# Targeted isolation of new antibacterial sesquiterpene coumarins from ammoniacum (Ferula communis L.)

Capucine Braillon, Elora Aubert, Régine Janet-Bintz, Megi Gjikolaj, Rania Hamidi, Léa Blondé, Jean Peluso, Pascal Villa, Véronique Pitchon, Pierre Fechter, et al.

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## To cite this version:

Capucine Braillon, Elora Aubert, Régine Janet-Bintz, Megi Gjikolaj, Rania Hamidi, et al. Targeted isolation of new antibacterial sesquiterpene coumarins from ammoniacum (Ferula communis L.). Fitoterapia, 2025, 186, pp.106833. 10.1016/j.fitote.2025.106833. hal-05390062

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Submitted on 1 Dec 2025

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Targeted isolation of new antibacterial sesquiterpene coumarins from ammoniacum (Ferula communis L.)

Capucine Braillon¹,², Elora Aubert¹,², Régine Janet-Bintz², Megi Gjikolaj¹, Rania Hamidi¹, Léa Blondé³, Jean Peluso³, Pascal Villa⁴, Véronique Pitchon⁵, Pierre Fechter², Catherine Vonthron-Sénécheau¹, Sergio Ortiz¹*.

1 UMR 7200 Laboratoire d'Innovation Thérapeutique (LIT), CNRS, Strasbourg Drug Discovery and Development Institute (IMS), Université de Strasbourg, 67400 Illkirch-Graffenstaden, France

2 UMR 7242 Biotechnologie et Signalisation Cellulaire (BSC), CNRS Université de Strasbourg, 67400 Illkirch-Graffenstaden, France

3 Plateforme de Bio-essais pharmacologiques et pharmacocinétiques en cytomique (eBioCyt), UPS 1401, Université de Strasbourg, 67400 Illkirch-Graffenstaden, France

4 PCBIS Plateforme de chimie biologie intégrative de Strasbourg, UAR 3286 CNRS/Université de Strasbourg, F-67000 Strasbourg, France

5 UMR 7044, Archéologie et Histoire Ancienne : Méditerranée-Europe (ARCHIMEDE), CNRS, Université de Strasbourg, 67000 Strasbourg, France
**Abstract**

The use of plant-based remedies in traditional medicine to combat bacterial infections has been scientifically studied. As part of an ongoing research for the discovery of new therapeutic options to treat bacterial infections, Ammoniacum gum resin from *Ferula communis* L. roots was explored. Ammoniacum appeared in many traditional formulations of Arab, Indian and Western medicines and its use has been reported throughout the centuries. This present study aimed to further explore this gum-resin using a molecular-networking-guided method for the accelerated discovery of new antibacterial compounds. HPLC-PDA-HRMS/MS molecular-networking-based dereplication strategy highlighted the presence of known sesquiterpene coumarins among potential new derivatives. By comparison of MS/MS fragmentation spectra between known and unknown compounds, a hydroxycinnamic sesquiterpene coumarin structure was predicted as a potential new compound. Mass-guided isolation followed by structural elucidation allowed us to corroborate the predicted structure of this new derivative identified as o-coumaric-ω-hydroxyferulenol (10). The new derivative showed MIC90 values of 1.84-6.17 µM against cutaneous Gram-positive bacteria with a bactericidal effect measured by MBC/MCI ratio evaluation and flow cytometry methodology. Furthermore, the new compound showed no *in vitro* cytotoxicity against eukaryotic cells. This study confirms the interest to analyse with modern techniques ancient traditional remedies in the exploration of new bioactive compounds with therapeutical potential.

**Key words:**

Molecular Networking; sesquiterpene coumarins; *Ferula*; targeted isolation; bactericidal activity

**Graphical abstract:**

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# 1. Introduction

The isolation of novel bioactive compounds from medicinal plants remains a significant challenge in natural product research. Despite the remarkable chemical diversity of plant metabolites, researchers frequently encounter the isolation of known compounds, limiting the identification of new bioactive structures. The application of advanced dereplication techniques to identify known compounds allow to prioritize the fractionation and purification of extracts bearing potential new structures. Moreover, the measure of fragmentation spectra at high resolution allows the determination of characteristic fragmentation patterns of unknown compounds, predicting the presence of chemical features in their structures [1]. The fragmentation patterns can be analysed by molecular network approach, organizing the ionized compounds in clusters by spectral fragmentation similarity. Furthermore, adding bioactivity information to a network (multi-informative molecular network), obtained for a set of fractions from an active extract, can highlight the presence of new bioactive compounds and target their isolation.

The escalating prevalence of antibiotic-resistant pathogens has significantly compromised the efficacy of existing treatments for dermal infections. Notably, methicillin-resistant *Staphylococcus aureus* (MRSA) has emerged as a predominant Gram-positive pathogen in skin infections, exhibiting high resistance rates to commonly used antibiotics such as penicillin, clindamycin, and erythromycin [2]. These developments underscore the urgent need for novel antimicrobial agents with enhanced efficacy and safety profiles to ensure effective management of skin-related bacterial infections.

Ammoniacum gum-resin is a white/yellowish viscous exudate from *Ferula communis* L. (Apiaceae) roots scarification founded principally in the Mediterranean area. The resin has been used by local population as expectorant, antispasmodic and antimicrobial ingredient in several medicinal preparations. Phytochemical studies have indicated that the chemical constituents of ammoniacum gum-resin primarily consist of monoterpenes, sesquiterpenes, furanocoumarines and sesquiterpene coumarins. Pharmacological research has demonstrated that ammoniacum gum-resin presents important antibacterial, free radical scavenging, cytotoxicity, anticonvulsant activities [3–5].

As part of an ongoing search for new bioactive compounds from Medieval Arabic Pharmacopeia [6,7], a phytochemical and bioactivity study was conducted on *F. communis* L. gum-resin extract. In this framework, a multi-informative molecular network strategy was carried out to targeting new antimicrobial compounds. This study reports the targeted isolation of a new sesquiterpene coumarin (SC) with significant and selective antibacterial activity against Gram-positive cutaneous bacteria. Moreover, its antibacterial activity was characterized by measuring its minimal bactericide concentration and by flow cytometry methodology.

# 2. Results and discussion

To support the construction of a multi-informative molecular network of ammoniacum and guide the targeted isolation of novel bioactive constituents, extraction procedures were performed prior to chemical analyses and fractionation steps. Two successive maceration extraction procedures were performed, first in ethyl acetate and then in methanol. The ethyl acetate extract demonstrated *in vitro* antibacterial activity specifically against Gram-positive bacteria, with minimum inhibitory concentrations ranging from 0.98 to 5.72 µg/mL **(Table 1)**. Furthermore, the extracts were evaluated against two erythromycin-resistant strains available in our laboratory, with the objective of assessing the relevance of pursuing antibacterial investigations against multidrug-resistant bacteria. Notably, the ethyl acetate extract demonstrated significant activity against both strains (1.46–2.93 µg/mL; **Table 1**), highlighting the therapeutic potential of ammoniacum. In contrast, no activity was observed against the Gram-negative strain *Escherichia coli*. These findings are consistent with previous studies reporting
selective antibacterial effects of non-polar extracts of ammoniacum against Gram-positive microorganisms [8].

