Abstract
In the present work the composition of biogenic volatile organic compounds (BVOCs) and the essential oil (EO) of Helichrysum araxinum Takht. ex Kirp. aerial parts, together with the antimicrobial activity, were investigated. The results showed the prevalence of sesquiterpene hydrocarbons in both spontaneous emissions as well as in the EO. The main compounds of BVOCs were γ-curcumene (10.7%), γ-muurolene (9.2%), and β-selinene (8.5%). This latter constituent also showed a similar amount in the EO and represented the most abundant compounds together with α-selinene (8.0%). It is Interesting to note the same percentage of monoterpene hydrocarbons (MHs) in both the aroma profile and the EO (18.0%) with the same most abundant compounds: β-pinene (6.3% in BVOCs vs. 5.1% in EO, respectively) and limonene (4.5% in VOCs vs. 4.9% in EO, respectively). With regard to the antimycotic activity, the EO showed to be inactive against the tested strains, while a moderate antibacterial activity was shown against Staphylococcus isolates.
1 Introduction
Helichrysum genus, Gol-e-Bimarg as it is called in the Persian traditional language, belongs to the Asteraceae family with 600 species mainly distributed in Africa and Madagascar [1]. Plants of this genus are known for their richness in secondary metabolites, including flavonoids, acetophenones, phloroglucinols, pyrones, triterpenoids, and sesquiterpenes [2]. These secondary metabolites, used as a biochemical defense mechanism against bacteria and fungi, have recently been of great interest [3], [4], [5], [6]. Helichrysum araxinum Takht. ex Kirp. is a native species of Turkey where it grows in rocky limestone slopes, forest clearings and steppes at 900–2500 m above sea level. It is a strongly suffruticose plant, 18–5-cm high, from subglaborous to thin lanate, with numerous erect twiggy stems and median cauline leaves. It shows very close and neat corymb with turbinate-cylindrical straw-colored capitula, each 5–6 mm long, from three to 10 in each corymb. All flowers are hermaphrodite [7].
The literature reports many medicinal benefits of the different Helichrysum species. The most recent report dates back to more than 10 years ago (2008) when Lourens et al. [8], in his review on the traditional use of the South African Helichrysum species, cited their use in treating gall bladder disorders, due to its bile regulation and diuretic effects. The H. araxinum herbal products were also used as relief for stomach ache, for their anti-infective, hepaprotective, cholagogic and choleretic effects; to stimulate the secretion of gastric juices, and for the treatment of coughs and diabetes mellitus. Furthermore, its antimicrobial [9] and cytotoxic properties [10] are well known.
Phytochemical investigation on H. araxinum reported in the literature has shown the presence of some flavonoids such as apigenin, luteolin, naringenin, astragalin, helichrysins A and B, isosalipurposide, apigenin 4′- and 7-glucosides, and quercetin 3-glucoside in the capitula, while kaempferol, quercetin, astragalin, and quercetin-3-glucoside were found in the leaves [11].
The present work investigates the chemical characterization by gas chromatography-mass spectrometry (GC-MS) of the spontaneous volatile emission of H. araxinum aerial parts and the essential oil (EO) composition for the first time. Furthermore, the in vitro antimicrobial activity of its EO against fungi and bacteria has also been reported.
2 Experimental
2.1 Plant material
Helichrysum araxinum Takht. ex Kirp., grown in pots under uniform environmental condition at the Centro di Ricerca Orticoltura e Florovivaismo (CREA), Sanremo, Italy, was collected in 2018 and identified by one of us (C. Cervelli). A voucher specimen was deposited at the Herbarium of Giardini Botanici Hanbury (La Mortola, Ventimiglia, Italy) (HMGBH.e/9006.2019.001).
2.2 EO extraction
Helichrysum araxinum air-dried aerial parts (50 g) were subjected to hydro-distillation for 2 h, using a Clevenger apparatus. The obtained oil was dehydrated over anhydrous magnesium sulfate, then diluted to 0.5% in n-hexane high-performance liquid chromatography (HPLC) grade prior to GC-MS injection. The injections were performed immediately after extraction.
