# Massularosides A-D, new triterpenoid saponins from the stem bark of Massularia acuminata with an N-acetylglucosamine residue and their antifungal properties

Charles Roberto Bilongo, Lin Marcellin Messi, Jean Faustin Atangana, Andreas Dietl, Joséphine Ngo Mbing, Dieudonné Emmanuel Pegnyemb, Mohamed Haddad, Olivier Placide Note, Olivier Potterat

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Charles Roberto Bilongo, Lin Marcellin Messi, Jean Faustin Atangana, Andreas Dietl, Joséphine Ngo Mbing, et al.. Massularosides A-D, new triterpenoid saponins from the stem bark of Massularia acuminata with an N-acetylglucosamine residue and their antifungal properties. Fitoterapia, 2025, 186, 10.1016/j.fitote.2025.106866. hal-05527422

HAL Id: hal-05527422

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Research Paper

# Massularosides A-D, new triterpenoid saponins from the stem bark of *Massularia acuminata* with an *N*-acetylglucosamine residue and their antifungal properties

Charles Roberto Bilongo<sup>a,b</sup>, Lin Marcellin Messi<sup>b,c</sup>, Jean Faustin Atangana<sup>d</sup>, Andreas Dietl<sup>e</sup>, Joséphine Ngo Mbing<sup>a</sup>, Dieudonné Emmanuel Pegnyemb<sup>a</sup>, Mohamed Haddad<sup>f</sup>, Olivier Placide Note<sup>a,d,*</sup>, Olivier Potterat<sup>e,**</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

**Keywords:**

Rubiaceae

*Massularia acuminata*

Triterpenoid saponins

NMR

Antifungal activity

## ABSTRACT

Four triterpenoid saponins named massularosides A-D (1–4), sharing a common α-L-rhamnopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-glucopyranosyl moiety, were isolated from the stem bark of *Massularia acuminata*. All four compounds are newly identified natural products, although compound 1 was previously chemically synthesized. The structures of the isolates were established by high-resolution ESI mass spectrometry (HRESIMS), 1D (¹H and ¹³C-DEPTq) and 2D (COSY, ROESY, HSQC and HMBC) NMR spectroscopy and by comparison with literature data to be 3-O-[α-L-rhamnopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-glucopyranosyl] oleanolic acid (1); 3-O-[α-L-rhamnopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-glucopyranosyl]-28-O-[β-D-glucopyranosyl] oleanolic acid (2); 3-O-[α-L-rhamnopyranosyl-(1 → 3)-[β-D-glucopyranosyl-(1 → 6)]-2-acetamido-2-deoxy-β-D-glucopyranosyl]-28-O-[β-D-glucopyranosyl] oleanolic acid (3) and 3-O-[β-D-glucopyranosyl (1 → 4)-α-L-rhamnopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-glucopyranosyl]-28-O-[β-D-glucopyranosyl] oleanolic acid (4), respectively. The antifungal activity of the butanol-soluble fraction, saponins-enriched fractions and compounds 1 and 3 was evaluated against a series of plant pathogenic fungi and oomycetes. The butanol-soluble fraction (>60 % inhibition at 125 µg/mL) and two saponins-enriched fractions (>65 % inhibition at 62.5 µg/mL) showed inhibitory activity against the oomycetes *Pythium myriotylum* and *Phytophthora megakarya*. Compound 1 inhibited the growth of *P. myriotylum* with a minimum inhibitory concentration (MIC) of 125 µg/mL.

## 1. Introduction

The Rubiaceae family is widely distributed in tropical and subtropical regions and holds the fourth position in terms of diversity among Angiosperm families [1], with around 13,000 species (trees, shrubs and herbaceous plants) grouped into 637 genera [2,3]. Among these, *Massularia acuminata* (G. Don) Bullock ex Hoyle is a small tree that grows up to 9 m high and thrives in the understory of the closed forest across West Africa, including Cameroon and Nigeria. The plant is commonly called

"Maiden breast plant" due to the shape of its fruit [4]. In some localities in Africa, this plant is also referred to as "Pako Ijebu", especially in the Southwest region of Nigeria, or "Oyébé" in the Southern region of Cameroon where the young twigs are used as chewing sticks for the oral cavity [5]. *M. acuminata* is used in African and Cameroonian traditional medicine for the treatment of several diseases such as oral thrush, tumours, and microbial infections. The leaves can also be used to prepare tea for treatment of malaria and stomach issues [6]. Previous phytochemical investigations of *M. acuminata* revealed the presence of several

<footnote><sup>a</sup> Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon</footnote>

<footnote><sup>b</sup> Institut de Recherche Agricole pour le Développement (IRAD), BP 2067 Yaoundé, Cameroon</footnote>

<footnote><sup>c</sup> Department of Chemistry - Biology, Faculty of Science, University of Bertoua, P.O. Box 416, Bertoua, Cameroon</footnote>

<footnote><sup>d</sup> Department of Chemistry, Higher Teacher Training College, University of Bertoua, P.O. Box 416, Bertoua, Cameroon</footnote>

<footnote><sup>e</sup> Division of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland</footnote>

<footnote><sup>f</sup> UMR 152 Pharma Dev, Université de Toulouse, IRD, UPS, France</footnote>

<footnote>* Corresponding author at: Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon.</footnote>

<footnote>** Corresponding author at: Division of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.</footnote>

<footnote>E-mail addresses: oliviernote1@yahoo.fr (O.P. Note), olivier.potterat@unibas.ch (O. Potterat).</footnote>
secondary metabolites including oleanane-type saponins [7] with various biological properties such as antifungal, cytotoxic, anti-oxidative, and antimicrobial activities [8,9]. As part of our ongoing search for antifungal saponins from Cameroonian medicinal plants, we have re-investigated *M. acuminata*. This paper reports on the isolation and chemical characterization of four triterpenoid saponins sharing a rare *N*-acetylglucosaminyl residue.

As saponins with an *N*-acetyl glucosaminyl residue have shown promising activity against phytopathogens [10], enriched saponin fractions and selected isolates were also evaluated for their antifungal properties against phytopathogenic fungi and oomycetes.

