1. Artemisia afra extraction:
1.1 Plant preparation:
Burundi at Rumonge South Burundi. The leaves were harvested before blooming and dried under shade before being sent to Nairobi in paper bags, then kept at room temperature until extraction.
1.2 Extraction Process
Samples of dried and powdered aerial part of A. afra weighing each 125 gm were extracted with 600 mL of Ethanol (100%), Hexane (85%), DCM (99.9%) and in water (800mL) in a flat bottomed flask and mixed on an orbital shaker. After gentle maceration for 48 hours, the extracts were filtered through Whatman filter paper n°1. The filtrate was concentrated under reduced pressure using a rotary evaporator at 20 rpm and 40°C bath temperature. Finally, concentrated extracts were collected in vials and placed in a water bath at 40°C to evaporate the remaining solvents and stored at room temperature for complete dryness.
2. Gene expression study for Fab_Z and Fab_I from P. falciparum after the exposition of the parasites to the crude extracts.
2.1Culture preparation and incubation
Cultures of Plasmodium falciparum (W2 and D6) with a parasitaemia of 4% were incubated with Artemisia afra extracts collected from Burundi, e.g.: Burundi ethanolic, hexane and dichloromethane extract to run an inhibition test.
Artemisia afra solution was made for all the extracts. 100 mg of extracts were dissolved in 200ul of DMS; double distilled water was used to dilute the extracts. 20ml of double distilled water was added to the final concentration of 5ug/ul; then the solution was sterilized by filtering it through a microfilter of 0.45um pore size.
- 6ml of W2 cultures were incubated with 666ul of A. afra extracts (DCM, ETOH, and Hexane plant extracts) at a final concentration of 0.5µg/µl
- 6ml of W2 culture as negative control incubated with 666ul CMS with DCMS (final DMSO concentration was 0.1%)
- 6ml of D6 cultures were incubated with 666ul of A. afra extracts (DCM, ETOH, and Hexane plant extracts) at a final concentration of 0.5µg/µl
6ml of D6 culture as negative control were incubated with 666ul CMS and DMSO (final concentration of DMSO was 0.1%).
The parasites were then kept for 2 days in the incubation room. After the inhibition test was done, the parasite was kept at -80°C. The samples were used later for RNA extraction to run gene expression of FAB_Z and FAB_I in each sample.
2.2 RNA extraction:
After the inhibition test was done, the total RNA for all samples (W2 and D6) was extracted by following the method of trizol reagent Invitrogen Company (http://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf).
The parasites were thawed in ice, the whole blood was centrifuged, and the supernatant was discarded. The pellet was lysed using 1ml Trizol, then Incubated at 37°C for 5 min to ensure the complete deproteinization of nucleic acids. 200ul of Chloroform was added, the tube was shaken vigorously by hand followed or using a vortex then centrifuged for 15 min at 12,000g at 4oC. The upper aqueous phase was carefully removed and placed in a new tube. Isopropanol was added (to precipitate the RNA) then mixed and centrifuged at 12,000g at 4oC during 15mn. The supernatant was discarded and the pellet was suspended with 1ml of 75% ethanol then vortex briefly and centrifuge for 10mn at 7,500 g 4°C. The supernatant was discarded and the pellet air-dried for 10mn. The RNA was resuspended with 40 ul of RNAs-free water and incubated in a heat block for 10 minutes at 60°C. The quantity and the quality of the RNA were determined respectively using Nano_Drop. The purity of the RNA sample was defined by the A260/A280 ratio. A ratio between 1.8 and 2.1 was indicative of highly purified RNA. The concentration of the extracted RNA was determined using the following equation:
RNA concentration (μg/μl) = (A260 * 40 * D)/1,000 where D = dilution factor
2.3 cDNA synthesis
The RNA samples were normalized by adding RNA free water to get a concentration of 50ug/ul of RNA for all samples; then the RNA was converted into cDNA by reverse transcription (RT). The oligo (DTs) primers were used for the reverse transcription. cDNA synthesis kit from Solis BioDyne was used for the reaction. The samples were incubated in a PCR machine at 50°C for 45 mn to allow the reverse transcription reaction to take place and to be complete, then in 85°C during 5min to inactivate the enzyme and stop the reaction. The cDNA samples were then kept at -20°C awaiting further analysis.
