Biological Activities and Chemical Composition of Santolina africana Jord. et Fourr. Aerial Part Essential Oil from Algeria: Occurrence of Polyacetylene Derivatives

The chemical composition of 18 oil samples of Santolina africana isolated from aerial parts at full flowering, collected in three locations in eastern Algeria was determined by GC(RI), GC/MS and 13C-NMR analysis. The major components were: germacrene D, myrcene, spathulenol, α-bisabolol, β-pinene, 1,8-cineole, cis-chrysanthenol, capillene, santolina alcohol, camphor, terpinen-4-ol and lyratol. The chemical composition appeared homogeneous and characterized by the occurrence of four derivatives which exhibited a conjugated alkene dialkyne moiety. They were identified for the first time in an essential oil from S. africana. The collective oil sample exhibited moderate antimicrobial and antioxidant activities whereas the anti-inflammatory activity presented a real potential. IC50 value of Santolina africana essential oil (0.065 ± 0.004 mg/mL) is 5-fold higher than IC50 value of NDGA used as positive control.


Introduction
The Santolina genus belongs to the Asteraceae family and is represented by more than 10 species widely distributed in Mediterranean area [1]. In all Santolina species,  [2,3] that grows naturally in forests and steppe pastures. This species is a bushy, green or ashy sub-shrub. The stems are woody, with floriferous branches erect in tuft, bare and thickened at the apex. The lower leaves are linear-cylindrical with short and obtuse segments. The bracts are ovate-oblong. The outer corollas are tube-styled ovary. The flowerheads are discoidal, yellow, homogamous [2].

Chemical Composition of Essential Oil
Aerial parts oils samples were submitted to GC-FID analysis, to determine the retention indices (RIs) of the EO components on two columns of different polarity and to GC/MS analysis. Further analysis by 13 C-NMR confirmed the identification of the main components. To allow the identification of four polyacetylene derivatives present at moderate or low levels, a composite sample (F1 to F6) was submitted to column chromatography (CC) over silica gel. Nine fractions were obtained and analyzed by GC-FID, GC/MS and 13 C-NMR. In total, 91 components accounting for 92.4% and 96.1% of the whole oil chemical composition were identified (Table S1, Figure 2), including forty-three monoterpenes, thirty-one sesquiterpenes, five phenylpropanoids, six polyacetylene derivatives and six others. The composition of S. africana EOs is generally homogeneous; the oils were found to possess little differences in the chemical composition but considerable variation in the levels of the individual components. All the samples were characterized by high proportions of monoterpenes (51.5- (1.8-7.3%) and lyratol (0.1-6.7%). Other two oxygenated monoterpenes: lyratal (tr-2.7%) and chrysanthenone (tr-4.5%), three sesquiterpene hydrocarbons: α-curcumene (0.3-3.2%), γ-curcumene (0.1-2.6%) and bicyclogermacrene (0.1-6.3%) as well as two oxygenated sesquiterpenes: β-elemol (up to 3.5%) and β-eudesmol (tr-3.0%) were present in appreciable amounts. Then, one sample for each location (B1, F5 and H1) and five other samples which exhibited various compositions (B3, B4, B5, B6 and H6) were presented in Table S1.

Identification of Polyacetylene Derivatives
In this study, the identification of four compounds proposed by the MS library was achieved. These compounds were presumed to be two pairs of stereoisomers (m/z = 200 and m/z = 198) corresponding to spiroacetalenol derivatives. Indeed, polyacetylene compounds are commonly found in the Asteraceae family [30]. The identification of these compounds was achieved by 13 C-NMR spectroscopy after fractionation (fractions Fr4 and Fr6, see Experimental part) by comparison of their spectral data with those reported in the literature. (E)-and (Z)-tonghaosu (m/z = 200) were identified by comparison with data previously described by Chanotiya et al. [31]. Figure S1). In the 13 C-NMR spectrum of Fr4 in which the E/Z ratio was close to 8/1 (46.6%/6.3%), a series of 13 peaks corresponding to the Z isomer was observed. It is the first time that the four spiroacetalenol derivatives [30] were identified in an EO from S. africana. The contents of (E)-2-(2 ,4 -hexadiynylidene)-1,6-dioxaspiro[4.4]-nona-3,7-diene and (E)-tonghaosu reached 7.3% (B3) and 3.8% (B6), respectively. These compounds were previously reported in EO from Chrysanthemum coronarium L. (aerial parts) [33] and in some solvent extracts from C. leucanthemum (roots) [34], C. coronarium (aerial parts) [35] and S. chamaecyparissus (leaves and buds) [36].

