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

Anaïs Pannequin, Joëlle Quetin-Leclercq, Jean Costa, Aura Tintaru, Alain Muselli

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Anaïs Pannequin, Joëlle Quetin-Leclercq, Jean Costa, Aura Tintaru, Alain Muselli. First Phytochemical Profiling and In-Vitro Antiprotozoal Activity of Essential Oil and Extract of Plagiochila porelloides. Molecules, 2023, 28 (2), pp.616. 10.3390/molecules28020616. [https://doi.org/10.3390/molecules28020616](https://doi.org/10.3390/molecules28020616)

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# First phytochemical profiling and *in-vitro* antiprotozoal activity of essential oil and extract of *Plagiochila porelloides*

Anaïs Pannequin¹, Joëlle Quetin-Leclercq², Jean Costa¹, Aura Tintaru³* and Alain Muselli¹*

¹ Université de Corse, UMR CNRS 6134, Laboratoire Chimie des Produits Naturels, BP 52, 20250 Corte, France; pannequin_a@univ-corse.fr (A.P.); costa_d@univ-corse.fr (J.C.); muselli_a@univ-corse.fr (A.M.)

² UCLouvain, Louvain Drug Research Institute, Pharmacognosy Group, Avenue E. Mounier 72, B-1200 Brussels, Belgium; joelle.leclercq@uclouvain.be (J.Q.-L.)

³ Aix Marseille Univ, CNRS, Centre Interdisciplinaire de Nanoscience de Marseille, UMR7325, 13288 Marseille, France; aura.tintaru@univ-amu.fr (A.T.)

* Correspondence: aura.tintaru@univ-amu.fr (A.T.); muselli_a@univ-corse.fr (A.M.)

**Abstract:** 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, namely (3,7,11)-trimethyl-dodec-4E,6E,10-atrien-3-ol 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.

**Citation:** Lastname, F.; Lastname, F.; Lastname, F. Title. *Molecules* **2022**, *27*, x. https://doi.org/10.3390/xxxxx

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**Copyright:** © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

**Keywords:** Bryophytes, *Plagiochila porelloides*, humbertiane, farnesane, sesquiterpenes, antiprotozoal

## 1. 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 being the first vegetables 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 worldwide disseminated. Among those, 1 800 species are located in Europe and almost 75% were identified on the 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 economically exploited, yet. To our knowledge, the Bryophytes of Corsica have been the subject of only one phytochemical study carried out earlier by our group [3,4]. Nevertheless, even if Bryophytes are currently poorly investigated, based on the results reported by the few studies conducted on this topic [5–10], they are depicted as “the pharmacy of tomorrow”. For all these reasons, the characterization of the molecular constituents of essential oils, volatile fractions, and extracts produced from the Bryophytes, combined with their screening of active compounds, constitute an interesting scientific challenge to be performed at the scale of Corsican region.

*Plagiochila* species is the largest genus in the Marchantiophyta, with at least 1 600 varieties [11]. This genus is characterized not only by morphological diversity but also by a huge chemical variety. From the composition of solvent extracts of almost sixty species of *Plagiochila*, the author distinguished twelve types [11] as following I: 2,3-secoaromadendrane sesquiterpene-type (subdivided depending on the degree of oxidation and acetylation of 2,3-secoaromadendrane); II: bibenzyl-type; III: cuparane-herbertane sesquiterpene-type; IV: bibenzyl-cuparane-herbertane-type; V: gymnomitrane (barbatane)-bicyclogermacrane sesquiterpene-type; VI: bicyclogermacrane-spathulenol sesquiterpene-type; VII: pinguisane sesquiterpene-type; VIII: 2,3-secoaromadendrane-sesquiterpene lactone-type; IX: cyclic bis-bibenzyl-2,3-secoaromadendrane-type; X: sesquiterpene lactone-type; XI: epiverrucosane type and XII: fusicoccane-labdane type. According to two studies concerning compounds extracted by diethyl oxide, *P. porelloides* can be integrated to type I. The first report of *P. porelloides* compounds led to the isolation 3α-acetoxybicyclogermacrene, bicyclogermacrene, plagiochilines A, -C, -D et -H as main constituents from Swiss species [[12]]. The second investigation reported on the isolation of three sesquiterpene esters from a German sample, derived from mono esterification of a 2,3-secoaromadendrane-type sesquiterpenoid by different fatty acids, in addition to spathulenol [13].

The volatiles constituents of the *Plagiochila* genus have been poorly studied. To our knowledge, only four studies have been carried out on the chemical composition of the essential oils prepared from six *Plagiochila* species: *P. biffaria* [14,15], *P. maderensis*, *P. retrorsa*, *P. stricta* [14], *P. asplenioides* [16] and *P. ovalifolia* [17] (see Table 1 and

Figure 1). The bibliographic data highlighted a chemical variability according to each studied species. However, the richness in hydrocarbon monoterpenes and oxygenated sesquiterpenes is the common point of all reported varieties. Moreover, all the investigated species constituted a source of new compounds. In *P. bifaria*, three eudesmane type sesquiterpenes, eudesm-4-en-6-one, eudesm-4(15)-en-6-one, 7-hydroxyeudesm-4-en-6-one were isolated and identified as new natural products [15]. In *P. asplenioides*, one aromadendrane sesquiterpene, aromadendra-1(10),3-diene, two aromatic sesquiterpene hydrocarbons, bisabola-1,3,5,7(14)-tetraene and bisabola-1,3,5,7-tetraene, three sesquiterpene oxides, muurolan-4,7-peroxide, plagiochilines W and X were described for the first time [16].
<figure><img src="image_3.png" /><figcaption>Figure 1. Structures of main components reported from the essential oils of *Plagiochila* species. (Numbers refers to table 1).</figcaption></figure>

