Chemical composition, antimicrobial and antioxidant activities of the essential oils from Senecio longiscapus Bojer leaves (Asteraceae)

The present work aims to study the chemical composition and the antimicrobial and antioxidant properties of the essential oil of Senecio longiscapus (SLEO) leaves. SLEO was extracted from fresh leaves by hydrodistillation with a yield of 3%. It is clear, yellow, with a relative density of 0.7466 at 20°C, a refractive index of 1.4959, an optical rotation of +3°47, an acid number of 1.53 and an ester number of 12.49. Gas chromatography/mass spectrometry (GC/MS) analysis of the SLEO identified 17 components, representing more than 99.09% of the overall composition. The main component of SLEO was sabinene (53.28%) and elemicin (15%), β-pinene (9.85%), methyleugenol (5.58%), α-pinene (4.84%) and mircene (2.37%) were the major components. At 7.48 mg/disk, SLEO inhibited the growth of all germs tested including four GRAM (+) and five GRAM (-) bacteria and one yeast. The zones of inhibition (ZI) ranged from 12 mm ( Yersinia enterolitica ) to 40 mm ( Bacillus subtilis ). The antioxidant activity of SLEO by the DPPH method was IC50 = 4.601 µg/ml. When administered orally at doses as high as 5 g/kg body weight, SLEO was not toxic to mice. Its non-toxicity, antimicrobial and antioxidant activities could make SLEO an alternative in the treatment of infectious diseases.


Introduction
The growing interest in essential oils is reflected in the extensive research being carried out around the world on aromatic plants.Aromatic plants produce essential oils (EO) that have great therapeutic power and interesting biological activities such as antibacterial, antifungal, larvicidal, insecticidal and antioxidant properties [1].Asteraceae (Compositeae) is one of the plant families that are good producers of essential oils.In Madagascar, this family is represented by 550 species of which 500 are endemic [2].
Senecio is the largest and most complex genus in the family of the Asteraceae having more than 1500 species distributed widely throughout the world [3,4].In Madagascar, the Senecio genus is represented by 85 species and 78 of which are endemic [5].Present in various formations in the Central Madagascar region, the Senecio genus is mainly found in open areas such as natural clearings and forest edges.The mountain environment with rocky soil is particularly favourable to its development with a rather remarkable microendemism [6].
The essential oils of the genus Senecio have been the subject of a large number of phytochemical studies and several biological activities have been demonstrated [7] but the studied species for essential oils constituents are still few and not more than 10 % of the species belonging to this genus [8].A number of studies have shown that several Senecio species have antimicrobial, antifungal and cytotoxic activities [3].
However, nearly thirty species of Senecio, because of their high pyrrolizidine alkaloids content, are very dangerous for both humans and animals [12,13].For examples, S. jacobaea, S. douglasii, S. riddellii caused the majority in the cattle losses in western US [12] and Senecio inaequidens was implicated in a livestock poisoning epidemic in South Africa [14].
We are interested in Senecio longiscapus which is an aromatic plant endemic to central Madagascar.According to our surveys of healers and traditional practitioners, Senecio longiscapus is used to treat sexually transmitted infections, stomach aches and intestinal parasites (vermifuge).In addition, the available literature on this plant has so far only reported botanical studies.
The main objectives of this study were to determine the composition and physicochemical characteristics of Senecio longiscapus leaf essential oil (SLEO) and to explore its potential antibacterial and antioxidant activities and toxicity.

Microbial strains
The microbial strains used were pathogens commonly sought in medical and food microbiological analysis and/or control.They include 4 Gram (-), 4 Gram (+) bacteria and one yeast (Table 1).

Animals
OF-1 strain Albino mice (Mus musculus), weighing 25 ± 2 g, were provided by the Pasteur Institute of Madagascar (IPM) breeding farm.

Extraction of the essential oil
The extraction of the essential oils from the fresh leaves of Senecio longiscapus was carried out by hydrodistillation using a Clevenger type apparatus [15].

Physico-chemical characterization
The physico-chemical parameters to be determined and the references used are presented in Table 2.

Essential oil analysis
The chemical composition of the essential oil was determined by gas chromatography/mass spectrometry (GC/MS) [16].The sample was analyzed using an AGILENT 5973 chromatograph (Network mass selective detector).It is equipped with a DBWAX column (0.25 mm x 30 m x 0.25 μm).The temperature of the column was programmed from 40°C to 250°C.The injector and detector temperatures are set at 280°C.Helium is the carrier gas used, with a flow rate of 1 ml/minute, the volume of sample injected being 1 μl.The peaks obtained were identified using AMDIS software Version 2.69 (Automated Mass Spectral Deconvolution and Identification System).

Assessment of antimicrobial activity
All the methods used for antimicrobial assay were detailed in our previous papers [17,18].

Assessment of antimicrobial activity
The sensitivity of microorganisms to the essential oil was determined by the agar diffusion method or aromatogram.Sterile paper disks (6 mm in diameter BioMérieux, REF 549916) were soaked with pure essential oil and placed on the surface of the inoculated Mueller-Hinton Agar (Scharlau®).The Petri dishes were incubated at 37°C for 24 h and the zones of inhibition were measured.The sensitivity to the essential oil was classified according to the diameter of the zones of inhibition as: not sensitive (-) for diameters less than 8 mm; sensitive (+) for diameters 9-14 mm; very sensitive (++) for diameters 15-19 mm and extremely sensitive (+++) for diameters larger than 20 mm [19].
Antibiotic and antifungal used as references in this study were respectively Neomycin 30 μg/disk and Miconazole 500 μg/disk.

