*2.2. Plant Material*

The plant material used in this study was collected from the Cape Flats Nature Reserve, University of the Western Cape, South Africa. A voucher specimen was identified at the Compton Herbarium, Kirstenbosch by Dr. Christopher Cupido (South African National Biodiversity Institute, Kirstenbosch), with herbarium number NBG1465544-0.

#### *2.3. Extraction and Purification of Chemical Constituents*

The aerial parts of the fresh plant material (2.5 kg) were blended and extracted with methanol (4.5 L) at room temperature (25 ◦C) for 24 h. The methanol extract was filtered and evaporated to dryness under reduced pressure at 40 ◦C to yield 97.77 g (3.9%). The total extract (97 g) was applied to a silica gel column (30 × 18 cm) and eluted using a gradient of hexane (Hex) and ethyl acetate (EtOAc) in order of increasing polarity: 94 fractions (500 mL each) were collected and combined according to their TLC profiles to yield 21 fractions, labeled I–XXI.

The main fraction XVII (500 mg) was subjected to a successive silica gel column using a Hex/EtOAc gradient (7:3; 100%) then Sephadex (using 95% aqueous ethanol) to produce 18-acetoxy-12-methoxy carnosic acid (compound **1**, 44.1 mg). The main fraction XIII (1.4 g) was chromatographed on silica gel using a Hex/EtOAc gradient (9:1; 7:3; 100%) then HPLC, using a gradient solvent system of MeOH and de-ionized water (70:30 to 100% MeOH in 45 min), producing compound **3** (Rt 23.62 min, 9 mg), compound **4** (Rt 25.19 min, 11.17 mg), compound **5** (Rt 32.17 min, 46.3 mg), and compound **6** (Rt 12.75 min, 40 mg). Main fraction XI was subjected to a successive silica gel column, under the same condition, then HPLC to produce compound 2 (Rt 19.67 min, 9.9 mg).

Main fraction X was subjected to a silica gel column under the same condition then chromatographed on a sephadex column (5 % aqueous ethanol) to produce compound **8** (34.6 mg), compound **7** (9.6 mg), compound **9** (12.5 mg), and compound **10** (54 4 mg).

#### *2.4. Alpha-Glucosidase Inhibitory Activity*

Alpha-glucosidase inhibitory activity of the isolated compounds was carried out according to the standard method, with a slight modification [24]. In a 96-well plate, the reaction mixture containing 50 μL of phosphate bu ffer (100 mM, pH = 6.8), 10 μL alpha-glucosidase (1 U/mL), and 20 μL of varying concentrations of isolated compounds was pre-incubated at 37 ◦C for 15 min. Next, 20 μL of p-NPG (5 mM) was added as a substrate and incubated further at 37 ◦C for 20 min. The reaction was stopped by adding 50 μL of sodium carbonate Na2CO3 (0.1 M). The absorbance of the released p-nitrophenol was measured at 405 nm using a Multiplate Reader (Multiskan thermo scientific, version 1.00.40, Vantaa, Finland). Acarbose at various concentrations was included as a standard. Each experiment was

performed in triplicates. The results were expressed as a percentage inhibition, which was calculated using Formula (1).

$$\text{Inhibitory activity (\%)} = (1 - \text{A/B}) \times 100\tag{1}$$

where A is the absorbance in the presence of the test substance and B is the absorbance of the control.

#### *2.5. Alpha-Amylase Inhibitory Activity*

The alpha-amylase inhibitory activity of the extract and fractions was carried out according to the standard method, with a slight modification [24]. In a 96-well plate, the reaction mixture containing 50 μL of phosphate buffer (100 mM, pH = 6.8), 10 μL alpha amylase (2 U/mL), and 20 μL of varying concentrations of isolated compounds was pre-incubated at 37 ◦C for 20 min. Next, 20 μL of 1% soluble starch (100 mM phosphate buffer pH 6.8) was added as a substrate and incubated further at 37 ◦C for 30 min. A volume of 100 μL of the color reagen<sup>t</sup> (DNS) was added and then boiled for 10 min. The absorbance of the resulting mixture was measured at 540 nm using a Multiplate Reader (Multiskan thermo scientific, version 1.00.40). Acarbose at various concentrations was used as a standard. Each experiment was performed in triplicates. The results were expressed as the percentage inhibition, which was calculated using Formula (2).

$$\text{Inhibitory activity (\%)} = (1 - \text{A/B}) \times 100\tag{2}$$

where A is the absorbance in the presence of the test substance and B is the absorbance of the control.