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<table><caption>**Table 1.** Main compounds of *P. biffaria*, *P. maderensis*, *P. retrorsa*, *P. stricta*, *P. aspenoides* and *P. ovalifolia* [14],[15], [16],[17].</caption><thead><tr><th>Species</th><th>Hydrocarbon terpenes</th><th>Oxygenated terpenes</th></tr></thead><tbody><tr><td>P. biffaria [14]</td><td></td><td>(-)-5R,7R,10S-eudesm-4(15)-en-6-one (9-19%, 1)</td></tr><tr><td></td><td></td><td>Methyl everninate (1-35%, 2)</td></tr><tr><td></td><td></td><td>peculiaroxide (13-16%, 3)</td></tr><tr><td>P. madernensis [14]</td><td>terpinolene (34-60%, 4)</td><td></td></tr><tr><td>P. retrorsa [14]</td><td>β-phellandrene (16-46%, 5)</td><td>peculiaroxide (9-12%, 3)</td></tr><tr><td>P. retrorsa [14]</td><td>allo-ocimene (15%, 6)</td><td>peculiaroxide (12%, 3)</td></tr><tr><td></td><td>neo-allo-ocimene (10%, 7)</td><td></td></tr><tr><td></td><td>terpinolene (13%, 4)</td><td></td></tr><tr><td>P. stricta [14]</td><td>allo-ocimene (7-19%, 7)</td><td>peculiaroxide (11-21%, 3)</td></tr><tr><td></td><td>bicyclogermacrene (4-17%, 8)</td><td>Spathulenol (2-14%, 9)</td></tr><tr><td></td><td>neo-allo-ocimene (4-11%, 7)</td><td></td></tr><tr><td>P. biffaria [15]</td><td></td><td>ent-eudesm-4-en-6-one (10), ent-eudesm-4(15)-en-6-one (1)</td></tr><tr><td></td><td></td><td>ent-7-hydroxyeudesm-4-en-6-one (11),</td></tr><tr><td>P. asplenioides [16]</td><td>(-)-selina-5,7(11)-diene (8%, 12)</td><td>plagio-4,7-peroxide (20%, 13)</td></tr><tr><td></td><td></td><td>maalian-5-ol (19%, 14)</td></tr><tr><td>P. ovalifolia [17]</td><td></td><td>ent-4β,10α 10α-dihydroxyaromadendrane (15)</td></tr><tr><td></td><td></td><td>Acetoxyisoplagiochilide (16)</td></tr><tr><td></td><td></td><td>Maalian-5-ol (13)</td></tr><tr><td></td><td></td><td>plagiochiline C (17)</td></tr><tr><td></td><td></td><td>Plagiochiline N (18)</td></tr></tbody></table>

The aim of the present work was to investigate the volatile metabolites of *P. porelloïdes* prepared by hydro-distillation (essential oil and hydrosol), hexane and diethyl oxide cold-extractions and microwave assisted extractions using a combination of techniques involving liquid Column Chromatography, GC/FID (using retention indices), GC-MS (EI), HRMS and NMR spectroscopy (¹H-, ¹³C- and 2D-NMR). As liverwort chemicals were generally very complex mixtures, our study deals firstly with the identification of components not recorded in MS-libraries; in addition, we are also reporting the isolation and structure determination of three unknown natural products showing a farnesane and humbertiane skeletons, respectively. Finally, the cytotoxicity and some anti-protozoal activities of the bulk extracts were investigated *in vitro*, to evaluate their potential pharmacological properties.

## 2. Results

In order to carry out a more exhaustive study of the volatile metabolites of *P. porelloïdes*, samples were prepared by multiple extraction procedures from dried plant material: essential oil and hydrosol were obtained by hydrodistillation, solvent extracts by cold maceration and assisted microwave 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 form the in-house library were considered as definitely identified 115 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. 116 117 118 119

Our study allowed identifying 50 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<sub>H</sub> 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*. 120 121 122 123

**2.1. Resolution of ambiguous identifications of sesquiterpenes** 124

Preparative liquid chromatography of *P. polleroïdes* essential oil was performed to obtained rich-sesquiterpene fractions. As GC-MS identification of sesquiterpenes in complex mixture can be a complex task [18], unambiguous identification of components 45, 48, 50, 51, 54 and 55 were definitively established using NMR-Extraction procedure [19]. 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 ¹³C-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 2). 125 126 127 128 129 130

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Structures of *P. porelloides* essential oil components identified by GC-MS and ensured by NMR. (Numbers refers to table 4). 131</figcaption></figure>

The compounds 23, 27 and 36 were concentrated in the hydrocarbon fraction of the essential oil and identified as aristolene 23, β-barbatene 27 and bicyclogermacrene 36 (Figure 2). 135 136

**2.2. Structural elucidations of new natural compounds** 138

Column chromatography of *P. porelloides* EXTO carried out using a gradient of polarity with hexane and diisopropyl oxide, was producing two fractions in which 58 (65%) was 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 C₁₅H₂₆O, (detected ion C₁₅H₂₆ONa⁺ (m/z)exp 245.1879 and (m/z)th 245.1876, error +1.2 ppm). The formula indicated a saturation degree of 3. The ¹H-NMR (CDCl₃, 300K) spectrum of 58 (Table 2,) showed the presence of five methyl groups δ 1.3, 1.6, 1.7, 1.8 (s, H₁₃, H₁₅, H₁₂ and H₁₄, respectively) and δ 0.9 (t, J= 7.44 Hz, H₁) and four olefinic protons δ 5.1 (t, J= 6.0 Hz, H₁₀), δ 5.6 (d, J=15.3 Hz, H₄), δ 5.9 (d, J= 10.8 Hz, H₆) and δ 6.5 (dd, J= 15.3, 10.8 Hz, H₅). The ¹³C-NMR spectrum contained 15 resonances: five methyls, three methylenes, four methines and three quaternary carbons. The occurrence of a quaternary carbon at 73.38 ppm suggested the presence of a tertiary alcohol. Spectral data were similar to those of nerolidol, which suggests a farnesane-type compound. The HSQC and HMBC correlations (Figure 3, (A)) and NOE correlations (Figure 3, (B)) indicated that the oxygenated carbon (C₃) was correlated with the methyl (C₁₃), the methylene group (C₂) and the methine (C₄). The signal between 5.5 and 6.6 ppm of ¹H-NMR spectrum showed coupling of methine protons (H₄, H₅ and H₆)(). This correlation indicated the presence of a conjugated ethylenic system. The signal of H₅ is observed as a doublet of doublets pattern due to its interaction with H₆ and H₄; the ³J 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153
corresponding coupling constant values are 10.8 and 15.3 Hz, respectively. The coupling constant ³JH₅,H₆, indicated the E-configuration of the diene fragment [20]. In addition, the coupling constant ³JH₄,H₅ (15.6 Hz) corresponds to an E-configuration of the C₄-C₅ double bond, confirmed by NOE correlation among H₅ and H₁₃. The configuration of the C₆-C₇ double bond, was established from NOE correlation between H₆ → H₈ and H₅ → H₁₄, respectively, suggesting an E configuration. Finally, the structure of compound 58 was determined as (3,7,11)-trimethyl-dodec-4E,6E,10-atrien-3-ol.

