Phenolic Compounds with Antioxidant Activity from Anthemis tinctoria L. (Asteraceae) Paraskevi Papaioannoua, Diamanto Lazaria,*, Anastasia Kariotib, Christos Soulelesa, Jörg Heilmannc, Dimitra Hadjipavlou-Litinad, and Helen Skaltsab,* a Laboratory of Pharmacognosy, School of Pharmacy, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece. Fax: +2310997662. E-mail: [email protected] b Department of Pharmacognosy & Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, 15771 Athens, Greece c Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece d Institute of Pharmacy, Department of Pharmaceutical Biology, University of Regensburg, Universitätsstrasse 31, D-93053, Regensburg, Germany * Authors for correspondence and reprint requests Z. Naturforsch. 62c, 326Ð330 (2007); received January 8, 2007 From the aerial parts of Anthemis tinctoria L. subsp. tinctoria var. pallida DC. (Asteraceae), one new cyclitol glucoside, conduritol F-1-O-(6Ј-O-E-p-caffeoyl)--d-glucopyranoside (1), has been isolated together with four flavonoids, nicotiflorin (2), isoquercitrin (3), rutin (4) and patulitrin (5). The structures of the isolated compounds were established by means of NMR, MS, and UV spectral analyses. Methanolic extract and pure isolated compounds were examined for their free radical, scavenging activity, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free stable radical, and for their inhibitory activity toward soybean lipoxygenase, using linoleic acid as substrate. Compounds 1 and 5 showed a strong scavenging effect in the DPPH radical assay. In addition 5 also exhibited high inhibitory activity on soybean lipoxy- genase. Key words: Anthemis tinctoria subsp. tinctoria pallida, Asteraceae, Flavonoids, Conduritol F Introduction Materials and Methods The genus Anthemis comprises about 130 spe- General experimental procedures cies predominately distributed around the Medi- α terranean (Heywood and Humphries, 1978). The The [ ]D values were obtained in MeOH at species of the Anthemis genus are widely used in 20 ∞C on a Perkin-Elmer 341 polarimeter. IR spec- pharmaceutics, cosmetics and food industry. The tra were obtained on a Perkin-Elmer Paragon 500 flowers of the genus have well-documented use as instrument. UV spectra were recorded on Shim- antiseptic and healing herbs, the main components adzu UV-160A and Hitachi U-2000 spectropho- being natural flavonoids and essential oils. In Eu- tometers according to Mabry et al. (1970). The 1H rope extracts, tinctures, tisanes (teas), and salves NMR spectra (400 MHz) and 13C NMR spectra are widely used as anti-inflammatory, antibacte- (50 and 100 MHz) were recorded in CD3OD using rial, antispasmodic, and sedative agents. Extracts Bruker DRX 400 and Bruker AC 200 spectrome- are used to allay pain and irritation, clean wounds ters. Chemical shifts are reported in δ (ppm) val- and ulcers, and aid prevention as well as therapy ues relative to TMS. COSY, HMQC, HSQC, of irradiated skin injuries, treatment of cystitis and HMBC and NOESY (mixing time 950 ms) were dental afflictions (Mann and Staba, 1986). Con- performed using standard Bruker microprograms. tinuing our chemotaxonomic examinations of the High resolution ESI mass spectral data were re- Greek flora belonging to Asteraceae and our corded on a TSQ 7000 mass spectrometer. Vac- search for new compounds of pharmacological in- uum-liquid chromatography (VLC): silica gel 60H terest, we now report the investigation of the aer- (Merck Art. 7736). Column chromatography: silica ial parts of Anthemis tinctoria L. subsp. tinctoria gel 60 (Merck Art. 9385), gradient elution with the var. pallida DC. solvent mixtures indicated in each case; Sephadex 0939Ð5075/2007/0500Ð0326 $ 06.00 ” 2007 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D P. Papaioannou et al. · Anthemis tinctoria and Antioxidant Activity 327 1 13 LH-20 (Pharmacia), elution with MeOH. Absorb- Table I. H (CD3OD, 400 MHz, J in Hz) and C (50 MHz) NMR data of compound 1 (at 295 K). ents for TLC: Merck RP 18 F254s, Art. 5685; Merck silica gel 60 F254s, Art. 5554; Merck cellulose, Art. δ δ Position H C 5716. Detection on TLC plates (silica gel): UV light, vanillin-H2SO4 spray reagent; Neu spray re- Conduritol F agent on cellulose (Neu, 1957). 