<table><caption>**Table 1** Antibacterial activity -MIC<sub>90</sub>- of extract (µg/mL) and isolated compounds (µM) against Gram positive and negative strains.</caption><thead><tr><th>Bacterial strain</th><th>Ethyl acetate extract</th><th>Methanol extract</th><th>Compound 10</th><th>Ferulenol</th><th>C+</th></tr></thead><tbody><tr><td><i>S. aureus</i> (HG001)</td><td>5.12 ± 1.85</td><td>250 ± 0.00</td><td>2.46 ± 1.06</td><td>8.46 ± 0.00</td><td>1.34 ± 0.00</td></tr><tr><td><i>S. epidermidis</i> (ATCC 35983)</td><td>0.98 ± 0.00</td><td>125 ± 0.00</td><td>3.69 ± 0.00</td><td>2.92 ± 1.26</td><td>1.34 ± 0.00</td></tr><tr><td><i>S. hominis</i> (CI)</td><td>2.32 ± 1.37</td><td>125 ± 0.00</td><td>3.09 ± 1.06</td><td>3.63 ± 1.26</td><td>1.34 ± 0.00</td></tr><tr><td><i>S. haemoliticus</i> (CI)</td><td>5.72 ± 1.99</td><td>250 ± 0.00</td><td>2.46 ± 1.06</td><td>11.38 ± 5.05</td><td>11.72 ± 9.99</td></tr><tr><td><i>S. epidermidis</i> (CI)</td><td>1.46 ± 0.56</td><td>125 ± 0.00</td><td>1.84 ± 0.00</td><td>3.63 ± 1.26</td><td>> 170.32</td></tr><tr><td><i>C. striatum</i> (CI)</td><td>2.93 ± 1.07</td><td>125 ± 0.00</td><td>6.17 ± 2.52</td><td>4.37 ± 0.00</td><td>> 170.32</td></tr><tr><td><i>E. coli</i> (MG1655)</td><td>> 500</td><td>> 500</td><td>> 448.90</td><td>> 341.06</td><td>85.16 ± 0.00</td></tr></tbody></table>

CI: clinical isolated strain; C+: positive control performed using erythromycin.

Following confirmation of the antibacterial activity of the crude extract, a fractionation process was carried out using normal-phase open column chromatography, yielding eighteen fractions (F1–F18). The antibacterial properties of each fraction were subsequently evaluated, and their chemical profiles were analysed using HPLC-DAD-HRMS/MS in positive ionization mode. The majority of constituents were identified as coumarin derivatives, as evidenced by their characteristic absorbance maxima at 270 and 310 nm, and a prominent fragment ion at *m/z* 175, corresponding to the 4-hydroxy-3-methylcoumarin moiety (Table 2) [9], accompanied by the loss of sesquiterpene chains composed of 15 carbon atoms. *F. communis* shows two different chemotypes, defined as “poisonous” and “non-poisonous” based on the main constituents revealed by phytochemistry studies and characterized as prenylated coumarins or daucane esters [10]. Sesquiterpene coumarins such as ferulenol are the most important constituents isolated from “poisonous” ammoniacum with reported bioactivities confirming the chemotype of the gum-resin used in this present study. They were mainly characterized as coumarin with a sesquiterpene side chain coupled in C-7 but recent studies also reported the existence of cyclized SCs with pyrane [3,2-C] structure [11,12].

