**2.4 Results and discussion**

*Essential Oils - Oils of Nature*

**2. Material and methods**

**2.2 Material used for bioassay**

Laboratory tests showed that *T. articulata* trunk wood and its root burl are durable

Trunk wood and root burl samples of *T. articulata* were collected from sweepings of craft processing workshops in Khemisset Region (central plate of Morocco), while samples of *C. atlantica* sawdust were collected from wood sawmill in the region of Azrou (Middle Atlas Mountains of Morocco). Sawdust was then sieved into particles of 1 mm size and triplicate samples of 250 g were subjected to hydro-

The chemical analysis and component identification of EOs were performed by gas chromatography (GC-FID) and by gas chromatography coupled with a mass spectroscopy (GC-MS). The identification of EO components was achieved by comparison of their retention indices (RI) relative to (C8-C22) *n*-alkanes with those of known compounds, and by comparison of similar mass spectra using Wiley/NBS mass spectral library of the GC-MS data system and other published mass spectra [19]. The percentage compositions of samples were calculated according to the area

Bioactivity of EOs extracted from *T. articulata* and *C. atlantica* woods was assessed in bioassay against the following wood-decaying basidiomycetes fungi: *Gloeophyllum trabeum* (BAM Ebw.109 strain), *Oligoporus placenta* (FPRL. 280 strain), *Coniophora puteana* (BAM Ebw. 15 strain), and *Trametes versicolor* (CTB 863 A strain). Fungi strains originated from the mycological collection of the Laboratory of Botany, Mycology and Environment, Faculty of Sciences in Rabat,

Bioassays of EOs were performed by direct contact of fungi strains on agar medium according to the method reported by Remmal et al. [20]. Oils were first diluted in a sterile solution of tap water-agar at 0.2% in order to obtain a homogeneous mixture, then distributed in test tubes containing 13.5 ml of sterilized malt-agar medium (20 g/l malt extract and 15 g/l agar), and kept at 45°C in a water bath. To obtain the final oil concentrations in the culture medium ranging from 1/250 to 1/5000 v/v, aseptic volumes of 1.5 ml of different dilutions were then added to those tubes before pouring EO-medium mixtures into Petri dishes. Additional control dishes containing only 13.5 ml of culture medium and agar

A lot of waste as slabs and sawdust results from wood processing of those species timbers. However, only sawdust accounts for about 8% of *C. atlantica* sawn timber that contains appreciable amounts of extractives [9–12]. Many recent works highlighted that essential oils (EOs) extracted from *T. articulata* and *C. atlantica* woods possess numerous biocidal activities [12–15]. Nevertheless, only few attempts to investigate the capability of EOs to protect woods against fungi decay have been previously undertaken [16–18]. The relationship between natural durability of *T. articulata* and *C. atlantica* woods and the bioactivity of their EOs was not yet established. Therefore, the present study is devoted to investigate this relationship.

against wood-decaying fungi [5, 6] as well as *C. atlantica* heartwood [6–8].

**2.1 Material used for EOs extraction and chemical analysis**

distillation for 4 hours to obtain pure essential oils (EOs).

of the chromatographic peaks using the total ion current.

**218**

Morocco.

**2.3 Experimental**

According to the bioassay conducted on oils extracted from *T. articulata* and *C. atlantica* woods, a significant inhibitory effect on the four tested wood-decaying fungi was observed (**Table 1**) with different levels of inhibition. *T. articulata* root burl wood EOs showed, however, a strong inhibitory action against those fungi strains with oil dilutions over 1/4000 v/v. *G. trabeum* fungus was the most sensitive to the inhibitory effect of this essential oil since it was inhibited by concentrations between 1/5000 for *T. articulata* root burl oil and 1/1000 v/v for Atlas cedar oil. *O. placenta* was the most resistant strain since its growth inhibition was not reached until 1/400 concentrations for *C. atlantica* oil and 1/800 for *T. articulata* trunk wood oil (**Table 1**).

