MATERIALS AND METHODS

Fresh H. stoechas (150 plants) were collected from Green Mountain area in Lybia during spring of 2006 then transferred to the laboratory of Floriculture and Medicinal Plants Department, Faculty of Agriculture, Moshtohor, Benha University, Benha, Egypt. Plants were dried and their apical parts were prepared for extraction and investigations.

Extraction of essential oils: The essential oils were extracted from apical area of plants by steam distillation method for 2½ h (Blazques et al., 1990).

Preparation of ethanol extract: Fifty gram of dried apical parts of plant were soaked in ethyl alcohol 95% for 48 h, then the preparation was squeezed through double layers of muslin and the supernatant was transferred to glass funnel with Whatman filter paper No. 1. The ethanolic extract was concentrated under vacuum (Karam et al., 1999).

Chemical constituents: The chemical components of essential oils and ethanolic extract of H. stoechas were determined and separated using Shimadzu UV-260 spectrophotometer, infrared spectrophotometer Pye Unicam SP-1000 and Silica gel (70-230 mesh, Merck) was used for thin liquid chromatography.

Antimicrobial activity: Antimicrobial activity of essential oils and ethanolic extract were tested in duplicate with some gram-positive and gram-negative pathogenic bacteria, i.e., Staphylococcus aureus, Staphylococcus epidermis, Staphylococcus citrus, Sterptococcus pneumonae, Escherchia coli, Enterobacter coleacae, Klebsiella pneumonae, Pseudomonas aeruginosa and three types of fungi i.e., Candida albicans, Candida tropicolis and Torulopsis glabrata. The tested bacteria were kindly supplied by Diary Research Laboratory, Food Science Department, Faculty of Agriculture, Moshtohor, Benha University, while the tested fungi were supplied by Plant Pathology Laboratory, Agriculture Botany Department, Faculty of Agriculture, Moshtohor, Benha University.

Central wells were made using sterilized cork porer (φ 6 mm) in the center of each plate and filled with the tested plant extract (200 μmL) and then inoculated under sterilized conditions with both tested bacteria and fungi. Bacterial plates were incubated at 37°C for 72 h and fungal plates were incubated at 25°C for full growth of control plate (5-7 days).

The anti-microbial activity of each test solution was estimated by measuring the zone of inhibition (clearing) around the central well. The diameter of the disc was subtracted from the measured clear zone (Levy, 2001).

RESULTS AND DISCUSSION

Chemical constituents
Essential oils: The essential oils isolated from flowers and leaves of H. stoechas were obtained in yield 0.7% (v/w). The major components in oil were alpha-pinene (59%), limonen (16.7%), alpha-bisabolol (9.6%), beta-carophyllene 4% and alpha-humlene (2.5%) (Table 1). These results were in agreement with that reported by Rios et al. (1991), Vermin and Poite (1998). Tsoukatou et al. (1999), Ascensao et al. (2001) and Carini et al. (2001).

The oils also contain other components in small amounts as geraniol and camphen which has bacteriostatic activity against gram positive and gram negative bacteria. This result was similar to that reported by Rios et al. (1990) and Chinou et al. (1997). Phloroglucinol and acetophenone derivatives were also identified from oil isolated from flowers and leaves of plants.

Ethanol extract: The chemical analysis of ethanolic extract obtained from H. stoechas revealed the identification of ten constituents in the extract including the three naturally occurring isomers of caffeoylquinic acid (37%), 2 isomeric dicaffeoylquinic acids (26.3%), 2 isomeric naringenin glucosides (2.5%) and tetrahydroxychalcone-glucoside (1%) (Table 1). These results agreed with those reported by Mericli et al. (1992) and Ali-Shtayeh et al. (1998). The chemical analysis revealed the presence of other constituents which couldn’t be quantified.

Table 1:	Chemical analysis of essential oil and ethanolic extract isolated from Helichrysum stoechas

Table 2:	Antimicrobial activity of essential oils extracted from Helichrysum stoechas
- = no effect, + = active with diameter of inhibition zone of 5-12 mm, + + = inhibition zone 13-20 mm, + + + = inhibition zone more than 20 mm

Antimicrobial activity
Essential oils: Data in Table 2 revealed that the essential oils extracted from leaves and flowers of H. stoechas had different inhibitory activities against some pathogenic bacteria and fungi. The essential oils had potent inhibition activity on Staphyllococcus aureus, Staphyllococcus epidermis and Klebsiella pneumonae (zone of inhibition varied from 21.5-24 m), while oil had moderate inhibitory activity on Eschershia coli (zone of inhibition 15.5 mm). Mild inhibitory activity of essential oil was observed on Enterbacter coleaca (5.5 mm), while it had no effect on other pathogenic organisms. The essential oil had moderate antifungal activity on Candida albicans only without any effect on other tested fungi.

These results agreed with that reported by Tsoukatou et al. (1999) and Roussis et al. (2002). The antimicrobial activity of essential oil extracted from H. stoechas might be attributed to that, the oil contain phloroglucinol and acetophenone derivatives which had antimicrobial activity against some types of pathogenic bacteria and some pathogenic fungi (Tomas et al., 1990).

The essential oils recorded in the present study were comparable with those of Lourens et al. (2004). The occurrence of terpenes such as α-pinene and sesquiterpenes such as β-caryophyllene exhibited activity in the in vitro 5-lipoxygenase assay (Baylac and Racine, 2003). In addition, administration by inhalation suggests that the volatile aromatic compounds may play a role in anti-infective therapy and several studies indicate significant antimicrobial properties for Helichrysum oils (Hutchings and Van Staden, 1994; Roussis et al., 2000; Van Vuuren et al., 2006).

Ethanol extract: The antimicrobial activities of ethanolic extract extracted were illustrated in Table 2. The ethanolic extract had potent inhibition activity on most gram-negative tested bacteria i.e., Klebsiella pneumonae, Enterobacter coleacae, Pseudomonas aerugenosa and Escherchia coli (zone of inhibition 22-25 mm). On the other hand, the ethanolic extract had moderate antibacterial activity on Staphyllococcus aureus while it had no effect on other tested bacteria and pathogenic fungi. These results were similar to that reported by Ali-Shtayeh et al. (1998).

The constituents of ethanol extract in present study were previously recorded in aqueous extract from H. stoechas (Carini et al., 2001) and other Helichrysum species (Tepe et al., 2005; Lall et al., 2006). These constituents were shown to display radical scavenging properties, with potency comparable to that of Trolox, the water-soluble analogue of vitamin E since many respiratory conditions and skin ailments are associated with inflammation (and hence release of free radicals) the presence of these anti-oxidant agents could explain the effectiveness of H. stoechas in the treatment of these conditions.

In conclusion, the present investigation indicates the potency of H. stoechas as antimicrobial and antioxidant plant. However, the observation that some organisms tolerate essential oils while were susceptible to ethanol extract (vice versa) robustly supports the traditional medicinal uses of H. stoechas as the whole crude extract.