## 2. Experimental section

### 2.1. General experimental procedures

For Medium Pressure Liquid Chromatography (MPLC), a system consisting of a Buchi Pump manager C-615, Pump module C-605 and borosilicate glass columns (460 × 25 mm and 460 × 15 mm) was used (Büchi Labortechnik, Flawil, Switzerland). Both MPLC and Vacuum-Liquid Chromatography (VLC) were carried out on silica gel 60 (40–63 µm, Merck), while column chromatography (CC) was carried out on Diaion HP-20 (Sigma-Aldrich, St.-Louis, USA) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Thin layer chromatography (TLC) was performed on pre-coated silica gel plates (60 F254, Merck) with *n*-butanol-acetic acid-water (60,15:25) as eluent and detection with vanillin reagent (10 % H₂SO₄ and 1 % vanillin in ethanol).

NMR spectra including ¹H, ¹³C-DEPTq, COSY, HSQC, HMBC and ROESY were measured on a Bruker Avance III 500 MHz spectrometer equipped with a 5 mm BBO probe (Bruker Switzerland AG, Fällanden, Switzerland) at 23 °C. Chemical shifts (δ) are reported in ppm and were referenced to the solvent signal. Coupling constants (J) are given in Hz. Data were analysed using MestReNova (version 14.2.0–26,256, Mestrelab Research SL, Santiago de Compostela, Coruna, Spain) and Spectrus Processor (version 2024.2.0, ACD/Labs, Toronto, Canada) softwares.

High-resolution mass spectra (HRMS) were recorded on a UHPLC system (Agilent Technologies, Santa Clara, CA, USA) connected to a Q-Exactive HF Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with a HESI-II source. 1 µL samples (ca. 200 ng/µL in methanol) were separated on an Acquity BEH C18 column (2.1 × 50 mm I.D.; 1.7 µm, Waters Corp., Milford, MA, USA) that was equipped with a 5 mm pre-column of the same phase at a flow rate of 0.6 mL/min and a column oven temperature of 30 °C. The mobile phase consisted of water (A) and acetonitrile (B) each containing 0.1 % formic acid. The gradient was defined as follows: 5 % B (1 min); 5–100 % B (7 min); 100 % B (4 min); 100–5 % B (3.9 min). The HESI-II source was operated at a source voltage of 3.5 kV, sheath gas flow rate of 55 units, auxiliary gas flow rate of 15 units, auxiliary gas temperature of 250 °C, sweep gas flow rate of 0, capillary temperature of 350 °C and an S-Lens RF level of 45. Mass scans from *m/z* 150–1400 were recorded in positive ion mode.

Optical rotation data were recorded on a Jasco P-2000 spectropolarimeter (Portmann Instruments AG, Biel-Benken, Switzerland) equipped with a 1 dm path-length sample cell at a constant temperature of 25 °C and a wavelength of 589 nm (Na-D line). Samples were dissolved in HPLC-grade methanol at a concentration of 1.0 mg/mL and 30 measurements were averaged. Specific optical rotation values ([(α)]<sup>25</sup><sub>D</sub>) are given in deg. mL g<sup>-1</sup> dm<sup>-1</sup>, and concentrations (c) are given in g/ 100 mL.

### 2.2. Plant material

The stem bark of *Massularia acuminata* (G.Don) Bullock ex Hoyle was harvested in December 2021 at Mbalmayo (Centre region of Cameroon) under the guidance of Mr. Victor Nana, botanist at the National

Herbarium of Cameroon (NHC), where a voucher specimen (44,578/ HNC) was deposited.

### 2.3. Extraction and isolation

The air-dried, powdered stem bark of *M. acuminata* (1500 g) was extracted with 5 L of ethanol-H₂O (80:20) by maceration at room temperature during 72 h. The resulting hydroalcoholic solution was then evaporated to dryness under reduced pressure to yield a brown residue (135 g). This residue was suspended in 300 mL of H₂O and then partitioned by solvents of increasing polarity starting with *n*-hexane (500 mL × 2), followed with ethyl acetate (500 mL × 2) and water-saturated *n*-butanol (500 mL × 4). The *n*-BuOH soluble phase was evaporated to dryness affording 40 g of a brown gum residue. A portion of this residue (38 g) was suspended in 150 mL of water and then submitted to column chromatography (CC) using Diaion HP-20 resin (column size 40 × 4.3 cm, i.d.), eluting with H₂O-MeOH (80:20; 50:50 and 0:100) to afford three main fractions, Frs. A (13 g), B (7 g) and C (11 g), after TLC monitoring. The 100 % MeOH fraction (Fr. C) was submitted to VLC using silica gel, eluted with CH₂Cl₂-MeOH (80:20) and CH₂Cl₂-MeOH-H₂O (80:20:2, 70:30:5) to give three subfractions labelled Frs. C1 (2.7 g), C2 (3.2 g) and C3 (2.9 g). Each of the subfractions was submitted to subsequent MPLC (2 bars; 6 mL/min) on silica gel using CH₂Cl₂-MeOH (80:20) for Fr. C1, CH₂Cl₂-MeOH-H₂O (80:20:2) for Fr. C2, and a step gradient (CH₂Cl₂-MeOH-H₂O 80:20:2; 70:30:3; 70:30:5) for Fr. C3. Subfraction Fr. C1 gave compound 1 (16.0 mg) while subfraction Fr. C2 afforded compound 2 (6.3 mg). Compounds 3 (14.9 mg) and 4 (5.3 mg) were obtained from Fr. C3 after further purification through MPLC (CH₂Cl₂-MeOH-H₂O 70:30:3; 2 bars; 6 mL/min) on silica gel and CC on Sephadex LH-20 eluted with MeOH.

## 2.4. Spectroscopic data of compounds

### 2.4.1. Massularoside A (1)

White amorphous powder; [α]<sup>25</sup><sub>D</sub>-0.1 (c 0.1, MeOH), (Zeng et al., 2014, [α]<sup>25</sup><sub>D</sub>-1.11 (c 0.36, MeOH)); ¹H NMR (C₅D₅N, 500 MHz) and ¹³C NMR (C₅D₅N, 125 MHz) data, see Tables 1 and 2; HRESIMS *m/z*: 806.5069 [M + H]<sup>+</sup> (calcd for C₄₄H₇₂NO₁₂ 806.5054, Δ 1.9 ppm).

