LWT - Food Science and Technology 47 (2012) 64e70 Contents lists available at SciVerse ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt Comparative analysis of phenolic profile, antioxidant, anti-inflammatory and cytotoxic activity of two closely-related Plantain species: Plantago altissima L. and Plantago lanceolata L.   Ivana N. Beara a, *, Marija M. Lesjak a, Dejan Z. Or ci c a, Natasa Ð. Simin a, Dragana D. Cetojevi c-Simin b, c a Biljana N. Bo zin , Neda M. Mimica-Duki c a b c Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia Oncology Institute of Vojvodina, Institutski put 4, 21204 Sremska Kamenica, Serbia Department of Pharmacy, Faculty of Medicine, Hajduk Veljkova 3, 21000 Novi Sad, Serbia a r t i c l e i n f o a b s t r a c t Article history: Received 14 July 2011 Received in revised form 30 December 2011 Accepted 2 January 2012 The present study was designed to define the phenolic profile, antioxidant, anti-inflammatory and cytotoxic activity of Plantago altissima L., which has never been studied before and to compare it with closely-related, renowned, well-studied Plantago lanceolata L. The presence and content of 44 phenolics in methanol extracts were studied using LCeMS/MS. A similar qualitative composition including dominant compounds as p-hydroxybenzoic, vanillic, gallic and chlorogenic acid, besides apigenin, luteolin and luteolin-7-O-glucoside was found between both extracts. Antioxidant activity of extracts was determined using several assays. All results of these tests were comparable to butylated hydroxytoluene, a well-known synthetic antioxidant. Anti-inflammatory potential was studied by means of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibitory activity. Activity of P. altissima towards COX-1/12-LOX inhibition (IC50 ¼ 4.4 and 3.6 mg/mL, respectively) was inferior to activity of P. lanceolata (IC50 ¼ 2.0 and 0.8 mg/mL, respectively). Treatment of four cell lines resulted in a considerable dose-dependent inhibition of cell growth, where P. lanceolata exerted a stronger effect (IC50 ¼ 172.3, 142.8, 405.5 and 551.7 mg/mL for HeLa, MCF7, HT-29 and MRC-5 cell lines, respectively). To conclude, P. altissima showed certain bio-potential, but was clearly inferior to P. lanceolata. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: Plantago Phenolics LCeMS/MS Antioxidant Anti-inflammatory Cytotoxic 1. Introduction The largest genus of the cosmopolitan family, Plantaginaceae is the genus Plantago L. and it comprises about 275 species. Extensive traditional use and modern medicinal application of several Plantago species is a consequence of their remarkable variety of curative properties: astringent, styptic, antimicrobial, expectorant, diuretic ci c, 2002; Samuelsen, 2000; and demulcent (Huang et al., 2009; Jan Tucakov, 1997; Wichtl, 1994; World Health Organisation, 1999). In addition, some studies confirm that certain Plantago species reveal considerable bioactivity, such as cytotoxic effects on cancer cell lines (Gálvez, Martín-Cordero, López-Lázaro, Cortés, & Ayuso, 2003), antiinflammatory (Samuelsen, 2000), immunoregulatory (Huang et al., Abbreviations: COX-1, cyclooxygenase-1; 12-LOX, 12-lipoxygenase; 12-HHT, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid; 12-HETE, 12(S)-hydroxy(5Z,8Z,10E,14Z)-eicosatetraenoic acid; SRB, sulforhodamine B; dw, dry weight. * Corresponding author. Tel.: þ381 21 4852755; fax: þ381 21 454065. E-mail address: ivana.beara@dh.uns.ac.rs (I.N. Beara). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2012.01.001 2009), antioxidant (Gálvez et al., 2003; Heimler, Isolani, Vignolini, Tombelli, & Romani, 2007) and antispasmodic effects (Fleer & Verspohl, 2007). Furthermore, some Plantago species are also included in the diet. Usually, they are consumed as fresh salads, soups, side dish or they can be used as herbal tea. The seeds of some species can be cooked and used as starch, or can be ground into a powder and added to flour when making bread and cakes (Heimler et al., 2007; Jan ci c, 2002; Tucakov, 1997; Tull, 2003). Although previous studies of some plantains indicate the high potential of these species as a source of biologically active compounds and healing agents, the majority of Plantago