<caption>**Table 2.** Full NMR data of (3,7,11)-trimethyl-dodec-4E,6E,10-atrien-3-ol 58 (500MHz, 300K and CDCl₃)</caption>

(3,7,11)-trimethyl-dodec-4E,6E,10-atrien-3-ol 58

<table><thead><tr><th colspan="3">¹³C NMR</th><th colspan="3">¹H NMR</th><th colspan="3">¹H-¹H and ¹H-¹³C 2D correlations</th></tr><tr><th></th><th>δ (ppm)</th><th>Type</th><th>δ (ppm)</th><th>Mult</th><th>J (Hz)</th><th>HMBC*</th><th>NOESY</th><th>COSY</th></tr></thead><tbody><tr><td>1</td><td>8.37</td><td>CH₃</td><td>0.9</td><td>t</td><td>7.4</td><td>H₂</td><td>H₂</td><td>-</td></tr><tr><td>2</td><td>35.43</td><td>CH₂</td><td>1.6</td><td>dd</td><td>7.44 &amp; 1.6</td><td>H₁ H₁₃</td><td>H₁ H₁₃</td><td>-</td></tr><tr><td>3</td><td>73.38</td><td>C-O</td><td>-</td><td>-</td><td>-</td><td>H₁ H₂ H₄ H₅ H₁₃</td><td>-</td><td>-</td></tr><tr><td>4</td><td>137.80</td><td>CH=</td><td>5.6</td><td>d</td><td>15.3</td><td>H₂ H₆ H₁₃</td><td>H₆ H₁₃</td><td>H₆</td></tr><tr><td>5</td><td>123.99</td><td>CH=</td><td>6.5</td><td>dd</td><td>15.3 &amp; 10.8</td><td>H₆ H₁₃</td><td>H₁₃ H₁₄</td><td>H₁₄</td></tr><tr><td>6</td><td>124.13</td><td>CH=</td><td>5.9</td><td>d</td><td>10.8</td><td>H₄ H₅ H₈ H₁₄</td><td>H₈ / H₉</td><td>H₈ / H₉</td></tr><tr><td>7</td><td>138.75</td><td>C=</td><td>-</td><td>-</td><td>-</td><td>H₅ H₈ H₁₄</td><td>-</td><td>-</td></tr><tr><td>8</td><td>39.97</td><td>CH₂</td><td>2.1</td><td>m</td><td>-</td><td>H₉ H₁₄</td><td>H₆</td><td>-</td></tr><tr><td>9</td><td>26.62</td><td>CH₂</td><td>2.1</td><td>m</td><td>-</td><td>H₈</td><td>H₁₀ H₁₄ H₁₅</td><td>H₁₄ H₁₅</td></tr><tr><td>10</td><td>124.03</td><td>CH=</td><td>5.1</td><td>t</td><td>6.0</td><td>H₈ H₁₂ H₁₅</td><td>H₉ H₁₂ H₁₅</td><td>H₁₂</td></tr><tr><td>11</td><td>131.71</td><td>C=</td><td>-</td><td>-</td><td>-</td><td>H₁₂ H₁₅</td><td>-</td><td>-</td></tr><tr><td>12</td><td>25.71</td><td>CH₃</td><td>1.7</td><td>s</td><td>-</td><td>H₁₀ H₁₅</td><td>H₁₀</td><td>-</td></tr><tr><td>13</td><td>27.58</td><td>CH₃</td><td>1.3</td><td>s</td><td>-</td><td>H₄</td><td>H₁</td><td>-</td></tr><tr><td>14</td><td>16.75</td><td>CH₃</td><td>1.8</td><td>s</td><td>-</td><td>H₆</td><td>H₅</td><td>-</td></tr><tr><td>15</td><td>17.70</td><td>CH₃</td><td>1.6</td><td>s</td><td>-</td><td>H₁₀ H₁₂</td><td>H₁₀</td><td>-</td></tr></tbody></table>

*HMBC correlation are reported as C->H

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<figure><img src="image_5.png" /><figcaption>**Figure 3.** Long range ¹³C-¹H HMBC (A) and NOE (B) correlations observed in compound 58</figcaption></figure>

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<figure><img src="image_6.png" /><figcaption>**Figure 4.** H-H coupling pattern of the ethylenic system of 58</figcaption></figure>

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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 amounted for 70% of the FID-response. ¹³C-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 2-hydroxy isopropyl group, characteristic of tertiary alcohol [21]. 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 ¹³C-NMR spectra of **F27**. 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: 5 methyl groups, 3 methylenes, 4 methynes of which two ethylenics at δc 124.88 and 129.59 ppm and 3 quaternary carbons at δc 74.38, 137.34 and 134.16 ppm (Table 3). 2D HSQC, HMBC and COSY experiments confirmed the structure of p-menthane framework displaying a side chain composed of five carbon atoms, three of which have sp³ hybridization and two of sp² type, forming a double bond (). 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 ¹H and ¹³C 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 Δδ relative to the other (7.4 ppm and 6.3 ppm, respectively), suggesting steric γ-effects generated by double bond stereochemistry. As ¹H-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. Concerning **42a**, the sp² methine proton δH11 at 5.15 ppm showed a strong NOE connectivity to H13 and H14, while the H3 had a strong NOE cross peak to the allylic proton of the methyl group C15 at δc 16.21 ppm. This clearly indicated that the double bond in the side chain of **42a** have 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

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bond brought by **42b** side chain was found as Z stereochemistry; these was assigned according to correlations between H₃ → H₁₃, and H₁₁ → H₁₅, respectively ().

