CHEMICAL PROFILE OF Nepeta cataria L. var. Citriodora (B ecker ) ESSENTIAL OIL AND in vitro EVALUATION OF BIOLOGICAL ACTIVITIES

Essential oil (EO) obtained by hydrodistillation in a Clevenger -type apparatus from aerial parts of Nepeta cataria L. var. citriodora (Becker), cultivated in Serbia was subjected to gas chromatography-mass spec troscopy (GC-MS) to determine the composition. Furthermore, N. cataria var. citriodora essential oil was tested to determine its antimicrobial, antioxidant, antihyperglycemic and anti-inflammatory activities in vitro . The antimicrobial activity was tested by broth microdilution method against 16 bacterial strains from American Type Culture Collection (ATCC). Four common tests for measuring in vitro antioxidant activity were used: 2, 2-diphenyl-1-picrylhydrazyl assay (DPPH), reducing power (RP), 2,2-azino-bis-3-ethylbenzo -thiazoline-6-sulfonic acid (ABTS) and β -carotene bleaching assay (BCB). Antihyperglycemic activity was examined by using α -glucosidase inhibitory potential (AHgA), while anti-inflammatory activity (AIA) was determined by protein denaturation bioassay, using egg albumin. In total, 36 compounds were isolated and detected by GC-MS technique in N. cataria var. citriodora EO. The EO is mainly comprised of oxygenated monoterpenes (93.1%), and the main compounds were two monoterpenoid alcohols, nerol (38.5%) and gera niol (24.9%), followed by two aliphatic aldehyde, geranial (14.6%) and neral (11.0%). Antimicrobial activity of this EO shows growth inhibition of all tested bacteria strains, and exhibited good antioxidant, antihyper glycemic and anti-inflammatory activities. The EO obtained from N. cataria var. citriodora grown in Serbia shows valuable biological activity, indicating its potential for use as a supplement in everyday diet and as a natural preservative in food industry.


INTRODUCTION
. Chemical profile of Nepeta cataria L. var. citriodora (Becker) essential oil and in vitro evaluation of biological activities. Acta Sci. Pol. Hortorum Cultus, 21(4), 67-74. https://doi.org/10. 24326/ asphc.2022.4.7 Due to their complex chemical composition, they possess a large spectrum of biological activities, such as antimicrobial, antioxidant, anti-inflammatory and immunostimulatory [Mucha and Witkowska 2021]. The global trend towards natural preservatives being used as alternatives to synthetic ones in food applications is supported by legislative actions and consumer concerns. In recent years, food industries have been using different EOs because of their ability to control the growth of pathogenic microorganisms and delay deterioration of food products [Prakash and Kiran 2016]. Furthermore, EOs have been considered to be natural antioxidants with high potential as additives in food to prevent degenerative diseases caused by oxidative stress [Abd Rashed et al. 2021].
Nepeta cataria L. (Lamiaceae), catnip or catmint, is a perennial herbaceous plant, which is characterized by nepetalactones in the EO. However, there are varieties of N. cataria which are characterized by being completely devoid of or producing little amounts of nepetalactones, and the main EO constituents are nerol, citronellal, neral and caryophyllene oxide. Because of these components, these varieties have a minty-lemony flavor, resemble true catnip, but are not attractive to cats. These varieties are called N. cataria L. var. citriodora (Becker) or lemon catnip [Gomes et al. 2020]. Because of its flavor, it is an appealing raw material in food industry and soft drinks, as well as vegetable and fruit canned food [Frolova et al. 2019]. Due to this, N. cataria var. citriodora is a valuable dietary supplement and a natural food additive. Furthermore, it is a good source for industrial production of citral (mixture of geranial and neral) widely used in the industry for improving organoleptic properties in pharmaceuticals, cosmetics, toothpastes, chewing gum and cigarettes [Pucci et al. 2020]. Investigations show that N. cataria var. citriodora can be used as a constituent of phytopharmaceutical preparations with a mild sedative, antispasmodic, antioxidant and anti-inflammatory activity [Modnicki et al. 2007, Ricci et al. 2010, Bernardi et al. 2011]. In addition, citral, main constituent of N. cataria var. citriodora EO, is known to possess antimicrobial, antioxidant, hypolipidemic and hepatoprotective effects, but it is also effective in cancer prevention and treatment [Onawunmi 1989, Uchida et al. 2017, Pucci et al. 2020. Taking in account global demand for citral, N. cataria var. citri-odora is occurring as new promising source of this compound, easy for commercial cultivation in temperate region [Aćimović et al. 2021].
The aim of this investigation was to determine the EO composition of N. cataria var. citriodora grown in Serbia and to evaluate its biological effects through antibacterial tests (against 16 bacterial strains that cause skin and food borne illness), antioxidant activity (DPPH • and ABTS •+ assays, reducing power and β-carotene bleaching assay), antihyperglycemic (AHgA) and anti-inflammatory (AIA) activity.