representatives have not been described thus far in terms of phytochemical composition and biological activity. Therefore, in this study, we assessed phenolic profile, antioxidant, anti-inflammatory and anti-cancer activity of the methanolic extracts of Plantago altissima L. and Plantago lanceolata L. To the best of our knowledge, P. altissima has not been examined with respect to any biological activity, but several phenolic compounds, such as flavonoids (Grubesi c & VladimirKne zevi c, 2004) have been described previously, but other data is I.N. Beara et al. / LWT - Food Science and Technology 47 (2012) 64e70 limited. P. altissima and P. lanceolata, a well-known remedy and food, belong to the same subgenus, Albicans (Rønsted, Franzyk, Mølgaard, Jaroszewski, & Jensen, 2003) and yet, they can be easily mistaken for each other due to their minor morphological differences, mostly in the plants’ height (Tutin et al., 1976). Consequently, the aim of this study was not only to examine the unexplored P. altissima, but also to compare its phenolic profile and biological activities with renowned P. lanceolata. Therefore, an LCeMS/MS technique was applied to evaluate quantitative content of numerous phenolics, including 14 phenolic acids, 25 flavonoids, 3 coumarins and 2 lignans. The antioxidant potential of extracts was determined using various assays  related to free radical (DPPH), reactive oxygen (HO, O2 ) and  reactive nitrogen species (NO ) scavenging ability, in addition to the potential of lipid peroxidation (LP) inhibition and reducing power (FRAP assay). Anti-inflammatory activity was determined by means of inhibition of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12LOX) enzymes, which are involved in the metabolism of arachidonic acid. To establish the anti-inflammatory activity of extracts, LCeMS/ MS technique was used for quantification of COX-1 and 12-LOX metabolites 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid (12HHT) and 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12HETE), respectively. Finally, cell growth activity of extracts was evaluated in vitro in a panel of four human cell lines: HeLa (cervix epitheloid carcinoma), MCF7 (breast adenocarcinoma), HT-29 (colon adenocarinoma) and MRC-5 (human fetal lung). Presented results were obtained using SRB (Sulforhodamine B) assay (Skehan et al., 1990). 2. Material and methods 2.1. Chemicals and reagents All standards of phenolic compounds were purchased from SigmaeAldrich Chem (Steinheim, Germany), Fluka Chemie GmbH (Buchs, Switzerland) or from ChromaDex (Santa Ana, USA). Reagents used for antioxidant and anti-inflammatory assays were purchased from suppliers listed in Beara et al. (2009), Beara et al. (2010) and Lesjak et al. (2011). DMEM (Dulbecco’s modified Eagle’s medium) and FCS (fetal calf serum) were obtained from PAA Laboratories GmbH (Pashing, Austria). Penicillin and streptomycin were purchased from Galenika (Belgrade, Serbia). All other reagents used in this study were of analytical grade. Platelet concentrate was kindly provided by The Institute for Blood Transfusion of Vojvodina, Novi Sad, Serbia. 2.2. Plant material and extract preparation The aerial parts of P. altissima were collected in June 2009 in Novi Sad, Serbia and P. lanceolata in June 2009 from the mountain of Fruska Gora, Serbia. The voucher specimens (P. altissima, No. 21804; P. lanceolata, No. 2-1829) were prepared and identified by Goran Ana ckov, PhD, and deposited at the Herbarium of Department of Biology and Ecology (BUNS Herbarium), Faculty of Sciences, University of Novi Sad, Serbia. Air-dried and smoothly grounded herbal samples weighing 30 g were extracted by maceration with 300 mL methanol/water (4:1) during 72 h at room temperature. After filtration, solvent was evaporated in vacuo at 45  C and crude residue was dissolved in hot, distilled water (1 g/mL). With the aim of removing non-polar compounds, the extracts were washed exhaustively with petroleum ether (fraction 40e60  C) and concentrated to dryness under vacuum, yielding 4.71 g and 3.03 g for P. altissima and P. lanceolata extracts, respectively. Dried extracts were dissolved in methanol/ water (4:1) to obtain 200 mg/mL or in dimethyl sulfoxide (DMSO) to obtain 200 and 100 mg/mL stock solutions for evaluation of the 65 antioxidant, anti-inflammatory and cytotoxic activity, respectively. Also, dried extracts were dissolved in a mixture of formic acid/ water (1:199) and methanol (in ratio of 7:3) for HPLCeMS analysis to obtain 0.2 mg/mL stock solutions. 