Isolation, characterization, and biological evaluation of a potent anti-malarial drimane sesquiterpene from Warburgia salutaris stem bark

The fine stem bark powder (1 kg) was subjected to maceration with dichloromethane (1:5 w/v) for 3 days with periodic shaking. The maceration extract was filtered through Whatman (No. 1) filter paper and concentrated under reduced pressure at 45 °C. Silica gel column chromatography (60 × 1000 mm; Merck silica gel, 60: 0.063–0.200 mm) was used to isolate the compound from the DCM extract. The column was eluted with hexane: ethyl acetate gradient and the ratio 8:2 yielded 35 fractions. The fractions were grouped according to the thin layer chromatography (silica gel 60 aluminium sheets, F 254—Merck, Whitehouse Station, New Jersey, USA) profiles, into seven combined fractions (Cfrs). The spots were fixed on the TLC plates with a 20% H₂SO₄ in methanol mixture and viewed under UV light (254 nm). The combined fractions were then dried under a fume-hood overnight; Cfrs 2–4 produced a white amorphous powder (NN-01). NN-01 was recrystallized from methanol to give white needles (mpt. 95–98 °C); [α]_D = − 11(0.1M in CHCl₃); HREI-MS: MF = C₁₇H₂₄O₅, [M+Na]⁺ ion at m/z 331.1619 (calcd: 331.1521).

The structure of NN-01 was determined using combined spectroscopic methods (FT-IR, Nuclear Magnetic Resonance (NMR) (¹H, ¹³C, COSY, DEPT, NOESY, HMBC, and HSQC, and HREI-MS). Its FT-IR spectrum contained characteristic functional group frequencies: IR ν max (3450, 2942, 2848, 1743, 1717, 1691 cm⁻¹). Its spectroscopic data (Table 1) are very similar to those of cinnamodial (1) [9]. However, its melting point (95–98 °C) was different from the melting point of cinnamodial (1) (135–137 °C) [9]. This difference in melting point led to the X-ray crystallographic study of NN-01 which confirmed its structure as iso-mukaadial acetate (2) (see Fig. 1).

The HepG2 (liver hepatocellular carcinoma) cells were obtained from the Highveld Biologicals (Pty) Ltd, Kelvin, SA, while the HEK293 (Human embryonic kidney) cells were provided by the University of the Witwatersrand, Medical school, Antiviral Gene Therapy Unit, SA. The modified method of Mosman [22] was used. The MTT assay is used to measure the cells metabolic activity, by reducing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into formazan salt using the enzyme succinate-tetrazolium reductase within the cell. The cells were developed to semi-confluency, in flasks for tissue culture (25 cm²), in Eagle’s minimal essential medium containing, 100 U/ml penicillin; 100 μg/ml streptomycin as well as 10% fetal bovine serum. Cells were then seeded at a density of 21 × 10³ cells in each well, with medium (100 µl) in a 96 well plate. A 24 h incubation period (37 °C; 5% CO₂) followed, after which fresh medium (100 µl) was added upon the removal of the old medium. Varying concentrations (50 µg/ml, 100 µg/ml and 150 µg/ml) of the compound were added in triplicates onto the cells then further incubated for 48 h at 37 °C. Cells without the experimental compound were used as the positive controls (100% viability).

The used medium was removed, after 48 h of incubation and, 100 µl new medium and 5 mg/ml MTT in PBS added, and this went into incubation (37 °C) for another 4 h. MTT and medium were then discarded, and 200 µl of DMSO was added per well, for dissolving the formazan. Absorbance of resulting mixture was then read with a Mindray 96A microplate reader (Vacutec, Hamburg, Germany) using a wavelength of 570 nm (detection λ) and that of 630 nm as the reference wavelength of nonspecific signals. The percentage viability of the cell was linked to absorbance and worked out in reference to the positive control using the equation: [(OD₅₇₀ Treated)/(Control)]× 100. All experimentations were done in triplicates.

