Spectral and chemometric analyses reveal antioxidant properties of essential oils from four Cameroonian Ocimum

Industrial Crops and Products 80 (2016) 101–108

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Spectral and chemometric analyses reveal antioxidant properties of essential oils from four Cameroonian Ocimum Jean Baptiste Fokou Hzounda a,b,c,∗ , Pierre Michel Dongmo Jazet a,d , Gabriel Lazar c , Dumitra R˘aducanu e , Iuliana Caraman c , Emmanuel Bassene b , Fabrice Fekam Boyom a , Iuliana Mihaela Lazar c a AntiMicrobial Agents Unit, Laboratory for Phytobiochemistry and Medicinal Plants Studies, Department of Biochemistry, University of Yaoundé I, PO Box 812, Yaoundé, Cameroon b Laboratory of Pharmacognosy and Botany, Department of Pharmacy, Cheikh Anta Diop University, PO Box 5005 Fann, Dakar, Senegal c Department of Environmental and Mechanical Engineering, Vasile Alecsandri University of Bacau, Calea Marasesti 157, Bacau 600115 Romania d Laboratory of Biochemistry, Department of Biochemistry, University of Douala, PO Box 24157, Douala, Cameroon e Department of Biology, Ecology and Environmental Protection, Vasile Alecsandri University of Bacau, Calea Marasesti 157, Bacau 600115 Romania

a r t i c l e

i n f o

Article history: Received 15 April 2015 Received in revised form 28 September 2015 Accepted 30 September 2015 Keywords: Ocimum Essential oil FTIR-ATR Flaxseed oil Antioxidant Chemotype

a b s t r a c t Antioxidants used to protect unstable unsaturated fatty acids from oxidation are toxic or lose their potential in fats. The aim of this work was to investigate the protective effect of Ocimum essential oils against auto-oxidation of flaxseed oil. Essential oils were extracted from leaves of cultivated Ocimum basilicum, Ocimum canum, Ocimum gratissimum and wild type Ocimum urticaefolium. Chemical profiles of the essential oils were determined using Fourier Transformed Infrared-Attenuated Total Reflectance (FTIR-ATR) coupled with chemometric analyses. The antioxidant effect was measured on flaxseed oil under accelerated oxidation for 15 days. The structural change of the flaxseed oil was investigated by spectrophotometric and titrimetric methods. Linalool/Eugenol, Eucalyptol, Thymol/␥-Terpinene, and Eugenol/ ␤-Bisabolene were found to be the respective chemotypes of Ocimum basilicum, O. canum, O. gratissimum and O. urticaefolium. The essential oil from O. urticaefolium showed the highest protective effect at 5 ␮l/ml, and was comparable to that of Butylated Hydroxy-toluene (BHT) (100 ␮g/ml) (p ≤ 0.05). The results achieved are promising and supported further studies of the tested essential oils for application to prevent oil oxidation during storage. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Polyunsaturated fatty acids can undergo oxidation by interacting with atmospheric oxygen during all steps from processing to storage. Oxidation usually results in undesirable flavor and toxic end-products (Farag et al., 1989). To avoid oxidation, many antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and Tert-butylhydroquinone (TBHQ) are usually added in the preparations. However, these antioxidants mostly of synthetic origin have shown questionable safety in con-

Abbreviation: FTIR-ATR, attenuated total reflectance-Fourier transformed infrared; BHT, tert-butyl-4-hydroxytoluene; BHA, tert-butyl-4-hydroxyanisole; TBHQ, tert-butylhydroquinone; HNC, National Herbarium of Cameroon; SRF/Cam, Forestry Research Service of Cameroon; FWHM, full width half maximum; SD, standard deviation; sqf, sufficient quantity for. ∗ Corresponding author. E-mail address: [email protected] (J.B.F. Hzounda). http://dx.doi.org/10.1016/j.indcrop.2015.09.077 0926-6690/© 2015 Elsevier B.V. All rights reserved.

