitochondrial (3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide assay. In addition, the macronutrients content, lipophilic com- pounds (fatty acids, tocopherols), and amino acids were also determined. C. myconis flowers were used in the senescence stage, which was previously identified as the stage that presented maximal phenolic content and highest antioxidant activity. In contrast, stems and leaves were employed due to their high biomass proportion. Regarding cytotoxicity, mitochondrial and lysosomal damage was only significant when HepG2 cells were exposed to the highest extract concentrations (stems and leaves, 0.9 mg/ml; senescent flowers, 0.3 mg/ml). Chemically, the senescent flowers were mostly characterized by their high levels of fat, amino acids (especially threonine), oleic acid, β-, and γ-tocopherol, while stems and leaves contained high concentrations of carbohydrates, linolenic acid, and α-tocopherol. In general, these results provide information regarding the threshold concentrations of C. myconis extracts that might be used in different applications without toxicity hazards. Introduction yellow flowering between March and August. Bessada et al. (2015)notedthatAsteraceae species possessed Natural medicinal plants are used by the majority of potentialtoactasasourceofbioactivecompounds the world’s population (Bernal et al., 2011)andthese with application in medicine, pharmaceutical, cos- so-called phytomedicines are playing an increasing metic,andfoodindustries.However,anddespite greater role in human health care. Medicinal plants their potential applications and uses in traditional are emerging as a strong alternative to synthetic pro- medicine (e.g., in the treatment of gastrointestinal ducts, not only in traditional medicine but also in a and liver disorders and malaria), there are few studies number of food and pharmaceutical products due to regarding the effectiveness and safety of Asteraceae their nutritional properties and bioactivity species. Therefore, evaluating the potential toxicity of (Krishnaiah et al., 2011;Bessadaetal.,2015). these plant extracts, particularly their cytotoxicity and Further, medicinal plants may provide beneficial genotoxicity, needs to be considered (Franco et al., health effects due to the presence of different bioactive 2015). compounds and nutraceuticals (Dias et al., 2014). C. myconis was previously characterized for its Coleostephus myconis (L.) Rchb.f. (family phenolic profile and antioxidant activity in differ- Asteraceae) is a species with ruderal growth and per- ent extracts obtained from the green parts (stems sistence in abandoned soils, known for its plentiful and leaves) and flowers at different ripening CONTACT João C. M. Barreira [email protected] REQUIMTE/LAQV, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, no 228, 4050-313 Porto, Portugal. © 2017 Taylor & Francis 642 S. M. F. BESSADA ET AL. stages. The highest antioxidant activity and phe- flowers. For each plant part, three independent nolic content was detected in senescent flowers, samples were used; each of the samples was further particularly those obtained from the hydro-etha- analyzed thrice. The vegetal material was then nolic extracts (Bessada et al., 2016). However, the frozen, lyophilized (48 h, −78°C, 0.015 mbar) cytotoxic potential of hydro-ethanolic extracts of (Telstar Cryodos-80, Terrassa, Barcelona), reduced C. myconis senescent flowers and green parts to powder and mixed to obtain homogenized sam- remains to be evaluated in a HepG2 (human hepa- ples, and stored in plastic tubes at room tempera- tocellular carcinoma) cell line, which is a reference ture for subsequent use. cell line for toxicity evaluation (Ju et al., 2015). In addition, the same botanical tissues were charac- Macronutrients Analysis terized regarding their macronutrient composi- tion, lipophilic compounds (fatty acids [FA] and Macronutrients were analyzed following the tocopherols), and amino acids composition. Association of Analytical Communities methods (AOAC, 2012). Moisture content was instrumen- tally determined using an infrared moisture analy- Materials and Methods zer (SMO 01, Scaltec Instruments, Heiligenstadt, Standards and Reagents Germany). Ash content was determined by incin- erating the sample in a muffle furnace at 550°C. All chemicals and solvents were of analytical grade Protein content (N × 6.25) was determined using and purchased from common sources. Tocol the Kjeldahl procedure. Total fat was determined (2-methyl-2-(4,8,12-trimethyl-tridecyl)-chroman- by Soxhlet extraction with petroleum ether. Total 6-ol) was purchased from Matreya Inc. carbohydrate content was determined by differ- (Pennsylvania, USA). The mixture of methyl esters ence. Energy was calculated according to the gen- of FA standards (FAME) Supelco37 was obtained eral Atwater factors (Atwater & Benedict, 1902): from Supelco (Bellefonte, PA, USA), while the Energy (kcal) = 4 × (g protein) + 3.75 × (g carbo- mixture of L-amino acids LAA21 and norleucine hydrate) + 9 × (g fat). The results were expressed was acquired from Sigma (St. Louis, MO, USA). as g/100 g of dry mass (dm). For the chromatographic analysis, acetonitrile (high-performance liquid chromatography [HPLC] grade) was obtained from Merck KGaA Amino Acids Profile (Darmstadt, Germany). Neutral red dye, 3-(4,5- The amino acids histidine (His), arginine (Arg), dimethylthiazolyl-2)-2,5-diphenyltetrazolium br- aspartic acid + glutamic acid (Asp + Glu), threo- omide (MTT), and Triton X-100 were purchased nine (Thr), glycine (Gly), alanine (Ala), proline from Sigma-Aldrich Co. Dimethyl sulfoxide, acetic (Pro), valine + methionine (Val + Met), phenyla- acid (glacial) 100%, and ethanol absolute were lanine (Phe), isoleucine (Ile), leucine (Leu), lysine bought from Merck KGaA. The HepG2 cell line (Lys), and tyrosine (Tyr) were analyzed by HPLC was obtained from European Collection of Cell using a fluorescence detector (FD), after subjecting Cultures. Cell culture media components were all the samples to acidic hydrolysis (HCl 6 mol/l, 110° ™ Invitrogen andacquiredfromThermoFisher C, 24 h) and derivatization with dansyl chloride Scientific Inc. Water was obtained from a Milli-Q (Rodrigues et al., 2015). water purification system (TGI Pure Water The chromatographic analysis was carried out Systems, USA). in an integrated HPLC system (Jasco, Tokyo, Japan), equipped with two Jasco PU-980 Plus HPLC pumps, an AS-2057 Plus automated injec- Samples tor (20 μl loop), and a FP-2020 Plus FD, pro- After taxonomical identification, C. myconis plants grammed for excitation at 335 nm and emission were collected in the Northwest of Portugal (Riba at 514 nm. The compounds separation was de Mouro, Minho) in June, 2014, and further achieved in a reversed-phase column (Luna 5u divided into green parts (stems and leaves) and C18, 4.6 × 250 mm,
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