Chemical composition variations, allelopathic, and antioxidant activities of Symphyotrichum squamatum (Spreng.) Nesom essential oils growing in heterogeneous habitats

2.1 Plant materials

The aerial parts of S. squamatum were collected during March (flowering stage) from two different habitats in Egypt. The first sample was collected from the Mediterranean coastal belt at Gamsa City, Al-Dakahlia Governorate, Egypt (31°26′35.4″N 31°33′35.5″E), and was called coastal sample. The second sample was collected at the same time from an abandoned area near Mansoura University, Mansoura, Egypt (31°02′22.2″N 31°21′09.7″E). This location is in the middle of the Nile Delta and was called inland sample. The collected samples were cleaned from dust and dried at room temperature (25 °C ± 2). The dried plant materials were ground into a fine powder using a grinder (IKA® MF 10 Basic Microfine Grinder Drive, Breisgau, Germany) and stored in paper bags until further analyses. Voucher specimen with code: Mans.0010272703 was added in the herbarium of Botany Department, Faculty of Science, Mansoura University, Egypt.

2.2 Extraction and analysis of the EOs

The EOs were separately extracted from 300 g of aerial parts of S. squamatum from each sample/habitat (coastal and inland) immediately after preparation by hydrodistillation using a Clevenger-type apparatus for three h. The oil layer of the oil was separated using diethyl ether and dried with anhydrous sodium sulfate (0.5 g). This extraction was repeated twice afforded two samples of EOs for each location. The extracted EOs were stored in sealed air-tight glass vials at 4 °C until further analysis.

The EOs components of the extracted samples were analyzed separately and identified following GC-MS analysis. The GC-MS analysis was performed via chromatography-mass spectrometry instrument at the Department of Medicinal and Aromatic Plants Research, National Research Center, Egypt. The GC-MS systems consist of TRACE GC Ultra Gas Chromatographs (THERMO Scientific Corp., USA), accompanied by thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer; Model ISQ spectrometer). The GC-MS system contained a TR-5 MS column with a dimension of 30 m × 0.32 mm ID × 0.25 μm. Helium gas was used as a carrier gas with a flow rate of 1.0 mL min⁻¹ and a split ratio of 1:10. The temperature cycle was as follows: 60 °C for one min and then rising at 4.0 °C per min until 240 °C and held for one min. The detector and injector were held at 210 °C. An aliquot of one μL of the diluted samples (1:10 hexane, v/v) was always injected. Mass spectra were derived by electron ionization (EI) at 70 eV, using a spectral range of m/z 40–450.

2.3 Identification of EOs constituents

The chemical compounds were identified according to their retention indices (relative to n-alkanes C₈-C₂₂), comparison with the authentic constituents available in our laboratories, and comparison of their mass spectra with that of Wiley spectral libraries databases (NIST AMDIS). The compound percentage was calculated based on the peak area derived from gas chromatography.

2.4 Antioxidant activity

The antioxidant scavenging activity of the extracted EOs from coastal and inland samples was evaluated according to their ability to scavenge the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical. In accordance with Sharma and Bhat (2009), a reaction mixture of two mL of DPPH (0.15 mM) and equal amount of various concentrations (50, 100, 200, 300, 400, 500, and 600 μL L⁻¹) of either EOs, in methanol, or ascorbic acid (standard antioxidant) was prepared in test tubes. The test tubes were shaken vigorously and incubated in dark condition at 25 °C for 30 min. The absorbance was measured by a spectrophotometer (Spectronic® 21D model) at 517 nm. The antioxidant scavenging activity was expressed in percentage as follows: $$\mathrm{R}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\%=[1-(\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}{\mathrm{e}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}/\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}{\mathrm{e}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}})]\times 100$$

The IC₅₀ (the concentration of the EOs required to scavenge 50% of the initial concentration of DPPH) was also calculated graphically.

2.5 Allelopathic bioassay

The allelopathic activity of the extracted EOs was evaluated against B. pilosa as one of the dangerous noxious weeds. Ripe seeds of B. pilosa weed were collected in June from the garden of Mansoura University, Mansoura, Egypt (30°38′33.5″N 31°59′50.4″E). The collected seeds were surface sterilized by 0.3% NaOCl for three min. To examine the allelopathic effect of the EOs, various concentrations (100, 200, 400, 600, 800, and 1000 µL L⁻¹) were prepared in Tween® 80 (Sigma–Aldrich, Germany) as an emulsifier. Twenty B. pilosa seeds were placed in Petri dishes (9 cm) lined with a filter paper Whatman No. 1. and five mL of each EOs concentration or tween (as control treatment) was added. The Petri dishes were sealed with Parafilm® tape and incubated in a growth chamber at 27 °C light regime conditions of 16 h/8h light/dark for five days (Abd El-Gawad, 2016). The shoot and root lengths of all seedlings per each plate were measured, and the allelopathic inhibition of root and shoot growth was calculated, with respect to control, as follows: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=100\times \frac{(\mathrm{N}\mathrm{o}/\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{N}\mathrm{o}/\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t})}{\mathrm{N}\mathrm{o}/\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}$$

