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Antioxidant activity and phenol content of Crithmum maritimum L. leaves
Plant Physiology and Biochemistry 47 (2009) 37–41
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Antioxidant activity and phenol content of Crithmum maritimum L. leaves Laetitia Meot-Duros*, Christian Magne´ Laboratoire d’Ecophysiologie et de Biotechnologie des Halophytes et des Algues Marines, EA 3877 (LEBHAM), Institut Universitaire Europe´en de la Mer, Universite´ de Bretagne ˆle Brest Iroise, Place Nicolas Copernic, 29280 Plouzane´, France Occidentale, Technopo
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2007 Available online 2 October 2008
Sea fennel (Crithmum maritimum L.) is an edible halophyte with various economical interests because of its high secondary metabolite content. However, little is known about water-soluble compounds in that species. Here, we have studied major solutes in C. maritimum leaves. Among these solutes, carbohydrates (sucrose, glucose) were the most abundant, followed by organic acids (malate and quinate) and a phenolic compound never described in a halophyte before: chlorogenic acid (CGA). Total phenols and chlorogenic acid contents were followed throughout one year, as well as antioxidant activity, in two populations of C. maritimum growing in contrasting habitats: sand hills and cliffs. Sea fennel leaves appeared to be rich in phenolic compounds, particularly in chlorogenic acid. On that point, differences between the two populations were found, sand hill plants accumulating more CGA than those growing on cliffs. Moreover, the former presented a higher radical-scavenging activity, and the two observations were positively correlated. These results indicate that sea fennel can be considered as a valuable source of antioxidant products, especially of chlorogenic acid. Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Antioxidant activity Crithmum maritimum Phenolic content Chlorogenic acid
1. Introduction Plant phenolic compounds are secondary metabolites with interesting properties for animal or human health. The beneficial effects of those molecules are related to their antioxidant activity [1], particularly their ability to scavenge free radicals, to donate hydrogen atoms or electrons, or to chelate metal cations [2]. Besides, phenolic compounds contribute largely to the colour and sensory characteristics of fruits and vegetables. In addition, they take part in growth and reproduction processes, and they provide protection against pathogens and predators [2]. At the cellular level, they participate in cell protection against the harmful action of reactive oxygen species (ROS), mainly oxygen free radicals, produced in response to environmental stresses such as salinity, drought, high light intensity or mineral nutrient deficiency [3], because of the imbalance between the production and scavenging of ROS in chloroplasts [4]. These cytotoxic activated oxygen species can seriously disrupt normal metabolism through oxidative damage to lipids, proteins and nucleic acids [5]. Accordingly, plants containing high concentrations of antioxidants show considerable
Abbreviations: ABTS, 2,20 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid); CGA, chlorogenic acid; DPPH, 1,1-diphenyl-2-picrylhydrazyl; GAE, gallic acid equivalent; ROS, reactive oxygen species. * Tel.: þ33 (0)2 98 49 86 69; fax: þ33 (0)2 98 49 87 72. E-mail addresses: [email protected] (L. Meot-Duros), christian.magne@ univ-brest.fr (C. Magne´). 0981-9428/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.09.006
resistance to the oxidative damage caused by the ROS, as shown in the case of salt stressed plants [3, 6]. Halophytic plants are naturally adapted to high salinity conditions in the soil and/or atmosphere [7]. They constitute an excellent model to investigate the different mechanisms of plant responses to salt stress in order to improve salt tolerance [8]. Recently, they have gained further interest since some of them might be considered as valuable sources of compounds for agro-food industry, cosmetics or medicine [9, 10]. These compounds include minerals, fibres, oil, phenolics and vitamins [11]. Crithmum maritimum L. (Apiaceae), commonly known as sea fennel or rock samphire, is a facultative halophyte growing on maritime cliffs and sometimes in sand. It is widely distributed along coastal areas of the Mediterranean Sea and of the Atlantic Ocean, and it has long been used as food ingredient and in folk medicine [12]. Its succulent leaves and young branches are pickled in vinegar and used as condiments. This perennial plant represents various economical interests, due to its high contents in flavonoids, carotenoids, vitamin C and substances with medicinal and antimicrobial properties [13]. Although the apolar components present in essential oil of sea fennel have been largely studied [14, 15], little is known about the main water-soluble compounds in this species. We have recently identified free quinic acid (Ce´rantola et al., unpublished results), a carbohydrate-derived secondary metabolite previously reported in other members of Apiaceae, as one of the major solutes of C. maritimum. Here we report the presence of chlorogenic acid and its possible consequences on plant extract bioactivity.
