1. Introduction
Plants species belonging to
Helichrysum Mill. genus (Asteraceae) have long been known for their healing properties, and preparations based on
Helichrysum species have been and continue to be used around the world [
1]. The pharmaceutical, cosmetic and perfume industries have taken a strong interest in
Helichrysum species because of the specific essential oil (EO) aroma and composition [
2]. Extracts from
Helichrysum species possess a wide range of pharmacological activities such as antioxidant, antimicrobial, antiatherosclerotic, antiproliferative, antidiabetic, neuroprotective and antiinflammatory activities [
3,
4,
5,
6]. There are 16 species in the
Helichrysum genus spread across Europe [
7]. Two species,
H. arenarium (L.) Moench. and
H. plicatum DC. [
8], are naturally occurring in Bulgaria, while
H. italicum (Roth) G. Don. is an introduced cultivated species in this country. Products derived from
H. italicum are widely used in the traditional medicine, cosmetics and the food industry and are particularly popular in the Mediterranean countries [
9,
10]. In recent years, there has been increasing interest in products from
H. italicum. As a result, the species has been commercially cultivated in France [
11], Portugal [
12], Bosnia and Herzegovina [
2,
13], Italy [
14,
15], Serbia [
10] and recently in Bulgaria. There has been significant interest in the phytochemical composition and pharmacological activity of
H. italicum during the last decade and a half [
13,
15,
16,
17,
18].
Helichrysum arenarium has a long tradition as a medicinal plant in the European ethnomedicine [
19]. Medicines based on
Helichrysi flos were enlisted in the State Pharmacopoeia of the USSR [
20], Pharmacopoeia Helvetica [
21] the Polish Pharmacopoeia [
22], as well as in a herbal monograph on
H. arenarium [
23]. Because of its healing properties,
H. arenarium has been collected from its natural populations; hence, wild collection has the potential to disturb stable populations of this species. In some European and Asian countries, such as Sweden, Poland, Kazakhstan and Serbia, the species is protected and cultivated [
24,
25,
26]. In Bulgaria,
H. arenarium is protected according to the Biodiversity Act, included in Annex 4 of this act and in the List of Species of Medicinal Plants under special regimen of conservation [
27,
28].
Research studies related to its phytochemical composition have focused mainly on the content of phenols and flavonoids [
29,
30,
31,
32,
33,
34,
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
47,
48,
49,
50,
51,
52] (
Table 1). This is not accidental because phenolic compounds, including flavonoids (like in
Helichrysi flos), used in traditional medicine (biological source
H. arenarium) have been demonstrated to have cholagogue, choleretic, hepatoprotective and inhibitory effects on tumor necrosis; in addition, these compounds are used to make a detoxifying herbal drug [
19,
30,
40]. Studies on EO composition of
H. arenarium are limited and the existing data for EO composition diverge widely [
4,
30,
31,
32,
33,
35] (
Table 1). For example, in a study of the Hungarian population, the predominant EO constituents were linalool, carvacrol, anethole, anisaldehyde and thymol [
31,
32]; in Serbia, major EO constituents included diepi-
α-cedrene,
α-ylangene, cyclosativene and limonene [
4]; in Iran, spathulenol,
β-pinene, limonene, alpha-cadinol and borneol were observed [
43,
45]. The latter authors concluded that the observed differences in the EO composition were due to the different geographical habitats of the species [
4,
31,
32,
33,
43,
45]. So far, phytochemical studies of the Bulgarian wild populations of
H. arenarium have not been conducted. To preserve the natural population of the species, it may need to be cultivated ex situ through its development as a cultivated crop. Phytochemical studies are necessary for the selection of accessions possessing high content and desirable composition of the EO. Overall, information on the EO composition of
H. italicum cultivated in Bulgaria is limited.
Previous research showed that
H. italicum EO exhibited antioxidant, antimicrobial, antiviral, anti-inflammatory, and antiproliferative activity [
13].
H. italicum showed low or no activity against tested bacteria. However, for all Gram-negative bacteria (
E. coli, P. aeruginosa, Salmonella typhimurium, S. enteritidis, K. aerogenes and
P. hauseri) minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were higher than 454.5 µL/mL EO. For the Gram-positive bacteria (
B. cereus,
L. monocytogenes,
R. equi, and
S. epidermidis) MIC and MBC values was 454.5 µL/mL, while for other (
B. spizizenii, E. faecalis, L. innocua, L. ivanovii, and
S. aureus) MIC and MBC values were higher than 454.5 µL/mL of EO [
10].
