1.    Introduction

The genus Hedychium is part of the Zingiberaceae family and includes about 80 species distributed mainly in Asia. H. gardnerianum is a plant native to the Himalayas whose stem can be 2 meters long and its leaves 30 cm long. Its flowers can be pink or orange yellow [1-3]. It is considered an invasive plant in most of the Azores archipelago [1]. The rhizomes of the Hedychium species are known to be fleshy and aromatic. Some species are cultivated only to extract the fragrant essential oil from their rhizomes. Aerial parts of the Hedychium species can be used in the paper industry and its flowers as culinary ingredients [4]. The bloom of the Hedychium species is very brief and usually occurs during the monsoon. The chemical profile of Hedychium sp. EO is reported as complex and comprising monoterpene and sesquiterpene derivatives. The compounds α-pinene, β-pinene, eucalyptol, linalool and nerolidol, although quite ubiquitous, are described as markers of the genus Hedychium [4].

The genus Pittosporum belongs to the Pittosporaceae family and encompasses ca. 200 species [5]. Among these, Pittosporum senacia is a species found in the Indian Ocean Islands. The chemical composition of EO from the whole plant from Mauritius was described in 2020 and 2021 by Jugreet et al. [5, 6]. In their most recent article, the distillation yield for P. senacia was found to be 0.77%. The chemical composition mainly comprised monoterpene hydrocarbons (up to 71.9%), and particularly β-myrcene (62.2%). Other compounds found were germacrene D (7.8%), limonene (3.4%), and β-phellandrene (2.9%). In an earlier article from 1998, EO from the leaves of P. senacia coursii, endemic to Madagascar, was studied [7]. With a distillation yield of 0.67%, 95% of the chemical composition of the EO was determined by GC-FID and GC/MS. With monoterpene hydrocarbons accounting for only 20.4%, including β-myrcene (6.3%), α-terpinene (4.6%) and camphene (3%), the main compounds were found to be sesquiterpene hydrocarbons and derivatives (69.5%) such as α-cadinol (19%), α-muurolol (15.9%), and δ-cadinene (11.3%). In the same study, the antimicrobial activity of this EO against Staphylococcus aureus was shown to outperform streptomycin, while it was in the same order of magnitude as streptomycin against Streptococcus faecalis [7].

The genus Psidium belongs to the Myrtaceae family which contains ca. 5500 species [8, 9]. This genus is known for presenting rich essential oil bearing plants [9]. Psidium cattleianum is a species found in Oceania, Brazil, North America and the Caribbean [8]. The shrub is typically between 1 and 4 meters in height. The EO from the leaves of P. cattleianum has been described as having antifungal activities against Candida spp. and antibacterial activities against several strains such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa in qualitative analyses with the antibiotic ampicillin as control [8].

The chemical composition of EO from P. cattleianum leaves has been studied with plants coming mainly from Brazil [8-12]. Older studies describe P. cattleianum EO harvested in Hawaii [13], Cuba [14], and California [15].

As part of a project dedicated to the chemical and sensory evaluation of plants from Reunion Island for applications in perfumery, the chemical composition and olfactory properties of four essential oils (EOs) for which literature data are scarce or non-existent were studied. The four species, from the Zingiberaceae, Pittosporaceae, and Myrtaceae botanical families illustrated in Fig. 1 are listed hereafter:

- Longose (Hedychium gardnerianum Sheppard ex Ker-Gawl) – flowers,

- Yellow ginger (Hedychium flavescens Carey ex Roscoe) – leaves,

- Bois de joli coeur (Pittosporum senacia Putt.) – leaves,

- Chinese guava (Psidium cattleianum Afzel. Ex Sabine) – floral tops.

The aim of these studies was to determine the chemical composition of these essential oils and evaluate their olfactory profile for potential applications in perfumery.

2.    Materials and methods
2.1 Plant materials
H. gardnerianum, H. flavescens, P. senacia and P. cattleianum were collected in 2020 on Reunion Island, in the wild between 800 and 1300 meters of altitude and their essential oils were obtained from OLICA (19 Rue Fangourin, Saint-Leu 97424, La Réunion, FRANCE). The four pictures of the species in Fig. 1 are licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license without modification. Authors are (1) and (4) B. Navez, (2) Forest and Kim Starr, (3) Change-ecorce.

Figure 1. The four species studied: (1) H. gardnerianum, flowers; (2) H. flavescens, leaves; (3) P. senacia, leaves; (4) P. cattleianum, floral tops.

