# Chemical Constituents of the Bark ofZanthoxylum gilletii (Rutaceae) and Their In Vitro Antiplasmodial and Molecular Docking Studies

Authors: Liliane Clotilde Dzouemo; Gervais Mouthé Happi; Sikiru Akinyeye Ahmed; Willifred Dongmo Tekapi Tsopgni; Michael Nde Akuma; Shina Salau; Emmanuel Ngeufa Happi; Jean Duplex Wansi
Journal: Journal of Chemistry
 (2022)

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## Abstract

The phytochemical investigations of the methanol extract ofZanthoxylum gilletii bark led to the isolation of thirteen compounds identified as two alkaloids including one acridone 5-hydroxynoracronycine (1) and one benzo [c] phenanthridine decarine (2), three lignans trans- and cis-fagaramide (3 and 4) and sesamin (5), two coumarins scoparone (6) and scopoletin (7), three pentacyclic triterpenoids fridelin (8), lupeol (9) and erythrodiol-3-O-palmitate (10), one phenolic compound vanillic acid (11) as well as two common steroids stigmasterol (12), and its derivative stigmasterol-3-O-β-D-glucopyranoside (13). The structures of all the isolated compounds were elucidated by means of their spectroscopic and spectrometric data (1D, 2D-NMR, MS) as well as the comparison of these data with those reported in the literature. Except for compounds 9 and 11–13, all the other isolated compounds are reported for the first time from Z. gilletii but have been already obtained from other Zanthoxylum species and in the Rutaceae family. Compounds 1, 3–5, and 9 were tested in vitro for their antiplasmodial potencies against Plasmodium falciparum 3D7, and the results revealed that all the tested compounds displayed an inhibition between 51.89% and 54.69% while only the mixture of 3 + 4 gave an IC50 lower than 10 000 nM (IC50 = 1333 nM). Furthermore, all the compounds have been evaluated in silico for their ability to inhibit the Plasmodium falciparum dihydroorotate dehydrogenase 5TBO. Sesamin (5) showed the greatest affinity to the antiplasmodium receptor than artemether® and chloroquine®. Further recorded data from their ADMET study, as well as their chemotaxonomy, are also discussed herein. The present study provides further information to enrich the chemistry of Z. gilletii and its qualification as an important source for good candidates in new antiplasmodial drug development.


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## Body

## 1. Introduction

Malaria is a parasitic disease with a great incidence in the public health sector in many countries in tropical areas of the world [1]. A recent report by the World Health Organization (WHO) indicated that 228 million cases of malaria have been recorded in the world with 405 thousand deaths including almost 94% occurring in Africa. More specifically, fifteen African countries accounted for 80% of global malaria deaths while 2,974,819 confirmed cases and an estimation of 14,841 deaths have been reported in Cameroon during the year 2020 [2, 3]. The disease is caused by the parasite Plasmodium transmitted through the bites of the mosquitoes Anopheles [1, 4]. It is well reported that medicinal plants represent an important source of bioactive compounds that can be considered as good candidates in the development of new potent drugs. For instance, the well-known antimalarial drug artemisinin was initially obtained from the plant Artemisia annua while quinine was isolated from Cinchona officinalis [5]. Unfortunately, the observed rise of resistance of P. falciparum to the available antimalarial drugs keeps actual the search for new affordable and potent compounds that can be developed as drugs. Zanthoxylum gilletii (Rutaceae) also called Z. macrophylla, Fagara tessmanii, or F. macrophylla is a tree encountered in the tropical rainforest that can reach 10 to 35 m high [6, 7]. The plant is one of the 2000 species that constituted the genus Zanthoxylum and its synonyms in the genus Fagara which are widely used in African folk medicine for the treatment of several diseases [8]. The stem bark of Z. gilletii is used in Cameroon and Madagascar for the treatment of microbial infection, cancer, inflammation, hypertension, and related disorders while its bark is used in Kenya and Ivory Coast against malaria [8]. The use of Z. gilletii in the management of malaria has been further supported by the good antimalarial activity of its ethanolic extract against a panel of Plasmodium falciparum strains with an IC50 value of 5 μg/mL [9]. As a continuity of our program in searching for antiplasmodial agents from Cameroonian medicinal plants [1, 10], we carried out phytochemical investigations of the bark of Z. gilletii. In addition, the in silico study of the isolated compounds as potential inhibitors of the 5TBO receptor (Plasmodium falciparum dihydroorotate dehydrogenase) as well as their ADMET evaluation and chemotaxonomy have been done, and the results are herein discussed. This study provides additional compounds to enrich the chemistry of Z. gilletii, its chemotaxonomic classification in the family Rutaceae as well as it further supports its use in folk medicine by the local population for the management of malaria and related symptoms.


## 2. Materials and Methods

### 2.1. General Instrumentation

EI-MS were recorded on a Finnigan MAT 95 spectrometer (70 eV) (Thermo Fisher Scientific, Darmstadt, Germany) with perfluorokerosene as a reference substance for EI-HR-MS. The spectrometer operated in positive and negative modes (m/z: 50–1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 1 ppm deviation using Na formate as a calibrant. The following parameters were used for experiments: spray voltage of 4.5 kV and the capillary temperature of 200°C. Nitrogen was used as sheath gas (4 l/min). The 1H- and 13C-NMR spectra were recorded at 500 MHz and 125 MHz, respectively, on Bruker AMX 500 NMR spectrometers (Bruker, Rheinstetten, Germany). Chemical shifts are reported in δ (ppm) using TMS as an internal standard, and coupling constants (J) were measured in Hz. Column chromatography was carried out on silica gel (70–230 mesh (Merck, Darmstadt, Germany). Thin-layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 aluminium foil (Merck, Darmstadt, Germany), and spots were detected using ceric sulphate spray reagent. All reagents used were of analytical grade.


### 2.2. Plant Material

The stem bark ofZanthoxylum gilletii (De Wild.) P. G. Waterman was harvested at Bana-Tentcheu (GPS coordinates: Latitude 5°07′57″N, Longitude 10°17′41″E, Elevation: 1412 m), West region, Cameroon, in April 2021. Some leave and bark samples have been used for the identification of the plant by Mr. Victor Nana in comparison with the plant material available in the National Herbarium of Cameroon database where a specimen was registered under the voucher number 38960 HNC.


