# Anti-Toxoplasma gondii screening of eight species used in Côte d'Ivoire, and bio-guided isolation of three previously undescribed nor-sesquiterpenoids and one benzyl glycoside from Elaeis guineensis leaves

Nangouban Ouattara, Mansour Issoufou-Tiny, Abdulmagid Alabdul Magid, Sandie Escotte-Binet, Philomène Akoua Yao-Kouassi, Isabelle Villena, Laurence Voutquenne-Nazabadioko

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Nangouban Ouattara, Mansour Issoufou-Tiny, Abdulmagid Alabdul Magid, Sandie Escotte-Binet, Philomène Akoua Yao-Kouassi, et al.. Anti-Toxoplasma gondii screening of eight species used in Côte d'Ivoire, and bio-guided isolation of three previously undescribed nor-sesquiterpenoids and one benzyl glycoside from Elaeis guineensis leaves. Phytochemistry, 2025, 234, pp.114442. 10.1016/j.phytochem.2025.114442. hal-04951281v2

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# Anti-Toxoplasma gondii screening of eight species used in Côte d'Ivoire, and bio-guided isolation of three previously undescribed nor-sesquiterpenoids and one benzyl glycoside from Elaeis guineensis leaves

Nangouban Ouattara<sup>a,b,d,*</sup> ID, Mansour Issoufou-Tiny<sup>a</sup> ID, Abdulmagid Alabdul Magid<sup>a</sup>, Sandie Escotte-Binet<sup>b,c</sup> ID, Philomène Akoua Yao-Kouassi<sup>d,e</sup>, Isabelle Villena<sup>b,c</sup>, Laurence Voutquenne-Nazabadioko<sup>a</sup>

## ARTICLE INFO

### Keywords:

Toxoplasma gondii

Vero cell

Cytotoxicity

Bioguided fractionation

NMR

Dereplication

Medicinal plants

Elaeis guineensis

Nor-sesquiterpene

Megastigman derivatives

Benzyl glycoside

## ABSTRACT

The apicomplexan parasite *Toxoplasma gondii* causes toxoplasmosis, a ubiquitous and cosmopolitan parasitosis, generally asymptomatic and potentially dangerous for the fetus and highly immunocompromised patients. Pyrimethamine and sulfadiazine, supplemented with folic acid, are the drugs of choice to treating the disease, but they produce severe side effects and treatments fail due to drug resistance. New anti-*Toxoplasma* compounds are needed, and natural compounds can be a good source for obtaining them. The antiparasitic activity of 40 polar and non-polar extracts of eight antiparasitic medicinal plants used in Côte d'Ivoire, and selected based on ethnopharmacological survey, were evaluated *in vitro* against *T. gondii*. Among them, the hydromethanolic extract of the *Elaeis guineensis* leaves exhibited the best parasite growth inhibition (94% ± 0.07) at 25 µg/mL without being cytotoxic at the same dose. The fractionation of this extract did not allow the recovery of antitoxoplasmic activity in its individualized fractions. The ¹³C-NMR based dereplication of this extract and the purification of two fractions highlighted the presence of saccharides (3 and 8–11), cardanols (12–13), terterpene (14), sterol (15), nor-sesquiterpene (16), megastigman derivatives (7 and 17–19), glycosylated benzyl (20–21), flavonoids (4–6 and 22), pseudoalkaloid (23), and allyl glycoside (24). Nine compounds 1–9 were identified during the dereplication process, whereas fifteen compounds 10–24 were identified after purification and four of them were undescribed (16–18 and 20). A synergistic effect between these metabolites is thought to be responsible for the anti-*Toxoplasma* activity. However, further studies should be carried out to confirm and explain this synergistic or antagonistic effect between the identified compounds.

## 1. Introduction

*Elaeis guineensis* Jacq. (Arecaceae) is the West African oil palm tree, reaching 20–25 m height, with a stipe measuring between 22 and 75 cm in diameter. Its leaves are pinnate and the petiole is robust and thorny. Flavonoids, tannins, saponins, phenolic compounds, alkaloids, and tocotrienols were previously identified in the leaves (Che et al., 2021; Yin et al., 2013; Horvath et al., 2006). Infusion of the leaves, either alone or in alcohol, is employed as an anti-parasitic remedy, particularly

against malaria in Côte d'Ivoire. Nevertheless, oxidised palm oil has been demonstrated to cause kidney, lung, liver and heart toxicity (Ebong et al., 1999). The hydromethanolic extracts of the leaves exhibit antimalarial activity against *P. falciparum* (Annan et al., 2012), and antimicrobial activities (Ebong et al., 1999; Yin et al., 2013).

Toxoplasmosis is a ubiquitous disease of parasitic origin caused by an obligate intracellular eukaryote *Toxoplasma gondii*, whose final host is a felid (Frenkel, 1974). Although most human infections are uncomplicated, the parasite can persist and the infection can be reactivated in

<footnote><sup>a</sup> Université de Reims Champagne-Ardenne, CNRS, ICMR 7312, 51097, Reims, France</footnote>

<footnote><sup>b</sup> Université de Reims Champagne Ardenne, ESCAPE UR 7510, 51097, Reims, France</footnote>

<footnote><sup>c</sup> Centre National de Référence de la Toxoplasmose, CHU Reims, 51092, Reims, France</footnote>

<footnote><sup>d</sup> Laboratoire de Constitution et Réaction de la Matière, UFR Sciences des Structures de la Matière et de Technologie, Université Félix HOUPHOUET-BOIGNY 22, BP 582, Abidjan, Côte d'Ivoire</footnote>

<footnote><sup>e</sup> Université Polytechnique de San Pedro, BP V1800, San Pedro, Côte d'Ivoire</footnote>

<footnote>* Corresponding author. Université de Reims Champagne-Ardenne, CNRS, ICMR 7312, 51097, Reims, France.
E-mail address: nangouban.ouattara@u-bordeaux.fr (N. Ouattara).</footnote>
immunosuppressed patients leading to cerebral or generalized toxoplasmosis in immunocompromised patients (Robert-Gangneux et al., 2022). In add, when infection is acquired during pregnancy, it can cause congenital toxoplasmosis with serious consequences for the fetus (Wong and Remington, 1994; McLeod et al., 2009; Codiacconi et al., 2020) or the infant. *T. gondii* could also cause severe ocular disease in immunocompetent patients (Holland, 2004; Kim et al., 2013) and in congenital toxoplasmosis (Bosch-Driessen et al., 2002). It is estimated in a recent meta-analysis that about 30–35% of the world’s population is infected with *T. gondii* (Bigna et al., 2020). Toxoplasmosis is ranked as the third largest contributor to the health burden caused by foodborne illness in Europe (Sousa et al., 2023). In Côte d'Ivoire, the latest studies Epidemiological studies have shown that seroprevalence has decreased from 80% in the 1960s to 45 % in 2014 (Bonouman-Ira et al., 2017). The current treatment of acute toxoplasmosis is the combination of sulfadiazine and pyrimethamine, which act synergistically to block the folate pathway, involved in DNA synthesis with the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes. Unfortunately, this inhibition also creates a deficiency in the host leading to adverse effects (hematopoiesis and foetological disorders) (Dujovne et al., 1967).

In addition to these side effects, there is drug resistance (Anacleto-Santos et al., 2023); therapeutic failures (Derouin, 2001; Baatz et al., 2006), and lack of a human vaccine shows that it is of great importance to identify novel potent candidates that would be well-tolerated and act on both tachyzoites and cysts (Mamaghani et al., 2023). The antiparasitic properties of natural products, such as plant derived substances, have been extensively investigated (Sepulveda-Arias et al., 2015). A review carried out between the period 1981–2019 stated that out of 20 antiparasitic drugs proven as a treatment, there were, nine derived from natural products and three were derived from synthetic pharmacophoric sources of natural products (Newman and Cragg, 2020). In recent years, several studies have been conducted *in vitro* and or *in vivo* that have shown that plant species could effectively inhibit the growth of *T. gondii* (Anacleto-Santos et al., 2023; Darme et al. 2021, 2022).

In this study, eight plant species were selected, based on a literature survey on plants most widely used by traditional healers in Côte d'Ivoire to treat bacterial and parasitic diseases, and for which anti-Toxoplasma activity had never been evaluated. These included: *Combretum micranthum* G. Don (Combretaceae) leaves, *Elaeis guineensis* Jacq. (Arecaceae) leaves, *Erigeron floribundus* Kunth Sch.Bip (Asteraceae) aerial parts, *Oldfieldia africana* Benth. & Hook. F. (Picrodendraceae) stem bark, *Octoknema borealis* Hutch. & Dalziel (Olacaceae) leaves and stem bark, *Omphalocarpum ahia* A. Chev. (Sapotaceae) leaves and stem bark, *Omphalocarpum elatum* Miers stem bark, and whole plant of *Tristemma coronatum* Benth (Melastomataceae). *C. micranthum*, commonly called Kinkeliba, is used in West Africa as a traditional medication for the treatment of malaria, diabetes, high blood pressure and inflammatory diseases. Its leaves extracts showed antitoxoplasmosis (Sepulveda-Arias et al., 2015), antibacterial (Banfi et al., 2014), antiviral activity (Ferrea et al., 1993), and protective effect against kidney damage (Osonwa et al., 2012). *E. floribundus*, a terrestrial herbaceous plant, is used in Côte d'Ivoire for the treatment of skin disorders, dyspepsia, abdominal pains, various diseases of microbial origin (Koné et al., 2002), in AIDS therapy, rheumatism, gout, cystitis, nephritis, dysmenorrhoea, dental pain, headache (Valnet, 1983; Burkill, 1985). *The aerial parts extracts showed* antimicrobial (Atindehou et al., 2002), antifungal (Tra Bi et al., 2008), anthelminthic (Diehl et al., 2004), anti-*Trypanosoma brucei* (Pretrelli et al., 2016), and antimalarial effect (Tepongning et al., 2018). *O. africana* is a large tree considered in Côte d'Ivoire as a powerful fetish tree with efficacious medicinal virtues. The bark is antiseptic and hæmostatic. A decoction of the bark is added to baths and draughts to treat blennorrhoea and act as a pelvic decongestant (Burkill, 1985). The stem bark extracts showed antimalarial (Rusaati et al., 2021), antibacterial and antifungal activities (Berinyuy et al., 2014). The stem bark of

*O. borealis*, a large tree, are used in traditional medicine for the treatment of pulmonary troubles whereas the leaves are used as febrifuges (Abbiw, 1990; Burkill, 1985). *O. ahia* is an evergreen medium-sized tree. It is used in folklore medicine for the treatment of pain, inflammation, bacterial and parasitic diseases (Armah et al., 2016; Burkill, 1997). The stem bark extracts showed antibacterial, anti-leishmania and anti-inflammatory activities (Alake et al., 2021). *O. elatum* is a large tree from West African, used traditionally for the treatment of sterility of males and increases lactation in women. A decoction of the bark, combined with the fruits of peppers (*Capsicum annuum*) and *Solanum anguivi*, is used to treat malaria. A decoction of the young leaves is used for the treatment of cough. Its stem bark extracts showed high *in vitro* anthelmintic (Nyasangox et al., 2018), anti-inflammatory and antimicrobial activities (Sandjo et al., 2014). *T. coronatum* is a semi-prostrate herb or shrub to nearly 1 m high in the ground vegetation of the closed forest, from Guinea to Togo. Leaves are used in folklore medicine as pain-killers, sedatives, for arthritis and rheumatism (Burkill, 1985), against typhoid fever, hemorrhoids, infertility, and skin diseases (Nguenang et al., 2018). Its leaves extracts showed antibacterial activity against a strain of *Pseudomonas aeruginosa* and strains of *Staphylococcus aureus*, as well as antifungal activity against *Candida* species, such as *C. albicans*, *C. tropicalis* and *C. glabrata* (Ahoua et al., 2015).

In this study, *n*-heptane (*n*-Hept), ethyl acetate (EtOAc), dichloromethane (DCM) and aqueous methanol 80% (MeOH 80%) extracts were prepared from the ten selected plants and tested *in vitro* at 25 µg/mL to evaluate their activity against *T. gondii*, and cytotoxicity on normal Vero cell line. In addition, a¹³C-NMR-based dereplication workflow combined with a bioactivity-guided fractionation process was applied on the most active extract (MeOH 80% extract of the leaves of *E. guineensis*) to identify the compounds being involved in the biological effect against *T. gondii*. This paper describes the isolation and characterization of three nor-sesquiterpenes and one benzyl glycoside previously undescribed.

## 2. Results and discussions

### 2.1. Identification of compounds from Elaeis guineensis

Chemical profiling of the six fractions HP₁A-HP₅ by ¹³C-NMR based dereplication was initiated in order to identify the main chemicals that could potentially be involved in the activity of the MeOH 80% extract of *E. guineensis* leaves (Fig. 1). Nine compounds, including two saccharides (1–2), three flavonoids (4–6), one megastigman derivative (7), three aliphatic acid derivatives (3 and 8–9) were identified. The correlated chemical shifts of cluster 1 in fraction HP₁A were assigned to glucopyranose (1) and α-galactopyranosyl-(1 → 3)-β-galactopyranoside (2) (Agrawal, 1992). Similarly, cluster 2 in fraction HP₁B proposed 1-monopalmitin (3) (Hu et al., 2022). The cluster 3 in fraction HP₃ proposed vicenin-2 (4) (Islam et al., 2014) and vitexin-2'-O-β-D-glucopyranoside (5) (Pauli and Junior, 1995). The cluster 4 in fraction HP₃ proposed vitexin (6) (Kim et al., 2005). The cluster 5 in fraction HP₄ proposed roseoside (7) (Matsunami et al., 2010) whereas cluster 6 in fraction HP₅ proposed elaidic acid methyl ester (8) and linoelaidic acid methyl ester (9) (Alexandri et al., 2017) (Fig. 1). The identification of these compounds was confirmed by comparison with literature data and by analysis of HR-ESI-MS spectra of HP₁A-HP₅.

The HP₃ fraction was purified giving four flavonoids (4–6 and 22), four megastigman derivatives (7 and 17–19), two benzyl glycosides (20–21), one pseudo alkaloid (23) and one allyl glycoside (24). Their structures were elucidated through the use of 1D and 2D-NMR experiments, in conjunction with the analysis of HR-ESI-MS data and by comparison with previously reported values. Three compounds 17–18 and 20 were undescribed and the know compounds are vicenin-2 (4) (Islam et al., 2014), vitexin-2'-O-glucoside (5) (Pauli and Junior, 1995), vitexin (6) (Kim et al., 2005), scutellarein-8-C-β-D-glucoside (22) (Kim et al., 2015), roseoside (7) (Matsunami et al., 2010), byzantionoside B (19) (Matsunami et al., 2010), hydrangeifolin I (21) (Hamerski et al.,
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Heatmap of peak intensities of ¹³C NMR (rows) and fractions (columns) from the MeOH 80% extract of *E. guineensis*. This representation allows the visualization of the carbon skeleton of the major compounds.</figcaption></figure>

2005), 2-ethyl-3-methylmaleimide N-β-D-glucopyranoside (23) (Krajewski et al., 1996) and erigeside F (24) (Yamamura et al., 1998) (Fig. 2). Likewise, HP₅ fraction was purified giving four aliphatic acid derivatives (8–11), two cardanol derivatives (12–13), one triterpene (14), one sterol (15) and one undescribed nor-sesquiterpene (16). The known compounds are elaidic acid methyl ester (8), linoelaidic acid methyl ester (9), palmitic acid (10), palmitic acid methyl ester (11) (Alexandri et al., 2017), 3-[12(E)-pentadecenyl]phenol (12), 3-[14 (E)-heptadecenyl]phenol (13) (Liu and Abreu, 2006), ursolic acid (14) (Mahato and Kundu, 1994), and stigmasta-4,24(28)-dien-3-one (15) (Sheu et al., 1999) (Fig. 2).