2.2 GC/MS-FID analyses
The analyses of the four EOs chemical compositions were performed on an Agilent GC 7820 chromatograph coupled to an Agilent 5977B electron ionization mass spectrometer and equipped with a flame ionization detector. The column used was Agilent HP5-MS capillary column (30 m x 0.25 mm i.d., film thickness 0.25 μm). The sample of EO was diluted to 1% v/v in ethyl acetate. One microliter of the sample was injected with a split ratio of 1/10. Carrier gas was helium with a flow rate of 1.2 mL/min. The temperatures of injector and source were 250 °C and 230 °C, respectively. The oven temperature was programmed to stay 4 min at 40°C, then to rise from 40 to 200°C with an increase of 2 °C/min and finally from 200 to 270 °C with an increase of 8 °C/min. For the MS, ionization energy was 70 eV and the range was from 35 to 350 m/z. The FID detector was set at 270 °C with an air flow of 400 mL/min and a hydrogen flow of 40 mL/min.

2.3 Characterization of the EOs
GC/MS was used for identification and GC-FID for quantification. The identification of EO constituents was carried out by matching the retention index (RI) determined against a series of C7-C40 alkanes as well as by matching the EI-MS mass spectra obtained with various databases (NIST20, Wiley6n and internal database).

2.4 GC/MS-O analyses
GC/MS-O analyses were performed on a Perkin Elmer Clarus 690 chromatograph coupled with a Perkin Elmer Clarus SQ8T mass selective detector and equipped with a Perkin Elmer sniffing port. The column was a Perkin Elmer Elite 5-MS (30m x 0.32mm i.d., film thickness 0.25μm). The sample of EO was diluted to 10% v/v in ethyl acetate. One microliter of the sample was injected with a split of 72 mL/min (inlet pressure: 23 psi). The split between the MS and the Olfactometry detector was 1:7 (v/v). The oven temperature was programmed to stay 2 min at 60 °C, to rise from 60 to 200 °C with an increase of 5 °C/min, then from 200 to 280 °C with an increase of 8 °C/min and finally held at 280 °C for 5 min. The solvent delay was 5.5 min. Four panelists carried out the GC/MS-O analysis in duplicate, on each of the four EOs. Compounds were annotated by matching the EI-MS mass spectra obtained with databases (NIST20).

3.    Results and discussion
3.1 Hedychium gardnerianum and Hedychium flavescens
Essential oils obtained from the rhizomes of the two species H. gardnerianum and H. flavescens have been described in the literature [2-4, 16-18]. For example, Ray et al. studied 10 species of Hedychium, including H. gardnerianum and H. flavescens, cultivated under the same conditions in India [4]. After harvesting, the EO of each plant was collected from the rhizomes and analyzed by GC/MS and GC-FID. The distillation yield of H. gardnerianum and H. flavescens species was about 0.20%, which is lower than other species of the genus such as H. thyrsiforme (0.75%) and H. gracile (0.65%), and higher than others such as H. flavum (0.10%) and H. greenii (0.05%). The EO of H. gardnerianum was described as dark yellow and that of H. flavescens as dark brown. The chemical compositions of the two EOs were determined, accounting for 75.9% and 92.8% of oils of H. gardnerianum and H. flavescens, respectively. Both were composed mainly of monoterpene derivatives, mostly β-pinene and α-pinene (28.03% and 21.15% for H. gardnerianum and 29.76% and 13.17% for H. flavescens, respectively). Eucalyptol, one of the main compounds of H. flavescens (12.80%), was found in a much smaller fraction in H. gardnerianum (0.42%) [4].

The EO of H. gardnerianum flowers has been very little studied; to the best of our knowledge, only two papers published in 2002 and 2003 described the EO obtained from this part of the plant [1, 2] and EO extracted from the leaves of H. flavescens has never been described. Medeiros et al. studied the chemical composition of EO from H. gardnerianum flowers from three locations on San Miguel Island in the Azores [1]. The composition of these was determined using GC/MS analysis alone, accounting for 87%, 94% and 91% of the total EO from the three locations, respectively. Although they came from the same island, significant differences in the relative percentages of compounds were observed, although GC/MS alone can hardly provide reliable quantitative information. The main compounds of the three EOs were monoterpene hydrocarbons with α-pinene (8.38-18.13%), β-pinene (5.06-11.99%), p-cymene (3.85-8.16%), γ-terpinene (3.15-14.43%) and the sesquiterpene derivatives α-cadinol (6.42-14.59%), β-caryophyllene (7.04-8.89%), δ-cadinene (4.89-8.76%) and τ-muurolol (2.64-5.86%). The chemical composition of EO from H. gardnerianum flowers from Fiji was studied by Smith et al. [2]. Up to 75% of its chemical composition was characterized by GC/MS-FID analyses. β-Caryophyllene (17.4%) and β-pinene (17.0%) were found to be the two main compounds.

3.2 Hedychium gardnerianum flowers EO analysis
The sample of H. gardnerianum flowers EO was found to be chemically very complex (Fig. 2). As shown in Table 1, a hundred compounds were detected by monodimensional gas chromatography and those identified accounted for more than 87% of the EO. The sample was mainly composed of sesquiterpene derivatives including β-farnesene (12.05%), α-cadinol (9.71%), α-farnesene (7.09%), τ-muurolol (5.89%) and δ-cadinene (5.83%). The monoterpene derivatives identified accounted for ca. 20% with α-pinene, β-pinene and humulene as main representatives (4.48%, 4.46% and 3.93%, respectively). The sample also contained diterpene derivatives including kaur-16-ene (2.42%).

