Chemical variability and antioxidant activity of Cedrus atlantica Manetti essential oils isolated from wood tar and sawdust

2.1 Plant material

The Cedar Wood was collected in the Atlas Mountain of Ifrane region-Morocco (Itzer and Senoual forests), in the month of April 2018. The authentication of the plant material was done by Dr. Guedira Abdelhamid, a botanist at the Forest Research Center of Morocco, and voucher of specimens (CA0418) was added to the herbarium of the Botany Department.

A part of each tree's wood stump was selected and dried at room temperature in the dark. Sawdust was obtained by grinding the parts obtained from the cedar stumps, while the Cedar tar is obtained by carbonization of the stump parts of the tree (the young trunk and branches do not give an oil) using “double pot” process. Although certified since ancient times, the “double pot” process is the most common, in which the wood for distillation is introduced into a pot with a pierced bottom, equipped with a lid, then placed over the opening of another pot, and all the joints are then fought. The whole thing is then placed in a pit; the upper pot must rise above the surface of the ground, around which a fire is lit. Under the effect of heat (final temperature of about 500 °C) and the absence of oxygen, the material to be distilled exudes and the distillate flows inside the lower pot: this is the vegetable tar (Burri, 2010).

2.2 Isolation of essential oils by hydro-distillation

To obtain the essential oil, 200 g of each sample (sawdust and tar) were immersed in 1 L of distilled water, and subjected to a hydrodistillation for about 4 hours using a Clevenger type apparatus (IsoLab Laborgerate, GmbH, Wertheim, Germany) according to the European Pharmacopeia method (European Pharmacopeia, Council of Europe. Strasbourg, 3rd ed. (1997) 121). The EOs extracted were dried under anhydrous sodium sulfate, filtered and finally stored in sealed vials at 4 °C. The yield of EOs (%, v/w) was determined and expressed on a dry weight basis using the following formula: $$YieldofEO\left(\%\right)=\frac{\mathit{volume}\mathit{of}\mathit{EO}\mathit{Obtained}\left(ml\right)}{\mathit{masse}\mathit{of}\mathit{dry}\mathit{matter}\left(g\right)}x100$$

2.3 Gas chromatography-mass spectrometry (GC - MS) analysis

The chromatographic analysis was performed at Hassan-II Agronomic and Veterinary Institute of Morocco. The GC–MS analysis was carried out with a Hewlett-Packard Gas Chromatographer (HP 6890) and a mass spectrometer (HP5973 series), equipped with an HP-5MS (5% phenyl-methyl siloxane) capillary column (30 m × 0.25 mm, film thickness: 0.25 m). The column was used at an injector temperature of 250 °C with the oven temperature programming ranges from 50 to 250 °C, for 5 min, with a gradient of 4° C/min, and then finally kept isothermal for 10 min. Nitrogen was used as the carrier gas at a rate of 1.7 mL/min. Essential oil (1 µl) was injected in a split mode ratio of 1:50. Mass spectra were obtained by electron ionization (EI) at an ionization energy of 70 eV, using a spectral scan range of 40–450 m/z. The ChemStation data analysis software was used to acquire mass spectra and total ions gas chromatography (GC-TIC) profiles. The components characterization was established by determination of their Kovats retention indices (KI) according to Van Den Dool equation (Van Den Dool and Kratz, 1963), and by matching the recorded spectra with a computed data library (Wiley 09, Nist 2011), according to a homologous series of n-alkanes (C₇-C₃₀) (Jaber et al., 2021). This characterization is completed by comparing the fragmentation patterns of mass spectra with those reported in the literature (Adams, 2007).

2.4 Antioxidant activities

2.5 Statistical analysis

Three replicates of each sample were used for statistical analysis, and the results are expressed as mean ± significant difference (±SD). One-way ANOVA and least significant difference (LSD) post hoc Tukeys honest significance test were used for comparing the antioxidant effects of different samples (p < 0.05). The data obtained were subjected to statistical analysis performed with SPSS Statistics 25 software. The Principal Components Analysis, the Hierarchical Clustering Analysis, the Heatmap (realized by ORIGINPRO 2020), was used to comprehend the similarity between the essential oils and different components.