### 2.3. Extraction and Isolation

The yellowish bark ofZ. gilletii (3.8 kg) has been dried and grounded to obtain a powder that was twice extracted with methanol at room temperature for 48 h, each, to afford 148.3 g of crude extract after removing solvent. After keeping a small amount for biological tests and further chemical analyses, 130 g of crude extract was dissolved in water and successively partitioned with ethyl acetate (EtOAc) and n-butanol to give two main fractions A (78.4 g) and B (46.8 g). The ethyl acetate soluble fraction A has been further chromatographed over a silica gel column (230–400 mesh, 5.0 × 75.0 cm) eluting with increasing polarity of ethyl acetate in n-hexane (n-Hex) followed by the gradient of methanol in ethyl acetate. A total of 563 subfractions (ca. 200 mL each) were collected and combined into 6 series A1‒A6 based on their TLC profiles.Further purification of series A1 (14.2 g,n-Hex/EtOAc 9 : 1) using column chromatography led to the precipitation of four compounds at four different successive polarities including compound 8 (3.3 mg) at n-Hex/EtOAc 39 : 1, compound 12 (20.6 mg) at n-Hex/EtOAc 19 : 1, compound 10 (4.2 mg) at n-Hex/EtOAc 37 : 3, and compound 9 (47.8 mg) at n-Hex/EtOAc 9 : 1.Likewise, the second series A2 (12.7 g,n-Hex/EtOAc 8 : 2) was also subjected to column chromatography over silica gel eluting with a gradient of EtOAc in n-Hex from 9 : 1 to 3 : 1 and allowed the collection of 132 subfractions (100 mL each) and the filtration of four distinct pure compounds 1 (19.3 mg), 2 (3.6 mg), 5 (32.4 mg), and 11 (2.4 mg) from the subfractions obtained at the polarity between n-Hex/EtOAc 17 : 3 and n-Hex/EtOAc 8 : 2.A third series A3 (8.2 g,n-Hex/EtOAc 7 : 3) has been further purified using the same chromatographic technique to afford 115 subfractions from which the unseparated mixture of compounds 3 and 4 (8.3 mg) was obtained at n-Hex/EtOAc 3 : 1, and compound 6 (3.2 mg) was obtained after column chromatography using Sephadex LH20 of the subfractions 64–86 (2.1 g, n-Hex/EtOAc 7 : 3) eluting isocratically with methanol.Finally, the series A4 (10.3 g,n-Hex/EtOAc 1 : 1) and A5 (11.4 g, EtOAc) were mixed and chromatographed using a gradient of methanol in dichloromethane to obtain compounds 7 (2.8 mg) and 13 (28.7 mg), while the last series A6 (18.1 g, AE/MeOH 5%) has been mixed to the n-BuOH soluble main fraction B and subjected to column chromatography using the increasing polarity of methanol in EtOAc to afford compound 13 (3.8 mg), and some precipitates are insoluble in the organic solvent.


### 2.4.Plasmodium falciparum Culture and Growth Inhibition Assay

Plasmodium falciparum 3D7 (chloroquine-sensitive) strain was obtained from the Biodefense and Emerging Infections (BEI) Research Resources (Manassas, VA) and maintained using a modified Trager and Jensen method. The biological assay has been done following a modified method of Yemback et al. [11]. Briefly, parasites were cultured in fresh O+ human red blood cells at 3% (v/v) hematocrit in RPMI 1640 culture media containing GlutaMAX and NaHCO3 (Gibco, UK) and supplemented with 25 mM HEPES (Gibco, UK), 1X hypoxanthine (Gibco, USA), 20 µg/mL gentamicin (Gibco, China), and 0.5% Albumax II (Gibco, USA). When needed, parasites were synchronized at the ring stage by sorbitol treatment and cultured through one cycle before treatment.Stock solutions (10 mM) were prepared in incomplete RPMI 1640 and mixed with parasite cultures (1% parasitemia and 1.5% hematocrit) in 96-well plates to a final drug concentration of 10µM for hit identification studies, or 10–0.078 µM for activity confirmation assays. The final dimethyl sulfoxide (DMSO) concentration per 100 μL culture per well was 0.1%. Chloroquine and artemisinin at a range of 1–0.0078 µM each were used as negative growth control (positive test control), while the solvent-treated culture (0.1% DMSO) was used as positive growth control (negative test control). Following 72 h incubation at 37°C in a 5% CO2 incubator, parasite growth was assessed by an SYBR green I-based DNA quantification assay. In brief, a 3x concentrated SYBR Green lysis buffer was added to each plate well containing parasitized erythrocytes and kept in the dark for about 30 minutes. Fluorescence was measured using a Fluoroskan Ascent multiwell plate reader with excitation and emission wavelengths at 485 and 538 nm, respectively. Mean half-maximal inhibitory concentrations (IC50 values) were derived by plotting percent growth against log drug concentration and fitting the response data to a variable slope sigmoidal curve function using GraphPad Prism v8.0.


### 2.5. Computational Methodology

#### 2.5.1. Computational Tools

Allin silico studies applied to this work were achieved using the following computational tool:(a)
ACDChemSketch® version 2.0, year 2020 used for modeling, first-hand, chemical structures to be fed for docking screening [12](b)
Autodock Vina® tool version 1.2.0, year 2010 used for the virtual screening procedure [13](c)
BIOVIA® Discovery Studio version 2021 used for the determination of binding pockets and for visualizing/analyzing the docked results [14](d)
Absorption Distribution Metabolism Excretion and Toxicity of Structure Activity-Relationship (ADMETSAR): a web server used to evaluate the ADMET properties, biological activity, and drug-likeness [15–18]


#### 2.5.2. Molecular Docking

(1) Ligand Preparations. In a quest to predict the antiplasmodial activity of the plant samples, compounds present in Z. gilletii were modeled using the ACDChem ChemSketch® version 2.0, year 2020 [12] computational tool and fed into Autodock vina® [13] for binding efficiency screening.(2) Receptor preparations. The antiplasmodium receptor of PDB CODE, 5TBO, was used as the target receptor for this study. The receptor was obtained from the (RCSB) database (https://www.rcsb.org/pdb). The resolution of receptor 5TBO (2.15 Å) approximately lies about the 2.0 Å recommended resolution for a receptor of good quality [19]. Water molecules around the crystal structure and other associated inhibitors were removed from the downloaded protease to avoid undesired interferences and unwanted molecular interactions.(3) Virtual screening. The virtual screening was done on Z. gilletii containing thirteen compounds using the Autodock vina® tool version 1.2.0, year 2010 [13] and Biovia Discovery Studio version 2021 [14]. The screening was guided using Chloroquine® and Artemether®, prominently used antiplasmodium therapeutics, as standards. Auto Dock Tools version 4.0 (ADT) was used to generate the autodock file (.pdbqt file) in preparation for docking. Other adjustments performed on both crystal structures include removing any extra water molecules, adding polar hydrogens, merging no-polar hydrogens, and adding Kollman charges. These adjustments are targeted at improving the affinity of the binding site. In modern computer-assisted drug design (CADD), the study of a potent drug is not complete without the activity study of the drug which is deducible from the compound’s inhibition constant. A potent compound is expected to have inhibition constant values ranging between 0.01 and 1 μM for a potent drug; therefore, this parameter alone is enough to screen compounds in the drug discovery journey [19].


