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Tocopherols, fatty acids and sterols in seeds of four Sardinian wild Euphorbia species
Fitoterapia 75 (2004) 50–61
Tocopherols, fatty acids and sterols in seeds of four Sardinian wild Euphorbia species R. Brunia, M. Muzzolia, M. Ballerob, M.C. Loib, G. Fantinc, F. Polid, G. Sacchettia,* a
Dipartimento delle Risorse Naturali e Culturali, Universita` degli Studi di Ferrara, Corso Porta Mare 2, Ferrara I-44100, Italy b Dipartimento di Scienze Botaniche, Universita` degli Studi di Cagliari, Viale S. Ignazio 13, Cagliari I-09123, Italy c Department of Chemistry, University of Ferrara, Via Luigi Borsari 46, Ferrara I-44100, Italy d Department of Evolutionary and Experimental Biology, University of Bologna, Via Irnerio 42, Bologna I-40126, Italy Received 21 February 2003; accepted 28 August 2003
Abstract Sardinian wild Euphorbia pithyusa, E. semiperfoliata, E. dendroides and E. characias seed oils were analyzed for their fatty acids, unsaponifiable and tocopherol content. Total tocopherol content showed a wide variability, ranging from 939 mgykg in E. semiperfoliata seeds to its absence in E. characias. The results on tocopherol content were statistically correlated with both 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test and the b-carotene bleaching antioxidant test. All seeds were rich in linolenic acid, while no uncommon fatty acids were detected. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Euphorbia spp.; Seed lipids; Tocopherols; Antioxidant activity
1. Introduction Mediterranean Euphorbia species have been the object of various studies and they have been proposed as potential renewable sources of unsaturated w1,2x and *Corresponding author. Tel.: q39-0532-293781; fax: q39-0532-208561. E-mail address: [email protected] (G. Sacchetti). 0367-326X/04/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2003.07.009
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uncommon fatty acids w3,4x or fine chemicals from the unsaponifiable fraction w5x. However, the major problem concerning these species is the high content of skin irritants, which preclude a wider range of applications w6x. Euphorbia pithyusa and E. semiperfoliata are endemic plants of the islands of Sardinia and Corsica, while E. dendroides and E. characias are species widespread in the western Mediterranean region w7x. All these Euphorbia species are known for their high phytochemical diversity w8–10x enhanced by the ecological isolation of Sardinia which makes this island a pivotal center of chemical and botanical bio-diversity in southern Europe. Due to the irritant and pharmacological activities of these species w11x much attention has been focused on the phytochemistry of the whole plant w12,13x and of its latex in particular w8,9x. However, except for the fatty acid composition of E. characias and E. dendroides seeds w14,15x, little attention has been focused on their oil-seed lipid composition and chemodiversity. Thus, in order to extend the knowledge of phytochemical biodiversity of Euphorbia species which grow wild in Sardinia, the seed oil composition of four Euphorbia spp. was studied. Particular emphasis was placed on tocopherol content and antioxidant activity. 2. Materials and methods 2.1. Plant material Euphorbia characias L. and E. pithyusa L. subsp. cupanii (Guss.) A.R. Sm. seeds were collected in March and June 2001 at Marganai Massif, Iglesias, (Cagliari), Italy. E. semiperfoliata Viv. seeds were collected during July 2001 at Arzana, (Nuoro), Italy. E. dendroides L. seeds were collected in San Elia Promontory, (Cagliari), during the same period. All accessions were identified by Prof Mauro Ballero, Department of Plant Sciences, University of Cagliari. Voucher specimens of the four species (CAG 1216; CAG 1212; CAG 1217; CAG 1215, respectively) were deposited at the Herbarium of the Department of Plant Sciences, University of Cagliari. 2.2. Fatty acid extraction The four Sardinian Euphorbia spp. seeds were ground using a blade grinder and forced through a 0.2 mm mesh-sieve. Care was taken to ensure that, during grinding, the temperature never exceeded 30 8C. After grinding the flour was stored in the dark at y20 8C. The fatty acid content was evaluated from extracts obtained by placing 5 g of each Euphorbia species seed flour in a flask with 100 ml of hexane and treating for 1 h in an ultrasound bath. Then, the samples were filtered and centrifuged for 20 min at 3000 rev.ymin. The supernatant was recovered, dried with a rotavapor and stored at y20 8C until the gas chromatographic analysis was performed.
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2.3. Fatty acid profile (FAP) The FAP was obtained via gas-chromatographic analyses of the fatty acids methyl esters (FAME). The FAME were prepared by trans-methylation using sodium methoxide in the presence of MeOAc w16x. Analyses were performed employing a Fisons 9160 9000 Series GC equipped with a Fisons EL980 amplifier, a FID detector and a Mega Megawax column (Mega, Legnano, Italy—i.d.s0.32 mm, length 30 m, film thicknesss0.45 mm). Operating conditions: injection temperature, 280 8C; detector temperature, 280 8C; split ratio 1:40; carrier gas, helium, at a flow rate of 2 mlymin. Oven temperature was initially 60 8C and then raised to 230 8C at a rate of 2 8Cymin, followed by 15 min at 230 8C. One microliter of each sample was injected. The fatty acid standards were obtained from Alltech (Deerfield, IL, USA). 2.4. Tocopherol extraction and analyses The flour (5 g) of each of the four Euphorbia species was placed in a flask containing 100 ml of MeOH and stirred for 24 h in the dark at a constant temperature of 25 8C. The samples were filtered and centrifuged for 20 min at 3000 rev.ymin. The supernatant was recovered, dried with a rotavapor and weighed. The dry extract was then placed in 100 ml of hexane and set in an ultrasound bath (Branson 5200, Danbury, CT, USA) for 30 min at a constant temperature of 25 8C to facilitate solubilisation. The sample was centrifuged at 3000 rev.ymin for 20 min. The supernatant recovered was dried with a rotavapor and stored at y20 8C until the analyses were performed. HPLC analysis: modular Jasco HPLC unit (Tokyo, Japan) consisting of a PU980 pump, a LG-1580-02 ternary gradient unit, a DG-980-503-line degaser, a UVy VIS 975 detector set at 295 nm, linked to an injection valve with a 20 ml sampler loop. A Lichrosorb Silica-gel Si 60 (5 mm and 25=0.4 cm; Teknokroma, Barcelona, Spain) column was used and the mobile phase was 0.05% iPrOHyhexane at a flow rate of 1 mlymin. The injection volume was 80 ml. Chromatograms were recorded and a, b, g, d-Tocopherol peaks from samples were identified by comparing their spectra with those of pure standards (Matreya Inc., Pleasant Gap, PA 16823, USA). The peak areas were determined by integration using dedicated Borwin software (Borwin ver. 1.22, JMBS Developments, Grenoble, France). Each extract analysis was performed in triplicate. 2.5. Unsaponifiable analysis Samples of the four Euphorbia seed extracts were saponified. Forty milligrams of each sample were well mixed in 5 ml 1 M MeOH–KOH with the aid of sonication and then horizontally shaken at 30 8C for 24 h. The unsaponifiable content was then extracted three times adding 1 ml of H2O, 2 ml of n-hexane and 0.1 ml of EtOH. The aqueous fractions were collected and extracted with the same mixture as above. All unsaponifiable fractions were collected and taken to dryness