2.6 Statistical analysis

The data of the allelopathic bioassay of the EOs were expressed as the percentage of inhibition with respect to control and were subjected to Generalized Linear Models (GLM) analysis, using the STATISTICA (version 7) software system (StatSoft, Inc. Tulsa, Oklahoma, USA, www.statsoft.com). The data of antioxidant, in triplicates, were subjected to one-way ANOVA followed by Duncan's test at probability level 0.05 using CoStat (version 6.311, CoHort Software, USA, www.cohort.com). The data of the EOs composition derived from GC-MS analysis of the coastal and inland samples from the Egyptian ecospecies as well as the data of the EOs reported from the Japanese and Turkish ecospecies were subjected to Pearson similarity correlation. The dataset consists of the concentrations (%) of 99 chemical compounds that were identified in the three investigated ecospecies. Also, the dataset was submitted to principal component analysis (PCA) to determine whether a significant difference exists between different ecospecies, based on the EOs composition. The software XLSTAT (version 2018, Addinsoft, NY, USA, www.xlstat.com) was used in the analysis of Pearson correlation and PCA.

3.1 Chemical constituents of S. squamatum EOs

The hydro-distillation extraction of the aerial plant parts of coastal and inland S. squamatum yielded 0.017% and 0.014% (v/w) of the EOs, respectively. The Japanese ecospecies of S. squamatum yielded more EOs (0.03%) than that of the Egyptian ecospecies (Miyazawa and Kameoka, 1977). The yield was more pronounced in the coastal sample, where the habitat is affected by salinity from the Mediterranean Sea. Bourgou et al. (2010) reported an increase of the EOs yield in Nigella sativa by increasing the salinity.

An overall of sixty compounds of EOs from both coastal and inland samples, representing 100% of the total mass, were characterized based on GC-MS analysis. Forty-six compounds were identified from the coastal sample and forty-nine from the inland sample of S. squamatum (Table 1). Among the identified compounds, sesquiterpenes represented the main component comprising 69.77% and 88.68% of the total EOs from the coastal and inland sample, respectively (Fig. 1). Sesquiterpene hydrocarbons were characterized as major components of EOs of the inland sample (47.39%), while the EOs of the coastal sample has 27.13% as sesquiterpene hydrocarbons of the total mass. On the other hand, the oxygenated sesquiterpenes were the major components in the coastal sample (61.55%) while they were represented by 22.38% in the inland sample (Fig. 1). These findings were in consonance with that of the Turkish ecospecies which attained high content of sesquiterpenes in its EOs (Ayaz et al., 2017). It is pertinent to mention here that sesquiterpenes have been reported as dominant group of the EOs of other related species of Aster such as A. albanicus (Nemanja et al., 2015), A. handelii (Xiao-ping and Xiaoping, 2006), A. lanceolatus (Dias et al., 2009), and A. tataricus (Choi, 2012).

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Fig. 1

Percentage of various classes of the chemical compounds of EOs from coastal and inland samples of Egyptian S. squamatum as well as Japanese (Miyazawa and Kameoka, 1977) and Turkish ecospecies (Ayaz et al., 2017).

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Contrarily, the EOs of the Japanese ecospecies was characterized by the dominance of monoterpenes (Miyazawa and Kameoka, 1977) which represented 84.88% of the total mass. Also, our results showed that the Egyptian ecospecies have a relatively high content of monoterpenes, but the coastal habitat sample attained higher content than that of the inland sample. Moreover, other species of Aster have been reported to contain monoterpenes as the major class of their EOs such as A. ageratoides (Miyazawa et al., 2008), A. poliothamnus (Tu et al., 2006), A. spathulifolius (Kim et al., 2014a), and A. scaber (Lee et al., 2012). In the present study, diterpenoids and hydrocarbons were determined as minor classes of the EOs in both inland and coastal samples.

In the EOs of the coastal sample, humulene epoxide, (-)-spathulenol, (-)-caryophyllene oxide, germacrene D, and α-humulene were identified as major compounds representing 59.72% of the EOs total mass. However, β-pinene, germacrene D, α-humulene, α-muurolene, humulene epoxide, (-)-caryophyllene oxide, and β-cadinene were the major compounds of the inland sample of the Egyptian S. squamatum ecospecies, where they represented 63.70% of its EOs (Table 1). Other minor compounds are listed in detail in Table 1.

