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Antioxidant properties of marigold extracts
Food Research International 37 (2004) 643–650 www.elsevier.com/locate/foodres
Antioxidant properties of marigold extracts Gordana S. Cetkovi c *, Sonja M. Djilas, Jasna M. Canadanovi c-Brunet, Vesna T. Tumbas Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia and Montenegro Received 26 August 2003; accepted 28 January 2004
Abstract The influence of methanolic and water extracts of growing wild marigold, Calendula arvensis L. (GWM) and cultivated marigold, Calendula officinalis L. (CM), in a concentration range of 0.10–0.90 mg/ml, was evaluated on three different free-radical species: 2,2diphenyl-1-picrylhydrazyl free radical (DPPH), hydroxyl radical and lipid peroxyl radical using electron spin resonance (ESR) spectroscopy. These extracts of CM and GWM, scavenged all types of investigated radicals in dependence on their applied concentrations. Generally, CM extracts possessed better scavenging and antioxidant activity than GMW extracts, while methanolic extracts exhibited lower activities than water extracts. Water extracts of CM had the best antioxidant properties; 0.75 mg/ml extracts completely eliminated hydroxyl radical, which was generated in Fenton system. The same concentration of this extract scavenged 92% DPPH and 95% peroxyl radical during lipid peroxidation. Antioxidant properties were in correlation with the contents of total phenolic compounds (14.49–57.47 mg/g) and flavonoids (5.26–18.62 mg/g) in extracts. The formation of o-semiquinone radicals from rutin and caffeic acid in lipid peroxidation system proved the mechanism (hydrogen donating and/or one-electron reduction) of free-radical scavenging activity. The ESR data demonstrate that methanolic and water extracts of CM possess similar free radicals scavenging and antioxidative activity as synthetic antioxidants BHA. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Growing wild and cultivated marigold; Free radicals; Polyphenols; Electron spin resonance
1. Introduction The potential toxicity of synthetic antioxidants (butylated hydroxyanisole-BHA, butylated hydroxytoluene-BHT, tertiary butylhydroquinone, esters of 3,4,5trihydroxybenzoic acid, etc.) (Ito, Fukushima, & Tsuda, 1985) has aroused an increased interest in preparing antioxidants from natural sources such as herbs, spices, seeds, cereals, fruits, and vegetables by extraction, fractionation and purification (Dillard & German, 2000; Wang & Lin, 2000). Numerous investigations have proved that medicinal herbs contain diverse classes of compounds such as polyphenols, tocopherols, alkaloids, tannins, carotenoids, terpenoids, etc. (Velioglu, Mazza, Gao, & Oo-
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Corresponding author. Tel.: +381-21-450-648; fax: +381-21-450413. E-mail address: [email protected] (G.S. Cetkovi c). 0963-9969/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.01.010
mah, 1998; Yoshida et al., 1989). Among them, flavonoids and phenolic acids are particularly attractive as they are known to exhibit various beneficial pharmacological properties such as vasoprotective, anticarcinogenic, antineoplastic, antiviral, anti-inflammatory, as well as antiallergic and antiproliferative activity on tumor cells (Carr, Zhu, & Frei, 2000; Kuhnau, 1976; Middleton & Kandaswami, 1992). Some of these properties have been related to the action of these compounds as antioxidants, free-radical scavengers, quenchers of singlet and triplet oxygen, and inhibitors of peroxidation. Antioxidant activity of phenolic compounds is correlated to some structure–activity relationships, such as redox properties and the number and arrangement of the hydroxyl groups (Cotelle et al., 1996). Marigold is a herb of ancient medicinal repute. In traditional and homeopathic medicine it has been used for skin complaints, wounds and burns, conjunctivitis and poor eyesight, menstrual irregularities, varicose
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veins, hemorrhoids, duodenal ulcers, etc. (Wichtl, 1994). Marigold grows as a wild and common garden plant throughout Europe and North America. The yellow or golden-orange flowers of marigold are used as spice, tea and medicine. They may be used as fresh or dried, and can be made into tea, tinctures, ointments and creams. The pharmacological activity of marigold is related to the content of several classes of secondary metabolites such as essential oils, flavonoids, sterols, carotenoids, tannins, saponins, triterpene alcohols, polysaccharides, a bitter principle, mucilage, and resin (Vidal-Ollivier et al., 1989). Bilia, Salvini, Mazzi, and Vincieri (2001) found that marigold flowers contain rutin, isoquercitrin, quercetin-3-O-rutinosylrhamnoside, isorhamnetin-3-Orutinosylrhamnoside, isorhamnetin-3-O-glucosylglucoside, and isorhamnetin-3-O-glucoside. According to the fact that marigold contains polyphenols, the assessment of its antioxidant properties is of great interest in the understanding of positive effect of these compounds especially in phytotherapy. The task of this study is to investigate the influence of the methanolic and water extracts of growing wild marigold, Calendula arvensis L. (GWM) and cultivated marigold, Calendula officinalis L. (CM) on stable 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical, reactive hydroxyl radical (scavenging activity, SA) and lipid peroxyl radicals (antioxidant activity, AA) using ESR spectroscopy.
2.2. Marigold extracts preparation Dried flowers of GWM and CM (5 g) were extracted with 70% methanol or distilled water (250 ml) in a shaker incubator at 25 °C for 24 h. The extracts were filtered, and obtained filtrates were concentrated under the reduced pressure to dryness. The yields (g), averages of triplicate analysis, of obtained extracts were:
GWM CM
Methanolic extract 1.2964 0.0095 1.3772 0.0101
Water extract 1.3528 0.0099 1.3876 0.0102
2.3. Total phenolic compounds in extracts Total phenolic compounds in extracts were determinated spectrophotometrically using the Folin–Ciocalteu reagent and the results are expressed as chlorogenic acid equivalents per g dry weight (Singleton, Orthofer, & Lamuela-Raventos, 1999). 2.4. Total flavonoids in extracts Total flavonoids in extracts (expressed as mg rutin per g dry weight) were estimated spectrophotometrically according to Markham (1989). 2.5. Thin-layer chromatography (TLC) and scavenging screening test of extracts
2. Materials and methods 2.1. Chemicals and plant materials Methanol, ethyl acetate, hydrogen peroxide, acetic and formic acid were obtained from ‘‘Zorka’’ Sabac (Serbia). DPPH, 5,5-dimethyl-1-pyrroline-N -oxide (DMPO) and a-phenyl-N tert-butylnitrone (PBN), Folin–Ciocalteu reagent, BHA, standards of flavonoids (kaempferol, quercetin and rutin) and phenolic acids (chlorogenic, caffeic, p-coumaric and vanillic acids) were purchased from Sigma Chemical Co. (USA). These chemicals were of analytical reagent grade. Other used chemicals and solvents were of the highest analytical grade. Methyl esters of sunflower oil were supplied by Fluka Chemie A.G. (Buchs, Switzerland). Plants were purchased from local herbal drugstore. Flowers of GWM were collected in the period May– June, 2001, in the region of mountain Zlatibor (Serbia), while flowers of CM were harvested during May, 2001, in the experimental garden in Zabalj, flat ground region of Vojvodina (Serbia). Voucher specimens of the collected plants (C. arvensis No. 1048 and C. officinalis No. 1053) were confirmed and deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Medicine, University of Novi Sad.