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under vacuum. The residue was silanized for GC-analyses with 2 ml of silanizing mixture (pyyHMDSyTMCS 5:2:1). After 1 h, the samples were dried under N2 stream at 70 8C and dissolved with 0.3 ml of n-hexane. Before the recovery of the surnatant, the samples were sonicated for 5 min and centrifuged for 10 min at 4000 rev.ymin. The GC apparatus conditions were the same as reported above except for the oven temperature, which was initially 230 8C and then raised to 320 8C with a rate of 5 8Cymin. 2.6. GC-MS analysis GC-MS analysis was performed on a Fisons 8060 8000 series GC equipped with a MEGA SE54 column (Mega, Legnano, Italy—i.d.s0.32 mm, length 25 m, film thicknesss0.15 mm) and coupled to a Fisons HD800 mass spectrometer with a MassLab data system. Helium was used as carrier gas at an inlet pressure of 30 kPa. The injector temperature was 300 8C and the samples were injected at the same conditions reported above for GC analyses. The mass spectra were recorded at an electron energy of 70 eV and the ion source temperature was 300 8C. Qualitative analysis was based on a comparison of the retention times and of the mass spectra with the corresponding data in the literature w17,18x. All sterols were identified in comparison with data from the literature, spectra of pure reference standards and with entries in nbs75k HP Chemstation library. 2.7. Scavenging activity on DPPH radical The antioxidant activity of the Euphorbia species seed extracts was assessed by the scavenging effect on DPPH radical (1,1-diphenyl-2-picrylhydrazyl) w19x. Samples of the same four Euphorbia seed extracts employed for tocopherol analyses were added to a MeOH solution of DPPH (1=10y4 M) and kept in the dark for 30 min. The absorbance of samples was measured at the spectrophotometer (Helios Gamma, Thermo Spectronic, Mercers Row, Cambridge, UK) at 517 nm against a blank of MeOH. The DPPH inhibition percentage was calculated according to Yen and Duh w20x. 2.8. b-Carotene bleaching test Approximatively 10 mg trans-b-carotene (type 1 synthetic, Sigma, St. Louis, MO, USA) was dissolved in 10 ml of CHCl3 and 0.2 ml of the solution was placed into a boiling flask containing 20 mg linoleic acid and 200 mg Tween-40 (Sigma). After removal of CHCl3, 50 ml of distilled water was added to the flask and vigorously shaken. Five milliliters of this emulsion were added to tubes containing the putative antioxidants (0.2 ml) w21x. The tubes were stoppered and placed in a water bath at 50 8C. Spectrophotometric readings at 470 nm were taken after 60 and 120 min of incubation. Controls consisted of butylated-hydroxy-anisole (BHA; positive control) and a linoleic acid and b-carotene emulsion (negative control).
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Relative antioxidant activities (RAA) were calculated with the following formula w22x: RAAsAbs of sampleyAbs of BHA. 2.9. Statistical analysis For each or the data collected, relative standard deviation was given. Analyses of variance were then performed with the aim to determine if the differences recorded for the chemical characterization among the species were significant. Correlations analyses (Pearson coefficient) between the concentrations of individual tocopherols, total tocopherol content, oil content, individual fatty acids, antioxidants results were also performed. All computations were made using the statistical software SPSS w23x. 3. Results and discussion 3.1. Fatty acids The fatty acid composition of the seeds is shown in Table 1. The oil content exhibited a wide variability, ranging from 12.32"1.42% in E. pithyusa to 4.10"0.47% in E. dendroides. Only E. characias and E. pithyusa subsp. cupanii seeds oil showed a similar fatty acid profile. For both of these species the predominant fatty acid compound was linolenic and, consequently, the SyU ratio showed low values (E. characias 0.21; E. pihytusa subsp. cupanii 0.08), whereas E. dendroides and E. sempierfoliata seed oils showed important differences. In fact, E. semiperfoliata seed oil is mainly composed of saturated fatty acids, with a clear predominance of stearic acid (52.79"6.08%) and a low amount of linolenic acid. Thus, the high SyU ratio (2.96) makes this oil strongly different, chemically, from the others. Finally, E. dendroides seed oil has a fatty acid composition similar to E. characias and E. pithyusa subsp. cupanii, but a higher SyU ratio (0.45) due to the consistently large presence of stearic acid (19.78"2.28%). The different values recorded for both linoleic and linolenic acids in the four Euphorbia species seeds confirmed the highly significant chemical diversity of the four species (Fs147.37 for linoleic acid; Fs995.11 for linolenic acid; d.o.fs8, P-0.001 for all computations), already evidenced for other chemicals in the Euphorbia genus w8–10x. Moreover, these results slightly differ from previously reported data regarding wholeplant extracts w12,13x. In fact, all the samples generally showed a higher presence of unsaturated fatty acids (linoleic and linolenic acid) and a lower content of palmitic acid. When compared with related published data w14,15x few, but noteworthy differences emerged. However, the fact that the correlation between the yield oil percentage and the unsaturated fatty acids is always positive suggests that the four Sardinian Euphorbia species accumulating the highest amount of oil in their seeds show preference for synthetizing unsaturated fatty acids.
Samples E. characias E. dendroides E. pithyusa subsp. cupanii E. semiperfoliata
Yield
Myristic
Palmitic
Stearic
9.17"1.05 0.66"0.07 13.34"1.54 2.02"0.23 4.10"0.47 0.92"0.11 7.97"0.92 19.78"2.28
Oleic
Linoleic
Linolenic
Arachidic
Behenic
SyU Ratio
8.13"0.94 22.24"2.56 52.02"5.99 1.78"0.21 – 0.21 5.84"0.67 16.61"1.91 46.38"5.34 0.86"0.09 1.53"0.18 0.45
12.32"1.42 – 6.41"0.74 1.09"0.13 12.26"1.41 21.23"2.44 59.01"6.79 – – 0.08 8.41"0.97 3.26"0.37 10.91"1.26 52.79"6.08 4.23"0.49 7.48"0.86 13.39"1.54 3.71"0.43 3.69"0.42 2.96
Mean ("S.E.; ns3) values.
