Chemical Constituents in Leaves and Aroma Products of Nicotiana rustica L. Tobacco

Nicotiana rustica L. (Aztec tobacco) is the only Nicotiana species, except common tobacco (N. tabacum L.), which is cultivated for tobacco products. The leaves of N. rustica, however, accumulate various specialized metabolites of potential interest. Therefore, the objective of this study was to evaluate certain classes of metabolites (by HPLC and GC-MS) in the leaves, the essential oil (EO), concrete and resinoid of N. rustica. Three pentacyclic triterpenes were identified in the leaves (by HPLC): betulin (252.78 μg g−1), betulinic (182.53 μg g−1) and oleanolic (69.44 μg g−1) acids. The dominant free phenolic acids in the leaves (by HPLC) were rosmarinic (4257.38 μg g−1) and chlorogenic (1714.40 μg g−1), and conjugated forms of vanillic (3445.71 μg g−1), sinapic (1963.11 μg g−1) and syringic (1784.96 μg g−1). The major flavonoids in the leaves were luteolin (960.44 μg g−1), apigenin (880.66 μg g−1) and hyperoside (780.72 μg g−1). The GC-MS profiling of the EO identified 19 components and the major ones were phytol (43.68 %), solanone (5.54 %), cis-5-butyl-4-methyldihydrofuran-2(3H)-one (5.23 %), dihydro-β-ionone (4.25 %), α-ionene (3.54 %) and β-damascenone (3.03 %). The major volatiles in the concrete were isoamyl alcohol (28.82 %), oxynicotine (9.02 %), phytol (7.80 %), 4-methyl-1-penthanol (6.33 %), cotinine (5.55 %) and 3-metyl-3-penthanol (4.09 %). Resinoid composition was dominated by nicotine (39.75 %), phytol (11.23 %), eicosane (4.88 %), diethyl phthalate (4.19 %), dibutyl phthalate (3.48 %) and solanone (3.27 %). Concrete and resinoid showed weak antibacterial activity. These results create grounds for considering N. rustica as a source to obtain aroma or other bioproducts.

