Antioxidant and Toxic Activity of Helichrysum arenarium (L.) Moench and Helichrysum italicum (Roth) G. Don Essential Oils and Extracts

Helichrysum arenarium (L.) Moench (sandy everlasting) is the only species from genus Helichrysum Mill that grows spontaneously in Lithuania. The chemical composition of the essential oils (EOs) from inflorescences and leaves of H. arenarium wild plants was analysed by GC-MS. Palmitic (≤23.8%), myristic (≤14.9%) and lauric (6.1%) acids, n-nonanal (10.4%), and trans-β-caryophyllene (≤6.5%) were the major constituents in the EOs. For comparison, the main components in EO from flowers (commercial herb material) of H. italicum were γ-curcumene (21.5%), β-selinene (13.6%), α-selinene (8.1%), β-eudesmol (8.3%), and α-pinene (6.5%). Composition of H. arenarium methanolic extracts was investigated by HPLC-DAD-TOF. The main compounds were the following: luteolin-7-O-glucoside, naringenin and its glucoside, apigenin, chlorogenic acid, arenol, and arzanol. Antioxidant activity of EOs and extracts was tested by DPPH● and ABTS●+ assays. Sandy everlasting extracts exhibited significantly higher radical scavenging activities (for leaves 11.18 to 19.13 and for inflorescences 1.96 to 6.13 mmol/L TROLOX equivalent) compared to those of all tested EOs (0.25 to 0.46 mmol/L TROLOX equivalent). Antioxidant activity, assayed electrochemically by cyclic and square wave voltammetry correlated with total polyphenolic content in extracts and radical scavenging properties of EOs and extracts. The toxic activity of EOs of both Helichrysum species was evaluated using a brine shrimp (Artemia salina) bioassay. H. italicum inflorescence EO was found to be toxic (LC50 = 15.99 µg/mL) as well as that of H. arenarium (LC50 ≤ 23.42 µg/mL) oils.


Introduction
The genus Helichrysum Mill (sect. Stoechadina, tribe Gnaphalieae) containing approximately 600 species is distributed throughout the entire world. The Helichrysum species have been used in many folk medicines, as flavouring spices or as food supplements, and for ornamental, cosmetic, and pharmaceutical purposes as well.
H. italicum (Roth) G. Don is a typical endemic Mediterranean species, subdivided into several subspecies, such as italicum; michrophyllum (Willd. Most of the reports concerning the volatile chemistry of the genus Helichrysum have described the composition of essential oils (EOs) of Mediterranean taxa. Variability in chemical composition of H. italicum EOs and other extracts is highly affected by genetic factors, phenological stage of development, differences in eco-climatic characteristic of growing habitats, and extract preparation techniques, as reviewed by Maksimović et al. [4] and references therein. Recent
Detailed chemical composition of H. arenarium and H. italicum EOs is given in Table S3 of Supplementary Materials. Fifty-three and fifty-five identified constituents comprised 96.4 ± 1.52% and 99.1 ± 0.44% of the total amounts of H. arenarium inflorescence and leaf oils, respectively. Thirty-two identified compounds comprised 89.4 ± 0.15% of total Italian immortelle EO.

Chemical Composition of Methanolic Extracts
Up to 29 compounds were identified tentatively in H. arenarium inflorescence and leaf extracts ( Table 2). Due to numerous detailed publications on H. italicum extracts [3,4,41,[43][44][45] and herein cited, the composition of extracts of Italian immortelle commercial herbs was not investigated in the present study.

Total Phenolic Content
Total phenolic content was about 1.6-fold higher in the H. arenarium leaf methanolic extract (1062.82 ± 12.36 mg/L of gallic acid equivalent), compared to that in flower (652.56 ± 5.87 mg/L) extract.