<table><caption>**Table 3.** Full NMR data of *p*-menth-1-en-3-[2-methylbut-1-enyl]-8-ol isomers 42a and 42b (in CDCl₃, at 500MHz and 300K)</caption><thead><tr><th rowspan="2"></th><th colspan="6">42a</th><th colspan="6">42b</th></tr><tr><th colspan="2">13C NMR</th><th colspan="2">1H NMR</th><th colspan="2">2D NMR</th><th colspan="2">13C NMR</th><th colspan="2">1H NMR</th><th colspan="2">2D NMR</th></tr><tr><th></th><th>δ (ppm)</th><th>DEPT</th><th>δ(ppm)</th><th>Mult (J, Hz)</th><th>HMBC*</th><th>NOESY</th><th>δ (ppm)</th><th>DEPT</th><th>δ(ppm)</th><th>Mult (J, Hz)</th><th>HMBC*</th><th>NOESY</th></tr></thead><tbody><tr><td>1</td><td>134.16</td><td>C</td><td>-</td><td>-</td><td>H7</td><td></td><td>134.29</td><td>C</td><td>-</td><td>-</td><td>H7</td><td></td></tr><tr><td>2</td><td>124.88</td><td>CH</td><td>4.95</td><td>d (1.26)</td><td>H7</td><td>H3 H7<br>H2 H5</td><td>125.19</td><td>CH</td><td>4.95 s</td><td>d (1.26)</td><td>H7</td><td>H3 H7</td></tr><tr><td>3</td><td>37.18</td><td>CH</td><td>3.08</td><td>m</td><td></td><td>H9 H11<br>H15</td><td>36.93</td><td>CH</td><td>3.08</td><td>m</td><td>H5</td><td></td></tr><tr><td>4</td><td>50.48</td><td>CH</td><td>1.5</td><td>m</td><td>H11 H2<br>H10 H9</td><td></td><td>H5 H6</td><td>50.54</td><td>CH</td><td>1.34</td><td>m</td><td>H9 H10 H5 H6</td></tr><tr><td>5</td><td>25.37</td><td>CH2</td><td>1.83/1.31</td><td>m</td><td>H3 H4<br>H6</td><td></td><td></td><td>25.29</td><td>CH2</td><td>1.83/1.31</td><td>m</td><td></td></tr><tr><td>6</td><td>30.7</td><td>CH2</td><td>2.03</td><td>m</td><td>H2 H7</td><td></td><td></td><td>30.65</td><td>CH2</td><td>1.96</td><td>m</td><td>H5 H7 H5</td></tr><tr><td>7</td><td>23.31</td><td>CH3</td><td>1.67</td><td>s</td><td>H2</td><td>H6</td><td></td><td>23.33</td><td>CH3</td><td>1.64</td><td>s</td><td>H7</td></tr><tr><td>8</td><td>74.38</td><td>C-OH</td><td>-</td><td>-</td><td>OH H10<br>H9</td><td></td><td>74.38</td><td>C-OH</td><td>-</td><td>-</td><td>H9 H10</td><td></td></tr><tr><td>9</td><td>25.78</td><td>CH3</td><td>1.17</td><td>s</td><td>H10</td><td>H3</td><td>25.71</td><td>CH3</td><td>1.17</td><td>s</td><td>H10</td><td>H3</td></tr><tr><td>10</td><td>28.93</td><td>CH3</td><td>1.24</td><td>s</td><td>OH H9</td><td>H4 H5<br>H4 H6</td><td>28.99</td><td>CH3</td><td>1.23</td><td>s</td><td>H8 H9</td><td>H4 H5</td></tr><tr><td>11</td><td>129.59</td><td>CH</td><td>5.15</td><td>dd (10.1<br>and 1.26)</td><td>H13 H15</td><td>H13 H14<br>OH</td><td>130.69</td><td>CH</td><td>5.11</td><td>d(10.1)</td><td>H13 H15</td><td>H15</td></tr><tr><td>12</td><td>137.34</td><td>C</td><td>-</td><td>-</td><td>H13 H15<br>H14</td><td></td><td>137.4</td><td>C</td><td>-</td><td>-</td><td>H13 H14<br>H15</td><td></td></tr><tr><td>13</td><td>32.5</td><td>CH2</td><td>2.03</td><td>q (7.32)</td><td>H11 H14<br>H15</td><td>H14 H15</td><td>25.11</td><td>CH2</td><td>2.19/2.09</td><td>m</td><td>H14 H15</td><td>H3 H14</td></tr><tr><td>14</td><td>12.55</td><td>CH3</td><td>1.02</td><td>t (7.32)</td><td>H13</td><td>H13</td><td>12.85</td><td>CH3</td><td>1.06</td><td>t (7.23)</td><td>H13</td><td>H13</td></tr><tr><td>15</td><td>16.21</td><td>CH3</td><td>1.73</td><td>d (0.88)</td><td>H11 H13</td><td>H3 H9<br>H13 H14</td><td>22.98</td><td>CH3</td><td>1.74</td><td>d (1.47)</td><td>H11 H13</td><td>H3 H9<br>H13 H14</td></tr><tr><td></td><td></td><td>OH</td><td>2.6</td><td>OH</td><td></td><td></td><td></td><td>OH</td><td>2.6</td><td>OH</td><td></td><td></td></tr></tbody></table>

<footnote>* HMBC correlations are reported as C->H</footnote>
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Structure of **42a** and schematic representation of HMBC correlations</figcaption></figure>

202

203

204

<figure><img src="image_8.png" /><figcaption>**Figure 6.** Zoom of NOESY spectrum showing specific correlations used to resolve stereochemistry of the acyclic double bond in **42a**, **b**</figcaption></figure>

205

206

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 [22]. In addition, two analogous structures were isolated after fungal transformation of α-farnesene while the NMR data (¹H and ¹³C) reported in the literature [23], differed significantly from the experimental data described here for both new sesquiterpene alcohols.

207

208

209

210

211

**2.3.** *P. porelloides* volatile components: chemical compositions of specific plant extracts

212

The chemical compositions of essential oil (EO), hydrosol extract (HY), both hexane and diethyle oxide extracts (EXT<sub>H</sub> and EXT<sub>O</sub>), volatile fraction (VF) as well as microwave extract (MW) were investigated by GC/RI, GC-MS and ¹³C-NMR (Table 4).

