Essential oils of basil chemotypes: Major compounds, binary mixtures, and antioxidant activity

Accepted Manuscript Essential oils of basil chemotypes: major compounds, binary mixtures, and antioxidant activity Hyrla Grazielle Silva de Araújo Couto, Arie Fitzgerald Blank, Ana Mara de Oliveira e Silva, Paulo Cesar de Lima Nogueira, Maria de Fátima ArrigoniBlank, Daniela Aparecida de Castro Nizio, Jessika Andreza de Oliveira Pinto PII: DOI: Reference:

S0308-8146(19)30740-X https://doi.org/10.1016/j.foodchem.2019.04.078 FOCH 24679

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

6 August 2018 15 April 2019 22 April 2019

Please cite this article as: de Araújo Couto, H.G.S., Blank, A.F., de Oliveira e Silva, A.M., de Lima Nogueira, P.C., de Fátima Arrigoni-Blank, M., de Castro Nizio, D.A., de Oliveira Pinto, J.A., Essential oils of basil chemotypes: major compounds, binary mixtures, and antioxidant activity, Food Chemistry (2019), doi: https://doi.org/10.1016/ j.foodchem.2019.04.078

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Essential oils of basil chemotypes: major compounds, binary mixtures, and antioxidant activity

Hyrla Grazielle Silva de Araújo Coutoa*, Arie Fitzgerald Blanka, Ana Mara de Oliveira e Silvab, Paulo Cesar de Lima Nogueirac, Maria de Fátima Arrigoni-Blanka, Daniela Aparecida de Castro Nizioa, Jessika Andreza de Oliveira Pintoa

a

Laboratory of Plant Genetic Resources and Essential Oils, Department of Agronomic Engineering,

Federal University of Sergipe, Avenida Marechal Rondon s/n, Rosa Elze, CEP 49100-000, São Cristóvão, state of Sergipe, Brazil. b

Laboratory of Bromatology, Department of Nutrition, Federal University of Sergipe, Avenida

Marechal Rondon s/n, Rosa Elze, CEP 49100-000, São Cristóvão, state of Sergipe, Brazil. c

Research Laboratory in Organic Chemistry, Department of Chemistry, Federal University of

Sergipe, Avenida Marechal Rondon s/n, Rosa Elze, CEP 49100-000, São Cristóvão, state of Sergipe, Brazil.

Essential oils of basil chemotypes: antioxidant activity

*Corresponding author Telephone 55-79-31946981 Fax 55-79-31946474 E-mail address: [email protected] (H.G.S.A. Couto)

1

Abstract: The antioxidant potential of the essential oils (EO) of 24 basil genotypes was assessed by 4 distinct in vitro evaluation methods. Different combinations of the major compounds found in the EO were also tested to identify those combinations responsible for the antioxidant activity of the volatile oils and verify the occurrence of synergism or antagonism between them. Results indicate that 9 EO exhibited promising antioxidant potential, with at least 52.68% of inhibition of the linoleic acid peroxidation at 10 µL/mL and 76.34% of inhibition of the DPPH• radical at 1 µL/mL. The major compound eugenol had the highest antioxidant activity. The antioxidant activity of these EO cannot be explained solely by the presence of the major compounds. Despite the influence of eugenol, the antioxidant activity is also related to the synergism between other minor compounds found in the EO. This fact confers a potent antioxidant activity to some basil EO.

Keywords: Ocimum, volatile oil, eugenol, synergism, antagonism.

Chemical compounds studied in this article: 1,8 cineole (PubChem CID: 2758); eugenol (PubChem CID: 3314); geranial (PubChem CID: 638011); linalool (PubChem CID: 6549); methyl chavicol (PubChem CID: 8815); (PubChem CID: 637520); neral (PubChem CID: 643779).

1. Introduction The demand for natural and safe foods that use fewer preservatives has increased due to health concerns and the trend of a healthy lifestyle (Zhang et al., 2016). Therefore, essential oils and herbs extracts have been proven as an alternative of scientific interest owing to their various application purposes, including the conservation of fresh and processed foods (Yuan et al., 2016). The antioxidant activity is one of the most investigated characteristics of essential oils. The use of synthetic antioxidants has led to carcinogenicity, which consequently arose the interest in substituting these chemicals by natural additives with antioxidant capacity, such as essential oils 2

(Filip et al., 2016). Essential oils (EO) are composed of volatile compounds, produced by the secondary metabolism of aromatic plants. These arouse great interest because they present various biological activities such as antioxidant, antifungal and antibacterial (Burt, 2004; Koroch et al., 2007). Essential oils can be obtained from various parts of plants such as leaves, flowers, roots, among others (Aquino et al., 2010; Joshi, 2013; Duskova et al., 2016).

Basil (Ocimum basilicum L.) belongs to the family Lamiaceae and is originated from Southeast Asia and Central Africa. The species was introduced to Brazil by European immigrants and occurs sub-spontaneously throughout the country (Lorenzi & Matos, 2008). O. basilicum has multiple applications in culinary, perfumery, and pharmacy. It can be used as a medicinal and ornamental plant and as a flavoring ingredient in the food industry. The different application purposes of basil are mainly related to the biological properties of its essential oils, which includes insecticidal, acaricidal, bactericidal, and antioxidant activities (Al Abbasy et al., 2015; Santos et al., 2012; Li & Chang, 2016). The composition of essential oils produced by plants is influenced by several factors, such as seasonality, temperature, luminosity, water and nutrients availability, pests attack, diseases, and genetic constitution (Gobbo-Neto & Lopes, 2007). The variation in the chemical composition of essential oils may affect their biological activity (Morais, 2009). Some studies have reported the antioxidant activity of essential oils, extracts, and leaves of basil cultivars (Filip et al., 2016; Flanigan & Niemeyer, 2014; Pandey et al., 2016). However, the research on major compounds combinations provides information about the interactions between them and assists the development of more effective formulations with standard concentrations. Different interactions between major compounds may result in synergistic, additive, or antagonistic effects (Katiki et al., 2017). The activity resulting from these combinations will be greater/lesser than or equal to that observed for the pure compound. In recent years a variety of techniques have been used to estimate the antioxidant power of substances and foods. Among these techniques, the most used are: DPPH•(2,2-diphenyl-1picrylhydrazyl) radical scavenging assay, ferric reducing antioxidant power (FRAP), ABTS+• 3

radical scavenging, cupric reducing antioxidant capacity (CUPRAC), oxygen radical absorption capacity (ORAC), and inhibition of the β-Carotene/linoleic acid system peroxidation (Chen et al., 2013; Shahidi & Zhong, 2015; Sridhara & Charles, 2019). The variety of methods, is due to the different mechanisms of antioxidant activity. These methods can act primarily by transferring hydrogen atoms, electron donation, and by the ability to chelate transition metals (Prior et al., 2005; Brewer 2011). This work aimed to evaluate the antioxidant activity of essential oils of basil cultivars and hybrids and their individual and mixed (binary and tertiary mixtures) major compounds, as well as verify the principal component related to this activity. So, four methods (DPPH, FRAP, ABTS + and β-carotene) were used to provide additional information on the antioxidant capacity of basil essential oils and their major compounds.