Antioxidant activity determination
The antioxidant capacity was evaluated by the method using free radical scavenging against DPPH (2, 2-Diphenyl-1-Pycryl Hydrazyl).The method was detailed in previous paper [20].

Toxicity determination
A volume of 0.3 ml of EO per 25 ± 2 g of body weight was administered to mice by oral route by means of an intubation cannula with a curved distal.Four batches of 5 male mice were used.Another one receiving physiological serum (NaCl 0.9%) was used as control.The mice were observed for 24 h.

SLEO physico-chemical parameters
The extraction yield and the physico-chemical parameters of SLEO are shown in Table 3.

SLEO antimicrobial activity
SLEO antimicrobial activity was tested at 745.2 mg/ml against nine strains of bacteria and one yeast.The IZs obtained are presented in the Table 5.Based on the standard of Ponce et al, [19], SLEO was active on all microbial strains tested with IZs ranging from 12 mm (Shigella flexneri) to 40 mm (Bacillus cereus).

SLEO antioxidant activity
The antioxidant activities of SLEO and ascorbic acid are presented in Table 7 and figure 3.For both SLEO and ascorbic acid, scavenging activity against DPPH increased with concentrations and the maximum of activity was reached at 112, 25 µg/ml.The IC50s for SLEO and ascorbic acid were 4.601 µg/ml and 3.245 µg/ml respectively.

SLEO toxicity
Four increasing doses of SLEO (1117.7,2235.5, 4471.0 and 8942 mg/kg) were administered orally to 4 homogeneous batches of 5 mice of 25 g±2.Another batch of 5 mice serving as a control received physiological serum (NaCl 0.9%).No symptoms of intoxication were observed with the 4 doses tested during 24 h.

Discussion
The yields of essential oils are extremely variable depending on the plants considered, but they are generally very low, below 1% [21].The extraction yield of SLEO was much higher (3%).
Seventeen (17) components representing 99.09% of the overall composition were detected in SLEO.This quantity was close to that observed for example in S. giganteus (18) [22], S. argophylloides (16) and S. viridis (15) [23].As shown in Table 8, the main components and their proportion (%) in relation to the overall oil vary according to the species of Senecio.Sabinene, with a percentage of 53.28%, was the main component of SLEO.Sabinene does not seem to be common in the genus Senecio EOs.It is also the main component of the EO of S. graveolens aerial part (23.8%) [10], but is present at much lower percentages in the EOs of S. polyanthemoides leaves (3.2%) [26], S. flammeus (2.6%) [9], S. mustersii aerial part (1.6%) [10] and S. graveolens leaves (0.8%) [27].
SLEO showed an antioxidant activity that increased with concentration and was maximal at 112.5 µg/ml.Its efficacy was about 70% of that of ascorbic acid.For comparison, the IC50s of the EOs from different parts of some Senecio species determined by the DPPH method assay and using ascorbic acid as standard reference are presented in Table 9.Based on the size of the difference between the EO and ascorbic acid IC50 values, SLEO has an antioxidant activity comparable to that of S. nudicaulis, higher than that of S. graciliflorus but significantly lower than that of S. glaucus.
As mentioned in Table 9, several Senecio species are known to be dangerous to humans and animals.Therefore, before considering the exploitation of the interesting biological properties already identified, it was necessary to evaluate the toxicity of SLEO.Acute toxicity tests on mice showed that at a dose as high as 5 g/kg body weight by the oral route, SLEO did not cause any symptoms of intoxication for 24 hours.This result is in agreement with our field surveys that no adverse effects due to the use of S. longiscapus have been reported.This is an important advantage as it does not limit the uses of SLEO, especially for therapeutic purposes.However, this result should be complemented by further toxicity tests.Other known biological properties of components that are among the main components in SLEO should be explored.Its non-toxicity, good antimicrobial and antioxidant activities could make SLEO an alternative in the treatment of infectious diseases.

Conclusion
The chemical composition and physicochemical characteristics of the essential oil of Senecio longiscapus leaves are well established.Two important pharmacological properties, antibacterial and antioxidant, and the absence of toxicity of the essential oil have been demonstrated.The results obtained on the antimicrobial activity of SLEO provided scientific basis of the use of Senecio longiscapus in traditional medecine.

Compliance with ethical standards
The plant was identified by comparison of an herbarium made from the collected material with the voucher specimen n°3553 of the Botanical and Zoological Park of Tsimbazaza (Antananarivo) made by Hildebrandt in 1933.

Figure 3
Figure 3 Effects of concentrations on the scavenging activity of SLEO and ascorbic acid against DPPH

Table 1
List of bacterial strains used

Table 3
Extraction yield and physico-chemical parameters of SLEO

Table 4
SLEO major components

Table 7
SLEO and ascorbic acid antioxidant activities *: Each value was the average of the values of 3 tests

Table 8
Number of components detected and major components in different Senecio species

Table 9
Comparison of the IC50s of EOs of some Senecio species and ascorbic acid determined by the DPPH scavenging method