#### 2.5.3. ADMET Studies

For predicting the toxicity level of the screened compounds,in silico toxicity studies were performed using the ADMETlab version 2.0 web server online [20]. Additionally, from the server, the absorption (human intestinal absorption), distribution (blood-brain barrier; water solubility), metabolism (cytochrome inhibition: CYP450), excretion (half-life), toxicity (hERG inhibition; carcinogenicity), and drug-likeness were computed. The results are summarized in Table 1.Table 1
ADMET score of qualified compounds fromZ. gilletii with respect to the selected standards.
CompoundsHIABBBWater solubility (mol/L)CYP450 inhibitionhERG inhibitionDrug likeness (Lipinski)T(1/2)Carcinogenicity1Yes (>60%)NoSoluble (−3.615)2/5InactiveYes (5/5)Short (0.255)Yes2Yes (>90%)NoModerately soluble (−6.520)3/5InactiveYes (5/5)Short (0.192)Yes3Yes (>90%)YesSoluble (−4.305)4/5InactiveYes (5/5)Long 0.547Yes5Yes (>90%)NoModerately soluble (−6.605)3/5InactiveYes (5/5)Short 0.137Yes8Yes (>90%)YesModerately soluble (−6.781)1/5InactiveYes (5/5)Very short (0.041)No9Yes (>90%)NoModerately soluble (−6.667)0/5InactiveYes (5/5)Very short (0.037)No12Yes (>90%)NoPoorly soluble (−7.008)1/5InactiveYes (5/5)Very short (0.030)No13Yes (>60%)NoSoluble (−4.793)0/5InactiveYes (5/5)Very short (0.043)NoChloroquineYes (>90%)YesSoluble (−3.902)(2/5)InactiveYes (5/5)Very short (0.070)NoArtemetherYes (>90%)NoSoluble (−4.677)(0/5)InactiveYes (5/5)Very short (0.089)No


## 2.1. General Instrumentation

EI-MS were recorded on a Finnigan MAT 95 spectrometer (70 eV) (Thermo Fisher Scientific, Darmstadt, Germany) with perfluorokerosene as a reference substance for EI-HR-MS. The spectrometer operated in positive and negative modes (m/z: 50–1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 1 ppm deviation using Na formate as a calibrant. The following parameters were used for experiments: spray voltage of 4.5 kV and the capillary temperature of 200°C. Nitrogen was used as sheath gas (4 l/min). The 1H- and 13C-NMR spectra were recorded at 500 MHz and 125 MHz, respectively, on Bruker AMX 500 NMR spectrometers (Bruker, Rheinstetten, Germany). Chemical shifts are reported in δ (ppm) using TMS as an internal standard, and coupling constants (J) were measured in Hz. Column chromatography was carried out on silica gel (70–230 mesh (Merck, Darmstadt, Germany). Thin-layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 aluminium foil (Merck, Darmstadt, Germany), and spots were detected using ceric sulphate spray reagent. All reagents used were of analytical grade.


## 2.2. Plant Material

The stem bark ofZanthoxylum gilletii (De Wild.) P. G. Waterman was harvested at Bana-Tentcheu (GPS coordinates: Latitude 5°07′57″N, Longitude 10°17′41″E, Elevation: 1412 m), West region, Cameroon, in April 2021. Some leave and bark samples have been used for the identification of the plant by Mr. Victor Nana in comparison with the plant material available in the National Herbarium of Cameroon database where a specimen was registered under the voucher number 38960 HNC.


## 2.3. Extraction and Isolation

The yellowish bark ofZ. gilletii (3.8 kg) has been dried and grounded to obtain a powder that was twice extracted with methanol at room temperature for 48 h, each, to afford 148.3 g of crude extract after removing solvent. After keeping a small amount for biological tests and further chemical analyses, 130 g of crude extract was dissolved in water and successively partitioned with ethyl acetate (EtOAc) and n-butanol to give two main fractions A (78.4 g) and B (46.8 g). The ethyl acetate soluble fraction A has been further chromatographed over a silica gel column (230–400 mesh, 5.0 × 75.0 cm) eluting with increasing polarity of ethyl acetate in n-hexane (n-Hex) followed by the gradient of methanol in ethyl acetate. A total of 563 subfractions (ca. 200 mL each) were collected and combined into 6 series A1‒A6 based on their TLC profiles.Further purification of series A1 (14.2 g,n-Hex/EtOAc 9 : 1) using column chromatography led to the precipitation of four compounds at four different successive polarities including compound 8 (3.3 mg) at n-Hex/EtOAc 39 : 1, compound 12 (20.6 mg) at n-Hex/EtOAc 19 : 1, compound 10 (4.2 mg) at n-Hex/EtOAc 37 : 3, and compound 9 (47.8 mg) at n-Hex/EtOAc 9 : 1.Likewise, the second series A2 (12.7 g,n-Hex/EtOAc 8 : 2) was also subjected to column chromatography over silica gel eluting with a gradient of EtOAc in n-Hex from 9 : 1 to 3 : 1 and allowed the collection of 132 subfractions (100 mL each) and the filtration of four distinct pure compounds 1 (19.3 mg), 2 (3.6 mg), 5 (32.4 mg), and 11 (2.4 mg) from the subfractions obtained at the polarity between n-Hex/EtOAc 17 : 3 and n-Hex/EtOAc 8 : 2.A third series A3 (8.2 g,n-Hex/EtOAc 7 : 3) has been further purified using the same chromatographic technique to afford 115 subfractions from which the unseparated mixture of compounds 3 and 4 (8.3 mg) was obtained at n-Hex/EtOAc 3 : 1, and compound 6 (3.2 mg) was obtained after column chromatography using Sephadex LH20 of the subfractions 64–86 (2.1 g, n-Hex/EtOAc 7 : 3) eluting isocratically with methanol.Finally, the series A4 (10.3 g,n-Hex/EtOAc 1 : 1) and A5 (11.4 g, EtOAc) were mixed and chromatographed using a gradient of methanol in dichloromethane to obtain compounds 7 (2.8 mg) and 13 (28.7 mg), while the last series A6 (18.1 g, AE/MeOH 5%) has been mixed to the n-BuOH soluble main fraction B and subjected to column chromatography using the increasing polarity of methanol in EtOAc to afford compound 13 (3.8 mg), and some precipitates are insoluble in the organic solvent.