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Table 1 Fatty acid profile (%) of four Euphorbia species seeds
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Table 2 Unsaponifiable fraction (%) of four Euphorbia species seeds E. characias E. dendroides E. pithyusa subsp. cupanii E. semiperfoliata Yield (wt.%) Hydrocarbons Fatty alcohols Cholesterol Campesterol b-Sitosterol D5-Avenasterol Lanosterol isomer Lanosterol b-Amyrin Cycloartanol 24-Methylen-cycloartenol Others
18.05"2.07 19.83"2.28 15.68"1.81 – 1.72"0.19 11.62"1.34 – – 10.92"1.26 3.42"0.39 16.16"1.86 12.77"1.47 7.88"0.91
16.1"1.84 17.78"2.05 17.37"2.01 0.94"0.11 – 12.89"1.48 – – 10.14"1.17 4.24"0.49 23.61"2.72 8.62"0.99 4.41"0.51
10.23"1.15 4.78"0.55 16.1"1.85 – 4.65"0.54 25.8"2.97 7.39"0.85 – – – 26.6"3.06 14.7"1.69 –
13.54"1.49 15.94"1.84 45.66"5.26 0.47"0.05 0.93"0.11 7.31"0.84 – 2.68"0.31 1.09"0.13 1.68"0.19 16.38"1.88 2.24"0.26 5.62"0.65
Mean ("S.E.; ns3) values.
3.2. Unsaponifiable fraction GC-MS of trimethylsilated unsaponifiable fraction of E. characias, E. pithyusa subsp. cupanii, E. dendroides and E. semiperfoliata seeds readily separated phytosterols and triterpenic alcohols (Table 2) showing qualitative and quantitative differences among the samples. A considerable amount of hydrocarbons and fatty alcohols was also detected, especially in E. semiperfoliata. The triterpenic alcohol cycloartanol was the main sterolic constituent in all samples, but its abundance in E. pithyusa subsp. cupanii was almost twice higher than that found in E. characias and E. semiperfoliata. 24-Methylenecycloartanol was found to be minoritary in E. semiperfoliata seeds and abundant up to 14.7"1.69% in E. pithyusa subsp. cupanii. Cholesterol was detected only in E. semiperfoliata and E. dendroides seeds. In comparison with previously published papers w12,13x, E. pithyusa subsp. cupanii seeds showed a higher qualitative homogenity in chemical composition, lacking both b-amyrin, lanosterol, cholesterol that were found in other seeds. On the contrary, a significant amount of D5-avenasterol was detected. Lanosterol, a sterol compound rarely found in plant seeds, was abundant in E. characias and E. dendroides seeds, but scarce in E. semiperfoliata seed oil. 3.3. Tocopherols and antioxidant activity The content of the single tocopherols and the quantitative ratio among them in seed oils of different plant species is known to be representative, and related to the similar habitats where these species grow. Moreover, the qualitative and quantitative evaluation of tocopherols in Brassicaceae, Orobanchaceae and Boraginaceae suggested a chemotaxonomic importance of these vitamin E isomers among the plants belonging to these families w24–26x. All the Euphorbia species seeds examined, with the sole exception of E. characias, showed a consistent presence of tocopherols
Samples
a-Tocopherol
b-Tocopherol
g-Tocopherol
d-Tocopherol
Total tocopherols
E. E. E. E.
ND 130.23"13.24 337.94"34.36 877.14"89.17
ND ND 51.88"5.27 12.77"1.30
ND 20.78"2.11 156.64"15.92 31.38"3.19
ND ND 13.76"1.34 17.56"1.78
ND 151.01"15.35 560.23"56.96 938.87"95.45
characias dendroides pithyusa subsp. cupanii semiperfoliata ND: Not Detected. Mean ("S.E.; ns3). * mgykg seeds.
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Table 3 Tocopherol content of Euphorbia species seeds*
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Table 4 Inhibition (%) of the 1,1-diphenyl-2-picrylhydrazyl radicals
E. characias E. dendroides E. pithyusa subsp. cupanii E. semiperfoliata BHAa a
50 (mgyml)
100 (mgyml)
200 (mgyml)
500 (mgyml)
1.42"0.05 3.46"0.07 29.37"1.9 6.32"0.1 95.61"3.5
2.95"0.07 8.35"0.09 43.38"2.7 15.87"1.3 95.00"3.3
4.79"0.08 16.10"1.5 91.40"3.5 29.59"1.9 95.71"3.7
6.42"0.09 19.57"1.15 92.36"3.9 43.79"2.5 95.87"4.1
Positive control: butylated hydroxy anisole.