sumers (Farag et al., 1989). Although ␣-tocopherol is said to be the most efficient peroxide scavenger of oils, report have revealed that it is less active in vegetable oils (Kamal-Eldin, 2006), emphasizing the urgent need for alternatives that are natural, effective and non-toxic. The flaxseed oil is the richest natural polyunsaturated vegetable oil and also the most susceptible to oxidation (Popa et al., 2012), and given its particular chemical composition, it is a good model to study the effect of potential antioxidant. FTIR-ATR is a non-destructive method that has been previously used to assess the structural change of flaxseed oil (Araújo et al., 2011; Rohman et al., 2011; Stenberg et al., 2005; Van de Voort et al., 1994). However, the protective effect of plant extracts has not yet been reported on these matrices using FTIR-ATR coupled with chemometric analysis. Plants of the genus Ocimum include aromatic species that are mostly found in tropical and subtropical zones of the world. Ocimum basilicum, Ocimum canum and Ocimum gratissimum are widely cultivated and used in Cameroon as spices in food and soups.

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The wide use of O. gratissimum in the preparation of pig had led to the denomination of “pig spice”. The essential oils from these three plants have shown antioxidant activity (Adesegun et al., 2013; Hzounda et al., 2014a; Prabhu et al., 2009; Tamil Selvi et al., 2015). Ocimum urticaefolium was recently described for its antifungal potential (Hzounda et al., 2014a), and it is used in Cameroon for medicinal purposes. In the best of our knowledge, no report has been published on the FTIR-ATR analysis of these essential oils as well as their antioxidant effect on polyunsaturated lipids. In this paper, we report the protective effect of essential oils from O. basilicum, O. canum, O. gratissimum, and O. urticaefolium growing in Cameroon on flaxseed oil oxidation. 2. Material and methods 2.1. Essential oil extraction Fresh leaves of O. basilicum L., O. gratissimum L., and O. canum L. were collected at Nkolomdom II, Yaoundé-Cameroon on August 08th, 2012. O. urticaefolium was collected on the Bamboutos Mountains in a Bamenda neighbourhood (Bali)-Cameroon on August 23rd, 2012. Plants were identified at the National Herbarium of Cameroon, Yaoundé where voucher specimens are deposited with the respective reference numbers 428,782 HNC, 5817/SRF/Cam, 15866/SRF/Cam, and 49,085 HNC. Plant samples were immediately taken to the laboratory after collection for extraction by hydrodistillation using a Clevenger-type apparatus as previously described (Hzounda et al., 2014a).

Fig. 1. Finger print region of Ocimum basilicum essential oil a.u: arbitrary unit.

2.2. Flaxseed oil Flaxseed oil was purchased from a market in Bacau, Romania. This oil contained 0% proteins, carbohydrate and cholesterol; 10% of saturated fatty acids; 18% of monounsaturated fatty acids; and 64% polyunsaturated fatty acids. 2.3. Attenuated total reflectance-Fourier transformed mid-infrared analysis of essential oils The spectra acquisition of the essential oils was performed on Tensor 27 Fourier transformed infrared spectrophotometer. Golden Gate Single Reflection Diamond ATR was used for sampling. The environmental conditions were maintained constant (the temperature at 25 ◦ C and humidity 30%). 10 ␮l of each essential oil was deposited directly on the ATR sampling device without any treatment. The spectra were recorded from 4000 to 550 cm−1 with a resolution of 4 cm−1 and 260 scan for sample and background. The scan velocity was 10 kHz and the interferogram size 14,220 points. Before each essential oil scan, the device was cleaned with 70% ethanol and the background of air was taken. The spectra were acquired with OPUS software and Origin 5.1 software was used for spectra manipulation.

Fig. 2. Finger print region of Ocimum canum essential oil. a.u:arbitrary unit.

