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Does antioxidant properties of the main component of essential oil reflect its antioxidant properties? The comparison of antioxidant properties of essential oils and their main components a
a
Andrzej L. Dawidowicz & Małgorzata Olszowy a
Faculty of Chemistry, Maria Curie Sklodowska University, Pl. Marii Curie Sklodowskiej 3, 20-031 Lublin, Poland Published online: 21 May 2014.
To cite this article: Andrzej L. Dawidowicz & Małgorzata Olszowy (2014): Does antioxidant properties of the main component of essential oil reflect its antioxidant properties? The comparison of antioxidant properties of essential oils and their main components, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.918121 To link to this article: http://dx.doi.org/10.1080/14786419.2014.918121
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.918121
Does antioxidant properties of the main component of essential oil reflect its antioxidant properties? The comparison of antioxidant properties of essential oils and their main components
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Andrzej L. Dawidowicz* and Małgorzata Olszowy Faculty of Chemistry, Maria Curie Sklodowska University, Pl. Marii Curie Sklodowskiej 3, 20-031 Lublin, Poland (Received 20 February 2014; final version received 22 April 2014) This study discusses the similarities and differences between the antioxidant activities of some essential oils: thyme (Thymus vulgaris), basil (Ocimum basilicum), peppermint (Mentha piperita), clove (Caryophyllus aromaticus), summer savory (Satureja hortensis), sage (Salvia hispanica) and lemon (Citrus limon (L.) Burm.) and of their main components (thymol or estragole or menthol or eugenol or carvacrol or camphor or limonene) estimated by using 2,20 -Diphenyl-1-picrylhydrazyl, 2,20 -azinobis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt and b-carotene bleaching assays. The obtained data show that the antioxidant properties of essential oil do not always depend on the antioxidant activity of its main component, and that they can be modulated by their other components. The conclusions concerning the interaction of essential oil components depend on the type of method applied for assessing the antioxidant activity. When comparing the antioxidant properties of essential oils and their main components, the concepts of synergism, antagonism and additivity are very relevant. Keywords: essential oils; antioxidant activity; herbs; DPPH assay; ABTS assay; b-carotene bleaching assay
1. Introduction Hazardous action of free radicals results from their oxidation activity towards biomolecules, e.g. proteins, amino acids, lipids or DNA. This process frequently leads to cell injury and its death (Fridovich 1999; Ignarro et al. 1999; McCord 2000; Zheng & Storz 2000). The initiated autooxidation process of lipids has also been recognised as a major process of food deterioration (Yanishlieva et al. 1999; Tepe et al. 2005). During this process, the sensory and nutritional quality of foods is lost. The negative activity of free radicals is eliminated and reduced by the application of antioxidants, i.e. compounds inhibiting the oxidation process in living organisms and in fat-based foods, particularly in meat and dairy products or fried foods (Politeo et al. 2007). Hence, the importance of antioxidants for human health and food industry is obvious. A lot of antioxidants are used in medicine and other industries. In general, they are divided into two groups: natural and synthetic. The second group is widely used as food additives to provide protection against oxidative degradation of foods. Recently performed toxicological studies have shown, however, that some synthetic antioxidant compounds, e.g. butylhydroxytoluene, butylhydroxyanisole, propyl gallate or tert-butylhydroquinone, cause side effects (Zhang et al. 2006). Butylhydroxytoluene and butylhydroxyanisole, for instance, were
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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A.L. Dawidowicz and M. Olszowy
Table 1. The composition of essential oils from selected herbs.
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Compounds Basil 1. Linalool 2. Estragolea 3. Methyleugenol 4. a-Bisabolene 5. Widdrol Clove 1. Eugenola 2. Caryophyllene 3. Eugenyl acetate Peppermint 1. Limonene 2. Eucalyptol 3. Menthone 5. (þ)-Isomenthone 7. Menthola 8. 4-Terpineol 9. a-Cubebene Summer savory 1. a-Pinene 2. b-Myrcene 3. o-Cymene 4. D -Limonene 5. d-Terpinene 6. Carvacrola 7. Caryophyllene 8. a-Bisabolene Thyme 1. a-Thujene 2. a-Pinene 3. Camphene 4. 1-Octen-3-ol 5. b-Myrcene 6. p-Cymene 7. d-Terpinene 8. Borneol 9. 4-Terpineol 10. Thymol methylether 11. Carvacrol methylether 12. Thymola S. hispanica 1. a-Pinene 2. Camphene 3. b-Pinene 4. b-Myrcene 5. Eucalyptol 6. cis-Ocimene 7. Linalool 8. Camphora 9. Borneol Lemon 1. a-Pinene 2. Sabinene 3. b-Pinene
RIb 1101 1204 1402 1507 1593
Composition (%)
(1098) (1195) (1401) (1509) (1597)
6.21 69.19 12.24 1.42 0.97
1362 (1356) 1416 (1418) 1519 (1524)
76.00 19.07 3.61
1035 1041 1163 1172 1182 1191 1393
(1031) (1033) (1154) (1164) (1173) (1189) (1390)
0.66 2.65 8.71 1.40 59.20 1.19 1.14
939 (939) 990 (991) 1022 (1022) 1030 (1031) 1063(1062) 1302 (1298) 1428 (1418) 1518 (1509)
0.60 1.00 2.40 7.37 18.48 67.50 0.57 0.56
930 939 958 980 990 1030 1064 1164 1185 1244 1253 1292
(931) (939) (953) (978) (991) (1026) (1062) (1165) (1189) (1235) (1244) (1290)
0.73 0.62 0.51 0.82 1.02 16.47 7.78 0.54 0.62 1.00 0.58 55.12
939 958 987 990 1036 1041 1102 1153 1166
(939) (953) (980) (991) (1033) (1040) (1098) (1143) (1165)
16.33 3.38 2.04 1.50 14.30 11.17 0.57 29.16 6.96
939 (939) 979 (976) 987 (980)
Concentration (mg/mL essential oil)
634.25 ^ 12.68
751.10 ^ 15.02
532.80 ^ 10.66
574.87 ^ 11.49
505.27 ^ 10.10
248.80 ^ 4.97
1.50 0.80 11.89 (Continued)
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Table 1. (Continued) Compounds 4. b-Myrcene 5. o-Cymene 6. D -Limonenea 7. g-Terpinene 8. g-Terpineol 9. Neral (b-citral) 10. Geranial (a-citral) a
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b
RIb 990 1022 1030 1063 1191 1244 1273
(991) (1022) (1031) (1062) (1189) (1244) (1270)
Composition (%) 1.11 1.90 70.70 6.87 0.76 0.90 1.50
Concentration (mg/mL essential oil)
601.61 ^ 12.03
Compounds which were identified by using reference compounds. The values in parenthesis present literature RI (Adams 1995).
recognised as promoters that are involved in liver damage and carcinogenesis (Ames 1983; Namiki 1990; Gu¨lc in et al. 2004). Such findings cause growing interest of researchers and consumers in the antioxidant properties of natural compounds. For this reason, natural antioxidants are extensively studied for their capacity to protect organisms and cells from the damage induced by oxidative stress, by ageing, degenerative diseases and cancer (Pokorny 1991; Briskin 2000). In the course of studies undertaken to find safe and potent natural antioxidants, a huge number of compounds and extracts from aromatic, spicy, medicinal and other plants have been examined (Madsen & Bertelson 1995; Barrata et al. 1998; Miguel 2010). Some attention has also been paid to essential oils, known since the middle ages not only due to their pleasant or unpleasant aroma but also due to their antibacterial, antifungal and anti-inflammatory activities (Bakkali et al. 2008; Misharina & Samusenko 2008). Essential oils are very complex mixtures containing about 20 –60 or more components at very different concentrations. In each such complex mixture, one or two components occur at higher concentration (20 –70%) than others, and these major components determine the biological properties of an essential oil. The question asked here is whether this rule also applies to the antioxidant properties of essential oils. This study attempts to answer it by comparing the antioxidant properties of some essential oils with the antioxidant properties of their main components. The oils from the following herbs and fruits were applied in the experiments: thyme (Thymus vulgaris), basil (Ocimum basilicum), peppermint (Mentha piperita), clove (Caryophyllus aromaticus), summer savory (Satureja hortensis), sage (Salvia hispanica) and lemon (Citrus limon (L.) Burm). In these experiments, the amount of the main component of the essential oil (thymol or carvacrol or estragole or eugenol or menthol or camphor or limonene) in the measuring system was the same as its amount in the system when the essential oil solution was examined. The antioxidant activity of methanolic solutions of the examined essential oils and their main components were established by using 2,20 -Diphenyl-1-picrylhydrazyl (DPPH), 2,20 azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and b-carotene bleaching assays.