Table 5 b-Carotene bleaching testa
E. characias E. dendroides E. pithyusa subsp. cupanii E. semiperfoliata BHA Negative controlc
RAAb 60 min
RAA 120 min
0.65"0.05 0.76"0.05 0.97"0.06 0.94"0.06 1 0.33"0.03
0.58"0.04 0.66"0.05 0.84"0.05 0.87"0.05 1 0.31"0.03
a
Concentration of the seeds extracts: 2 mgyml. Relative Antioxidant Activities. c Negative control: linoleic acid and b-carotene emulsion. b
with important qualitative and quantitative differences among the species (Table 3). All the differences recorded among the four Euphorbia species seeds for the total tocopherol content and for the individual tocopherols were highly significant (Fs 242436.6 for the total tocopherol content, Fs353147.5 for a-tocopherol, Fs 2756.271 for b-tocopherol, Fs14508.41 for g-tocopherol, Fs305.31 for d-tocopherol; d.o.fs6, P-0.001 for all computations). In fact, E. semiperfoliata showed a total tocopherol content of 93% and 40% higher than E. dendroides and E. pithyusa subsp. cupanii, respectively. Concerning the detection of tocopherols in E. pithyusa subsp. cupanii and E. semiperfoliata seeds, all the four vitamin E isomers were found, while in E. dendroides seed oil only a- and g-tocopherol were detected. From a quantitative point of view, the amount of single compounds with respect to the total tocopherol content was very different in all the Euphorbia species seeds. In fact, in E. semiperfoliata and E. dendroides, a-tocopherol accounted for 93.43% and 86.24% of the total tocopherol amount, respectively. In E. pithyusa subsp. cupanii seed oil, a-tocopherol was present in a lower amount (60.32% of the total tocopherols) but at the same time g-tocopherol was an important fraction (27.96%). In fact, E. pithyusa subsp. cupanii seeds presented the highest amount of g-topherol between the Euphorbia spp., 86% more than E. dendroides and 79% more than E. semiperfoliata. However, the tocopherols content was always positively correlable with the antioxidant tests performed. In fact, on both 1,1-diphenyl-2-picrylhydrazyl (DPPH)
Table 6 Pearson correlation coefficients between the individual tocopherols, the total tocopherol content*, oil content (%), fatty acids (%), antioxidants values at each concentration for both DPPH and b-carotene bleaching test in the four Euphorbia species bT
gT
dT
TTC Yield C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 50a 0.82 y0.37 y0.44 0.48 0.66 y0.29
y0.04 0.85 y0.39 y0.88 y0.62 y0.29 0.78 0.22 y0.71 y0.33 0.79 0.26 y0.23 0.52 0.07 y0.57 y0.19 0.72 y0.17 y0.76 y0.03 y0.35 0.75 0.32 0.36 0.97 y0.83 y0.96 0.16 y0.41 y0.1 y0.81 y1.00 0.77
y0.84 0.33 0.40 y0.51 y0.69 0.27 y1.0 y0.32 y0.98 0.81 0.97
0.67 y0.48 y0.58 0.34 0.49 y0.19 0.94 0.65 0.84 y0.78 y0.80 y0.92
0.82 y0.31 y0.34 0.48 0.69 y0.41 0.96 0.10 1.00 y0.82 y0.99 y0.96 0.80
0.15 0.99 1.00 0.56 0.37 0.74 y0.45 y0.68 y0.35 0.81 0.28 0.41 y0.57 y0.36
200a
500a
60b
120b
0.29 0.99 0.99 0.66 0.50 0.71 y0.32 y0.69 y0.21 0.72 0.14 0.27 y0.47 y0.22 0.99
0.25 0.99 1.00 0.63 0.47 0.71 y0.36 y0.70 y0.25 0.75 0.18 0.31 y0.51 y0.26 0.99 1.00
0.40 0.75 0.77 0.98 0.71 0.71 0.97 0.69 0.67 0.74 0.87 0.93 y0.60 0.86 0.88 0.69 0.39 0.48 y0.20 0.29 0.31 y0.67 y0.53 y0.50 y0.09 0.41 0.41 0.64 0.16 0.18 0.02 y0.47 y0.46 0.16 y0.33 y0.35 y0.37 0.05 0.11 y0.11 0.40 0.39 0.96 0.67 0.66 0.99 0.77 0.75 0.99 0.74 0.73 0.83 0.82 0.85
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aT 0.23 0.16 0.89 0.97 0.16 bT 0.99 0.63 0.45 0.79 gT 0.55 0.38 0.70 dT 0.97 0.55 TTC 0.32 Yield C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 50a 100a 200a 500a 60b
100a
Significant values (P-0.05) are given in bold *mgykg seeds. Ns12. a Values referred to the concentration employed in DPPH radical scavenging test; b values referred to detection time in b-carotene bleaching test; a, b, g, d-Tsa, b, g, d-Tocopherol; TTC: Total Tocopherol Content. 59
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and b-carotene bleaching tests E. semiperfoliata and E. pithyusa subsp. cupanii gave the best results (Tables 4 and 5). E. pithyusa subsp. cupanii extract exibited a scavenging activity on the DPPH radical, with values comparable to the positive control at 200 and 500 mgyml. E. semiperfoliata seed extracts showed a dose-dependent antioxidant activity, with a scavenging action of nearly 50%. E. dendroides extract activity was low, while E. characias seed extracts did not show any scavenging activity. In the b-carotene bleaching test the E. pithyusa subsp. cupanii and E. semiperfoliata seed extracts gave the best results, with relative antioxidant activity (RAA) values almost equivalent to the positive control butylated-hydroxy-anisole (BHA) and only slightly reduced after 120 min (12.0% E. pithyusa subsp. cupanii; 7.4% E. semiperfoliata). As expected, as a consequence of their lower tocopherols content, E. characias and E. dendroides showed a protective activity 20–30% worse. Interestingly, E. pithyusa subsp. cupanii, which showed a consistently lower content of a-tocopherol than E. semiperfoliata, revealed a higher scavenging activity against DPPH radical (Table 4). These results seem to reveal a more important role of b- and g-tocopherol in scavenging activity in the DPPH test. In the b-carotene bleaching test (Table 5), these kinds of qualitative correlations were not so evident. In fact, E. semiperfoliata and E. pithyusa subsp. cupanii had a similar scavenging activity. The quantitative correlation between the individual tocopherols and the antioxidant activity in both performed tests seems to be more pertinent than the correlation between the total tocopherol content and the same radical-scavenging results (Table 6). In fact, all the individual tocopherols in the four Euphorbia species are positively correlated with the antioxidant activity results, usually with significant Pearson coefficient values. While the total tocopherol content shows an anomalous negative—but significant—correlation with radical scavenging activity (Table 6). The same considerations could be made between individual tocopherols— or total tocopherol—content and the fatty acids profile. It is known that the fatty acids profile, especially the unsaturated fraction, is positively correlated with the tocopherols. In the Euphorbia seed oils considered, this kind of positive correlation always exists, but only for b- and g-tocopherol, i.e. those vitamin E isomers, which always exhibited a significant positive correlation with the antioxidant tests results. Acknowledgments Investigation supported from Ministero dell’Istruzione, dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST) of Italy. A special thanks goes to Orna Crinion for advice on language translation. References w1 x w2 x w3 x w4 x w5 x
Villalobos MJ, Correal Castellanos E. Grasas Aceites (Seville) 1992;43:39. Agarwal R, Mustafa J, Gupta A, Osman SM. Fett Wiss Technol 1995;97:526. Vogel R, Pascual-Villalobos MJ, Roebbelen G. Angew Bot 1993;67:31. Vioque J, Pastor JE, Vioque E. Grasas Aceites (Seville) 1994;45:161. Hoffmann JJ. Critical reviews in plant sciences. Boca Raton: CRC Press, 1983.