TLC was performed on 20 20 cm plates precoated with microcrystalline cellulose (Camag, Muttanez, Switzerland). Volumes of 1 ll of 1% methanolic solution of standards and investigated extracts were spotted on the plates. TLC analysis was performed with ethyl acetate:formic acid:acetic acid:water in the volume ratio 100:11:11:26 as mobile phase. Spots were observed under UV light at 366 nm and sprayed with 0.1% methanolic solution of DPPH radical (Dapkevicius et al., 2002). 2.6. DPPH radical assay Blank probe was obtained by mixing 600 ll 0.4 mM methanolic solution of DPPH and 200 ll of methanol. A volume of x ll of 1% methanolic solution of investigated extract was added to a mixture of (200 x) ll of methanol and 600 ll of 0.4 mM methanolic solution of DPPH radical (probe). The range of the investigated extract concentrations was 0.10–0.90 mg/ml. After that the mixture was stirred for 2 min and transferred to a quartz flat cell ER-160FT. The ESR spectra were recorded on an ESR spectrometer Bruker 300E (Rheinstetten, Germany) under the following conditions: field modulation 100 kHz, modulation amplitude 0.256 G, receiver gain 2 104 , time constant 40.96 ms, conver-
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sion time 327.68 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23 °C. The SA value of the extract was defined as: SA ¼ 100 ðho hx Þ=ho (%); where ho and hx are the height of the second peak in the ESR spectrum of DPPH radicals of the blank and the probe, respectively. 2.7. Hydroxyl radical assay Hydroxyl radicals were obtained by the Fenton reaction in the system: 0.2 ml 10 mM H2 O2 , 0.2 ml 10 mM FeCl2 4H2 O and 0.2 ml 0.3 M DMPO as spin trap (blank). The influence of methanolic and water extracts on the formation and transformation of hydroxyl radicals was investigated by adding the extracts to the Fenton reaction system in the range of concentrations 0.10–0.90 mg/ml. ESR spectra were recorded after 5 min, with the following spectrometer settings: field modulation 100 kHz, modulation amplitude 0.512 G, receiver gain 2 105 , time constant 81.92 ms, conversion time 163.84 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23 °C. The SA value of the extract was defined as: SA ¼ 100 ðho hx Þ=ho (%), where ho and hx are the height of the second peak in the ESR spectrum of DMPO-OH spin adduct of the blank and the probe, respectively.
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x-band frequency 9.64 GHz, power 20 mW, temperature 23 °C. The AA value of the extract was defined as: AA ¼ 100 ðho hx Þ=ho (%) where ho and hx are the height of the first peak in the ESR spectrum of PBN-OOL spin adduct of the blank and the probe, respectively. Magnetic field scanning determinations were calibrated using FremyÕs salt (peroxyllamine disulphonate). Splitting constants were calculated from computer-generated second derivatives of the spectra, after optimizing signal-to-noise ratios and were verified by computer simulations. 2.9. Statistical analysis The StudentÕs t-test and ANOVA were used to determine the statistical difference. The criterion for statistical significance was p < 0:05, unless indicated otherwise. Data presented in graphs show the calculated means of three replications with vertical bars as standard deviation. Statistical analysis was carried out using Microsoft Excel 2000.
3. Results 3.1. Characterization of the phenolic compounds and the flavonoids in extracts
2.8. Peroxyl radical assay Methyl esters of sunflower oil were exposed to air for 72 h at room temperature to allow their peroxidation. The oil prepared in this way contained about 60% hydroperoxide-enriched methyl linoleate. Blank probe was obtained by mixing 0.6 g of hydroperoxide enriched methyl linoleate of sunflower oil, 0.1 ml of 0.1 mM FeCl2 4H2 O and 0.085 g of PBN as spin trap. The influence of all investigated extracts on the formation and transformation of peroxyl radicals was investigated by adding the extracts to the blank (before adding FeCl2 4H2 O) in the range of concentrations 0.10–0.90 mg/ml. ESR spectra were recorded after 24 h, with the following spectrometer settings: field modulation 100 kHz, modulation amplitude 1.021 G, receiver gain 5 105 , time constant 327.68 ms, conversion time 1310.72 ms, center field 3440.00 G, sweep width 100.00 G,
The contents of total phenolic compounds and flavonoids in methanolic and water extracts of GWM and CM are presented in Table 1. The obtained results showed that the contents of total phenolic compounds and flavonoids in methanolic and water extracts of CM were significantly higher (p < 0:05) than in the corresponding extracts of GWM. However, the contents of total phenolic compounds in methanolic and water extracts of GWM were not significantly different (p > 0:05), and neither were the contents of flavonoids (p > 0:05). The same conclusion holds for both types of CM extracts. The results of TLC analyses showed that flavonoids (kaempferol, quercetin and rutin, as well as some other glycosides) and phenolic acids (chlorogenic, caffeic, p-coumaric and vanillic) were present in the methanolic and water extracts of GWM and CM.
Table 1 Contents of total phenolic compounds and flavonoids in methanolic and water extracts of GWM and CM Extract
Total phenolic compounds (mg/g)
Total flavonoids (mg/g)
Methanolic extract of GWM Water extract of GWM Methanolic extract of CM Water extract of CM
14.49 0.38 15.12 0.40 55.07 1.11 57.47 1.12
5.26 0.36 5.31 0.36 18.48 0.70 18.62 0.73
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The scavenging screening test of the methanolic and water extracts of both types of marigold was assessed by spraying the TLC plates with a solution of purple colored stable DPPH. The reduction of the color could be observed visually as a yellowish spot on a purple background (results are not shown).