## 2.4.Plasmodium falciparum Culture and Growth Inhibition Assay

Plasmodium falciparum 3D7 (chloroquine-sensitive) strain was obtained from the Biodefense and Emerging Infections (BEI) Research Resources (Manassas, VA) and maintained using a modified Trager and Jensen method. The biological assay has been done following a modified method of Yemback et al. [11]. Briefly, parasites were cultured in fresh O+ human red blood cells at 3% (v/v) hematocrit in RPMI 1640 culture media containing GlutaMAX and NaHCO3 (Gibco, UK) and supplemented with 25 mM HEPES (Gibco, UK), 1X hypoxanthine (Gibco, USA), 20 µg/mL gentamicin (Gibco, China), and 0.5% Albumax II (Gibco, USA). When needed, parasites were synchronized at the ring stage by sorbitol treatment and cultured through one cycle before treatment.Stock solutions (10 mM) were prepared in incomplete RPMI 1640 and mixed with parasite cultures (1% parasitemia and 1.5% hematocrit) in 96-well plates to a final drug concentration of 10µM for hit identification studies, or 10–0.078 µM for activity confirmation assays. The final dimethyl sulfoxide (DMSO) concentration per 100 μL culture per well was 0.1%. Chloroquine and artemisinin at a range of 1–0.0078 µM each were used as negative growth control (positive test control), while the solvent-treated culture (0.1% DMSO) was used as positive growth control (negative test control). Following 72 h incubation at 37°C in a 5% CO2 incubator, parasite growth was assessed by an SYBR green I-based DNA quantification assay. In brief, a 3x concentrated SYBR Green lysis buffer was added to each plate well containing parasitized erythrocytes and kept in the dark for about 30 minutes. Fluorescence was measured using a Fluoroskan Ascent multiwell plate reader with excitation and emission wavelengths at 485 and 538 nm, respectively. Mean half-maximal inhibitory concentrations (IC50 values) were derived by plotting percent growth against log drug concentration and fitting the response data to a variable slope sigmoidal curve function using GraphPad Prism v8.0.


## 2.5. Computational Methodology

### 2.5.1. Computational Tools

Allin silico studies applied to this work were achieved using the following computational tool:(a)
ACDChemSketch® version 2.0, year 2020 used for modeling, first-hand, chemical structures to be fed for docking screening [12](b)
Autodock Vina® tool version 1.2.0, year 2010 used for the virtual screening procedure [13](c)
BIOVIA® Discovery Studio version 2021 used for the determination of binding pockets and for visualizing/analyzing the docked results [14](d)
Absorption Distribution Metabolism Excretion and Toxicity of Structure Activity-Relationship (ADMETSAR): a web server used to evaluate the ADMET properties, biological activity, and drug-likeness [15–18]


### 2.5.2. Molecular Docking

(1) Ligand Preparations. In a quest to predict the antiplasmodial activity of the plant samples, compounds present in Z. gilletii were modeled using the ACDChem ChemSketch® version 2.0, year 2020 [12] computational tool and fed into Autodock vina® [13] for binding efficiency screening.(2) Receptor preparations. The antiplasmodium receptor of PDB CODE, 5TBO, was used as the target receptor for this study. The receptor was obtained from the (RCSB) database (https://www.rcsb.org/pdb). The resolution of receptor 5TBO (2.15 Å) approximately lies about the 2.0 Å recommended resolution for a receptor of good quality [19]. Water molecules around the crystal structure and other associated inhibitors were removed from the downloaded protease to avoid undesired interferences and unwanted molecular interactions.(3) Virtual screening. The virtual screening was done on Z. gilletii containing thirteen compounds using the Autodock vina® tool version 1.2.0, year 2010 [13] and Biovia Discovery Studio version 2021 [14]. The screening was guided using Chloroquine® and Artemether®, prominently used antiplasmodium therapeutics, as standards. Auto Dock Tools version 4.0 (ADT) was used to generate the autodock file (.pdbqt file) in preparation for docking. Other adjustments performed on both crystal structures include removing any extra water molecules, adding polar hydrogens, merging no-polar hydrogens, and adding Kollman charges. These adjustments are targeted at improving the affinity of the binding site. In modern computer-assisted drug design (CADD), the study of a potent drug is not complete without the activity study of the drug which is deducible from the compound’s inhibition constant. A potent compound is expected to have inhibition constant values ranging between 0.01 and 1 μM for a potent drug; therefore, this parameter alone is enough to screen compounds in the drug discovery journey [19].


### 2.5.3. ADMET Studies

For predicting the toxicity level of the screened compounds,in silico toxicity studies were performed using the ADMETlab version 2.0 web server online [20]. Additionally, from the server, the absorption (human intestinal absorption), distribution (blood-brain barrier; water solubility), metabolism (cytochrome inhibition: CYP450), excretion (half-life), toxicity (hERG inhibition; carcinogenicity), and drug-likeness were computed. The results are summarized in Table 1.Table 1
ADMET score of qualified compounds fromZ. gilletii with respect to the selected standards.
CompoundsHIABBBWater solubility (mol/L)CYP450 inhibitionhERG inhibitionDrug likeness (Lipinski)T(1/2)Carcinogenicity1Yes (>60%)NoSoluble (−3.615)2/5InactiveYes (5/5)Short (0.255)Yes2Yes (>90%)NoModerately soluble (−6.520)3/5InactiveYes (5/5)Short (0.192)Yes3Yes (>90%)YesSoluble (−4.305)4/5InactiveYes (5/5)Long 0.547Yes5Yes (>90%)NoModerately soluble (−6.605)3/5InactiveYes (5/5)Short 0.137Yes8Yes (>90%)YesModerately soluble (−6.781)1/5InactiveYes (5/5)Very short (0.041)No9Yes (>90%)NoModerately soluble (−6.667)0/5InactiveYes (5/5)Very short (0.037)No12Yes (>90%)NoPoorly soluble (−7.008)1/5InactiveYes (5/5)Very short (0.030)No13Yes (>60%)NoSoluble (−4.793)0/5InactiveYes (5/5)Very short (0.043)NoChloroquineYes (>90%)YesSoluble (−3.902)(2/5)InactiveYes (5/5)Very short (0.070)NoArtemetherYes (>90%)NoSoluble (−4.677)(0/5)InactiveYes (5/5)Very short (0.089)No


## 2.5.1. Computational Tools

Allin silico studies applied to this work were achieved using the following computational tool:(a)
ACDChemSketch® version 2.0, year 2020 used for modeling, first-hand, chemical structures to be fed for docking screening [12](b)
Autodock Vina® tool version 1.2.0, year 2010 used for the virtual screening procedure [13](c)
BIOVIA® Discovery Studio version 2021 used for the determination of binding pockets and for visualizing/analyzing the docked results [14](d)
Absorption Distribution Metabolism Excretion and Toxicity of Structure Activity-Relationship (ADMETSAR): a web server used to evaluate the ADMET properties, biological activity, and drug-likeness [15–18]