Spectrophotometric (DPPH • and ABTS •+ ) Assays
The antioxidant activity of H. arenarium and H. italicum EOs and H. arenarium extracts tested by DPPH • assay is presented in Table 3. The activity of EOs was similar, practically not varying with species, plant organs, and year of raw material collection. A significant difference was observed between sandy everlasting methanolic extracts and EOs and between methanolic extracts themselves. The activity of leaf extract (19.13 ± 0.04 mmol/L, TROLOX equivalent) was three-fold higher compared to that of inflorescence extract (6.13 ± 0.04 mmol/L). ABTS •+ assay data showed the same tendency as the DPPH • results: activity of sandy everlasting EOs was similar as well, and activity of methanolic leaf extracts (11.18 ± 0.002 mmol/L, TROLOX equivalent) was also significantly higher (about 6-fold), compared to that of flowers (1.96 ± 0.01 mmol/L) ( Table 4). Electrochemical techniques cyclic and square wave voltammetry (differing in modes of potential application to the working electrode) are widely used to obtain information about redox active substances in solutions [86]. The working metal or carbon-based electrode is immersed in the sample and its potential scanned to the positive direction. During this forward scan, the potential of the working electrode gradually becomes more positive and, therefore, the oxidizing power of the electrode increases. As soon as the potential of the electrode reaches the oxidation potential of the electroactive sample constituent, oxidation of the compound occurs: the lower the potential of oxidation, the more powerful the reducing, i.e., antioxidant, properties of the compound. The oxidation (anodic) peak potential value (Epa) depends on the chemical structure of the electroactive substance, electrode material, composition, and pH value of the solution. The magnitude of the oxidation (anodic) peak current (Ipa) at Epa is related to the concentration of the electroactive compound. During the reverse scan, reduction currents are registered. The presence of reduction (cathodic) peaks Ipc at reduction (cathodic) potentials Epc in the reverse scan provides information about the reversibility of the redox reaction of the oxidized compounds generated in the forward scan.
Low-cost chemically inert carbon paste electrodes are simply prepared from graphite powder and a liquid inert binder. Carbon paste electrodes have more advantages, such as low background currents, rapid renewal of the surface, and easy modification [87].
To test the antioxidant properties of EOs obtained from inflorescences H. italicum, and inflorescences and leaves of H. arenarium, a strategy of modification of bulk carbon paste with these oils was used. Our former investigation of EO isolated from Eupatorium cannabinum [88] has shown that this approach is suitable to detect easily oxidizable (E pa in the region 0.04 to 0.1 V) compounds, i.e., possible antioxidants, by means of cyclic or square wave voltammetry. However, in the case of investigated EOs from both Helichrysum species, cyclic or square wave voltammograms (not shown) did not reveal any oxidation currents throughout the potential region-0.2 to 1.0 V.
Contrary to the results of EO tests, cyclic voltammograms of carbon paste electrode in H. italicum and H. arenarium extracts (methanolic extracts were diluted with phosphate buffer pH 7.3 before measurements) showed the presence of anodic currents at potentials above 0.1 V (Figure 1). However, the presence of electroactive substances with E pa1 at about 0.28 V and E pa2 at about 0.47 V were more clearly observed in the case of extract of H. italicum inflorescences (Figure 1, dash-dot line). In the reverse scan, an increase of cathodic current below 0.35 V was recorded, indicating that certain oxidized compounds were reduced.      (Table 5). Italian immortelle inflorescence and sandy everlasting leaf and flower EOs were found to be toxic, and were not statistically differentiated (p > 0.05). Toxicity tests of methanolic extracts (up to 300 µL) of both Helichrysum species performed by the same method showed that the extracts were non-toxic.
A comparison of the results of chemical composition obtained in the present study with those from other countries is quite complicated because of the limited number of published papers on this topic (for more details, see Table S2 in Supplementary Materials). Some compositional similarities were observed for plant material of Lithuanian origin and for H. arenarium of Caucasian origin cultivated in Hungary (Soroksár) and the Polish commercial sample [78,79]. The latter inflorescence oils contained significant amounts of aliphatic acids (up to 34.6%). Methyl palmitate (28.5%), dodecanoic (11.9%), decanoic acid (9.8%), and octanoic acids (6.0%) have been determined as predominant compounds in the Hungarian commercial sample [78]. Prevalent amounts of methyl palmitate (≤28.5%), capric (≤19.8%), lauric (≤14.6%), pelargonic (≤6.9%), and caprylic acid (6.0%) have been determined in plants cultivated in Hungary [79]. Methyl pentadecanoate (31.0%) and oleic acid (30.3%) along with ethyl hexadecanoate (20.2%) and linoleic acid (18.9%) were determined as major constituents, respectively, in flower EO of H. arenarium plants cultivated in Italy and plants grown naturally in Turkey [83,84] (see Table S2, Supplementary Materials).
