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Ermina Cilović Kozarević1
, Aida Smajlagić2*, Merima Ibišević1, Darja Husejnagić3Jelena Arsenijević4and Zoran Maksimović5
1Faculty of Pharmacy, University of Tuzla, Urfeta Vejzagića 8, 75000 Tuzla, Bosnia and Herzegovina
2Faculty of Natural Sciences and Mathematics, University of Tuzla, Urfeta Vejzagića, Tuzla Department of Chemistry, Bosnia and Herzegovina
3Faculty of Natural Sciences and Mathematics, University of Tuzla, Urfeta Vejzagića, Tuzla Department of Biology, Bosnia and Herzegovina
4Department of Plant Physiology, Institute for Biological Research "Siniša Stankovic"-National Institute of Republic of Serbia, University of Belgrade, Bulevar Despota Stefana, Belgrade, Serbia
5University of Belgrade - Faculty of Pharmacy, Department of Pharmacognosy, Vojvode Stepe, Belgrade, Serbia.
Corresponding Author E-mail:aidataletovic88@gmail.com
Article Publishing History
Article Received on : 21 Nov 2024
Article Accepted on :
Article Published : 04 Feb 2025
Plant oils have attracted interest for centuries as natural remedies in treatment of various diseases. The Inula verbascifolia (Willd.) Hausskn.isgrowing wild plant in Bosnia and Herzegovina. Aromatic natural oils are one of the most significant sources of natural organic components. The natural vegetable oil of the selected plant (Inula verb.) was obtained by the hydrodistillation method. A comparison was made between the fragrant vegetable oil from the aerial parts of the plant in the flowering period (summer) and after the flowering period (autumn). In this study, chemical compounds were tested, comparing the content and composition of natural oils from the plant Inula verbascifolia. The aerial parts of the plant contained a fragrant and yellow essential oil. The identified 125 constituents accounted for 86.87% and 88.38% of the oil. The dominant compounds of both EOs were tridecanal, (3Z)- hexenyl benzoate, α-murolol, hexadecanoic acid, linalool and undecanal. Since essential aromatic oils possess a number of antimicrobial properties, an analysis of antimicrobial activity was also performed in this work. The antimicrobial activity of a mixture of EOs was determined on selected ATCC strains of microorganisms. Results of antimicrobial activity indicated that all used the microorganisms were sensitive to the EO. No data about antimicrobial activity of Inula verbascifolia has been published yet.
KEYWORDS:Antimicrobial activity; Essential oils; Inula verbascifolia; isolation
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Copy the following to cite this article:Kozarević E. C, Smajlagić A, Ibišević M, Husejnagić D, Arsenijević J, Maksimović Z. Significant Chemical Compounds, Antimicrobial Activity of the Essential Oils From I. Verbascifolia (Wild). Hausskn. Orient J Chem 2025;41(1).
Kozarević E. C, Smajlagić A, Ibišević M, Husejnagić D, Arsenijević J, Maksimović Z. Significant Chemical Compounds, Antimicrobial Activity of the Essential Oils From I. Verbascifolia (Wild). Hausskn. Orient J Chem 2025;41(1). Available from: https://bit.ly/42HFawy
Introduction
Eucalyptus (Inula verbascifolia) is a plant that is found in parts of Italy, Dalmatia, Balkans and parts Crete, Asia Minor, Pontus region and Syria. It characterized by greyish leaves with yellow flowers. It is up to 50 cm tall (Figure 1). It grows in coastal cliffs in the coastal area. It is also found on dry stony pastures and rocks. The plant species Inula verbascifolia contains secondary metabolites such as essential oils, terpenes1,2. It also contains polyphenolic compounds and exhibit antioxidant properies3. The variability of the composition and content of EOs in aromatic plants depends on the individual genetic variations, developmental stages and environmental factors. Environmental factors include soil composition, heat, sunlight, humidity4,5. It is well known that the genus Inula has different biological activities. Gokbulut et al., 2013 investigated antioxidant properties, Bae et al., 2019 beside antioxidant investigated hepatoprotective properties while Bar-Shalom et al., 2019 reported anticancer properties of the genus Inula6,7,8. Seka et al., 2014 published etnopharmacological and medicinal use of genus Inula such as antimicrobial, anti-inflammatory, cytotoxic and insecticidal activities9. As far as we know, no results have been reported on the antimicrobial activity of essential oils from parts of the Inula verbascifolia. In this study, the aspect is based on the compounds present in the essential oil from parts of Bosnia and Herzegovina during the flowering period and after the flowering period. The study is also based on the antimicrobial activity of the corresponding ATCC strains.
