Antibacterial activity and essential oil composition of Calendula arvensis L

Essential oil composition and antibacterial activity of Calendula arvensis L. were investigated. The essential oil of aerial part was obtained through hydro-distillation using a Clevenger type apparatus with 0.38 (v/w) yield. The essential oil components were determined by GC-MS analyses. Thirty-six components were identified in the essential oil that represented 91.8 % of the oil. The major components of the essential oil were δ-cadinene (14.8 %), epi-cubebol (10.7 %), α-cadinol (8.5 %), cubenol (7.7 %), cubebol (7.2 %), 1-epi-cubenol (5.4 %) and ledene (5.1 %). Antibacterial activity of essential oil was observed against Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 14579, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 by using a broth microdilution. The essential oil showed weak inhibitory activity against E. coli and B. cereus at 8 mg/mL. The oil didn‘t show any antibacterial activity against S. aureus and P. aeruginosa. These results revealed that the oil was rich in oxygenated sesquiterpenes as well as had a weak antimicrobial activity. ARTICLE HISTORY Received: January 20, 2020 Revised: March 09, 2020 Accepted: August 09, 2020


INTRODUCTION
Calendula L. is a member of Calenduleae tribe of Asteraceae family. Calendula L. genus is illustrated in Turkey by three species which are C. arvensis M.Bieb., C. officinalis L., and C. suffruticosa Vahl [1]. Calendula species are used as an antipyretic and anti-inflammatory remedy in Italian folk medicine [2]. In European folk medicine, Calendula species are considered as an immune tonic that aids in prevention of sickness in winter [3]. C. arvensis is known by its local name as "Portakal nergisi" in Turkey and is used externally to treat varicose veins, eczema, fungus, warts and wounds [4]. C. arvensis has sedative, antibacterial, analgesic, lymphagogue, demulcent, choleretic, vulnerary anti-tumor, mild anodyne anti-inflammatory, antioxidants, anti-parasitic, antiviral, and antiseptic activities [5]. C. officinalis has immunostimulant, hepatoprotective, anti-HIV, antitumor, anti-inflammatory, spasmogenic and spasmolytic effects [6]. C. suffruticosa has mostly antioxidant and antifungal activity, especially its ethanolic extract [7]. Previously, the essential oils and hydrosol extract from aerial parts of C. arvensis L. were examined by using GC-FID and GC/MS. The main compounds were zingiberenol 1 (8.7-29.8%), eremoligenol (4.2-12.5%), β-curcumene (2.1-12.5%), CONTACT: Hüseyin Servi  servi@hotmail.com.tr  Department of Pharmaceutical Botany, Faculty of Pharmacy, Altınbaş University, Istanbul, Turkey ISSN-e: 2148-6905 /© IJSM 2020 zingiberenol 2 (4.6-19.8%) and (E, Z)-farnesol (3.5-23.4%). The antioxidant activity of essential oil and extract was checked by using DPPH, FRAP and β-carotene methods. The hydrosol extract showed high antioxidant activity. The antifungal activity of the essential oil and hydrosol extract was studied against Penicillium expansum and Aspergillus niger. The extract showed the highest inhibitory activity against P. expansum and A. niger [8]. The essential oil of aerial parts of C. arvensis L. was studied by GC-FID and GC-MS methods. The essential oil was obtained by hydrodistillation (HD) and microwave distillation (MD). The antimicrobial activities of the essential oils, hexane, ether and methanolic extracts of the C. arvensis L. were studied. The essential oil (HD) and methanolic extract had tolerable antibacterial activity against Staphylococcus aureus and Bacillus cereus. All the extracts were reported to have good antituberculosis activity against Mycobacterium smegmatis [9].
The first purpose of this research was to obtain essential oil from aerial parts of C. arvensis by using the hydrodistillation method as well as to determine antibacterial activity. And the second purpose was to determine the variation in the volatile oil composition of C. arvensis and to show that essential oil differences are related to geographical regions.

Plant Material
Plant material (natural) was collected in flowering periods from Istanbul (İkitelli-Başakşehir) province of Turkey in April 2017. Voucher specimen was deposited in the Herbarium of Marmara University (Voucher no: MARE20229), Turkey.

