In Vitro and In Silico Evaluation of the Efficacy and Safety of a Ghanaian Herbal Medicine for the Treatment of Onchocerciasis and African Trypanosomiasis

In Vitro and In Silico Evaluation of the Efficacy and Safety of a Ghanaian Herbal Medicine for the Treatment of Onchocerciasis and African Trypanosomiasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In Vitro and In Silico Evaluation of the Efficacy and Safety of a Ghanaian Herbal Medicine for the Treatment of Onchocerciasis and African Trypanosomiasis Barbara Zenabu Anibea, Eric Coffie, Daniel Moscoh Ayine-Tora, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7189198/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background This study investigated the anti-onchocercal and antitrypanosomal properties of a Ghanaian herbal medicine (NTD-O2) and its medicinal plant constituents, Xylopia aethiopica fruits and Bambusa vulgaris leaves, with the aim of addressing the therapeutic potential and safety of a herbal medicine against Onchocerciasis and Animal African Trypanosomiasis in Ghana. Methods Extracts from NTD-O2 and the medicinal plants were tested against Onchocerca ochengi and Trypanosoma brucei brucei in vitro . Bioassay-guided fractionation, spectroscopic and spectrometric techniques identified bioactive compounds, while in silico methods explored their possible mechanisms of action. Results NTD-O2 extracts achieved 100% inhibition of adult male O. ochengi worm motility, with moderate activity against adult female worms. Additionally, the extracts demonstrated promising antitrypanosomal activity (IC 50 = 9.44 µg/mL and 10.68 µg/mL) against the positive control, diminazene aceturate (IC 50 = 0.13 ± 0.02 µg/mL). The active compound found in the NTD-O2 extract was bis(4-methylheptyl) phthalate. On the contrary, the compounds isolated from X. aethiopica – ent-kaur-16-en-19-oic acid, xylopic acid, and ent-kaur-16-en-15-one-19-oic acid – and the long chain carbonyl compounds from B. vulgaris were inactive (IC 50 = > 100 ± 0.46 µg/mL). These results were corroborated by in silico analysis. Conclusion The findings highlight significant variability in the chemical composition and bioactivity of the herbal medicine NTD-O2 and its plant constituents. Given the health risks linked to the ingestion of phthalate derivatives, it is essential to conduct regular assessments of the quality and safety of herbal medicines to ensure consumer protection. Herbal medicine safety Xylopia aethiopica Bambusa vulgaris Onchocerciasis Animal Trypanosomiasis phthalate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Rooted in indigenous knowledge and cultural beliefs, herbal medicine (HM) has been the cornerstone of human healthcare for centuries and continues to offer readily available and affordable healthcare to approximately 80% of the population in developing countries. Practically, for most indigenous people living in remote rural areas with limited access to conventional medical services, HM is the only treatment option. 1 Besides, over the past three decades, many developed countries have widely embraced the use of HM to promote healthier living. 2 – 4 While HM is perceived to have less adverse effects, they are commonly used as self-care treatments and are unregulated, posing huge safety risks for consumers. In response to these challenges, coupled with the recognition of their enormous value for achieving universal healthcare, the World Health Organization (WHO) established technical guidelines and standards to ensure the safety, efficacy and quality control of HM. 5 – 8 HM is a significant aspect of Ghana’s cultural heritage and its development over the years is well documented. 9 – 13 Since 2012, the practice has been approved by the government for integration into the mainstream healthcare system, 14 resulting in a wide range of HM products on the Ghanaian market for treating a variety of disease conditions. The Ghana Food and Drugs Authority (FDA) is mandated to approve and register such products based on efficacy, safety and clinical data. The rational use of HMs has largely been supported by research output on the scientific validation of their efficacy claims. However, the challenge is that many of the FDA-approved products cover common ailments such as malaria, anaemia, cough, and asthma, unlike remedies for conditions like the neglected tropical diseases (NTDs) due to lack of access to efficacy testing platforms. 12 Hence, many people affected by NTDs are exposed to unapproved HM, a situation with the potential for serious safety and efficacy implications and therefore requires urgent scientific validation for better healthcare outcomes. Onchocerciasis, commonly known as river blindness, is a parasitic disease caused by the filarial nematode Onchocerca volvulus and transmitted through the bite of infected female blackflies ( Simulium spp .). 15 The disease is responsible for a range of skin and eye-related morbidities including irreversible blindness. 16 Onchocerciasis is reported among the NTDs that remain endemic in several Ghanaian communities after more than 18 rounds of annual mass drug administration with ivermectin. 17 Our interaction with the Ghana Federation of Traditional Medicines Practitioners Association (GHAFTRAM) revealed the existence and high patronage of HM by affected people in NTD-endemic communities. 18 As part of efforts to contribute to the promotion and development of these remedies, we sought to conduct a comparative investigation of a HM used in treating onchocerciasis (NTD-O2) and its medicinal plant constituents. Thus, the constituents of NTD-O2 and its component medicinal plants ( Xylopia aethiopica fruits and Bambusa vulgaris leaves) were extracted and separately screened for anti-onchocercal activity against Onchocera ochengi . Bioassay-guided fractionation was used to identify the bioactive constituents. Structural elucidation techniques involving spectroscopic and spectrometric methods were used to characterize the chemical constituents. The possible mechanisms of action of the compounds were further determined by in silico techniques. Animal African trypanosomiasis (AAT), unlike human trypanosomiasis (sleeping sickness,) continues to be highly neglected primarily due to limited resources and global attention directed towards human disease control. While AAT causes huge economic loses in livestock production, coupled with the direct effects on human health, the financial incentives for developing veterinary drugs for livestock disease are often low, resulting in limited treatment options. 19 According to Tweneboah et al. 20 , information on the current situation of AAT in Ghana is scanty due to neglect even at the national level. In our earlier work, 18 we demonstrated that a chemical constituent of NTD-B4, a herbal medicine used against schistosomiasis, was effective against Trypanosoma brucei brucei compared to the standard antitrypanosomal drug, diminazene aceturate. Hence, NTD-O2 extracts and compounds as well as those from the medicinal plants were subjected to antitrypanosomal activity screening against Trypanosoma brucei brucei . To ensure herbal medicines can serve as viable alternatives to conventional treatments for parasitic infections, regular screening is essential. This process guarantees quality assurance, efficacy, and safety, thereby enhancing their credibility and protecting consumers. Material and methods Herbal Medicine and Plant Materials NTD-O2 is a Ghanaian herbal medicine used in the treatment of onchocerciasis. It was prepared from two medicinal plants, the dry fruits of Xylopia aethiopica and the fresh leaves of Bambusa vulgaris and provided by GHAFTRAM for the study. In addition to the herbal medicine (NTD-O2), the constituent plant materials were also collected and analyzed separately. Dry X. aethiopica fruits were sourced from Nima Market, Accra, and fresh B. vulgaris leaves from the University of Ghana campus. Both were identified by Mr. Patrick Ekpe and assigned voucher numbers BZA 001 and BZA 002 at the Ghana Herbarium, Department of Plant and Environmental Biology, University of Ghana. Extraction and Isolation NTD-O2 was subjected to the protocol described by Twumasi et al. (2020) 18 with slight modification. Briefly, a total of 3.8 litres of NTD-O2 was extracted in 500 mL aliquots with equal volumes of HPLC-grade dichloromethane (DCM) and n-butanol (n-BuOH) sequentially. For each aliquot, the procedure was repeated three times to yield the corresponding crude extracts NTD-O2-DCM (7 g) and NTD-O2-BuOH (25 g). The NTD-O2-BuOH extract was then sequentially fractionated with hexane, DCM, Ethylacetate (EtOAc) and Methanol (MeOH). Each fraction was concentrated under vacuum and refrigerated at 4°C for about 72 h to facilitate precipitation of solids. The EtOAc fraction gave a yellow-brown solid (O2-F3-S, 5 g) and was purified over a column of 60 g of silica gel (130–270 Å mesh, SIGMA-ALDRICH) eluted with petroleum ether (PE) and EtOAc to yield compound 1 (O2-F3-S, 8 mg) and compound 2 (O2-F3-ML, 20 mg). Dried powder (1 kg) of X. aethiopica fruits and fresh leaves of B. vulgaris (674.0 g) were separately extracted continuously via cold percolation with Petroleum Ether (PE) for 72 hours. The solvent was removed under reduced pressure to give an oily crude extract for X. aethiopica (XA/PE, 30.8 g) and greenish paste crude for B. vulgaris (BL/PE, 10 g). XA/PE (10 g) was chromatographed over a column of silica gel (120 g) and eluted with PE/EtOAc (v/v = 10:0, 9:1, 8:2, 6:4, 1:1, 0:10) and EtOAc/MeOH (9.5:0.5). A total of 315 eluates (v = 20 mL) were obtained and pooled together based on TLC profile into 13 fractions (1–13). Upon evaporation under vacuum, fractions 1–12 were oily while fraction 13 was a powdery solid. Following refrigeration at 4°C for about 72 h, fractions 2–5 generously precipitated colourless crystals (BZA 01, 5 g), fractions 11–12 precipitated another set of colourless crystals (BZA 02, 4.7 g), while fraction 9 precipitated a greenish powder (BZA 05, 200 mg). B. vulgaris (BL/PE, 7 g) was also subjected to silica gel column chromatography using a similar protocol as described above to yield 2 compounds, a white powder (BL 2, 10 mg) and yellow gum (BL 4, 3 mg). Thin-layer chromatography (TLC) was conducted on silica gel 60 F 254 (Merck) pre-coated aluminium foil slides, visualized with UV light (254 and 364 nm) and anisaldehyde spray reagent heated to 110°C. Structure elucidation techniques 1 H and 13 C NMR spectra were recorded on a Brüker, 500 MHz spectrometer in CDCl 3, with chemical shifts referenced to tetramethyl silane. LC-MS analysis was performed using an Agilent HPLC system equipped with a Kinetex Core C18 column (2.6 µM, 3 x 50 mm, 100 Å) at 40°C. The mobile phase consisted of acetonitrile-ethyl acetate 9:1 eluted under gradient conditions at 0.7 mL/min. Injection volume was 2 µL, and the mass spectra were obtained using electrospray ionization (ESI) and atmospheric pressure chemical ionization. GC-MS analysis was conducted on a Perkin Elmer GC Clarus 580 with electron impact ionization at 70 eV and helium gas at 1 mL/min. National Institute Standard and Technology (NIST) library was employed in compound identification. Infra-red spectral data was acquired neat on a Perkin Elmer FTIR spectrometer using the attenuated total reflectance technique. MS analyses of samples were performed using an Agilent 1100 Autosampler instrument with ESI at 3.5 kV and a Thermo Q-Exactive orbitrap instrument. X-ray crystallography measurements were done on a Brüker ECO D8 diffractometer, with data collection at 296.15 K and the structure solved with the olex2.solve and refined with and refined with the ShelXL. In vitro anti-onchocercal assay Stock solution (20 mg/mL) of NTD-O2-DCM and NTD-O2-BuOH in DMSO were tested on the worms and larvae of O. ochengi as previously described. 18 , 23 Auranofin (10 µM) was used as the positive control while 2% of DMSO served as the negative control. The viability of the adult female worms was evaluated biochemically by visually assessing the percentage inhibition of formazan production after incubating the nodules in 500 µL of 0.5 mg/mL MMT. 24 A loss of the characteristic blue coloration in the worms indicated worm death. A test sample was classified as active if it inhibited more than 90% of female worm motility or formazan production, moderately active if it caused 50–89% inhibition, and inactive if inhibition was below 50%. In vitro Anti-trypanosomal assay Bloodstream forms of T. brucei brucei were used in this study due to their similarity to the human-infective forms, T. b. rhodesiense and T. b. gambiense . 25 The protocol employed is as previously described. 