The present study analyzed the EO composition of the Bulgarian population of H. arenarium and compared it with the EO of H. italicum, which already has an established international market. The working hypothesis was that EO composition of H. italicum and H. arenarium would be similar.
4. Materials and Methods
4.1. Plant Material
The materials utilized in this study were aerial parts in full flowering. The plant materials of
H. arenarium were collected from three locations (numbered 689; 691; 699) of population Pobitite kamani, near Varna town (
Figure 2A) (43.228196 N; 27.705116 E; 114 masl) with an official permit (#790/19.04.2019 of MOCB). The collected samples were air-dried at room temperature until a constant weight. Voucher specimens of
H. arenarium were deposited at the Herbarium of the Agricultural University, Plovdiv, Bulgaria (SOA) [
65].
Samples of
H. italicum introduced from Bosnia, France and Corsica and grown side by side in Bulgaria were obtained from experimental fields at the Institute of Roses, Essential and Medical Plants in Kazanlak, Bulgaria (
Figure 2B). These plantations were established via vegetative propagation/rooting of fresh green cuttings prepared from the original imported plants. The plants originating from Bosnia and Herzegovina and Corsica were imported to Bulgaria as seedlings in trays, while the plants from France were imported and grown from collected seeds.
4.2. Essential Oil (EO) Extraction
The EO of all samples was extracted via hydrodistillation in a 2 L Clevenger-type apparatus (Laborbio Ltd. Sofia, Bulgaria,
laborbio.com, accessed on 12 June 2021). Since the samples from
H. arenarium were much smaller, the EO of
H. arenarium was isolated from 45 g of flowering aerial parts of each accession by hydrodistillation in a Clevenger-type distillation unit for 2 h plus in 0.8 L of water. The duration of the hydrodistillation was 2 h and all samples were extracted in two replicates.
Samples from the introduced and grown in Bulgaria materials of H. italicum were 1000 g of fresh aboveground plant material in the full flowering stage. In addition, to obtain larger EO samples for biological activity testing, steam distillation was performed in 5 L metal cylindrical containers using 1.5 L of water under the grate on which the raw material was placed. The steam distillation time was 1.5 h. The EO extraction was done at the Institute of Roses, Essential and Medical Plants in Kazanlak, Bulgaria, and each extraction was performed in two replicates. After isolation of each subsample, EO volume and weight were measured, and the EO samples were stored in a freezer at 4 °C for further analyses.
4.3. Gas Chromatography (GC) Flame Ionization Detection (FID) and Gas Chromatography–Mass Spectroscopy (MS) Analyses of the Essential Oils (EO)
The chemical profiles of the
H. italicum and
H. arenarium EO, in two replications, were determined by GC-FID and GC/MS techniques using a 7890A gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA), according to the methods described in our previous study [
66]. The GC-MS analysis was performed on a 7890A gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) coupled directly to an Agilent mass selective detector (MSD-5975C). The system was equipped with a HP-5ms fused silica capillary column (5% phenyl 95% dimethylpolysiloxane, 30 m × 0.32 mm i.d., film thickness 0.25 μm, Agilent Technologies, USA). The oven temperature was programmed from 40 °C to 300 °C at a rate of 5 °C/min, and held for 10 min. The temperatures of the injector, the MS quadrupole and the ion source were 250 °C, 150 °C and 230 °C, respectively. The MSD transfer line was maintained at 270 °C.
All mass spectra were acquired in the EI mode (scan range of m/z 50–500 at 1 s/decade; ionization energy of 70 eV). Split ratio was 1:10. The constituents present in the EO samples were identified by comparing their linear retention indices, estimated using a mixture of a homologous series of aliphatic hydrocarbons from C8 to C40 and MS fragmentation patterns with those from an Adams mass spectra library and NIST′08 (National Institute of Standards and Technology).
