The Essential Oil Composition of Helichrysum italicum (Roth) G. Don: Inﬂuence of Steam, Hydro and Microwave-Assisted Distillation

: italicum (Roth) G. Don (Asteraceae), also known as immortelle, usually grows in the Mediterranean area. The composition of the essential oil (EO) of immortelle is a mixture of various aromatic substances, mainly monoterpenes and sesquiterpenes. Distillation is the most widely used method for extraction of EO immortelle, although the yield is very low (<1%). In this work, we aim to investigate how the use of different distillation methods affects the yield and chemical composition of immortelle EO. For this purpose, we applied two conventional methods: steam distillation (SD) and hydrodistillation (HD), and a modern (environmentally friendly) technique—microwave-assisted distillation (MAD). Wild immortelles from four different locations in Croatia were collected and carefully prepared for extraction. Each sample was then analyzed by gas chromatography–mass spectrometry (GC-MS). GraphPad Prisma statistical software was used to study the statistics between different groups of connections and analyze the data on the number of connections. The results show that HD gives a signiﬁcantly higher yield (0.31 ± 0.09%) compared to MAD (0.15 ± 0.03%) and SD (0.12 ± 0.04%). On the other hand, the highest number of chemical compounds was identiﬁed with MAD (95.75 ± 15.31%), and most of them are subordinate compounds with complex structures. SD isolated EOs are rich in derived acyclic compounds with the highest percentage of ketones. The results show that the application of different distillation methods signiﬁcantly affects the composition of the obtained immortelle EO, considering the yield of EO, the number of isolated, derived and non-derived compounds, chemotypes and compounds with simple (acyclic) and complex structures.


Introduction
Helichrysum italicum (Roth) G. Don is known as immortelle or curry plant and belongs to the family Asteraceae. It usually grows on dry, sandy and rocky soils in the Mediterranean region at a wide range of altitudes: up to 2200 m above sea level [1,2]. In the Croatian flora, it is present along the coastal belt and on the islands in the form of two subspecies: microphyllum and italicum [3]. H. italicum is a 30-70 cm tall shrub with flowers grouped in yellow heads [4]. On the branched stems, the 1-3 cm long leaves are alternate and twisted at the edges. The flowers and leaves are traditionally used for therapeutic purposes to relieve respiratory, digestive, and skin problems [5].
The collection of above-ground parts of wild immortelle ( Figure 1A) was carried out in July 2018 at four different sites in the coastal area of Dalmatia (Croatia). The selected sites (see Table 1) were far from major settlements, industry and main roads. At least 20 individual plants were collected at each site. Plants that were damaged or dirty were not harvested. The collection was done according to botanical [19] and legal regulations (permit for collecting non-timber forest products 112/2018). The identification of the plant material was performed by Professor Goran T. Anačkov, and the specimens were confirmed and deposited in the Herbarium of the Department of Biology and Ecology (BUNS Herbarium) of the Faculty of Natural Sciences of the University of Novi Sad. Drying of the plant material was carried out for 15 days at room temperature in the dark. After drying, the plant material was cut into 1-3 cm pieces, homogenized ( Figure 1B) and stored in the dark until extraction.

Steam Distillation (SD)
SD was performed with water-soaked plant material to allow more efficient penetration of steam through the plant material (76 ± 37 g). The steam was generated with a steam generator connected to the distillation apparatus by a pipe system. The temperature of the steam was determined by the boiling point of water at atmospheric pressure. After 2.5 h Separations 2022, 9,280 3 of 13 of collecting the distillate, EO was collected in a small amount of n-pentane to verify the separation of EO from water. The EOs obtained were dried with anhydrous sodium sulfate and then stored at 4 • C in the dark.
Separations 2022, 9, x FOR PEER REVIEW 3 of 15 plant material was carried out for 15 days at room temperature in the dark. After drying, the plant material was cut into 1-3 cm pieces, homogenized ( Figure 1B) and stored in the dark until extraction.