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The ESR spectra of DPPH radicals in the blank and in probes with water and methanolic extracts of GWM and CM are characterized by their five lines of relative intensities 1:2:3:2:1 and hyperfine splitting constant aN ¼ 9.03 G (Fig. 1). The DPPH radicals scavenging activity of all extracts increased dose-dependently at mass concentrations ranging from 0.10 to 0.90 mg/ml (Fig. 2). With increasing concentrations, scavenging activity of methanolic and water extracts of CM increased from 15.63% to 95.34% and from 28.32% to 100%, respectively. The difference between SA of these extracts was statistically significant (p < 0:01). All concentrations tested for both extracts of GWM possessed consistently lower SA
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Fig. 2. The SA of different concentrations of the methanolic (A) and water (B) extracts of GWM and CM on DPPH radicals. Bars sharing the same letter for GWM (a–h) and CM (A–G) extracts are not significantly different (p > 0:01) from one another.
(p < 0:05). For example, water extract of GWM at the concentration of 0.90 mg/ml scavenged about 60% DPPH radical, while the same concentration of water extract of CM completely eliminated DPPH radical. Both types of GWM extracts at the concentrations less than 0.45 mg/ml had no significant SA. BHA used as control scavenged 69.58% of DPPH radicals at 0.45 mg/ml. 3.3. Hydroxyl radical scavenging activity of extracts
Fig. 1. ESR spectra of the DPPH radicals: (a) in the absence of extracts (blank); (b) in the presence of 0.45 mg/ml of water extract of GWM; (c) in the presence of 0.45 mg/ml of water extract of CM.
As shown in Fig. 3(a), the reaction of Fe2þ and H2 O2 in the presence of spin trapping agent DMPO generated a 1:2:2:1 quartet of lines in the ESR spectrum with the hyperfine coupling parameters (aN and aH ¼ 14.9 G). The ESR spectra of DMPO-OH spin adducts formed in the presence of 0.45 mg/ml water extracts of GWM and CM are shown in Fig. 3(b) and (c). The influence of the different concentrations of methanolic and water extracts of GWM and CM on formation and transformation of hydroxyl radical produced in the Fenton reaction is shown in Fig. 4. The addition of the methanolic and water extracts of GWM and CM to the reaction system (from 0.10 to 0.90 mg/ml) resulted in a dose-dependent inhibition of the ESR signal intensity of DMPO-OH spin adduct. As can be seen from Fig. 4(B), water extract of CM (p < 0:01)
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Fig. 3. ESR spectra of DMPO-OH spin adducts: (a) in the absence of extracts (blank); (b) in the presence of 0.45 mg/ml of water extract of GWM; (c) in the presence of 0.45 mg/ml of water extract of CM.
Fig. 4. Scavenging activity of different concentrations of methanolic (A) and water (B) extracts of GWM and CM on hydroxyl radicals generated in the Fenton reaction. Bars sharing the same letter for GWM (a–f) and CM (A–G) extracts are not significantly different (p > 0:01) from one another.
exhibited the strongest SA (the values ranging from 35.62% to 100%). But generally, both types of CM extracts showed significantly higher SA (p < 0:01) than the extracts of GWM. For example, the addition of 0.45 mg/ ml water extract of CM markedly scavenged the hydroxyl radical, SA ¼ 88.08% (Fig. 3(b) and (c)), while the same concentration of water extract of GWM produced only SA ¼ 34.74% (Figs. 3(b) and 4(B)). The total elimination of hydroxyl radical (SA ¼ 100%) was obtained in the presence of 0.75 mg/ml of water extract or 0.90 mg/ml of methanolic extract of CM, while this was not registered in the case of GWM extracts. 0.45 mg/ml of BHA scavenged 71.34% of hydroxyl radicals.
The highest antioxidant activity on peroxyl radical (AA ¼ 100%) was observed for water extract of CM at 0.90 mg/ml, while the same concentration of water extract of GWM exhibited a significantly (p < 0:05) lower activity (AA ¼ 69.28%). As in the case of SA on DPPH and hydroxyl radical, higher concentrations of water and methanolic extracts of both types of marigold were more effective in reducing the ESR signal of PBN-OOL spin adducts. There is a significant difference between the antioxidant activity of methanolic and water extracts of GWM and CM (p < 0:05). BHA eliminated 64.81% of peroxyl radicals at 0.45 mg/ml.
3.4. Lipid peroxyl radical antioxidant activity of extracts 4. Discussion The typical ESR spectrum of the PBN-peroxyl radical (PBN-OOL) spin adduct was detected in catalytic oxidation system of hydroperoxide-enriched methyl esters of sunflower oil in the absence and the presence of extracts (Fig. 5). Hyperfine coupling parameters (aN ¼ 14.75G and aH ¼ 2.8 G) are typical of the PBNOOL spin adduct. The AA of different concentrations of the methanolic and water extracts of GWM and CM on peroxyl radicals is presented in Fig. 6.
Herbals and especially herbal extracts, which contain different classes of polyphenol, are very attractive not only in the modern phytotherapy but also for the food industry. Therefore, in this study we investigated antioxidant properties of methanolic and water extracts of GWM and CM. Good activity in the scavenging screening test was the first indication of the possible scavenging activity of the investigated extracts. Spraying the plates with DPPH
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(B) Fig. 5. ESR spectra of PBN-OOL spin adduct formed during catalytic oxidation of hydroperoxide-enriched methyl esters of sunflower oil: (a) in the absence of extract (blank); (b) in the presence of 0.45 mg/ml water extract of GWM; (c) in the presence of 0.45 mg/ml water extract of CM.
reagent revealed the presence of a number of phenolic compounds with scavenging activity in all the investigated extracts. According to the ESR data all marigold extracts scavenged DPPH, hydroxyl and peroxyl radicals in a concentration dependent manner. Scavenging and antioxidant activities were in correlation with the contents of total phenolic compounds and flavonoids in investigated extracts. The methanolic and water extracts of CM had the excellent ability to remove a stable DPPH radical, from the investigated system, while this ability, in case of GWM extracts, was lower. The activity of all the types of extracts can therefore be attributed to the hydrogendonating ability and direct scavenging activity of the active constituents (Brand-Williams, Cuvelier, & Berset, 1995). Hydroxyl radicals are the major active oxygen species causing lipid oxidation and enormous biological damage (Aruoma, Kaur, & Halliwell, 1991; Milic, Djilas, & Canadanovi c-Brunet, 1998; Milic, Djilas, Canadanovi cBrunet, & Milic, 2000). In our experimental investigations the methanolic and water extracts of CM showed at all concentrations significantly higher lipid peroxyl radical antioxidant activity during catalytic oxidation of
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Fig. 6. Effect of different concentrations of methanolic (A) and water (B) extracts of GWM and CM on peroxyl radical in catalytic oxidation system of hydroperoxide-enriched methyl esters of sunflower oil. Bars sharing the same letter for GWM (a–g) and CM (A–E) extracts are not significantly different (p > 0:01) from one another.
