Antimicrobial Activity of Essential Oil and Furanocoumarin Fraction of Three Heracleum Species

Home , Furanocoumarin

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 2 pp. 723ñ728, 2017 ISSN 0001-6837 Polish Pharmaceutical Society

ANTIMICROBIAL ACTIVITY OF ESSENTIAL OIL AND FURANOCOUMARIN FRACTION OF THREE HERACLEUM SPECIES

JOANNA POLITOWICZ1*, ELØBIETA G BAROWSKA2, JAROS£AW PRO∆K”W3, STANIS£AW J. PIETR2 and ANTONI SZUMNY1

1Wroclaw University of Environmental And Life Sciences, Department of Chemistry, C.K. Norwida 25, 50-375 Wroc≥aw, Poland 2Wroclaw University of Environmental And Life Sciences, Department of Plant Protection, Grunwaldzka 53, 50-375 Wroc≥aw, Poland 3Wroclaw University of Environmental And Life Sciences, Department of Plant Biology, Koøuchowska 5b, 51-631 Wroc≥aw, Poland

Keywords: essential oil, antimicrobial activity, furanocoumarin, Heracleum

The genus Heracleum L. belongs to the family Two of them, H. mantegazzianum and H. per- Apiaceae and consists of about 60-70 species, that sicum, are widely used in folklore medicine for the occur mainly in the temperate zone of Eurasia (1-3). treatment of many disorders and have pharmacolog- In Europe there are about 9-11 species (4). In the ical activities: antibacterial, cardiovascular, antican- paper we describe 3 taxa belonging to the genus. didal, analgesic, cytotoxic and anti-inflammatory Three of them are called together as ìgiant (6). Moreover, the fruits of H. persicum are used as Heracleumsî (hogweeds) and are alien species for a spice and flavoring ingredient in food products (7). Europe, and simultaneously, they are commonly dis- H. mantegazzianum is used as an ornamental plant tributed and becoming invasive there, i.e., Heracleum and also for animal feeding in North America and sosnowskyi Manden., H. mantegazzianum Somm. & Europe (8). Lev. and H. persicum Desf. ex Fisch. The two first The aim of this study was to investigate the species listed are native for the Caucasus mountains chemical composition of fruit essential oils and while H. persicum is native mainly for Iran, East furanocoumarin fractions of H. sosnowskyi, H. man- Turkey, North Iraq, North-East Syria, South tegazzianum and H. persicum. Moreover, the antimi- Azerbaijan and South Armenia. They were intro- crobial activity of furanocoumarin fractions from duced from Asia to Europe for decorative purposes or three of the above mentioned Heracleum species as fodder plants (1, 5). The distinguishing character- was examined. istics of the three ìgiant hogweedsî are commonly known, but especially H. sosnowskyi and H. man- EXPERIMENTAL tegazzianum can be mistaken. However, Jakubska- Busse et al. (3) show how to distinguish them using Plant material attached photographs in their article. All three species The fruits of Heracleum sosnowskyi, H. man- belong to the section Pubescentia Mand., thus there tegazzianum and H. persicum were collected from are close relationships among them. Namely, accord- wild-grown plants in Wroc≥aw, in August 2015 and ing to a neighbor-joining analysis of AFLP data, H. identified by Jaros≥aw ProÊkÛw. The voucher speci- mantegazzianum is most closely related to H. sos- mens have been deposited in the Herbarium of nowskyi, and subsequently both species are related to Department of Plant Biology, at the Institute of H. persicum (it means that the latter one is less relat- Biology, Wroc≥aw University of Environmental and ed to them both; see Fig. 3 in Jahodova et al. (5)). Life Sciences, Wroc≥aw, Poland for further reference.

* Corresponding author: e-mail: [email protected]

723 724 JOANNA POLITOWICZ et al.