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L. Meot-Duros, C. Magne´ / Plant Physiology and Biochemistry 47 (2009) 37–41
2. Materials and methods
2.7. TLC determination of chlorogenic acid and quantification by HPLC
2.1. Chemicals Folin-Ciocalteu phenol reagent, FeCl3, gallic acid, chlorogenic acid, 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were purchased from Sigma–Aldrich. Methanol, chloroform, acetic acid and ethyl acetate were from Carlo Erba Reagenti. All other reagents were of analytical grade. 2.2. Plant material Aerial parts of C. maritimum were collected at random from five plants growing along the shoreline at Pointe du Toulinguet (Brittany, France). Here, two sampling sites were studied. The first one consisted of sand hills and was subjected to sea spray. The second site was below cliffs, protected from sea spray and rich in organic matter. Plants were sampled at four dates over 1 year: in May 2006 (spring), August 2006 (summer), November 2006 (autumn) and February 2007 (winter). 2.3. Soil analysis Soil samples were taken at the root environment of each plant (10 cm depth). The following analyses were performed: organic matter content by wet combustion and total nitrogen by the Kjeldahl method. In the soluble extracts (1:5, soil to distilled water), pH were determined by the potentiometric method, Naþ and Kþ by flame spectrophotometry, Ca2þ and Mg2þ by atomic absorption spectrophotometry. 2.4. Preparation of the methanol extracts The collected samples were washed with deionized water, rapidly soaked, stored at 25 C and then freeze dried. The dry material was ground to a fine powder. For the extraction, about 200 mg of powder were homogenized with 5 ml water/methanol (1:1) under magnetic stirring at 4 C for 20 min. After centrifugation of the mixture (15 min at 4 C, 4000 g), the resulting pellet was extracted twice following the same protocol. The supernatants were collected, pooled and filtrated over glass wool. The obtained extract was concentrated by rotary evaporation at 30 C. The residue was dissolved in deionized water and used for the following analyses. 2.5. Determination of major organic solutes in Crithmum maritimum One millilitre of the crude extract was concentrated by rotary evaporation at 30 C, and the dry residue was solubilized in D2O for 1 H NMR analysis. NMR spectra were obtained using a Bru¨ker DRX400 spectrometer (400 MHz), equipped with a 5 mm Dual 1H probehead. A typical 1D 1H NMR spectrum consisted of 32 scans. Determination of major solutes present in sea fennel was made on NMR spectra in comparison with external standards. 2.6. Assay of total phenols The concentration of total phenols was determined with FolinCiocalteu reagent following the colorimetric method adapted by Sanoner et al. [16]. Measurements were carried out in triplicate and calculations were based on a calibration curve obtained with gallic acid. The levels of total phenols were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE g1 DW).
Thin-layer chromatography was performed on 10 cm 20 cm silica gel 60 Alugram SIL G/UV254 (Macherey-Nagel, Germany). Plates were developed in methanol/chloroform/water (65:35:10) to the top, and dried at room temperature. Detection was made by spraying a FeCl3 solution (4% in absolute ethanol). Phenols with one hydroxyl function gave a blue spot, those with two hydroxyl groups turn green, the other phenolics appeared red or brown. Free chlorogenic acid in the extracts was determined with an analytical HPLC system consisting of a Waters Series highperformance liquid chromatograph equipped with a 717plus autosampler, a 600E pump and the Millennium Chromatography Manager software. The detection system consisted of a diode array detector tuned at 320 nm. Chlorogenic acid separation was achieved on a Waters C18 ODS 2 reverse-phase column (150 mm 4.6 mm i.d.), maintained at room temperature. The mobile phase, degassed by vacuum filtration through a Millipore HA membrane, was water/ethyl acetate/acetic acid (95.6:4.1:0.3, v/ v). The flow rate, in isocratic mode, was 0.8 ml min1. All quantitations were based on peak area compared to that of pure chlorogenic acid and the samples were analyzed in triplicate. The chlorogenic acid content was expressed as milligrams of chlorogenic acid per gram of dry weight (mg CGA g1 DW). 2.8. Assessment of antioxidant activity 2.8.1. Scavenging activity of DPPH radical The scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was determined by the method described by Marwah et al. [17]. Briefly, the reaction medium contained 2 ml of 100 mM DPPH$ violet solution in ethanol and 2 ml of plant extract (or water for the control). The reaction mixture was incubated in the dark for 15 min and the absorbance was recorded at 517 nm. The assay was carried out in triplicate. The decrease in absorbance on addition of test samples was used to calculate the antiradical activity, as expressed by the inhibition percentage (%IP) of DPPH radical, following the equation:
%IP ¼ ½ðAc As Þ=Ac 100 where Ac and As are the absorbencies of the control and of the test sample after 15 min, respectively. From a plot of concentration against %IP, a linear regression analysis was performed to determine the IC50 (extract concentration resulting in a 50% inhibition) value for each sample. 2.8.2. Scavenging activity of ABTS radical cation The ABTS radical cation (ABTS$þ)-scavenging activity was measured according to the method described by Re et al. [18]. ABTS was dissolved in water to a 7 mM concentration. The ABTS radical cation was produced by adding to the ABTS stock solution 2.45 mM potassium persulphate (final concentration). The completion of radical generation was obtained in the dark at room temperature for 12–16 h. This solution was then diluted with ethanol to adjust its absorbance at 734 nm to 0.70 0.02. To determine the scavenging activity, 1 ml of diluted ABTS$þ solution was added to 10 ml of plant extract (or water for the control), and the absorbance at 734 nm was measured 6 min after the initial mixing, using ethanol as the blank. The percentage of inhibition was calculated by the equation:
Inhibition percentageð%IPÞ ¼ ½ðAc As Þ=Ac 100 where Ac and As are the absorbencies of the control and of the test sample, respectively.
L. Meot-Duros, C. Magne´ / Plant Physiology and Biochemistry 47 (2009) 37–41
From a plot of concentration against %IP, a linear regression analysis was performed to determine the IC50 value for each plant extract. 2.8.3. Evaluation of antioxidant activity by phosphomolybdenum reagent The total antioxidant capacity of the plant extracts was evaluated by the method of Prieto et al. [19]. A 0.1 ml aliquot of plant extract was mixed with 1 ml of the reagent solution (28 mM sodium phosphate and 4 mM ammonium molybdate in 0.6 M sulphuric acid). Tubes were capped and incubated at 95 C for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm and standard curve was performed with ascorbic acid solution. The antioxidant capacity of the samples was expressed as milligrams of ascorbic acid equivalents per gram of dry weight. 2.9. Statistical analysis All determinations were run in triplicate on at least two different experiments, and the results were reported as the mean and standard deviation. Statistical variance analysis of independent data with six replicates was performed using ANOVA and compared with least significant differences (LSD) at the 5% level. All statistical analyses were performed with the STATISTICA software.