2.4. Chemometric analysis The spectra were first smoothened on 15 points using the Savitzky–Golay algorithm and each spectrum was divided into three regions. The first region from 550 cm−1 to 1600 cm−1 , corresponding to the fingerprint region, was submitted to the second derivative and the amplitude of the absorption up to 0.005 arbitrary units (a.u.) was taken into consideration for peaks’ characteristics. The regions from 2500 cm−1 to 3200 cm−1 and from 3200 cm−1 to 3600 cm−1 were decomposed into individual peaks using a Gaussian function (Lazar et al., 2012, 2013). The decomposition was considered good when, at least seven fit converged to the same decomposition. The goodness of the decomposition was assessed

Fig. 3. Finger print region of Ocimum gratissimum essential oil. a.u: arbitrary unit.

through chi-square analyses, the full width half maximum (FWHM) and then the decomposition fit was considered acceptable when the adjusted R2 was equal to 0.99.

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compound. In addition, the peak center from 3030 to 2995 cm−1 was assigned to unconjugated HC CH and that from 3660 to 3066 cm−1 to OO H compound (Mayo et al., 2004; Sadoudi et al., 2014; Stenberg et al., 2005; Stuart, 2004; Stuart and Ando, 1997; Van de Voort et al., 1994). Each of these selected regions was submitted to decomposition using a Gaussian algorithm. Each spectrum from negative control was first analyzed to select the day with the highest change compared to the first day. From this day, the percentage of formation of each of the assigned compounds was obtained using the following formula: %I = (

A0 − Ai ) × 100 A0

(1)

where %I is the percentage of oxidation inhibition, A0 is the area of the negative control at day 0, Ai is the absorbance of the samples at day i.

Fig. 4. Finger print regions of Ocimum urticaefolium essential oils. a.u: arbitrary unit.

2.5. Protective effect on flaxseed oil 15 ml of flaxseed oil were mixed with different volumes of essential oil to make two concentrations (5 ␮l/ml and 0.1 ␮l/ml) and stored in an unlit oven at 60 ◦ C for 15 days. Aliquots were collected on days 0, 3, 6, 9, and 15 for analyses. BHT (100 ␮g/ml and 12.5 ␮g/ml) was used as positive control. 2.6. Spectroscopic analysis of the flaxseed oil deterioration The structural changes were assessed by mid-infrared analysis. To achieve this, 10 ␮l of each sample was placed without any dilution on the ATR eye and three spectra were recorded for each concentration. The range of the infrared scan was the same as for the essential oil analysis, but the number of scans was fixed at 50 rather than 260 for the essential oil. 2.6.1. Chemometric analyses of the flaxseed oil deterioration All the spectra for each day were extracted from the spectra of negative control at day 0, and the resulted spectra were smoothened as for that of the essential oil. The regions were assigned as 769 to 646 cm−1 for cis HC CH , 1066 to 914 cm−1 for trans HC CH , and 1739 to 1724 cm−1 for C O in aldehyde

2.6.2. Conjugated-diene and -triene 300 ␮l of each preparation was collected and dissolved in 3 ml of pure chloroform. After homogenization, all samples were scanned from 500 to 200 nm on ultraviolet–visible spectrophotometer Carry 100 equipped with Varian software. The following formulas were used to calculate the presence of conjugated-diene and conjugatedtriene (Angerosa et al., 2006): K = K226 −

1 2

(K226 + K236 )

for conjugated-diene and K = K262 −

1 2

(K262 + K274 )



(2)

 (3)

for conjugated-triene (Sapino et al., 2005), where Ki is the absorbance at wavelength i. 2.6.3. Peroxide value The peroxide value was measured at day 15 for essential oils and BHT. Briefly, 300 ␮l of each sample were collected and dissolved in a solution of acetic acid-chloroform (3/2) and polysorbate 80 (sqf 5%). After thorough mixing, 50 ␮l of saturated potassium iodide solution were added. The tubes were gently mixed and allowed for reaction for 2 min. 5 ml of distilled water were then added into the tubes followed by vigorous shaking. 50 ␮l of starch (10 g/L) were subsequently added in each tube and mixed again. The prepared solution was titrated with sodium thiosulfate (0.02 N in distilled water). The following formula was used to calculate the peroxide value (IFRA, 2011): PV(mM) = V × C × 50, (4) with V = consumption of 0.02 M sodium thiosulfate solution, and C = molar concentration of sodium thiosulfate solution. 2.7. Statistical analyses The data was statistically analyzed using Kruskal–Wallis test for classification of the spectrum and ANOVA for data comparison using OriginPro 8.5 3. Results and discussion 3.1. Essential oils characterization

Fig. 5. Evolution of the spectrum according to time.