Chemical Variability
The 18 samples were submitted to statistical analyses: the principal components analysis (PCA, covariance) ( Figure 3, Table S1), in which the plan defined by the two axes F1 and F2 described 51.05% of the total variance of the population (the two axes F1 and F2 accounted for 31.70% and 19.35%. respectively). It may be noted that the composition of all samples was qualitatively quite similar. Although the compositions of the individual samples varied substantially for various components, it was not possible to distinguish groups within the essential oil samples. Therefore, one main group (16 samples) and differentiated two atypical compositions (B5 and B6, Figure 3) were observed. Indeed, B5 and B6 were discriminated by a high percentage of sesquiterpene hydrocarbons (bicyclogermacrene, (E)-α-bisabolene, γ-curcumene) and particularly germacrene D, 25.3% (B5) and 20.2% (B6) vs. 0-7.5% for the other samples. All the samples from the Fesdis location were homogeneous, while the composition of the samples from the Hamla location appeared much less homogeneous. Conversely, it appeared that the Bouilef samples, located between the two others locations (Fesdis and Hamla) were different, so two samples (B3 and B4) were aggregated to those of the Fesdis location, whereas the two samples B1 and B2 were quite similar to the Hamla samples ( Figure 3).

Antimicrobial Activity
The antimicrobial activity of the EO of S. africana isolated from the aerial part at full flowering was assayed against four bacteria, two yeasts and three filamentous fungi, using the agar disc diffusion method (Table 1).

Antimicrobial Activity
The antimicrobial activity of the EO of S. africana isolated from the aerial part at full flowering was assayed against four bacteria, two yeasts and three filamentous fungi, using the agar disc diffusion method (Table 1).  The oil was considered active when the diameter of inhibition zone was equal to or greater than 13 mm [24]. The agar diffusion method showed that the oil was effective against Staphylococcus aureus, the two yeasts and the three filamentous fungi with diameters of inhibition zone ranging from 13.0 mm to 19.7 mm. The most potent activity was demonstrated against Staphylococcus aureus and Aspergillus fumigatus with inhibition zones of 19.7 mm and 17.5 mm respectively. In contrast, the growth of Bacillus cereus, Klebsiella pneumoniae and Escherichia coli were not inhibited by the EO. This assumption is in accordance with previous studies on the Santolina genus. Indeed, the S. africana EO which contained acenaphtane (25.23%), calarene (21.54%), ocimene (17.44%) as its major components, exhibited a moderate or low activity against the same microorganisms with diameters of inhibition zone ranging from 6.50 mm to 20.15 mm. Bacillus subtilis and Staphylococcus aureus were the most susceptible to this EO, with inhibition zones of 20.15 and 19.5 mm, respectively [7].

Antioxidant Activity
The results of the DPPH • free radical scavenging test were presented in Table 2 and Figure S2. The EO of the aerial parts of S. africana have a high antioxidant activity at concentrations of 32, 64, 128 and 256 mg/mL, with inhibition percentages ranged between 85.54 ± 2.17% and 100 ± 0.00% and an IC 50 value of 1.51 ± 0.04 mg/mL. However, the antioxidant potential of S. africana EO was found to be low than that of ascorbic acid (positive control), with a percentage inhibition of 100%, at a concentration of 2 mg/mL and a significantly lower IC 50 value of 0.02 ± 0.0005 mg/mL. Values are means of triplicates ± standard deviation; * NDGA: Nordihydroguaiaretic Acid; # mg/mL.