Compound 16 has the molecular formula C₁₃H₁₆O₄, deduced from the protonated molecular ion at m/z 237.1125 [M + H]+(calcd for C₁₃H₁₇O₄, 237.1127), in positive mode HR-ESI-MS. The ¹H NMR and ¹H-¹H COSY spectra displayed signals of three methyl groups at δ<sub>H</sub> 1.25, 1.26 and 1.74, two methylene protons at δ<sub>H</sub> 2.44 (1H, d, *J* = 17.2 Hz),

2.55 (1H, d, *J* = 17.2 Hz), 2.67 (1H, d, *J* = 18.4 Hz), 3.07 (1H, d, *J* = 18.4 Hz) and one ethylenic proton at δ<sub>H</sub> 6.95 (1H, br. s) (Table 1). This, in a combination with signals in ¹³C NMR and HSQC spectra of two ketones at δ<sub>C</sub> 195.7 and 201.0, four ethylenic carbons at δ<sub>C</sub> 103.1, 131.9, 142.8 and 160.0, two methylene carbons at δ<sub>C</sub> 39.9 and 50.5, two quaternary carbons at δ<sub>C</sub> 39.0 and 83.8, and three methyl carbons at δ<sub>C</sub> 17.5, 21.9 and 23.2, suggest a nor-sesquiterpene derivative (ITO et al., 1997) (Fig. 2). The ketone at δ<sub>C</sub> 201.0 (C-3) correlates in HMBC with the methylene protons H<sub>αβ</sub>-2 at δ<sub>H</sub> 2.67 and 3.07, coupled together with a geminal coupling constant of *J* = 18.4 Hz, which indicates that these protons were between two quaternary carbons. HMBC correlations were observed from H<sub>2αβ</sub>-2 to carbons at δ<sub>C</sub> 39.0 (C-1), 83.8 (C-6) and 131.9 (C-4), from C-1 to the methylene protons H<sub>αβ</sub>-7 at δ<sub>H</sub> 2.44 (d, *J* = 17.2 Hz) and 2.55 (d, *J* = 17.2 Hz), and the methyl protons H-13 (δ<sub>H</sub> 1.25) and H-12 (δ<sub>H</sub> 1.26), and from H<sub>αβ</sub>-7 to the second ketone at δ<sub>C</sub> 195.7 (C-8) (Fig. 3). Similarly, HMBC correlations were observed between

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Structures of compounds purified from HP₃ and HP₅ fractions of *E. guineensis*.</figcaption></figure>
<table><caption>**Table 1**

$^{13}$C and $^{1}$H-NMR spectroscopic data for compounds 16–18 and 20 in CDCl$_{3}$ or MeOH-d$_{4}$.a,b</caption><thead><tr><th rowspan="2">Position</th><th colspan="2">16</th><th colspan="2">17</th><th colspan="2">18</th><th colspan="2">20</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>1</td><td>39.0</td><td>–</td><td>46.2</td><td>–</td><td>43.4</td><td>–</td><td>113.1</td><td>–</td></tr><tr><td>2</td><td>39.9</td><td>3.07 d (18.4)</td><td>35.8</td><td>1.62 dt (13.7, 6.5)<sup>eq</sup></td><td>48.2</td><td>2.18 dt (17.5, 1.9)<sup>ex</sup></td><td>151.3</td><td>–</td></tr><tr><td></td><td></td><td>2.67 d (18.4)</td><td></td><td>1.76 dd (13.7, 7.3)<sup>ex</sup></td><td></td><td>2.59 d (17.5)<sup>eq</sup></td><td></td><td>–</td></tr><tr><td>3</td><td>201.0</td><td>–</td><td>64.5</td><td>3.80 m</td><td>211.2</td><td>–</td><td>145.7</td><td>–</td></tr><tr><td>4</td><td>131.9</td><td>6.95 br. s</td><td>37.5</td><td>1.64 dd (13.8, 6.8)<sup>eq</sup></td><td>49.2</td><td>2.23 dt (17.5, 1.9)<sup>ex</sup></td><td>122.3</td><td>7.39 dd (8.2, 1.5)</td></tr><tr><td></td><td></td><td></td><td></td><td>2.11 dd (13.8, 6.8)<sup>ex</sup></td><td></td><td>2.61 d (17.5)<sup>eq</sup></td><td></td><td>–</td></tr><tr><td>5</td><td>142.8</td><td>–</td><td>85.5</td><td>–</td><td>84.2</td><td>–</td><td>118.6</td><td>6.91 t (8.1)</td></tr><tr><td>6</td><td>83.8</td><td>–</td><td>57.5</td><td>2.49 d (9.6)</td><td>53.3</td><td>1.76 dd (6.7, 1.9)</td><td>123.2</td><td>7.57 dd (8.1, 1.5)</td></tr><tr><td>7</td><td>50.5</td><td>2.55 d (17.2)</td><td>120.3</td><td>5.77 dd (15.4, 9.6)</td><td>20.8</td><td>1.52 m</td><td>170.4</td><td>–</td></tr><tr><td></td><td></td><td>2.44 d (17.2)</td><td></td><td></td><td></td><td>1.90 m</td><td></td><td>–</td></tr><tr><td>8</td><td>195.7</td><td>–</td><td>142.4</td><td>5.87 dd (15.4, 5.4)</td><td>36.2</td><td>1.72 m</td><td>51.6</td><td>3.98 s</td></tr><tr><td>9</td><td>103.1</td><td>–</td><td>67.3</td><td>4.31 q (6.4)</td><td>74.5</td><td>1.74 m</td><td></td><td></td></tr><tr><td>10</td><td>17.5</td><td>1.74 s</td><td>22.3</td><td>1.28 d (6.2)</td><td>18.9</td><td>3.91 m</td><td></td><td></td></tr><tr><td>11</td><td>160.0</td><td>–</td><td>17.8</td><td>1.10 s</td><td>19.5</td><td>1.24 d (6.2)</td><td></td><td></td></tr><tr><td>12</td><td>23.2*</td><td>1.26 s*</td><td>180.0</td><td>–</td><td>78.0</td><td>3.58</td><td></td><td></td></tr><tr><td>13</td><td>21.9*</td><td>1.25 s*</td><td>22.2</td><td>1.36 s</td><td>23.8</td><td>1.34 s</td><td></td><td></td></tr><tr><td>1'</td><td></td><td></td><td></td><td></td><td>glc</td><td></td><td>glc</td><td></td></tr><tr><td>2'</td><td></td><td></td><td></td><td></td><td>100.9</td><td>4.33 d (7.8)</td><td>101.4</td><td>4.89 d (7.7)</td></tr><tr><td>3'</td><td></td><td></td><td></td><td></td><td>73.7</td><td>3.17 dd (9.3, 7.8)</td><td>73.4</td><td>3.54 m</td></tr><tr><td>4'</td><td></td><td></td><td></td><td></td><td>76.8</td><td>3.36 t (9.3)</td><td>76.3</td><td>3.48 m</td></tr><tr><td>5'</td><td></td><td></td><td></td><td></td><td>70.5</td><td>3.23 t (9.3)</td><td>70.1</td><td>3.40 m</td></tr><tr><td>6'</td><td></td><td></td><td></td><td></td><td>75.9</td><td>3.42 ddd (7.4, 5.4, 2.2)</td><td>76.7</td><td>3.36 m</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>67.3</td><td>3.59 dd (11.9, 5.4)</td><td>66.3</td><td>3.61 m</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>3.96 dd (11.9, 2.2)</td><td></td><td>4.02 d (10.8, 1.6)</td></tr><tr><td>1''</td><td></td><td></td><td></td><td></td><td>api</td><td></td><td>rham</td><td></td></tr><tr><td>2''</td><td></td><td></td><td></td><td></td><td>109.5</td><td>5.07 d (2.6)</td><td>100.7</td><td>4.71 d (1.5)</td></tr><tr><td>3''</td><td></td><td></td><td></td><td></td><td>76.6</td><td>3.89 d (2.6)</td><td>70.7</td><td>3.27 t (6.9)</td></tr><tr><td>4''</td><td></td><td></td><td></td><td></td><td>79.1</td><td>–</td><td>71.4</td><td>3.82 dd (3.4, 1.6)</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>73.5</td><td>3.75 d (9.5)</td><td>72.6</td><td>3.36 m</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>3.95 d (9.5)</td><td></td><td></td></tr><tr><td>5''</td><td></td><td></td><td></td><td></td><td>64.2</td><td>3.59 m</td><td>68.4</td><td>3.64 m</td></tr><tr><td>6''</td><td></td><td></td><td></td><td></td><td></td><td></td><td>16.5</td><td>1.21 d (6.2)</td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Main HMBC correlations of compound 16.</figcaption></figure>

methyl protons H-13 and carbons C-12 and C-6. The ethylenic proton H-4 at δ$_{H1}$ 6.95 (δ$_{C4}$ 131.9) showed HMBC correlations with two ethylenic carbons at δ$_{C}$ 142.8 (C-5) and 160.0 (C-11), attached to a hydroxyl. Finally, the methyl protons at δ$_{H}$ 1.74 (H-10), were correlated in the HMBC with the quaternary ethylenic carbon C-9 at δ$_{C}$ 103.1 (Fig. 3). The absolute configurations of C-6 was established (6S) by comparing the experimental ECD spectra of 16 with the published (–)-(6S)- eucomegastigmane A (Yan et al., 2017). The $^{20}$[α]$_{D}$ –0.3° (c 0.1; MeOH) confirm the stéréochemistry. Thus, the structure of compound 16 is (–)-(6S)-4,5,6,7-tetrahydro-5,9-dihydroxy-4,4,8-trimethyl-2(3H),7-naphthalendione which named (–)-(6S)-elaeisdione.

Compound 17 has the molecular formula C$_{13}$H$_{20}$O$_{4}$, deduced from the protonated molecular ion having lost one or two water molecules at m/z 223.1333 [M - H$_{2}$O + H]+(calcd C$_{13}$H$_{19}$O$_{3}$, 223.1312) or 205.1223 [M - 2H$_{2}$O + H]+(calcd C$_{13}$H$_{17}$O$_{2}$ 205.1226) (Hu and al., 2022), in positive mode HR-ESI-MS. Analysis of the $^{1}$H NMR, $^{13}$C NMR, COSY, ROESY, HSQC and HMBC spectra led to the identification of a cucubalactone derivative (Cheng et al., 2002) (Figs. 2 and 4), a

nor-sesquiterpene megastigman. The $^{13}$C-NMR spectrum displayed thirteen signals: three methyls (δ$_{C}$ 17.8, 22.2, 22.3), two methylenes (δ$_{C}$ 35.8, 37.5), three methynes (δ$_{C}$ 57.5, 64.5, 67.3), two ethylenic carbons (δ$_{C}$ 120.3, 142.4), two quaternary signals (δ$_{C}$ 46.2, 85.5) and one carbonyl at δ$_{C}$ 180.0. The signals at δ$_{C}$ 64.5, 67.3, and 85.5 were characterized as carbons bearing an oxygen atom. The similarity of the NMR data of 17 and cucubalactone showed that they had an identical carbon skeleton (Table 1). The main difference in the $^{13}$C-NMR spectra was the presence of one supplementary hydroxy substituent at C-6 in cucubalactone (Cheng et al., 2002). The specific chemical shift of the tertiary carbon C-3 at 64.5 ppm (δ$_{H3}$ 3.80) is indicative of the presence of a tertiary alcohol in a ring. This correlated in HMBC with the methylene protons H$_{αβ}$-2 at δ$_{H}$ 1.62 (dt, *J* = 13.7, 6.5 Hz) and 1.76 (dd, *J* = 13.7, 7.3 Hz) and H$_{αβ}$-4 at δ$_{H4}$ 1.64 (dd, *J* = 13.8, 6.8 Hz), and 2.11 (dd, *J* = 13.8, 6.8 Hz), allowing their respective positioning. Similarly, the protons H$_{αβ}$-4 correlated in HMBC with the methyl C-13 (δ$_{C}$ 22.2), carbons C-2 (δ$_{C}$ 35.8), C-6 (δ$_{C}$ 57.5), C-3 (δ$_{C}$ 64.5) and C-5 (δ$_{C}$ 85.5). The protons H$_{2αβ}$-2 showed HMBC correlation with the methyl C-11 (δ$_{C}$ 17.8), carbons C-4 (δ$_{C}$ 37.5), C-1 (δ$_{C}$ 46.2), C-3, C-6 and the carbonyl C-12 at δ$_{C}$ 180.0 ppm (Fig. 4). The lactone function between C-12 and the quaternary carbon C-5 (δ$_{C}$ 85.5) was corroborated by their specific chemical shifts (Cheng et al., 2002). HMBC correlations from C-5 to protons H-6, H$_{2}$-4, H-13 and H-7 suggested the position of the lateral chain. The ethylenic proton H-7 at δ$_{H}$ 5.77 (1H, dd, *J* = 15.4, 9.6 Hz) is coupled in COSY spectrum with the vicinal protons H-6 (δ$_{H}$ 2.49, d, *J* = 9.6 Hz) and H-8 (δ$_{H}$ 5.87, dd, *J* = 15.4, 5.4 Hz), which correlated in COSY with it vicinal proton H-9 at δ$_{H}$ 4.31 (1H, q, *J* = 6.4 Hz/δ$_{C}$ 67.3). The coupling constant *J* = 15.4 Hz indicated that this ethylenic group was in the trans configuration (Fig. 4). Carbon C-9 correlated in HMBC with the methyl protons H-10 at δ$_{H}$ 1.28 (3H, d, *J* = 6.2 Hz/δ$_{C10}$ 22.3). Finally, the

<footnote>a in ppm, *J* in brackets in Hz.</footnote>

<footnote>b NMR spectra recorded at 500 or 600 MHz ($^{1}$H) and at 125 or 150 MHz ($^{13}$C).</footnote>
<figure><img src="image_6.png" /><figcaption>Fig. 4. Main HMBC, COSY and ROESY correlations of compounds 17–18 and 20.</figcaption></figure>

stereochemistry of carbons C-3, C-6 and C-9 was established by the ROESY correlations between H-3 with H<sub>eq</sub>-4, H-6 with H-11, H-6 with H-13 and H-7 with H-9 or H-8 with H-9, showing that they belong to the same chemical environment, respectively. Thus, the structure of compound **17** was elucidated to be 1β,5β-dimethyl-3β-hydroxy-6α-(3-hydroxy-1-butenyl)-12-oxobicyclo-[3.2.1]-13-oxaoctane, which was named 6-dehydroxy-cucubalactone or elaeisdiol.

Compound **18** has the molecular formula C₂₄H₄₀O₁₂ deduced from the sodium ion at *m/z* 543.2418.