The spectra obtained from the essential oils were grouped on component and the results showed that only one component which explains 68% of the variation was formed with equal correlation coefficient. The following equation was automatically generated

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Table 1 Characteristics of Cis and Trans double bounds of the untreated flaxseed oil. Days

3

6

Type of bound

transa

cisb

transa

cisb

transa

cisb

transa

Wave number ± SEc Area ± SEc SIGMA FWHMd Height Reduced Chi2 Adj R2

986.03 ± 0.86 0.013 ± 0.00 9.17 21.60 5.69E-04 1.53E-11 0.99972

698.56 ± 1.25 −0.002 ± 0.00 3.28 7.73 −2.28E-04 2.56E-09 0.96127

985.45 ± 0.27 0.049 ± 0.00 9.95 23.44 0.00198 2.59E-11 0.99994

704.03 ± 19.66 −0.018 ± 0.00 7.91 18.63 −9.18E-04 4.56E-09 0.98831

991.85 ± 0.7 0.163 ± 0.02 9.09 21.41 0.00715 3.62E-08 0.99669

708.56 ± 0.51 −0.276 ± 0.02 12.99 30.58 -0.00847 3.76E-08 0.9961

984.92 ± 2.93 0.498 ± 0.12 1.48E + 01 34.81 0.01343 5.17E-08 0.99817

a b c d

9

15

vibration of the C H when the double bound is on trans configuration. vibration of C H when the double bound is on cis configuration. Standard error. Full width at half maximum.

by STATGRAPHIC for this component. 0.528324 × O.basilicum + 0.495122 × O.canum + 0.513282 × O.gratissimum + 0.460727 × O.urticaefolium.

The values of the variables (weights or correlation coefficients) in the equation are standardized by subtracting their means and dividing by their standard deviations. This equation revealed that the correlation coefficients from the four essential oils were very close. The formation of one component from the essential oils’ spectra showed consistency given that all the plants are from the same genus. Moreover, with respect to the phylogenetic characterization, the genes encoding the production of the enzymes that actually synthetize the terpenoids present in the chemical profiles of these ´ oils (Hzounda et al., 2014a) are the same (Carovic-Stanko et al., 2010; Nagegowda, 2010; Singh et al., 2004). The analysis of variance of the smoothed spectra revealed that the spectra were significantly different from each other (p = 0.000). This highlights the fact that the essential oils are from plant belonging to the same genus but different species. The characteristic peaks from finger print region are shown in Figs. 1–4 for O. basilicum, O. canum, O. gratissimum and O. urticaefolium respectively.

The principal components of this region lead to two component which can be described by the following equations automatically generated by STATGRAPHIC. Component 1: 0.689228 × O. basilicum + 0.2226 × O. canum + 0.219936 × O. gratissimum + 0.653484 × O. urticaefolium Component 2: −0.0903921 × O. basilicum − 0.798907 × O. canum + 0.567871 × O. gratissimum + 0.17635 × O. urticaefolium The values of the weights in the equation are standardized by subtracting their means and dividing by their standard deviations. The first component is dominated by O. basilicum (0.689228) and O. urticaefolium (0.653484) while the second is dominated by O. canum (−0.798907) and O. gratissimum (0.567871). In fact, the weights (or correlation coefficients) associated to these essential oils in each component are the higher. This finding confirms that all the essential oils are from completely different plants species, as otherwise mentioned by Naumann et al., (2014) who recently used FTIR-ATR to discriminate plants from Solanaceae taxa. These essential oils were not previously analyzed using this approach therefore their chemotypes were determined by assignment of the bands at the fingerprint regions according to the available data from literature (Kulkarni et al., 2013; Movasaghi et al., 2008; Schulz and Baranska, 2007; Schulz et al., 2004, 2005, 2003; Stuart, 2004; Stuart and Ando, 1997). The chemotypes for