2. Materials and Methods

2.1. Chemicals Tween 20, Iron(III) chloride hexahydrate, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), potassium persulfate, linoleic acid (≥99%), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), (±)-6-Hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid (Trolox) (97%), and the standard compounds linalool, 1,8 cineole, methyl chavicol, and eugenol were obtained from Sigma-Aldrich. Sodium acetate, Glacial acetic acid, β-Carotene, Chloroform, and Absolute ethyl alcohol were obtained from Synth®, Êxodo®, Fluka™, and Neon, respectively.

2.2. Plant material and essential oils extraction Twenty-four basil genotypes were used, which included 20 commercial cultivars and 4 experimental hybrids obtained from the Basil Breeding Program of the Federal University of Sergipe (UFS). The commercial cultivars consisted of ‘Maria Bonita’, developed by the Federal 4

University of Sergipe (Blank et al., 2007), 14 cultivars purchased from the Company Richters (Ocimum basilicum ‘Anise’, Ocimum basilicum ‘Ararat’, Ocimum basilicum ‘Edwina’, Ocimum basilicum ‘Dark Opal’, Ocimum basilicum ‘Genovese’, Ocimum basilicum ‘Green Globe’, Ocimum basilicum ‘Italian Large Leaf’, Ocimum basilicum ‘Magical Michael’, Ocimum basilicum ‘Mrs. Burns’, Ocimum basilicum ‘Napoletano’, Ocimum basilicum ‘Nufar F1’, Ocimum basilicum ‘Osmin’, Ocimum basilicum ‘Purple Ruffles’, and Ocimum x citriodorum ‘Sweet Dani’), and 5 cultivars purchased from the company ISLA (Ocimum basilicum ‘Grecco a Palla’; Ocimum basilicum ‘Italian Large Leaf (ISLA)’; Ocimum basilicum ‘Italian Large Red Leaf’; Ocimum basilicum ‘Red Rubin Purple Leaf’, and Ocimum basilicum ‘Limoncino’). The experimental hybrids were ‘Cinnamon’ x ‘Maria Bonita’; ‘Genovese’ x ‘Maria Bonita’; ‘Sweet Dani’ x ‘Cinnamon’; and ‘Sweet Dani' x ‘Genovese.' All genotypes were cultivated in the Experimental Farm "Campus Rural da UFS," located in the municipality of São Cristóvão, state of Sergipe, Brazil (lat. 10°55'27"S; long. 37°12'01"W; alt. 46 m asl.), in the rainy season, which comprises the months from April to July. According to Köppen, the climate of the region is classified as As (tropical savanna climate) (Alvares et al., 2013), with an average annual temperature of 25.2°C. The soil of the experimental area is characterized as Red Yellow Podzolic. The fertilization was performed with 800 kg/ha of mono-ammonium phosphate, 300 kg/ha of potassium chloride, and 5 t/ha of cattle manure. Drip irrigation was applied when necessary. Plants were manually harvested in June (Pinto et al., 2018). Essential oils of basil plants were obtained by drying the leaves in a forced air circulation oven, at 40 ºC, for 5 days. Essential oils were extracted by hydrodistillation in an adapted Clevenger apparatus (Ehlert et al., 2006), using samples of 50 g of dried leaves. Afterward, essential oils were stored in amber flasks at -20 °C until the chemical composition analysis.

2.3. Chemical composition of essential oils

5

The chemical composition of the essential oils was analyzed using a GC-MS/FID (QP2010 Ultra, Shimadzu Corporation, Kyoto, Japan) equipped with an autosampler AOC-20i (Shimadzu), as described in Silva et al. (2018). Individual compounds of the essential oil were identified, as explained in Silva et al. (2018).

2.4. Antioxidant activity Initially, the total antioxidant potential of the essential oil of the 20 basil cultivars and 4 basil hybrids was investigated using the following methods: DPPH•(2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay; ferric reducing antioxidant power (FRAP); ABTS+• radical scavenging; and inhibition of the β-Carotene/linoleic acid system peroxidation. Subsequently, major compounds were mixed, simulating the chemical composition of the essential oils of 9 cultivars that exhibited high antioxidant potential in the 4 methods initially tested. Mixtures were obtained from commercially purchased standard compounds (Sigma Chemical Co., USA), by combining them with the same proportion as that found in the essential oils selected for this step. These mixtures were denominated as “essential oils simulation.” After preparation, the antioxidant activity of these 9 mixtures was evaluated by the same methods previously mentioned and compared with the antioxidant activity of the respective essential oils. Then, compounds linalool, 1,8 cineole, eugenol, and methyl chavicol were tested separately, and so were their binary and tertiary mixtures. All compounds were tested at the same concentration (10 µL/mL), separately and in mixtures. Thus, 5 binary mixtures (mixture 1: 1,8 cineole + linalool; mixture 2: 1,8 cineole + eugenol; mixture 3: linalool + eugenol, mixture 4: 1,8 cineole + methyl chavicol, and mixture 5: linalool + methyl chavicol) and 2 tertiary mixtures (mixture 6: 1,8 cineole + linalool + eugenol, and mixture 7: 1,8 cineole + linalool + methyl chavicol) were tested. The antioxidant activity of essential oils, simulations, and binary and tertiary mixtures was evaluated using 4 methods, which will be described below. These methods are based on different

6

mechanisms and can provide complementary information about the antioxidant capacity of the samples studied.

2.4.1. DPPH· radical scavenging capacity A 50 μL aliquot of the essential oil/simulation/mixture sample at 1 µL/mL concentration (diluted in absolute ethyl alcohol) was added to 150 μL of DPPH• methanolic solution at 6x10-5 mol/l. The control sample of the DPPH assay consisted of 50 μL of absolute ethyl alcohol (reagent used to dilute essential oil samples) and 150 μL of DPPH methanolic solution at 6x10-5 mol/l. The analysis was followed by a control treatment without antioxidant, containing 50 μL of ethanol. The reduction of the DPPH• radical was measured in a spectrophotometer at 515nm, after 30 minutes of rest at room temperature (Brand-Williams et al., 1995). The decrease in the absorbance values of the samples was correlated with that of the control, and the scanning percentage of the DPPH• radical was expressed by the following equation: (Equation 1) In which Abscontrol is the absorbance of the control, and Abssample is the absorbance of the sample solution. Results were expressed as DPPH-scavenging activity, and the assays were performed in triplicate.

2.4.2. Ferric-reducing antioxidant power (FRAP) The methodology described by Pulido et al. (2000), with adaptations, was used to determine the reductive potential of the samples. A 9 μL aliquot of the essential oil/simulation/mixture sample at a concentration of 1 µL/mL (diluted in absolute ethyl alcohol) was transferred to 96-well microplates, which were added with 27 μL of distilled water and 270 μL of the FRAP reagent (corresponding to 25 mL of 0.3 M acetate buffer; 2.5 mL of a 10 mM TPTZ solution; and 2.5 mL of a 20 mM ferric chloride aqueous solution). Microplates were maintained at 37 °C for 30 min. The reading was carried out at 595 nm. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic 7

acid) was dissolved in absolute ethyl alcohol, and results were expressed in μMol of Trolox equivalents/mL of essential oil, by the standard curve constructed, with concentrations from 25 to 2000 μM of Trolox.