[M + Na]+(calcd C₂₄H₄₀O₁₂Na, 543.2417), in positive mode HR-ESI-MS. The ¹H NMR and ¹³C<sub>J-modulated</sub> NMR spectral data of compound **18** showed similarity to that of compound **17** (Table 1). The difference was presence of a ketone at C-3 (δ<sub>C</sub> 211.2) rather than a hydroxyl, an oxygen-linked methylene forming a cyclised ether-oxy function at C-12 (δ<sub>H</sub> 3.58/δ<sub>C</sub> 78.0) rather than a lactone group, the loss of the ethylenic group in C-7 position, and the presence of a sugar moiety. HMBC correlations from the methyl protons H-13 with quaternary carbon C-5 (δ<sub>C</sub> 84.2), C-3 and C-4, and from methyl protons H-11 with carbons C-5, C-3, C-12, and C-6 (δ<sub>C</sub> 53.3) provided compelling evidence for the cyclised form. Correlations in COSY between H-7 and its vicinal H-6 and H-8, between H-8 and its vicinal H-7 and H-9, and between H-9 and its vicinal H-8 and H-10 justified their respective positions. The stereochemistry of carbons C-1 and C-6 was established by the ROESY correlations between H-12 with H<sub>eq</sub>-2, and H-6, alpha oriented, and between methyl H-11 with protons H-7b and H-8b. The chemical shift of C-9 (δ<sub>H</sub> 3.91/δ<sub>C</sub> 74.5) indicate a carbon bearing a hydroxyl and is indicative for 9S (ca. 74 ppm) configuration as observed for Δ7,8-type of 9-hydroxymegastigmane 9-O-β-D-glucopyranoside like in byzantionoside B (**19**) (Matsunami et al., 2010). The remaining ¹H and ¹³C NMR resonances were assignable to two sugar moieties. The signals for an anomeric proton at δ<sub>H</sub> 4.33 (1H, d, *J* = 7.8 Hz, H-1'), two oxymethylene protons at δ<sub>H</sub> 3.59 (1H, dd, *J* = 11.9, 5.4 Hz) and 3.96 (1H, dd, *J* = 11.9, 2.2 Hz) (H<sub>2</sub>-6'), an oxymethylene carbon at δ<sub>C</sub> 67.3 (C-6'), five oxymethine carbons at δ<sub>C</sub> 70.5 (C-4'), 73.7 (C-2'), 75.9 (C-5'), 76.8 (C-3') and 100.9 (C-1'), and proton coupling constants above 7 Hz, were consistent with those of a β-glucopyranosyl moiety (Table 1). The large coupling constants from anomeric proton (>7 Hz) and the axial correlations observed between H-1'/H-3' and H-1'/H-5' in the ROESY spectrum, indicated their β-configurations. A second anomeric proton at δ<sub>H</sub> 5.07 (1H, d, *J* = 2.6 Hz, H-1') was correlated in COSY with an oxymethylene protons at δ<sub>H</sub> 3.89 (1H, d, *J* = 2.6 Hz, H<sub>β</sub>-2''), and the signals of two oxymethylenes carbon at δ<sub>C</sub> 64.2 (C-5'') and 73.5 (C-4''), are consistent with an apiofuranose. HMBC correlations from H-9 to the anomeric carbon C-1' (δ<sub>C</sub> 100.9) and from H-1'' (δ<sub>H</sub> 5.07) to the carbon C-6' indicate the positions of the sugar chain and the interglycosidic linkage. This is confirmed by the ROESY correlations between H-1'/H-9 and

H-1''/H-6'. Thus, compound **18** was elucidated as (1S,6R, 9R)-6-[3-hydroxybutyl]-1,5-dimethyl-12-oxabicyclo-[3.2.1]-octan-3-one-9-O-

α-apiofuranoside-(1 → 6)-O-β-glucopyranoside name elaeisoside.

Compound **20** has the molecular formula C₂₄H₂₈O₁₃, deduced from the sodium molecular ion at *m/z* 499.1424 [M + Na]+(calcd C₂₀H₂₈O₁₃Na, 499.1428), in positive mode HR-ESI-MS. The ¹H NMR and ¹³C NMR spectral data of compound **20** showed similarity to that of 2,3-dihydroxybenzoic acid methyl ester 3-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (Xu et al., 2009) (Table 1). Analysis of the COSY, ROESY, HSQC and HMBC spectra indicated that in contrast to the compound described by Xu et al., the second glucose unit is replaced by rhamnopyranose in **20**. It is characterised by its methyl group at δ<sub>H</sub> 1.21 (3H, d, *J* = 6.2 Hz) and δ<sub>C</sub> 16.5 (C-6''), and its anomeric proton at δ<sub>H</sub> 4.71 (1H, d, *J* = 1.5 Hz) and carbon at δ<sub>C</sub> 100.7 (C-1''). The coupling constant of H-1'' (³J<sub>H-1''/H-2''</sub> = 1.5 Hz) and the correlations observed between H-1''/H-2'' in the ROESY spectrum of **20**, indicated the α-anomer configuration of the rhamnose unit. The structural elucidation of benzyl was readily established from the specific chemical shifts of an identified aromatic ring [δ<sub>H-5</sub> 6.91/δ<sub>C-5</sub> 118.6, δ<sub>H-4</sub> 7.39/δ<sub>C-4</sub> 122.3, δ<sub>H-6</sub> 7.57/δ<sub>C-6</sub> 123.2] (Table 1). HMBC correlations from H-6 to δ<sub>C</sub> 151.3 (C-2) and 170.4 (C-7), H-5 to δ<sub>C</sub> 113.1 (C-1) and δ<sub>C</sub> 145.7 (C-3) allowed their respective positioning. The ester function was justified by the HMBC crosspeak between C-7 and H<sub>3</sub>-8 (δ<sub>H</sub> 3.98/δ<sub>C</sub> 51.6). The interglycosidic linkage was established by HMBC correlations from H-1' (δ<sub>H</sub> 4.89) to carbon C-3 (δ<sub>C</sub> 145.7) and from H-1'' (δ<sub>H</sub> 4.71) to carbon C-6' (δ<sub>C</sub> 66.3) and by ROESY correlation between H-1''/H-6'. Thus, the structure of **20** was elucidated as methyl ester of 2,3-dihydroxybenzoic acid 3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside (**20**) (Figs. 2 and 4).

## 2.2. Biological screening of crude extracts on *T. gondii*

The *n*-Hept, DCM, EtOAc and 80%MeOH extracts of the ten selected drugs were evaluated for their cytotoxic activity against Vero cells. Among the 40 tested extracts, six were found to be cytotoxic at the concentration of 25 µg/mL, namely the *n*-Hept extract of *T. coronatum*, the DCM extracts of *E. floribundus* and leaves of *O. ahia*, the EtOAc extracts of leaves and bark of *O. borealis* and the MeOH 80% extract of *O. Elatum* (Table 2). The toxicity induced by these extracts on cells leads to cell death, and therefore would have as an immediate consequence preventing cell invasion by tachyzoites or parasite multiplication. Consequently, very few parasites will be marked by antibodies in the enzyme-linked immunosorbent assay (ELISA), which will not reflect the actual inhibition of parasite multiplication by the product. Therefore, only the non-cytotoxic extracts were then evaluated for their
<table><caption>**Table 2**

*In vitro* cytotoxicity of selected plant extracts on Vero cells.</caption><thead><tr><th>Plant species</th><th>Plant part</th><th>Extract</th><th>Cytotoxic activity against Vero cells</th><th>Anti-toxoplasma activity against T. gondii</th></tr></thead><tbody><tr><td rowspan="4">C. micranthum</td><td rowspan="4">leaf</td><td>n-Hep</td><td>3% ± 0.02</td><td>1% ± 0.01</td></tr><tr><td>DCM</td><td>2% ± 0.01</td><td>79% ± 0.18</td></tr><tr><td>EtOAc</td><td>3% ± 0.03</td><td>64% ± 0.23</td></tr><tr><td>MeOH</td><td>3% ± 0.04</td><td>5% ± 0.04</td></tr><tr><td rowspan="4">E. guineensis</td><td rowspan="4">leaf</td><td>n-Hep</td><td>2% ± 0.03</td><td>29% ± 0.16</td></tr><tr><td>DCM</td><td>2% ± 0.01</td><td>1% ± 0.01</td></tr><tr><td>EtOAc</td><td>6% ± 0.06</td><td>9% ± 0.09</td></tr><tr><td>MeOH</td><td>5% ± 0.04</td><td>94% ± 0.07</td></tr><tr><td rowspan="4">E. floribundus</td><td rowspan="4">aerial parts</td><td>n-Hep</td><td>3% ± 0.03</td><td>13% ± 0.13</td></tr><tr><td>DCM</td><td>56% ± 0.9</td><td>nd<sup>a</sup></td></tr><tr><td>EtOAc</td><td>3% ± 0.02</td><td>25% ± 0.18</td></tr><tr><td>MeOH</td><td>4% ± 0.04</td><td>3% ± 0.03</td></tr><tr><td rowspan="4">O. africana</td><td rowspan="4">stem bark</td><td>n-Hep</td><td>10% ± 0.02</td><td>67% ± 0.08</td></tr><tr><td>DCM</td><td>10% ± 0.05</td><td>87% ± 0.09</td></tr><tr><td>EtOAc</td><td>3% ± 0.02</td><td>7% ± 0.07</td></tr><tr><td>MeOH</td><td>7% ± 0.03</td><td>14% ± 0.09</td></tr><tr><td rowspan="4">O. ahia</td><td rowspan="4">leaf</td><td>n-Hep</td><td>6% ± 0.03</td><td>7% ± 0.03</td></tr><tr><td>DCM</td><td>82% ± 0.17</td><td>nd<sup>a</sup></td></tr><tr><td>EtOAc</td><td>16% ± 0.14</td><td>91% ± 0.10</td></tr><tr><td>MeOH</td><td>10% ± 0.04</td><td>6% ± 0.08</td></tr><tr><td rowspan="4">O. ahia</td><td rowspan="4">Stem bark</td><td>n-Hep</td><td>4% ± 0.02</td><td>10% ± 0.09</td></tr><tr><td>DCM</td><td>7% ± 0.02</td><td>96% ± 0.05</td></tr><tr><td>EtOAc</td><td>5% ± 0.07</td><td>23% ± 0.17</td></tr><tr><td>MeOH</td><td>3% ± 0.02</td><td>1% ± 0.01</td></tr><tr><td rowspan="4">O. borealis</td><td rowspan="4">leaf</td><td>n-Hep</td><td>7% ± 0.02</td><td>11% ± 0.12</td></tr><tr><td>DCM</td><td>4% ± 0.03</td><td>5% ± 0.04</td></tr><tr><td>EtOAc</td><td>32% ± 0.10</td><td>nd<sup>a</sup></td></tr><tr><td>MeOH</td><td>11% ± 0.06</td><td>1% ± 0.01</td></tr><tr><td rowspan="4">O. borealis</td><td rowspan="4">Stem bark</td><td>n-Hep</td><td>10% ± 0.02</td><td>19% ± 0.08</td></tr><tr><td>DCM</td><td>3% ± 0.02</td><td>2% ± 0.02</td></tr><tr><td>EtOAc</td><td>23% ± 0.07</td><td>nd<sup>a</sup></td></tr><tr><td>MeOH</td><td>7% ± 0.05</td><td>0% ± 0.00</td></tr><tr><td rowspan="4">O. elatum</td><td rowspan="4">Stem bark</td><td>n-Hep</td><td>5% ± 0.02</td><td>47% ± 0.38</td></tr><tr><td>DCM</td><td>7% ± 0.03</td><td>57% ± 0.13</td></tr><tr><td>EtOAc</td><td>11% ± 0.01</td><td>13% ± 0.19</td></tr><tr><td>MeOH</td><td>31% ± 0.05</td><td>nd<sup>a</sup></td></tr><tr><td rowspan="4">T. coronatum</td><td rowspan="4">whole plant</td><td>n-Hep</td><td>23% ± 0.05</td><td>nd<sup>a</sup></td></tr><tr><td>DCM</td><td>15% ± 0.5</td><td>72% ± 0.36</td></tr><tr><td>EtOAc</td><td>12% ± 0.03</td><td>40% ± 0.31</td></tr><tr><td>MeOH</td><td>7% ± 0.01</td><td>3% ± 0.04</td></tr></tbody></table>

*In vitro* anti-*Toxoplasma* activity of extracts exhibiting less than 20% cytotoxicity at 25 µg/mL (N = 6).

Positive *anti-toxoplasmic* activity control (pyrimethamine): 70–90% parasite growth inhibition rate at 1 µM.

Positive cytotoxicity control (pyrimethamine): 60–70% cytotoxicity at 20 µg/mL (i.e. 80 µM).

a nd = not determined.

antitoxoplasmic activity *in vitro* at 25 µg/mL (Table 2). The results revealed that eight of the 34 non-cytotoxic extracts showed more than 60% parasite growth inhibition at 25 µg/mL. These extracts were the DCM and EtOAc extracts of *C. micranthum*, the MeOH 80% extract of *E. guineensis*, the n-Hept and DCM of *O. africana*, the EtOAc of the leaves and DCM of the stem bark of *O. ahia*, and the DCM extract of *T. coronatum* with a parasite growth inhibition rate of 79%, 64%, 94%, 67%, 87%, 91%, 96%, and 72%, respectively.

Previous study on *C. micranthum* leaves showed that its aqueous extract had moderate antitoxoplasmic activity with IC₅₀ of 254 µg/mL (Benoit-Vical et al., 2000) and 217 µg/mL (Sepulveda-Arias et al., 2015)

against RH tachyzoites of *T. gondii* in the RMC5 cells. However, our work showed also that DCM and EtOAc extracts of this plant possessed anti-toxoplasmic activity at 25 µg/mL on Vero cells with 79% ± 0.18, and 64% ± 0.23 growth inhibition, respectively. The use in traditional medicine of the genus *Omphalocarpum* as an antiparasitic is more highlighted in the literature. This fact has been corroborated by our results which supported the traditional use of the stem bark of *O. ahia* as an antiparasitic (Armah et al., 2016) based on the strong inhibition of *T. gondii* growth at 25 µg/mL of the DCM extracts from stem bark (96% ± 0.05) and EtOAc from leaves (91% ± 0.10). Whereas DCM extract of *O. elatum* was moderately active against *T. gondii* tachyzoites (57% ± 0.13). Our work showed for the first time that the stem bark of *Oldfieldia africana* and all plant of *Tristemma coronatum* were active against *T. gondii* at 25 µg/mL (Table 2). The traditional antiparasitic use of *E. guineensis* leaves in Côte d'Ivoire that had been observed for the 70% hydroethanolic extract against *P. falciparum* (another *Apicomplexa* parasite) with IC₅₀ 1.195 µg/mL (Annan et al., 2012), was supported by the strong inhibition (94% ± 0.07) of *T. gondii* growth at 25 µg/mL by the hydromethanolic 80% extract. This extract was therefore selected for further study to identify the compounds being involved in the biological effect against *T. gondii*.