Although it is known that there are research results on the action of natural oils, isolation of compounds and antimicrobial activity, the mechanism of action itself has not been fully explained. Essential oils due to their lipophilic nature, pass through the cell membrane and affect changes in ion concentration, pH gradient. They also damage lipids and proteins in the bacterial cell. It all leads to cell damage and cell lysis10.
Figure 1: Inula verbscifolia (orginal photooriginal picture from the picking site)Click here to View Figure
Materials and Methods
The floral parts of the plant I. verbascifolia were taken in the area Bosnia and Hercegovina
( Podveležje, Mostar City) (N43°18’36.0″ E17°54’40.6″) during and after flowering period in 2020 and were air-dried. Typical specimens, identified by Prof. Cilovic Kozarevic, at the University of Tuzla, Department of Pharmaceutical Botany, Bosnia and Herzegovina.
Reagents and Chemicals
The chemicals used in this research are Streptomycin and Iodonitrotetrasolium chloride (Sigma-Aldrich), Ampicillin and Ketoconazole, purchased with the support of Serbia.
The process of distillation of essential oil
The aerial parts of I. verbascifolia were crushed. According to Ph. Eur. certain amount of the plant was subjected to distillation on an apparatus for determining essential oils (Clevenger Apparatus). Natural essential oils were obtained by distillation, dried with anhydrous Na2SO4 and left in the refrigerator (-20°C) until analysis.
Analysis of chemical compounds
The analysis of essential oils was done on a GC-MS/FID. Peaks of the individual chemical compounds within the obtained chromatogram were identified. After identification, their retention indices (RI) were compared with the baseline data. Analyses were performed at Agilent GC (6890N). Capillary column HP-5MS was used (length 30m, 250μm, film thickness 0.25μm).
GC was equipped with an Agilent MSD-5975 and FID. The temperature of FID was 300ºC. Helium gas was used as a carrier in a constant flow regime of 1.0 mL min-1. The injection volume of EOs dissolved in ethanol was 1 µL at temperature 200ºC. The oven was programmed at a temperature from 60°C to 280ºC from 3°C/min-1 and then at 280°C for 5 min. The MSD was operated from 35 to 55 m/z with transfer line at 250°C. NIST, literature and other spectral libraries were used for data analysis. The results are expressed as a percentage (%) of each component in the EO.
Antimicrobial activity assessment
Three reference bacterial strains Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and fungus Candida albicans were used to determine antimicrobial activity assessment of essential oils made of I. Verbascifolia aerial parts mixture.
The MO reference strains were made in the laboratory Mycology of the Institute for Biological Research “Siniša Stanković”, at the University of Belgrade in Serbia.
Minimum inhibitory concentration and minimum bactericidal /fungicidal concentration was determined utilizing microdilution method, using 96-well microtiter plates11. Namely, microorganisms were cultivated overnight on selective media, and then inoculated into bacteria and fungus on microtiter plates. Serial dilutions of the EO were made and then added to the nutrient medium.
As a negative control medium without EO was used, and a positive controls we used the commercial antimicrobial agents streptomycin, ampicillin, and ketoconazole. Microplates were incubated with a sterile film cover for bacteria on 24 h at 37°C and fungus on 72 h at 28°C.
The lowest concentration after incubation that showed no growth of microorganisms was determined as MIC. The minimum bactericidal/fungicidal concentration was determined by serial reinoculation of 10µL of the wells without growth into 100µL of sterile nutrient medium.
After reincubation (24 h/37°C for bacteria,72 h/28°C for fungus), 40 µL of microbial growth indicator, purple p-iodonitrotetrasolium chloride (0.2 mg/mL), was added to the wells, which were then incubated for 30 min. The results were compared with the control wells.