2.3 Headspace solid phase microextractions (HS-SPME)
The headspace from fresh aerial parts of H. araxinum (about 2 g) was sampled by solid phase microextraction (SPME). The adsorption of volatiles was performed with a Supelco polydimethylsiloxane fiber assembly (100-μm coating thickness, Supelco, St. Louis, MO, USA) preconditioned according to the manufacturer’s instructions. After the equilibration time, the septum of each vial was perforated by the holder (syringe), then the fiber was exposed to the headspace of the sample for 30 min at room temperature. Once the sampling was complete, the fiber was retracted into the holder and directly injected in the GC-MS apparatus for separation and analysis.
2.4 GC-MS analyses
The GC-MS analysis was carried out with an Agilent 7890D gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS (Agilent Technologies Inc.) capillary column (30 m×0.25 mm; coating thickness 0.25 μm) and an Agilent 5977B single quadrupole mass detector (Agilent Technologies Inc.).
The GC oven temperature was set to rise from 60 to 240 °C at a rate of 3 °C/min. The split ratio was adjusted at 1:25. The carrier gas helium was at 1 mL/min; an injection of 1 μL (0.5% HPLC grade n-hexane solution). The acquisition parameters were as follows: full scan; scan range: 30–300 m/z; scan time: 1.0 s.
2.5 Peak identification
Identification of the EO components was carried out either through the comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) with a series of n-hydrocarbons, and by a computer matching against commercial (NIST 14 and ADAMS 2007) and laboratory-developed mass spectra library built up from pure substances and components of known oils and MS literature data [12], [13], [14], [15], [16], [17].
2.6 Antimycotic activity
The antimycotic activity of the H. araxinum EO was checked against different fungal species. In detail, two feline clinical isolates of Microsporum canis and Trichophyton mentagrophytes, a clinical isolate from turtles of Fusarium solani and two environmental fungi, Aspergillus niger and Aspergillus flavus. All the microorganisms were used for in vitro sensitivity assays and were maintained on malt extract agar (MEA) at room temperature until use.
The minimum inhibitory concentration (MIC) value was calculated by a microdilution test, performed as recommended by the Clinical and Laboratory Standards Institute (CLSI M38-A2) for molds (2008) [18], starting from a 5% EO dilution. Five percent, 2.5%, 1.5%, and 1% dilutions were carried out. All assays were performed in triplicate.
2.7 Antibacterial activity
The EO was tested against three field bacterial strains: Escherichia coli (Gram-negative), Staphylococcus aureus and Staphylococcus pseudointermedius (Gram-positive). The strains have been previously isolated from canine clinical specimens, typed and stored at −80 °C in glycerol broth.
The antibacterial activity of the EO, diluted at 10% in dimethyl sulfoxide (DMSO, Oxoid Ltd., Basingstoke, Hampshire, UK) was tested using the Kirby-Bauer agar disc diffusion method [19]. A commercial disk impregnated with chloramphenicol (30 μg) (Oxoid) and a paper disk impregnated with 10 μL of DMSO were included as positive and negative controls, respectively. All tests were performed in triplicate. Successively, the MIC was determined with the broth microdilution method following the guidelines of the CLSI [20] with some modifications as previously reported [21].
3 Results and discussion
The complete identification of the spontaneous emission as well as the EO composition is reported in Table 1 . Sesquiterpene hydrocarbons represented the most abundant chemical class of compounds in both biogenic volatile organic compounds (BVOCs) and EO, accounting for 79.5% and 53.1%, respectively. The identified compounds belonging to this class are very different in the SPME and EO. γ-Curcumene (10.7%), γ-muurolene (9.2%), β- and α-selinene (8.5 and 7.3%, respectively) as well as guaia-6,9-diene (7.3%) were the most abundant sesquiterpene hydrocarbons in the VOC emission of H. araxinum. These compounds were also the most represented in the EO, where β- and α-selinene together with γ-curcumene accounted for over 23%. Conversely, γ-muurolene was significantly less represented in the EO (0.4%).