213

214

215

216
<table><caption>**Table 4.** Chemical compositions of *P. porelooides* samples from Corsica</caption><thead><tr><th rowspan="2">No<sup>1</sup></th><th rowspan="2">Compounds</th><th rowspan="2">L RI<sub>A</sub><sup>2</sup></th><th rowspan="2">RI<sub>A</sub><sup>3</sup></th><th rowspan="2">RI<sub>P</sub><sup>4</sup></th><th rowspan="2">EO</th><th rowspan="2">HY</th><th colspan="2">EXT</th><th rowspan="2">MW</th><th rowspan="2">VF</th><th rowspan="2"></th></tr><tr><th>EXT<sub>H</sub></th><th>EXT<sub>O</sub></th></tr></thead><tbody><tr><td>1</td><td>hexanal</td><td>770</td><td>771</td><td>819</td><td>t</td><td>-</td><td>t</td><td>t</td><td>-</td><td>t</td><td>IR, MS</td></tr><tr><td>2</td><td>heptanal</td><td>876</td><td>876</td><td>1079</td><td>t</td><td>-</td><td>t</td><td>t</td><td>-</td><td>t</td><td>IR, MS</td></tr><tr><td>3</td><td>α-pinene</td><td>931</td><td>931</td><td>1032</td><td>t</td><td>-</td><td>t</td><td>t</td><td>-</td><td>1.0</td><td>IR, MS</td></tr><tr><td>4</td><td>camphene</td><td>943</td><td>944</td><td>1066</td><td>t</td><td>-</td><td>t</td><td>t</td><td>-</td><td>0.1</td><td>IR, MS</td></tr><tr><td>5</td><td>6-methylhept-5-en-2-one</td><td>963</td><td>954</td><td>1343</td><td>0.1</td><td>-</td><td>t</td><td>-</td><td>-</td><td>0.1</td><td>IR, MS</td></tr><tr><td>6</td><td>oct-1-en-3-ol</td><td>959</td><td>962</td><td>1453</td><td>0.4</td><td>1.9</td><td>-</td><td>t</td><td>-</td><td>t</td><td>IR, MS</td></tr><tr><td>7</td><td>octen-3-one</td><td>963</td><td>980</td><td>1260</td><td>t</td><td>-</td><td>-</td><td>t</td><td>-</td><td>0.1</td><td>IR, MS</td></tr><tr><td>8</td><td>octan-3-ol</td><td>982</td><td>982</td><td>1401</td><td>t</td><td>-</td><td>-</td><td>t</td><td>-</td><td>-</td><td>IR, MS</td></tr><tr><td>9</td><td>phenylaldehyde</td><td>1013</td><td>1010</td><td>1616</td><td>t</td><td>-</td><td>-</td><td>-</td><td>-</td><td>t</td><td>IR, MS</td></tr><tr><td>10</td><td>β-phellandrene</td><td>1021</td><td>1021</td><td>1219</td><td>t</td><td>-</td><td>-</td><td>0.1</td><td>-</td><td>0.4</td><td>IR, MS</td></tr><tr><td>11</td><td>nonanal</td><td>1083</td><td>1083</td><td>1396</td><td>0.1</td><td>-</td><td>t</td><td>-</td><td>-</td><td>-</td><td>IR, MS</td></tr><tr><td>12</td><td>octen-3-yle acetate</td><td>1094</td><td>1094</td><td>1377</td><td>0.1</td><td>-</td><td>t</td><td>0.6</td><td>0.2</td><td>2.4</td><td>RI, MS, Ref</td></tr><tr><td>13</td><td>decanal</td><td>1186</td><td>1185</td><td>1496</td><td>t</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>RI, MS, Ref</td></tr><tr><td>14</td><td>bicycloelemene</td><td>1334</td><td>1334</td><td>1552</td><td>0.3</td><td>-</td><td>0.9</td><td>1.1</td><td>0.4</td><td>2.0</td><td>RI, MS, Ref</td></tr><tr><td>15</td><td>maali-1,3-diene</td><td>1347</td><td>1346</td><td>1532</td><td>0.8</td><td>-</td><td>0.5</td><td>0.6</td><td>-</td><td>1.5</td><td>RI, MS, Ref</td></tr><tr><td>16</td><td>anastrepene</td><td>1373</td><td>1369</td><td>-</td><td>0.2</td><td>-</td><td>0.4</td><td>1.2</td><td>0.8</td><td>1.4</td><td>RI, MS, Ref</td></tr><tr><td>17</td><td>isoledene</td><td>1372</td><td>1373</td><td>-</td><td>0.1</td><td>-</td><td>0.1</td><td>0.2</td><td>-</td><td>0.2</td><td>IR, MS</td></tr><tr><td>18</td><td>α-copaene</td><td>1379</td><td>1376</td><td>1498</td><td>0.2</td><td>-</td><td>0.4</td><td>0.4</td><td>-</td><td>1.7</td><td>IR, MS</td></tr><tr><td>19</td><td>β-elemene</td><td>1388</td><td>1388</td><td>1592</td><td>0.2</td><td>-</td><td>0.4</td><td>0.6</td><td>-</td><td>1.4</td><td>IR, MS</td></tr><tr><td>20</td><td>african-3-ene</td><td>1391</td><td>1390</td><td>-</td><td>-</td><td>-</td><td>0.0</td><td>0.3</td><td>-</td><td>1.1</td><td>RI, MS, Ref</td></tr><tr><td>21</td><td>α-barbatene</td><td>1414</td><td>1409</td><td>1565</td><td>1.8</td><td>-</td><td>1.3</td><td>2.5</td><td>0.4</td><td>4.7</td><td>RI, MS, Ref</td></tr><tr><td>22</td><td>tritomarene</td><td>1416</td><td>1413</td><td>1410</td><td>0.2</td><td>-</td><td>-</td><td>0.5</td><td>-</td><td>1.1</td><td>RI, MS, Ref</td></tr><tr><td>23</td><td>aristolene</td><td>1420</td><td>1419</td><td>1581</td><td>2.6</td><td>-</td><td>5.0</td><td>1.5</td><td>4.1</td><td>7.3</td><td>RI, MS, NMR</td></tr><tr><td>24</td><td>γ-maaliene</td><td>1428</td><td>1425</td><td>1613</td><td>1.2</td><td>-</td><td>2.9</td><td>0.2</td><td>3.0</td><td>0.5</td><td>RI, MS, Ref</td></tr><tr><td>25</td><td>calarene</td><td>1439</td><td>1438</td><td>1603</td><td>0.6</td><td>-</td><td>0.5</td><td>0.6</td><td>0.5</td><td>1.0</td><td>IR, MS</td></tr><tr><td>26</td><td>α-maaliene</td><td>1440</td><td>1440</td><td>-</td><td>0.5</td><td>-</td><td>t</td><td>0.6</td><td>0.8</td><td>-</td><td>RI, MS, Ref</td></tr><tr><td>27</td><td>β-barbatene</td><td>1445</td><td>1445</td><td>1663</td><td>28.7</td><td>-</td><td>23.1</td><td>19.5</td><td>32.6</td><td>39.5</td><td>RI, MS, NMR</td></tr><tr><td>28</td><td>aromadendrene</td><td>1447</td><td>1458</td><td>1601</td><td>0.3</td><td>-</td><td>0.4</td><td>0.5</td><td>-</td><td>0.6</td><td>RI, MS, Ref</td></tr><tr><td>29</td><td>α-acoradiene</td><td>1464</td><td>1460</td><td>-</td><td>0.6</td><td>-</td><td>0.2</td><td>0.3</td><td>-</td><td>0.9</td><td>RI, MS, Ref</td></tr><tr><td>30</td><td>β-acoradiene</td><td>1465</td><td>1462</td><td>-</td><td>0.1</td><td>-</td><td>0.4</td><td>0.2</td><td>-</td><td>0.4</td><td>RI, MS, Ref</td></tr><tr><td>31</td><td>γ-curcumene</td><td>1475</td><td>1471</td><td>-</td><td>0.1</td><td>-</td><td>0.7</td><td>0.5</td><td>-</td><td>0.4</td><td>RI, MS, Ref</td></tr><tr><td>32</td><td>β-chamigrene</td><td>1478</td><td>1473</td><td>-</td><td>1.3</td><td>-</td><td>0.5</td><td>0.8</td><td>-</td><td>1.5</td><td>RI, MS, Ref</td></tr></tbody></table>
