*2.7. Statistical Analysis*

All the measurements were done in triplicate and IC50 values were calculated using GraphPad Prism 5 version 5.01 (Graph pad software, Inc., La Jolla, CA, USA.) statistical software.

#### **3. Results and Discussion**

#### *3.1. Chemical Characterization of the Isolated Compounds*

Chromatographic purification of a methanolic extract of *S. africana-lutea* was done using different techniques, including semi prep-HPLC yielded pure terpenoids (Figure 1), four of which were reported for the first time.

**Figure 1.** Chemical structures of the isolated compounds (**1**–**10**) from *S. africana-lutea*.

Compound **1** was isolated as amorphous yellowish-brown powder. The high-resolution mass spectrometry (HRMS) data indicated a molecular ion peak at [M]+ 403.2115 *<sup>m</sup>*/*<sup>z</sup>*, suggesting a possible chemical formula of C23H32O6. Its IR spectrum exhibited bands at 1725 and 3447 cm<sup>−</sup><sup>1</sup> forester and hydroxyl groups. The UV spectrum showed two absorptions at 210 and 280 nm. The 1H NMR spectra (Table 1, Supplementary Materials) showed signals of three methyls at 1.19 ppm (*d*, *J* = 6.8 Hz), 1.17 (*d*, *J* = 6.8 Hz), and 1.06 (s); an aromatic proton at 6.46 (s); an oxygenated methylene proton at 3.97, 4.24 (*d*/each, *J* = 11.4 Hz); a multiplet signal at 3.17 (*sept*, *J* = 6.8 Hz); in addition to acetoxyl and a methoxyl groups at 1.86, 3.75. The 13C NMR, distortionless enhancement by polarization transfer (DEPT-135), and heteronuclear multiple quantum coherence (HSQC) spectra indicated the presence of 23 carbons (Table 2) classified as four methyls, including a methoxy (61.1), two methylene, six methines, one of which was aromatic (117.8), and eight quaternary carbons, including a carbonyl (181.7) and five aromatic carbons (126.8, 133.8, 148.9, 143.7, and 139.6) in addition to the acetoxyl group (172.5, 20.7). The NMR data indicated an abietane diterpene similar to carnosic acid, previously isolated from the same source [14], with the only difference being the presence of extra acetoxyl and methoxyl groups. The methoxyl group was allocated at C-12 from the heteronuclear multiple bond correlation (HMBC) spectra, which showed correlations (among others) between the methoxyl protons and C-12 (143.7). The acetoxyl group was allocated at C-19 due to the presence of a methylene signal at 3.97 and 4.24, and both protons showed HMBC correlations with the acetoxyl's carbonyl group, C-4/C-5, and C-3. Other 2D data [heteronuclear multiple bond correlation (HMBC) and nuclear overhauser effect spectroscopy (NOESY)] as shown in Figure 2 confirmed the structure of compound **1** as 19-acetoxy-12-methoxycarnosic acid. The absolute configuration of the compound is proposed to belong to the normal abietane skeleton based on a biosynthetic basis, because normal abietane diterpene derivatives were previously isolated from the same source and directly related to the isolated compound [14,28].

The HRMS of compound **2** showed the molecular ion peak at [M]+ 417.1907 *<sup>m</sup>*/*<sup>z</sup>*, corresponding to the molecular formula of C23H30O7. Its IR spectrum exhibited bands at 1727 and 3447 cm<sup>−</sup><sup>1</sup> for ester and hydroxyl groups. The UV spectrum showed two peaks at 212 and 287 nm. Analysis of the 1H NMR data (Table 1) showed the presence of an isopropyl group [a proton at δH = 3.04 (*sept*, *J* = 6.5 Hz); two methyls at 1.24 (d/both, *J* = 6.5 Hz)]; an aromatic proton (δH = 6.79, s); three separated protons attached

to three oxygenated carbons at 4.66 (*dd, J* = 3.7, 12.1 Hz), 4.74 (*d*, *J* = 3.2 Hz), and 4.29 (*d*, *J* = 3.2 Hz); in addition to four methyl signals at 1.01 and 0.99 (s each), 2.07 (s, acetoxy), and 3.66 (s, methoxy). The 13C NMR, DEPT-135 and HSQC showed 23 carbons classified as four methyls at (16.2, 26.7, 22.3, 22.4), an acetoxy (21.2), a methoxy (58.4), two methylene groups (25.3, 24.3), six methines, (one of which was aromatic), nine quaternary carbons, including two carbonyls (170.7, 178.1), and five aromatics (126.6, 123.4, 141, 141.8, and 134.7). According to HSQC, the proton resonating at δH 4.66 attached to carbon at 74.0 showed HMBC correlations with carbons at 174 (CO acetoxy), 77.4 (C-7), 51.5 (C-5), and 46.5 (C-10), which indicates the presence of a lactone ring at position six. Additionally, the proton at 4.29 (H-7) showed HMBC correlations with carbons at 74.0 (C-6), 126.6 (C-8), 123.4 (C-9), and 120.4 (C-14) as shown in Table 2. On the other hand, the spectroscopic data of compound **2** showed a close similarity with methoxyrosmanol, the only di fference being the presence of an extra acetoxyl signal (2.07/170.7). The position of the acetate group was located at C-3 (from HMBC and NOESY correlations). Other 2D spectra confirmed the structure of compound **2** as 3β-acetoxy-7 α-methoxyrosmanol [29–31].

