Chemical Composition and Antimicrobial Activities of Helichrysum amorginum Cultivated in Greece
Chinou, Ioanna B
The chemical composition of the essential oil obtained from the aerial parts of the cultivated Helichrysum amorginum was investigated by GC and GC/MS. From the 29 identified constituents representing 85.0% of the total oil, spathulenol (36.6%) and [beta]-pinene (12.5%) were the major components. Furthermore, it was found that the oil exhibited a moderate antimicrobial activity against four Gram-negative bacteria, two Gram-positive and three pathogenic fungi.
Key Word Index
Helichrysum amorginum, Asteraceae, essential oil composition, spathulenol, [beta]-pinene, antimicrobial activity.
The genus Hclichrysum belonging to the family Asteraceae (1), is represented in Greece by 10 species among which six are endemic. The studied species in this report is Helichrysum amorpinum Boiss. et Orph. which is a dwarf shrub with yellow flowers that grows on dry cliffs. The plant material was offered for this study by the monastery of Hozoviotissa, on the Greek island Amorgos (Aegean Region), where H. amorginum is endemic as well as cultivated taxon.
The oil of H. amorginum growing wild on the island of Amorgos has been identified and reported previously (2) and in comparison with the oil obtained from the cultivated plant studied here, it appears to be quite different. Geraniol (32.1%), geranyl acetate (20.8%) and neryl acetate (17.5%) were identified as the main volatile components of the wild species, while spathulenol (36.6%), [beta]-pinene (12.5%), [alpha]-pinene (4.3%) and myrtenal (5.5%) were the major constituents of the cultivated oil.
The recent reports on oil compositions of the genus Helichrysum come from the Mediterranean taxa H. italicum (3), H. picardii (4),H. italicum ssp. serotinum, H. stoechas ssp. stoechas (5), H. rupestre and H. ainbiguum (6), as well as the Greek Helichrysum H. italicum ssp. italicum, H. italicum ssp. microphyllum, H. stoechas ssp. harrelieri, H. orientale, H. amorginum, H. taenari, H. heldreichii, and H. doerfleri species (2,7,8).
In the framework of our chemical and biological investigations on the volatile metabolites of Greek endemic and Mediterranean plant species, we report in this study the chemical constituents of the oil obtained from the aerial parts of the cultivated taxon H. amorginum.
Plant material: The plant H. aiuorginiDH was collected during its flowering period at the monastery of Hozoviotissa (June-July 1998) and was identified by D. Perdctzoglou. Voucher specimen has been deposited at the Herbarium ol the Laboratory of Pharmacognosy.
Isolation of the oil: The plant material (200 g) was subjected to liydrodistillation for 3 h in 1 Lt H,O, in a modified Cleveuger apparatus with a water-cooledoil receiverto reduce hydrodistillation overheating artifacts. The oil was collected in pesticide analysis grade Et,O and was subsequently dried over anhydrous sodium sulphate and then stored at 4°-6°C until analyzed. The oil was light yellow with a distinct sharp odor and a yield oi 0.23 (v/w).
GC and GC/MS analyses: The GC analyses were performed on a SRI 8610C gas Chromatograph equipped with a column injector (200°C) and a flame ionization detector (250°C). The capillary column used was a DB-5 (30 m x 0.32mm). The initial temperature of the column was 60°C and was heated to 280°C with a 3°C/min rate. The GC/MS analyses were performed on a Hewlett Packard 5973-6890 system operating on EI mode (equipped with a split/splitless injector (200°C) 1/10 split ratio, HP 5MS 30 m x 0.25 mm, 0.25 µm film thickness capillary column). The initial temperature of the column was 60°C and was heated to 280°C with a 3°C/min rate. In both analyses, helium was used as a carrier gas (2mL/ min). The identification of the compounds was based on comparison of their retention times with those of authentic samples and/or by comparison of their mass spectra with those of the NBS/NIST and Wiley Libraries (9). The retention indices were obtained according to Van den Dool (10).
Antimicrobial assays: The antimicrobial activities were determined using the dilution technique (11), by measuring the minimum inhibitory concentration (MIC) of the oils against two Gram-positive bacteria: Staplujlococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 12228), four Gram-negative bacteria: Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Klebsiella pneunwniae (ATCC 13883) andPseuclonionas ueruginosa (ATCC 227853), and three pathogenic fungi: Candida albicans, C. tropicalis and Torulopsis glabrata. The minimum inhibitory concentrations (MICs) were measured for [beta]-pinene, spathulenol and apinene (Table II). Standard antibiotics (netilmicin, amoxicillin and clavulanic acid) were used in order to control the sensitivity of the tested bacteria while 5-flucytocine, amphotericin B and intraconazole were used in order to control the tested fungi. Technical data have been described previously (11).
Results and Discussion
GC and GC/MS analyses of the oils led to the identification of the main components with their percentages and retention indices, listed in Table I. Twenty-nine constituents were identified and quantified from the oil of the cultivated H. amorgimim representing 85.0% of the total oil. The major constituents were spathulenol (36.6%), [beta]-pinene (f2.5%), [alpha]-pinene (4.3%) and myrtenal (5.5%).
In the antibacterial screening (Table II), the oil exhibited a moderate spectrum of activities against all tested bacteria (MIC values 6.50-20 mg/mL) while it was inactive against the pathogenic fungi. In contrast, the oil of the wild plant had shown a significant activity against all the tested bacteria (MIC values 0.75-7.5 mg/mL). For the screening, pure spathulenol, [alpha]-pinene as well as [beta]-pinene, were assayed on the same cultures under identical conditions to compare their activities with those of the investigated oils. The antimicrobial properties of the oil of the wild H. amorginum is suspected to be associated, to a considerable degree, to the existence of geraniol (2), while the activity of the present study is thought to be associated with [alpha]-pinene and spathulenol, which appear to possess similar activities against the tested microorganisms.
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Ioanna B. Chinou*, Christos Bougatsos and Dimitrios Perdetzoglou
Department of Pharmacognosy, School of Pharmacy, University of Athens, University Campus of Zografou 157 71
* Address for correspondence
Received: October 2000
Revised: December 2000
Accepted: January 2001
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