## 2.3. Bioguided screening of MeOH extract of Elaeis guineensis on T. gondii

Based on the results of the antitoxoplasmic activity screening, an extraction was carried out on 1.5 kg of the air-dried and powdered *E. guineensis* leaves. The hydromethanolic extract was fractionated on a Diaion HP-20 resin column to obtain HP₁–HP₅ fractions. The

<table><caption>**Table 3**

Cytotoxicity and anti-*Toxoplasma* activities of fractions obtained from MeOH 80% extract of *E. guineensis* against *T. gondii* in Vero cells at 25 µg/mL (N = 10).</caption><thead><tr><th>Fractions from<br>hydromethanolic<br>extract of E. guineensis</th><th>Molecular family</th><th>Cytotoxic<br>activity<br>against Vero<br>cells</th><th>Anti-<br>Toxoplasma<br>gondii activity</th></tr></thead><tbody><tr><td>MeOH 80% extract</td><td>-</td><td>5% ± 0.04</td><td>94% ± 0.07</td></tr><tr><td>HP₁A</td><td>Saccharides</td><td>7% ± 0.09</td><td>2% ± 0.02</td></tr><tr><td>HP₁B</td><td>Glycerolipids</td><td>4% ± 0.08</td><td>5% ± 0.07</td></tr><tr><td>HP₂</td><td>Flavonoids</td><td>6% ± 0.07</td><td>6% ± 0.10</td></tr><tr><td>HP₃</td><td>Flavonoids,<br>terpenoids</td><td>2% ± 0.04</td><td>8% ± 0.12</td></tr><tr><td>HP₄</td><td>Flavonoids,<br>terpenoids and<br>sterols</td><td>3% ± 0.02</td><td>1% ± 0.02</td></tr><tr><td>HP₅</td><td>Terpenoids,<br>flavonoids and fatty<br>acids</td><td>67% ± 0.08</td><td>nd<sup>a</sup></td></tr><tr><td>CPC₁</td><td>Fatty acids and<br>glycerolipids</td><td>81% ± 0.07</td><td>nd<sup>a</sup></td></tr><tr><td>CPC₂</td><td>Terpenoids and<br>sterols</td><td>77% ± 0.04</td><td>nd<sup>a</sup></td></tr><tr><td>CPC₃</td><td>Terpenoids and<br>sterols</td><td>47% ± 0.07</td><td>nd<sup>a</sup></td></tr><tr><td>CPC₄</td><td>Sesquiterpenoids</td><td>9% ± 0.17</td><td>22% ± 0.015</td></tr><tr><td>CPC₅</td><td>Sesquiterpenoids</td><td>8% ± 0.09</td><td>8% ± 0.07</td></tr><tr><td>CPC₆</td><td>Saccharides</td><td>5% ± 0.04</td><td>0% ± 0.0</td></tr><tr><td>CPC₇</td><td>Saccharides</td><td>4% ± 0.04</td><td>15% ± 0.15</td></tr><tr><td>CPC₈</td><td>Flavonoids</td><td>2% ± 0.04</td><td>1% ± 0.01</td></tr><tr><td>CPC₉</td><td>Sesquiterpenoids<br>and glycosides</td><td>3% ± 0.02</td><td>1% ± 0.01</td></tr><tr><td>CPC₁₀</td><td>Sesquiterpenoids<br>and glycosides</td><td>5% ± 0.10</td><td>11% ± 0.13</td></tr><tr><td>CPC₁₁</td><td></td><td>1% ± 0.01</td><td>7% ± 0.07</td></tr></tbody></table>

Positive *anti-toxoplasmic* activity control (pyrimethamine): 70–90% parasite growth inhibition rate at 1 µM.

Positive cytotoxicity control (pyrimethamine): 60–70% cytotoxicity at 20 µg/mL (i.e. 80 µM).

a nd = not determined.
cytotoxicity of the MeOH 80% extract and obtained fractions was evaluated at 25 µg/mL on Vero cells (Table 3) and showed that the less polar fraction HP₅ is cytotoxic with a growth inhibition rate of 67%. Therefore, HP₅ was excluded from the antiparasitic screening. The HP₁–HP₄ fractions were evaluated for their anti-Toxoplasma activity at the concentration 25 µg/mL, but none showed anti-Toxoplasma activity (Table 3). It seems that the fractionation of the hydromethanolic extract led to the loss of the antitoxoplasmic activity. A chemosensitivity on the HP₅ fraction was carried out to determine the concentration at which it is no longer cytotoxic, in order to measure its anti-toxoplasmic activity. The cytotoxicity of HP₅ was evaluated on Vero cells at 25, 12.5, 6.25, 3.125 and 1.563 µg/mL and the IC₅₀ was found to be 16.0 µg/mL. The concentration which induced less than 12 % of Vero cells growth inhibition was 6.25 µg/mL. Unfortunately, HP₅ showed less than 1% *T. gondii* growth inhibition at this concentration.

It is important to point out that, despite the historical effectiveness of bioguided fractionation of plant extracts, loss of activity and failure to isolate active compounds during this process can occur (Caesar and Cech, 2019; Nothias et al., 2018). The main reasons of these pitfalls may be the degradation of compounds during the purification process, low concentration of bioactive compounds making their isolation difficult, and/or potential synergistic effects (Nothias et al., 2018). This can be avoided by “synergy-directed fractionation” (Junio et al., 2011) which combines chromatographic separation and synergy testing in combination with a known active constituent in the original extract. Several studies have shown that the overall activity of plant extracts can result from mixtures of compounds with synergistic, additive, or antagonistic activity (Wagner and Ulrich-Merzenich, 2009).

While there are multiple possible explanations for this failure, it is true that in some cases loss of activity occurs because multiple constituents are required to observe the biological effect.

The first explanation could be the irreversible adsorption of compounds on the column packing (Qiu et al., 2013). To test this purpose, the MeOH 80% extract was fractionated by centrifugal partition chromatography (CPC), a solid support free liquid–liquid separation technique involving the distribution and transfer of solutes between at least two immiscible liquid phases according to their partition coefficients. This method avoids irreversible adsorption of analytes due to the absence of solid support. The CPC fractionation of the MeOH 80% extract led to eleven fractions which were evaluated for their cytotoxicity on Vero cells at 25 µg/mL showing that the less polar fractions (CPC₁–CPC₃) possessed cytotoxic activity (Table 3). The non-cytotoxic fractions were evaluated for their antiparasitic activity, unfortunately revealing that CPC₄–CPC₁₁ fractions possessed less than 50% anti-Toxoplasma activity at 25 µg/mL. Thus, the loss of activity of the MeOH 80% extract is not due to irreversible adsorption of compounds to the column. This suggests that the anti-Toxoplasma activity of the MeOH 80% extract could result from mixtures of compounds with synergistic, additive, or antagonistic activity.

The second hypothesis was that there was a synergistic interaction between the metabolites of the MeOH 80% extract. It is known that when two or more drugs are given in combination, the effect can be superadditive (synergistic); that is, they can demonstrate action beyond that is expected of their individual power and effectiveness. Unlike synergy, some drug combinations may show sub-additivity (antagonism) or simple additivity (Tallarida, 2011). Thus, the idea that HP₅, the only cytotoxic fraction at 25 µg/mL, could be responsible of the anti-Toxoplasma activity of the MeOH 80% extract of *E. guineensis* at lower concentrations when mixed with another fraction was verified. So, five combinations were reconstituted HP₁A + HP₅, HP₁B + HP₅, HP₂ + HP₅, HP₃ + HP₅ and HP₄ + HP₅, respecting their ratios in the MeOH 80% extract (Table 4). These combinations were evaluated for their cytotoxicity at the concentration of 25 µg/mL on Vero cell line (Table 4) and, surprisingly, none of these combinations showed more than 20% cytotoxic activity at the concentration tested. The combination tested HP₁B + HP₅ (25 µg/mL), in which the HP₅ concentration was 13.88 µg/mL

<table><caption>**Table 4**

Cytotoxicity and anti-Toxoplasma activities of some fractions and combinations of MeOH 80% extract of *E. guineensis* (N = 6).</caption><thead><tr><th>Fractions</th><th>Concentration in µg/mL</th><th>Cytotoxicity against Vero cells</th><th>Anti-Toxoplasma activity at 25 µg/mL</th><th>Anti-Toxoplasma activity at 100 µg/mL</th></tr></thead><tbody><tr><td>HP1A</td><td>23.5</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP5</td><td>1.5</td><td>0 ± 0.00</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP1A + HP5</td><td>25</td><td>3% ± 0.01</td><td>0 ± 0.00</td><td>0 ± 0.00</td></tr><tr><td>HP1B</td><td>11.1</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP5</td><td>13.9</td><td>40% ± 0.01</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP1B + HP5</td><td>25</td><td>19% ± 0.01</td><td>64% ± 0.05</td><td>92% ± 0.02</td></tr><tr><td>HP2</td><td>14.2</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP5</td><td>10.8</td><td>30% ± 0.01</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP2 + HP5</td><td>25</td><td>7% ± 0.05</td><td>67% ± 0.03</td><td>85% ± 0.03</td></tr><tr><td>HP3</td><td>19</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP5</td><td>6</td><td>15% ± 0.01</td><td>1% ± 0.00</td><td>nd<sup>a</sup></td></tr><tr><td>HP3 + HP5</td><td>25</td><td>2% ± 0.01</td><td>50% ± 0.08</td><td>83% ± 0.05</td></tr><tr><td>HP4</td><td>16.9</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP5</td><td>8.1</td><td>20% ± 0.01</td><td>nd<sup>a</sup></td><td>nd<sup>a</sup></td></tr><tr><td>HP4 + HP5</td><td>25</td><td>10% ± 0.07</td><td>74% ± 0.02</td><td>92% ± 0.03</td></tr></tbody></table>

Positive anti-toxoplasmic activity control (pyrimethamine): 70–90% parasite growth inhibition rate at 1 µM.

Positive cytotoxicity control (pyrimethamine): 60–70% cytotoxicity at 20 µg/mL (i.e. 80 µM).

<sup>a</sup> nd = not determined.

showed a toxicity of 19% while HP₅ tested alone showed toxicity of 40% at this concentration. The other combinations tested HP₂ + HP₅, HP₃ + HP₅, and HP₄ + HP₅ also showed that these combinations were less cytotoxic than HP₅ alone at the same concentration for HP₅ (Table 4). These results suggested a sub-additive (antagonist) effect concerning the cytotoxic activity on Vero cell line.

These five combinations were evaluated for their anti-Toxoplasma activity at the concentration 25 µg/mL. The HP₃ + HP₅ combination in which the concentration of HP₅ was 5.95 µg/mL showed an anti-Toxoplasma effect of 50%, resulting in synergistic activity since the anti-Toxoplasma activity of HP₅ alone at the same concentration was 1%. The most active combination was HP₄ + HP₅ with 74% antiparasitic activity (Table 4).

## 2.4. Discussion of the anti-toxoplasmic activity of compounds identified in *E. guineensis*

The activity observed in these plants could be attributed to a synergistic effect between the phytochemical constituents, including aliphatic acids, terpenoids, sterols and flavonoids which could have antiparasitic activities. However, further studies should be carried out to confirm and explain this synergistic or antagonistic effect between the identified compounds. All CPC fractions (CPC₁–CPC₁₁) were analyzed by ¹H and ¹³C NMR spectroscopy in order to identify the main class of compounds (Table 3) and compared to HP₁A–HP₅ fractions. No additional compounds were identified.

HP₅ contained long-chain fatty acids identified as palmitic acid (10), palmitic acid methyl ester (11), elaidic acid methyl ester (8), and linoelaidic acid methyl ester (9). Three of them (8–9 and 11) belong to fatty acid methyl esters (FAMES) which have been reported to exhibit antibacterial properties. Some authors have even described that FAMES could be the next generation of antibiotic agents acting through membrane disruption and reactive oxygen species production (Casillas-Vargas et al., 2021). Fatty acids are a broad group of amphipathic molecules with a terminal carboxyl group that, based on the number of double bonds, are classified into saturated and unsaturated
fatty acids. These molecules play a crucial role in the structure of cell membranes, energy production, signaling, and regulation of different biochemical pathways (De Carvalho and Caramujo, 2018). Most likely, fatty acids like as those in the active fraction HP₅ affected the lipid bilayer integrity or even the inner membrane complex of *T. gondii*. Both structures are vital for invasion and replication of the parasite (Choi et al., 2019). Similar compounds, such as hexadecanoic acid methyl ester, have been shown to affect the integrity of bacterial cell membranes (Shaaban et al., 2021). In addition to affecting cell membranes, inhibition of bacterial fatty acid synthesis is suggested to be linked to these molecules (Abubacker and Deepalakshmi, 2013; Zheng et al., 2005). *T. gondii* acyl-CoA diacylglycerol acyltransferase (TgDGAT) is an integral membrane protein localized in the cortical and perinuclear endoplasmic reticulum of the parasite and is responsible for triacylglycerol synthesis (Anacleto-Santos et al., 2023). Some studies have reported that oleate, palmitoleate, and linoleate impair parasite replication by interfering with TgDGAT function (Nolan et al., 2018). Therefore, *T. gondii* growth can be limited by controlling the parasite lipidomics, as previously reported (Anacleto-Santos et al., 2023; Smith et al., 2021). Based on these reports, the fatty acids in HP₅ could disrupt the parasite's membranes or inhibit enzymes required for *T. gondii* fatty acid synthesis.

Like fatty acids, allyl glycoside such as erigeside F (24) isolated from HP₃ may also exhibit selective toxicity towards the parasite by inhibiting fatty acid synthesis within the parasite (enoyl-acyl carrier protein reductase).

Among drug discoveries with antibacterial potentials, 75% were terpenes, including mono, di- and triterpenes (Zacchino et al., 2017). Although the exact mechanism of action of terpenes remains unknown, it is believed that they can inhibit both oxygen uptake and oxidative phosphorylation processes (Grin et al., 1999). Terpenes have been extensively reported to exhibit antibiotic, antiviral, and antiparasitic properties (Bisht et al., 2021). The growth inhibitory activity of ursolic acid, a triterpenoid detected in HP₅, against *T. gondii* has already been reported with IC₅₀ of 5.9 µM (Choi and Lee, 2018, 2019; Deng et al., 2019). Triterpenoids isolated from the barks of *Quercus crispula*, 29-nor-lupane-3,20-dione, oleanolic acid acetate, and ursolic acid acetate, showed anti-*Toxoplasma* activity with an IC₅₀ between 6.8 and 24.4 µM (Endo et al., 2019). A pentacyclic triterpenoid from *Olea europaea*, maslinic acid, was evaluated on *T. gondii* tachyzoites and showed an IC₅₀ of 58.2 µM. It seems that this terpenoid disrupts the ultrastructure and motility of the parasite due to inhibition of protease activity (De Pablos et al., 2010). Betulone, a lupane-type triterpene extracted from the bark of *Alnus glutinosa* showed anti-*Toxoplasma* activity *in vitro* (IC₅₀ 2.7 µM) (Darme et al., 2021). Their ability to affect lipid membranes has also been reported (Mahizan et al., 2019). Some pentacyclic triterpenes have been shown to inhibit the expression of COX-2 (De-Almeida et al., 2019), which reduces *T. gondii* infection and upregulates the proinflammatory immune response in mice (Pereira et al., 2019). COX-2 is a crucial factor in *T. gondii* propagation in human trophoblasts; therefore, its inhibition can induce a proinflammatory response capable of controlling parasite proliferation (De Souza et al., 2021). Ursolic acid identified in HP₅ is also pentacyclic; therefore, it could affect the host Th1 immune response. A sterol, β-sitostenone, analogue of stigmasta-4,24 (28)-dien-3-one (15) was evaluated on *T. gondii* tachyzoites and showed IC₅₀ of 17.3 µM (Darme et al., 2022).