Results and Discussion
Results of isolation and identification of chemical compounds from natural essential oils
After the analysis, the results showed that the aerial parts of the I. verbascifolia plant contain 0.01% essential oil during the flowering period (summer) where 62 components were identified, which accounts for 86.87% of the oil presence. Also, the results were analysed after flowering period (in autumn). At that time, 60 components were identified, which makes up 88.38% of the oil, and the aboveground parts of the plant contained 0.005% EO, which are shown in Table 1. Essential oils isolated in summer at room temperature were solid, and at body temperature in a liquid aggregate state, while the EO isolated in autumn was in a solid aggregate state. Both EOs were yellow and fragrant.
The EOs of aerial parts of I. verbascifolia were abundant in the presence of high concentration of non-terpene (other) compounds and constituted 58.62 – 62.30% of the oils, followed by oxygenated sesquiterpnes (16.07-16.95%), oxygenated monoterpenes (6.59-12.71%) and finally sesquiterpene hydrocarbons (0.98-1.03%) in Table 1.
Table 1: Present compounds in essential oil I. verbascifolia collected in summer and autumn
No Retention period Constituent RIL RIE EO IVA_S (%) EO IVA_A (%) 2 12,857 Linalool 1095 1101.4 3.06 5.43 3 12,983 n-nonanal 1100 1104.3 0.61 0.78 4 15,878 Menthol 1167 1173.9 – 0.23 5 16,013 Octanoicacid 1176 1176.9 0.29 0.26 6 16,637 α-terpineol 1186 1191.7 0.49 0.87 7 16,925 Dodecane 1200 1198.5 0.46 – 8 17,219 n-decanal 1201 1205.4 0.76 1.44 9 17,713 methyl ether 1219 1216.9 – 0.43 10 18,202 Nerol 1227 1228.4 – 0.59 11 19,331 Geraniol 1249 1254.7 0.39 0.99 12 20,138 n-decanol 1266 1272.4 0.28 0.64 13 20,261 nonanoic acid 1275 1276.6 0.69 1.01 15 21,197 Tridecane 1300 1298.3 0.24 – 16 21,603 n-undecanal 1305 1308.0 3.38 6.17 17 21,963 (2E,4E)-decadienal 1315 1316.4 0.50 0.39 18 22,454 isobutyl benzoate 1327 1328.1 0.29 – 19 23,707 Eugenol 1356 1357.6 – 0.35 20 24,372 decanoic acid 1370 1373.7 1.70 1.48 22 24,839 (E)-β-damascenone 1383 1384.6 – 0.29 24 25,178 (Z)-jasmone 1392 1392.3 0.41 0.34 25 25,656 methyl eugenol 1403 1403.9 0.89 0.32 28 25,811 n-dodecanal 1408 1408.1 1.46 2.56 29 26,292 (E)-caryophyllene 1417 1420.2 0.36 – 31 26,776 Dictamnol 1428 1432.6 – 0.27 33 27,581 geranyl acetone 1453 1452.5 0.62 2.14 34 27,914 Dehydroaromadendrane 1460 1461.1 – 0.20 35 28,252 undecanoic acid 1467 1469.4 1.54 – 37 28,504 n-dodecanol 1469 1475.5 – 3.46 39 28,838 ar-curcumen 1479 1484.1 – 0.35 41 28,972 (E)-β-ionone 1487 1487.4 0.62 1.53 44 29.466 benzyl tiglate 1497 1499.9 0.58 – 46 29,858 n-tridecanal 1509 1509.9 6.53 10.06 47 30,08 γ-cadinene 1513 1515.8 0.45 0.23 49 30,885 italicene ether 1536 1536.5 – 0.23 50 31,958 (E)-nerolidol 1561 1564.0 0.55 0.38 52 32,25 (3Z)-hexenyl