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TLC confirmed the presence of CGA in the leaf extract of C. maritimum. Only one green spot appeared on the plant extract chromatogram, with a Rf of 0.71, corresponding with chlorogenic acid standard. 3.3. Total phenols and chlorogenic acid levels Total phenol content in the leaves of Crithmum maritimum growing on sand hills and below cliffs was followed over one year (Fig. 2). During spring, summer and winter, there was no difference between the two sets of plants. The level of phenolic compounds increased between spring and summer, from 23 to 33 mg GAE g1 DW. Autumn was characterized by a large (3-fold) decrease of phenol levels in plants growing below cliffs. A similar drop in phenol concentration was found in plants grown on sand hills during winter, so that no significant differences in phenolic levels between the two sets of plants were observed at that period. As stated from the NMR analysis, Crithmum maritimum species appeared to be rich in chlorogenic acid. However, CGA level in plants from sand hills differed significantly from that in cliff plants (Fig. 3). Indeed, plants growing in sand presented a high CGA concentration throughout the growing season (18.8– 27.9 mg CGA g1 DW), whereas plants growing in cliffs produced much less CGA (3– 10 mg CGA g1 DW). 3.4. Antioxidant activity
3. Results 3.1. Soil composition Mineral and organic composition of soils sampling at the root environment of Crithmum maritimum plants are given in Table 1. Cliffs soil appeared to be richer in organic matter, total nitrogen and organic carbon than soil taken in sand hills. Cliffs were also characterized by a higher level of sodium, potassium and magnesium, whereas sand hills soil was richer in calcium. 3.2. Major organic solutes in methanolic extracts of sea fennel The extraction step of dry powder of Crithmum maritimum leaves allowed procurement of a crude methanol/water extract representing about 45% of the initial dry mass. This extract, analyzed by 1H NMR, enabled the determination of major solutes present in sea fennel. The 1H NMR spectrum of methanolic extract of sea fennel showed the predominance of soluble carbohydrates, including sucrose and glucose, and that of an organic acid: malate (Fig. 1). Noteworthy on this NMR spectrum was the presence of characteristic sets of signals in the 1.8–2.2 ppm and 6.3–7.6 ppm regions, assigning a hydroxycinnamic compound: chlorogenic acid. The first set of signals could also correspond to free quinic acid, a component of chlorogenic acid moiety.
Table 1 Chemical composition of soil sampled in sand hills and cliffs
Organic carbon Total N Organic matter pH Na K Ca Mg
Sand hills
Cliffs
0.33 0.07 0.21 0.05 0.58 0.13 9.4 0.1 500 10 215 1.5 5134 61 286 2
1.13 0.29 0.48 0.05 1.94 0.50 7.6 0.3 991 19 321 3 4232 55 806 4
Organic carbon, organic matter and total N are expressed in % dry soil; Naþ, Kþ, Ca2þ and Mg2þ are expressed in mg/kg dry soil. Means S.E. of three replicates are indicated.
3.4.1. DPPH radical scavenging activity Antiradical properties of Crithmum maritimum leaves are given in Table 2. DPPH scavenging activity in sea fennel varied over the year and also according to the population. During spring, sand hill plants presented a low IC50 contrary to cliff plants (0.811 and 1.211 mg ml1, respectively), which indicates a higher antiradical activity. Summer was characterized by a very low IC50 (0.25 mg ml1) for plants living in the two habitats. Such a strong antiradical activity was still found during autumn in sand hill plants, but not in cliff plants whose IC50 rose to 1.05 mg ml1. In winter, a 7-fold increase of the DPPH IC50 of sand hill plants was found, so that antiradical activity of the two sets of plants became close. 3.4.2. ABTS radical scavenging activity Leaves of sea fennels growing below maritime cliffs were characterized by a strong ABTS scavenging activity during spring and summer (Table 2), with IC50 of about 0.10– 0.17 mg ml1. Thereafter, scavenging activity differed greatly in the two sets of plants: ABTS IC50 of sand hill plants remained low (increasing just a little in winter) whereas it increased sharply in cliff plants. As a result, ABTS scavenging activity was 4 times higher in sand hill plants than in cliff plants. 3.4.3. Total antioxidant capacity Leaves of Crithmum maritimum from the two stations presented a strong total antioxidant activity throughout the year (Table 2). This activity was the highest in spring and summer for plants living in cliffs. Autumn was characterized by the lowest antioxidant activity for cliff plants, and the highest for sand hill plants. 4. Discussion Crithmum maritimum exhibited an original water-soluble composition. Besides soluble carbohydrates (sucrose, glucose) and free malate, noteworthy was the abundance of two compounds never reported in halophytic species: free quinic and chlorogenic acids. Recent studies showed the presence of free quinate and/or chlorogenate in other members of the Apiaceae family. Thus, free
L. Meot-Duros, C. Magne´ / Plant Physiology and Biochemistry 47 (2009) 37–41
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Suc Suc
Glc Glc
Glc
Mal CGA CGA/Qui
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5 ppm
1
Fig. 1. H NMR spectrum of Crithmum maritimum leaf methanolic extract. CGA, chlorogenic acid; Glc, glucose; Mal, malic acid; Qui, quinic acid; Suc, sucrose.