1 Introduction leaves, from 3.4 to 8.32 %. Sisson and Severson (1990) in a study on the alkaloid levels and composition of 64 Nicotiana species, reported 5.4 µg g −1 total-alkaloid content in freeze-dried green leaves from greenhouse-grown plants (of which 98.2 % were nicotine, 0.5 % nornicotine, 0.8 % anabasine, 0.5 % anatabine), and considerably higher content in leaves from field-grown plants at 25.6 µg g −1 (97.1 % nicotine, 0.7 % nornicotine, 0.5 % anabasine, 1.7 % anatabine). Kurucu et al. (1998) found substantial differences in the alkaloid levels (nicotine, nornicotine and anabasine) of N. rustica leaves depending on plant cultivation conditions (for example, nicotine levels varying from 7.71 % to 2.12 % in the tobacco from two different regions of Turkey). In a comprehensive review on the secondary metabolites from the genus Nicotiana and their biological activities, Jassbi et al. (2017) pointed out that nicotine biosynthesis and aerial accumulation in Nicotiana species may increase substantially in response to environmental and other factors, such as herbivore attack and leaf damage. Stanfill et al. (2015) determined 5.09 % nicotine level in a Brazilian nasal snuff (rapé) product made entirely of N. rustica tobacco without flavorings, while according to Furbee (2009) N. rustica leaves contained up to 18 % nicotine. Studies of floral volatiles from N. rustica L. (Raguso, Levin, Foose, Holmberg & McDade, 2003;Schlotzhauer, Horvat, Chortyk, Nottingham & Jackson, 1995) observe that nicotine has been present in major amounts in flower oil as well (over 20 %), a coherent result of the well-documented intensive synthesis of this secondary metabolite by the species (Jassbi et al., 2017;Sisson & Severson, 1990). Cured leaves of N. rustica L. are also a traditionally recognized source for obtaining citric acid, as its average content of 7 to 8 % may reach as high as 18 % if appropriate cultivation and postharvest practices are applied (Tatarchenko et al., 2003). In terms of the assessment of N. rustica L. leaf quality as a material for smoking and smokeless tobacco products, a number of basic indices of the chemical composition have been determined, e.g. ash (13.7 -19.4 %), reducing substances (1.20 -1.80 %), soluble carbohydrates (in trace amounts), total nitrogen (4.10 -4.60 %), ammo-IJFS April 2020 Volume 9 pages 146-159 nia (0.71 -0.79 %) and organic acids (15.90 -24.40 %) (Tatarchenko et al., 2003). Hristeva and Nikolov (2006) evaluated nine varieties of N. rustica L. as genetic donors in selection procedures aimed at enhancing the smoking quality of common tobacco, and provided the following data about their chemical characteristics: ash (13.08 -15.87 %), reducing substances (6.77 -15.00 %), soluble carbohydrates (6.13 -14.06 %), total nitrogen (1.51 -2.12 %), total alkaloids (0.78 -2.87 %), nicotine (0.30 -1.44 %), chlorogenic acid (0.43 -0.83 %) and rutin (0.73 -1.31 %). In the last two decades the focus of phytochemical research on tobacco has been on the analysis of biologically active secondary metabolites such as plant volatiles, terpenes, carotenoids, sterols, saponins, phenolics and secondary alkaloids, and the results reveal the clear potential of N. tabacum L. and some other Nicotiana species in this aspect (Andersson, Wennstrom & Gry, 2003;Budzianowski, 2014;Chowański et al., 2016;Jassbi et al., 2017;Kodama, Fujimori & Kato, 1984;Rodgman & Perfetti, 2016;Zhou, Li, Feng & Li, 2013). The distinctive phytochemical profile of Nicotiana species justifies their processing by different extraction techniques (traditional, accelerated, microwave or ultrasound assisted solvent extraction, supercritical extraction with liquefied gasses and subcritical water extraction) (Huie, 2002). Extracts from N. rustica L. leaves and flowers revealed biological and pharmacological activities, such as antimicrobial, anti-proteolytic, antioxidant and insecticidal (Bakht, Azra & Shafi, 2013;Digrak, Alma & Ilçim, 2001;Ibrahim, Aliyu, Abusufiyanu, Bashir & Sallau, 2011;Jassbi et al., 2017). Driven by these contemporary trends in tobacco science, in 2015 the Tobacco and Tobacco Products Institute in Bulgaria set up a research programme for the investigation of tobacco species that are not common to Bulgaria, including N. rustica L. and N. alata Link & Otto. Therefore, the objective of the current study is to evaluate certain classes of biologically active secondary metabolites in the leaves of N. rustica L. tobacco, experimentally grown in Bulgaria, and to identify the chemical composition of different aroma products (essential oil, concrete and resinoid).  -September 2016) was characterized by an average temperature of 22 o C and an average rainfall of 44.5 mm, during which two additional irrigations were carried out. Fresh leaves were picked by hand at maturity. The leaves were stringed and sun-cured in the open air for about two weeks, until the characteristic leaf coloration (green with bronze or brown hues) developed and the mid-rib became brittle. Until processing, leaf material was stored in cardboard boxes in an air-conditioned warehouse. For the preparation of analytical samples, the leaves were ovendried (40 o C; 6 h) and ground in a laboratory mill. A portion of the ground sample, intended for polyphenol and triterpene analysis, was further powdered by a laboratory homogenizer. The moisture content (%) was determined by drying to constant weight at 103 ± 2 o C, and all results have been presented on a dry weight (DW) basis.

Chemicals
HPLC grade methanol and acetonitrile, as well as phenolic acid and flavonoid standards were purchased from Sigma (Sigma-Aldrich Chemie GmbH, Germany).