Fig. 6. Quantity of trans and cis saturation and unconjugated double bound in presence of different concentration of essential oil and Butylated hydroxytoluene. OB01: Ocimum basilicum at 0.1 ␮l/ml; OB5: Ocimum basilicum at 5 ␮l/ml; OC01: Ocimum canum at 0.1 ␮l/ml; OC5: Ocimum canum at 5 ␮l/ml; OG01: Ocimum gratissimum at 0.1 ␮l/ml; OG5: Ocimum gratissimum at 5 ␮l/ml; OU01: Ocimum urticaefolium at 0.1 ␮l/ml; OU5: Ocimum urticaefolium at 5 ␮l/ml; BHT12.5: butylated hydroxytoluene at 12.5 ␮g/ml; BHT100: butylated hydroxytoluene at 100 ␮g/ml; a, b, c, d, e: statistical difference for trans double bound; ␣, ␤, ␥, ␦, : statistical difference cis double bounds and 1, 2, 3, 4: statistical difference for unconjugated double bound.

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Fig. 7. Spectrophotometric monitoring of conjugated diene and triene. OB01: Ocimum basilicum at 0.1 ␮l/ml; OB5: Ocimum basilicum at 5 ␮l/ml; OC01: Ocimum canum at 0.1 ␮l/ml; OC5: Ocimum canum at 5 ␮l/ml; OG01: Ocimum gratissimum at 0.1 ␮l/ml; OG5: Ocimum gratissimum at 5 ␮l/ml; OU01: Ocimum urticaefolium at 0.1 ␮l/ml; OU5: Ocimum urticaefolium at 5 ␮l/ml; BHT12.5 = butylated hydroxytoluene at 12.5 ␮g/ml; BHT100: butylated hydroxytoluene at 100 ␮g/ml, ␣, ␤, ␥, , ␨: statistical difference at p ≤ 0.05 for conjugated triene and a, b, c, d, e, f: statistical difference at p ≤ 0.05 for conjugated diene.

Fig. 8. Inhibition of Malonyl Dialdehyde formation. OB01: Ocimum basilicum at 0.1 ␮l/ml; OB5: Ocimum basilicum at 5 ␮l/ml; OC01: Ocimum canum at 0.1 ␮l/ml; OC5: Ocimum canum at 5 ␮l/ml; OG01: Ocimum gratissimum at 0.1 ␮l/ml; OG5: Ocimum gratissimum at 5 ␮l/ml; OU01: Ocimum urticaefolium at 0.1 ␮l/ml; OU5: Ocimum urticaefolium at 5 ␮l/ml; BHT12.5: butylated hydroxytoluene at 12.5 ␮g/ml; BHT100: butylated hydroxytoluene at 100 ␮g/ml; a, b, c, d, e, f: statistical difference at p ≤ 0.05.

each essential oil were confirmed based on their previous analyses by GC and GC/MS (Hüe et al., 2015; Hzounda et al., 2014a,b). The clearest bands appeared at 918, 993, 1234, 1267, 1373, and 1514 cm−1 for O. basilicum (Fig. 1). The spectrum contained all the key peaks reported for linalool (Sandasi et al., 2011) and all the major peaks attributed to eugenol (Wang and Sung, 2011) as evidenced in Fig. 1. Therefore, linalool/eugenol was suggested for this essential oil in accordance with previously published findings(Hüe et al., 2015Hü; Hzounda et al., 2014a). The most important bands for O. canum appeared at 843, 887, 984, 1053, 1080, 1167, 1213, 1234, 1306, 1358, 1377, 1441, 1466 cm−1 (Fig. 2). Four of the most clear bands, appearing at 843, 984, 1213 and 1377 cm−1 were attributed to the 1,8-cineol as previously shown for other essential oils (Schulz et al., 2005). The bands at 887, 1053, 1080, 1167, 1234, 1306, 1358, 1441, 1466 cm−1 were not useful for this analysis (Stuart, 2004; Stuart and Ando, 1997), and might correspond to some volatile terpenoids (Schulz and Baranska, 2007; Schulz et al., 2005, 2003). All these observations are consistent with the chemotype obtained by GC and GC/MS (Hüe et al., 2015; Hzounda et al., 2014a).