### 2.4.2. Massularoside B (2)

White amorphous powder; [α]<sup>25</sup><sub>D</sub>-17.1 (c 0.1, MeOH); ¹H NMR (C₅D₅N, 500 MHz) and ¹³C NMR (C₅D₅N, 125 MHz) data, see Tables 1 and 2; HRESIMS *m/z*: 968.5597 [M + H]<sup>+</sup> (calcd for C₅₀H₈₂NO₁₇ 968.5582, Δ 1.5 ppm).

### 2.4.3. Massularoside C (3)

White amorphous powder; [α]<sup>25</sup><sub>D</sub>-20.4 (c 0.1 MeOH); ¹H NMR (C₅D₅N, 500 MHz) and ¹³C NMR (C₅D₅N, 125 MHz) data, see Tables 1 and 2; HRESIMS *m/z* 1130.6071 [M + H]<sup>+</sup> (calcd for C₅₆H₉₃NO₂₂ 1130.6111, Δ 3.5 ppm).

### 2.4.4. Massularoside D (4)

White amorphous powder; [α]<sup>25</sup><sub>D</sub>-9.1 (c 0.1 MeOH); ¹H NMR (C₅D₅N, 500 MHz) and ¹³C NMR (C₅D₅N, 125 MHz) data, see Tables 1 and 2; HRESIMS *m/z* 1130.6062 [M + H]<sup>+</sup> (calcd for C₅₆H₉₃NO₂₂ 1130.6111, Δ 4.3 ppm).

