First Phytochemical Profiling and In-Vitro Antiprotozoal Activity of Essential Oil and Extract of Plagiochila porelloides

Volatiles metabolites from the liverwort Plagiochila porelloides harvested in Corsica were investigated by chromatographic and spectroscopic methods. In addition to already reported constituents, three new compounds were isolated by preparative chromatography and their structures were elucidated by mass spectrometry (MS) and NMR experiments. Hence, an atypic aliphatic compound, named 1,2-dihydro-4,5-dehydronerolidol and two isomers, (E) and (Z), possessing an unusual humbertiane skeleton (called p-menth-1-en-3-[2-methylbut-1-enyl]-8-ol) are newly reported and fully characterized in this work. The in vitro antiprotozoal activity of essential oil and extract of P. porelloides against Trypanosoma brucei brucei and Leishmania mexicana mexicana and cytotoxicity were determined. Essential oil and Et2O extract showed a moderate activity against T. brucei with IC50 values: 2.03 and 5.18 μg/mL, respectively. It is noteworthy that only the essential oil showed a high selectivity (SI = 11.7). Diethyl oxide extract exhibited moderate anticancer (cancerous macrophage-like murine cells) activity and also cytotoxicity (human normal fibroblast) with IC50 values: 1.25 and 2.96 μg/mL, respectively.


Introduction
Bryophytes are largely located in various ecosystems characterized by humid climates, such as lakes, rivers, swings, etc., where the water, an essential element for their development and sexual reproduction, is abundantly present. Considered as the oldest green plants, they are the first vegetables that adapted to terrestrial life 500 million years ago [1]. Furthermore, following their taxonomy, the bryophytes are classified among pteridophytes and algae, and could by sub-divided into three coordinate phyla: liverworts (Marchantiophyta or Hepaticae), mosses (Bryophyta) and hornworts (Anthocerotophyta). Nowadays, approximatively 25,000 species of bryophytes were identified and disseminated worldwide. Among those, 1800 species are located in Europe and almost 75% were identified in French territory [1].
In particular, Corsica has more than 500 species of bryophytes spread over all the vegetation levels of the island [2]. Most of the bryophytes live in fresh and humid places but they are also found in dry and open habitats. They are also present in running water, around streams and lakes, in marshes and bogs. In addition, these plants that contribute significantly to flora diversity and play an essential role in the functioning of many ecosystems (peat bogs, forests, etc.). Bryophytes constitute an important plant biomass available throughout the year, which is not yet economically exploited. To our knowledge, the bryophytes of Corsica have been the subject of only one phytochemical study carried out Table 1. Summary of principal volatile molecules reported for essential oils of different Plagiochila species [13][14][15][16].

Structure of the Main Volatile Compounds
est people, and are especially common in tropical areas. They include Human African Trypanosomiasis (or African Sleeping Sickness, HAT) and Leishmaniasis caused by Trypanosoma brucei (T.b) and some twenty species of Leishmania, respectively [19]; some forms are lethal for humans. Another common characteristic of these diseases is the absence of an efficient treatment which would not cause toxicity, resistance or other side-effects. Several essential oils are known to possess antimicrobial properties and could also be considered as a source of new antiparasitic compounds [20]. Table 1. Summary of principal volatile molecules reported for essential oils of different Plagiochila species [13][14][15][16].
Finally, the cytotoxicity and some antiprotozoal activities of the bulk extracts were investigated in vitro, to evaluate their potential pharmacological properties. Generally, for in vitro screening phase, a molecule is considered to have strong antiparasitic activity if its median inhibitory concentration (IC50) is below 1 µg·mL −1 . For complex mixtures such as essential oils, the activity becomes interesting when its IC50 is less than 12.5 µg·mL −1 . When the selectivity index (SI) is greater than five, then the sample is considered a hit and is therefore likely to proceed to the in vivo stage [18]. Neglected tropical diseases (NTDs) are a group of communicable diseases that prevail in tropical and subtropical conditions in about 150 countries and affect more than one billion people, mainly in the world's poorest people, and are especially common in tropical areas. They include Human African Trypanosomiasis (or African Sleeping Sickness, HAT) and Leishmaniasis caused by Trypanosoma brucei (T.b) and some twenty species of Leishmania, respectively [19]; some forms are lethal for humans. Another common characteristic of these diseases is the absence of an efficient treatment which would not cause toxicity, resistance or other side-effects. Several essential oils are known to possess antimicrobial properties and could also be considered as a source of new antiparasitic compounds [20].