In a general overview of the chemical composition of EOs of the two samples, we observed that the two samples include most of the components with different percentages. All the major components of the two samples especially germacrene D, β-pinene, spathulenol, and caryophyllene oxide were already reported as major compounds in the EOs of other species of Aster such as A. subulatus, A. albanicus, A. spathulifolius and A. lanceolatus (Ayaz et al., 2017; Dias et al., 2009; Kim et al., 2014a; Nemanja et al., 2015).

3.2 Correlation between S. squamatum ecospecies based on EOs composition

The analysis of PCA showed that the two samples of the Egyptian S. squamatum, as well as the Turkish and Japanese plants, were more comparable in the composition of the EOs (Fig. 2). Specifically, more correlation was found between the Egyptian inland S. squamatum of the present study and the Japanese ecospecies. These two samples were also correlated to the β-pinene, D-limonene, β-cadinene, and trans-caryophyllene. Also, β-pinene represented 40.9% and 24.2% of the total EOs from the Japanese and Egyptian inland S. squamatum, respectively.

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Fig. 2

Principal component analysis (PCA) based on the chemical composition of the EO derived from Egyptian ecospecies of S. squamatum collected from Mediterranean coastal desert (coastal) and inland abandoned habitat (inland) as well as Turkish and Japanese ecospecies.

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On the other hand, the other two samples (Egyptian coastal and Turkish S. squamatum) were more correlated to each other, and showed a close relation to elemol, (-)-caryophyllene oxide, (-)-spathulenol, humulene epoxide, α-humulene, α-muurolene, and germacrene D. This correlation could be ascribed to the similarity in the climate of both geographical regions of the Mediterranean climate (Ayaz et al., 2017). The formation of EOs in plants is profoundly affected by climatic conditions, particularly temperature, solar radiation, day length, and water supply (Baser and Buchbauer, 2015). Moreover, the variation in the EOs can be attributed to the habitat structure, population genetics, soil type, and stress condition (Abd El-Gawad, 2016; Khazaie et al., 2008).

Pearson correlation coefficient analysis revealed a significant positive correlation between the inland sample and the Japanese ecospecies (0.786) as well as the coastal sample (0.376) (Table S1). However, the Japanese ecospecies showed a negative correlation with the Turkish ecospecies (−0.023).

Generally, the rest of major compounds didn’t show a specific correlation to any samples (Fig. 2). The present data revealed that the chemotypes of S. squamatum maintain their typical pattern despite ecological or climatic differences. Environmental factors induce the production of specific chemicals pattern by activation of the gene expression. This means that the potential to produce bioactive compounds is genetically coded (Franz, 1993).

On the other hand, our results of soil analysis revealed a significant variation in the organic carbon and salinity between the two locations (Table S2). Salinity stress is one of the most important factors that can affect the composition of EOs in plants. Changes in the composition of the EOs in response to salinity have been reported in various plants such as Salvia officinalis (Taarit et al., 2010), Nigella sativa (Bourgou et al., 2010), and Ocimum basilicum (Tarchoune et al., 2013). Germacrene D and caryophyllene oxide (major compounds in the present study) were induced in Ocimum basilicum due to salinity (Hassanpouraghdam et al., 2011).

3.3 Antioxidant activity of S. squamatum EOs

The EOs from the aerial parts of S. squamatum showed moderate radical scavenging activity of the DPPH in a concentration-dependent manner (Table 2). The EOs from the coastal sample of S. squamatum revealed more antioxidant activity than that of the inland sample, where they attained an IC₅₀ value of 382.53 μL L⁻¹ and 559.63 μL L⁻¹, respectively. However, the IC₅₀ value for ascorbic acid (standard antioxidant) was 107.81 μL L⁻¹ (Table 2). The significant variation in the antioxidant activity between the two samples could be ascribed to the variation in the composition of the EOs.

The EOs are considered as an important class of the allelochemicals that are enhanced in the plant under stress conditions such as salinity stress. Thus, as we expect, the extracted EOs sample from the coastal area of the Mediterranean Sea to show more antioxidant activity. Under salt stress condition, the reactive oxygen species (ROS) are generated in plant cells, and in consequence, the antioxidant defense systems (enzymatic and non-enzymatic) are triggered (El-Shora and Abd El-Gawad, 2015a, 2015b). It is worth mentioning here that plants produce a vast array of chemical compounds specific to their habitats. These compounds play a significant role in stress amelioration, enhancement of the defense system, as well as communication with other organisms including herbivores, insects, pathogens, and other plants (Jones and Dangl, 2006).