Table 2 Peroxide vibration in flaxseed oil with and without antioxidant on day 15. Peroxyde vibration Wave number SEk Control OB01a OB5b OC01c OC5d OG01e OG5f OU01g OU5h BHT12,5i BHT100j a b c d e f g h i j k l

3446.49 3397.79 3456.46 3411.86 3431.63 3449.74 3441.77 3429.13 3392.52 3403.25 3403.15

± ± ± ± ± ± ± ± ± ± ±

1.08 4.12 4.92 4.8 2.81 1.2 3.55 11.63 1.46 3.86 2.23

Ocimum basilicum at 0.1 ␮l/ml. Ocimum basilicum at 5 ␮l/ml. Ocimum canum at 0.1 ␮l/ml. Ocimum canum at 5 ␮l/ml. Ocimum gratissimum at 0.1 ␮l/ml. Ocimum gratissimum at 5 ␮l/ml. Ocimum urticaefolium at 0.1 ␮l/ml. Ocimum urticaefolium at 5 ␮l/ml. Butylated hydroxytoluene at 12.5 ␮g/ml. Butylated hydroxytoluene at 100 ␮g/ml. Standard Error. Full width at half maximum.

Area ± SEk 0.56 4.19 1.58 2.05 8.54 0.03 5.72 2.6 17.32 3.34 3.80

± ± ± ± ± ± ± ± ± ± ±

0.05 1.02 0.34 0.07 0.25 0.00 0.24 0.12 0.47 0.64 0.58

SIGMA

FWHMl

Height

Red Chi2

Adj. R2

45.91 80.72 86.61 98.25 104.54 18.44 105.19 96.23 100.02 80.16 84.61

108.12 190.08 203.94 231.36 246.16 43.43 247.72 226.59 235.53 188.76 199.24

0.005 0.021 0.007 0.008 0.033 0.0006 0.022 0.011 0.069 0.016 0.018

4.95E-09 7.45E-08 2.38E-08 1.58E-08 3.31E-07 4.55E-09 1.24E-07 1.49E-08 9.00E-08 4.75E-08 3.58E-08

0.99857 0.999 0.99786 0.9982 0.99801 0.99984 0.9982 0.99915 0.99984 0.99894 0.99928

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Fig. 9. Determination of the peroxide value by titrimetric approach. OB01: Ocimum basilicum at 0.1 ␮l/ml; OB5: Ocimum basilicum at 5 ␮l/ml; OC01: Ocimum canum at 0.1 ␮l/ml; OC5: Ocimum canum at 5 ␮l/ml; OG01: Ocimum gratissimum at 0.1 ␮l/ml; OG5: Ocimum gratissimum at 5 ␮l/ml; OU01: Ocimum urticaefolium at 0.1 ␮l/ml; OU5: Ocimum urticaefolium at 5 ␮l/ml; BHT12.5: butylated hydroxytoluene at 12.5 ␮g/ml; BHT100: butylated hydroxytoluene at 100 ␮g/ml; a, b, c, d, e, f: statistical difference at p ≤ 0.05.