Moreover, our results (Table 1) differed drastically from those obtained for sandy everlasting EOs of Serbian [56], Iranian [64], and Chinese origin [74], where monoterpene and sesquiterpene hydrocarbons were found to be the main components (details in Supplementary Materials, Table S2).
The data obtained clearly demonstrated remarkable differences in the chemical composition of the oils of Lithuanian H. arenarium L. from the sandy everlasting EOs of from other countries.
Italian immortelle inflorescence EO obtained from commercial herbs used in this study was characterized by principal compounds sesquiterpene hydrocarbons: γ-curcumene and monoterpene α-pinene (6.5 ± 1.50%). Thirty-two identified compounds comprised 89.4 ± 0.15% of the total H. italicum EO (Table S3 in Supplementary Materials). The chemical composition of the investigated EO differed from H. italicum EOs of neryl acetate chemotype revealed in numerous studies (Table S1 in Supplementary Materials). On the other hand, γor ar-curcumene, αor β-selinene were common constituents in Italian immortelle EOs.
Chemical composition of the EO obtained from a commercial inflorescence sample of Italian immortelle (H. italicum) differed drastically from EOs of Lithuanian wild sandy everlastings (H. arenarium) as well. Aliphatic acids predominated in H. arenarium oils, while sesquiterpene hydrocarbons were a major fraction in the H. italicum inflorescence EO. It should be mentioned that appreciable amounts of ester bonded acids and volatile carboxylic acids (after derivatization procedure) have been identified in EOs of H. italicum of Croatian origin [89]. Differences in chemical composition have demonstrated clear intraspecific variation of the different Helichrysum (H. italicum and H. arenarium) species.
According to the available literature data, around 100 compounds from different classes, such as phenolic acids, flavonoids, phthalides, arzanol derivatives and other pyrones, coumarins, sterols, lignans, etc., have been identified in various sandy everlasting extracts [45,49,63]. Even more, some compounds have been isolated and identified for the first time in H. arenarium extracts [67,68,73,[75][76][77]. Twenty-nine compounds were identified tentatively in H. arenarium flower and leaf extracts in the present research, and most of them have been already identified in H. arenarium extracts or in other Helichrysum species. Fifteen constituents (bitalin A, dihydrosyringin, chlorogenic acid, unknown 1, caffeic acid, two naringenin glucoside isomers, syringin, dicaffeoylquinic acid, luteolin glycoside, apigenin-7-O-gentiobioside or apigenin-7,4'-di-O-β-glucoside, apigenin, arenol, arzanol, and isosalipurposide) provided m/z ions on both ionization types, while some constituents were detected only by positive or by negative ionization. Because of very detailed and numerous published data on H. italicum extracts, the chemical composition of commercial Italian immortelle extract was not investigated in the present study.
The DPPH • and ABTS •+ assays are most frequently used to evaluate the ability of antioxidants to scavenge free radicals. As determined by these tests, the activity of EOs from both Helichrysum species ranged from 0.25 ± 0.00 to 0.46 ± 0.01 for TROLOX (mmol/L) equivalent (Tables 3 and 4) and practically did not vary with species, plant organs, and year of raw material collection.
Significant difference was observed between sandy everlasting EOs and methanolic extracts, the activity of leaf extract being 3-fold higher compared to that of inflorescence extract (DPPH • assay, Table 3). ABTS •+ assay results showed the same tendency: activity of sandy everlasting flowers and leaf EOs was similar as well, activity of methanolic leaf extracts was also significantly higher (about six-fold) compared to that of flowers (Table 4).
The low ability to scavenge free radicals suggested that EOs of different Helichrysum species or plant parts did not have significant amounts of specific compounds that could successfully scavenge free radicals. For comparison, radical scavenging activity of Rhododendron tomentosum EOs was 48.19 ± 0.1 mmol/L (TROLOX equivalent) [90].
There are no available data on antioxidant activity of EOs from H. arenarium. Data on radical scavenging potential of extracts from sandy everlasting are limited [45,[52][53][54][55]57,60,63]. Helichrysi flos water extracts exhibited antioxidant and antilipoperoxidant properties; the plant extracts diminished enzymatically induced lipid peroxidation and reduced NADPH cytochrome c reductase activity in liver microsomes [52]. The hydrogen-donating ability and the reducing power of the lyophilized water extracts from inflorescences of sandy everlasting were determined spectrophotometrically; their OH • scavenging activity in the H 2 O 2 /OH • -luminol-microperoxidase system was measured by a chemiluminometric method [53]. One lyophilizate proved to be more effective in scavenging DPPH radicals compared with silibinin used as a standard [53]. Various extracts (methanolic, ethanolic and 70% v/v, ethanolic extracts, before and after acid hydrolysis) of Romanian sandy everlasting (collected from Botos , ani county), containing high amounts of polyphenols were tested for antioxidant properties [63]. The highest activity (5.82 TROLOX equivalent/mL extract) was found for the 70% (v/v) ethanolic extract of the flowers of H. arenarium and the lowest in the absolute ethanol-mediated extraction samples (3.71 TROLOX equivalent/mL). Antioxidative properties of infusions from H. italicum and H. arenarium plants were compared [45]. H. italicum infusions exhibited stronger radical scavenging activity than that of H. arenarium plants; and it was dependent on the morphological type of the plant, on the harvesting time and plant organ. Radical scavenging activity measured by the DPPH • test in infusions prepared from green parts of H. arenarium was stronger than that from flowers [45].
