Abstract: The oils obtained by hydrodistillation and steam distillation of the aerial part of Stachys pubescence Ten. was analyzed by GC and GC/MS. Water distilled essential oil of the aerial part of S. pubescence, was rich in fatty acids like hexadecanoic acid and linoleic acid and also benzaldehyde and spathulenol whereas the steam distilled oil of the plant contained hexadecanoic acid, spathulenol and eugenol. Both of oils were rich in fatty acids (36.6 and 27.9%, respectively). Moreover, the content of oxygenated mono and sesquiterpenss were defined higher in steam distilled oil than hydrodistiled oil (24.5, 17.2 and 6.1, 15.5%, respectively). In conclusion it seems that oxygenated terpenoids were trended to steam distillation method more than hydrodistilation, respectively.
INTRODUCTION
The genus Stachys L. (Lamiaceae) comprises more than 300 species in the world. In Iran, Stachys genus is represented by about 34 species including Stachys pubescence as an endemic species (Mozaffarian, 1998; Rechinger and Hedge, 1982). Flavonoids (Skaltsa et al., 2007; Delazar et al., 2005), Phenyl ethanoid glycosides (Nazemiyeh et al., 2006; Miyase et al., 1996; Nishimura et al., 1991), terpenes (Paternostro et al., 2000; Khanavi et al., 2003, 2004, 2008), iridoids (Meremeti et al., 2004) and saponins (Yamamoto et al., 1994) are the main compounds occurring in this genus.
There are also several reports about the major compounds of the oil of various Stachys species, for instance the oil of S. turcomanica is rich in germacrene D (17.4%), 7-epi-α-selinene (10.5%) and β-elemene (9.2%) (Firouznia et al., 2009). Moreover, main compounds of S. trinervis oil were reported previously as α-pinene (42.68%) and δ-2-carene (31.90%) while in S. subaphylla oil major constituents were identified as δ-2-carene (23.93%), α-pinene (19.29%) and sabinene (19.11% (Khanavi et al., 2008).
In Iran, the aerial parts of S. inflata are traditionally used for the treatment of infection, asthma, rheumatic and other inflammatory disorders (Maleki et al., 2001). Several studies have shown that Stachys species have identified with various effects such as anti-inflammatory (Khanavi et al., 2005; Kukic et al., 2007; Rezazadeh et al., 2005; Sharifzadeh et al., 2005), antinephritic (Hayashi et al., 1994), hypotensive (Takeda et al., 1997), anxyolytic (Rabbani et al., 2003), antimicrobial (Dulger et al., 2005; Skaltsa et al., 1999), antiallergic reactions (Shin, 2004; Kim et al., 2003), antioxidant (Meremeti et al., 2004; Haznagy-Radnai et al., 2006; Vundac et al., 2007), cytotoxic effects (Khanavi et al., 2012) as well as an effect on hyaluronidase activity (Takeda et al., 1985).
The aim of this study is to determine the hydrodistilled and steam-distilled essential oils composition of S. pubescence and carry out a comparative evaluation between these two methods.
MATERIALS AND METHODS
Plant material: The aerial part of Stachys pubescence was collected from Khalkhal, Province of Ardabil, Iran in June 2009 during the flowering stage. Voucher specimen has been deposited at the Institute of Medicinal Plants (ACECR), Karaj, Iran.
Isolation of the volatile oils: The air-dried aerial part of this species was subjected to separate hydrodistillation using a Clevenger-type apparatus for 3 h and also was submitted for 3 h to steam distillation. After decanting and drying of the oils on anhydrous sodium sulfate, they were sealed in dark vials until usage.
Gas chromatography/mass spectrometry: Analysis of the water and steam distilled oils from S. pubescence were analyzed by means of GC and GC/MS. The GC analysis was performed on a Shimadzu 15 A gas chromatograph equipped with a split/splitless injector (250°C) and a flame ionization detector (250°C). N2 was used as carrier gas (1 mL min-1) and the capillary column used was DB-5 (50 mx0.2 mm; film thickness 0.32 μm). The column temperature was kept at 60°C for 3 min and then heated to 220°C with a 5°C min-1 rate and kept constant at 220°C for 10 min.