1 4.18 dd (J = 4.6, 4.1) 76.2 2 3.50 dd (J = 9.9, 3.7) 72.3 3 3.67 dd (J = 9.9, 7.5) 74.8 Plant material 4 3.94 br d (J = 7.4) 74.0 5 5.79 dd (J = 10.0, 2.5) 135.8 Aerial parts of Anthemis tinctoria L. subsp. tinc- 6 5.89 ddd (J = 9.9, 4.6, 1.6) 126.0 toria var. pallida DC. were collected on Mount Glucose Ј Pelion in June 2002 and authenticated by Dr. Th. 1 4.46 d (J = 7.9) 103.8 2Ј 3.28 dd (J = 7.8, 9.1) 74.7 Constantinidis (Institute of Systematic Botany, 3Ј 3.39 t (J = 7.8) 78.0 Agricultural University of Athens). A voucher 4Ј 3.39 t (J = 7.8) 71.9 specimen is deposited in the Herbarium of Labo- 5Ј 3.58 ddd (J = 9.9, 6.2, 2.1) 75.9 ratory of Pharmacognosy, University of Athens 6Јa 4.56 dd (J = 12.0, 2.1) 64.7 6Јb 4.27 dd (J = 12.0, 6.2) under the number Skaltsa & Lazari 135. Caffeoyl group 1Љ Ð 128.1 Extraction and isolation 2Љ 7.05 d (J = 2.1) 115.9 3Љ Ð 147.6 Air-dried powdered aerial parts of the plant 4Љ Ð 149.8 (0.74 kg) were extracted at room temperature with 5Љ 6.78 d (J = 8.3) 117.1 6Љ 6.96 dd (J = 1.6, 8.3) 123.7 cyclohexane/Et2O/MeOH (1:1:1 v/v/v). The ex- 7Љ 7.60 d (J = 15.8) 147.4 tract was washed with brine, the aqueous layer re- 8Љ 6.30 d (J = 16.2) 115.2 extracted with EtOAc, and the organic layer dried 9Љ Ð 169.8 with Na2SO4 and concentrated under reduced pressure. In continuation, the plant was exhaus- tively extracted with methanol at room tempera- λ ε (7.9 mg). Ð UV (MeOH): max = 290.5 nm (log = ture. The residue of EtOAc (5.4 g) was prefrac- ε α 20 3.80), 325 nm (log = 3.78). Ð [ ]D Ð11.73∞ tionated by VLC on silica gel (8.5 ¥ 6.0 cm), using (MeOH, c 0.09). Ð ESI-HRMS (pos.): m/z = hexane/EtOAc/Me CO mixtures of increasing po- + 2 471.1498 [M+H] (required for C21H26O12 larity as eluents to give eight fractions of 500 mL 471.1503). Ð 1H and 13C NMR spectral data: see each: A (hexane/EtOAc, 75:25 v/v), B (hexane/ Table I. EtOAc, 50:50 v/v), C (hexane/EtOAc, 25:75 v/v), D (EtOAc, 100%), E (EtOAc/Me2CO, 90:10 v/v), Scavenging activity on DPPH radical F (EtOAc/Me2CO, 75:25 v/v), G (Me2CO, 100%) and H (MeOH, 100%). Fraction H (3.9 g) was sub- The free radical scavenging activity of the ex- jected to further chromatographic separations as tracts and isolates was performed using the DPPH method, as previously described (Kontogiorgis described below. VLC of fraction H (CH2Cl2/ MeOH, 10:0 to 0:10 v/v) followed by several CC and Hadjipavlou-Litina, 2003). Briefly, 1 mL on silica gel and Sephadex LH-20, allowed the iso- (0.1 mm) solution of DPPH in ethanol was added μ lation of 2 (34.5 mg). The methanol extract was to an equal volume of the tested extract (20 Lor μ concentrated and 22.0 g of the residue (36.9 g) 200 L in DMSO) and compounds (final concen- tration 0.1 mm and 0.2 mm) and left at room tem- were subjected to VLC on silica gel using CH2Cl2/ MeOH mixtures of increasing polarity as eluents perature for 20 and 60 min. After incubation the to give several fractions. VLC of fraction N (4.1 g) absorbance was recorded at 517 nm (CH2Cl2/MeOH, 70:30 Ð CH2Cl2/MeOH, 60:40), followed by several CC on silica gel and Sephadex Soybean lipoxygenase inhibition LH-20, allowed the isolation of 1 (7.9 mg), 2 The bioassay was performed according to a pre- (9.4 mg), 3 (2.4 mg), 4 (17.5 mg) and 5 (4.5 mg). viously described procedure (Kontogiorgis and Hadjipavlou-Litina, 2003). All samples (extract Conduritol F-1-O-(6Ј-O-E-p-caffeoyl)--d-glu- and isolates) were initially dissolved in DMSO copyranoside (1): Amorphous yellow powder (approx. 50 mg in 2 mL DMSO for plant extract). 328 P. Papaioannou et al. · Anthemis tinctoria and Antioxidant Activity The incubation mixture consisted of several ali- Compound 1 was obtained as an amorphous yel- quots of the test sample, 100 μL of sodium linole- lowish powder and was identified as conduritol F- ate (0.1 mm) and 0.2 mL of the enzyme solution 1-O-(6Ј-O-E-p-caffeoyl)--d-glucopyranoside by (1/3 ¥ 10Ð4, w/v in saline). After incubation at 1D and 2D NMR spectroscopic analyses and by room temperature for 3 min the conversion of the MS spectrometry. Its ESI-HRMS spectrum ex- sodium linoleate to 13-hydroperoxylinoleic acid hibited a pseudomolecular ion [M+H]+ at m/z was recorded at 234 nm and compared with an ap- 471.1498, compatible with the molecular formula propriate standard inhibitor (quercetin). The same C21H26O12. In agreement, 21 carbon signals were procedure was followed for the pure compounds observed in the 13C NMR spectrum. Besides the 6 13 in order to determine the IC50 value of each com- carbon signals of a sugar moiety, the C NMR pound.