Previous studies by our team [5, 8] showed that *T. articulata* and *C. atlantica* woods were classified as very durable to durable (DC 1 and 2) and means of mass loss of test specimens was below 5.20% compared to those of Scot pine wood (control) (40.70%). According to their durability indexes (*X*) determined by NF EN 350-1 and CEN/TS 15083-1 standards [22, 23] and the biological risks defined by EN 335-2 standard [24], natural durability levels of those woods against wooddecaying fungi allow them to access high-risk classes of biological attacks 4 and 5 for an end-use without preservative treatment regarding decay fungi [25, 26]. Compared to similar studies on Moroccan coniferous woods (**Figure 1**), the natural durability of native Atlas cedar wood is similar to that of *C. atlantica* heartwood (DC 1 and 2) originated from a south Italian plantation [7], whereas *Pinus halepensis* and *P. pinaster* woods were considered as less durable (DC 4) [6, 27]. Generally, pine woods contain less active extractives than those of *Cupressaceae*. Adamopoulos et al. [28] reported that the weakness of natural durability of both heartwood and sapwood of *Pinus leucodermis* is related to low presence of bioactive extractives that can inhibit the brown-rot fungus, *Coniophora puteana*.

In addition, other works by our team [8, 29] revealed that EOs of *C. atlantica* wood is dominated by ketones (52.05%) and alcohols (26.58%), while those of thuya are dominated by alcohols (about 55–78%) and sesquiterpenes (13–22%) (**Table 2**). Major components of *C. atlantica* oil are, respectively, E-*γ*-atlantone, E-*α*-atlantone, 5-isocedranol, 9-iso-thujopsanone, cedranone, Z-*α*-atlantone, cedroxyde, and 14-hydroxy-*δ*-cadinene [8] (**Table 3**).


#### **Table 1.**

*Minimal inhibitory concentrations (MIC) (v/v) determined for essential oils of thuya and Atlas cedar woods by bioassay conducted on malt-agar medium on wood-decaying fungi.*

**Figure 1.** *Natural durability of studied woods against wood-decaying fungi.*


#### **Table 2.**

*Chemical groups (in%) identified, by GC-MS, in essential oils of Tetraclinis articulata and Cedrus atlantica woods.*

EOs of *T. articulata* woods are rich in thymol, 3-tert-butyl-4-methoxyphenol, cedrol, and *α*-cedrene (**Table 3**). The oil of *T. articulata* trunk wood contains phenols such as *α*- and *β*-acorenol, cedrol, and totarol, along with terpenes as *α*-cedrene that can protect this wood from fungi decay. The strong inhibitory effect of *T. articulata* root burl oil is probably due to its oxygenated fraction rich in phenols as thymol, 3-tert-butyl-4-methoxyphenol, and cedrol. A previous work already highlighted this significant antibacterial activity at low concentrations [30]. Abundance of tropolones and phenols in *Cupressaceae* woods may explain their high natural durability grades against wood-decaying fungi as reported by Haluk and Roussel [16] and by more recent works [13, 30, 31]. Early investigations found that thujaplicines (tropolones) of *Thuja plicata* possesses strong inhibitory effect against the wood blue stain fungi and many wooddecaying fungi [32].

Natural durability of *C. atlantica* wood can be correlated to bioactivity of its oils essentially rich in sesquiterpene ketones as atlantones [9, 14]. In the literature,

**221**

**Table 3.**

*atlantica woods.*

*Durable Woods and Antifungal Activity of Their Essential Oils: Case of* Tetraclinis articulata*…*

**Chemical group Component KI TTW TRB ACW** Alcohols Thymol 1290 8.20 39.80 —

Esters Hexyl isobutyrate 1150 — — 1.38

Ketones Camphor 1143 0.11 — 1.28

Oxides italicene oxide 1538 0.91 0.59 —

Terpenes *α*-Cedrene 1409 17.59 7.77 —

Deodarone (Dihydro-2,2,6 trimethyl-6-(4-methyl-3 cyclohexen-1-yl)-2H-pyran-3(4H)-

one)

3-Tert-butyl-4-methoxyphenol 1491 16.50 24.70 — Turmerol 1578 — — 3.45 Cedrol 1596 14.80 6.35 1.90 *α*-Acorenol 1633 4.20 1.10 *β*-Acorenol 1637 4.15 1.30 — Himachalol 1647 — 0.30 2.45 5-Isocedranol 1669 — — 11.70 *β*-Santalol 1741 — — 2.00 E-Z-Farnesol 1742 — — 1.10 Totarol 2314 2.60 1.50 —

