Hepatoprotective, Antioxidant, and Anticancer Effects of the Tragopogon Porrifolius Methanolic Extract

Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2015, Article ID 161720, 10 pages http://dx.doi.org/10.1155/2015/161720 Research Article Hepatoprotective, Antioxidant, and Anticancer Effects of the Tragopogon porrifolius Methanolic Extract Clara Tenkerian,1 Mirvat El-Sibai,1 Costantine F. Daher,1 and Mohamad Mroueh2 1 Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, P.O. Box 36, Byblos 1401, Lebanon 2Department of Pharmaceutical Sciences, School of Pharmacy, Lebanese American University, P.O. Box 36, Byblos 1401, Lebanon Correspondence should be addressed to Mohamad Mroueh; [email protected] Received 26 November 2014; Accepted 2 January 2015 Academic Editor: Felice Senatore Copyright © 2015 Clara Tenkerian et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tragopogon porrifolius (Asteraceae), commonly referred to as white salsify, is an edible herb used in Lebanese folk medicine to treat cancer and liver dysfunction. In this study, we investigated the antioxidant activity of Tragopogon porrifolius methanolic extract, both in vitro and in vivo, in addition to its hepatoprotective and anticancer activities. Total phenolic and flavonoid contents were measured and found to be 37.0 ± 1.40 mg GAE/g and 16.6 ± 0.42 mg QE/g dry weight, respectively. In vitro antioxidant assays 2+ revealed an FRAP value of 659 ± 13.8 mol Fe /g of extract and DPPH IC50 value 15.2 g/mL. In rats subjected to CCl4-induced hepatotoxicity, significant increase in CAT, SOD, and GST levels was detected. The highest dose of the extract (250 mg/kg) recorded a fold increase of 1.68 for SOD, 2.49 for GST, and 3.2 for CAT. The extract also showed substantial decrease in AST (57%), ALT (56%), and LDH (65%) levels. Additionally, the extract caused a dose-dependent decrease in cell viability and proliferation. In conclusion, the methanolic extract of T. porrifolius displayed a relatively high antioxidant activity both in vitro and in vivo as well as hepatoprotective potential against liver toxicity in rats and anticancer effect on MDA-MB-231 and Caco-2 cells. 1. Introduction diseases, including cancer [2, 3]. DNA damage induced by both exogenous and endogenous free radicals is well Oxygen free radicals or reactive oxygen species (ROS), ∙− documented and widely accepted to be a major cause of including the superoxide radical O2 , hydrogen peroxide ∙ genomic instability and cancer. Human studies have shown H2O2, and the highly reactive hydroxyl radical OH, are that oxidative DNA damage is an important carcinogenic and extremely reactive molecules and can oxidize lipids, proteins, mutagenic factor in the sense that it favors the acquisition and even DNA. They are normally present in a balance of mutations and contributes to cellular transformation and with antioxidants molecules that improve the body’s cellular cancer cell survival [2]. defense system against oxidative damage. Antioxidants help According to the World Health Organization [5], 80% of maintain lower levels of free radicals; thus they perform the population of developing countries in Asia and Africa rely beneficial physiological roles1 [ , 2]. The antioxidant defense on traditional medicine for primary health care. Resorting system of the body can be in the form of low molecular weight to traditional medicine in order to discover plants with antioxidants such as vitamins E and C which block free radi- therapeutic properties has proven valuable in the search cals, or in the form of enzymes such as superoxide dismutase, for new bioactive compounds [6]. In fact, in recent years, catalase, and the glutathione system (glutathione, glutathione concerns over harmful side effects of synthetic compounds reductase, peroxidase, and transferase) that reduce the levels have shifted the focus to natural plant resources, which of reactive oxygen species [3, 4]. When an imbalance occurs represent an abundant source of biologically active molecules between free radicals and the antioxidant defense system, this [7]. Taking into account that only 1% of the estimated 500,000 may result in oxidative stress, which has been shown to be plant species on Earth have been investigated, the need for involved in the initiation and progression of various human novel medicinal bioactive compounds is substantial [7]. 