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Cuperus FP, Boswinkel G, Derksen JTP. J Am Oil Chem Soc 1996;73:1635. Pignatti S. Flora d’Italia. Bologna: Edagricole, 1982. Appendino G, Belloro E, Tron GC, Jakupovic J, Ballero M. J Natl Prod 1999;62:1399. Appendino G, Belloro E, Tron GC, Jakupovic J, Ballero M. Fitoterapia 2000;71:134. Appendino G, Jakupovic S, Tron GC, Jakupovic J, Milon V, Ballero M. J Natl Prod 1999;61:749. Bruni A, Ballero M, Poli F. J Ethnopharmacol 1997;57:97. Conti L, Marchetti M, Soccolini F, Usai M. Phytochemistry 1990;29:77. Conti L, Marchetti M, Usai M, Botteghi C. Phytochemistry 1988;27:791. www.ncaur.usda.gov. NCAUR database. United States Department of Agriculture. Ferlay V, Mallet G, Masson A, Ucciani E, Gruber M. Oleagineux 1993;48:91. Christie WW. J Lipid Res 1982;23:1072. Carpenter DR, Hammerstone JF, Romanczyk LJ, Aikten WM. J Am Oil Chem Soc 1994;71:845. Dutta PC, Normen L. J Chromatogr A 1998;816:177. Wang M, Li J, Rangarajan M, Shao Y, LaVoie EJ, Huang TC, Ho CT. J Agric Food Chem 1998;46:4869. Yen GC, Duh PD. J Agric Food Chem 1994;42:629. Taga MS, Miller EE, Pratt DE. J Am Oil Chem Soc 1984;61:928. Dapkevicius A, Venskutonis R, van Beek TA, Linssen JPH. J Sci Food Agric 1998;77:140. Norusis MS. SPSS, Ed. Chicago: SPSS, 1999. Velasco L, Goffman FD. Phytochemistry 1999;52:423. Goffman FD, Thies W, Velasco L. Phytochemistry 1999;50:793. Velasco L, Goffman FD, Pujadas-Salva` AJ. Phytochemistry 2000;54:295.
Tocopherols, fatty acids and sterols in seeds of four Sardinian wild Euphorbia species R. Brunia, M. Muzzolia, M. Ballerob, M.C. Loib, G. Fantinc, F. Polid, G. Sacchettia,* a
Dipartimento delle Risorse Naturali e Culturali, Universita` degli Studi di Ferrara, Corso Porta Mare 2, Ferrara I-44100, Italy b Dipartimento di Scienze Botaniche, Universita` degli Studi di Cagliari, Viale S. Ignazio 13, Cagliari I-09123, Italy c Department of Chemistry, University of Ferrara, Via Luigi Borsari 46, Ferrara I-44100, Italy d Department of Evolutionary and Experimental Biology, University of Bologna, Via Irnerio 42, Bologna I-40126, Italy Received 21 February 2003; accepted 28 August 2003
Abstract Sardinian wild Euphorbia pithyusa, E. semiperfoliata, E. dendroides and E. characias seed oils were analyzed for their fatty acids, unsaponifiable and tocopherol content. Total tocopherol content showed a wide variability, ranging from 939 mgykg in E. semiperfoliata seeds to its absence in E. characias. The results on tocopherol content were statistically correlated with both 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test and the b-carotene bleaching antioxidant test. All seeds were rich in linolenic acid, while no uncommon fatty acids were detected. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Euphorbia spp.; Seed lipids; Tocopherols; Antioxidant activity
1. Introduction Mediterranean Euphorbia species have been the object of various studies and they have been proposed as potential renewable sources of unsaturated w1,2x and *Corresponding author. Tel.: q39-0532-293781; fax: q39-0532-208561. E-mail address: [email protected] (G. Sacchetti). 0367-326X/04/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2003.07.009
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uncommon fatty acids w3,4x or fine chemicals from the unsaponifiable fraction w5x. However, the major problem concerning these species is the high content of skin irritants, which preclude a wider range of applications w6x. Euphorbia pithyusa and E. semiperfoliata are endemic plants of the islands of Sardinia and Corsica, while E. dendroides and E. characias are species widespread in the western Mediterranean region w7x. All these Euphorbia species are known for their high phytochemical diversity w8–10x enhanced by the ecological isolation of Sardinia which makes this island a pivotal center of chemical and botanical bio-diversity in southern Europe. Due to the irritant and pharmacological activities of these species w11x much attention has been focused on the phytochemistry of the whole plant w12,13x and of its latex in particular w8,9x. However, except for the fatty acid composition of E. characias and E. dendroides seeds w14,15x, little attention has been focused on their oil-seed lipid composition and chemodiversity. Thus, in order to extend the knowledge of phytochemical biodiversity of Euphorbia species which grow wild in Sardinia, the seed oil composition of four Euphorbia spp. was studied. Particular emphasis was placed on tocopherol content and antioxidant activity. 2. Materials and methods 2.1. Plant material Euphorbia characias L. and E. pithyusa L. subsp. cupanii (Guss.) A.R. Sm. seeds were collected in March and June 2001 at Marganai Massif, Iglesias, (Cagliari), Italy. E. semiperfoliata Viv. seeds were collected during July 2001 at Arzana, (Nuoro), Italy. E. dendroides L. seeds were collected in San Elia Promontory, (Cagliari), during the same period. All accessions were identified by Prof Mauro Ballero, Department of Plant Sciences, University of Cagliari. Voucher specimens of the four species (CAG 1216; CAG 1212; CAG 1217; CAG 1215, respectively) were deposited at the Herbarium of the Department of Plant Sciences, University of Cagliari. 2.2. Fatty acid extraction The four Sardinian Euphorbia spp. seeds were ground using a blade grinder and forced through a 0.2 mm mesh-sieve. Care was taken to ensure that, during grinding, the temperature never exceeded 30 8C. After grinding the flour was stored in the dark at y20 8C. The fatty acid content was evaluated from extracts obtained by placing 5 g of each Euphorbia species seed flour in a flask with 100 ml of hexane and treating for 1 h in an ultrasound bath. Then, the samples were filtered and centrifuged for 20 min at 3000 rev.ymin. The supernatant was recovered, dried with a rotavapor and stored at y20 8C until the gas chromatographic analysis was performed.
