Expression of the Fab enzymes (Fab I and Fab Z) from Plasmodium falciparum after exposure to Artemisia afra plant extracts and drugs screening

The emergence and spread of drug resistance of the malaria parasite to the main treatment emphasize the need to develop new antimalarial drugs. In this context, the fatty acid biosynthesis (FAS_II) pathway of the malaria parasite is one of the ideal targets due to its crucial role in parasite survival. In this study, we report the expression and the affinity binding of Fab_I and Fab_Z after exposure to the parasite with different extracts of the Artemisia afra. The parasites were exposed for 2 days to different extracts. Gene expression was done to determine the level of expression of the fab enzymes after treatments. A GCMS was run to determine the different compounds of the plant extracts, followed by a virtual screening between the fab enzymes and the active compounds using Pyrex. The results showed different expression patterns of the Fab enzymes. Fab_I expression was downregulated in the W2 and D6 strains by the ethanolic extract but was increased by Hexane and DCM extracts. A different expression pattern was observed for Fab_Z. It was all upregulated except in the D6 strain when exposed to the ethanolic and hexane extracts. Virtual screening showed an affinity with many compounds. Hits compounds with high binding energy were detected. 11alphaHydroxyprogesterone and Aspidospermidin-17-ol were found to have high binding energy with Fab_I respectively (− 10.7 kcal/mol; − 10.2 kcal/mol). Fab_Z shows also high affinity with 11alpha-Hydroxyprogesterone (− 10 kcal/mol) and Thiourea (− 8.4 kcal/mol). This study shows the potential of A. afra to be used as a new source of novel antimalarial compounds.


Introduction
Malaria is still a big concern in the African health system due to its high number of death yearly. Africa is the most affected, where more than 90% of the death occur in those sub-Saharan countries. Today we are facing a big challenge concerning the spread of the resistance to main drugs wildly spread in South East Asia, a threat that can drive into Africa. New indigenous gene mutations of the Plasmodium falciparum parasite were discovered in Rwanda in 2020. The mutation concerns a new indigenous lineage, the pfkelch13 R561H, and can drive Artemisinin resistance (Uwimana et al. 2020). These raise the need to develop new drugs that can overcome that resistance. Researchers are increasingly focusing on using medicinal plants as a polytherapy that works better in overcoming parasite resistance. Artemisia afra is one of those promising medicinal plants with a high antimalarial effect and is not toxic in high dosage. This plant was growing wildly from the cape to the Eastern part of Africa and was used for decades by traditional healers. It is one of the best-known medicinal plants. The list of uses covers a wide range of ailments from coughs, colds, fever, loss of appetite, colic, headache, earache, and intestinal worms to malaria (Van Wyk et al. 1997;Gathirwa et al. 2007; Van der Kooy et al. 2008). In this project, different strains of malaria parasite W2 (CQ resistant) and D6 (CQ sensitive) were exposed to different extracts of Artemisia afra collected from Burundi, to monitor the expression level of different genes Fab I and Fab Z that belong to the Fab enzymes, type II pathway implied in the fatty acid biosynthesis of malaria's parasite. Those Fab enzymes are crucial for the parasite's survival and spreading mostly during the liver stage, and are targets for the development of new drugs candidate. Studying their expression when exposing the parasites to the plant extracts can bring a lot of information about using those genes as a target for new drugs.

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.

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 (800 mL) in a flat bottomed flask and mixed on an orbital shaker. After gentle maceration for 48 h, 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.

Culture 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 200 ul of DMS; double distilled water was used to dilute the extracts. 20 ml of double distilled water was added to the final concentration of 5 ug/ul; then the solution was sterilized by filtering it through a microfilter of 0.45 um pore size. The parasites were then kept for 2 days in the incubation room at 37 °C in a gas mixture, 3% CO2, 5% O2 and 92% N2. After the inhibition test, the parasite was kept at − 80 °C to conserve RNA integrity. The samples were used later for RNA extraction to run gene expression of FAB_Z and FAB_I in each sample.

