3.2.2. Antioxidant Activity

The in vitro antioxidant activity of the isolated compounds of the methanolic extract of *S. africana-lutea* were investigated by evaluating their ferric-ion reducing antioxidant power (FRAP), trolox equivalent absorbance capacity (TEAC), and oxygen radical absorbance capacity (ORAC) activities. The TEAC and FRAP are assays based on a single electron transfer (SET) mechanism, in which the antioxidant transfers an electron to the corresponding cationic radical to neutralize it [36], while ORAC is based on a hydrogen atom transfer (HAT) mechanism, in which the antioxidant exhibits the potential health-beneficial roles via transferring hydrogen atom(s) to the reactive species, thereby deactivating them [37]. The results demonstrated that compounds **1** and **5** exhibited strong activity on ORAC (2588.2 ± 10.1; 2357.2 ± 0.1) μM TE/g, respectively. Compounds **5** and **6** showed moderate activities on TEAC (862.2 ± 1.4; 705.5 ± 2.0) μM TE/g, whereas compounds **5** and **2** demonstrated significant inhibitory activity on FRAP (2262.9 ± 11.0; 2200.9 ± 14.2) μM AAE/g when compared to the reference antioxidant epigallocatechingallate (EGCG), as shown in Table 4. Phenolic compounds have been reported to be responsible for the antioxidant activity of numerous plant species by stabilization of radicals by donating electrons or by metal ion complexation, among other mechanisms. Nevertheless, other aspects can be considered, for example, the presence of vicinal hydroxyl groups is essential in a pronounced antioxidant activity [38]. Abietane diterpenes are known for having strong antioxidant activity due to the presence of ortho-dihydroxyl groups/vicinal hydroxyls in the aromatic ring that serve as hydrogen and/or electron donating agents to the corresponding reactive species leading to the formation of the stable quinone derivatives [39]. It has been shown that the phenolic group at the 11 position would be more implicated in the antioxidant activity [40]. In general, phenolic compounds are expected to transfer electrons or donate protons to the reactive radicals because of the resonance stability of the phenoxy radical [41].

**Table 4.** Antioxidant activities of the isolated compounds.


Not active (NA) at the test concentrations; epigallocatechingallate (EGCG). Trolox equivalent absorbance capacity (TEAC); oxygen radical absorbance capacity (ORAC); ferric-ion reducing antioxidant power (FRAP). The results are expressed as mean ± SEM for *n* = 3.

The structure-activity relationship (SAR) of compound **5** could be related to the presence of ortho-dihydroxyl groups/vicinal hydroxyls in the aromatic ring as well as the presence of the free hydroxyl groups at the 19 position in its chemical structure, which are responsible for its high activity, demonstrated when compared to compound **6**. However, the substitution of the free OH at the 12 position by a methoxyl group is directly related to the decrease of the activity observed. The presence of an acetoxy group in compound **1** could be responsible for its high capacity of hydrogen atom transfer, demonstrated in the ORAC assay. However, the antioxidant activity of compound **2** demonstrated in FRAP is high because of the high-stress lactone ring, which may open during the course of the chemical reaction leading to an extension of the conjugation and formation of the p-quininoidal structure.

Compound **1**: Yellow, amorphous solid; [α]28D +70.43 (0.1, MeOH); UV (MeOH) λmax (log ε) 210 (4.31), 280 (3.79); nm; IR (KBr) νmax 3447, 3320, 2954, 1725, 1570, 1381, 1250, 1039cm–1; 1H; and 13C NMR data, see Tables 1 and 2; positive-ion high-resolution electrospray ionisation mass spectrometry (HRESIMS) [M−H]<sup>+</sup> *403.2115* (calcd for C23H32O6, 404.2199).

Compound **2**: Red brownish powder; [α]28D +22.92 (0.2, MeOH); UV (MeOH) λmax (log ε) 212 (4.32), 287 (4.09); nm; IR (KBr) νmax 3447, 3300, 2958, 1727, 1575, 1246, 1034 cm–1; 1H; and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS [M−H]<sup>+</sup> *417.1890* (calcd for C23H32O6, 418.1932).

Compound **3**: Brown amorphous powder; [α]28D +49.04 (0.07, MeOH); UV (MeOH) λmax (log ε) 210 (4.31), 295 (3.92); nm; IR (KBr) νmax 3450, 2962, 1769, 1634, 1435, 1239, 1034 cm–1; 1H; and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS [M−H]<sup>+</sup> *417.1907* (calcd for C23H32O6, 418.20).

Compound **4**: Yellow amorphous powder; [α]28D -53.27 (0.03, MeOH); UV (MeOH) λmax (log ε) 210 (4.31), 281 (4.13); nm; IR (KBr) νmax 3330, 2950, 1750, 1617, 1446, 1243, 1050 cm–1; 1H; and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS [M−H]<sup>+</sup> 401.1609 (calcd for C23H32O6, 402.2042).
