Phytochemical Characterization and Biological Activities of Nepeta cadmea Boiss.

﻿ The genus Nepeta (Lamiaceae) is represented by 39 species, in total 50 taxa, 19 of which are endemic, in Turkey. Nepeta species are known as “kedinanesi” in Turkish and has many traditional usages such as antispasmodic, diuretic, antiseptic, antitussive, and antiasthmatic activities. The aerial parts of N. cadmea were hydrodistillated for 3 hours by using Clevenger apparatus to gather essential oil. To determine its chemical characterization, the essential oil was analyzed by GC-FID and GC-MS, simultaneously. 4aα,7α,7aβ-Nepetalactone (74.0%), 4aα,7α,7aα-nepetalactone (4.5%) and caryophyllene oxide (2.5%) were found as major components for essential oil. The potential in vitro antibacterial activity of the essential oil was evaluated using the broth microdilution assay. A panel of human pathogenic strains Escherichia coli NRRL B-3008, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 13311, Bacillus cereus NRRL B-3711 and Streptococcus sanguinis ATCC 10556 were used. Minimal Inhibitory Concentrations (MIC) of the samples were determined, where in ciprofloxacin was used as a positive control in the assay. MIC values were found 2500, 1000, 600, 600, 600 μg/mL against E. coli , P. aeruginosa , S. typhimurium , B. cereus and S. sanguinis , respectively. Compared the literature, it was seen that the essential oil had lower effective against these strains.


INTRODUCTION
The genus Nepeta L. (Lamiaceae) comprises by nearly 250 species throughout the world [1]. Nepeta species are generally distributed in Europe, South-West and Central Asia, North America, North Africa and the Mediterranean regions [2]. According to the recent researches, the genus contains 39 species, in total 50 taxa, 19 of which are endemic, in Turkey [3][4][5]. The plant is distributed the West Anatolia, South Anatolia, and South west Anatolian regions of Turkey [6]. As in most of the members of Lamiaceae, many Nepeta species have traditional usages against the common colds stomachache and as stimulants [7,8]. In addition, several Nepeta species have traditional use as diuretic, diaphoretic, antitussive, antispasmodic, antiasthmatic, febrifuge, soothing, nervous disorders, depression, spices and herbal tea [9][10][11].
CONTACT: Mehmet Çiçek  mcicek@pau.edu.tr  Department of Molecular Biology and Genetics, Faculty of Science and Art, Pamukkale University, Denizli, Turkey ISSN-e: 2148-6905 /© IJSM 2020 Some of the Nepeta species are good nectar sources for bees [12]. The endemic species N. cadmea Boiss., known as "honaz pisikotu" in Turkish [5], is herbaceous, perenial, often aromatic, rich in essential oils content, stems erect and several branched 30-120 cm, densely glandular hairs. Leaves triangular-ovat 3-6 x 1-3 cm. green, inflorescence clearly distant, many flovered verticillaster, calyx tubular, 9-12 mm, corolla with a yellow hood and white lower lip, 12-15 mm [3]. The essential oils of N. cadmea species collected from two different regions in Turkey were exhibited antimicrobial activity against some gram (+) and gram (-) bacteria by using disc diffusion method [13]. In addition, recent antimicrobial activity studies on Nepeta species have been reported to be a natural preservative and a strong antimicrobial agent on food products. [14,15]. In this work, we aimed to investigate chemical composition of essential oil and in vitro biological activities of endemic N. cadmea species in Turkey.

Plant Material and Isolation of Essential Oil
Aerial parts of N. cadmea were collected from natural habitat in Denizli province and identified by Mehmet Çiçek. The voucher specimen has been deposited in the herbarium of the Faculty of Pharmacy, Ankara University in Ankara, Turkey (AEF). Collection locality: C2 Denizli: above Çamlık forest, old Kızılcabölük road, oak areas above second fountain, 880 m, 14.06.2017, M. Çiçek 2017-4-16 (Voucher number: AEF 28879). The aerial parts of N. cadmea were hydrodistillated for three hours using Clevenger-type apparatus to obtain essential oil.