## 2.5. Acid hydrolysis and sugar analysis

Each compound (1–4, 0.4–1.0 mg) was hydrolyzed with 2 M HCl (0.5 mL) for 15 h at 100 °C. The hydrolysate was extracted with ethyl acetate (EtOAc, 2 × 0.5 mL). The aqueous phase was dried under nitrogen and then under high vacuum. The residue was treated with 250 µL of a mixture of (+)-2-butanol and acetyl chloride 10:0.5 (v/v) at 55 °C for 15 h. After drying under nitrogen, the residue was derivatized with 250 µL of a mixture of trifluoroacetic anhydride (TFAA) and EtOAc 3:4
<table><caption>**Table 1**
NMR spectroscopic data (500 MHz for ¹H and 125 MHz for ¹³C, C₅D₅N) for the aglycone moiety of compounds 1–4 (δ in ppm and J in Hz)ª.</caption><thead><tr><th rowspan="2">C</th><th rowspan="2"></th><th colspan="2">1</th><th colspan="2">2</th><th colspan="2">3</th><th colspan="2">4</th></tr><tr><th>δ<sub>C</sub></th><th>δ<sub>H</sub></th><th>δ<sub>C</sub></th><th>δ<sub>H</sub></th><th>δ<sub>C</sub></th><th>δ<sub>H</sub></th><th>δ<sub>C</sub></th><th>δ<sub>H</sub></th></tr></thead><tbody><tr><td rowspan="2">1</td><td rowspan="2">CH<sub>2</sub></td><td>38.8</td><td>1.34</td><td>38.9</td><td>1.34</td><td>38.8</td><td>1.33</td><td>38.8</td><td>1.33</td></tr><tr><td></td><td>0.87</td><td></td><td>0.77</td><td></td><td>0.78</td><td></td><td>0.78</td><td></td></tr><tr><td rowspan="2">2</td><td rowspan="2">CH<sub>2</sub></td><td>26.6</td><td>2.14</td><td>26.6</td><td>2.14</td><td>26.6</td><td>2.12</td><td>26.6</td><td>2.12</td></tr><tr><td></td><td>1.70</td><td></td><td>1.72</td><td></td><td>1.68</td><td></td><td>1.68</td><td></td></tr><tr><td>3</td><td>CH</td><td>89.5</td><td>3.22 (dd, J = 11.7; 4.4)</td><td>89.6</td><td>3.21 (dd, J = 11.8; 4.5)</td><td>89.6</td><td>3.21 (dd, J = 12.1; 4.6)</td><td>89.6</td><td>3.20 (dd, J = 12.1; 4.6)</td></tr><tr><td>4</td><td>C</td><td>39.5</td><td>/</td><td>39.5</td><td>/</td><td>39.5</td><td>/</td><td>39.5</td><td>/</td></tr><tr><td>5</td><td>CH</td><td>56.0</td><td>0.75 (br d, J = 12.1)</td><td>56.0</td><td>0.74 (br d, J = 12.3)</td><td>56.0</td><td>0.72 (br d, J = 12.1)</td><td>56.1</td><td>0.72 (br d, J = 12.1)</td></tr><tr><td rowspan="2">6</td><td rowspan="2">CH<sub>2</sub></td><td>18.9</td><td>1.48</td><td>18.9</td><td>1.46</td><td>18.8</td><td>1.43</td><td>18.8</td><td>1.43</td></tr><tr><td></td><td>1.29</td><td></td><td>1.26</td><td></td><td>1.29</td><td></td><td>1.29</td><td></td></tr><tr><td rowspan="2">7</td><td rowspan="2">CH<sub>2</sub></td><td>33.5</td><td>1.45</td><td>33.4</td><td>1.44</td><td>33.4</td><td>1.43</td><td>33.4</td><td>1.43</td></tr><tr><td></td><td>1.28</td><td></td><td>1.33</td><td></td><td>1.32</td><td></td><td>1.32</td><td></td></tr><tr><td>8</td><td>C</td><td>40.0</td><td>/</td><td>40.2</td><td>/</td><td>40.2</td><td>/</td><td>40.2</td><td>/</td></tr><tr><td>9</td><td>CH</td><td>48.3</td><td>1.61</td><td>48.4</td><td>1.58</td><td>48.3</td><td>1.57</td><td>48.3</td><td>1.57</td></tr><tr><td>10</td><td>C</td><td>37.3</td><td>/</td><td>37.3</td><td>/</td><td>37.3</td><td>/</td><td>37.2</td><td>/</td></tr><tr><td rowspan="2">11</td><td rowspan="2">CH<sub>2</sub></td><td>23.8</td><td>1.99</td><td>23.8</td><td>1.98</td><td>23.7</td><td>1.97</td><td>23.8</td><td>1.97</td></tr><tr><td></td><td>1.88</td><td></td><td>1.88</td><td></td><td>1.86</td><td></td><td>1.86</td><td></td></tr><tr><td>12</td><td>CH</td><td>122.8</td><td>5.48 (br s)</td><td>123.2</td><td>5.43 (s)</td><td>123.2</td><td>5.41 (s)</td><td>123.2</td><td>5.43 (s)</td></tr><tr><td>13</td><td>C</td><td>145.2</td><td>/</td><td>144.5</td><td>/</td><td>144.5</td><td>/</td><td>144.5</td><td>/</td></tr><tr><td>14</td><td>C</td><td>42.5</td><td>/</td><td>42.5</td><td>/</td><td>42.4</td><td>/</td><td>42.4</td><td>/</td></tr><tr><td rowspan="2">15</td><td rowspan="2">CH<sub>2</sub></td><td>28.6</td><td>2.15</td><td>28.6</td><td>2.16</td><td>28.5</td><td>2.16</td><td>28.6</td><td>2.15</td></tr><tr><td></td><td>1.20</td><td></td><td>1.18</td><td></td><td>1.19</td><td></td><td>1.19</td><td></td></tr><tr><td rowspan="2">16</td><td rowspan="2">CH<sub>2</sub></td><td>24.0</td><td>1.90</td><td>24.1</td><td>1.89</td><td>24.0</td><td>1.89</td><td>24.0</td><td>1.90</td></tr><tr><td></td><td>1.88</td><td></td><td>1.87</td><td></td><td>1.87</td><td></td><td>1.87</td><td></td></tr><tr><td>17</td><td>C</td><td>46.8</td><td>/</td><td>46.6</td><td>/</td><td>46.6</td><td>/</td><td>46.5</td><td>/</td></tr><tr><td>18</td><td>CH</td><td>42.3</td><td>3.33 (dd, J = 13.7; 4.6)</td><td>42.1</td><td>3.22 (dd, J = 13.7; 4.5)</td><td>41.9</td><td>3.23 (dd, J = 13.5; 4.6)</td><td>42.0</td><td>3.23 (dd, J = 13.6; 4.6)</td></tr><tr><td rowspan="2">19</td><td rowspan="2">CH<sub>2</sub></td><td>47.0</td><td>1.81</td><td>47.3</td><td>1.76</td><td>46.5</td><td>1.74</td><td>47.3</td><td>1.74</td></tr><tr><td></td><td>1.27</td><td></td><td>1.24</td><td></td><td>1.23</td><td></td><td>1.23</td><td></td></tr><tr><td>20</td><td>C</td><td>31.3</td><td>/</td><td>31.1</td><td>/</td><td>31.1</td><td>/</td><td>31.1</td><td>/</td></tr><tr><td rowspan="2">21</td><td rowspan="2">CH<sub>2</sub></td><td>34.6</td><td>1.46</td><td>34.2</td><td>1.36</td><td>34.3</td><td>1.33</td><td>34.3</td><td>1.33</td></tr><tr><td></td><td>1.20</td><td></td><td>1.08</td><td></td><td>1.08</td><td></td><td>1.08</td><td></td></tr><tr><td rowspan="2">22</td><td rowspan="2">CH<sub>2</sub></td><td>33.2</td><td>2.05</td><td>32.9</td><td>2.06</td><td>32.9</td><td>2.05</td><td>32.9</td><td>2.04</td></tr><tr><td></td><td>1.83</td><td></td><td>1.83</td><td></td><td>1.82</td><td></td><td>1.83</td><td></td></tr><tr><td>23</td><td>CH<sub>3</sub></td><td>17.3</td><td>0.95 (s)</td><td>17.3</td><td>0.96 (s)</td><td>17.2</td><td>0.96 (s)</td><td>17.3</td><td>0.96 (s)</td></tr><tr><td>24</td><td>CH<sub>3</sub></td><td>28.5</td><td>1.16 (s)</td><td>28.5</td><td>1.15 (s)</td><td>28.5</td><td>1.15 (s)</td><td>28.5</td><td>1.15 (s)</td></tr><tr><td>25</td><td>CH<sub>3</sub></td><td>15.7</td><td>0.76 (s)</td><td>15.8</td><td>0.81 (s)</td><td>15.8</td><td>0.83 (s)</td><td>15.8</td><td>0.81 (s)</td></tr><tr><td>26</td><td>CH<sub>3</sub></td><td>17.7</td><td>0.99 (s)</td><td>17.8</td><td>1.11 (s)</td><td>17.8</td><td>1.10 (s)</td><td>17.8</td><td>1.11 (s)</td></tr><tr><td>27</td><td>CH<sub>3</sub></td><td>26.5</td><td>1.30 (s)</td><td>26.5</td><td>1.27 (s)</td><td>26.4</td><td>1.25 (s)</td><td>26.4</td><td>1.26 (s)</td></tr><tr><td>28</td><td>C</td><td>180.6</td><td>/</td><td>176.8</td><td>/</td><td>176.9</td><td>/</td><td>176.8</td><td>/</td></tr><tr><td>29</td><td>CH<sub>3</sub></td><td>24.1</td><td>1.02 (s)</td><td>24.0</td><td>0.90 (s)</td><td>24.0</td><td>0.89 (s)</td><td>24.0</td><td>0.90 (s)</td></tr><tr><td>30</td><td>CH<sub>3</sub></td><td>33.6</td><td>0.96 (s)</td><td>33.5</td><td>0.92 (s)</td><td>33.4</td><td>0.89 (s)</td><td>33.5</td><td>0.92 (s)</td></tr></tbody></table>

Assignments are based on HMBC, HSQC, COSY and ¹³C-DEPTq experiments. a) Overlapped ¹H NMR signals are reported without multiplicity.

(v/v) at 55 °C for 1 h. GC-MS analysis was performed as previously described [11]. D-glucosamine ($t_R$ = 20.06/24.26 min) and L-rhamnose ($t_R$ = 10.47 min) were identified in the hydrolysate of compound 1, while D-glucosamine, L-rhamnose and D-glucose ($t_R$ = 15.11/17.12 min) were detected in the hydrolysates of 2–4. The two peaks observed for D-glucose and D-glucosamine are due to the anomeric forms after derivatization.