Chemical composition of VOCs and EO of H. araxinum aerial parts.
| Compounds | L.R.I.a | L.R.I.b | Class | SPME | EO | |
|---|---|---|---|---|---|---|
| Relative percentage (%) | ||||||
| 1 | Bornylene | 908 | 908c | MH | 0.1±0.01 | 0.1±0.02 |
| 2 | α-Pinene | 939 | 939 | MH | 3.2±0.24 | 3.7±0.16 |
| 3 | α-Fenchene | 950 | 953 | MH | – | 1.0±0.08 |
| 4 | Camphene | 954 | 954 | MH | 1.3±0.88 | 0.6±0.03 |
| 5 | β-Pinene | 979 | 979 | MH | 6.3±1.15 | 5.1±0.16 |
| 6 | Myrcene | 991 | 991 | MH | 0.2±0.10 | 0.2±0.01 |
| 7 | (E)-3-Hexen-1-ol acetate | 1005 | 1002 | NT | 0.3±0.07 | – |
| 8 | α-Terpinene | 1017 | 1017 | MH | 0.6±0.21 | 0.5±0.02 |
| 9 | p-Cymene | 1025 | 1025 | MH | 0.1±0.01 | 0.2±0.02 |
| 10 | Limonene | 1029 | 1029 | MH | 4.5±0.15 | 4.9±0.21 |
| 11 | γ-Terpinene | 1060 | 1060 | MH | 1.0±0.37 | 0.9±0.03 |
| 12 | Terpinolene | 1089 | 1089 | MH | 0.7±0.25 | 0.8±0.03 |
| 13 | Exo-fenchol | 1122 | 1122 | OM | 0.1±0.09 | 0.5±0.02 |
| 14 | 3-Caraneol | 1143 | 1125d | OM | – | 0.2±0.02 |
| 15 | Camphene hydrate | 1148 | 1150 | OM | – | 0.2±0.01 |
| 16 | Borneol | 1169 | 1169 | OM | 0.3±0.04 | 1.2±0.03 |
| 17 | 4-Terpineol | 1177 | 1177 | OM | 0.4±0.07 | 0.7±0.01 |
| 18 | α-Terpineol | 1189 | 1189 | OM | 0.6±0.08 | 1.9±0.01 |
| 19 | Methyl 8-methyl-nonanoate | 1277 | 1265 | NT | 0.1±0.01 | 0.2±0.01 |
| 20 | Bornyl acetate | 1289 | 1289 | OM | 0.3±0.06 | 0.5±0.02 |
| 21 | Perilla alcohol | 1297 | 1295 | OM | – | 0.4±0.03 |
| 22 | p-Mentha-1,4-dien-7-ol | 1330 | 1333 | OM | – | 0.1±0.00 |
| 23 | 7-Epi-silphiperfol-5-ene | 1348 | 1348 | SH | 0.3±0.01 | 0.1±0.01 |
| 24 | Neryl acetate | 1362 | 1362 | OM | 0.2±0.03 | 0.3±0.01 |
| 25 | α-ylangene | 1375 | 1375 | SH | 1.3±0.03 | 0.4±0.03 |
| 26 | Di-epi-α-cedrene-(I) | 1382 | 1385 | SH | 0.7±0.11 | 0.3±0.01 |
| 27 | Modephene | 1385 | 1384 | SH | 3.6±0.11 | 1.4±0.07 |
| 28 | α-Isocomene | 1388 | 1388 | SH | 1.9±0.13 | 0.9±0.03 |
| 29 | (+)-Sativene | 1396 | 1392 | SH | 0.1±0.08 | – |
| 30 | Italicene | 1406 | 1406 | SH | 2.4±0.23 | 1,2±0.01 |
| 31 | (±)-β-Isocomene | 1412 | 1412 | SH | 0.3±0.03 | – |
| 32 | β-Isocomene | 1412 | 1407 | SH | – | 0.2±0.02 |
| 33 | Cis-α-bergamotene | 1413 | 1413 | SH | 1.1±0.22 | 0.3±0.01 |
| 34 | β-Caryophyllene | 1420 | 1419 | SH | 1.3±0.24 | 0.2±0.01 |
| 35 | Trans-α-bergamotene | 1435 | 1435 | SH | 1.2±0.31 | 0.5±0.01 |
| 36 | Aromadendrene | 1440 | 1441 | SH | 3.3±0.29 | – |
| 37 | γ-Patchoulene | 1441 | 1441 | SH | – | 2.0±0.06 |
| 38 | Guaia-6,9-diene | 1443 | 1447 | SH | 7.3±0.50 | 4.5±0.18 |
| 39 | β-Gurjunene | 1447 | 1434 | SH | 3.1±0.22 | 1.6±0.06 |
| 40 | (E)-β-Farnesene | 1457 | 1457 | SH | 0.6±0.03 | – |
| 41 | α-Elemene | 1462 | 1469 | SH | – | 0.1±0.01 |
