Sutherlandia frutescens is a well-known and much used, commercially cultivated indigenous medicinal plant in southern Africa. S. frutescens contains several essential bioactive compounds with clinically proven pharmacological activities (Haraguchi 2001:1; Ojewole 2008:126; Prevoo, Swart & Swart 2008:118). This makes the plant attractive as a medicine for various ailments and diseases. Sutherlandia is recommended by the South African Department of Health as a supporting treatment for people living with Acquired Immune Deficiency Syndrome (AIDS) (Mills et al. 2005:1; SA Healthinfo 2009:1; Seier et al. 2002:1). It is also prescribed for treatment of cancer, tuberculosis (TB), diabetes, anxiety and clinical depression (Mills et al. 2005:1; SA Healthinfo 2009:1; Van Wyk 1997).

TB is a highly infectious disease caused by Mycobacterium tuberculosis and it kills millions of people annually. It is also one of the common co-infections in people living with Human Immunodeficiency Virus (HIV) and/or AIDS, thus worsening the HIV and/or AIDS pandemic. The drugs that are currently used to treat TB have to be taken over a long period, and because of this people prematurely stop treatment leading to the development of resistance. Also, there is a growing increase in drug-resistant strains which are considered a significant health threat (WHO 2007a, 2007b). World-wide efforts are being made to discover quicker acting drugs which are also effective against the drug-resistant strains. While Sutherlandia is prescribed for people with TB, it is still not known if the extracts have antimycobacterial activity and what compounds in this plant might act against M. tuberculosis and its mode of action. This study was aimed at investigating the activity of Sutherlandia frutescens extracts against Mycobacterium smegmatis. M. tuberculosis grows extremely slowly and represents a biological hazard in the laboratory. M. smegmatis contains a similar cell-wall structure to M. tuberculosis and is commonly used in laboratory experiments for research analysis due to the fact that it is a ‘fast grower’ and non-pathogenic (Reyrat & Kahn 2001). Furthermore the highly infectious nature of M. tuberculosis restricts its use for large-scale screening of probable drug candidates. M. smegmatis has been found to display a drug-sensitivity profile similar to M. tuberculosis (Chaturvedi et al. 2007). The plant extracts were primarily screened against this rapidly growing and non-pathogenic mycobacterial strain with a view to identifying active compounds which could later be tested against M. tuberculosis’s shikimate kinase. This study set out to provide preliminary data on in vitro anti-mycobacterial activity as a first step to further investigations.

Of the three extractants used in this study, ethanol was the best extractant; it was able to extract the most mass from the ground plant material throughout the three harvest periods. Distilled water extracted the least amount of plant material. A TLC was used to profile the crude extracts and the fractions for all the compounds found in S. frutescens. The plates developed in CMWF1 separated more compounds than those developed in CMWF2 and BAW (Figure 1). Also the ethanol and the DCM:MeOH 1:1 extracts had more compounds compared to the water extract and the same observation was made of the UV active bands.

Each crude extract was assayed against M. smegmatis to determine its MIC values. Table 5 shows that DCM:MeOH 1:1 extracts had the greatest activity with MIC values ranging from as low as 0.28 mg/mL to 1.04 mg/mL and water extracts had the least. The same observation was made by Katerere and Eloff (2005) where the hexane, DCM and ethylacetate extracts produced better antimicrobial activities (MIC values of 2.50 mg/mL, 1.25 mg/mL and 0.31 mg/mL, respectively) against Staphylococcus aureus which is a Gram positive microorganism that cannot be compared to Mycobacterium spp.; their water, ethanol and acetone extracts had MIC values as high as 10 mg/mL. Despite the fact that traditional healers frequently use water to make decoctions and adaptogenic tonics, the water extract was found to be the least active and these findings could be attributed to the inability of water to extract non-polar compounds (Masoko & Nxumalo 2013). There was no activity found in bioautography (result not shown). Cases where MIC values indicated antimycobacterial activity, while bioautography could not detect any, could be explained by the possibility of evaporation of active compounds during removal of the TLC eluents as was mentioned earlier or by the disruption of synergism between active constituents caused by TLC (Masoko & Eloff 2005). The fact that no compound isolated on bioautograms had antimycobacterial activity shows that it is not a feasible project to try to isolate antimycobacterial compounds from this plant.