## 2.6. Assay for antifungal activity

### 2.6.1. Microorganisms and culture conditions

Isolates of Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Pythium myriotylum, Phytophthora infestans, Phytophthora megakarya and Botrytis cinerea were supplied by the Regional Biocontrol and Applied Microbiology Laboratory of the Institute of Agricultural Research for Development in Cameroon. The organisms were maintained on potato dextrose agar (PDA) or Sabouraud dextrose agar plates incubated at 25–28 °C for 5–7 days to promote sporulation and active mycelial growth. Zoospore suspensions of P. megakarya and P. myriotylum for MIC determination were prepared by flooding plates with sterile distilled water, gently scraping the surface to release spores, and filtering through sterile gauze to remove mycelial fragments. The concentration of zoospores was determined using a Malassez hemocytometer under a light microscope, and suspensions were adjusted to 1 × 10⁵ spores/mL for each species [12,13].

### 2.6.2. Antifungal assay

Antifungal activity of fractions was assessed against A. niger, A. flavus, F. oxysporum, P. myriotylum, P. infestans, P. megakarya and B. cinerea. Medium supplementation was according to the protocol described by Behiry et al., 2023 [14]. M. acuminata fractions were dissolved in 2 % dimethyl sulfoxide (DMSO). The mycelial explants of the seven pathogens (5 mm) were taken from 7-day cultures and transferred individually to the centre of a Petri dishes containing PDA culture medium supplemented with different concentrations of fractions (2 mg/mL, 1 mg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, and 62.5 µg/mL). The commercial product Bankit® 25 SC Azoxystrobin (Syngenta, Mexico City, Mexico) was used as positive control. The inoculated dishes were sealed with cling film and incubated at 25 °C for 8 days. All tests were carried out in triplicate. The diameters of mycelial growth were measured and the percent inhibition was calculated by the formula given below:

$$ \text{Inhibition (\%)} = \frac{\text{Dc} - \text{Dt}}{\text{Dc}} \times 100 $$

Dc: Average diameter of mycelial growth in the unsupplemented Petri dish,

Dt: Average diameter of mycelial growth in the supplemented petri dish.

MIC values were determined for 1 and 3 against P. megakarya and P. myriotylum. Test compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare 100 mg/mL stock solutions. For MIC testing, serial
<caption>**Table 2**</caption>

NMR spectroscopic data (500 MHz for ¹H and 125 MHz for ¹³C, C₅D₅N) for the sugar moieties of compounds **1–4** (δ in ppm and J in Hz)ª.

<table><thead><tr><th></th><th></th><th>1</th><th></th><th>2</th><th></th><th>3</th><th></th><th>4</th></tr><tr><th></th><th></th><th>δ<sub>C</sub></th><th>δ<sub>H</sub></th><th>δ<sub>C</sub></th><th>δ<sub>H</sub></th><th>δ<sub>C</sub></th><th>δ<sub>H</sub></th><th>δ<sub>C</sub></th><th>δ<sub>H</sub></th></tr></thead><tbody><tr><td colspan="10"><strong>3-O-Sugars</strong></td></tr><tr><td colspan="10"><strong>GlcNAc</strong></td></tr><tr><td>1</td><td>CH</td><td>104.5</td><td>5.07 (d, J = 8.4)</td><td>104.5</td><td>5.08 (d, J = 8.4)</td><td>104.4</td><td>5.07 (d, J = 7.6)</td><td>104.4</td><td>5.05 (d, J = 8.3)</td></tr><tr><td>2</td><td>CH</td><td>58.0</td><td>4.46</td><td>58.1</td><td>4.46</td><td>58.1</td><td>4.45</td><td>58.1</td><td>4.43</td></tr><tr><td>3</td><td>CH</td><td>82.5</td><td>4.60</td><td>82.5</td><td>4.61</td><td>82.5</td><td>4.60</td><td>81.4</td><td>4.59</td></tr><tr><td>4</td><td>CH</td><td>74.2</td><td>4.32</td><td>74.2</td><td>4.32</td><td>74.2</td><td>4.15</td><td>74.5</td><td>4.18</td></tr><tr><td>5</td><td>CH</td><td>78.5</td><td>3.92</td><td>78.5</td><td>3.91</td><td>78.8</td><td>3.90</td><td>78.5</td><td>3.88</td></tr><tr><td>6</td><td>CH<sub>2</sub></td><td>63.0</td><td>4.50 4.39</td><td>63.0</td><td>4.50 4.39</td><td>69.7</td><td>4.72 4.36</td><td>63.0</td><td>4.54 4.37</td></tr><tr><td></td><td>CO</td><td>170.3</td><td>/</td><td>170.3</td><td>/</td><td>170.3</td><td>/</td><td>170.3</td><td>/</td></tr><tr><td></td><td>CH<sub>3</sub></td><td>23.8</td><td>2.13</td><td>23.8</td><td>2.13</td><td>23.8</td><td>2.12</td><td>23.8</td><td>2.11</td></tr><tr><td></td><td>NH</td><td>/</td><td>9.16 (d, J = 9.0)</td><td>/</td><td>9.17 (d, J = 8.9)</td><td>/</td><td>9.16 (d, J = 9.0)</td><td>/</td><td>9.18 (d, J = 8.9)</td></tr><tr><td colspan="10"><strong>Rha</strong></td></tr><tr><td>1</td><td>CH</td><td>103.7</td><td>5.90 (br s)</td><td>103.7</td><td>5.91 (d, J = 1.6)</td><td>103.6</td><td>5.90 (br s)</td><td>102.8</td><td>5.88 (br s)</td></tr><tr><td>2</td><td>CH</td><td>73.0</td><td>4.55</td><td>73.0</td><td>4.56</td><td>73.6</td><td>4.74</td><td>72.5</td><td>4.71</td></tr><tr><td>3</td><td>CH</td><td>70.8</td><td>4.16</td><td>70.8</td><td>4.16</td><td>70.7</td><td>4.16</td><td>70.6</td><td>4.18</td></tr><tr><td>4</td><td>CH</td><td>73.1</td><td>4.75</td><td>73.1</td><td>4.76</td><td>73.1</td><td>4.76</td><td>85.6</td><td>4.44</td></tr><tr><td>5</td><td>CH</td><td>70.6</td><td>4.94</td><td>70.6</td><td>4.96</td><td>70.5</td><td>4.94</td><td>68.7</td><td>5.00</td></tr><tr><td>6</td><td>CH<sub>3</sub></td><td>18.9</td><td>1.69 (d, J = 6.3)</td><td>18.9</td><td>1.69 (d, J = 6.3)</td><td>18.9</td><td>1.69 (d, J = 6.1)</td><td>18.8</td><td>1.73 (d, J = 6.1)</td></tr><tr><td colspan="10"><strong>Glc I</strong></td></tr><tr><td>1</td><td>CH</td><td></td><td></td><td></td><td></td><td>105.7</td><td>5.05 (d, J = 7.8)</td><td>107.1</td><td>5.25 (d, J = 7.7)</td></tr><tr><td>2</td><td>CH</td><td></td><td></td><td></td><td></td><td>75.5</td><td>4.01</td><td>76.9</td><td>4.12</td></tr><tr><td>3</td><td>CH</td><td></td><td></td><td></td><td></td><td>78.7</td><td>4.21</td><td>79.0</td><td>4.23</td></tr><tr><td>4</td><td>CH</td><td></td><td></td><td></td><td></td><td>71.8</td><td>4.22</td><td>71.3</td><td>4.31</td></tr><tr><td>5</td><td>CH</td><td></td><td></td><td></td><td></td><td>78.3</td><td>4.11</td><td>76.9</td><td>4.11</td></tr><tr><td>6</td><td>CH<sub>2</sub></td><td></td><td></td><td></td><td></td><td>63.0</td><td>4.30 4.25</td><td>62.5</td><td>4.43 4.22</td></tr><tr><td colspan="10"><strong>28-O-Sugar</strong></td></tr><tr><td colspan="10"><strong>Glc</strong></td></tr><tr><td>1</td><td>CH</td><td></td><td></td><td>96.1</td><td>6.36 (d, J = 8.1)</td><td>96.0</td><td>6.27 (d, J = 8.1)</td><td>96.1</td><td>6.35 (d, J = 8.1)</td></tr><tr><td>2</td><td>CH</td><td></td><td></td><td>74.5</td><td>4.22</td><td>74.2</td><td>4.32</td><td>74.5</td><td>4.22</td></tr><tr><td>3</td><td>CH</td><td></td><td></td><td>79.3</td><td>4.28</td><td>79.0</td><td>4.21</td><td>79.2</td><td>4.56</td></tr><tr><td>4</td><td>CH</td><td></td><td></td><td>71.4</td><td>4.38</td><td>71.2</td><td>4.35</td><td>71.4</td><td>4.37</td></tr><tr><td>5</td><td>CH</td><td></td><td></td><td>79.7</td><td>4.04</td><td>79.1</td><td>4.20</td><td>79.7</td><td>4.05</td></tr><tr><td>6</td><td>CH<sub>2</sub></td><td></td><td></td><td>62.5</td><td>4.46 4.42</td><td>62.9</td><td>4.49 4.37</td><td>62.9</td><td>4.50 4.38</td></tr></tbody></table>