Steam Distillation (SD)
SD was performed with water-soaked plant material to allow more efficient penetration of steam through the plant material (76 ± 37 g). The steam was generated with a steam generator connected to the distillation apparatus by a pipe system. The temperature of the steam was determined by the boiling point of water at atmospheric pressure. After 2.5 h of collecting the distillate, EO was collected in a small amount of n-pentane to verify the separation of EO from water. The EOs obtained were dried with anhydrous sodium sulfate and then stored at 4 °C in the dark.

Hydrodistillation (HD)
HD was performed in a Clevenger apparatus. Dried plant material (112 ± 35 g) was immersed in water in a distillation flask. The distillation lasted for 2.5 h. The essential oils were collected in n-pentane, which was also dried with anhydrous sodium sulfate and stored in the dark at 4 °C until use.

Microwave-Assisted Distillation (MAD)
MAD was performed in a microwave extraction system (Milestone "ETHOS X") in a laboratory oven (1900 W maximum). Dried plant material (140 ± 15 g) was soaked in water before being placed in a distillation flask. Distillation lasted 35 min at atmospheric pressure and the maximum power was 500 W (98 °C) at a wave frequency of 2.45 GHz. The system for cooling the steam was located above the oven. The distillate was collected with an n-pentane trap in a side tube, dried over anhydrous sodium sulphate, and stored at 4 °C until analysis.

Hydrodistillation (HD)
HD was performed in a Clevenger apparatus. Dried plant material (112 ± 35 g) was immersed in water in a distillation flask. The distillation lasted for 2.5 h. The essential oils were collected in n-pentane, which was also dried with anhydrous sodium sulfate and stored in the dark at 4 • C until use.

Microwave-Assisted Distillation (MAD)
MAD was performed in a microwave extraction system (Milestone "ETHOS X") in a laboratory oven (1900 W maximum). Dried plant material (140 ± 15 g) was soaked in water before being placed in a distillation flask. Distillation lasted 35 min at atmospheric pressure and the maximum power was 500 W (98 • C) at a wave frequency of 2.45 GHz. The system for cooling the steam was located above the oven. The distillate was collected with an n-pentane trap in a side tube, dried over anhydrous sodium sulphate, and stored at 4 • C until analysis.

GC-MS Analysis
Analysis of the obtained EO samples was performed using a gas chromatograph (GC) model 7890A (Agilent Technologies, Santa Clara, CA, USA) in conjunction with a selective mass detector (MS) 5975C (Agilent Technologies). The injection temperature was set at 250 • C, and the injection volume was 1 µL. A nonpolar HP-5MS column (30 m × 0.25 mm × 0.25 µm) was used for chromatographic separation of the compounds, with the stationary phase consisting of 5% phenylmethylpolysiloxane. The carrier gas was high purity helium with a flow rate of 1 mL min-1. The oven temperature was programmed as follows: Hold at 70 • C for 2 min, heat to 200 • C at 3 • C min −1 , and hold for 18 min. The split ratio was 1:50, the ionization energy was 70 eV, the ion source temperature was Separations 2022, 9, 280 4 of 13 230 • C, the MS quad temperature was 150 • C, and the transfer line was 280 • C. The mass scan interval was 30-350 mass units. The retention indices were determined relative to the retention time of the n-alkanes (C 9 -C 25 ). Compounds were identified based on the retention indices and comparison of the spectra with spectra from the Wiley 9 and NIST 14 databases and with previously published work. The amount of each compound was calculated by integrating the area under the peak [9].

Statistical Analysis
Statistical analysis was performed using GraphPad Prisma software (version 9). The data distribution was examined with the Shapiro-Wilk test, while the two-way test ANOVA with Tukey post hoc test was used to examine the statistical significance between the different groups of preparations. For data analysis of the yield of EO, the one-way ANOVA with Tukey post hoc test was used. Kruskal-Wallis and Dunn tests were used to analyze the data for the number of compounds identified. Results are presented as mean ± standard deviation. Only results with a calculated probability p of less than 0.05 are considered statistically significant. Symbols: * for p < 0.05, ** for p < 0.001, *** for p < 0.0001, and **** for p < 0.00001.

