Chemical composition and microbiological evaluation of essential oil from Hyssopus officinalis L. with white and pink flowers

Abstract Hyssopus officinalis L. is a common plant that is most usually found in three color forms - f. cyaneus (blue), f. ruber Mill. (purple/pink) and f. albus Alef (white). In the present work, we evaluated the chemical composition and antimicrobial activity of essential oils obtained from Polish-sourced white- and pink-flowered H. officinalis. Gas chromatography-mass spectrometry analysis of the essential oil has shown that both forms of color have a different content of main components. The principle essential oil component of white-flowered H. officinalis L. was pinocamphone (51%), while pink-flowered H. officinalis L. contained almost equal amounts of pinocamphone (28.8%) and isopinocamphone (21.9%). Of note, the essential oil of the pink form was more active against Grampositive bacteria, especially against Bacillus subtilis.


Introduction 1
Hyssop (Hyssopus officinalis L.), while grown in many countries around the world, is a plant native to southwestern Asia and southern Europe. Due to its camphor aroma and bitter taste, it is often used as a kitchen spice. In both the fresh and the dried state, it is also a supplement to salads, meats, vegetables, cottage cheese and pâté. Moreover, in the production of vermouth and bitter liqueurs (Chartreuse and Benedictine) hyssop supplies the unique spicy taste [1,2].
The aim of this work was to evaluate the chemical composition and antimicrobial activity of essential oil obtained from Poland white and pink-flowered H. officinalis L. The present work is a continuation of previous research [7,30]

Extraction of essential oils 2.2
Essential oil hydro-distillation was carried out using a Deryng apparatus according to the previously described procedure [31]. Herein, 40 g of air-dried plant material plus 500 ml distilled water underwent 3 h distillation. Subsequently, anhydrous sodium sulfate was used to remove the water after extraction. The hyssop essential oils (HEO) were then collected into dark glass bottles and stored in a refrigerator at 4 o C. The distillation of the essential oil was repeated 3 times.

Gas chromatography 2.3
Chromatographic analysis was carried out according to the previously described procedure [32]. In so doing, a GC-MS: ITMS Varian 4000 GC-MS/MS (Varian, USA) equipped with a CP-8410 auto-injector and a 30 m x 0.25 mm VF-5ms column (Varian, USA) was used. In this study, film thickness was 0.25 μm, carrier gas was He 0.5 ml/min, injector and detector temperature were, respectively, at 250 and 200°C; split ratio was 1:50; and inject volume was 5 μl. A temperature gradient was first applied (50°C) for 1 minute, then increased by 4°C/min to 250°C, with 250°C held for 10 minutes. The ionization energy was 70 eV; the range recorded was 35-1000 m/z and the scan rate was 0.80 s per scan. Data acquisition and processing, and instrumental control were performed by the Varian MS Workstation Version 6.42.
Subsequently, a GC/FID GC Varian 3800 (Varian, USA) equipped with a CP-8410 auto-injector and a 30 m x 0.25 mm DB-5 column (J&W Scientific, USA) was employed. The film thickness was 0.25 μm, the carrier gas was He 0.5 ml/min, injector and detector FID temperatures were 260°C; split ratio was 1:100; and injection volume was 5 μl. A temperature gradient was then applied (50°C for 1 minute, then increased by 4°C/min to 250°C, with 250°C held for 10 minutes).
Qualitative analysis was carried out via MS spectra, these were compared with the spectra library by means of the NIST MS Search Program (NIST 08, 2005), as well as with the data available in literature [33]. Identity of the compounds was confirmed by their retention indices, wherein the retention indices were determined in relation to a homologous series of n-alkanes (C 10 -C 40 ) under the same operating conditions. Retention indices were also compared with literature data [34][35][36][37]. The percentages of main components of the essential oil were presented assuming that the sum of peak areas for all identified constituents is 100%.