18 Briefly, crude extracts were dissolved in 100% DMSO to make a 20 mg/mL stock, then diluted to 2 mg/mL in sterile distilled water for testing. Isolated compounds (BZA 01, BZA 02, BZA 05 from X. aethiopica ; BL 2 and BL 4 from B. vulgaris ) were prepared similarly. Trypanosome cells in media without treatment served as the negative control, while diminazene aceturate (DA) was the positive control. Wild-type bloodstream forms of T. b. brucei (GuTat 3.1 strain) were cultured in Hirumi’s Modified Iscove’s Medium-9 (HMI-9), 26 supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum (Gibco). The cultures were maintained at 37°C with 5% CO 2 . An Alamar blue assay was used to test the crude extracts and compounds. 27 Serial dilutions (100 to 0.1953 µg/mL) were prepared in 96-well plates. Log-phase trypanosomes were added, and the plates were incubated for 72 hours at 37°C in 5% CO 2 . A final concentration of 44 µM of resazurin sodium salt (Sigma-Aldrich) in phosphate buffered saline was then added to each well, plates were incubated for additional 5 h and the absorbance measured at 570 nm using the Varioskan Lux Elisa plate reader (ThermoFisher Scientific, USA). Each test was done in triplicate, with three technical replicates. Statistical analysis Plate readouts from the Alamar blue assay were evaluated using non-linear regression analysis (log10 inhibitor versus response-variable slope) to assess growth inhibition. This analysis was performed using GraphPad Prism version 8.0.1. The IC 50 values of the test samples were determined from three biological replicates, each conducted in triplicate. In silico studies In silico studies were performed on compounds isolated from X. aethiopica and B. vulgaris in the current study and those reported in literature to validate the outcomes of the in vitro anti-onchocercal and antitrypanosomal assays. These studies included predictions of pharmacodynamics (PD) and pharmacokinetics (PK), as well as molecular modelling of compounds. Selection of Protein Targets For O. ochengi , the typical protein targets selected were glutathione S-transferase and glutamate-gated chloride ion channel. In the case of T. brucei brucei , ornithine decarboxylase was chosen as the protein target. 28 – 31 Ornithine Decarboxylase is a crucial enzyme-dependent growth factor, playing a vital role for the parasite through the formation of polyamine putrescine, a molecule important in the pathway for the production of trypanothione. 32 Prediction of the PD and PK of the Compounds The SwissADME server was used to predict PD and PK properties of the isolated compounds. 33 Measurement of PK Properties and Drug-Likeness The SwissADME server was further utilized to determine the physicochemical descriptors of the compounds, as well as to define their PK properties and drug-like nature. The Brain or Intestinal Estimated Permeation (BOILED-Egg) model was employed to intuitively evaluate passive human gastrointestinal absorption (HIA) and blood-brain barrier (BBB) penetration. This model was also used to compute the lipophilicity and polarity of the compounds. Modelling of compounds The compounds, both isolated in the current study and those reported in literature, were docked to the crystal structure of the Glutathione S-Transferase (Protein Data Bank (PDB) ID: 1TU8, resolution 1.80 Å) 32 and Glutamate-gated chloride channel (GluCl) (PDB: 3RHW, resolution 3.26 Å), which are protein targets for onchocerciasis. Additionally, these compounds were docked against ornithine decarboxylase (PDB ID: 1F3T, resolution 2.00 Å), 34 a protein target for trypanosomiasis. The crystal structures of co-crystallized ligands were obtained from PDB. 35 , 36 These structures were prepared by removing the co-crystallized ligands and adding the hydrogen atoms using the Scigress version FJ 2.6 program. The binding pocket center of the Glutathione S-Transferase was identified by fitting the atoms of the co-crystallised ligand 5-hexyl Glutathione (x = 2.570, y = 0.814, z = 49.968) with a radius of 10 Å. Similarly, the binding pocket centre of glutamate-gated chloride channel was defined by the position of the atoms of co-crystallized ligand ivermectin (x = 12.675, y = 95.052, z = 24.998) with a radius of 10 Å. The binding pocket centre of ornithine decarboxylase was identified by the position of the phosphorus atom of the co-crystallised ligand pyridoxal-5'-phosphate (coordinates: x = 26.206, y = 15.614, z = 3.800) and a radius of 10 Å. To validate the predicted binding modes and relative energies of the ligands, the GoldScore (GS) 37 , ChemScore (CS) 38 , 39 , ChemPLP 40 and Astex statistical potential (ASP) 41 scoring functions were implemented using the GOLD v5.4 software suite. Results and discussion In vitro anti-onchocercal and antitrypanosomal activity of NTD-O2 The results of the in vitro anti-onchocercal and antitrypanosomal assays are summarized in Table 1 . In primary screens against O. ochengi , both the dichloromethane extract of NTD-O2 (NTD-O2-DCM) and the butanol extract (NTD-O2-BuOH) at 200 µg/mL, as well as the positive control, auranofin at 10 µM showed 100 ± 0% inhibition of the adult male worm. This indicates that the crude extracts of the herbal medicine at the specified concentration were as effective as the control against the adult male worm. However, while auranofin maintained the same level of activity against the adult female worm, the extracts demonstrated moderate activity, with 61.0 ± 1.8% and 56.6 ± 4.4% inhibition, respectively. Due to the moderate activity against the female worm, which is a critical criterion for advancing to secondary screens, the extracts were not selected for further testing against the parasites. In contrast, the antitrypanosomal assay results were promising for both extracts, with IC 50 values of 10.68 ± 2.54 µg/mL for NTD-O2-DCM and 9.44 ± 1.88 µg/mL for NTD-O2-BuOH against T. b. brucei . The positive control, diminazene aceturate, recorded an IC 50 value of 0.13 ± 0.02 µg/mL (Table 1 ). While the extracts were less potent than diminazene aceturate, their IC 50 values are still within a range that suggests possible optimization or application in combination therapies to enhance efficacy. Table 1 Anti-onchocercal and antitrypanosomal screening of NTD-O2 and its medicinal plant isolates No. Test sample Cytotoxic Anti-onchocercal activity against adult worms of O. ochengi Antitrypanosomal activity against bloodstream forms T. b. brucei %Inhibition of males (5 days) %Killing of females (7 days) IC 50 (µg/mL) Mean ± SD NTD-O2 1 NTD-O2-DCM No 100 ± 0 61.0 ± 1.8 10.68 ± 2.54 2 NTD-O2-BuOH No 100 ± 0 56.6 ± 4.4 9.44 ± 1.88 3 Auranofin (10 µM) N/A 100 ± 0 100 ± 0 N/A 4 Diminazene aceturate N/A N/A N/A 0.13 ± 0.02 Isolates from NTD-O2-BuOH 1 O2-F3-S N/A 100 ± 0.46 2 O2-F3-ML N/A 1.1 ± 0.3 3 Diminazene aceturate N/A 0.18 ± 0.01 Isolates from medicinal plants 1 BZA 01 N/A > 100 ± 0.46 2 BZA 02 N/A ND 3 BZA 05 N/A > 100 ± 0.46 4 BL2 N/A > 100 ± 0.46 5 BL4 N/A > 100 ± 0.46 6 Diminazene aceturate N/A 0.18 ± 0.01 N/A = Not applicable; N/D = Not determined; Calculated adult worm activity responses were done in quadruplicates, IC 50 values represent mean averages of 3 biological replicates. The higher activity results of the antitrypanosomal assay informed a bioassay-guided fractionation and isolation of compounds from NTD-O2 and its medicinal plants, X. aethiopica and B. vulgaris . NTD-O2-BuOH was prioritized over NTD-O2-DCM due to its higher IC 50 value. Antitrypanosomal activity of isolates from NTD-O2-BuOH NTD-O2-BuOH was sequentially fractionated with hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) to obtain the corresponding fractions. Isolates from the hexane and DCM fractions encountered solubility issues in dimethyl sulfoxide (DMSO), precluding their assay results. Additionally, attempts to purify the MeOH fraction were unsuccessful. Hence, only isolates O2-F3-S and O2-F3-ML from the EtOAc fraction were evaluated for their antitrypanosomal activity. O2-F3-S exhibited limited bioactivity, with an IC 50 value of 100 ± 0.46 µg/mL. In contrast, O2-F3-ML demonstrated significant antitrypanosomal activity, with an IC 50 value of 1.1 ± 0.3 µg/mL. When compared to the standard antitrypanosomal agent, diminazene aceturate, which had an IC 50 of 0.18 ± 0.01 µg/mL, O2-F3-ML shows a comparable efficacy. The cytotoxicity (CC 50 ) of O2-F3-ML was determined to be 100 µg/mL, whereas diminazene aceturate exhibited a CC 50 of 36 ± 2 µg/mL against the standard phenylarsine oxide, which has a CC 50 value of 1.0 ± 0.1 µg/mL. This indicates that the cytotoxicity of O2-F3-ML is moderate. Consequently, the selective index (SI) was calculated to be 89 ± 25 for O2-F3-ML and 203 ± 22 for diminazene aceturate. These findings suggest that O2-F3-ML possesses notable antitrypanosomal activity and hence was subjected to structure elucidation. Structure Elucidation of O2-F3-ML Sample O2-F3-ML was obtained as a yellowish oil with a retention factor (Rf) of 0.75 on TLC developed in hexane and acetone (7:3). The IR data gave absorptions at 2958 cm- 1 , 2927 cm − 1 and 2858 cm − 1 (C-H), 1726 cm − 1 (C = O) and 1610 cm − 1 (aromatic C = C). The 1 H-NMR spectrum ( Supplementary information Figure S1 ) exhibited a typical AA'BB' system at H-4/H-4' (δ H 7.53, dd, J = 3.3 and 5.7 Hz, 2H) and H-3/H-3' (δ H 7.70, d, J = 3.3 Hz, 2H) supporting an ortho-disubstituted benzene ring with identical substituents in both positions. Signals were seen at δ H 0.90 (d, J = 5.5 Hz, 3H) for the secondary methyl protons H-12/H-12' and δ H 0.91 (t, J = 13.8 Hz, 3H) for the terminal methyl protons H-11/H-11'. A string of multiplets at δ H 1.31, δ H 1.35, δ H 1.42, δ H 1.68 and δ H 4.22, was suggestive of methylene groups, with the latter linked to the ester. The 13 C NMR and DEPT 135 spectra ( Supplementary information Figures S1 , S2 ) revealed resonances for methyl signals δ C 11.1 (C-12/C-12') and δ C 14.2 (C-11/C-11'), methylene carbon signals at δ C 23.1 (C-10/C-10'), δ C 23.9 (C-9/C-9'), δ C 29.0 (C-8/C-8'), δ C 30.5 (C-7/C-7'), and an sp 3 methine carbon signal at δ C 38.9 (C-6/C-6'). The sp 3 oxygenated carbon (C-5/C-5') appeared at δ C 68.4. Further, there were aromatic methine carbon peaks at δ C 128.9 (C-4/C-4'), δ C 131.0 (C-3/C-3') along with a quaternary aromatic carbon peak at δ C 132.6 (C-2/C-2') and a carbonyl carbon at δ C 167.9 (C-1/C-1') due to an ester moiety. HMBC spectral data provided correlations between the methyl protons at δ H 0.90 (H-12) and δ C 23.1 (C-10), 23.9 (C-9) and 29.0 (C-8). Correlations H-5/C-8, C-1 established the ester linkage and H-12/C-6, C-7, C-9; H-5/C-6 confirmed the branched position of the side chain. The mass spectrum from the GC-MS analysis displayed fragment peaks at m/z 29, 43, 57, 71, 83, 149, 167, and 279, characteristic of alkyl phthalates ( Supplementary information Figure S4 ). The molecular ion peak occurred at m/z 390 and the base peak at m/z 149, representing a protonated phthalate anhydride. Based on these assignments, the structure of O2-F3-ML was deduced as bis(4-methylheptyl) phthalate (Fig. 1 ). Phthalates and their derivatives are widely recognized as environmental contaminants due to their extensive industrial use. Prolonged or excessive exposure to these compounds has been associated with significant health risks in humans, including endocrine disruption, metabolic disorders, cancer, reproductive health issues and developmental challenges in children. 42 , 43 In contrast, phthalates have also been reported in natural sources such as plants, bacteria, and fungi, demonstrating diverse biological potential such as antitumor, cytotoxic, allopathic, larvicidal, antiviral and anti-inflammatory properties. 