Composition, Antioxidant Potential, and Antimicrobial Activity of Helichrysum plicatum DC. Various Extracts

Helichrysum plicatum DC. is widely used in folk medicine in treating a variety of health disorders. The aim of this study was to examine the influence of different extraction solvents on the chemical composition, antioxidant potential, and antimicrobial activities of H. plicatum. Aerial parts were separately extracted with ethanol, dichloromethane, and sunflower oil. The oil extract (OE) was re-extracted with acetonitrile. A total of 142 compounds were tentatively identified in ethanolic (EE), dichloromethane (DCME), and acetonitrile (ACNE) extracts using HPLC-DAD/ESI-ToF-MS. The dominant compound class in all extracts were α-pyrones, alongside flavonoids in EE, terpenoids in DCME and ACNE, and phloroglucinols in DCME. The antioxidant potential of the extracts was assessed by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay. EE and DCME possessed the most potent radical scavenging capacity. Antimicrobial activity was investigated on eight bacterial, two yeast, and one fungal species. All extracts exhibited high antifungal and notable antibacterial activities compared to control substances, with DCME being the most potent. DCME exhibited stronger antimicrobial activity against P. aeruginosa than the standard chloramphenicol.

Helichrysum plicatum DC. is a herbaceous perennial plant, which is native to the Balkan and Anatolian Peninsulas and Iran [13]. It has been used in folk medicine for wound-healing and the treatment of gastric and hepatic disorders, diabetes, and kidney stones [14][15][16]. Chemically, this species responsible for the therapeutic effects of the plant. H. plicatum polar extracts are reported to possess antimicrobial, antidiabetic, spasmolytic, nephroprotective, and antimutagenic activities [13,[17][18][19][20][21].
It is apparent that extracts from the same plant will differ in their composition of bioactive compounds depending on the extraction solvent. Since, with the exception of the essential oil, only the phenolic composition of polar extractives has been investigated in H. plicatum to date, the aim of this study was to perform a more detailed chemical analysis of the plant and to examine the influence of solvents of different polarity on the extraction yield, composition of extracts, antioxidant potential, and antimicrobial activities.

Phytochemical Profile
Aerial parts of wild H. plicatum at the full blooming stage were extracted with solvents of different polarity. The yields were 7.14%, 2.88%, and 0.62% for ethanol (EE), dichloromethane (DCME), and acetonitrile oil (ACNE) extracts, respectively. The phytochemical content of the extracts was assessed by HPLC-DAD/ESI-ToF-MS. The peaks in chromatograms were tentatively identified on the basis of the exact molecular masses and formulas, UV spectra, and literature data (Tables S1-S3, Supplementary material). The total ion chromatograms (TIC) in the positive (for EE and DCME) and negative (for EE, DCME and ACNE) modes and HPLC-DAD chromatograms of the investigated extracts are shown in the Supplementary material ( Figures S1-S3).

Antioxidant Potential
The antioxidant activity of H. plicatum extracts was assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay (Table 2). EE, DCME, and ACNE demonstrated significant DPPH scavenging activity. The analysed extracts could not be compared since the polar extract (EE) was analysed by the polar DPPH method (DPPH in methanol) and nonpolar extracts (DCME, ACNE, and OE) were analysed by the nonpolar DPPH method (DPPH in toluene). However, after comparison with standards analysed by the same method, we concluded that the DCME extract exhibited a stronger antioxidant potential than the BHT standard (toluene), while EE exhibited slightly lower activity than BHT (methanol) and Trolox.

Antimicrobial Activity
The antimicrobial activity of H. plicatum was investigated against five Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Proteus hauseri, Klebsiella pneumoniae, Salmonella enterica subsp. enterica), three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes), two yeasts (Candida albicans, Saccharomyces cerevisiae), and one fungal strain (Aspergillus brasiliensis) (Tables 3-5). All the extracts displayed notable antibacterial activity in the range of 0.157-2.5 mg/mL. DCME was more active than the chloramphenicol standard against Pseudomonas aeruginosa. In addition, DCME demonstrated the best antibacterial activity among the extracts, as indicated by the lowest minimal inhibitory concentration (MIC) values. The tested extracts exhibited better or the same antifungal activity as compared to the commercial drug nystatin. Once more, DCME had the lowest MIC values.

