Phytoconstituents and in vitro Evaluation of Antioxidant Capacities of Cotula Cinerea (Morocco) Methanol Extracts

SHORT REPORT Rec. Nat. Prod. 9:4 (2015) 572-575 Phytoconstituents and in vitro Evaluation of Antioxidant Capacities of Cotula Cinerea (Morocco) Methanol Extracts Farid Khallouki,1, 3 Khalid Sellam,2 Rabia Koyun,3 Irvila Ricarte,3 Chakib Alem,1 Lhoussaine Elrhaffari2 and Robert W. Owen3* 1 Biochemistry of Natural Products Laboratory, Faculty of Sciences & Techniques, 52000 Errachidia, Morocco 2 Microbiology Laboratory, Department of Biology, Faculty of Sciences & Techniques, 52000 Errachidia, Morocco 3 Division of Preventive Oncology, National Center for Tumor Diseases, Im Neuenheimer Feld, 581, 69120, Heidelberg, Germany (Received October 02, 2014; Revised March 23, 2015; Accepted March 27, 2015) Abstract: The purpose of this study was to determine the phytochemical content of Cotula cinerea to establish principal components which may consolidate its use as a medicinal plant in the southeast of Morocco. The amount of total phenolic compounds as determined by analytical HPLC in methanol extracts was 79.23 ± 2.5 mg/g dry matter. The major phenolic compounds identified by HPLC-ESI-MS were neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5dicaffeoylquinic acid and luteolin-4´-O-glucoside. All compounds displayed very strong antioxidant capacities in the DPPH, FRAP and ORAC assays. The data indicates that methanol extracts of C. cinerea via their antioxidant capacities, may be effective disease prevention potions in traditional African medicine which is probably related to the significant content of echinoids and flavonoids. Keywords: Cotula cinerea; chlorogenic acids; flavonoids; antioxidant capacity. © 2015 ACG Publications. All rights reserved. 1. Plant Source The aerial parts of C. cinerea were collected in Errachidia (Morocco), during the flowering period (April/June, 2009). The plant was previously identified and authenticated by Dr Ben Tatou at the Scientific Institute, Rabat, Morocco and a voucher specimen (FSTBIO 04) is deposited at the herbarium of the Faculty of Science and Techniques, Errachidia. The dried plant material was stored in the dark, at room temperature (25°C) before extraction. 2. Previous Studies C. cinerea (Compositae, subfamily Asteroideae, Tribe Anthemideae, subtribe Cotulinae) is a fragrant, perennial herb with yellow flowers, which grows in the southeast of Morocco and Algeria. It is known locally as “guertoufa”. Traditionally, the leaves of this plant are still used for colic, cough, bronchopulmonary cooling, diarrhoea and digestive disorders [1], and an analgesic effect has also been previously described [2]. To date, no information with regard to the phytochemical content of C. cinerea has been published except its essential oil composition (trans-thujone as a major component) * Corresponding author: E- Mail: (Robert W. Owen), Phone +49-6221-42-3317 The article was published by Academy of Chemistry of Globe Publications www.acgpubs.org/RNP © Published 06/01/2015 EISSN:1307-6167 Capacities of Cotula Cinerea (Morocco) methanol extracts 573 [3] and the purpose of this study was to determine the phytochemical content of C. cinerea to establish principal components which may consolidate its use as a medicinal plant in the southeast of Morocco. 