hydroperoxide-enriched methyl esters of sunflower oil (p < 0:01) than the same type of GWM extracts. Generally, in all investigated cases, lipid peroxyl radical AA was lower than hydroxyl radical SA but higher than DPPH radical SA (p < 0:01). The probable causes of these differences are: (i) extracts are very complex mixtures of a number of different compounds with distinct polarity, sometimes showing synergistic actions, (ii) the kinetic constants of reactions between hydroxyl radical and polyphenols are generally higher than for the reactions between peroxyl radicals or DPPH radicals and polyphenols, (iii) the different system and spin traps are used to measure lipid peroxyl radical AA and hydroxyl SA. Phenolic compounds are known as powerful chainbreaking antioxidants (Shahidi & Wanasundara, 1992). They scavenge lipid peroxyl radicals and thereby break radical chain sequences through the same mechanism as scavenging activity on hydroxyl radical: (i) hydrogen atom transfer from the antioxidant to the lipid peroxyl radical, resulting in formation of a stable antioxidant radical and relatively stable cis,trans-lipidhydroperoxide (LOO + ArOH ! ArO + LOOH); (ii) deactivating lipid peroxyl radicals by single electron transfer (LOO +
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ArOH ! LOO + ArOHþ ; LOO + ArOþ ! ArO + LOOH); (iii) chelating transition metals to suppress the initiation of radical formation during catalytic oxidation of lipids. The formed phenoxyl radical (ArO ) is relatively stable, and it only reacts slowly with the substrate (LOOH), but rapidly with another lipid peroxyl radical (Ou, Huang, Hampsch-Woodill, Flanagan, & Deemer, 2002). Significantly higher SA values for OH radical assay than for DPPH radicals for both types of GWM and CM extracts (p < 0:01) can be explained by the fact that some natural compounds are good iron chelators. It was suggested that flavonoid compounds (with o-diphenolic groups in the 3,4-dihydroxy position in ring B and the ketol structure, 4-oxo, 3-OH or 4-oxo, 5-OH in the C ring of the flavonols) and phenolic acids (with o-dihydroxyl groups) might be exerting their protective effects through chelation of metal ions in the course of the Fenton reaction, or by altering the iron redox chemistry (Morel, Lescoat, Cillard, & Cillard, 1994; Ruiz-Larrea, Leal, Martin, Martinez, & Lacort, 1995). The phenolic acids, present in our marigold extracts, may exist in free, esterified and glycosidic forms. Ohnishi et al. (1994) reported that caffeic and chlorogenic acids possess stronger peroxyl radical antioxidant activity then dl-a-tocopherol or ascorbic acid at the same concentrations. They also proposed that these phenolic acids may directly react with another peroxyl radical, scavenging and converting it into a much less active product with quinonic structure. Thus, the diphenolics, chlorogenic and caffeic acids, apparently have higher radical scavenging ability than monophenolics (p-coumaric acid), consistent with the chemical criteria applied to diphenolics (Cuvelier, Richard, & Berset, 1992). The scavenging properties of antioxidant compounds (flavonoid and phenolic acids) are often associated with their ability to form stable radicals. For example, rutin and caffeic acid with o-dihydroxyl group in B ring, can scavenge radicals effectively and usually give rise to semiquinone free radicals, which were identified by ESR analyses (Milic et al., 1998). These radicals are insufficiently reactive and they can disappear by several mechanisms (Bors, Heller, Michel, & Saran, 1990). It has been reported (Calliste, Trouillas, Allais, Simon, & Duroux, 2001), that phenolic acids and their glycosides, aglicon, monoglycosyl or diglycosyl flavonoids are distributed in the different solvents as a function of polarity. Based on this reason, water extracts contain the most polar compounds such as triglycosyl flavonoids and high molecular weight compounds. These facts might explain the stronger scavenging and antioxidant activity of water extracts compared with methanolic extracts of GWM and CM. More work should be done to characterize individual phenolic compounds of the extracts of both types of
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marigold in order to assign particular antioxidant effects to individual compounds of the extracts. In the future studies it would be desirable to employ experimental conditions that can more specifically reflect the various gastric/intestinal microenvironments that are pertinent to the uptake of plant secondary metabolites in the human system.
Acknowledgements This research is part of the project ‘‘Biologically Active Constituents of Growing wild Plants as Natural Sources for Pharmacy, Cosmetics and Foodstuff Industry’’ (Project No. 1862), which is financially supported by the Ministry of Science, Technologies and Development of the Republic of Serbia.