Based on molecular formula, fragmentation patterns, and comparison with phytochemical data previously reported for *F. communis*, the major compounds were classified into three groups of sesquiterpene coumarins: hydroxyferulenol derivatives (compounds **1–6**), *R*-substituted hydroxyferulenols (compounds **7–9**, **15**), and ferprenin-type derivatives (compounds **11–13**, **17–21**), as detailed in Table 2. The hydroxyferulenol derivatives (**1–6**) exhibited a characteristic pseudo-molecular ion [*M*–*H*<sub>2</sub>O+*H*]<sup>+</sup> at *m/z* 365 and fragmented to produce the diagnostic *m/z* 175 ion. The *R*-substituted derivatives were distinguished by a fragment at *m/z* 387 (*m/z* 365 + *Na*<sup>+</sup>), with molecular ions observed at *m/z* 425 (compounds **7–8**, acetyl-hydroxyferulenol), *m/z* 419 (compound **9**, methyl-hydroxyferulenol) and *m/z* 487 (compound **15**, benzyl-hydroxyferulenol). Compounds **11–13** and **17–21** displayed a pseudo-molecular ion [*M*+*H*]<sup>+</sup> at *m/z* 365 and also yielded a fragment at *m/z* 175, comparable to the hydroxyferulenol series. Their molecular formula, C<sub>24</sub>H<sub>28</sub>O<sub>3</sub>, suggests the presence of an additional unsaturation compared to ferulenol (**14**), likely attributable to cyclization within the sesquiterpene backbone, consistent with the ferprenin scaffold previously described in *F. communis*
and *F. fukanensis* [12]. The presence of ferulenol was confirmed by co-injection with a commercial
standard.
<table><caption>**Table 2** Identification of chemical constituents present in ethyl acetate extract of ammoniacum in positive mode by comparison of MS/MS data.</caption><thead><tr><th>Code</th><th>RT (min)</th><th>UV (λ<sub>max</sub>)</th><th>m/z</th><th>Adduct</th><th>Chemical Formula</th><th>Δppm</th><th>ΔmDa</th><th>Fragments (m/z)</th><th>Putative Identification</th><th>Ref</th></tr></thead><tbody><tr><td rowspan="3">1</td><td rowspan="3">14.5</td><td rowspan="3">281.308</td><td>365.2130</td><td>[M-H<sub>2</sub>O+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>30</sub>O<sub>4</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td rowspan="3">175.0392; 109.1016</td><td rowspan="3">hydroxyferulenol derivative I</td><td rowspan="3">[10]</td></tr><tr><td>383.2260</td><td>[M+H]<sup>+</sup></td></tr><tr><td>787.4244</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="2">2</td><td rowspan="2">14.6</td><td rowspan="2">281.308</td><td>365.2130</td><td>[M-H<sub>2</sub>O+H]<sup>+</sup></td><td rowspan="2">C<sub>24</sub>H<sub>30</sub>O<sub>4</sub></td><td rowspan="2">3.64</td><td rowspan="2">1.33</td><td rowspan="2">175.0387</td><td rowspan="2">hydroxyferulenol derivative II</td><td rowspan="2">[10]</td></tr><tr><td>787.4244</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="2">3</td><td rowspan="2">14.8</td><td rowspan="2">281.308</td><td>365.2130</td><td>[M-H<sub>2</sub>O+H]<sup>+</sup></td><td rowspan="2">C<sub>24</sub>H<sub>30</sub>O<sub>4</sub></td><td rowspan="2">3.64</td><td rowspan="2">1.33</td><td rowspan="2">175.0387; 93.0694</td><td rowspan="2">hydroxyferulenol derivative III</td><td rowspan="2">[10]</td></tr><tr><td>787.4244</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="3">4</td><td rowspan="3">16.5</td><td rowspan="3">283.306</td><td>365.2130</td><td>[M-H<sub>2</sub>O+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>30</sub>O<sub>4</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td rowspan="3">175.0387</td><td rowspan="3">hydroxyferulenol derivative IV</td><td rowspan="3">[10]</td></tr><tr><td>383.2260</td><td>[M+H]<sup>+</sup></td></tr><tr><td>787.4244</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td>5</td><td>17.0</td><td>283.306</td><td>365.2115</td><td>[M-H<sub>2</sub>O+H]<sup>+</sup></td><td>C<sub>25</sub>H<sub>30</sub>O<sub>4</sub></td><td>7.25</td><td>2.87</td><td>175.0388; 135.1148</td><td>hydroxyferulenol derivative V</td><td>[10]</td></tr><tr><td rowspan="3">6</td><td rowspan="3">17.6</td><td rowspan="3">283.306</td><td>381.2049</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>30</sub>O<sub>4</sub></td><td rowspan="3">-4.42</td><td rowspan="3">-1.68</td><td rowspan="3">107.0471; 175.0392</td><td rowspan="3">hydroxyferulenol derivative VI</td><td rowspan="3">[10]</td></tr><tr><td>403.1892</td><td>[M+Na]<sup>+</sup></td></tr><tr><td>783.3959</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="2">7</td><td rowspan="2">25.5</td><td rowspan="2">282.308</td><td>447.2148</td><td>[M+Na]<sup>+</sup></td><td rowspan="2">C<sub>26</sub>H<sub>32</sub>O<sub>5</sub></td><td rowspan="2">0.13</td><td rowspan="2">0.06</td><td rowspan="2">387.1789; 229.0866</td><td rowspan="2">acetyl hydroxy ferulenol derivative I</td><td rowspan="2">[10]</td></tr><tr><td>425.2317</td><td>[M+H]<sup>+</sup></td></tr></tbody></table>
<table><tr><td></td><td></td><td></td><td>843.3978</td><td>[2M+Na]<sup>+</sup></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">8</td><td rowspan="3">26.5</td><td rowspan="3">282.308</td><td>447.2151</td><td>[M+Na]<sup>+</sup></td><td rowspan="3">C<sub>26</sub>H<sub>32</sub>O<sub>5</sub></td><td rowspan="3">0.8</td><td rowspan="3">0.36</td><td>387.1939;</td><td>acetyl hydroxy ferulenol derivative II</td><td rowspan="3">[10]</td></tr><tr><td>425.2318</td><td>[M+H]<sup>+</sup></td><td>175.0358</td><td></td></tr><tr><td>871.4325</td><td>[2M+Na]<sup>+</sup></td><td></td><td></td></tr><tr><td>9</td><td>28.1</td><td>282.308</td><td>419.2188</td><td>[M+Na]<sup>+</sup></td><td>C<sub>25</sub>H<sub>32</sub>O<sub>4</sub></td><td>-2.45</td><td>-1.03</td><td>387.1806</td><td>methyl hydroxy ferulenol derivative I</td><td>[10]</td></tr><tr><td rowspan="3">10</td><td rowspan="3">29.3</td><td rowspan="3">278.310</td><td>551.2442</td><td>[M+Na]<sup>+</sup></td><td rowspan="3">C<sub>33</sub>H<sub>36</sub>O<sub>6</sub></td><td rowspan="3">-2.29</td><td rowspan="3">-1.21</td><td>387.1939;</td><td>n.i.</td><td rowspan="3"></td></tr><tr><td>529.2578</td><td>[M+H]<sup>+</sup></td><td>365.2113;</td><td></td></tr><tr><td>1079.5005</td><td>[2M+Na]<sup>+</sup></td><td>175.0387</td><td></td></tr><tr><td rowspan="3">11</td><td rowspan="3">33.2</td><td rowspan="3">282.305</td><td>365.2130</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>28</sub>O<sub>3</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td>175.0387</td><td>ferprenin derivative I</td><td rowspan="3">[12,13]</td></tr><tr><td>387.1939</td><td>[M+Na]<sup>+</sup></td><td></td></tr><tr><td>751.4136</td><td>[2M+Na]<sup>+</sup></td><td></td></tr><tr><td rowspan="3">12</td><td rowspan="3">33.6</td><td rowspan="3">282.305</td><td>365.2130</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>28</sub>O<sub>3</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td>175.0383</td><td>ferprenin derivative II</td><td rowspan="3">[12,13]</td></tr><tr><td>387.1939</td><td>[M+Na]<sup>+</sup></td><td></td></tr><tr><td>751.4136</td><td>[2M+Na]<sup>+</sup></td><td></td></tr><tr><td rowspan="3">13</td><td rowspan="3">34.4</td><td rowspan="3">282.305</td><td>365.2130</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>28</sub>O<sub>3</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td>175.0390</td><td>ferprenin derivative III</td><td rowspan="3">[12,13]</td></tr><tr><td>387.1939</td><td>[M+Na]<sup>+</sup></td><td></td></tr><tr><td>751.4136</td><td>[2M+Na]<sup>+</sup></td><td></td></tr><tr><td rowspan="2">14</td><td rowspan="2">39.2</td><td rowspan="2">282.308</td><td>367.2296</td><td>[M+H]<sup>+</sup></td><td rowspan="2">C<sub>24</sub>H<sub>30</sub>O<sub>3</sub></td><td rowspan="2">6.21</td><td rowspan="2">2.28</td><td>175.0393</td><td>ferulenol</td><td rowspan="2">[9]</td></tr><tr><td>389.2137</td><td>[M+Na]<sup>+</sup></td><td></td></tr></table>
<table><tr><td></td><td></td><td></td><td>733.4528</td><td>[2M+H]<sup>+</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td>487.2500</td><td>[M+H]<sup>+</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">15</td><td rowspan="2">40.8</td><td rowspan="2">282.308</td><td>509.2365</td><td>[M+Na]<sup>+</sup></td><td rowspan="2">C<sub>31</sub>H<sub>34</sub>O<sub>5</sub></td><td rowspan="2">3.18</td><td rowspan="2">1.55</td><td rowspan="2">387.1939</td><td rowspan="2">benzyl hydroxy ferulenol</td><td rowspan="2">[15]</td></tr><tr><td>995.4922</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="3">16</td><td rowspan="3">41.4</td><td rowspan="3">282.308</td><td>467.2826</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>29</sub>H<sub>38</sub>O<sub>5</sub></td><td rowspan="3">6.1</td><td rowspan="3">2.85</td><td rowspan="3">387.1957; 175.0317</td><td rowspan="3">isovaleryl coladonin derivative I</td><td rowspan="3">[14]</td></tr><tr><td>489.2621</td><td>[M+Na]<sup>+</sup></td></tr><tr><td>955.5516</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="3">17</td><td rowspan="3">43.5</td><td rowspan="3">282.308</td><td>365.2130</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>28</sub>O<sub>3</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td rowspan="3">175.0391</td><td rowspan="3">ferprenin derivative IV</td><td rowspan="3">[12,13]</td></tr><tr><td>387.1939</td><td>[M+Na]<sup>+</sup></td></tr><tr><td>751.4136</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="3">18</td><td rowspan="3">44.1</td><td rowspan="3">282.308</td><td>365.2130</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>28</sub>O<sub>3</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td rowspan="3">197.0192</td><td rowspan="3">ferprenin derivative V</td><td rowspan="3">[12,13]</td></tr><tr><td>387.1939</td><td>[M+Na]<sup>+</sup></td></tr><tr><td>751.4136</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="3">19</td><td rowspan="3">49.01</td><td rowspan="3">282.308</td><td>365.213</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>28</sub>O<sub>3</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td rowspan="3">n.f.</td><td rowspan="3">ferprenin derivative VI</td><td rowspan="3">[12,13]</td></tr><tr><td>387.1939</td><td>[M+Na]<sup>+</sup></td></tr><tr><td>751.4136</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td rowspan="3">20</td><td rowspan="3">49.35</td><td rowspan="3">282.308</td><td>365.213</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>28</sub>O<sub>3</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td rowspan="3">n.f.</td><td rowspan="3">ferprenin derivative VII</td><td rowspan="3">[12,13]</td></tr><tr><td>387.1939</td><td>[M+Na]<sup>+</sup></td></tr><tr><td>751.4136</td><td>[2M+Na]<sup>+</sup></td></tr></table>
<table><tr><td rowspan="3">21</td><td rowspan="3">51.69</td><td rowspan="3">282.308</td><td>365.213</td><td>[M+H]<sup>+</sup></td><td rowspan="3">C<sub>24</sub>H<sub>28</sub>O<sub>3</sub></td><td rowspan="3">3.64</td><td rowspan="3">1.33</td><td rowspan="3">n.f.</td><td rowspan="3">ferprenin derivative VIII</td><td rowspan="3">[12,13]</td></tr><tr><td>387.2052</td><td>[M+Na]<sup>+</sup></td></tr><tr><td>751.4136</td><td>[2M+Na]<sup>+</sup></td></tr><tr><td>22</td><td>55.52</td><td>208</td><td>769.4169</td><td>[M+H]<sup>+</sup></td><td>C<sub>43</sub>H<sub>60</sub>O<sub>12</sub></td><td>0.78</td><td>0.6</td><td>n.f.</td><td>n.i.</td><td></td></tr><tr><td>23</td><td>56.05</td><td>208</td><td>769.4169</td><td>[M+H]<sup>+</sup></td><td>C<sub>43</sub>H<sub>60</sub>O<sub>12</sub></td><td>0.78</td><td>0.6</td><td>n.f.</td><td>n.i.</td><td></td></tr><tr><td>24</td><td>57.75</td><td>208</td><td>769.4169</td><td>[M+H]<sup>+</sup></td><td>C<sub>43</sub>H<sub>60</sub>O<sub>12</sub></td><td>0.78</td><td>0.6</td><td>n.f.</td><td>n.i.</td><td></td></tr></table>