quinic acid was found in carrot [20], and CGA was reported in fennel [21], celery [22] and carrot [23]. Therefore, the two molecules could play a chimiotaxonomic role in that family. Crithmum maritimum had relatively high phenolic contents compared to other crop species. Chu et al. [24] reported a level of about 6 mg GAE g1 DW in different vegetables, and Zhou and Yu [25] found 13 and 10.6 mg GAE g1 DW in spinach and broccoli, respectively. These levels correspond to the smallest one measured in sea fennel in our study. In particular, sea fennel appeared as an Apiacea rich in phenols. Literature is scarce about the levels of phenolic compounds in halophytes. In a recent study by Ksouri et al. [6], the Brassicaceae Cakile maritima L. was found to accumulate phenolics (between 31 and 67 mg GAE g1 DW), those levels being similar or higher than those found in sea fennel. Hydroxycinnamic acids like chlorogenic acid are widely spread phenolics in plants [26]. Among the most CGA-rich species are Coffea species, where CGA accumulates in green beans [27] and contributes to coffee beverage bitterness. In our study, chlorogenic acid was constitutively present in sea fennel leaves and markedly accumulated during the growing season in sand hill plants. Even in resting plants (in winter), CGA concentration reached 3 mg g1 DW, this level being 3 times higher than that the maximum level measured in carrot [26], and similar to that in fennel [21]. Therefore, C. maritimum appeared to be the most CGA-rich Apiaceae, with between 1.9% and 2.8% DW in plants living in sand hills. Sea fennel can thus be considered as a valuable source of chlorogenic acid. In this study, plants from sand hills accumulated more CGA (up to 9-fold) than plants growing below cliffs. This might be related to environmental factors which differed in the two habitats. Plants living on sand hills were likely more stressed than those below cliffs because off nutrient deficiency. Accordingly, a wide range of environmental stresses, including boron and nitrogen deficit, have been showed to increased CGA levels in plants [28, 29]. Moreover, plants
in sand suffered from both water stress (due to the nature of sandy substrate) and ionic stress (due to sea sprays), contrary to plants living below cliffs. Therefore, it is likely that plants living in the stressful sand hill environment produced more ROS, which in turn activates the synthesis of antioxidants such as CGA or other radicalscavenging systems to prevent cell death. Accordingly, anti-radical tests showed that plants from sand hills presented a stronger radical-scavenging activity (DPPH and ABTS IC50 were up to 7 and 4 times lower, respectively, than those for cliff plants). This direct link between elevated CGA levels and oxidative stress tolerance was already described in Solanaceae by Niggeweg et al. [30]. DPPH radical-scavenging activity differed between the two populations, but also with the season. Leaf extracts had a stronger radical scavenging activity in summer, when the natural irradiance was at its maximum and resulted in the production of ROS in plants. Moreover, DPPH and ABTS radical-scavenging activities presented a good correlation with the level of phenolic compounds (r ¼ 0.84 and 0.79, respectively), and in particular with CGA (r ¼ 0.72 and 0.56, respectively). Thus, both types of plants showed the highest concentration in total phenols in summer when the strongest antiradical activity was found. Conversely, in winter, plants from the two habitats had the lowest levels of radical-scavenging activity, of total phenols and of CGA. Unlike radical-scavenging activity, total antioxidant activity was poorly correlated with the content of phenolic compounds (r ¼ 0.37). Conveniently, the global antioxidant property of a plant extract is generally considered as the result of the combined activity of a wide range of compounds, including phenolics, peptides, organic acids and other components [31]. Our results suggest that sea fennel can be considered as a valuable source of antioxidant products, especially of chlorogenic acid. This compound, in addition to its strong antioxidant capacity, is known to have antiviral, anti-inflammatory and immune properties [32, 33], so that sea fennel might find a number of industrial applications, particularly in the medicinal field.
Total phenolic level (mg GAE.g-1 DW)
a 30
a
sand hill cliffs
b
c d
20 e
e
e
10 0 spring
summer 1
autumn
winter
Fig. 2. Total phenol level (mg GAE g DW) in the leaves of Crithmum maritimum L. growing in sand hills and below cliffs. Measurements were carried out in triplicate. Means and standard deviations are represented, and different letters above the bars indicate significantly different (P < 0.05) means.
Chlorogenic acid level (mg.g-1 DW)
40 40
sand hill cliffs
a
30 b
b
20 c 10
d d
0
spring
summer 1
autumn
d e winter
Fig. 3. Chlorogenic acid level (mg g DW) in the leaves of Crithmum maritimum L. growing in sand hills and below cliffs. Measurements were carried out in triplicate. Means and standard deviations are represented, and different letters above the bars indicate significantly different (P < 0.05) means.
L. Meot-Duros, C. Magne´ / Plant Physiology and Biochemistry 47 (2009) 37–41 Table 2 Total antioxidant activity and radical-scavenging activity of Crithmum maritimum L. leaf extracts against DPPH$ and ABTS$þ, expressed in mg eq ascorbate g1 DW and mg ml1, respectively
Sand hills
Cliffs
Spring Summer Autumn Winter Spring Summer Autumn Winter
DPPH scavenging activity
ABTS scavenging activity
Antioxidant capacity
(IC50, mg ml1)
(IC50, mg ml1)
(mg eq g1 DW)
0.811 0.07 b 0.248 0.00 a 0.146 0.02 a 1.111 0.01 d 1.211 0.01 d 0.245 0.01 a 1.049 0.01 d 0.928 0.07 c
0.131 0.005 bc 0.112 0.004 ab 0.095 0.006 a 0.171 0.009 e 0.157 0.011 de 0.139 0.014 bcd 0.405 0.011 f 0.418 0.074 f
5.65 0.26 cd 5.76 0.17 a 6.02 0.70 cd 4.09 1.51 e 6.78 0.14 c 7.04 0.65 b 4.03 0.59 e 5.19 0.59 de
Measurements were carried out in triplicate. Means and standard deviations are indicated. Values for each radical scavenging activity followed by the same letter were not significantly different (P < 0.05).
Acknowledgements This research was partly supported by the Brest Metropole Oceane (BMO) through a Ph.D fellowship (L.M.). We thank N. Kervarec and S. Ce´rantola (Service de Re´sonance Magne´tique Nucle´aire, Universite´ de Bretagne Occidentale) for their technical assistance in obtaining NMR spectra. Contribution N 1093 of the IUEM, European Institute for marine Studies (Brest, France).