MATERIAL AND METHODS
Plant material. Nepeta cataria var. citriodora were cultivated at the Institute of Field and Vegetable Crops Novi Sad, at the Department for Vegetables and Alternative Crops Bački Petrovac (45°21'N; 19°35'E). The above ground flowering parts of plants were harvested at full flowering stage during July, 2019. The above ground parts were harvested manually, and the biomass was placed at room temperature until constant weight. Voucher specimens were confirmed and deposited at the Herbarium BUNS, the University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, under the acquisition number 2-1401.
Essential oil (EO) isolation. Air-dried aerial parts of N. cataria var. citriodora were submitted to hydrodistillation using the Clevenger apparatus. The obtained EO was separated from aqueous layer and dried over anhydrous sodium sulphate and stored in a dark glass vial at 4°C for further analysis. This process was performed in three replications, and average content of EO was 0.24%.
Gas chromatography-mass spectrometry analysis (GC-MS). The EO was analyzed using an HP 5890 gas chromatograph coupled to an HP 5973 MSD and fitted with a capillary column HP-5MS. The components were identified based on their linear retention index relative to C 8 -C 32n -alkanes, comparison with data reported in the literature (Wiley and NIST databases). Quantification was done by external standard method using calibration curves generated by running GC analysis of representative authentic compounds.
Determination of antimicrobial activities. Antimicrobial activity of essential oil was tested against . Chemical profile of Nepeta cataria L. var. citriodora (Becker) essential oil and in vitro evaluation of biological activities. Acta Sci. Pol. Hortorum Cultus, 21(4), 67-74. https://doi.org/10. 24326/ asphc.2022.4.7 16 bacterial strains from American Type Culture Collection (ATCC) by broth microdilution method according to Varga et al. [2019]. Tested bacterial strains with ATCC numbers are given in table 2. As a control, a gentamicin strip test was used for determination of minimal inhibitory concentration (MIC).
In vitro anti-inflammatory activity assay. Anti-inflammatory activity (AIA) was determined by protein denaturation bioassay using egg albumin (from fresh hen's egg) according to Ullah et al. [2014], using diclofenac sodium as control.
In vitro antioxidant, α-glucosidase and anti--inflammatory activities of N. cataria var. citriodora are shown in Table 3. Employing the DPPH method, the N. cataria var. citriodora EO showed antioxidant activity of 86.33 μmol TE 100 mL -1 EO, reducing power of 16.28 μmol TE 100 g -1 , and scavenging capacity of ABTS radicals of 299.98 μmol TE 100 mL -1 EO. Furthermore, the activity obtained by β-carotene bleaching assay was found to be 118.63 μmol TE 100 mL -1 EO. In this study, N. cataria var. citriodora EO was less efficient in the antihyperglycemic assay (AhgA = 79.08%) than the positive control, acarbose. Based on the obtained results for N. cataria var. citriodora EO at a concentration of 250 mg mL -1 , it was capable of inhibiting the denaturation of the proteins for 51.36%. In this study, diclofenac sodium was used as a standard anti-inflammatory drug, which showed EC 50 value of 1.14 mg mL -1 .