Isolation of EOs
In this work, we compared how the use of different distillation methods affects the yield and composition of immortelle EO. Wild immortelle was used for SD, HD and MAD. The yield of essential oils of immortelle obtained by each distillation method was calculated from the mass of plant material used during the distillation process and the mass of EO in pure and concentrated states. HD gave a significantly higher yield (0.31 ± 0.09%) of EO compared to SD (0.12 ± 0.04%) and tended to give a higher amount of oil compared to MAD (0.15 ± 0.03%). Considering the duration of the distillation method used, both HD and SD had the same distillation time (2.5 h), but HD isolated twice the amount of EO compared to SD. MAD, on the other hand, had a much shorter distillation time (35 min), but produced a similar amount of EO as SD. The yields of EO obtained by all three methods are consistent with those previously reported for immortelle, where yields of less than 1% were also reported [6,18].
After isolation of the essential oils, GC-MS analysis was performed. A total of 120 compounds were identified in 12 samples of immortelle EOs. The MAD method yielded the EO with the highest number of identified compounds (73-106), which was significantly higher compared to the samples obtained with SD (55-79), but close to HD, which yielded EO with (90-96). SD isolated samples of immortelle EO in which the lowest number of compounds was identified. One possible reason is that SD is limited compared to the other two distillation methods because steam cannot penetrate dry cell membranes [20], which limits the isolation of volatile compounds to the plant surface. Table 2 shows the chemical composition of EOs of immortelle for each sample. In samples from different sites obtained by the same distillation method, the same compounds are present in different proportions. The differences are due to genetic, environmental, and climatic factors that affect plant growth and biosynthesis of the compounds [21,22].
A total of 37 common compounds were identified in all samples, regardless of the distillation method and plant material used, and are highlighted in Table 2. Figure 2A shows the statistics of the compounds identified in at least one sample with a proportion greater than 5%. The most important compound in the essential oils of immortelle was neryl-acetate, which was the most frequently isolated in the isolates from SD (about 10%), but did not show statistically significant differences from the other distillation methods. The high content of neryl-acetate was also reported by other authors for Helichrysum italicum Roth. G. Don ssp. Italicum [18,23] and related species [24]. Other compounds with high abundance were α-pinene, Italidione I, II and III, γ-curcumene, ar-curcumene, αand β-selinene, and rosifoliol. Italidione I was significantly increased in SD compared to the other methods used, and Italidione II was significantly increased in SD compared to MAD. MAD isolated significantly more α-pinene compared to SD and tended to isolate more than HD. In the samples obtained with HD, γ-curcumen was not detected, while SD and MAD isolated a relatively high amount of γ-curcumen. The absence of γ-curcumene in the samples obtained by HD can be explained by its photosensitivity [25], which is enhanced in the aqueous medium by the effect of high temperature. In addition, we tested whether the chemotype of EO changed. SD and HD produced EOs with a nerol-rich chemotype, whereas MAD isolated an EO with a mixed chemotype that had similar amounts of nerol, curcumene, and selinene compounds in the isolates ( Figure 2B). Since the starting material was the same for all three methods, these changes could only have been caused by the distillation methods themselves.
neryl-acetate, which was the most frequently isolated in the isolates from SD (about 10%), but did not show statistically significant differences from the other distillation methods. The high content of neryl-acetate was also reported by other authors for Helichrysum italicum Roth. G. Don ssp. Italicum [18,23] and related species [24]. Other compounds with high abundance were α-pinene, Italidione I, II and III, γ-curcumene, ar-curcumene, α-and β-selinene, and rosifoliol. Italidione I was significantly increased in SD compared to the other methods used, and Italidione II was significantly increased in SD compared to MAD. MAD isolated significantly more α-pinene compared to SD and tended to isolate more than HD. In the samples obtained with HD, γ-curcumen was not detected, while SD and MAD isolated a relatively high amount of γ-curcumen. The absence of γ-curcumene in the samples obtained by HD can be explained by its photosensitivity [25], which is enhanced in the aqueous medium by the effect of high temperature. In addition, we tested whether the chemotype of EO changed. SD and HD produced EOs with a nerol-rich chemotype, whereas MAD isolated an EO with a mixed chemotype that had similar amounts of nerol, curcumene, and selinene compounds in the isolates ( Figure 2B). Since the starting material was the same for all three methods, these changes could only have been caused by the distillation methods themselves.