2.3. LCeMS/MS analysis of the selected phenolics The Agilent 1200 series liquid chromatograph, consisting of vacuum degasser, binary pump, autosampler and thermostated column compartment was used for separation of all analytes, whose detection was carried out by means of Agilent series 6410B triple-quadrupole mass spectrometer with electrospray ionization (ESI). MassHunter ver. B.03.01. software (Agilent Technologies) was used for instruments control and data analysis. The injection volume for all samples was 5 mL. The binary mobile phase consisted of formic acid/water (1:1999; A) and methanol (B) and was delivered at a flow rate of 1 mL/min. Gradient elution was performed using the following solvent gradient: starting with 70% A/30% B, reaching 30% A/70% B in 6.00 min, then 100% B at 9.00 min, holding until 12.00 min, with post-time of 3 min. The separation was achieved using a Zorbax Eclipse XDB-C18 RR 4.6 mm  50 mm  1.8 mm (Agilent Technologies) reversed-phase column held at 45  C. The eluate was forwarded, without flow splitting, into an ESI ion source with following settings: drying gas (N2) temperature, 350  C; flow, 9 L/min; nebulizer gas pressure, 40 psi; capillary voltage, 4 kV, negative polarity. All compounds were quantified in dynamic MRM mode (multiple reaction monitoring mode). Compound-specific, optimized MS/MS parameters are given in Table 1. Extracts used for LCeMS/MS quantification were dissolved in starting mobile phase solvent to the concentration of 0.2 mg/mL. All used standards were dissolved in DMSO to prepare stock solutions of 10 mg/mL. The mix of stock solutions was prepared, with concentration of each compound being 100 mg/mL. The mix was subsequently serially diluted, giving working standard solutions with concentration ranging from 25.0 to 0.0015 mg/mL, which were used for construction of the calibration curves. Concentrations of standard compounds in extracts were determined from the peak areas by using the equation for linear regression obtained from the calibration curves (R2 gt; 0.995). 2.4. Antioxidant activity Assays considering reduction of DPPH radical, hydroxyl-radical, superoxide anion and NO scavenger capacity, lipid peroxidation and reducing power (FRAP assay) of plant extracts were performed according to the earlier reported procedures (Beara et al., 2009; Lesjak et al., 2011). 2.5. COX-1 and 12-LOX assay Ex vivo COX-1 and 12-LOX assay was undertaken according to prior reported method (Beara et al., 2010). 2.6. Cell growth activity 2.6.1. Grow and culture of the cell lines For the estimation of cell growth effects, human cell lines HeLa (cervix epitheloid carcinoma; ECACC No. 93021013), MCF7 (breast adenocarcinoma; ECACC No. 86012803), HT-29 (colon adenocarcinoma; ECACC No. 91072201) and MRC-5 (human fetal lung; ECACC No. 84101801) were used. Cell lines were grown in DMEM with 45 mg/mL glucose, supplemented with 100 mL/mL heat inactivated FCS, 100 IU/mL of penicillin and 100 mg/mL of streptomycin. All investigated cell lines grow attached to the surface. They were cultured in 25 cm2 flasks at 37  C in atmosphere of 5% CO2 and 100% 66 I.N. Beara et al. / LWT - Food Science and Technology 47 (2012) 64e70 Table 1 LCeMS/MS data for standard compounds. Compound Compound-specific MS/MS parameters Retention time (min) Fragmentor voltage (V) Precursor ion (m/z) Product ion (m/z) Collision energy (V) Gallic acid Catechin Protocatechuic acid Chlorogenic acid Epigallocatechin gallate Epicatechin 2,5-Dihydroxybenzoic acid p-Hydroxybenzoic acid Aesculetin Caffeic acid Vanillic acid Syringic acid p-Coumaric acid Umbelliferone Scopoletin Ferulic acid Vitexin Sinapic acid Luteolin-7-O-glucoside Hyperoside Quercetin-3-O-glucoside Rutin Apiin o-Coumaric acid Myricetin Quercitrin Kaempferol-3-O-glucoside Apigenin-7-O-glucoside Secoisolariciresinol 3,4-Dimethoxycinnamic acid Baicalin Daidzein Matairesinol Quercetin Naringenin Cinnamic acid Luteolin