2. Results and discussion The compounds identified in the essential oils obtained from basil, clove, peppermint, summer savory, thyme, sage and lemon along with their peak percent concentrations and Kovats indices on ZB5-MS column are given in Table 1. The table contains only components that exceed 0.5% in the examined essential oils. According to the listed data, each of the examined oils contains one main component: eugenol, carvacrol, menthol, thymol, estragole, camphor and limonene in clove, summer savory, peppermint, thyme, basil, S. hispanica and lemon, respectively. Except
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camphor in S. hispanica essential oil, the content of the other main component in each given essential oil is greater than 50%. The structures of the main components are shown in Figure 1. The comparison of IC50 values of the examined materials (compounds or mixtures) is the simplest way to find the relation between their antioxidant properties. Table 2 lists the IC50 values for all essential oils and their main components estimated by using DPPH, ABTS and bcarotene bleaching assays. According to these data, all main components of the examined essential oils, except estragole (the main component of basil essential oil), exhibit higher antioxidant properties than the corresponding essential oils. It is worth noticing, however, that the comparison of IC50 for mixture and its main component shows the difference in the antioxidant capacity of these substances and does not allow to answer the question whether the major component of the mixture determines its antioxidant properties. The corresponding measuring systems contain different amounts of the mixture main component. Free radical-scavenging capacities of all essential oils and their main components, examined by using DPPH assay are presented in Figure 2. In these experiments, the amount of the main essential oil component (thymol or carvacrol or estragole or eugenol or menthol or camphor or limonene) in the measuring system was the same as in the system when the essential oil solution was examined. As observed in the figure, in the case of clove and summer savory essential oil, their antioxidant activity is very similar to the activity of their main components, eugenol and carvacrol, respectively (for clove and eugenol inhibition percent (I%) is about 82 ^ 1.9 and for summer savory and carvacrol about 53 ^ 1.5). The data for these essential oils show that the antioxidant properties of the essential oil depend in fact on the antioxidant properties of its main component. The other components of clove and summer savory essential oil do not exhibit antioxidant activities and do not influence the activity of eugenol and carvacrol, respectively. Quite a different situation is observed in the case of peppermint and basil essential oils. Their main components, menthol and estragole, do not exhibit antioxidant properties as estimated by the DPPH method (for menthol I% ¼ 3 ^ 0.3 and for estragole 5 ^ 0.4). It is not strange, as
Figure 1. Chemical structure of the main components of the examined essential oils.
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Table 2. IC50 values [mg/mL] of examined essential oils and their main components. Method of antioxidant activity determination
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Essential oil/main component Clove Eugenol Summer Savory carvacrol Lemon Limonene Savory Camphor Thyme Thymol Basil Estragole
DPPH
ABTS
b-Carotene
0.3257 ^ 0.009 0.1967 ^ 0.005 0.5088 ^ 0.013 0.4080 ^ 0.012 6.6768 ^ 0.133 1.8680 ^ 0.056 6.0854 ^ 0.183 4.7842 ^ 0.120 0.4029 ^ 0.011 0.2540 ^ 0.005 0.5798 ^ 0.013 5.7650 ^ 0.173
0.1595 ^ 0.005 0.1492 ^ 0.003 0.3671 ^ 0.011 0.1173 ^ 0.002 2.9670 ^ 0.089 2.9330 ^ 0.088 6.0854 ^ 0.152 1.2245 ^ 0.034 0.5758 ^ 0.011 0.3651 ^ 0.008 0.2133 ^ 0.006 3.4590 ^ 0.104
0.1848 ^ 0.005 0.0658 ^ 0.002 0.4356 ^ 0.013 0.2168 ^ 0.004 6.8595 ^ 0.206 1.2577 ^ 0.038 3.1256 ^ 0.078 1.0053 ^ 0.028 0.1913 ^ 0.004 0.1638 ^ 0.004 0.1616 ^ 0.005 5.7689 ^ 0.173
both compounds are not donors of proton and electron required for the neutralisation of DPPH radical. Hence, the antioxidant properties of peppermint and basil essential oils (I% about 89.1 ^ 2.7 and I% about 46.1 ^ 1.3; respectively) result from the properties of other components occurring in them (e.g. 4-terpineol, methyleugenol). As peppermint essential oil exhibits the lowest antioxidant activity of all the examined compounds, it was not diluted before measuring the remaining oils. Considering the ratio between the antioxidant properties of Salvia and lemon essential oils and their main components, lower antioxidant properties of the main components than of the essential oils were found (for Salvia and camphor I% ¼ 13 ^ 0.7 and 10 ^ 0.4, respectively, and for lemon and limonene ¼ 13.6 ^ 0.9 and 8.0 ^ 0.6, respectively). It suggests that the antioxidant properties of these oils are significantly affected not only by main components but also by other components (borneol, caryophyllene, citral). Greater antioxidant properties of the main components than the corresponding oil are observed for thymol (I% is about 63 ^ 1.7) and thyme essential oil (I% is about 57 ^ 1.6). The obtained results indicate the antagonistic influence of other thyme essential oil constituents on
Figure 2. Free radical-scavenging capacities of the essential oils and their main components examined by using DPPH assay.
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the antioxidant properties of thymol. Considering all the presented cases, it is difficult to state unequivocally that the main components of essential oil determine its antioxidant properties. The ABTS method is similar to the DPPH method in the evaluation of antioxidant properties, because in both the methods the antioxidant activity is estimated from the rate of scavenging process of colour radicals (Dawidowicz & Olszowy 2012). According to the results from Osman et al. (2006), ABTS cation radical is more reactive and its neutralisation path is different from that of the DPPH radical. Free radical-scavenging capacities of all the essential oils and their main components examined by using ABTS assay are presented in Figure 3. The analysis of the data shows that the antioxidant properties of the examined essential oils and their main components are slightly different from those established by DPPH method; however, the relationship between the antioxidant properties of the main component and the corresponding essential oil is similar. The most visible difference in the antioxidant properties estimated by DPPH and ABTS method is for thymol and thyme essential oil. The antioxidant activity of thymol is significantly greater than the antioxidant properties of thyme essential oil in ABTS method than in DPPH (cation radical I% for thymol and thyme is about 62 ^ 1.4 and 38 ^ 0.95, respectively). The results obtained in the ABTS method also confirm that the antioxidant properties of the essential oil do not always depend on the properties of its main components. Figure 4 presents the I% for all the examined essential oils and their main components determined by b-carotene bleaching assay. As it is seen, the relations between the antioxidant activity of a given essential oil and its main component are in a few cases different from those established by DPPH and ABTS methods. It is not strange if we take into account that the antioxidant activity in b-carotene bleaching method is determined by comparing two competitive chemical reactions in which the examined antioxidant and/or model antioxidant bcarotene take part (Moon & Shibamato 2009). This method assesses the ability of radical neutralisation by homolytic degradation of OZH bond in hydroxyl group occurring in an antioxidant, whereas in DPPH and ABTS method, the radical scavenging is preceded by dissociation of this group. It is worth mentioning that not only b-carotene but also other compounds possessing double bonds (e.g. p-cymene, borneol, b-myrcene, linalool, sabinene), that take part in b-carotene bleaching assay, are also able to form radical adduct with peroxyl radical and exhibit antioxidant properties.
