Volatolomics of Three South African Helichrysum Species Grown in Pot under Protected Environment

Helichrysum decorum DC, Helichrysum lepidissimum S. Moore, and Helichrysum umbraculigerum are three species traditionally used in the South African medicine. The present work deals with the investigation of the spontaneous emission and the essential oils obtained from these plants cultivated in open field under uniform conditions. Fractions of the volatile organic compounds of the three species were rich in monoterpene hydrocarbons, representing more than 70% of the total composition. Pinene isomers were the most representative compounds: β-pinene in H. decorum (53.0%), and α-pinene in H. lepidissimum (67.9%) and H. umbraculigerum (54.8%). These latter two species evidenced an important amount of sesquiterpene hydrocarbons (SH) especially represented by γ-curcumene (H. lepidissimum) and α- and β-selinene (H. umbraculigerum). On the contrary, in the EOs, sesquiterpenes compounds prevailed, representing more than 64% of the identified fraction to reach more than 82 and 87% in H. umbraculigerum and H. lepidissimum, respectively. Although the chemical classes and their relative abundances were comparable among the three species, the individual compounds of EOs showed large differences. In fact, caryophyllene oxide (26.7%) and γ-curcumene (17.4%) were the main constituents in H. decorum, and H. lepidissimum respectively, while neo-intermedeol (11.2%) and viridiflorol (10.6%) characterized H. umbraculigerum.


Introduction
The genus Helichrysum, belonging to the family Asteraceae, comprises more than 500 species, of which almost half are indigenous to South Africa [1][2][3]. Different species of Helichrysum are widely used in the traditional local medicine, thanks to the variety of secondary metabolites that the plants belonging to this genus can produce [3]. Their aerial parts are employed as herbal teas for the treatment of respiratory issues, digestive problems, as diuretic and anti-inflammatory agents, and for other purposes [4,5]. Several Helichrysum species are appreciated for their aroma profile, strictly connected to the presence of essential oils, produced and stored in the glandular trichomes located in almost all the vegetative epigeal parts of the plant [4]. The essential oil plays an important role for the taxonomic attribution of these species [6], as well as for their biological activities [7]. Helichrysum species are, indeed, characterized by a huge genetic variability, due to their polymorphisms, as a consequence of different environmental and growing conditions. It was observed that both the different morphological characters of the plants and the chemotypes of their EOs are attributable to the genetic heritage as well, and therefore the chemical composition can be used for the taxonomic identification [8]. In the last decades, it is no coincidence that the essential oils obtained from different species of this genus received increasing interest, for both their chemical composition and their biological activities [2,7,9].
Continuing our research on the utilization of Helichrysum spp. indigenous of South Africa, in collaboration with Centro di Ricerca Orticoltura e Florovivaismo (CREA-OF) located in Sanremo (Italy), three new Helichrysum species were investigated. Helichrysum lepidissimum S. Moore and Helichrysum umbraculigerum Less. are biennial or perennial herb shrubs, Helichrysum decorum DC is a plant growing in sandy grassland or open woodland from sea level to 900 m. The African Zulu inzagomas (diviners) smoked/inhaled or burned unspecific parts of the plant, which resulted in a trance state [3]. Growing on rocky grounds in submontane areas, H. lepidissimum is a perennial shrub [10] from which Mkhize (2015) isolated lepidissipyrone [11]. This compound showed a structure similar to arzanol, isolated from H. italicum ssp. microphyllum, and it was known for its antioxidant, antiinflammatory and anti-HIV activities. According to Lourens et al., 2008 the powder and ointment prepared from this species are used as a body ointment in traditional usage [3]. H. umbraculigerum, instead, is a perennial erected plant reported in several studies as the main natural source of cannabigerol [12].
Despite their important traditional uses, investigations on these Helichrysum species are lacking, and the studies reported in the literature only cite them without any other research on their biological activity or on their secondary metabolites content. This work aims to evaluate the chemical composition of both the spontaneous volatile emissions and the essential oils of the three South African species of Helichrysum cultivated at the CREA-OF (Italy). To the best of our knowledge these investigations have never been previously reported in the literature.

Volatiles Organic Compounds (VOCs)
Thirty-six compounds were detected by GC-MS methods in the spontaneous volatile emissions, with a percentage of identification ranging between 99.5% to 100% of the whole volatilome (Table 1). H. umbraculigerum was the richest plant for variety of compounds emitted (21) compared to H. decorum (16) and H. lepidissimum (15). Interestingly, only four constituents were shared by the samples, and two of them (β-pinene and α-pinene) were major ones.