The compound was tested against a NF54 Plasmodium falciparum strain (chloroquine sensitive); the tests were done in triplicate. Continuous in vitro cultures of asexual erythrocytes stage of the parasite were maintained following the modified method of Trager and Jensen [23]. The viability of the parasite was evaluated by looking at the parasite lactate dehydrogenase activity, which was conducted following a modified method described by Makler et al. [24]. Test sample was made to a stock solution of 20 mg/ml in DMSO (100%). The stock solution was preserved at − 20 °C. Dilutions of the stock were made on the day of experimentation. Artesunate and chloroquine were the drugs of reference in the experiment.

Full dose–response was conducted on the test compound to find concentrations at which 50% of parasitic growth is inhibited (IC₅₀ value). Primary compound concentration for the trial was at 100 μg/ml, thereafter successively diluted to concentration twice as much in medium to produce ten measures with varying concentrations; minimum concentration being 0.2 μg/ml. Chloroquine initial concentration was at 1000 ng/ml. Solvent concentrations in which the infected red blood cells were subjected to, had no significant consequence on the viability of parasite. A non-linear dose–response curve fitting analysis was used to calculate the IC₅₀ values, with the version4.0 Graph Pad Prism software (Graph Pad Prism, Inc: San Diego, CA, USA, 1994–2003).

Many malaria-endemic areas are developing countries with infected people who cannot afford anti-malarial drugs and, therefore, rely on traditional medicine and medicinal plants as primary health care for the treatment of malaria [2]. Discovering novel phytochemical compounds for use in drug development, is the main goal of ethno-pharmacology [26]. Bioassay guided separation of the crude extract led to the isolation, purification and characterization of iso-mukaadial acetate. It was the first time that this compound was isolated from this plant, and the first time that any anti-malarial activity is being reported. The anti-malarial and cytotoxic activity results are depicted in Table 2.

In drug discovery, a compound is considered a success if it has an IC₅₀ ≤ 1 µg/ml and a selectivity index that is at least ten times more active on the parasite than it is on human cell-lines [27]. Iso-mukaadial acetate shows good in vitro anti-malarial activity. There has been no report of any other isolated anti-malarial compound from W. salutaris in literature. Katiyar et al. suggests that natural sources have a potential of delivering natural compounds as base for synthetic analogues in drug discovery [26]. It is noteworthy to state that the high antiplasmodial activity of iso-mukaadial acetate makes it eligible for structural modification studies to reduce cytotoxicity while retaining or improving antiplasmodial activity.

Plasmodium berghei has been proven to be most effective in early investigation of anti-malarial activity for new test compounds [28]. Compounds with more than 30% parasite inhibition and that increase the rodents’ survival time [29], when compared to the absolute control are considered effective in standard screening tests. The study results showed varied antiplasmodial activity with the 5-day treatment groups showing optimum parasite inhibition, for both the crude extract and iso-mukaadial acetate, than those of the 1-day treatment. The results are depicted in Figs. 2, 3, 4, 5.

The reduce red blood cell count and haematocrit in the study is consistent with previous malaria studies of severe malarial anaemia. A study showed that red blood cell destruction and decrease in red blood cell production in response to parasite invasion may lead to reduced haematocrit and haemoglobin levels in the blood of infected animals [30]. However, mechanisms by which haematology parameters were affected are not described in this study. Further plasma and serum studies may provide mechanisms by which observed malaria-induced haematology changes occurred. The study groups are represented as, UMA-Uninfected iso-mukaadial acetate treated; UCE-Uninfected crude extract treated; IC-infected without treatment; ICQ-infected chloroquine treated; ICE1-infected crude extract 1-day treatment; ICE2-infected crude extract 5-day treatment; IMA1-infected iso-mukaadial acetate 1-day treatment; IMA2-infected iso-mukaadial acetate 5-day treatment. Column statistics was carried out for all haematology data.

The difference in weight was measured at 2-day intervals to determine whether the W. salutaris crude extract and iso-mukaadial acetate prevent weight loss, commonly observed in malaria. Effective anti-plasmodial products are expected to have protective effects against the loss of weight commonly observed in malaria infection, in this study, these protective effects were observed in the iso-mukaadial acetate and W. salutaris groups as shown in Fig. 6.