GC/MS analysis was performed using a Hewlett-Packard 5973 mass selective detector connected with a HP 6890 gas chromatograph. The separation was achieved by use of a HP5MS (5% Phenylmethylsiloxane) capillary column (60 mx0.25 mm; film thickness 0.25 μm). The column temperature was held at 60°C for 3 min and programmed up to 220°C at a rate of 5°C min-1 and then kept constant at 220°C for 3 min. Helium was used as the carrier gas (1 mL min-1). MS were taken at 70 eV. Identification of oils compounds were made by comparing their mass spectra and Retention Indices (RI) with those given in the literature and those authentic samples (Adams, 1995). Relative percentage amounts were calculated from peak area using a Simadzu CR4A chromatopac software which was adjusted for that.
Identification of the compounds: Retention indices of components were calculated by using retention times of n-alkans that were injected after the oil at the same chromatographic conditions. The compounds were identified by comparison of their mass spectra and Retention Indices (RI) with those reported in the literature (Adams, 1995; Davies, 1990) and of the authentic samples or by comparison with those held in a computer library (Wiley 275.L).
RESULTS AND DISCUSSION
Table 1 shows the constituents of hydrodistilled and steam distilled essential oils of S. pubescence. Both oils were light yellow with a distinct sharp odour in a yielding of 0.2% (w/w) for hydrodistillation and 0.3% (w/w) for steam distillation, respectively.
The 36 components were detected in the hydrodistilled oil of S. pubescence representing 89.1% of the total oil. The major constituents were hexadecanoic acid (24.6%) and linoleic acid (12.0%), whereas the steam distilled oil of the plant contained 23 compounds (90.2%), with hexadecanoic acid (21.5%), spathulenol (11.6%) and eugenol (11.1%) as the main constituents.
From Table 1 it is evident that the composition of the oils obtained by hydrodistillation and steam distillation of S. pubescence are different quantitatively but the total amount of the non-terpenoid fraction in the hydrodistilled and steam distilled oils of the plant (53.9 and 37.4%) were higher than monoterpenes (10 and 24.5%) and sesquiterpenes (22.1 and 17.2%) and some of the identified components in steam distilled oil were not found in the water distilled oil. Also, oxygenated sesquiterpenes were higher in steam distilled oil (17.2% against 15.5%). This pattern was observed in oxygenated monoterpenes (24.5 against 6.1%) too.
Previous investigation on the oil of some species of Stachys showed various compositions. Sesquiterpene hydrocarbons were the predominant fraction in the oils of S. scardica, S. cretica ssp. cretica, S. germanica ssp. heldreichii and S. laxa with germacrene D as the major compound (Skaltsa et al., 2003; Sajjadi and Mehregan, 2003) where as spathulenol is the main compound of S. spinolosa and S. byzantina and α-copaene and β-caryophyllene in S. euboica were the main components (Skaltsa et al., 2003; Khanavi et al., 2004). Also, the results of our previous researches showed that monoterperne hydrocarbons with δ-2-carene and α-pinene are major compounds of S. trinervis and S. subaphylla (Khanavi et al., 2008).
| Table 1: | Camparative chemical composition (%) of Stachys pubescence oil obtained by hydrodistillation and steam distillation |
Although, most species of Stachys have rather low amounts of aliphatic and non- terpenoid fractions, some other species of Stachys, as the same of our results, have shown relatively high amounts of fatty acids and aliphatic esters (Skaltsa et al., 2003).
According to our result it is evident that the composition of the oils obtained by hydrodistillation and steam distillation of S. pubescence are different qualitatively and quantitatively. Some of the identified components in water distilld oil were not found in the steam distilled oil. Also it seems that in steam distillated oils, oxygenated terpenoids are higher than in hydrodistilled whereas non-terpenoids are the main fraction in hydrodistilled oil of this species.
CONCLUSION
According to mentioned results, two different ways of essential oil isolation caused definite variations in major terpenoids. It seems that oxygenated terpenoids are trended toward steam distillation more than hydrodistilation, respectively. Further investigation is needed to confirm these issues but if this pattern of compound isolation were repeated in some other plants it might be a new way of producing some essential oils enriched oxygenated compounds.
ACKNOWLEDGMENTS
We are grateful to Mr. Ajani and Institute of Medicinal Plants (ACECR), for identification of the plant.