Phytochemical profile of three Ballota species essential oils and evaluation of the effects on human cancer cells

Abstract Three Ballota species, Ballota undulata, Ballota saxatilis and Ballota nigra ssp. foetida, were investigated for their cytotoxicity against two human cancer cells, hepatoma HepG2 cell line and breast cancer MCF-7 cell line, and for their antioxidant activity. The chemical composition of essential oils was studied by GC and GC–MS. Sesquiterpenes were the main constituents. The most antiproliferative essential oil against HepG2 cells was B. undulata with a percentage of inhibition of 81.36 ± 3.54 at a concentration of 100 μg/mL, while against MCF-7 cells essential oil from B. saxatilis was the most active with a percentage of inhibition of 24.18 ± 1.13 at a concentration of 100 μg/mL. The antioxidant activity was investigated by DPPH test for all the oils. B. undulata showed the highest antiradical effect, with IC50 value of 529.7 ± 37.4 μg/mL.


Introduction
The genus Ballota includes perennial herbs of the Lamiaceae family widespread in Europe. Ballota species have been used in folk medicine as antiulcer, antispasmodic, diuretic,

Chemical composition of the essential oils
The chemical composition of the essential oils from the three Ballota species (B. undulata, B. saxatilis and B. nigra ssp. foetida) was performed through analytical gas chromatography and GC-MS analysis (Table 1). Seventy-three compounds were identified in the oil from B. nigra ssp. foetida (Bn), that accounted for 95.1% of the total oil. The sesquiterpenes germacrene D (23.1%), (E)-β-caryophyllene (20.3%) and caryophyllene oxide (6.2%) were the main constituents, that together accounted for about the half of the total oil (49.6%). on the whole, sesquiterpenes (69.9%) and particularly sesquiterpene hydrocarbons (55.9%) prevailed over all the other components. other fractions were carbonylic compounds (8.5%) and monoterpenes (7.7%). our results are essentially in agreement with previous studies on the essential oils of this subspecies, reporting germacrene D, (E)-β-caryophyllene and caryophyllene oxide as main compounds of the oil but in different percentages (Bader et al. 2003;Fraternale et al. 2009;Fraternale & Ricci 2014). Differently, α-pinene and β-pinene were the main components identified in the oil of B. nigra L. from Iran analysed by Jamzad et al. (2011).
Eighty-five compounds were identified in B. saxatilis (Bs), accounting for the 95.0% of the oil. Linalool (11.2%), (E)-β-caryophyllene (8.8%), caryophyllene oxide (6.3%) and (E)-2-hexenal (5.6%) were the main compounds. The amounts of sesquiterpenes (24%) and monoterpenes   Bader et al. 2003). Particularly, the oil from our sample has in common with the Turkish oil the abundance of caryophyllene and caryophyllene oxide. However, its composition is more similar to the oil previously described by Bader et al. (2003) that reports linalool as main compound. Differently, phenols, carbonylic compounds and fatty acids and esters that are well represented in our sample are scarce or totally absent in the oil from Jordan. Ninety-one components were found in B. undulata (Bu), that constituted the 91.7% of the oil, in which prevailed the two sesquiterpene hydrocarbons germacrene D (16.0%) and bicyclogermacrene (10.4%). as for Bn, sesquiterpenes (46.6%) and particularly sesquiterpene hydrocarbons (39.3%) prevailed over the other fractions; fatty acids and esters (15.8%) were also well represented. The only previous report on the essential oil of this species collected in Jordan (Bader et al. 2003) shows similar results except from the presence of fatty acids and esters and hydrocarbons in our sample on respect to the Jordan sample.

Radical scavenging activity
The antioxidant potential of essential oils from Ballota species was determined by DPPH test ). The reduction of DPPH absorption is indicative of the capacity of the oils to scavenge free radicals, independently of any enzymatic activity. The scavenging effects of essential oils on DPPH were examined at different concentrations (0.025, 0.05, 0.1, 0.25, 0.5, 1 and 2 mg/ml). The absorbance decreases as a result of a colour change from purple to yellow as the radical is scavenged by antioxidants. all essential oils were able to reduce the stable free radical DPPH to the yellow-coloured 1,1-diphenyl-2-picrylhydrazyl (Table 2). Bu showed the highest antiradical effect with IC 50 value of 529.7 ± 37.4 μg/mL (Figure 1), while Bs and Bn showed lower activity (32.02 ± 3.40% and 48.22 ± 1.08% of inhibition at 2 mg/mL, respectively). The antioxidant activity of different Ballota species was previously investigated by Citoğlu and co-workers, among them B. saxatilis subsp. saxatilis exhibited remarkable antisuperoxide anion formation (Citoğlu et al. 2004). The antioxidant activity determined for B. nigra as inhibition of Cu(2+)-induced LDL peroxidation was attributed to phenylpropanoid derivatives . No antioxidant activity was previously demonstrated for B. undulata. Qazan demonstrated that B. undulata hydroalcoholic extract (70% EtoH) possesses active hypolipidaemic constituents when orally administered to the atherogenic rabbits (Qazan 2008).

Antiproliferative activity on HepG2 and MCF-7 cells
The three Ballota species were evaluated for their in vitro antiproliferative properties against two human cancer cell lines: hepatoma HepG2 cell line and breast cancer MCF-7 cell line.
The two human tumour cell lines were capable of attachment to form a homogeneous monolayer on the plastic substratum of the culture wells, as is ideal for the MTT assay. The MTT test is a simple bioassay used for primary screening of crude plant extracts and isolated compounds ). For each cell line, there was a linear relationship between cell number and absorbance, measured at 550 nm in both control and drug-treated wells  ( Figure 2). after 48 h of treatment, the antiproliferative activity was determined. The results on the growth of the human tumour cell lines are given in Table 3 and Table 4. The most antiproliferative essential oil against HepG2 cells was B. undulata (Bu) with a percentage of inhibition of 81.36 ± 3.54 at a concentration of 100 μg/mL (Figure 3) while against MCF-7   cells essential oil from B. saxatilis (Bs) was the most active with a percentage of inhibition of 24.18 ± 1.13 at a concentration of 100 μg/mL. No previous study was conducted on the evaluation of antiproliferative activity on cancer cells of Ballota species. Cell type cytotoxic specificity is observed in some plant extracts. This specificity of plant extracts is likely due to the presence of different classes of compounds in the extract, as it has been documented in the case of known classes of compounds (Cragg et al. 1994). Previous studies showed that sesquiterpenes, particularly germacrene, the main compound identified in the B. undulata (Bu) essential oil, could be responsible, at last in part, for cytotoxic activity against different human cancer cell lines (Grecco Sdos et al. 2015).

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