Benzyl benzoate 1762 — — 1.16 Z-*β*-Santalol acetate 1823 — — 1.15 Z-Ternine 1838 — — 1.25

Cedranone 1620 — — 4.13 9-Iso-thujopsanone 1637 — — 4.45

E *γ*-Atlantone 1701 — — 19.73 Z *α*-Atlantone 1713 — — 4.02 E *α*-Atlantone 1773 0.74 — 16.86

Cedroxide 1704 — — 2.38

*β*-Cedrene 1434 4.00 1.80 *α*-Himachalene 1447 — 1.07 — Thujupsadiene 1462 — 1.10 *α*-Acoradiene 1464 2.33 — *β*-Acoradiene 1465 2.26 — *β*-Himachalene 1499 — 1.25 0.79 *α*-Dehydro-ar-himachalene 1511 — — 1.13

*γ*-Cadinene 1513 1.18 0.31

*KI, Kovàts Index; TTW, Thuya Trunk Wood; TRB, Thuya Root Burl; ACW, Atlas Cedar Wood.*

*The main components (in%) identified, by GC-MS, in essential oils of Tetraclinis articulata and Cedrus* 

*γ*-Dehydro-ar-himachalene 1529 — — 1.57 14 Hydroxy-murolene 1775 — — 1.00 14 Hydroxy *δ*-cadinene 1799 1.21 0.24 1.94

1694 — — 1.07

*DOI: http://dx.doi.org/10.5772/intechopen.87214*


*Durable Woods and Antifungal Activity of Their Essential Oils: Case of* Tetraclinis articulata*… DOI: http://dx.doi.org/10.5772/intechopen.87214*

#### **Table 3.**

*Essential Oils - Oils of Nature*

**Figure 1.**

**Table 2.**

*woods.*

*Natural durability of studied woods against wood-decaying fungi.*

**220**

decaying fungi [32].

EOs of *T. articulata* woods are rich in thymol, 3-tert-butyl-4-methoxyphenol, cedrol, and *α*-cedrene (**Table 3**). The oil of *T. articulata* trunk wood contains phenols such as *α*- and *β*-acorenol, cedrol, and totarol, along with terpenes as *α*-cedrene that can protect this wood from fungi decay. The strong inhibitory effect of *T. articulata* root burl oil is probably due to its oxygenated fraction rich in phenols as thymol, 3-tert-butyl-4-methoxyphenol, and cedrol. A previous work already highlighted this significant antibacterial activity at low concentrations [30]. Abundance of tropolones and phenols in *Cupressaceae* woods may explain their high natural durability grades against wood-decaying fungi as reported by Haluk and Roussel [16] and by more recent works [13, 30, 31]. Early investigations found that thujaplicines (tropolones) of *Thuja plicata* possesses strong inhibitory effect against the wood blue stain fungi and many wood-

*Chemical groups (in%) identified, by GC-MS, in essential oils of Tetraclinis articulata and Cedrus atlantica* 

**Chemical group Thuya trunk wood Thuya root burl Atlas cedar wood** Alcohols 54.80 78.50 26.60 Esters — — 5.70 Ketones 1.40 0.30 52.00 Oxydes 0.90 0.60 3.40 Terpenes 31.20 14.10 8.00 Others 1.75 1.15 — Global 90.04 94.65 95.69

Natural durability of *C. atlantica* wood can be correlated to bioactivity of its oils essentially rich in sesquiterpene ketones as atlantones [9, 14]. In the literature,

*The main components (in%) identified, by GC-MS, in essential oils of Tetraclinis articulata and Cedrus atlantica woods.*

African padauk (*Pterocarpus soyauxii*) medium-heavy heartwood manifests a remarkable decay resistance attributed to its specific extractive compounds [33]. Moreover, extractive compounds obtained from black locust heartwood were able to increase the native durability of European beech against wood-decaying fungi from class 5 (not durable) to class 3 (moderately durable) [34].