n.f.: not fragmented. n.i.: not identified.
Regarding the antibacterial activity of fractions, F4 to F8 and F14-F15 manifested significant activity with MIC₉₀ values varying in a range from 2.0 µg/mL to 27.3 µg/mL (Table S1). Among them, the highest antibacterial activity was measured in fraction F4 and F5, followed by the slow increase of the MIC₉₀ in fractions F6 to F8. When adding these data to the obtained molecular network (colour gradient depending on the measured MIC₉₀ value), two cluster were identified as bearing compounds present in active fractions (Figure 1, Figure S1). Cluster A features ferulenol derivatives (1-6, 14) present in active, mild and inactive fractions. High similarity between fragmentation spectra of ferulenol and compounds 1-6 on cluster A limited the propagation of annotation of compounds present in active extracts.

<figure><img src="image_4.png" /><figcaption>**Figure 1**</figcaption></figure>

Multi-informative molecular network of fractions of ethyl acetate extract of *F. communis*. Node size is correlated to the sum of peak areas. Whole molecular network built on ESI-HRMS/MS data, acquired in a positive mode. Cluster **A** combines sesquiterpene coumarins derived from ferulenol, while cluster **B** includes R-substituted hydroxyl ferulenol aromatic derivatives.
Cluster B was characterized as comprising *R*-substituted hydroxyl ferulenol derivatives. Among these, compound **15** (*m/z* 509.2236), identified as *ω*-benzoyloxyferulenol [15] was associated with an additional, previously uncharacterized compound (compound **10**, *m/z* 551.2442) detected in biologically active fractions (F14-F15). Comparative analysis of their MS/MS fragmentation spectra (Fig. 2) revealed a common ferulenol core, as evidenced by a shared major fragment ion at *m/z* 387. In addition, a diagnostic fragment at *m/z* 175 supported the presence of a coumaric structural moiety. The mass difference between the sodium adduct of compound **10** ([M+Na]⁺, *m/z* 551.2442) and the major fragment ion (*m/z* 387) corresponds to a neutral loss of 164.0503 Da, consistent with a C₉H₈O₃ moiety (theoretical *m/z* 164.0473). This mass shift suggests the elimination of a substituent requiring six degrees of unsaturation, consistent with the presence of a coumaric acid residue.

<figure><img src="image_5.png" /><figcaption>**Figure 2**</figcaption></figure>

Comparison of fragmentation spectra between **A. ω-benzoyloxyferulenol (15)** and **B. Compound 10**.
Data were acquired on ESI-HRMS/MS on a positive mode.