42 In vitro antitrypanosomal activity of the medicinal plant isolates Compounds BZA 01, BZA 05, BL 2 and BL 4 recorded an IC 50 value of > 100 ± 0.46 µg/mL and hence, did not demonstrate any antitrypanosomal activity. BZA 02, on the other hand, could not be assayed due to challenges with solubility in DMSO. Structure elucidation of the medicinal plant isolates BZ 01 BZA 01 was obtained as colourless crystals that appeared as an orange spot when visualized in anisaldehyde with Rf of 0.65 on TLC developed in PE: EtOAc (10:1). The IR spectrum showed characteristic peaks at 1726.56 cm − 1 for carbonyl and 1687.91 cm − 1 for unsaturation (C = C). The 13 C NMR and DEPT 135 spectra ( Supplementary information Figures S5, S6 ) exhibited 20 carbon signals sorted by the DEPT experiment into 5 quaternary carbons (including a carboxylic carbon at δ C 185.1 (C-3) and an alkene carbon (C-16) at δ C 155.8), 3 methine carbons, 2 tertiary methyl carbons and 10 methylene carbons, which were consistent with a kaurenoic acid skeleton. In the 1 H NMR spectrum ( Supplementary information Figure S7 ), signals for the olefinic protons H20a (d, δ H 4.80, 1H) and H-20b (d, δ H 4.74, 1H) and tertiary methyls H-4 (s, δ H 1.24, 3H) and H-7 (s, δ H 0.95, 3-H) supported the presence of kaurenoic acid. HMBC correlations between C-3/H-4, H-5 established the position of the carboxylic acid while the exocyclic alkene was fixed by correlations from C-15, C-16, C-17/H-20a, H-20b. In addition, the crystal structure revealed an empirical formula of C 20 H 30 O 2 and M = 302.43 g/mol: C 20 H 30 O 2 had an orthorhombic, space group P2 1 2 1 2 1 (no. 19), a = 12.3081(7) Å, b = 23.8205(14) Å, c = 24.0947(14) Å, V = 7064.2(7) Å 3 , Z = 4, T = 296.15 K, µ(MoKα) = 0.071 mm − 1 , Dcalc = 1.134 g/cm 3 , 323447 reflections measured (4.09° ≤ 2Θ ≤ 57.286°), 18029 unique ( R int = 0.1149, R sigma = 0.0468). The final R 1 was 0.0728 (I > 2σ(I)) and wR 2 was 0.1523 (Fig. 3 ). Consequently, the structure of BZA 01 was determined as ent-kaur-16-en-19-oic acid, previously isolated from X. aethiopica . 21 BZ 02 BZA 02 was also obtained as colourless crystals. On TLC developed in PE: EtOAc (10:2), the Rf was 0.40 and appeared purple in anisaldehyde spray reagent. The IR spectrum revealed absorption bands of hydroxyl (3280.90 cm − 1 ), carbonyl (1724.65 cm − 1 ) and olefinic C = C stretch (1687.91 cm − 1 ). 1 H and 13 C NMR ( Supplementary information Figures S8, S9 ) data were similar with those of BZA 01, indicating structural similarities. The significant difference was an additional quaternary carbon peak, δ C 171.8 (C-21) in BZA O2 due to an ester carbonyl peak at δ C 171.8 (C21) and the acetyl carbon at δ C 21.7 (C-22). The position of the ester group at C-19 was supported by the HMBC correlations of H-20a and H-20b to C-16, C-18 and C-19, suggesting that BZA 02 is xylopic acid, also previously isolated from X. aethiopica . 22 X-ray crystallography data gave an empirical formula of C 22 H 32 O 4 and M = 360.47 g/mol. BZA 02 came out as an orthorhombic shaped crystal with space group P2 1 2 1 2 1 , a = 11.0960(5) Å, b = 11.8948(7) Å, c = 14.9745(8) Å, V = 1976.40(18) Å3, Z = 4, T = 296.15 K, µ(MoKα) = 0.082 mm-1, Dcalc = 1.211 g/cm3, 20732 reflections measured (5.02° ≤ 2Θ ≤ 53.028°), 4083 unique (Rint = 0.0979, Rsigma = 0.0772) as well as a final R1 was 0.0460 (I > 2σ(I)) and wR2 was 0.0980 which were used to calculate and confirm the structure of BZA 02 as xylopic acid (Fig. 3 ). BZ 05 BZA 05 was isolated as a fine green powder and exhibited IR and NMR spectra closely resembling those of BZA 01. However, notable differences included a characteristic IR absorption band at 1724.36 cm⁻¹, indicative of a ketone, and a significant downfield shift in the 13 C NMR spectrum for carbon C-15 — from δ C 49.1 in BZA 01 to δ C 210.5 in BZA 05 — consistent with ketone functionality ( Supplementary Information Figure S10 ). HMBC correlations between C-15 and H-20a, H-20b and H-14 were supportive of the assignment. The proposed structure was corroborated with the MS data analysis which exhibited an M + 1 peak at m/z 317.21 accompanied with fragments at m/z 299.20 and 271.2 ( Supplementary Information Figure S11 ). Hence, the structure BZA 05 was deduced as ent-kaur-16-en-15-one-19-oic acid, with a molar mass of 316 g/mol (Fig. 5 ). BL 2 and BL 4a Compound BL 2 was isolated as white powder. Its IR spectrum exhibited characteristic absorption bands for C–H stretching at 2914.8 cm − 1 and 2848.4 cm − 1 , a carbonyl group at 1733.8 cm − 1 , and an olefinic C = C stretch at 1683.9 cm − 1 . In the 13 C NMR spectrum ( Supplementary Information Figure S12) , signals corresponding to carbonyl carbons were observed at δ C 195.0 (aldehyde) and δ C 173.7 (ester). Olefinic carbons resonated at δ C 155.2, 143.6, 134.9, and 124.1. An oxygenated carbon appeared at δ C 64.1, while the remaining signals, ranging from δ C 39.3 to δ C 14.0, indicated the presence of a long alkyl chain. This was further supported by a series of multiplets in the upfield region of the 1 H NMR spectrum (δ H 2.34–0.88). An aldehydic proton was observed at δ H 9.36, and signals at δ H 6.44 and 5.12 were attributed to olefinic protons ( Supplementary Information Figure S13) . Compound BL 4 , a yellow gum, showed IR absorption bands at 2916.4 cm − 1 and 2849 cm − 1 (C–H stretching) and a carbonyl absorption at 1702.4 cm − 1 . The 13 C NMR spectrum ( Supplementary Information Figure S14) displayed a carbonyl resonance at δ C 180.5, with additional signals between δ C 34.5 and 14.5, while the 1 H NMR spectrum ( Supplementary Information Figure S15) showed multiplets in the δ H 2.34–0.88 range. These spectral features are consistent with the structure of a fatty acid. Molecular Modelling To validate the results of the in vitro antitrypanosomal screening, in silico analysis was conducted to provide further understanding of the biological mechanisms involved. Physicochemical descriptors such as MW (g/mol), log P , HD, HA. RB, Lipinski, Ghose and Veber of the isolated compounds - bis(4-methylheptyl) phthalate, ent-kaur-16-en-19-oic acid, xylopic acid and ent-kaur-16-en-15-one-19-oic acid were determined as shown in ( Supplementary information Table S1 ) to predict their individual ADME parameters. HA, HD, RB, log P and Lipinski values were within the drug-like chemical space. Likewise, MW was within the Known Drug Space ( Supplementary information Table S2 ). Co-crystallized ligands, including ivermectin (targeting glutathione S-transferase) and 5-hexyl glutathione (targeting the glutamate-gated chloride channel) for onchocerciasis, as well as pyridoxal-5-phosphate and doxycycline (targeting ornithine decarboxylase) for trypanosomiasis, were used as references to predict their functional scores against the respective target sites. Subsequently, the compounds were docked, together with and a wide range of compounds reported in the literature to have been isolated from X. aethiopica ( Supplementary information Table S4 ). Modelling against glutathione S-transferase (Onchocerciasis) The co-crystallized ligand of glutathione S-transferase ( Supplementary Information Figure S16 ) was first docked to generate root mean square deviation (RMSD) values for the heavy atoms. ASP obtained an average RMSD of 2.1366, PLP = 5.1415, CS = 1.6224, and GS = 1.9383. CS and GS showed stronger prediction power of scoring functions against the target ( Supplementary information Table S3 ). The modeling showed that the compounds occupy the hydrophobic binding pockets with a plausible binding mode. In comparison to the standard drug ivermectin, the binding affinity score for bis(4-methylheptyl) phthalate was remarkably high. Its carbonyl group formed a hydrogen bond with the side chain lysine 35 amino acid, and it also exhibited a π-π interaction with phenylalanine 8 (Fig. 5 ) . Ivermectin exhibited a similar binding mode. In contrast, the kaurene diterpenoids ent-kaur-16-en-19-oic acid, xylopic acid and ent-kaur-16-en-15-one-19-oic acid were inactive. Modelling against glutamate-gated chloride channel (Onchocerciasis) Firstly, the co-crystallized ligand of the glutamate-gated chloride channel ( Supplementary information Figure S17) was docked to generate average RMSD values for the heavy atoms as follows: ASP = 3.4741, PLP = 1.4928, CS = 1.4232, and GS = 1.7654, demonstrating stronger predictive power of the PLP and CS scoring functions against the target site ( Supplementary information Table S5 ). The compounds occupied the hydrophilic binding pockets as a plausible binding mode, with 5-hexyl glutathione, the co-crystallized ligand, exhibiting the highest score, closely followed by bis(4-methylheptyl) phthalate ( Supplementary information Table S6) . Binding revealed hydrogen bond interactions between their respective carbonyl groups and the side chain hydroxyl groups of threonine, glutamine, and serine amino acids, with a typical illustration with asparagine amino acid (Fig. 6 ) . Modelling against Ornithine Decarboxylase (Trypanosomiasis) The co-crystallized ligands, pyridoxal-5-phosphate and doxycycline were initially docked with Ornithine Decarboxylase ( Supplementary information Figure S18 ) to generate the RMSD values for the heavy atoms. ASP obtained an average RMSD of 0.4776, PLP = 1.5958, CS = 6.2696 and GS = 0.6362. In this case, GS and ASP exhibited a very strong prediction power of scoring functions as compared to PLP and CS against the target ( Supplementary information Table S7) . The modeled compounds again occupied the hydrophilic binding pockets with a plausible binding mode. As observed previously, bis(4-methylheptyl) phthalate demonstrated exceptionally high binding affinity with respect to the reference compounds, pyridoxal-5-phosphate and doxycycline (Supplementary information Table S8 ). It showed hydrogen bonding with the amine side chain of arginine while the phenyl moiety formed a π-π stack interaction with the imidazole side chain histidine (Fig. 7 ) . Conclusion The present study provides valuable insights into the pharmacological potential and safety considerations of the Ghanaian herbal formulation NTD-O2, traditionally used for the treatment of neglected tropical diseases. In vitro assays revealed that NTD-O2 possesses significant antitrypanosomal activity, with the isolated compound bis(4-methylheptyl) phthalate demonstrating potent efficacy against T. brucei . This activity was further supported by in silico molecular docking studies, which suggested favourable interactions with key parasitic targets, indicating a plausible mechanism of action. However, the identification of bis(4-methylheptyl) phthalate—a compound widely recognized as a synthetic plasticizer and environmental contaminant—raises important concerns regarding the authenticity and safety of the observed bioactivity. The absence of comparable activity in the individual plant constituents, Xylopia aethiopica and Bambusa vulgaris , suggests that the therapeutic effects may not be attributable to the herbal components themselves but rather to external contamination introduced during processing or packaging. This finding underscores the critical need for stringent quality control measures in the preparation and evaluation of herbal medicines. While the observed antitrypanosomal activity is promising, the potential toxicological implications of phthalate exposure cannot be overlooked. These results highlight the dual necessity of validating the efficacy of traditional remedies while ensuring their safety through comprehensive chemical profiling and contamination screening. Future research should focus on optimizing the efficacy and safety profiles of promising compounds and exploring their potential in combination therapies. Abbreviations Animal African trypanosomiasis AAT Astex statistical potential ASP ChemScore CS Diminazene aceturate DA Ghana Federation of Traditional Medicines Practitioners Association GHAFTRAM Ghana Food and Drugs Authority FDA Glutamate-gated chloride channel GluCl GoldScore GS Herbal medicine HM Human gastrointestinal absorption HIA Neglected tropical diseases NTDs Pharmacodynamics PD Pharmacokinetics PK Protein Data Bank PDB Root mean square deviation RMSD Selective index SI World Health Organization WHO Declarations Supplementary Information The online version contains supplementary material available at… Acknowledgements The authors express their gratitude to the Department of Chemistry for NMR and X-ray data acquisition and New Zealand eScience Infrastructure (NeSI) high-performance computing facilities as part of this research funded jointly by the collaborating institutions and through the Ministry of Business, Innovation and Employment Research Infrastructure program. URL: https://www.nesi.org.nz. The Trypanosoma brucei brucei cell line (GUTat 3.1 strain) was originally obtained from the Department of Parasitology, Noguchi Memorial Institute for Medical Research, University of Ghana. Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The dataset supporting the conclusions of this article is included within the article and its additional file. Competing interests The authors have no competing interests. Funding This research was supported by Worldwide Universities Network Research Development Fund 2017 from the Worldwide Universities Network (UK) and grant number 18-191 RG/CHE/AF/AC_G - FR3240303659 from The World Academy of Sciences. Authors' contributions BZA : Data acquisition and analysis; Writing – original draft, revision and final approval. 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Supplementary Files InVitroandInSilicoEvaluationoftheEfficacyandSafetyofaGhanaianHerbalMedicinefortheTreatmentofOnchocerciasisandAfricanTrypanosomiasisSupplementarydata.