Chemical Composition and Antioxidant Activity of ...

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Chemical Composition and Antioxidant Activity of Myrtus communis L. Floral Buds Essential Oil a

a

Ahmed Snoussi , Mohamed Moncef Chaabouni , Nabiha Bouzouita

a b

& Faten Kachouri

c a

Ecole Supérieure des Industries Alimentaires, 58 Avenue Alain Savary, 1003, Tunis, Tunisia b

University of Tunis El Manar, Laboratory of Structural Organic Chemistry, Faculty of Sciences of Tunis, 2092, El Manar, Tunisia c

Ecole Supérieure des Industries Alimentaires, 58 Avenue Alain Savary, 1003, Tunis, Tunisia Available online: 08 Dec 2011

To cite this article: Ahmed Snoussi, Mohamed Moncef Chaabouni, Nabiha Bouzouita & Faten Kachouri (2011): Chemical Composition and Antioxidant Activity of Myrtus communis L. Floral Buds Essential Oil, Journal of Essential Oil Research, 23:2, 10-14 To link to this article: http://dx.doi.org/10.1080/10412905.2011.9700440

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Chaabouni Snoussi et al.

Chemical Composition and Antioxidant Activity of Myrtus communis L. Floral Buds Essential Oil Ahmed Snoussi, Mohamed Moncef Chaabouni* and Nabiha Bouzouita Ecole Supérieure des Industries Alimentaires, 58 Avenue Alain Savary, 1003 Tunis, Tunisia University of Tunis El Manar, Laboratory of Structural Organic Chemistry, Faculty of Sciences of Tunis, 2092 El Manar, Tunisia

Faten Kachouri Ecole Supérieure des Industries Alimentaires, 58 Avenue Alain Savary, 1003 Tunis, Tunisia

Downloaded by [Tokyo Nogyo Daigaku] at 19:40 26 January 2012

Abstract The essential oil of Mytrus communis L. floral buds from Tunisia was analyzed by GC and GC/MS. Thirty-seven compounds were identified, representing 98.7% of the total oil. The major constituents were described as a-pinene (48.9%) and 1,8-cineole (15.3%). The antioxidant activity of the obtained essential oil was investigated by means of b-carotene bleaching (BCB) test and free radical scavenging assay using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). Results showed that Myrtus communis L. floral buds essential oil has a significant antioxidant effect when tested by each method respectively. The strong antioxidant activity of the studied oil can be attributed to the high content in hydrocarbon monoterpenes and oxygenated monoterpenes.

Key Word Index Floral buds essential oil, Myrtus communis L., chemical composition, antioxidant activity, a-pinene, 1,8-cineole.

Introduction Lipid oxidation is one of the major sources of change that occurs during the processing and storage of food. A number of food quality parameters such as nutrient content, safety, color, flavor and texture can be influenced by oxidative damage (1). The primary and secondary products of lipid oxidation are determinant to health. In the human body, the oxidative damage of biological molecules is involved in degenerative or pathological processes such as aging, coronary heart disease, cancer, arteriosclerosis and rheumatism (2,3). Hence, an external supply of antioxidants is necessary to avoid lipid deterioration in foods and living systems. The currently used artificial additives, such as butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), propyl gallate and tertiary butyl hydroxyl quinone (TBHQ), have been found to exhibit various health effects. The continuous use of such synthetic chemicals may cause health hazards such as teratogenic and carcinogenic effects in laboratory animals and primates (4). Therefore, interest in finding naturally occurring antioxidants that have the potential to protect human beings from damage induced by lipid oxidation has intensified. Myrtus communis L. (Myrtaceae), myrtle, is an evergreen shrub widespread in Mediterranean woodlands, maquis and garrigues. Essential oil from leaves, flowers and fruits of the

plant is widely used as a functional ingredient in the food, liquor and cosmetic industries. It tends to vary in composition and it is mainly used for the treatment of lung disorders. It has been found to also have antibacterial and antioxidant activities (5,6). A survey of the recent literature showed that data on the antioxidant activity of the known varieties is rather scarce (7,8,9). Therefore, the aim of the present study is to examine the chemical composition of the essential oils extracted from the floral buds of M. communis L. by GC and GC/MS and to evaluate its antioxidant activity using two different methods: BCB test and DPPH assay.