2.4.3. ABTS+• scavenging capacity The methodology described by Re et al. (1999) was used to determine the antioxidant capacity against the ABTS+• radical. The ABTS+• radical cation was initially formed from the reaction of 5 mL of a 7 mM ABTS+ solution in 88 μL of a 2.45 mM potassium persulfate solution (K2S2O8), incubated at room temperature, for 16 h, in the darkness. Afterward, the ABTS+ solution was diluted in ethanol until obtaining a solution of 0.70 ± 0.05 of absorbance, at 734 nm. A 3 μL sample of the essential oil at a concentration of 10 µL / mL (diluted in absolute ethyl alcohol) was added to 300 μL of the ABTS+• radical, in microplates, at room temperature and darkness. The experiment was performed in triplicate, and absorbance readings were carried out for 6 minutes, using a spectrophotometer, at 734nm. Results were expressed in μMol of Trolox equivalents per mL of oil, according to the standard curve constructed.

2.4.4. Inhibition of the β-carotene/linoleic acid system peroxidation The β-Carotene/linoleic acid system evaluates the capacity of an antioxidant to inhibit the linoleic acid peroxidation and prevent the β-Carotene decolorization. The evaluation followed the methodology described by Miller (1971), with some modifications. Initially, distilled water was saturated with oxygen for 30 minutes to prepare the emulsifying solution, containing 200 μL of βCarotene (2 mg/mL), 10 μL of linoleic acid, 50 μL of Tween 20, and 500 μL of Chloroform. Then, the chloroform was evaporated for 10 minutes in a hood. Afterward, the solution was added with 25 mL of saturated water. Microplates were added with 10 μL of the essential oil sample, at a concentration of 10 μL/mL (diluted in absolute ethyl alcohol), and 240 μL of the emulsifying solution. 8

The reading was performed at 470 nm of absorbance at time 0h. The microplate was incubated in the darkness, at 50 °C, for 2 hours, and subsequently, a new reading was performed at the same wavelength. Results were expressed as % of oxidation inhibition, by the formula:

(Equation 2) In which Abscontrol is the absorbance of the control, and Abssample is the absorbance of the sample solution.

2.5. Synergism/antagonism evaluation The antioxidant activity of the binary and tertiary mixtures was evaluated regarding the synergistic or antagonistic effects between the compounds. The result of the mixture corresponds to the antioxidant activity of the binary or tertiary mixture (A + B or A + B + C). The result of A, B, and C corresponds to the antioxidant activity detected for each compound individually, based on the methodologies described above. Mixtures that presented values higher than the values of the compounds alone were considered as synergism, mixtures that presented smaller results than the values of the compounds alone were considered antagonism. When there was no difference between the values of the mixtures and the compounds alone an indifferent effect was considered.

2.6. Statistical analysis The experiments for the antioxidant activity of the essential oils, simulations, and mixtures were carried out in a completely randomized design with three replications. The antioxidant activity of the essential oils selected at the initial stage was compared with that of their respective simulations, using a 9 x 2 factorial scheme, with 9 cultivars and 2 compounds sources (essential oil and simulation). Data were subject to analysis of variance (ANOVA), using the SISVAR® statistical software. Means were clustered according to the Scott-Knott’s test, at the 5% probability level, and expressed as mean ± SE (standard error). 9

Subsequently, cluster analysis and principal component analysis (PCA) were performed in the STATISTICA® software. The data corresponding to the % of major compounds (1,8 cineole, linalool, eugenol, methyl chavicol, neral, geranial, and (E)-methyl cinnamate) and antioxidant activities of the essential oils in the 4 methods were used to cluster the 24 basil genotypes by the Ward’s clustering method, based on their Euclidean distances. Also, a correlation analysis was performed between the compounds and the antioxidant activity of the essential oils in the different methods.

3. Results and discussion

3.1. Essential oils chemical composition The 24 genotypes showed 16 compounds in their composition: linalool, methyl chavicol, neral, geranial, eugenol, and (E)-methyl cinnamate were detected at higher amounts (Table 1). Different basil cultivars have the genetic ability to generate and maintain different sets of chemical compounds, leading to a wide variety of chemotypes within the same species. This phenomenon occurs because the culture is highly pollinated by bees. Currently, over 55 bee species have been reported as basil pollinators (Muniz et al., 2013; Avetisyan et al., 2017; Srivastava et al., 2018). Previous research has described the chemical diversity of basil. For instance, a study on the EO of basil plants from North India (Padalia et al., 2017) revealed methyl chavicol (56.1-89.7%) and linalool (1.0-33.7%) as the major compounds. Another study on basil cultivars also carried out in India showed 1,8 cineole (2.7-5.2%), linalool (23.5-40%), methyl chavicol (25-53%), eugenol (2.1-5.4%), and methyl eugenol (1.9-10%) as the major compounds (Kakaraparthi et al., 2015). Other countries, such as Oman, have reported linalool (69.87%), Geraniol (9.75%), methyl chavicol (6.02%), and 1,8 cineole (4.9%) as the major compounds of basil essential oils. In Brazil, 31 basil accessions and 7 basil cultivars were chemically characterized, revealing 8 chemical groups: Group 1 - EO with linalool and 1,8 cineole; Group 2 - EO with linalool, 10

Geraniol, and α-Trans-bergamotene; Group 3 - EO with linalool, methyl chavicol, methyl cinnamate, and β-Bourbonene; Group 4 - EO with linalool, methyl chavicol, Epi-α-cadinol, and αTrans-bergamotene; Group 5 - EO with linalool, methyl eugenol, α-Trans-bergamotene, and Epi-αcadinol; Group 6 - EO with linalool, Geraniol, and Epi-α-cadinol; Group 7 - EO with linalool and methyl chavicol; Group 8 - EO with Geranial and Neral (Costa et al., 2015).

3.2. Antioxidant activity screening of basil cultivars and hybrids The 4 methods tested (DPPH, FRAP, ABTS, and β-carotene) revealed significant differences between the antioxidant activities of the essential oils of the 24 samples analyzed. Nine cultivars stood out for their high antioxidant activity [Edwina, Dark Opal, Genovese, Green Globe, Italian Large Leaf, Magical Michael, Grecco a Palla, Italian Large Leaf (Isla), and Italian Large Red Leaf]. These nine cultivars differed statistically from the essential oils of the other 15 genotypes evaluated by the Scott-Knott’s test (p <0.05) (Table 2). The major compounds of these cultivars consist of the combinations of linalool, 1,8-cineole, and eugenol [Edwina, Italian Large Leaf, Grecco a Palla, Italian Large Leaf (Isla), and Italian Large Red Leaf]; linalool, 1,8 cineole, and methyl chavicol (Genovese and Green Globe); linalool and 1,8 cineole (Dark Opal); or linalool and eugenol (Magical Michael) (Table 1). Linalool and methyl chavicol have been reported as the major compounds of basil cultivars’ essential oils (Oliveira et al., 2013; Padalia et al., 2017). Eugenol (4.60%) and 1,8 cineole (4.04%) have also been reported in the chemical composition of some genotypes of Ocimum basilicum L. from Africa (Tshilanda et al., 2016). This variation in the chemical composition might be influenced by abiotic factors, such as temperature, sunlight intensity, water and nutrient availability, seasonality, and plant age (Cabello et al., 2014; Selmar & Kleinwächter, 2013). The clustering analysis was used to cluster basil cultivars by the similarities of their chemical composition, considering the major compounds, and their antioxidant activity profile detected in the DPPH, FRAP, ABTS, and β-Carotene methods (Figure 1A). Thus, two large clusters 11