The strong antifungal activity of *T. articulata* root burl wood EOs is probably related to their alcohol fraction, rich in thymol and 3-tert-butyl-4-methoxyphenol, which confer them this significant bioactivity. The combined action of two phenolic compounds, such as thymol and carvacrol, was previously reported [17, 35]. Action of phenolic compounds on fungi is primarily based on the inhibition of fungal enzymes containing SH group in their active site [36, 37]. The antifungal activity of the EOs of *C. atlantica* wood can be correlated to its sesquiterpene ketones, which are mainly atlantones (about 40.61%). Bioassays conducted with pure *α*-atlantones extracted from *Decalepis hamiltonii* revealed great inhibitory activity against pests and molds [38]. In our study, alcohols present in *C. atlantica* oil in significant amount (more than 26%), such as isocedranol, tumerol, himachalol, and cedrol, may also be involved in the inhibitory effect of this oil. Other compounds such as cadinenes (monoterpene hydrocarbon) could also have a great antimicrobial property [39]. However, synergistic action of two or more components of essential oils extracted from thuya and Atlas cedar woods can also be involved in the observed bioactivity reported in our study.

Antifungal activity of several thyme species oils was recently tested successfully against wood-decaying fungi especially those of *Thymus bleicherianus* [17]. Furthermore, EOs of *T. articulata* root burl showed an antibacterial activity two to six times greater compared to that of reference antibiotic and were more effective on *Staphylococcus aureus* (Gram+ ) and *Escherichia coli* (Gram<sup>−</sup>) with significant bacteriostatic and bactericidal effects [40]. Regarding inhibition mechanisms of active compounds of EOs, it was reported that volatile alcohols can act on both lytic and synthetic enzyme pathways and growth inhibition consequently occurred after breaking off natural extension hyphae of fungi [41, 42].

Active compounds contained in oils of *T. articulata* and *C. atlantica* woods, which were successfully tested against wood-decaying fungi, may protect and ensure good natural durability levels for these woods ranging from very durable (DC1) to durable (DC2) classes.

Findings of this study could allow us to consider recovering wastes from *T. articulata* and *C. atlantica* wood processing for the extraction of bioactive oils and use them as biocide especially in preservative treatment of less-durable woods such as those of pines. Also, these results are in favor of more protection for these threatened species and for the rehabilitation in their natural environment. Natural compounds of *T. articulata* and *C. atlantica* oils can then replace the use of current petrochemical compounds that are becoming more and more criticized for their harmful effect on human health and the environment.

### **3. Conclusion**

The present study has clarified the relationship between the natural durability of *T. articulata* and *C. atlantica* woods and the antifungal activity of their EOs. According to the bioassay conducted on those oils, a significant inhibitory activity was obtained on the four wood-rotting fungi tested, mainly for oils extracted from *T. articulata* root burl. Stronger inhibitory effect was then reached by dilutions over 1/5000 v/v for this oil rich in phenols. The durability classes of *T. articulata*

**223**

**Author details**

Abdelwahed Fidah1

and Abderrahim Famiri1

1 Forest Research Center, Rabat, Morocco

provided the original work is properly cited.

*Durable Woods and Antifungal Activity of Their Essential Oils: Case of* Tetraclinis articulata*…*

and *C. atlantica* woods positively correlated with bioactivity of their oils against the three wood-decaying fungi specified by the CEN/TS 15083-1 standard. Active EOs extracted from wastes of these woods can be valorized as wood-preservative

This project is supported by Forest Research Center in Rabat (High Commission

for Water and Forests and Fight against Desertification) in collaboration with

Faculty of Sciences in Rabat (Mohammed V University), Morocco.

\*, Mohamed Rahouti<sup>2</sup>

2 Faculty of Sciences, Mohammed V University in Rabat, Morocco

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: fidah.abdelwahed@gmail.com

, Bousselham Kabouchi<sup>2</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.87214*

treatment.

**Acknowledgements**

*Durable Woods and Antifungal Activity of Their Essential Oils: Case of* Tetraclinis articulata*… DOI: http://dx.doi.org/10.5772/intechopen.87214*

and *C. atlantica* woods positively correlated with bioactivity of their oils against the three wood-decaying fungi specified by the CEN/TS 15083-1 standard. Active EOs extracted from wastes of these woods can be valorized as wood-preservative treatment.