Experimental Chemicals: 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene, BHT), b-carotene, linoleic acid, 2,2-diphenyl1-picrylhydrazyl (DPPH) were procured from Sigma-Aldrich Chemie (Steinheim, Germany). Analytical grade ethanol, chloroform, anhydrous sodium sulfate (Na2SO4) and Tween 40 were obtained from Merck (Darmstadt, Germany). a-Thujene, a-pinene, b-pinene, myrcene, a-terpinene, limonene, 1,8-cineole, g-terpinene, terpinolene, linalool, linalyl acetate, eugenol and methyl eugenol were purchased from Fluka Chemika. Plant material: Myrtus communis L. floral buds were

*Address for correspondence: [email protected]

Rec: Sept 2010 Acc: Oct 2010

1041-2905/11/0001-010$14.00/0—© 2011 Allured Business Media 10/Journal of Essential Oil Research

Vol. 23, March/April 2011

M. communis

collected from the region of Jabbalah in Ain-Draham (northwestern Tunisia) in June 2009. After the botanical identification according to the Tunisian flora (10), the plant material was selected and cleaned of impurities in the laboratory. Oil extraction: The plant material (150 g) was subjected to hydrodistillation using a Dean-Stark apparatus. The extraction was carried on until there was no significant increase in the volume of oil collection. The obtained essential oil was dried over anhydrous sodium sulfate and preserved in a sealed vial at 4°C until further analysis.

Gas chromatography analysis: The essential oil was analyzed using an Agilent HP 6890 gas chromatograph with a HP–5MS 5% phenylmethylsiloxane capillary column (30 m x 0.25 mm, 0.25µm film thickness) equipped with FID detector. Oven temperature was maintained at 40°C for 1 min initially and then raised at the rate of 2°C/min to 280°C. Injector and detector temperatures were set at 250°C and 280°C respectively. Helium was used as carrier gas at a flow rate of 0.9 mL/ min and the sample was injected in the split mode (1:10). The percentage composition of the oil was calculated from the elec-


Table I. Chemical composition of essential oil from M. communis L. floral buds. N

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

ethyl isobutyrate 761 - RI, MS tricyclene 924 926 RI, MS a-thujene 928 931 RI, MS, CoI a-pinene 938 939 RI, MS, CoI a-fenchene 948 951 RI, MS camphene 950 953 RI, MS sabinene 972 975 RI, MS b-pinene 980 980 RI, MS, CoI myrcene 991 991 RI, MS, CoI isobutyl 2-methylbutyrate 1010 1009 RI, MS d-3-carene 1012 1011 RI, MS a-terpinene 1018 1018 RI, MS, CoI p-cymene 1025 1026 RI, MS limonene 1032 1031 RI, MS, CoI 1,8-cineole 1033 1033 RI, MS, CoI 1040 1040 RI, MS (Z)-b-ocimene 1048 1050 RI, MS (E)-b-ocimene g-terpinene 1063 1062 RI, MS, CoI terpinolene 1093 1089 RI, MS, CoI linalool 1101 1099 RI, MS, CoI borneol 1163 1165 RI, MS terpinen-4-ol 1179 1177 RI, MS a-terpineol 1189 1189 RI, MS myrtenol 1202 1196 RI, MS geraniol 1257 1255 RI, MS linalyl acetate 1262 1275 RI, MS, CoI eugenol 1357 1356 RI, MS, CoI neryl acetate 1368 1365 RI, MS geranyl acetate 1384 1383 RI, MS b-elemene 1392 1391 RI, MS methyl eugenol 1404 1401 RI, MS, CoI b-caryophyllene 1419 1418 RI, MS g-elemene 1433 1437 RI, MS allo-aromadendrene 1460 1461 RI, MS spathulenol 1576 1576 RI, MS caryophyllene oxide 1584 1581 RI, MS humulene epoxide II 1603 1606 RI, MS Chemical classes Monoterpene hydrocarbons Oxygenated monoterpenes Total monoterponoids Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total sesquiterpenoids Aliphatic compounds Benzenoid compounds Total

a

RIb

RIc

Identificatione

Percentaged 0.2 0.1 0.5 48.9 0.2 0.1 0.4 0.1 0.1 0.5 1.6 0.4 2.0 6.5 15.3 0.1 2.1 2.5 2.7 3.1 0.2 0.3 0.8 2.7 2.3 1.8 1.1 0.2 0.3 0.1 0.6 0.2 0.1 0.1 0.3 0.1 0.1 68.3 27.0 95.3 0.4 0.6 1.0 0.7 1.7 98.7%