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2.3. Fatty acid profile (FAP) The FAP was obtained via gas-chromatographic analyses of the fatty acids methyl esters (FAME). The FAME were prepared by trans-methylation using sodium methoxide in the presence of MeOAc w16x. Analyses were performed employing a Fisons 9160 9000 Series GC equipped with a Fisons EL980 amplifier, a FID detector and a Mega Megawax column (Mega, Legnano, Italy—i.d.s0.32 mm, length 30 m, film thicknesss0.45 mm). Operating conditions: injection temperature, 280 8C; detector temperature, 280 8C; split ratio 1:40; carrier gas, helium, at a flow rate of 2 mlymin. Oven temperature was initially 60 8C and then raised to 230 8C at a rate of 2 8Cymin, followed by 15 min at 230 8C. One microliter of each sample was injected. The fatty acid standards were obtained from Alltech (Deerfield, IL, USA). 2.4. Tocopherol extraction and analyses The flour (5 g) of each of the four Euphorbia species was placed in a flask containing 100 ml of MeOH and stirred for 24 h in the dark at a constant temperature of 25 8C. The samples were filtered and centrifuged for 20 min at 3000 rev.ymin. The supernatant was recovered, dried with a rotavapor and weighed. The dry extract was then placed in 100 ml of hexane and set in an ultrasound bath (Branson 5200, Danbury, CT, USA) for 30 min at a constant temperature of 25 8C to facilitate solubilisation. The sample was centrifuged at 3000 rev.ymin for 20 min. The supernatant recovered was dried with a rotavapor and stored at y20 8C until the analyses were performed. HPLC analysis: modular Jasco HPLC unit (Tokyo, Japan) consisting of a PU980 pump, a LG-1580-02 ternary gradient unit, a DG-980-503-line degaser, a UVy VIS 975 detector set at 295 nm, linked to an injection valve with a 20 ml sampler loop. A Lichrosorb Silica-gel Si 60 (5 mm and 25=0.4 cm; Teknokroma, Barcelona, Spain) column was used and the mobile phase was 0.05% iPrOHyhexane at a flow rate of 1 mlymin. The injection volume was 80 ml. Chromatograms were recorded and a, b, g, d-Tocopherol peaks from samples were identified by comparing their spectra with those of pure standards (Matreya Inc., Pleasant Gap, PA 16823, USA). The peak areas were determined by integration using dedicated Borwin software (Borwin ver. 1.22, JMBS Developments, Grenoble, France). Each extract analysis was performed in triplicate. 2.5. Unsaponifiable analysis Samples of the four Euphorbia seed extracts were saponified. Forty milligrams of each sample were well mixed in 5 ml 1 M MeOH–KOH with the aid of sonication and then horizontally shaken at 30 8C for 24 h. The unsaponifiable content was then extracted three times adding 1 ml of H2O, 2 ml of n-hexane and 0.1 ml of EtOH. The aqueous fractions were collected and extracted with the same mixture as above. All unsaponifiable fractions were collected and taken to dryness
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under vacuum. The residue was silanized for GC-analyses with 2 ml of silanizing mixture (pyyHMDSyTMCS 5:2:1). After 1 h, the samples were dried under N2 stream at 70 8C and dissolved with 0.3 ml of n-hexane. Before the recovery of the surnatant, the samples were sonicated for 5 min and centrifuged for 10 min at 4000 rev.ymin. The GC apparatus conditions were the same as reported above except for the oven temperature, which was initially 230 8C and then raised to 320 8C with a rate of 5 8Cymin. 2.6. GC-MS analysis GC-MS analysis was performed on a Fisons 8060 8000 series GC equipped with a MEGA SE54 column (Mega, Legnano, Italy—i.d.s0.32 mm, length 25 m, film thicknesss0.15 mm) and coupled to a Fisons HD800 mass spectrometer with a MassLab data system. Helium was used as carrier gas at an inlet pressure of 30 kPa. The injector temperature was 300 8C and the samples were injected at the same conditions reported above for GC analyses. The mass spectra were recorded at an electron energy of 70 eV and the ion source temperature was 300 8C. Qualitative analysis was based on a comparison of the retention times and of the mass spectra with the corresponding data in the literature w17,18x. All sterols were identified in comparison with data from the literature, spectra of pure reference standards and with entries in nbs75k HP Chemstation library. 2.7. Scavenging activity on DPPH radical The antioxidant activity of the Euphorbia species seed extracts was assessed by the scavenging effect on DPPH radical (1,1-diphenyl-2-picrylhydrazyl) w19x. Samples of the same four Euphorbia seed extracts employed for tocopherol analyses were added to a MeOH solution of DPPH (1=10y4 M) and kept in the dark for 30 min. The absorbance of samples was measured at the spectrophotometer (Helios Gamma, Thermo Spectronic, Mercers Row, Cambridge, UK) at 517 nm against a blank of MeOH. The DPPH inhibition percentage was calculated according to Yen and Duh w20x. 2.8. b-Carotene bleaching test Approximatively 10 mg trans-b-carotene (type 1 synthetic, Sigma, St. Louis, MO, USA) was dissolved in 10 ml of CHCl3 and 0.2 ml of the solution was placed into a boiling flask containing 20 mg linoleic acid and 200 mg Tween-40 (Sigma). After removal of CHCl3, 50 ml of distilled water was added to the flask and vigorously shaken. Five milliliters of this emulsion were added to tubes containing the putative antioxidants (0.2 ml) w21x. The tubes were stoppered and placed in a water bath at 50 8C. Spectrophotometric readings at 470 nm were taken after 60 and 120 min of incubation. Controls consisted of butylated-hydroxy-anisole (BHA; positive control) and a linoleic acid and b-carotene emulsion (negative control).
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Relative antioxidant activities (RAA) were calculated with the following formula w22x: RAAsAbs of sampleyAbs of BHA. 2.9. Statistical analysis For each or the data collected, relative standard deviation was given. Analyses of variance were then performed with the aim to determine if the differences recorded for the chemical characterization among the species were significant. Correlations analyses (Pearson coefficient) between the concentrations of individual tocopherols, total tocopherol content, oil content, individual fatty acids, antioxidants results were also performed. All computations were made using the statistical software SPSS w23x. 3. Results and discussion 3.1. Fatty acids The fatty acid composition of the seeds is shown in Table 1. The oil content exhibited a wide variability, ranging from 12.32"1.42% in E. pithyusa to 4.10"0.47% in E. dendroides. Only E. characias and E. pithyusa subsp. cupanii seeds oil showed a similar fatty acid profile. For both of these species the predominant fatty acid compound was linolenic and, consequently, the SyU ratio showed low values (E. characias 0.21; E. pihytusa subsp. cupanii 0.08), whereas E. dendroides and E. sempierfoliata seed oils showed important differences. In fact, E. semiperfoliata seed oil is mainly composed of saturated fatty acids, with a clear predominance of stearic acid (52.79"6.08%) and a low amount of linolenic acid. Thus, the high SyU ratio (2.96) makes this oil strongly different, chemically, from the others. Finally, E. dendroides seed oil has a fatty acid composition similar to E. characias and E. pithyusa subsp. cupanii, but a higher SyU ratio (0.45) due to the consistently large presence of stearic acid (19.78"2.28%). The different values recorded for both linoleic and linolenic acids in the four Euphorbia species seeds confirmed the highly significant chemical diversity of the four species (Fs147.37 for linoleic acid; Fs995.11 for linolenic acid; d.o.fs8, P-0.001 for all computations), already evidenced for other chemicals in the Euphorbia genus w8–10x. Moreover, these results slightly differ from previously reported data regarding wholeplant extracts w12,13x. In fact, all the samples generally showed a higher presence of unsaturated fatty acids (linoleic and linolenic acid) and a lower content of palmitic acid. When compared with related published data w14,15x few, but noteworthy differences emerged. However, the fact that the correlation between the yield oil percentage and the unsaturated fatty acids is always positive suggests that the four Sardinian Euphorbia species accumulating the highest amount of oil in their seeds show preference for synthetizing unsaturated fatty acids.