It has been demonstrated that a sesquiterpene, kurubasch aldehyde, exerts a moderate inhibitory effect on the growth of *Plasmodium falciparum* (IC₅₀ = 76 µM) and slow-proliferating breast cancer cells MCF7 (78 µM). However, it was discovered to be a highly potent inhibitor of S180 cancer cell proliferation, with an IC₅₀ value of 7.4 µM (Traoré-Coulibaly et al., 2017). In a review published between 2015 and 2020, the authors conducted a comprehensive examination of the biological activity of sesquiterpenes and reported cytotoxicity, antibacterial, antifungal, anthelmintic and other activities. In light of these assertions, it would be useful to assess the anti-*Toxoplasma* activity of the

nor-sesquiterpenes (7 and 16–19) identified in *E. guineensis*, from which three of previously undescribed, as the presence of glycoside could influence their pharmacological properties. The mode of action of terpenes includes, among other mechanisms, the generation of reactive oxygen species (ROS) and other free radicals after an oxidative stress process. Based on these facts, it could be inferred that the anti-*Toxoplasma* effect of *E. guineensis* MeOH 80% extract could be linked to these mechanisms of action. Nevertheless, more studies need to be done in order to evaluate this extract as well as its possible characterized compounds on the complete life cycle of *T. gondii* and *in vivo* models.

*T. gondii* inhibits production of TNF-α and IL-12. TNF-α is a cytokine of inflammatory and immune response and together with IL-6 can enhance proliferation and differentiation of B lymphocytes (Lang et al., 2007). TNF-α activates eosinophil cytotoxicity toward protozoa and induces secretion of acute phase proteins via IL-6 production. TNF-α and IFN-γ have antiproliferative properties. In *toxoplasmosis*, TNF-α appears to be indispensable for macrophage activation and inhibition of parasite replication.

Several studies reported the anti-inflammatory properties of *E. guineensis* leaf extracts (Tow et al., 2021; Yin et al., 2013; Soundararajan and Sreenivasan, 2012). *E. guineensis* extracts contained flavonoids which have antioxidant, apoptosis-inducing, and anti-inflammatory properties (Hassanpour and Doroudi, 2023) and also decreases oxidative stress (Bubols et al., 2013; Miller, 1996). Flavonoid's mechanism is based on the activation of nuclear factor Nrf2 (Adetunji et al., 2023; Mendonca and Soliman, 2020), which activates other cellular defense mechanisms by Phase II initiation of detoxifying and antioxidant. However, there is a paucity of information in the literature regarding their cytotoxicity and anti-parasitic activity. Furthermore, *in vitro* studies have demonstrated that the combination of pyrimethamine with taxifolin (dihydroquercetin) exhibits significant inhibitory activity against *T. gondii* RH strain tachyzoites, with an IC₅₀ of 1.39 µg/mL (p < 0.05). Notably, this combination is not cytotoxic for HFF and Hek-293 infected cell lines (Abugri et al., 2018). Similarly, Dégbé et al. (2018) demonstrated that the ethanolic extract of *Vernonia amygdalina* leaves exhibited a proportional content of phenols, tannins, flavonoids, and polysaccharides. The extract demonstrated the highest efficacy against the *T. gondii* RH strain tachyzoites and the lowest cytotoxicity on the HFF cell (Dégbé et al., 2018). These results suggest that the C-glycoside flavonoids isolated in the HP₃ fraction may modulate or enhance the antitoxoplasmic activity of terpenes, sterols, or other compounds in a mixture.

### 3. Conclusions

This study revealed the *in vitro* anti-*Toxoplasma* activities of the DCM and EtOAc extracts of the stem bark of *C. micranthum*, of the MeOH 80% extract of the leaves of *E. guineensis*, n-Hep and DCM extracts from the stem bark of *O. africana*, EtOAc extract from the leaves and DCM extract from the stem bark of *O. ahia*, from the DCM extract of the stem bark of *O. elatum* and the DCM extract of the aerial parts of *T. coronatum*. Further research will need to be investigated to identify their active components and to search for original compounds to treat toxoplasmosis.

Four undescribed compounds 16–18 and 20 and twenty known compounds were isolated from the leaves of *E. guineensis*. Among the compounds elucidated by dereplication or after purification, only vicenin-2 (4) and vitexin (6) were previously identified in *E. guineensis* by UHPLC-UV/PDA (Che et al., 2021; Tow et al., 2021). The MeOH 80% extract of *E. guineensis* is active against *T. gondii* RH and is not cytotoxic to Vero cells at concentrations at 25 µg/mL that killed up to 94% of the parasites. The fractionation of the MeOH 80% extract did not allow recovery of antitoxoplasmic activity in its individualized fractions. Its efficacy could be related to the mechanisms of action of fatty acids, flavonoids and terpenes; nevertheless, as different molecules are found in this extract, a synergistic or antagonistic effect cannot be discarded. It is therefore crucial to evaluate the components of the MeOH 80% extract
as well as their mode of action, both in the parasite and host cells, to validate the vital targets (preferably those not supplied by the host) leading to the identification of a main molecule which will probably allow the design of analogous triterpenoids or FAMES with anti-Toxoplasma activity. Finally, these results demonstrated that *E. guineensis* metabolites can be considered as possible candidates to obtain lead compounds against *T. gondii* and that natural compounds can be a good source for the development of new antiparasitic drugs. However, further biological studies will be required in order to promote the antiparasitic efficacy of these molecules.

# 4. Materials and methods

## 4.1. Instrumentation

1D and 2D-NMR spectra were recorded in CD₃OD on a Bruker Avance III 500 spectrometer (¹H at 500 MHz and ¹³C at 125 MHz) or on Bruker Avance DRX III 600 spectrometer equipped with a TCI cryo-probe, and using standard Bruker microprograms (Karlsruhe, Germany). The ¹³C NMR spectra were acquired at 150.91 MHz using a standard zgpg pulse sequence with an acquisition time of 0.9 s, a relaxation delay of 3 s. HR-ESI-MS were obtained from Micromass Q-TOF micro-instrument (Manchester, UK). The centrifugal partition chromatography (CPC) equipment consists of a 303 mL FCPE300® column (Rousselet Robatel Kromaton, Annonay, France) containing 7 circular partition disks etched with a total of 231 partition twin cells (≈1 mL per twin cell), a Knauer Preparative 1800 V7115 pump (Berlin, Germany), a UVD 170 S detector set at 210, 254, 280 and 366 nm (Dionex, Sunnivale, CA, USA) and a Pharmacia Superfrac collector (Uppsala, Sweden). Flash chromatography was performed on a Grace Reveleris system equipped with dual UV and ELSD detection using Grace cartridges (silica gel or RP-C₁₈), and the monitored wavelengths were at 205, 254, and 366 nm. Semi-preparative HPLC was performed on an apparatus equipped with an ASI-100 Dionex autosampler, an Ultimate 3000 pump ThermoFisher Scientific, a diode array detector UVD 340S and a Chromeleon software (Dionex, ThermoFisher Scientific, France). RP-C₁₈ column (Phenomenex 250 × 15 mm, Luna 5 µm, Interschim, France) was used for the semi-preparative HPLC with a binary gradient eluent (H₂O pH 2.4 with TFA; CH₃CN) and a flow rate of 4.7 mL/min; RP-C₁₈ column (Phenomenex 250 × 21,1 mm, Luna 5 µm, Interschim, France) was used for the preparative HPLC with a binary gradient eluent (H₂O pH 2.4 with TFA; CH₃CN) and a flow rate of 20 mL/min. The chromatogram was monitored at 205, 254, 300, and 360 nm. Analytical TLC was performed using silica gel plates (Merck Kieselgel 60 F254) and RP-C₁₈ plates (Kieselgel 60 F254) and visualized at 254 and 366 nm and by spraying the dried plates with vanillin and 50 % of H₂SO₄, followed by heating.

## 4.2. Chemicals, reagents, parasites and cell culture

All solvents for extraction, fractionation and solubilized were purchased from Carlo Erba Reactifs SDS (Val de Reuil, France). The RH strain (genotype I) of *T. gondii* was provided by the Toxoplasma Biological Resource Center (BRC Toxoplasma, France). Tachyzoites of *T. gondii* the RH strain were cultured on Vero cells monolayers (ATCC, CCL81) at 37 °C, 5% CO₂ in a humidified incubator. Cells and parasites were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Investrogen, France) supplemented with 5% fetal bovine serum (SVF) for cells cultures or 2% (v/v) SVF for parasites cultures (Biowest, France) and supplemented with 1% antibiotics (penicillin-streptomycin) (GIBCO). All tested compounds were isolated by semi-preparative HPLC and compound purity was confirmed to be ≥ 95% prior to biological assays using analytical HPLC and quantitative NMR spectroscopy.

## 4.3. Plant material

The selected plants were all harvested in February 2021 in their

natural habitats in Côte d'Ivoire. Botanical determination was performed by Mr Téré H.G. and authentication was made by Pr. Tiebre S.-M. from the National Floristic Centre (NFC) of university Félix HOUPHOUET-BOIGNY Cocody Abidjan (Côte d'Ivoire), in which, voucher specimens were deposited (Table 5).

## 4.4. Preparation of the 40 crude extracts

Air-dried plant material of each species (10 g) was ground (0.2 mm sieve). The plant material was extracted successively with *n*-Hept, DCM, EtOAc and MeOH 80%, at room temperature for 2 × 24 h, each 100 mL. The filtrates were taken to dryness under vacuum at 40 °C and the residues were stored at room temperature until testing.

## 4.5. Extraction and fractionation of *E. guineensis* leaves

Air-dried and powdered leaves of *E. guineensis* (1.5 kg) were extracted, at room temperature, for 2 × 24 h, with *n*-Hept, DCM, EtOAc and MeOH 80%, successively, each 15 L, to give after solvent removal under vacuum the corresponding extracts *n*-Hept (25.9 g), DCM (13.6 g), EtOAc (7.1 g) and MeOH 80% (44.6 g). A part of the MeOH 80% extract (30 g) was fractionated by Diaion HP-20 resine chromatography (5 × 45.5 cm), eluted successively with H₂O, 25%, 50%, 75% of MeOH in H₂O and finally with MeOH; each 3 L, to obtain fractions: HP₁ (20.5 g), HP₂ (1.5 g), HP₃ (3.8 g), HP₄ (2.5 g) and HP₅ (1.2 g), respectively. Fraction HP₁ was solubilized in 200 mL of H₂O and partitioned with *n*-butanol (3 × 250 mL) to obtain the HP₁A (H₂O soluble fraction, 19.6 g) and HP₁B (*n*-BuOH soluble fraction, 0.9 g).

## 4.6. Chemical profiling of the six fractions by ¹³C-NMR based dereplication

Briefly, fractions HP₁–HP₅ were chemically profiled based on ¹³C NMR data as described in a previous work (Hubert et al., 2014; Darme et al., 2021). All ¹³C NMR spectra of the fraction series were processed and submitted to Hierarchical Clustering Analysis (HCA) for the recognition of ¹³C NMR metabolite fingerprints. In this way, similarity measurements between ¹³C NMR signals belonging to individual structures within the fraction series were visualized as “chemical shift clusters” on a HCA correlation heat map given in Fig. 1. As a result, 9 major chemical shift clusters colored in red were revealed on the heat map, corresponding to the major metabolites of the MeOH 80% extract (Fig. 1). With the help of *acd_ lotus/nmrshiftdb2* (Nuzillard, 2023) and NP-MRD databases containing chemical shift values of natural metabolites, the corresponding chemical structures were identified.

## 4.7. Purification of HP₃ and HP₅ and identification of its major components

Fraction HP₃ (2 g) was separated into 16 subfractions named HP₃.₁–HP₃.₁₆ using silica gel flash chromatography in solid state mode and eluted with CHCl₃/MeOH/H₂O (from 95/5/0 to 70/30/5, v/v/v). Subfraction HP₃.₂ (17 mg) was purified by semi-prep HPLC, eluted with a gradient from 90% to 100% of CH₃CN in water as a mobile phase, in 26 min to afford compounds **17** (t<sub>R</sub> 21.0 min, 1 mg) and **19** (t<sub>R</sub> 23.5 min, 1 mg). Subfraction HP₃.₆ (17 mg) was purified under the same experimental conditions as subfraction HP₃.₂ in semi-prep HPLC, to obtain compounds **23** (t<sub>R</sub> 7.1 min, 1.5 mg) and **24** (t<sub>R</sub> 20.0 min, 1.1 mg). Subfraction HP₃.₈ (85 mg) was purified by preparative HPLC, eluted with a gradient from 90% to 100% of CH₃CN in water as a mobile phase, in 26 min to afford compound **7** (5.3 mg). The subfractions HP₃.₁₀, HP₃.₁₁, HP₃.₁₄, HP₃.₁₅ and HP₃.₁₆ were purified under the same experimental conditions as subfraction HP₃.₈ in preparative HPLC. Purification of subfraction HP₃.₁₀ (233 mg) resulted in the isolation of compounds **18** (20.6 mg) and **20** (15.7 mg). Purification of subfraction HP₃.₁₁ (382 mg) resulted in the isolation of compound **21** (24.3 mg). Purification of
<table><caption>**Table 5**

Plant species analyzed for their anti-Toxoplasma activity.</caption><thead><tr><th>Botanical name (family)</th><th>Part used</th><th>Collection area</th><th>Collection date</th><th>Voucher no</th></tr></thead><tbody><tr><td>Combretum micranthum G. Don (Combretaceae),</td><td>Leaf</td><td>Waninou, north-west Côte d'Ivoire<br>GPS: 10.06763; 12.84672</td><td>16/02/2021</td><td>UCJ002988</td></tr><tr><td>Elaeis guineensis Jacq. (Arecaceae)</td><td>Leaf</td><td>Adiopodoumé, south Côte d'Ivoire<br>GPS: 5.33003; 4.13064</td><td>29/02/2021</td><td>UCJ0141165</td></tr><tr><td>Erigeron floribundus (Kunth) Sch.Bip. (Asteraceae)</td><td>Aerial parts</td><td>Zagné, south-west Côte d'Ivoire<br>GPS: 6.21500; 7.48823</td><td>25/02/2021</td><td>UCJ003610</td></tr><tr><td>Oldfieldia africana Benth. & Hook. f. (Picrodendraceae)</td><td>Stem bark</td><td>Zagné, south-west Côte d'Ivoire<br>GPS: 6.21500; 7.48823</td><td>23/02/2021</td><td>UCJ006232</td></tr><tr><td>Octoknema borealis Hutch. & Dalziel (Olacaceae)</td><td>Leaf and stem bark</td><td>Azaguié, south-east Côte d'Ivoire<br>GPS: 5.78954; 4.14758</td><td>07/02/2021</td><td>UCJ013216</td></tr><tr><td>Omphalocarpum ahia A. Chev. (Sapotaceae)</td><td>Leaf and stem bark</td><td>Keibly, south-west Côte d'Ivoire<br>GPS: 5.99994; 7.7524</td><td>24/02/2021</td><td>WAG1830636</td></tr><tr><td>Omphalocarpum elatum Miers (Sapotaceae)</td><td>Stem bark</td><td>Azaguié, south-east Côte d'Ivoire<br>GPS: 5.78954; 4.14758</td><td>07/02/2021</td><td>UCJ016568</td></tr><tr><td>Tristemma coronatum Benth. (Melastomataceae)</td><td>Whole parts</td><td>Yacassé-Mé, south-east Côte d'Ivoire<br>GPS: 5.76860; 3.99075</td><td>07/02/2021</td><td>CSRS004223</td></tr></tbody></table>

subfraction HP$_{3-14}$ (210 mg) resulted in the isolation of compound 5 (12.8 mg). Purification of subfraction HP$_{3-15}$ (266 mg) resulted in the isolation of compounds 22 (22.4 mg) and 6 (6.3 mg). Purification of subfraction HP$_{3-16}$ (302 mg) resulted in the isolation of compound 4 (16.4 mg).