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Sep, 2025 Reviews received at journal 10 Sep, 2025 Reviews received at journal 02 Sep, 2025 Reviews received at journal 26 Aug, 2025 Reviews received at journal 23 Aug, 2025 Reviewers agreed at journal 23 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers invited by journal 17 Aug, 2025 Editor invited by journal 15 Aug, 2025 Editor assigned by journal 15 Aug, 2025 Submission checks completed at journal 06 Aug, 2025 First submitted to journal 06 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7189198","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":504601096,"identity":"cbb895ad-dc38-4680-af18-ef93e6d3e49e","order_by":0,"name":"Barbara Zenabu Anibea","email":"","orcid":"","institution":"University of Ghana","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"Zenabu","lastName":"Anibea","suffix":""},{"id":504601097,"identity":"c40ead78-db20-4f2c-a960-4669085469fb","order_by":1,"name":"Eric Coffie","email":"","orcid":"","institution":"University of 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16:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7189198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7189198/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89849350,"identity":"df34f255-9a88-4581-8393-12a2c0233f12","added_by":"auto","created_at":"2025-08-25 17:05:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":101712,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure and HMBC representation of Bis(4-methylhepthyl)phthalate\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7189198/v1/dfcaff1e943609713c16a6be.png"},{"id":89850334,"identity":"8775a15e-90d0-4f1c-9550-9a3984179ce5","added_by":"auto","created_at":"2025-08-25 17:21:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":421695,"visible":true,"origin":"","legend":"\u003cp\u003eChemical and Crystal structure, and HMBC representation of ent-kaur-16-en-19-oic acid\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7189198/v1/546d5a73831aeac451ae0ccb.png"},{"id":89850018,"identity":"550ca4dd-9047-4a94-8e73-37400a2e7277","added_by":"auto","created_at":"2025-08-25 17:13:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":514231,"visible":true,"origin":"","legend":"\u003cp\u003eChemical and Crystal structure, and HMBC representation of Xylopic acid\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7189198/v1/665fdb0946b844914ded09ef.png"},{"id":89849357,"identity":"4633f697-8c8a-4735-b89a-8d1134f162a7","added_by":"auto","created_at":"2025-08-25 17:05:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":114299,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of ent-kaur-16-en-15-one-19-oic acid and HMBC representation of ent-kaur-16-en-15-one-19-oic acid\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7189198/v1/bafc4fd414d2d7a9bd01f449.png"},{"id":89849352,"identity":"01e89cf1-f326-47b1-b82e-4e823cba29ce","added_by":"auto","created_at":"2025-08-25 17:05:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":828320,"visible":true,"origin":"","legend":"\u003cp\u003eThe docked configuration of bis(4-methylheptyl) phthalate in the binding site of glutathione S-transferase as predicted by ChemPLP (\u003cstrong\u003eA\u003c/strong\u003e) The ligand occupies the binding pocket. The protein surface is rendered. Blue depicts a hydrophilic region on the surface; brown depicts hydrophobic region and white shows neutral areas. (\u003cstrong\u003eB\u003c/strong\u003e) Hydrogen bonds are shown as green lines between bis(4-methylheptyl) phthalate and the amino acid Lysine 35 whiles ℼ-ℼ interaction between the molecule and phenylalanine 8.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7189198/v1/17b12c08e3e548722479a1f6.png"},{"id":89850336,"identity":"6cd47e04-5aac-437b-bb2a-552212993c6d","added_by":"auto","created_at":"2025-08-25 17:21:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":931473,"visible":true,"origin":"","legend":"\u003cp\u003eThe docked configuration of bis(4-methylheptyl) phthalate in the binding site of glutamate-gated Cl ion channel as predicted by ChemPLP (A). The ligand occupies the binding pocket. The protein surface is rendered. Blue depicts a hydrophilic region on the surface; brown depicts hydrophobic region and white shows neutral areas. (B) Hydrogen bonds are shown as green lines between bis(4-methylheptyl) phthalate and the amino acid Asparagine 264.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7189198/v1/f5d63ec2bfcb10c0047ba25d.png"},{"id":89850031,"identity":"5dc8c5c9-36f8-4661-9e16-8cf0ec1ecd2b","added_by":"auto","created_at":"2025-08-25 17:13:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":695191,"visible":true,"origin":"","legend":"\u003cp\u003eThe docked configuration of bis(4-methylheptyl) phthalate in the binding site of Ornithine decarboxylase as predicted by Goldscore (A). The ligand occupies the binding pocket. The protein surface is rendered. Blue depicts a hydrophilic region on the surface; brown depicts hydrophobic region and whites shows neutral areas. (B) Hydrogen bonds are shown as green lines between bis(4-methylheptyl) phthalate and the amino acids Arginine 227 whiles ℼ-ℼ interaction is shown with histidine.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7189198/v1/198aeb02957013aa65a42571.png"},{"id":89850020,"identity":"c7954665-2f4e-4e39-a5b5-f2c7483fbd04","added_by":"auto","created_at":"2025-08-25 17:13:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1685702,"visible":true,"origin":"","legend":"","description":"","filename":"InVitroandInSilicoEvaluationoftheEfficacyandSafetyofaGhanaianHerbalMedicinefortheTreatmentofOnchocerciasisandAfricanTrypanosomiasisSupplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-7189198/v1/f845e297cc88335bb2cb6cad.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Vitro and In Silico Evaluation of the Efficacy and Safety of a Ghanaian Herbal Medicine for the Treatment of Onchocerciasis and African Trypanosomiasis","fulltext":[{"header":"Background","content":"\u003cp\u003eRooted in indigenous knowledge and cultural beliefs, herbal medicine (HM) has been the cornerstone of human healthcare for centuries and continues to offer readily available and affordable healthcare to approximately 80% of the population in developing countries. Practically, for most indigenous people living in remote rural areas with limited access to conventional medical services, HM is the only treatment option.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Besides, over the past three decades, many developed countries have widely embraced the use of HM to promote healthier living.\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e While HM is perceived to have less adverse effects, they are commonly used as self-care treatments and are unregulated, posing huge safety risks for consumers. In response to these challenges, coupled with the recognition of their enormous value for achieving universal healthcare, the World Health Organization (WHO) established technical guidelines and standards to ensure the safety, efficacy and quality control of HM.\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eHM is a significant aspect of Ghana\u0026rsquo;s cultural heritage and its development over the years is well documented.\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Since 2012, the practice has been approved by the government for integration into the mainstream healthcare system,\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e resulting in a wide range of HM products on the Ghanaian market for treating a variety of disease conditions. The Ghana Food and Drugs Authority (FDA) is mandated to approve and register such products based on efficacy, safety and clinical data. The rational use of HMs has largely been supported by research output on the scientific validation of their efficacy claims. However, the challenge is that many of the FDA-approved products cover common ailments such as malaria, anaemia, cough, and asthma, unlike remedies for conditions like the neglected tropical diseases (NTDs) due to lack of access to efficacy testing platforms.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Hence, many people affected by NTDs are exposed to unapproved HM, a situation with the potential for serious safety and efficacy implications and therefore requires urgent scientific validation for better healthcare outcomes.\u003c/p\u003e\u003cp\u003eOnchocerciasis, commonly known as river blindness, is a parasitic disease caused by the filarial nematode \u003cem\u003eOnchocerca volvulus\u003c/em\u003e and transmitted through the bite of infected female blackflies (\u003cem\u003eSimulium spp\u003c/em\u003e.).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The disease is responsible for a range of skin and eye-related morbidities including irreversible blindness.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Onchocerciasis is reported among the NTDs that remain endemic in several Ghanaian communities after more than 18 rounds of annual mass drug administration with ivermectin.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Our interaction with the Ghana Federation of Traditional Medicines Practitioners Association (GHAFTRAM) revealed the existence and high patronage of HM by affected people in NTD-endemic communities.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e As part of efforts to contribute to the promotion and development of these remedies, we sought to conduct a comparative investigation of a HM used in treating onchocerciasis (NTD-O2) and its medicinal plant constituents. Thus, the constituents of NTD-O2 and its component medicinal plants (\u003cem\u003eXylopia aethiopica\u003c/em\u003e fruits and \u003cem\u003eBambusa vulgaris\u003c/em\u003e leaves) were extracted and separately screened for anti-onchocercal activity against \u003cem\u003eOnchocera ochengi\u003c/em\u003e. Bioassay-guided fractionation was used to identify the bioactive constituents. Structural elucidation techniques involving spectroscopic and spectrometric methods were used to characterize the chemical constituents. The possible mechanisms of action of the compounds were further determined by \u003cem\u003ein silico\u003c/em\u003e techniques.\u003c/p\u003e\u003cp\u003eAnimal African trypanosomiasis (AAT), unlike human trypanosomiasis (sleeping sickness,) continues to be highly neglected primarily due to limited resources and global attention directed towards human disease control. While AAT causes huge economic loses in livestock production, coupled with the direct effects on human health, the financial incentives for developing veterinary drugs for livestock disease are often low, resulting in limited treatment options.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e According to Tweneboah et al.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, information on the current situation of AAT in Ghana is scanty due to neglect even at the national level. In our earlier work,\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e we demonstrated that a chemical constituent of NTD-B4, a herbal medicine used against schistosomiasis, was effective against \u003cem\u003eTrypanosoma brucei brucei\u003c/em\u003e compared to the standard antitrypanosomal drug, diminazene aceturate. Hence, NTD-O2 extracts and compounds as well as those from the medicinal plants were subjected to antitrypanosomal activity screening against \u003cem\u003eTrypanosoma brucei brucei\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTo ensure herbal medicines can serve as viable alternatives to conventional treatments for parasitic infections, regular screening is essential. This process guarantees quality assurance, efficacy, and safety, thereby enhancing their credibility and protecting consumers.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHerbal Medicine and Plant Materials\u003c/h2\u003e\u003cp\u003eNTD-O2 is a Ghanaian herbal medicine used in the treatment of onchocerciasis. It was prepared from two medicinal plants, the dry fruits of \u003cem\u003eXylopia aethiopica\u003c/em\u003e and the fresh leaves of \u003cem\u003eBambusa vulgaris\u003c/em\u003e and provided by GHAFTRAM for the study. In addition to the herbal medicine (NTD-O2), the constituent plant materials were also collected and analyzed separately. Dry \u003cem\u003eX. aethiopica\u003c/em\u003e fruits were sourced from Nima Market, Accra, and fresh \u003cem\u003eB. vulgaris\u003c/em\u003e leaves from the University of Ghana campus. Both were identified by Mr. Patrick Ekpe and assigned voucher numbers BZA 001 and BZA 002 at the Ghana Herbarium, Department of Plant and Environmental Biology, University of Ghana.