However, the general profile of the EOs of the two samples of S. squamatum was comparable, though the composition is so different. The sample of the coastal location has a high content of oxygenated compounds, particularly sesquiterpenes, whereas it was triple fold for the inland sample (Fig. 1). This explains the more antioxidant activity of the coastal sample compared to the inland one. Therefore, the antioxidant activity of the coastal sample of S. squamatum might be attributed to the synergistic or singular effect of the major compound(s) identified in this sample such as humulene epoxide, (-)-spathulenol, (-)-caryophyllene oxide, germacrene D, and α-humulene. The synergistic activity of the terpenoid compounds was reported for other terpenoid compounds in other plants (Chaubey, 2012; Mitić et al., 2018).

On the other hand, the antioxidant activity of the inland sample could be attributed to β-pinene, germacrene D, α-humulene, α-muurolene, humulene epoxide, (-)-caryophyllene oxide, and β-cadinene. Nevertheless, the other minor compounds could play a significant role in antioxidant activity, even at low concentration (Carrillo and Tena, 2006). It is possible that the minor constituents may be implicated in synergism with the other active compounds (Hou et al., 2007; Lattaoui and Tantaoui-Elaraki, 1994).

Caryophyllene oxide was reported as an antioxidant compound in the EOs of Cullen plicata and Allophylus africanus (Abd El-Gawad, 2016; Balogun et al., 2014). Usually, the EOs rich in caryophyllene and its isomers α-humulene and β-caryophyllene possess biological activity (Sabulal et al., 2006). The α-humulene and β-pinene have been reported as antimicrobial agents (Jirovetz et al., 2006), while α-humulene, caryophyllene oxide were showed cytotoxic activity (Hou et al., 2007). Therefore, these major compounds could be responsible for the observed antioxidant activity of the EOs of S. squamatum.

3.4 Allelopathic activity of S. squamatum EOs

The EOs of S. squamatum aerial parts showed significant allelopathic activity against the root and shoot growth of the noxious weed Bidens pilosa (Fig. 3 & Table S3). The inhibition was dose-dependent. The root system of B. pilosa was inhibited more than shoot system under the effect of the EOs. This inhibition could be ascribed to the direct contact of the radicle with the EOs or even due to the higher permeability of the root cells (Abd El-Gawad et al., 2018). The present results showed significant variation in the allelopathic activity between the coastal and inland samples of S. squamatum (Fig. 3). This variation could be attributed to variation in the content of the EOs which is affected indirectly either by the soil properties (Table S2) or the climatic conditions. Soil analysis of the coastal desert sample revealed significantly high salinity compared to the inland sample due to the effect of the Mediterranean Sea. Also, salinity has been reported as a stress factor responsible for the induction of the EOs in plants (Hassanpouraghdam et al., 2011).

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Fig. 3

Inhibitory effect of the EOs from the aerial parts of S. squamatum collected from inland and costal deserts on the shoot (A), and root (B) growth of B. pilosa.

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The variation in the allelopathic activity is consonant with the antioxidant activity, and it might be correlated to the variation in composition of the EOs between the two samples. The variations in soil factors, light, temperature, nutrients, water content, daylength, genetic pool, and harvesting time have been reported to influence the EOs composition and hence affect biological activities such as allelopathy (Abd El-Gawad et al., 2019).

The allelopathic activity of the EOs against B. pilosa was studied for other donor species such as Xanthium strumarium (Abd El-Gawad et al., 2019), Cullen plicata (Abd El-Gawad, 2016), Artemisia scoparia (Kaur and Batish, 2010), Tagetes minuta (Arora et al., 2017), Eucalyptus citriodora (Setia et al., 2007). The activity of the EOs from these plants against B. pilosa could be ranked in the following order: E. citriodora > C. plicata > X. strumarium > T. minuta > S. squamatum (present study) > A. scoparia. This variation in the allelopathic activity might be ascribed to the variation in the EOs composition.

The allelopathic activity of the EOs from S. squamatum could be ascribed to the activity of the major compounds which act either singular or synergistic. Most of the major compounds of the EOs were reported as allelochemicals such as germacrene D (Dali and Xinru, 1996), spathulenol (Nishimura and Mizutani, 1995), caryophyllene oxide (Abd El-Gawad, 2016), humulene epoxide (Tellez et al., 2000), and pinene (Wang and Zhu, 1996).

Although S. squamatum produce a little amount of the EOs, it showed promising biological activities (Antioxidant and allelopathic). Also, this plant has a wide range of distribution in Egypt, so significant biomass can be obtained easily and could be integrated in EOs production. As a weed, this may be a potential way to control this plant as well as to become a good resource for bioactive compounds.