Genistein Kaempferol Apigenin Isorhamnetin Chrysoeriol Baicalein Amentoflavone 0.58 0.74 0.79 0.80 0.81 0.95 1.03 1.08 1.13 1.18 1.24 1.31 1.69 1.73 1.77 1.90 1.90 1.92 2.13 2.16 2.25 2.33 2.60 2.62 2.67 2.75 2.80 2.81 2.90 2.99 3.40 3.43 3.66 3.74 3.87 3.91 4.03 4.12 4.55 4.71 4.79 4.82 5.15 5.78 90 150 105 100 165 150 100 80 105 100 100 90 90 120 80 90 200 100 230 200 210 135 250 100 150 190 190 135 130 110 140 145 130 130 130 100 135 145 130 130 160 125 165 220 169 289 153 353 457 289 153 137 177 179 167 197 163 161 191 193 431 223 447 463 463 609 563 163 317 447 447 431 361 207 445 253 357 301 271 147 285 269 285 269 315 299 269 537 125 245 109 191 169 245 109 93 133 135 108 182 119 133 176 134 311 193 285 300 300 300 269 119 179 300 284 268 165 103 269 208 122 151 151 103 133 133 285 117 300 284 269 375 10 10 9 10 16 10 9 10 15 10 15 7 9 19 8 11 22 17 30 30 30 42 36 5 20 27 30 41 26 7 22 31 24 15 16 5 25 32 0 25 21 20 0 35 humidity, sub-cultured twice a week and a single cell suspension was obtained using 1 mg/mL trypsin with 0.4 mg/mL EDTA. For the analysis of cell growth effects serial dilutions in 9 mg/mL NaCl were used. Samples were filtered through a 0.22 mm microfilters to obtain sterility. Concentrations of samples were in the range of 62.5e1000 mg/mL. Concentration of DMSO in cell culture was 50 mL/mL. 2.6.2. Sulforhodamine B (SRB) assay Cell lines were harvested and plated into 96-well microtiter   c-Simin plates at seeding density of 4  103 cells per well (Cetojevi et al., in press), in a volume of 180 mL, and preincubated in complete medium supplemented with 50 mL/mL FCS, at 37  C for 24 h. Serial dilutions and solvent were added (20 mL/well) to achieve required final concentrations and control. Microplates were then incubated at 37  C for an additional 48 h. Cell growth was evaluated by the colourimetric SRB assay according to Skehan et al. (1990). Cells were fixed with 0.5 g/mL TCA (1 h, 4  C), washed with distilled water and stained with 4 mg/mL SRB (30 min, room temperature). The plates were then washed with acetic acid/water (1:99) to remove unbound dye. Protein-bound dye was extracted with 10 mmol/L TRIS base. Absorbance was measured on a microplate reader at 540/620 nm. 2.7. Statistical analysis Percent of inhibition achieved by different concentration of extracts was calculated by the following equation in performed antioxidant assays: I(%) ¼ (A0 A)/A0  100, where A0 was the absorbance of the control reaction and A was the absorbance of the examined samples, corrected for the value of blank probe. Percent of COX-1 and 12-LOX inhibition achieved by different concentrations of extracts was calculated by the following equation: I(%) ¼ 100  (R0 R)/R0, where R0 and R were response ratios (metabolite peak area/internal standard peak area) in the control reaction and in the examined samples, respectively. Both R and R0 were corrected for the value of blank probe. Effect on cell growth was calculated as: I(%) ¼ 100  At/Ac, where At is the absorbance of the test sample and Ac is the absorbance of the control, both obtained after subtracting absorbencies at reference wave length. Corresponding inhibition-concentration curves were drawn using Origin software, version 8.0 and IC50 values (concentration of I.N. Beara et al. / LWT - Food Science and Technology 47 (2012) 64e70 extract that inhibited DPPH, O2 , HO and NO radical, MDA, COX-1 and 12-LOX metabolites formation and cell growth by 50%) were determined. For each assay and extract composition determinations, all of the results were expressed as mean  SD of three or eight (cytotoxic activity) different trials. A comparison of the group means and the significance between the groups were verified by one-way ANOVA. Statistical significance was set at p < 0.05. 