Although DPPH • and ABTS •+ scavenging assays in vitro are very popular, critical evaluation of these assays [91] pointed to their main limitation, i.e., these compounds are not found in living organisms. Besides, the following other considerations argue against direct polyphenol reactions with radicals in vivo: low concentrations of polyphenols in tissues, high level of metabolism, and biotransformation that polyphenols undergo in the organism, slow action (minutes or hours) as a radical scavenger of an antioxidant must be irrelevant in vivo in cells or even in situ in foods and etc. As an alternative to radical-scavenging assays, quick, simple, and inexpensive electrochemical techniques such as cyclic voltammetry, differential pulse or square wave voltammetry have been employed for evaluation of antioxidant properties of beverages, plant extracts, or individual polyphenols [92][93][94][95][96]. Electrochemical approaches are based on the chemico-physical properties of the compounds and, thus, can be considered a direct test for antioxidant properties.
Similar voltammetric profiles (Figures 1 and 2) indicate that the extracts could contain the same compounds. Substances with pH-dependent oxidation potentials falling in the potential region from 0.2 to 0.5 V at pH 7 are possibly compounds containing a flavonoid structure with catechol or galloyl groups [92][93][94][95]97,98]. According to the results presented in Table 2, probable candidates are chlorogenic acid, caffeic acid, kaempherol, luteolin, and apigenin. Direct comparison of obtained E pa values with literature data is rather complicated since the conditions of the experiment (electrode material, ionic strength of the solution, the presence of organic solvent, concentration of electroactive substance, etc.) may cause peak shifts [97]. Higher values of I pa in voltammograms for leaf extracts (Figures 1 and 2, dashed lines) compared to those for inflorescence extracts (Figures 1 and 2, solid lines) correlated with higher total polyphenolic content in leaf extract. In the case of EOs, the non-appearance of anodic currents suggested the absence of easily oxidizable compounds that correlated with weak radical scavenging properties of H. arenarium EOs.
In vivo toxicity tests were performed using brine shrimp (Artemia salina) larvae [99]. LC 50 values varied from 15.99 to 23.42 µg/mL for H. italicum and H. arenarium EOs, respectively (Table 5). According to Meyer's and Clarkson's toxicity criterion, plant extracts with LC 50 < 1000 µg/mL are considered as toxic and extracts with LC 50 of 0-100 µg/mL are highly toxic, respectively [100]. Regarding the toxicity classification with Meyer's and Gosselin, Smith and Hodge's criteria, the investigated EOs of H. italicum and H. arenarium could be attributed to the toxic/moderately toxic class [100]. Although the chemical composition of the EOs of two Helichrysum species was different and LC 50,95 values varied, their toxic activities appeared to be similar (p > 0.05) when data were analysed statistically. To the best of our knowledge, researches devoted to evaluation of in vivo toxic capacity of both Helichrysum species (H. arenarium and H. italicum) are scarce. H. italicum EO did not show a mutagenic effect against larvae of Drosophila melanogaster Meigen (Diptera: Drosophilidae); however, co-incubation of the larvae with EO and urethane showed significant reduction of mutations caused by urethane [101]. EOs from H. italicum possessed high larvicidal activity against the mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) [9] and displayed contact toxicity against the stored food insect Sitophilus zeamais Motsch (Coleoptera: Curculionidae) [47]. Investigation of in vitro phytotoxic activity of EO from H. italicum ssp. italicum on germination of radish and garden cress [7] showed that EO was active (probably due to the presence of bioactive sesquiterpenes) against radicle elongation of radish. Antifungal and antibacterial activity of H. arenarium and H. italicum EOs was among the most investigated properties of these oils [15,21,22,24,27,32,33,[35][36][37][38]40,43,[56][57][58][61][62][63][64]66]. The activity ranged from weak to strong, depending on EO and fungi/bacteria species.
The toxic activity of H. arenarium and H. italicum EOs was investigated for the first time in this study.
In vivo toxic activity of different amounts of methanolic extracts (30, 50, 100, and 300 µL) of both Helichrysum species was investigated by the same Artemia salina larvae method [99]. Mortality of brine shrimp (Artemia salina) larvae was observed neither after 24 h nor after 48 h. It appeared that the extracts were not only non-toxic but, conversely, could possibly be used as a nutrient medium for shrimps. Commercial sample of dried H. italicum inflorescences was purchased from Farmalabor (Farmacisti associate, Canosa di Puglia, Italy) producer.