were formed, the first one consisting of 8 cultivars [Edwina, Magical Michael, Dark Opal, Genovese, Italian Large Red Leaf, Italian Large Leaf (Isla), Italian Large Leaf, and Grecco a Palla] and the second one consisting of 16 cultivars (Anise, Purple Ruffles, Ararat, Napoletano, Green Globe, Mrs Burns, Limocino, Cinnamon x Maria Bonita, Sweet Dani x Cinnamon, Sweet Dani, Nufar F1, Sweet Dani x Genovese, Red Rubin Purple Leaf, Maria Bonita, Genovese x Maria Bonita, and Osmin). The first cluster contained the cultivars with the best antioxidant performance in the 4 methods evaluated. This cluster is mainly characterized by the cultivars with essential oils rich in 1,8 cineole, linalool, and eugenol. Cluster 2 consists of cultivars with essential oils containing as major compounds 1,8 cineol, linalool, methyl chavicol, citral (neral and geranial), and/or (E) methyl cinnamate. Linalool and citral (neral and geranial) are non-phenolic terpenes found in several essential oils, and previous studies have evaluated their antioxidant capacity involving oxidizable substrates (measurement of autoxidation rate). Research on oxidizable substrates, such as Cumene (Isopropyl benzene), revealed that these compounds reduced the oxidation rate to a critical limit concentration (4% v/v for linalool and 0.12% v/v for citral) (Baschieri et al., 2017).

3.3. Antioxidant activity of essential oils and their respective simulations The comparison of the antioxidant activities between the essential oils of the 9 cultivars and their simulations, obtained by the mixture of commercially purchased standard compounds, showed that only the essential oil of cultivar Italian Large Leaf (Isla) and its simulation were not statistically different from each other in all tests performed (Table 3). This essential oil has linalool (61.15%), 1,8 cineole (12.33%), and eugenol (7.54%) as major compounds (Table 1). The absence of significant difference between the essential oil and its simulation (using standard compounds at the same proportions as those found in the metabolite) suggest that the antioxidant activity of this volatile oil is conferred by the major compounds mentioned above. 12

Other cultivars evaluated in this study also have these same compounds; however, the differences in concentrations and the occurrence of other minor compounds might have potentiated the antioxidant activity of the essential oils when compared with their simulations. In many cases, the major compound cannot be confirmed as the only responsible for the biological activity of the essential oil since this phenomenon is the result of the interaction between different compounds (Frutuoso et al., 2013). Simulations without eugenol (cultivars Dark Opal, Genovese, and Green Globe) did not exhibit antioxidant activity. Nevertheless, the essential oils of these cultivars had antioxidant activity and differed statistically from their simulations (Table 3). This effect can be attributed to the contribution of other non-phenolic components found at lower concentrations in the essential oil, which is a non-elucidated topic (Baschieri et al., 2017). In general, the essential oils exhibited antioxidant activities higher than or equal to those detected in the respective simulations, constituted only by the major compounds. This fact has also been verified in coriander, whose essential oil had higher antioxidant activity than the major compound linalool (Duarte et al., 2016). Results of several studies have arisen the interest in understanding the action of essential oils and the complex interactions between their compounds. These interactions, which result in synergistic and antagonistic effects, make essential oils potentially useful for the food industry (Amorati et al., 2013). Synergism is the interaction between two or more antioxidant or antimicrobial substances; it potentiates the effect of the mixture and increases the effects when compared with those of the same substances alone. Conversely, antagonism occurs when the interaction between substances have a lesser effect than the sum of their individual effects (Chou, 2006; Milos & Makota, 2012; Skroza et al., 2015). The additive interaction occurs when the effect of the mixture of compounds equals the sum of the effects of the same compounds alone. These results justify the interest in evaluating the antioxidant activity of the major compounds found in these essential oils, either alone or in binary and tertiary mixtures. 13

3.4.

Antioxidant

activity

of

individual

and

mixed

major

compounds

and

their

synergistic/antagonistic effects Eugenol and its mixtures (1,8 cineole + eugenol; linalool + eugenol; and 1,8 cineole + linalool + eugenol) showed higher mean values in all the tests performed and differed statistically (α = 0.05) from the other mixtures and compounds (Table 4). The FRAP method had three exceptions (1,8 cineole + linalool; 1,8 cineole + eugenol; and linalool + eugenol), which showed indifferent effects. In this case, the antioxidant activity of the mixture corresponds to the sum of the effects of each compound alone. Antagonistic activity was detected in most of the binary and tertiary mixtures, in all the antioxidant methods used. Interestingly, no synergism was observed in any of the mixtures, which reinforces the results obtained in the previous tests, where the antioxidant activity of basil essential oils was strongly linked to the presence and activity of eugenol and its interaction with other minor compounds. The principal component analyses (PCA) (Figure 1B) revealed that the first and second principal components represent 63.2% of the total variance. The first principal component is responsible for 39.78% of the total variance and correlated positively with the methods DPPH (r = 0.93), FRAP (r = 0.89), ABTS (r = 0.94), and β-carotene (r = 0.93) and with the compound eugenol (r = 0.67). The second principal component accounted for 23.42% of the total variance and correlated positively with 1,8 cineole (r = 0.70) and linalool (r = 0.56) and negatively correlated with neral (r = -0.96) and geranial (r = -0.96). These results show that the mentioned methods (DPPH, FRAP, ABTS, β-carotene, and eugenol) were the most important for the clustering of cultivars of group 1. Compounds 1,8 cineole and linalool also contributed to this clustering for they are in the same quadrant (Figure 1B). Among the major compounds used in the correlation analysis, eugenol had the highest correlation with the antioxidant activities of the essential oils, considering the 4 methods [DPPH (r = 0.52), FRAP (r = 0.54), ABTS (r = 0.61), and β-carotene (r = 0.58)].

14

These results suggest that the antioxidant activities of the essential oils are strongly related to the presence of eugenol (Table 5). Eugenol is a phenolic compound of the class of phenylpropanoids. Some studies have stated that the antioxidant activity of phenolic compounds is due to their chemical structure, which allows them to donate hydrogen to free radicals and stabilize by dislocating the electrons in the aromatic ring by resonance (Ang et al., 2015). The antioxidant activity of eugenol has been reported in previous studies. However, some works have shown that the antioxidant activity is related to a limiting value, i.e., the increase in eugenol concentration may increase the antioxidant activity of a given sample up to a specific limiting concentration (Goñi et al., 2016). Moreover, the Food and Drug Administration (FDA) affirms that the use of eugenol in food is considered as safe only if it administered within the established limits, meaning that exceeding this limit can cause health damage and even be fatal (FDA, 2016).