Extraction procedure of volatile aroma com- Clara, CA, USA) with the ZB-5 column (30 m × pounds 0.25 µm film × 0.25 mm i.d.,). The GC conditions Hydrodistillation using a Deryng apparatus were the same as those for GC-MS. was used for the isolation of the essential oil of three Heracleum species. A suspension of 10 g of materi- Antimicrobial activity al was placed in a 250 mL round flask together with The furanocoumarin fractions from three 100 mL of distilled water. The sample flask was Heracleum species were tested against a panel of heated for 2 h after reaching the boiling point. The pathogens including Gram-positive strains: vapors were condensed by means of a cold refriger- Staphylococcus aureus PCM 2054, S. pseudinter- ant. After 120 min of the process, the essential oil medius KP-Spi1 (isolated from dog), Streptococcus was transferred into 2.5 mL vials and kept at -15OC agalactiae KP-Sag1 (isolated from dog), Bacillus until gas chromatography-mass spectrometry (GC- subtilis PCM 1949 and Gram-negative strains: MS) analyses were performed. Analyses were run in Escherichia coli PCM 2057, Pectobacterium triplicate. atrosepticum IOR-1826 (plant pathogens) as well as the yeast Candida albicans KP-Ca1. These strains Extraction of furanocumarin fractions came from the following collections: KP ñ Fruits were collected and dried in the shadow Department of Pathology (University of Environ- for 12 days. The amount of 30 g crushed plant mate- mental and Life Sciences, Wroc≥aw, Poland), PCM rial was extracted by hexane in room temperature ñ Polish Collection of Microorganisms (Institute of for 24 h. The extracts were then filtered by Immunology and Experimental Therapy, Polish Whatman no. 1 filter paper. The solvent from the Academy of Sciences, Wroc≥aw, Poland), IOR ñ extracts was removed by using rotary vacuum evap- Culture Collection of Plant Pathogens at Institute of orator with the water bath temperature of 40OC. For Plant Protection (Poznan, Poland). essential oil removal, residues were submitted to The antimicrobial activity of furanocoumarin freeze-drying (24 h) process. fractions of Heracleum spp. was evaluated by the disc diffusion technique by determination of growth Chromatographic analyses inhibition zones (9). Briefly, a volume of 100 µL of The chemical composition of the essential oil suspension of the test microorganisms containing was analyzed using a gas chromatograph (GC) cou- 1.5 ◊ 108 cfu/mL of bacteria or 1.5 ◊ 106 cfu/mL was pled to a mass spectrometer (MS) detector (Saturn spread on Mueller Hinton agar (MHA, Sigma- 2000 MS Varian Chrompack, CA, USA) with ZB-5 Aldrich) or Sabouraud dextrose agar medium (SDA, (Phenomenex, CA, USA) column (30 m ¥ 0.25 µm HiMedia Laboratories), respectively. The turbidity film ¥ 0.25 mm i.d.). The MS was equipped with an of the tested strains was standardized to 0.5 ion-trap analyzer set at 1508 for all analyses with an McFarland (spectrophotometer VIS-723G, Ray- electron multiplier voltage of 1350 V. Scanning (1 leigh, Beijing). Paper discs (Whatman no. 1, scan/s) was performed in the range of 35-500 m/z England, 6 mm diameter) were placed on the agar using electron impact ionization at 70 eV. The surface and impregnated with 20 µL of stock solu- analyses were carried out using helium as a carrier tions of furanocoumarin fractions (35 or 70 µg/mL). gas at a flow rate of 1.0 mL/min, in split mode 20, A negative control was prepared using solvent (10% and with the following program for the oven tem- DMSO) employed to dissolve the plant crude perature: 60OC at the beginning and hold 3 min; extracts. In addition, filter discs impregnated with 3OC/min to 120OC; and 15OC/min to 300OC with hold tetracycline or nystatin (35 or 70 µg/mL) were used for 2 min. The injector was held at 250OC. as positive reference control for bacteria and fungi, The compounds were identified by using 3 respectively. The plates, after remaining at 4OC for 2 independent analytical methods: retention indices h, were incubated at 37OC (24 h) for bacterial strains (RI), retention times of authentic chemical-stan- (for P. atrosepticum at 28OC) and at 30OC (48 h) for dards, and mass spectra of compounds and their yeasts. Antimicrobial activity was evaluated by comparison with NIST14 spectral library collection. measuring the diameters of inhibition zones in mil- The retention index standards used in this study con- limeters. The experiment was done in triplicate and sisted of a mixture of aliphatic hydrocarbons rang- mean values are presented in Table 3. The antimi- ing from C-5 through C-30 dissolved in hexane crobial activity of essential oils (1% and 10% v/v) (Aldrich). The quantification was carried out by gas was prepared in the same technique. chromatography analysis (GC, FID, carrier gas H2) The data were subjected to analysis of variance on Agilent Technologies 7890N (GC System, Santa using the Tukey test (p < 0.05) using STATISTICA Antimicrobial activity of essential oil and furanocoumarin fraction... 725

Table 1. The percentage aroma composition and coumarin fraction of the essential oil of H. mantegazzianum, H. persicum and H. sosnowskyi.