| 42 | 9-Epi-(E)-caryophyllene | 1466 | 1466 | SH | – | 0.1±0.01 |
| 43 | α-Acoradiene | 1466 | 1466 | SH | 0.1±0.09 | – |
| 44 | γ-Gurjunene | 1473 | 1477 | SH | 0.8±0.08 | – |
| 45 | 4-Epi-α-acoradiene | 1475 | 1475 | SH | – | 0.1±0.01 |
| 46 | Aristolochene | 1476 | 1479 | SH | 0.3±0.05 | 0.2±0.02 |
| 47 | γ-Muurolene | 1480 | 1480 | SH | 9.2±1.22 | 0.4±0.02 |
| 48 | γ-Curcumene | 1483 | 1483 | SH | 10.7±1.40 | 6.7±0.38 |
| 49 | 4,11-Selinadiene | 1485 | 1485 | SH | – | 5.6±0.18 |
| 50 | β-Selinene | 1486 | 1490 | SH | 8.5±0.72 | 8.5±0.25 |
| 51 | Valencene | 1496 | 1496 | SH | 4.5±0.83 | – |
| 52 | δ-Selinene | 1497 | 1493 | SH | 4.7±0.35 | 6.0±0.25 |
| 53 | α-Selinene | 1498 | 1498 | SH | 7.3±0.76 | 8.0±0.38 |
| 54 | α-Muurolene | 1500 | 1500 | SH | 0.3±0.02 | 0.1±0.05 |
| 55 | Epizonarene | 1501 | 1502 | SH | 0.1±0.08 | – |
| 56 | γ-Cadinene | 1513 | 1514 | SH | – | 0.2±0.01 |
| 57 | β-Curcumene | 1516 | 1516 | SH | 0.3±0.09 | 0.2±0.02 |
| 58 | 7-Epi-α-selinene | 1517 | 1522 | SH | 0.2±0.07 | 0.3±0.01 |
| 59 | β-Cadinene | 1518 | 1517 | SH | 2.2±0.43 | 1.3±0.05 |
| 60 | δ-Cadinene | 1523 | 1523 | SH | 0.3±0.01 | 0.2±0.06 |
| 61 | Selina-3,7(11)-diene | 1542 | 1547c | SH | 0.9±0.23 | 0.40.01 |
| 62 | (4aR,8aS)-4a-Methyl-1-methylene-7-(propan-2-ylidene) decahydronaphthalene | 1544 | 1544c | SH | 0.6±0.22 | 0.7±0.00 |
| 63 | α-Calacorene | 1546 | 1546 | SH | – | 0.4±0.01 |
| 64 | Epi-globulol | 1585 | 1585 | OS | – | 0.2±0.04 |
| 65 | Viridiflorol | 1591 | 1593 | OS | – | 1.0±0.02 |
| 66 | Guaiol | 1596 | 1601 | OS | – | 0.6±0.04 |
| 67 | Humulene-1,6-dien-3-ol | 1619 | 1619c | OS | – | 1.1±0.03 |
| 68 | Selina-6-en-4-ol | 1624 | 1624c | OS | – | 0.7±0.11 |
| 69 | Selina-6-en-4α-ol | 1636 | 1636c | OS | – | 5.8±0.26 |
| 70 | Cubenol | 1642 | 1647 | OS | – | 1.7±0.13 |
| 71 | Hinesol | 1642 | 1642 | OS | – | 0.3±0.13 |
| 72 | Agarospirol | 1645 | 1648 | OS | – | 0.4±0.04 |
| 73 | β-Eudesmol | 1649 | 1651 | OS | – | 1.4±0.10 |
| 74 | α-Eudesmol | 1654 | 1654 | OS | – | 1.6±0.14 |
| 75 | Neointermedeol | 1660 | 1660 | OS | – | 3.5±0.11 |
| 76 | Intermedeol | 1667 | 1667 | OS | – | 0.1±0.07 |
| 77 | β-Bisabolol | 1671 | 1675 | OS | – | 0.4±0.05 |
| 78 | Ylangenal | 1675 | 1674c | OS | – | 0.1±0.11 |
| 79 | α-Bisabolol | 1686 | 1685 | OS | – | 0.3±0.07 |
| 80 | Juniper camphor | 1693 | 1700c | OS | – | 0.3±0.02 |
| 81 | Trans-α-bergamotol | 1700 | 1691 | OS | – | 0.9±0.02 |
| 82 | β-Santalol | 1715 | 1716 | OS | – | 0.1±0.08 |
| 83 | Cis-lanceol | 1763 | 1761 | OS | – | 0.1±0.01 |
| 84 | 3,5,6,7,8,8a-Hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-2(1H)naphthalenone | 1781 | 1773c | OS | – | 0.1±0.02 |
| Yield of EO (% w/w) | 1.4±0.01 | |||||
| Chemical classes | SPME | EO | ||||
