GC/MS analysis and antimicrobial activity of essential oils of Telekia speciosa (Schreb.) Baumg

1University of Tuzla Faculty of Pharmacy, Urfeta Vejzagića 8, 75000 Tuzla, Bosnia and Hercegovina 2University of Tuzla Faculty of Technology, Urfeta Vejzagića 8, 75000 Tuzla, Bosnia and Hercegovina 3Institute for Biological Research "Siniša Stanković"-National Institute of Republic of Serbia, University of Belgrade, Department of Plant Physiology, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia 4University of Belgrade Faculty of Pharmacy, Department of Pharmacognosy, Vojvode Stepe 450, 11221 Belgrade, Serbia *Corresponding author: ermina.cilovic@untz.ba


INTRODUCTION
Telekia speciosa (Schreb.) Baumg. (Asteraceae) is a perennial herbaceous plant widespread in Eastern and Central Europe and the Balkan Peninsula. It is up to 200 cm high and it has alternating, wide, whole leaves and large heterogeneous yellow flowers with a diameter of 5-8 cm which can be individual or in cluster inflorescence. It inhabits wet and shady positions in mountain woodlands (Chalchat et al., 2004;Domac, 1994), and somewhere it is planted as a decorative plant. It is closely related to genus Inula, including Inula helenium L., a well-known medicinal plant (Stojakowska et al., 2011). The underground parts of T. speciosa are traditionally used as a remedy for bronchial asthma in Balkan countries (Serbia, Bosnia and Herzegovina). For example, smoke after burning underground parts is inhaled for the cure of asthma (Redzić, 2007).
The underground parts of T. speciosa contain essential oil, bitter compounds and inulin (Marković et al., 2010). Phytochemical investigations have revealed T. speciosa as a rich source of sesquiterpene lactone -isoalantolactone, especially in its underground parts which amount is equivalent to I. helenium underground parts (Radulović et al., 2010;Stojakowska et al., 2011;2015a). The aerial parts extracts have been found to contain fatty acids, namely palmitic, linoleic, oleic, and caproic acids, and sterols (Deliorman et al., 2002;Orhan and Sener, 2003). Aerial parts of T. speciosa accumulated miscellaneous sesquiterpene lactones, mainly of guaiane, pseudoguaiane, xanthane, and eudesmane type (Stojakowska et al., 2015b). Isoalantolactone, a bioactive compound present in T. speciosa underground parts and aerial parts essential oils, has been found to have various pharmacological activities including anti-inflammatory, antimicrobial, and anticancer properties, with no significant toxicity. Alantolactone and isoalantolactone have been extensively investigated on several cancer cell lines, such as colon, melanoma, ovary, prostate, lung, and leukemia (Rasul et al., 2013). A previous study also revealed that extracts from both leaves and flowers of T. speciosa showed high antiproliferative activity against the cancer cell lines tested (Yuan et al., 2018). In addition, several in vitro and in vivo studies evaluated antimicrobial properties of isoalantolactone against methycillin-resistant Staphylococcus aureus (MRSA) and α-toxin, a product of most S. aureus microorganisms and essential for the pathogenesis of pneumonia (Qiu et al., 2011;Zhou et al., 2020). Unlike T. speciosa, for which, as far as we know, there are no available data on antimicrobial activity, essential oil of I. helenium underground parts was active against several Gram-positive and Gram-negative bacteria and Candida strains (Deriu et al., 2008). Increasing bacterial resistance to antibiotics, antimicrobials, and antifungal agents is a growing concern facing the medical, pharmaceutical, sanitation, and food industries (Krist et al., 2015). An alternative to reduce the use of synthetic chemicals is the search for antimicrobials from medicinal plants. In this context, plants constituents, such as essential oils and their main components, terpenoids, have attracted considerable interest (Swamy et al., 2016). The aim of the present study was to analyze the essential oils of both, aerial and underground parts of T. speciosa and to evaluate antimicrobial activity of essential oils.

Plant material
Aerial and underground parts of T. speciosa were collected at the location of Karaula, Olovo municipality (Bosnia and Herzegovina) (N44°10'22.3", E18°38'58.6") during the flowering period in July 2018. The plant material was identified according to Flora Croatica (Domac, 1994) by authors and the voucher specimen was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, the University of Tuzla. The plant material was cleaned, cut, and air-dried.

Isolation and GC-FID/MS analyses of volatiles
The dried aerial and underground parts of T. speciosa were chopped and subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The obtained essential oils were separated, dried over anhydrous sodium sulfate, and stored at -20°C until the analysis. The constituents of essential oils were determined by the GC-FID/MS method, as described previously (Cilović et al., 2019).