Essential Oil Chemical Composition and Yield
The complete chemical composition and the hydrodistillation yields of the essential oils (EOs) obtained from the dried aerial parts of H. decorum, H. lepidissimum and H. umbraculigerum are reported in Table 2. Altogether 112 compounds were identified, representing 92.5 to 97.1% of the total chemical composition. H. umbraculigerum presented the highest number of constituents (47 vs. 41 in both H. decorum and H. lepidissimum), as in VOC analysis. Remarkable was the fact that these oils, apart from 3 minor constituents, had no compounds in common. Moreover, the essential oil yield of H. lepidissimum was 0.6% w/w, while for the other two species it was so low that it could not be determined. In general, the species of this genus are known to produce low amounts of essential oil [7].   Concerning the chemical composition, sesquiterpenes were the most represented class of compounds in all the EOs. The oxygenated form prevailed, accounting for 48.5, 60.4 and 55.2% in H. decorum, H. lepidissimum and H. umbraculigerum, respectively, while the hydrocarbon form ranged from 17.5% in H. decorum to 26.5 and 26.9% in H. lepidissimum and H. umbraculigerum, respectively. These three EO samples showed great differences in their compositions. Caryophyllene oxide was the main constituent in H. decorum (26.7%), followed by β-caryophyllene (8.4%). Despite the oxygenated sesquiterpenes (OS) dominated the H. lepidissimum EO, γ-curcumene, a sesquiterpene hydrocarbons, was the main constituent of this oil (17.4%), followed by β-bisabolol (12.5%), epi-globulol (7.4%), and rosifoliol (7.2%). The H. umbraculigerum essential oil instead was characterized by a predominance of OS, i.e., neo-intermedeol (11.2%) and viridiflorol (10.6%), followed by SH α-selinene (9.2%) and β-selinene (6.2%).
The presence of high percentages of caryophyllene oxide is not very common in the genus Helichrysum, even though this compound was reported by Rabehaja, D.J.R. et al. for the Malagasy H. benthamii Viguier & Humbert (4.0%) [17]. These authors also evidenced a similar behaviour with the same species concerning the predominance of sesquiterpenes, with prevalence of the oxygenated ones (73.5%) in H. hirtum Humbert. Caryophyllene oxide was also detected in other South African species, such as H. cymosum (L.) D. Don subsp. cymosum studied by Giovanelli et al. [4]. γ-Curcumene, a sesquiterpene hydrocarbon typical of the EO of H. italicum (Roth) G. Don was reported as the main component of Serbian samples [18]. This chemical compound, together with rosifoliol, was detected in appreciable percentages in the EO of H. italicum Don. subsp. microphyllum (Willd.) Cambess, also known as H. italicum subsp. tyrrhenicum, which is widely employed in aromatherapy [19]. Aćimović at al. [18], in fact, evidenced that Helichrysum chemotypes containing γ-curcumene as main component could be used in perfumery industries thanks to their appreciated fragrances, as well as in food and pharmaceutical industries as natural preservatives.
Noteworthy is the appreciable percentage of oxygenated diterpenes in the H. lepidissimum EO (5.6%) with geranyl linalool as unique identified compound. This class of constituent was also found in other Helichrysum species EOs, even though in higher percentages [7,25].

Plant Material
The South African Helichrysum plants studied in the present work (see Table 3 (Table 3).

Plant Material
The South African Helichrysum plants studied in the present work (see Table 3) belong to the collection of Centro di Ricerca Orticoltura e Florovivaismo (CREA-OF), located in Sanremo, Italy. The seeds were purchased from specialized companies in sailing seeds of African plant species (Silver Hill-PO Box 53108, Kenilworth, 7745 Cape Town, South Africa and B&TWorld Seeds-Paguignan, 34210 Aigues Vives, Gard, France). The plants were grown under the same edaphic substrate (perlite (2:1 v/v added with 4 g/L slow-release fertilizer) and climatic conditions (Csa in Köppen-Geiger climate classification with an average annual temperature of 16 °C and an annual rainfall of about 700 mm; frosts are light and very rare). After clonal propagation, the plants grew in pots in the open air and were periodically watered. Flowering took place after one year. A voucher sample of each plant was deposited at the herbarium of the Hanbury Botanical Gardens (La Mortola-Ventimiglia, Imperia, Italy) ( Table 3). Table 3. Botanical description of the three analyzed South African Helichrysum species.

H. decorum DC
Voucher: HMGBH.e/9006.2020.002 • Biennial or perennial herb up to 1.3 m tall grows in rough grassland or scrub, often on forest margins or in damp gullies and along streambanks • Stem stout: usually simple thinly greyish-white woolly, leafy.
• Radical leaves rosetted in the first year of growth, wanting at flowering, elliptic, narrowed to a broad clasping base, apex obtuse or subacute, apiculate, both surfaces thinly greyish-white woolly. Cauline leaves diminishing in size upwards, oblong-lanceolate or elliptic-lanceolate, apex usually acute, base clasping, upper surface glandular-setose, thinly cobwebby, lower thinly greyish-white woolly.