Phytochemical Profile
The predominant compounds in EE were flavonoids and their glycosylated derivatives. Luteolin (compound 45, Table 1), naringenin (62), apigenin (48), quercetin (66), kaempferol (69), as well as naringenin-, apigenin-, and kaempferol-glucosides (58-60, 43, 44, 64) have been previously reported in H. plicatum [14,107]. These compounds are probably the main contributors to the therapeutic effects of the plant, which are used for treating hepatic and gastric disorders. In addition, the same flavonoids found in H. arenarium, which are reported to exhibit choleretic, cholagogue, hepatoprotective, and detoxifying activities, are also present in H. plicatum [107]. Furthermore, the phenolic-rich extracts of H. plicatum, with the glycosides of naringenin, apigenin, quercetin, and kaempferol as the main compounds, followed by their aglycones and chlorogenic acid, exhibited potent cytotoxic activities against human cancer cell lines [108]. Flavonoids and other phenolic constituents of water and ethanol extracts are also thought to be responsible for the antidiabetic and spasmolytic effects of this plant [17,18]. DCME and ACNE were characterized by the presence of α-pyrones. Numerous diverse pyrones were isolated from Helichrysum spp., although they were poorly analysed in H. plicatum. Plicatipyrone (10) was previously isolated from this species [22]. Akaberi et al. [1] listed several different groups of α-pyrone derivatives that are distributed in Helichrysum spp., namely monomers, glycosylated forms, homo-, and heterodimers. The α-pyrone profile tentatively determined in H. plicatum in the present study corresponds to α-pyrones found in other Helichrysum species. Vrkoč et al. [27] characterized the yellow pigment from the flowers of H. arenarium as a mixture of two heterodimers-phloroglucinyl α-pyrones arenol (11) and homoarenol (later named arzanol [109], 13). Arzanol is probably the most investigated α-pyrone from Helichrysum spp. It has been reported to possess a variety of pharmacological activities, including antioxidant, anti-inflammatory, and anti-HIV [109][110][111].

Antioxidant Potential
The results obtained in this work are in agreement with literature data indicating that the methanolic and ethanolic extracts of H. plicatum exhibit dose-dependent antioxidant activity. Antioxidant activity was attributed to the phenolic content of the extracts, mostly phenolic acids and flavonoids [87,108]. These compounds act by inhibiting enzymes or chelating trace elements involved in reactive oxygen species generation or by reducing highly-oxidizing free radicals through hydrogen atom donation [117,118]. The phenolic radicals generated in the process of free radical scavenging can be stabilized via intramolecular hydrogen bonds and by electron delocalization in the aromatic ring.
Comparison of the EC 50 values with literature data obtained by different protocols and DPPH concentrations is not consistent. Kadifkova Panovska and Kulevanova [119] reported EC 50 values in the range 6-11 mg/mL for H. plicatum methanol and ethanol extracts with 100 mM DPPH, while 37-88 µg/mL EC 50 values with 0.08 mM DPPH were reported by Bigović et al. [108] and 235-918 µg/mL with 0.1 mM DPPH by Acet et al. [120]. Even though 0.1 mM DPPH was used in the present study, the results are still difficult to compare with Acet et al. because of the different mixing volumes of extracts and DPPH solutions, as well as the different incubation times. Nevertheless, compared to Trolox, the results presented herein are similar to the results from a previous study [108], whereas compared to BHT, the extracts from the present study (EE, DCME, and ACNE) were more potent in radical scavenging than the methanol and ethanol extracts of H. plicatum investigated by Kadifkova Panovska and Kulevanova [119].
The most potent radical scavenging capacity in the present study was exhibited by EE and DCME, which are both rich in α-pyrones. Rosa et al. [110] showed that the α-pyrones arzanol and helipyrone achieved remarkable efficacy in scavenging lipid peroxyl radicals. Since arzanol (Figure 2) showed powerful scavenging activity against linoleic acid peroxyl radicals in the linoleic acid autoxidation test and methylarzanol was only slightly active, the authors concluded that the α-pyrone enolic hydroxyl, which can mimic a phenolic hydroxyl in assays of antioxidant activity, is responsible for the observed radical scavenging properties [110]. Furthermore, arzanol exhibited antioxidant properties at non-cytotoxic concentrations [121], which qualifies it as a candidate for investigation as a possible food additive. The presence of flavonoids probably also contributed to the significant antioxidant potential of EE.
compared to BHT, the extracts from the present study (EE, DCME, and ACNE) were more potent in radical scavenging than the methanol and ethanol extracts of H. plicatum investigated by Kadifkova Panovska and Kulevanova [119].
The most potent radical scavenging capacity in the present study was exhibited by EE and DCME, which are both rich in α-pyrones. Rosa et al. [110] showed that the α-pyrones arzanol and helipyrone achieved remarkable efficacy in scavenging lipid peroxyl radicals. Since arzanol ( Figure  2) showed powerful scavenging activity against linoleic acid peroxyl radicals in the linoleic acid autoxidation test and methylarzanol was only slightly active, the authors concluded that the αpyrone enolic hydroxyl, which can mimic a phenolic hydroxyl in assays of antioxidant activity, is responsible for the observed radical scavenging properties [110]. Furthermore, arzanol exhibited antioxidant properties at non-cytotoxic concentrations [121], which qualifies it as a candidate for investigation as a possible food additive. The presence of flavonoids probably also contributed to the significant antioxidant potential of EE.