Results and Discussion
To carry out a more exhaustive study of the volatile metabolites of P. porelloides, samples were prepared by four different extraction procedures, starting each time from new dried plant material. In this context, essential oil and hydrosol were obtained by hydrodistillation, solvent extracts by cold maceration and assisted microware extractions as well as the volatiles were sampled using SPME. The identification of components involved a methodology first based on the comparison of RI and MS data with those contained in the in-house library or commercial libraries. After this preliminary analysis, components matched by standards from the in-house library were considered as definitely identified while components matched only by commercial library database needed identification-confirmation. In the present work, several components remained unidentified. So, preparative liquid chromatography and additional NMR experiments were carried out to achieve an unambiguous compound identification, as well as the complete NMR assignment.
Our study allowed for the identification of 58 compounds representing 76.9% of the essential oil (EO) and 52.6% of the hydrosol extract (HY), 82.6% and 77.9% of hexane and diethyl oxide solvent extracts (EXT H and EXT O ), 89.4% of microwave extract (MW) and 90.3% volatile fraction (VF). Among them, the presence of three unknown sesquiterpenoids was revealed in the diethyl oxide extract and the essential oil of P. porelloides.

Resolution of Ambiguous Identifications of Sesquiterpenes
Preparative liquid chromatography of P. polleroides essential oil was performed to obtained rich-sesquiterpene fractions. As GC-MS identification of sesquiterpenes in complex mixture can be a complex task [21], unambiguous identification of components 45, 48, 50, 51, 54 and 55 were definitively established using NMR-Extraction procedure [22]. Among these, the presence of the isomers spathulenol 48, globulol 50 and viridiflorol 51 with close RI and mass spectra was confirmed by comparison of their 13 C-NMR data with those described in the literature. The same procedure allowed the identification of 4-epi-maaliol 45, rosifoliol 54, maalian-5-ol 55 ( Figure 1). To carry out a more exhaustive study of the volatile metabolites of P. porelloides, samples were prepared by four different extraction procedures, starting each time from new dried plant material. In this context, essential oil and hydrosol were obtained by hydrodistillation, solvent extracts by cold maceration and assisted microware extractions as well as the volatiles were sampled using SPME. The identification of components involved a methodology first based on the comparison of RI and MS data with those contained in the in-house library or commercial libraries. After this preliminary analysis, components matched by standards from the in-house library were considered as definitely identified while components matched only by commercial library database needed identificationconfirmation. In the present work, several components remained unidentified. So, preparative liquid chromatography and additional NMR experiments were carried out to achieve an unambiguous compound identification, as well as the complete NMR assignment.
Our study allowed for the identification of 58 compounds representing 76.9% of the essential oil (EO) and 52.6% of the hydrosol extract (HY), 82.6% and 77.9% of hexane and diethyl oxide solvent extracts (EXTH and EXTO), 89.4% of microwave extract (MW) and 90.3% volatile fraction (VF). Among them, the presence of three unknown sesquiterpenoids was revealed in the diethyl oxide extract and the essential oil of P. porelloides.