<table><tbody><tr><td>33</td><td>α-curcumene</td><td>1473</td><td>1481</td><td>1738</td><td>0.1</td><td>-</td><td>0.1</td><td>0.4</td><td>-</td><td>0.7</td><td>RI, MS, Ref</td></tr><tr><td>34</td><td>β-selinene</td><td>1483</td><td>1485</td><td>-</td><td>1.1</td><td>-</td><td>0.0</td><td>0.1</td><td>-</td><td>-</td><td>RI, MS, Ref</td></tr><tr><td>35</td><td>β-maaliene</td><td>1480</td><td>1481</td><td>-</td><td>0.1</td><td>-</td><td>0.1</td><td>0.3</td><td>-</td><td>-</td><td>RI, MS, Ref</td></tr><tr><td>36</td><td>bicyclogermacrene</td><td>1494</td><td>1494</td><td>1744</td><td>8.2</td><td>-</td><td>16.4</td><td>14.1</td><td>17.8</td><td>14.2</td><td>RI, MS, NMR</td></tr><tr><td>37</td><td>204[M]+; 107(100); 93(90)</td><td>1498</td><td>1497</td><td>-</td><td>1.5</td><td>-</td><td>0.4</td><td>0.9</td><td>-</td><td>1.0</td><td></td></tr><tr><td>38</td><td>ledene</td><td>1494</td><td>1498</td><td>1706</td><td>t</td><td>-</td><td>-</td><td>0.9</td><td>-</td><td>0.4</td><td>RI, MS</td></tr><tr><td>39</td><td>α-chamigrene</td><td>1503</td><td>1500</td><td>-</td><td>t</td><td>-</td><td>-</td><td>0.4</td><td>-</td><td>0.4</td><td>RI, MS, Ref</td></tr><tr><td>40</td><td>γ-cadinene</td><td>1507</td><td>1514</td><td>1775</td><td>t</td><td>-</td><td>-</td><td>0.6</td><td>-</td><td>0.7</td><td>RI, MS</td></tr><tr><td>41</td><td>α-alaskene</td><td>1512</td><td>1513</td><td>-</td><td>t</td><td>-</td><td>-</td><td>0.5</td><td>-</td><td>0.4</td><td>RI, MS, Ref</td></tr><tr><td>42a</td><td>p-menth-1-en-3-[2-methyl-1E-butenyl]-8-ol</td><td>-</td><td>1536</td><td>1917</td><td>1.2</td><td>3.4</td><td>0.3</td><td>1.2</td><td>-</td><td>-</td><td>RI, MS, NMR</td></tr><tr><td>42b</td><td>p-menth-1-en-3-[2-methyl-1Z-butenyl]-8-ol</td><td>-</td><td>1536</td><td>1917</td><td>3.8</td><td>-</td><td>0.6</td><td>-</td><td>-</td><td>-</td><td>RI, MS, NMR</td></tr><tr><td>43</td><td>tamariscol</td><td>1535</td><td>1536</td><td>1917</td><td>1.2</td><td>-</td><td>0.6</td><td>1.1</td><td>-</td><td>-</td><td>RI, MS</td></tr><tr><td>44</td><td>204[M]+; 161(100); 91(56)</td><td>-</td><td>1532</td><td>1917</td><td>3.7</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>45</td><td>4-epi-maaliol</td><td>-</td><td>1544</td><td>-</td><td>0.7</td><td>-</td><td>0.6</td><td>0.3</td><td>-</td><td>-</td><td>RI, MS, NMR</td></tr><tr><td>46</td><td>222[M]+; 107(100); 135(51)</td><td>-</td><td>1545</td><td>1832</td><td>0.9</td><td>4.7</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>47</td><td>pallustrol</td><td>1567</td><td>1558</td><td>1923</td><td>t</td><td>-</td><td>-</td><td>t</td><td>-</td><td>-</td><td>RI, MS, Ref</td></tr><tr><td>48</td><td>spathulenol</td><td>1557</td><td>1561</td><td>2090</td><td>0.7</td><td>9.3</td><td>1.1</td><td>0.6</td><td>3.5</td><td>-</td><td>RI, MS, NMR</td></tr><tr><td>49</td><td>204[M]+; 107(100); 135(56)</td><td>-</td><td>1567</td><td>-</td><td>0.9</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>50</td><td>globulol</td><td>1571</td><td>1578</td><td>2077</td><td>4.4</td><td>17.2</td><td>1.9</td><td>1.3</td><td>0.2</td><td>0.9</td><td>RI, MS, NMR</td></tr><tr><td>51</td><td>viridiflorol</td><td>1591</td><td>1585</td><td>2085</td><td>5.8</td><td>4.2</td><td>1.9</td><td>-</td><td>-</td><td>0.3</td><td>RI, MS, NMR</td></tr><tr><td>52</td><td>222[M]+; 107(100)</td><td>-</td><td>1588</td><td>-</td><td>1.2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>53</td><td>238[M]+; 149(100)</td><td>-</td><td>1591</td><td>1920</td><td>5.21</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>54</td><td>rosifoliol</td><td>1599</td><td>1587</td><td>2108</td><td>3.8</td><td>7.3</td><td>5.9</td><td>6.1</td><td>0.4</td><td>-</td><td>RI, MS, NMR</td></tr><tr><td>55</td><td>maalian-5-ol</td><td>1607</td><td>1595</td><td>2051</td><td>5.4</td><td>9.4</td><td>2.2</td><td>1.9</td><td>7.6</td><td>1.2</td><td>RI, MS, NMR</td></tr><tr><td>56</td><td>204[M]+; 107(100); 135(76)</td><td>-</td><td>1611</td><td>1938</td><td>1.4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>57</td><td>222; 107(100); 105(75)</td><td>-</td><td>1613</td><td>-</td><td>1.3</td><td>4.2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>58</td><td>(3,7,11)-trimethyl-dodec-4E,6E,10-atrien-3-ol</td><td>-</td><td>1616</td><td>2136</td><td>-</td><td>-</td><td>13.3</td><td>15.7</td><td>17.2</td><td>-</td><td>RI, MS, NMR</td></tr><tr><td colspan="12">Total identified</td><td>76.9</td><td>52.6</td><td>82.6</td><td>77.9</td><td>89.4</td><td>90.3</td></tr><tr><td colspan="12">Classes of compounds (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="12">Hydrocarbon compounds</td><td>49.3</td><td>-</td><td>54.4</td><td>49.2</td><td>60.4</td><td>85.4</td></tr><tr><td colspan="12">Oxygenated compounds</td><td>27.5</td><td>72.3</td><td>28.1</td><td>28.7</td><td>29.1</td><td>4.9</td></tr><tr><td colspan="12">Monoterpene hydrocarbons</td><td>-</td><td>-</td><td>-</td><td>0.1</td><td>-</td><td>1.5</td></tr><tr><td colspan="12">Monoterpene oxygenated</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td colspan="12">Sesquiterpene hydrocarbons</td><td>49.3</td><td>-</td><td>54.4</td><td>49.1</td><td>60.4</td><td>83.8</td></tr><tr><td colspan="12">Sesquiterpene oxygenated</td><td>26.9</td><td>70.5</td><td>28.1</td><td>28.1</td><td>28.8</td><td>2.4</td></tr><tr><td colspan="12">Other</td><td>0.6</td><td>1.9</td><td>-</td><td>0.6</td><td>0.2</td><td>2.5</td></tr></tbody></table>
It has to be mentioned that earlier studies focused on different liverworts reported also an antiprotozoal activity, notably for alpha-eudesmol [29] and marchantin A [30]