DISCUSSION
Nepeta cataria is characterized by chemical polymorphism [Baranauskiene et al. 2003]. There are several main chemotypes that were previously described. The one with nepetalactones (several steroisomers) as dominant compounds in the EO, which could be used as an insect repellent, for pharmaceutical preparations, as well as herbal tea and for culinary purposes, while another one containing citral (isomeric mixture of geranial and neral) i.e. var. citriodora, which is interesting in perfume industry due to the EO [Klimek and Modnicki 2005]. However, EO composition depends on many factors, such as the region, variety and cultivar, hybridization and mutation, climatic and stress conditions, harvest time and vegetation period (first or second cut), plant part and postharvest processing (fresh or dry), isolation technique and conditions, etc. [Aćimović et al. 2021].
Further, comprehensive report of antimicrobial activity of N. cataria EO against wider range of microorganisms (11 bacteria, 12 fungi and a yeast) indicated possibilities for application in food and pharmaceutical industry [Adiguzel et al. 2009]. Previous research, as well as this study, shoved that N. cataria EO is effective against bacteria P. aeruginosa which cause various types of infections [Kim et al. 2006]. Since P. aeruginosa represent one of the most resistant bacteria, this data will be valuable for new scientific studies in the future. According to literature, the antioxidant activity of N. cataria has been studied by several authors and different methods. The results show that ethanol extract of N. cataria possesses scavenging DPPH radical in range between 0.449 and 0.491 mM TE g -1 depending on harvest time [Duda et al. 2015]. Furthermore, scavenging DPPH radical potential of N. cataria water extract was 220.2 μmol TE g -1 DW, while synthetic ABTS radical in water extract expressed activity of 292.0 μmol TE g -1 DW [Mihaylova et al. 2013]. Methanolic extract of N. cataria reduced stable DPPH radicals with an IC 50 value of 171.98 µg ml -1 , while EO with dominant nepetalactones remained inactive [Adiguzel et al. 2009]. The fresh raw material of N. cataria from Poland was characterized with the ability to reduce free radicals (% DPPH) between 2.45 and 4.57% depending on crop age [Wieteska et al. 2018].
Experiments conducted in vitro using α-glucosidase activity tests showed that different extracts of N. cataria have significant beneficial effect on glycemic control, as well as in normalization of liver and pancreas function, so it may be applied for reducing complications against diabetes mellitus [Aly et al. 2010]. Furthermore, monoterpenes such as geraniol, nerol, citral, linalool, etc., have positive influence on glucose metabolism may have potential in the control of type 2 diabetes mellitus [Tan et al. 2016].
Investigations show that N. cataria EO in concentration of 0.0005 mL kg -1 in female rats expressed peripheric anti-inflammatory properties by reducing the induced edema after carrageenan injection [Ricci et al. 2010]. Furthermore, anti-inflammatory effect could be related to the free radical scavenging activity and depends on a synergic action of all the components [Miceli et al. 2005]. It is known that nerol, which can be found in many EOs, possesses potential anti-inflammatory and antinociceptive activities, supporting the application of this plants in treating various diseases associated with inflammation and pain [Bounihi et al. 2013].

CONCLUSIONS
The main compounds of N. cataria var. citriodora EO were nerol, geraniol, geranial and neral. EO of N. cataria var. citriodora shows significant antibacterial activity against all investigated bacterial strains, the most interesting being the antibacterial effect against P. aeruginosa, which is one of the most resistant bacteria. In view of the biological activity, examined essential oil might be considered a valuable natural constituent with antioxidant, antihyperglycemic, and anti-inflammatory properties, and potential application in food products.