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. sos- nowskyi. 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