<table><caption>**Table 5.** The cytotoxicity (W138 and J774) and *in-vitro* activity of *P. porelloides* EO and EXTo against *Leishmania mexicana* (Lmm) and *Trypanosoma brucei* brucei (Tbb).</caption><thead><tr><th rowspan="3">Sample</th><th colspan="3">Cytotoxicity</th><th colspan="3">Antiprotozoal assay</th><th colspan="3">Selectivity indices</th></tr><tr><th colspan="3">IC<sub>50</sub> ± SD in µg/mL (µM for Pure Compound)</th><th colspan="3">SI = IC<sub>50</sub> (W138)/IC<sub>50</sub> (J774 or parasite)</th><th colspan="3">SI = IC<sub>50</sub> (W138)/IC<sub>50</sub> (J774 or parasite)</th></tr><tr><th>WI38</th><th>J774</th><th>Lmm</th><th>Tbb</th><th>J774</th><th>Lmm</th><th>Tbb</th><th>J774</th><th>Lmm</th><th>Tbb</th></tr></thead><tbody><tr><td>EO</td><td>23.85±4.39</td><td>28.81±0.45</td><td>15.99±0.85</td><td>2.03±0.12</td><td>0.8</td><td>1.5</td><td>11.7</td><td>0.8</td><td>1.5</td><td>11.7</td></tr><tr><td>EXTo</td><td>2.96±0.17</td><td>1.25±0.08</td><td>17.73±1.14</td><td>5.18±0.81</td><td>2.4</td><td>0.2</td><td>0.6</td><td>2.4</td><td>0.2</td><td>0.6</td></tr><tr><td>Camptothecin</td><td>0.031±0.002</td><td>0.01±0.001</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pentamidine</td><td></td><td></td><td>0.07±0.004</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Suramine</td><td></td><td></td><td></td><td>0.03±0.004</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

WI38: non cancer human fibroblasts; J774: cancerous macrophage-like murine cells; Tbb: Trypanosoma brucei brucei (bloodstream forms); Lmm: Leishmania mexicana promastigotes; Selectivity index calculated for antiparasitic activities compared to WI38 cytotoxicity.

## 3. Materials and Method

### 3.1. 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 [31] and voucher specimens were deposited in the herbarium of University of Corsica, Corte (France).

### 3.2. Essential oil and hydrosol isolation

After 15 day 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* [32]. Hydrosol (300 mL) obtained by HD was submitted to Liquid/liquid Extraction (LLE) in order to obtain an liquid extract (HY, 57.3 mg). LLE was performed three times using 50 mL of diethyle oxide.

The essential-oil yield (0.2 %) were expressed in % (w/dw) based on the weight of the dried plant material.

### 3.3. SPME experiments

Volatile fraction (VF) emitted by the plant sample were extracted with HS-SPME method. Bryophytes samples (1 g) were crushed and disposed into 20 mL headspace vials. The vial was 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 vial for 60 min. Later, the volatiles sampled by the solid-phase fiber was analysed after desorption into the gas chromatography-mass spectrometry (GC-MS) injection port (5 min) in splitless mode. Each sample was done in triplicates.
**3.4. Solvent extractions** 289

Dried plant materials (200 g) were mechanically powdered and extracted with hexane and diethyl oxide at room temperature for 24 hours each in order to give after filtration and concentration in vacuum, both extract called EXTH (400 mg) and EXTO (640 mg), respectively.

**3.5. Microwave-assisted extractions** 293

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. Solvent was then filtered on activated carbon and concentrated under vacuum. Resulting extract was next taken up in absolute ethanol and centrifuged (20 min at 6000 rpm) and the supernatant was collected and concentrated to finally obtain the MAE extract.