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExtraction and Isolation\u003c/h3\u003e\n\u003cp\u003eNTD-O2 was subjected to the protocol described by Twumasi et al. (2020)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e with slight modification. Briefly, a total of 3.8 litres of NTD-O2 was extracted in 500 mL aliquots with equal volumes of HPLC-grade dichloromethane (DCM) and n-butanol (n-BuOH) sequentially. For each aliquot, the procedure was repeated three times to yield the corresponding crude extracts NTD-O2-DCM (7 g) and NTD-O2-BuOH (25 g). The NTD-O2-BuOH extract was then sequentially fractionated with hexane, DCM, Ethylacetate (EtOAc) and Methanol (MeOH). Each fraction was concentrated under vacuum and refrigerated at 4\u0026deg;C for about 72 h to facilitate precipitation of solids. The EtOAc fraction gave a yellow-brown solid (O2-F3-S, 5 g) and was purified over a column of 60 g of silica gel (130\u0026ndash;270 \u0026Aring; mesh, SIGMA-ALDRICH) eluted with petroleum ether (PE) and EtOAc to yield compound \u003cb\u003e1\u003c/b\u003e (O2-F3-S, 8 mg) and compound \u003cb\u003e2\u003c/b\u003e (O2-F3-ML, 20 mg).\u003c/p\u003e\u003cp\u003eDried powder (1 kg) of \u003cem\u003eX. aethiopica\u003c/em\u003e fruits and fresh leaves of \u003cem\u003eB. vulgaris\u003c/em\u003e (674.0 g) were separately extracted continuously via cold percolation with Petroleum Ether (PE) for 72 hours. The solvent was removed under reduced pressure to give an oily crude extract for \u003cem\u003eX. aethiopica\u003c/em\u003e (XA/PE, 30.8 g) and greenish paste crude for \u003cem\u003eB. vulgaris\u003c/em\u003e (BL/PE, 10 g). XA/PE (10 g) was chromatographed over a column of silica gel (120 g) and eluted with PE/EtOAc (v/v\u0026thinsp;=\u0026thinsp;10:0, 9:1, 8:2, 6:4, 1:1, 0:10) and EtOAc/MeOH (9.5:0.5). A total of 315 eluates (v\u0026thinsp;=\u0026thinsp;20 mL) were obtained and pooled together based on TLC profile into 13 fractions (1\u0026ndash;13). Upon evaporation under vacuum, fractions 1\u0026ndash;12 were oily while fraction 13 was a powdery solid. Following refrigeration at 4\u0026deg;C for about 72 h, fractions 2\u0026ndash;5 generously precipitated colourless crystals (BZA 01, 5 g), fractions 11\u0026ndash;12 precipitated another set of colourless crystals (BZA 02, 4.7 g), while fraction 9 precipitated a greenish powder (BZA 05, 200 mg). \u003cem\u003eB. vulgaris\u003c/em\u003e (BL/PE, 7 g) was also subjected to silica gel column chromatography using a similar protocol as described above to yield 2 compounds, a white powder (BL 2, 10 mg) and yellow gum (BL 4, 3 mg). Thin-layer chromatography (TLC) was conducted on silica gel 60 F\u003csub\u003e254\u003c/sub\u003e (Merck) pre-coated aluminium foil slides, visualized with UV light (254 and 364 nm) and anisaldehyde spray reagent heated to 110\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eStructure elucidation techniques\u003c/h3\u003e\n\u003cp\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra were recorded on a Br\u0026uuml;ker, 500 MHz spectrometer in CDCl\u003csub\u003e3,\u003c/sub\u003e with chemical shifts referenced to tetramethyl silane. LC-MS analysis was performed using an Agilent HPLC system equipped with a Kinetex Core C18 column (2.6 \u0026micro;M, 3 x 50 mm, 100 \u0026Aring;) at 40\u0026deg;C. The mobile phase consisted of acetonitrile-ethyl acetate 9:1 eluted under gradient conditions at 0.7 mL/min. Injection volume was 2 \u0026micro;L, and the mass spectra were obtained using electrospray ionization (ESI) and atmospheric pressure chemical ionization. GC-MS analysis was conducted on a Perkin Elmer GC Clarus 580 with electron impact ionization at 70 eV and helium gas at 1 mL/min. National Institute Standard and Technology (NIST) library was employed in compound identification. Infra-red spectral data was acquired neat on a Perkin Elmer FTIR spectrometer using the attenuated total reflectance technique. MS analyses of samples were performed using an Agilent 1100 Autosampler instrument with ESI at 3.5 kV and a Thermo Q-Exactive orbitrap instrument. X-ray crystallography measurements were done on a Br\u0026uuml;ker ECO D8 diffractometer, with data collection at 296.15 K and the structure solved with the olex2.solve and refined with and refined with the ShelXL.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanti-onchocercal assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStock solution (20 mg/mL) of NTD-O2-DCM and NTD-O2-BuOH in DMSO were tested on the worms and larvae of \u003cem\u003eO. ochengi\u003c/em\u003e as previously described.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Auranofin (10 \u0026micro;M) was used as the positive control while 2% of DMSO served as the negative control. The viability of the adult female worms was evaluated biochemically by visually assessing the percentage inhibition of formazan production after incubating the nodules in 500 \u0026micro;L of 0.5 mg/mL MMT.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e A loss of the characteristic blue coloration in the worms indicated worm death. A test sample was classified as active if it inhibited more than 90% of female worm motility or formazan production, moderately active if it caused 50\u0026ndash;89% inhibition, and inactive if inhibition was below 50%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eAnti-trypanosomal assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBloodstream forms of \u003cem\u003eT. brucei brucei\u003c/em\u003e were used in this study due to their similarity to the human-infective forms, \u003cem\u003eT. b. rhodesiense\u003c/em\u003e and \u003cem\u003eT. b. gambiense\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The protocol employed is as previously described.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Briefly, crude extracts were dissolved in 100% DMSO to make a 20 mg/mL stock, then diluted to 2 mg/mL in sterile distilled water for testing. Isolated compounds (BZA 01, BZA 02, BZA 05 from \u003cem\u003eX. aethiopica\u003c/em\u003e; BL 2 and BL 4 from \u003cem\u003eB. vulgaris\u003c/em\u003e) were prepared similarly. Trypanosome cells in media without treatment served as the negative control, while diminazene aceturate (DA) was the positive control. Wild-type bloodstream forms of \u003cem\u003eT. b. brucei\u003c/em\u003e (GuTat 3.1 strain) were cultured in Hirumi\u0026rsquo;s Modified Iscove\u0026rsquo;s Medium-9 (HMI-9),\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum (Gibco). The cultures were maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. An Alamar blue assay was used to test the crude extracts and compounds.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Serial dilutions (100 to 0.1953 \u0026micro;g/mL) were prepared in 96-well plates. Log-phase trypanosomes were added, and the plates were incubated for 72 hours at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. A final concentration of 44 \u0026micro;M of resazurin sodium salt (Sigma-Aldrich) in phosphate buffered saline was then added to each well, plates were incubated for additional 5 h and the absorbance measured at 570 nm using the Varioskan Lux Elisa plate reader (ThermoFisher Scientific, USA). Each test was done in triplicate, with three technical replicates.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003ePlate readouts from the Alamar blue assay were evaluated using non-linear regression analysis (log10 inhibitor versus response-variable slope) to assess growth inhibition. This analysis was performed using GraphPad Prism version 8.0.1. The IC\u003csub\u003e50\u003c/sub\u003e values of the test samples were determined from three biological replicates, each conducted in triplicate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003estudies\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e studies were performed on compounds isolated from \u003cem\u003eX. aethiopica\u003c/em\u003e and \u003cem\u003eB. vulgaris\u003c/em\u003e in the current study and those reported in literature to validate the outcomes of the \u003cem\u003ein vitro\u003c/em\u003e anti-onchocercal and antitrypanosomal assays. These studies included predictions of pharmacodynamics (PD) and pharmacokinetics (PK), as well as molecular modelling of compounds.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSelection of Protein Targets\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003eO. ochengi\u003c/em\u003e, the typical protein targets selected were glutathione S-transferase and glutamate-gated chloride ion channel. In the case of \u003cem\u003eT. brucei brucei\u003c/em\u003e, ornithine decarboxylase was chosen as the protein target.\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Ornithine Decarboxylase is a crucial enzyme-dependent growth factor, playing a vital role for the parasite through the formation of polyamine putrescine, a molecule important in the pathway for the production of trypanothione.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePrediction of the PD and PK of the Compounds\u003c/h2\u003e\u003cp\u003eThe SwissADME server was used to predict PD and PK properties of the isolated compounds.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMeasurement of PK Properties and Drug-Likeness\u003c/h3\u003e\n\u003cp\u003eThe SwissADME server was further utilized to determine the physicochemical descriptors of the compounds, as well as to define their PK properties and drug-like nature. The Brain or Intestinal Estimated Permeation (BOILED-Egg) model was employed to intuitively evaluate passive human gastrointestinal absorption (HIA) and blood-brain barrier (BBB) penetration. This model was also used to compute the lipophilicity and polarity of the compounds.\u003c/p\u003e\n\u003ch3\u003eModelling of compounds\u003c/h3\u003e\n\u003cp\u003eThe compounds, both isolated in the current study and those reported in literature, were docked to the crystal structure of the Glutathione S-Transferase (Protein Data Bank (PDB) ID: 1TU8, resolution 1.80 \u0026Aring;)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and Glutamate-gated chloride channel (GluCl) (PDB: 3RHW, resolution 3.26 \u0026Aring;), which are protein targets for onchocerciasis. Additionally, these compounds were docked against ornithine decarboxylase (PDB ID: 1F3T, resolution 2.00 \u0026Aring;),\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e a protein target for trypanosomiasis. The crystal structures of co-crystallized ligands were obtained from PDB.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e These structures were prepared by removing the co-crystallized ligands and adding the hydrogen atoms using the Scigress version FJ 2.6 program. The binding pocket center of the Glutathione S-Transferase was identified by fitting the atoms of the co-crystallised ligand 5-hexyl Glutathione (x\u0026thinsp;=\u0026thinsp;2.570, y\u0026thinsp;=\u0026thinsp;0.814, z\u0026thinsp;=\u0026thinsp;49.968) with a radius of 10 \u0026Aring;. Similarly, the binding pocket centre of glutamate-gated chloride channel was defined by the position of the atoms of co-crystallized ligand ivermectin (x\u0026thinsp;=\u0026thinsp;12.675, y\u0026thinsp;=\u0026thinsp;95.052, z\u0026thinsp;=\u0026thinsp;24.998) with a radius of 10 \u0026Aring;. The binding pocket centre of ornithine decarboxylase was identified by the position of the phosphorus atom of the co-crystallised ligand pyridoxal-5'-phosphate (coordinates: x\u0026thinsp;=\u0026thinsp;26.206, y\u0026thinsp;=\u0026thinsp;15.614, z\u0026thinsp;=\u0026thinsp;3.800) and a radius of 10 \u0026Aring;. To validate the predicted binding modes and relative energies of the ligands, the GoldScore (GS)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, ChemScore (CS)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, ChemPLP\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and Astex statistical potential (ASP)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e scoring functions were implemented using the GOLD v5.4 software suite.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanti-onchocercal and antitrypanosomal activity of NTD-O2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results of the \u003cem\u003ein vitro\u003c/em\u003e anti-onchocercal and antitrypanosomal assays are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In primary screens against \u003cem\u003eO. ochengi\u003c/em\u003e, both the dichloromethane extract of NTD-O2 (NTD-O2-DCM) and the butanol extract (NTD-O2-BuOH) at 200 \u0026micro;g/mL, as well as the positive control, auranofin at 10 \u0026micro;M showed 100\u0026thinsp;\u0026plusmn;\u0026thinsp;0% inhibition of the adult male worm. This indicates that the crude extracts of the herbal medicine at the specified concentration were as effective as the control against the adult male worm. However, while auranofin maintained the same level of activity against the adult female worm, the extracts demonstrated moderate activity, with 61.