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. therm ofish er. com/ conte nt/ sfs/ manua ls/ trizol_ reage nt. pdf).
The parasites were thawed in ice, the whole blood was centrifuged, and the supernatant was discarded. The pellet was lysed using 1 ml Trizol, then Incubated at 37 °C for 5 min to ensure the complete deproteinization of nucleic acids. 200 ul 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 4 °C. 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 4 °C for 15 mn. The supernatant was discarded and the pellet was suspended with 1 ml of 75% ethanol then vortex briefly and centrifuge for 10 mn at 7500g 4 °C. The supernatant was discarded and the pellet air-dried for 10 mn. The RNA was resuspended with 40 ul of RNAs-free water and incubated in a heat block for 10 min 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:

cDNA synthesis
The RNA samples were normalized by adding RNA free water to get a concentration of 50 ug/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 Bio-Dyne 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 at 85 °C for 5 min to inactivate the enzyme and stop the reaction. The cDNA samples were then kept at − 20 °C awaiting further analysis.

FAB_Z FAB_I primers
Primers for FAB_Z and Fab_I were created using Prime 3 (http:// bioin fo. ut. ee/ prime r3-0. 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 100 uM was prepared for each primer using PCR water buffer. 10 ul from the stock solution was added to 90 ul of PCR water to prepare a working solution of 10 uM.

Conventional PCR for FAB_I and FAB_Z
The designed primers were tested during the conventional PCR, and their parameters were set up. 5 × FIREpol Master Mix ready to load with syber green dye was used for the conventional PCR. 4 ul of a master mix containing DNA polymerase, 5 × reaction buffer, 12.5 mM Mgcl 2 , 1 mM dNTPs, blue and yellow dye added into labelled PCR tubes 1 ul of primers solutions were added.
cDNA samples were thawed, and 2 ul were added to 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 found the right one that amplified 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 5 min and then 40 cycles of denaturation at 95 °C for 1 min, annealing 60 °C for 1 min, extension 72 °C for 1 min, followed by the final extension at 72 °C for 10 min, and then held at 4 °C. After gel electrophoresis was made to ensure that the primers got correctly amplified with those parameters set up.

Housekeeping gene for P. falciparum
To run gene expression of Fab enzymes: Fab_Z (Betahydroxyacyl-Acyl-carrier Protein Dehydratase) and Fab _I (Enoyl Acyl-Carrier-Protein Reductase), we needed a housekeeping gene as a reference. Actin was chosen as the housekeeper. 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.

• Actin Primers:
Forward AGC AGC AGG AAT CCA CAC AA Reverse TGG TTG ATG GTG CAA GGG TT Product size: 164 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 5 min and then 40 cycles of denaturation at 95 °C for 1 min, annealing 60 °C for 1 min, extension 72 °C for 1 min, followed by the final extension at 72 °C for 10 min, and then held at 4 °C. After gel electrophoresis was made to make sure that the primers got correctly amplified.

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 2 × 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 photographed ( Fig. 1).

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. 5 × HOT FIREPol EvaGreen qPCR mix plus (No Rox) from Solis Bio-Dyne was used for the quantitative real-time PCR. The qPCR master mix composition was Hot FIREpol DNA polymerase, 5 × Evagreen qPCR buffer, 12.5 mM Mgcl 2 , dNTPs, Evagreen dye, No ROX dye. 96 wells plates for real-time PCR were used to set the reaction. In each well, 10 μl of total volume solution was prepared, each containing 2 μl of master mix for qPCR, 0.5 μl for the reverse primers, 0.5 μl for the forward primers, 2 ul for cDNA and 5 μl 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.

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 37 C 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.
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 filtered through 0.45 µm PTFE filters and then transferred to sample vials for GCMS analysis.
GCMS method A Shimadzu QP 2010-SE GCMS coupled with an auto-sampler was used for the analysis. Ultrapure He (99.999%) was used as the carrier gas at a flow rate of 1 ml/minute. A BPX5 non-polar capillary column, 30 m; 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 min). The total run-time was 30 min. 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 min was used.