Retention Heracleum Compound indices mantegazz. persicum sosnowskyi Exp. a Lit. a Area (%)b* Area (%)b* Area (%)b* 1-Hexanol 856 854 0.06 0.30 0.06 Isopropyl 3-methylbutanoate 882 877 0.21 1.00 0.07 Isobutyl isobutanoate 899 900 0.02 0.06 0.02 Butyl isobutanoate 940 936 0.06 0.19 0.05 Isopropyl 3-methyl-2-butenoate 948 940 0.19 0.48 0.15 1-Heptanol 957 953 0.02 0.01 0.02 Sabinene 966 966 0.01 0.03 0.01 Butyl butanoate 981 978 2.47 ± 0.06 3.73 ± 0.06 2.26 ± 0.06 Isobutyl 2-methylbutanoate 989 990 0.07 0.06 0.06 Hexyl acetate 994 995 1.57 ± 0.03 1.39 ± 0.03 1.20 ± 0.03 p-Cymene 1009 1014 1.94 ± 0.04 1.32 ± 0.03 1.60 ± 0.04 Butyl 2-methylbutanoate 1027 1030 0.20 0.38 0.09 Butyl 3-methylbutanoate 1031 1029 0.11 0.32 0.38 2-Methylbutyl butanoate 1043 1044 0.11 0.09 0.07 γ-Terpinene 1048 1050 0.13 0.24 0.17 1-Octanol 1056 1057 1.80 ± 0.04 2.18 ± 0.05 2.15 ± 0.05 Hexyl propionate 1087 1086 0.30 1.36 0.13 Hexyl isobutanoate 1133 1132 4.86 ± 0.12 3.16 ± 0.11 3.85 ± 0.11 1-Nonanol 1159 1159 0.09 0.01 0.07 Hexyl butanoate 1174 1176 10.85 ± 0.27 40.37 ± 1.00 11.51 ± 0.27 Decanal 1177 1180 1.11 ± 0.03 1.27 ± 0.03 9.51 ± 0.24 2-Decanol 1184 1186 0.27 0.48 0.22 Octyl acetate 1192 1193 51.54 ±1.28 16.60 ± 0.41 43.44 ± 1.08 Hexyl 2-methylbutanoate 1219 1223 6.98 ± 0.17 5.86 ± 0.15 6.47 ± 0.17 Hexyl 3-methylbutanoate 1223 1224 1.65 ± 0.04 1.53 ± 0.04 1.33 ± 0.03 Octyl isobutanoate 1330 1329 1.57 ± 0.04 1.22 ± 0.03 1.80 ± 0.04 Hexyl hexanoate 1370 1371 0.88 ± 0.02 3.45 ± 0.09 0.83 ± 0.02 Octyl butanoate 1373 1374 0.62 ± 0.02 2.00 ± 0.04 0.57 ± 0.01 Octyl 2-methylbutanoate 1416 1422 1.80 ± 0.01 2.23 ± 0.05 1.55 ± 0.01 Octyl hexanoate 1563 1569 0.84 0.21 0.31 p-Octylanisole 1637 1630 1.41 ± 0.03 1.16 ± 0.03 1.03 ± 0.03 Angelicin 1805 1805 56.17 ± 1.40 24.15 ± 0.60 6.02 ± 0.16 Psoralene 1835 1836 3.20 ± 0.11 6.45 ± 0.16 0.52 ± 0.01 Methoxsalen 2027 2034 0.45 ± 0.01 2.03 ± 0.05 0.09 ± 0.01 Bergaptene 2070 2080 3.92 ± 0.09 9.33 ± 0.23 15.20 ± 0.38 Isobergaptene 2116 - 14.59 ± 0.36 15.94 ± 0.37 21.15 ± 0.52 Isopimpinellin 2238 2245 10.90 ± 0.24 26.01 ± 0.65 37.81 ± 0.94 Pimpinellin 2272 - 8.32 ± 0.22 14.59 ± 0.36 18.52 ± 0.46 Imperatorin 2400 2379 2.41 ± 0.06 1.47 ± 0.03 0.66 ± 0.01 a Exp. = experimental, Lit. = literature (17, 18). b Average of the three replicates. * Mean ± SD (standard deviation). 726 JOANNA POLITOWICZ et al.