2.4 FAB_Z FAB_I primers
Primers for FAB_Z and Fab_I were created using Prime 3 (http://bioinfo.ut.ee/primer3-0.4.0/) and ordered online. The primers were designed to avoid hairpins and self-annealing with a GC content around 40% - 50% and an annealing temperature of 60°C.
A stock solution of 100uM was prepared for each primer using PCR water buffer. 10ul from the stock solution was added to 90 ul of PCR water to prepare a working solution of 10uM.
OLIGO start lenght tm gc% any_th 3'_th hairpin seq
LEFT PRIMER 511 20 58.93 50.00 0.00 0.00 0.00 TTTGCTGGAGTGGATGGAGT
RIGHT PRIMER 687 24 59.91 41.67 0.00 0.00 0.00 CGATAAGGCAAACGTCATTTCTGA
Product size: 177 bp
OLIGO start lenght tm gc% any_th 3'_th hairpin seq
LEFT PRIMER 333 22 60.36 50.00 0.00 0.00 0.00 CGGGTGGGGTATTGCTAAAGAA
RIGHT PRIMER 510 20 59.31 50.00 1.61 0.00 0.00 AGAAGCGTCAAAGGGTAGCA
PRODUCT SIZE: 178 bp
2.5 Conventional PCR for FAB_I and FAB_Z
The designed primers were tested during the conventional PCR, and their parameters were set up. 5x FIREpol Master Mix ready to load with syber green dye was used for the conventional PCR. 4ul of a master mix containing DNA polymerase, 5x reaction buffer, 12.5 mM Mgcl2, 1mM dNTPs, blue and yellow dye added into labelled PCR tubes 1ul of primers solutions were added
cDNA samples were thawed and 2ul were added in each PCR tube and topped up with water till 20 ul of the final volume. The sample was placed into the PCR thermocycler, and the run was set with different parameters until we find the right one that amplify better the genes. The following amplification program was finally used for the two primers (FAB_Z and FAB_I): Initial denaturation at 95°C for 5min and then 40 cycles of denaturation at 95°C for 1 min, annealing 60°C for 1min, extension 72°C for 1min, followed by the final extension at 72°C for 10min, and then held at 4°C. After gel electrophoresis was made to make sure that the primers got correctly amplified with those parameters set up.
2.6 Housekeeping gene for P. falciparum
To run gene expression of Fab enzymes: Fab_Z (Beta-hydroxyacyl-Acyl-carrier Protein Dehydratase) and Fab _I (Enoyl Acyl-Carrier-Protein Reductase) we needed a housekeeping gene as a reference which is necessary to calculate the expression level of the other genes during real-time PCR. In our study, Actin was chosen as the housekeeping gene for Plasmodium falciparum. Primers were made to amplify the actin gene during real-time PCR. Prime 3 was used to make the primers and parameters were set to fit the same parameters as for Fab_z and Fab _I.
A conventional PCR was run first to test the primers chosen for actin as amplified. The following parameters were used: Initial denaturation at 95°C for 5min and then 40 cycles of denaturation at 95°C for 1 min, annealing 60°C for 1min, extension 72°C for 1min, followed by the final extension at 72°C for 10min, and then held at 4°C. After gel electrophoresis was made to make sure that the primers got correctly amplify
2.7 Gel electrophoresis for PCR products
After running the conventional PCR for the genes and the housekeeping gene, a gel was prepared for the PCR product. Agarose gel (2%) in 2x TBE buffer was prepared. Ethidium bromide was included in the gel. Eight μl of each amplification reaction was loaded onto the gel. The gel was visualized on a UV trans-illuminator and photograph gel.