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Antioxidant activity and phenol content of Crithmum maritimum L. leaves Laetitia Meot-Duros*, Christian Magne´ Laboratoire d’Ecophysiologie et de Biotechnologie des Halophytes et des Algues Marines, EA 3877 (LEBHAM), Institut Universitaire Europe´en de la Mer, Universite´ de Bretagne ˆle Brest Iroise, Place Nicolas Copernic, 29280 Plouzane´, France Occidentale, Technopo
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2007 Available online 2 October 2008
Sea fennel (Crithmum maritimum L.) is an edible halophyte with various economical interests because of its high secondary metabolite content. However, little is known about water-soluble compounds in that species. Here, we have studied major solutes in C. maritimum leaves. Among these solutes, carbohydrates (sucrose, glucose) were the most abundant, followed by organic acids (malate and quinate) and a phenolic compound never described in a halophyte before: chlorogenic acid (CGA). Total phenols and chlorogenic acid contents were followed throughout one year, as well as antioxidant activity, in two populations of C. maritimum growing in contrasting habitats: sand hills and cliffs. Sea fennel leaves appeared to be rich in phenolic compounds, particularly in chlorogenic acid. On that point, differences between the two populations were found, sand hill plants accumulating more CGA than those growing on cliffs. Moreover, the former presented a higher radical-scavenging activity, and the two observations were positively correlated. These results indicate that sea fennel can be considered as a valuable source of antioxidant products, especially of chlorogenic acid. Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Antioxidant activity Crithmum maritimum Phenolic content Chlorogenic acid
1. Introduction Plant phenolic compounds are secondary metabolites with interesting properties for animal or human health. The beneficial effects of those molecules are related to their antioxidant activity [1], particularly their ability to scavenge free radicals, to donate hydrogen atoms or electrons, or to chelate metal cations [2]. Besides, phenolic compounds contribute largely to the colour and sensory characteristics of fruits and vegetables. In addition, they take part in growth and reproduction processes, and they provide protection against pathogens and predators [2]. At the cellular level, they participate in cell protection against the harmful action of reactive oxygen species (ROS), mainly oxygen free radicals, produced in response to environmental stresses such as salinity, drought, high light intensity or mineral nutrient deficiency [3], because of the imbalance between the production and scavenging of ROS in chloroplasts [4]. These cytotoxic activated oxygen species can seriously disrupt normal metabolism through oxidative damage to lipids, proteins and nucleic acids [5]. Accordingly, plants containing high concentrations of antioxidants show considerable
Abbreviations: ABTS, 2,20 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid); CGA, chlorogenic acid; DPPH, 1,1-diphenyl-2-picrylhydrazyl; GAE, gallic acid equivalent; ROS, reactive oxygen species. * Tel.: þ33 (0)2 98 49 86 69; fax: þ33 (0)2 98 49 87 72. E-mail addresses: [email protected] (L. Meot-Duros), christian.magne@ univ-brest.fr (C. Magne´). 0981-9428/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.09.006
resistance to the oxidative damage caused by the ROS, as shown in the case of salt stressed plants [3, 6]. Halophytic plants are naturally adapted to high salinity conditions in the soil and/or atmosphere [7]. They constitute an excellent model to investigate the different mechanisms of plant responses to salt stress in order to improve salt tolerance [8]. Recently, they have gained further interest since some of them might be considered as valuable sources of compounds for agro-food industry, cosmetics or medicine [9, 10]. These compounds include minerals, fibres, oil, phenolics and vitamins [11]. Crithmum maritimum L. (Apiaceae), commonly known as sea fennel or rock samphire, is a facultative halophyte growing on maritime cliffs and sometimes in sand. It is widely distributed along coastal areas of the Mediterranean Sea and of the Atlantic Ocean, and it has long been used as food ingredient and in folk medicine [12]. Its succulent leaves and young branches are pickled in vinegar and used as condiments. This perennial plant represents various economical interests, due to its high contents in flavonoids, carotenoids, vitamin C and substances with medicinal and antimicrobial properties [13]. Although the apolar components present in essential oil of sea fennel have been largely studied [14, 15], little is known about the main water-soluble compounds in this species. We have recently identified free quinic acid (Ce´rantola et al., unpublished results), a carbohydrate-derived secondary metabolite previously reported in other members of Apiaceae, as one of the major solutes of C. maritimum. Here we report the presence of chlorogenic acid and its possible consequences on plant extract bioactivity.
