Essential oil constituents, phenolic content and antioxidant activity of Lavandula stricta Delile growing wild in southern Iran

Abstract Lavandula stricta belongs to the Lamiaceae family and is considered as an endemic medicinal plant in southern Iran. Essential oil composition, total phenolic content and antioxidant activity from two different populations of L. stricta were studied for the first time. A GC and GC/MS analysis of essential oil isolated from the aerial part of L. stricta identified 31 constituents; the major constituents were α-pinene (58.34–63.52%), linalool (8.85–9.36%), 3-methyl butyl 2-methyl butanoate (7.45–7.70%), sabinene (2.84–3.56%), limonene (2.87–3.21%) and myrcene (2.25%). The total phenolic content of methanolic extracts was determined with the Folin–Ciocalteu reagent and the antioxidant activity of methanolic extract and essential oil were determined with the 2, 2-diphenyl-1-picryl hydrazyl free radical scavenging assay, respectively. Total phenols varied from 61.05 to 64.45 mg GAE/g dry weight, and IC50 values in the radical scavenging assay ranged from 334.11 to 395.23 μg/mL in methanolic extracts and 420–475 μg/mL in essential oil.


Introduction
The genus Lavandula (lavender) belonging to the Lamiaceae family constitutes about 30 species of herbaceous and often aromatic, widely distributed in Mediterranean, Sahara-Arabian and Iran-Turanian regions (Feinbrun-dothan 1978). Recent studies show that the essential oil of some Lavandula species has shown a wide range of physiological properties, such as analgesic, sedative, carminative, anti-flatulence, anti-colic, disinfectant, bactericide, fungicide, anti-inflammatory and anti-depressant activity (Field et al. 2008;Smigielski et al. 2008;Zuzarte et al. 2009). Lavandula stricta is a perennial and bushy aromatic herb (35-50cm high) with violet-coloured inflorescences that grow naturally in the alpine region of Genow and Rodan in Hormozgan province, South Iran. The aerial parts of L. stricta are commonly used in traditional and folklore medicine for treatment of cramps, muscle pains and head cold among native people (Alizadeh 2014). A previous literature review on essential oil composition in Lavandula species showed that these are rich in terpenoids, such as linalool, linalyl acetate, 1, 8-cineole, fenchone and other terpenoids. However, the chemical composition and the amount of components vary among and within the Lavandula essential oils (Table S3). Prusinowska et al. 2015 showed linalool (29-39.2%), α-terpineol (7.1-12.7%), borneol (4.8-9.3%) and terpinen-4-ol (4.8-6.9%) were the major constituents of Lavandula angustifolia essential oil in Poland. In another study, linalool (34-47%), linalyl acetate (17-34%), camphor (4-9%) and eucalyptol (3-7%) were reported as the main components of Lavandula intermedia essential oil in Spain (Carrasco et al. 2015). Ait Said et al. 2015 reported carvacrol (48.9%), E-caryophyllene (10.8%) and caryophyllene oxide (7.7%) were the main components of the essential oil of Lavandula coronopifolia growing in Morocco. To the best of our knowledge, there are no published reports on phytochemical constituents of Iranian L. stricta described here. This paper represents the first report of Iranian L. stricta bioactive compounds at two different natural habitats in Iran.

