Isolation of alpha-linolenic acid from Sutherlandia frutescens and its inhibition of Mycobacterium tuberculosis’ shikimate kinase enzyme

The shikimate enzyme is one of the most vital enzymes involved in the metabolic pathways of Mycobacterium tuberculosis. The principle of the shikimate kinase inhibition assay is primarily based on the enzyme shikimate kinase phosphorylating shikimic acid to form shikimate-3-phosphate and a resultant ADP as shown in Fig. 1. In the presence of a good inhibitor less or none of this resultant ADP will be produced, an appropriate inhibitor would bind where a phosphate is supposed to bind on the shikimic acid and therefore subsequently restricting its phosphorylation and hence producing no ADP. The strongest inhibitor produces less ADP. After performing a series of shikimate kinase inhibition assays on the crude extracts it was noticed that the DCM: MeOH 1:1 extract was showing more potential in terms of inhibition of the shikimate kinase enzyme’s activity and hence had a fairly low average of ADP produced and also as seen on Table 4 had an IC₅₀ of 0.1 ug/ml (Fig. 2) which was lower than the H₂O (5.1 ug/ml) (Fig. 3) and the ethanol (1.7 ug/ml) extracts (Fig. 4). This realization led to the decision to performing more tests with this extract.

Percentage inhibition obtained from single point inhibitions of the 20 sub-fractions produced via preparative HPLC. Sample 1,13,14,18 and 20 shows almost 100 % inhibition while sample 7 has almost 0 % inhibition against shikimate kinase (Fig. 5). Of the 20 sub-fractions that were collected, sub-fraction 8 was shown to be one of the most active against the shikimate kinase enzyme (Fig. 6), hence the decision to pursue its isolation and characterization. Figure 6 shows that at just after 10 min, a large peak of a compound (sub-fraction 8) with a molecular ion peak at (m/z) 277.4 was collected and this compound ionized in the negative electrospray ionization (ES-). This compound had a bright yellow colour and was sticky upon evaporation with the rotavap. The TLC plate on Fig. 7 also showed a single band, meaning that separation did occur and that a single compound was found in this sub-fraction 8. The most useful tool in identification of a compound in chemistry is the SDBS assimilations software; this software was very helpful in determining the structure and molecular formula of the isolated compound by using its proton and carbon NMR spectra (Table 5). The protons and the carbons of the isolated compound exactly matched those assimilated with from the software hence the software was able to identify the compound’s chemical formula as C₁₈H₃₀O₂ giving a total molecular mass of 278 u.

Its IUPAC name and structure were determined using Chem Draw ultra 8 software and these are represented on Fig. 8. The IUPAC name of the isolated compound was found to be (9Z, 12Z, 15Z)-octadeca-9,12,15-trienoic acid and its common name is alpha linolenic acid. IC₅₀ of the compound was also determined and recorded to be 3.7 ug/ml (Fig. 9). Alpha-linolenic (ALA) is an essential omega-3 fatty acid that cannot be synthesized by the human body and hence has to be supplied by dietary sources such as fish, plants, walnuts, grape seed (canola), several legumes, flaxseed, and green leafy vegetables and vegetable oils [14, 15] but has not been reported to have been isolated from S. frutescens before. Stark et al. [14] mentioned that potential benefits of ALA include cardio-protective effects, modulation of the inflammatory response, and a positive impact on both central nervous system function and behavior. According to Stark et al. [14], as a result of many studies they have conducted in their study, ALA is not only essential for dietary requirements but also has therapeutic properties. Several studies have proven amongst other free fatty acids that alpha linolenic acid is active against a wide range of microorganisms including bacteria such as S. aureus and Bacillus subtilis [16], Helicobacter pylori, [17] viruses such as hepatitis C virus (HCV) [18] and fungi such as Rhizoctonia solani and Crinipellis perniciosa [19]. According to Desbois and Smith, [20] their antibacterial mode of action is not clearly understood but may also result from the inhibition of enzyme activity, impairment of nutrient uptake, generation of peroxidation and auto-oxidation degradation products or direct lysis of bacterial cells. The broad spectrum of activity they possess, the non-specific mode of action and their safety makes them attractive as antibacterial agents for various applications in medicine, agriculture and food preservation.

This is a novel study. Firstly, this is the first report on identifying ALA from S. frutescens. Secondly, this is the first report showing ALA from S. frutescens inhibiting shikimate kinase enzyme, an important drug target for M. tuberculosis. Thirdly, this is the first time that ADP quantification method for shikimate kinase inhibition assay using HPLC was used. In other words, a reliable HPLC based M. tuberculosis shikimate kinase inhibition assay was developed in this study. Several studies have used liquid chromatography-Mass spectrometry (LC-MS) to determine M. tuberculosis shikimate kinase enzyme activity by quantifying shikimate-3-phosphate which is a resulting product of the phosphorylation of shikimate [21‐23]. The advantage of using the ADP method is that one can see the viability of the enzyme by tracing the production of ADP thus using the HPLC machine not only ensures the collection of qualitative data but also the quantitative data. The ADP method used in this study could be useful for scientists to study other potential M. tuberculosis shikimate kinase inhibitors. Kinases have a conserved phosphoryl transfer mechanism [24‐26] thus to find a unique inhibitor and a way of determining inhibition of kinases advance kinases kinetic studies.