Figure 3. Free radical-scavenging capacities of the essential oils and their main components examined by using ABTS assay.
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Figure 4. Free radical-scavenging capacities of the essential oils and their main components examined by bcarotene bleaching assay.
The results obtained from b-carotene bleaching assay, as well as from DPPH and ABTS methods, do not allow us to state unequivocally that the main components of an essential oil determine its antioxidant properties. In order to confirm the last statement, we decided to compare the antioxidant activity of systems containing essential oil or its main component (used in the same amount as it occurs in the measuring system with essential oil) or essential oil with addition of known amount of the main component. Three essential oils and their major components were chosen for these experiments: clove essential oil and eugenol, Salvia essential oil and camphor, basil essential oil and estragole. The antioxidant activity of these systems, estimated by using ABTS assay, is presented in Figure 5. The analysis of these results shows that the addition of the main component to the essential oil causes:
Figure 5. Free radical-scavenging capacities of the chosen essential oils, their main components and essential oils with addition of the main component examined by using ABTS assay.
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A.L. Dawidowicz and M. Olszowy . the increased antioxidant properties of essential oil (cf. clove and clove with eugenol); . the decreased antioxidant properties of essential oil (cf. Salvia and camphor); . negligible differences in antioxidant properties of essential oil (cf. basil and estragole).
Hence, the main component does not always determine the antioxidant activity of the examined essential oils. The antioxidant activity of the main compound can be modulated by other components of essential oils.
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3. Experimental section 3.1. Materials Thyme, peppermint, summer savory, basil, sage, clove buds and lemon essential oils were obtained by means of steam distillation. Thyme, peppermint and summer savory, cultivated in eastern Poland, were purchased from a local herb planter. Basil, sage, clove buds and lemons were purchased at a local market. The herbs were air-dried, cut and stored at þ 88C. The lemon peel was cut and stored under the same conditions. Immediately before the isolation of essential oil, an appropriate amount of plant material was ground and its exactly weighed portion was subjected to distillation process. DPPH, b-carotene, linoleic acid, Tween 20, ABTS, potassium persulfate (di-potassium peroxydisulfate), thymol, estragole, menthol, eugenol, carvacrol, camphor and limonene were purchased from Sigma Aldrich (Poznan´, Poland). Methanol was purchased from the Polish Chemical Plant – POCh (Gliwice, Poland). Water was purified on a Milli-Q system from Millipore (Bedford, MA, USA). 3.2. Steam distillation Steam distillation process was performed for 3 h applying the Deryng apparatus, a Clevengertype (WPL, Gliwice, Poland) apparatus described in detail in the Polish Pharmacopea V, which contained a plant sample (50 g) and 600 mL of water. The distillation time was measured after the fall of the first drop of the distillate. The separated essential oils were dried by freezing and, after filtration, stored at þ 48C until further experiments. 3.3. Solutions of essential oils and essential oil main components The solutions of essential oil from thyme or clove or summer savory or basil or sage or lemon were prepared by dissolving 60 mL of a given essential oil in 5 mL of methanol. Before antioxidant measurements, the obtained solutions were diluted 1:20 in the same solvent. Due to the weak antioxidant properties of peppermint essential oil, this oil was applied in its natural form (without prior dilution). To estimate the IC50 values of the examined essential oils and their major components, stock solutions differing in concentrations of these antioxidants were prepared. To provide the same amount of the main essential oil component (thymol or carvacrol or estragole or eugenol or menthol or camphor or limonene) in the measuring system as it is present in the system when essential oil solution is applied, the examined essential oils were analysed by means of GC and the concentration of their main component was quantified precisely. Following the obtained results, methanolic solutions of adequate concentrations of main components were prepared. 3.4. Chromatographic analysis Qualification of the essential oil components in the prepared samples was performed using GC/ MS QP2010 (Shimadzu, Kyoto, Japan). A ZB5-MS fused silica capillary column (30 m
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£ 0.25 mm i.d., 0.25 mL film thickness) (Phenomenex, Shim-Pol, Izabelin, Poland) was used. Helium (grade 5.0) was used as carrier gas. Sample of 1 mL was injected by an AOC-20i-type autosampler. The injector temperature was 3108C. The following temperature program was applied: 1 min at 508C and then a linear increase in temperature up to 3108C at the rate of 68C/ min. The mass spectrometer was operated in EI mode at 70 eV; the ion source temperature was 2208C. The mass spectra were measured in the range of 35 –360 amu. Qualitative analysis was carried out comparing the retention indices (RI) and MS spectra for the obtained peaks with the analogous data from Adams (4th ed.) (Adams 1995), FFNSC 1.3 and NIST’05 databases. The identity of the main essential oil components was additionally confirmed by comparing their RI and MS spectra of their peaks with analogous data obtained for standards. Quantification of the essential oils was performed using a gas chromatograph with a flame ionisation detector-GC-FID model GC-2010 (Shimadzu, Kyoto, Japan). A sample of 1 mL was injected by AOC-20i-type autosampler into a ZB5-MS fused silica capillary column (30 m £ 0.25 mm i.d., 0.25 mL film thickness) (Phenomenex). The temperature program during GCFID separation was the same as for GC/MS. The quantity of the main components in examined solution was determined using the external standard method and calibration solutions of the main component standards. The quantities of the other essential oil components were given as peak percent and should be regarded as tentative. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were defined as the analyte concentration producing signal-to-noise ratio equal to 3:1 and 10:1, respectively. The estimated LOD and LOQ limits of the main essential oil components (mg/mL) were as follows: 0.34 and 1.15 for thymol, 0.26 and 0.88 for carvacrol, 0.54 and 1.8 for estragole, 0.87 and 2.91 for eugenol, 0.47 and 1.58 for menthol, 0.15 and 0.54 for camphor and 0.02 and 0.06 for limonene (LOD and LOQ, respectively). 3.5. Antioxidant activity The antioxidant activity of the examined solution of essential oils and its main components were determined by using DPPHz, ABTS and b-carotene bleaching assays. The scavenging of radicals by potential antioxidant was measured for all these methods. 3.5.1. DPPH method An aliquot (2940 mL) of methanolic DPPHz solution (24 mg/mL) was mixed in a 4 mL test tube with essential oil solution or main essential oil component solution (60 mL). In the case of peppermint, DPPHz solution (2400 mL) was mixed with methanol (500 mL) and pure essential oil or menthol solution (100 mL). Before measurement, each mixture was vigorously shaken during 30 s and immediately transferred into a quartz cuvette (1 cm £ 1 cm £ 3.5 cm). The decrease in absorbance at 516 nm was registered in continuous manner during 60 min employing a UV Probe-1800 spectrophotometer (Shimadzu). Subsequent readings were taken at regular intervals (60 s). The I% was calculated according to the following equation: At I ð%Þ ¼ 1 2 £ 100%; At¼0 where At¼0 and At are the values of absorbance of DPPHz at 0 min and at time equal to (t) min, respectively. 3.5.2. ABTS assay Generation of ABTS radical cation was performed according to Re et al. (1999). The ABTSzþ solution was prepared by the reaction of 5 mL of a 7 mM aqueous ABTS solution and 88 mL of
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A.L. Dawidowicz and M. Olszowy
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140 mM (2.45 mM final concentration) potassium persulfate (K2S2O8) solution. The mixture was incubated in the dark for 16 h. The formed radical cation was then diluted in methanol until the initial absorbance value of 0.7 at 744 nm was reached. A volume of 2000 mL of the prepared ABTS radical cation solution was mixed in a 4 mL test tube with essential oil solution or main essential oil component solution (20 mL). In the case of peppermint, pure essential oil was used (20 mL). To estimate the influence of the reinforcement of the essential oil in its main component the measuring systems were composed of . 1990 mL of ABTS radical cation solution, 20 mL of methanolic essential oil solution (c ¼ 0.5 mg/mL) and 10 mL of methanol, when antioxidant properties of initial clove or basil or Salvia essential oil solution were examined; . 1990 mL of ABTS radical cation solution, 20 mL of methanolic essential oil solution (c ¼ 0.5 mg/mL) and 10 mL of methanolic solution of eugenol or estragole or camphor (c ¼ 0.5 mg/mL) when antioxidant properties of reinforced clove, basil and Salvia essential oil solution were examined. The reaction mixture was stirred vigorously for 30 s and poured into cuvettes (1 cm £ 1 cm £ 3.5 cm). The decrease in absorbance was recorded in a continuous manner during 60 min at 744 nm employing UV Probe-1800 spectrophotometer (Shimadzu). I% was calculated from the following equation:
I ð%Þ ¼
At 12 £ 100%; At¼0
where At¼0 and At are the values of absorbance of ABTSzþ at 0 min and at time equal to (t) min, respectively.