Resolution of Ambiguous Identifications of Sesquiterpenes
Preparative liquid chromatography of P. polleroides essential oil was performed to obtained rich-sesquiterpene fractions. As GC-MS identification of sesquiterpenes in complex mixture can be a complex task [21], unambiguous identification of components 45, 48, 50, 51, 54 and 55 were definitively established using NMR-Extraction procedure [22]. Among these, the presence of the isomers spathulenol 48, globulol 50 and viridiflorol 51 with close RI and mass spectra was confirmed by comparison of their 13 C-NMR data with those described in the literature. The same procedure allowed the identification of 4-epimaaliol 45, rosifoliol 54, maalian-5-ol 55 ( Figure 1

Structural Elucidations of New Natural Compounds
Column chromatography of P. porelloides EXTO was carried out using a gradient of polarity with hexane and diisopropyl oxide, which produced two fractions in which 58 (65%) were isolated from the polar fraction. EI mass spectra of 58 exhibited a base peak at m/z 107 and a signal at m/z 222, which could be attributed to the molecular ion. ESI (+)-

Structural Elucidations of New Natural Compounds
Column chromatography of P. porelloides EXT O was carried out using a gradient of polarity with hexane and diisopropyl oxide, which produced two fractions in which 58 (65%) were isolated from the polar fraction. EI mass spectra of 58 exhibited a base peak at m/z 107 and a signal at m/z 222, which could be attributed to the molecular ion. ESI (+)-HRMS measurements confirms the molecular formula C 15 Figure 2B and Figure  S5) indicated that the oxygenated carbon (C 3 ) was correlated with the methyl (C 13 ), the methylene group (C 2 ) and the methine (C 4 ). The signal between 5.5 and 6.6 ppm of 1 H-NMR spectrum showed coupling of methine protons (H 4 , H 5 and H 6 ) ( Figure 3). This correlation indicated the presence of a conjugated ethylenic system. The signal of H 5 is observed as a doublet of doublets pattern due to its interaction with H 6 and H 4 ; the corresponding 3 J coupling constant values are 10.8 and 15.3 Hz, respectively. The coupling constant 3 J H5,H6 , indicated the E-configuration of the diene fragment [23]. In addition, the coupling constant 3 J H4,H5 (15.6 Hz) corresponds to an E-configuration of the C 4 -C 5 double bond, confirmed by NOE correlation among H 5 and H 13 . The configuration of the C 6 -C 7 double bond was established from NOE correlation between H 6 → H 8 and H 5 → H 14 , respectively, suggesting an E configuration. Finally, the structure of compound 58 was determined as 1,2-dihydro-4,5-dehydronerolidol. Table 2. Full NMR data of 1,2-dihydro-4,5-dehydronerolidol (58) (500 MHz, 300 K and CDCl 3 ).