Samples E. characias E. dendroides E. pithyusa subsp. cupanii E. semiperfoliata
Yield
Myristic
Palmitic
Stearic
9.17"1.05 0.66"0.07 13.34"1.54 2.02"0.23 4.10"0.47 0.92"0.11 7.97"0.92 19.78"2.28
Oleic
Linoleic
Linolenic
Arachidic
Behenic
SyU Ratio
8.13"0.94 22.24"2.56 52.02"5.99 1.78"0.21 – 0.21 5.84"0.67 16.61"1.91 46.38"5.34 0.86"0.09 1.53"0.18 0.45
12.32"1.42 – 6.41"0.74 1.09"0.13 12.26"1.41 21.23"2.44 59.01"6.79 – – 0.08 8.41"0.97 3.26"0.37 10.91"1.26 52.79"6.08 4.23"0.49 7.48"0.86 13.39"1.54 3.71"0.43 3.69"0.42 2.96
Mean ("S.E.; ns3) values.
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Table 1 Fatty acid profile (%) of four Euphorbia species seeds
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Table 2 Unsaponifiable fraction (%) of four Euphorbia species seeds E. characias E. dendroides E. pithyusa subsp. cupanii E. semiperfoliata Yield (wt.%) Hydrocarbons Fatty alcohols Cholesterol Campesterol b-Sitosterol D5-Avenasterol Lanosterol isomer Lanosterol b-Amyrin Cycloartanol 24-Methylen-cycloartenol Others
18.05"2.07 19.83"2.28 15.68"1.81 – 1.72"0.19 11.62"1.34 – – 10.92"1.26 3.42"0.39 16.16"1.86 12.77"1.47 7.88"0.91
16.1"1.84 17.78"2.05 17.37"2.01 0.94"0.11 – 12.89"1.48 – – 10.14"1.17 4.24"0.49 23.61"2.72 8.62"0.99 4.41"0.51
10.23"1.15 4.78"0.55 16.1"1.85 – 4.65"0.54 25.8"2.97 7.39"0.85 – – – 26.6"3.06 14.7"1.69 –
13.54"1.49 15.94"1.84 45.66"5.26 0.47"0.05 0.93"0.11 7.31"0.84 – 2.68"0.31 1.09"0.13 1.68"0.19 16.38"1.88 2.24"0.26 5.62"0.65
Mean ("S.E.; ns3) values.
3.2. Unsaponifiable fraction GC-MS of trimethylsilated unsaponifiable fraction of E. characias, E. pithyusa subsp. cupanii, E. dendroides and E. semiperfoliata seeds readily separated phytosterols and triterpenic alcohols (Table 2) showing qualitative and quantitative differences among the samples. A considerable amount of hydrocarbons and fatty alcohols was also detected, especially in E. semiperfoliata. The triterpenic alcohol cycloartanol was the main sterolic constituent in all samples, but its abundance in E. pithyusa subsp. cupanii was almost twice higher than that found in E. characias and E. semiperfoliata. 24-Methylenecycloartanol was found to be minoritary in E. semiperfoliata seeds and abundant up to 14.7"1.69% in E. pithyusa subsp. cupanii. Cholesterol was detected only in E. semiperfoliata and E. dendroides seeds. In comparison with previously published papers w12,13x, E. pithyusa subsp. cupanii seeds showed a higher qualitative homogenity in chemical composition, lacking both b-amyrin, lanosterol, cholesterol that were found in other seeds. On the contrary, a significant amount of D5-avenasterol was detected. Lanosterol, a sterol compound rarely found in plant seeds, was abundant in E. characias and E. dendroides seeds, but scarce in E. semiperfoliata seed oil. 3.3. Tocopherols and antioxidant activity The content of the single tocopherols and the quantitative ratio among them in seed oils of different plant species is known to be representative, and related to the similar habitats where these species grow. Moreover, the qualitative and quantitative evaluation of tocopherols in Brassicaceae, Orobanchaceae and Boraginaceae suggested a chemotaxonomic importance of these vitamin E isomers among the plants belonging to these families w24–26x. All the Euphorbia species seeds examined, with the sole exception of E. characias, showed a consistent presence of tocopherols
Samples
a-Tocopherol
b-Tocopherol
g-Tocopherol
d-Tocopherol
Total tocopherols
E. E. E. E.
ND 130.23"13.24 337.94"34.36 877.14"89.17
ND ND 51.88"5.27 12.77"1.30
ND 20.78"2.11 156.64"15.92 31.38"3.19
ND ND 13.76"1.34 17.56"1.78
ND 151.01"15.35 560.23"56.96 938.87"95.45
characias dendroides pithyusa subsp. cupanii semiperfoliata ND: Not Detected. Mean ("S.E.; ns3). * mgykg seeds.
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Table 3 Tocopherol content of Euphorbia species seeds*
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Table 4 Inhibition (%) of the 1,1-diphenyl-2-picrylhydrazyl radicals
E. characias E. dendroides E. pithyusa subsp. cupanii E. semiperfoliata BHAa a
50 (mgyml)
100 (mgyml)
200 (mgyml)
500 (mgyml)
1.42"0.05 3.46"0.07 29.37"1.9 6.32"0.1 95.61"3.5
2.95"0.07 8.35"0.09 43.38"2.7 15.87"1.3 95.00"3.3
4.79"0.08 16.10"1.5 91.40"3.5 29.59"1.9 95.71"3.7
6.42"0.09 19.57"1.15 92.36"3.9 43.79"2.5 95.87"4.1
Positive control: butylated hydroxy anisole.