## 2.5.2. Molecular Docking

(1) Ligand Preparations. In a quest to predict the antiplasmodial activity of the plant samples, compounds present in Z. gilletii were modeled using the ACDChem ChemSketch® version 2.0, year 2020 [12] computational tool and fed into Autodock vina® [13] for binding efficiency screening.(2) Receptor preparations. The antiplasmodium receptor of PDB CODE, 5TBO, was used as the target receptor for this study. The receptor was obtained from the (RCSB) database (https://www.rcsb.org/pdb). The resolution of receptor 5TBO (2.15 Å) approximately lies about the 2.0 Å recommended resolution for a receptor of good quality [19]. Water molecules around the crystal structure and other associated inhibitors were removed from the downloaded protease to avoid undesired interferences and unwanted molecular interactions.(3) Virtual screening. The virtual screening was done on Z. gilletii containing thirteen compounds using the Autodock vina® tool version 1.2.0, year 2010 [13] and Biovia Discovery Studio version 2021 [14]. The screening was guided using Chloroquine® and Artemether®, prominently used antiplasmodium therapeutics, as standards. Auto Dock Tools version 4.0 (ADT) was used to generate the autodock file (.pdbqt file) in preparation for docking. Other adjustments performed on both crystal structures include removing any extra water molecules, adding polar hydrogens, merging no-polar hydrogens, and adding Kollman charges. These adjustments are targeted at improving the affinity of the binding site. In modern computer-assisted drug design (CADD), the study of a potent drug is not complete without the activity study of the drug which is deducible from the compound’s inhibition constant. A potent compound is expected to have inhibition constant values ranging between 0.01 and 1 μM for a potent drug; therefore, this parameter alone is enough to screen compounds in the drug discovery journey [19].


## 2.5.3. ADMET Studies

For predicting the toxicity level of the screened compounds,in silico toxicity studies were performed using the ADMETlab version 2.0 web server online [20]. Additionally, from the server, the absorption (human intestinal absorption), distribution (blood-brain barrier; water solubility), metabolism (cytochrome inhibition: CYP450), excretion (half-life), toxicity (hERG inhibition; carcinogenicity), and drug-likeness were computed. The results are summarized in Table 1.Table 1
ADMET score of qualified compounds fromZ. gilletii with respect to the selected standards.
CompoundsHIABBBWater solubility (mol/L)CYP450 inhibitionhERG inhibitionDrug likeness (Lipinski)T(1/2)Carcinogenicity1Yes (>60%)NoSoluble (−3.615)2/5InactiveYes (5/5)Short (0.255)Yes2Yes (>90%)NoModerately soluble (−6.520)3/5InactiveYes (5/5)Short (0.192)Yes3Yes (>90%)YesSoluble (−4.305)4/5InactiveYes (5/5)Long 0.547Yes5Yes (>90%)NoModerately soluble (−6.605)3/5InactiveYes (5/5)Short 0.137Yes8Yes (>90%)YesModerately soluble (−6.781)1/5InactiveYes (5/5)Very short (0.041)No9Yes (>90%)NoModerately soluble (−6.667)0/5InactiveYes (5/5)Very short (0.037)No12Yes (>90%)NoPoorly soluble (−7.008)1/5InactiveYes (5/5)Very short (0.030)No13Yes (>60%)NoSoluble (−4.793)0/5InactiveYes (5/5)Very short (0.043)NoChloroquineYes (>90%)YesSoluble (−3.902)(2/5)InactiveYes (5/5)Very short (0.070)NoArtemetherYes (>90%)NoSoluble (−4.677)(0/5)InactiveYes (5/5)Very short (0.089)No


## 3. Results and Discussion

### 3.1. Phytochemical Study

Thirteen compounds (Figure1) have been isolated and characterized from the chemical investigation of the methanol extract of the bark of Z. gilletii. Based on their spectroscopic (1H and 13C) NMR and spectrometric (EI-MS) data (see Table 1S on supplementary data), as well as the comparison of these data with those reported in the literature, the isolated compounds have been identified as 5-hydroxynoracronycine (1) [21], decarine (2) [22], trans-fagaramide (3) [23], cis-fagaramide (4) [24], sesamin (5) [25, 26], scoparone (6) [27], scopoletin (7) [28], fridelin (8) [29], lupeol (9) [30, 31], erythrodiol-3-O-palmitate (10) [32], vanillic acid (11) [23], stigmasterol (12), and stigmasterol-3-O-β-D-glucopyranoside (13) [31, 33].Figure 1
Chemical structures of compounds1–13 isolated from Z. gilletii.


### 3.2.In Vitro Antiplasmodial Activity

Among the isolated compounds, those in large amounts (compounds1, 3 + 4, 5 and 9) were submitted for in vitro antiplasmodial activity against the chloroquine-sensitive P. falciparum 3D7 (Pf 3D7) using chloroquine (IC50 = 24.74 nM) and artemisinin (IC50 = 17.76 nM) as reference drugs. All the compounds displayed good inhibition rates measured as 1 (54.69%), 3 + 4 (53.48%), 5 (51.89%), and 9 (52.64%). However, the most potent compound was the mixture 3 + 4 with an IC50 value of 1333 nM while compounds 1, 5, and 9 were less or not active with IC50 values greater than 10,000 nM (Table 2). The inhibition level greater than 50% for all the compounds showed that the Z. gilletii extract is an important source of antiplasmodial compounds supporting the use of the plant in traditional medicine for the treatment of malaria and deserves further investigations in checking of synergistic effects of those compounds as well as the characterization of further constituents that can help in the development of new potent antimalarial drugs.Table 2
Antiplasmodial activity of compounds againstP.  falciparum 3D7.
Samples% InhibitionaIC50 (in nM)154.69>10,0003 + 453.481333551.89>10,000952.64>10,000Chloroquine61.8624.74Artemisinin60.0017.76aTested concentration: 10 μM.