Table 1. Chemical composition of Hedychium gardnerianum flowers essential oil. 

Our results are in agreement with the article by Medeiros et al. concerning the study of the Eos of longose leaves and flowers (H. gardnerianum) from the island of San Miguel (Azores) [1]. In their study, the main compounds were α-pinene, β-pinene, p-cymene, γ-terpinene, β-caryophyllene, β-cadinene and α-cadinol. These compounds were all present in the sample studied herein. However, β-farnesene, the main compound in our sample, was identified to a much lesser extent (ca. 3%) in the study by Medeiros et al. This could suggest a particular role of farnesene in plant defense particularly in the Reunion Island territory for a compound known to be associated with insect attraction [19].

Figure 2. Chromatogram of Hedychium gardnerianum flowers essential oil.

3.3 Hedychium flavescens leaves EO analysis
The EO sample of H. flavescens leaves was found to be relatively simple and to contain mainly three compounds: β-pinene (47.81%), α-pinene (18.74%) and β-caryophyllene (17.47%), which together accounted for 84.02% of the sample (Fig. 3). As shown in Table 2, in total, 96.26% of the EO was identified. In addition of these three compounds, monoterpene and sesquiterpene derivatives were found in smaller proportions such as the monoterpene hydrocarbons D-limonene (0.60%), β-myrcene (0.16%), γ-terpinene (0.05%) and sesquiterpene hydrocarbons bicyclogermacrene (2.65%), germacrene B (1.29%) and β-cadinene (1.14%).

Figure 3. Chromatogram of Hedychium flavescens leaves essential oil.

To our knowledge, the chemical composition of the EO obtained solely from the leaves of H. flavescens have not been published so far. In the most recent article on the EO of H. flavescens rhizomes, 78.05% of a sample obtained from a plant grown in India could be identified [4]. The main compounds described were β-pinene (24.77%) and α-pinene (11.17%), both present in large proportions in our sample (47.81 and 18.74%, respectively), together with β-caryophyllene and bicyclogermacrene.

Table 2. Chemical composition of Hedychium flavescens leaves essential oil. 

 3.4 Pittosporum senacia leaves EO analysis
The relatively simple composition of the EO from Pittosporum senacia leaves was determined for more than 99% of the total EO (Fig. 4). As presented in Table 3, its chemical profile essentially contained four compounds: the C9 linear alkane hydrocarbon nonane (36.24%) and three monoterpene derivatives, β-pinene (25.11%), β-myrcene (19.19%) and α-pinene (7.36%), which together accounted for 87.9%. Moreover, the chemical profile included monoterpene derivatives such as limonene (1.46%), (Z)- and (E)-β-ocimene (0.01% and 0.09%, respectively), sesquiterpene derivatives like germacrene D (3.68%), humulene (0.40%), as well as other alkanes like decane (0.09%) and undecane (0.56%).

Figure 4. Chromatogram of Pittosporum senacia leaves essential oil.

Table 3. Chemical composition of Pittosporum senacia leaves essential oil. 

3.5 Psidium cattleianum floral tops EO analysis
In the most recent study, a distillation yield of 0.83% was reported for EO from P. cattleianum leaves [8]. A total of 68 compounds were observed within the oil, 60 of which could be identified. β-Caryophyllene was the main compound found in 14.7%, followed by eucalyptol (11.7%) and γ-muurolene (5.6%). The second most recent paper, published in 2019, describes the EO of the leaves of P. cattleianum. The EO was mainly composed of sesquiterpene and monoterpene derivatives (47.8% and 28.7%, respectively). A total of 46 compounds were identified with β-caryophyllene (23.4%), caryophyllene oxide (11.4%) and α-pinene (11.3%) as the main compounds [9]. Our results are in agreement with this study (Fig. 5), whose main compounds described were β-caryophyllene and α-pinene at 23.42% and 11.31%, respectively, caryophyllene oxide being present at 1.67%. Indeed, the chemical profile of the EO sample from P. cattleianum floral tops was identified at 88.41%. As shown in Table 4, nearly 70% of the composition consisted of sesquiterpene derivatives including the main compound, β-caryophyllene, present at 43.68% In addition, fifteen monoterpene derivatives have been observed and identified. The four main monoterpenes were β-myrcene (4.67%), α-pinene (4.50%), (Z)-β-ocimene (3.23%) and terpinolene (2.98%).

Figure 5. Chromatogram of Psidium cattleianum leaves essential oil.

Table 4. Chemical composition of Psidium cattleianum leaves essential oil.