Essential Oil Isolation
The essential oils were isolated by hydro-distillation of dried material (50 g each) in a Clevenger-type apparatus for 2 h, as per the European Pharmacopoeia. The ratio of plant material to water was 1:20. A yellow-grey, greasy mass with a sweet characteristic odor was obtained. Hydrodistillation yielded 1.1% (v/w, on a dry weight basis) of EO from H. italicum inflorescences. Yield of the H. arenarium EOs slightly ranged and was less than 0.5% (v/w). The obtained oils were dried over anhydrous sodium sulphate, kept in closed dark vials, and stored in a refrigerator; the samples were diluted with a mixture of pentane and diethyl ether (1:1) before analysis.

Preparation of Extracts
Samples of air-dried inflorescences and leaves were ground into a homogenous powder and protected from light and humidity until analysis. Preparation of extract was made according to Pharmacopeia requirements. 2 g of crushed herbal material (flowers and leaves) and 40 mL of solvent (mixture of water and methanol (1:1)) were used for extraction. Extraction procedure was performed in an ultrasonic bath at room temperature for 30 min. The mixture was filtrated through a filter paper for qualitative analysis (pore size 11 µm).

GC (Flame-Ionization Detector FID) Analysis
Quantitative analyses of the essential oils were carried out on HP 5890II chromatograph equipped with an FID (Hewlett Packard, Palo Alto, CA, USA), using DB-5 ((5%phenyl)-methylpolisiloxane; 50 m × 0.32 mm × 0.25 µm) and HP-FFAP (polyethylene glycol 30 m × 0.25 mm i.d., film thickness 0.25 µm) capillary columns (Agilent, J&W Scientific, Santa Clara, CA, USA). The GC oven's temperature was programmed as follows: increased from 50 • C (isothermal for 1 min) to 160 • C (isothermal for 2 min) at a rate of 5 • C/min, then increased to 250 • C at a rate of 10 • C/min; the final temperature was kept for 4 min. The temperature of the injector and detector was maintained at 250 • C. The flow rate of the carrier gas (hydrogen) was 1 mL/min. At least 2 repetitions (n ≥ 2) per analysis were performed.