### 3.3. Molecular Docking

The binding affinities of all the thirteen compounds isolated fromZ. gilletii as well as the selected standards (Chloroquine® and Artemether®) are given in Table 3 and Figure 2, while Figures 3–8 (also see Table 2S on supplementary data) show their binding interactions at the 5TBO receptor’s binding site. The result shows that eight out of the thirteen identified compounds (1, 2, 3, 5, 8, 9, 12, 13) were found to have a greater affinity to the antiplasmodium receptor than Artemether® while eleven compounds (1–9, 12-13) were found to possess a greater affinity to the antiplasmodium receptor than Chloroquine®. This can also infer that the plant Z. gilletii has a high potency as an antiplasmodial agent. It can also be mentioned that since both standards used for this study (Chloroquine® and Artemether®) resulted in a maximum number of two conventional hydrogen bonds (Figure 8) while most selected compounds exhibited more, and with shorter bond length, it is understandable why the identified compounds have greater binding affinity to the antiplasmodium receptor than the standards.Table 3
Binding energy of isolated compounds and standards to the anti-plasmodium (5TBO) receptor.
CompoundsBinding energy (∆G) kcal/mol1−8.82−9.53−8.74−7.35−10.06−7.37−7.48−8.39−8.710−7.111−6.312−8.613−8.6Chloroquine−7.3Artemether−8.3Figure 2
Graphical illustration of binding energy of the isolated compounds and standards to the anti-plasmodium (5TBO) receptor.Figure 3
Docking poses of alkaloids1 (a) and 2 (b) at the 5TBO receptor binding site.
(a)(b)Figure 4
Docking poses of lignans3 (a), 4 (b) and 5 (c) at the 5TBO receptor binding site.
(a)(b)(c)Figure 5
Docking poses of coumarins6 (a) and 7 (b) at the 5TBO receptor binding site.
(a)(b)Figure 6
Docking poses of triterpenoids8 (a), 9 (b) and 10 (c) at the 5TBO receptor binding site.
(a)(b)(c)Figure 7
Docking poses of vanillic acid11 (a), the steroids 12 (b) and 13 (c) at the 5TBO receptor binding site.
(a)(b)(c)Figure 8
Structures of the standard drugs (a) and their docking poses (b) with the 5TBO receptor.
(a)(b)


### 3.4. ADMET Studies

The compounds displaying a good affinity (binding energy) to the 5TBO receptor binding site were further analysed for their ADMET properties, and the prediction results are presented in Table1. The human intestinal absorption (HIA) of an oral drug is the essential prerequisite for its apparent efficacy. Furthermore, the close relationship between oral bioavailability and intestinal absorption has also been proven, and HIA can be seen as an alternative indicator for oral bioavailability to some extent. A drug molecule with an absorbance of less than 30% is poorly absorbed [20]. The results from Table 1 show that compounds 1–3, 5, 8-9, 12-13 as well as both standard drugs (chloroquine and artemether) are strongly absorbed by the intestine.Water solubility is one important condition that a drug must satisfy before thein vitro drug optimization process [20]. In this regard, the results show that all the identified compounds except compound 12 are water-soluble with −3.615, −4.305, and −4.793 for compounds 1, 3, and 13, respectively, which are more soluble than the selected standards, chloroquine (−3.902) and artemether (−4.677).Cytochrome P450 inhibitors are responsible for the biotransformation of a drug; a potential drug should not alter their kinetics [34]. The results show that although compounds 1 (2/5), 2 (3/5), 3 (4/5), and 5 (3/5) violated the minimum inhibition allowed as reported by Falade et al. [19], compounds 8 (0/5), 9 (0/5), 12 (1/5), and 13 (0/5) show an improved value of less inhibition compared to chloroquine (2/5).The human either-a-go-go related inhibition (hERG) is a measure of electrical pulse transmission in the human heart (iodine-channel blocking). A potential drug should not block the activity of the hERG inhibitors [34]. Table 1 shows that all compounds present themselves as inactive against blocking the heart functions, as the standards. Carcinogenicity results also show that compounds 8, 9, 12, and 13 are noncarcinogenic to mammals. However, compounds 1, 2, 3, and 5 are seen to be toxic.Brain-blood barrier (BBB) prediction test suggests that compounds1, 2, 5, 9, 12, and 13 do not have the ability to cross the BBB; hence, their modes of action do not involve neurological engineering, such as appetite reduction as in chloroquine. It also suggests that the six (6) compounds will pose little or no side effects on administration as compared to chloroquine. However, compounds 3 and 8 have a tendency to cross the BBB.A good drug must also be able to stay long (activity) enough in the body before excretion [35]. The half-life of a drug is the description of how long a drug will stay active in the body before excretion. Table 1 indicates that all identified compounds can exhibit a better excretion than the standard drugs.In addition, Table1 also describes the drug likeliness based on Lipinski’s rules of five (5) criteria [36]. The rule states that for a compound that must stand as a potential drug, its structural properties must first satisfy the following rules: molecular weight ≤500; MiLOGP ≤4.15; number of oxygen ≤10; number of hydrogen ≤5; number of hydroxide ≤5.Results show that all identified compounds satisfy this drug condition and hence are more likely to be applied as a drug.


### 3.5. Chemotaxonomic Significance

The purification of theZ. gilletii extract via successive column chromatography resulted in the isolation and identification of thirteen specialized metabolites including 5-hydroxynoracronycine (1), decarine (2), trans-fagaramide (3), cis-fagaramide (4), sesamin (5), scoparone (6), scopoletin (7), fridelin (8), lupeol (9), erythrodiol-3-O-palmitate (10), vanillic acid (11), stigmasterol (12), and its derivative stigmasterol-3-O-β-D-glucopyranoside (13).Except compound10 which is obtained here for the first time from the genus Zanthoxylum and in general from the family Rutaceae, all the other compounds have been previously reported from the genus Zanthoxylum or from the genus Fagara which contains several species which are synonyms of those classified in the genus Zanthoxylum. For instance, trans-fagaramide (3), scopoletin (7), and vanillic acid (11) have been previously reported from the bark of Fagaramacrophylla and Fagara tessmannii [23, 37]. These two species are both synonyms of Z. gilletii, the plant we have investigated. This result partially supports the taxonomy of our plant and its similarity with the two Fagara species based on their chemical constituents. Further evidence has been given by the identification of the other metabolites: the acridone alkaloids have been mainly isolated from the Rutaceae plants and more especially from the plants from the genus Citrus. Unsurprisingly, the acridone 5-hydroxynoracronycine (1) is one of the major compounds along with sesamin (5) which were already reported from the plant species Z. poggei and Z. dinklagei [21, 26], respectively, as well as for the first time to the best of our knowledge from Z. gilletii. Another lignan named cis-fagaramide (4) has been also isolated during our investigations but has been already identified by Cheng et al. in [16] during their works on Z. schinifolium. The alkaloid decarine (2) is obtained for the first time from Z. gilletii while it has been found in Z. madagascariense [22]. Furthermore, the coumarin scopoletin (6) is widely encountered in many plant species and has been detected and characterized from the species Z. rhetsa, Z. schinifolium, and Z. leprieurii [27, 38, 39]. The two triterpenoids fridelin (8) and lupeol (9) are also mainly distributed in several plants and within the family Rutaceae, and they have been isolated from Z. schinofolium, Z. tessmannii, or Citrus aurantium [24, 29, 40]. However, the third triterpene identified as erythrodiol-3-O-palmitate (10) has not been previously obtained from the family Rutaceae as indicated earlier. Compound 10 is a derivative of β-amyrin belonging to the oleanane-type triterpenoids [41]. The literature survey indicates that some congeners of compound 10 including β-amyrin or β-amyrin acetate have already been reported from the Zanthoxylum genus. Indeed, β-amyrin was obtained in Z. simulans and Z. nitidum [42, 43], while β-amyrin acetate was reported from the species Z. schinifolium [24]. This evidence strongly supported the possibility of obtention of erythrodiol-3-O-palmitate (10) which is very structurally close to β-amyrin acetate and differs from it by just the length of the fatty acid attached to C-3 as well as the oxidation of the methyl CH3-28 to a primary alcohol function CH2OH [41]. Erythrodiol-3-O-palmitate (10) has been previously obtained from Trilepsium madagascariense (Moraceae) [32] and is reported for the first time from Z. gilletii and the family Rutaceae. Finally, the steroids stigmasterol (12) and stigmasterol-3-O-β-D-glucopyranoside (13) are usually obtained from several plant materials. Thus, all the chemical compounds isolated from Z. gilletii provide clear insights supporting the taxonomy of the plant and enrich the knowledge of its chemistry.