benzoate 1565 1571.4 7.97 2.39 53 32,501 n-hexyl benzoate 1579 1578,3 1.02 – 54 32,569 ar-turmerol 1582 1579.9 – 0.67 55 32,781 caryophyllene oxide 1582 1585.3 2.67 1.72 57 33.248 n-hexadecane 1600 1597,3 0.36 – 58 33,747 humulene epoxide II 1608 1611.0 0.54 – 59 33,755 Tetradecanal 1611 1611.1 – 0.56 60 34,369 Benzophenone 1626 1627.8 – 0.41 61 34.429 1-epi-Cubenol 1627 1629,6 0.27 – 62 34,441 Muurola-4,10(14)-dien-1-β-ol 1630 1630.1 0.27 0.38 64 34,784 Caryophyll -4(12),8(13)-dien-5-α-ol 1639 1639.0 0.30 – 65 34.759 Caryophyll -4(12),8(13)-dien-5-β-ol 1639 1638.7 0.86 – 67 34,993 α-murolol 1644 1645.0 7.89 8.41 69 35.174 (Z)-methyl jasmonate 1648 1650.1 0.31 – 72 35,466 α-cadinol 1652 1657.9 2.98 3.21 73 35,586 14-hydroxy-(Z)-caryophyllene 1666 1661.5 0.20 – 75 36,03 tridecanoic acid 1678 1673.5 1.47 4.53 76 36,012 14-hydroxy-9-epi-(E)-caryophyllene 1668 1674.0 0.42 – 77 36,138 hexyl salicylate 1674 1676.7 – 0.21 78 36,138 Cadalene 1675 1676.7 0.22 0.20 79 36,261 n-tetradecanol 1676 1679.6 – 0.42 81 36,925 n-heptadecane 1700 1697.5 – 0.29 84 37,458 n-pentadecanal 1711 1713.1 0.67 0.34 85 37,741 3-methoxy-cuminyl isobutyrate n/a 1721.1 0.46 – 87 38,154 Isobicyclogermacrenal 1733 1733.0 – 0.31 88 38,679 Fukinone 1756 1748.0 – 0.48 90 39.281 benzyl benzoate 1759 1765,4 3.95 – 92 39,444 tetradecanoic acid 1769 1770.1 – 2.94 94 40,414 Octadecane 1800 1798.3 – 0.20 95 40,742 dehydrofukinone 1813 1808.1 – 0.28 96 41,972 hexahydrofarnesyl acetone 1845 1844.6 1.52 0.56 97 42.300 phenyl ethyl octanoate 1854 1854,8 0.60 – 98 42,567 pentadecanoic acid 1869 1862.7 0.51 0.58 99 42.766 benzyl salicylate 1864 1868,9 1.47 – 103 44,389 (5E,9E)-farnesyl acetone 1913 1918.3 0.45 0.45 106 46,072 hexadecanoic acid 1975 1971.7 6.74 6.95 107 46.662 1-eicosene 1987 1991.0 0.36 – 111 49,451 n-octadecanol 2077 2081.7 0.22 0,25 112 49,977 n-heneicosane 2100 2099.0 0.27 – 113 50,479 (E)-phytol 2111 2116.2 – 0.33 115 51,425 9(Z),12(Z)-Octadecadienoic acid 2140 2148.6 – 1.56 116 53.528 (E)-phytol acetate 2218 2222.1 0.63 – 117 55.602 n-tricosane 2300 2297.3 0.87 – 119 58.268 n-tetracosane 2400 2395.8 0.36 – 120 60,943 n-pentacosane 2500 2498.7 2.48 0.31 121 63.327 n-hexacosane 2600 2595.1 0.24 – 122 65,84 n-heptacosane 2700 2698.9 3.26 1.31 123 68,159 n-octacosane 2800 2799.1 0.42 0.26 124 70,425 n-nonacosane 2900 2902.3 5.85 3.97 125 74,759 Untriacontane 3100 3100.8 0.64 0.48 Total identified 86.87 88.38 Oxygenated monoterpenes 6.59 12.71 Sesquiterpene hydrocarbons 1.03 0.98 Oxygenated sesquiterpenes 16.95 16.07 Other 62.30 58.62EO IVA_S – Essential oil of Inula verbascifolia aerial parts in summer
EO IVA_A – Essential oil of Inula verbascifolia aerial parts in autumn
RIE – retention index experimental
n/a –data is not available
„-„ – not detected
Bold components are presented in amounts over 1%.