3.1 Essential oils yield

The EO yield of 8.6% and 11% was obtained from the C. atlantica wood tar collected respectively in the Itzer and Senoual forests, which is noticeably higher than that obtained from sawdust of C. atlantica from the same origin with a yield of 3.51% and 5.98% (Table 1). Several studies in the literature have investigated to analyze the cedar wood EO, in which the yields vary greatly depending on the forest source, and the part of the tree used. Indeed, the study reported by Fidah et al. (2016) showed an average yield of sawdust essential oil of 3.4%, which is in accordance with the result of our study. However, no information is available on the characterization of the essential oil obtained from C. atlantica wood tar, except a few studies reported by Nam et al. (2015) and Kılıç et al. (2017), that analyzed tar oil obtained by dry distillation of species of stumps.

The variation in essential oils yield could be attributed to various factors such as the geographical origin and the time of harvest. In fact, photochemistry is considerably affected by the number of hours of light and sunshine that influence the accumulation of some compounds over a specific period of time in response to environmental conditions (Houicher et al., 2018). In addition to these environmental factors, the mechanism of essential oil production, which occurs via secondary metabolism, undergoes variation according to the plant’s genetic constitution (da Silva de Souza et al., 2017). Indeed, the yield of EOs extracted from tars is higher than that of oils extracted from sawdust; this variation can be related to the influence of pyrolysis process parameters such as temperature, heating rate and particle size on the products yield (Crespo et al., 2017). Reactions involved during the pyrolysis process produces condensable vapors and gaseous products, further condensation of these vapors produces bio-oil (Tar). The study reported by Varma et al. (2019) revealed that with increasing reaction temperature (temperature range and/or heating rate), vapor formation increases, therefore, vapor condensation also increases, which result to high bio-oil yield.

3.2 Chemical composition of essential oils tested

Gas chromatography-mass spectrometry analysis of the EOs derived from wood tar and sawdust of C. atlantica revealed the identification of almost 71 compounds. These components respectively represent 95.52%, 94.20%, 89.52% and 95.75% of the total chromatographic peaks. They are mainly composed of sesquiterpenes (24%, 26%, 45.5%, and 42%) and the oxygenated hydrocarbons (24%, 26%, 13.63%, and 9.7%) (Fig. 1) with the predominance of the sesquiterpene hydrocarbon β-himachalene (24–44%) in the different essential oils studied. Table 2 depicts the identified compounds and their relative abundance as well as their Kovats Indices values. EO obtained from the cedar wood (sawdust) (Itzer and Senoual forests) present a good concentration of β-himachalene (27.67–44.23%), the other important compounds were α-himachalene (12.2–16.69%), trans-Cadina-1(6),4-diene (11.27–8.45%), 6-camphenol (4.54–3.16%), cedryl acetate (8.54–4.19%), 11α H-Himachal-4-en-1β-ol (9.42–1.32%). While samples coming from the cedar wood tar in the same forests mainly contain the β-himachalene (24.25–24.05%), α-himachalene (13.76–1.15%), trans-cadina-1(6), 4-diene (7.65–7.87%), 6-camphenol (7.44–8.76%), cis-sabinene hydrate (6.17–5.92%), and methyl-1, 4 cyclohexadiene (9.06–13.56%). A comparative study of the phytochemical profiling of the four essential oils shows a similarity between the different compounds obtained from the wood of C. atlantica. On the other hand, there is heterogeneity between the chemical compounds of these essential oils and those obtained after the thermal conversion of the wood. Sawdust EO from Senoual forest showed almost a two-time higher content of β-himachalene in comparison to other EOs. α-himachalene was lower in wood tar essential oil from Senoual forest followed by three other EOs. In fact, cedryl acetate, 11α H-Himachal-4-en-1β-ol was found only in two EO from sawdust. Cis-Sabinene hydrate was identified only in the essential oils of wood tar. These results were in agreement with previous reports on EOs obtained from Moroccan Cedar Wood (Chalchat et al., 1994; Aberchane et al., 2004; Uehara et al., 2017), which identified approximately forty compounds strongly dominated by α-himachalene (12.74%, 11.6%, 16.92%) and β-himachalene (33.45%, 33.8%, 43.18%). Several reports investigated the chemical composition of cedar wood EO in different countries, including Lebanon (Saab et al., 2005), France (Paoli et al., 2011), Morocco (Uehara et al., 2017), and Algeria (Boudarene et al., 2004), significant chemical variations have been observed in these studies. The chemical composition of the samples from Lebanon and France, in which himachalol and α-pinene have been found as major components, was quite different from those of Moroccan and Algerian sawdust essential oils containing mostly himachalene isomers. It has been reported that the chemical profile of essential oils might vary with the orientation of biosynthesis towards the preferential formation of specific products under the influence of the seasons, the phonologic stages of the plant, the composition of the soil, the time of collection and the geographical origin (Vasconcelos et al., 2020). This qualitative and quantitative variability can be attributed to the genetic properties of the plant, which regulate the biosynthesis of secondary metabolites in correlation with the genetic, epigenetic and genomic properties of the plant's genes or their surrounding regions. This chemical variability is also attributed to the organ used to extract the essential oil, as well as to the reactions involved in the thermal decomposition of the wood, in which important parameters such as reaction temperature, heating rate, and reaction pressure have a significant influence on the compositions of pyrolysis products. (Urbizu-González et al., 2017; Efika et al., 2018; Elhidar et al., 2019; Magris et al., 2019; Rezgui et al., 2020). The study reported by (Chen et al., 2021) revealed that changes in the composition of wood tar in response to increased temperature of the pyrolysis process promoted diversity of some chemical compounds.