## 3.1. Phytochemical Study

Thirteen compounds (Figure1) have been isolated and characterized from the chemical investigation of the methanol extract of the bark of Z. gilletii. Based on their spectroscopic (1H and 13C) NMR and spectrometric (EI-MS) data (see Table 1S on supplementary data), as well as the comparison of these data with those reported in the literature, the isolated compounds have been identified as 5-hydroxynoracronycine (1) [21], decarine (2) [22], trans-fagaramide (3) [23], cis-fagaramide (4) [24], sesamin (5) [25, 26], scoparone (6) [27], scopoletin (7) [28], fridelin (8) [29], lupeol (9) [30, 31], erythrodiol-3-O-palmitate (10) [32], vanillic acid (11) [23], stigmasterol (12), and stigmasterol-3-O-β-D-glucopyranoside (13) [31, 33].Figure 1
Chemical structures of compounds1–13 isolated from Z. gilletii.


## 3.2.In Vitro Antiplasmodial Activity

Among the isolated compounds, those in large amounts (compounds1, 3 + 4, 5 and 9) were submitted for in vitro antiplasmodial activity against the chloroquine-sensitive P. falciparum 3D7 (Pf 3D7) using chloroquine (IC50 = 24.74 nM) and artemisinin (IC50 = 17.76 nM) as reference drugs. All the compounds displayed good inhibition rates measured as 1 (54.69%), 3 + 4 (53.48%), 5 (51.89%), and 9 (52.64%). However, the most potent compound was the mixture 3 + 4 with an IC50 value of 1333 nM while compounds 1, 5, and 9 were less or not active with IC50 values greater than 10,000 nM (Table 2). The inhibition level greater than 50% for all the compounds showed that the Z. gilletii extract is an important source of antiplasmodial compounds supporting the use of the plant in traditional medicine for the treatment of malaria and deserves further investigations in checking of synergistic effects of those compounds as well as the characterization of further constituents that can help in the development of new potent antimalarial drugs.Table 2
Antiplasmodial activity of compounds againstP.  falciparum 3D7.
Samples% InhibitionaIC50 (in nM)154.69>10,0003 + 453.481333551.89>10,000952.64>10,000Chloroquine61.8624.74Artemisinin60.0017.76aTested concentration: 10 μM.


## 3.3. Molecular Docking

The binding affinities of all the thirteen compounds isolated fromZ. gilletii as well as the selected standards (Chloroquine® and Artemether®) are given in Table 3 and Figure 2, while Figures 3–8 (also see Table 2S on supplementary data) show their binding interactions at the 5TBO receptor’s binding site. The result shows that eight out of the thirteen identified compounds (1, 2, 3, 5, 8, 9, 12, 13) were found to have a greater affinity to the antiplasmodium receptor than Artemether® while eleven compounds (1–9, 12-13) were found to possess a greater affinity to the antiplasmodium receptor than Chloroquine®. This can also infer that the plant Z. gilletii has a high potency as an antiplasmodial agent. It can also be mentioned that since both standards used for this study (Chloroquine® and Artemether®) resulted in a maximum number of two conventional hydrogen bonds (Figure 8) while most selected compounds exhibited more, and with shorter bond length, it is understandable why the identified compounds have greater binding affinity to the antiplasmodium receptor than the standards.Table 3
Binding energy of isolated compounds and standards to the anti-plasmodium (5TBO) receptor.
CompoundsBinding energy (∆G) kcal/mol1−8.82−9.53−8.74−7.35−10.06−7.37−7.48−8.39−8.710−7.111−6.312−8.613−8.6Chloroquine−7.3Artemether−8.3Figure 2
Graphical illustration of binding energy of the isolated compounds and standards to the anti-plasmodium (5TBO) receptor.Figure 3
Docking poses of alkaloids1 (a) and 2 (b) at the 5TBO receptor binding site.
(a)(b)Figure 4
Docking poses of lignans3 (a), 4 (b) and 5 (c) at the 5TBO receptor binding site.
(a)(b)(c)Figure 5
Docking poses of coumarins6 (a) and 7 (b) at the 5TBO receptor binding site.
(a)(b)Figure 6
Docking poses of triterpenoids8 (a), 9 (b) and 10 (c) at the 5TBO receptor binding site.
(a)(b)(c)Figure 7
Docking poses of vanillic acid11 (a), the steroids 12 (b) and 13 (c) at the 5TBO receptor binding site.
(a)(b)(c)Figure 8
Structures of the standard drugs (a) and their docking poses (b) with the 5TBO receptor.
(a)(b)