For O. gratissimum (Fig. 3), the most important bands appeared at 586, 781, 810, 945, 1057, 1088, 1155, 1223, 1288, 1381, 1419, 1464, 1514, 1581, 1618 cm−1 . The key band of thymol appeared at 804 cm−1 corresponding to C H wagging (Schulz et al., 2005), but this band can switch to 810 cm−1 ; in addition, the presence of bands at 946, 1088 and 1289 cm−1 is specific for thymol in thymol-type essential oil (Rodríguez-Solana et al., 2014; Schulz et al., 2003). Therefore, the bands at 810, 945, 1087 and 1289 cm−1 were attributed to thymol in O. gratissimum. The bands at 781 cm−1 and 945 cm−1 were attributed to ␥-terpinene, based on previous studies on other essential oils (Schulz and Baranska, 2007; Schulz et al., 2003). The other identified bands were not useful for this analysis. In conclusion, the thymol/␥-terpinene chemotype was suggested for this essential oil as previously described (Hüe et al., 2015; Hzounda et al., 2014a,b). For O. urticaefolium (Fig. 4), the most important bands were obtained at 795, 818, 914, 993, 1036, 1126, 1151, 1180, 1205, 1236, 1267, 1331, 1427, 1464, 1512, 1589, 1639 cm−1 . Also, all the absorption peaks attributed to eugenol (Wang and Sung, 2011) were identified in the spectrum. The bands at 795 (C H wagging), 1371 ( CH3 symmetric deformation), 1427 ( CH2 stretching in ring), 1463 ( C H deformation in ring) and 1639 ( C C stretching) were attributed to ␤-sabinene. This attribution was done according to the literature (Mayo et al., 2004; Movasaghi et al., 2008; Schulz et al., 2005, 2003; Stuart, 2004; Stuart and Ando, 1997) and in correlation with the data from GC and GC/MS. The attribution also took into account the flexibility of the appearance of the band as observed previously for pure thymol and thymol in essential oil (Rodríguez-Solana et al., 2014; Schulz et al., 2003).

3.2. Protective effect on flaxseed oil As shown in Fig. 5, the oils underwent structural modification due to the test conditions. From this spectrum the region from 3660 to 3066 cm−1 was assigned to the peroxide formation; the region from 3030 to 2995 cm−1 to the non-conjugated C H quantification; the region from 1800 to 1675 cm−1 was assigned to the aldehyde formation; the region from 1066 to 914 cm−1 to trans C C quantification and, the region from 769 to 646 cm−1 for the quantification of cis C C. The accelerated oxidation process leads to structural change of the compounds in the flaxseed oil. This observation is consistent with the findings of other authors. In fact, Movasaghi et al. (2008) heating flaxseed oil at 60 ◦ C for 15 days showed a considerable oxidation of flaxseed oil at day 15. Russin et al. (2006) and Araújo et al. (2011) also observed that flaxseed oil undergoes structural

changes such as loss of cis double bond, gain of trans double bond and increase in aldehyde and peroxide formation.

3.2.1. Analysis of cis and trans double bond on untreated flaxseed oil During heating, the flaxseed oil losses its Cis-double bonds as shown in Table 1 by areas of the peaks. As indicated in Table 1, day 15 corresponds to the highest loss in cis double bonds with high appearance of trans double bonds. The results for this day were used for evaluating the effect of the essential oils on flaxseed oil submitted to the accelerated oxidation process. Fig. 6 demonstrates that essential oils and BHT protect flaxseed oil against oxidation (p ≤ 0.05) given the variation in double bounds. Analysis revealed that the least effective oil was that of O. basilicum that exerted an overall not statistically different activity from the negative control (p ≤ 0.05). The most active was the oil of O. urticaefolium as at 5 ␮l/ml this essential oil succeeded to prevent any change in cis double bonds and unconjugated double bonds. At this concentration, this essential oil also inhibited 80% of the formation of trans double bonds. The spectrophotometric method was consistent with this observation only for conjugated triene. In fact, all the essential oils as well as Butyl hydroxyl toluene reduced the formation of conjugated-triene (p ≤ 0.05). All the antioxidants showed a significant difference compared to the negative control, and no statistical difference was observed between O. gratissimum oil at the tested concentration and BHT at 100 ␮g/ml (p ≤ 0.05). O. urticaefolium and O. basilicum at 5 ␮l/ml were statistically comparable to BHT at 12.5 ␮g/ml. For conjugated diene, this method revealed apparent stimulation of the production of conjugated diene by all the essential oils and BHT (Fig. 7). The high appearance of conjugated double bonds in treated flaxseed oil could be due to the conjugated double bonds from the antioxidants themselves. In fact, compounds as Eugenol and buthylated hydroxytoluene play their antioxidant effect by undergoing oxidation themselves and lead to the delocalization of the electron in the ring with formation of conjugated diene (Bondet et al., 1997) and this mechanism can be extrapolated to other phenolic compounds as Thymol. With the exception for O. basilicum oil, all the essential oils showed better effect with regards to conjugated bonds compared to BHT. This observation corroborate previous findings (Movasaghi et al., 2008) where authors showed that Zataria multiflora and Bunium persicum essential oils were most potent than BHT and TBHQ. Also, Rohman et al. (2011) using citric acid as antioxidant to inhibit the production of conjugated-diene and -triene showed low reduction compared to the mixture BHT and BHA.