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8% and 56.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4% inhibition, respectively. Due to the moderate activity against the female worm, which is a critical criterion for advancing to secondary screens, the extracts were not selected for further testing against the parasites.\u003c/p\u003e\u003cp\u003eIn contrast, the antitrypanosomal assay results were promising for both extracts, with IC\u003csub\u003e50\u003c/sub\u003e values of 10.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54 \u0026micro;g/mL for NTD-O2-DCM and 9.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 \u0026micro;g/mL for NTD-O2-BuOH against \u003cem\u003eT. b. brucei\u003c/em\u003e. The positive control, diminazene aceturate, recorded an IC\u003csub\u003e50\u003c/sub\u003e value of 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g/mL (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While the extracts were less potent than diminazene aceturate, their IC\u003csub\u003e50\u003c/sub\u003e values are still within a range that suggests possible optimization or application in combination therapies to enhance efficacy.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnti-onchocercal and antitrypanosomal screening of NTD-O2 and its medicinal plant isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTest sample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCytotoxic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eAnti-onchocercal activity against adult worms of \u003cem\u003eO. ochengi\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAntitrypanosomal activity against bloodstream forms \u003cem\u003eT. b. brucei\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%Inhibition of males\u003c/p\u003e\u003cp\u003e(5 days)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%Killing of females\u003c/p\u003e\u003cp\u003e(7 days)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/mL)\u003c/p\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eNTD-O2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNTD-O2-DCM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e61.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNTD-O2-BuOH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e56.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAuranofin\u003c/p\u003e\u003cp\u003e(10 \u0026micro;M)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDiminazene aceturate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003eIsolates from NTD-O2-BuOH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO2-F3-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO2-F3-ML\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDiminazene aceturate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003eIsolates from medicinal plants\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBZA 01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBZA 02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBZA 05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBL2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBL4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDiminazene aceturate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eN/A\u0026thinsp;=\u0026thinsp;Not applicable; N/D\u0026thinsp;=\u0026thinsp;Not determined; Calculated adult worm activity responses were done in quadruplicates, IC\u003csub\u003e50\u003c/sub\u003e values represent mean averages of 3 biological replicates.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe higher activity results of the antitrypanosomal assay informed a bioassay-guided fractionation and isolation of compounds from NTD-O2 and its medicinal plants, \u003cem\u003eX. aethiopica\u003c/em\u003e and \u003cem\u003eB. vulgaris\u003c/em\u003e. NTD-O2-BuOH was prioritized over NTD-O2-DCM due to its higher IC\u003csub\u003e50\u003c/sub\u003e value.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAntitrypanosomal activity of isolates from NTD-O2-BuOH\u003c/h2\u003e\u003cp\u003eNTD-O2-BuOH was sequentially fractionated with hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) to obtain the corresponding fractions. Isolates from the hexane and DCM fractions encountered solubility issues in dimethyl sulfoxide (DMSO), precluding their assay results. Additionally, attempts to purify the MeOH fraction were unsuccessful. Hence, only isolates O2-F3-S and O2-F3-ML from the EtOAc fraction were evaluated for their antitrypanosomal activity.\u003c/p\u003e\u003cp\u003eO2-F3-S exhibited limited bioactivity, with an IC\u003csub\u003e50\u003c/sub\u003e value of 100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u0026micro;g/mL. In contrast, O2-F3-ML demonstrated significant antitrypanosomal activity, with an IC\u003csub\u003e50\u003c/sub\u003e value of 1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;g/mL. When compared to the standard antitrypanosomal agent, diminazene aceturate, which had an IC\u003csub\u003e50\u003c/sub\u003e of 0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u0026micro;g/mL, O2-F3-ML shows a comparable efficacy.\u003c/p\u003e\u003cp\u003eThe cytotoxicity (CC\u003csub\u003e50\u003c/sub\u003e) of O2-F3-ML was determined to be 100 \u0026micro;g/mL, whereas diminazene aceturate exhibited a CC\u003csub\u003e50\u003c/sub\u003e of 36\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;g/mL against the standard phenylarsine oxide, which has a CC\u003csub\u003e50\u003c/sub\u003e value of 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;g/mL. This indicates that the cytotoxicity of O2-F3-ML is moderate. Consequently, the selective index (SI) was calculated to be 89\u0026thinsp;\u0026plusmn;\u0026thinsp;25 for O2-F3-ML and 203\u0026thinsp;\u0026plusmn;\u0026thinsp;22 for diminazene aceturate. These findings suggest that O2-F3-ML possesses notable antitrypanosomal activity and hence was subjected to structure elucidation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStructure Elucidation of O2-F3-ML\u003c/h2\u003e\u003cp\u003eSample O2-F3-ML was obtained as a yellowish oil with a retention factor (Rf) of 0.75 on TLC developed in hexane and acetone (7:3). The IR data gave absorptions at 2958 cm-\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003c/sup\u003e 2927 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2858 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C-H), 1726 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O) and 1610 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (aromatic C\u0026thinsp;=\u0026thinsp;C). The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR spectrum (\u003cb\u003eSupplementary information Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) exhibited a typical AA'BB' system at H-4/H-4' (δ\u003csub\u003eH\u003c/sub\u003e 7.53, dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.3 and 5.7 Hz, 2H) and H-3/H-3' (δ\u003csub\u003eH\u003c/sub\u003e 7.70, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.3 Hz, 2H) supporting an ortho-disubstituted benzene ring with identical substituents in both positions. Signals were seen at δ\u003csub\u003eH\u003c/sub\u003e 0.90 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.5 Hz, 3H) for the secondary methyl protons H-12/H-12' and δ\u003csub\u003eH\u003c/sub\u003e 0.91 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.8 Hz, 3H) for the terminal methyl protons H-11/H-11'. A string of multiplets at δ\u003csub\u003eH\u003c/sub\u003e 1.31, δ\u003csub\u003eH\u003c/sub\u003e 1.35, δ\u003csub\u003eH\u003c/sub\u003e 1.42, δ\u003csub\u003eH\u003c/sub\u003e 1.68 and δ\u003csub\u003eH\u003c/sub\u003e 4.22, was suggestive of methylene groups, with the latter linked to the ester. The \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR and DEPT 135 spectra (\u003cb\u003eSupplementary information Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2\u003c/b\u003e) revealed resonances for methyl signals δ\u003csub\u003eC\u003c/sub\u003e 11.1 (C-12/C-12') and δ\u003csub\u003eC\u003c/sub\u003e 14.2 (C-11/C-11'), methylene carbon signals at δ\u003csub\u003eC\u003c/sub\u003e 23.1 (C-10/C-10'), δ\u003csub\u003eC\u003c/sub\u003e 23.9 (C-9/C-9'), δ\u003csub\u003eC\u003c/sub\u003e 29.0 (C-8/C-8'), δ\u003csub\u003eC\u003c/sub\u003e 30.5 (C-7/C-7'), and an \u003cem\u003esp\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e methine carbon signal at δ\u003csub\u003eC\u003c/sub\u003e 38.9 (C-6/C-6'). The \u003cem\u003esp\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e oxygenated carbon (C-5/C-5') appeared at δ\u003csub\u003eC\u003c/sub\u003e 68.4. Further, there were aromatic methine carbon peaks at δ\u003csub\u003eC\u003c/sub\u003e 128.9 (C-4/C-4'), δ\u003csub\u003eC\u003c/sub\u003e 131.0 (C-3/C-3') along with a quaternary aromatic carbon peak at δ\u003csub\u003eC\u003c/sub\u003e 132.6 (C-2/C-2') and a carbonyl carbon at δ\u003csub\u003eC\u003c/sub\u003e 167.9 (C-1/C-1') due to an ester moiety.\u003c/p\u003e\u003cp\u003eHMBC spectral data provided correlations between the methyl protons at δ\u003csub\u003eH\u003c/sub\u003e 0.90 (H-12) and δ\u003csub\u003eC\u003c/sub\u003e 23.1 (C-10), 23.9 (C-9) and 29.0 (C-8). Correlations H-5/C-8, C-1 established the ester linkage and H-12/C-6, C-7, C-9; H-5/C-6 confirmed the branched position of the side chain. The mass spectrum from the GC-MS analysis displayed fragment peaks at m/z 29, 43, 57, 71, 83, 149, 167, and 279, characteristic of alkyl phthalates (\u003cb\u003eSupplementary information Figure S4\u003c/b\u003e). The molecular ion peak occurred at m/z 390 and the base peak at m/z 149, representing a protonated phthalate anhydride. Based on these assignments, the structure of O2-F3-ML was deduced as bis(4-methylheptyl) phthalate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePhthalates and their derivatives are widely recognized as environmental contaminants due to their extensive industrial use. Prolonged or excessive exposure to these compounds has been associated with significant health risks in humans, including endocrine disruption, metabolic disorders, cancer, reproductive health issues and developmental challenges in children.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e In contrast, phthalates have also been reported in natural sources such as plants, bacteria, and fungi, demonstrating diverse biological potential such as antitumor, cytotoxic, allopathic, larvicidal, antiviral and anti-inflammatory properties.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eantitrypanosomal activity of the medicinal plant isolates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCompounds BZA 01, BZA 05, BL 2 and BL 4 recorded an IC\u003csub\u003e50\u003c/sub\u003e value of \u0026gt;\u0026thinsp;100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u0026micro;g/mL and hence, did not demonstrate any antitrypanosomal activity. BZA 02, on the other hand, could not be assayed due to challenges with solubility in DMSO.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStructure elucidation of the medicinal plant isolates\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003eBZ 01\u003c/h2\u003e\u003cp\u003eBZA 01 was obtained as colourless crystals that appeared as an orange spot when visualized in anisaldehyde with Rf of 0.65 on TLC developed in PE: EtOAc (10:1). The IR spectrum showed characteristic peaks at 1726.56 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for carbonyl and 1687.91 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for unsaturation (C\u0026thinsp;=\u0026thinsp;C).\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR and DEPT 135 spectra (\u003cb\u003eSupplementary information Figures S5, S6\u003c/b\u003e) exhibited 20 carbon signals sorted by the DEPT experiment into 5 quaternary carbons (including a carboxylic carbon at δ\u003csub\u003eC\u003c/sub\u003e 185.1 (C-3) and an alkene carbon (C-16) at δ\u003csub\u003eC\u003c/sub\u003e 155.8), 3 methine carbons, 2 tertiary methyl carbons and 10 methylene carbons, which were consistent with a kaurenoic acid skeleton. In the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectrum (\u003cb\u003eSupplementary information Figure S7\u003c/b\u003e), signals for the olefinic protons H20a (d, δ\u003csub\u003eH\u003c/sub\u003e 4.80, 1H) and H-20b (d, δ\u003csub\u003eH\u003c/sub\u003e 4.74, 1H) and tertiary methyls H-4 (s, δ\u003csub\u003eH\u003c/sub\u003e 1.24, 3H) and H-7 (s, δ\u003csub\u003eH\u003c/sub\u003e 0.95, 3-H) supported the presence of kaurenoic acid. HMBC correlations between C-3/H-4, H-5 established the position of the carboxylic acid while the exocyclic alkene was fixed by correlations from C-15, C-16, C-17/H-20a, H-20b.