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Fig. 1

Percentage of various classes of the chemical compounds of EOs.

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3.3 Heatmap for the major components corresponding to different sawdust and tar essential oils extracted.

All major components of tar and sawdust essential oils from the two locations demonstrated variations in the identifying compounds in each essential oil using the heatmap with dendrogram, which is summarized in Fig. 2. The accentuated element is represented by a light orange color in the profile of the heatmap. Accordingly, β-himachelene, α-himachelene, and trans-cadina-1(6),4-diene peaked at the sawdust EO from Senoual. Furthermore, cedryl acetate and 11αH-himachal-4-en-1β-ol reached its highest percentage at the sawdust EO from Itzer. Methyl-1,4-cyclohexadiene, 6-camphenol, and cis-sabinene hydrate peaked at the tar EO from Senoual forest.

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Fig. 2

Heatmap analysis for the major EOs components.

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3.4 Principal components analysis (PCA) and hierarchical clustering analysis (HCA) for the major components of the four essential oils

PCA and HCA were applied as statistical tools to reveal the chemical variability and the interrelationships between the essential oils (Kalleli et al., 2019). PCA analysis showed the correlation between the compositions of all four-cedar wood EOs studied. The first, second, and third principal components (PC1, PC2, and PC3) explained 71.3%, 22.6%, and 6.1% of the total variance, respectively (Fig. 3b), thus representing 100% of the cumulative variance. The three-dimensional axial system generated by the PCA of the essential oils showed that there are two main groups. Thus α-himachalene, β-himachalene, trans-cadina-1 (6),4-diene, 11αH-himachalene-4-en-1β-ol and cedryl acetate were the principal components that contributed to the clustering of the samples obtained from sawdust EOs. 6-camphenol, cis-sabinene hydrate, and methyl-1,4-cyclohexadiene were the primary components that contributed to the clustering of the samples obtained from tar EOs. The most important difference in the chemical composition of essential oils were found after the thermal decomposition of the wood. This is not surprising because the proportions and composition of the four major pyrolysis products (coal, oils, tars, and gases) vary according to the reaction conditions. Some parameters modify notably, the proportion and the composition of the pyrolysis process of wood, such as the nature of the wood (species), the moisture content of the wood, the size of the species of wood, the heating rate, and the final reaction temperature (Efika et al., 2018; Varma et al., 2019).

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Fig. 3

Multivariate analysis for essential oils: (a) HCA dendrogram; (b) PCA score plot across dimensions of the first three principal components.

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The results obtained by PCA are confirmed by the application of HCA as an unsupervised form recognition method. The dendrogram obtained by HCA, using the group average algorithm (Square Euclidean distance) displayed in Fig. 3a, allowed classifying the studied populations into two main clusters. The first group includes the two EOs from cedar tars, while the populations of cedar sawdust represent the second group.

3.5 The antioxidant capacity

The stable DPPH radical scavenging model is a widely used method for measuring the free radical scavenging property of various samples (Li et al., 2020). The essay is considered when the purple-colored free radical DPPH is reduced to a stable yellow colored diamagnetic molecule due to a reaction with the hydrogen-donating scavenger (Arika et al., 2019). Antioxidant activities are given in Table 3. In all four experiments, with increases in concentration of EO, the scavenging of free radicals of these compounds increased. From the inhibition rate curves, we have graphically deduced the IC₅₀ values of EOs shown in Table 4. The capacity of EO from wood tar (IC₅₀ (EO.Tar.IT) = 0.126 mg/mL, IC₅₀ (EO.Tar.SN) = 0.143 mg/mL) was stronger than that from sawdust (IC₅₀ (EO.Saw.IT) = 16.264 mg/mL, IC₅₀ (EO.Saw.SN) = 15.559 mg/mL). However, the IC₅₀ value for ascorbic acid (standard antioxidant) was 0.084 mg/mL.