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Markham, K. R. (1989). Flavones, flavonols and their glycosides. In J. B. Harborne & P. M. Dey (Eds.), Plant phenolics: Vol. 1. Methods in plant biochemistry (pp. 193–237). London: Academic Press. Middleton, E., & Kandaswami, C. (1992). Effects of flavonoids on immune and inflammatory functions. Biochemical Pharmacology, 43, 1167–1179. Milic, B. Lj., Djilas, S. M., & Canadanovi c-Brunet, J. M. (1998). Antioxidative activity of phenolic compounds on the metal-ion breakdown of lipid peroxidation system. Food Chemistry, 61, 443–447. Milic, B. Lj., Djilas, S. M., Canadanovi c-Brunet, J. M., & Milic, N. B. (2000). Radicali liberi in biologia, medicina e nutrizione. In F. Capasso, R. De Pasquale, G. Grandolini, & N. Mascolo (Eds.), Farmacognosia (pp. 449–462). Berlin: Springer. Morel, I., Lescoat, G., Cillard, P., & Cillard, J. (1994). Role of flavonoids and iron chelation in antioxidant action. Methods in Enzymology, 234, 437–443. Ohnishi, M., Morishita, H., Iwahashi, H., Toda, S., Shirataki, Y., Kimura, M., & Kido, R. (1994). Inhibitory effect of chlorogenic acid on linoleic acid peroxidation and heamolysis. Phytochemistry, 36, 579–583. Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J. A., & Deemer, E. (2002). Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: A comparative study. Journal of Agriculture and Food Chemistry, 50, 3122–3128. Ruiz-Larrea, M. B., Leal, A. M., Martin, C., Martinez, R., & Lacort, M. (1995). Effects of estrogens on the redox chemistry of iron: A
possible mechanism of the antioxidant action of estrogens. Steroids, 60, 780–783. Shahidi, F., & Wanasundara, P. K. J. P. D. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition, 32, 67–103. Singleton, V. L., Orthofer, R., & Lamuela-Raventos, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Methods in Enzymology, 299, 152–178. Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity in fruits and total phenolic in selected fruits, vegetables, and grain products. Journal of the Science of Food and Agriculture, 46, 4113–4117. Vidal-Ollivier, E., Elias, R., Faure, F., Babadjamian, A., Crespin, F., Balansard, G., & Boudon, G. (1989). Flavonal Glycosides from Calendula officinalis flowers. Planta Medica, 55, 73–74. Wang, S. Y., & Lin, H.-S. (2000). Antioxidant activity in fruits and leaves of blackberry, raspberry, and strawberry varies with cultivar and developmental stage. Journal of the Science of Food and Agriculture, 48, 140–146. Wichtl, M. (1994). Herbal drugs and phytopharmaceuticals (p. 446). Stuttgart: Medpharm Scientific Publisher. Yoshida, T., Mori, K., Hatano, T., Okumura, T., Uehara, I., Komagoe, K., Fujita, Y., & Okuda, T. (1989). Studies on inhibition mechanism of autoxidation by tannins and flavonoids. V. Radicalscavenging effects of tannins and related polyphenols on DPPH radical. Chemical and Pharmaceutical Bulletin, 37, 1919–1921.
Antioxidant properties of marigold extracts Gordana S. Cetkovi c *, Sonja M. Djilas, Jasna M. Canadanovi c-Brunet, Vesna T. Tumbas Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia and Montenegro Received 26 August 2003; accepted 28 January 2004
Abstract The influence of methanolic and water extracts of growing wild marigold, Calendula arvensis L. (GWM) and cultivated marigold, Calendula officinalis L. (CM), in a concentration range of 0.10–0.90 mg/ml, was evaluated on three different free-radical species: 2,2diphenyl-1-picrylhydrazyl free radical (DPPH), hydroxyl radical and lipid peroxyl radical using electron spin resonance (ESR) spectroscopy. These extracts of CM and GWM, scavenged all types of investigated radicals in dependence on their applied concentrations. Generally, CM extracts possessed better scavenging and antioxidant activity than GMW extracts, while methanolic extracts exhibited lower activities than water extracts. Water extracts of CM had the best antioxidant properties; 0.75 mg/ml extracts completely eliminated hydroxyl radical, which was generated in Fenton system. The same concentration of this extract scavenged 92% DPPH and 95% peroxyl radical during lipid peroxidation. Antioxidant properties were in correlation with the contents of total phenolic compounds (14.49–57.47 mg/g) and flavonoids (5.26–18.62 mg/g) in extracts. The formation of o-semiquinone radicals from rutin and caffeic acid in lipid peroxidation system proved the mechanism (hydrogen donating and/or one-electron reduction) of free-radical scavenging activity. The ESR data demonstrate that methanolic and water extracts of CM possess similar free radicals scavenging and antioxidative activity as synthetic antioxidants BHA. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Growing wild and cultivated marigold; Free radicals; Polyphenols; Electron spin resonance
1. Introduction The potential toxicity of synthetic antioxidants (butylated hydroxyanisole-BHA, butylated hydroxytoluene-BHT, tertiary butylhydroquinone, esters of 3,4,5trihydroxybenzoic acid, etc.) (Ito, Fukushima, & Tsuda, 1985) has aroused an increased interest in preparing antioxidants from natural sources such as herbs, spices, seeds, cereals, fruits, and vegetables by extraction, fractionation and purification (Dillard & German, 2000; Wang & Lin, 2000). Numerous investigations have proved that medicinal herbs contain diverse classes of compounds such as polyphenols, tocopherols, alkaloids, tannins, carotenoids, terpenoids, etc. (Velioglu, Mazza, Gao, & Oo-
*
Corresponding author. Tel.: +381-21-450-648; fax: +381-21-450413. E-mail address: [email protected] (G.S. Cetkovi c). 0963-9969/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.01.010
mah, 1998; Yoshida et al., 1989). Among them, flavonoids and phenolic acids are particularly attractive as they are known to exhibit various beneficial pharmacological properties such as vasoprotective, anticarcinogenic, antineoplastic, antiviral, anti-inflammatory, as well as antiallergic and antiproliferative activity on tumor cells (Carr, Zhu, & Frei, 2000; Kuhnau, 1976; Middleton & Kandaswami, 1992). Some of these properties have been related to the action of these compounds as antioxidants, free-radical scavengers, quenchers of singlet and triplet oxygen, and inhibitors of peroxidation. Antioxidant activity of phenolic compounds is correlated to some structure–activity relationships, such as redox properties and the number and arrangement of the hydroxyl groups (Cotelle et al., 1996). Marigold is a herb of ancient medicinal repute. In traditional and homeopathic medicine it has been used for skin complaints, wounds and burns, conjunctivitis and poor eyesight, menstrual irregularities, varicose
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veins, hemorrhoids, duodenal ulcers, etc. (Wichtl, 1994). Marigold grows as a wild and common garden plant throughout Europe and North America. The yellow or golden-orange flowers of marigold are used as spice, tea and medicine. They may be used as fresh or dried, and can be made into tea, tinctures, ointments and creams. The pharmacological activity of marigold is related to the content of several classes of secondary metabolites such as essential oils, flavonoids, sterols, carotenoids, tannins, saponins, triterpene alcohols, polysaccharides, a bitter principle, mucilage, and resin (Vidal-Ollivier et al., 1989). Bilia, Salvini, Mazzi, and Vincieri (2001) found that marigold flowers contain rutin, isoquercitrin, quercetin-3-O-rutinosylrhamnoside, isorhamnetin-3-Orutinosylrhamnoside, isorhamnetin-3-O-glucosylglucoside, and isorhamnetin-3-O-glucoside. According to the fact that marigold contains polyphenols, the assessment of its antioxidant properties is of great interest in the understanding of positive effect of these compounds especially in phytotherapy. The task of this study is to investigate the influence of the methanolic and water extracts of growing wild marigold, Calendula arvensis L. (GWM) and cultivated marigold, Calendula officinalis L. (CM) on stable 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical, reactive hydroxyl radical (scavenging activity, SA) and lipid peroxyl radicals (antioxidant activity, AA) using ESR spectroscopy.