3.5.3. b-Carotene bleaching assay The estimation of the reacted b-carotene concentration in the examined systems was performed by the slightly modified Dapkevicius method (Gulluce et al. 2007). The stock solution of bcarotene/linoleic acid emulsion in water was prepared as follows: the mixture composed of 25 mL linoleic acid, 185 mL Tween 20 (200 mg) and 1 mL b-carotene solution (containing 0.5 mg of b-carotene in 1 mL of chloroform) underwent the vacuum evaporation process to remove chloroform. The obtained residue was dispersed in 100 mL of distilled water saturated with oxygen (30 min at 100 mL/min) and vigorously shaken. The absorbance of the final emulsion was measured at 470 nm using UV Probe-1800 spectrophotometer (Shimadzu). As a blank, emulsion without b-carotene was applied in the measurements. Aliquot (2.5 mL) of the emulsion with b-carotene was placed in a glass optical cuvette (1 cm £ 1 cm £ 3.5 cm) containing 350 mL of the essential oil solution or the main essential oil component solution. In the case of peppermint, pure essential oil was used (350 mL). The zero reading for the reaction mixture was taken at 470 nm immediately after the mixing. Subsequent readings were taken at regular intervals (10 min) until carotene in the control sample was decolourised (180 min). The control sample was prepared in the same way as the reacting mixture, the only difference being the replacement of the essential oil solution or the main component solution in a given solvent by pure solvent. Emulsion without b-carotene (2.5 mL) mixed with 350 mL of methanol was used to zero the spectrophotometer.
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The antioxidant activity was expressed as I% relative to the control using the following equation: AA ¼ 100 £
DRC 2 DRS DRc
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where AA is the antioxidant activity; DRC is the degradation rate of b-carotene in the control sample ¼ {[ln (a/b)]/t} and DRS is the degradation rate of b-carotene in the sample with antioxidant ¼ {[ln (a/b)]/t} – a is the absorbance at time 0; b is the absorbance at defined time (for example at 10, 20, . . . , 180 min) and t is the time. 3.6. Statistical analysis Results are presented as mean values ^ SD. In order to determine the reproducibility of the measurements, each antioxidant activity assay was carried out three times. RSD of all the measurements were lower than 10%. P , 0.01 was assumed as the statistical difference between the experimental points. 4. Conclusion Essential oils from different plants have attracted much interest due to their antioxidant, antitumour, antibacterial, antifungal and insecticidal properties. In general, the major components are found to reflect quite well the biological features of these oils. The presented results show that it is not always true in the case of the antioxidant properties of essential oils. The antioxidant activity of the main compound can be modulated by other components of essential oils. When comparing the antioxidant properties of essential oils and their main components, the concepts of synergism, antagonism and additivity are really relevant. It should be stressed, however, that the presented conclusions concerning the interaction of essential oil components depend on the type of method applied for assessing antioxidant activity. References Adams RP. 1995. Identification of essential oil components by gas chromatography/mass spectrometry. Carol Stream, IL: Allured Publishing Corporation. Ames BM. 1983. Dietary carcinogens and anticarcinogens: oxygen radical and degenerative diseases. Science. 221:1256–1263. Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils – A review. Food Chem Toxicol. 46:446–475. Baratta MT, Dorman HJD, Deans SG, Figueiredo AC, Barroso JG, Ruberto G. 1998. Antimicrobial and antioxidant properties of some commercial essential oils. Flavour Frag J. 13:235–244. Briskin DA. 2000. Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiol. 124:507–514. Dawidowicz AL, Olszowy M. 2012. Mechanism change in estimating of antioxidant activity of phenolic compounds. Talanta. 97:312– 317. Fridovich I. 1999. Fundamental aspects reactive oxygen species, or what’s the matter with oxygen. Ann NY Acad Sci. 893:13–18. ¨ I˙. 2004. Comparison of antioxidant activity of clove (Eugenia Gu¨lc in I`, S¸at ˙IG, Beydemir S¸, Elmastas¸ Ku¨freviog˘lu O caryophylata Thunb) buds and lavender (Lavandula stoechas L.). Food Chem. 87:393–400. Gulluce M, Sahin F, Sokmen M, Ozer H, Daferera D, Sokmen A, Polissiou M, Adiguzel A, Ozkan H. 2007. Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longfolia L. ssp. longifolia. Food Chem. 103:1449–1456. Ignarro LJ, Cirino G, Castini A, Napoli C. 1999. Nitric signaling molecule in the vascular system: an overview. J Cardiovasc Pharmacol. 34:879–886. Madsen HL, Bertelsen G. 1995. Spices as antioxidants. Trends Food Sci Tech. 6:271–277. McCord JM. 2000. The evolution of free radicals and oxidative stress. Am J Med Sci. 108:652–659.
Downloaded by [Florida Institute of Technology] at 23:06 20 August 2014
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Miguel MG. 2010. Antioxidant activity of medicinal and aromatic plants. A review. Flavour Frag. 25:291– 312. Misharina TA, Samusenko AL. 2008. Antioxidant properties of essential oils from lemon, grapefruit, coriander, clove and their mixture. Appl Biochem Microbiol. 45:482–486. Moon J, Shibamoto T. 2009. Antioxidant assays for plant and food components. J Agric Food Chem. 57:1655–1660. Namiki M. 1990. Antioxidants/antimutagens in food. Crit Rev Food Sci Nutr. 29:273–300. Osman AM, Wong KKY, Fernyhough A. 2006. ABTS radical- driven oxidation of polyphenols: isolation and structural elucidation of covalent adducts. Biochem Biophys Res Commun. 346:321–329. Pokorny J. 1991. Natural antioxidants for food use. Trends Food Sci Tech. 2:223–227. Politeo O, Jukic M, Milos M. 2007. Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Food Chem. 101:379–385. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. 1999. Antioxidant activity applying improved ABTS radical cation decolorization assay. Free Radi Biol Med. 26:1231– 1237. Tepe B, Daferera D, Sokmen A, Sokmen M, Polissiou M. 2005. Antimicrobial and antioxidant activities of the essential oil and various extracts of Salvia tomentosa Miller (Lamiaceae). Food Chem. 90:333–340. Yanishlieva NV, Marinova EM, Gordon MH, Raneva VG. 1999. Antioxidant activity and mechanism of action of thymol and carvacrol in two lipid systems. Food Chem. 64:59–66. Zhang H, Chen F, Wang X, Yao HY. 2006. Evaluation of antioxidant activity of parsley (Petroselinum crispum) essential oil and identification of its antioxidant constituents. Food Res Int. 39:833–839. Zheng M, Storz G. 2000. Redox sensing by prokaryotic transcription factors. Biochem Pharmacol. 59:1–6.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Does antioxidant properties of the main component of essential oil reflect its antioxidant properties? The comparison of antioxidant properties of essential oils and their main components a
a
Andrzej L. Dawidowicz & Małgorzata Olszowy a
Faculty of Chemistry, Maria Curie Sklodowska University, Pl. Marii Curie Sklodowskiej 3, 20-031 Lublin, Poland Published online: 21 May 2014.