| Monoterpene hydrocarbons (MH) | 18.0±7.57 | 18.0±0.74 | ||||
| Oxygenated monoterpenes (OM) | 1.9±0.31 | 6.0±0.10 | ||||
| Sesquiterpene hydrocarbons (SH) | 79.5±7.90 | 53.1±1.95 | ||||
| Oxygenated sesquiterpenes (OS) | – | 20.7±1.30 | ||||
| Non-terpene derivatives (NT) | 0.4±0.06 | 0.2±0.01 | ||||
| Total identified (%) | 99.8±0.10 | 98.0±0.50 | ||||
Data are reported as mean values (n=3; ±standard deviation [SD]); L.R.I. aLinear retention time experimentally determined; L.R.I. bLinear retention time reported by Adams [22]; cLinear retention time reported by NIST [23]; dLinear retention index in chemspider (www.chemspider.com). VOCs, volatile organic compounds.
Antimycotic and antibacterial activity of the H. araxinum EO.
| Fungi strains | Bacterial strains | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| M. canis | T. mentagrophytes | A. flavus | A. niger | T. solani | S. aureus | S. intermedius | E. coli | |||
| MIC (%) | MIC (%) | Disk (mm) | MIC (%) | Disk (mm) | MIC (%) | Disk (mm) | ||||
| >5 | >5 | >5 | >5 | >5 | 5 | 8 | 5 | 8 | Non-effective | |
MIC, mean inhibitory concentration.
Oxygenated sesquiterpenes showed a more relevant relative abundance in the EO composition. Among them, selina-6-en-4α-ol, cubenol, α- and β-eudesmol showed the highest contents (5.8%, 1.7%, 1.6% and 1.4%, respectively), whereas their presence was not detected in the spontaneous emission.
Monoterpene hydrocarbons were present in the same relative abundance both in BVOCs and in the EO (about 18.0%), with β-pinene (6.3% vs. 5.1%, respectively), limonene (4.5% vs. 4.9%, respectively), and α-pinene (3.2% vs. 3.7%, respectively) as the main constituents.
To the best of our knowledge there are no reports in the literature on the spontaneous emission of the studied Helichrysum species. The only study on the EOs from leaves, stems and flowers of Iranian H. araxinum [11] showed limonene as the most abundant compound in all the plant parts (29.2, 23.6, and 21.2%, respectively), together with α-pinene, a monoterpene hydrocarbon, present in leaves and stems (14.4% and 13.4%, respectively). On the contrary, the EO analyzed in the current study, showed a very low amount of these two constituents (only 4.9% of the total composition of limonene followed by 4% of α-pinene).
The published EO composition of H. araxinum flowers [11] was, instead, dominated by α-cadinol which represented over 18%, followed by borneol (11.9%), δ-cadinene (9.0%), bornyl acetate (8.0%), and α-humulene (7.3%). Except for borneol and bornyl acetate, which were present in very low amounts (1.2% and 0.5%, respectively), the other three compounds were not detected in the EO studied herein.