HPLC analysis of polyphenols in the leaves
Portions of 0.5 g homogenized leaf samples were taken and subjected to triple sonicated extraction with 70 % methanol at 70 o C for 3 hours. The combined extract was evaporated at 60 o C, the residue was dissolved in methanol and the solution (filtered through a 0.45 µm syringe filter) was transferred to the HPLC unit. For the extraction of conjugated phenolics, 2M HCl in methanol was used, and the other conditions were the same (Marchev, Georgiev, Ivanov, Badjakov & Pavlov, 2011). The HPLC system used for the determination of phenolic acids and flavonoids consisted of a Waters 1525 Binary Pump (Waters, Milford, MA, USA), UV-VIS detector (Waters 2487 Dual λ Absorbance Detector (Waters, Milford, MA, USA) and SUPELCO Discovery HS C18 column (5 µm, 250 mm Ö 4.6 mm, operated at a temperature of 26 o C), and Breeze 3.30 software. The determination of phenolic acids was done according to Marchev, Georgiev, Ivanov et al. (2011). The mobile phases were: Phase A 2 % acetic acid and Phase B 0.5 % acetic acid:acetonitrile = 1:1 (v/v). The gradient elution profile was (Phase B): 0 -30 min increase from 5 % to 35 % at 0.8 mL min −1 ; 30 -45 min from 35 % to 70 % at 0.4 mL min −1 ; 45 -50 min from 70 % to 80 % at 1.2 mL min −1 ; 50 -60 min from 80 % to 100 % at 1.2 mL min −1 ; 60 -65 min decrease from 100 % to 5 % at 0.8 mL min −1 and 65 -70 min maintained to equilibrate the column. The standards used for building the calibration curves were gallic, protocat-echuic, salicylic, chlorogenic, vanillic, caffeic, syringic, ferulic, sinapic, p-coumaric and cinnamic acids. Detection was performed at wavelengths of 280 nm and 320 nm. The mobile phases in the gradient elution of flavonoids were: Phase A 2 % acetic acid and Phase B methanol. The gradient elution profile was set up as follows (Phase B): 0 -10 min increase from 30 % to 50 % at 1.0 mL min −1 ; 10 -15 min held at the same flow rate; 15 -16 min increase to 52 % at a flow rate of 0.8 mL min −1 ; 16 -30 min increase to 80 % at the same flow rate; 30 -35 min decrease to 30 % at 1.0 mL min −1 and then maintained to 40 min to equilibrate the column (Marchev, Georgiev, Ivanov et al., 2011). Myricetin, kaempferol, quercetin, hesperidine and apigenin were used as standards for plotting the calibration curves. Detection wavelengths were 308 nm and 380 nm. The determination of the quercetin glycosides rutin and hyperoside was carried out under the following conditions: 2 % acetic acid (Phase A) and acetonitrile (Phase B) used as mobile phases, and elution gradient set up to the profile of 0-15 min 20 % Phase B; 15-17 min 50 % Phase B and 17-20 min 20% Phase B (Ivanov et al., 2014). Rutin and hyperoside were used as standards to build the calibration curves. The detection was carried out at 370 nm.

HPLC analysis of triterpenes in the leaves
Samples of 1.0 g finely ground tobacco were subjected to threefold extraction with acetone (each for 30 min), in an ultrasonic bath, at hydro module 1:20 (w/v) and temperature 45 o C. The solvent in the combined extract was evaporated at 60 o C, using a rotary vacuum evaporator, and the residue was transferred to 1 mL methanol, filtered through a 22 µm filter and analyzed by HPLC. The identification of triterpenes was carried out on the same HPLC system as described for polyphenols. The mobile phase was an aqueous solution of potassium dihydrogen phosphate (pH 2.8): methanol = 12:88 (v/v), the flow rate was: 0 -18 min at 0.8 mL min −1 ; 18 -19 min at 0.6 mL min −1 ; 19 -30 min at 0.6 mL min −1 ; 30 -31 min at 0.8 mL min −1 and 31 -40 min at 0.8 mL min −1 . The detection wavelength IJFS April 2020 Volume 9 pages 146-159 was 210 nm. The determination of triterpenes was against a standard curve, with carnosic acid, betulin, betulinic, ursolic and oleanolic acid (97 %) (Extrasynthese, France) used as standards  2.3 GC-MS profiling of aroma products (essential oil, concrete and resinoid) One and the same plant material can be processed by different methods, thus obtaining aroma products with specific chemical composition, olfactory, biological and other properties. Common aroma products obtained from essential oil-bearing and medicinal plants include essential oils, concretes, absolutes, resinoids, oleoresins and tinctures (Bauer, Garbe & Surburg, 2001). The flow chart ( Fig. 1) illustrates the basic principles of obtaining the aroma products investigated in this study, essential oil, concrete and resinoid. Essential oil was obtained by hydrodistillation of 100 g dry tobacco in a laboratory Clevengertype glass apparatus of the British Pharmacopoeia, modified by Balinova and Diakov (Stoyanova, Georgiev & Atanasova, 2007 One µL of the sample diluted with n-hexane (10 %, v/v) was injected into the GC-MS system at split ratio 30:1. The GC analysis was carried out using an Agilent 7890A GC system and FID temperature of 270 o C. In order to obtain the same elution order with GC-MS, simultaneous triplicate injections were done by using the same column and the same operational conditions. The identification of compounds was made by comparing their mass spectra with those from mass spectra libraries (Adams, 2001) and by comparing the literature and estimated Kovat's (retention) indices that were determined using mixtures of homologous series of normal alkanes from C 8 to C 40 in hexane, under the conditions described above. The percentage ratio of volatile components was computed using the normalization method of the GC/FID peak areas.