Discussion
The differences obtained prompted us to examine what were the main changes in the composition of EO when different distillation methods were used. EO are complex mixtures of volatile compounds consisting mainly of two groups, terpenes and phenolic compounds [8]. All the essential oils we obtained were very rich in terpenes, which accounted for more than 60% of all EO compositions. The differences in the proportion of terpene compounds are shown in Figure 3A. Derived monoterpenes and non-derived sesquiterpenes accounted for the majority of EO compositions, 52.4 ± 11.2% at SD, 46.5 ± 5.2% at HD, and 60.30 ± 3.4% at MAD. Sesquiterpenes are the most represented in all three methods, except for two samples obtained by SD (PL-SD and KO-SD). Statistically, MAD isolated more sesquiterpenes than HD and tended to isolate more than SD, while SD isolated significantly more other (non-terpene) compounds than MAD. When sesquiterpenes were further partitioned, the difference in sesquiterpene content came from subordinate sesquiterpenes. The high content of sesquiterpenes in the immortelle EO can be explained by the growing season and the time of harvest of the plant material. In the early stages of plant development, monoterpenes are the predominant group of compounds, while sesquiterpenes are present during and after flowering [28]. Compared to monoterpenes, sesquiterpenes have a higher boiling point, so they are less sensitive to temperature changes and their content is usually higher in summer [21]. Immortelle EOs obtained with MAD contained a significantly higher proportion of subordinate sesquiterpenes compared to other samples. When EOs obtained by different distillation methods were compared, no significant differences were found in the proportion of subordinate monoterpenes, monoterpene derivatives, and sesquiterpene derivatives.
We also compared the presence of heteroatoms (mainly oxygen) in the compounds and defined these compounds as derived in the text. The results show a significant difference in the derivation of compounds, with MAD isolating almost twice as many underived compounds compared to SD and HD, while SD and HD produced more derived compounds than MAD ( Figure 3B). Comparing the specific functional groups of the derived compounds, alcoholic compounds were present in a significantly higher proportion in the samples obtained from SD than in the samples obtained from MAD. The percentage of ketone compounds showed a significant difference between all distillation methods used, with SD isolating the most ketones, followed by HD. MAD isolated the least amount of ketones. Regarding the content of esters and other compounds (ethers, aldehydes, phenols, furans, and epoxides), no significant differences were observed in the samples studied. There are two possible explanations for this phenomenon: heat penetration into the material is more efficient at MAD than at HD, while it is least efficient at SD [20,29,30]. If the derived compounds occur closer to the surface of the plant material, then the derived compounds are more abundant in SD than in MAD. The problem with this explanation is that MAD did not have the highest yield or the most identified compounds in the EOs. In addition, the compounds isolated from MAD are not the sum of the compounds isolated from SD and HD and the compounds isolated from the deeper parts of the plant, but MAD isolated other compounds that led to the chemotype change. Another possibility is that the oxygenated compounds were formed during the distillation process. At SD and especially at HD, EO is exposed to water and heat for a long time (2.5 h), while at MAD, the interaction of water and EO is relatively less and shorter (no additional water, everything comes from the material itself and the distillation takes much less time (35 min)).