GC-FID and GC-MS Analyzes
The essential oil was analyzed by GC using a Hewlett Packard 6890 system (SEM Ltd, Istanbul, Turkey) and an HP Innowax fused silica capillary column (FSC) (60 m x 0.25 mm ∅, with 0.25 µm film thickness) was used with nitrogen at 1 mL/min. Initial oven temperature was 60°C for 10 min, and increased at 4°C/min to 220°C, then kept constant at 220°C for 10 min and increased at 1°C/min to 240°C. Injector temperature was set at 250°C. Percentage compositions of the individual components were obtained from electronic integration using flame ionization detection (FID, 250°C) Relative percentages of the separated compounds were calculated from FID chromatograms. GC-MS analysis was performed with a Hewlett-Packard GCD, system (SEM Ltd, Istanbul, Turkey) and Innowax FSC column (60 m x 0.25 mm, 0.25 µm film thickness) was used with Helium. GC oven temperature conditions were as described above, split flow was adjusted at 50 mL/min, the injector temperature was at 250 °C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 425 as previously reported [16]. Identification of the essential oil components were carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial [17,18] and in-house "Başer Library of Essential Oil Constituents" built up by genuine compounds and components of known oils, as well as MS literature data [19] was used for the identification as also previously reported in detail [16].

Antimicrobial Activity
The standard antibiotic ciprofloxacin was suplied from commercial sources like Sigma-Aldrich (St. Louis, USA) was evaluated for its antibacterial properties, which were in pharmaceutical grade or highest possible purity. Microorganisms strains used for the evaluation of antimicrobial activity were obtained from the American Type Culture Collection (ATCC) and Agricultural Research Service Culture Collection (NRRL) in lyophilized form. The microorganisms were stored at -85°C in glycerol until inoculation and purity testing.
The potential in vitro antimicrobial activity of the essential oil was evaluated using the broth microdilution assay. A panel of human pathogenic strains Escherichia coli NRRL B-3008, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 13311, Bacillus cereus NRRL B-3711 and Streptococcus sanguinis ATCC 10556 were used. Minimal Inhibitory Concentrations (MIC) of the samples were determined where in ciprofloxacin was used as a positive control in the experiments.
Antimicrobial activity of the essential oil was evaluated by broth microdilution assay according to a modified Clinical and Laboratory Standards Institute (CLSI) method. E. coli NRRL B-3008, P. aeruginosa ATCC 27853, S. typhimurium ATCC 13311, B. cereus NRRL B-3711 and S. sanguinis ATCC 10556 were used as test microorganisms. The essential oil (20-0.019 mg/mL) was dissolved in sterile dimethyl sulfoxide (DMSO) for the initial stock solution. 100 µL of essential oil was applied to 96-well microplates and 2 fold serial dilutions were performed. After the dilutions, 50 µL aliquots of tubidometrically adjusted microorganisms were inoculated (105-106 CFU/mL) on to the plates. After incubation at 37°C for 24 h the first well was treated with 20 µL of resazurin, which insured on all microplates the MIC, where the lowest concentration of the samples prevented visible growth. The standard antibiotic ciprofloxacin (128-0.25 µg/mL) were used as standard controls. Solvent and microbial controls were also added to the assay plate. Antibacterial assays were repeated at least three times for all the test samples [20].

Antimicrobial activity
The in vitro antimicrobial activity of the essential oil of N. cadmea was evaluated against two gram-positive bacteria namely B. cereus and S. sanguinis, and also three gram-negative bacteria namely E. coli, Pseudomonas aeruginosa and S. typhimurium by broth microdilution method. As a result, it was found that essential oil of N. cadmea have different antimicrobial activity. Antibacterial activity values of the essential oil of N. cadmea were found between 2500-600 μg/ml. MIC values were found as 2500, 1000, 600, 600, 600 μg/mL against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Bacillus cereus and Streptococcus sanguinis, respectively. The results were given in Table 2.
Literature survey showed to be found the study of antimicrobial activity on a few of Nepeta species. In a study conducted by Saraç and Uğur [13], an antimicrobial activity test was performed on the essential oil of N. cadmea species by using disc diffusion method. As a result, some gram-positive and gram-negative bacteria were found to exhibit activity against. Zomorodian et al. [14] studied on antimicrobial activity of the essential oil of N. cataria using different microorganisms with the broth microdilution method. They concluded that the essential oil of N. cataria can be used as a natural protective agent on food products. Yavuz et al. [15] reported that the methanol extract of N. nuda can be evaluated as a potent good antimicrobial agent on 5 bacteria. Çelik et al. [23]  To the best of our knowledge, the study presented herein is the first report on the antimicrobial activity of the essential oil of N. cadmea by using broth microdilution method.

CONCLUSION
In the present study, we concluced that the essential oil of N. cadmea was exhibited different activity against E. coli NRRL B-3008, P. aeruginosa ATCC 27853, S. typhimurium ATCC 13311, B. cereus NRRL B-3711 and S. sanguinis ATCC 10556. To the best of our knowledge, this is the first report on the antimicrobial activity by microdilution. However, it will be useful to investigate different antibacterial and antifungal tests in later studies. We also plan to compare different Nepeta species and compare them. In the future, we suggest that essential oils obtained from plants can be used as antimicrobial agents in search of infectious diseases, treatment and new drugs.