Biochemical response of Cupressus sempervirens to cement dust: Yields and chemical composition of its essential oil

1 Introduction

Terpenes are the most functionally and structurally diverse group of plant secondary metabolites. They are thought to play a pivotal role in essential physiological and biological processes such as respiration, photosynthesis and regulation of growth and development (Phillips et al., 2008). They are also involved in plant–environment interactions by playing roles in pollinator attraction, defence and plant–plant communications (Mahmoud and Croteau, 2002). Because of their odoriferous principles and their intriguing biological activities such as antioxidant, antimicrobial, antifungal, antiviral, anti-inflammatory, anti-carcinogenic and antimalarial among others (Bakkali et al., 2008), terpenes find extensive industrial applications as flavouring agents, topical medicine, perfumes and cosmetics (Aharoni et al., 2005). With regard to extensive demand and commercial interests of essential oils, numerous aromatic species have been exhaustively studied and substantial insight into the chemical composition and the metabolism of their essential oils has been published. Member of the Cupressaceae family are among the potential producers of essential oils and some of them namely Cupressus lusitanica and Cupressus sempervirens have been studied in detail (Cool et al., 1998; De Alwis et al., 2009). The basic constituent of the oil of C. sempervirens is α-pinene, to which are attributed the antioxidant (Wang et al., 2008), antimicrobial (Jiang et al., 2011; Ojeda-Sana et al., 2013), antifungal (Chang et al., 2008), anti-inflammatory (Bae et al., 2012), antimalarial, anticoagulative (Yang et al., 2011), phytotoxic (Abrahim et al., 2003), cytotoxic (Matsuo et al., 2011) and myorelaxant (Lima et al., 2010) properties.

As commonly happens for many species, the yield and chemical composition of the essential oil bearing plants have been shown to be strongly influenced by the genetic (Hosni et al., 2010, 2013) and environmental factors such as salinity, moisture, temperature, light intensity, nutrition and photoperiod (Banchio et al., 2005; Harrathi et al., 2012). However, the effect of air pollution in general and cement dust in particular on the essential oil constitution and production has not received much attention. This “silent threat” is generally associated with disruption of physiological and biochemical processes, particularly those including the biosynthesis of essential oils (Kupcinskiene et al., 2008; Dziri and Hosni, 2012). These authors reported that the exposure to cement dust resulted in profound qualitative and quantitative changes in the chemical composition of the essential oils of Pinus sylvestris and Pinus halepensis needles. From compositional stand point, long term exposure to cement dust was associated with higher concentration of γ-terpinene and caryophyllene oxide in P. sylvestris needles (Kupcinskiene et al., 2008), while a significant decrease in all components of the oil of P. halepensis was observed in the needles collected from plants grown in heavy polluted sites (Dziri and Hosni, 2012). Based on these antecedents, it seems that the impact of cement dust on essential oil constituents depend mostly on plant species.

Keeping in view these aspects, the present study was conducted to assess the effects of cement dust on yields and chemical composition of the essential oil from C. sempervirens foliage. These results should contribute to better understanding of the biochemical response of this species to environmental contamination by cement dust and will help to estimate the suitability of using C. sempervirens in creating green areas around cement factories.

2 Materials and methods

2.1

2.1 Sampling sites

The study area is located at the close vicinity of the cement factory of Bir Mcherga (north-eastern Tunisia) (Fig. 1). The experimental design was adopted from Bačić et al. (1999) and Yilmaz and Zengin (2004). The geographic positions of the polluted sites (site 1, 2) and the unpolluted site are determined with Magellan Pro handheld global positioning system (GPS) and they are as follows: Site 1 situated at the distance of 50 m from the cement factory (latitude 36°, 31′, 1,97″ (N); longitude 10°, 01′, 18,33″ (E); altitude 157.35 m), and site 2 situated at 800 m from the cement factory (latitude 36°, 30′, 43,140″ (N); longitude 10°, 01′, 8,23″ (E); altitude 142.3 m). Environmental monitoring studies show that the main pollutants in the aforementioned sites are CaO, (SO_x), (NO_x), (CO_x) and fluorides (RNEE 2001). These pollutants are emitted in large part by the cement factory and to a less extent by the road traffic. The pH of the soil ranged from 8.7 to 8.4 in site 1 and site 2, respectively. The unpolluted site is situated at 5 km in the east of the cement factory: Ecole Supérieure d′Agriculture de Mograne (latitude 36°, 25′, 38″ (N); longitude 10°, 05′, 41″ (E); altitude 149 m), at the western foot of the Zaghouan mountain. It’s only affected by local emission caused by home furnace and the low intensive road traffic (Dziri and Hosni, 2012). The pH of the soil was 7.6. The climate of Bir Mcherga is mild and continental with hot and dry summer while, it is cold and humid in winter. The most frequent annual wind direction is the east wind.