Fraction HP$_{5}$ (1.1 g) was separated into 37 subfractions named HP$_{5-1}$-HP$_{5-37}$ using silica gel flash chromatography eluted with n-Hept/acetone/CHCl$_{3}$/MeOH (from 95/5/0/0 to 0/0/1/1, v/v/v/v). The combined subfractions HP$_{5-1}$ and HP$_{5-2}$ (21 mg) were purified by semi-prep HPLC, eluted with a gradient from 80% to 100% of CH$_{3}$CN in water as a mobile phase, in 10 min, then in isocratic elution with CH$_{3}$CN for 10 min to afford compounds 14 (t$_{R}$ 12.9 min, 1 mg), 8 (t$_{R}$ 15.1 min, 1 mg) and 9 (t$_{R}$ 18.6 min, 1.3 mg). Subfraction HP$_{5-3}$ (20 mg) was purified by semi-prep HPLC, eluted with a gradient from 80% to 100% of CH$_{3}$CN in water as a mobile phase, in 5 min, then in isocratic elution with CH$_{3}$CN for 20 min to afford compounds 15 (t$_{R}$ 16.5 min, 2.1 mg), 13 (t$_{R}$ 17.2 min, 1 mg) and 12 (t$_{R}$ 23.7 min, 1 mg). Subfraction HP$_{5-4}$ (13 mg) was purified by semi-prep HPLC, eluted with a gradient from 80% to 88% of CH$_{3}$CN in water as a mobile phase, in 10 min to afford compounds 10 (t$_{R}$ 7.4 min, 1 mg) and 11 (t$_{R}$ 8.9 min, 1 mg). Similarly, subfraction HP$_{5-4}$ was purified by semi-prep HPLC, eluted with a gradient from 80% to 88% of CH$_{3}$CN in water as a mobile phase, in 7 min, then in isocratic elution with CH$_{3}$CN for 3 min at 88% of CH$_{3}$CN and in isocratic elution with CH$_{3}$CN for 5 min to afford compound 16 (t$_{R}$ 3,3 min, 1 mg).

### 4.7.1. (–)-(6S)-elaedisdione (16)

yellowish solid; $^{1}$H NMR and $^{13}$C NMR (CDCl$_{3}$), see Table 1; $^{20}$[α]$_{D}$ -0.3° (c 0.1; MeOH), IR $v_{max}$

(cm$^{-1}$): OH (3357); CH$_{3}$ (2953-2823); 2x C=O (1713; 1669); HR-ESI-MS *m/z* 237.1125 [M + H]+(calcd C$_{13}$H$_{17}$O$_{4}$, 237.1127).

### 4.7.2. Elaeisdiol or 6-dehydroxy-cucubalactone (17)

yellowish solid; UV (MeOH) $\lambda_{max}$ nm (log $\epsilon$): 220.0 (5. 00); $^{20}$[α]$_{D}$ -5.0° (c 0.1; MeOH); $^{1}$H NMR and $^{13}$C NMR (CD$_{3}$OD), see Table 1; IR $v_{max}$ (cm$^{-1}$): OH (3550); CH$_{3}$ (2960-2870); COOC (1830); HR-ESI-MS *m/z* 223.1333 [M - H$_{2}$O + H]+(calcd C$_{13}$H$_{19}$O$_{3}$, 223.1312) or 205.1223 [M - 2H$_{2}$O + H]$^{+}$

(calcd C$_{13}$H$_{17}$O$_{2}$, 205.1226).

### 4.7.3. Elaeisoside (18)

yellowish solid; UV (MeOH) $\lambda_{max}$ nm (log $\epsilon$): 280.0 (3. 11), 220.0 (5.00); $^{20}$[α]$_{D}$ +25.2° (c 0.1; MeOH); $^{1}$H NMR and $^{13}$C NMR (CD$_{3}$OD), see Table 1; IR $v_{max}$ (cm$^{-1}$): OH (3371); CH$_{3}$ (2932-2910); C=O (1700); CH$_{2}$-O-C (1012); HR-ESI-MS *m/z* 543.2418 [M + Na]+(calcd C$_{24}$H$_{40}$O$_{12}$Na, 543.2417).

### 4.7.4. 2,3-Dihydroxybenzoic acid 3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside (20)

yellowish solid; UV (MeOH) $\lambda_{max}$ nm (log $\epsilon$): 278.0 (4. 82), 220.0 (5. 00); $^{20}$[α]$_{D}$ -2,4° (c 0.1; MeOH); $^{1}$H NMR and $^{13}$C NMR (CD$_{3}$OD), see Table 1; IR $v_{max}$ (cm$^{-1}$): OH (3357); COOC (1673); CH$_{3}$ (2927); OH-phenol (1457); HR-ESI-MS *m/z* 499.1424 [M + Na]+(calcd C$_{20}$H$_{28}$O$_{13}$Na, 499.1428).

### 4.8. Centrifugal partition chromatography (CPC) fractionation of MeOH 80% extract

A three-phase solvent system composed of n-Hept/MtBE/CH$_{3}$CN/water in the ratio 1/1/1/1 (v/v/v/v) (500 mL, of each) was used. The solvent mixture was thoroughly equilibrated in a separatory funnel. After separation of the n-heptane-rich upper phase (UP$_{0}$), one equivalent volume of MTBE (500 mL) was added to the mixture of middle (MP$_{0}$) and lower (LP$_{0}$) phases in order to slightly reduce the polarity of the remaining two-phase solvent system. After decantation, the final middle and lower phases (MP and LP) were separated. The column was filled with the LP at the minimal rotation speed of 200 rpm. The rotation speed was then increased to 1400 rpm. The sample solution (1.5 g), dissolved in a mixture of LP/MP/UP$_{0}$ (45/10/5 v/v/v), was loaded into the column by progressively pumping the less polar UP$_{0}$ in the ascending mode. The flow rate of the mobile phase was then maintained at 10 mL/min until the end of the experiment. The UP$_{0}$ was pumped for 30 min after the release of the dead volume to ensure the elution of all hydrophobic compounds. The moderately polar MP was then pumped for 30 min to elute compounds with a medium hydrophobicity. Finally, in order to recover the most hydrophilic compounds retained at the head of the column, the role of the two liquid phases was switched by pumping the aqueous LP as the mobile phase in the descending mode at 10 mL/min. Fractions of 20 mL were collected. The separation was monitored by UV at 210, 254, 280 and 366 nm. All fractions were analyzed by TLC and HPLC and then pooled, giving 11 fractions (CPC$_{1}$–CPC$_{11}$).

### 4.9. Solubilization of extracts and fractions

All extracts and fractions were solubilized in dimethyl sulfoxide (DMSO). The final DMSO concentration was less than 0.5% in the final mixture (DMSO/culture medium, v/v). DMSO is commonly used for *in vitro* and *in vivo* experiments and is an aprotic amphiphilic molecule containing a highly polar domain and two non-polar methyl groups (MacGregor, 1967). This characteristic allows it to be soluble in polar or non-polar solvents. This also makes it capable of solubilizing many organic compounds polar and non-polar (Balakin et al., 2004; Santos et al., 2003).
## 4.10. Cytotoxic activity

The *in vitro* cytotoxicity of all 40 crude extract, HP1-HP5 and CPC1-CPC11 fractions was assessed on Vero cell cultures using the UptiBlue viable cell count assay (Interchim, France). The crude extracts were prepared at 250 µg/mL in dimethyl sulfoxide (DMSO)/culture medium, v/v, and kept at -30 °C, for a final sample tested at 25 µg/mL. A suspension of 20,000 Vero cells were cultured in 96-well plate with IMDM supplemented with 5% SVF (v/v). After 4 h of incubation, crude extracts or fractions to test were deposited in wells at 25 µg/mL. After 72 h of incubation at 37 °C and 5% CO2, wells were washed with phosphate buffer saline and fresh IMDM culture medium supplemented with 5% SVF and 10% UptiBlue was added. After an additional 3 h of incubation, the plate was measured with spectrophotometer with two wavelength, 570 nm corrected at 600 nm (FLUOstar Omega microplate reader, BMG Labtech, France). A cytotoxicity threshold was arbitrarily defined at cell viability of 80%.

## 4.11. Anti-Toxoplasma activity

All the experiments were performed at least in triplicate. The crude extracts were prepared at 250 µg/mL in dimethyl sulfoxide (DMSO)/ culture medium, v/v, and kept at -30 °C, for a final sample tested at 25 µg/mL 20,000 Vero cells were filled in 96-well plates with IMDM supplemented with 2% SVF (v/v), then incubated for 3 h at 37 °C and 5% CO2. Then, 50,000 parasites of the RH strain contained in 50 µL of culture medium were deposited, followed by 3 h of incubation in the same conditions. This time allowed the parasites to sediment and invade the Vero cells. Host cells were infected in a parasite-to-cell ratio of 5:2. Wells were then washed with PBS and 100 µL and 25 µL of sample solution was deposited per well in duplicate to obtain a final concentration of 25 µg/mL or 100 µg/mL. Dilutions were performed with culture medium. Finally, the plates were incubated for 72 h at 37 °C and 5% CO2 before being fixed with cold methanol for 5 min at -20 °C and then parasite multiplications were revealed by enzyme immunoassay.

Of the 96 wells, eight wells were reserved as a control of the Vero alone representing 100% cell viability, then six reserved wells were inoculated with *T. gondii* as a parasite growth control under untreated conditions, and finally, two final wells were inoculated under the same conditions as described above with pyrimethamine at 1 µg/mL as a control.

The IC50 assays were processed in the same conditions as previously described (Darme et al., 2022). Cells were then rehydrated with 100 µL of PBS for 10 min at room temperature. The plate was treated with 100 µL of a 3% H2O2 solution for 10 min at 37 °C to destroy the endogenous peroxidases of the Vero cells and then was washed with PBS and filled with 60 µL of primary polyclonal anti-*T. gondii* antibody (Bio-rad Rabbit Anti-*T. gondii* 9070-0556) diluted to 1/1000th in a conjugate buffer. After 1 h of incubation at 37 °C, wells were washed four times with wash buffer. Subsequently, 60 µL of secondary goat antibody anti-rabbit IgG coupled with horseradish peroxidase (Bio-rad Goat Anti-Rabbit IgG (H + L) HRP conjugate 170-6515) diluted to 1/2000th in the conjugate buffer were added, and incubated for 1 h at 37 °C. The wells were washed four more times and 200 µL of OPD (o-phenylenediamine dihydrochloride) substrate (P8412 Sigma-Aldrich, France) was added in each well. After 15 min at room temperature protected from light, the enzymatic reaction was stopped by the addition of 50 µL of hydrochloric acid (HCl) 3 M. To finish, 200 µL of each well were transferred to a new well and a spectrophotometer reading was performed at 492 nm, corrected at 630 nm.

Each experiment was performed with three controls: control with host cells alone, control with host cells and parasites without the drug for parasite multiplication, and control with host cells with parasites and pyrimethamine at 1 µM. The intensity of the staining is proportional to the amount of parasites present in the well.

The anti-*Toxoplasma* activity of samples was determined using the

formula: 100 - [Δ<sub>Ods</sub>/Δ<sub>Odv</sub>] × 100; Δ<sub>Odv</sub> = OD at 492 nm - OD at 630 nm for Vero cells invaded by tachyzoites without sample; Δ<sub>Ods</sub> = OD at 492 nm - OD at 630 nm for Vero cells invaded by tachyzoites and treated with sample.

## 4.12. Statistical analysis

Statistical significance in plaque assay, proliferation, and fungal growth inhibition assay was evaluated by two-tailed unpaired tests using an Excel spreadsheet. Statistical data are expressed as the mean value±standard error.

### CRediT authorship contribution statement

**Nangouban Ouattara:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Project administration, Methodology, Investigation, Formal analysis, Data curation.
**Mansour Issoufou-Tiny:** Investigation.
**Abdulmagid Alabdul Magid:** Writing – review & editing, Writing – original draft, Supervision, Methodology.
**Sandie Escotte-Binet:** Validation, Supervision, Investigation, Data curation.
**Philomène Akoua Yao-Kouassi:** Supervision, Resources, Funding acquisition, Formal analysis.
**Isabelle Villena:** Supervision, Resources, Project administration, Funding acquisition.
**Laurence Voutquenne-Nazabadioko:** Writing – original draft, Visualization, Resources, Project administration, Funding acquisition.

### Declaration of competing interest

The authors declare that they have no conflicts of interest associated with this publication.

### Acknowledgements

The authors thank the ICMR-UMR-7312 CNRS and the UR7510 ESCAPE of the University of Reims Champagne-Ardenne (France), the National Reference center on toxoplasmosis in the CHU of Reims, the UFR Sciences of Structures of Matter and Technology of the Université of Félix-Houphouët-Boigny (Côte d'Ivoire), the Ministry of Higher Education and Scientific Research of Ivory Coast for the financial support.

## Appendix. ASupplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.phytochem.2025.114442.

### Data availability

Data will be made available on request.

### References

Abbiw, D.K., 1990. Useful Plants of Ghana. West African Uses of Wild and Cultivated Plants. Intermediate Technology Publications, London, pp. 154–157. https://doi.org/10.3362/9781780443737. Royal Botanic Gardens, Kew.

Abubacker, M.N., Deepalakshmi, T., 2013. *In vitro* antifungal potentials of bioactive compound methyl ester of hexadecanoic acid isolated from *Annona muricata* Linn. (Annonaceae) leaves. *Biosci Biotechnol Res Asia* 10, 879–884. https://doi.org/10.13005/bbra/1211.

Abugri, D.A., Witola, W.H., Russell, A.E., Troy, R.M., 2018. *In vitro* activity of the interaction between taxifolin (dihydroquercetin) and pyrimethamine against Toxoplasma gondii. *Chem. Biol. Drug Des.* 91 (1), 194–201. https://doi.org/10.1111/cbdd.13070.

Adetunji, J.A., Fasae, K.D., Awe, A.I., Paimo, O.K., Adegoke, A.M., Akintunde, J.K., Sekhoacha, M.P., 2023. The protective roles of citrus flavonoids, naringenin, and naringin on endothelial cell dysfunction in diseases. *Heliyon* 9 (6), e17166. https://doi.org/10.1016/j.heliyon.2023.e17166.

Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 31 (10), 3307–3330. https://doi.org/10.1016/0031-9422(92)83678-R.
Ahoua, A.R.C., Konan, A.G., Bonfoh, B., Koné, M.W., 2015. Antimicrobial potential of 27 plants consumed by chimpanzees (Pan troglodytes versus Blumenbach) in Ivory Coast. BMC Compl. Alternative Med. 15 (1), 383. https://doi.org/10.1186/s12906-015-0918-7.

Alake, J., Akte, S.A., Ahlidja, W., Armah, F.A., Amponah, I.K., 2021. Indigenous knowledge affords antimicrobial plants for the management of infections. Int J Trop Dis Health 42 (3), 12–21. https://doi.org/10.9734/IJTDH/2021/v42i330411.

Alexandri, E., Ahmed, R., Siddiqui, H., Choudhary, M.I., Tsiafoulis, C.G., Gerothanassis, I.P., 2017. High resolution NMR spectroscopy as a structural and analytical tool for unsaturated lipids in solution. Molecules 5 (10), 1663. https://doi.org/10.3390/molecules22101663, 22.

Anaceto-Santos, J., Calzada, F., López-Camacho, P.Y., López-Pérez, T. de J., Carrasco-Ramírez, E., Casarrubias-Tabarez, B., Fortoul, T.I., Rojas-Lemus, M., López-Valdés, N., Rivera-Fernández, N., 2023. Evaluation of the anti-*Toxoplasma gondii* efficacy, cytotoxicity and GC/MS profile of *Pleopeltis crassinervata* active subfractions. Antibiotics 12 (5), 889. https://doi.org/10.3390/antibiotics12050889.