To cite this article: Andrzej L. Dawidowicz & Małgorzata Olszowy (2014): Does antioxidant properties of the main component of essential oil reflect its antioxidant properties? The comparison of antioxidant properties of essential oils and their main components, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.918121 To link to this article: http://dx.doi.org/10.1080/14786419.2014.918121
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.918121
Does antioxidant properties of the main component of essential oil reflect its antioxidant properties? The comparison of antioxidant properties of essential oils and their main components
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Andrzej L. Dawidowicz* and Małgorzata Olszowy Faculty of Chemistry, Maria Curie Sklodowska University, Pl. Marii Curie Sklodowskiej 3, 20-031 Lublin, Poland (Received 20 February 2014; final version received 22 April 2014) This study discusses the similarities and differences between the antioxidant activities of some essential oils: thyme (Thymus vulgaris), basil (Ocimum basilicum), peppermint (Mentha piperita), clove (Caryophyllus aromaticus), summer savory (Satureja hortensis), sage (Salvia hispanica) and lemon (Citrus limon (L.) Burm.) and of their main components (thymol or estragole or menthol or eugenol or carvacrol or camphor or limonene) estimated by using 2,20 -Diphenyl-1-picrylhydrazyl, 2,20 -azinobis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt and b-carotene bleaching assays. The obtained data show that the antioxidant properties of essential oil do not always depend on the antioxidant activity of its main component, and that they can be modulated by their other components. The conclusions concerning the interaction of essential oil components depend on the type of method applied for assessing the antioxidant activity. When comparing the antioxidant properties of essential oils and their main components, the concepts of synergism, antagonism and additivity are very relevant. Keywords: essential oils; antioxidant activity; herbs; DPPH assay; ABTS assay; b-carotene bleaching assay
1. Introduction Hazardous action of free radicals results from their oxidation activity towards biomolecules, e.g. proteins, amino acids, lipids or DNA. This process frequently leads to cell injury and its death (Fridovich 1999; Ignarro et al. 1999; McCord 2000; Zheng & Storz 2000). The initiated autooxidation process of lipids has also been recognised as a major process of food deterioration (Yanishlieva et al. 1999; Tepe et al. 2005). During this process, the sensory and nutritional quality of foods is lost. The negative activity of free radicals is eliminated and reduced by the application of antioxidants, i.e. compounds inhibiting the oxidation process in living organisms and in fat-based foods, particularly in meat and dairy products or fried foods (Politeo et al. 2007). Hence, the importance of antioxidants for human health and food industry is obvious. A lot of antioxidants are used in medicine and other industries. In general, they are divided into two groups: natural and synthetic. The second group is widely used as food additives to provide protection against oxidative degradation of foods. Recently performed toxicological studies have shown, however, that some synthetic antioxidant compounds, e.g. butylhydroxytoluene, butylhydroxyanisole, propyl gallate or tert-butylhydroquinone, cause side effects (Zhang et al. 2006). Butylhydroxytoluene and butylhydroxyanisole, for instance, were
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Table 1. The composition of essential oils from selected herbs.
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Compounds Basil 1. Linalool 2. Estragolea 3. Methyleugenol 4. a-Bisabolene 5. Widdrol Clove 1. Eugenola 2. Caryophyllene 3. Eugenyl acetate Peppermint 1. Limonene 2. Eucalyptol 3. Menthone 5. (þ)-Isomenthone 7. Menthola 8. 4-Terpineol 9. a-Cubebene Summer savory 1. a-Pinene 2. b-Myrcene 3. o-Cymene 4. D -Limonene 5. d-Terpinene 6. Carvacrola 7. Caryophyllene 8. a-Bisabolene Thyme 1. a-Thujene 2. a-Pinene 3. Camphene 4. 1-Octen-3-ol 5. b-Myrcene 6. p-Cymene 7. d-Terpinene 8. Borneol 9. 4-Terpineol 10. Thymol methylether 11. Carvacrol methylether 12. Thymola S. hispanica 1. a-Pinene 2. Camphene 3. b-Pinene 4. b-Myrcene 5. Eucalyptol 6. cis-Ocimene 7. Linalool 8. Camphora 9. Borneol Lemon 1. a-Pinene 2. Sabinene 3. b-Pinene
RIb 1101 1204 1402 1507 1593
Composition (%)
(1098) (1195) (1401) (1509) (1597)
6.21 69.19 12.24 1.42 0.97
1362 (1356) 1416 (1418) 1519 (1524)
76.00 19.07 3.61
1035 1041 1163 1172 1182 1191 1393
(1031) (1033) (1154) (1164) (1173) (1189) (1390)
0.66 2.65 8.71 1.40 59.20 1.19 1.14
939 (939) 990 (991) 1022 (1022) 1030 (1031) 1063(1062) 1302 (1298) 1428 (1418) 1518 (1509)
0.60 1.00 2.40 7.37 18.48 67.50 0.57 0.56
930 939 958 980 990 1030 1064 1164 1185 1244 1253 1292
(931) (939) (953) (978) (991) (1026) (1062) (1165) (1189) (1235) (1244) (1290)
0.73 0.62 0.51 0.82 1.02 16.47 7.78 0.54 0.62 1.00 0.58 55.12
939 958 987 990 1036 1041 1102 1153 1166
(939) (953) (980) (991) (1033) (1040) (1098) (1143) (1165)
16.33 3.38 2.04 1.50 14.30 11.17 0.57 29.16 6.96
939 (939) 979 (976) 987 (980)
Concentration (mg/mL essential oil)
634.25 ^ 12.68
751.10 ^ 15.02
532.80 ^ 10.66
574.87 ^ 11.49
505.27 ^ 10.10
248.80 ^ 4.97
1.50 0.80 11.89 (Continued)
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Table 1. (Continued) Compounds 4. b-Myrcene 5. o-Cymene 6. D -Limonenea 7. g-Terpinene 8. g-Terpineol 9. Neral (b-citral) 10. Geranial (a-citral) a
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b
RIb 990 1022 1030 1063 1191 1244 1273
(991) (1022) (1031) (1062) (1189) (1244) (1270)
Composition (%) 1.11 1.90 70.70 6.87 0.76 0.90 1.50
Concentration (mg/mL essential oil)
601.61 ^ 12.03
Compounds which were identified by using reference compounds. The values in parenthesis present literature RI (Adams 1995).
recognised as promoters that are involved in liver damage and carcinogenesis (Ames 1983; Namiki 1990; Gu¨lc in et al. 2004). Such findings cause growing interest of researchers and consumers in the antioxidant properties of natural compounds. For this reason, natural antioxidants are extensively studied for their capacity to protect organisms and cells from the damage induced by oxidative stress, by ageing, degenerative diseases and cancer (Pokorny 1991; Briskin 2000). In the course of studies undertaken to find safe and potent natural antioxidants, a huge number of compounds and extracts from aromatic, spicy, medicinal and other plants have been examined (Madsen & Bertelson 1995; Barrata et al. 1998; Miguel 2010). Some attention has also been paid to essential oils, known since the middle ages not only due to their pleasant or unpleasant aroma but also due to their antibacterial, antifungal and anti-inflammatory activities (Bakkali et al. 2008; Misharina & Samusenko 2008). Essential oils are very complex mixtures containing about 20 –60 or more components at very different concentrations. In each such complex mixture, one or two components occur at higher concentration (20 –70%) than others, and these major components determine the biological properties of an essential oil. The question asked here is whether this rule also applies to the antioxidant properties of essential oils. This study attempts to answer it by comparing the antioxidant properties of some essential oils with the antioxidant properties of their main components. The oils from the following herbs and fruits were applied in the experiments: thyme (Thymus vulgaris), basil (Ocimum basilicum), peppermint (Mentha piperita), clove (Caryophyllus aromaticus), summer savory (Satureja hortensis), sage (Salvia hispanica) and lemon (Citrus limon (L.) Burm). In these experiments, the amount of the main component of the essential oil (thymol or carvacrol or estragole or eugenol or menthol or camphor or limonene) in the measuring system was the same as its amount in the system when the essential oil solution was examined. The antioxidant activity of methanolic solutions of the examined essential oils and their main components were established by using 2,20 -Diphenyl-1-picrylhydrazyl (DPPH), 2,20 azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and b-carotene bleaching assays.