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Figure 1 Location of the study area (left) and the sampling sites in the polluted zone (right).

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At each study site, leaves of C. sempervirens were collected from 15 trees. To avoid daily variations of the essential oil components, the sampling was done as far as possible in the same time of the day (11 h–13 h). The harvested material was dried at room temperature (20 ± 2 °C) for one week, ground by using a Retsch blender mill (Normandie-Labo, Normandy, France), sifted through 0.5 mm mesh screen to obtain a uniform particle size and subsequently assayed for their essential oil composition.

3 Results and discussion

From the foliage of C. sempervirens, pale yellowish oils were obtained with yields of 1.36%, 1.1% and 0.72% for sites 1, 2 and the control, respectively (Fig. 2). These results suggest that the essential oil were intensively synthesized in dusted plants which can be viewed as an adaptive mechanism to cope with the deleterious effects of cement dusts (Dziri and Hosni, 2012) . At this point, it is to be expected that the higher proportion of carbon was allocated to the synthesis and storage of secondary metabolites presumably involved in the chemical defence particularly against cement dust-induced oxidative stress as previously reported in Schinus areira (Wannaz et al., 2003), Lemna minor, Ceratophyllum submersum, Potamogeton natans (Erdal and Demirtas, 2010) and Pinus halepensis (Dziri and Hosni, 2012).

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Figure 2 Yields (% dw) of essential oils of C. sempervirens leaves collected from unpolluted (control) and polluted sites (site 1 and 2).

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The identified components and their percentages are given in Table 1, where they are listed in order of their elution from the Rtx-1 column. A total of 54 compounds, covering more than 93% of the total integrated GC peak area were identified. Comparison of the analytical data revealed remarkable qualitative and quantitative differences between plants from polluted and unpolluted (control) sites. The most complex composition (54 compounds) was recorded for the oil collected from the heavily polluted site (Site 1), whereas, plant essential oils from site 2 and control site were less complex (15 and 26 compounds for site 2 and control site, respectively). Differences in the number of identified compounds suggest that numerous metabolic pathways were elicited in the secondary metabolism of C. sempervirens under atmospheric pollution caused by cement dusts.

Considering the main chemical classes, the most plentiful compounds were monoterpene hydrocarbons (85.5–95.5%) with α-pinene (48.05–63.75%) and δ-3-carene (17.54–24.43%) being the main components. The former compound was particularly abundant in plants from site 1 while the second compound was abundant in plants from the control site. Remarkable increase in the amount of some monoterpene hydrocarbons such as sabinene, p-cymene and limonene were observed in the plant growing closest to the cement factory. The reciprocal trend was observed for α-thujene, camphene, β-pinene, (E)-β-ocimene, γ-terpinene and p-cymenene.

For oxygenated monoterpenes, the highest contribution of this fraction was found in plants from the unpolluted site (1.95%) while the lowest was observed in plants from site 2 (0.24%). The same trend was also observed for sesquiterpene hydrocarbons which were more abundant in plants from the unpolluted site (2.7%). In contrast, oxygenated sesquiterpenes with spathulenol as the most prominent compounds were particularly abundant in the oils from plants collected in the heavily polluted site 1. Other components mainly 1-octen-3-ol and undecan-2-ol showed the same pattern being more represented in the oils from plants of the polluted sites. In order to shed more light on the chemical variability of the essential oils acquired at different sites (Dziri and Hosni, 2012), a PCA was applied to all identified constituents. The scatter plot, obtained by PCA (Fig. 3a, b) showed a great variability in the essential oil chemical composition, with 100% cumulative variance in the two factorial plans. The first principal component (PC 1), accounting for 66.75% of total variance allows distinguishing the oil from the heavily polluted site (site 1) which is characterized by its relatively higher contents of sesquiterpenes (α-humulene, spathulenol, etc.), diterpene manoyl oxide and other compounds namely 1-octen-3-ol which could be considered as indicators of cement dust pollution. The second PC 2, accounting for further 33.25% allowed clear separation of the oil from site 2, characterized by its higher amounts of monoterpene hydrocarbons (tricyclene, α-pinene, p-cymene, δ-3-carene and limonene) and other components such as undecan-2-ol.

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Figure 3 Principal component analysis (PCA) of A: site of collect and, B: the leaf essential oils of C. sempervirens.