2.2. Marigold extracts preparation Dried flowers of GWM and CM (5 g) were extracted with 70% methanol or distilled water (250 ml) in a shaker incubator at 25 °C for 24 h. The extracts were filtered, and obtained filtrates were concentrated under the reduced pressure to dryness. The yields (g), averages of triplicate analysis, of obtained extracts were:
GWM CM
Methanolic extract 1.2964 0.0095 1.3772 0.0101
Water extract 1.3528 0.0099 1.3876 0.0102
2.3. Total phenolic compounds in extracts Total phenolic compounds in extracts were determinated spectrophotometrically using the Folin–Ciocalteu reagent and the results are expressed as chlorogenic acid equivalents per g dry weight (Singleton, Orthofer, & Lamuela-Raventos, 1999). 2.4. Total flavonoids in extracts Total flavonoids in extracts (expressed as mg rutin per g dry weight) were estimated spectrophotometrically according to Markham (1989). 2.5. Thin-layer chromatography (TLC) and scavenging screening test of extracts
2. Materials and methods 2.1. Chemicals and plant materials Methanol, ethyl acetate, hydrogen peroxide, acetic and formic acid were obtained from ‘‘Zorka’’ Sabac (Serbia). DPPH, 5,5-dimethyl-1-pyrroline-N -oxide (DMPO) and a-phenyl-N tert-butylnitrone (PBN), Folin–Ciocalteu reagent, BHA, standards of flavonoids (kaempferol, quercetin and rutin) and phenolic acids (chlorogenic, caffeic, p-coumaric and vanillic acids) were purchased from Sigma Chemical Co. (USA). These chemicals were of analytical reagent grade. Other used chemicals and solvents were of the highest analytical grade. Methyl esters of sunflower oil were supplied by Fluka Chemie A.G. (Buchs, Switzerland). Plants were purchased from local herbal drugstore. Flowers of GWM were collected in the period May– June, 2001, in the region of mountain Zlatibor (Serbia), while flowers of CM were harvested during May, 2001, in the experimental garden in Zabalj, flat ground region of Vojvodina (Serbia). Voucher specimens of the collected plants (C. arvensis No. 1048 and C. officinalis No. 1053) were confirmed and deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Medicine, University of Novi Sad.
TLC was performed on 20 20 cm plates precoated with microcrystalline cellulose (Camag, Muttanez, Switzerland). Volumes of 1 ll of 1% methanolic solution of standards and investigated extracts were spotted on the plates. TLC analysis was performed with ethyl acetate:formic acid:acetic acid:water in the volume ratio 100:11:11:26 as mobile phase. Spots were observed under UV light at 366 nm and sprayed with 0.1% methanolic solution of DPPH radical (Dapkevicius et al., 2002). 2.6. DPPH radical assay Blank probe was obtained by mixing 600 ll 0.4 mM methanolic solution of DPPH and 200 ll of methanol. A volume of x ll of 1% methanolic solution of investigated extract was added to a mixture of (200 x) ll of methanol and 600 ll of 0.4 mM methanolic solution of DPPH radical (probe). The range of the investigated extract concentrations was 0.10–0.90 mg/ml. After that the mixture was stirred for 2 min and transferred to a quartz flat cell ER-160FT. The ESR spectra were recorded on an ESR spectrometer Bruker 300E (Rheinstetten, Germany) under the following conditions: field modulation 100 kHz, modulation amplitude 0.256 G, receiver gain 2 104 , time constant 40.96 ms, conver-
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sion time 327.68 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23 °C. The SA value of the extract was defined as: SA ¼ 100 ðho hx Þ=ho (%); where ho and hx are the height of the second peak in the ESR spectrum of DPPH radicals of the blank and the probe, respectively. 2.7. Hydroxyl radical assay Hydroxyl radicals were obtained by the Fenton reaction in the system: 0.2 ml 10 mM H2 O2 , 0.2 ml 10 mM FeCl2 4H2 O and 0.2 ml 0.3 M DMPO as spin trap (blank). The influence of methanolic and water extracts on the formation and transformation of hydroxyl radicals was investigated by adding the extracts to the Fenton reaction system in the range of concentrations 0.10–0.90 mg/ml. ESR spectra were recorded after 5 min, with the following spectrometer settings: field modulation 100 kHz, modulation amplitude 0.512 G, receiver gain 2 105 , time constant 81.92 ms, conversion time 163.84 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23 °C. The SA value of the extract was defined as: SA ¼ 100 ðho hx Þ=ho (%), where ho and hx are the height of the second peak in the ESR spectrum of DMPO-OH spin adduct of the blank and the probe, respectively.
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x-band frequency 9.64 GHz, power 20 mW, temperature 23 °C. The AA value of the extract was defined as: AA ¼ 100 ðho hx Þ=ho (%) where ho and hx are the height of the first peak in the ESR spectrum of PBN-OOL spin adduct of the blank and the probe, respectively. Magnetic field scanning determinations were calibrated using FremyÕs salt (peroxyllamine disulphonate). Splitting constants were calculated from computer-generated second derivatives of the spectra, after optimizing signal-to-noise ratios and were verified by computer simulations. 2.9. Statistical analysis The StudentÕs t-test and ANOVA were used to determine the statistical difference. The criterion for statistical significance was p < 0:05, unless indicated otherwise. Data presented in graphs show the calculated means of three replications with vertical bars as standard deviation. Statistical analysis was carried out using Microsoft Excel 2000.