Diphenyl-pacrylhydrazyl radical-scavenging compounds appeared as yellow spots against a purple background. When reverse-phase TLC plates were used with DPPH as detecting agent, the developing colour was stable, enabling the identification of radical-scavenging activity after a few minutes (Figure 2). This method can also be utilised for the bioassay-guided isolation of unidentified natural antioxidants and may be used for selection of potential antioxidants from a group of structurally diverse compounds. The current application also demonstrates the versatility and adaptability of a standard HPTLC system to serve an additional purpose in the drug discovery arena. Nonetheless, no antimycobacterial activity was established from extracts of S. frutescens. The study suggests that there is no correlation between the antioxidant activity and antibacterial activity of the extracts from S. frutescens. In a study by Katerere and Eloff (2005), a significant amount of antioxidant activity was also recorded for the more polar solvent (e.g. water) used in their extraction process, thereby suggesting that bacterial oxidative status or metabolism of reactive oxygen species may be involved in superoxide dismutase functions (Langford, Williams & Kroll 1991).

HPLC analysis is an analytical procedure that is more sensitive and selective in the area of natural products, not only to profile chemical constituents of plants but also to quantify isolated substances (Marques et al. 2013). The water and ethanol crude extracts were analysed, using HPLC-MS to obtain their chromatographic profiles. The resulting profiles of these extracts in both ESI^- and ESI⁺ modes (electrospray ionisation negative and positive modes) are shown in Figures 3 and 4. The chromatographic profile of the ethanol extract on Figure 4 shows a high amount of compounds with a negative mode of ionisation than those having a positive ES. This is an exact opposite compared to the water extract (Figure 3) which contains more compounds in its ES+ mode than its ES-. Fu et al. (2008, 2010) have isolated some of the most vital compounds found in S. frutescens which are cycloartane glycosides and flavonol glycosides. In a study by Fu et al. (2008), four new isolated cycloartane glycosides, named sutherlandiosides A,B,C and D, were isolated, with sutherlandioside B being the major glycoside of this plant and also responsible for most of the plant’s bioactivities. Sutherlandioside A and B both had a chemical formula of (C₃₆H₆₀O₁₀) and provided a pseudo ion peak at m/z 675.4042 [M + Na]⁺; the only difference was that there was a β-glucopyranosyl element moiety in sutherlandioside B. Sutherlandioside C had a chemical formula of (C₃₆H₅₈O₁₀) and a molecular weight of 650 u, and D was the same as C except that it had a chemical formula of (C₃₆H₅₈O₉) and that it lacked one oxygen molecule. From the chromatographic files in this study the glycoside-rich area is found from just above 14 min to just below 16 min, and also this region is seen in the negative mode electrospray ionisation. These sutherlandiosides are triterpenoids, hence could be seen elevated in the ethanol extract rather than the water extract; this is probably due to the fact that triterpenoids dissolve better in alcohol than water. Fu et al. (2010) also isolated flavonol glycosides and they were named sutherlandins A, B, C and D; their pseudo molecular peaks ranged from m/z 767 to 747 and they are more evident and in abundance in Figure 3 on the water extract. This is probably due to the fact that flavonoids are more polar and hence were able to dissolve best in water. In addition to the compounds already mentioned, the chromatographic profiles also show a lot of compounds present in S. frutescens that have not yet been characterised nor isolated.

In conclusion, this study and several studies mentioned above have shown that S. frutescens has activity against a range of microorganisms and this research supports the usage of this plant by traditional healers for various ailments, including the treatment and supporting treatment of TB. Further studies to test the extracts against M. tuberculosis’ shikimate kinase enzyme and to isolate the antimycobacterial compounds will be conducted.