The GC analysis was performed on an Agilent GC-7890A gas chromatograph (Agilent Technologies, USA) equipped with a flame ionization detector (FID) and HP-5 silica fused capillary column (30 m length × 0.32 mm i.d. × 0.25 µm film thickness) under the same conditions as described above. The FID temperature was maintained at 280 °C for the oil analyses. The relative composition of the investigated samples was calculated on the basis of the GC-FID peak areas (measured using the HP-5 ms column) without using a correction factor.
The GC-FID analysis of the EO was performed with a gas chromatograph 7890A gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) coupled to a flame ionization detector (FID) and HP-5 silica fused capillary column (30 m length × 0.32 mm i.d. × 0.25 µm film thickness). The oven temperature was programmed as mentioned above. The detector and injector temperatures were 280 °C and 220 °C, respectively. The carrier gas was helium at a flow rate of 1 mL/ min. Essential oil samples (1 μL) were injected using the split mode. The percentage composition of EO samples was calculated using the peak normalization method.
4.4. Method for Testing Antimicrobial Activity
The EO of
H. italicum (plant material originated in Bosnia, France and Corsica) was tested against nine microorganisms with an agar disc diffusion method according to in our previous study [
66]. In this study, 0.1 mL of microbial suspension was spread on the Mueller Hinton Agar (MHA, Oxoid, UK) for bacteria and Sabouraud Dextrose agar (SDA, Oxoid, UK) for yeasts. Six mm diameter filter paper discs were used for testing. The filter paper was impregnated with 15 μL of EO and placed on MHA, SDA, respectively, with a microbial inoculum. The MHA was maintained at 4 °C for 2 h and then at 37 °C for 24 h and SDA was maintained at 4 °C for 2 h and then at 25 °C for 24 h. After a 24 h incubation period, the diameter of the inhibition zones was measured (in mm). Chloramphenicol (30 µg, Oxoid, UK) and fluconazole (25 µg, Oxoid, UK) served as positive antimicrobial controls. Antimicrobial activity was measured in triplicate.
Microorganisms
Nine strains of microorganisms were used to determine antimicrobial activity of the EOs, including three Gram-positive bacteria (SA-Staphylococcus aureus subs. aureus CCM 4223, EF-Enterococcus faecalis CCM 4224, SP-Streptococcus pneumonia CCM 4501), Gram-negative bacteria (PA-Pseudomonas aeroginosa CCM 1959, YE-Yersinia enterocolitica CCM 5671, SE-Salmonella enterica subsp. enterica CCM 3807), and yeasts (CA-Candida albicans CCM 8186, CK-C. krusei CCM 8271, CT-C. tropicalis CCM 8223 (CT)). The microorganisms were obtained from the Czech Collection of Microorganisms (Brno, Czech Republic).
4.5. Statistical Analyses of the Data
One-way analysis of variance was conducted to determine the effect of (1) collection location of H. arenarium on the concentration (%) of α-pinene, sabinene, β-pinene, D-limonene, trans-verbenol, 1-terpinen-4-ol, n-tetradecane, β-gurjunene, germacrene D, germacra-4(15),5,10(14)-trien-1, monoterpenes, sesquiterpenes, long-chain alkane and diterpenoids, and (2) country of origin of H. italicum (Bosnia, France and Corsica) on the concentration (%) of α-pinene, D-limonene, 2-methyl butyl-2-methyl butyrate, isoamyl tiglate, 1-terpinen-4-ol, nerol, neryl acetate, α-copaene, italicene, α-cis-bergamotene, β-caryophyllene, p-cymen-7-ol acetate, α-guaiene, γ-curcumene, β-himachalene, β-curcumene, germacrene D-4-ol, γ-eudesmol, tau.-muurolol, β-eudesmol, monoterpenes, sesquiterpenes, ester and long-chain alkane.
One-way analysis of variance was also conducted to determine if there were significant differences among the three locations where H. italicum was collected in terms of nine antimicrobial activities (SA, EF, SP, PA, YE, SE, CA, CK and CT).
For each response variable, the validity of model assumptions was verified by examining the residuals as described in Montgomery [
67]. When the effect was either marginally significant (0.05 <
p-value < 0.1) or significant (
p-value < 0.05), multiple means comparison was completed using Fisher’s LSD at the 5% level of significance, and letter groupings were generated. The analysis was completed using the GLM Procedure of SAS [
68].