Annan, K., Sarpong, K., Asare, C., Dickson, R., Amponah, K.I., Gyan, B., Ofori, M., Gbedema, S.Y., 2012. In vitro anti-plasmodial activity of three herbal remedies for malaria in Ghana : *Adenia cissampeloides* (Planch.) Harms., *Termina liaivorensis* A. Chev., and *Elaeis guineensis* Jacq. Pharmacogn. Res. 4 (4), 225–229. https://doi.org/10.4103/0974-8490.102270.

Armah, F.A., Annan, K., Dickson, R.A., Mensah, A.Y., Ameyaw, E.O., Anning, A.A., 2016. Anti-leishmanial, anti-inflammatory and antioxidant potential of *Ophalocarpum ahia* A. Chev. Der Pharm Lett. 8 (2), 161–168.

Atindehou, K.K., Koné, M., Terreaux, C., Traore, D., Hostettemann, K., Dosso, M., 2002. Evaluation of the antimicrobial activity of medicinal plants from the Ivory Coast. Phytother Res. 16, 497–502. https://doi.org/10.1002/ptr.970.

Baatz, H., Mirshahi, A., Puchta, J., Gümbel, H., Hattenbach, L.-O., 2006. Reactivation of toxoplasma retinochoroiditis under atovaquone therapy in an immunocompetent patient. Ocul. Immunol. Inflamm. 14 (3), 185–187. https://doi.org/10.1080/09273940600659740.

Balakin, K.V., Ivanenkov, Y.A., Skorenko, A.V., Nikolsky, Y.V., Savchuk, N.P., Ivashchenko, A.A., 2004. In silico estimation of DMSO solubility of organic compounds for bioscreening. SLAS Discovery 9 (1), 22–31. https://doi.org/10.1177/1087057103260006.

Banfi, S., Caruso, E., Orlandi, V., Barbieri, P., Cavallari, S., Vigano, P., Clerici, P., Chiodaroli, L., 2014. Antibacterial activity of leaf extracts from *Combretum micranthum* and *Guiera senegalensis* (Combretaceae). Res. J. Microbiol. 9, 66–81. https://doi.org/10.3923/jm.jm.2014.66.81.

Benoit-Vical, F., Santillana-Hayat, M., Kone-Bamba, D., Mallie, M., Derouin, F., 2000. Anti-*Toxoplasma* activity of vegetal extracts used in West African traditional medicine. Parasite 7 (1), 3–7. https://doi.org/10.1051/parasite/2000071003.

Berinyuy, F., Ngoupayo, J., Kwetche, P.R.F., Kuate, T.T., Kuamouo, J., Fokam, M.A., Thaleu, B.N., 2014. Phytochemical screening and antimicrobial activity of extracts from the stem bark of *Oldfieldia africana* Benth & Hook (Euphorbiaceae) : preliminary study. Int.J.Curr.Res.Chem.Pharma.Sci. 1 (7), 101–109.

Bigna, J.J., Tochie, J.N., Tounouga, D.N., Bekolo, A.O., Ymele, N.S., Youda, E.L., Sime, P. S., Nansseu, J.R., 2020. Global, regional, and country seroprevalence of Toxoplasma gondii in pregnant women : a systematic review, modelling and meta-analysis. Sci. Rep. 10 (1), 12102. https://doi.org/10.1038/s41598-020-69078-9.

Bisht, B.S., Bankoti, H., Bharti, T., 2021. A review on therapeutic uses of terpenoids. J. Drug Deliv. Therapeut. 11, 182–185. https://doi.org/10.22270/jddt.v11i1-b.4523.

Bonouman-Ira, Bosson-Vanga, A.H., Offianan, A.T., Angora, K.E., Djanai, A.J., 2017. Séroprévalence de la toxoplasmose chez les femmes enceintes reçues au laboratoire de parasitologie de l'Institut Pasteur de Côte d'Ivoire. Séroprévalence de Toxoplasmosis among pregnant in the Laboratory of Institut Pasteur de Côte d'Ivoire. Rev int sc méd - RISM 19 (4), 267–271.

Bosch-Driessen, L.E.H., Berendschot, T.T.J.M., Ongkosuwito, J.V., Rothova, A., 2002. Ocular toxoplasmosis. Ophthalmology 109 (5), 869–878. https://doi.org/10.1016/S0161-6420(02)00990-9.

Bubols, G.B., Vianna, Dda R., Medina-Remon, A., von Poser, G., Lamuela-Raventos, R.M., Eifler-Lima, V.L., Garcia, S.C., 2013. The antioxidant activity of coumarins and flavonoids. Mini Rev. Med. Chem. 13 (3), 318–334. https://doi.org/10.2174/138955713804999775.

Burkill, H.M., 1985. The Useful Plants of West Africa, second ed. Royal Botanic Garden, pp. 458–459.

Burkill, H.M., 1997. The Useful Plants of West Tropical Africa, second ed. vol. 3. Royal Botanic Gardens, Kew-London. Families J.-L.

Caesar, L.K., Cech, N.B., 2019. Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2. Nat. Prod. Rep. 36, 869–888. https://doi.org/10.1039/c9np00011a.

Casillas-Vargas, G., Ocasio-Malavé, C., Medina, S., Morales-Guzmán, C., Del Valle, R.G., Carballeira, N.M., Sanabria-Ríos, D.J., 2021. Antibacterial fatty acids: an update of possible mechanisms of action and implications in the development of the next-generation of antibacterial agents. Prog. Lipid Res. 82, 101–1093.

Che, Zain M.S., Osman, M.F., Lee, S.Y., Shaari, K., 2021. UHPLC-UV/PDA method validation for simultaneous quantification of luteolin and apigenin derivatives from *Elaeis guineensis* leaf extracts: an application for antioxidant herbal preparation. Molecules 19 (4), 1084. https://doi.org/10.3390/molecules26041084, 26.

Cheng, Y.-X., Zhou, J., Deng, S.-M., Tan, N.-H., 2002. New norsequiterpenoids from cucubatus baccifer. Planta Med. 68 (1), 91–94. https://doi.org/10.1055/s-2002-00545.

Choi, W.H., Lee, I.A., 2018. Evaluation of anti-*Toxoplasma gondii* effect of ursolic acid as an novel Toxoplasmosis inhibitor. Pharmaceuticals 11 (2), 43. https://doi.org/10.3390/ph11020043.

Choi, W.H., Lee, I.A., 2019. The mechanism of action of ursolic acid as a potential anti-toxoplasmosis agent, and its immunomodulatory effects. Pathogens 8 (2), 61. https://doi.org/10.3390/pathogens8020061.

Choi, J.-W., Lee, J., Lee, J.-H., Park, B.-J., Lee, E.J., Shin, S., Cha, G.-H., Lee, Y.-H., Lim, K., Yuk, J.-M., 2019. Omega-3 polyunsaturated fatty acids prevent *Toxoplasma gondii* infection by inducing autophagy *via* AMPK activation. Nutrients 11, 2137. https://doi.org/10.3390/nu11092137.

Darme, P., Spalenka, J., Hubert, J., Escotte-Binet, S., Debelle, L., Villena, I., Sayagh, C., Borie, N., Martinez, A., Bertaux, B., 2021. Investigation of antiparasitic activity of ten European tree bark extracts on *Toxoplasma gondii* and bioguided identification of triterpenes in *Alnus glutinosa* barks. Antimicrob. Agents Chemother. 66, e01098. https://doi.org/10.1128/AAC.01098-21, 21.

Codiacconi, C., Picone, O., Lambert, V., Maurice, P., Pomar, L., Winer, N., Guibaud, L., Lavergne, R., Saliou, A., Quinio, D., Benachi, A., Noel, C., Ville, Y., Cuillier, F., Pomares, C., Ferret, N., Filisetti, D., Weingertner, A., Vequeau-Goua, V., et al., 2020. Ultrasound features of fetal toxoplasmosis: a contemporary multicenter survey in 88 fetuses. Prenat. Diagn. 40 (13), 1741–1752. https://doi.org/10.1002/pd.5756.

Darme, P., Escotte-Binet, S., Cordonnier, S., Remy, S., Hubert, J., Sayagh, C., Borie, N., Villena, I., Voutquenne-Nazabadioko, L., Dauchez, M., Baud, S., Renault, J.H., Aubert, D., 2022. Anti-*Toxoplasma gondii* effect of lupane-type triterpenes from the bark of black alder (*Alnus glutinosa*) and identification of a potential target by reverse docking. Parasite 29, 7. https://doi.org/10.1051/parasite/2022008.

De Carvalho, C.C.R., Caramujo, M.J., 2018. The various roles of fatty acids. Molecules 23, 2583. https://doi.org/10.3390/molecules23102583.

De Pablos, L.M., González, G., Rodrigues, R., García Granados, A., Parra, A., Osuna, A., 2010. Action of a pentacyclic triterpenoid, maslinic acid, against *Toxoplasma gondii*. *J. Nat. Prod.* 73, 831–834. https://doi.org/10.1021/pn900749b.

De Souza, G., Silva, R.J., Milión, I.C.B., Rosini, A.M., de Araújo, T.E., Teixeira, S.C., Oliveira, M.C., Franco, P.S., da Silva, C.V., Mineo, J.R., et al., 2021. Cyclooxygenase (COX)-2 modulates *Toxoplasma gondii* infection, immune response and lipid droplets formation in human trophoblast cells and villous explants. Sci. Rep. 11, 12709. https://doi.org/10.1038/s41598-021-92120-6.

De-Almeida, S.C.X., da-Silva, A., Sousa, N.R.T., Amorim, I.H.F., Leite, B.G., Neves, K.R.T., Costa, J.G.M., Felipe, C.F.B., de-Barros, Viana G.S., 2019. Antinociceptive and anti-inflammatory activities of a triterpenene-rich fraction from *Himatanthus drasticus*. *Braz. J. Med. Biol. Res.* 52, e7798. https://doi.org/10.1590/1414-431X20197798.

Dégbé, M., Debierre-Grockiego, F., Tété-Bénissan, A., Débare, H., Aklikokou, K., Dimier-Poisson, I., Gbeassor, M., 2018. Extracts of *Tectona grandis* and *Vernonia amygdalina* have anti-*Toxoplasma* and proinflammatory properties *in vitro*. Parasite 21, 11. https://doi.org/10.1051/parasite/2018014.

Deng, Y., Wu, T., Zhai, S.-Q., Li, C.-H., 2019. Recent progress on anti-*Toxoplasma* drugs discovery: design, synthesis and screening. Eur. J. Med. Chem. 183, 111711. https://doi.org/10.1016/j.ejmech.2019.111711.

Derouin, F., 2001. Anti-toxoplasmosis drugs. Curr. Opin. Invest. Drugs 2 (10), 1368–1374. London, England. https://europepmc.org/article/MED/11890349.

Diehl, M.S., Atindehou, K.K., Téré, H., Betschart, B., 2004. Prospect for anthelminthic plants in the Ivory Coast using ethnobotanical criteria. J. Ethnopharmacol. 95, 277–284. https://doi.org/10.1016/j.jep.2004.07.012.

Dujovne, C.A., Chan, K.C., Zimmerman, H.J., 1967. Sulfonamide hepatic injury. N. Engl. J. Med. 277 (15), 785–788. https://doi.org/10.1056/NEJM196710122771503.

Ebong, P.E., Owu, D.U., Isong, E.U., 1999. Influence of palm oil (*Elaeis guineensis*) on health. Plant Foods Hum. Nutr. 53 (3), 209–222. https://doi.org/10.1023/A:1008089715153.

Endo, M., Shigetomi, K., Mitsuhashi, S., Igarashi, M., Ubukata, M., 2019. Isolation, structure determination and structure-activity relationship of anti-toxoplasma triterpenoids from Quercus crispula Blume outer bark. J. Wood Sci. 65 (1), 3. https://doi.org/10.1186/s10086-019-1782-8.

Ferrea, G., Canessa, A., Sampietro, F., Cruciani, M., Romussi, G., Bassetti, D., 1993. *In vitro* activity of a *Combretum micranthum* extract against herpes simplex virus types 1 and 2. Antivir. Res. 21 (4), 317–325. https://doi.org/10.1016/0166-3542(93)90010-G.

Frenkel, J.K., 1974. Breaking the transmission chain of *Toxoplasma*: a program for the prevention of human toxoplasmosis. Bull. N. Y. Acad. Med. 50 (2), 228–235.

Grin, S.G., Wyllie, J.L., Markham, D.N., 1999. Leach, the role of structure and molecular properties of terpenoids in determining their antimicrobial activity. Flavour Fragrance J. 14, 322–332. https://doi.org/10.1002/(SICI)1099-1026(199909/10)14:53.0.CO;2-4.

Hamerski, L., Bomm, M.D., Silva, D.H.S., Young, M.C.M., Furlan, M., Eberlin, M.N., Castro-Gamboa, I., Cavalheiro, A.J., da Silva Bolzani, V., 2005. Phenylpropanol glucosides from leaves of Coussarea hydrangeifolia (Rubiaceae). Phytochemistry 66 (16), 1927–1932. https://www.sciencedirect.com/science/article/pii/S0031942612005002943.

Hassanpour, S.H., Doroudi, A., 2023. Review of the antioxidant potential of flavonoids as a subgroup of polyphenols and partial substitute for synthetic antioxidants. Avicenna J Phyomed. 13 (4), 354–376. https://doi.org/10.22038/AJP.2023.21774.

Holland, G.N., 2004. Ocular toxoplasmosis: a global reassessment: part II: disease manifestations and management. Am. J Ophthalmol. 137 (1), 1–17. https://doi.org/10.1016/j.ajo.2003.10.032.

Horvath, G., Wessjohann, Bigirimana, J., Jansen, M., Guisez, Y., Caubergs, R., Horemans, N., 2006. Differential distribution of tocopherols and tocotrienols in photosynthetic and non-photosynthetic tissues. Phytochemistry 67 (12), 1185–1195. https://doi.org/10.1016/j.phytochem.2006.04.004.

Hu, Y., Liu, X., Wu, X., Zhang, Z., Wu, D., Chen, C., Su, W., Zhang, L., Li, J., Wang, H.D., 2022. Several natural phytochemicals from Chinese traditional fermented food-pickled *Raphanus sativus* L.: purification and characterization. Food Chem. X 16 (15), 100390. https://doi.org/10.1016/j.fochx.2022.100390.
Hubert, J., Nuzillard, J.-M., Purson, S., Hamzaoui, M., Borie, N., Reynaud, R., Renault, J.-H., 2014. Identification of natural metabolites in mixture : a pattern recognition strategy based on ¹³C NMR. Anal. Chem. 86 (6), 2955-2962. https://doi.org/10.1021/ac403223f.

Islam, M.N., Ishita, I.J., Jung, H.A., Choi, J.S., 2014. Vicenin 2 isolated from *Artemisia capillaris* exhibited potent anti-glycation properties. Food Chem. Toxicol. 69, 55-62. https://doi.org/10.1016/j.fct.2014.03.042.

Ito, N., Etoh, T., Hagiwara, H., Kato, M., 1997. Novel synthesis of degradation products of carotenoids, megastigmatrienone analogs and blumenol-A. J. Chem. Soc., Perkin Trans. 1: Organic and Bio-Organic Chemistry 10, 1571-1579. https://doi.org/10.1039/A607028K.