Atom
No. 13      Compounds 42a and 42b were isolated from the fraction F27 (12 mg) obtained from P. porelloides essential oil by column chromatography using hexane and diisopropyl oxide (90:10). Both apolar and polar GC chromatograms of F27 exhibited one major signal which amounted for 70% of the FID-response. The 13 C-NMR spectra of F27 exhibited 29 signals including one carbon atom at δc 74.4 ppm with double relative intensity. These observations support the hypothesis of the occurrence of a mixture of diastereoisomeres. While the molecular ion of 42 has not been observed, the EI-MS spectra showed a base peak at m/z 107; the other intense fragment ion, detected at m/z 59 suggests the presence of a 2hydroxy isopropyl group, characteristic of tertiary alcohol [24]. ESI (+)-HRMS measurements allowed to determine the molecular formula C15H26O, (detected ion C15H26ONa + (m/z)exp 245.1878 and (m/z)th 245.1876, error +0.8 ppm). According to the δC intensities, two sets of resonances with a ratio (2:1) assigned to 42a and 42b, respectively, could be extracted from the 13 C-NMR spectra of F27 ( Figure S7). An Attached Proton Test (APT) experiment confirms the molecular formula C15H26O for both isomers and the assignment of the 15 carbon signals was carried out as follow: five methyl groups, three methylenes, four methynes of which two ethylenics at δC 124.88 and 129.59 ppm and three quaternary carbons at δC 74.38, 137.34 and 134.16 ppm (Table 3). Two-dimensional HSQC, HMBC and COSY experiments ( Figures S8 and S9) confirmed the structure of p-menthane framework displaying a side chain composed of five carbon atoms, three of which have sp 3 hybridization and two of sp 2 type, forming a double bond (Figure 4). The isopropyl alcohol group was confirmed by HMBC assignment, where appeared the correlations between protons of both methyl terminal groups (δH at 1.17 and 1.24 ppm, respectively) and the quaternary carbon atom at δC 74.38 ppm. Moreover, our 1 H and 13 C NMR data are in good agreement with those of α-terpineol, an alcohol monoterpene with p-menth-1-en-8-ol structure. Regarding the side chain assignment, the δCH2 and one δCH3 of one isomer exhibited excessive Compounds 42a and 42b were isolated from the fraction F27 (12 mg) obtained from P. porelloides essential oil by column chromatography using hexane and diisopropyl oxide (90:10). Both apolar and polar GC chromatograms of F27 exhibited one major signal which amounted for 70% of the FID-response. The 13 C-NMR spectra of F27 exhibited 29 signals including one carbon atom at δ c 74.4 ppm with double relative intensity. These observations support the hypothesis of the occurrence of a mixture of diastereoisomeres. While the molecular ion of 42 has not been observed, the EI-MS spectra showed a base peak at m/z 107; the other intense fragment ion, detected at m/z 59 suggests the presence of a 2-hydroxy isopropyl group, characteristic of tertiary alcohol [24]. ESI (+)-HRMS measurements allowed to determine the molecular formula C 15 H 26 O, (detected ion C 15 H 26 ONa + (m/z) exp 245.1878 and (m/z) th 245.1876, error +0.8 ppm). According to the δ C intensities, two sets of resonances with a ratio (2:1) assigned to 42a and 42b, respectively, could be extracted from the 13 C-NMR spectra of F27 ( Figure S7). An Attached Proton Test (APT) experiment confirms the molecular formula C 15 H 26 O for both isomers and the assignment of the 15 carbon signals was carried out as follow: five methyl groups, three methylenes, four methynes of which two ethylenics at δ C 124.88 and 129.59 ppm and three quaternary carbons at δ C 74.38, 137.34 and 134.16 ppm (Table 3). Two-dimensional HSQC, HMBC and COSY experiments ( Figures S8 and S9) confirmed the structure of p-menthane framework displaying a side chain composed of five carbon atoms, three of which have sp 3 hybridization and two of sp 2 type, forming a double bond (Figure 4). The isopropyl alcohol group was confirmed by HMBC assignment, where appeared the correlations between protons of both methyl terminal groups (δ H at 1.17 and 1.24 ppm, respectively) and the quaternary carbon atom at δ C 74.38 ppm. Moreover, our 1 H and 13 C NMR data are in good agreement with those of α-terpineol, an alcohol monoterpene with p-menth-1-en-8-ol structure. Regarding the side chain assignment, the δ CH2 and one δ CH3 of one isomer exhibited excessive ∆δ relative to the other (7.4 ppm and 6.3 ppm, respectively), suggesting steric γ-effects generated by double bond stereochemistry. As 1 H-coupling constants of the allylic system were not sufficiently to resolve the stereochemistry, NOESY experiments were acquired to elucidate the spatial proximities determined by the double bond for each isomer ( Figure S10). Concerning 42a, the sp 2 methine proton δ H 11 at 5.15 ppm showed a strong NOE connectivity to H 13 and H 14 , while the H 3 had a strong NOE cross peak to the allylic proton of the methyl group C 15 at δ C 16.21 ppm. This clearly indicated that the double bond in the side chain of 42a has an E configuration. These spectral features require a structure of p-menth-1-en-3-[2-(E)-methylbut-1-enyl]-8-ol for 42. Contrary, the double bond brought by the 42b side chain was found as Z stereochemistry; these was assigned according to correlations between H 3 → H 13 , and H 11 → H 15 , respectively ( Figure 5). Table 3. Full NMR data of p-menth-1-en-3-[2-methylbut-1-enyl]-8-ol isomers 42a and 42b (in CDCl 3 , at 500 MHz and 300 K).

Atom
No.
42a 42b 13    Both alcohol isomers 42a and 42b possess a humbertiane skeleton, a relatively rare sesquiterpene pattern. To our knowledge, only four isomeric isohumbertiols, structurally related to the alcohols 42a and 42b, were identified from the wood of Humbertia madagascariensis Lam [25]. In addition, two analogous structures were isolated after fungal transformation of α-farnesene while the NMR data ( 1 H and 13 C) reported in the literature [26], differed significantly from the experimental data described here for both new sesquiterpene alcohols.