3. Results and discussion 3.1. LCeMS/MS analysis of the selected flavonoids The quantification of the chosen phenolics in both P. altissima and P. lanceolata extracts was performed using the LCeMS/MS technique. The MRM mode was applied as the preferred acquisition method for accurate quantification. This type of analysis provides high sensitivity and specificity, due to the fact that only ions specific to targeted analytes are monitored. Corresponding chromatograms are shown in Fig. 1, while overall data concerning the content of the standard compounds are presented in Table 2. The results of analysis showed that both extracts are rich in phenolic acids. Considerable amounts of benzoic acid derivatives phydroxybenzoic and 3,4,5-trihydroxybenzoic (gallic acid) were 67 found in P. altissima and P. lanceolata extracts. The most dominant benzoic acid derivative found in both P. altissima and P. lanceolata extracts was vanillic acid and determined amounts were significantly different (343.9 and 411.5 mg/g of dw, respectively). This has previously been reported for P. lanceolata (Fons, Rapior, Gargadennec, Andary, & Bessière, 1998). Furthermore, a significant difference in the content of cinnamic acid was ascertained, the highest being in P. lanceolata (209.8 mg/g of dw), whilst the content in P. altissima was markedly lower (56.2 mg/g of dw). In accordance with previous studies of P. lanceolata, our study also detected the presence of caffeic acid in both extracts (Maksyutina, 1971). Nevertheless, the most dominant hydroxycinnamic acid derivative found was chlorogenic acid with significant difference in its content (5.5 and 7.2 mg/g of dw in P. altissima and P. lanceolata, respectively). Since the analysis of flavonoid profile points out that the Plantago genus mainly contains flavones (Rønsted et al., 2003), our research was focused on this class of flavonoid compounds. Consequently, the most dominant flavonoids in both P. altissima and P. lanceolata extracts were apigenin, luteolin and luteolin-7-Oglucoside. Results obtained for luteolin and luteolin-7-O-glucoside in P. lanceolata were in agreement with data previously reported (Kawashty, Gamal-El-Din, Abdalla, & Saleh, 1994). A previous study has detected the presence of baicalein and baicalin in Plantago Fig. 1. LCeMS/MS chromatograms of P. altissima (a) and P. lanceolata (b) Extracts, 1: gallic acid, 2: protocatechuic acid, 3: chlorogenic acid, 4: 2,5-Dihydroxybenzoic acid, 5: p-hydroxybenzoic acid, 6: aesculetin, 7: caffeic acid, 8: vanillic acid, 9: syringic acid, 10: p-coumaric acid, 11: scopoletin, 12: ferulic acid, 13: vitexin, 14: sinapic acid, 15: luteolin-7-Oglucoside, 16: hyperoside, 17: quercetin-3-O-glucoside, 18: rutin, 19: apiin, 20: quercitrin, 21: kaempferol-3-O-glucoside, 22: apigenin-7-O-glucoside, 23: quercetin, 24: naringenin, 25: cinnamic acid, 26: luteolin, 27: kaempferol, 28: apigenin, 29: isorhamnetin, 30: chrysoeriol, 31: amentoflavone. 68 I.N. Beara et al. / LWT - Food Science and Technology 47 (2012) 64e70 Table 2 Determined concentrations of selected phenolics in examined Plantago extracts. Compound Phenolic acids p-Hydroxybenzoic acid 2,5-Dihydroxybenzoic acid Protocatechuic acid Vanillic acid Gallic acid Syringic acid Cinnamic acid o-Coumaric acid p-Coumaric acid Caffeic acid Ferulic acid 3,4-Dimethoxycinnamic acid Sinapic acid Chlorogenic acid Flavonoids Apigenin Apigenin-7-O-glucoside Apiin Vitexin Amentoflavone Baicalein Baicalin Daidzein Genistein Isorhamnetin Kaempferol Kaempferol-3-O-glucoside Chrysoeriol Luteolin Luteolin-7-O-glucoside Quercetin Quercitrin Quercetin-3-O-glucoside Hyperoside Rutin Myricetin Naringenin Epicatechin Catechin Epigallocatechin gallate Coumarins Umbelliferone Aesculetin Scopoletin Lignans Matairesinol Secoisolariciresinol Table 3 IC50 Values for evaluated antioxidant assays of examined extracts and BHT. IC50 values for scavenging activity of radical species Content of selected phenolics in examined Plantago extracts (mg/g of dw) Extract P. altissima P. lanceolata 231.80  5.12 b 21.25  0.27 b 95.22  0.26 a 343.87  3.10 a 294.68  5.96 b 62.86  1.22 b 56.25  1.89 a Nd 153.61  3.56 b 121.70  2.67 b 90.10  1.42 b Nd 3.30  0.23 a 5549.10  60.57 a 149.46  2.35 a 16.20  0.24 a 103.48  1.08 b 411.52  3.27 b 212.01  4.22 a 30.16  0.82 a 209.78  8.23 b Nd 87.67  1.44 a 90.46  2.98 a 57.01  2.32 b Nd 3.62  0.15 a 7115.62  178.34 b P. altissima 10.74  0.81 c 312.52  15.85 b 27.22  1.67 b 1.39  0.10 c P. lanceolata 4.20  0.19 a 236.12  9.35 a 23.85  0.79 b 0.19  0.02 b Standard, BHT 8.28  0.50 b 233.68  19.28 a N.a. a N.a. a 195.51  13.94 a 8.03  0.61 a 1.02  0.03 a 0.32  0.01 a 4.19  0.09 a Nd Nd Nd Nd 1.45  0.07 0.70  0.01 