Antimicrobial activity of aroma products
The antimicrobial activity was determined against the Gram-positive bacteria  (Zaika, 1988). Blank dishes, with only solvent applied were also included as a negative control, in order to make the necessary corrections due to solvent activity. All tests were performed in triplicate.

Statistical analysis
All experiments were performed at least three times. All data were presented as mean ± standard deviation (SD). Statistical significance was assessed by Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison. Differences between means were considered statistically significant if p < 0.05.

Basic chemical indices of tobacco
Leaf chemical composition is one of the decisive criteria in the evaluation of the quality of any tobacco type cultivated for commercial purposes (i.e. with the purpose to yield leaf material with certain, typical chemical, physical and sensory features). The assessment of the quality level and the typical nature of tobacco leaf material through its chemical composition are based on the complex consideration of some recognized, key indicators (Leffingwell, 1999;Stanfill et al., 2015). These include: the total alkaloid content (expressed as nicotine, free and/or ionized); the total content of reducing sugars (or alternatively of soluble carbohydrates); the total nitrogen content (or alternatively protein nitrogen); and ash (i.e. the total mineral matter, expressed as pure ash, corrected for sand and other stuck impurities), and are determined by standardized methods (by the ISO or national bodies). As it has been stated previously, N. rustica has been historically cultivated for tobacco products intended for human consumption, and is still an important cash crop in certain regions of the world. On the other hand, the species remains relatively IJFS April 2020 Volume 9 pages 146-159 uncommon to Bulgaria and there is scarce data about its leaf chemical composition relevant to this particular geographical origin. Therefore, the first step in the current study was the determination of the basic leaf chemical indices of N. rustica tobacco experimentally grown in the country, in an attempt to assess its quality and compliance with the tobacco type profile (Table  1). An analysis of the numerical data in Table 1 reveals that the chemical composition of the studied plant material can be considered as typical for N. rustica, with a chemical profile described by some authors (Tatarchenko et al., 2003) as resembling that of cigar tobacco which combines high alkaloid, nitrogen and ash levels with very low or trace amounts of soluble carbohydrates. Nevertheless, there were some characteristic leaf features, obviously influenced by environmental and other factors of the growing area or by the applied agricultural practices, which affected plant metabolism. The content of nicotine was reasonably high (3.09 %), but still closer to the lower limit of the characteristic 2 to 8 % nicotine range reported for commercial N. rustica tobacco (Tatarchenko et al., 2003). Similarly, the total nitrogen content was insufficiently high (2.68 %), being about a half of that found in the leaves of high quality N. rustica (Tatarchenko et al., 2003). On the other hand, leaves accumulated substantially higher amounts of reducing carbohydrates (3.82 %) than the typical range cited for the species by the same authors (0.5 -1 %). These findings suggest that, in case of an interest in a commercially-oriented production of N. rustica in the country, the growing conditions (e.g. agricultural practices, soil, plant nutrition, curing and breeding) should be optimized so as to realise the full potential of the species in terms of leaf quality. It should be regarded, as well, that some of the analyzed leaf chemical indicators represent plant metabolites that are extractible and biologically active, and their levels would contribute to the composition and properties of plant-derived crude or processed extracts.

Triterpenes and polyphenols
The analysis of the two groups of biologically active metabolites, triterpenes and polyphenols, res- ± standard deviation ulted in the qualitative and quantitative profile of N. rustica L. leaves as presented in Table 2 and Table 3. Three pentacyclic triterpenes of the oleanane type were identified in the leaves, among which dominated the terpene alcohol betulin (50.08 % of the total triterpenoid content). The presence of ursolic and carnosic acids were not detected. All three triterpene secondary metabolites have well documented biological and pharmacological activities (Parikh et al., 2014;Patlolla & Rao, 2012). In the group of free phenolic acids, cinnamic acid derivatives comprising mainly rosmarinic and chlorogenic acid were dominant. In the group of conjugated phenolic acids (determined after acid hydrolysis), hydroxybenzoic acid derivatives (vanillic acid, syringic acid) were dominant. The dominant free and conjugated flavonols were myrecitin and quercetin, respectively. Flavons were mainly in a conjugated form. In the group of quercetin glycosides, only the free form of hyperosid was identified, while the conjugated form of hesperetin was dominant in flavon glycosides. This profile of polyphenol metabolites in the leaves of N. rustica L. substantially differs from the profile established in N. tabacum L. leaves from Bulgaria, reflecting species distinctiveness.

