{'fraction_non_alphanumeric': 0.037142857142857144, 'fraction_numerical': 0.02378737541528239, 'mean_word_length': 4.829202168861348, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Chemical composition and in vitro antioxidant and antibacterial activity of Heracleum transcaucasicum and Heracleum anisactis roots essential oil

Introduction: In vitro antioxidant and antibacterial activity and volatile compositions of two Heracleum species (Apiaceae) including Heracleum transcaucasicum and Heracleum anisactis roots Essential Oil (EO) were investigated. Methods: The volatile compositions of EOs were analyzed by GC/Mass spectroscopy. To detect the antioxidant activity of essential oils TLC-bioautography and DPPH radical scavenging assay by spectrophotometry was performed. Additionally, the antibacterial activity of two essential oils were studied and compared against four pathogenic bacteria by agar disc diffusion method and MIC values of the EOs were determined using the broth dilution method. Results: Myristicin was the dominant component in both EOs. It was identified as 96.87% and 95.15% of the essential oil composition of H. transcaucasicum and H. anisactis roots, respectively. The TLC-bioautography showed antioxidant spots in both EOs and IC50 of H. anisactis and H. transcaucasicum EO was found to be 54 μg × ml (-1) and 77 μg × ml (-1), respectively. Regarding the antimicrobial assay, H. anisactis EO exhibited weak to moderate antibacterial activity against gram-positive bacteria and also Escherichia coli, whereas the essential oil from H. transcaucasicum was inactive. Conclusion: Based on the results from this study, both tested EOs mainly consist of myristicin. Despite the presence of myristicin with known antibacterial property, the EO from H. transcacausicum showed no antibacterial activity. Thus it is supposed that the biological activity of plants is remarkably linked to the extracts’ chemical profile and intercomponents’ synergistic or antagonistic effect could play a crucial role in bioactivity of EOs and other plant extracts.


Introduction
Essential oils of various species of edible and medicinal plants have been extensively used in folk medicine, food flavoring, and perfumery, cosmetic and pharmaceutical industries. Variation in chemical composition of essential oils in particular and extracts of medicinal plants may be observed due to the origin, the environmental conditions, and the developmental stage of collected plant materials. The genus Heracleum is one of the largest genera of Umbelliferae (Apiaceae). This genus is widely distributed in Asia 1 and represented by 10 species in the flora of Iran 2 from which H. transcaucasicum and H.anisactis are two abundant species in Azerbaijan region. 3,4 Heracleum species are well known as essential oil rich plants and medicinal plants as well. In Iranian folk medicine, fruits, stems and leaves of Heracleum species are used as spices, carminative and antiseptic. 4 The ripe fruits, leaves and young stems of H. persicum (Golpar) are also used as antiseptic, carminative, digestive and analgesic in the Iranian folk medicine. 3 A great number of studies refer to the compositions of Heracleum species essential oils. Various studies on Heracleum species have reported variations in the essential oil composition according to the stage of growing and different parts of the plant [5][6][7] and biological activities. The main volatile substances in the majority of EOs from Heracleum species are aliphatic esters such as octyl acetate and hexyl butyrate, hexyl-2-methylbutanoate and hexyl isobutyrate. (E)-anethole β-pinene, ρ-cymene, terpinolene, α-caryophylene, α-bergamotene, α-farnesene, zingiberene, spathulenol, cis-anethole, stragole, 2,5-dimethyl styrene and β-springene were reported in leaves EO. 8,9 The major constituents of H. persicum roots essential oil were identified as viridiflorol (23.05%), elemol (3.63%), β-maliene (3.07%), spathulenol (3.34%) and 2-tetradecanol (3.38%). 10 The main components of stem oil before flowering were (e)-anethole (47.0%), terpinolene (20.0%), [gamma]-terpinene (11.6%) and limonene (11.5%), while stem oil at the full flowering stage contained (e) -anethole (60.2%), terpinolene (11.3%) and [gamma]-terpinene (7.1%). The unripe and ripe seeds oil of H. persicum contained aliphatic esters, aliphatic alcohols and monoterpenes esters and monoterpenes such as hexyl butyrate, octyl acetate and hexyl isobutyrate. 11,12 The antimicrobial activity and other biological activities of an essential oil are mainly attributed to its major components, although sometimes the synergistic or antagonistic effect of one compound in minor percentage of mixture plays a key role in activity. Therefore, EOs exhibit various biological activities based on variations in their individual components. 13 In the framework of our investigation on the chemical compositions of H. transcaucasicum and H. anisactis, we reported the composition and antibacterial activity of EOs of aerial parts. 14 In the present study, we examined the essential oils of their roots as well as their antioxidant and antimicrobial activities against four pathogenic bacteria, which have not been the subject of previous investigations.