Spontaneous Emission Analysis and EO Extraction
Living fresh plant material (almost 1 g) was the subject of the HS-SPME (head space-solid phase microextraction) analyses which was performed using 100 µm polydimethylsiloxanes (PDMS) fiber manufactured by Supelco Ltd. (Bellefonte, PA, USA). As recommended by the manufacturer's instruction, prior to the analyses, the fiber was conditioned at 250 °C for 30 min in the injector of a gas chromatograph. The plant material was placed in a 50 mL glass vial, covered with an aluminum foil, and then left for 60 min (equilibration time). Exposition of the fiber in the headspace phase of the samples took place for 15 min at a temperature of 23 °C. Subsequently, the fiber was transferred to the injector of the gas chromatograph (temperature 250 °C), where the analytes were thermally desorbed [27]. The composition of the compounds desorbed from SPME fiber was examined using GC-MS.

Essential Oil Hydrodistillation
The essential oil was obtained from the dried aerial parts of the three species of Helichrysum by hydrodistillation with a Clevenger-type apparatus, performed for 2 h at 100 °C, according to the method reported in the European Pharmacopoeia [28]. The hydrodistillation was carried out in triplicates, on 50 g of plant material and the collected essential oil was refrigerated at 4 °C and maintained far from light sources until analyses.

Gas Chromatography-Mass Spectrometry Analyses
The essential oils were diluted to 0.5% in HPLC-grade n-hexane before the injection in the GC-MS apparatus. The GC/EI-MS analyses were performed with an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS capillary column (30 m × 0.25 mm; coating thickness 0.25µm) and an Agilent 5977B single quadrupole mass detector.
The analytical conditions were set as follows: oven temperature ramp from 60 to 240 °C at 3 °C/min; injector temperature, 220 °C; transfer line temperature, 240 °C; carrier gas helium, 1 mL/min. The injection volume was 1 µL, with a split ratio of 1:25. The acquisition parameters were: full scan; scan range: 30-300 m/z; scan time: 1.0 sec.
The Identification of the constituents was based on a comparison of the retention

Spontaneous Emission Analysis and EO Extraction
Living fresh plant material (almost 1 g) was the subject of the HS-SPME (head spacesolid phase microextraction) analyses which was performed using 100 µm polydimethylsiloxanes (PDMS) fiber manufactured by Supelco Ltd. (Bellefonte, PA, USA). As recommended by the manufacturer's instruction, prior to the analyses, the fiber was conditioned at 250 • C for 30 min in the injector of a gas chromatograph. The plant material was placed in a 50 mL glass vial, covered with an aluminum foil, and then left for 60 min (equilibration time). Exposition of the fiber in the headspace phase of the samples took place for 15 min at a temperature of 23 • C. Subsequently, the fiber was transferred to the injector of the gas chromatograph (temperature 250 • C), where the analytes were thermally desorbed [27]. The composition of the compounds desorbed from SPME fiber was examined using GC-MS.

Essential Oil Hydrodistillation
The essential oil was obtained from the dried aerial parts of the three species of Helichrysum by hydrodistillation with a Clevenger-type apparatus, performed for 2 h at 100 • C, according to the method reported in the European Pharmacopoeia [28]. The hydrodistillation was carried out in triplicates, on 50 g of plant material and the collected essential oil was refrigerated at 4 • C and maintained far from light sources until analyses.

Gas Chromatography-Mass Spectrometry Analyses
The essential oils were diluted to 0.5% in HPLC-grade n-hexane before the injection in the GC-MS apparatus. The GC/EI-MS analyses were performed with an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent HP-5MS capillary column (30 m × 0.25 mm; coating thickness 0.25 µm) and an Agilent 5977B single quadrupole mass detector.
The analytical conditions were set as follows: oven temperature ramp from 60 to 240 • C at 3 • C/min; injector temperature, 220 • C; transfer line temperature, 240 • C; carrier gas helium, 1 mL/min. The injection volume was 1 µL, with a split ratio of 1:25. The acquisition parameters were: full scan; scan range: 30-300 m/z; scan time: 1.0 s. The Identification of the constituents was based on a comparison of the retention times with those of the authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons. Computer matching was also used against commercial (NIST 14 and ADAMS 2007) and laboratory-developed mass spectra libraries built up from pure substances and components of commercial essential oils of known composition and MS literature data [14,[29][30][31][32][33].

Conclusions
The present study represents a contribution to increasing the knowledge about the chemical composition of the HSs and the essential oils of three South African Helichrysum species that were not studied yet. It should be a starting point for future investigations, which can lead to a more informed employment of these plants, as they are already used in the traditional local medicine. The studied species showed huge differences in the chemical composition of both the spontaneous emissions and the EOs.
The chemical differences of the aroma profile of the studied samples together with their habitus can be exploited for the ornamental use of these plants.