Assignments are based on HMBC, HSQC, COSY, ROESY and ¹³C-DEPTq experiments. a) Overlapped ¹H NMR signals are reported without multiplicity.

two-fold dilutions were made in Sabouraud dextrose broth (SDB) to achieve final concentrations from 1000 µg/mL (1 mg/mL) down to 15.625 µg/mL. The final DMSO concentration did not exceed 1 %. A 1 % DMSO control was included with DMSO added to SDB at the same final concentration as in the highest compound concentration, with inoculum but no test compound, to assess any potential inhibitory effect of DMSO on fungal growth [13].

A sterile 96-well microtiter plate was used for the broth micro-dilution assay. In each well, 100 µL of the appropriate compound dilution in SDB was dispensed, followed by 100 µL of the calibrated zoospore suspension of either *P. megakarya* or *P. myriotylum*, for a final volume of 200 µL per well. Each concentration was tested in triplicate for both species. Controls included a positive control (SDB + azoxystrobin, no compound, no DMSO), a 1 % DMSO control (SDB + 1 % DMSO + azoxystrobin, no compound) and a negative control (SDB, no compound, no inoculum). Plates were sealed and incubated at 25–28 °C for 48–72 h without shaking. After incubation, wells were examined visually for turbidity or mycelial mat formation. MIC values were defined as the lowest concentration (µg/mL) of the test compound that completely inhibited visible growth compared to the positive control. For confirmation, aliquots from wells with no visible growth were subcultured onto fresh SDB agar plates and incubated for an additional 48 h to check for viability [13]. Results are reported as mean MIC values from at least three independent experiments.

### 3. Results and discussion

Air-dried powdered stem bark of *M. acuminata* was extracted with 80 % (v/v) aqueous ethanol by maceration at room temperature for 72 h. After evaporation of the solvent, the resulting dark residue was suspended in water and partitioned successively with *n*-hexane, ethyl acetate and water-saturated *n*-butanol (*n*-BuOH). The *n*-BuOH soluble phase was evaporated to dryness affording a fraction which was submitted to column chromatography (CC) on Diaion HP-20 resin to yield saponin-enriched fractions. Further fractionations by a combination of vacuum-liquid chromatography (VLC) and medium pressure liquid chromatography (MPLC) on silica gel followed by CC on Sephadex LH-20 afforded four saponins (1–4) as amorphous powders (Fig. 1).

The molecular formula of compound **1** was determined as C₄₄H₇₁NO₁₂ from the protonated molecular ion peak at *m/z* 806.5069 [M + H]⁺ (calcd. for C₄₄H₇₂NO₁₂ 806.5054) observed in positive ion mode HRESIMS. The ¹H NMR spectrum revealed the presence of seven angular methyl signals at δ<sub>H</sub> 0.76 (s, H-25), 0.95 (s, H-23), 0.96 (s, H-30), 0.99 (s, H-26), 1.02 (s, H-29), 1.16 (s, H-24), and 1.30 (s, H-27), which correlated in the HSQC spectrum with the carbons at δ<sub>C</sub> 15.7 (C-25), 17.3 (C-23), 33.6 (C-30), 17.7 (C-26), 24.1 (C-29), 28.5 (C-24), and 26.5 (C-27), respectively. Moreover, an oxymethine at δ<sub>H</sub> 3.22 (dd, J = 11.7, 4.4 Hz, H-3) / δ<sub>C</sub> 89.5 (C-3), three olefinic carbons including a methine (δ<sub>H</sub> 5.48, br s, H-12) / δ<sub>C</sub> 122.8 (C-12), a quaternary carbon (δ<sub>C</sub> 145.2, C-13), and a carbonyl (δ<sub>C</sub> 180.6, C-28) were observed. These data enabled the identification of the aglycone moiety as oleanolic acid, which was also supported by comparison with glaberrimosides A–C,
<figure><img src="image_3.png" /><figcaption>Fig. 1. Structures of compounds 1–4.</figcaption></figure>

previously reported in literature [15]. The signal at δC 180.6 was attributable to a free carboxylic acid while the glycosylation induced shift observed for C-3 (δC 89.5) suggested that compound **1** was a monodesmosidic triterpenoid saponin with an osidic chain attached to this position. The α-orientation of H-3 was supported by its coupling constants (J = 11.7, 4.4 Hz).

