Chemical Composition of Aerial Parts Essential Oils from Six Endemic Malagasy Helichrysum Species

The essential oils of six endemic Malagasy Helichrysum species were investigated by GC (RI), GC–MS and 13C NMR spectrometry. In total, 153 compounds were identified accounting for 90.8% to 99.9% of the total composition. The main constituents were α-pinene for H. benthamii, 1,8-cineole for H. dubardii, (E)-β-caryophyllene for H. indutum, and H. bojerianum. H. diotoides essential oil was characterized by the presence of two lilac alcohols and four lilac acetates whereas H. hirtum essential oil exhibited an atypical composition with 7β-H-silphiperfol-5-ene, 7-epi-subergorgiol, and 7-epi-silphiperfol-5-en-13-oic acid as major components.


Introduction
Madagascar, a big island in the Indian Ocean, is one of the countries in the world having particular hotspot biodiversity. Together with this biological richness, medicinal plants hold an important place in the everyday life of Malagasy people. The medicinal plants inventoried in Madagascar consist of 3245 species, of which 60% are endemic. Croton L. and Helichrysum Mill. are the most represented genera.
In this study, we were interested in six species: Helichrysum dubardii R. Vig. and Humbert, H. benthamii R. Vig. and Humbert, H. hirtum Humbert, H. indutum Humbert, H. bojerianum DC., H. diotoides DC [11]. Helichrysum dubardii and H. benthamii consist of subshrub plants with ericoid growth form, the leaves are deltoid, erect and applied on the twigs. H. dubardii leaves have only a single midrib vein, glabrous on the upper side, silvery white on the lower side. The bractal appendages of the inflorescences are yellowish white. In H. benthamii, the leaves have one to three veins arising from the base, covered with a dense gray tomentum above, loose underneath. The bractal appendages of the flowers are sulfur yellow [12]. H. hirtum and H. indutum are subshrub plants, leaves evenly distributed on the stem while the flowers have white bractal appendages. The first species has twigs covered with glandular hairs interspersed with fine cottony hairs and glands; the leaves are sessile, oblong. and with five veins arising from the base. The second species is covered with homogeneous fine cottony hairs sometimes dotted with sessile glands [12]. H. bojerianum and H. diotoides are also subshrub plants, with leaves evenly distributed on the stem. The bractal appendages of the inflorescences are sulfur yellow. H. bojerianum is covered with an ashy white aranose tomentum. The leaves are elliptical, acute sessile, with three veins arising from the base while H. diotoides is covered with a grayish aranose tomentum. The leaves are deltoid and sessile.
The aim of this work was to study for the first time, the chemical composition of the leaf essential oil extracted from these six endemic species growing wild in the center of Madagascar: H. dubardii, H. benthamii, H. hirtum, H. indutum, H. bojerianum, and H. diotoides.

Results
Twelve oil samples obtained by hydrodistillation (yields: 0.11-0.26%) of aerial parts of six Helichrysum species growing wild in Madagascar were analyzed by gas chromatography (GC) in combination with retention indices on two columns of different polarity, by gas chromatography coupled with mass spectroscopy (GC-MS) and by carbon-13 nuclear magnetic resonance ( 13 C NMR). Due their complexity, H. hirtum and H. dubardii essential oils were also fractionated on silica gel column chromatography (CC). In total, 153 compounds were identified accounting for 90.8% to 99.9% of the total composition (Table 1). Camphene Thuja-2,4(10)-diene Sabinene Lilac alcohol A (2S,2 S,5 S) Verbenone Lilac alcohol B (2R,2 S,5 S) Nerol Carvone Piperitone Geraniol Isopiperitenone Bornyl acetate 1266 1577 - Myrtenyl acetate Eugenol Cyclosativene Aromadendrene Caryolan Humulène oxide II * 1591 2031 Cubenol Intermedeol  Order of elution and relative percentages of individual components are given on an apolar column (BP-1) excepted those with an asterisk (*) percentages on polar column (BP-20); RI a , RI p : retention indices measured on apolar and polar capillary columns respectively; percentages in bold: components identified by a combination of GC(RI), GC-MS and 13 C NMR; 13 C NMR (italic): compounds identified by 13

Helichrysum Hirtum Essential Oil
The chromatographic profile of H. hirtum oil samples (Hh1, Hh2 and Hh3) varied drastically from the others and was characterized by the presence of many oxygenated sesquiterpenes. In the process of analyzing the chemical composition of the essential oils, we noticed that several compounds remained undetermined, providing very unsatisfactory matching with commercial or in-house MS libraries. Then, the EO was fractionated by silica gel column chromatography (CC), using a gradient of solvents. These compounds could however be identified from the fraction of CC by applying our in-house 13 C NMR computerized methodology [14,15].
We detailed the identification by 13 C NMR of 7-epi-silphiperfol-5-en-13-oic acid (152) and silphiperfol-5-en-13-oic acid (153) in the sample Hh1 (Table 2). For these two compounds: -all the expected signals were observed; -the chemical shift variations between the reference spectrum (Marco et al. [16]) and the recorded spectrum of the sample Hh1 were low. Indeed, they were less than or equal to 0.08 ppm for at least 12 signals out of 15. Only the carbons of the acid function or near the acid function (i.e., C5, C6, C13) exhibited a higher chemical shift variation; -a DEPT sequence confirmed the number of hydrogens linked to each carbon.
It should be point out that the chemical shift values of carbons, measured on spectra recorded using high field spectrometers, were given with two decimal places. Nevertheless, it occasionally Plants 2020, 9, 265 9 of 14 arose that chemical shift values were given with only one decimal. In such a case, although it is not mathematically correct, comparison of data given with one decimal and those given with two decimals unambiguously allowed identification of compounds. Plants 2020, 9, 265 10 of 15 mathematically correct, comparison of data given with one decimal and those given with two decimals unambiguously allowed identification of compounds.  H. faradafini, H. bracteiferum, and H. gymnocephalum are actually the most produced in Madagascar and used as expectorant and as a preventative or curative remedy for treating coughs, colds, and bronchitis [2].