\u003c/p\u003e\u003cp\u003eIn addition, the crystal structure revealed an empirical formula of C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and \u003cem\u003eM\u003c/em\u003e\u0026thinsp;=\u0026thinsp;302.43 g/mol: C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e had an orthorhombic, space group P2\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e (no. 19), \u003cem\u003ea\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.3081(7) \u0026Aring;, \u003cem\u003eb\u003c/em\u003e\u0026thinsp;=\u0026thinsp;23.8205(14) \u0026Aring;, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24.0947(14) \u0026Aring;, \u003cem\u003eV\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7064.2(7) \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eZ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4, \u003cem\u003eT\u003c/em\u003e\u0026thinsp;=\u0026thinsp;296.15 K, \u0026micro;(MoKα)\u0026thinsp;=\u0026thinsp;0.071 mm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eDcalc\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.134 g/cm\u003csup\u003e3\u003c/sup\u003e, 323447 reflections measured (4.09\u0026deg; \u0026le; 2Θ\u0026thinsp;\u0026le;\u0026thinsp;57.286\u0026deg;), 18029 unique (\u003cem\u003eR\u003c/em\u003e\u003csub\u003eint\u003c/sub\u003e = 0.1149, R\u003csub\u003esigma\u003c/sub\u003e = 0.0468). The final \u003cem\u003eR\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e was 0.0728 (I\u0026thinsp;\u0026gt;\u0026thinsp;2σ(I)) and \u003cem\u003ewR\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e was 0.1523 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Consequently, the structure of BZA 01 was determined as ent-kaur-16-en-19-oic acid, previously isolated from \u003cem\u003eX. aethiopica\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eBZ 02\u003c/h2\u003e\u003cp\u003eBZA 02 was also obtained as colourless crystals. On TLC developed in PE: EtOAc (10:2), the Rf was 0.40 and appeared purple in anisaldehyde spray reagent. The IR spectrum revealed absorption bands of hydroxyl (3280.90 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), carbonyl (1724.65 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and olefinic C\u0026thinsp;=\u0026thinsp;C stretch (1687.91 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (\u003cb\u003eSupplementary information Figures S8, S9\u003c/b\u003e) data were similar with those of BZA 01, indicating structural similarities. The significant difference was an additional quaternary carbon peak, δ\u003csub\u003eC\u003c/sub\u003e 171.8 (C-21) in BZA O2 due to an ester carbonyl peak at δ\u003csub\u003eC\u003c/sub\u003e 171.8 (C21) and the acetyl carbon at δ\u003csub\u003eC\u003c/sub\u003e 21.7 (C-22). The position of the ester group at C-19 was supported by the HMBC correlations of H-20a and H-20b to C-16, C-18 and C-19, suggesting that BZA 02 is xylopic acid, also previously isolated from \u003cem\u003eX. aethiopica\u003c/em\u003e. \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eX-ray crystallography data gave an empirical formula of C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and M\u0026thinsp;=\u0026thinsp;360.47 g/mol. BZA 02 came out as an orthorhombic shaped crystal with space group P2\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e, a\u0026thinsp;=\u0026thinsp;11.0960(5) \u0026Aring;, b\u0026thinsp;=\u0026thinsp;11.8948(7) \u0026Aring;, c\u0026thinsp;=\u0026thinsp;14.9745(8) \u0026Aring;, V\u0026thinsp;=\u0026thinsp;1976.40(18) \u0026Aring;3, Z\u0026thinsp;=\u0026thinsp;4, T\u0026thinsp;=\u0026thinsp;296.15 K, \u0026micro;(MoKα)\u0026thinsp;=\u0026thinsp;0.082 mm-1, Dcalc\u0026thinsp;=\u0026thinsp;1.211 g/cm3, 20732 reflections measured (5.02\u0026deg; \u0026le; 2Θ\u0026thinsp;\u0026le;\u0026thinsp;53.028\u0026deg;), 4083 unique (Rint\u0026thinsp;=\u0026thinsp;0.0979, Rsigma\u0026thinsp;=\u0026thinsp;0.0772) as well as a final R1 was 0.0460 (I\u0026thinsp;\u0026gt;\u0026thinsp;2σ(I)) and wR2 was 0.0980 which were used to calculate and confirm the structure of BZA 02 as xylopic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eBZ 05\u003c/h2\u003e\u003cp\u003eBZA 05 was isolated as a fine green powder and exhibited IR and NMR spectra closely resembling those of BZA 01. However, notable differences included a characteristic IR absorption band at 1724.36 cm⁻\u0026sup1;, indicative of a ketone, and a significant downfield shift in the \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectrum for carbon C-15 \u0026mdash; from δ\u003csub\u003eC\u003c/sub\u003e 49.1 in BZA 01 to δ\u003csub\u003eC\u003c/sub\u003e 210.5 in BZA 05 \u0026mdash; consistent with ketone functionality (\u003cb\u003eSupplementary Information Figure S10\u003c/b\u003e). HMBC correlations between C-15 and H-20a, H-20b and H-14 were supportive of the assignment. The proposed structure was corroborated with the MS data analysis which exhibited an M\u0026thinsp;+\u0026thinsp;1 peak at m/z 317.21 accompanied with fragments at m/z 299.20 and 271.2 (\u003cb\u003eSupplementary Information Figure S11\u003c/b\u003e). Hence, the structure BZA 05 was deduced as ent-kaur-16-en-15-one-19-oic acid, with a molar mass of 316 g/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eBL 2 and BL 4a\u003c/h2\u003e\u003cp\u003e\u003cb\u003eCompound BL 2\u003c/b\u003e was isolated as white powder. Its IR spectrum exhibited characteristic absorption bands for C\u0026ndash;H stretching at 2914.8 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2848.4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a carbonyl group at 1733.8 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and an olefinic C\u0026thinsp;=\u0026thinsp;C stretch at 1683.9 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In the \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectrum (\u003cb\u003eSupplementary Information Figure S12)\u003c/b\u003e, signals corresponding to carbonyl carbons were observed at δ\u003csub\u003eC\u003c/sub\u003e 195.0 (aldehyde) and δ\u003csub\u003eC\u003c/sub\u003e 173.7 (ester). Olefinic carbons resonated at δ\u003csub\u003eC\u003c/sub\u003e 155.2, 143.6, 134.9, and 124.1. An oxygenated carbon appeared at δ\u003csub\u003eC\u003c/sub\u003e 64.1, while the remaining signals, ranging from δ\u003csub\u003eC\u003c/sub\u003e 39.3 to δ\u003csub\u003eC\u003c/sub\u003e 14.0, indicated the presence of a long alkyl chain. This was further supported by a series of multiplets in the upfield region of the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectrum (δ\u003csub\u003eH\u003c/sub\u003e 2.34\u0026ndash;0.88). An aldehydic proton was observed at δ\u003csub\u003eH\u003c/sub\u003e 9.36, and signals at δ\u003csub\u003eH\u003c/sub\u003e 6.44 and 5.12 were attributed to olefinic protons (\u003cb\u003eSupplementary Information Figure S13)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCompound BL 4\u003c/b\u003e, a yellow gum, showed IR absorption bands at 2916.4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2849 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026ndash;H stretching) and a carbonyl absorption at 1702.4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectrum (\u003cb\u003eSupplementary Information Figure S14)\u003c/b\u003e displayed a carbonyl resonance at δ\u003csub\u003eC\u003c/sub\u003e 180.5, with additional signals between δ\u003csub\u003eC\u003c/sub\u003e 34.5 and 14.5, while the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectrum (\u003cb\u003eSupplementary Information Figure S15)\u003c/b\u003e showed multiplets in the δ\u003csub\u003eH\u003c/sub\u003e 2.34\u0026ndash;0.88 range. These spectral features are consistent with the structure of a fatty acid.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMolecular Modelling\u003c/h2\u003e\u003cp\u003eTo validate the results of the \u003cem\u003ein vitro\u003c/em\u003e antitrypanosomal screening, \u003cem\u003ein silico\u003c/em\u003e analysis was conducted to provide further understanding of the biological mechanisms involved. Physicochemical descriptors such as MW (g/mol), log \u003cem\u003eP\u003c/em\u003e, HD, HA. RB, Lipinski, Ghose and Veber of the isolated compounds - bis(4-methylheptyl) phthalate, ent-kaur-16-en-19-oic acid, xylopic acid and ent-kaur-16-en-15-one-19-oic acid were determined as shown in (\u003cb\u003eSupplementary information Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) to predict their individual ADME parameters. HA, HD, RB, log \u003cem\u003eP\u003c/em\u003e and Lipinski values were within the drug-like chemical space. Likewise, MW was within the Known Drug Space (\u003cb\u003eSupplementary information Table S2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eCo-crystallized ligands, including ivermectin (targeting glutathione S-transferase) and 5-hexyl glutathione (targeting the glutamate-gated chloride channel) for onchocerciasis, as well as pyridoxal-5-phosphate and doxycycline (targeting ornithine decarboxylase) for trypanosomiasis, were used as references to predict their functional scores against the respective target sites. Subsequently, the compounds were docked, together with and a wide range of compounds reported in the literature to have been isolated from \u003cem\u003eX. aethiopica\u003c/em\u003e (\u003cb\u003eSupplementary information Table S4\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eModelling against glutathione S-transferase (Onchocerciasis)\u003c/h2\u003e\u003cp\u003eThe co-crystallized ligand of glutathione S-transferase (\u003cb\u003eSupplementary Information Figure S16\u003c/b\u003e) was first docked to generate root mean square deviation (RMSD) values for the heavy atoms. ASP obtained an average RMSD of 2.1366, PLP\u0026thinsp;=\u0026thinsp;5.1415, CS\u0026thinsp;=\u0026thinsp;1.6224, and GS\u0026thinsp;=\u0026thinsp;1.9383. CS and GS showed stronger prediction power of scoring functions against the target (\u003cb\u003eSupplementary information Table S3\u003c/b\u003e). The modeling showed that the compounds occupy the hydrophobic binding pockets with a plausible binding mode. In comparison to the standard drug ivermectin, the binding affinity score for bis(4-methylheptyl) phthalate was remarkably high. Its carbonyl group formed a hydrogen bond with the side chain lysine 35 amino acid, and it also exhibited a π-π interaction with phenylalanine 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Ivermectin exhibited a similar binding mode. In contrast, the kaurene diterpenoids ent-kaur-16-en-19-oic acid, xylopic acid and ent-kaur-16-en-15-one-19-oic acid were inactive.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eModelling against glutamate-gated chloride channel (Onchocerciasis)\u003c/h2\u003e\u003cp\u003eFirstly, the co-crystallized ligand of the glutamate-gated chloride channel (\u003cb\u003eSupplementary information Figure S17)\u003c/b\u003e was docked to generate average RMSD values for the heavy atoms as follows: ASP\u0026thinsp;=\u0026thinsp;3.4741, PLP\u0026thinsp;=\u0026thinsp;1.4928, CS\u0026thinsp;=\u0026thinsp;1.4232, and GS\u0026thinsp;=\u0026thinsp;1.7654, demonstrating stronger predictive power of the PLP and CS scoring functions against the target site (\u003cb\u003eSupplementary information Table S5\u003c/b\u003e). The compounds occupied the hydrophilic binding pockets as a plausible binding mode, with 5-hexyl glutathione, the co-crystallized ligand, exhibiting the highest score, closely followed by bis(4-methylheptyl) phthalate (\u003cb\u003eSupplementary information Table S6)\u003c/b\u003e. Binding revealed hydrogen bond interactions between their respective carbonyl groups and the side chain hydroxyl groups of threonine, glutamine, and serine amino acids, with a typical illustration with asparagine amino acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eModelling against Ornithine Decarboxylase (Trypanosomiasis)\u003c/h2\u003e\u003cp\u003eThe co-crystallized ligands, pyridoxal-5-phosphate and doxycycline were initially docked with Ornithine Decarboxylase (\u003cb\u003eSupplementary information Figure S18\u003c/b\u003e) to generate the RMSD values for the heavy atoms. ASP obtained an average RMSD of 0.4776, PLP\u0026thinsp;=\u0026thinsp;1.5958, CS\u0026thinsp;=\u0026thinsp;6.2696 and GS\u0026thinsp;=\u0026thinsp;0.6362. In this case, GS and ASP exhibited a very strong prediction power of scoring functions as compared to PLP and CS against the target (\u003cb\u003eSupplementary information Table S7)\u003c/b\u003e. The modeled compounds again occupied the hydrophilic binding pockets with a plausible binding mode. As observed previously, bis(4-methylheptyl) phthalate demonstrated exceptionally high binding affinity with respect to the reference compounds, pyridoxal-5-phosphate and doxycycline \u003cb\u003e(Supplementary information Table S8\u003c/b\u003e). It showed hydrogen bonding with the amine side chain of arginine while the phenyl moiety formed a π-π stack interaction with the imidazole side chain histidine (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study provides valuable insights into the pharmacological potential and safety considerations of the Ghanaian herbal formulation NTD-O2, traditionally used for the treatment of neglected tropical diseases. \u003cem\u003eIn vitro\u003c/em\u003e assays revealed that NTD-O2 possesses significant antitrypanosomal activity, with the isolated compound bis(4-methylheptyl) phthalate demonstrating potent efficacy against \u003cem\u003eT. brucei\u003c/em\u003e. This activity was further supported by \u003cem\u003ein silico\u003c/em\u003e molecular docking studies, which suggested favourable interactions with key parasitic targets, indicating a plausible mechanism of action.