Chemical Profile and Antimicrobial Activity of the Essential Oils of Helichrysum arenarium (L.) Moench. and Helichrysum italicum (Roth.) G. Don

This study compared the essential oils (EO) composition of Helichrysum arenarium (Bulgarian populations) with that of the cultivated species H. italicum. The EO composition of H. arenarium and H. italicum were analyzed via gas chromatography. In general, 75 components were identified in H. arenarium EO and 79 in H. italicum EO. The predominant constituents in H. arenarium EO were α-pinene (34.64–44.35%) and sabinene (10.63–11.1%), which affirmed the examined population as a new chemical type. Overall, the main EO constituents of H. italicum originating in France, Bosnia and Corsica were neryl acetate (4.04–14.87%) and β-himachalene (9.9–10.99%). However, the EOs profile of H. italicum introduced from the above three countries differed to some extent. D-limonene (5.23%), italicene, α-guaiene and neryl acetate (14.87%) predominated in the H. italicum introduced from France, while α-pinene (13.74%), δ-cadinene (5.51%), α-cadinene (3.3%), β-caryophyllene (3.65%) and α-calacorene (1.63%) predominated in plants introduced from Bosnia. The EOs of the plants introduced from France and Corsica had similar chemical composition and antimicrobiological activity.


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 Table 1. Literature data of phytochemical research on Helichrysum arenarium.
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.

Helichrysum italicum
The ANOVA results that identified significant (bold) and nonsignificant differences among the mean constituents of H. italicum are shown in Table 5. The chemical composition of EO of H. italicum is presented in Supplementary Table S2, Tables 6 and 7. The Gas chromatography-mass spectrometry (HS/GC-MS) analyses showed 79 EO constituents (Supplementary Table S2). Generally, sesquiterpenes were predominant in the EO from all three locations: 45.23% (France), 47.9% (Corsica) and 54.8% (Bosnia). β-Himachalene was found to be present in the three analyzed samples in the range of 9.9% to 10.9% (Table  6), although EO of each of those samples possessed a specific profile. For example, italicene and α-guaiene were present in higher quantity in the plants introduced from Corsica and France (Table 6). The EO of plants introduced from Bosnia was characterized by higher content of δ-cadinene (5.51%), α-cadinene (3.3%), β-caryophyllene (3.65%) and αcalacorene (1.63%) compared with the other samples (Supplementary Table S2). The structural formulas of some of the main compounds are presented on Figure 1.

Helichrysum italicum
The ANOVA results that identified significant (bold) and nonsignificant differences among the mean constituents of H. italicum are shown in Table 5. The chemical composition of EO of H. italicum is presented in Supplementary Table S2, Tables 6 and 7. The Gas chromatography-mass spectrometry (HS/GC-MS) analyses showed 79 EO constituents (Supplementary Table S2). Generally, sesquiterpenes were predominant in the EO from all three locations: 45.23% (France), 47.9% (Corsica) and 54.8% (Bosnia). β-Himachalene was found to be present in the three analyzed samples in the range of 9.9% to 10.9% (Table 6), although EO of each of those samples possessed a specific profile. For example, italicene and α-guaiene were present in higher quantity in the plants introduced from Corsica and France (Table 6). The EO of plants introduced from Bosnia was characterized by higher content of δ-cadinene (5.51%), α-cadinene (3.3%), β-caryophyllene (3.65%) and α-calacorene (1.63%) compared with the other samples (Supplementary Table S2). The structural formulas of some of the main compounds are presented on Figure 1.   Monoterpenes represented the second major class of the H. italicum EO. In the EO of plants introduced from France, monoterpenes were observed in the greatest quantity (37.3%), with D-limonene (5.23%) and neryl acetate (14.87%) being the predominant monoterpenes ( Table 6). The main EO constituents of plants introduced from Bosnia were α-pinene (13.74%) and p-cymen-7-ol acetate (5.27%) ( Table 6).

Antimicrobial Activity of the H. italicum EO
The H. italicum EO of plants introduced from Bosnia, Corsica, and France were tested for antimicrobial activity against nine microorganisms using the disc diffusion method. Antimicrobial activity of different microorganisms and location ranged from 2.33 to 14.67 mm. Overall, the EO of H. italicum from all locations was more effective against S. aureus and ranged between 9.33 and 14.67 mm (Table 8). Moderate antimicrobial effect was found against C. krusei and C. tropicalis. The lowest antimicrobial activity was found against Y. eneterocolitca. In general, the tested EOs were more effective against Gram-positive bacteria.
Analyses of the composition of the EO from Central European samples were conducted by several authors [31,33,35]. These analyses also reported differences between EO obtained from different geographic locations. Samples from the Caucasus region analyzed by Czinner et al. [31] showed the presence of 1.5% of β-asarone, which was not found in the samples from Central Europe.