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2. Materials and methods
2.7. TLC determination of chlorogenic acid and quantification by HPLC
2.1. Chemicals Folin-Ciocalteu phenol reagent, FeCl3, gallic acid, chlorogenic acid, 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were purchased from Sigma–Aldrich. Methanol, chloroform, acetic acid and ethyl acetate were from Carlo Erba Reagenti. All other reagents were of analytical grade. 2.2. Plant material Aerial parts of C. maritimum were collected at random from five plants growing along the shoreline at Pointe du Toulinguet (Brittany, France). Here, two sampling sites were studied. The first one consisted of sand hills and was subjected to sea spray. The second site was below cliffs, protected from sea spray and rich in organic matter. Plants were sampled at four dates over 1 year: in May 2006 (spring), August 2006 (summer), November 2006 (autumn) and February 2007 (winter). 2.3. Soil analysis Soil samples were taken at the root environment of each plant (10 cm depth). The following analyses were performed: organic matter content by wet combustion and total nitrogen by the Kjeldahl method. In the soluble extracts (1:5, soil to distilled water), pH were determined by the potentiometric method, Naþ and Kþ by flame spectrophotometry, Ca2þ and Mg2þ by atomic absorption spectrophotometry. 2.4. Preparation of the methanol extracts The collected samples were washed with deionized water, rapidly soaked, stored at 25 C and then freeze dried. The dry material was ground to a fine powder. For the extraction, about 200 mg of powder were homogenized with 5 ml water/methanol (1:1) under magnetic stirring at 4 C for 20 min. After centrifugation of the mixture (15 min at 4 C, 4000 g), the resulting pellet was extracted twice following the same protocol. The supernatants were collected, pooled and filtrated over glass wool. The obtained extract was concentrated by rotary evaporation at 30 C. The residue was dissolved in deionized water and used for the following analyses. 2.5. Determination of major organic solutes in Crithmum maritimum One millilitre of the crude extract was concentrated by rotary evaporation at 30 C, and the dry residue was solubilized in D2O for 1 H NMR analysis. NMR spectra were obtained using a Bru¨ker DRX400 spectrometer (400 MHz), equipped with a 5 mm Dual 1H probehead. A typical 1D 1H NMR spectrum consisted of 32 scans. Determination of major solutes present in sea fennel was made on NMR spectra in comparison with external standards. 2.6. Assay of total phenols The concentration of total phenols was determined with FolinCiocalteu reagent following the colorimetric method adapted by Sanoner et al. [16]. Measurements were carried out in triplicate and calculations were based on a calibration curve obtained with gallic acid. The levels of total phenols were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE g1 DW).
Thin-layer chromatography was performed on 10 cm 20 cm silica gel 60 Alugram SIL G/UV254 (Macherey-Nagel, Germany). Plates were developed in methanol/chloroform/water (65:35:10) to the top, and dried at room temperature. Detection was made by spraying a FeCl3 solution (4% in absolute ethanol). Phenols with one hydroxyl function gave a blue spot, those with two hydroxyl groups turn green, the other phenolics appeared red or brown. Free chlorogenic acid in the extracts was determined with an analytical HPLC system consisting of a Waters Series highperformance liquid chromatograph equipped with a 717plus autosampler, a 600E pump and the Millennium Chromatography Manager software. The detection system consisted of a diode array detector tuned at 320 nm. Chlorogenic acid separation was achieved on a Waters C18 ODS 2 reverse-phase column (150 mm 4.6 mm i.d.), maintained at room temperature. The mobile phase, degassed by vacuum filtration through a Millipore HA membrane, was water/ethyl acetate/acetic acid (95.6:4.1:0.3, v/ v). The flow rate, in isocratic mode, was 0.8 ml min1. All quantitations were based on peak area compared to that of pure chlorogenic acid and the samples were analyzed in triplicate. The chlorogenic acid content was expressed as milligrams of chlorogenic acid per gram of dry weight (mg CGA g1 DW). 2.8. Assessment of antioxidant activity 2.8.1. Scavenging activity of DPPH radical The scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was determined by the method described by Marwah et al. [17]. Briefly, the reaction medium contained 2 ml of 100 mM DPPH$ violet solution in ethanol and 2 ml of plant extract (or water for the control). The reaction mixture was incubated in the dark for 15 min and the absorbance was recorded at 517 nm. The assay was carried out in triplicate. The decrease in absorbance on addition of test samples was used to calculate the antiradical activity, as expressed by the inhibition percentage (%IP) of DPPH radical, following the equation:
%IP ¼ ½ðAc As Þ=Ac 100 where Ac and As are the absorbencies of the control and of the test sample after 15 min, respectively. From a plot of concentration against %IP, a linear regression analysis was performed to determine the IC50 (extract concentration resulting in a 50% inhibition) value for each sample. 2.8.2. Scavenging activity of ABTS radical cation The ABTS radical cation (ABTS$þ)-scavenging activity was measured according to the method described by Re et al. [18]. ABTS was dissolved in water to a 7 mM concentration. The ABTS radical cation was produced by adding to the ABTS stock solution 2.45 mM potassium persulphate (final concentration). The completion of radical generation was obtained in the dark at room temperature for 12–16 h. This solution was then diluted with ethanol to adjust its absorbance at 734 nm to 0.70 0.02. To determine the scavenging activity, 1 ml of diluted ABTS$þ solution was added to 10 ml of plant extract (or water for the control), and the absorbance at 734 nm was measured 6 min after the initial mixing, using ethanol as the blank. The percentage of inhibition was calculated by the equation:
Inhibition percentageð%IPÞ ¼ ½ðAc As Þ=Ac 100 where Ac and As are the absorbencies of the control and of the test sample, respectively.
L. Meot-Duros, C. Magne´ / Plant Physiology and Biochemistry 47 (2009) 37–41
From a plot of concentration against %IP, a linear regression analysis was performed to determine the IC50 value for each plant extract. 2.8.3. Evaluation of antioxidant activity by phosphomolybdenum reagent The total antioxidant capacity of the plant extracts was evaluated by the method of Prieto et al. [19]. A 0.1 ml aliquot of plant extract was mixed with 1 ml of the reagent solution (28 mM sodium phosphate and 4 mM ammonium molybdate in 0.6 M sulphuric acid). Tubes were capped and incubated at 95 C for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm and standard curve was performed with ascorbic acid solution. The antioxidant capacity of the samples was expressed as milligrams of ascorbic acid equivalents per gram of dry weight. 2.9. Statistical analysis All determinations were run in triplicate on at least two different experiments, and the results were reported as the mean and standard deviation. Statistical variance analysis of independent data with six replicates was performed using ANOVA and compared with least significant differences (LSD) at the 5% level. All statistical analyses were performed with the STATISTICA software.