Chemical composition of aroma products (essential oil, concrete and resinoid)
Beside the identification of phytochemicals in the cured leaves of N. rustica L. tobacco from IJFS April 2020 Volume 9 pages 146-159 ± standard deviation Bulgaria, our goal was also the characterization of the chemical composition of the essential oil (obtained by hydrodistillation) and that of two concentrated, ready-to-use aroma products obtained by solvent extraction, concrete and resinoid. We hypothesized that the comparative assessment of these three aroma and biologically active products would reveal distinct differences in their volatile profiles, due to the influence of temperature, pH of the medium, extractant nature and other factors relating to the transformations of plant metabolites during hydrodistillation and extraction. The essential oil of N. rustica was a light brown liquid, and the final concentrated extraction products (concrete and resinoid) were dark brown waxy masses, all with specific tobacco odor. In these characteristics they did not differ from the respective aroma products obtained from common tobacco described elsewhere (Georgiev & Stoyanova, 2006;Popova et al., 2015). The yield of concrete was 1.50 ± 0.01 % (w/w) and that of resinoid was 15.62 ± 0.09 % (w/w). These results were sufficiently high, in a parallel with the yields of concrete (0.25 -3.0 %) and resinoid (8 -27 %) from common aromatic plants (Georgiev & Stoyanova, 2006). On a yield basis, the results suggest the potential of N. rustica leaves as a source to obtain aroma products by solvent extraction. The yield of essential oil, however, was considerably lower (0.09 ± 0.00 %, v/w), thus supporting some previous observations about only trace amounts of essential oil in N. rustica leaves (Tatarchenko et al., 2003).
For comparison, leaves of common tobacco were found to contain between 0.2 % and 1.5 % essential oil (Alagić, "Selekcija", Palić, Stojanović & Nikolić, 2002;Tatarchenko et al., 2003;Zhang, Gao, Zhang, Liu & Ye, 2012), and in single cases of highly aromatic Bulgarian oriental varieties up to 2-2.5 % (Georgiev & Stoyanova, 2006). Data from the identification of the volatile composition of the essential oil, concrete and resinoid are presented in Table 4. GC-MS analysis identified 19 components in the essential oil of N. rustica L. leaves (constituting 75.49 % of the total oil content). Six of the identified volatiles were in quantities over 3 %: (E)-phytol (43.68 %), solanone (5.54 %), cis-5butyl-4-methyldihydrofuran-2(3H)-one (5.23 %), dihydro-β-ionone (4.25 %), α-ionene (3.54 %) and β-damascenone (3.03 %). These results reveal an essential oil that is not significantly different from the one of Oriental type N. tabacum L. (Popova et al., 2015). N. rustica L., however, has not been referenced much as a source of essential oil or other aroma products, and it is hard to make parallels to previous results about leaf oil chemical composition. The established low oil accumulation also probably does not favor N. rustica as a promising aromatic plant. Overall, data in Table 4 showed that, in terms of aroma-active compounds, N. rustica essential oil could not be associated with distinct major compounds, responsible for its aroma profile. As previously reported, tobacco essential oil (N. tabacum L.) is different from other plant essential oils, which always contain a set of special profile-shaping components (Alagić et al., 2002;Zhang et al., 2012). One possible explanation of these results may be related to the particular distillation conditions of tobacco oil (i.e. distillation from a strongly acidified medium, pH 2.00), which stimulate the acidic hydrolysis of certain classes of plant metabolites (including carotenoids). Nineteen compounds were identified in the concrete, representing 73.83 % of its composition. Six of them were in quantities over 3 % and could be considered as major components: isoamyl alcohol (28.82 %), oxynicotine (9.02 %), (E )phytol (7.80 %), 4-methyl-1-penthanol (6.33 %), cotinine (5.55 %) and 3-metyl-3-penthanol (4.09 %). It is hard to draw comparisons, as to the In the resinoid, a total of 16 compounds were identified (85.44 % of its composition). The major constituents (over 3 %) were: nicotine (39.75 %), (E)-phytol (11.23 %), eicosane (4.88 %), diethyl phthalate (4.19 %), dibutyl phthalate (3.48 %) and solanone (3.27 %). The technology of obtaining, together with the high native alkaloid level in the leaves, make N. rustica L. resinoid a higher-nicotine product, with a more specific possible application. For example, a suitable one could be its inclusion in hair treatment products, as there is evidence for a favorable effect of tobacco alkaloids on hair growth (Murkute, Sahu, Mali & Rangari, 2010). The distribution of the identified chemical con-stituents of essential oil, concrete and resinoid, by groups of chemicals (percentage of the identified) reveal the distinctive profile of each of the three aroma products. The essential oil was dominated by oxygenated diterpenes (57.86 %), followed by oxygenated monoterpenes (11.70 %) and nitrogenous compounds (7.51 %). The most abundant chemical constituents of the concrete belonged to the group of oxygenated aliphatics (64.72 %), followed by nitrogenous compounds (22.05 %) and oxygenated diterpenes (10.56 %). The major part of the resinoid was constituted by nitrogenous compounds (52.49 %, and mainly alkaloids), followed by diterpene (13.14 %), aromatic (8.98 %) and oxygenated aliphatic (8.84 %) compounds. The results show that some of the aroma-active classes of compounds (e.g. oxygenated terpenes and aromatic compounds) were found in the oil, but not in the concrete and res-IJFS April 2020 Volume 9 pages 146-159 IJFS April 2020 Volume 9 pages 146-159 inoid (or present in much smaller amounts). Apparently this distribution is related to the different pathway of obtaining the respective aroma products and reflects the influence of factors such as temperature (being respectively 30 o C in concrete extraction, 70 o C in resinoid extraction and steam temperature in essential oil distillation), duration of treatment (1.5 h in the extraction of concrete and 3 h in the hydrodistillation of the oil), and solvent polarity (nonpolar solvent in the case of concrete and polar for resinoid). The identification of individual aromaactive compounds in the essential oil, such as α-ionene, dihydro-β-ionone and β-damascenone, seems to be related to the degradation of leaf carotenoids, as influenced by the high temperature and occurrence of acidic hydrolysis during hydrodistillation, previously reported for common tobacco and other plant materials (Leffingwell, 1999;Winterhalter & Rouseff, 2001). Such carotenoid-related compounds were not identified in N. rustica concrete and resinoid, whereas the latter contained more hydrocarbons and their oxygenated derivatives due to the selective extraction by the solvents, which gave them the characteristic waxy texture.