2. Results and discussion The compounds identified in the essential oils obtained from basil, clove, peppermint, summer savory, thyme, sage and lemon along with their peak percent concentrations and Kovats indices on ZB5-MS column are given in Table 1. The table contains only components that exceed 0.5% in the examined essential oils. According to the listed data, each of the examined oils contains one main component: eugenol, carvacrol, menthol, thymol, estragole, camphor and limonene in clove, summer savory, peppermint, thyme, basil, S. hispanica and lemon, respectively. Except
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camphor in S. hispanica essential oil, the content of the other main component in each given essential oil is greater than 50%. The structures of the main components are shown in Figure 1. The comparison of IC50 values of the examined materials (compounds or mixtures) is the simplest way to find the relation between their antioxidant properties. Table 2 lists the IC50 values for all essential oils and their main components estimated by using DPPH, ABTS and bcarotene bleaching assays. According to these data, all main components of the examined essential oils, except estragole (the main component of basil essential oil), exhibit higher antioxidant properties than the corresponding essential oils. It is worth noticing, however, that the comparison of IC50 for mixture and its main component shows the difference in the antioxidant capacity of these substances and does not allow to answer the question whether the major component of the mixture determines its antioxidant properties. The corresponding measuring systems contain different amounts of the mixture main component. Free radical-scavenging capacities of all essential oils and their main components, examined by using DPPH assay are presented in Figure 2. In these experiments, the amount of the main essential oil component (thymol or carvacrol or estragole or eugenol or menthol or camphor or limonene) in the measuring system was the same as in the system when the essential oil solution was examined. As observed in the figure, in the case of clove and summer savory essential oil, their antioxidant activity is very similar to the activity of their main components, eugenol and carvacrol, respectively (for clove and eugenol inhibition percent (I%) is about 82 ^ 1.9 and for summer savory and carvacrol about 53 ^ 1.5). The data for these essential oils show that the antioxidant properties of the essential oil depend in fact on the antioxidant properties of its main component. The other components of clove and summer savory essential oil do not exhibit antioxidant activities and do not influence the activity of eugenol and carvacrol, respectively. Quite a different situation is observed in the case of peppermint and basil essential oils. Their main components, menthol and estragole, do not exhibit antioxidant properties as estimated by the DPPH method (for menthol I% ¼ 3 ^ 0.3 and for estragole 5 ^ 0.4). It is not strange, as
Figure 1. Chemical structure of the main components of the examined essential oils.
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Table 2. IC50 values [mg/mL] of examined essential oils and their main components. Method of antioxidant activity determination
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Essential oil/main component Clove Eugenol Summer Savory carvacrol Lemon Limonene Savory Camphor Thyme Thymol Basil Estragole
DPPH
ABTS
b-Carotene
0.3257 ^ 0.009 0.1967 ^ 0.005 0.5088 ^ 0.013 0.4080 ^ 0.012 6.6768 ^ 0.133 1.8680 ^ 0.056 6.0854 ^ 0.183 4.7842 ^ 0.120 0.4029 ^ 0.011 0.2540 ^ 0.005 0.5798 ^ 0.013 5.7650 ^ 0.173
0.1595 ^ 0.005 0.1492 ^ 0.003 0.3671 ^ 0.011 0.1173 ^ 0.002 2.9670 ^ 0.089 2.9330 ^ 0.088 6.0854 ^ 0.152 1.2245 ^ 0.034 0.5758 ^ 0.011 0.3651 ^ 0.008 0.2133 ^ 0.006 3.4590 ^ 0.104
0.1848 ^ 0.005 0.0658 ^ 0.002 0.4356 ^ 0.013 0.2168 ^ 0.004 6.8595 ^ 0.206 1.2577 ^ 0.038 3.1256 ^ 0.078 1.0053 ^ 0.028 0.1913 ^ 0.004 0.1638 ^ 0.004 0.1616 ^ 0.005 5.7689 ^ 0.173
both compounds are not donors of proton and electron required for the neutralisation of DPPH radical. Hence, the antioxidant properties of peppermint and basil essential oils (I% about 89.1 ^ 2.7 and I% about 46.1 ^ 1.3; respectively) result from the properties of other components occurring in them (e.g. 4-terpineol, methyleugenol). As peppermint essential oil exhibits the lowest antioxidant activity of all the examined compounds, it was not diluted before measuring the remaining oils. Considering the ratio between the antioxidant properties of Salvia and lemon essential oils and their main components, lower antioxidant properties of the main components than of the essential oils were found (for Salvia and camphor I% ¼ 13 ^ 0.7 and 10 ^ 0.4, respectively, and for lemon and limonene ¼ 13.6 ^ 0.9 and 8.0 ^ 0.6, respectively). It suggests that the antioxidant properties of these oils are significantly affected not only by main components but also by other components (borneol, caryophyllene, citral). Greater antioxidant properties of the main components than the corresponding oil are observed for thymol (I% is about 63 ^ 1.7) and thyme essential oil (I% is about 57 ^ 1.6). The obtained results indicate the antagonistic influence of other thyme essential oil constituents on
Figure 2. Free radical-scavenging capacities of the essential oils and their main components examined by using DPPH assay.
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the antioxidant properties of thymol. Considering all the presented cases, it is difficult to state unequivocally that the main components of essential oil determine its antioxidant properties. The ABTS method is similar to the DPPH method in the evaluation of antioxidant properties, because in both the methods the antioxidant activity is estimated from the rate of scavenging process of colour radicals (Dawidowicz & Olszowy 2012). According to the results from Osman et al. (2006), ABTS cation radical is more reactive and its neutralisation path is different from that of the DPPH radical. Free radical-scavenging capacities of all the essential oils and their main components examined by using ABTS assay are presented in Figure 3. The analysis of the data shows that the antioxidant properties of the examined essential oils and their main components are slightly different from those established by DPPH method; however, the relationship between the antioxidant properties of the main component and the corresponding essential oil is similar. The most visible difference in the antioxidant properties estimated by DPPH and ABTS method is for thymol and thyme essential oil. The antioxidant activity of thymol is significantly greater than the antioxidant properties of thyme essential oil in ABTS method than in DPPH (cation radical I% for thymol and thyme is about 62 ^ 1.4 and 38 ^ 0.95, respectively). The results obtained in the ABTS method also confirm that the antioxidant properties of the essential oil do not always depend on the properties of its main components. Figure 4 presents the I% for all the examined essential oils and their main components determined by b-carotene bleaching assay. As it is seen, the relations between the antioxidant activity of a given essential oil and its main component are in a few cases different from those established by DPPH and ABTS methods. It is not strange if we take into account that the antioxidant activity in b-carotene bleaching method is determined by comparing two competitive chemical reactions in which the examined antioxidant and/or model antioxidant bcarotene take part (Moon & Shibamato 2009). This method assesses the ability of radical neutralisation by homolytic degradation of OZH bond in hydroxyl group occurring in an antioxidant, whereas in DPPH and ABTS method, the radical scavenging is preceded by dissociation of this group. It is worth mentioning that not only b-carotene but also other compounds possessing double bonds (e.g. p-cymene, borneol, b-myrcene, linalool, sabinene), that take part in b-carotene bleaching assay, are also able to form radical adduct with peroxyl radical and exhibit antioxidant properties.