Discussion
In a recent review, Rafidison et al., highlighed the actual state of Malagasy medicinal plants and particularly the pharmacological and ethnobotanical investigations. Croton and Helichrysum are the most cited genera. Even more, H. faradafini is present in the top 20 most cited species. Concerning essential oils, H. faradafini, H. bracteiferum, and H. gymnocephalum are actually the most produced in Madagascar and used as expectorant and as a preventative or curative remedy for treating coughs, colds, and bronchitis [2].

Discussion
In a recent review, Rafidison et al., highlighed the actual state of Malagasy medicinal plants and particularly the pharmacological and ethnobotanical investigations. Croton and Helichrysum are the most cited genera. Even more, H. faradafini is present in the top 20 most cited species. Concerning essential oils, H. faradafini, H. bracteiferum, and H. gymnocephalum are actually the most produced in Madagascar and used as expectorant and as a preventative or curative remedy for treating coughs, colds, and bronchitis [2].
H. bojerianum and H. diotoides EOs exhibited a different chemical composition. Even if, the percentage of (E)-β-caryophyllene was low in H. bojerianum and H. diotoides EOs (16.1% and 15.0% respectively), both oils can be classified as sesquiterpene hydrocarbon-rich oil (76.8% and 58.6% respectively). However, the H. bojerianum EO appeared original by the presence of six lilac derivatives (two alcohols and four acetates) at an appreciable ratio around 15%.
Finally, H. hirtum EO can be classified as sesquiterpene hydrocarbon-rich oil (91.0%) but the chemical composition differed drastically from the others by the (i) absence of monoterpene hydrocarbons (only traces of α-pinene and 0.1% of p-cymenene), (ii) a very low amount of oxygenated monoterpenes (1.3%), (iii) the presence of several sesquiterpenes exbibiting silphiperfolane and presilphiperfolane skeletons. To our knowledge, the presence of silphiperfolane and presilphiperfolane derivatives has never been reported in Helychrysum EOs but these skeletons were reported in asteraceae family (Petasites, Matricaria, Sphaeranthus, Otanthus) [23].
This original chemical composition can be an important feature for marketing. Taking into account that the wild populations of H. hirtum were distributed in a limited area (Tapia or Uapaca bojeri Forest, highlands of Madagascar-around Arivonimamo), over-exploitation has greatly increased the vulnerability of H. hirtum [24]. Therefore, the protection of H. hirtum populations should be a high priority now and domestication can be considered as an excellent alternative to supply the continuous market needs by producing high quality and stable raw material, and at the same time, alleviating the pressure on natural resources from overharvesting.

NMR Analysis
13 C NMR analyses were performed on an AVANCE 400 Fourier Transform spectrometer (Bruker, Wissembourg, France) operating at 100.623 MHz for 13 C, equipped with a 5 mm probe, in CDCl 3 , with all shifts referred to internal tetramethylsilane (TMS). 13 C NMR spectra were recorded with the following parameters: pulse width (PW): 4 µs (flip angle 45 • ); acquisition time: 2.73 s for 128 K data table with a spectral width (SW) of 220.000 Hz (220 ppm); CPD mode decoupling; digital resolution 0.183 Hz/pt. The number of accumulated scans ranged from 2000-3000 for each sample (around 40 mg of oil in 0.5 mL of CDCl 3 ). Exponential line broadening multiplication (1.0 Hz) of the free induction decay was applied before Fourier Transformation.

Identification of Individual Components
Identification of the components was based: (i) on comparison of their GC retention indices (RIs) on polar and apolar columns, determined relative to the retention times of a series of n-alkanes with linear interpolation ("Target Compounds" software of PerkinElmer), with those of authentic compounds and (ii) on comparison of the signals in the 13 C NMR spectra of EOs with those of reference spectra compiled in the laboratory spectral library, with the help of a laboratory-made software [13][14][15]. In the investigated samples, individual components were identified by NMR at contents as low as 0.5%. Several compounds were identified by comparison of 13 C NMR chemical shifts with those reported in the literature, for instance 7-epi silphiperfol-5-en-13-oic acid and silphiperfol-5-en-13-oic acid [16], beyerene [17], δ-terpineol [18], 7-epi-silphiperfolenal and 7-episubergorgiol [19], 13-hydroxysilphiperfol-6-ene [20], pogostol [21], 14-hydroxy-α-humulene [22], and lilac alcohol B [13].

Conclusions
This study provides useful scientific data to promote in situ conservation and to select chemical profiles for eventual production. Our results confirmed that Malagasy Helichrysum EOs exhibited an important chemical variability and these data are useful for projects of biodiversity conservation.