4. Conclusions

In all the antioxidant activity tests performed in this work, eugenol had higher antioxidant activity and higher mean values when tested alone, statistically differing from the other compounds. All essential oils and all tested mixtures with eugenol in their composition showed higher antioxidant activity in all methods. This result suggests that the antioxidant activities can be related to the presence of eugenol. The higher antioxidant activity exhibited by the essential oils when compared with their simulations (constituted only by the major compounds) evidences that this activity is not related only to the presence of the major compounds, i.e., it may also be related to the synergism between major and minor compounds found in the essential oils. Thus, further in vivo studies on basil essential oils with high antioxidant potential should be developed, aiming at replacing synthetic preservatives. 15

4. Acknowledgments This study was financed in part by the Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brasil (CNPq), the Fundação de Apoio à Pesquisa e a Inovação Tecnológica do Estado de Sergipe (Fapitec/SE) - Brasil, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES - Finance Code 001), and the Financiadora de Estudos e Projetos - Brasil (FINEP).

5. Conflict of interest The authors declare no conflict of interest.

6. References Al Abbasy, D. W., Pathare, N., Al-Sabahi, J. N., Khan, S. A. (2015). Chemical composition and antibacterial activity of essential oil isolated from Omani basil (Ocimum basilicum Linn.). Asian Pacific Journal of Tropical Disease, 5(8), 645-649. Alvares, C. A., Stape, J. L., Sentelhas, P. C., Gonçalves, J. L. M., Sparovek, G. (2013). Köppen's climate classification map for Brasil. Meterologische Zeitschrift, 22(6), 711-728. Avetisyan, A., Markosian, A., Petrosyan, M., Sahakyan, N., Babayan, A., Aloyan, S., & rchounian, A. (2017). Chemical composition and some biological activities of the essential oils from basil Ocimum different cultivars. BMC complementary and alternative medicine, 17(1), 60. Amorati, R., Foti, M. C., Valgimigli, L. (2013). Antioxidant Activity of Essential Oils. Journal of Agricultural and Food Chemistry, 61(46), 10835-10847. Ang, L. Z. P., Hashim, R., Sulaiman, S. F., Coulibaly, A. Y., Sulaiman, O., Kawamura, F., Salleh, K. M. (2015). In vitro antioxidant and antidiabetic activites of Gluta torquata. Industrial Crops and Products, 76, 755-760. Aquino, L. C. L., Santos, G. G., Trindade, R. C.; Alves, J. A. B.; Santos, P. O.; Alves, P. B.; Blank, 16

A. F.; Carvalho, L. M. (2010). Atividade Antimicrobiana Dos Óleos Essenciais De ErvaCidreira E Manjericão Frente a Bactérias De Carnes Bovinas. Alimentos e Nutrição, 21, 529– 535. Baschieri, A., Ajvazi, M. D., Tonfack, J. L. F., Valgimigli, L., Amorati, R. (2017). Explaining the antioxidant activity of some common non-phenolic components of essential oils. Food Chemistry, 232, 656-663. Blank, A. F., Souza, E. M., Arrigoni-Blank, M. F., Paula, J. W. A., Alves, P. B. (2007). Maria Bonita: Cultivar de manjericão tipo linalol. Pesquisa Agropecuaria Brasileira, 42(12), 18111813. Brand-Williams, Cuvelier, M. E., Berset, C. (1995). Use of a Free Radical Method to Evaluate Antioxidant Activity. Food Sci. Technol., 28, 25-30. Brewer, M. S. (2011). Natural antioxidants: Sources, compounds, mechanisms of action, and potential applications. Comprehensive Reviews in Food Science and Food Safety, 10, 221–247. Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in foods—a review. International Journal of Food Microbiology, 94 (3), 223–253. Cabello, J. V., Lodeyro, A. F., Zurbriggen, M. D. (2014). Novel perspectives for the engineering of abiotic stress tolerance in plants. Current Opinion in Biotechnology, 26, 62-70. 74. Chen, Z., Bertin, R., Froldi, G. (2013). EC50 estimation of antioxidant activity in DPPH assay using several statistical programs. Food Chemistry, 138, 414–420. Chou, T. (2006). Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies. Pharmacological Reviews, 58(3), 621-681. Costa, A. S., Arrigoni-Blank, M. F., Carvalho Filho, J. L. S., Santana, A. D. D., Santos, D. A., Alves, P. B., Blank, A. F. (2015). Chemical diversity in basil (Ocimum sp.) germplasm. The Scientific World Journal, Article ID 352638, 1-9. Duarte, A., Luís, Â., Oleastro, M., Domingues, F. C. (2016). Antioxidant properties of coriander 17

essential oil and linalool and their potential to control Campylobacter spp. Food Control, 61, 115-122. Dusková, E., Dusek, K., Indrák, P., Smékalová, K. (2016). Postharvest changes in essential oil content and quality of lavender flowers. Industrial Crops and Products, 79 225–231. Ehlert, P. A. D., Blank, A. F., Arrigoni-Blank, M. F., Paula, J. W. A., Campos, D. A., Alviano, C. S. (2006). Tempo de hidrodestilação na extração de óleo essencial de sete espécies de plantas medicinais. Revista Brasileira de Plantas Medicinais, 8(2), 79-80. FDA,

(2016).

U.S.

Food

and

Drug

Administration

[WWW

Document].

https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=182.20 Filip, S., Vidović, S., Vladić, J., Pavlić, B., Adamović, D., Zeković, Z. (2016). Chemical composition and antioxidant properties of Ocimum basilicum L. extracts obtained by supercritical carbon dioxide extraction: Drug exhausting method. The Journal of Supercritical Fluids, 109, 20-25. Flanigan, P. M., Niemeyer, E. D. (2014). Effect of cultivar on phenolic levels, anthocyanin composition, and antioxidant properties in purple basil (Ocimum basilicum L.). Food Chemistry, 164, 518-526. Frutuoso, A. E., Teotonio, N., Leda, T., Lemos, G. De, Coelho, E. L., Maria, D., Teixeira, A. (2013). Óleos essenciais aplicados em alimentos: Uma revisão. Revista Brasileira de Pesquisa em Alimentos, 4, 69-81. Gobbo-Neto, L., Lopes, N. P. (2007). Plantas medicinais: Fatores de influência no conteúdo de metabólitos secundários. Quimica Nova, 30(2), 374-381. Goñi, M. L., Gañán, N. A., Strumia, M. C., Martini, R. E. (2016). Eugenol-loaded LLDPE films with antioxidant activity by supercritical carbon dioxide impregnation. Journal of Supercritical Fluids, 111, 28-35. Hidalgo, M., Sánchez-Moreno, C., Pascual-Teresa, S. (2010). Flavonoid-flavonoid interaction and its effect on their antioxidant activity. Food Chemistry, 121(3), 691-696. 18

Joshi, R. K. (2013). Chemical constituents and antibacterial property of the essential oil of the roots of Cyathocline purpúrea. Journal of Ethnopharmacology, 145, 621–625. Kakaraparthi, P. S., Srinivas, K. V. N. S., Kumar, J. K., Kumar, A. N., Kumar, A. (2015). Composition of herb and seed oil and antimicrobial activity of the essential oil of two varieties of Ocimum basilicum harvested at short time. Journal Plant Development, 22, 59–76. Katiki, L. M., Barbieri, A. M. E., Araujo, R. C., Veríssimo, C. J., Louvandini, H., Ferreira, J. F. S. (2017). Synergistic interaction of ten essential oils against Haemonchus contortus in vitro. Veterinary Parasitology, 243(June), 47-51. Koroch, A. R., Zygadlo, J. A., Juliani, H. R. (2007). Bioactivity of Essential oils and their componentes.