Anti-Inflammatory Activity
The anti-inflammatory potential of S. africana EO was evaluated by determining its ability to inhibit lipoxygenases (LOX). Indeed, LOXs are a non-heme iron-containing dioxygenases that convert linoleic, arachidonic and other polyunsaturated fatty acid into biologically active metabolites involved in the inflammatory and immune responses. Several inflammatory processes such as arthritis, bronchial asthma and cancer are associated with an important production of leukotrienes catalysed by LOX pathway from arachidonic acid [37][38][39][40]. The inhibition of the LOX pathway with inhibitors of LOX would prevent the production of leukotrienes and therefore could constitute a therapeutic target for treating of human inflammation-related diseases. Thus, the search for new LOX inhibitors appears us critical because many of which exhibit significant anti-inflammatory activity.
The ability of S. africana EO to inhibit soybean lipoxygenase was determined as an indication of potential anti-inflammatory activity. S. africana EO exhibited an inhibition of LOX activity ( Table 2). The percentage of inhibition increases with the concentration of S. africana EO, i.e., 23.4% at 0.015 mg/mL to 57.6% at 0.075 mg/mL of EO. No LOX activity could be detected in the presence of 0.1 mg/mL of S. africana EO, suggesting almost complete inhibition of LOX activity. The IC 50 values (concentration at which 50% of the lipoxygenase was inhibited) were determined for the S. africana EO and for the non-competitive inhibitor of lipoxygenase, the nordihydroguaiaretic acid (NDGA) ( Table 2), usually used as reference in anti-inflammatory assays [38][39][40]. Data showed that the IC 50 value of S. africana essential oil (0.065 ± 0.004 mg/mL) is 5-fold higher than IC 50 value of NDGA (0.013 ± 0.003 mg/mL).
In previous studies, Derouiche et al. [7] reported a percentage inhibition of the free radical DPPH of the S. africana flower EO of about 13.80% at a concentration of 0.1 M, a value much lower than ascorbic acid (more than 70% of inhibition) used as a positive control. Nouasri et al. [17] evaluated the antioxidant activity of the essential oil of the aerial parts of S. chamaecyparissus using two methods, the DPPH • free radical scavenging test and the β-carotene bleaching test. They reported that S. chamaecyparissus EO had low antioxidant capacity to reduce DPPH • radical with an IC 50 of about 43.01 ± 8.04 mg/mL, compared to BHT (IC 50 = 0.072 ± 0.001 mg/mL) and ascorbic acid (IC 50 = 0.004 ± 0.001 mg/mL). The β-carotene bleaching test revealed that the EO had a moderate activity with a percentage inhibition of the oxidation of linoleic acid of the order of 47.00 ± 3.13%, a value that is higher than that of ascorbic acid tested (11.05%), but much lower than BHT (96.92%).
The measurement of antioxidant activity has revealed that aerial parts of S. africana EO exhibited an antioxidant activity that could have an eventual possibility to be used in the food industry, as a natural antioxidant agent, for the preservation of foodstuffs, or in the field of health, for the prevention of various diseases.
Concerning the anti-inflammatory activity, the low ratio between the two values of IC 50 (S. africana EO vs. NDGA) makes it possible to consider the S. africana EO as a high inhibitor of the LOX activity [48]. Thus, according to the results, S. africana EO exhibits a high inhibition of LOX activity, suggesting an anti-inflammatory potential.

Plant Material
Aerial parts of Santolina africana were collected during the flowering period in May 2016 in three locations in the Batna province (Eastern Algeria): Fesdis (Fesdis: F1-6; Bouilef: B1-6) and Oued Chaaba (Hamla: H1-6) (Figure 1). Identification of the plant material was performed by Dr. Babali B., (Laboratory of Ecology and Management of Natural Ecosystems, University of Tlemcen, Imama Tlemcen, Algeria). A voucher specimen has been deposited at the Laboratory of Natural Products (Department of Biology, University of Tlemcen, Algeria), under the accession n • A. 2844. The essential oil was obtained by hydrodistillation of dried aerial parts (around 150-280 g) for 2 h. Yields have been calculated from dry material.

Gas Chromatography (GC) Analysis
GC analyses were performed on a Clarus 500 FID gas chromatograph (PerkinElmer, Courtaboeuf, France) equipped two fused silica gel capillary columns (50 m × 0.22 mm, film thickness 0.25 µm), BP-1 (polydimethylsiloxane) and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 to 220 • C at 2 • C/min and then held isothermal at 220 • C for 20 min, injector temperature: 250 • C; detector temperature: 250 • C; carrier gas: hydrogen (1.0 mL/min); split: 1/60. The relative proportions of the oil constituents were expressed as percentages obtained by peak area normalization, without using correcting factors. Retention indices (RIs) were determined relative to the retention times of a series of n-alkanes with linear interpolation ('Target Compounds' software of PerkinElmer).

NMR Analysis
13 C-NMR analyses were performed on an AVANCE 400 Fourier Transform spectrometer (Bruker, Wissembourg, France) operating at 100.623 MHz for 13 C, equipped with a 5 mm probe, in CDCl 3 , with all shifts referred to internal tetramethylsilane (TMS). 13 C-NMR spectra were recorded with the following parameters: pulse width (PW): 4 µs (flip angle 45 • ); acquisition time: 2.73 s for 128 K data table with a spectral width (SW) of 220.000 Hz (220 ppm); CPD mode decoupling; digital resolution 0.183 Hz/pt. The number of accumulated scans ranged 2000-3000 for each sample (around 40 mg of oil in 0.5 mL of CDCl 3 ). Exponential line broadening multiplication (1.0 Hz) of the free induction decay was applied before Fourier transformation.

