## 2 Materials and Methods
### Fractionation and Isolation of Bioactive Compounds from P. obliquum  
_P. obliquum_ leaf powder (500 g) from SANBI and Nelspruit were extracted separately with 5L of acetone and vigorously shaken for 8 h on a Labotec shaking machine. A Buchner funnel was used to filter the supernatant through Whatman No. 1 filter paper, and a Buchi Rotavapor R-114 (Labotec) was used to evaporate the solvent under vacuum. The concentrated extract was then transferred to a pre-weighed beaker. The same technique was carried out again on the plant material. The extracted mass was then measured after the extract had been allowed to dry at room temperature, yielding 36.6 g (Nelspruit) and 42.17 g (SANBI). The solvent-solvent extraction/fractionation of plant extracts protocol was developed by the National Cancer Institute, modified by eliminating the carbon tetrachloride extraction step. Five solvent-solvent fractions containing compounds with different polarities from _P. obliquum_ acetone leaf extract were then obtained [25; 26]. To obtain the initial CHCl\({}_{3}\) and H\({}_{2}\)O fractions, the acetone extract was reconstituted in 500 mL of CHCl\({}_{3}\): H\({}_{2}\)O (1:1) in a separatory funnel and the two layers were partitioned. The H\({}_{2}\)O and _n_-BuOH fractions were then obtained by combining the H\({}_{2}\)O fraction with an equivalent volume of _n_-BuOH. The _n_-hexane fraction was obtained by extracting the initial CHCl\({}_{3}\) fraction with an equal volume of _n_-hexane and a 10% H\({}_{2}\)O-methanol mixture after the initial CHCl\({}_{3}\) fraction had been dried in a vacuum rotary evaporator. To create the 35% H\({}_{2}\)O-MeOH fraction and the CHCl\({}_{3}\) fraction, the 10% H\({}_{2}\)O-MeOH fraction was first diluted to 35% H\({}_{2}\)O-MeOH. Therefore, five fractions were obtained: H\({}_{2}\)O, n-BuOH, 35% H\({}_{2}\)O-MeOH, CHCl\({}_{3}\), and _n_-hexane fractions.  
Column chromatography was used to isolate the bioactive chemicals from the SANBI CHCl\({}_{3}\) fraction, with silica gel serving as the stationary phase. A uniform slurry made from about 1000 g of silica gel (Merck) and _n_-hexane was then loaded into a glass column with the dimensions of 40 cm in height and 4.5 cm in diameter. The dried CHCl\({}_{3}\) fraction (36.47 g) was combined with 50 g of silica gel prior to the addition of 100 mL of acetone, before drying for approximately 2-3 h at room temperature. The column bed was then overlaid with the dried CHCl\({}_{3}\) fraction. _n_-Hexane with ethyl acetate (EtOAc) was added in increasing percentages (5%) to 100% to yield various fractions that were collected.  
After the fractions had dried, white precipitates were observed in some honey jars from 70% to 60% _n_-hexane fractions and were then washed with acetone to remove impurities and yielded 50 mg of pure white crystals of obliquumol.  
By constantly purifying the dried fractions collected at \(n\)-hexane concentrations ranging from 95 to 85% with EtOAc, a 140 mg combination of a mixture of lupeol and \(\beta\)-amyrin was obtained.  
Fractions collected from 30-5% hexane were combined, since they contained similar compounds based on TLC finger printing. The 1.2 g yield from the combined fractions from the first column was dissolved in acetone, combined with 1 g of silica gel, and allowed to dry at room temperature. The sample was then deposited into a silica gel bed with dimensions of 40 cm in height by 2.5 cm in diameter, and it was eluted using a solution of 70% to 30% EtOAc and \(n\)-hexane. About 50 mg of \(O\)-methylalloptaeroxylin was then isolated. Nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) were used to analyse the samples and the structures of the isolated compounds were elucidated. The 1-dimensional (1D) NMR (\({}^{1}\)H, \({}^{13}\)C, and dept-135) and 2-dimensional (2D) NMR (COSY, HMBC, HSQC, and NOESY) spectra were used. The data was also compared with the literature to conclusively interpret the structures.