The EO did not show any antimycotic activity at the tested dilution and the MIC value was fixed at >5%. The EO resulted not active against E. coli with both the methods employed. It showed moderate antibacterial activity when tested against the staphylococcal strains. In fact, the agar disk diffusion method showed a growth inhibitory zone of 8 mm with both S. aureus and S. pseudointermedius isolates, and 5% (v/v) MIC values were determined with them (Table 2).
The H. araxinum EO showed a moderate activity against mold. Moreover, our results are in agreement with those found by other authors who analyzed the antibacterial activity of Helichrysum italicum EO and found that Gram-negative were more resistant than Gram-positive bacteria [24], [25], [26].
Cui et al. [27] found H. italicum oil active against E. coli and S. aureus. The antibacterial mechanism of this oil, which had an amount of γ-curcumene (11.64%) similar to our oil (10.7%), was related to the disruption of cell membranes with consequent losses of intracellular constituents.
From the literature, the EO of Piper reticulatum studied by Santana and his collaborators [28], showed a similar chemical composition of H. araxinum EO investigated herein; in fact, sesquiterpene hydrocarbons were the main class of compounds especially represented by β-selinene (19.0 %), β-elemene (16.1 %), and α-selinene (15.5 %). The antifungal activity of that oil was tested on A. flavus and T. mentagrophytes and the obtained results confirmed the inactivity of these oils on these fungi, in agreement with our results.
References
1. Najar B, Cervelli C, Ferri B, Cioni PL, Pistelli L. Essential oils and volatile emission of eight South African species of Helichrysum grown in uniform environmental conditions. S Afr J Bot 2019;124:178–87.10.1016/j.sajb.2019.05.015Search in Google Scholar
2. Maffei Facino R, Carini M, Franzoi L, Pirola O, Bosisio E. Phytochemical characterization and radical scavenger activity of flavonoids from Helichrysum italicum G. Don (Compositae). Pharmacol Res 1990;22:709–21.10.1016/S1043-6618(05)80097-0Search in Google Scholar
3. Sharifi-Rad M, Roberts TH, Matthews KR, Bezerra CF, Morais-Braga MF, Coutinho HD, et al. Ethnobotany of the genus Taraxacum – phytochemicals and antimicrobial activity. Phytother Res 2018;32:2131–45.10.1002/ptr.6157Search in Google Scholar
4. Stojanović-Radić Z, Pejcić M, Stojanović N, Sharifi-Rad J, Stanković N. Potentail of Ocimum basilicum L. and Salvia officinalis L. essential oils against biofilms of P. aeruginosa clinical isolates. Cell Mol Biol (Noisy-le grand) 2016;62:27–33.Search in Google Scholar
5. Salehi B, Sharopov F, Martorell M, Rajkovic J, Ademiluyi AO, Sharifi-Rad M, et al. Phytochemicals in Helicobacter pylori infections: what are we doing now? Int J Mol Sci 2018;19:Pii:E2361.10.3390/ijms19082361Search in Google Scholar
6. Sharifi-Rad J, Ayatollahi SA, Varoni E, Salehi B, Kobarfard F, Sharifi-Rad M, et al. Chemical composition and functional properties of essential oils from Nepeta schiraziana Boiss. Farmacia 2017;65:802–12.Search in Google Scholar
7. Davis PH. Flora of Turkey and the East Aegean Island. Vol. 5. Edinburgh 1975:80–97.Search in Google Scholar
8. Lourens AC, Viljoen AM, van Heerden FR. South African Helichrysum species: a review of the traditional uses, biological activity and phytochemistry. J Ethnopharmacol 2008;119:630–52.10.1016/j.jep.2008.06.011Search in Google Scholar
9. Albayrak S, Aksoy A, Sagdic O, Hamzaoglu E. Compositions, antioxidant and antimicrobial activities of Helichrysum (Asteraceae) species collected from Turkey. Food Chem 2009;119:114–22.10.1016/j.foodchem.2009.06.003Search in Google Scholar