Antimicrobial activity of essential oils
The antimicrobial activity (AMA) of T. speciosa essential oils were tested against six strains of bacteria, i.e. Gram-positive Staphylococcus aureus (ATCC 6538 and clinical isolate) and Bacillus cereus (clinical isolate), and Gram-negative Pseudomonas aeruginosa (ATCC 27853 and clinical isolate) and Escherichia coli (ATCC 35210), as well as against two strains of fungus Candida albicans (ATCC 10231 and clinical isolate). The microorganisms were obtained from the Mycology Laboratory of the Institute for Biological Research "Siniša Stanković", University of Belgrade, Serbia. The antibacterial assay was done by microdilution method (Cazella et al., 2019) utilizing 96-well microtiter plates to determine the minimal inhibitory concentration (MIC) and minimal bactericidal /fungicidal concentration (MBC/MFC). The inoculum was cultivated in a solid medium to verify the absence of contaminations, and for validation. Nutrient media were Tryptic Soy Broth (TSB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi. The microorganism suspensions (inocula) were adjusted with sterile saline until the concentration of 1.0 x 10 5 CFU mL -1 . The inoculum was prepared daily and stored at 4°C until its utilization. The essential oil was added to the nutrient medium for the growth of microorganisms. Then the microorganism inocula were added and the plates were incubated for 24 h at 37°C for bacteria and 72 h at 28°C for fungi. The lowest concentration without visible microbial biomass growth under a binocular magnifying glass was defined as MIC. Determining the absence of growth of microorganisms, i.e. determination MBC/MFC, was performed by serial reinoculation of 10 µL of inoculated medium from wells where no growth of the microorganism was recorded in 100 µL of sterile nutrient medium and reincubation for 24 h at 37°C for bacteria and for 72 h at 28°C for fungi. The results were confirmed after adding 40 µL of purple p-iodonitrotetrasolium chloride (Sigma), microbial growth indicator solution (0.2 mg mL -1 distilled water) to each well and incubation for 30 minutes at 37°C (Tsukatani et al., 2012). Comparison of color intensity was performed with control wells in which unhindered growth of microorganisms was enabled, and commercial antimicrobial agents streptomycin (Sigma), ampicillin (Panfarma, Belgrade, Serbia), and ketoconazole (Zorka farma, Šabac, Serbia) were used as positive controls. Inoculated medium without added essential oil was used as the negative control. The antimicrobial tests were carried out in triplicate. The results were expressed in values of arithmetical average ± standard deviation.