The ¹H NMR spectrum of compound **1** also showed two anomeric protons at δH 5.07 (d, *J* = 8.4 Hz) and 5.90 (br s), with their corresponding carbons signals observed in the ¹³C-DEPTq spectrum at δC 104.5 and 103.7, respectively. Acid hydrolysis afforded D-glucosamine and L-rhamnose, which were identified by GC-MS analysis after derivatisation with (+)-2-butanol and acetyl chloride. The beta configuration of the glucosamine residue as well as the alpha configuration of the rhamnosyl residue were determined from the coupling constants of the anomeric protons and the ¹³C NMR data [16]. Full NMR assignments of each sugar moiety were based on extensive analysis of 1D and 2D NMR spectra (Table 2). Characteristic signals at δH 2.13 (s, COCH3) / δC 23.8 and δH 9.16 (d, *J* = 9.0 Hz, NH) identified N-acetylglucosamine (2-acetamido-2-deoxy-β-D-glucose, GlcNAc). A methyl group at δH 1.69 (d, *J* = 6.3 Hz, CH3) / δC 18.9 confirmed the presence of a rhamnose unit [16,17]. The sequence of the oligosaccharide chain was established on the basis of COSY, HMBC and ROESY experiments. A cross-peak in the HMBC spectrum between H-3 of the aglycone (δH 3.22) and C-1 of GlcNAc (δC 104.5), as well as a ROESY correlation between H-1 of GlcNAc (δH 5.07) and H-3 of the oleanolic acid moiety (δH 3.22), indicated that the GlcNAc unit was linked to the aglycone at position C-3. The HMBC correlation observed between H-1 of Rha (δH 5.90) and C-3 of GlcNAc (δC 82.5) in conjunction with the COSY spin system from H2-6 to H-3 within the GlcNAc moiety revealed the 1 → 3 linkage between the two sugar units. This was further supported by the ROESY correlation observed between H-1 of Rha (δH 5.90) and H-3 of GlcNAc (δH 4.60) (Fig. 2). The structure of compound **1** was thus established to be 3-O-[α-L-rhamnopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-glucopyranosyl] oleanolic acid and named massularoside A. This compound has already been synthesized [18], but it is isolated for the first time from a natural source in this study.

The HRESIMS of compound **2** showed a protonated molecular ion peak at m/z 968.5597 (M + H)⁺ (calcd for C₅₀H₈₂NO₁₇ 968.5582) which was in accordance with the molecular formula C₅₀H₈₁NO₁₇. Analysis of the ¹H and ¹³C NMR spectra of **2** and comparison with compound **1** and literature data allowed the identification of oleanolic acid as aglycone (Table 1). The difference between the masses of compounds **1** and **2** (162 amu) corresponded to a hexosyl unit. Extensive analysis of the 1D and 2D NMR spectra of compound **2** revealed that the sugar chain

<figure><img src="image_4.png" /><figcaption>Fig. 2. Key COSY, HMBC and ROESY correlations observed for compound **1**.</figcaption></figure>

attached at C-3 was identical to the one attached at this position in compound **1**. In addition, by comparing the NMR spectra of compound **2** with those of compound **1**, six additional oxygenated carbon signals (five tertiary and one secondary) including one anomeric carbon at δC 96.1, were found in the ¹³C-DEPTq NMR spectrum of **2**, while one additional anomeric proton at δH 6.36 (d, *J* = 8.1 Hz) was observed in the ¹H NMR spectrum. These data indicated the presence of one additional glucopyranosyl unit (Glc) in compound **2** which was confirmed by GC-MS analysis after acid hydrolysis. Moreover, the chemical shift of the carboxyl group at C-28 (δC 176.8) suggested glycosylation at this position. This was confirmed by the chemical shift of the anomeric carbon of Glc at δC 96.1 which is characteristic of a glucose unit directly attached at C-28 of a triterpene [10]. Finally, the HMBC correlation between the anomeric proton H-1 at δH 6.36 and C-28 confirmed that the glucopyranosyl sugar unit was linked to C-28 (Fig. 3). Therefore, the structure of compound **2** was determined to be 3-O-[α-L-rhamnopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-glucopyranosyl]-28-β-D-glucopyranosyl oleanolic acid, named as massularoside B.

The HRESIMS of compound **3** exhibited a protonated molecular ion peak at m/z 1130.6071 (M + H)⁺ (calcd for C₅₆H₉₂NO₂₂ 1129.6111) corresponding to the molecular formula C₅₆H₉₁NO₂₂. NMR signals of the aglycone part (Table 1) were almost superimposable on those of **2**, indicating that compound **3** also contains oleanolic acid as aglycone. Compared to compound **2**, a mass difference of 162 amu suggested the presence of an additional hexosyl unit. Acid hydrolysis afforded D-glucosamine, L-rhamnose and D-glucose.