Results and discussion
The yield and composition of essential oils, isolated by hydrodistillation from the aerial parts of L. stricta are shown in Table S2. The yields of the essential oils, of L. stricta based on dry weight, ranged from 0.21 to 0.36% (w/w) (Table S2). In total, 31 constituents were identified in L. stricta essential oil grown in Genow region accounting for 99.86%. The major constituents were α-pinene (58.34%), linalool (9.36%), 3-methyl butyl 2-methyl butanoate (7.45%), sabinene (3.56%), limonene (2.87%) and myrcene (2.25%). The essential oil of L. stricta in Genow region contained hydrocarbon monoterpenes (73.69%), oxygenated monoterpenes (25.81%) and sesquiterpene hydrocarbons (0.36%). In L. stricta essential oil grown in Rodan region, 31 compounds (99.95% of the total oil) were identified and the main components were α-pinene (63.52%), linalool (8.85%), 3-methyl butyl 2-methyl butanoate (7.70%), limonene (3.21%) and sabinene (2.84%); monoterpene hydrocarbons (75.05%) and oxygenated monoterpenes (24.64%) constituted the most abundant fractions of the oil (Table S2). Generally, α-pinene was found to be the major monoterpene hydrocarbon in the essential oil of L. stricta in Genow (58.34%) and Rodan (63.52%) habitats. Our results are in agreement with those of others reporting monoterpenes as the main constituents in some Lavandula species (Table S3). We selected several reports of some Lavandula species from different countries for comparison with the chemical constituents in essential oil (Table S3). A comparison of our result with the previous study on the chemical constituents of the Lavandula species suggests that the major constituents in L. stricta essential oil are clearly different from those in other Lavandula species. Linalool, linalyl acetate, 1,8-cineole, fenchone and camphor are the main oxygenated monoterpenes components in most Lavandula species, but in our study, α-pinene as hydrocarbon monoterpene is the main constituent in L. stricta essential oil (Table S3). This difference in the volatile constituents of the plant species could be attributed to genetic (species and subspecies), geographical origins, environmental and climatic conditions, harvest time and essential oil extraction and quantification methods (Loziene & Venskutonis 2005;Sefidkon et al. 2006;Alizadeh 2015).
The total phenolic content of the methanolic extracts of L. stricta was measured by the Folin-Ciocalteu reagent and expressed as gallic acid equivalents/g dry weight. According to Table S4, the total phenolic content was 64.45 and 61.05 mg GAE/g dry weight in Genow and Rodan regions, respectively.
Our results show that L. stricta has a high phenolic content compared with a previous report by Messaoud et al. 2012 in L. coronopifolia (31.3), Lavandula multifida (30.8) and Lavandula stoechas (25.2 mg GAEg −1 ) extracts. Also, our results are found to be higher than those reported in the methanol extract of L. angustifolia from Lithuania (Miliauskas et al. 2004), Lavandula officinalis water extract from Iran (Bouayed et al. 2007), hexane and aqueous fraction in L. intermedia from Spain (Torras-Claveria et al. 2007) and in ethanol or water extracts of L. stoechas from Turkey (Gulcin et al. 2004). The antioxidant activities of the methanolic extract and essential oil of L. stricta were assessed by the dPPH free radical scavenging methods. The IC 50 values were 324.11 and 395.23 μg/mL for methanolic extract and 420-475 μg/mL in essential oil in Genow and Rodan regions, respectively. The antioxidant activity of standard antioxidants was 24.70, 35.79 and 184.15 μg/mL for gallic acid, quercetin and vitamin E., respectively (Table S4). Previous study in some Lavandula species indicated the different species have high antioxidant activity; the antioxidant activity of methanolic extract and essential oil of L. coronopifolia (15.8-162.2 μg/mL), L. stoechas (34.2-2321.7 μg/mL) and L. multifida (19.3-201.6 μg/mL) grown wild in Tunis was reported (Messaoud et al. 2012). Torras-Claveria et al. 2007 reported the antioxidant activity of different solvent and fractions of Lavandula x intermedia ranged between 40.63 and 421.48 μg/mL. In another study, the antioxidant activity of five Lavandula species L. angustifolia ssp. angustifolia, L. angustifolia ssp. angustifolia Munstead, L. angustifolia ssp. angustifolia Hidcote Blue, L. angustifolia ssp. pyrenaica and Lavandula hybrid was 95.60, 101.40, 96.53, 110.36 and 73.53 μg/mL, respectively (Robu et al. 2012). Also, Blažeković et al. 2010 reported the IC 50 values for L. x intermedia 'Budrovka' extracts ranged from 15.06 to 45.25 μg/mL, while those obtained for extracts of L. angustifolia were 10.62-33.95 μg/mL. The antioxidant activity of essential oils may be primarily attributed to the concentration of their phenolic constituents, such as thymol, carvacrol and methyl thymol (Lin et al. 2009). It seems that the low level of phenolic components, such as thymol, carvacrol and Methyl thymol in L. stricta essential oil, reduces the antioxidant property in essential oil.
As seen in Table S4, the extract and essential oil from plants in Genow region have higher phenolic content and were most effective in scavenging the dPPH radical, compared to those in Rodan region. Thus, our results show that a correlation between the antioxidant activities and the total phenolic contents was revealed. These results suggest that the major part of the antioxidant activity in L. stricta results from the phenolic compounds. This result is in line with that reported by dorman and Hiltunen (2004) in Satureja hortensis, Alizadeh et al. 2011 in Origanum majorana, andMessaoud et al. 2012 in Lavandula species, who found similar correlations between total phenolic content and antioxidant activity of various medicinal and aromatic plants. Furthermore, it should be taken into consideration that antioxidant activity might be pertained to the chemical structure of phenolic compounds, as well as synergistic or antagonistic effect of compounds present in the crude extract (Messaoud et al. 2012;Gharibi et al. 2013).

Conclusion
In this study, the essential oil, phenolic content and antioxidant activity of two populations of L. stricta from Iran were reported for the first time. Thirty-one constituents were identified in the essential oil of L. stricta at two different natural habitats. The main components of the oil were α-pinene, linalool, 3-methyl butyl 2-methyl butanoate, sabinene, limonene and myrcene which are mostly monoterpene hydrocarbons and oxygenated monoterpenes. The methanolic extract of two populations of L. stricta showed high amount of phenolic content and moderate antioxidant activity compared with synthetic antioxidant.