Content and composition of essential oils
The aerial parts of T. speciosa contained 0.02 % (v/w) of yellow, liquid fragrant essential oil. The identified 49 constituents accounting for 85.05 % of the oil are presented in Table 1. The essential oil of aerial parts (AEO) of T. speciosa was characterized by the presence of a high concentration of oxygenated sesquiterpenes (49.14 %). The major components were (E)nerolidol (11.54 %), caryophyllene oxide (10.54 %), and (2Z,6E)farnesol (4.52 %). Sesquiterpene hydrocarbons constituted 7.77 % of the oil with the dominant compound β-caryophyllene (4.90 %). Nerol (4.77 %) was the major representative of oxygenated monoterpenes, which constituted 15.77 % of the oil. Non-terpene (other) compounds presented an appreciable amount of essential oil (12.37 %) with dominant (E)-phytol (4.14 %). The amount of essential oil found in the underground parts (UEO) of T. speciosa (0.31 %, v/w) was higher than in the aerial parts. The oil was yellowish semi-solid mass, with aromatic odor. By cooling and standing it crystallizes in the form of opaque needle-like crystals. The identified 35 constituents accounting for 97.26 % of the oil are presented in Table 1. This essential oil was characterized by the presence of a high concentration of oxygenated sesquiterpenes (90.33 %) with isoalantolactone (IAL) being the major component (83.41 %). Alantolactone, an oxygenated sesquiterpene, was also presented, but in a substantially lower amount (2.56 %). Sesquiterpene hydrocarbons constituted 3.13 %, while oxygenated monoterpenes constituted 3.80 % of the oil, with thymol derivative 10-isobutyryloxi-8,9-epoxithymol isobutyrate (1.42 %), found in appreciable amount. In the previous investigations, aerial parts collected during the flowering period of T. speciosa from Bosnia and Herzegovina and Serbia contained slightly higher amounts of essential oil than in the current study, i.e. 0.04 % and 0.06 % (v/w), respectively. The major components of those oils were oxygenated sesquiterpenes (E,Z)-farnesol (12.0 % in the essential oil from Serbia), (E)-nerolidol (10.2-10.3 %), caryophyllene oxide (4.5-8.2 %), (Z,E)-farnesol (7.7 % in the essential oil from Bosnia and Herzegovina), β-caryophyllene (5.4 %), similar to the currently analyzed AEO (Cilović et al., 2019;Radulović et al., 2010). It is worth noting that T. speciosa leaf essential oil, originating from Poland, as dominant constituents also contained a farnesol isomer (i.e. (E,E)-farnesol, 21.2 %) and (E)-nerolidol (17.9 %), while flower essential oil was abundant with isoalantolactone (23.0 %) and 10-isobutyryloxy-8,9-epoxythymol isobutyrate (20.5 %) along with other several thymol derivatives (Wajs-Bonikowska et al., 2012). These two compounds were present in several times lower amounts in the currently analyzed oil, which could be explained by the fact that it was obtained from the complete aerial parts in which flowers represented only one minor part. Based on the previously and currently analyzed essential oil from T. speciosa aerial parts it could be concluded that the characteristic compounds for analyzed oil are (E)-nerolidol, caryophyllene oxide, different stereoisomers [(E,Z)-, (Z,E)-, (E,E)-] of farnesol, β-caryophyllene, and other compounds present in lower amounts.
In the previous investigations, underground parts collected during the flowering period of T. speciosa from Bosnia and Herzegovina and Poland contained almost equal or somewhat higher amounts of essential oil than in the current study, i.e. 0.29 % and 0.41 % (v/w), respectively. Only underground parts of T. speciosa collected during the flowering period from Montenegro contained higher amounts of essential oil than in the current study, i.e. 1.7 %. The dominant compound in all those samples was isoalantolactone (62.3 % -95 %). Beside isoalantolactone, other oxygenated sesquiterpene alantolactone (2.4 % in the essential oil from Bosnia and Herzegovina), and thymol derivatives 10-isobutyryloxi-8,9-epoxithymol isobutyrate (2.9-3.4 %), 9-isobutyryloxythymol isobutyrate (2.1 % in the essential oil from Poland) were also present in the previously analyzed essential oils (Chalchat et al., 2004;Cilović et al., 2019;Wajs-Bonikowska et al., 2012). Results about the chemical composition of T. speciosa underground parts essential oil obtained in this study are in accordance with the published literature data. Taking into account previous data and the currently analyzed oil underground parts of T. speciosa may be considered as a potential raw material for isoalantolactone isolation.