Antimicrobial activity
Two of the obtained aroma products, concrete and resinoid, were tested for antimicrobial activity against a set of test-microorganisms. They inhibited only the growth of the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. Their activity, however, was weak to moderate, with diameters of the inhibition zones from 9.8±0.01 mm to 11.5±0.03 mm. The rest of the tested bacteria, yeasts and molds were not sensitive to the concrete and resinoid from N. rustica L. leaves. Similar findings of limited or no inhibition activity of ethanol and n-hexane extracts from N. rustica L. leaves on bacteria were reported by Bakht et al. (2013).

Conclusions
The study reveals the potential of N. rustica L. leaves as a plant material that is rich in biologically active compounds, expanding therefore its importance beyond the production of smoking or smokeless tobacco products. The findings justify its consideration as a valuable source for obtaining aroma products of the types traditionally used in cosmetics or other areas (essential oil, concrete, resinoid). Different classes of secondary metabolites with biological activity, alkaloids, triterpenes, phenolic acids and flavonoids, have been identified in the leaves of the tobacco species that is not traditional to Bulgaria. The analysis of the chemical composition of the distilled leaf essential oil and the two concentrated extraction products (concrete and resinoid) revealed their specific volatile profile. To the best of our knowledge, these are the first data about obtaining concrete and resinoid from N. rustica L. and their chemical identification, especially with regard to the one grown in Bulgaria. The results from the study create grounds for directing the respective aroma products to specific applications and for future investigations aimed at expanding the species' importance.