According to the compound structure, the identified compounds were classified into simple (acyclic) compounds and compounds with complex structures (containing at least one ring in the structure). The results are shown in Figure 3C. Immortelle EO samples obtained from SD contain a significant proportion of acyclic compounds compared to MAD, while MAD isolates a significantly higher proportion of complex compounds compared to the other two methods. In addition, compounds with complex structures were divided into monocyclic, bicyclic, and tricyclic compounds. MAD isolated statistically significant differences only for bicyclic compounds compared to HD, but on average it isolated more monocyclic, bicyclic, and tricyclic compounds compared to the other two methods used. It has been previously reported that cyclic monoterpenes can be converted to acyclic monoterpenes under the influence of heat [31]. This is the explanation for the formation of oxygenated compounds, which are more abundant in SD and HD compared to MAD. In SD and HD, the plant materials are heated in an aqueous environment, which increases the probability of addition and decyclization reactions between isolated compounds. Under the distillation conditions of MAD, heat and water could not react with volatile compounds, which reduced the structural complexity of the compounds and the amount of derived compounds. , 9, x FOR PEER REVIEW 10 of 15 divided into monocyclic, bicyclic, and tricyclic compounds. MAD isolated statistically significant differences only for bicyclic compounds compared to HD, but on average it isolated more monocyclic, bicyclic, and tricyclic compounds compared to the other two methods used. It has been previously reported that cyclic monoterpenes can be converted to acyclic monoterpenes under the influence of heat [31]. This is the explanation for the formation of oxygenated compounds, which are more abundant in SD and HD compared to MAD. In SD and HD, the plant materials are heated in an aqueous environment, which increases the probability of addition and decyclization reactions between isolated compounds. Under the distillation conditions of MAD, heat and water could not react with volatile compounds, which reduced the structural complexity of the compounds and the amount of derived compounds.  and complex (at least one ring in the structure) compounds identified in EOs. Complex compounds were further divided into monocyclic, bicyclic and tricyclic compounds. * for p < 0.05, ** for p < 0.001, *** for p < 0.0001, and **** for p < 0.00001.
In summary, SD isolated EOs rich in derived acyclic compounds with the highest amount of ketones. MAD isolated EOs rich in derived compounds with complex structures. HD had intermediate results in terms of functional groups and structural complexity of the isolated compounds. This can probably be caused by the transformation of the compounds.
This study has shown that the application of different distillation methods significantly affects the composition of the obtained Immortelle EO, considering the yield of EO, a range of isolated, derived and non-derived compounds, chemotypes and compounds with simple (acyclic) and complex structures. The differences in yield and composition obtained by different extraction methods play an important role in the choice of extraction method in practice.

Conclusions
Three different types of distillation methods were used: SD, HD, and MAD, to investigate how the use of different distillation methods affects the yield and chemical composition of Immortelle EO. HD gives a significantly higher yield (0.31 ± 0.09%) of EO compared to SD (0.12 ± 0.04%) and MAD (0.15 ± 0.03%). A total of 120 compounds were identified in 12 samples of immortelle EOs, most of which were terpenes, accounting for more than 60% of all EO compositions.
Using the same plant starting material, we found differences in the chemotype of EO. SD and HD produced EOs of the nerol-rich chemotype, whereas MAD isolated EO of the mixed chemotype, which had similar amounts of nerol, curcumene, and selinene compounds in the isolates. In addition, the traditionally used SD produced EOs rich in oxygenated acyclic compounds, which are highly sought after in the perfume industry. HD produced more EO than the other methods with a similar composition to SD. MAD produced EOs rich in non-derived compounds with complex structure and had the highest compound diversity of all three methods.
Considering the common standard quality markers EO of immortelle (neryl-acetate and α-pinene), all three distillation methods yielded EO. Considering the further use and desired chemical composition EO, it is necessary to consider the appropriate distillation methods.