\u003c/p\u003e\u003cp\u003eHowever, the identification of bis(4-methylheptyl) phthalate\u0026mdash;a compound widely recognized as a synthetic plasticizer and environmental contaminant\u0026mdash;raises important concerns regarding the authenticity and safety of the observed bioactivity. The absence of comparable activity in the individual plant constituents, \u003cem\u003eXylopia aethiopica\u003c/em\u003e and \u003cem\u003eBambusa vulgaris\u003c/em\u003e, suggests that the therapeutic effects may not be attributable to the herbal components themselves but rather to external contamination introduced during processing or packaging. This finding underscores the critical need for stringent quality control measures in the preparation and evaluation of herbal medicines.\u003c/p\u003e\u003cp\u003eWhile the observed antitrypanosomal activity is promising, the potential toxicological implications of phthalate exposure cannot be overlooked. These results highlight the dual necessity of validating the efficacy of traditional remedies while ensuring their safety through comprehensive chemical profiling and contamination screening. Future research should focus on optimizing the efficacy and safety profiles of promising compounds and exploring their potential in combination therapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAnimal African trypanosomiasis AAT\u003c/p\u003e\n\u003cp\u003eAstex statistical potential ASP\u003c/p\u003e\n\u003cp\u003eChemScore CS\u003c/p\u003e\n\u003cp\u003eDiminazene aceturate DA\u003c/p\u003e\n\u003cp\u003eGhana Federation of Traditional Medicines Practitioners Association GHAFTRAM\u003c/p\u003e\n\u003cp\u003eGhana Food and Drugs Authority FDA\u003c/p\u003e\n\u003cp\u003eGlutamate-gated chloride channel GluCl \u003c/p\u003e\n\u003cp\u003eGoldScore GS\u003c/p\u003e\n\u003cp\u003eHerbal medicine HM\u003c/p\u003e\n\u003cp\u003eHuman gastrointestinal absorption HIA\u003c/p\u003e\n\u003cp\u003eNeglected tropical diseases NTDs\u003c/p\u003e\n\u003cp\u003ePharmacodynamics PD \u003c/p\u003e\n\u003cp\u003ePharmacokinetics PK\u003c/p\u003e\n\u003cp\u003eProtein Data Bank PDB\u003c/p\u003e\n\u003cp\u003eRoot mean square deviation RMSD \u003c/p\u003e\n\u003cp\u003eSelective index SI\u003c/p\u003e\n\u003cp\u003eWorld Health Organization WHO\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The online version contains supplementary material available at\u0026hellip;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to the Department of Chemistry for NMR and X-ray data acquisition and New Zealand eScience Infrastructure (NeSI) high-performance computing facilities as part of this research funded jointly by the collaborating institutions and through the Ministry of Business, Innovation and Employment Research Infrastructure program. URL: https://www.nesi.org.nz. The \u003cem\u003eTrypanosoma brucei brucei\u003c/em\u003e cell line (GUTat 3.1 strain) was originally obtained from the Department of Parasitology, Noguchi Memorial Institute for Medical Research, University of Ghana.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article is included within the article and its additional file.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Worldwide Universities Network Research Development Fund 2017 from the Worldwide Universities Network (UK) and grant number 18-191 RG/CHE/AF/AC_G - FR3240303659 from The World Academy of Sciences.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBZA\u003c/strong\u003e: Data acquisition and analysis; Writing \u0026ndash; original draft, revision and final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEC\u003c/strong\u003e: Data acquisition and analysis; Writing \u0026ndash; revision and final approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDM\u003c/strong\u003e: Supervision, Data acquisition and analysis; Writing \u0026ndash;revision and final approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTMG\u003c/strong\u003e: Supervision, Data acquisition and analysis; Writing \u0026ndash;revision and final approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDOS\u003c/strong\u003e: Funding acquisition, Conceptualization, Supervision, Data analysis. Writing \u0026ndash; original draft, revision and final approval\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization (WHO), Traditional Medicine \u003cem\u003eEB124.R9 \u003c/em\u003e2009.\u003c/li\u003e\n\u003cli\u003eBodeker G, Kronenberg F. A public health agenda for traditional, complementary, and alternative medicine. Am J Public Health. 2002;92(10):1582\u0026ndash;91 \u003c/li\u003e\n\u003cli\u003eErnst E. Prevalence of use of complementary/alternative medicine: a systematic review. \u003cem\u003eBull World Health Organ\u003c/em\u003e. 2000;78(2):252\u0026ndash;7 \u003c/li\u003e\n\u003cli\u003eNissen N. Practitioners of Western herbal medicine and their practice in the UK: beginning to sketch the profession. \u003cem\u003eComplement Ther Clin Pract\u003c/em\u003e. 2010;16(4):181\u0026ndash;6 \u003c/li\u003e\n\u003cli\u003eFlynn MAT, Maloff DA, Maloff B, Mutasingwa D, Wu M, Ford C, et al. 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Docking and scoring in virtual screening for drug discovery: methods and applications. \u003cem\u003eNat Rev Drug Discov\u003c/em\u003e. 2004;3(11):935\u0026ndash;49.\u003c/li\u003e\n\u003cli\u003eJones G, Willett P, Glen RC, Leach AR, Taylor R. Development and validation of a genetic algorithm for flexible docking. \u003cem\u003eMol Biol\u003c/em\u003e. 1997;267:22.\u003c/li\u003e\n\u003cli\u003eEldridge MD, Murray CW, Auton TR, Paolini GV, Mee RP. Empirical scoring functions: The development of a scoring function to estimate the binding affinity of ligands in receptor complexes. \u003cem\u003eJ Comput Aided Mol Des\u003c/em\u003e. 1997;11:21.\u003c/li\u003e\n\u003cli\u003eMiller PS, Smart TG. Binding, activation and modulation of Cys-loop receptors. \u003cem\u003eTrends Pharmacol Sci\u003c/em\u003e. 2010;31(4):161\u0026ndash;74.\u003c/li\u003e\n\u003cli\u003eKorb O, St\u0026uuml;tzle T, Exner TE. Empirical scoring functions for advanced protein-ligand docking with PLANTS. \u003cem\u003eJ Chem Inf Model\u003c/em\u003e. 2009;49(1):84\u0026ndash;96.\u003c/li\u003e\n\u003cli\u003eMooij WT, Verdonk ML. General and targeted statistical potentials for protein-ligand interactions. \u003cem\u003eProteins\u003c/em\u003e. 2005;61(2):272\u0026ndash;87.\u003c/li\u003e\n\u003cli\u003eYasmin F, Nazli Z-i-H, Shafiq N, Aslam M, Bin Jardan YA, Nafidi H-A, Bourhia M. Plant-based bioactive phthalates derived from \u003cem\u003eHibiscus rosa-sinensis\u003c/em\u003e: As in vitro and in silico enzyme inhibition. \u003cem\u003eACS Omega\u003c/em\u003e. 2023;8(36):32677\u0026ndash;89. https://doi.org/10.1021/acsomega.3c03342\u003c/li\u003e\n\u003cli\u003eOmidpanah S, Saeidnia S, Saeedi M, Hadjiakhondi A, Manayi A. Phthalate contamination of some plants and herbal products. \u003cem\u003eBol Latinoam Caribe Plant Med Aromat\u003c/em\u003e. 2018;17(1):61\u0026ndash;7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Herbal medicine, safety, Xylopia aethiopica, Bambusa vulgaris, Onchocerciasis, Animal Trypanosomiasis, phthalate","lastPublishedDoi":"10.21203/rs.3.rs-7189198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7189198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThis study investigated the anti-onchocercal and antitrypanosomal properties of a Ghanaian herbal medicine (NTD-O2) and its medicinal plant constituents, \u003cem\u003eXylopia aethiopica\u003c/em\u003e fruits and \u003cem\u003eBambusa vulgaris\u003c/em\u003e leaves, with the aim of addressing the therapeutic potential and safety of a herbal medicine against Onchocerciasis and Animal African Trypanosomiasis in Ghana.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eExtracts from NTD-O2 and the medicinal plants were tested against \u003cem\u003eOnchocerca ochengi\u003c/em\u003e and \u003cem\u003eTrypanosoma brucei brucei in vitro\u003c/em\u003e. Bioassay-guided fractionation, spectroscopic and spectrometric techniques identified bioactive compounds, while \u003cem\u003ein silico\u003c/em\u003e methods explored their possible mechanisms of action.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eNTD-O2 extracts achieved 100% inhibition of adult male \u003cem\u003eO. ochengi\u003c/em\u003e worm motility, with moderate activity against adult female worms. Additionally, the extracts demonstrated promising antitrypanosomal activity (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.44 \u0026micro;g/mL and 10.68 \u0026micro;g/mL) against the positive control, diminazene aceturate (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g/mL). The active compound found in the NTD-O2 extract was bis(4-methylheptyl) phthalate. On the contrary, the compounds isolated from \u003cem\u003eX. aethiopica\u003c/em\u003e \u0026ndash; ent-kaur-16-en-19-oic acid, xylopic acid, and ent-kaur-16-en-15-one-19-oic acid \u0026ndash; and the long chain carbonyl compounds from \u003cem\u003eB. vulgaris\u003c/em\u003e were inactive (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u0026micro;g/mL). These results were corroborated by \u003cem\u003ein silico\u003c/em\u003e analysis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe findings highlight significant variability in the chemical composition and bioactivity of the herbal medicine NTD-O2 and its plant constituents. Given the health risks linked to the ingestion of phthalate derivatives, it is essential to conduct regular assessments of the quality and safety of herbal medicines to ensure consumer protection.\u003c/p\u003e","manuscriptTitle":"In Vitro and In Silico Evaluation of the Efficacy and Safety of a Ghanaian Herbal Medicine for the Treatment of Onchocerciasis and African Trypanosomiasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 17:05:04","doi":"10.21203/rs.3.rs-7189198/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-11T13:24:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-10T12:29:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-02T14:02:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-27T03:55:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-23T14:08:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305907937753061951549864706323239823840","date":"2025-08-23T12:01:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201574243326842270952753630172903520340","date":"2025-08-18T06:01:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76456253564338176504020815476655946661","date":"2025-08-18T04:36:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152679833509574371863406471255468334024","date":"2025-08-18T04:17:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-18T02:52:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-15T14:31:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-15T14:28:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-06T17:56:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2025-08-06T17:52:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d51d9d0-9ed2-4584-a9f0-b348008bc3b4","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T23:08:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-25 17:05:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7189198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7189198","identity":"rs-7189198","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}