To confirm this structural prediction, a targeted purification was performed, resulting in the isolation of compound **10** (*m/z* 529.2594 ion, corresponding to a molecular formula of C₃₃H₃₆O₆). The ¹H spectra (Figure S2) integrated for 36 protons (Figure 3, Table 3) among which were identified 3 singlets methyls (δ<sub>H</sub> 1.70, 1.51, 1.59 ppm) and four aromatic protons (δ<sub>H</sub> 7.94, 7.11, 7.35 and 7.13 ppm, Table 3). ¹H-¹H COSY correlation (Figure S4) between these four protons confirmed the presence of an aromatic group disubstituted. ¹H-¹³C HMBC correlations (Figure S5) between H-5/C-4 (δ<sub>H</sub> 7.94 ppm/δ<sub>C</sub> 174.3 ppm), H-1'/C-4 (δ<sub>H</sub> 3.20 ppm/δ<sub>C</sub> 174.3 ppm) and H-1'/C-2 (δ<sub>H</sub> 3.20 ppm/δ<sub>C</sub> 169.1 ppm) confirmed the presence of a coumarin group substituted in position 3. This substitution was identified as a sesquiterpene chain, confirmed on ¹H-¹H COSY correlations (H-1'/H-2', H-4'/H-5', H-5'/H-6', H-8'/H-9', H-9'/H-10') and on ¹H-¹³C HMBC correlations (H-1'/C-3', H-5'/C-3', H-5'/C-7', H-9'/C-7', H-9'/C-11' and H-12'/C-10'). E conformation of the double bonds were confirmed by NOE correlations (Figure 3, Figure S7). These data were consistent with the reported data of ω-hydroxyferulenol, a sesquiterpene coumarin isolated from *F. communis* [16]. This similarity confirmed the ferulenol skeleton highlighted by the HRMS/MS fragmentation pattern based on the *m/z* 365,2121 fragment. However, they differed at C-12' chemical shift (δ<sub>C</sub> 71.1 for Compound 10). ¹H spectra revealed the supplementary presence of 6 protons between δ<sub>H</sub> 7.97-6.56 ppm, compared with spectra of ω-hydroxyferulenol. ¹H-¹H COSY correlation between H-2''/H-3'' with a constant coupling of 16.1 Hz confirmed the presence of an E-conformation ethylenic bond. The conformation was corroborated by NOE correlations between H-3'' and H-9''. Moreover, *o*-hydroxylphenyl group was identified based on ¹H-¹H-COSY (H-3/H-4, H-4/H-5, and H-5/H-6 with constant coupling of 8.0 Hz, Figure S4) and ¹H-¹³C HMBC spectra (H-2/C-6, H-3/C-6 and H-5/C-6 correlations, Figure S5). Additionally, ¹H-¹³C HMBC correlations between H-12'/C1'', H-2''/C1'', H-3''/C-4'' and H-3''/C-5'' allow to identify and to place an *o*-coumaric acid substitution on C-12' position.
Based on its 1D and 2D NMR characteristics, Compound **10** was thus identified as a *o*-coumaric-*ω*-hydroxy-ferulenol which corroborates the previous predicted structure by HRMS/MS spectra analysis.

<figure><img src="image_6.png" /><figcaption>**Figure 3**</figcaption></figure>

Key ¹H-¹H COSY NOESY and ¹H-¹³C HMBC correlations of Compound **10**.

<caption>**Table 3**</caption>

¹H and ¹³C NMR resonance assignments of Compound **10** in CD₃OD, 500 and 125 MHz.

<table><thead><tr><th>Position</th><th>1H δ (ppm) (J in Hz)</th><th>13C δ (ppm)</th></tr></thead><tbody><tr><td>2</td><td>-</td><td>169.1</td></tr><tr><td>3</td><td>-</td><td>102.1</td></tr><tr><td>4</td><td>-</td><td>174.3</td></tr><tr><td>5</td><td>7.94 (dd, J = 8.0, 1.7 Hz, 1H)</td><td>125.7</td></tr><tr><td>6</td><td>7.11 (m, 1H)</td><td>123.8</td></tr><tr><td>7</td><td>7.35 (td, J = 8.0, 1.7 Hz, 1H)</td><td>131.2</td></tr><tr><td>8</td><td>7.13 (m, 1H)</td><td>116.9</td></tr><tr><td>9</td><td>-</td><td>154.5</td></tr><tr><td>10</td><td>-</td><td>123.6</td></tr><tr><td>1'</td><td>3.20 (d, J = 6.7 Hz, 2H)</td><td>23.8</td></tr><tr><td>2'</td><td>5.23 (td, J = 6.7, 0.96 Hz, 1H)</td><td>125.5</td></tr><tr><td>3'</td><td>-</td><td>134.7</td></tr><tr><td>4'</td><td>1.89 (m, 2H)</td><td>40.9</td></tr><tr><td>5'</td><td>1.99 (m, 2H)</td><td>27.7</td></tr><tr><td>6'</td><td>5.05 (td, J = 6.9, 0.96 Hz, 1H)</td><td>126.1</td></tr><tr><td>7'</td><td>-</td><td>135.1</td></tr><tr><td>8'</td><td>1.91 (m, 2H)</td><td>40.1</td></tr><tr><td>9'</td><td>2.04 (m, 2H)</td><td>27.3</td></tr><tr><td>10'</td><td>5.37 (td, J = 7.1, 0.96 Hz, 1H)</td><td>130.3</td></tr></tbody></table>
<table><tr><td>11'</td><td>-</td><td>131.3</td></tr><tr><td>12'</td><td>4.48 (s, 2H)</td><td>71.1</td></tr><tr><td>13'</td><td>1.70 (s, 3H)</td><td>16.5</td></tr><tr><td>14'</td><td>1.51 (s, 3H)</td><td>16.1</td></tr><tr><td>15'</td><td>1.59 (s, 3H)</td><td>14.1</td></tr><tr><td>1"</td><td>-</td><td>169.5</td></tr><tr><td>2"</td><td>6.56 (d, J = 16.1 Hz, 1H)</td><td>118.3</td></tr><tr><td>3"</td><td>7.97 (d, J = 16.6 Hz, 1H)</td><td>142.4</td></tr><tr><td>4"</td><td>-</td><td>122.6</td></tr><tr><td>5"</td><td>-</td><td>158.3</td></tr><tr><td>6"</td><td>6.80 (d, J = 8 Hz, 1H)</td><td>117.1</td></tr><tr><td>7"</td><td>7.14 (m, 1H)</td><td>132.6</td></tr><tr><td>8"</td><td>6.77 (t, J = 7.5 Hz, 1H)</td><td>120.7</td></tr><tr><td>9"</td><td>7.43 (dd, J = 8.0, 1.5 Hz, 1H)</td><td>130.1</td></tr></table>

Compound **10**, along with the reference compound ferulenol, was evaluated for its antimicrobial activity against the same panel of bacterial strains previously used (Table 1). Compound **10** and ferulenol exhibited noteworthy antibacterial activity, particularly against Gram-positive strains, with minimum inhibitory concentrations (MIC<sub>90</sub>) ranging from 2.92 to 11.38 µM (Table 4). One of the highest efficacy was observed against a clinical isolate of *S. epidermidis*, which notably displayed resistance to the erythromycin control (MIC<sub>90</sub> > 170.32 µM) (Table 1). In the literature, ferulenol also demonstrated potent antimycobacterial activity, with MIC values as low as 5.46 µM against *Mycobacterium* spp. Furthermore, its combination with isonicotinic acid hydrazide resulted in a synergistic effect, significantly enhancing the antimycobacterial activity and reducing the MIC values [15]. Beyond its effect on *Mycobacterium*, ferulenol was active against several Gram-positive genera including *Bacillus*, *Staphylococcus*, *Streptococcus*, and *Enterococcus* spp., with MIC values reported at 1.72 µM [17].