3. Results 3.1. Characterization of the phenolic compounds and the flavonoids in extracts
2.8. Peroxyl radical assay Methyl esters of sunflower oil were exposed to air for 72 h at room temperature to allow their peroxidation. The oil prepared in this way contained about 60% hydroperoxide-enriched methyl linoleate. Blank probe was obtained by mixing 0.6 g of hydroperoxide enriched methyl linoleate of sunflower oil, 0.1 ml of 0.1 mM FeCl2 4H2 O and 0.085 g of PBN as spin trap. The influence of all investigated extracts on the formation and transformation of peroxyl radicals was investigated by adding the extracts to the blank (before adding FeCl2 4H2 O) in the range of concentrations 0.10–0.90 mg/ml. ESR spectra were recorded after 24 h, with the following spectrometer settings: field modulation 100 kHz, modulation amplitude 1.021 G, receiver gain 5 105 , time constant 327.68 ms, conversion time 1310.72 ms, center field 3440.00 G, sweep width 100.00 G,
The contents of total phenolic compounds and flavonoids in methanolic and water extracts of GWM and CM are presented in Table 1. The obtained results showed that the contents of total phenolic compounds and flavonoids in methanolic and water extracts of CM were significantly higher (p < 0:05) than in the corresponding extracts of GWM. However, the contents of total phenolic compounds in methanolic and water extracts of GWM were not significantly different (p > 0:05), and neither were the contents of flavonoids (p > 0:05). The same conclusion holds for both types of CM extracts. The results of TLC analyses showed that flavonoids (kaempferol, quercetin and rutin, as well as some other glycosides) and phenolic acids (chlorogenic, caffeic, p-coumaric and vanillic) were present in the methanolic and water extracts of GWM and CM.
Table 1 Contents of total phenolic compounds and flavonoids in methanolic and water extracts of GWM and CM Extract
Total phenolic compounds (mg/g)
Total flavonoids (mg/g)
Methanolic extract of GWM Water extract of GWM Methanolic extract of CM Water extract of CM
14.49 0.38 15.12 0.40 55.07 1.11 57.47 1.12
5.26 0.36 5.31 0.36 18.48 0.70 18.62 0.73
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The scavenging screening test of the methanolic and water extracts of both types of marigold was assessed by spraying the TLC plates with a solution of purple colored stable DPPH. The reduction of the color could be observed visually as a yellowish spot on a purple background (results are not shown).
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The ESR spectra of DPPH radicals in the blank and in probes with water and methanolic extracts of GWM and CM are characterized by their five lines of relative intensities 1:2:3:2:1 and hyperfine splitting constant aN ¼ 9.03 G (Fig. 1). The DPPH radicals scavenging activity of all extracts increased dose-dependently at mass concentrations ranging from 0.10 to 0.90 mg/ml (Fig. 2). With increasing concentrations, scavenging activity of methanolic and water extracts of CM increased from 15.63% to 95.34% and from 28.32% to 100%, respectively. The difference between SA of these extracts was statistically significant (p < 0:01). All concentrations tested for both extracts of GWM possessed consistently lower SA
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Fig. 2. The SA of different concentrations of the methanolic (A) and water (B) extracts of GWM and CM on DPPH radicals. Bars sharing the same letter for GWM (a–h) and CM (A–G) extracts are not significantly different (p > 0:01) from one another.
(p < 0:05). For example, water extract of GWM at the concentration of 0.90 mg/ml scavenged about 60% DPPH radical, while the same concentration of water extract of CM completely eliminated DPPH radical. Both types of GWM extracts at the concentrations less than 0.45 mg/ml had no significant SA. BHA used as control scavenged 69.58% of DPPH radicals at 0.45 mg/ml. 3.3. Hydroxyl radical scavenging activity of extracts
Fig. 1. ESR spectra of the DPPH radicals: (a) in the absence of extracts (blank); (b) in the presence of 0.45 mg/ml of water extract of GWM; (c) in the presence of 0.45 mg/ml of water extract of CM.
As shown in Fig. 3(a), the reaction of Fe2þ and H2 O2 in the presence of spin trapping agent DMPO generated a 1:2:2:1 quartet of lines in the ESR spectrum with the hyperfine coupling parameters (aN and aH ¼ 14.9 G). The ESR spectra of DMPO-OH spin adducts formed in the presence of 0.45 mg/ml water extracts of GWM and CM are shown in Fig. 3(b) and (c). The influence of the different concentrations of methanolic and water extracts of GWM and CM on formation and transformation of hydroxyl radical produced in the Fenton reaction is shown in Fig. 4. The addition of the methanolic and water extracts of GWM and CM to the reaction system (from 0.10 to 0.90 mg/ml) resulted in a dose-dependent inhibition of the ESR signal intensity of DMPO-OH spin adduct. As can be seen from Fig. 4(B), water extract of CM (p < 0:01)
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Fig. 3. ESR spectra of DMPO-OH spin adducts: (a) in the absence of extracts (blank); (b) in the presence of 0.45 mg/ml of water extract of GWM; (c) in the presence of 0.45 mg/ml of water extract of CM.
Fig. 4. Scavenging activity of different concentrations of methanolic (A) and water (B) extracts of GWM and CM on hydroxyl radicals generated in the Fenton reaction. Bars sharing the same letter for GWM (a–f) and CM (A–G) extracts are not significantly different (p > 0:01) from one another.
exhibited the strongest SA (the values ranging from 35.62% to 100%). But generally, both types of CM extracts showed significantly higher SA (p < 0:01) than the extracts of GWM. For example, the addition of 0.45 mg/ ml water extract of CM markedly scavenged the hydroxyl radical, SA ¼ 88.08% (Fig. 3(b) and (c)), while the same concentration of water extract of GWM produced only SA ¼ 34.74% (Figs. 3(b) and 4(B)). The total elimination of hydroxyl radical (SA ¼ 100%) was obtained in the presence of 0.75 mg/ml of water extract or 0.90 mg/ml of methanolic extract of CM, while this was not registered in the case of GWM extracts. 0.45 mg/ml of BHA scavenged 71.34% of hydroxyl radicals.
The highest antioxidant activity on peroxyl radical (AA ¼ 100%) was observed for water extract of CM at 0.90 mg/ml, while the same concentration of water extract of GWM exhibited a significantly (p < 0:05) lower activity (AA ¼ 69.28%). As in the case of SA on DPPH and hydroxyl radical, higher concentrations of water and methanolic extracts of both types of marigold were more effective in reducing the ESR signal of PBN-OOL spin adducts. There is a significant difference between the antioxidant activity of methanolic and water extracts of GWM and CM (p < 0:05). BHA eliminated 64.81% of peroxyl radicals at 0.45 mg/ml.
3.4. Lipid peroxyl radical antioxidant activity of extracts 4. Discussion The typical ESR spectrum of the PBN-peroxyl radical (PBN-OOL) spin adduct was detected in catalytic oxidation system of hydroperoxide-enriched methyl esters of sunflower oil in the absence and the presence of extracts (Fig. 5). Hyperfine coupling parameters (aN ¼ 14.75G and aH ¼ 2.8 G) are typical of the PBNOOL spin adduct. The AA of different concentrations of the methanolic and water extracts of GWM and CM on peroxyl radicals is presented in Fig. 6.