Antimicrobial activity 2.4
Essential oils from the two color forms of H. officinalis were screened for antibacterial and antifungal activities by way of the micro-dilution broth method, using Mueller-Hinton broth either as Mueller-Hinton broth with 5% lysed sheep blood for growth of non-fastidious and fastidious bacteria, respectively, or as Mueller-Hinton broth with 2% glucose for growth of fungi. Minimal inhibitory concentration (MIC) of tested essential oils were evaluated for the panel of the reference microorganisms which belonged to the American Type Culture Collection (ATCC), including Gramnegative bacteria ( The essential oils dissolved in dimethylosulfoxide (DMSO), were first diluted to the concentration (10 mg/ml) in the aforementioned and recommended broths for determining the antibacterial and antifungal activity. Subsequently, using the same media, serial two-fold dilutions were made in order to obtain final concentrations of plant extracts ranging from 0.156 to 10 mg/ml. The sterile 96-well polystyrene microtitrate plates (Nunc, Denmark) were prepared by dispensing 200 µl of appropriate dilution of plant material in culture broth per well. The inocula were then prepared with fresh microbial cultures in sterile 0.85% NaCl to match the turbidity of 0.5 McFarland standard, and 2 µl were added to the wells to obtain a final density of 1.5 x 10 6 CFU/ml and 5 x 10 4 CFU/ ml for bacteria and yeasts, respectively. After incubation (35°C for 18-24 h), the MICs were assessed visually as the lowest oil concentration showing complete inhibition in the growth of the tested microorganisms. Appropriate DMSO (at a final concentration 10%), a positive control (containing inoculum without plant material) and negative control (containing plant material without inoculum) were included on each microplate. Reference compounds -vancomycin, ciprofloxacin for bacteria and fluconazole for yeasts were purchased from Sigma-Aldrich, (St. Louis, USA).
Minimal bactericidal concentration (MBC) or minimal fungicidal concentration (MFC) was obtained by subculturing 5 µl from each well that showed thorough growth inhibition, from the last positive one and from the growth control onto recommended agar plates. The plates were incubated at 35 o C for 24 h and the MBC/MFC was defined as the lowest concentration of essential oil without growth of microorganisms. Each experiment was repeated in triplicate.

Statistical analysis 2.5
The average values of the component contents were calculated using Excel (Microsoft, USA). Ethical approval: The conducted research is not related to either human or animals use.

