CHEMICAL CONSTITUENTS AND BIOLOGICAL POTENTIAL OF ESSENTIAL OILS OF HELICHRYSUM ITALICUM (

SUMMARY The chemical constituents of essential oils isolated from the Helichrysum italicum (Roth) G. Don by steam-distillation were analyzed by GC-MS. The oils were predominantly composed of sesquiterpene hydrocarbons with 52.35%, with γ-curcumene as major constituent (14,11%), β-selinene (11,31%) and α-curcumene (10,42%). The antimicrobial activity of the essential oils was evaluated against Gram-positive and Gram-negative bacteria and fungi. Results showed that the oils exhibited antibacterial activities and that Staphylococcus aureus , Listeria monocytogenes and Bacillus subtilis are very sensitive.


INTRODUCTION
The interest in herbal remedies has been significantly increased in the last few decades.In the worldwide, all the natural resources including medicinal plants, fungi and algae are screened for their biological activities (Enazi et al. 2018).Medicinal plants play an important role in the discovery and isolation of new drugs.The essential oils are known as a secondary plant metabolite which form part of naturopathic therapy, are widely known for their antimicrobial properties (Rapper et al. 2013;Hosseini et al. 2016).Various biological characteristics, such as digestive, anti-inflammatory, sedative, antioxidant, antimicrobial, antiviral, and also cytotoxic activities have been attributed to the essential oils (Carvalho et al. 2018).
Numerous members of the Asteraceae family are important crop species of cut flowers and ornamentals, as well as being medicinal and aromatic plants, many of which produce essential oils used in pharmaceutical industries (Abad et al. 2013).Helichrysum is one of the important genera for medical purposes.This genus is currently widely distributed in Africa, Madagascar, the Mediterranean basin, Macaronesia, central Asia and India (Juliano et al. 2018) includes more than a thousand taxa that have a higher occurrence in the Mediterranean areas of Europe (Viegas et al. 2014;Facino et al. 1988).Almost 25 species are native of Mediterranean area and the most widespread species is Helichrysum italicum (Roth) G. Don (Guinoiseau et al. 2013).H. italicum is a typically Mediterranean plant.It is a small aromatic shrub with yellow flowers, up to 40-50 cm high, growing on dry cliffs and sandy soil.It is widespread along the East coast and on the islands of the Adriatic sea (Mastelic et al, 2005).The genus Helichrysum has an important source of secondary metabolites such as flavonoids, phytocannabinoids, triterpenoids, diterpenoids, steroids, organic acids, phloroglucinol and acetophenone derivatives (Guinoiseau et al. 2013).
The aim of this study was to determine the chemical composition and antimicrobial activity of H. italicum oils from Montenegro.

MATERIAL AND METHODS
Plant material of Helichrysum italicum was collected from several sites in the south of Montenegro; Isolation of the essential oils the dried plant samples was subjected to steam distillation for 3 hours.Samples oils were dried over anhydrous sodium sulphate and stored at low temperature before analysis.The analyses were performed on a gas chromatograph-mass spectrometer, GC-MS QP 2010 plus, Shimadzu, equipped with split-splitless injector and a ZB-5MS capillary column (30m × 0.25 mm; 0.25 µm film thickness).The chromatographic conditions were as mentioned in the preceding paragraph.Injector was heated at 260 °C, detector (MSD) was heated at 260 °C, while the column temperature was linearly programmed from 35 to 270 °C (5.0 °C min-1).The EI MS spectra (70 eV) were obtained in the scan mode in m/z range 50-500.

Antimicrobial activity
To assess the antimicrobial properties of essential oil of Helichrysum italicum, nine strains of pathogenic microorganisms were used in the study (Table 1).All microorganisms were derived from the culture collection of the Institute of the Health of Montenegro and Department of Biology, Faculty of Sciences and Mathematics, University of Montenegro.Bacteria were subcultured from nutrient agar slopes into nutrient broth and in Sabouraud dextrose broth for Candida albicans.The resulting bacterial broth was used as the inoculum in microbial analysis.Cell numbers of the inoculum were standardized at 10 5 cell/mL.Incubation lasted 18 h at 37°C for bacteria and 48 h at 26°C for C. albicans.
Essential oils dilutions were prepared directly in the Mueller-Hinton broth and tested in concentrations range from final concentrations of 71, 35, 14, 7, 3.5, 1.4 µl/ml.
The minimal inhibition concentration (MIC) values were determined for the microbial strains to the essential oils of Helichrysum italicum.The MIC is defined as the lowest concentration at which the microorganism does not demonstrate visible growth.Tests were carried out in duplicate The main components in H. italicum oils are γ-curcumene (14,11%), βselinene (11,31%) and α-curcumene (10,42%).From Table 2 it can be seen that in the essential oils from H. italicum dominates sesquiterpene hydrocarbons with 52.35%.Other components that are present in the essential oil are: α-selinene (6.07%), neryl acetate (3,91%) and α-copaene (3,68%).The monoterpene 1,8cineole is represented in essential oil with 0,22%.The twenty three compounds were identified, representing about 67.32% of the total oil.According to the literature, essential oil of H. Italicum has significant chemical and biological potential.However, the composition of essential oils varies considerably depending on the method of extraction, time of maturation, soil composition and climatic conditions.

Antimicrobial potential
The data of the antimicrobial activity assessed by dilution method showed that in general the antimicrobial activities of the tested essential oil were varied on the type of microorganism (Table 3).The antimicrobial activity of the essential oil of H. italicum from Montenegrian origin has been evaluated for the first time

CONCLUSIONS
Our results revealed that γ-curcumene, β-selinene and α-curcumene are the major components of H. italicum essential oil from Montenegro.The oils possess rather a significant activity against microorganisms S. aureus, L. monocytogenes and B. subtilis.Investigations by other authors have indicated that H. italicum oils from the Mediterranean region has significant biological potential, but also significantly differs from the area and ecological conditions.Since these first reports of the biological activity of H. italicum from the area of Montenegro research should be continued and extended to biotesting.Mastelic, J., Politeo, O., Jerkovic,I., Radosevic, N. (2005) Research work Paolini et al, 2006 reported that in composition of essential oils of H. italicum dominated monoterpenes such as neryl acetate, neryl propanoate and α-pinene.According research Morone-Fortunato et al. 2010 in essential oils of H. italicum are a large proportion of sesquiterpenes.The samples from Italian essential oils of H. italicum contained mainly γ-curcumene, β-selinene and α-selinene (Morone-Fortunato et al. 2010).

Table 1 .
Microbial strains and reference number ATCC The results of the chemical analyses of essential oils of.H. italicum investigated are presented in Table2.

Table 2 .
Chemical composition (% of compound) of the essential oils from H.

Table 3 .
Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of H. italicum tested Essential Oils against microorganisms using dilution method Staphylococcus aureus and Listeria monocytogenes (with an MIC of <1.4 μg /mL), and Bacillus subtilis (with an MIC of 1.4 μg /mL) were the most sensitive bacteria.Investigation according Nostro et al (2001) reported that essential oil of H. italicum to inhibit the growth of Staphylococcus aureus in a concentration dependent manner, with no difference in sensitivity between methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus strains.