3. Present Study The powdered aerial parts of C. cinerea (20 g) were extracted with hexane (1x3h) to remove lipid followed by methanol (3x3h) in a Soxhlet apparatus. The methanol extracts were concentrated to dryness, and the residues stored at 4 °C. Reverse-phase analytical HPLC, HPLC-ESI-MS and semipreparative HPLC were conducted as described previously [4-6]. Full details of the analytical methods are given in the Supporting information file. Finally, antioxidant activity of the purified compounds was assessed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing ability of plasma (FRAP) and oxygen radical absorbance capacity (ORAC) assays as described previously [7]. Identification of phenolic compounds in solvent extracts of C. cinerea: The major phenolic compounds identified in the solvent extracts of C. cinerea were echinoid (chlorogenic acid isomers and dicaffeoyl quinic acid isomers) and a flavonoid as identified by HPLC-ESI-MS-MS (Table 1). An analytical HPLC chromatogram at 340 nm of a methanol extract is depicted in Fig. 1. along with the extracted negative-ion chromatograms [M – H]– at m/z 353, 515 and 447 representing the chlorogenic acid isomers, the dicaffeoylquinic acid isomers and luteolin-4´-O-glucoside respectively. Mass spectra are given in the Supporting information file. The concentrations in mg/g dry matter were neochlorogenic acid (7.9), chlorogenic acid (11.9), cryptochlorogenic acid (7.1), 3,4-dicaffeoylquinic acid (6.8), 3,5-dicaffeoylquinic acid (7.9), 4,5-dicaffeoylquinic acid (7.1) and luteolin-4´-O-glucoside (30.6). The structures are shown in Fig. 2. All compounds displayed very strong antioxidant capacities in the DPPH, FRAP and ORAC assays: e.g. far stronger (x 3.3 on average) in the ORAC assay, than the water soluble derivative of vitamin E, Trolox (Table 2). These principal components may contribute to the effectivity of C. cinerea extracts in traditional African medicine. Figure 1. Reverse-phase HPLC (UV, 340 nm) and extracted ion chromatograms of a methanol extract of C. cinerea 1: neochlorogenic acid; 2: chlorogenic acid; 3: cryptochlorogenic acid; 4: 3,4-dicaffeoylquinic acid; 5: 3,5-dicaffeoylquinic acid; 6: luteolin-4´-O-glucoside; 7: 4,5-dicaffeoylquinic acid. Khallouki et.al., Rec. Nat. Prod. (2015) 9:4 572-575 574 Table 1. HPLC-ESI-MS data of the phenolic compounds detected in the methanol extracts of C. cinerea. [M – H]– No Phenolic compound I Neochlorogenic acid (M = 354) 353.1 II Chlorogenic acid (M = 354) 353.1 III Cryptochlorogenic acid (M = 354) 353.1 IV 3,4-Dicaffeoylquinic acid (M = 516) 515.0 V 3,5-Dicaffeoylquinic acid (M = 516) 515.1 VI VII Luteolin-4´-O-glucoside (M = 448) 4,5-Dicaffeoylquinic acid (M = 516) 447.1 515.0 COOH OH COOH OH O OH OH 191.1 = [M – H]–-caffeic acid 179.1 = [M – H]–-quinic acid 191.1 = [M – H]–-caffeic acid 179.1 = [M – H]–-quinic acid 191.1 = [M – H]–-caffeic acid 179.1 = [M – H]–-quinic acid 353.1 = [M – H]–-caffeic acid 191.1 = [M – H]–-2 x caffeic acid 179.1 = [M – H]–-caffeic acid + quinic acid 353.1 = [M – H]–-glucose 191.1 = [M – H]–-2 x caffeic acid 179.1 = [M – H]–-caffeic acid + quinic acid 285.1 = [M – H]–-glucose 353.1 = [M – H]–-glucose 191.1 = [M – H]–-2 x caffeic acid 179.1 = [M – H]–-caffeic acid + quinic acid COOH OH O HO OH O C HO C O OH O H H HPLC-ESI-MS fragmentation (neg. ion m/z) O C H H H H II I OH OH III OH OH OH OH COOH OH COOH OH H O OH O O C O C O C O H H O OH C O H H H H H V IV OH OH OH OH OH OH OH OH COOH OH OH O HO O O HO HO HO O C O O O C OH H VI H H OH H C OH O OH VII OH OH OH Figure 2. Structures of the phenolic c (...truncated)