46.25  2.73 b 13.18  0.18 a 232.01  9.87 b 101.81  4.20 a 2.26  0.07 b 0.57  0.04 a 79.29  4.67 b Nd 46.81  1.30 a Nd 1.57  0.16 b Nd Nd Nd 184.38  6.22 a 10.69  0.70 b 3.48  0.05 b 0.58  0.08 b 10.10  0.65 b Nd Nd Nd Nd Nd Nd 5.95  0.34 a 14.96  0.94 a 146.16  5.26 a 119.15  3.56 b 1.00  0.01 a 6.46  0.25 b 34.67  1.55 a 2.65  0.12 45.37  0.95 a Nd 0.42  0.07 a Nd Nd Nd Nd 15.77  1.22 b 0.22  0.01 a Nd 8.88  0.43 a 0.68  0.01 b Nd Nd Nd Nd Values are means  SD of three measurements. Means within each row with different letters (a,b) differ significantly (p < 0.05). Nd, not detected. species (Samuelsen, 2000), but these compounds were not detected in the extracts examined here. A significant difference was found in the content of kaempherol-3-O-glucoside in both species (46.2 mg/g of dw for P. altissima: 5.9 mg/g of dw for P. lanceolata). The presence of rutin in P. altissima was not detected in a previous phytochemical study (Grubesi c & Vladimir-Kne zevi c, 2004) where the TLC method was applied for flavonoid analysis, while the application of highly-sensitive LCeMS/MS analysis in our research allowed the detection of low concentrations of this compound. Finally, certain attempts to detect selected compounds from other classes of flavonoids such as flavanones (naringenin) or flavanoles (catechin, epicatechin and epigallocatechin gallate) were made, but only naringenin was detected in traces. Interestingly, two coumarins, aesculetin and scopoletin were found to be present, while lignans, matairesinol and secoisolariciresinol were not detected. DPPH (mg/mL) HO (mg/mL) O2 (mg/mL) NO (mg/mL) Values are means  SD of three measurements. Means within each column with different letters (aec) differ significantly (p < 0.05). N.a., 50% inhibition not achieved. The phenolic profile of P. altissima and P. lanceolata, presented here is in accordance with previous implications that the Plantago genus contains numerous flavones (Rønsted et al., 2003) and the results obtained confirm that these two species, according to the dominant presence of this subgroup of flavonoids, could be classified into flavone (flavone glycosides) chemotypes. Additionally, P. altissima and P. lanceolata are phylogenetically closely related, but some differences in the flavonoid content of both are obvious, mostly regarding the quantitative rather than the qualitative variations. To date, there has been not much data concerning the chemical composition of P. altissima. The presence of foremost phenolics in both species, such as chlorogenic acid, apigenin, luteolin or luteolin-7-O-glycoside detected in this study can be particularly valuable as a base for the further research of this species and their content can implicate numerous potential applications of the extracts examined here. For instance, levels of chlorogenic acid established in this study can indicate the potential use of these plant species as a source of this compound, or could be of use in medicine, due to the valuable properties of chlorogenic acid to humans, including antioxidant, hepatoprotective or hypoglycemic activity (Marques & Farah, 2009). Nevertheless, considerable content of determined flavonoids, which are natural polyphenol products with particularly wide and potent biological activity, can implicate antioxidant, antispasmodic, cytotoxic or anti-inflammatory activity of investigated species (Beara et al., 2009; Havsteen, 2002). 3.2. Antioxidant activity Antioxidant activity is manifested in a wide variety of actions, such as inhibition of oxidising enzymes, chelation of transition metals, transfer of hydrogen or single electron to radicals, singlet oxygen deactivation, or enzymatic detoxification of reactive oxygen species, the total antioxidant activities should be evaluated through different methods in order to extensively characterise the antioxidant potential of pure compounds or extracts. Therefore, the extracts of P. altissima and P. lanceolata, as well as standard antioxidant 3,5-di-tert-butyl-4-hydroxytoluene (BHT), were examined with regard to scavenging capacity towards DPPH, hydroxyl, superoxide anion, and nitric oxide radicals, lipid peroxidation and reducing power. Antioxidant activities of both extracts were concentration dependent, and determined IC50 values are shown in Tables 3 and 4. The IC50 value was not determined for BHT towards Table 4 IC50 Values for lipid peroxidation assay and reducing power of examined extracts and BHT. Extract IC50 values, lipid peroxidation (mg/mL) Reducing power, FRAP (mg of AAE/g of dw) P. altissima P. lanceolata Standard, BHT 177.71  9.5 c 24.83  1.77 b 21.29  0.40 a 66.70  3.33 c 109.80  6.57 b 25.32  2.5 a Values are means  SD of three measurements. Means within each column with different letters (aec) differ significantly (p < 0.05). 