Antimicrobial activity of essential oils
Both investigated essential oils AEO and UEO exerted antimicrobial activity against all tested microorganisms (Tables 2 and  3). As shown in Table 2, bacterio-and fungistatic effects of UEO were more prominent then those of AEO, but lower then the control antibiotics streptomycin and ampicillin and antifungal ketoconazole. Inhibition of growth of tested microorganisms by T. speciosa UEO was achieved with MICs ranging from 1.0 to 11.0 mg mL -1 , while MICs of AEO varied from 4.0 to 30.0 mg mL -1 . Similarly, bactericide and fungicide effects were observed in the presence of lower concentrations of UEO (MBC/MFC 4.0-15.0 mg mL -1 ) in comparison to AEO (MBC/MFC 7.0-90.0 mg mL -1 ) (Table 3). However, both essential oils were less effective than the antimicrobial drugs that were used as positive controls. Among tested bacteria, ATCC strains of S. aureus and E. coli were the most susceptible strains to T. speciosa UEO (MIC 1.1, and 1.0 mg mL -1 , respectively; MBC 7.0 mg mL -1 for both microorganisms), while both standard and clinical strains of P. aeruginosa were the most (and equally) resistant in the presence of this oil (MIC and MBC were 7.0 mg mL -1 , and 15.0 mg mL -1 , respectively). There was no apparent difference in susceptibility toward UEO between Gram-positive and Gram-negative bacteria. On the other hand, AEO exhibited stronger effects on the tested Gram-negative bacteria than on Gram-positive ones.
In addition, even though AEO showed generally lower activity in comparison to UEO, the standard strain of P. aeruginosa (the only one among the tested strains) was more susceptible to AEO (MIC 4.0 mg mL -1 ) than to UEO (MIC 7.0 mg mL -1 ). The most resistant strain to T. speciosa AEO was a clinical isolate of B. cereus (MIC 30.0 mg mL -1 ; MBC 90.0 mg mL -1 ).
Clinical isolate of C. albicans was highly susceptible to UEO (MIC 1.0 mg mL -1 ), but not to AEO (MIC 11.0 mg mL -1 ). Both EOs showed similar and relatively low anticandidal activity on standard strain of this fungus. The analyzed essential oils did not show a large difference in AMA against clinical and ATCC strains of microorganisms, in contrast, to control antibiotics (streptomycin, ampicillin) and antifungal ketoconazole which showed a lower effect on clinical than on standard strains, except in the case of C. albicans. Clinical isolate of this fungus is more than 10 times susceptible to UEO compared to the standard strain. The potential of major constituents in the currently analyzed oils to act as antimicrobials or to contribute to the antimicrobial effects of essential oils was confirmed in several previous studies. Qiu et al. (2011) investigated the activity of IAL on S. aureus and α-toxin. α-Toxin is a product of most S. aureus microorganisms and is essential for the pathogenesis of pneumonia. MIC value of IAL for S. aureus was more than 1.024 mg mL -1 and IAL inhibited the expression of α-toxin, in S. aureus at very low concentrations. Results were confirmed in vitro and in vivo. Also IAL, in combination with penicillin G, exhibited significant synergism against 21 β-lactamase-positive S. aureus strains (including MRSA). MIC values of the penicillin G alone and in combination with IAL against tested bacterial S. aureus and MRSA strains were reduced three to even twenty-six times (Zhou et al., 2020). Nerolidol, the major compound in the currently analyzed T. speciosa AEO, is common component found in the essential oil of various medicinal plants. Many studies showed its antimicrobial activity. The mixture of (Z,E)-nerolidol exhibited potent antimicrobial activity against S. aureus and 20 strains of MRSA (Hada et al., 2003). (E)-Nerolidol also exhibited antimicrobial activity against S. aureus with MIC values ranging from 125 to 500 µg mL -1 (Braca et al., 2008). In another study MIC values for nerolidol against Streptococcus mutans, Salmonela enterica, and Aspergillus niger were 25.0, from 3.9 to 15.6, and 62.5 µg mL -1 , respectively (Chan et al., 2016). For (E,E)-farnesol, alicyclic sesquiterpene alcohol present in many essential oils as well as in T. speciosa AEO, good antimicrobial and fungistatic activity is reported (Krist et al., 2015). Even though MIC values for caryophyllene oxide and βcaryophyllene, to the best of knowledge, were not yet published, the AMA of essential oil from leaves of Croton heliotropiifolius which contained β-caryophyllene as dominant compound inhibited the growth of several Gram-positive and Gram-negative bacteria. Its antibacterial activity was characterized as weak to moderate against the analyzed strains (de In the context of the current investigation, it is worth noting that there is a great similarity in the chemical composition of the essential oil of T. speciosa underground parts with previously analyzed essential oil of Inula helenium L. root which exhibited substantial antimicrobial activity against several bacterial and fungal strains. The dominant principles of I. helenium root essential oil were three isomeric eudesmane-type sesquiterpene lactones: alantolactone (51.3-55.8 %), isoalantolactone (26.1-36.9 %), and diplophyllin (5.1 %). It was experimentally confirmed that essential structural parts responsible for antimicrobial activity of I. helenium root essential oil are eudesmane core olefinic bonds, alongside the α,β-methylenelactone ring of sesquiterpene lactones. Bacterial and fungal strains used in this research were similar to those in the current study. Clinical isolate of B. cereus and standard strain of S. aureus were among the most susceptible strains to analyzed I. helenium L. root essential oil (MIC 0.017 and 0.6 mg mL -1 , respectively). The most resistant strains were clinical isolate of P. aeruginosa and standard strain of E. coli (MIC 14.8 mg mL -1 for both). Analyzed essential oil showed generally better AMA against Gram-positive strains regarding Gram-negative ones.
T. speciosa UEO obtained best results of antibacterial activity also for the standard strain of S. aureus, clinical isolate of B. cereus, and unlike I. helenium root essential oil, for the standard strain of E. coli. Clinical isolate of P. aeruginosa was among the most resistant strains to this essential oil, too. The oil showed also good fungicidal activity against clinical isolate of C. albicans, while standard strain was among the most resistant ones.

CONCLUSION
In the current study, Telekia speciosa (Schreb.) Baumg. was characterized by the presence of a high concentration of oxygenated sesquiterpenes with isoalantolactone as the predominant constituent of essential oil underground parts, and (E)nerolidol and caryophyllene oxide as the major constituents of essential oil aerial parts. According to this, underground parts may be a potential raw material for isoalantolactone isolation. T. speciosa essential oil was, to the best of our knowledge, investigated for antimicrobial activity against pathogens for the first time in this study. Essential oil of underground parts achieved better antimicrobial activity compared to the essential oil of aerial parts with the most susceptible standard strains of S. aureus and E. coli, and clinical isolate of C. albicans. The obtained results are important from the aspect of T. speciosa application as an antimicrobial agent. Traditionally its underground parts are used in bronchial asthma therapy and have a great potential to be used as a new therapeutic drug with recommendation for further pharmacological and toxicological investigations.