Major selected monoterpenes α-pinene and 1,8-cineole found in Salvia lavandulifolia (Spanish sage) essential oil as regulators of cellular redox balance

Figures & data

Figure 1. GC Chromatogram of Salvia lavandulifolia essential oil.

Figure 2. (A) Effect of monoterpenes on cell viability. U373-MG cells were treated with α-pinene and 1,8-cineole (a range of concentrations from 10 to 400 μM) for 24 h. Cell viability was measured using MTT assay. Experiments were performed in triplicate and results are expressed as mean ± S.D. *p < 0.05 versus control. (B) Cytoprotective effect of monoterpenes. U373-MG cells were pretreated with α-pinene and 1,8-cineole (a range of concentrations from 10 to 100 μM) for 24 h, previous to the treatment with H₂O₂ (1 mM) for 30 min. Cell viability was measured using the MTT assay. Experiments were performed in triplicate and results are expressed as mean ± S.D. *p < 0.05 versus control, # p < 0.05 versus H₂O₂-treated cells. (C) Effect of monoterpenes on cell morphology. U373-MG cells were pretreated with α-pinene and 1,8-cineole (10 and 25 μM) for 24 h, previous to the treatment with H₂O₂ (1 mM) for 30 min. Representative images are shown.

Table 1. Chemical composition of the essential oil of S. lavandulifolia.

Compound	Retention index	Percentage (%)
α-Pinene	926	18.39
Camphene	942	6.19
α-Terpinene	1008	0.15
1,8-Cineole	1023	19.57
t-β-Ocimene	1029	0.08
β-Phellandrene	1031	12.82
γ-Terpinene	1053	1.11
t-Sabinene-hydrate	1058	0.30
Thujone	1103	0.08
Camphor	1133	15.52
Borneol	1154	0.22
Isoborneol	1156	0.56
d-Terpineol	1178	5.37
Terpineol	1206	0.41
Myrtenol	1213	0.08
Eugenol	1343	0.43
Cuminaldehyde	1239	0.09
Geranyl acetate	1372	3.03
β-Caryophyllene	1441	2.17
α-Humulene	1469	0.34
α-Bisabolol	1443	0.24
α-Farnesene	1477	0.51
d-Cadinol	1505	0.35
t-Cadinol	1508	0.34
Cadinene	1513	0.86
Ledol	1541	7.81
Bisabolene	1555	1.54
Viridiflorol	1579	0.08
Amount of identified compounds (%)		98.64

Table 2. (A) Antiradical activity measured using the oxygen radical absorbance (ORAC) method. (B) Effect of monoterpenes on lipid peroxidation.

Compounds	ORAC value (µmol Trolox/mg)
α-Pinene	0.030	± 0.003
1,8-Cineole	0.045	± 0.003
Camphor	0.012	± 0.001
	TBARS (pmol/mg protein)
Control	1.198	± 0.20
H2O2 (1 mM)	3.880	± 0.05*
α-Pinene (10 µM + H2O2)	2.559	± 0.40^#
α-Pinene (25 µM + H2O2)	2.392	± 0.08^#
1,8-Cineole (10 µM + H2O2)	3.058	± 0.40^#
1,8-Cineole (25 µM + H2O2)	2.868	± 0.50^#

U373-MG cells were pretreated with α-pinene and 1,8-cineole (10 and 25 μM) for 24 h, previous to the treatment with H₂O₂ (1 mM) for 30 min. Lipid peroxidation was measured using TBARS assay. Data are expressed as mean ± SD. Results are representative of three independent experiments. *p < 0.05 versus control, #p < 0.05 versus H₂O₂-treated cells.

Figure 3. Effect of monoterpenes on intracellular ROS production. U373-MG cells were pretreated with α-pinene and 1,8-cineole (10 and 25 μM) for 24 h, previous to the treatment with H₂O₂ (1 mM) for 30 min. The intracellular ROS production was measured using the dichlorofluorescein assay. Data are expressed as percentage of ROS production, mean ± SD, #p < 0.05, versus H₂O₂-treated cells.

Figure 4. (A) Effect of monoterpenes on protein expression of antioxidant enzymes. U373-MG cells were pretreated with α-pinene and 1,8-cineole (10 and 25 μM) for 24 h, previous to the treatment with H₂O₂ (1 mM) for 30 min. The levels of protein expression of CAT, SOD, GR, GPx, and HO-1 were measured by Western blots. (B) Effect of monoterpenes on Nrf2 protein expression. U373-MG cells were pretreated with α-pinene and 1,8-cineole (10 and 25 μM) for 24 h, previous to the treatment with H₂O₂ (1 mM) for 30 min. Nuclear and cytoplasmic Nrf2 protein expression were measured by Western blots. Data are expressed as mean ± SD. Results are representative of three independent experiments. *p < 0.05 versus control, # p < 0.05 versus H₂O₂.

Table 3. Effect of monoterpenes on caspase-3 activity.

Compounds	Caspase-3 (%)
Control	100.0 ± 4.4
H2O2 (1 mM)	201.4 ± 2.8*
α-Pinene (10 µM + H2O2)	141.5 ± 1.9^#
α-Pinene (25 µM + H2O2)	133.8 ± 1.1^#
1,8-Cineole (10 µM + H2O2)	113.3 ± 1.9^#
1,8-Cineole (25 µM + H2O2)	163.4 ± 1.5

U373-MG cells were pretreated with α-pinene and 1,8-cineole (10 and 25 μM) for 24 h, previous to the treatment with H₂O₂ (1 mM) for 30 min. Data are expressed as mean ± SD. Results are representative of three independent experiments. *p < 0.05 versus control, # p < 0.05 versus H₂O₂-treated cells.