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TLC confirmed the presence of CGA in the leaf extract of C. maritimum. Only one green spot appeared on the plant extract chromatogram, with a Rf of 0.71, corresponding with chlorogenic acid standard. 3.3. Total phenols and chlorogenic acid levels Total phenol content in the leaves of Crithmum maritimum growing on sand hills and below cliffs was followed over one year (Fig. 2). During spring, summer and winter, there was no difference between the two sets of plants. The level of phenolic compounds increased between spring and summer, from 23 to 33 mg GAE g1 DW. Autumn was characterized by a large (3-fold) decrease of phenol levels in plants growing below cliffs. A similar drop in phenol concentration was found in plants grown on sand hills during winter, so that no significant differences in phenolic levels between the two sets of plants were observed at that period. As stated from the NMR analysis, Crithmum maritimum species appeared to be rich in chlorogenic acid. However, CGA level in plants from sand hills differed significantly from that in cliff plants (Fig. 3). Indeed, plants growing in sand presented a high CGA concentration throughout the growing season (18.8– 27.9 mg CGA g1 DW), whereas plants growing in cliffs produced much less CGA (3– 10 mg CGA g1 DW). 3.4. Antioxidant activity
3. Results 3.1. Soil composition Mineral and organic composition of soils sampling at the root environment of Crithmum maritimum plants are given in Table 1. Cliffs soil appeared to be richer in organic matter, total nitrogen and organic carbon than soil taken in sand hills. Cliffs were also characterized by a higher level of sodium, potassium and magnesium, whereas sand hills soil was richer in calcium. 3.2. Major organic solutes in methanolic extracts of sea fennel The extraction step of dry powder of Crithmum maritimum leaves allowed procurement of a crude methanol/water extract representing about 45% of the initial dry mass. This extract, analyzed by 1H NMR, enabled the determination of major solutes present in sea fennel. The 1H NMR spectrum of methanolic extract of sea fennel showed the predominance of soluble carbohydrates, including sucrose and glucose, and that of an organic acid: malate (Fig. 1). Noteworthy on this NMR spectrum was the presence of characteristic sets of signals in the 1.8–2.2 ppm and 6.3–7.6 ppm regions, assigning a hydroxycinnamic compound: chlorogenic acid. The first set of signals could also correspond to free quinic acid, a component of chlorogenic acid moiety.
Table 1 Chemical composition of soil sampled in sand hills and cliffs
Organic carbon Total N Organic matter pH Na K Ca Mg
Sand hills
Cliffs
0.33 0.07 0.21 0.05 0.58 0.13 9.4 0.1 500 10 215 1.5 5134 61 286 2
1.13 0.29 0.48 0.05 1.94 0.50 7.6 0.3 991 19 321 3 4232 55 806 4
Organic carbon, organic matter and total N are expressed in % dry soil; Naþ, Kþ, Ca2þ and Mg2þ are expressed in mg/kg dry soil. Means S.E. of three replicates are indicated.
3.4.1. DPPH radical scavenging activity Antiradical properties of Crithmum maritimum leaves are given in Table 2. DPPH scavenging activity in sea fennel varied over the year and also according to the population. During spring, sand hill plants presented a low IC50 contrary to cliff plants (0.811 and 1.211 mg ml1, respectively), which indicates a higher antiradical activity. Summer was characterized by a very low IC50 (0.25 mg ml1) for plants living in the two habitats. Such a strong antiradical activity was still found during autumn in sand hill plants, but not in cliff plants whose IC50 rose to 1.05 mg ml1. In winter, a 7-fold increase of the DPPH IC50 of sand hill plants was found, so that antiradical activity of the two sets of plants became close. 3.4.2. ABTS radical scavenging activity Leaves of sea fennels growing below maritime cliffs were characterized by a strong ABTS scavenging activity during spring and summer (Table 2), with IC50 of about 0.10– 0.17 mg ml1. Thereafter, scavenging activity differed greatly in the two sets of plants: ABTS IC50 of sand hill plants remained low (increasing just a little in winter) whereas it increased sharply in cliff plants. As a result, ABTS scavenging activity was 4 times higher in sand hill plants than in cliff plants. 3.4.3. Total antioxidant capacity Leaves of Crithmum maritimum from the two stations presented a strong total antioxidant activity throughout the year (Table 2). This activity was the highest in spring and summer for plants living in cliffs. Autumn was characterized by the lowest antioxidant activity for cliff plants, and the highest for sand hill plants. 4. Discussion Crithmum maritimum exhibited an original water-soluble composition. Besides soluble carbohydrates (sucrose, glucose) and free malate, noteworthy was the abundance of two compounds never reported in halophytic species: free quinic and chlorogenic acids. Recent studies showed the presence of free quinate and/or chlorogenate in other members of the Apiaceae family. Thus, free
L. Meot-Duros, C. Magne´ / Plant Physiology and Biochemistry 47 (2009) 37–41
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Suc Suc
Glc Glc
Glc
Mal CGA CGA/Qui
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5 ppm
1
Fig. 1. H NMR spectrum of Crithmum maritimum leaf methanolic extract. CGA, chlorogenic acid; Glc, glucose; Mal, malic acid; Qui, quinic acid; Suc, sucrose.