Flavours

and

Fragrances:

Chemistry,

Bioprocessing

and

Sustainability, Springer-Verlag, Berlin Heidelberg, 87-115. Li, Q. X., Chang, C. L. (2016). Chapter 25 - Basil (Ocimum basilicum L.) Oils. In Essential Oils in Food Preservation, Flavor and Safety (pp. 231-238). Lorenzi, H.; Matos, F. J. A. (2008). Plantas medicinais no Brasil: nativas e exóticas. Nova Odessa: Instituto Plantarum, 2008. 544 p. Miller, H. E. (1971). A simplified method for the evaluation of antioxidants. Journal of the American Oil Chemists Society, 48(2), 91-91. Milos, M., Makota, D. (2012). Investigation of antioxidant synergisms and antagonisms among thymol, carvacrol, thymoquinone and p-cymene in a model system using the Briggs-Rauscher oscillating reaction. Food Chemistry, 131(1), 296-299. Morais, L. A. S. (2009). Influência Dos Fatores Abióticos Na Composição Química Dos Óleos Essenciais. Horticultura Brasileira, 27(2), 4050-4063. Muniz, J. M., Pereira, A. L. C., Valim, J. O., Campos, W. G. (2013). Patterns and mechanisms of temporal resource partitioning among bee species visiting basil (Ocimum basilicum) flowers. Arthropod-Plant Interactions, 7(5), 491-502. Oliveira, R. A., Moreira, I. Oliveira, F. F. (2013). Linalool and methyl chavicol present basil 19

(Ocimum sp.) cultivated in Brazil. Revista Brasileira de Plantas Medicinais, 15(2), 309-311. Padalia, R. C., Verma, R. S., Upadhyay, R. K., Chauhan, A., Singh, V. R. (2017). Productivity and essential oil quality assessment of promising accessions of Ocimum basilicum L. from north India. Industrial Crops and Products, 97, 79-86. Pandey, V., Patel, A., Patra, D. D. (2016). Integrated nutrient regimes ameliorate crop productivity, nutritive value, antioxidant activity and volatiles in basil (Ocimum basilicum L.). Industrial Crops and Products, 87, 124-131. Pinto, J. A. O., Blank, A. F., Nogueira, P. C. L., Arrigoni-Blank, M. F., Andrade, T. M., Sampaio, T. S., Pereira, K. L. G. (2019). Chemical charaterization of the essential oil leaves of basil genotypes cultivated in different seasons. Boletín Latinoamericano y del Caribe de Plantas Medicinais y Aromáticas. 18 (1), 58-70. Pinto, J. A. O., Blank, A. F., Andrade, T. M., Sá Filho, J. C. F., Nascimento, L. F. A., Silva, D. C., Arrigoni-Blank, M. F (2018). Cropping season affect the performance of basil cultivars and hybrids. Bioscience Journal, 34(3), 640-647. Prior, R. L., Wu, X., Schaich, K. (2005). Standard methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 53, 4290–4302. Pulido, R., Bravo, L., Saura-Calixto, F. (2000). Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. Journal of Agricultural and Food Chemistry, 48(8), 3396-3402. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C. (1999). Antioxidant Activity Applying an Improved Abts Radical Cation Decolorization Assay. Free Radical Biology and Medicine, 26(9), 1231-1237. Sacchetti, G., Maietti, S., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., Bruni, R. (2005). Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chemistry, 91(4), 621-632. 20

Santos, F. C. C., Vogel, F. S. F., Monteiro, S. G. (2012). Efeito do óleo essencial de manjericão (Ocimum basilicum L.) sobre o carrapato bovino Rhipicephalus (Boophilus) microplus em ensaios in vitro. Semina:Ciencias Agrarias, 33(3), 1133-1140. Selmar, D., Kleinwächter, M. (2013). Stress enhances the synthesis of secondary plant products: The impact of stress-related over-reduction on the accumulation of natural products. Plant and Cell Physiology, 54(6), 817-826. Shahidi, F., Zhong, Y. (2015). Measurement of antioxidant activity. Journal of Functional Foods, 18, 757–781. Silva, D. C., Blank, A. F., Nizio, D. A. C., Sampaio, T. S., Nogueira, P. C. L., Arrigoni-Blank, M. F. (2018). Chemical diversity of essential oils from native populations of Eplingiella fruticosa. Crop Breeding and Applied Biotechnology, 18, 205-214. Skroza, D., Generalić Mekinić, I., Svilović, S., Šimat, V., Katalinić, V. (2015). Investigation of the potential synergistic effect of resveratrol with other phenolic compounds: A case of binary phenolic mixtures. Journal of Food Composition and Analysis, 38, 13-18. Sridhara, K., Charles, A. L. (2019). In vitro antioxidant activity of Kyoho grape extracts in DPPH• and ABTS• assays: Estimation methods for EC50 using advanced statistical programs. Food Chemistry, 275(1), 41-49. Srivastava, A., Gupta, A. K., Sarkar, S., Lal, R. K., Yadav, A., Gupta, P., Chanotiya, C. S. (2018). Genetic and chemotypic variability in basil (Ocimum basilicum L.) germplasm towards future exploitation. Industrial Crops and Products, 112, 815-820. Tshilanda, D. D., Babady, P. B., Onyamboko, D. N. V., Tshiongo, C. M. T., Tshibangu, D. S. T., Ngbolua, K. te N., Tasalu, P. V., Mpiana, P. T. (2016). Chemo-type of essential oil of Ocimum basilicum L. from DR Congo and relative in vitro antioxidant potential to the polarity of crude extracts. Asian Pacific Journal of Tropical Biomedicine, 6(12), 1022-1028. Yuan, G., Chen, X., Li, D. (2016). Chitosan films and coatings containing essential oils: The antioxidant and antimicrobial activity, and application in food systems. Food Research 21

International, 89, 117-128. Zhang, Y., Liu, X., Wang, Y., Jiang, P., Quek, S. (2016). Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Food Control, 59, 282-289.

22

Figure legend:

Fig. 1. Two-dimensional dendogram representing the similarity between 24 basil cultivars and hybrids (A) and principal component analysis (PCA) involving the major compounds and the antioxidant activity of the essential oils evaluated by the DPPH, FRAP, ABTS, and β-carotene methods (B).