Junio, H.A., Sy-Cordero, A.A., Ettefang, K.A., Burns, J.T., Micko, K.T., Graf, T.N., Richter, S.J., Cannon, R.E., Oberlies, N.H., Cech, N.B., 2011. Synergy-directed fractionation of botanical medicines: a case study with goldenseal (hydrastis canadensis). J. Nat. Prod. 74, 1621-1629. https://doi.org/10.1021/np200336g.

Kim, J.H., Lee, B.C., Kim, J.H., Sim, G.S., Lee, D.H., Lee, K.E., Yun, Y.P., Pyo, H.B., 2005. The isolation and antioxidative effects of vitexin from Acer palmatum. Arch Pharm. Res. (Seoul) 28 (2), 195-202. https://doi.org/10.1007/BF02977715.

Kim, S.J., Scott, I.U., Brown, G.C., Brown, M.M., Ho, A.C., Ip, M.S., Frecchia, M., 2013. Interventions for *Toxoplasma retinochoroiditis*. Ophthalmology 120 (2), 371-378. https://doi.org/10.1016/j.ophtha.2012.07.061.

Kim, H.M., Lee, D.G., Lee, S., 2015. Plant-derived molecules from *Sauropsia grandifolia* as inhibitors of aldose reductase. Journal of the Korean Society for Applied Biological Chemistry 58 (3), 365-371. https://doi.org/10.1007/s13765-015-0051-9.

Koné, M., Atindehou Kamanzi, D., Traoré, D., 2002. Plantes et médecine traditionnelle dans la région de Ferkessédougou (Côte d'Ivoire). West-African annals of botany 2, 13-23.

Krajewski, D., Tóth, G., Schreier, P., 1996. 2-Ethyl-3-methylmaleimide N-β-d-glucopyranoside from the leaves of mangosteen (Garcinia mangostana). Phytochemistry 43 (1), 141-143. https://doi.org/10.1016/00319422(96)00276-2.

Lang, C., Groß, U., Lüder, C.G.K., 2007. Subversion of innate and adaptive immune responses by *Toxoplasma gondii*. Parasitol. Res. 100 (2), 191-203. https://doi.org/10.1007/s00436-006-0306-9.

Liu, Y., Abreu, P.J.M., 2006. Long chain alkyl and alkenyl phenols from the roots of Ozoroa insignis. J. Braz. Chem. Soc. 17, 527-532. https://doi.org/10.1007/s00044-016-1521-2.

MacGregor, W.S., 1967. The chemical and physical properties of DMSO. Ann. N. Y. Acad. Sci. 141 (1), 3-12. https://doi.org/10.1111/j.1749-6632.1967.tb34860x.

Mahato, S.B., Kundu, A.P., 1994. ¹³C NMR spectra of pentaacyclic triterpenoids-A compilation and some salient features. Phytochemistry 37, 1517-1575. https://doi.org/10.1016/S0031-9422(00)89569-2.

Mahizan, N.A., Yang, S.K., Moo, C.L., Song, A.A., Chong, C.M., Chong, C.W., Abushelaibi, A., Lim, S.E., Lai, K.S., 2019. Terpene derivatives as a potential agent against antimicrobial resistance (AMR) pathogens. Molecules 24, 2631. https://doi.org/10.3390/molecules24142631.

Mamaghani, A.J., Fathollahi, A., Arab-Mazar, Z., kohansal, K., Fathollahi, M., Spotin, A., Bashiri, H., Bozorgomid, A., 2023. *Toxoplasma gondii* vaccine candidates : a concise review. Ir. J. Med. Sci. 192 (1), 231-261. https://doi.org/10.1007/s11845-022-02998-9.

Matsunami, K., Otsuka, H., Takeda, Y., 2010. Structural revisions of blumenol C glucoside and byzantionoside B. Chem. Pharm. Bull. 58 (3), 438-441. https://doi.org/10.1024/cpb.58.438.

McLeod, R., Kieffer, F., Sautter, M., Hosten, T., Pelloux, H., 2009. Why prevent, diagnose and treat congenital toxoplasmosis? Mem Inst Oswaldo Cruz 104 (2), 320-344. https://doi.org/10.1590/S0074-02762009000200029, 2009.

Mendonca, P., Soliman, K.F.A., 2020. Flavonoids activation of the transcription factor Nrf2 as a hypothesis approach for the prevention and modulation of SARS-CoV-2 infection severity. Antioxidants 24 (8), 659. https://doi.org/10.3390/antioxidation96509.

Miller, A., 1996. Antioxidant flavonoids: structure, function and clinical usage. Alternative Med. Rev. 1 (2), 103-111.

Newman, D.J., Cragg, G.M., 2020. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J. Nat. Prod. 83 (3), 770-803. https://doi.org/10.1021/acs.jnatprod.9b01285.

Nguenang, G.S., Mbaveng, A.T., Fankam, A.G., Manekeng, H.T., Nayim, P., Wamba, B.E. N., Kuete, V., 2018. *Tristemna hirtum* and five other Cameroonian edible plants with weak or no antibacterial effects modulate the activities of antibiotics against Gram-negative multidrug-resistant phenotypes. Sci. World J. 22, 7651482. https://doi.org/10.1155/2018/7651482, 2018.

Nolan, S.J., Romano, J.D., Kline, J.T., Coppens, I., 2018. Novel approaches to kill *Toxoplasma gondii* by exploiting the uncontrolled uptake of unsaturated fatty acids and vulnerability to lipid storage inhibition of the parasite. Antimicrob. Agents Chemother. 62, e00347. https://doi.org/10.1128/aac.00347-18, 18.

Nothias, L.F., Nothias-Esposito, M., Da Silva, R., Wang, M., Protsyuk, I., Zhang, Z., Sarvepalli, A., Leyssen, P., Touboul, D., Costa, J., Paolini, J., Alexandrov, T., Litaudon, M., Dorrestein, P.C., 2018. Bioactivity-based molecular networking for the discovery of drug leads in natural product bioassay-guided fractionation. J. Nat. Prod. 81, 758-767. https://doi.org/10.1021/acs.jnatprod.7b00737.

Nuzillard, J.-M., 2023. Use of carbon-13 NMR to identify known natural products by querying a nuclear magnetic resonance database—an assessment. Magn. Reson. Chem. 61 (11), 582-588. https://doi.org/10.1002/mrc.5386.

Nyasangox, T., Fru, C.G., Sithole, S., Mukanganyama, S., 2018. The effects of phytosterols isolated from *Omphalocarpus elatii* (Sapotaceae) on mouse peritoneal cells. J. Drug Discov. Dev. Deliv. 5 (1), 1032.

Osonwa, U.E., Umeyor, C.E., Okon, J.E., Joel, E., Agboke, A., Udosen, V.M., 2012. Physicotechnical properties of tablets formulated with ethanolic extract of fresh leaves of *Combrebium micranthum* G. Don. J. Appl. Pharmaceut. Sci. 2 (12), 125-129. https://doi.org/10.7324/JAPS.2012.21223.

Pauli, G.F., Junior, P., 1995. Phenolic glycosides from *Adonis aleppica*. Phytochemistry 38, 1245-1250. https://doi.org/10.1016/0031-9422(94)00635-7.

Pereira, A.C.A., Silva, R.J., Franco, P.S., de Oliveira Gomes, A., Souza, G., Milian, I.C.B., Ribeiro, M., Rosini, A.M., Guirelli, P.M., Ramos, E.L.P., 2019. Cyclooxygenase (COX)-2 inhibitors reduce *Toxoplasma gondii* infection and upregulate the pro-inflammatory immune response in *Calomys callosus* rodents and human monocyte cell line. Front. Microbiol. 10, 225. https://doi.org/10.3389/fmicb.2019.00225.

Petrelli, R., Orsando�, G., Sorci, L., Maggi, F., Ranbarian, F., Biapora Nya, P.C., Petrelli, D., Vitali, L.A., Lupidi, G., Quassinti, L., Bramucci, M., Hofer, A., Cappellacci, L., 2016. Biological activities of the essential oil from *Erigeron floribundus*. Molecules 21 (8). https://doi.org/10.3390/molecules21081065. Article 8.

Qiu, F., Cai, G., Jaki, B.U., Lankin, D.C., Franzblau, S.G., Pauli, G.F., 2013. Quantitative purity-activity relationships of natural products: the case of anti-tuberculosis active triterpenes from *Oplopanax horridus*. J. Nat. Prod. 76, 413-419. https://doi.org/10.1021/np3007809.

Robert-Gangneux, F., Aubert, D., Toxoplasmosis, Villena, I. 2022. A widespread zoonosis diversely affecting humans and animals. Editeur: A Sing. Zoonoses: Infections Affecting Humans and Animals Pour. Springer, Cham. https://doi.org/10.1007/978-3-030-85877-3-14-1. ISBN 978-3-030-85877-3.

Rusaati, B.I.W., Gendusa, A.P., Joo, S.H., Park, J.W., Masumbuko, N.C., Iragi, K.G., Ngbolua, K.-T.-N., Furaha, M.A., Rolly, N.K., Kang, J.W., 2021. A systematic review of antimalarial medicinal plants in Democratic Republic of the Congo. Bois Forets Tropiques 347, 11-27. https://doi.org/10.19182/bft2021.347.a31882.

Sandjo, L.P., Fru, C.G., Kuete, V., Nana, F., Yeboah, S.O., Mapitse, R., Abegaz, B.M., Efferth, T., Opatz, T., Ngadjui, B.T., 2014. Elatumic acid: a new uursolic acid congener from *Omphalocarpus elatii* Miers (Sapotaceae). Z. Naturforsch., C: J. Biosci. 69 (7-8), 276-282. https://doi.org/10.5560/znc.2014-00500.

Santos, N.C., Figueira-Coelho, J., Martins-Silva, J., Saldanha, C., 2003. Multidisciplinary utilization of dimethyl sulfoxide : pharmacological, cellular, and molecular aspects. Biochem. Pharmacol. 65 (7), 1035-1041. https://doi.org/10.1016/s0006-2952(03)00002-9.

Sepulveda-Arias, J.C., Veloza, L.A., Mantilla-Muriel, L.E., 2015. Anti-*Toxoplasma* activity of natural products: a review. Recent Pat Anti infect Drug Discov 9 (3), 186-194. https://doi.org/10.2174/1574891x10666150401120321.

Shaaban, M.T., Ghaly, M.F., Fahmi, S.M., 2021. Antibacterial activities of hexadecanoic acid methyl ester and green-synthesized silver nanoparticles against multidrug-resistant bacteria. J. Basic Microbiol. 61, 557-568. https://doi.org/10.1002/jbm202100061.

Sheu, J.H., Wang, G.H., Sung, P.J., Duh, C.Y., 1999. New cytotoxic oxygenated fucosterols from the brown alga *Turbinaria conoides*. J. Nat. Prod. 62 (2), 224-227. https://doi.org/10.1021/jn980233s.

Smith, N.C., Goulart, C., Hayward, J.A., Kupz, A., Miller, C.M., van Dooren, G.G., 2021. Control of human toxoplasmosis. Int. J. Parasitol. 51, 95-121. https://doi.org/10.1016/j.ijpara.2020.11.001.

Soundararajan, V., Sreenivasan, S., 2012. Antioxidant activity of *Elaeis guineensis* leaf extract: an alternative nutraceutical approach in impeding aging. APCBEE Procedia 2, 153-159. https://doi.org/10.1016/j.apcbee.2012.06.028.

Sousa, S., Fernandes, M., Correia da Costa, J.M., 2023. Serotyping, a challenging approach for *Toxoplasma gondii* typing. Front. Med. 10. https://doi.org/10.3389/fmed.2023.1111509.

Tallarida, R.J., 2011. Quantitative methods for assessing drug synergism. Genes Cancer 2 (11), 1003-1008. https://doi.org/10.1177/1947601912440575.

Tepongning, R.N., Mbah, J.N., Avouloul, F.L., Jerme, M.M., Ndanga, E.K., Fekam, F.B., 2018. Hydroethanolic extracts of *Erigeron floribundus* and *Adarizadira indica* reduced Plasmodium berghei parasitemia in balb/c mice. Evid Based Complement Alternat Med 21, 5156710. https://doi.org/10.1155/2018/5156710.

Tow, W.K., Goh, A.P., Sundralingam, U., Palanisamy, U.D., Sivasothy, Y., 2021. Flavonoid composition and pharmacological properties of *Elaeis guineensis* Jacq. leaf extracts: a systematic review. Pharmaceuticals 24 (10), 961. https://doi.org/10.3390/ph14100961, 14.

Tra Bi, F.H., Koné, M.C., Kouamé, N.F., 2008. Antifungal activity of *Erigeron floribundus* (asteraceae) from Côte d'Ivoire, West Africa. Trop. J. Pharmaceut. Res. 7, 975-979. https://doi.org/10.4314/tjpr.v7i2.14681.

Traoré-Coulibaly, M., YougBaRé, S., OuédRaOgO, J.B., Guissou, I.P., Nacoulma, O.G., Guiguéméd, T.R., Christensen, S.B., 2017. Activité antiplasmodique de Chanthium multiflorum (Schumach. & Thonn.) Hiern (Rubiaceae) et de Trichilia emetica Vahl. (Méliaceae) utilisées dans le traitement du paludisme. Sciences de la Santé 40 (2).

Valnet, J., 1983. Traitement des maladies par les plantes. Maloine, Paris, pp. 500-512.

Wagner, H., Ulrich-Merzenich, G., 2009. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine 16, 97-110. https://doi.org/10.1016/j.phymed.2008.12.018.

Wong, S.-Y., Remington, J.S., 1994. Toxoplasmosis in pregnancy. Clin. Infect. Dis. 18 (6), 853-862. https://doi.org/10.1093/clinids/18.6.853.

Xu, M., Yang, C.R., Zhang, Y.J., 2009. Minor antifungal aromatic glycosides from the roots of Gentiana rescens (Gentianaceae). Chin. Chem. Lett. 20 (10), 1215-1217. https://doi.org/10.1093/jclet/2009.05.006.

Yamamura, S., Ozawa, K., Ohtani, K., Kasai, R., Yamasaki, K., 1998. Antihistaminic flavones and aliphatic glycosides from Mentha spicata. Phytochemistry 48 (1), 131-136. https://doi.org/10.1016/S00319422(97)01112-6.

Yan, J., Shi, X., Donkor, P.O., Zhu, H., Gao, X., Ding, L., Qiu, F., 2017. Nine pairs of megastigmate enantiomers from the leaves of *Eucommia ulmoides* Oliver. J. Nat. Med. 71, 780-790. https://doi.org/10.1007/s11418-017-1102-9.
Yin, N.S., Abdullah, S., Phin, C.K., 2013. Phytochemical constituents from leaves of *elaies guineensis* and their antioxidants and antimicrobial activities. Int. J. Pharm. Pharmaceut. Sci. 5, 137-140. Suppl 4.

Zacchino, S.A., Butassi, E., Cordisco, E., Svetaz, L.A., 2017. Hybrid combinations containing natural products and antimicrobial drugs that interfere with bacterial and

fungal biofilms. *Phytomedicine* 37, 14–26. https://doi.org/10.1016/j.phymed.2017.10.021.

Zheng, C.J., Yoo, J.-S., Lee, T.-G., Cho, H.-Y., Kim, Y.-H., Kim, W.-G., 2005. Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids. FEBS Lett. 579, 5157–5162. https://doi.org/10.1016/j.febslet.2005.08.028.