Dominant components in both EOs were linalool (3.06-5.43%), undecanal (3.38-6.17%), dodecanal (1.46-2.56%), tridecanal (6.53-10.06%), (3Z)-hexenyl benzoate (2.39-7.97%),
α-murolol (7.89-8.41%), α-cadinol (2.98-3.21%), tridecanoic acid (1.47-4.53%), hexadecanoic acid (6.74-6.95%), n-nonacosane (3.97-5.85%).Benzyl benzoate was presented only in EO of I. verbascifoliaaerial parts in summer in amount 3.95%, while n-dodecanol and tetradecanoic acid were presented only in EO ofI. verbascifoliaaerial parts in autumn in amounts 3.46% and 2.94%, respectively. As far as we know, studies about essential oils for wild growing plant I. verbascifolia were done only in Croatia and two locations in Greece. Dominant components in I. verbascifolia aerial parts essential oil from Croatia (Badija island) are Hexadecanoic acid with 10.4%, followed by 9(Z),12(Z)-Octadecadienoic acid (6.5%), tetradecanal (4.5%), Germacrene D (4.4%) and δ-cadinene (3.3%)12. Dominant components in I. verbascifolia aerial parts essential oil from Greece location Viotia were: Methyl salicylate (23.4%),cis-Chrysanthenol (17.3%), β-caryophyllene (13.2%), linalool (7.1%), tridecanal (5.3%) while dominant components in I. verbascifolia aerial parts essential oil from Greece location Attiki were: Linalool (21.2%), epi–α-Cadinol (19.5%), (Z)-Nuciferol (16.6%), α-murolol (9.4%),(3Z)-hexenyl benzoate (6.4%)1. In comparison with EOs from Croatia and from two locations in Greece, the composition of I. Verbascifolia aerial parts EO from Bosnia and Herzegovina presents a combination of the above mentioned Eos constituents. Based on the obtained results, after comparing the flowering period of the plant (summer) and after flowering period of the plant (autumn), the presence of the compound (3Z)-hexenyl benzoate is reduced in autumn by 5.58%, while the presence of the other listed dominant compounds: linalool, n-undecanal, n-tridecanal, tridecanoic acid is increased in autumn by the maximum 3.53%.
Results of antimicrobial activity assessment
Table 2 shows the results of the obtained antimicrobial activity values.
Table 2: The values of essential oil antimicrobial activity (I.verbascifolia)
Strain ATCC MIC (mg/mL) MBC/MFC (mg/mL) Staphylococcus aureus 6538 4 7 Pseudomonas aeruginosa 27853 3 4 Escherichia coli 35210 7 15 Candida albicans 10231 11 15Minimum inhibitory concentration (MIC)
Minimum bactericidal concentration (MBC); Minimum fungicidal concentration (MFC)
EO made from I. verbascifolia parts mixture showed the strongest antimicrobial effect on Pseudomonas aeruginosa and Staphylococcus aureus with MIC values 3 mgmL-1and 4 mgmL-1, respectively. Inhibition of growth has also been reported for E.coli and Candida albicans with slightly higher MIC and MBC/MBC values. After the results obtained, it can be observed that Inula verbascifolia essential oil possesses antimicrobial activity against the microorganisms used, only in different concentrations. Based on research, we know that data on the antimicrobial activity of Inula verbscifolia essential oil are not available.
Conclusions
Aromatic essential oils are known to be used in medicine, the pharmaceutical industry and the food industry. It is precisely because of their aromatic properties that they were chosen for research. The EOs from I.verbascifolia aerial parts during and after flowering period was isolated. The chemical composition of EOs were determined. They were abundant in the presence of high concentration of non-terpene (other) compounds. After the research studies, it is noticed that the results obtained with the essential oil of the plant Inula verbascifolia are new and therefore special. Antimicrobial activity assessment of I. verbascifolia aerial parts EO have been done for the first time, as far as we know. The obtained antimicrobial tests showed all positive results and that the essential oil inhibits the growth of microorganisms.
Acknowledgement
Much of the research has been done at the Institute for Biological Research “Siniša Stanković”, University of Belgrade. The authors are grateful.
Conflicts of Interest
The mentioned authors have no conflicts of interest regarding the publication of this paper.
Funding Sources
The author(s) received no financial support for the research, authorship, and/or publication of this article.
References
Tzakou, O.; Petropoulou, A.; Harvala, C.; Constantinidis, Th.; Volatile Compounds of Two Members of Inula verbascifolia group: I. verbascifolia (Wild.) Hausskn. ssp. parnassica (Boiss. Et Heldr.) Tutin and I. verbascifolia ssp. Methanea (Hausskn.) Tutin. J Essent Oil Res,2001, 13: 364-366.
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