## 3.4. ADMET Studies

The compounds displaying a good affinity (binding energy) to the 5TBO receptor binding site were further analysed for their ADMET properties, and the prediction results are presented in Table1. The human intestinal absorption (HIA) of an oral drug is the essential prerequisite for its apparent efficacy. Furthermore, the close relationship between oral bioavailability and intestinal absorption has also been proven, and HIA can be seen as an alternative indicator for oral bioavailability to some extent. A drug molecule with an absorbance of less than 30% is poorly absorbed [20]. The results from Table 1 show that compounds 1–3, 5, 8-9, 12-13 as well as both standard drugs (chloroquine and artemether) are strongly absorbed by the intestine.Water solubility is one important condition that a drug must satisfy before thein vitro drug optimization process [20]. In this regard, the results show that all the identified compounds except compound 12 are water-soluble with −3.615, −4.305, and −4.793 for compounds 1, 3, and 13, respectively, which are more soluble than the selected standards, chloroquine (−3.902) and artemether (−4.677).Cytochrome P450 inhibitors are responsible for the biotransformation of a drug; a potential drug should not alter their kinetics [34]. The results show that although compounds 1 (2/5), 2 (3/5), 3 (4/5), and 5 (3/5) violated the minimum inhibition allowed as reported by Falade et al. [19], compounds 8 (0/5), 9 (0/5), 12 (1/5), and 13 (0/5) show an improved value of less inhibition compared to chloroquine (2/5).The human either-a-go-go related inhibition (hERG) is a measure of electrical pulse transmission in the human heart (iodine-channel blocking). A potential drug should not block the activity of the hERG inhibitors [34]. Table 1 shows that all compounds present themselves as inactive against blocking the heart functions, as the standards. Carcinogenicity results also show that compounds 8, 9, 12, and 13 are noncarcinogenic to mammals. However, compounds 1, 2, 3, and 5 are seen to be toxic.Brain-blood barrier (BBB) prediction test suggests that compounds1, 2, 5, 9, 12, and 13 do not have the ability to cross the BBB; hence, their modes of action do not involve neurological engineering, such as appetite reduction as in chloroquine. It also suggests that the six (6) compounds will pose little or no side effects on administration as compared to chloroquine. However, compounds 3 and 8 have a tendency to cross the BBB.A good drug must also be able to stay long (activity) enough in the body before excretion [35]. The half-life of a drug is the description of how long a drug will stay active in the body before excretion. Table 1 indicates that all identified compounds can exhibit a better excretion than the standard drugs.In addition, Table1 also describes the drug likeliness based on Lipinski’s rules of five (5) criteria [36]. The rule states that for a compound that must stand as a potential drug, its structural properties must first satisfy the following rules: molecular weight ≤500; MiLOGP ≤4.15; number of oxygen ≤10; number of hydrogen ≤5; number of hydroxide ≤5.Results show that all identified compounds satisfy this drug condition and hence are more likely to be applied as a drug.


## 3.5. Chemotaxonomic Significance

The purification of theZ. gilletii extract via successive column chromatography resulted in the isolation and identification of thirteen specialized metabolites including 5-hydroxynoracronycine (1), decarine (2), trans-fagaramide (3), cis-fagaramide (4), sesamin (5), scoparone (6), scopoletin (7), fridelin (8), lupeol (9), erythrodiol-3-O-palmitate (10), vanillic acid (11), stigmasterol (12), and its derivative stigmasterol-3-O-β-D-glucopyranoside (13).Except compound10 which is obtained here for the first time from the genus Zanthoxylum and in general from the family Rutaceae, all the other compounds have been previously reported from the genus Zanthoxylum or from the genus Fagara which contains several species which are synonyms of those classified in the genus Zanthoxylum. For instance, trans-fagaramide (3), scopoletin (7), and vanillic acid (11) have been previously reported from the bark of Fagaramacrophylla and Fagara tessmannii [23, 37]. These two species are both synonyms of Z. gilletii, the plant we have investigated. This result partially supports the taxonomy of our plant and its similarity with the two Fagara species based on their chemical constituents. Further evidence has been given by the identification of the other metabolites: the acridone alkaloids have been mainly isolated from the Rutaceae plants and more especially from the plants from the genus Citrus. Unsurprisingly, the acridone 5-hydroxynoracronycine (1) is one of the major compounds along with sesamin (5) which were already reported from the plant species Z. poggei and Z. dinklagei [21, 26], respectively, as well as for the first time to the best of our knowledge from Z. gilletii. Another lignan named cis-fagaramide (4) has been also isolated during our investigations but has been already identified by Cheng et al. in [16] during their works on Z. schinifolium. The alkaloid decarine (2) is obtained for the first time from Z. gilletii while it has been found in Z. madagascariense [22]. Furthermore, the coumarin scopoletin (6) is widely encountered in many plant species and has been detected and characterized from the species Z. rhetsa, Z. schinifolium, and Z. leprieurii [27, 38, 39]. The two triterpenoids fridelin (8) and lupeol (9) are also mainly distributed in several plants and within the family Rutaceae, and they have been isolated from Z. schinofolium, Z. tessmannii, or Citrus aurantium [24, 29, 40]. However, the third triterpene identified as erythrodiol-3-O-palmitate (10) has not been previously obtained from the family Rutaceae as indicated earlier. Compound 10 is a derivative of β-amyrin belonging to the oleanane-type triterpenoids [41]. The literature survey indicates that some congeners of compound 10 including β-amyrin or β-amyrin acetate have already been reported from the Zanthoxylum genus. Indeed, β-amyrin was obtained in Z. simulans and Z. nitidum [42, 43], while β-amyrin acetate was reported from the species Z. schinifolium [24]. This evidence strongly supported the possibility of obtention of erythrodiol-3-O-palmitate (10) which is very structurally close to β-amyrin acetate and differs from it by just the length of the fatty acid attached to C-3 as well as the oxidation of the methyl CH3-28 to a primary alcohol function CH2OH [41]. Erythrodiol-3-O-palmitate (10) has been previously obtained from Trilepsium madagascariense (Moraceae) [32] and is reported for the first time from Z. gilletii and the family Rutaceae. Finally, the steroids stigmasterol (12) and stigmasterol-3-O-β-D-glucopyranoside (13) are usually obtained from several plant materials. Thus, all the chemical compounds isolated from Z. gilletii provide clear insights supporting the taxonomy of the plant and enrich the knowledge of its chemistry.


## 4. Concluding Remarks

Thirteen compounds (1–13) have been isolated and characterized from the phytochemical investigations of the methanol extract of the bark from Z. gilletii, a Cameroonian medicinal plant. The compounds have been sorted into six main classes of compounds including two alkaloids (1 and 2), three lignans (3–5), two coumarins (6 and 7), three pentacyclic triterpenoids (8–10), one phenolic compound (11) as well as two steroids (12 and 13). The chemotaxonomic significance of the isolated compounds strengthened the classification of the plant in the Rutaceae family and provided additional compounds to enrich the chemistry of the species Z. gilletii (Rutaceae). More interestingly, all the compounds tested in vitro showed good inhibition rates of Pf 3D7 with values greater than 50% while the molecular docking of the isolated compounds showed that a total of eleven compounds displayed a good affinity with the 5TBO receptor binding site compared to the standard drugs chloroquine and artemether. Likewise, the ADMET study indicated that isolated compounds exhibited good and favourable parameters that qualify them as good candidates for drug development. The present study, in addition to supporting the use of the plant in traditional medicine for the treatment of malaria, gives significant insights for further in vitro and in vivo pharmacological investigations in the discovery of new antimalarial drugs.



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*Source: 1111817-2022-11-22.xml*