GC-MS Analysis
Analyses were performed on a chromatograph Shimadzu GC-2010 PLUS (Shimadzu, Kyoto, Japan) interfaced to a Shimadzu GC-MS-QP2010 ULTRA mass spectrometer (Shimadzu, Kyoto, Japan) and fitted with a capillary column Rxi-5MS (Restek, Bellefonte, PA, USA), (5%-phenyl)-methylpolisiloxane 33 m × 0.25 mm i.d., film thickness 0.25 µm). The conditions of chromatographic separation were the same as for the GC (FID) analysis. The temperature of the injector and detector was 250 • C. The flow rate of carrier gas (helium) was 1 mL/min, split 1:20. At least 2 repetitions (n ≥ 2) per analysis were performed. The temperature of ion source was 220 • C. Mass spectra in electron mode were generated at 70 eV, 0.97 scans/second, mass range 33-400 m/z.

Identification of Individual Components
The percentage composition of the essential oils was computed from GC peak areas without correction factors. Qualitative analysis was based on comparison of retention indexes on both columns (polar and non-polar), co-injection of some reference terpenoids (α-, β-pinene, 1,8-cineole, linalool, camphor, β-caryophyllene, α-humulene and caryophyllene oxide), and C8-C28 n-alkane series; and mass spectra with corresponding data in the literature [85] and computer mass spectra libraries (Flavour and Fragrance of Natural and Synthetic Compounds 2 (FFNSC 2), Wiley and NIST). Identification was approved when the computer match with mass spectral libraries was with probabilities above 90%. The relative proportions of the oil constituents were expressed as percentages obtained by peak area normalization, all relative-response factors being taken as one.

HPLC-DAD-MS (TOF) Analysis
Methanolic extracts from H. arenarium inflorescences and leaves were analyzed by HPLC technique using a system HPLC/Diode Array Detector (DAD)/Time of Flight (TOF) (Agilent 1260 Infinity (Agilent Technologies, Waldbronn, Germany) and Agilent 6224 TOF (Agilent Technologies, Santa Clara, CA, USA)) equipped with a reverse phase column ZORBAX Eclipse XDB (C18, 5 µm particle size, 150 × 4.6 mm, Agilent Technologies, Santa Clara, CA, USA). The column temperature was maintained at 35 • C. Gradient system was applied: A (deionized water, containing 0.1% of formic acid) and B (acetonitrile, containing 0.1% of formic acid). Chromatographic separation was performed at a flow rate of 0.5 mL/min in the HPLC system by the following stepwise gradient elution method: initial 95%(A)/5%(B); from 0 to 20 min from initial ration to 0% DAD range was set from 190 to 600 nm with selected scans at 254, 280, 320, 365, 380, 430, 480, and 515 nm, using 360 nm as reference abundance.