2.8 Real-time PCR
After a successful amplification for all the genes (Fab_I and Fab_Z) and the housekeeping gene (Actin), a Real-time PCR was run to study the expression of the genes Fab_I and Fab_Z when exposed to our active extracts Artemisia afra. 5x HOT FIREPol EvaGreen qPCR mix plus (No Rox) from Solis BioDyne was used for the quantitative real-time PCR. The qPCR master mix composition was Hot FIREpol DNA polymerase, 5x Evagreen qPCR buffer, 12.5mM Mgcl2, dNTPs, Evagreen dye, No ROX dye. 96 wells plate for real-time PCR were used to set the reaction (Figure 3.1). In each well, 10 μl of total volume solution were prepared each containing 2μl of master mix for qPCR, 0.5 μl for the reverse primers, 0.5μl for the forward primers, 2ul for cDNA and 5μl of water. Each sample was tested in triplicate. Light Cycler 96 software was used to set the parameters for Real-time qPCR and to visualize the results
3. Virtual screening with Artemisia afra compounds found during GCMS and the Fab enzymes
3.1 Gas Chromatography-Mass Spectrometry (GCMS) Analysis of Artemisia afra extracts
Artemisia afra from Burundi was collected and extracted with three solvents: Dichloromethane, Ethanol, and Hexane. The extracts were dried using a rotary evaporator and kept in the oven at 37C until complete evaporation of the solvents. Pure artemisinin crystal was used as a standard to test any presence of the compound in Artemisia afra extracts.
3.1.2 Sample Preparation
Samples were dissolved and diluted in suitable organic solvents i.e. dichloromethane and ethanol extracts were dissolved in methanol solvent, while hexane extract was dissolved in hexane solvent) and passed through carbon black to remove waxes and chlorophylls. The samples were then filtered through 0.45 µm PTFE filters then transferred to sample vials for GCMS analysis.
3.1.3 GCMS Method
A Shimadzu QP 2010-SE GCMS coupled to an auto sampler was used for the analysis. Ultrapure He (99.999 %) was used as the carrier gas at a flow rate of 1ml / minute. A BPX5 non-polar capillary column, 30m; 0.25 mm ID; 0.25 µm film thickness, was used for separation. The GC was programmed as follows: 60 ˚C; 10 ˚C /min to 250 ˚C (10 minutes). The total run-time was 30 minutes. Only 1 µL of the sample was injected. The injection was done in split mode, 10:1. The injection was done at 200 C. The interface temperature was set at 250 C. The EI ion source was set at 200 C. Mass analysis was done in full scan mode, 50-550 m/z. A solvent delay time of 2 minutes was used.
3.2 Drug screening with PyRx
After GCMS a library of compounds was prepared based on the GCMS results of Artemisia afra extracts from Burundi. The library was screened against two macromolecules (FAB_I FAB_Z) using Autodock Vina in PyRx 0.8 version (Troot et al., 2010). PyRx is a Virtual Screening software for Computational Drug Discovery that can be used to screen libraries of compounds against potential drug targets. The software is open access and is available online at http://pyrx.sourceforge.net
3.2.1 Preparation of the library of small molecules
The 3D structures of all the compounds from GCMS were searched from three chemical structure databases and downloaded. Three databases were used: ChemSpider (http://www.chemspider.com/), PubChem from NCBI (https://pubchem.ncbi.nlm.nih.gov/search/), and ChEMBL (https://www.ebi.ac.uk/chembl/) from the European Bioinformatics Institute (EBI). The small molecules (Compounds) were downloaded and visualized with Pymol then save in the same file in PDB format.
3.2.2 3D structure of Plasmodium falciparum Fab enzymes: Fab Z and Fab I
The 3D structure of the Fab enzymes: Fab_Z and Fab_I were downloaded from the RCSB protein database (PDB), (https://www.rcsb.org/). Fab_Z (PDB entry: 3AZA), (Maity et al., 2011); Fab_I (PDB entry: 3LT0) (Maity et al., 2010). The macromolecules were crystallized with their ligands. The macromolecules were open in txt format with notepad then all the ligands were removed to free the interaction sites.
3.2.3 Virtual screening with PyRx: Protocol
PyRx has the two virtual screening software Autodock 4.0 and Autodock vina. Autodock Vina was used during the drug screening because AutoDock Vina significantly improves the average accuracy of the binding mode predictions compared to AutoDock 4 and is faster (Troot et al., 2010).