Results and discussion 3
The HEO content obtained via hydrodistillation was (on average) 0.7% of total dry weight for white-flowered plants (HWF) and 0.5% of total dry weight for pink-flowered plants (HPF). In the oil of the HWF form, 44 components were identified, and in HPF -49 components. The percentages of chemical constituents of essential oils are shown in Table 1.
Main HEO component-content may vary depending on the plant developmental stage, the age of the plant, the manner and place of cultivation and date of harvest, the plant part harvested and way of distillation, as well as the phenotype. During the development of the plant, the main constituents of the essential oil vary: pinocamphone dominates before flowering, and, during flowering, isopinocamphone [37]. Regarding the age of the plant, first year plants exhibit higher pinocamphone content, while isopinocamphone content dominates in three-year-old plants [22]. As to the manner of cultivation, Tavakoli and Aghajani [38] saw that the main ingredients of H. officinalis (pinocamphone or isopinocamphone) varied depending on the degree of irritation. Regarding altitude of cultivation, Fraternale et al. [39], in assessing the influence of climate on the content of the main components, noted that the oil obtained from the raw material growing below the 100 m above sea level contour contained a higher percentage of pinocamphone, while the oil obtained from the 1000 m above sea level contour contained a higher level of isopinocamphone. Moreover, the oil obtained from plants planted below the 100 m contour contained a much lower percentage of linalool and camphor compared to the oil obtained from plants grown at higher altitudes. Regarding the plant parts, the main components of the HEO were isopinocamphone (49.7-57.7%) in all parts of the plant, while β-pinene and pinocarvone concentrations were variable for each organ, and pinocamphone content never exceeded 1.5% [39]. As to place of occurrence, according to literature, the main HEO component content may vary depending on the place of occurrence, even in a small area. In the study of Bernotiene & Butkiene [27], isopinocamphone (33.6 and 16.8%) was the main constituent in two sites, and pinocarvone (21.1 and 28.1%) was the main component in another two sites. Wesolowska et al. [40], when comparing methods of obtaining HEO (hydrodistillation and steam distillation) did not find significant differences in the isopinocamphone content (the main ingredient). This varied, however, from 40.07% to 45.45% depending on the method. With regard to phenotype, the chemical composition of the three forms of H. officinalis growing in Yugoslavia was found to Unauthenticated Download Date | 8/15/18 5:11 PM be predominantly pinocamphone, isopinocamphone and pinocarvone [3]. Phenotypic variability in the composition of essential oils was also found in the hyssop growing in Moldova. Herein, the principal HEO components in the examined forms were isopinocamphone and pinocamphone [4]. In the present study, pinocamphone (51%) was the dominating compound in the essential oil of white flowers, while in pink flowers, the content of pinocamphone and isopinocamphone was comparatively 28.7% and 21.9%, respectively.
A comparison of the chemical composition of the essential oils from white-and pink-flowered H. officinalis with literature data suggests that the forms present in Poland are f. albus Alef. and f. ruber Mill. However, further taxonomic studies are needed.  Table 2).
The essential oils from pink-flowered H. officinalis was more active against bacteria Gram-positive: Staphylococcus aureus, S. epidermidis, Streptococcus pyogenes, S. pneumoniae and S. mutans. Moreover, it reveals exceptional activity against Bacillus subtilis. The activity of the essential oil against bacteria Gram-negative and yeast of the Candida spp. were similar. Tested essential oils showed lower activity compared to the reference components.
The antimicrobial properties of essential oils depend on a whole group of compounds having very different chemical structures, therefore, the essential oil antimicrobial effects can be explained through several diverse mechanisms. Probably the high lipophilicity of essential oils is very significant because it determines the cell and mitochondrial membrane penetrative qualities (considered as 'good'), the disruption of the structure and functions of the membranes, as well as the enhanced permeability [41]. Furthermore, in studies conducted by Hristova et al. [42], pinocamphnone, isopinocamphone, α− and β−pinene HEO show synergistic effects against Candida spp. The essential oil obtained from the hyssop showed a stronger activity than did pure ingredients. Dehghanzadeh et al. [43] analyzed the composition and antimicrobial properties of the essential oil obtained from H. officinalis growing in Iran, and found it chemically different from the previously described hyssop oils. GC/MS analysis showed that the main components of this oil were thymol> β-bisabolol> carvacrol. This oil showed high antibacterial activity against Klebsiella sp. and Erwinia amylovora (responsible for the occurrence of fire blight on pear and apple crops) and no activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Xannthomonas axonopodis pv citri, Acidovorax sp. Streptomyces scabies and Pseudomonas florescence. Antibacterial and antioxidant properties of the essential oil obtained from H. officinalis grown in Turkey were studied by Kizil et al. [10]. They showed that the chemotype is rich in isopinocamphone> β-pinene> terpinen-4-ol, and was active against Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Escherichia coli, while there was no activity against Pseudomonas aeruginosa.
As revealed, hyssop-derived essential oils have different antimicrobial activity. However, the use of pure oil may be limited due to its neurotoxicity [44], and HEO oil is contraindicated in patients with epilepsy and during pregnancy.

Conclusions 4
An analysis of essential oils of the two flower forms of Hyssopus officinalis L. grown in Poland with white and pink flowers showed different chemotypes. A comparison of the obtained results and the literature data indicate that the essential oil from white-flowered plants contains mainly pinocamphone and β-pinene, whereas oil obtained from pink-flowered plants contained almost equal amounts of pinocamphone and isopinocamphone.

Conflict of interest:
Authors state no conflict of interest.