23

Table 1. Content of major compounds of essential oils of 20 cultivars and 4 hybrids of basil. Cultivars / hybrids

Compounds 1,8-Cineole

Linalool

Methyl chavicol

Eugenol

E-methyl cinnamate

Neral

Geranial

RRIo

1015

1080

1183

1338

1369

1224

1253

RRIl

1026

1096

1195

1356

1376

1235

1264

Anise

7.78

-

81.02

-

-

-

-

Ararat

3.15

15.67

67.69

-

-

-

-

Edwina

6.93

72.76

-

5.66

-

-

-

Dark Opal

17.97

54.64

-

-

-

-

-

Genovese

6.35

57.33

27.43

-

-

-

-

Green Globe

5.14

22.95

58.40

-

-

-

-

Italian Large Leaf

8.80

63.80

-

11.29

-

-

-

Magical Michael

0.65

63.78

-

20.13

-

-

-

Mrs. Burns

0.62

38.31

-

-

-

22.21

27.35

Napoletano

6.45

26.13

54.48

-

-

-

-

Nufar F1

6.51

66.25

11.86

-

-

-

-

Osmin

15.61

58.44

-

8.41

-

-

-

Purple Ruffles

10.54

18.37

57.46

-

-

-

-

-

0.36

1.40

-

-

37.03

49.07

Grecco a Palla

4.13

24.10

-

29.85

-

-

-

Italian Large Leaf (Isla)

12.33

61.15

-

7.54

-

-

-

Italian Large Red Leaf

10.49

64.31

-

12.57

-

-

-

Red Rubin Purple Leaf

13.80

60.95

-

5.76

-

-

-

-

8.54

-

-

-

21.68

27.59

Maria Bonita

4.76

78.10

-

-

-

-

-

Híbrido Cinnamon x Maria Bonita

2.37

33.73

-

-

38.76

-

-

Híbrido Genovese x Maria Bonita

5.02

67.65

-

-

-

9.16

11.45

Híbrido Sweet Dani x Cinnamon

5.40

16.90

0.99

-

61.54

1.09

0.95

Híbrido Sweet Dani x Genovese

6.16

58.72

16.62

-

-

4.07

4.98

Sweet Dani

Limoncino

Source: extracted from Pinto et al. (2019). RRIo: Relative Retention Index - observed; RRIl: Relative Retention Index- literature (Adams, 2007).

24

Table 2. Antioxidant activity of 20 cultivars and 4 hybrids of basil, according to methods of DPPHscavenging activity, ABTS free radical scavenging, ferric-reducing antioxidant power (FRAP), and β-carotene. DPPH

FRAP

ABTS

β-carotene

(% of inhibition)

(µMol/mL)

(µMol/mL)

(% of inhibition)

Anise

10.5 ± 3.58h

0.05 ± 0.04g

0.90 ± 0.06d

2.05 ± 0.00f

Ararat

52.88 ± 1.20d

0.52 ± 0.01d

1.18 ± 0.11d

16.09 ± 0.02e

Edwina

78.22 ± 1.22b

0.55 ± 0.01c

2.14 ± 0.14b

39.87 ± 0.03d

Dark Opal

83.48 ± 0.23a

0.57 ± 0.02c

2.34 ± 0.12a

52.96 ± 0.02c

Genovese

81.84 ± 0.39a

0.61 ± 0.06c

2.07 ± 0.31b

52.68 ± 0.00c

Green Globe

70.32 ± 0.29c

0.57 ± 0.05c

1.90 ± 0.23c

51.66 ± 0.04c

Italian Large Leaf

84.71 ± 0.10a

0.56 ± 0.02c

2.42 ± 0.17a

78.42 ± 0.02a

Magical Michael

76.34 ± 0.41b

0.62 ± 0.01c

1.94 ± 0.09c

41.33 ± 0.03d

Mrs. Burns

11.55 ± 0.66h

0.26 ± 0.01e

0.97 ± 0.12d

9.14 ± 0.01f

Napoletano

45.97 ± 0.67e

0.61 ± 0.02c

1.24 ± 0.18d

22.28 ± 0.01e

Nufar F1

16.68 ± 3.48g

0.48 ± 0.08d

1.02 ± 0.17d

18.03 ± 0.02e

Osmin

29.76 ± 0.69f

0.66 ± 0.04b

1.04 ± 0.28d

21.64 ± 0.03e

Purple Ruffles

5.70 ± 0.80i

0.12 ± 0.03f

0.97 ± 0.02d

8.47 ± 0.02f

Sweet Dani

67.94 ± 2.00c

0.67 ± 0.02b

1.72 ± 0.33c

64.27 ± 0.00b

Grecco a Palla

84.12 ± 0.26a

0.78 ± 0.03a

2.63 ± 0.14a

79.59 ± 0.01a

Italian Large Leaf (Isla)

80.57 ± 0.90b

0.70 ± 0.05b

2.11 ± 0.22b

61.59 ± 0.04b

Italian Large Red Leaf (Richters)

79.52 ± 1.10b

0.76 ± 0.01a

2.14 ± 0.29b

53.83 ± 0.03c

7.48 ± 1.31i

0.23 ± 0.07e

0.91 ± 0.08d

10.00 ± 0.01f

15.10 ± 0.74g

0.29 ± 0.09e

1.10 ± 0.14d

7.87 ± 0.01f

Maria Bonita

0.69 ± 0.52j

0.03 ± 0.06g

0.85 ± 0.10d

12.40 ± 0.01f

Híbrido Cinnamon x Maria Bonita

1.83 ± 1.38j

0.05 ± 0.02g

0.79 ± 0.05d

5.08 ± 0.01f

Híbrido Genovese x Maria Bonita

0.06 ± 0.37j

0.12 ± 0.03f

0.78 ± 0.07d

18.87 ± 0.01e

Híbrido Sweet Dani x Cinnamon

1.69 ± 0.97j

0.15 ± 0.03f

0.94 ± 0.19d

21.76 ± 0.01e

Híbrido Sweet Dani x Genovese

3.23 ± 0.24j

0.24 ± 0.02e

0.99 ± 0.13d

14.53 ± 0.02e

Cultivars / hybrids

Red Rubin Purple Leaf Limoncino

* Means followed by different letters in the columns differ from each other by the Scott-Knott’s test (p <0.05).