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In general, our results revealed significant changes in qualitative and quantitative chemical composition of the essential oils of C. sempervirens in response to cement dusts. The obvious variations in the percent contribution of group components as well as individual component imply that the biosynthesis and storage of monoterpene hydrocarbons in plants exposed to cement dusts receive relatively high priority (Dziri and Hosni, 2012). Support to this assumption is given by Judzentiene et al. (2007) and Kupcinskiene et al. (2008), who reported that the exposure of Scots pine (P. sylvestris) to cement dusts resulted in increased amount of monoterpene hydrocarbons. They also showed that the magnitude of variations in monoterpene content depends on the distance from the cement factory and the age of plant. A redirection of the secondary metabolism of Aleppo pine (P. halepensis) towards the biosynthesis of monoterpene hydrocarbons in response to cement dust has been recently reported (Dziri and Hosni, 2012). Such metabolic feature can be explained by the fact that the biosynthesis of monoterpenes requires relatively low molecular resources (precursors) and enzymatic activity, which make it energetically feasible compared to that of sesquiterpenes which requires more biological precursors, enzymatic reactions namely oxidations, reductions, condensations, isomerization, cyclizations, etc. (Baby et al., 2010).

In addition to this metabolic scheme, the dynamic accumulation of monoterpenes in dusted plants might be a direct consequence of the inhibition of emission of biogenic volatile compound following the stomatal closure, leading hence to an increased amount of stored monoterpene in the storage cavities of C. sempervirens, considered as a monoterpene-storing species and a stronger emitter of biogenic volatile compounds (Loreto and Schnitzler, 2010).

From a physiological perspective, this chemical changes in the metabolism of terpenes may be interpreted as an attempt to cope with the deleterious effects induced by cement dusts. Confirmation of this assumption is reflected in the opposite changes in the amount of the main components α-pinene and δ-3-carene which has been considered as defensive chemicals against oxidative stress induced by air pollution (Shpak et al., 2007). These authors also reported increased amount of β-pinene, δ-3-carene and terpinolene in P. sylvestris growing in radionuclide contaminated areas, which is in good agreement with our results. The same species when exposed to cement dusts exhibited higher amount of δ-3-carene, γ-terpinene, terpinolene and α-terpinene (Kupcinskiene et al., 2008). The protective mechanism mediated by monoterpene has also been reported in S. areira exposed to gaseous SO₂ (Wannaz et al., 2003). It has been proposed that monoterpenes were involved in stabilizing cell membranes via lipid–lipid, lipid–protein or protein–protein interactions and/or by scavenging the reactive oxygen species (ROS) conferring hence, better protection from environmental constraints (i.e. ozone, atmospheric CO₂, higher temperature, drought, salinity, UV radiations, and air pollution among others) (Hartikainen et al., 2009; Holopainen, 2011).

However, because of the extremely complicated environmental situations in the area surrounding the cement factory (high CO₂ concentration, high concentration of SO_x and NO_x, alkaline soils etc.), it is not always clear which pollutant induces changes in the chemical composition of the essential oil. Nevertheless, literature data could provide key elements to explain at least partly the results obtained herein. For example, Räisänen et al. (2008) demonstrated that the long term exposure to elevated CO₂ concentrations and higher temperatures resulted in remarkable increase of α-pinene and decrease of δ-3-carene in P. sylvestris. Similar results were also found one year earlier in the same species growing in radionuclides contaminated areas (Shpak et al., 2007). They attributed this trend to the increased reactivity of δ-3-carene in oxidative processes, which increase with increasing contamination. Growing under elevated ozone (O₃), plant of European aspen (Populus tremula) showed increased amount of α-pinene (Hartikainen et al., 2009). Three years later, the same authors have evaluated the impact of elevated temperature and ozone on the emission of volatile organic compounds (VOCs) in silver birch (Betula pendula) and have pinpointed that the increased emission of VOCs in response to elevated ozone was the direct consequence of the enhancement of the transcription of genes encoding terpenes synthesis namely 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and isopentenyl diphosphate (IPP) isomerase (Hartikainen et al., 2012). In contrast, the emission of monoterpenes was completely repressed when the ozone concentrations surpass the threshold required (150–300 ppm) (Loreto and Schnitzler, 2010).

Taken together, it appears that the response of a plant to contamination depends on the plant species, nature of pollutant and the residence time of the pollutant.

4 Conclusions

This is the first study concerning the impact of cement dust on the yield and chemical composition of the essential oil of C. sempervirens. Our results clearly indicate that cement dust induced a remarkable increase in the yields of the essential oils and induces qualitative and quantitative changes in their chemical composition. The cement dust related increase in the yield is potentially associated with increase of the monoterpene, particularly α-pinene content. In addition, from an ecological point of view, C. sempervirens could represent an excellent alternative for the creation of green areas around cement factory due to its relative tolerance to air pollution caused by cement dust. From a commercial standpoint, these results may be significant given the economic importance of monoterpenes for the fragrance, flavour and pharmaceutical industries.