Biological activities and volatile constituents of Daucus muricatus L. from Algeria

Background In order to find new bioactive natural products, the antimicrobial and antioxidant activities of essential oil components extracted from the separated organs of the Algerian medicinal and aromatic plant Daucus muricatus L. were studied. Results The chemical composition of essential oils obtained by hydrodistillation (HD) was investigated using Gas Chromatography–Retention Indices (GC-RI) and GC–Mass Spectrometry (GC-MS). Two types of essential oils were produced by D. muricatus: (i) The oil from roots is mainly composed by nonterpenic oxygenated compounds (59.8 g/100 g), and (ii) the aerial part oils (i.e., the leaves, stems, flowers, and umbels) was mainly composed by terpenic hydrocarbon compounds (62.3–72.2 g/100 g). The chemical composition of the volatile fraction isolated from different organs of Daucus muricatus were studied by HS–SPME/GC–RI and GC–MS after optimization of Solid Phase MicroExtraction parameters. For all organs studied, the main volatiles emitted by the plant were hydrocarbon compounds (60.7–82.2 g/100 g). Only quantitative differences between the volatiles of the separated organs studied were observed. In addition, the activity of the oil of D. muricatus against eight bacterial strains and one yeast was investigated. The oil from roots revealed active against S. aureus, while the essential oil obtained from the aerial parts was active against the yeast C. albicans. Conclusions Daucus muricatus essential oil seems be a promising source of natural products with potential antimicrobial activity.


Background
Daucus is a genus belonging to the Apiaceae family and consists of about 600 species that are widely distributed around the world. In Algeria, the Daucus genus is represented by more than 27 species living in dry and uncultivated areas [1], and they are mostly found from Tlemcen to Mascara [1,2]. The most prevalent of the species is Daucus carota L. (carrot) reported with eight subspecies throughout Algeria [1]. Daucus muricatus L., synonym of Artedia muricata L., Caucalis muricata L., and Platyspermum muricatum Hoffm., is widely distributed in Algeria, Spain, Portugal, Corsica, Sardinia, Sicily, Italy, the Aegean Islands, and Turkey [2]. Daucus muricatus is an annual plant 30-50 cm high, dark green, bristling at the base, with a stem thickened at the nodes and branches spreading erect. The leaves are soft and lanceolate in their periphery in segments cut into narrow strips with white flowers. The umbels opposite the leaves at the end are contracted, the fruit are large, elliptical and compressed, armed with spines expanded and confluent at the base [1,2]. Several investigations deal with the chemical composition of essential oils of the Daucus species . While no study has investigated D. muricatus essential oils, most of them have reported the chemical composition of essential oils from D. carota and its subspecies [3,4,[6][7][8][9][10][11][12][13][14][15][16]20,22,23,[25][26][27]. However, only three studies have reported the chemical composition of essential oils from Daucus species from Algeria. The first reported the chemical composition of the essential oil of D. reboudii Coss. [17], and the other two reported the chemical composition of the oil from D. crinitus Desf. [18,19]. Previous reports showed that the chemical composition of Daucus species was more dominated by monoterpene hydrocarbon compounds such α-pinene and sabinene [3,4,10,14,15], and occasionally by phenylpropanoids compounds such as apiol, myristicin, and isochavicol [3,[14][15][16]. Several studies recently investigated the biological activity of Daucus essential oils [6,10,12,19,20]. However, there remain many species and subspecies of Daucus that have not yet been examined.
As part of our ongoing chemical investigation of the essential oils from the Algerian Daucus genus [18] and our search for active natural products to fight nosocomial infections, we investigated for the first time the chemical composition and biological activities of Daucus muricatus L. through the study of: (i) the volatile components of D. muricatus roots, leaves, stems, flowers, and umbels extracted by hydrodistillation and by solid phase microextraction (SPME) using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), (ii) the antibacterial activity of D. muricatus essential oil against nine species of microorganisms involved in nosocomial infections using paper disc diffusion and agar dilution methods.