Table 5 b-Carotene bleaching testa
E. characias E. dendroides E. pithyusa subsp. cupanii E. semiperfoliata BHA Negative controlc
RAAb 60 min
RAA 120 min
0.65"0.05 0.76"0.05 0.97"0.06 0.94"0.06 1 0.33"0.03
0.58"0.04 0.66"0.05 0.84"0.05 0.87"0.05 1 0.31"0.03
a
Concentration of the seeds extracts: 2 mgyml. Relative Antioxidant Activities. c Negative control: linoleic acid and b-carotene emulsion. b
with important qualitative and quantitative differences among the species (Table 3). All the differences recorded among the four Euphorbia species seeds for the total tocopherol content and for the individual tocopherols were highly significant (Fs 242436.6 for the total tocopherol content, Fs353147.5 for a-tocopherol, Fs 2756.271 for b-tocopherol, Fs14508.41 for g-tocopherol, Fs305.31 for d-tocopherol; d.o.fs6, P-0.001 for all computations). In fact, E. semiperfoliata showed a total tocopherol content of 93% and 40% higher than E. dendroides and E. pithyusa subsp. cupanii, respectively. Concerning the detection of tocopherols in E. pithyusa subsp. cupanii and E. semiperfoliata seeds, all the four vitamin E isomers were found, while in E. dendroides seed oil only a- and g-tocopherol were detected. From a quantitative point of view, the amount of single compounds with respect to the total tocopherol content was very different in all the Euphorbia species seeds. In fact, in E. semiperfoliata and E. dendroides, a-tocopherol accounted for 93.43% and 86.24% of the total tocopherol amount, respectively. In E. pithyusa subsp. cupanii seed oil, a-tocopherol was present in a lower amount (60.32% of the total tocopherols) but at the same time g-tocopherol was an important fraction (27.96%). In fact, E. pithyusa subsp. cupanii seeds presented the highest amount of g-topherol between the Euphorbia spp., 86% more than E. dendroides and 79% more than E. semiperfoliata. However, the tocopherols content was always positively correlable with the antioxidant tests performed. In fact, on both 1,1-diphenyl-2-picrylhydrazyl (DPPH)
Table 6 Pearson correlation coefficients between the individual tocopherols, the total tocopherol content*, oil content (%), fatty acids (%), antioxidants values at each concentration for both DPPH and b-carotene bleaching test in the four Euphorbia species bT
gT
dT
TTC Yield C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 50a 0.82 y0.37 y0.44 0.48 0.66 y0.29
y0.04 0.85 y0.39 y0.88 y0.62 y0.29 0.78 0.22 y0.71 y0.33 0.79 0.26 y0.23 0.52 0.07 y0.57 y0.19 0.72 y0.17 y0.76 y0.03 y0.35 0.75 0.32 0.36 0.97 y0.83 y0.96 0.16 y0.41 y0.1 y0.81 y1.00 0.77
y0.84 0.33 0.40 y0.51 y0.69 0.27 y1.0 y0.32 y0.98 0.81 0.97
0.67 y0.48 y0.58 0.34 0.49 y0.19 0.94 0.65 0.84 y0.78 y0.80 y0.92
0.82 y0.31 y0.34 0.48 0.69 y0.41 0.96 0.10 1.00 y0.82 y0.99 y0.96 0.80
0.15 0.99 1.00 0.56 0.37 0.74 y0.45 y0.68 y0.35 0.81 0.28 0.41 y0.57 y0.36
200a
500a
60b
120b
0.29 0.99 0.99 0.66 0.50 0.71 y0.32 y0.69 y0.21 0.72 0.14 0.27 y0.47 y0.22 0.99
0.25 0.99 1.00 0.63 0.47 0.71 y0.36 y0.70 y0.25 0.75 0.18 0.31 y0.51 y0.26 0.99 1.00
0.40 0.75 0.77 0.98 0.71 0.71 0.97 0.69 0.67 0.74 0.87 0.93 y0.60 0.86 0.88 0.69 0.39 0.48 y0.20 0.29 0.31 y0.67 y0.53 y0.50 y0.09 0.41 0.41 0.64 0.16 0.18 0.02 y0.47 y0.46 0.16 y0.33 y0.35 y0.37 0.05 0.11 y0.11 0.40 0.39 0.96 0.67 0.66 0.99 0.77 0.75 0.99 0.74 0.73 0.83 0.82 0.85
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aT 0.23 0.16 0.89 0.97 0.16 bT 0.99 0.63 0.45 0.79 gT 0.55 0.38 0.70 dT 0.97 0.55 TTC 0.32 Yield C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C22:0 50a 100a 200a 500a 60b
100a
Significant values (P-0.05) are given in bold *mgykg seeds. Ns12. a Values referred to the concentration employed in DPPH radical scavenging test; b values referred to detection time in b-carotene bleaching test; a, b, g, d-Tsa, b, g, d-Tocopherol; TTC: Total Tocopherol Content. 59
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and b-carotene bleaching tests E. semiperfoliata and E. pithyusa subsp. cupanii gave the best results (Tables 4 and 5). E. pithyusa subsp. cupanii extract exibited a scavenging activity on the DPPH radical, with values comparable to the positive control at 200 and 500 mgyml. E. semiperfoliata seed extracts showed a dose-dependent antioxidant activity, with a scavenging action of nearly 50%. E. dendroides extract activity was low, while E. characias seed extracts did not show any scavenging activity. In the b-carotene bleaching test the E. pithyusa subsp. cupanii and E. semiperfoliata seed extracts gave the best results, with relative antioxidant activity (RAA) values almost equivalent to the positive control butylated-hydroxy-anisole (BHA) and only slightly reduced after 120 min (12.0% E. pithyusa subsp. cupanii; 7.4% E. semiperfoliata). As expected, as a consequence of their lower tocopherols content, E. characias and E. dendroides showed a protective activity 20–30% worse. Interestingly, E. pithyusa subsp. cupanii, which showed a consistently lower content of a-tocopherol than E. semiperfoliata, revealed a higher scavenging activity against DPPH radical (Table 4). These results seem to reveal a more important role of b- and g-tocopherol in scavenging activity in the DPPH test. In the b-carotene bleaching test (Table 5), these kinds of qualitative correlations were not so evident. In fact, E. semiperfoliata and E. pithyusa subsp. cupanii had a similar scavenging activity. The quantitative correlation between the individual tocopherols and the antioxidant activity in both performed tests seems to be more pertinent than the correlation between the total tocopherol content and the same radical-scavenging results (Table 6). In fact, all the individual tocopherols in the four Euphorbia species are positively correlated with the antioxidant activity results, usually with significant Pearson coefficient values. While the total tocopherol content shows an anomalous negative—but significant—correlation with radical scavenging activity (Table 6). The same considerations could be made between individual tocopherols— or total tocopherol—content and the fatty acids profile. It is known that the fatty acids profile, especially the unsaturated fraction, is positively correlated with the tocopherols. In the Euphorbia seed oils considered, this kind of positive correlation always exists, but only for b- and g-tocopherol, i.e. those vitamin E isomers, which always exhibited a significant positive correlation with the antioxidant tests results. Acknowledgments Investigation supported from Ministero dell’Istruzione, dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST) of Italy. A special thanks goes to Orna Crinion for advice on language translation. References w1 x w2 x w3 x w4 x w5 x
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