Essential oil extraction
Air-dried roots of H. transcaucasicum and H. anisactis were subjected to hydro-distillation using a Clevengertype apparatus. The yields of essential oils were respectively 1.7% v/w and 1.3% in terms of dried starting material. The essential oils were preserved in sealed vials in a refrigerator prior to analysis.

Gas Chromatography-Mass Spectrometry (GC-MS)
Essential oils were analysed on an Agilent 6890 GCquadrupole MS system (Agilent Technologies, Palo Alto, USA) with fused capillary column HP-5 (phenyl methyl siloxane, 30 m × 0.25 mm; 0.25 μm film thickness, Agilent Technologies) and a flame ionization detector (FID) which was operated in EI mode at 70 eV. Injector was set at 210 °C. Essential oils were dissolved in n-hexane (1:4) and 1 µl of each essential oil solution was injected with a split ratio of 1:25. Analyses were performed with the column held initially at 60 °C for 2 min and then increased by 4 °C/min up to 240 °C. Helium was employed as carrier gas (0.8 ml/min). The MS operating parameters were as follows: ionization potential, 70 eV; transfer line temperature 270 °C; ion source temperature 230 °C; quadrupole 150 °C; solvent delay 2 min; scan speed 2000 amu/s; and scan range 30-600 amu. The relative amount of individual components of the total oil is expressed as percentage peak area relative to total peak area. Qualitative identification of the different constituents was performed by comparison of their relative retention times and mass spectra with those of authentic reference compounds, or by mass spectra. 15

Identification of the compounds
The identification of compounds was based on direct comparison of the retention times and mass spectral data with those for the standards and by computer matching with the Wiley 229, Nist 107, Nist 21 Library, as well as by comparing the fragmentation patterns of the mass spectra with those reported in the literature. 15 For quantification purpose, relative area percentages were obtained by FID without the use of correction factors.

Determination of in vitro antioxidant activity
To detect the activity of individual components of each EOs, TLC-bioautography with 1, 1-Diphenyl-2picrylhydrazyl (DPPH, Sigma-Aldrich Quımica S.A., Madrid, Spain) radical as a detection reagent was carried out. The components of EOs were separated on pre-coated Silica gel aluminum plate F 254 with 0.2 mm thickness (Merck-Germany) using Toluene-Ethylacetate in the ratio of 93:7 as the mobile phase 16 and DPPH solution (0.04% in MeOH) for detection. Then the plate was allowed to dry at room temperature. The antioxidant components were visualized by spraying the plate with DPPH reagent and were left 15 min at room temperature. The separated antioxidant spots were appeared as yellow on a purple background. [17][18][19] The TLC plate was observed with visible light (Fig. 1). The free radical scavenging capacities of the EOs were measured from the bleaching of the purple-colored methanol solution of DPPH (0.008 mg%). The stock concentration 1 mg/ml of the EOs was prepared followed by dilution in order to obtain concentrations of 5×10 -1 , 2.5×10 -1 , 1.25×10 -1 , 6.25×10 -2 , 3.13×10 -2 and 1.56×10 -2 mg/ ml. The obtained concentrations in equal volumes of 2 ml were added to 2 ml of a 0.008% of DPPH solution. After a 30 min incubation period at 25 ºC, the absorbance at 517 nm was determined against a blank. Tests were carried out in triplicate where the average absorption was noted for each concentration. 20 Antibacterial activity of essential oils was firstly screened by the disc diffusion method. One hundred μl of the bacterial suspensions were spread over the plates containing Mueller-Hinton agar plus 4% polysorbate 20 using a sterile cotton swab in order to get a uniform microbial growth on both control and test plates. The bacterial suspension was spread on the surface of the plates in a way that nothing remained afloat. The essential oils were dissolved in 10% aqueous dimethyl sulfoxide (DMSO) and sterilized by filtration through a 0.45 μm membrane filter. Sterilized discs (Whatman no.1, 6 mm diameters) were impregnated with 50 μL of different concentrations (1:1, 1:5, 1:10) of the respective EOs in two steps; firstly the dry disks were placed on the surface of the agar plate. Then 30 μL of each sample was poured directly on the center of the disk using sampler and after 15 min, the remaining 20 μL was poured on the same disk. Paper disc moistened with 10% aqueous DMSO in the same way as test samples was placed on the seeded plate as a vehicle control. A standard disc containing Amikacin (30 mg) was used as reference control. The plates were incubated for 30 min at refrigerator and then they were incubated at 37 °C for 18 h. The refrigeration was needed for slowing down the growth of the bacteria and consequently for giving the oil enough time to diffuse before bacterial growth. After the incubation period, the zone of inhibition was measured with a calliper. All experiments were performed in triplicate, and mean values were calculated.