Determination of Total Phenolic Content (TPC)
The total phenolic content of H. arenarium methanol/water (1:1) extracts (from leaves and inflorescences) was determined using Folin-Ciocalteu assay [102]. 20 µL of methanolic/water extract and 1580 µL of distilled water was added to 100 µL Folin Ciocalteu reagent and 300 µL of Na 2 CO 3 (20% w/v). The mixture was left in the darkness at room temperature for 2 h. The absorbance at 765 nm wavelength was measured using a spectrophotometer (UV/Vis Lambda 25, Perkin Elmer, Buckinghamshire, UK). The results are expressed as mg/L gallic acid equivalent. Calibration curve used for calculations ( Figure S2 in Supplementary Materials) was obtained using different concentrations of gallic acid 0.00; 50; 100; 150; 250 and 500 mg/L. All measurements were done in triplicate.
4.9. Antioxidant Activity 4.9.1. Spectrophotometric Assays Antioxidant Capacity ABTS •+ Assay Antioxidant capacity ABTS •+ assay was applied for H. arenarium inflorescence and leaf EOs and methanolic extracts. The stock solution containing ABTS •+ (2,2'-azino-bis(3ethylbenzotiazoline-6-sulfonic acid) diammonium salt) and potassium persulfate (K 2 S 2 O 8 ) was prepared dissolving these materials in a mixture of methanol and water (80:20) and left in the darkness for 12 h [33]. The working solution was prepared by diluting stock solution with a mixture of methanol and water (80:20) to obtain an absorbance value of 0.730 ± 0.02 at 734 nm. The absorbance was measured using the spectrophotometer (UV/Vis Lambda 25, Perkin Elmer, Buckinghamshire, UK). Essential oils and extracts for analysis were diluted with a mixture of methanol and water (80:20); 0.1 mL of prepared sample was allowed to react with 3.9 mL of working ABTS •+ solution for 15 min in the darkness. Thereafter, the absorbance of the reacted mixture was measured. The results are expressed in mmol/L TROLOX equivalent. All measurements were done in triplicate. DPPH • Assay DPPH • assay was applied for H. arenarium (inflorescence and leaf) and H. italicum inflorescence EOs and H. arenarium inflorescence and leaf methanolic extracts. 6 × 10 −5 M stock solution of DPPH • was obtained by dissolving 2,2-diphenyl-1-picrylhydrazyl with methanol. The working solution was prepared by diluting stock solution with methanol to obtain an absorbance value of 0.730 ± 0.02 at 515 nm. Essential oils and extracts for analysis were diluted with a mixture of methanol and water (80:20); 0.1 mL of the prepared sample was allowed to react with 3.9 mL of working DPPH • solution for 30 min in the darkness [33]. Thereafter, the absorbance of reacted mixture was measured. The results are expressed in mmol/L TROLOX equivalent. All measurements were done in triplicate.
TROLOX Equivalent ABTS •+ and DPPH • Assays 5 mg of TROLOX ((±)-6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic acid) was dissolved in a mixture of methanol and water (70:30) and diluted to 100 mL. To obtain standard calibration curves the solutions of five concentrations (200, 100, 50, 25, and 12.5 mmol/L) were prepared from this solution. 0.1 mL of each TROLOX solution was allowed to react with 3.9 mL of working solution of ABTS •+ and DPPH • . Absorbance values were measured after 15 and 30 min at 734 and 515 nm, respectively. Linear calibration curves ( Figure S3 in Supplementary Materials) were obtained, and their parameters were used for further calculations of antioxidant activity. All measurements were done in triplicate.

Electrochemical (Cyclic and Square Wave Voltammetry) Analysis
Cyclic and square wave voltammetry analysis at carbon paste electrode was applied for H. arenarium (inflorescence and leaf) and H. italicum inflorescence methanolic extracts. For EOs of both Helichrysum species, modified carbon paste electrode was used in this method.
Amperometric measurements were performed with BAS-Epsilon Bioanalytical system (West Lafayette, IN, USA). A conventional three-electrode cell contained carbon paste or essential oil-modified carbon paste electrode as a working electrode, platinum as an auxiliary electrode, and Ag/AgCl, 3 N NaCl as a reference electrode.
Carbon paste electrode was prepared by thoroughly mixing 200 mg of graphite powder with 100 µL of paraffin oil. EO-modified carbon paste electrode was prepared by mixing 100 mg of graphite, 50 µL of EO, and 50 µL of paraffin oil. The paste was packed into the cavity of a homemade electrode consisting of a plastic tube (2.9 mm) and a copper wire serving as an electrode contact. The surface of the electrode was thereafter smoothened on a white paper.
Phosphate buffer prepared from 0.025 M KH 2 PO 4 contained 0.1 M KCl. The pH value was adjusted with KOH.
Cyclic voltammograms were recorded in the potential region-0.2 to 1.0 V at a potential scan rate 100 mV/s. Square wave voltammograms were recorded under the following conditions: step potential 4 mV, amplitude 50 mV, frequency 25 Hz.