5.5 software for Windows. All data were presented of the plant, but also by exposure to ìvaporî hover- as the mean values ± standard deviation (SD). ing over the plant on hot days (13). Analysis using GC-MS led to identification of RESULTS AND DISCUSSION 31 compounds (amounting from 95.5% to 98.5% of the total oil). Aliphatic esters (83.6-91.9%) were the Hydrodistillation applying Deryng apparatus main group of compounds identified in the essential was used to extract essential oils from the fruits of oil of all three Heracleum species. In addition to three wild growing Heracleum species (H. man- aliphatic esters, alcohols (4.0-4.6%) and terpenes tegazzianum, H. persicum and H. sosnowskyi). It is (2.2-3.0%) constituted a very low amount of essen- worth mentioning that this study presents compara- tial oil. Hexyl isobutanoate, hexyl butanoate, octyl tive chemical composition of the essential oil and acetate and hexyl 2-methylbutanoate were the most furanocoumarin fraction obtained from three abundant compounds in all three species, which was Heracleum species, which were collected in Poland. previously reported (14). The major group of com- Table 1 shows total volatile components, their reten- pounds that appeared in the studied H. mantegazz- tion indices and relative percentages of the individ- ianum and H. sosnowskyi essential oil are aliphatic ual compounds of the essential oil isolated from the esters, which constitute 91.9 and 88.6% of the total three Heracleum species. The essential oil yields essential oil, respectively. Itís major constituents varied and ranged from 3.6 to 4.5% (v/w, based on were butyl butanoate (2.3 and 2.5%), hexyl the dry weight of the plant material). Surprisingly, butanoate (15.5 and 13.9%), octyl acetate (51.0 and the highest yield of essential oils was from H. man- 53.5%) and hexyl 2-methylbutanoate (6.4 and 7.0%) tegazzianum (4.5%) and the lowest from H. per- in the fruit essential oil, respectively. In addition, for sicum (3.6%). Authors, who examined the essential the third examined Heracleum species which is oil of H. persicum, H. mantegazzianum also report- commonly consumed in Iran, the chemical composi- ed high essential oil yields (3.5 and 4.0%) for the tion is also well known (15). The qualitative analy- fruits of two Heracleum species, respectively (10- sis was the same as in the case of two other 12). Noteworthy is the absence of furanocumarins Heracleum species, but we observed a higher value (expected to be phytotoxic) in the analyzed essential of hexyl butanoate (40.4%) and a lower value of oils. This fact is in contrast to the widespread belief, octyl acetate (20.6%) in the essential oil. In the as well as in some papers, that burns could be caused study presented by Hemati et al. (16), only the quan- by furanocumarins not only through direct contact tity of the above mentioned major compounds

Table 2. Antimicrobial activity of furanocoumarin fractions of Heracleum species using disc diffusion method. H. sosnowskyi H. mantegazzianum H. persicum Antibiotics Microorganisms (70 mg mL-1) (70 mg mL-1) (70 mg mL-1) (35 mg mL-1) Zone of inhibition in mm S. aureus PCM 2054 3.7 ± 0.6b 4.3 ± 0.6b 5 ± 0b 28.3 ± 0.8a * (13%) (15%) (18%) (100%) S. pseudintermedius KP-Spi1 1.7 ± 0.6b 0 ± 0b 0 ± 0b 25.7 ± 0.3a * (6%) (0%) (0%) (100%) S. agalactiae KP-Sag1 8.7 ± 0.3b 10.3 ± 0.3b 8.8 ± 0.1b 25.0 ± 1.0a * (35%) (41%) (35%) (100%) B. subtilis PCM1949 0 ± 0b 0 ± 0b 0 ± 0b 38.0 ± 1.3a * (0%) (0%) (0%) (100%) E. coli PCM 2057 7.7 ± 0.4b 2.7 ± 0.4c 7.7 ± 1.0b 21.0 ± 0.5a * (37%) (13%) (37%) (100%) P. atrosepticum IOR-1826 8.5 ± 0.5b 2.2 ± 0.5c 6.3 ± 0.8b 24.3 ± 0.8a * (35%) (9%) 26%) (100%) C. albicans KP-Ca1 2.0 ± 0.5c 6.0 ± 0.5b 5.3 ± 0.6b 17.3 ± 1.2a * (12%) (35%) (31%) (100%)

