Pharmacological assessment of the antiprotozoal activity, cytotoxicity and genotoxicity of medicinal plants used in the treatment of malaria in
4.9 Human S9 liver fraction treatment of extracts
Human S9 liver fraction was incorporated into the Salmonella reverse mutagenicity assay to also assess the human metabolic activation (or deactivation) of potentially mutagenic plant extracts. Pooled hepatic S9 fraction represents the post-mitochondrial supernatant fraction from homogenized human liver, and S9 fractions from the same batch only were used for the experiments (Lot# SLBR5681V,
153 S2442, Sigma Aldrich, always stored at í70°C). A sterile 0.1 M ß-nicotinamide adenine dinucleotide phosphate disodium salt (NADP, Sigma Aldrich) solution was freshly prepared and kept on ice. A 1 M glucose-6-phosphate solution and a MgCl2 (123 mg/mL) – KCl (81.4 mg/mL) salt solution were aseptically prepared. Subsequently, a 4% S9 mixture was freshly prepared by first mixing 69.125 mL of sterile H2O, 87.5 mL of sterile 0.2 M phosphate buffer (pH 7.4), 875 μL of glucose-6-phosphate solution, 3.5 mL of MgCl2 – KCl salt solution, and 7 mL of NADP solution, and then adding 7.0 mL of pooled human S9 liver fraction. The S9 mixture was kept on ice and quickly introduced to the following steps.
Sample extracts and controls were treated with the S9 mixture via a pre-incubation assay. A total of 50 μL of extract solution and 50 μL DMSO were added to 500 μL S9 mixture, vortexed, and incubated at 37°C with constant shaking at 100 rpm for exactly 30 min. The mutagenic substance 2-aminofluorene (2-AF, 1 mg/mL DMSO) was used as a positive control for both Salmonella strains.
After incubation and metabolic activation of samples, the samples were introduced to the Salmonella reverse mutagenicity assay by adding 2.0 mL TBA top agar and 100 μL of aliquot of the test strain suspension (overnight culture) as described in section 4.8. Each plate contained 20 μL human liver S9 fraction. Further information is given in Supplementary Table S4. Figure 2 was created using biorender.com software.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Author Contributions
F.S. wrote the manuscript. F.S. and L.P. designed the overall strategy of the study. F.S. and G.A.
collected and processed the plant material. G.A. prepared herbarium voucher specimens and taxonomically identified the plant species. F.S. performed the extraction procedures and created the extract library. F.S. and O.F.O. prepared the extract solutions for all assays and performed the heme biocrystallization inhibition assay. G.C. and L.P. performed the antiplasmodial and the cytotoxicity experiments. F.S., A.N. and O.F.O. conducted the genotoxicity experiments. F.S., O.F.O., A.N. and L.P. analyzed the data. J.S. contributed toward the background on P. hadiensis in Wakiso district.
L.A.G. provided oversight of lab work and directed the study. All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by two grants from the BMBF—German Federal Ministry of Education and Research (13FH026IX5, 13FH066PX5; PI: L.A.G. and Co-I: F.S.). We acknowledge support for the Article Processing Charge from the German Research Foundation (DFG, 414051096) and the Open Access Publication Fund of Neubrandenburg University of Applied Sciences (HSNB). The content is solely the responsibility of the authors and does not necessarily reflect the official views of the DFG, HSNB, or BMBF.
This is a provisional file, not the final typeset article 154 Acknowledgments
Greatest thanks to the Ugandan traditional healers in the Greater Mpigi region and neighboring regions who provided the ethnobotanical information that forms the foundation of this study and who provided guidance during the collection of plant materials. Thanks to research assistant Tidjani Cisse for assisting during the extractions of plant material. Thanks to student assistant Kristine Kossol for assistance in graphic design. The authors acknowledge An Matheeussen, Natascha Van Pelt and Pim-Bart Feijens for their excellent technical assistance with the in vitro biological evaluation. Thanks to Dr. Akram Salam for fruitful discussions and advice after drafting the manuscript.
Data Availability Statement
The data presented in this study are available in this article and in the Supplementary Material.
Ethnobotanical data are available in Schultz, F.; Anywar, G.; Wack, B.; Quave, C. L.; Garbe, L.-A., Ethnobotanical study of selected medicinal plants traditionally used in the rural Greater Mpigi region of Uganda. J. Ethnopharmacol. 2020, 256, 112742.
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