Chemical composition, antioxidant, antibacterial and cytotoxic activities of essential oil of Leontopodium leontopodioides (Willd.) Beauverd

Abstract The essential oil (EO) from the aerial parts of Leontopodium leontopodioides (Willd.) Beauverd was obtained by hydrodistillation and analysed by GC–FID and GC–MS. Sixty-five compounds were identified which represent 96.2% of the total composition of the EO. The major components of the EO were palmitic acid (11.6%), n-pentadecanal (5.7%), linalool (3.8%), β-ionone (3.3%), hexahydrofarnesyl acetone (3.2%), bisabolone (3.2%) and β-caryophyllene (3.2%). The EO exhibited an excellent antibacterial activity against Staphylococcus aureus and Bacillus subtilis according to the MIC values tested by micro-dilution method. It also exhibited a significant cytotoxicity against HepG2 and MCF-7 cell lines with the IC50 values of 67.44 and 70.49 μg/mL according to the MTT assay. However, the antioxidant activity test revealed that the EO exhibited a weak DPPH radical-scavenging activity. In conclusion, the EO of L. leontopodioides could be regarded as a bioactive natural product and deserves further study for its potential therapeutic effects.


Introduction
The Leontopodium genus (family Asteraceae) is comprised of about 58 species, which are distributed in Asia and Europe. In total, 37 species were found in China (Chen and Bayer 2011). The aerial parts of Leontopodium leontopodioides (Willd.) Beauverd are widely used in the traditional Chinese herbal formulas to treat albuminuria, hematuria and vaginitis (Li et al. 2012). Previous phytochemical investigations showed that flavones and flavonoid glycosides were the main components of L. leontopodioides (Pan et al. 2009;Zhang et al. 2011). The Asteraceae family is well known as a source of essential oils, and the essential oils extracted from many species of this family possessed pharmacological properties such as antimicrobial activity (Küçükbay et al. 2012), schistosomicidal activity (Aguiar et al. 2013) and anticholinesterase (Pan et al. 2009).
There is no previous report on the chemical composition and biological activities of the essential oil from L. leontopodioides, therefore we reported the chemical composition of the essential oil from aerial parts of L. leontopodioides for the first time. Furthermore, we evaluated the antibacterial, cytotoxic and antioxidant properties of the EO.

Antioxidant activity
The effect of the L. leontopodioides EO on DPPH radical scavenging, evaluated on the basis of the 50% inhibition concentration (IC 50 ) value, was compared to BHT. The results are shown in Table S2. It revealed that the EO exhibits a weak DPPH radical-scavenging activity, with an IC 50 value of 0.971 mg/mL, which was much lower than that of BHT (0.045 mg/mL).

Antibacterial activity
The EO of L. leontopodioides was tested against two Gram-positive and two Gram-negative bacterial strains. The antibacterial activities were qualitatively assessed by the presence or absence of inhibition zone diameters, and quantitatively evaluated by the MIC and MBC values. The results are shown in Table S3.
The EO showed an excellent ability to inhibit the growth of Staphylococcus aureus (MIC = 0.039 mg/mL, MBC = 0.039 mg/mL) and Bacillus subtilis (MIC = 0.313 mg/mL, MBC = 0.625 mg/ mL). Although the previous studies revealed the strong antibacterial activity of linalool, β-ionone and β-caryophyllene (Mendiola et al. 2008;Park et al. 2012;Dahham et al. 2015), the antibacterial activity of EOs should not be only attributed to the major components, but should take the synergistic effects between the minor and major constituents into consideration.
However, the EO of L. leontopodioides was not active against the tested Gram-negative bacteria (MIC values > 1 mg/mL) (Jani et al. 2017).

Cytotoxic activity
The EO was tested in vitro by MTT assay against two cancer cell lines, namely HepG2 cells (liver hepatocellular cell line) and MCF-7 (human breast adenocarcinoma cell line). The results are presented in Table S4. The growth of both cell lines was inhibited by the EO in a concentration-dependent manner. Likewise, cytotoxicity of the EO was increased as a function of duration of exposure, which suggested a time-dependent effect of the EO. The IC 50 values for the cytotoxic effects of the EO on HepG2 and MCF-7 cells were calculated to be 67.44 and 70.49 μg/mL for 72 h, respectively. These IC 50 values are commonly considered as active (Bézivin et al. 2003). In general, the cytotoxic activity of EOs is mostly due to the presence of phenols, aldehydes and alcohols (Bruni et al. 2004;Sacchetti et al. 2005;Bakkali et al. 2008) as well as the synergistic effects of these active chemicals with the other main constituents (Lei et al. 2008;Sun et al. 2015). Owing to the great number of constituents, EOs seem to have no specific cellular targets (Carson et al. 2002). Furthermore, β-caryophyllene, one of the main components of the L. leontopodioides EO, has been reported to possess the potential ability to suppress tumour motility, cell invasion and tumour aggregation (Dahham et al. 2015).

Conclusion
In this study, chemical composition and biological activities of the essential oil from the aerial parts of L. leontopodioides were reported firstly. Apart from its low antioxidant activity, the EO possessed a potent antibacterial activity against S. aureus and B. subtilis. Furthermore, the results also demonstrated that the EO exhibited a significant cytotoxicity against cancer cell lines. Therefore, the EO from the aerial parts of L. leontopodioides could be regarded as a bioactive natural product, and is worthy of further investigation for its potential use.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This research was financially supported by the Shandong Science and Technology Program [grant number ZR2014HM018].