In the Ferric ion Reducing Antioxidant Power (FRAP) assay the antioxidant is able to give an electron to the free radicals, which produces the neutralization of the radical. The reducing capacity of the EO was assessed when Fe³⁺ is converted to Fe²⁺. In this experiment, the intensity of Perl’s Prussian blue color was measured at 700 nm, and the higher increase in Prussian blue color intensity revealed a strong antioxidant activity (Ashraf et al., 2020). It was found that the reducing power of the EO increased with increasing concentration (Table 3). EO isolated from wood tar had the highest reducing activity (Lower EC₅₀) (EC₅₀ (EO.Tar.SN) = 0.410 mg/mL; EC₅₀ (EO.Tar.IT) = 0.830 mg/mL), followed by those from sawdust (EC₅₀ (EO.Saw.SN) = 1.99 mg/mL; EC₅₀ (EO.Saw.IT) = 2.219 mg/mL). Ascorbic acid was used as standard (EC₅₀ (Ascorbic acid) = 0.007 mg/mL). This result was also confirmed by the result of the DPPH assay.

To our knowledge, antioxidant activity of C. atlantica EO from tar and sawdust has not been reported previously. However, some researchers have already studied the antioxidant capacity of extracts, EOs and wood tar from aerial parts of C. atlantica Manetti. Belkacem et al. (2021) and Fadel et al. (2016), reported that EO and organic extract of aerial parts of C. atlantica growing in Algeria possess good ferric ion reducing power (EC₅₀ = 0.325 mg/mL), and DPPH radical scavenging activity (IC₅₀ = 0.0013 mg/mL). These values are significantly lower than the IC₅₀ value (15.559 mg/mL) observed in this study, and slightly lower than that reported by Naimi et al. (2015), which investigated the antioxidant activity of the flavonoid extract of C. atlantica with an IC₅₀ value of 0.400 mg/mL. The antioxidant activity of C. atlantica wood tar EO still not evaluated in previous studies, however, a study reported by Skanderi and Chouitah (2020) investigated the antioxidant activity of Atlas cedar tar growing in Algeria, and reported an EC₅₀ value of 0.075 mg/mL.

The observed antioxidant properties in this study could be mainly attributed to phenolic compounds, which enhance the rate of hydrogen transfer to free radicals and the inhibitory strength detected in essential oils (Rezgui et al., 2020). The high antioxidant activity in the tars EOs could be related to the presence of methyl-1, 4-cyclohexadiene of which, the C-H reaction mechanism can be described in part as a process where the C-H hydrogen is transported as a proton to the radical DPPH with its accompanying electron from the π-electron diene system (Foti et al., 2008). The oxygenated monoterpene (6-camphenol) and the monoterpene (cis-sabinene-hydrate) could also be taken into account for the antioxidant activity of EO from wood tar. Studies have recently shown that monoterpenes constitute a large group of antioxidant molecules due to their functional groups (alcohols), and to the conjugated double bonds of the terpene structure. The minor components may also contribute to the activity of EO individually and/or synergistically (Karolina et al., 2019; Zielinska-baljet and Joanna, 2020).

In order to better understand the correlation between the IC₅₀/EC₅₀ and chemical composition of EOs obtained, PCA analysis was carried out using a correlation matrix of all of the major phytochemical compounds and presented in Fig. 4. The two principal components (PC1 and PC2) explaining 93.9% of the total data variance. The points with greater IC₅₀ values (poor antioxidant activity) were observed on the right side of Fig. 4 and associated with α-himachalene, β-himachalene, trans-cadina-1 (6),4-diene, 11αH-himachalene-4-en-1β-ol and cedryl acetate, it can be seen that the antioxidant capacity and these components showed significantly positive correlations with PC1. Points with lower IC₅₀ values (strong antioxidant activity) were observed on the left side of Fig. 4, and were associated with 6-camphenol, cis-sabinene hydrate, and methyl-1,4-cyclohexadiene, and showed significantly negative correlation with PC1.

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Fig. 4

Biplot of first and second principal components from PCA of C. atlantica EOs components in association with the IC₅₀ values.

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