The anti-Gram-positive activity highlighted before was deeper investigated using a *S. epidermidis* (ATCC 35983) strain in an MBC (Minimal Bactericidal Concentration) assay and a flow cytometry analysis. The MBC of ferulenol and Compound **10** were determined at 8.54 µM and 5.92 µM respectively, as these lowest concentrations totally inhibited the growth of bacteria after sub-culture on agar media (Table 4, Figure S7). Because the MBC/MIC ratio<4, the effect of both ferulenol and Compound **10** were defined as bactericidal [18]. To further confirm the bactericidal effect of ferulenol and the new derivative Compound **10**, a Propidium Iodide (PI)-uptake was carried out. PI is a fluorophore that can enter cells through disrupted membranes and bind to DNA. This binding will lead to a fluorescence emission at 617 nm indicating a dead cell. Untreated bacteria as negative control were mostly alive with 3.0 % of cells above the PI fluorescence gate while heat-killed bacteria showed more than 81.6 % of dead/injured cells. In addition to the MBC assay, PI-uptake results also confirmed the bactericidal activity of both ferulenol and the new Compound **10** (Figure 4). The 72.9 % of dead-cells after Compound **10** exposition at 4 times CMI showed that there was a significant increase in the
fluorescence compared to heat-treated bacteria. The increase in PI fluorescence was similar to that observed following ferulenol treatment. (Figure S8). The results indicated that both ferulenol and Compound 10 could cause disruption of the outer membrane of S. epidermidis in a dose-dependent manner. These findings indicated that Compound 10-treatment caused, at least at high concentration the bacteria to lose cell membrane integrity, confirming its bactericidal activity.

<table><caption>**Table 4:** MIC, MBC and MBC/MIC ratio of isolated SCs against S. epidermidis ATCC35983.</caption><thead><tr><th>Compound</th><th>MIC (µM)</th><th>MBC (µM)</th><th>MBC/MIC ratio</th></tr></thead><tbody><tr><td>Compound 10</td><td>3.69</td><td>5.92</td><td>1.6</td></tr><tr><td>Ferulenol 14</td><td>2.92</td><td>8.54</td><td>2.9</td></tr><tr><td>C+</td><td>1.71</td><td>1.71</td><td>1</td></tr></tbody></table>

C+: positive control performed using ofloxacin

<figure><img src="image_7.png" /><figcaption>**Figure 4**</figcaption></figure>

Bactericidal effect of ferulenol and Compound 10 changes in permeability cells in S. epidermidis ATCC 35983 with different sesquiterpene coumarins concentrations (% of altered cells, corresponding to % of PI-positive cells). Bars representing the mean and error bars representing standard deviation of three independent experiments. C- a is bacterial growth positive control, while C+ represents heat-inactivated bacteria as positive control of the growth inhibition, similar to colistin used at 4 times MIC.

In the literature, ferulenol was identified as a potent inhibitor of malate:quinone oxidoreductase (MQO) in Campylobacter jejuni, an essential enzyme involved in bacterial energy metabolism [19]. Interestingly, a Mycobacterium tuberculosis strain lacking MQO was still able to grow normally under standard in vitro conditions but showed reduced colony-forming units burden in macrophages and in mice lungs in vivo [20]. Coumarin moieties exhibit antibacterial activity by targeting several key bacterial enzymes and cellular structures. The main molecular targets include DNA gyrase, topoisomerase II, fatty acid synthesis enzymes (FabI and FabK) and bacterial membranes. Meanwhile, linear sesquiterpenes derivatives such as nerolidol and farnesol can inhibit bacterial efflux pumps (e.g., NorA, Tet(K), MsrA, MepA) in resistant S. aureus strains [21,22]. Future experiments must be
conducted to identify the molecular targets on bacteria of the bactericidal activity of coumarin-sesquiterpene derivatives such as ferulenol and Compound **10**.

<caption>**Table 5**</caption>

*In vitro* cytotoxic activity -CC₅₀- of isolated SCs (in µM).

<table><thead><tr><th rowspan="2">Compounds</th><th colspan="2">Tested cell lines</th><th rowspan="2">SI (CC<sub>50</sub>/MIC)</th></tr><tr><th>HepG2</th><th>HaCaT</th></tr></thead><tbody><tr><td>Compound 10</td><td>> 189.39 ± 0.00<br>(> 100.00 ± 0.00 µg/mL)</td><td>> 189.39 ± 0.00<br>(> 100.00 ± 0.00 µg/mL)</td><td>> 51.32</td></tr><tr><td>Ferulenol 14</td><td>26.11 ± 0.41<br>(9.57 ± 0.15 µg/mL)</td><td>16.72 ± 0.68<br>(6.13 ± 0.25 µg/mL)</td><td>5.73</td></tr><tr><td>C+</td><td>0.46 ± 0.05<br>(0.16 ± 0.02 µg/mL)</td><td>0.34 ± 0.14<br>(0.12 ± 0.05 µg/mL)</td><td>-</td></tr></tbody></table>

C+: positive control of cytotoxicity performed using camptothecin. SI: Selectivity Index between CC₅₀ obtained against HaCaT cell line and *S. epidermidis* ATCC35983. "-": not determined.

To evaluate the effects on human cell, a viability test was performed on hepatocarcinoma and keratinocyte lines. Ferulenol showed cytotoxicity activity against both cell lines with CC₅₀ values of 26.11 ± 0.41 µM on HepG2 cells and 16.72 ± 0.68 µM on HaCaT cells, while Compound **10** showed no effects (Table 5). Compound 10 showed a Selectivity Index (SI) of 51.32, indicating a strong selectivity of its antibacterial activity over eukaryotic cells [23]. Ferulenol has been shown to significantly decrease proliferation on MCF-7, HL-60, Caco-2 and SK-OV-3 at 1 and 0.1 µM [24]. Ferulenol affects mitochondrial function, leading to decreased mitochondrial dehydrogenase activity and a reduction in mitochondrial membrane potential, which are indicative of its role in promoting cell death [25]. Our results highlight the importance of assessing how ω-substitution of coumarin-sesquiterpene affects mitochondrial membrane potential and cytotoxicity.