Results and discussion
Essential oil chemical composition GC-RI and GC-MS analysis of D. muricatus essential oils obtained from the roots, stems, leaves, flowers, and umbels that accounted for 92.8, 94.7, 94.5, 95.4, and 95.7 g/100 g of the oils, respectively and allowed the identification of 99 compounds. Their retention indices, yields, and relative concentrations expressed in g/100 g of essential oil are shown in Table 1. Among these, 39 monoterpenes, 32 sesquiterpenes, 22 nonterpenic compounds, three diterpenes, two phenylpropanoids, and one C 13 -isopropenoid were identified. All components were identified by comparison of their EI-MS and GCretention indices with those of our laboratory-produced "Arômes" library, with the exception of nine components that were identified by comparison with spectral data and retention indices from the literature (see Table 1). Two types of essential oils were produced by D. muricatus. The root oils are mainly composed by oxygenated compounds (59.8 g/100 g), and the aerial part oils (i.e., the leaves, stems, flowers, and umbels) were dominated by the occurrence of hydrocarbon compounds (62.3-72.2 g/100 g). The main components of root oil were nonterpenic aliphatic compounds that accounted for 56.7 g/100 g, such as eicosane (18.6 g/ 100 g), undecan-2-one (10.2 g/100 g), and tridecanol (6.4 g/100 g). Conversely, the main components of aerial organs of D. muricatus were monoterpene hydrocarbons (52.0-58.5 g/100 g). For all organs studied, limonene (21.9-24.0 g/100 g) and α-pinene (9.9-21.8 g/100 g) were the main components. Their relative abundances were followed by that of sabinene (4.7-8.1 g/100 g) in stems, leaves and flowers, and trans-sabinyl acetate in umbels (12.1 g/100 g). On moving from the bottom to the top of the plant, we noted that the relative concentrations of nonterpenic compounds decreased as follows: 56.7 g/100 g in the roots, 12.5 g/100 g in the stems, 7.7 g/100 g in the leaves, 4.0 g/100 g in the flowers, and then 3.6 g/100 g in the umbels.

HS-SPME analysis
The volatiles emitted from the D. muricatus roots, leaves, stems, umbels, and flowers were investigated using HS-SPME under optimized parameters. The optimization of the HS-SPME sampling parameters was conducted using fresh plant material based on the sum of the total peak areas obtained using GC-FID. The maximum sum of the total peak area was acquired for an equilibrium and extraction temperature of 70°C, an equilibrium time of 60 min, and an extraction time of 30 min. The GC-RI and GC-MS analysis identified 78 components: 42 monoterpenes, 18 nonterpenic compounds, 16 sesquiterpenes, and two phenylpropanoids ( Table 2). Identification of 74 components was conducted by comparing their EI-MS and retention indices with those in our laboratory-produced "Arômes" library, and four components were identified by comparing their EI-MS data and their apolar retention indices with those reported in the literature and in commercial libraries. Regarding the organ contribution to the aromatic plant fingerprint, it should be noted that the volatile constituents were more abundant in the leaves than in the other parts of the plant. Our analysis showed that the chemical composition of the HS fractions obtained from different organs was qualitatively similar but differed by the relative amounts of the main components. Relative to D. muricatus oil, the main volatiles emitted by plant were hydrocarbon compounds (60.7-82.2%) for all organs studied. More precisely, the sum of hydrocarbon monoterpenes (33.6%-64.1%) and hydrocarbon nonterpenic compounds (6.4%-25.5%) was higher than that of oxygenated compounds, which never accounted for more than 23.9 g/100 g. The main volatile components of roots were terpinolene (10.2%), bornyl acetate (9.7%), p-cymene (9.1%), α-pinene (8.7%), and undecane (7.2%). The relative amounts of terpinolene (0.4%-1.3%) and p-cymene (3.1%-3.4%) were lower in the aerial organs, and the main volatile emitted by leaves, flowers and umbels was limonene (30.6%, 19.1%, and 28.1%, respectively). In the stems, limonene (13.3%) was present in lower amounts than undecane (16.9%), which was identified as a major component. In addition, undecane was produced in appreciable amounts in leaves, flowers, and umbels (6.1%, 3.6%, and 7.4%, respectively). They were  accompanied by α-pinene, which accounted for 8.1% in stems and always more than 13.1% in leaves, flowers and umbels. With these hydrocarbon compounds, we noted the occurrence of trans-sabinyl acetate, which had a relatively higher concentration in umbels (9.6%) than in the other aerial organs (2.6%-3.8%). The chemical differences observed between the essential oils and the volatile fractions extracted using HD and SPME, respectively, can be explained by the fact that the first technique is based on the liquid quasi-total extraction of plant volatiles, and the latter technique is controlled by a solid/gas equilibrium step. During hydrodistillation, the most volatile and water soluble compounds are lost in the gaseous phase and in the hydrolate, respectively, whereas, with HS extraction, it is the fiber affinity of each compound that monitors the sampling of the volatiles. As a consequence, it should be noted that 23 compounds (1a-1e, 5a, 8a, 9a, 13a, 14a, 19a, 20a, 21a, 22a, 22b, 27a, 27b, 29a, 38a, 47a, 51a, 54a, and 65a) were only identified in the volatile fractions extracted using HS-SPME.