quinic acid was found in carrot [20], and CGA was reported in fennel [21], celery [22] and carrot [23]. Therefore, the two molecules could play a chimiotaxonomic role in that family. Crithmum maritimum had relatively high phenolic contents compared to other crop species. Chu et al. [24] reported a level of about 6 mg GAE g1 DW in different vegetables, and Zhou and Yu [25] found 13 and 10.6 mg GAE g1 DW in spinach and broccoli, respectively. These levels correspond to the smallest one measured in sea fennel in our study. In particular, sea fennel appeared as an Apiacea rich in phenols. Literature is scarce about the levels of phenolic compounds in halophytes. In a recent study by Ksouri et al. [6], the Brassicaceae Cakile maritima L. was found to accumulate phenolics (between 31 and 67 mg GAE g1 DW), those levels being similar or higher than those found in sea fennel. Hydroxycinnamic acids like chlorogenic acid are widely spread phenolics in plants [26]. Among the most CGA-rich species are Coffea species, where CGA accumulates in green beans [27] and contributes to coffee beverage bitterness. In our study, chlorogenic acid was constitutively present in sea fennel leaves and markedly accumulated during the growing season in sand hill plants. Even in resting plants (in winter), CGA concentration reached 3 mg g1 DW, this level being 3 times higher than that the maximum level measured in carrot [26], and similar to that in fennel [21]. Therefore, C. maritimum appeared to be the most CGA-rich Apiaceae, with between 1.9% and 2.8% DW in plants living in sand hills. Sea fennel can thus be considered as a valuable source of chlorogenic acid. In this study, plants from sand hills accumulated more CGA (up to 9-fold) than plants growing below cliffs. This might be related to environmental factors which differed in the two habitats. Plants living on sand hills were likely more stressed than those below cliffs because off nutrient deficiency. Accordingly, a wide range of environmental stresses, including boron and nitrogen deficit, have been showed to increased CGA levels in plants [28, 29]. Moreover, plants
in sand suffered from both water stress (due to the nature of sandy substrate) and ionic stress (due to sea sprays), contrary to plants living below cliffs. Therefore, it is likely that plants living in the stressful sand hill environment produced more ROS, which in turn activates the synthesis of antioxidants such as CGA or other radicalscavenging systems to prevent cell death. Accordingly, anti-radical tests showed that plants from sand hills presented a stronger radical-scavenging activity (DPPH and ABTS IC50 were up to 7 and 4 times lower, respectively, than those for cliff plants). This direct link between elevated CGA levels and oxidative stress tolerance was already described in Solanaceae by Niggeweg et al. [30]. DPPH radical-scavenging activity differed between the two populations, but also with the season. Leaf extracts had a stronger radical scavenging activity in summer, when the natural irradiance was at its maximum and resulted in the production of ROS in plants. Moreover, DPPH and ABTS radical-scavenging activities presented a good correlation with the level of phenolic compounds (r ¼ 0.84 and 0.79, respectively), and in particular with CGA (r ¼ 0.72 and 0.56, respectively). Thus, both types of plants showed the highest concentration in total phenols in summer when the strongest antiradical activity was found. Conversely, in winter, plants from the two habitats had the lowest levels of radical-scavenging activity, of total phenols and of CGA. Unlike radical-scavenging activity, total antioxidant activity was poorly correlated with the content of phenolic compounds (r ¼ 0.37). Conveniently, the global antioxidant property of a plant extract is generally considered as the result of the combined activity of a wide range of compounds, including phenolics, peptides, organic acids and other components [31]. Our results suggest that sea fennel can be considered as a valuable source of antioxidant products, especially of chlorogenic acid. This compound, in addition to its strong antioxidant capacity, is known to have antiviral, anti-inflammatory and immune properties [32, 33], so that sea fennel might find a number of industrial applications, particularly in the medicinal field.
Total phenolic level (mg GAE.g-1 DW)
a 30
a
sand hill cliffs
b
c d
20 e
e
e
10 0 spring
summer 1
autumn
winter
Fig. 2. Total phenol level (mg GAE g DW) in the leaves of Crithmum maritimum L. growing in sand hills and below cliffs. Measurements were carried out in triplicate. Means and standard deviations are represented, and different letters above the bars indicate significantly different (P < 0.05) means.
Chlorogenic acid level (mg.g-1 DW)
40 40
sand hill cliffs
a
30 b
b
20 c 10
d d
0
spring
summer 1
autumn
d e winter
Fig. 3. Chlorogenic acid level (mg g DW) in the leaves of Crithmum maritimum L. growing in sand hills and below cliffs. Measurements were carried out in triplicate. Means and standard deviations are represented, and different letters above the bars indicate significantly different (P < 0.05) means.
L. Meot-Duros, C. Magne´ / Plant Physiology and Biochemistry 47 (2009) 37–41 Table 2 Total antioxidant activity and radical-scavenging activity of Crithmum maritimum L. leaf extracts against DPPH$ and ABTS$þ, expressed in mg eq ascorbate g1 DW and mg ml1, respectively
Sand hills
Cliffs
Spring Summer Autumn Winter Spring Summer Autumn Winter
DPPH scavenging activity
ABTS scavenging activity
Antioxidant capacity
(IC50, mg ml1)
(IC50, mg ml1)
(mg eq g1 DW)
0.811 0.07 b 0.248 0.00 a 0.146 0.02 a 1.111 0.01 d 1.211 0.01 d 0.245 0.01 a 1.049 0.01 d 0.928 0.07 c
0.131 0.005 bc 0.112 0.004 ab 0.095 0.006 a 0.171 0.009 e 0.157 0.011 de 0.139 0.014 bcd 0.405 0.011 f 0.418 0.074 f
5.65 0.26 cd 5.76 0.17 a 6.02 0.70 cd 4.09 1.51 e 6.78 0.14 c 7.04 0.65 b 4.03 0.59 e 5.19 0.59 de
Measurements were carried out in triplicate. Means and standard deviations are indicated. Values for each radical scavenging activity followed by the same letter were not significantly different (P < 0.05).
Acknowledgements This research was partly supported by the Brest Metropole Oceane (BMO) through a Ph.D fellowship (L.M.). We thank N. Kervarec and S. Ce´rantola (Service de Re´sonance Magne´tique Nucle´aire, Universite´ de Bretagne Occidentale) for their technical assistance in obtaining NMR spectra. Contribution N 1093 of the IUEM, European Institute for marine Studies (Brest, France).
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