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 (Trott and Olson 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 to access and is available online at http:// pyrx. sourc eforge. net 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. chems pider. com/), PubChem from NCBI (https:// pubch em. ncbi. nlm. nih. gov/ search/), and ChEMBL (https:// www. ebi. ac. uk/ chembl/) from the European Bioinformatics Insti- tute (EBI). The small molecules (Compounds) were downloaded, visualized with Pymol and then saved in the same file in PDB format.
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 opened in txt format with notepad then all the ligands were removed to free the interaction sites.
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 (Trott and Olson 2010).

Gel electrophoresis for PCR product
After parasite RNA extraction with trizol from the samples, we got different concentrations of RNA for each sample after measuring with a nanodrop. Before making the cDNA, RNA concentration was normalized to get an equal amount in all samples (experimental and controls). A conventional PCR was then run to determine the amplification parameters for the primers, the Fab enzyme genes and the housekeeping gene (Actin). A gel was run to visualize the amplicons (Fig. 1).
The results showed that the primers had well amplified the Fab enzymes genes (FabI and FabZ) and the housekeeping gene. Many parameters were set to get the best amplification. The amplification was optimum with these parameters: Initial denaturation at 95 °C for 5 min and then 40 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, extension at 72 °C for 1 min, followed by the final extension at 72 °C for 10 min, and then held at 4 °C.

Real-time amplification
The same parameter for the conventional PCR was reported to do the real-time PCR for the gene expression study. The target genes were well amplified as shown in Fig. 2 In blue colour, we have the amplification for the housekeeping gene (Actin), in yellow the amplification for Fab_I, and in red colour the amplification for Fab_Z. In grey is the amplification of the negative control and for the empty wells (drawn down of the picture). Actin has started to be amplified, followed by Fab_I, then by Fab_Z. Actin was well amplified. He had the best fluorescent curve of 5.7, followed by Fab_I, which was also well amplified with a fluorescent curve of 5.7. Fab Z fluorescent curve was 4.8. Still, the amplification was less compared to the other genes.

Gene expression for Fab I after exposition of the parasites to A. afra extracts
The fold change expression of Fab_I and Fab_Z was calculated from the Ct (cycle threshold is defined as the number of cycles required for the fluorescent signal to cross the threshold) values generated during the real-time PCR, and the result is shown in Tables 1 and 2 The fold change for the untreated sample is 1, for the treated samples when the fold change is greater than 1 it means that the targeted gene is upregulated and when it is lower than 1 it means that the targeted gene is downregulated. The Fab I gene from the W2 strain of P. falciparum was upregulated with the hexane extract and downregulated by the ethanolic and dichloromethane extracts of the plant. In the second train for D6, we have a different path; Fab I was upregulated by DCM extract and downregulated by hexane and ethanolic extracts. Fab_I appeared to be downregulated in the two strains by the ethanolic extract of Artemisia afra (see Table 1).
The logarithm 10 of the fold change for FabI in each treatment was calculated to make the histogram (Fig. 3). The histogram is centred in 0, the log tenfold change) of the untreated sample for D6 and W2. The up columns showed upregulation, and the down columns showed downregulation. Depending on the length of the column, we can see the level of expression on both sides when downregulated or upregulated. This figure shows Fab_I is upregulated in the hexane and DCM extract in the W2 and D6 strain. However, the upregulation appears to be three times higher in the D6 strain compared to the W2 strain. Fab I downregulation is higher when both strains expose the ethanolic extract.

Gene expression for Fab_Z after exposition to A. afra extracts
Gene expression was upregulated for Fab_Z in the W2 strain when exposed to the three different extracts. The expression was downregulated in the D6 strain, except for the DCM extract. The DCM extract upregulates Fab_Z in the two strains of P. falciparum.
The log10 of the fold difference was calculated (Fig. 4), and the histogram was made to show the expression of Fab_Z; this allowed a better view of the expression scheme for Fab_Z depending on the extracts used. The histogram is centred on 0. The up columns showed upregulation, and the down columns showed downregulation.
In this figure (Fig. 4), the downregulation of the enzyme Fab_Z by the ethanolic and the hexane extract in the D6 strain appear to be almost at the same level.

