Chemical Constituents of the Essential Oil of Stachys fruticulosa M. Bieb. From Iran

Background: Stachys (Lamiaceae) is a genus with varied effects and applications in traditional medicine. The essential oil (EO) of different species of genus Stachys has received much attention in different studies. According to previous evidence, the components of EO in different species have diversity although there is no previous data regarding investigating Stachys fruticulosa for EO. Considering the importance of the plants of genus Stachys, vast types of compounds in the EO of the genus, the aim of the present study was to evaluate the components of EO of S. fruticulosa M. Bieb. Methods: The EO of the aerial parts from S. fruticulosa was obtained by hydrodistillation and then the oil was analyzed by gas chromatography/mass spectrometry (GC/MS) and 95% of the oil (17 components) was identified accordingly. The identity of the components was assigned by comparing their mass spectra and retention indices with those of authentic samples. Results: Most oil components were α-terpinyl acetate (24.6%), 3-n-Butyl phthalide (20.5%), p-cymene (18.2%), and β-phellandrene (18.2%). Conclusion: The components of essential oil from S. fruticulosa were identified for the first time, and these substances may be responsible for the biological effects of these essential oils.

paper (7) reported the composition of the EO from the aerial parts (leaves) of S. byzantina from the north-west of Iran as well. The EO was extracted by hydrodistillation from the selected plants, and its chemical composition was determined by the GC-MS system. The mass fraction of the oil on the dry weight base was 0.4%. Altogether, 21 compounds were identified corresponding to 87.9% of the total oil. The main components were germacrene D (9.6%), menthone (6.9%), 1,8-cineole (14.8%), α-terpineol (7.8%), cubenol (9.9%), α-cadinol (6.8%), and linalool (12.9 %). Additionally, the EO composition of the aerial parts of S. parviflora was analyzed by GC-FID and GC-MS apparatus, followed by characterizing 23 compounds representing 99.9% of the oil. Muurolol (48.4%) and Z-caryophyllene (11.2%) were the major components of the oil, and oxygenated sesquiterpenes (71.4%) were the major fraction of the EO (8).
In similar research (9), the EO of S. lavandulifolia Vahl (Lamiaceae) was isolated by the hydrodistillation of the aerial parts of the plant with a yield of 0.25%. Then, the chemical composition of the volatile oil was analyzed by capillary GC and GC/MS. Finally, the main components were germacrene-D (13.2%), β-phellandrene (12.7%), β-pinene (10.2%), myrcene (9.4%), α-pinene (8.4%), and Z-β-ocimene (5.8%). Some biological and pharmacological activities were reported from Stachys species as well. According to previous reports, the hydroalcoholic extract of the aerial parts of other species (S. inflate) shows potent anti-inflammatory activity in the rat and the methanolic extract of the tuber of S. sieboldii has anti-anoxia actions in mice (10,11).
A literature survey has shown that S. fruticulosa has not previously been investigated for EO. But, the methanol extracts of its aerial parts were evaluated for their antioxidant activity and the total phenolic content using ferric ion reducing antioxidant power (FRAP) and Folin-Ciocalteu assays. The FRAP value and phenol content were reported 62.0945 ± 4.5272 mmol Fe 2+ /100 g dry weigh plant and 4450.368 ± 280.0766 mg gallic acid equivalent/100 g dry weigh plant, respectively (12). The obtained extracts from the aerial parts of S. fruticulosa were examined for its antibacterial activities against G + and Gstrains and the results revealed that S. fruticulosa methanolic extract inhibited the growth of Staphylococcus aureus and Bacillus cereus (13).

Plant Material and Isolation Procedure
The plant material was collected from the Savejbolagh area in the northwest of Tehran in July 2006. A voucher specimen was deposited in the Herbarium of the School of Pharmacy, Shahid Beheshti University of Medical Sciences. The aerial parts of the plant were air-dried and the oil was obtained by hydrodistillation-solvent extraction using a Clevenger-type apparatus for 4 hours and n-hexane.

Identification of Oil Components
The analytical GC method was carried out using a ThermoQuest 2000 GC coupled with the Thermo/Finnigan mass system and an RTx-1 glass capillary column (methyl phenyl siloxane 30 m X 0.32 mm, 0.25 μm film thickness), N 2 as the carrier gas with a flow rate of 1.5 mL/minute, the split ratio of 1:10, and a flame ionization detector. Temperature programming was performed from 50-250°C at 3°/minute with injector and detector temperatures to 270 °C . The quadrupole mass spectrometer operated at 70 eV ionization energy and electron ionization mass spectral spectra were obtained in the scan mode at the m/e range of 35-400 amu. Next, retention indices were determined by using the retention times of n-alkanes, which was injected after the oil under the same chromatographic conditions.
In addition, the retention indices for all components were determined using n-alkanes as the standard. Then, the constituents were identified by comparing retention indices (i.e., RRI & RTx-1) with those reported in the literature, along with comparing their mass spectra with those held in the Wiley library of mass spectra or with the published mass spectra (14).

Discussion
Stachys fruticulosa M. Bieb. was found to contain 17 compounds, in which monoterpene hydrocarbons (41.9%) and phthalides (23.2%) were the major classes. To the best of our knowledge, it is the first report on the chemical constituents of S. fruticulosa.
The main components of the oil from the other Stachys species were reported to be germacrene-D (6,7,10,11), muurolol (8), and α-pinene (5), which completely contradicts the findings of the present study, in which only 0.5% α-pinene was present in the oil. Hexadecanoic acid reported in the S. fruticulosa EO (1.2%) was found in S. palaestina (3) and S. pubescence as well (4).

Conclusion
Literature survey and investigation of the results of different studies was induced by considering the results of the present study and the comparison of the findings of other studies. Thus, it seems that many factors are involved in a wide diversity of chemical constituents of the EOs, and the investigation of volatile oils is a subject of pharmacognostical studies at all times.