Toxicity Test
Toxicity, usually expressed by LC 50 values, is the degree to which chemical compound or mixtures of chemicals can damage an organism, a tissue, or a cell. Toxic activity of H. arenarium (inflorescences and leaves, separately) and H. italicum (inflorescence) EOs was tested in vivo, using brine shrimp Artemia salina (larvae) [99]. The eggs of shrimps hatch within 48 h to provide larvae (nauplii) in sea water (31 g sea salt per litre of water) at 20-25 • C. Thereafter, different concentrations of sand everlasting and immortelle essential oils dissolved in dimethyl sulfoxide were added. Survivors were counted after 24 h. Lethality (LC 50 and LC 95 ) of nauplii was calculated (n ≥ 4, with 95% confidence interval). Curves showing dependence of brine shrimp (Artemia salina) larvae lethality (%) on Log of EO in saline water are presented in Figure S4 in Supplementary Materials. Control tests were performed both with salt water and salt water with added DMSO (5-50 µL).
In vivo toxic activity of different amounts of methanolic extracts (30, 50, 100 and 300 µL) of both Helichrysum species was investigated by the same Artemia salina larvae method [99]. Survivors were counted after 24 and 48 h. The control tests were performed both with salt water and salt water with methanol (30-300 µL).

Statistical Analysis
The collected data were subjected to a one-way analysis of variance (ANOVA); the results were expressed as mean values, range intervals, and standard deviation (SD) values, using XLSTAT (trial version, Addinsoft 2014, Paris, France).

Conclusions
Chemical composition of EOs obtained by hydrodistillation from inflorescences and leaves of H. arenarium wild plants growing in Eastern Lithuania was reported. EOs containing palmitic (≤23.8%), myristic (≤14.9%) and lauric (6.1%) acids, n-nonanal (≤10.4%), and trans-β-caryophyllene (≤6.5%) differed from previously investigated sandy everlastings of Lithuanian origin. The data obtained clearly demonstrated remarkable dissimilarities in the chemical composition of the oils of H. arenarium (L.) Moench of Lithuanian origin and essential oils of sandy everlastings from other countries.
Composition of H. arenarium methanolic extracts containing main compounds luteolin-7-O-glucoside, naringenin and its glucoside, apigenin, chlorogenic acid, arenol, and arzanol did not differ remarkably from previously investigated sandy everlasting extracts in other countries.
Low ability to scavenge free radicals (DPPH • and ABTS •+ assays) suggested that EOs of different Helichrysum species or plant parts did not have significant amounts of specific compounds that could successfully scavenge free radicals. Methanolic extracts of H. arenarium leaves and inflorescences exhibited the ability to scavenge radicals, the activity of leaf extract being higher (up by about six-fold) compared to that of inflorescences.
Radical scavenging activities of extracts correlated with total polyphenolic content. I pa values in voltammograms, i.e., electrochemical data, correlated with total polyphenolic content in H. arenarium extracts as well. The extracts showed a high level of total phenolic content and antioxidant potential, which make the studied plant species a potential source of natural antioxidants.
In the case of EOs, the non-appearance of anodic currents indicated the absence of easily oxidizable compounds that correlated with weak radical scavenging properties of H. arenarium and H. italicum EOs.
In vivo toxic activity of EOs and methanolic extracts of both Helichrysum species by brine shrimp bioassay was evaluated for the first time. H. italicum inflorescence and H. arenarium leaf and flower EOs was found to be toxic/moderately toxic, but did not differentiate statistically. Methanolic extracts of both species were not toxic.
Supplementary Materials: The following are available online. Table S1. Principal chemical composition (compounds ≥ 5.0%) of Helichrysum italicum (Roth) G. Don essential oils investigated in various countries over the last 10 years *, Table S2. Summary of previously reported data on chemical composition and bioactivity of essential oils of H. arenarium (L.) Moench) plants grown in different countries worldwide *, Table S3. Chemical composition of essential oils obtained from H. arenarium inflorescences and leaves and H. italicum inflorescences, Figure S1. Geographical indication of H. arenarium (L.) Moench sampling site in Eastern Lithuania (Utena district), Figure S2. Gallic acid standard calibration curve, Figure S3. TROLOX standard calibration curves: (a) DPPH˙assay; (b) ABTS˙+ assay, Figure S4. Dependence of brine shrimp (Artemia salina) larvae lethality (%) on Log of essential oil concentration in saline water.