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3.2.2. Analysis of malonyl-di-aldehyde formation The essential oils and BHT reduced the formation of aldehydetype molecule (P ≤ 0.05) (Fig. 8). At all concentrations, the antioxidant prevented the formation of aldehyde at more than 49% (P ≤ 0.05). O. gratissimum was the most efficient at 5 ␮l/ml as its effect was better than that of BHT at tested concentrations. These essential oils and BHT, reduced the formation of malonyl dialdehyde in the supplemented flaxseed oil. These results are similar to that obtained earlier (Zangiabadi et al., 2012). In fact, this author used thiobarbituric acid and showed that the essential oil from Zataria multiflora and Bunium persicum reduced the formation of malonyl dialdehyde and this effect was comparable to that of BHT. Using spectrophotometric analysis, Farag et al. (1989) also showed that the essential oil from Thymus (another plant from the Lamaceae family) had a good effect in the reduction of malonyldialdehyde. 3.2.3. Analysis of peroxide value The FTIR-ATR analysis of the peroxide bending region revealed an increased formation of peroxide type molecule in the negative control evidenced by the presence of a peak centered at 3446 cm−1 (Table 2). With the exception of O. gratissimum at the tested concentrations in the protected flaxseed oil, there were no peaks near 3444 cm−1 . The peaks at the hydroxyl bending region on the protected oil are summarized in Table 2. Results presented in Table 2 corroborate those obtained with the titrimetric method. From this approach, the essential oils and BHT showed an effect on the peroxide formation on the flaxseed oil at the tested concentrations (P ≤ 0.05). O. urticaefolium followed by O. basilicum both at 5 ␮l/ml and BHT at 100 ␮g/ml were the most active antioxidants (Fig. 9) (P ≤ 0.05). The areas of the peak from the protected oil are proportional to the concentration of the antioxidant, suggesting that the peroxide observed is not from the flaxseed degradation but likely from the added antioxidants. Molecules as eugenol and butylated hydroxyltoluene protect against oxidation by being oxidized to form more stable end-products (Bondet et al., 1997). The stable products can form a peroxide bond, but the geometrical conformation, as well as the presence of the inductor, lead to a switch in the stretching of the peroxide (Mayo et al., 2004). The difference in peroxide values observed between the titrimetric and FTIR-ATR approaches can be explained by the fact that the protocol used is more specific for the hydro-peroxide (R O O H) than for the general peroxide (R O O R) (IFRA, 2011).

4. Conclusion The results obtained from this study highlighted the main components of the essential oils from O. basilicum, O. canum, O. gratissimum and O. urticaefolium from Cameroon using a non destructive FTIR-ATR method. The spectra obtained thereof provide a tool for quality control of these essential oils. The results also revealed that these essential oils have significant protective effect against flaxseed oil accelerated oxidation with comparable profiles to that of butyl hydroxyl toluene. Further studies regarding the toxicity of these essential oils should be carried out in the perspective of their use as natural preservatives.

Acknowledgment The authors thank the “Agence Universitaire de la Francophonie (AUF) for financial support through the Eugene Ionescu Scholarship that allows the realization of this work.

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The authors also thank Ana Maria Macsim-Vasilachi, Madeleine Nina Ngo Mback love and Elena Goldan, for technical help during this work.

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