Figure 3. Free radical-scavenging capacities of the essential oils and their main components examined by using ABTS assay.
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Figure 4. Free radical-scavenging capacities of the essential oils and their main components examined by bcarotene bleaching assay.
The results obtained from b-carotene bleaching assay, as well as from DPPH and ABTS methods, do not allow us to state unequivocally that the main components of an essential oil determine its antioxidant properties. In order to confirm the last statement, we decided to compare the antioxidant activity of systems containing essential oil or its main component (used in the same amount as it occurs in the measuring system with essential oil) or essential oil with addition of known amount of the main component. Three essential oils and their major components were chosen for these experiments: clove essential oil and eugenol, Salvia essential oil and camphor, basil essential oil and estragole. The antioxidant activity of these systems, estimated by using ABTS assay, is presented in Figure 5. The analysis of these results shows that the addition of the main component to the essential oil causes:
Figure 5. Free radical-scavenging capacities of the chosen essential oils, their main components and essential oils with addition of the main component examined by using ABTS assay.
8
A.L. Dawidowicz and M. Olszowy . the increased antioxidant properties of essential oil (cf. clove and clove with eugenol); . the decreased antioxidant properties of essential oil (cf. Salvia and camphor); . negligible differences in antioxidant properties of essential oil (cf. basil and estragole).
Hence, the main component does not always determine the antioxidant activity of the examined essential oils. The antioxidant activity of the main compound can be modulated by other components of essential oils.
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3. Experimental section 3.1. Materials Thyme, peppermint, summer savory, basil, sage, clove buds and lemon essential oils were obtained by means of steam distillation. Thyme, peppermint and summer savory, cultivated in eastern Poland, were purchased from a local herb planter. Basil, sage, clove buds and lemons were purchased at a local market. The herbs were air-dried, cut and stored at þ 88C. The lemon peel was cut and stored under the same conditions. Immediately before the isolation of essential oil, an appropriate amount of plant material was ground and its exactly weighed portion was subjected to distillation process. DPPH, b-carotene, linoleic acid, Tween 20, ABTS, potassium persulfate (di-potassium peroxydisulfate), thymol, estragole, menthol, eugenol, carvacrol, camphor and limonene were purchased from Sigma Aldrich (Poznan´, Poland). Methanol was purchased from the Polish Chemical Plant – POCh (Gliwice, Poland). Water was purified on a Milli-Q system from Millipore (Bedford, MA, USA). 3.2. Steam distillation Steam distillation process was performed for 3 h applying the Deryng apparatus, a Clevengertype (WPL, Gliwice, Poland) apparatus described in detail in the Polish Pharmacopea V, which contained a plant sample (50 g) and 600 mL of water. The distillation time was measured after the fall of the first drop of the distillate. The separated essential oils were dried by freezing and, after filtration, stored at þ 48C until further experiments. 3.3. Solutions of essential oils and essential oil main components The solutions of essential oil from thyme or clove or summer savory or basil or sage or lemon were prepared by dissolving 60 mL of a given essential oil in 5 mL of methanol. Before antioxidant measurements, the obtained solutions were diluted 1:20 in the same solvent. Due to the weak antioxidant properties of peppermint essential oil, this oil was applied in its natural form (without prior dilution). To estimate the IC50 values of the examined essential oils and their major components, stock solutions differing in concentrations of these antioxidants were prepared. To provide the same amount of the main essential oil component (thymol or carvacrol or estragole or eugenol or menthol or camphor or limonene) in the measuring system as it is present in the system when essential oil solution is applied, the examined essential oils were analysed by means of GC and the concentration of their main component was quantified precisely. Following the obtained results, methanolic solutions of adequate concentrations of main components were prepared. 3.4. Chromatographic analysis Qualification of the essential oil components in the prepared samples was performed using GC/ MS QP2010 (Shimadzu, Kyoto, Japan). A ZB5-MS fused silica capillary column (30 m
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£ 0.25 mm i.d., 0.25 mL film thickness) (Phenomenex, Shim-Pol, Izabelin, Poland) was used. Helium (grade 5.0) was used as carrier gas. Sample of 1 mL was injected by an AOC-20i-type autosampler. The injector temperature was 3108C. The following temperature program was applied: 1 min at 508C and then a linear increase in temperature up to 3108C at the rate of 68C/ min. The mass spectrometer was operated in EI mode at 70 eV; the ion source temperature was 2208C. The mass spectra were measured in the range of 35 –360 amu. Qualitative analysis was carried out comparing the retention indices (RI) and MS spectra for the obtained peaks with the analogous data from Adams (4th ed.) (Adams 1995), FFNSC 1.3 and NIST’05 databases. The identity of the main essential oil components was additionally confirmed by comparing their RI and MS spectra of their peaks with analogous data obtained for standards. Quantification of the essential oils was performed using a gas chromatograph with a flame ionisation detector-GC-FID model GC-2010 (Shimadzu, Kyoto, Japan). A sample of 1 mL was injected by AOC-20i-type autosampler into a ZB5-MS fused silica capillary column (30 m £ 0.25 mm i.d., 0.25 mL film thickness) (Phenomenex). The temperature program during GCFID separation was the same as for GC/MS. The quantity of the main components in examined solution was determined using the external standard method and calibration solutions of the main component standards. The quantities of the other essential oil components were given as peak percent and should be regarded as tentative. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were defined as the analyte concentration producing signal-to-noise ratio equal to 3:1 and 10:1, respectively. The estimated LOD and LOQ limits of the main essential oil components (mg/mL) were as follows: 0.34 and 1.15 for thymol, 0.26 and 0.88 for carvacrol, 0.54 and 1.8 for estragole, 0.87 and 2.91 for eugenol, 0.47 and 1.58 for menthol, 0.15 and 0.54 for camphor and 0.02 and 0.06 for limonene (LOD and LOQ, respectively). 3.5. Antioxidant activity The antioxidant activity of the examined solution of essential oils and its main components were determined by using DPPHz, ABTS and b-carotene bleaching assays. The scavenging of radicals by potential antioxidant was measured for all these methods. 3.5.1. DPPH method An aliquot (2940 mL) of methanolic DPPHz solution (24 mg/mL) was mixed in a 4 mL test tube with essential oil solution or main essential oil component solution (60 mL). In the case of peppermint, DPPHz solution (2400 mL) was mixed with methanol (500 mL) and pure essential oil or menthol solution (100 mL). Before measurement, each mixture was vigorously shaken during 30 s and immediately transferred into a quartz cuvette (1 cm £ 1 cm £ 3.5 cm). The decrease in absorbance at 516 nm was registered in continuous manner during 60 min employing a UV Probe-1800 spectrophotometer (Shimadzu). Subsequent readings were taken at regular intervals (60 s). The I% was calculated according to the following equation: At I ð%Þ ¼ 1 2 £ 100%; At¼0 where At¼0 and At are the values of absorbance of DPPHz at 0 min and at time equal to (t) min, respectively. 3.5.2. ABTS assay Generation of ABTS radical cation was performed according to Re et al. (1999). The ABTSzþ solution was prepared by the reaction of 5 mL of a 7 mM aqueous ABTS solution and 88 mL of
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140 mM (2.45 mM final concentration) potassium persulfate (K2S2O8) solution. The mixture was incubated in the dark for 16 h. The formed radical cation was then diluted in methanol until the initial absorbance value of 0.7 at 744 nm was reached. A volume of 2000 mL of the prepared ABTS radical cation solution was mixed in a 4 mL test tube with essential oil solution or main essential oil component solution (20 mL). In the case of peppermint, pure essential oil was used (20 mL). To estimate the influence of the reinforcement of the essential oil in its main component the measuring systems were composed of . 1990 mL of ABTS radical cation solution, 20 mL of methanolic essential oil solution (c ¼ 0.5 mg/mL) and 10 mL of methanol, when antioxidant properties of initial clove or basil or Salvia essential oil solution were examined; . 1990 mL of ABTS radical cation solution, 20 mL of methanolic essential oil solution (c ¼ 0.5 mg/mL) and 10 mL of methanolic solution of eugenol or estragole or camphor (c ¼ 0.5 mg/mL) when antioxidant properties of reinforced clove, basil and Salvia essential oil solution were examined. The reaction mixture was stirred vigorously for 30 s and poured into cuvettes (1 cm £ 1 cm £ 3.5 cm). The decrease in absorbance was recorded in a continuous manner during 60 min at 744 nm employing UV Probe-1800 spectrophotometer (Shimadzu). I% was calculated from the following equation:
I ð%Þ ¼
At 12 £ 100%; At¼0
where At¼0 and At are the values of absorbance of ABTSzþ at 0 min and at time equal to (t) min, respectively.