25

Table 3. Content of major compounds, antioxidant activity of essential oils of basil cultivars and their respective simulations, according to the methods of DPPH free scavenging, ABTS free radical scavenging, ferric-reducing antioxidant power (FRAP), and β-carotene. Major compounds content in the

β-carotene (% of

DPPH FRAP (µMol/mL) ABTS (µMol/mL)

simulations* (%)

(% of inhibition)

inhibition)

Cultivars 1,8-

Methyl LinaloolEugenol

Cineole

Edwina

6,30

Dark Opal

17,97

Genovese

6,35

Green Globe

Italian

5,14

Essential Essential oil Simulation

chavicol

72,76

54,64

57,33

22,95

5,66

0,00

0,00

0,00

0,00

0,00

27,47

58,40

oil

63,80

11,29

0,00

Grecco a Palla

Italian

4,13

63,78

24,10

20,13

29,85

0,00

0,00

61,15

7,54

0,00

1.02 ±

0.67 ±

1.88 ±

1.44 ±

47.07

2.04dA

0.13cA

0.04eB

0.14bA

0.24cA

±3.46dA

0.24 ±

2.03 ±

0.00 ±

2.47 ±

0.20 ±

70.32 ±

0.01eB

0.23bA

0.01fB

0.07aA

0.13dB

1.43bA

0.00 ±

0.90 ±

0.00 ±

1.73 ±

0.06 ±

45.52 ±

0.00eB

0.22cA

0.01fB

0.05bA

0.06dB

1.41dA

0.00 ±

0.41 ±

0.00 ±

1.33 ±

0.04 ±

25.28 ±

0.00eB

0.07dA

0.01fB

0.09bA

0.04dB

4.39fA

69.58 ±

1.74 ±

1.64 ±

2.41±

1.63 ±

63.00 ±

1.68cB

0.69bA

0.22cA

0.07aA

0.21cB

5.17cA

78.61 ±

0.60 ±

2.66 ±

1.38 ±

2.15 ±

37.96 ±

0.97aA

0.08dB

0.26bA

0.11bB

0.26bA

3.05eA

81.38 ±

2.43 ±

4.04 ±

2.34 ±

2.91 ±

78.53

2.04aA

0.15aB

0.00aA

0.14aB

0.03aA

±4.47aA

63.68 ±

0.91 ±

0.91 ±

1.77 ±

1.74 ±

44.39 ±

0.64dA

0.03cA

0.03dA

0.06bA

0.14cA

3.20dA

74.00

0.81 ±

1.70 ±

1.77 ±

1.16 ±

46.01 ±

±0.48bA

0.06cB

0.13cA

0.58bA

1.04cA

3.10dB

37.39± 2.37dB

0.81± 2.19eB

62.58 ± 0.89bA

2.76± 0.70eB

39.44±12.96dA

0.65± 5.08eB

75.57 ± 3.05aA

0.00± 2.13eB

48.57 ± 0.73cB

44.71± 0.88cB

78.34 ± 0.42aA

64.63± 0.86aB

65.71 ±0.73bA

Leaf (Isla) Italian

44.22± 1.96cA

Large 10,49

64,31

12,57

0,00

Simulation oil

76.49 ± 1.81aA

Large 12,33

oil

58.89 ±

Leaf

Magical Michael 0,65

Essential Simulation

62.67 ± 1.00bA

Large 8,80

Essential Simulation

61.84 ± 2.16bB

Red Leaf

51.70A±5.87bA

Means followed by different uppercase letters in the rows, for the same method, and lowercase letters in the columns differ from each other by the Scott-Knott’s test (p <0.05). *Major compounds contents in the simulations are equivalent to the concentrations of the same compounds in the essential oils of the cultivars, according to Table 1.

26

Table 4. Antioxidant activity of major compounds present in essential oils of basil genotypes (Ocimum basilicum), tested separately and in mixtures, and their effects, according to the methods of DPPH free scavenging, ABTS free radical scavenging, ferric-reducing antioxidant power (FRAP), and β-carotene.

Compo Compound Compo und 1

2

und 3

DPPH (% of

FRAP Effect

inhibition) 1,8 cineole

-

0.00 ± 0.00b

-

0.61 ± 0.12b

-

-

cineole

2.58 ± 0.73b

-

chavicol

linalool

methyl-

linalool

-

0.07 ±

-

7.96 ± 4.69c

-

-

84.47 ±

-

0.06c -

2.94 ± 0.07a

-

0.17 ±

0.37a -

0.00 ± 0.00d

-

0.08c

nt

0.05c

stic

82.76 ±

Antagoni

4.04 ±

Indiffere

2.67 ±

Antagoni

81.62 ±

Antagoni

1.80a

stic

0.00a

nt

0.10a

stic

0.75a

stic

80.92 ±

Antagoni

4.04 ±

Indiffere

2.84 ±

Antagoni

82.19 ±

Antagoni

3.12a

stic

0.00a

nt

0.03a

stic

1.94a

stic

82.58 ±

Antagoni

4.04 ±

Indiffere

2.52 ±

Antagoni

78.21 ±

Antagoni

0.28a

stic

0.00a

nt

0.35b

stic

1.66b

stic

0.00 ±

Antagoni

0.16 ±

0.00c

stic

0.12c

0.00 ±

Antagoni

0.09 ±

0.00c

stic

0.01c

methyl- 0.00 ± 0.00b Antagoni

0.00 ±

Antagoni

0.14 ±

chavicol

0.00c

stic

0.03c

-

0.00 ± 0.00b Antagoni stic

-

chavicol 1,8

8.94 ± 1.49c

0.00c

eugenol

cineole

-

stic

eugenol

methyl-

inhibition)

0.08 ±

-

1,8

0.07 ±

Effect

Indiffere

eugenol

linalool

4.04 ±

0.08 ±

(% of

0.00 ±

-

cineole

-

0.00a

0.33 ± 0.02b Antagoni

linalool

1,8

0.00 ±

Effect

0.07c

0.05b -

linalool

cineole

-

-

1,8 cineole

84.14 ± 0.70a

-

1,8

-

0.00c

-

chavicol

0.00 ±

(µMol/m L)

0.00c

linalool

methyl-

Effect

L)

-

eugenol

(µMol/m

β-carotene

ABTS

0.00 ± 0.00b Antagoni stic

stic

Antagoni 8.78 ± 1.36c Antagoni stic

Antagoni 4.55 ± 0.61d Antagoni stic

Antagoni 1.95 ± 1.87d Antagoni stic

stic

Antagoni 3.17 ± 0.98d Antagoni stic

*Means followed by the different letters in the columns differ from each other by the Scott-Knott’s test (p<0.05).

27

stic

stic

28

Table 5. Correlation coefficients between the major compounds present in essential oils of basil and their antioxidant activity by the DPPH, FRAP, ABTS, and β-Carotene methods. Compounds

1,8-

Linalo

Methyl

Cineole

ol

chavicol

Nera Gerani Eugeno l

Linalool

0.38

Methyl chavicol

-0.02

-0.53

Neral

-0.52

-0.40

-0.24

Geranial

-0.52

-0.41

-0.24

1.00

Eugenol

0.04

0.20

-0.34

-

al

l

(E)- Methyl cinnamate

DPP FRA ABT H

P

S

-0.24

0.24 (E)-

Methyl

-0.15

-0.24

-0.17

cinnamate DPPH

-

-0.11

-0.17

-0.09

0.52

-0.34

-0.01

0.54

-0.36

0.88

-0.12

0.61

-0.28

0.95 0.79

0.01

0.58

-0.20

0.89 0.79 0.93

0.10 0.15

0.13

-0.08

0.11

FRAP

0.16

0.14

-0.15

0.02

ABTS

0.18

0.18

-0.20

0.13

β-carotene

0.13

0.15

-0.27

0.00

29

Highlights

Some basil cultivars have promising antioxidant potential Eugenol effectively contributes to the antioxidant activity of essential oils Eugenol presented a high correlation with the antioxidant activity methods applied No synergistic effect was detected among major compounds for antioxidant activity

30

31