GCMS results
After the GCMS, the results in Table 3 showed that the ethanolic and dichloromethane extract had a high content of Bicyclo [2.2.1]heptan-2-ol, 1,7,7-trimethyl-, (1S-endo)-. The hexane extract had the same compounds but at a low amount compared to ethanolic and DCM extract. The major compound in the hexane extract is Eucalyptol (Area 55.52). This compound is also found in DCM extract at a lower amount (area 19.77) and in ethanolic extract of A. afra (5.59). Hexane extract has fewer compounds compared to the others.

Drug screening with PyRx
After GCMS of the ethanolic, hexane and dichloromethane extracts in total, 51 molecules were detected. The affinity binding of the molecules was tested against Fab_I and Fab_Z with vina. After drug screening, the binding energy for each molecule was determined and expressed in Kcal/mol see Table 4. The results showed that many compounds have a high affinity with the Fab enzymes. The results showed some hit compounds for Fab_I and Fab_Z with high biding energy. 11alpha-Hydroxyprogesterone has high binding energy with FabI (− 10.7 kcal/mol) and Fab Z (− 10 kcal/mol). The second hit is Aspidospermidin, which also has high binding energy with FabI (10.2 kcal/ mol). The third is Thiourea,-which has a binding energy of − 9 kcal/mol for FabI and − 8.4 kcal/mol for Fab Z.
The binding site for Fab_I with 11 alpha-hydroxyprogesterone (hit1) and with Aspidospermidin (hit 2) was shown respectively in Figs. 5 and 6. The 3D structure of Fab_I was downloaded from PDB (Protein Data Bank), and the active sites were determined before docking. After the virtual screening, the ligands were found to bind inside the active site and were in interaction with many residues in the active site. Residue

Discussion
Artemisia afra is a big antimalarial plant, and it is as potent as Artemisia annua, where the main antimalarial drug, artemisinin is extracted. A lot of studies show his antimalarial activities. In vitro studies run by Gathirwa et al. in 2007 about methanolic and water extracts of A. afra collected from Kenya show strong IC50 respectively for MEOH 9.04 µg/ml (for D6); 3.98 µg/ml (for W2) and for the water extract 11.23 µg/ml (D6); 4.65 µg/ml (W2), during that study the in vivo antimalarial assay display also a huge chemosupression which was greater than 70% (Gathirwa et al. 2007). More recent studies using different solvent (DCM, Water, Hexane, and EtOH) extracts of a. afra from Burundi had displayed also big in vitro and in vivo antimalarial activity against W2 and D6, with some extracts having an IC50 lesser than 3 µg/ml and an ED50 of 6.43 mg/ml (Kane et al. 2019a, b).
In this study, an inhibition test was run by incubating during 48 h the parasite cultures with A. afra extracts to study the expression level of the P. falciparum Fab enzymes (Fab_I and Fab_Z). The fab enzymes play an essential role in the fatty acid synthesis pathway (FAS II). The prodigious proliferative capacity of malarial parasites necessitates access to an abundant source of fatty acids (FAs). These carboxylic acid-linked acyl chains are required for the production of lipid species that are essential for parasite membrane and lipid body biogenesis (Palacpac et al. 2004). It has been shown that FAS II can be exploited for antimalarial drug discovery (Waller et al. 2003;Perozzo et al. 2002). In our results, Fab_Z (D6 strain) was downregulated by the ethanolic and hexane extract of A. afra. Fab_Z is the primary dehydratase that participates in the elongation cycle of saturated as well as unsaturated fatty acid biosynthesis (Sharma et al. 2003). Downregulation of Fab_Z can lead to parasite death due to its crucial role in the fatty acid de novo biosynthesis. Fab_I also play a crucial role during the liver stage of malaria infection (Yu et al. 2008). A downregulation of Fab_I can lead to a failure of the parasite to complete the liver stage by an inability to form intrahepatic merozoites that normally initiate blood-stage infection. The results showed a downregulation of Fab_I by the ethanolic and the DCM extracts of Artemisia afra for the W2 strain. In the D6 strain, the gene is also downregulated by the hexane and     -2-ol, 1,7,7-trimethyl-, (1S-endo)-9.173 30.12 3,7-Octadiene-2,6-diol, 2,6-dimethyl-9.227 0.69 1,7-Octadiene-3,6-diol, 2,6-dimethyl-10.573 1.11 Bornyl acetate 10.794 0.96 3-Cyclohexene-1-methanol, 2-hydroxy-.alpha.,.alpha.,4-trimethyl-11.932 1.5 ethanolic extracts; this indicates that Artemisia afra could act as a drug with a prophylactic effect that can be able to stop the parasite in the earlier liver stage before the spread into the bloodstream. A study run in 2022 by Ashraf et al. came to strengthen this statement, where an in vitro exposition of Artemisia afra tea extract to the pre-erythrocytic (hepatic) stages of Plasmodium species (P. berghei and P. falciparum) led to a significant dose-dependent reduction in the size and number of hepatic schizonts. Artemisia afra (4 g/l) completely cleared parasites from the cultures after 4 days of treatment for P. falciparum and after 2 days of treatment for P. berghei with no cytotoxic effect (Ashraf et al. 2022).