± SD (standard deviation). * positive reference control nystatin (for fungi) and tetracycline (for bacteria) were used. () ñ the percent inhibition of microorganisms compared to suitable reference control is in brackets. Values followed by the same letter, within the same line, are not significantly different (p > 0.05 ), Tukeyís multiple-range test. Antimicrobial activity of essential oil and furanocoumarin fraction... 727 slightly varied. Those authors claimed the presence possess moderate antimicrobial activity. Similar of hexyl butanoate and octyl acetate at the level of results to our research were presented by Walasek et 56.5 and 16.5%, respectively. al. (22). They observed that some pure fura- No significant differences were found in the nocoumarins (pimpinellin, imperatorin, and phel- chemical composition of the examined fruit essen- lopterin) isolated from H. mantegazzianum and tial oil of H. sosnowskyi, H. persicum and H. man- plant extract have moderate inhibitory activity tegazzianum, which confirms the suggestions that against some Gram-positive bacteria (Staphylo- the species can be closely related. The only differ- coccus spp., Bacillus spp., Micrococcus spp.) and ence between these three species was the quantity of yeasts (C. albicans, C. parapsitosis). Also, the hexyl butanoate and octyl acetate. In the H. per- essential oil of H. lanatum from aerial parts was sicum essential oil we observed a high level of hexyl investigated by Kharkwal and coworkers (23). They butanoate and a low level of octyl acetate, contrary observed microbial activity against S. aureus, E. coli to H. sosnowskyi and H. mantegazzianum samples. and C. albicans with an inhibition zone of This fact was also confirmed by Jakubska-Busse et ~10.3ñ11.6 mm. Ahmadian-Attari and others (24) al. (3) who studied the chemical composition of the studied the antibacterial effect of hydroalcoholic essential oil of H. sosnowskyi and H. mantegazz- extracts of H. persicum. They observed inhibition of ianum. Heracleum species produce very interesting growth for B. cereus (25 mm zone of 25 mm), S. secondary metabolites which are widely investigat- aureus (zone of 18 mm) as well as E. coli and ed due to their biological activities (e.g., antibacter- Pseudomonas aeruginosa (zone of 11 mm). They ial, antifungicidal, insecticidal, cytotoxic, inflamma- tested 20 mg/mL of extract while we applied the tory, analgesic, antioxidant and antiviral (6, 7, 15, extracts from H. sosnowskyi and H. persicum in the 16). Usually, microbial activity of essential oils, concentration of 70 µg/mL and we observed 6.3ñ8.5 extracts, and pure compounds obtained from mm zones of inhibition growth of E. coli and P. Heracleum species is tested in the concentration atrosepticum. In our research, the furanocumarin ranging from 6 µg/mL to 10 mg/mL (6, 19, 20). The fractions from all Heracleum species were inactive biological effects depend on the qualitative and against B. subtilis. The essential oils didnít inhibit quantitative composition of the tested essential oils growth of all the examined strains. However, Habibi or plant extract. In our research, the essential oil of et al. (25) showed maximum inhibitory activity of Heracleum species didnít show antimicrobial activi- essential oil of H. rechingeri against B. subtilis with ty whereas furanocoumarin fractions showed anti- an inhibition zone of 20 mm. Akcin and others (26) bacterial effectiveness in the concentration of 70 showed that essential oil obtained from H. platytae- µg/mL. The results are presented in Table 2. The nium had no inhibitory activity against the tested results showed that the extracts from Heracleum bacteria (S. aureus, E. coli, Pseudomonas aerugi- species has moderate activity against the tested nosa), but nevertheless exhibited moderate to strong pathogens as compared with the reference drugs activity against Candida globrata with inhibition (tetracycline or nystatin, 35 µg/mL). The extracts zones varying between 12ñ32 mm. Firuzi and others inhibited growth of tested microorganisms at the (15) showed moderate microbial effects of essentail level of 6ñ41%, compared to the reference control ñ oil of Heracleum species against Staphylococcus, antibiotics (100%). The most sensitive of all the Bacillus, Escherichia, Salmonella, Pseudomonas, tested plant extracts was strain of S. agalactiae Candida, Aspergillus. They observed inhibition (zone inhibition 8.7ñ10.3 mm). The growth of this growth of tested microbes with zones 6ñ12 mm. streptococcus was inhibited at the level of 35ñ41%. Differences in antimicrobial activity of the Extracts of H. persicum and H. sosnowskyi showed tested plants are obviously related to differences in a similar inhibitory effect for Gram-negative rods E. their contents of active compounds (Table 1). The coli and P. atrosepticum (26ñ37%). Least activity main compounds of H. sosnowskyi were psoralene was exhibited against S. aureus with the smallest (56%) and isobergapten (~15%), extract of H. man- inhibition zone (3.7ñ5 mm). The extracts from all tegazzianum contained psoralene (24%) and isopim- the tested plant Heracleum species were inactive pinellin (26%) and H. persicum has mainly against endospore forming rods of B. subtilis and izopimpinellin (~38%) and isobergapten (21%). The cocci of S. pseudintermedius. Two of them, H. man- mentioned compounds are classified as active tegazzianum and H. persicum, showed antifungal antimicrobial compounds (6). The obtained results effect against C. albicans with inhibition zones of show that the antimicrobial activity is different 6.0 and 5.3 mm, respectively. 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