**3.6. Essential oil (EO) and diethyl oxide extract (EXTO) fractionations** 299

Essential oil (EO, 800 mg) and diethyle oxide extract (EXTO, 640 mg) of *P. Porelloides* was submitted to column chromatography (CC) on a silica-gel column (200-500 µm, 12 g, Clarisep® Bonna Agela Technologies, Willington, USA) with Combi Flash apparatus (Teledyne ISCO, Lincoln, USA) equipped with a fraction collector monitored by an UV detector. Using gradients of (v/v) hexane/diisopropyl oxide (HEX/DIPO), forty-seven fractions (2 hydrocarbon fractions and 45 oxygenated fractions) were eluted from EO and two (one hydrocarbon fraction and one oxygenated fraction) were eluted from EXTO.

**3.7. GC-FID Conditions** 306

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 rising 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 H₂ (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*<sub>R</sub>) of a series of n-alkanes (C5–C30; commercial solution obtained from Restek, Bellefonte, PA, USA) using the Van den Dool and Kraqtz equation [33].

**3.8. GC-MS Analysis** 317

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.

## 3.9. 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 (N₂) flow of 100 L.h⁻¹ heated at 35 °C. Accurate mass experiments were performed using reference ions from CH₃COONa 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.

## 3.10. 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 EXT₀. NMR experiments were acquired in CDCl₃ (EuroIsotop, Saint Aubin, France), at 300K using a Bruker Avance DRX 500 NMR spectrometer (Karlsruhe, Germany) operating at 500.13 MHz for ¹H and 125.77 MHz for ¹³C Larmor frequency with a double resonance broadband fluorine observe (BBFO) 5 mm probe head. ¹³C-NMR experiments were recorded using one-pulse excitation pulse sequence (90° excitation pulse) with ¹H decoupling during signal acquisition (performed with WALTZ-16); the relaxation delay has been set at 2 s. For each analyzed sample, depending on the compound concentration, 3k up to 5k free induction decays (FID) 64k complex data points were collected using a spectral width of 30000 Hz (240 ppm). Chemical shifts (δ in ppm) were reported relative to residual signal of CDCl₃ (δc 77.04 ppm). Complete ¹H and ¹³C assignments of the new compound were obtained using 2D gradient-selected NMR experiments, ¹H-¹H COSY (COrelation SpectroscopY), ¹H-¹³C HSQC (Heteronuclear Single Quantum Correlation), ¹H-¹³C HMBC (Heteronuclear Multiple Bond Coherence) and ¹H-¹H NOESY (Nuclear Overhauser Effect SpectroscopY), for which conventional acquisition parameters were used, as described in the literature [34].

## 3.11. Identification of components

Identification of individual components in essential oil, extracts or CC-fractions was based on a methodology involving integrated techniques, such as GC retention indices, GC-MS (EI) and NMR. Identification of volatiles sampled by SPME were carried only by GC retention indices and GC-MS (EI). The identification of individual components was based i) on the comparison of the retention indices (RIs) determined on the polar and nonpolar columns with those of authentic compounds or literature data [24,35] ii) on computer matching of the mass spectra with commercial MS-libraries and the mass spectra with those listed in our homemade MS-library built of mass spectra of authentic compounds or literature data [36,37] iii) comparing the ¹³C-NMR chemical shifts of CC-fraction components with those of reference spectra reported in the literature iv) NMR assignments using 1D and 2D data.

(3,7,11)-trimethyl-dodec-4E,6E,10-atrien-3-ol 58: Yellow oil (20 mg); RI apolar 1616, RI polar 2136; for ¹H and ¹³C NMR data (see Table 2); MS (EI; 70 eV) m/z (rel. Int): 222 [M⁺] (4), 107 (100), 41 (80), 135 (73), 69 (62), 91 (53), 93
(53), 105 (49), 43 (46), 79 (40), 204 (30), 119 (30), 161 (29), 77 (28), 55 (26). HRMS: detected ion m/z 245.1879 [MNa⁺] 360
(calc. for C₁₅H₂₆ONa⁺, error: +1.2ppm). 361

*p*-menth-1-en-3-[2-methylbut-1-enyl]-8-ol **42a,b**: Colorless oil (7mg); RI apolar 1528, RI polar 1917; for ¹H and ¹³C NMR data (see Table 3); MS (EI; 70 eV) m/z (rel. Int): 222 [M⁺] (1), 107 (100), 59 (31), 161 (31), 91 (31), 41 (28), 175 (26), 105 (25), 135 (23), 79 (23), 119 (22), 93 (21), 108 (19), 43 (16), 77 (16). HRMS: detected ion m/z 245.1878 [MNa⁺] (calc. for C₁₅H₂₆ONa⁺, error: +0.8ppm). 362
363
364
365

**3.12. Component Quantification** 366

The quantification of components was performed using the methodology reported by Bicchi [38] and adapted in our laboratory [39]. 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). 367
368
369
370

**3.13. Parasites, cells and media** 371

*Trypanosoma brucei brucei* (strain427) bloodstream forms were cultured in vitro in HMI9 medium containing 10% heat-inactivated foetal bovine serum [40]. *Leishmania mexicana mexicana* promastigotes (MHOM/BZ/84/BEL46) were cultivated in vitro in a semi-defined medium (SDM-79) [41] supplemented with 15% heat-inactivated foetal bovine serum. The human normal fibroblast cell line, WI-38, was cultivated in vitro in DMEM medium containing 4 mM L-glutamine, 1 mM sodium pyruvate supplemented with 10% heat-inactivated foetal bovine serum and penicillin-streptomycin (100 UI/ml to 100 µg/ml). All cells were incubated in a humidified atmosphere with 5% CO₂ at 37 °C except the *Leishmania promastigotes* which were incubated at 28 °C. 372
373
374
375
376
377
378

**3.14. In vitro test for antitrypanosomal and antileishmanial activity** 379

The *in vitro* test was performed as described previously [42] 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 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 performed in duplicate. 380
381
382
383
384
385
386

**3.15. Cytotoxicity assay** 387

The cytotoxicity of the essential oil and extracts on WI-38 and J774 cells was evaluated as previously described from the same stock solutions [43]. 388
389
390

**Supplementary Materials:** 391

**Author Contributions:** Conceptualization, A.P., A.T. and A.M.; methodology, A.P., A.T. and A.M.; formal analysis, A.P.; investigation, A.P. and J.Q.L.; resources, A.P.; writing—original draft preparation, A.P., A.T. and A.M.; 392
393
writing—review and editing, A.P., A.T. J.C. and A.M; project administration, A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:**

**Conflicts of Interest:** The authors declare no conflict of interest.

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