Compounds listed in order of elution from HP-5MS column. Retention indices relative to C8 – C22 n-alkanes on HP-5MS column. Retention indices according to Adams (1995) d Percentage (mean of three analyses) based on FID peak area e RI: Retention indices relative to C8 – C22 n-alkanes on HP-5MS column, MS: mass spectrum, CoI: co-injection with authentic compounds (Fluka Chemika). a

b c


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tronic integration of the FID peak areas. The data presented are mean percent values of three analyses. Major compounds were quantified with the use of response factors. Gas chromatography/mass spectrometry analysis: GC/ MS analysis of the oil was carried out on an Agilent HP 6890 gas chromatograph equipped with an Autospec M-610 mass detector in the electron impact mode (Ionization energy: 70 eV) operating under the same conditions as described above. Compounds identification: The components of the essential oil were identified by comparison of their retention indices (11) relative to C8-C22 n-alkanes injected at the same conditions of real samples. Also, mass spectral matching with reference compounds contained in Wiley 238.L and Adams (12) libraries and, whenever possible, co-injection with authentic compounds has been performed. Analyses were performed in triplicate. Determination of the antioxidant activity by the b-carotene bleaching test: The antioxidant activity of M. communis L. floral buds essential oil was assessed using the b-carotene bleaching test (13). Approximately 2 mg of b-carotene was dissolved in 10 mL chloroform. The carotene - chloroform solution, 1 mL, was mixed with 20 mg linoleic acid and 200 mg Tween 40. Chloroform was removed using a rotary evaporator at 40°C for 5 min, and to the residue, 50 mL of oxygenated distilled water was added, slowly with vigorous agitation, to form an emulsion. The emulsion (4 mL) was added to a tube containing 0.2 mL of the sample solution in ethanol (2 mg/mL). Butylated hydroxytoluene (BHT), a stable antioxidant, was used for comparative purpose. Control sample contained 0.2 mL ethanol instead. The blank consisted on an emulsion without b-carotene. The absorbance was immediately measured at 470 nm. The tubes were placed in a water bath at 50°C and the oxidation of the emulsion was monitored spectrophotometrically by measuring absorbance at 470 nm until the color of b-carotene disappeared in the control (t=120°C). Antioxidant activity coefficients (AAC) were calculated using the following equation: AAC = [AA(120) – AC(120) / AC(0) – AC(120)] x 1000

1

Where AA(120) is the absorbance of antioxidant at 120 min, AC(120) is the absorbance of the control at 120 min and AC(0) is the absorbance of the control at 0 min. Determination of antioxidant activity by the DPPH assay: To evaluate the free radical scavenging activity, the essential oil was allowed to react with a stable free radical, 2,2-diphenyl-b-picryl hydrazyl radical DPPH (14). The antioxidant activity of M. communis L. floral buds essential oil was measured in terms of hydrogen-donating or radical scavenging ability. A stock solution (20 mg/mL) of the essential oil was prepared in ethanol. Dilutions are made to obtain concentrations ranging from 1 to 0.001 mg/mL-1. Two mL of each diluted solutions were mixed with 2 mL of freshly prepared DPPH solution in ethanol (2.10-4 M) and allowed to stand for 30 min in the dark at room temperature. The absorbance of the solution was measured at 517 nm against ethanol as the blank. The radical scavenging activity was calculated using the following formula: %inhibition = [(AC(0) – AA(t))/AC(0)] x 100 Where AC(0) is the absorbance of the control at t = 30 min and AA(t) is the absorbance of the antioxidant at t = 30 min. BHT was used as a positive control. The sample concentration providing 50% inhibition (IC50) was calculated by plotting inhibition percentages against concentrations of the samples. The assay was carried out in triplicate and results were stated in mean ± standard deviation.

Results and discussion Chemical composition: The hydrodistillation of M. communis L. floral buds yielded yellow colored oil with a mildly menthol-like odor (yield: 0.2% w/w). GC and GC/MS analysis of the oil led to the identification and quantification of 37 components listed in order of elution from HP-5MS column in Table I and which accounted for 98.7% of the total oil. The essential oil was characterized by a high percentage of monoterpene hydrocarbons (68.3%), followed by oxygenated

2

Figure 1. Absorbance changes of b-carotene at 470 nm in the presence of M. communis L. floral buds essential oil and BHT. Figure 2. Different antioxidant activity coefficient (AAC). 12/Journal of Essential Oil Research


M. communis

Table II. DPPH scavenging activities of various concentrations of M. communis L. floral buds essential oil and BHT. Concentrations (mg.mL-1)


0.001 0.005 0.1 0.5 1 IC50 (µg.mL-1)