Comparing the EOs between H. arenarium and H. italicum
The species from the genus Helichrysum are widely used in traditional medicine worldwide and they are known as everlasting flowers [1]. As noted in the introduction, some Helichrysum species are widespread and others are cultivated in the Mediterranean, Iberian Peninsula and Eastern Europe [13,19,55]. The EO composition of H. italicum has been studied widely, while H. arenarium has been studied mainly for the content of phenols and flavonoids (Table 1). Data comparing the composition of the EO between the two species have not been published. The samples of H. arenarium collected and analyzed in this study contain a specific EO profile that differs from the EO composition of H. italicum. Monoterpenes (α-pinene, sabinene) were the predominant class of compounds in the EO of H. arenarium, while the EO of H. italicum was dominated by the class of sesquiterpenes (neryl acetate and β-himachalene). However, the EO samples of H. italicum from three regions differed from each other to some extent. D-limonene (5.23%), italicene, α-guaiene and neryl acetate (14.87%) predominated in the H. italicum plants introduced from France, while the H. italicum plants introduced from Bosnia had predominantly α-pinene (13.74%) and δ-cadinene (5, 51%). This difference in the EO composition between H. arenarium and H. italicum refutes our working hypothesis.

Antimicrobial Activity of the H. italicum EO
Djihane et al. [56] studied antimicrobial activity of H. italicum EO against Grampositive bacteria (G + ), Gram-negative (G − ) bacteria and fungi. Gram positive bacteria were more sensitive to the presence of EO than G − bacteria or fungi. The results from this study were similar with our results.
In the current study, fungi were the most resistant class of microorganisms to the presence of the EO. Oliva et al. [57] tested H. italicum EO against methicillin-sensitive Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Candida albicans (ATCC 14053) and the clinical strains of methicillin-resistant S. aureus, carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa. Interestingly, fungicidal/bactericidal potency against C. albicans and carbapenem-resistant A. baumannii was revealed at a concentration of 5% v/v. Staver et al. [58] conducted antimicrobial assays and showed that EO had weak to moderate antimicrobial potential with S. aureus and S. epidermidis as the most sensitive bacterial strains. In the study of Dzamic et al. [59], the most sensitive bacteria to H. italicum EO were Bacillus cereus and Salmonella typhimurium, while the most sensitive fungus was yeast, Candida albicans. Similar to our study, Mollova et al. [60] showed H. italicum EO from France had more pronounced antimicrobial activity against the G + bacteria Staphylococcus aureus, Bacillus subtilis, and the fungus Aspergillus brasiliensis, as well as a stronger antioxidant potential compared with the other EOs. The obtained results from this study are in good agreement with the findings of Cantore et al. [61] who reported that G + bacteria are more sensitive to plant EOs than G − bacteria. Mesic et al. [62] reported that immortelle essential oil inhibited only Gram-positive bacteria and possessed antifungal effects.
With respect to the antibacterial properties of H. italicum EO and its related constituents, Rossi et al. [63] demonstrated that the EO obtained from endemic plants of Corsica was more effective against the G + bacterium S. aureus than against the G − strains E. coli, Enterobacter aerogenes and P. aeruginosa. In our study, the most resistant microorganisms tested were G − bacteria. It is commonly known that G − bacteria are less susceptible to EO than G + bacteria, and this is directly connected to the bacterial cell wall structure. In G − bacteria, the cell wall is a complex envelope constituted by the cytoplasmic membrane, the periplasm and the outer membrane. Results reported in Cantore et al. [61] and Rossi et al. [63] are consistent with those in our study. Antimicrobial activity of H. italicum EO from Algeria with α-cedrene, α-curcumene and geranyl acetate as dominant compounds assayed by disk diffusion method inhibited growth of S. aureus, M. luteus, E. cereus, B. cereus, S. epidermidis, B. subtilis, P. aeruginosa, E. faecalis and P. mirabilis, but did not affect E. coli, K. pneumonia and L. monocytogenes. In addition, yeasts (C. albicans and S. cervisae), as well as fungi (F. solani, A. niger, A. alternata and A. rabiei), were also inhibited by H. italicum EO [64].

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 airdried 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.

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). 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 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.

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.

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.

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.
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].

Conclusions
This study assessed the chemical profile of Helichrysum arenarium (Bulgarian populations) with that of the cultivated species H. italicum introduced from three different countries and grown side by side in Bulgaria. The main components in H. arenarium EO were α-pinene (34.64-44.35%) and sabinene (10.63-11.1%), indicating a possible new chemotype not previously reported in the literature. The chemical profile of H. italicum EO originating in France, Bosnia and Corsica were neryl acetate (4.04-14.87%) and βhimachalene (9.9-10.99%); however, there were differences between the EO from plants introduced from the above countries. The H. italicum EO plants originating in France, Bosnia and Corsica were evaluated for antimicrobial activity and it was revealed that the EO of plants from France and Corsica had similar composition and antimicrobial activity.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/plants11070951/s1, Table S1: Constituents and concentrations of Helichrysum arenarium from Bulgaria. The min-max range represents the H. arenarium essential oil constituents from the three locations in Bulgaria. Table S2: Constituents and concentrations of Helichrysum italicum introduced from France, Corsica and Bosnia. The min-max range includes variation in concentrations of the essential oil constituents from all three locations; France, Corsica, and Bosnia.