Antimicrobial activity (assay disk)
Preliminary screening of the antimicrobial activity in vitro of the essential oils from D. muricatus species against nine pathogenic microorganisms were studied using the filter paper disc agar-diffusion technique. The results showed variation in the antimicrobial properties of the plant essential oil ( Table 3). The essential oil showed strong activity (inhibition zone >20 mm), moderate activity (inhibition zone <20-12 mm), and no inhibition (zone <12 mm). The highest activity (diameter of inhibition zone 22 mm) was demonstrated against S. aureus by the essential oil of the root, while the lowest (diameter of inhibition zone 6 mm) was demonstrated against E. coli by oil from the aerial parts. Other hand, C. albicans, B. cereus and L. monocytogenes were also prone to growth inhibition with diameter zones of inhibition ranging from 12 to 16 mm. Rest of the bacterial strains (B. subtilis, E. faecalis, P. aeruginosa, K. pneumonia and E. coli) showed no inhibition, with diameter of zones of inhibition ranging from 8 to 10 mm (Table 3).

Minimum inhibitory concentrations (MIC)
The in-vitro antibacterial activities of essential oil from the roots and aerial parts of D. muricatus against the employed bacteria were assessed qualitatively and quantitatively by the presence or absence of inhibition zones. The noted antibacterial and antifungal effects of the two are presented in Table 3. In general, the roots oil showed higher activity against bacteria than oil of aerial parts. The most prominent inhibitory action of roots oil was observed against S. aureus with a MIC of 0.8 μg/ml. However, B. cereus and L. monocytogenes showed moderate activity with MIC values of 65 μg/ml. As for the antifungal effect, the aerial parts oil was found to be effective against the pathogenic yeast C. albicans (MIC = 45 μg/ml) and an average activity antimicrobial on the B. cereus and L. monocytogenes with a MIC of 250 μg/ml. It should be noted that the highest tested concentration (6000 μg/ml) of had no effect on other growth of microorganisms. Various chemical compounds isolated by hydrodistillation of oils from D. muricatus   have direct activity against many species of bacteria, such as terpenes and a variety of aliphatic hydrocarbons (alcohols, aldehydes and ketones). The lipophilic character of their hydrocarbon skeleton and the hydrophilic character of their functional groups are of main importance in the antimicrobial action of essential oils components. Therefore, a rank of activity has been proposed as follows: phenols > aldehydes > ketones > alcohols > esters > hydrocarbons [28]. The activity of the roots oil could be explained at least partially by its a Order of elution is given on apolar column (Rtx-1). Numbers correspond to those in Table 1 content of undecan-2-one (10.2%). This ketone was previously proved to have antimicrobial and nematicidal activity [29,30]. The higher activity of the roots oil compared to the aerial parts oil could be attributed to this fact. Another major class of this oil, aliphatic alcohols was, likewise, previously reported as an antimicrobial compound and was reported to possess strong to moderate activities against several bacteria [31].

Conclusion
Volatiles isolated from separated organs of D. muricatus by HS-SPME and hydrodistillation were investigated using GC-RI and GC-MS. Concerning the essential oils, oil from D. muricatus roots was mainly composed of oxygenated compounds, while oil from aerial parts (i.e., the leaves, stems, flowers, and umbels) was dominated by hydrocarbon compounds. Moreover, the study of the volatiles sampled by HS-SPME showed that the chemical composition of the HS fractions obtained from different organs was qualitatively similar but differed by the relative concentrations of the main components. It is interesting to note that the sample preparation method impacted quantitatively on the GC profile of D. muricatus volatiles. The antimicrobial properties of D. muricatus essential oils tested on nine microorganisms species showed that oil from roots was active against S. aureus, while essential oil obtained from aerial parts was active against the yeast C. albicans.

Experimental
Plant material and oil isolation  [32] according to the European Pharmacopoeia and yielded 0.02% for roots and 0.03-0.12% for aerial parts w/w of oil.

HS-SPME conditions
The single organs of D. muricatus (stems, leaves, flowers, umbels and roots separately) were cut roughly with scissors (1-2 cm long) before subjection to HS-SPME. The SPME device (Supelco) coated with divinylbenzene/ carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 30 μm) was used for extraction of the plant volatiles. Optimization of conditions was carried out using fresh organs of the plant (1 g in a 20 mL vial) and based on the number and the sum of total peak areas measured on GC-FID. Temperature, equilibration time and extraction time were selected after nine experiments combining four temperatures (30, 50, 70 and 90°C), four equilibration times (20,40, 60 and 80 min) and three extraction times (15, 30 and 45 min). After sampling, SPME fibre was inserted into the GC and GC-MS injection ports for desorption of volatile components (5 min), both using the splitless injection mode. Before sampling, each fibre was reconditioned for 5 min in the GC injection port at 260°C. HS-SPME and subsequent analyses were performed in triplicate. The coefficient of variation (1.6% < CV < 17.8%) calculated on the basis of total area obtained from the FID-signal for the samples indicated that the HS-SPME method produced reliable results.

