Genus Hypericum L.includes great numberof herbal speciesof which H.perforatum L. is the most significant and the most investigated one. There are less data about other species of this genus, although they can be interesting both from scientific and practical point of view.

We have therefore focused our attention to Hypericum olympicum from the area of south-east Serbia from which we have isolated essential oil and examined its chemical composition and antimicrobial properties. The chemical composition of the essential oil was examined by GC-MS method. Forty components were detected and the most important 12 were identified.

We have applied dilution method on liquid nutritive media (Medium 3 - Difco I TSB-Torlak) with cellulose discs for investigation of antimicrobial action of the essential oil (MIC and MLC), by using the following 10 test cultures: Escherichia coli 95, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 10031, Salmonella enteritidis ATCC 13076, Sarcina lutea ATCC 9341, Bacillus subtilis ATCC 6633, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.

The essential oil shows the best antimicrobial action (MIC - 5 ml - MLC - 10 ml) in the following test cultures: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Bacillus subtilis ATCC 6633, Bacillus subtilis NCTC 8236, while the following test cultures are less sensitive: Klebsiiella pneumoniae and Sarcina lutea ATCC 9341.

Genus Hypericum L. comprises a large number of herbal species, among which the best known and the most investigated one, was herbal species H. perforatum L. (1,2,3). There have been few data about the other herbal species of genus Hypericum L., although they can be interesting, both from the scientific and the practical point of view (4).

In this paper, we investigated herbal species Hypericum olympicum L. from the region of the southeast Serbia. The results of those investigations of the chemical composition as well as antimicrobial activity of the essential oil have been given.

Herbal material (floral part of the plant) was collected on the Rujan mountain (Southeast Serbia) in July 1995 and dried, ground and sifted through a sieve with a hole of 1 mm.

Essential oil (0.32%) was isolated by hydrodistillation in a Clevenger type apparatus (5) and submitted to GC-MS analysis. For this purpose, HP-5890 gas chromatograph, equipped with SPB-5 fused silica capillary column (30 m x 0.25 mm i.d., 0.25 mm film thickness) and HP-5971 mass selective detector, was used. After injection, column firstly was kept at 50°C (2 min) and then heated (5°/min) up to 300°C. Carrier gas was helium. Electron impact mass spectra (70 eV) were acquired in scan mode and compared with those from the corresponding base of mass spectra (Wiley's library). Among forty detected components, the most important 12 were identified.

Microbiological activity of the essential oil was investigated by the assay of minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) with the application of the following 10 test microorganisms: Escherichia coli 95 (origin - Institute for Pharmacy Torlak, Belgrade), Salmonella enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae ATCC 10031, Sarcina lutea ATCC 9341, Bacillus subtilis ATCC 6633, Bacillus subtilis NCTC 8236, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404 (origin - Oxford, England) (6,7).

As nutritive media, the following have been used: Medium 3 (Difco Laboratories Detroit Michigan USA) for growing of gram positive and negative bacteria and Tripton soy broth (TSB-Torlak) for growing Candida albicans and Aspergillus niger. Medium for the total number of microorganisms (bacteria and fungus - UBMO, Torlak) with pH after sterilization 7.0 for the assay of total number of microorganisms in working inoculum and selective media for isolation and detection of test-microorganisms were: Endo agar, Torlak (for E.coli), SS or WB-agar, Torlak (for S. enteritidis),Cetrimid agar, Torlak (for Bacillus subtilis), Chapman medium, Torlak (for S.aureus and S. lutea), and Tripton soy agar (Torlak) for C. albicans and A. niger.

Nutritive media have been prepared, sterilized and pH has been checked according to the instruction of the manufacturer.

The procedure for the assay of antimicrobial activity consisted of the following. From the suspension of test-microorganisms, formed with 10 cm³ 0.9 % NaCl and 24 h culture, working inoculum has been prepared by injecting 0.1 cm³ of the suspension in 9.9 cm³ of Medium 3 (for bacteria) and Tripton soy broth (for fungi) in a luminary chamber for sterile work. On sterile cellulose discs (for easier visual following of turbidity) of the diameter 12.7 mm (Schleicher & Schül, Dassel, Germany), the quantities of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60 ml of the essential oil have been applied by micro-pipettes, which were soaked in the test-tubes of dilution sequence with 0.5 cm³ of the appropriate liquid medium, after which each of 1 cm³ of working inoculum for every test-microorganism, in which antimicrobial action was previously determined by the method of diffusion on solid nutritive media, that is the assay of the essential oil quantity that gives the inhibition zone of 1-2 mm in diameter (see Fig. 2).

Incubation was performed at 37°C both for bacteria and for fungi. 1 cm³ of working inoculum dilution of 10^-4 was covered by the medium for the total number of microorganisms in inoculum after 24-48 h at 35-37°C (CFU/ml). The changes in the clarity of inoculum (the essential oil activity) in test tubes were followed 72 h (3 days). Then, insemination was carried out from the test tubes without turbidity, each 0.1 cm³ on the medium for the total number of microorganisms, as well as the application of inoculum by eza on selective nutritive media (assay of MIC and MLC - see Fig. 3 and Table 2)

The results of the essential oil investigation of H. olympicum L. are given in the Table 1. and on the Fig. 1.
The results of the antimicrobial activity investigation are given in the Table 2 and in figures 2 and 3.

Figure 1. GCMS chromatogram of the essential oil of Hypericum olympicum L.
DISCUSSION

As shown in Table 1, and Figure 1, 40 components were detected in examined oil, 12 were identified, and the main componenents are: a-cubebene, d-cadinene, g-cadinene, b-farnezene and caryophyllene.

In order to examine antimicrobial activity we used dilution method, as a precise and relevant one for determination of MIC and MLC. The results presented in Table 2 and figures 2 and 3 reveal that H. olympycum L. essential oil has effect on test cultures (bacteria and fungi). Its antimicrobial activity on resistent fungus C. albicans has special importance.

As we in our previous studies (8,9,10,11) examined chemical composition and antimicrobial activity of several Hypericum species essential oils, under the same experimental conditions, it is possible to compare examined species.

In the framework of this paper, we expressed our general observation that the H. olympicum L. essential oil, although containing lower number of detected and identified components, showed the strongest antimicrobial activity.

The more detailed interpretation of these results will be the subject of our further work.

Figure 2. Schema of the diffusion method for the assay of antimicrobial activity of the
essential oils H. olympicum L.


Figure 3. Schema of the dilution method (with cellulose disc) for the assay of antimicrobial activity of the
essential oils H. olympicum L.
CONCLUSION

On the basis of the results obtained, we concluded following:

1. Forty components were detected in examined essential oil, 12 were identified and main components are: a-cubebene, d-cadinene, g-cadinene, b-farnezene and b-caryophyllene.

2. Dilution method with cellulose disc has given good and precise results of determination MIC and MLC (5-10ml, 10-20ml respectively), depending on kind of the test culture.

3. MIC and MLC values were constant during all days of the experiment (72 h) and recorded for all tested micro-organisms.

4. H. olympicum L. essential oil (vintage 1995) showed the strongest effect on Gram (-) bacteria Pseudomonas aeruginosa ATCC 9027, Salmonella enteritidis ATCC 13076, Klebsiella pneumoniae ATCC 10031and Gram (+) Staphylococcus aureus ATCC 6538, Bacillussubtilis ATCC 6633, Bacillus subtilis NCTC 8236, as well as on the yeast Candida albicans ATCC 10231.

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