Herbals and especially herbal extracts, which contain different classes of polyphenol, are very attractive not only in the modern phytotherapy but also for the food industry. Therefore, in this study we investigated antioxidant properties of methanolic and water extracts of GWM and CM. Good activity in the scavenging screening test was the first indication of the possible scavenging activity of the investigated extracts. Spraying the plates with DPPH
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reagent revealed the presence of a number of phenolic compounds with scavenging activity in all the investigated extracts. According to the ESR data all marigold extracts scavenged DPPH, hydroxyl and peroxyl radicals in a concentration dependent manner. Scavenging and antioxidant activities were in correlation with the contents of total phenolic compounds and flavonoids in investigated extracts. The methanolic and water extracts of CM had the excellent ability to remove a stable DPPH radical, from the investigated system, while this ability, in case of GWM extracts, was lower. The activity of all the types of extracts can therefore be attributed to the hydrogendonating ability and direct scavenging activity of the active constituents (Brand-Williams, Cuvelier, & Berset, 1995). Hydroxyl radicals are the major active oxygen species causing lipid oxidation and enormous biological damage (Aruoma, Kaur, & Halliwell, 1991; Milic, Djilas, & Canadanovi c-Brunet, 1998; Milic, Djilas, Canadanovi cBrunet, & Milic, 2000). In our experimental investigations the methanolic and water extracts of CM showed at all concentrations significantly higher lipid peroxyl radical antioxidant activity during catalytic oxidation of
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Fig. 6. Effect of different concentrations of methanolic (A) and water (B) extracts of GWM and CM on peroxyl radical in catalytic oxidation system of hydroperoxide-enriched methyl esters of sunflower oil. Bars sharing the same letter for GWM (a–g) and CM (A–E) extracts are not significantly different (p > 0:01) from one another.
hydroperoxide-enriched methyl esters of sunflower oil (p < 0:01) than the same type of GWM extracts. Generally, in all investigated cases, lipid peroxyl radical AA was lower than hydroxyl radical SA but higher than DPPH radical SA (p < 0:01). The probable causes of these differences are: (i) extracts are very complex mixtures of a number of different compounds with distinct polarity, sometimes showing synergistic actions, (ii) the kinetic constants of reactions between hydroxyl radical and polyphenols are generally higher than for the reactions between peroxyl radicals or DPPH radicals and polyphenols, (iii) the different system and spin traps are used to measure lipid peroxyl radical AA and hydroxyl SA. Phenolic compounds are known as powerful chainbreaking antioxidants (Shahidi & Wanasundara, 1992). They scavenge lipid peroxyl radicals and thereby break radical chain sequences through the same mechanism as scavenging activity on hydroxyl radical: (i) hydrogen atom transfer from the antioxidant to the lipid peroxyl radical, resulting in formation of a stable antioxidant radical and relatively stable cis,trans-lipidhydroperoxide (LOO + ArOH ! ArO + LOOH); (ii) deactivating lipid peroxyl radicals by single electron transfer (LOO +
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ArOH ! LOO + ArOHþ ; LOO + ArOþ ! ArO + LOOH); (iii) chelating transition metals to suppress the initiation of radical formation during catalytic oxidation of lipids. The formed phenoxyl radical (ArO ) is relatively stable, and it only reacts slowly with the substrate (LOOH), but rapidly with another lipid peroxyl radical (Ou, Huang, Hampsch-Woodill, Flanagan, & Deemer, 2002). Significantly higher SA values for OH radical assay than for DPPH radicals for both types of GWM and CM extracts (p < 0:01) can be explained by the fact that some natural compounds are good iron chelators. It was suggested that flavonoid compounds (with o-diphenolic groups in the 3,4-dihydroxy position in ring B and the ketol structure, 4-oxo, 3-OH or 4-oxo, 5-OH in the C ring of the flavonols) and phenolic acids (with o-dihydroxyl groups) might be exerting their protective effects through chelation of metal ions in the course of the Fenton reaction, or by altering the iron redox chemistry (Morel, Lescoat, Cillard, & Cillard, 1994; Ruiz-Larrea, Leal, Martin, Martinez, & Lacort, 1995). The phenolic acids, present in our marigold extracts, may exist in free, esterified and glycosidic forms. Ohnishi et al. (1994) reported that caffeic and chlorogenic acids possess stronger peroxyl radical antioxidant activity then dl-a-tocopherol or ascorbic acid at the same concentrations. They also proposed that these phenolic acids may directly react with another peroxyl radical, scavenging and converting it into a much less active product with quinonic structure. Thus, the diphenolics, chlorogenic and caffeic acids, apparently have higher radical scavenging ability than monophenolics (p-coumaric acid), consistent with the chemical criteria applied to diphenolics (Cuvelier, Richard, & Berset, 1992). The scavenging properties of antioxidant compounds (flavonoid and phenolic acids) are often associated with their ability to form stable radicals. For example, rutin and caffeic acid with o-dihydroxyl group in B ring, can scavenge radicals effectively and usually give rise to semiquinone free radicals, which were identified by ESR analyses (Milic et al., 1998). These radicals are insufficiently reactive and they can disappear by several mechanisms (Bors, Heller, Michel, & Saran, 1990). It has been reported (Calliste, Trouillas, Allais, Simon, & Duroux, 2001), that phenolic acids and their glycosides, aglicon, monoglycosyl or diglycosyl flavonoids are distributed in the different solvents as a function of polarity. Based on this reason, water extracts contain the most polar compounds such as triglycosyl flavonoids and high molecular weight compounds. These facts might explain the stronger scavenging and antioxidant activity of water extracts compared with methanolic extracts of GWM and CM. More work should be done to characterize individual phenolic compounds of the extracts of both types of
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marigold in order to assign particular antioxidant effects to individual compounds of the extracts. In the future studies it would be desirable to employ experimental conditions that can more specifically reflect the various gastric/intestinal microenvironments that are pertinent to the uptake of plant secondary metabolites in the human system.
Acknowledgements This research is part of the project ‘‘Biologically Active Constituents of Growing wild Plants as Natural Sources for Pharmacy, Cosmetics and Foodstuff Industry’’ (Project No. 1862), which is financially supported by the Ministry of Science, Technologies and Development of the Republic of Serbia.
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