Gas chromatography
GC analyses were carried out using a Perkin-Elmer (Waltham, MA, USA) Autosystem XL GC apparatus equipped with a dual flame ionization detection system and a fused-silica capillary columns (60 m x 0.22 mm I.D., film thickness 0.25 μm), Rtx-1 (polydimethylsiloxane). The oven temperature was programmed from 60°C to 230°C at 2°C/min and then held isothermally at 230°C for 35 min. Injector and detector temperatures were maintained at 280°C. Samples were injected in the split mode (1/50), using helium as the carrier gas (1 mL/min); the injection volume was 0.2 μL. Retention indices (RI) of the compounds were determined from a software from Perkin-Elmer. Component relative concentrations were calculated based on GC peak areas without using correction factors.

Gas chromatography-mass spectrometry
Samples were analyzed with a Perkin-Elmer Turbo mass detector (quadrupole), coupled to a Perkin-Elmer Autosystem XL, equipped with the fused-silica capillary

Component identification
Identification of the components was based (i) on the comparison of their GC retention indices (RI) on non polar and polar columns, determined relative to the retention time of a series of n-alkanes with linear interpolation, with those of authentic compounds or literature data [33,34]; and (ii) on computer matching with commercial mass spectral libraries [33][34][35] and comparison of spectra with those of our personal library.

Component quantification
Quantification of essential oil components was expressed using relative concentration in g/100 g of essential oil. The procedure included the calcul of FID response factors (RFs) relative to an internal standard. We carried out a methodology reported in the literature [36] and improved in our laboratory [37]. The application of this analytical procedure allowed the determination of the oil component relative concentrations expressed in g/100 g of essential oil. Relative amounts of individual components obtained during HS-SPME experiments, were calculated on the basis of their GC peak areas on the Rtx-1 capillary column, without FID response factor correction.

Bacterial and yeast strains and media
The bacterial strains used in this study, i.e. Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus (Gram positive), Escherichia coli and Klebsiella pneumoniae (gram negative) were isolated at the Medical Reanimation Department of the Hospital University Center of Tlemcen in Algeria. The yeast Candida albicans was isolated at the Dermatology Department of the same hospital. Bacterial strains preserved in nutrient agar at 4°C, were revivified in nutrient solution and incubated at 37 ± 1°C during 18 to 24 h. 0.1 mL of each culture was added to 10 mL BHIB (Brain Heart Infusion Broth, pronadisa Hispanalab). C. albicans preserved at 4°C in the Sabouraud agar supplemented with chloramphenicol was revivified in nutrient solution and incubated at 30 ± 1°C during 24 to 48 h. 0.1 mL of each culture was added to 10 mL sterile physiological water. For antimicrobial assay, bacterial strains were grown on Mueller-Hinton Agar (MHA, Pronadisa Hispanalab) while C. albicans was grown on Sabouraud Dextrose Agar + chloramphenicol (SDA, Merck). Bacterial and yeast inoculate reached microbial densities in the range 106 to 107 cfu/mL.

Antimicrobial activity Paper-disc diffusion method
Antibacterial activities of essential oil from root and all aerial parts of the plant were assessed using the paper disk agar diffusion method according to Rios [38]. Absorbent disks (Whatman disk 6-mm diameter) were impregnated with 20 μl of oil, to concentration of 5 mg mL-1, and then placed on the surface of inoculated plates (90 mm) and incubated at 37°C for 24 h. Negative controls were prepared using a disk impregnated with the same solvent as that used to dissolve the plant oils. Antimicrobial activity was assessed by measuring the inhibition zone. All the tests were performed in triplicate.

Dilution-agar method
A dilution agar method was used to determine the Minimum Inhibitory Concentrations (MIC). Stock solutions were obtained by dissolving extracts in dimethylsulfoxide (DMSO 1%). Serial dilutions were made to obtain concentrations ranging from 0 to 100 μg mL-1 of the essential oil. Each mixture was added to Mueller-Hinton agar for bacteria [39,40]. The Petri dishes contained a sterile solution of DMSO and the culture medium, respectively. After incubation at 37°C for 24 h for bacteria and at 30°C for 48 h for the yeast. The experiments were performed in triplicate.