P. porelloides Volatile Components: Chemical Compositions of Specific Plant Extracts
The chemical compositions of essential oil (EO), hydrosol extract (HY), both hexane and diethyl oxide extracts (EXTH and EXTO), volatile fraction (VF) as well as microwave extract (MW) were investigated by GC/RI, GC-MS and 13 C-NMR (Table 4). Both alcohol isomers 42a and 42b possess a humbertiane skeleton, a relatively rare sesquiterpene pattern. To our knowledge, only four isomeric isohumbertiols, structurally related to the alcohols 42a and 42b, were identified from the wood of Humbertia madagascariensis Lam [25]. In addition, two analogous structures were isolated after fungal transformation of α-farnesene while the NMR data ( 1 H and 13 C) reported in the literature [26], differed significantly from the experimental data described here for both new sesquiterpene alcohols.
The volatile metabolites of P. porelloides were very atypical but the most surprising was the exclusive presence of an oxygenated linear sesquiterpene 58 in the solvent extracts. It is likely that hydrodistillation conditions cause the degradation of 58 into several compounds. However, the presence of 58 in the microwave extract proves that the temperature is not the only experimental parameter responsible for the degradation process. It is very likely that the protic character of water plays an important role. The absence of 58 in the volatile fraction emitted by the plant material can be explained by the conditions used for SPME; particularly the selectivity of the adsorbent phase seems to be implicated. The SPME experiment carried out on the plant extract support this hypothesis, allowing to detect the entire components previously listed except 58 [28].
It is difficult to accurately distinguish between the real essential oil constituents and those issued by the compound degradation. As the plant resources are limited, hemisynthesis trials become too complicated, especially as bicyclogermacrene is known to degrade into viridiflorol and spathulenol [29,30]. However, in our work, these compounds are present in samples of cold and hot extractions, we think that these compounds may be naturally present in P. porelloides.

Evaluation of Biological Activity: Antitrypanosomal, Antileishmanial and Cytotoxic Activities
To complete our study, we tested the antiparasitic activity of P. porelloides EO and EXT O against two parasite models, Leishmania mexicana mexicana and Trypanosoma brucei brucei, respectively. In particular, Leishmania mexicana mexicana is responsible for leishmaniasis disease which drastically impacts the Corsican territory.
It could be noted that P. porelloides EO and EXT O could be considered as having a good activity with IC50 values ≤ 20 µg/mL of Leishmania mexicana mexicana and the best activity (<6 µg/mL) on Trypanosoma brucei brucei [27]. Concerning the selectivity which was assessed here on the human non cancer fibroblast cell line WI38, only the essential oil had a sufficient selectivity (SI: 11.7) to be a good candidate for bioguided analysis.
Concerning cytotoxic activities, we observed that diethyl oxide extract of P. porelloides could be considered as having a good potential with IC 50 value ≤ 5 µg/mL of WI38 and J774 (murine cancer macrophages) cells, but we did not find a clear selectivity on the cancer cell line. Nevertheless, this cytotoxicity may be interesting in the search for anticancer agents (Table 5). It must be mentioned that earlier studies focused on different liverworts reported also an antiprotozoal activity, notably for alpha-eudesmol [31] and marchantin A [32].

Plant Material
Fresh P. porelloides were harvested in 2019 in one location of Corsica (France). Botanical determination was performed according to the determination keys summarized in Bryophyte Flora [33] and voucher specimens were deposited in the herbarium of University of Corsica, Corte (France).

Essential Oil and Hydrosol Isolation
After 15 days of drying, plant material (500 g) was subjected to hydrodistillation (HD) for 5 h using a Clevenger-type apparatus according to the method recommended in the European pharmacopoeia [34]. Hydrosol (300 mL) obtained by HD was submitted to Liquid/Liquid Extraction (LLE) in order to obtain a liquid extract (HY, 57.3 mg). LLE was performed three times using 50 mL of diethyl oxide.
The essential-oil yield (0.2%) was expressed in % (w/dw) based on the weight of the dried plant material.