69 I.N. Beara et al. / LWT - Food Science and Technology 47 (2012) 64e70 Table 5 IC50 Values for COX-1 and 12-LOX assay of examined extracts and standard compounds. Extract P. altissima P. lanceolataa Standard Aspirina Quercetina IC50 values (mg/mL) COX-1 inhibition 12-LOX inhibition 4.42  0.33 c 2.00  0.34 b 3.59  0.30 c 0.75  0.08 b 0.001  0.00008 a Na Na 0.084  0.007 a Values are means  SD of three measurements. Means within each column with different letters (aec) differ significantly (p < 0.05). Na, not active in applied concentration range. a Reference Beara et al., 2010. the superoxide anion and nitric oxide radicals because BHT reached no more than 32 and 10% of inhibition, respectively. The concentration of BHT was in the range of 0.1e10.0 mg/mL, and the low inhibition potency can be explained by the low solubility of BHT in aqueous buffers. Overall, our results demonstrate that P. lanceolata and P. altissima showed potent antioxidant effects comparable with synthetic antioxidant BHT. In addition, our results show that antioxidant activity of P. lanceolata was markedly higher than that of P. altissima. This is the first reported antioxidant activity of P. altissima whereas that of P. lanceolata has been described previously (Gálvez, Martín-Cordero, Houghton, & Ayuso, 2005). The high antioxidant activity of the species examined here is probably attributed to the presence of aforementioned phenolics, since antioxidant effects have often been regarded to be dependent on the presence of this class of compounds. Furthermore, dietary intake of phenolics has been associated to reduce the risk of different diseases, such as cancer, cardiovascular disease, diabetes, or atherosclerosis (Beara et al., 2009), probably due to their potent antioxidant properties (Havsteen, 2002). Accordingly, the antioxidant activity of these Plantago species can contribute to their benefits as a food or food supplement. 3.3. Anti-inflammatory activity To evaluate the anti-inflammatory activity of P. altissima extract an optimized ex vivo test for determination of COX-1 and 12-LOX inhibition potency was performed. The extract demonstrated inhibitory activity towards COX-1 (IC50 ¼ 4.4 mg/mL) and 12-LOX (IC50 ¼ 3.6 mg/mL), although it was significantly lower than the activity of a well-known potent inhibitor of COX-1, aspirin (IC50 ¼ 0.001 mg/mL) and 12-LOX, quercetin (IC50 ¼ 0.084 mg/mL). In comparison to the previously reported activities of P. lanceolata (Beara et al., 2010), P. altissima expressed a lower activity (Table 5). Metabolism of arachidonic acid gives rise to pro-inflammatory agents (Smith, 1989) and these reactions are based on free radical mechanisms. Phenolics are renowned for their free radical scavenging capacity, therefore the high content of these compounds in our extracts may be the reason for potent inhibition of the COX-1 and 12-LOX pathways seen here. However, the complex cascade of arachidonic acid metabolism involves not only COX-1 and 12-LOX enzymes, but also a 15- and 5-lipoxygenase, cyclooxygenase-2, cytochrome P450 and epoxygenase. Accordingly, further investigation involving other inflammatory routes is required in order to determine whether the extracts of the examined species do posses anti-inflammatory activity in other steps of the pathway. Furthermore, the results obtained may form the basis of future studies towards cytotoxic activity in cancer cell lines, since it was found that some compounds, like 12-HETE, a product of 12-LOX, are involved in the progression of various cancers (Nie & Honn, 2002). Moreover, even though COX-1 is constitutively expressed in different tissues and is involved in normal cellular homeostasis, it can be overexpressed in diverse cancer cells, whilst the selective inhibitors of this enzyme can influence their growth, proliferation and apoptosis (Daikoku et al., 2005; Hong et al., 1999; McFadden, Riggs, Jackson, & Cunningham, 2006). 