Isolation of the Essential Oil
The volatile oil of C. arvensis aerial parts (436 g) was obtained by Clevenger apparatus (3 h) with the hydrodistillation method. C. arvensis aerial parts produced 0.38% (v/w) essential oil yields. The oil was hold in amber vials under -20°C until analyzed.

Gas Chromatography/Mass Spectrometry Analysis
The GC-MS analysis was employed with an Agilent 5975C Inert XL EI/CI MSD system in EI mode. Essential oil of aerial part was kept in n-hexane was injected (1 μL) in split mode. The temperatures of the injector and MS transfer line were adjusted at 250˚C. Innowax FSC column (60 m x 0.25 mm, 0.25 µm film thickness) and helium as carrier gas (1 mL/min) were utilized in GC/MS analyses. The temperature of oven was adjusted to 60˚C for 10 min. and increased to 220˚C at a rate of 4˚C/min. The temperature kept stable at 220˚C for 10 min. and then increased to 240˚C at a rate of 1˚C/min. Mass spectra were saved at 70 eV with the mass range m/z 35 to 425. The relative percentage quantities of the separated compounds were calculated from integration of the peaks in MS chromatograms (given in Figure 1). The analysis was realized in triplicate.

Antibacterial Assay
Antibacterial activity of the essential oil was studied against Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 14579, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922. The minimum inhibitory concentration (MIC) values were determined for the oil, on each organism by using microplate dilution method [24]. Stock solution of the oil (16 mg/mL) was prepared with %10 dimethyl sulfoxide (DMSO). Serial dilution of essential oil was done on 96-well microplates with using Mueller Hinton Broth (MHB). Bacteria were standardized in MHB according to McFarland No:0.5. Bacterial cultures were mixed with different concentrations of essential oils on microplates and were incubated 24 h at 37ºC. Minimum inhibitory concentrations (MIC: mg/mL) were detected at the minimum concentration where bacterial growth was not detected. All the experiments were performed in duplicate.
According to a study from Trabzon, C. arvensis was reported to contain α-selinene, αpinene, (Z)-sesquilavandulol, 7-epi-silphiperfol-5-ene, viridiflorene, and β-pinene as main compounds [9]. But these compounds did not detect in the essential oil of this study except for α-pinene (0.2%). The oil from Trabzon had monoterpene and sesquiterpene as major groups. In the present study, the oil from Istanbul has oxygenated sesquiterpenes as a dominant group and showed a dissimilar chemical profile from the previous study. The difference in the composition of both oils may be correlated with the geographical region, collection time and specific climate conditions. The oil from Trabzon showed moderate antibacterial activity against S. aureus and B. cereus but in the current study, the oil did not show activity against S. aureus. Both oil samples had activity against B. cereus at different MIC values. The antibacterial activity differences in previous research and present data could be related to different main compounds of oils.
Another study from Corsica-France had common main compounds with the current study. The main compounds of the oil from France were δ-cadinene (15.1%) and α-cadinol (12.4%) [33]. The similarities were observed between essential oil constituents of Corsica-France and Istanbul-Turkey plants. But there are quantitative dissimilarities in main compounds of both essential oils.

CONCLUSION
The present study indicated that C. arvensis oil was rich in oxygenated sesquiterpenes and had antibacterial activity against E. coli and B. cereus. The oil did not show activity against S. aureus and P. aeruginosa. This result indicates a higher concentration of the oil is required to inhibit the growth of these bacteria. The essential oil of the current work is different from previous researches as quantitative and qualitative composition. The variations of essential oil ingredients and composition may be correlated with environmental factors such as temperature, humidity, and photoperiod. The quantitative composition of the essential oils can be related to plant age and harvesting time. Further studies on essential oil of C. arvensis are required to isolate main compounds of oil that are responsible for its antibacterial activities.

Declaration of Conflicting Interests and Ethics
The authors declare no conflict of interest. This research study complies with research publishing ethics. The scientific and legal responsibility for manuscripts published in IJSM belongs to the author(s).