SPME Experiments
Volatile fractions (VF) emitted by the plant were extracted with the HS-SPME method. Bryophytes samples (1 g) were crushed and disposed into 20 mL headspace vials. The vials were sealed with a silicon septum placed in 70 • C dry bath, and equilibrated for 30 min. A 75 µm DVB/CAR/PDMS solid-phase fiber (Supelco, Bellefonte, PA, USA) was then plugged into the headspace of the vials for 60 min. Later, the volatiles sampled by the solid-phase fiber were analyzed after desorption into the gas chromatography-mass spectrometry (GC-MS) injection port (5 min) in splitless mode. Each sample was conducted in triplicate.

Solvent Extractions
Dried plant materials (200 g) were mechanically powdered and extracted with hexane and diethyl oxide at room temperature for 24 h each in order to give after filtration and concentration in vacuum, both extracts called EXT H (400 mg) and EXT O (640 mg), respectively.

Microwave-Assisted Extractions
Dried plant materials were mechanically powdered and extracted using Multiwave 3000 (Anton Paar, Gratz, Austria) apparatus provided with 16 ceramic vessels. For each vessel, 5 g of dried bryophyte were introduced with 40 mL of hexane and extraction was realized at 180 • C during 20 min. The solvent was then filtered on activated carbon and concentrated under vacuum. The resulting extract was next taken up in absolute ethanol and centrifuged (20 min at 6000× g rpm) and the supernatant was collected and concentrated to finally obtain the MAE extract.

GC-FID Conditions
Analyses were carried out using a Perkin-Elmer Clarus 600 Gas Chromatography (GC) apparatus (Walthon, MA, USA) equipped with a single injector and two flame ionization detectors (FIDs) for simultaneous sampling to two fused-silica capillary columns (60 m × 0.22 mm i.d., film thickness 0.25 µm; Restek, Bellefonte, PA, USA) with stationary phases of different polarity, i.e., a nonpolar Rtx-1 (polydimethylsiloxane) and a polar Rtx-Wax (polyethylene glycol). The oven temperature was programmed to rise from 60 to 230 • C at 2 • C min −1 and held isothermal at 230 • C for 30 min. The injector temperature was maintained at 280 • C and detector temperature at 280 • C, the carrier gas was H 2 (0.7 mL.min −1 ) and the samples were injected (0.1 µL of pure oil) in the split mode (1:80). Retention indices (RIs) of the compounds were determined relative to the retention times (t R ) of a series of n-alkanes (C5-C30; commercial solution obtained from Restek, Bellefonte, PA, USA) using the Van den Dool and Kraqtz equation [35].

GC-MS Analysis
Essential oils, extracts and fractions obtained by CC were investigated using a Perkin Elmer Turbo Mass quadrupole detector directly coupled to a Perkin Elmer Autosystem XL (Walton, MA, USA), equipped with the two same fused-silica capillary columns as described above. Both columns were used with the same MS detector. The analyses were consecutively carried out on the nonpolar and on the polar column. Hence, for each sample, two reconstructed ionic chromatograms (RIC) were provided, which were investigated consecutively. The GC conditions were the same as described above and the MS parameters as follows: ion-source temperature, 150 • C, ionization energy, 70 eV; electron ionization mass spectra acquired over a mass range of 35-350 amu during a scan time 1 s. The injection volumes for the essential oil and the fractions were 0.1 µL, and 0.2 µL, respectively.

High Resolution Mass Spectrometry Experiments
High resolution mass spectrometry experiments were performed with a Synapt G2 HDMS quadrupole/time-of-flight (Manchester, UK) equipped with an electrospray source operating in positive mode. Samples were introduced at 10 µL.min −1 flow rate (capillary voltage +2.8 kV, sampling cone voltage: varied between +20 V and +30 V) under a curtain gas (N 2 ) flow of 100 L.h −1 heated at 35 • C. Accurate mass experiments were performed using reference ions from CH 3 COONa internal standard. The samples were dissolved and further diluted in methanol (Sigma-Aldrich, St-Louis-MO, USA) doped with formic acid (1% v/v) prior to analysis. Data analyses were conducted using MassLynx 4.1 programs provided by Waters.