Analysis of the 1D and 2D NMR spectra of compound **3** indicated that a glucopyranosyl unit (Glc) was attached at position C-28 as in compound **2**. On the other hand, by comparing the NMR spectra of compound **3** with those of compound **2**, six additional carbon atom signals were found in the ¹³C-DEPTq NMR spectrum of compound **3**, including

<figure><img src="image_5.png" /><figcaption>Fig. 3. Key COSY, HMBC and ROESY correlations observed for compound **2**.</figcaption></figure>
one anomeric carbon (δ<sub>C</sub> 105.7), while an additional anomeric proton was observed in the ¹H NMR spectrum at δ<sub>H</sub> 5.05 (d, J = 7.8 Hz). These data confirmed the presence of one additional glucopyranosyl unit (Glc I) in compound 3. The downfield shift of C-6 of GlcNAc appearing at δ<sub>C</sub> 69.7 compared to δ<sub>C</sub> 63.0 in **1** and **2** suggested glycosylation at this position. The HMBC correlation between H-1 of Glc I at δ<sub>H</sub> 5.05 and C-6 of the GlcNAc at δ<sub>C</sub> 69.7 confirmed that the additional glucopyranosyl unit was linked to the GlcNAc at position C-6. This was also supported by the ROESY spectrum which showed a correlation between the anomeric proton of Glc I at δ<sub>H</sub> 5.05 and H-6 of GlcNAc at δ<sub>H</sub> 4.36 (Fig. 4). Hence, the structure of compound **3** was determined as 3-O-[α-L-rhamnopyranosyl-(1 → 3)-[β-D-glucopyranosyl-(1 → 6)]-2-acetamido-2-deoxy-β-D-glucopyranosyl]-28-β-D-glucopyranosyl oleanolic acid and named masularoside C.

A protonated molecular ion peak at m/z 1130.6062 [M + H]<sup>+</sup> (calcd for C<sub>56</sub>H<sub>92</sub>NO<sub>22</sub> 1130.6111) was observed in the HRESIMS of compound 4 which indicated that it was an isomer of **3**. Acid hydrolysis afforded D-glucosamine, L-rhamnose and D-glucose. Analysis of 1D and 2D NMR spectra revealed that compound **4** has a distinct sugar chain attached at C-3 of the aglycone compared to compound **3**, while both compounds share an identical β-glucopyranosyl residue at C-28 of the aglycone. The chemical shift of C-6 of GlcNAc appearing at δ<sub>C</sub> 63.0 like in compounds **1** and **2** (instead of δ<sub>C</sub> 69.7 in compound **3**), showed that the GlcNAc moiety was not substituted at this position. On the other hand, the downfield shift of C-4 of Rha at δ<sub>C</sub> 85.6 compared to compound **3** (δ<sub>C</sub> 73.1) suggested that the glucopyranosyl unit was linked to Rha at position C-4. This was confirmed by the HMBC correlation between the proton of the methyl group of Rha (H<sub>3</sub>-6) at δ<sub>H</sub> 1.73 and C-4 of the same sugar Rha at δ<sub>C</sub> 85.6 as well as the HMBC correlation between the anomeric proton H-1 of Glc I at δ<sub>H</sub> 5.25 and C-4 of Rha. In addition, a ROESY correlation between H-1 of Glc I at δ<sub>H</sub> 5.25 and H-4 of Rha δ<sub>H</sub> 4.44 ppm was observed (Fig. 5). Hence, the structure of compound **4** was determined to be 3-O-[β-D-glucopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-glucopyranosyl]-28-O-[β-D-glucopyranosyl] oleanolic acid, named as masularoside D.

The butanol-soluble fraction and two saponin fractions (Frs. C1 and C2, see 2.3) were evaluated for their antifungal properties against a series of phytopathogenic fungi and oomycetes. The butanol-soluble fraction (>60 % inhibition at 125 µg/mL) and the two saponin fractions (>67 % inhibition at 62.5 µg/mL) showed inhibitory activity against the oomycetes *Pythium myriotylum* and *Phytophtora megakarya*. In contrast, no activity was observed against *Aspergillus niger*, *A. flavus*, *Fusarium oxysporum*, *Botrytis cinerea*, and *Phytophtora infestans* (<50 % inhibition at 1.0 mg/mL). As a positive control, the commercial agricultural fungicide azoxystrobin exhibited over 83 % inhibition at a concentration of 225 µg/mL against all tested phytopathogens, except *P. infestans*, which showed 50.7 % inhibition. Compounds **1** and **3**, which were available in sufficient amounts were tested against *P. myriotylum* and *P. megakarya*. While both compounds were inactive against *P. megakarya* (MIC >1000 µg/mL), compound **1** (MIC 125 µg/mL), but not compound **3**, showed moderate inhibitory activity against *P. myriotylum*. The differential activity of **1** and **3** against *P. myriotylum* is in line with previous observations showing the strongest antifungal activities for monodesmosidic saponins [19]. Full bioactivity data are provided in the Supporting Information.

## 4. Conclusions

Compounds **1–4** represent the first saponins reported to contain a 3-O-[α-L-Rha-(1 → 3)-GlcNAc] oleanolic acid structural element, which can therefore be seen as a potential chemotaxonomic marker for *M. acuminata* and possibly the genus *Massularia*. Notably, two saponins previously isolated from *M. acuminata* also feature oleanolic acid as the aglycone but bear distinct glycosidic residues, including a ¹Rha-³Glc moiety in one of them [7,8]. The butanol-soluble fractions and enriched saponin fractions showed promising activity against two

<figure><img src="image_6.png" /><figcaption>Fig. 4. Key COSY, HMBC and ROESY correlations observed for compound 3.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 5. Key COSY, HMBC and ROESY correlations observed for compound 4.</figcaption></figure>

phytopathogenic oomycetes. Further studies will be needed to assess the potential of *M. acuminata* for the control of these organisms causing severe plant diseases.

## CRediT authorship contribution statement

**Charles Roberto Bilongo:** Writing – original draft, Investigation, Data curation. **Lin Marcellin Messi:** Writing – review & editing, Methodology, Formal analysis, Conceptualization. **Jean Faustin Atangana:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Andreas Dietl:** Writing – review & editing, Investigation, Formal analysis, Data curation. **Joséphine Ngo Mbing:** Investigation, Data curation. **Dieudonné Emmanuel Pegnyemb:** Project administration, Conceptualization. **Mohamed Haddad:** Visualization, Validation. **Olivier Placide Note:** Validation, Supervision, Project administration, Conceptualization. **Olivier Potterat:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that there is no conflicting interest.

## Acknowledgements

We gratefully acknowledge Prof. Robin Teufel (Division of Pharmaceutical Biology, University of Basel) for providing the technical infrastructure used for structure elucidation. Thanks are also due to Malik Rakhmanov (Division of Pharmaceutical Biology, University of Basel) for recording the HRESIMS data. L. M. Messi thanks the Royal Society of Chemistry for a RSC Research Fund grant (R23-2541923514).
# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fitote.2025.106866.

## Data availability

Data will be made available on request.

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