Antimicrobial Activity
There are several studies on H. plicatum antimicrobial activity, which was ascribed to the phenolic and flavonoid contents [13,120,122]. Fungi were more sensitive to H. plicatum extracts than bacteria, which corroborates our findings. In previous studies, Gram-positive bacteria were more sensitive to the tested extracts than Gram-negative bacteria, although this pattern was not observed in the present study. In general, polyphenols have been widely investigated for their antimicrobial activities, which can be attributed to both direct action against microorganisms as well as the suppression of microbial virulence factors [123]. In this study, all extracts contained a variety of phenolic compounds. Furthermore, ACNE and DCME were characterized by the presence of (poly)methoxylated flavones. Methoxylated flavones from H. nitens displayed antifungal activity against Cladosporium cucumerinum [42]. This group of compounds is reported to exhibit mild antibacterial but potent fungicidal properties [124,125]. They are externally located on leaf and stem surfaces where they presumably play a role in antimicrobial defence. Methylation decreases the antioxidant potential but increases the stability of flavones, their lipophilicity, and, consequently, the ability to permeate membranes [126]. The presence of polymethoxylated flavonoids was confirmed herein in DCME, and it can be assumed that nonpolar flavonoids were responsible for the pronounced antifungal activity of this extract.
The main compounds of DCME, the most active extract in the antimicrobial tests, were αpyrones, terpenoids, and phloroglucinols. The terpenoid components from H. italicum, along with flavonoids, were responsible for the antimicrobial properties of the plant [127]. Antimicrobial activity of α-pyrones from Helichrysum species has also been demonstrated. Four heterodimeric (italipyrone, plicatipyrone, arenol, and arzanol) and three homodimeric (helipyrone and two related compounds) α-pyrones from H. stoechas, which were identified in the H. plicatum investigated herein, displayed high activities against Gram-positive bacteria [26]. In another study, arzanol showed outstanding action against multidrug-resistant Staphylococcus aureus isolates, making it a good choice for further studies of suppression of antibiotic-resistant bacterial strains [23], while phloroglucinyl α-pyrones from H. decumbens displayed significant antifungal activity against Cladosporium herbarum [29].
The variety of compounds identified in H. plicatum most probably exert synergistic effects. Antioxidants and antimicrobials from natural sources have gained popularity over synthetic ones in