3.4. Cytotoxic activity The cytotoxicity of P. altissima and P. lanceolata extracts and podophyllotoxin, a potent cytotoxin, for human cell lines HeLa (cervix epitheloid carcinoma), MCF7 (breast adenocarcinoma), HT-29 (colon adenocarinoma) and MRC-5 (human fetal lung) is shown in Table 6. Treatment of four cell lines with both extracts and podophyllotoxin resulted in a considerable dose-dependent inhibition of cell growth, where HeLa and MCF7 were more sensitive to both extracts than the other two cell lines (MRC-5 and HT-29). Stronger inhibitory effects were observed with P. lanceolata, which exerted growth inhibition of HeLa, MCF7 and HT-29 cell lines by 50% at lower concentrations than P. altissima. Both extracts also affected growth of human lung normal cells (MRC-5), but reached IC50 at higher concentrations than for cancer cell lines. This suggests that these extracts are more cytotoxic towards cancer cells. In comparison to the highly potent cytotoxic agent, podophyllotoxin, both extracts had significantly lower activity. There have been no previous reports on the cytotoxic potential of P. altissima. However, results in this study are comparable to previous reports concerning the inhibitory effects of P. lanceolata extract on the growth of breast adenocarcinoma cells (Gálvez et al., 2003). For the first time, we have shown that this species has the potential to slow the growth of cervix epitheloid carcinoma and colon adenocarinoma cells using immortalised cell lines. Previous studies have demonstrated that chlorogenic, vanillic and gallic acid, luteolin, luteolin-7-O-glucoside and apigenin are potent anticancer agents (Gálvez et al., 2003; Marques & Farah, 2009; Patel, Shukla, & Gupta, 2007). These phenolics are present in significant amounts in P. altissima and P. lanceolata and their presence can be correlated with the anti-cancer properties seen in these extracts. Accordingly, the Plantago species examined in this study could be regarded as possible anti-cancer agents and possible source of new therapeutics. Table 6 IC50 Values of examined extracts and standard compounds for SRB assay standards concentration (mg/mL) required to inhibit cell growth by 50% (IC50). Extract P. altissima P. lanceolata Standard Podophyllotoxin IC50 (mg/mL) MRC-5 HeLa MCF7 HT-29 920.65  53.57 c 551.69  53.18 b 262.55  32.15 c 172.33  11.74 b 255.16  21.33c 142.78  3.50 b 511.99  20.00 c 405.50  47.82 b 0.0047  0.0008 a 0.0041  0.0003 a 0.0013  0.0002 a 0.0030  0.0005 a Values are means  SD of eight measurements. Means within each column with different letters (aec) differ significantly (p < 0.05). 70 I.N. Beara et al. / LWT - Food Science and Technology 47 (2012) 64e70 4. Conclusions As a result of the extensive use of Plantains as remedies and medicinal food, it is of great importance to investigate their chemical profile and bio-potential. Although some renowned species, such as Plantago major and P. lanceolata, are the subject of numerous studies that confirm their benefits, there are many Plantain species that are still unexplored. In this study, the phenolic profile, antioxidant, anti-inflammatory and cytotoxic activity of P. altissima are reported for the first time. Results obtained for P. altissima are compared to the well-known P. lanceolata, which can be easily mistaken for each other due to their morphological similarities. Both extracts showed notable phenolic content, as well as antioxidant, anti-inflammatory (COX-1 and 12-LOX inhibition potential) and cytotoxic activity. However, overall results indicate that P. lanceolata demonstrates superior bio-potential and its use as traditional remedy and functional food is validated. In general, this study showed that the unexplored P. altissima should not be carelessly mistaken with P. lanceolata, since the differences in the examined phenolics, mostly regarding quantitative content, and biological activities are clear. 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