### 3. Conclusion

In this study, we successfully validate a multi-informative Molecular Network approach as an effective strategy to prioritize the targeted isolation of antibacterial compounds from Ammoniacum gum resin extract. This methodology led to the isolation and structural elucidation of a previously undescribed o-coumaric-ω-hydroxyferulenol. This new compound exhibited potent antibacterial activity against Gram-positive bacteria, including the *S. aureus* HG001 strain, and demonstrated promising selectivity, with no cytotoxic effects observed on HaCaT or HepG2 cells in *in vitro* assays. This finding, not only confirm the effectiveness of HPLC-PDA-HRMS/MS molecular-networking-based dereplication strategy to target putative bioactive compounds and predict their structures but also highlight the therapeutic interest of sesquiterpene coumarins. Since substitution of the ω-position seems to significantly decrease the cytotoxicity of sesquiterpene coumarins, further works are strongly recommended to focus on the structure-activity relationship of ω-substituted analogues. To this purpose, total synthesis routes can be established, accessing to new derivatives to determine structure-activity relationships with reference and clinically isolated multidrug-resistant bacterial strains.

### 4. Experimental

#### 4.1 General experimental procedures

Nuclear magnetic resonance experiments were performed in a spectrometer Bruker 500 MHz (Fällanden, Switzerland) with methanol-d4 as solvent. Ethyl acetate, dichloromethane (DCM),
methanol (MeOH), HPLC grade methanol, HPLC grade acetonitrile, trifluoroacetic acid (TFA) and
cyclohexane were obtained from CARLO ERBA Reagents (Val-de-Reuil, France). Ethyl acetate,
dichloromethane and cyclohexane were distilled before use. Silica gel (43-60 nm) was purchased from
VWR International (Leuven, Belgium). MilliQ grade water was obtained from a Merck Millipore MilliQ
System (Molsheim, France).

## 4.2 Plant material and extraction

Dry root resin from *F. communis* L. was purchased from local pharmacy in Sidi Slimane (Morocco) in June 2023. The corresponding voucher (Code VON-EA-GA-001) was deposited at the Herbarium of the Laboratoire d'Innovation Thérapeutique (Université de Strasbourg). A 10 g sample of powdered ammoniacum was macerated in ethyl acetate (1% w/v) for 24h with. After filtration, same plant material was subjected to a second maceration in methanol (1% ww/v) for 24h. Filtered crude extracts were dried by rotary evaporator until a dry and sticky residues were obtained.

## 4.3 HPLC-PDA-HRMS/MS analysis

HPLC-PDA-HRMS/MS analyses were performed on an HPLC-PDA Agilent 1200 system coupled with an Agilent Accurate Mass QToF 6520 mass spectrometer (Agilent, USA), controlled with a Mass Hunter software. The chromatographic separation was done on a 5 µm particles Uptisphere C18, 250 x 4.6 mm column. Stock solutions of extract and fractions were prepared in HPLC grade methanol at 10 and 1 mg/mL concentration respectively and the injection volume was 10 µL. The mobile phase consisted of 0.1% of formic acid in water (solvent A) and 0.1% of formic acid in acetonitrile (solvent B). Elution of the mobile phase was performed with a flow rate of 0.8 mL/min in gradient mode: 0-10 min (60%, v/v, B); 10-25 min (60-75%, v/v, B); 25-35 min (75%, v/v, B); 35-40 (75-100%, v/v, B); 40-50 (100%, v/v, B); 50-51 (100-60%, v/v, B) and 51-56 (60%, v/v, B). Detection wavelengths for chromatograms were set between 190 and 500 nm. HRMS analyses were performed in ESI positive and negative modes with the following inlet conditions for both modes: ESI gas temperature of 340 °C, nebulizer of 30 psig, MS TOF fragmentor of 120 V, skimmet of 68 V and a collision energy of 30 eV. The MS/MS events were performed on the most abundant ions detected in full MS scans.

## 4.4 MS/MS data treatment

HRMS data (.d) files of extract and fractions obtained in positive mode (more informative set of data), were converted to .mzML files with MSConvert 3.0 software and then treated using an open source software MZmine 2.53. Noise level was set to 1.5×10² and 10 for mass detection at MS1 and MS2 levels, respectively. The ADAP chromatogram builder was used by setting to a minimum group size of 5 scans, group intensity threshold of 1.0×10³, minimum highest intensity of 1.0×10³, and *m/z* tolerance of 10 ppm. The intensity window S/N as an S/N estimator was used to deconvolute the chromatogram by ADAP wavelets algorithm with a signal to noise ratio set at 15, a minimum feature of 1.0×10³, a peak duration ranging from 0.02 to 0.6 min, a coefficient threshold at 10, and an RT wavelet range of 0.01–0.2 min “Isotope peak grouper” was used to detect isotope pattern forming peaks and to remove additional minor isotopic peaks using an *m/z* tolerance of 10 ppm, RT tolerance of 0.5 min (absolute), maximum charge set at 1. The most intense isotope was selected as the representative isotope. Then, the aligned list peak was gap-filled with RT range of 0.04 min and *m/z* tolerance of 8 ppm. The resulting mass list was filtered using “duplicate peak filter” and “feature list rows filter” to remove all duplicates and features and keep only MS/MS scans.

## 4.5 Mass spectral organization and dereplication
After MS data treatment, the corresponding .mgf file was exported to the GNPS website to construct a molecular network. The precursor ions mass tolerance was set to 2.0 Da with MS/MS fragmentation tolerance of 0.5 Da. A network was created by setting the edges to have a cosine value of 0.70 and more than 3 common peaks. The spectra in the network were then searched against GNPS's spectral libraries, under the same conditions. Additional putative identification of unmatched peaks was carried out by comparing available MS/MS fragmentation patterns in the literature and in open databases (https://lotus.naturalproducts.net/; https://coconut.naturalproducts.net/). Data were visualized using Cytoscape 3.8.0 software. Data of peak area was added to the network from.csv file, obtained from MZmine 2.53. Size of nodes was set proportionally to the total area of each peak, detected in the analysed extract.

## 4.6 Fractionation and isolation

A part of the dried residue (2 g) was subjected to a first fractionation using silica gel column chromatography with a vertical glass column (100 cm x 6 cm diameter) half-filled with a silica gel (43-60 nm, from VWR International, Leuven, Belgium). Elution was carried out using mobile phase composed of cyclohexane and ethyl acetate with an increasing polarity gradient (from 95:5 v/v to 60:40 v/v). Silica gel was finally cleaned from the last retained compounds by eluting EtOAc 100% and EtOAc/MeOH (90:10 v/v). Flow rate was manually controlled to collect and afford fractions F1 to F18. Molecular networking-selected fractions were purified by semipreparative-HPLC equipped with an Uptisphere Strategy C-18-3 column (250 x 21 mm, 10µm particles size, Interchim). Mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0,1% formic acid (B). The elution was carried out with a flow rate of 15 mL/min using the optimized solvent gradient described in section 4.3: 60% B (0-10 min), 75% B (25-35 min), 100% B (40-50 min) and 60% B (51-56 min) to isolate Compound **10**.