Antimicrobial Activity
There are several studies on H. plicatum antimicrobial activity, which was ascribed to the phenolic and flavonoid contents [13,120,122]. Fungi were more sensitive to H. plicatum extracts than bacteria, which corroborates our findings. In previous studies, Gram-positive bacteria were more sensitive to the tested extracts than Gram-negative bacteria, although this pattern was not observed in the present study. In general, polyphenols have been widely investigated for their antimicrobial activities, which can be attributed to both direct action against microorganisms as well as the suppression of microbial virulence factors [123]. In this study, all extracts contained a variety of phenolic compounds. Furthermore, ACNE and DCME were characterized by the presence of (poly)methoxylated flavones. Methoxylated flavones from H. nitens displayed antifungal activity against Cladosporium cucumerinum [42]. This group of compounds is reported to exhibit mild antibacterial but potent fungicidal properties [124,125]. They are externally located on leaf and stem surfaces where they presumably play a role in antimicrobial defence. Methylation decreases the antioxidant potential but increases the stability of flavones, their lipophilicity, and, consequently, the ability to permeate membranes [126]. The presence of polymethoxylated flavonoids was confirmed herein in DCME, and it can be assumed that nonpolar flavonoids were responsible for the pronounced antifungal activity of this extract.
The main compounds of DCME, the most active extract in the antimicrobial tests, were α-pyrones, terpenoids, and phloroglucinols. The terpenoid components from H. italicum, along with flavonoids, were responsible for the antimicrobial properties of the plant [127]. Antimicrobial activity of α-pyrones from Helichrysum species has also been demonstrated. Four heterodimeric (italipyrone, plicatipyrone, arenol, and arzanol) and three homodimeric (helipyrone and two related compounds) α-pyrones from H. stoechas, which were identified in the H. plicatum investigated herein, displayed high activities against Gram-positive bacteria [26]. In another study, arzanol showed outstanding action against multidrug-resistant Staphylococcus aureus isolates, making it a good choice for further studies of suppression of antibiotic-resistant bacterial strains [23], while phloroglucinyl α-pyrones from H. decumbens displayed significant antifungal activity against Cladosporium herbarum [29].
The variety of compounds identified in H. plicatum most probably exert synergistic effects. Antioxidants and antimicrobials from natural sources have gained popularity over synthetic ones in the last twenty years because they most likely exhibit fewer side effects. Arenarin, which represents a mixture of phenolic antibiotics from H. arenarium, a plant with a similar phytochemical content to H. plicatum, is used as a skin and eye antibacterial agent in Russia [23]. Owing to the important biological activities of H. plicatum extracts and isolated compounds, they could be considered for use in the pharmaceutical, cosmetic, and food industries.

Plant Material
A commercial sample of wild-growing plant material was obtained from the Institute for Medicinal Plants Research "Dr. Josif Pancić", Belgrade, Serbia. Wild-growing plant material was collected during the full-blooming stage in Macedonia in 2017.

Extraction Procedure
The dried and powdered plant material was extracted with 96% ethanol, dichloromethane, and sunflower oil in the dark for seven days. After filtration, solvent was removed from ethanol and dichloromethane extracts. In order to avoid ionization difficulties with the oil matrix, prior to HPLC-DAD/ESI-ToF-MS analyses, the oil extract was re-extracted with acetonitrile by overhead rotary mixer for 18 h.

HPLC-DAD/ESI-ToF-MS Analyses
Prior to injection, the test samples were dissolved in methanol (c ≈ 10 mg/mL) and filtered through a 0.45-µm filter.
An HPLC apparatus (Agilent 1100 Series, Agilent Technologies) with a degasser, autosampler, The charged molecular ions were obtained by electrospray ionization (ESI) at atmospheric pressure: the eluted compounds were mixed with nitrogen in the heated interface and the polarity was set to negative, with the following ES parameters: capillary voltage, 4000 V; gas temperature, 350 • C; drying gas flow rate, 12 L/min; nebulizer pressure, 45 psig (310.26 Pa); fragmentation voltage, 140 V, and masses were measured in the range 100-2500 m/z. MassHunter Workstation software was used for data recording and processing.