Screening of Antimicrobial Activity
The agar diffusion method [54] was used for the determination of antimicrobial activity of the EOs. Briefly, a suspension of the tested microorganisms (1 mL of a suspension at 10 6 cells/mL for bacteria and yeasts, 10 7 cells/mL for S. aureus and 10 4 spores/mL for filamentous fungi) was spread on the solid media plates, using Mueller-Hinton agar for bacteria, Sabouraud dextrose for yeasts and PDA for filamentous fungi. Filter paper discs (6 mm in diameter) were impregnated with 15 µL of the oil and 5 µL of DMSO and placed on the surface of inoculated plates. The activity was determined by measuring the inhibitory zone diameter in mm after incubation for 24 h at 37 • C for bacteria, 24-48 h at 30 • C for yeasts and 3 to 5 days at 25 • C for filamentous fungi. Fluconazole (FLU 25 µg/disc), nystatin (NY 30 µg/disc) were used as reference antifungal against yeasts and filamentous fungi and chloramphenicol (CHL 30 µg/disc), ciprofloxacin (CIP 10 µg/disc), gentamicin (GMN 10 µg/disc), vancomycin (VAN 30 µg/disc) were used as positive controls against bacteria. DMSO was used as negative control. Each test was performed in duplicate or in triplicate.

DPPH Radical Scavenging Activity
The antioxidant activity was measured on a sample of EO (Collective sample Fesdis F1-6). The antioxidant activity of S. africana EO was measured on the basis to scavenge of the 2.2-diphenyl-1-picrylhydrazil (DPPH • ) free radical, according to the experimental protocol of Blois [55]. A volume of 2.5 mL with various concentrations (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.015625 mg/mL) of the EO in absolute ethanol were added to 1 mL of an ethanolic solution of DPPH at 0.03 mg/mL. For each concentration, a blank was prepared. In parallel, a negative control is prepared by mixing 2.5 mL of absolute ethanol with 1 mL of ethanolic solution of DPPH. After incubation in the dark for 30 min at room temperature, the absorbance was measured against a blank at 517 nm. The activity of the EO was compared to ascorbic acid as a positive control. DPPH free radical scavenging activity in percentage (%) was calculated using the following formula: DPPH scavenging activity (%) = [(A control − A sample )/A control ] × 100 (1) with: A control is the absorbance of the negative control; A sample is the absorbance of the tested sample. The concentration of the EO required for the 50% reduction in the initial concentration of DPPH (IC 50 ) was calculated from the graph plotted of percentage inhibition against essential oil concentrations.

Anti-Inflammatory Capacity of Santolina africana Essential Oil
The anti-inflammatory capacity of S. africana essential oil (collective sample Fesdis F1-6) was evaluated by in vitro lipoxygenase inhibition assay [56][57][58]. The in vitro analysis for lipoxygenase inhibitory activity was performed using Lipoxidase type I-B (Lipoxygenase, LOX, EC 1.13.11.12) from Glycine max (soybean) purchased from Sigma-Aldrich Chimie (Saint-Quentin-Fallavier, France). It was determined by kinetic mode of spectrophotometric determination method, which was performed by recording the rate of change in absorbance at 234 nm. Indeed, the increase of absorbance at 234 nm due to formation of 13-hydroperoxides of linoleic acid (substrate used for LOX inhibition activity assay) [56][57][58].
A stock solution of LOX was prepared by dissolving around 5.7 units/mL of LOX in 0.2 M borate buffer pH 9.0 (1 unit corresponding to 1 µmol of hydroperoxide formed per min). Five concentrations of S. africana EO in dimethylsulfoxide (DMSO) were tested as inhibitor solution for LOX inhibition activity assay: 1.5, 2.5, 5.0, 7.5 and 10.0 mg/mL.
The LOX inhibition assays were performed by mixing 10 µL of LOX solution with 10 µL of inhibitor solution in 970 µL of boric acid buffer (0.2 M; pH 9.0) and incubating them briefly at room temperature. The reaction started by addition of 10 µL of substrate solution (Linoleic acid, 25 mM) and the velocity was recorded for 30 s at 234 nm. One assay was measured in absence of inhibitor solution and one assay was measured with DMSO mixed with distilled water (99.85% of DMSO in distilled water) which made it possible to eliminate the inhibition effect of DMSO. No inhibitor effect of DMSO on the LOX activity was detected and the LOX activity measured without inhibitor solution was considered as control (100% enzymatic reaction). All assays were performed on triplicate. The percentage of lipoxygenase inhibition was calculated according to the equation: Inhibition % = (V 0control − V 0sample ) × 100/V 0control (2) V 0control is the activity of LOX in absence of inhibitor solution and V 0sample is the activity of LOX in presence of inhibitor solution [58]. The IC 50 was calculated by the concentration of S. africana EO in DMSO inhibiting 50% of LOX activity.
Supplementary Materials: The following are available online, Table S1: Chemical composition of 18 samples of Santolina africana aerial part essential oil, Figure S1: Structure of polyacetylenes derivatives 88, 89, 90 and 91, Figure S2:

Conflicts of Interest:
The authors declare no conflict of interest.