Determinations of minimum inhibitory concentration
The agar dilution method was employed to determine minimum inhibitory concentration (MIC) of the EOs according to CLSI guidelines. The prepared inoculum was added to Muller Hinton broth plus 4% polysorbate 20 medium to reach the final concentration of 106 CFU/ ml. One milliliter of the inoculated medium was added to 8 sterile tubes including seven tests containing serially diluted EOs in 10% and a control tube with 10% aqueous DMSO without essential oil respectively. The tubes were incubated at 37 °C for 24 h. After that, from the content of each tube streak culture was performed onto Mueller Hinton Agar plates. The first concentration with no sign of bacterial growth on plates was considered as MIC. All experiments were performed in three separate occasions.

Results
The Pale yellow EOs were obtained in the yields of 1.7% and 1.3% (v/w) on a dry weight basis from H. transcaucasicum and H. anisactis roots, respectively. EOs were analyzed by GC and GC-MS and the chemical constituents of both essential oils were identified qualitatively and quantitatively. Eleven components (which accounted for 99.18% of total constituents in oil) were identified as constituents of H. transcaucasicum roots essential oil. The major components of this essential oil were myristicin (which accounted for 96.87% of total constituents in oil), followed by apiol (1.87%) and eugenol (0.12%). In a smaller percent, Eugenol methyl ether (0.05%) and Cisisomyristicin (0.05%) were identified as constituents of the oil ( Table 1). Analysis of H. anisactis roots essential oil revealed 17 components accounting for 96.89% of the total essential oil composition. The main component was found to be myristicin (95.15%) followed by isoelemicin (0.8%), nerolidol (0.44%), osthole (0.13%) and eugenol (0.08%) ( Table 2). Among components identified in EOs, eugenol was not reported, previously. Moreover, myristicin, an alkenylbenzene derivative, prevalent in  both of the oils was evident as a major component, while most components were only found as a trace (≤1%). According to the result of TLC-bioautography both EOs showed three distinctive yellowish antioxidant spots with RF values of 0.35, 0.6, 0.85 and 0.9, although antioxidant spots of H. anisactis were almost larger than those of H. transcaucasicum EO (Fig. 1). DPPH radical scavenging test on the two species of Heracleum with ultraviolet-visible spectrophotometry revealed that the concentration of H. anisactis after scavenging 50% of DPPH radicals (IC 50 ) was 54 µg × ml(-1) whereas the figure for H. transcaucasicum was 77 µg × ml(-1).

Assessment of antimicrobial activity
Firstly the EOs of the plants were screened for antimicrobial activity against the selected strains using the agar disc diffusion method. The results are presented in Table 3. According to the results, the EOs of H. anisactis exhibited weak to moderate antimicrobial activity against the gram positive strains and also E. coli as our gram negative indicator. As it is shown in Table 3, the maximum inhibition zone diameter was obtained from  Fig. 3 and Fig. 4). No sign of grown inhibition was observed in the case of all the bacterial strains when examined with the EO of H. transcacausicum. MIC values of the essential oil of H. anisactis were determined using the broth dilution method and the results are depicted in Table 3 as well. As can be seen in Table 3  The findings of the study on antibacterial activity of EOs indicated that the EO from H. anisactis roots showed moderate activity, whereas the H. transcaucasicum EO showed no activity. The essential oil of H. anisactis was found to be more active against gram positive bacteria. S. epidermidis was the most sensitive strain with the strongest inhibition zone of 13 ± 1.2 mm. This finding is in line with previous studies in that they indicated the higher sensitivity of the gram positive bacteria to the natural herbal extracts in comparison with their gram negative counterparts. It can be justified with the intrinsic resistance of gram negative bacteria with lipopolysaccharide outer layer. 24 These results also could be accounted for the synergism or antagonism effect of various components of EOs.

Conclusion
To our knowledge, this is the first report on the EO from H. anisactis and H. transcaucasicum roots. The present results demonstrate high antioxidant activity for both EOs and moderate antibacterial activity only for H. anisactis. Various compounds were detected in both EOs, from which myrisitin, anethole, elemicin, osthole, eugenol, nerolidol and hydrocarbon esters could be predominant contributors to the free radical scavenging and antibacterial activity of the EOs. Considering all results, it is supposed that particular bioactive functional properties of plants are highly affected by their chemical contents profile.