10. Bigović D, Šavikin K, Janković T, Menković N, Zdunić G, Stanojković T, et al. Antiradical and cytotoxic activity of different Helichrysum plicatum flower extracts. Nat Prod Commun 2011;6:819–22.10.1177/1934578X1100600617Search in Google Scholar
11. Oji KA, Shafaghat A. Constituents and antimicrobial activity of the essential oils from flower, leaf and stem of Helichrysum armeneium. Nat Prod Commun 2012;7:671–4.10.1177/1934578X1200700533Search in Google Scholar
12. Adams RP. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. Carol Stream, IL: Allured Publishing Corporation, 1995.Search in Google Scholar
13. Davies NW. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicon and carbowax 20M phases. J Chromatogr A 1990;503:1–24.10.1016/S0021-9673(01)81487-4Search in Google Scholar
14. Jennings W, Shibamoto T. Qualitative analysis of flavor and fragrance volatiles by glass capillary gas chromatography. Food/Nahrung. New York: Academic Press, 1980.Search in Google Scholar
15. Masada Y. Analysis of essential oils by gas chromatography and mass spectrometry. New York, NY: John Wiley & Sons, Inc., 1976.Search in Google Scholar
16. Stenhagen E, Abrahamsson S, McLafferty FW. Registry of mass spectral data. New York, NY: Wiley & Sons, 1974.Search in Google Scholar
17. Swigar AA, Silverstein RM. Monoterpenes. Milwaukee, WI: Aldrich Chemical Company, 1981.Search in Google Scholar
18. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved 541 standard, 2nd ed. CLSI document M38-A2. Wayne, PA: CLSI, 2008.Search in Google Scholar
19. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; approved standard – 11th ed. CLSI document. Wayne, PA: Clinical and Laboratory Standards Institute, 2012. M02-11.Search in Google Scholar
20. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A2. Villanova, PA: CLSI, 1990.Search in Google Scholar
21. Ebani VV, Nardoni S, Bertelloni F, Giovanelli S, Rocchigiani G, Pistelli L, et al. Antibacterial and antifungal activity of essential oils against some pathogenic bacteria and yeasts shed from poultry. Flavour Frag J 2016;31:302–9.10.1002/ffj.3318Search in Google Scholar
22. Adams RP. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy, 4th ed. Carol Stream, IL: Allured Publishing Corporation, 2007.Search in Google Scholar
23. NIST 14/EPA/NIH Mass Spectra Library; I. Hoboken, NJ: Wiley and Sons, Inc., 2014.Search in Google Scholar
24. Nardoni S, Giovanelli S, Pistelli L, Mugnaini L, Profili G, Pisseri F, et al. In vitro activity of twenty commercially available, plant-derived essential oils against selected dermatophyte species. Nat Prod Commun 2015;10:1473–8.10.1177/1934578X1501000840Search in Google Scholar
25. Stupar M, Gbric ML, Dzamic A Unkovic N, Ristic M, Vukojevic J. Antifungal activity of Helichrysum italicum (Roth) G. Don (Asteraceae) essential oil against fungi isolated from cultural heritage objects. Arch Biol Sci 2014;66:1539–45.10.2298/ABS1404539SSearch in Google Scholar
26. Bouzid D, Nouioua W, Soltani E, De Haro JP, Angeles Esteban M, Zerroug M. Chemical constituents of Helichrysum italicum (Roth) G. Don essential oil and their antimicrobial activity against Gram-positive and Gram-negative bacteria, filamentous fungi and Candida albicans. Saudi Pharm J 2017;25:780–7.10.1016/j.jsps.2016.11.001Search in Google Scholar PubMed PubMed Central
27. Cui H, Zhao C, Lin L. Antibacterial activity of Helichrysum italicum oil on vegetables and its mechanism of action. J Food Process Pres 2015;39:2663–72.10.1111/jfpp.12516Search in Google Scholar
28. Santana AI, Vila R, Cañigueral S, Gupta MP. Chemical composition and biological activity of essential oils of different species of piper from Panama. Planta Med 2017;82:986–91.10.1055/s-0042-108060Search in Google Scholar PubMed
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