## 4.7 Antibacterial assays

Antibacterial activities of crude extracts, fractions and isolated compounds were assessed against 6 Gram-positive strains; respectively *S. aureus* (HG001), *S. epidermidis* (ATCC 35983 and a clinical isolate -CI- from the HUS -Hopitaux Universitaires de Strasbourg- collection), *S. hominis* (CI, HUS collection), *S. haemoliticus* (CI, HUS collection), *C. striatum* (CI, HUS collection), and *E. coli* (MG1655) as a Gram-negative control. They were grown overnight at 37°C by shaking at 200 rpm in Luria Broth (LB) medium. A microdilution assay was performed to define the minimum inhibitory concentration (MIC) of each sample. Serial two-fold dilutions that ranged from 62.50 µg/mL to 0.12 µg/mL for extracts and fractions and from 50.0 µg/mL to 0.10 µg/mL for isolated compound were carried out in 96-well micro-plates containing 100 µL of LB medium. A 100 µL inoculum of 5*10⁵ CFU/mL of each strain was added to each well and incubated overnight at 37°C by shaking at 200 rpm. Medium with 4% DMSO and bacterial inoculum were used as positive control of bacterial growth while LB only wells were used as positive control of sterility. Wells containing bacteria and erythromycin (Sigma-Aldrich, Darmstadt, Germany) ranging from 50 µg/mL to 0.10 µg/mL were used as a positive controle of growth inhibition. MICs were evaluated by measuring OD600 values (Tecan M200Pro, Tecan Life, Männedorf). The MIC₉₀ was defined as the lowest extract concentration required to inhibit the growth of 90% of the bacterial population compared to the growth control. Experiments were repeated three times.

To determine the MBC value of ferulenol and Compound **10**, 20 µL of each well equal or two times greater and lower than MIC against *S. epidermidis* (ATCC 35983) was spread on LB-agar medium and incubated at 37°C for 24h. The lowest concentrations at which no CFU were observed after this subculture were taken as MBC values. Each well was sampled twice per assay and all assays were performed in duplicate and performed twice at different times.
## 4.8 Flow cytometry analysis

Propidium iodide (PI) uptake was measured by flow cytometry to evaluate the impact of compound treatment on bacterial cell viability. *Staphylococcus epidermidis* (ATCC 35983) was cultured overnight in fresh LB at 37°C and adjusted to an inoculum with an OD₆₀₀ of 0.3, corresponding to the early logarithmic growth phase. Heat-inactivated bacteria and colistin-treated bacteria (4×MIC) were used as positive controls for cell death. Stock solutions of each compound at 10 mM in DMSO were prepared and serially diluted two-fold, ranging from 5.0 to 0.3125 mM, in LB. This dilution series resulted in final concentrations in each well ranging from 4×MIC to 0.5×MIC. The 96-well microplates were incubated at 37°C with shaking. After 24 hours of treatment, cell death was assessed using PI, a DNA-intercalating agent that selectively penetrates bacteria with damaged membranes. This fluorescent dye allows analysis of membrane integrity for discriminating viable and dead cells. Following staining, the samples were analyzed using a Guava EasyCyte Plus Flow Cytometer with Guava ExpressPro software (Guava Technologies, Merck, Millipore), utilizing a 488 nm blue excitation laser and a red emission filter (680/30 nm). After exclusion of debris based on forward and side scatter (FSC/SSC) parameters, cut-off threshold was applied to a uniform bacteria population. A panel of several relevant controls were used to define PI-positive and PI-negative populations: an unstained bacterial sample (to assess autofluorescence), a PI-stained untreated sample (to represent viable cells), and positive controls such as heat-inactivated bacteria and colistin-treated bacteria (to represent dead cells). Experiment was conducted three independent times, with data collected from 1000 cells per well.

## 4.9 Cytotoxicity assay

Cytotoxic effects of the compounds were evaluated on HepG2 and HaCaT cell lines, representing hepatocarcinoma and keratinocyte models, respectively. Briefly, 180 µL of cell suspension on DMEM supplemented medium (10% FBS and 1% penicillin/streptomycin) at 27777 cell/mL of concentration were seeded and incubated in 96-well plates for 24 h at 37 °C with 5% CO₂. Then, stock solution of pure compounds at 20 mg/mL in DMSO were diluted in DMEM supplemented medium to obtain a range of concentration of 1000 – 0.5 µg/mL. From each solution, 20 µL were added per well and plates were incubated (37°C with 5% CO₂) for 72 h. After incubation, the medium was replaced with 100 µL of MTT solution (15 mg of MTT salt in 50 mL of DMEM supplemented medium). After 45 min of incubation, the MTT solution was replaced with 100 µL of DMSO and absorbance was measured spectrophotometrically at 570 nm to quantify the formazan formed by MTT reduction. Camptothecin was used as positive control. The assay was run in triplicate and repeated three times. The CC₅₀ values were measured, representing 50% of the cytotoxic effect of the compounds on the cell lines used.

## 4.10 Statistical analysis

The data were presented as the mean ± standard deviation (SD) of experiments performed independently three times for antibacterial, flow cytometry and cytotoxicity assays. Means and standard deviation (± SD) of the means were calculated with the program Microsoft Office 2019 Excel (Microsoft Corporation, USA).

## Credit author statement

**Capucine Braillon:** Methodology, Investigation, Formal analysis, Data curation, Writing - original draft, Writing - review and editing, **Elora Aubert:** Methodology, Investigation, Formal analysis, Data curation, **Régine Janet-Bintz:** Methodology, Formal analysis, Writing - review & editing, **Megi Gjikolaj:** Investigation, Formal analysis, **Rania Hamidi:** Investigation, Formal analysis, **Léa Blondé:** Investigation,
Methodology, Formal analysis, Data curation, Writing- review & editing, **Jean Peluso**: Investigation, Methodology, Formal analysis, Data curation, Writing- review & editing, **Pascal Villa**: Resources, **Véronique Pitchon**: Conceptualization, Investigation. **Pierre Fechter**: Supervision, Resources, Conceptualization, Writing - review & editing, **Catherine Vonthron-Sénécheau**: Supervision, Resources, Conceptualization, **Sergio Ortiz**: Methodology, Investigation, Data curation, Writing – original draft, Writing - review & editing, Supervision, Resources, Conceptualization

**Declaration of Competing Interest**

The authors declare that there are not conflicts of interest.

**Data availability**

No data was used for the research described in the article.

**Acknowledgments**

We are grateful to Dr. Delphine Garnier and Dr. Cheng Deng (PCBIS platform UAR 3286) for mass spectrometry and NMR analysis.

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