Composition and Antibacterial Activity of the Essential Oil of Phlomidoschema parviflorum from Iran

The monotypic genus Phlomidoschema Vved. belongs to the family Lamiaceae, subfamily Lamioideae and tribe Stachydeae [1]. Phlomidoschema parviflorum (Benth.) Vved. grows in restricted parts of Iran (Khorasan Province) as well as Afghanistan, west of Pakistan, Pamir Mountains, and Punjab of India. Morphologically, the species is characterized by perennial, multi-stemmed and subshrub habit and white subfloccose and canescent hairs on the leaves and stem [2]. The species is considered vulnerable and rare according to IUCN categories [3]. The infusion of the aerial flowering part of the plant is used locally for the treatment of gastrointestinal disorders, colic and stomachache. From the phylogenetic point of view, the genus is closely allied with the genus Stachys. The most relative and closest species of the genus Stachys to the genus Phlomidoschema was found to be S. lavandulifolia Vahl. [1]. As far as our literature survey could ascertain, there is no report on the oil composition and biological activity of P. parviflorum. So, in continuation of our research on the composition and biological activity of Iranian aromatic and essential oil-bearing plants of the mint family [4–10], we have investigated the composition and antibacterial activity of the essential oil of P. parviflorum. The aerial full flowering parts of Phlomidoschema parviflorum were collected from Khorasan-e Razavi Province, Ghayen towards Gonabad, Zirkuh region with an altitude of 1300–1350 m. A voucher specimen was identified and deposited (MPH-1220) in the Medicinal Plants and Drugs Research Institute Herbarium (MPH), Shahid Beheshti University of Tehran, Iran. Air-dried aerial parts (200 g) were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The oil was dried over anhydrous sodium sulfate and stored in sealed vials at 4C until analysis and tested. GC analysis was carried out on a Thermoquest-Finnigan Trace GC instrument equipped with a capillary DB-5 fused silica column (60 m 0.25 mm i.d., film thickness 0.25 m). The oven temperature was raised from 60C to 250C at a rate of 5C/min, then held at 250C for 10 min. Nitrogen was used as the carrier gas at a flow rate of 1.1 mL/min; the split ratio was adjusted to 1:50. The injector and detector (FID) temperatures were kept at 250C and 280C, respectively. GC-MS analysis was carried out on a Thermoquest-Finnigan Trace GC-MS instrument equipped with a DB-5 fused silica capillary column. Helium was used as the carrier gas at a flow rate of 1.1 mL/min with a split ratio of 1:50. The quadrupole mass spectrometer was scanned over 45–465 amu with an ionizing voltage of 70 eV. Gas chromatographic conditions were the same as given above for GC. The constituents of the oil were identified by calculation of their retention indices under temperature-programmed conditions for n-alkanes (C 6 –C 24 ), and the essential oil on a DB-5 column. Identification of individual compounds was made by comparison of their mass spectra with those of the internal reference mass spectra library or with authentic compounds and confirmed by comparison of their retention indices with those reported in the literature [11]. In vitro antibacterial activity of the essential oil was assessed against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 as models of Gram positive and Gram negative bacteria, respectively. The broth microdilution susceptibility method using 96 well trays was used to determine the minimum concentration of essential oil required for inhibition of visible growth (MIC) or killing (MBC) of the test strains. For this purpose, the standard protocol of CLSI (Clinical Laboratory and Standards Institute) was used with some modifications [12]. The inoculants of the bacterial strains were prepared from freshly cultured strains using sterile normal saline, which were adjusted to 0.5 McFarland standard turbidity and then further diluted (1:1000 for bacteria) by sterile Mueller-Hinton broth (MHB) just before adding to the wells containing a desired range of diluted essential oil.