GCMS of Artemisia afra extracts
GCMS was run in all three extracts: ethanolic, hexane and dichloromethane of Artemisia afra leaves from Burundi. 10 compounds were found in hexane extract; the major compound was Eucalyptol (55.5%). Cineole or Eucalyptol has mucolytic, bronchodilating and anti-inflammatory properties. It reduces the exacerbation rate in patients suffering from COPD (chronic obstructive pulmonary disease) and ameliorates symptoms in patients suffering from asthma and rhinosinusitis (Fischer and Dethlefsen 2013). In the dichloromethane extract, 30 compounds were detected by GCMS, and the major compounds were Borneol (33.5%) and Eucalyptol (21.9%). Borneol is a common ingredient in many traditional Chinese herbal formulas and has many uses. It aids the digestive system by stimulating the production of gastric juices; tones the heart and improves circulation; treat bronchitis, coughs, and colds; can relieve pain caused by rheumatic diseases and sprains; reduces swelling; relieves stress; and can be used as a tonic to promote relaxation and reduce exhaustion (http:// acupu nctur etoday. com/ herbc entral/ borne ol. php). In the methanolic extract, the major compound was also Borneol (Bicyclo [2.2.1] heptan-2-ol, 1, 7, 7-trimethyl-, (1S-endo) (40.6%). The majority of the compounds found in the ethanolic were also found in the dichloromethane and some in the hexane extract. The same major compound was found by Mwangi et al. (1995) when analyzing the essential oil from Artemisia afra by GCMS. The major constituents were 1, 8-cineole (67.4%), terpinen-4-ol (6.5%) and borneol (5.1%). The medicinal effect attributed to that plant can be because of the presence of all those active compounds. The first line against malaria is artemisinin and derivatives extracted from Artemisia annua. Artemisinin was not detected in all three extracts of Artemisia afra, so this means that the antimalarial activity lay on the other actives compounds of that plant.

Affinity binding with FabI and Fab Z
A library was created based on the compounds found during GCMS and was screened against the two fab enzymes (Fab I and FabZ) to test virtually their potential interactions using Autodock Vina with PyRx. Autodock Vina is a docking software that aims to predict the ligand-protein complex structure interaction by exploring the conformational space of the ligands within the binding site of the protein. A scoring function is then utilized to approximate the free energy of binding between the protein and the ligand in each docking pose (Lionta et al. 2014).
After the virtual screening, all compounds were found to display a binding affinity that varies from high to low binding energies with the Fab enzymes, this may indicate Bold values indicate the most abundant compounds after GCMS  effects of Aspidospermidin-and Thiourea, 1-(adamantane-1-carbonyl)-3-cyclohexyl. 11alpha-Hydroxyprogesterone has recently been patented for treating skin diseases, particularly psoriasis. It is an important pharmaceutical compound with anti-androgenic and blood-pressure-regulating activity (Nguyen et al. 2012). Those active compounds were also found to interact with most of the residue of the active side of Fab I. However, an in vitro test is necessary to confirm their antimalarial activity.

Conclusion
This study shows the high potential of using Artemisia afra as a source in the search for new antimalarial drugs. The FAS II pathway constitutes a prime target for developing prophylactic and curative drugs.