Antioxidant Assay
The antioxidant activity of the plant extracts was evaluated by the 2,2-diphenyl-1-picrylhydrazil (DPPH) assay [128], which is based on the depletion of the colour of the stable free radical DPPH in a reaction with potential antioxidants and measured by the decrease of absorbance at 517 nm. Concentrations of the extracts were in the range from 0.1 to 1.75 mg/mL. A volume of 200 µL was mixed with 1800 µL of a methanolic solution of DPPH (0.1 mM). The reaction mixture was shaken and then incubated in the dark for 30 min. After this period, the absorbance of the remaining DPPH radical was measured at 517 nm (A sample ). Blank probes were done in the same way, using 200 µL of methanol instead of the extract solution to obtain A blank . All determinations were performed in triplicate. The percentage of inhibition of the DPPH radical, I(%), by each sample was calculated according to the equation: The EC 50 (concentration of an extract that reduces the absorption of DPPH solution by 50%) was calculated from the curve of the dependence of I(%) from the concentration of each extract. DPPH scavenging activity was also determined for Trolox and butylated hydroxytoluene (BHT), which are known artificial antioxidants that were used as positive probes. Tests were performed in triplicate.
The same procedure was undertaken for the determination of the radical scavenging capacity of oil extracts, except that toluene instead of methanol was used as a solvent, and BHT dissolved in toluene as a positive probe. Antimicrobial activity was evaluated using the broth microdilution method according to the National Committee for Clinical Laboratory Standards [129]. The 96-well plates were prepared by dispensing 100 µL of Mueller-Hinton broth for bacteria and Sabouraud dextrose broth for yeasts and fungi into each well. Test extracts were dissolved in DMSO to a stock concentration of 20 mg/mL, then 100 µL from the stock solution of the tested extracts were added to the first row of the plate and double-diluted in the broth. The direct colony method was used in preparing the suspension of bacteria and yeasts in sterile 0.9% saline, while preparation of the suspension of fungal spores included gentle stripping of spores from agar slants with growing Aspergilli into sterile 0.9% saline. Suspension turbidity was conducted by comparison with 0.5 McFarland standard. After measuring the optical density OD 600 , the colony count was also checked after a series of dilutions of initial suspensions. Due to the visual detection of growth inhibition, the maximum concentrations of microorganisms were used. Ten µL of bacterial or yeast suspension or suspension of spores were added to each well to give a final concentration of 10 6 CFU/mL for bacteria and 10 5 CFU/mL for yeasts and fungi. In order to compare the activity of an extract with already existing, commercially available antimicrobial agents, broad-spectrum compounds commonly used in such assays served as positive controls [130]. Chloramphenicol served as the positive control for bacteria, while nystatin served as the positive control for yeasts and fungi. The inoculated plates were incubated at 37 • C for 24 h for bacteria and at 28 • C for 48 h for the yeasts and fungi. The MIC was determined as the lowest concentration that inhibited visible microbial growth.

Antimicrobial Assay
Minimum bactericidal (MBC) and minimum fungicidal concentrations (MFC) were determined by plating 10 µL of samples from wells where no colony growth was observed onto nutrient agar medium for bacteria and Sabouraud dextrose agar for yeasts and fungi. After the incubation period, the lowest concentration with no visible growth (no colony) was defined as the minimum microbicidal concentration.

Conclusions
In this study, the influence of three extraction solvents (ethanol, dichloromethane, and sunflower oil) on the composition, antioxidant, and antimicrobial activities of Helichrysum plicatum was investigated. The extracts comprised very diverse groups of secondary metabolites, including terpenoids and a myriad of (poly)phenolic compounds, flavonoids, α-pyrones, phloroglucinols, phenolic acids, acetophenones, phthalides, and other phenolic derivatives. EE and DCME, both rich in α-pyrones, possessed a marked antioxidant potential. DCME demonstrated the best antibacterial activity among the extracts. All extracts displayed significant antifungal capabilities, with MIC values lower than or equivalent to the MICs of the commercial antifungal agent nystatin. DCME had the lowest MIC values, which could be a consequence of the presence of nonpolar polymethoxylated flavonoids. Based on the results from this study, H. plicatum extracts could be considered for use as natural additives in food and cosmetic industries.