M. communis L. floral buds essential oil

BHT

13.61 ± 1.85 28.35 ± 2.25 45.27 ± 0.90 56.71 ±1.06 72.63 ± 1.71 240 ± 2.90

17.83 ± 1.65 33.25 ± 0.77 64.21 ± 0.53 76.00 ± 0.62 85.74 ± 0.40 20 ± 0.98

monoterpenes (27.0%), while the sesquiterpene hydrocarbons and their oxygenated derivates accounted only for 1.0%. It is worth mentioning the presence of aliphatic (0.7%) and benzenoid (1.7%) compounds. The major components were a-pinene (48.9%), 1,8–cineole (15.3%), limonene (6.5%), linalool (3.1%), terpinolene (2.7%), myrtenol (2.7%), g-terpinene (2.5%), geraniol (2.3%), (E)b-ocimene (2.1%), p-cymene (2.0%), linalyl acetate (1.8%), d-3-carene (1.6%) and eugenol (1.1%). The major compounds obtained in this study were also found to be the major components of the essential oil obtained from different parts of M. communis L. but in varying proportions. According to Bouzouita et al. (15) the main components of leaves essential oil were 1,8–cineole (61.0%) and a-pinene (23.7%). This variation in the distribution between the monoterpene hydrocarbons and the oxygenated monoterpenes in the plant material could be related to changes throughout the plant’s vegetative cycle along with the environmental factors such as geography, temperature, day length, nutrients, etc. (6). These factors influence the plant biosynthetic pathways and consequently the relative proportion of the main compounds. Myrtle essential oils can be separated into two groups, depending on the content in myrtenyl acetate (16,17). In the present study, the former compound was absent. This result is in agreement with those obtained by Messaoud et al. (18) and Jamoussi et al. (19) who reported the absence of myrtenyl acetate in the Tunisian myrtle oil. Moreover, these authors showed that there is a good correlation between these groups and the origin of the plants. Antioxidant activity of M. communis L. floral buds essential oil—b-carotene bleaching test: The antioxidant activity of M. communis L. floral buds essential oil was evaluated by the b-carotene bleaching test, in which the oxidation of linoleic acid generates peroxyl free radicals due to the abstraction of hydrogen atom from diallylic methylene groups of linoleic acid. The free radical then will oxidize the highly unsaturated b-carotene (20). The degradation rate of b-carotene depends on the presence of antioxidants in the studied oil and on their antioxidant activity. Figure 1 shows the effect of the oil at 100 ppm and 200 ppm in comparison with BHT at 200 ppm. It is obvious that both concentrations of essential oil showed appreciable antioxidant activity. The absorbance decreased rapidly in the control sample, without addition of antioxidant, whereas in the presence of the oil, they retained their color, and thus absorbance for a longer time. The overall antioxidant strength decreased to the following order: Essential oil 200 ppm > BHT 200 ppm > Essential oil 100 ppm. Vol. 23, March/April 2011

The antioxidant activity coefficient (AAC) values are summarized in Figure 2. Floral buds essential oil performed better in its effect on reducing the oxidation of b-carotene than BHT at the same concentration. At 200 ppm, the AAC of the essential oil and BHT were estimated respectively as 670.49 and 581.97. Free radical scavenging activity: The DPPH scavenging activities (% inhibition) of various concentrations of M. communis L. floral buds essential oil and BHT are summarized in Table II. DPPH is a molecule containing a stable free radical. In the presence of an electron-donating compound, the purple color typical of the free DPPH radical diminishes in intensity, a change that can be followed spectrophotometrically at 517 nm (21). Various concentrations of the oil were investigated for their DPPH radical scavenging activity and they were compared with that of BHT. The oil scavenged the DPPH radical in a dose-dependent manner. The effect increased from 13.61 ± 1.85% to 72.63 ± 1.71% as concentrations increased from 0.001 to 1 mg/mL-1. The IC50 values, which refer to the smallest concentration of antioxidants necessary for 50% of inhibition, were 240 ± 2.90 µg/mL-1 for the oil and 20 ± 0.98 µg/mL-1 for the BHT. The lower the IC50 value the more reactive the compound under study. As can be seen, BHT is more effective than the studied oil. Many authors have reported antioxidant and radicalscavenging properties of essential oils. The studied oil exhibits remarkable antioxidant properties when compared to other studies (5). This strong activity can be attributed to the presence of higher amount of 1,8-cineole. As far as the present study’s literature survey could ascertain, some plant essential oils have proved to have various biological effects, including antioxidant activity due to the presence of 1,8-cineole (22,23). It was found that 1,8-cineole is the most powerful scavenging constituent for the oil of M. aquatic (24). Other components, even in small amounts, could improve the antioxidant activity of the oil indicating a possible synergistic interaction of the constituents. Eugenol and methyl eugenol, with a contribution of 1.1% and 0.6% respectively of the total oil, were reported to play an important role in antioxidant effectiveness (25). Moreover, Wei and Shibamato (26) showed the presence of a significant antioxidant potential of essential oils rich in hydrocarbon monoterpens particularly a-pinene and limonene. In the present study, a-pinene is present with about the half of the total oil composition (48.9%).

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Conclusion The present study is the first report on chemical composition and antioxidant activity of the essential oil extracted from M. communis L. floral buds, though earlier studies have reported the chemical composition of essential oil from leaves and berries from various Mediterranean origins. The oil activity was high enough to be considered as potential natural antioxidant and could perhaps be formulated as a part of daily supplements or additives to prevent oxidative stress that contributes to many degenerative diseases. References


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