NMR Conditions
Nuclear Magnetic Resonance (NMR) spectra were recorded on the CC-fraction F27 obtained from the EO and the polar CC-fraction obtained from the EXT O . NMR experiments were acquired in CDCl 3 (EuroIsotop, Saint Aubin, France), at 300 K using a Bruker Avance DRX 500 NMR spectrometer (Karlsruhe, Germany) operating at 500.13 MHz for 1 H and 125.77 MHz for 13 C Larmor frequency with a double resonance broadband fluorine observe (BBFO) 5 mm probe head. 13 C-NMR experiments were recorded using one-pulse excitation pulse sequence (90 • excitation pulse) with 1 H decoupling during signal acquisition (performed with WALTZ-16); the relaxation delay was set at 2 s. For each analyzed sample, depending on the compound concentration, 3 k up to 5 k free induction decays (FID) 64 k complex data points were collected using a spectral width of 30,000 Hz (240 ppm). Chemical shifts (δ in ppm) were reported relative to residual signal of CDCl 3 (δ C 77.04 ppm). Complete 1 H and 13 C assignments of the new compound were obtained using 2D gradient-selected NMR experiments, 1 H-1 H COSY (COrrelation SpectroscopY), 1 H-13 C HSQC (Heteronuclear Single Quantum Correlation), 1 H-13 C HMBC (Heteronuclear Multiple Bond Coherence) and 1 H-1 H NOESY (Nuclear Overhauser Effect SpectroscopY), for which conventional acquisition parameters were used, as described in the literature [36].

Component Quantification
The quantification of components was performed using the methodology reported by Bicchi [40] and adapted in our laboratory [41]. Briefly, the compound quantification was carried out using peak normalization, including FID response factors relative to tridecane (0.7 g/100 g) used as internal standard, and expressed as normalized contents (% abundances).

In Vitro Test for Antitrypanosomal and Antileishmanial Activity
The in vitro test was performed as described previously [44]. Pentamidine isethionate salt (a commercial antileishmanial drug) and suramine sodium salt (a commercial antitrypanosomal drug) were used as positive controls in all experiments with an initial concentration of 10 µg/mL. First, stock solutions of crude extracts and compounds were prepared in DMSO at 20 mg/mL (and 2 mg/mL for positive controls). The solutions were further diluted in medium to give 0.2 mg/mL stock solutions. Essential oil and extracts were tested in eight serial three-fold dilutions (final concentration range: 100-0.05 µg/mL) in 96-well microtiter plates. All tests were repeated three times in duplicate.

Cytotoxicity Assay
The cytotoxicity of the essential oil and extracts on WI-38 and J774 cells was evaluated as previously described from the same stock solutions [45].

Conclusions
Three new phytochemicals were isolated and identified from Corsican Plagiochila porelloides. Both (E) and (Z) stereoisomers showing an unusual humbertiane skeleton, namely p-menth-1-en-3-[2-methylbut-1-enyl]-8-ol, could be fully characterized using combined analytical approach. Moreover, an atypic aliphatic compound, named 1,2-dihydro-4,5dehydronerolidol is also newly reported and fully characterized in this work. The in-vitro antiprotozoal activity of essential oil and extract of P. porelloides against Trypanosoma brucei brucei and Leishmania mexicana mexicana and cytotoxicity were assessed. Essential oil and diethyl oxide extract showed a moderate activity against T. brucei (IC50 values found were 2.03 and 5.18 µg/mL, respectively). It is noteworthy that only the essential oil sample has shown a high selectivity (SI = 11.7), whereas the diethyl oxide extract exhibited moderate anticancer (cancerous macrophage-like murine cells) activity and cytotoxicity (human normal fibroblast) with IC 50 values: 1.25 and 2.96 µg/mL, respectively.