2 Evidence-Based Complementary and Alternative Medicine Tragopogon porrifolius, Asteraceae family, is an edible of 1 M potassium acetate (CH3COOK), and 2.8 mL of deion- herb and is commonly known as salsify, oyster plant, and ized water. The mixture was incubated at room temperature vegetable oyster. T. porrifolius is widespread throughout the for 40 minutes and the absorbance was measured at 415 nm. Mediterranean region where it grows wild and is cultivated. The total flavonoid content was expressed as quercetin Allpartsoftheplantareedible,theroots,leafyshoots,and equivalents mg (QE)/g dry weight extract. openflowersareusedasbeingbothcookedandraw[8], and, in Lebanon, the shoots are more frequently consumed 2.4. HPLC Analysis of T. porrifolius Methanolic Extract. The thantheroots.Thenutritionalvalueofthisplanthasbeen phenolic acids and flavonoids were analyzed using Shimadzu attributed to its monounsaturated and essential fatty acids, HPLC system (Shimadzu Corp., Kyoto, Japan) consisting of vitamins, polyphenols, and fructooligosaccharides compo- LC 10-ADVP pump, SCL 10A system controller coupled with nents [9]. Recent studies in our laboratory revealed that a photo-diode array detector (SPD-M20A), FCV-10AL low intake of aqueous extract of T. porrifolius caused improve- pressure gradient, Rheodyne injector (Model 7125), DGU- ment of lipemia and increased satiety in rats with no visible 20A online degasser, Shim-pack VP-ODS column, (4.6 mm adverse effects [10]. Additionally, methanol, ethyl acetate, i.d. × 150 mm), and precolumn (10 × 4.6 mm i.d. 5 m) and chloroform extracts of aerial parts of T. porrifolius equipped with LC solution 1.23 SP1 software (Shimadzu, ∘ demonstrated anti-inflammatory effects in mice11 [ ]. Kyoto, Japan). The column was operated at 25 C. The mobile To the best of our knowledge, no studies were conducted phase consisted of water : acetic acid : methanol (10:2: on T. porrifolius to investigate its antioxidant, hepatopro- 88 v/v) as solvent A, and water : acetic acid : methanol (90 : 2 : tective, and anticancer activities. Therefore, this study was 8 v/v) as solvent B at a flow rate of 1.5 mL/min. The gradient carried out to evaluate the in vitro and in vivo antioxidant elution program was as follows: 0–15 min solvent A and 15– activities of the plant methanolic extract and its hepatopro- 30 min solvent followed by washout period for 10 min and the tective effect against CCl4-induced liver damage in rats, in wavelength of detection was set at 280 nm. The phenolic acids addition to its cancer activity against colon (Caco-2) and and flavonoids were identified by matching the retention time breast (MDA-MB-231) cancer cell lines. and their spectral characteristics with those of the standard compounds. 2. Materials and Methods All phenolic acids and flavonoid standards (gallic acid, chlorogenic acid, vanillic acid, syringic acid, caffeic acid, 2.1. Plant Material and Extraction. The T. porrifolius plant ellagic acid, myricetin, quercetin, luteolin, kaempferol, and material was collected from south of Lebanon and air-dried in apigenin) were purchased from Sigma-Aldrich Co. (St. Louis, the shade. The dried plant material was cut into small pieces MO,USA).AllHPLCsolventswerepurchasedfromMerck and soaked in methanol for 72 hours and the extract was then (Germany). The standard stock solutions (0.2 mg/mL) were filtered using Whatman no. 1 filter paper. This process was prepared by dissolving each standard in methanol and diluted repeated twice to ensure maximal extraction. The solvent was with the mobile phase in the range of 10–60 g/mL. removed under reduced pressure using a rotary evaporator ∘ (40 C, 337 mbar) and the residue was dried under vacuum ∘ (9.23% yield) and stored at −20 Cuntiluse. 2.5. In Vitro Antioxidant Assays 2.5.1. FRAP Assay. The FRAP value was calculated based 2.2. Determination of Total Phenolic Content. Total phenolic on the method of Benzie and Strain [14]usingferrous content was estimated by the Folin-Ciocalteu colorimetric sulphate (10–100 mM FeSO4⋅7H2O) as a standard. Briefly, method, based on the procedure of Singleton and Rossi [12] 20 L of either the extract or different concentrations of the using gallic acid (100–1000 mg/L in 80% methanol) as a stan- ferrous sulphate standard was added to 150 Lofthefreshly ∘ dard. Briefly, 50 L of the 1 : 5 diluted and filtered extract (at an prepared and prewarmed (at 37 C) FRAP reagent (300 mM original concentration of 100 mg/mL in methanol) was mixed acetate buffer at a pH of 3.6, 10 mM 2,4,6-tripyridyl-s-triazine with 450 Lofdistilledwaterand2.5mLof0.2NFolin- (TPTZ) in 40 mM HCl, and 20 mM ferric chloride solution Ciocalteu reagent. After 5 min, 2 mL of saturated sodium in a 10 : 1 : 1 ratio). The absorbance was measured after 8 carbonate(75g/L)wasaddedandthemixturewasincubated ∘ minutes in an ELISA microplate reader at 600 nm. The FRAP at 30 C for 90 minutes with intermittent shaking. The value (M ferric ions reduced to ferrous form per gram) of absorbance of the resulting blue-colored solution was mea- each sample was determined after subtracting the blank read- suredat765nmandthetotalphenoliccontentwasexpressed ing. as mg gallic acid equivalents (GAE)/g of dry weight extract. 2.5.2. DPPH Assay. The DPPH assay is based on the ability 2.3. Determination of Total Flavonoid Content. Total flavo- of this stable radical to react with hydrogen donors [15]. noid content was determined by the aluminum chloride Briefly, 50 L of the extract (10, 50, and 100 g/mL) and colorimetric method described by Chang et al.

Load more