3.5.3. b-Carotene bleaching assay The estimation of the reacted b-carotene concentration in the examined systems was performed by the slightly modified Dapkevicius method (Gulluce et al. 2007). The stock solution of bcarotene/linoleic acid emulsion in water was prepared as follows: the mixture composed of 25 mL linoleic acid, 185 mL Tween 20 (200 mg) and 1 mL b-carotene solution (containing 0.5 mg of b-carotene in 1 mL of chloroform) underwent the vacuum evaporation process to remove chloroform. The obtained residue was dispersed in 100 mL of distilled water saturated with oxygen (30 min at 100 mL/min) and vigorously shaken. The absorbance of the final emulsion was measured at 470 nm using UV Probe-1800 spectrophotometer (Shimadzu). As a blank, emulsion without b-carotene was applied in the measurements. Aliquot (2.5 mL) of the emulsion with b-carotene was placed in a glass optical cuvette (1 cm £ 1 cm £ 3.5 cm) containing 350 mL of the essential oil solution or the main essential oil component solution. In the case of peppermint, pure essential oil was used (350 mL). The zero reading for the reaction mixture was taken at 470 nm immediately after the mixing. Subsequent readings were taken at regular intervals (10 min) until carotene in the control sample was decolourised (180 min). The control sample was prepared in the same way as the reacting mixture, the only difference being the replacement of the essential oil solution or the main component solution in a given solvent by pure solvent. Emulsion without b-carotene (2.5 mL) mixed with 350 mL of methanol was used to zero the spectrophotometer.
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The antioxidant activity was expressed as I% relative to the control using the following equation: AA ¼ 100 £
DRC 2 DRS DRc
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where AA is the antioxidant activity; DRC is the degradation rate of b-carotene in the control sample ¼ {[ln (a/b)]/t} and DRS is the degradation rate of b-carotene in the sample with antioxidant ¼ {[ln (a/b)]/t} – a is the absorbance at time 0; b is the absorbance at defined time (for example at 10, 20, . . . , 180 min) and t is the time. 3.6. Statistical analysis Results are presented as mean values ^ SD. In order to determine the reproducibility of the measurements, each antioxidant activity assay was carried out three times. RSD of all the measurements were lower than 10%. P , 0.01 was assumed as the statistical difference between the experimental points. 4. Conclusion Essential oils from different plants have attracted much interest due to their antioxidant, antitumour, antibacterial, antifungal and insecticidal properties. In general, the major components are found to reflect quite well the biological features of these oils. The presented results show that it is not always true in the case of the antioxidant properties of essential oils. The antioxidant activity of the main compound can be modulated by other components of essential oils. When comparing the antioxidant properties of essential oils and their main components, the concepts of synergism, antagonism and additivity are really relevant. It should be stressed, however, that the presented conclusions concerning the interaction of essential oil components depend on the type of method applied for assessing antioxidant activity. References Adams RP. 1995. Identification of essential oil components by gas chromatography/mass spectrometry. Carol Stream, IL: Allured Publishing Corporation. Ames BM. 1983. Dietary carcinogens and anticarcinogens: oxygen radical and degenerative diseases. Science. 221:1256–1263. Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils – A review. Food Chem Toxicol. 46:446–475. Baratta MT, Dorman HJD, Deans SG, Figueiredo AC, Barroso JG, Ruberto G. 1998. Antimicrobial and antioxidant properties of some commercial essential oils. Flavour Frag J. 13:235–244. Briskin DA. 2000. Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiol. 124:507–514. Dawidowicz AL, Olszowy M. 2012. Mechanism change in estimating of antioxidant activity of phenolic compounds. Talanta. 97:312– 317. Fridovich I. 1999. Fundamental aspects reactive oxygen species, or what’s the matter with oxygen. Ann NY Acad Sci. 893:13–18. ¨ I˙. 2004. Comparison of antioxidant activity of clove (Eugenia Gu¨lc in I`, S¸at ˙IG, Beydemir S¸, Elmastas¸ Ku¨freviog˘lu O caryophylata Thunb) buds and lavender (Lavandula stoechas L.). Food Chem. 87:393–400. Gulluce M, Sahin F, Sokmen M, Ozer H, Daferera D, Sokmen A, Polissiou M, Adiguzel A, Ozkan H. 2007. Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longfolia L. ssp. longifolia. Food Chem. 103:1449–1456. Ignarro LJ, Cirino G, Castini A, Napoli C. 1999. Nitric signaling molecule in the vascular system: an overview. J Cardiovasc Pharmacol. 34:879–886. Madsen HL, Bertelsen G. 1995. Spices as antioxidants. Trends Food Sci Tech. 6:271–277. McCord JM. 2000. The evolution of free radicals and oxidative stress. Am J Med Sci. 108:652–659.
Downloaded by [Florida Institute of Technology] at 23:06 20 August 2014
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Miguel MG. 2010. Antioxidant activity of medicinal and aromatic plants. A review. Flavour Frag. 25:291– 312. Misharina TA, Samusenko AL. 2008. Antioxidant properties of essential oils from lemon, grapefruit, coriander, clove and their mixture. Appl Biochem Microbiol. 45:482–486. Moon J, Shibamoto T. 2009. Antioxidant assays for plant and food components. J Agric Food Chem. 57:1655–1660. Namiki M. 1990. Antioxidants/antimutagens in food. Crit Rev Food Sci Nutr. 29:273–300. Osman AM, Wong KKY, Fernyhough A. 2006. ABTS radical- driven oxidation of polyphenols: isolation and structural elucidation of covalent adducts. Biochem Biophys Res Commun. 346:321–329. Pokorny J. 1991. Natural antioxidants for food use. Trends Food Sci Tech. 2:223–227. Politeo O, Jukic M, Milos M. 2007. Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Food Chem. 101:379–385. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. 1999. Antioxidant activity applying improved ABTS radical cation decolorization assay. Free Radi Biol Med. 26:1231– 1237. Tepe B, Daferera D, Sokmen A, Sokmen M, Polissiou M. 2005. Antimicrobial and antioxidant activities of the essential oil and various extracts of Salvia tomentosa Miller (Lamiaceae). Food Chem. 90:333–340. Yanishlieva NV, Marinova EM, Gordon MH, Raneva VG. 1999. Antioxidant activity and mechanism of action of thymol and carvacrol in two lipid systems. Food Chem. 64:59–66. Zhang H, Chen F, Wang X, Yao HY. 2006. Evaluation of antioxidant activity of parsley (Petroselinum crispum) essential oil and identification of its antioxidant constituents. Food Res Int. 39:833–839. Zheng M, Storz G. 2000. Redox sensing by prokaryotic transcription factors. Biochem Pharmacol. 59:1–6.