Compound **3** showed NMR data similar to compound **2**. The 1H NMR showed the absence of the C-3 methine signal at 4.7 and the appearance of the signal of methylene protons (4.05 s) attached to an acetoxyl group (from HMBC spectra). The acetoxyl group is attached to C19 because the NOESY and HMBC correlations showed cross-peaks between the acetoxy methyl/C-19, in addition to CH2-19/C3; C4 and C5. Other 2D data confirmed the structure of compound **3** as 19-acetoxy-7 α-methoxyrosmanol [28].

The NMR of compound **4** (Tables 1 and 2) showed a close similarity with carnosol [32] and compounds **2** and **3**, except for the absence of the C-6 α(OH)/C-7 α(OH) system appearing at the C-20/C-7 α lactone ring, in addition to a methoxy group, which was located at C-12, as shown in Figure 1. The HMBC spectra showed a correlation between H6/C7; C5; C4; C8 and H7/C6; C8; C13; C9. Other 2D data confirmed the structure of compound **4** as 19-acetoxy-12-methoxy carnosol.


**Table 1.** NMR spectroscopic data assignments (400 MHz) for compounds **1**–**6** (δ in ppm, m, J in Hz) in CDCl3.

Singlet (s); doublet (d); septuplet (sept); multiplet (m); broad doublet (br d); doublet doublet (dd); \* not well defined; doublet doublet doublet (ddd).


**Table 2.** NMR spectroscopic data assignments (100 MHz) for compounds **1**–**6** (δ in ppm) in CDCl3.

**Figure 2.** Key 1H−1H correlation spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser e ffect spectroscopy (NOESY) correlations of **1** and **2**.

Compound **5** was isolated as a white powder. The HRMS data indicated an ion peak at [M]+ 345.1694 *<sup>m</sup>*/*<sup>z</sup>*, suggesting a possible chemical formula of C20 H28 O5. Its IR spectrum exhibited bands at 1675 cm<sup>−</sup><sup>1</sup> for a lactone-carbonyl, as well as at 3500 and 3210 cm<sup>−</sup><sup>1</sup> attributed to hydroxyl groups. The UV spectrum showed two peaks at 208 and 284 nm. The NMR spectra of compound **5** (Tables 1 and 2) showed similar signals to compound **1**, the di fference between them being the absence of the acetoxyl and methoxy1 groups and the appearance of the dioxygenated-carbon signal at 102.8 (C-19), attached to a singlet proton at 5.6, which forms a lactone ring with C-20 (180.1). In particular, the HMBC showed a correlation between a proton at 5.6 (H-19) and the C-20 (180.1)/C-4 (37.0)/C-5 (50.2). Other 2D spectra in comparison with literature data confirmed the structure of compound **5** as clinopodiolide A [33]. On the other hand, compound **6** showed typical NMR signals similar to the ones of compound **5** (Tables 1 and 2), except for the presence of an extra methoxyl signal, which was placed on C-12 from

the HMBC correlations. Other 2D spectra in comparison with literature data confirmed the structure of compound **6** as clinopodiolide B [33]. The occurrence of the lactol moiety at C-19–C-20 is very unusual in nature and it has been recently isolated for the first time from *Salvia clinopodioide*. To the best of our knowledge, these compounds (**5** and **6**) have been isolated for the first time from *S. africana lutea*.

#### *3.2. In Vitro Bioactivity*

#### 3.2.1. Alpha-Glucosidase and Alpha-Amylase Activities

Alpha-glucosidase is an enzyme located in the brush border of the small intestine epithelium, which catalyzes the breaking down of the reaction of disaccharides and starch to glucose. Glucosidase inhibitors reduce the rate of carbohydrate digestion and delay the carbohydrate absorption from the alimentary tract [29]. Alpha-amylase is one of the main enzymes in humans that is directly involved in the breakdown of starch to simpler sugars [30].

It hydrolyses complex polysaccharides to produce oligosaccharides and disaccharides, which are then hydrolyzed by alpha-glucosidase to monosaccharide, which are absorbed through the small intestines into the hepatic portal vein and increase postprandial glucose levels. The inhibitory mechanisms of these enzymes are characterized by delaying carbohydrate digestion and reducing the rate of glucose absorption [34]. The bio-evaluation of natural resources for the antidiabetic properties has been intensified, and a grea<sup>t</sup> deal of research is being carried out to identify plants with potent anti-diabetic activity with emphasis on the inhibition of the two enzymes, alpha-glucosidase and alpha-amylase. In this study, the inhibitory activity of the isolated compounds from *S. africana lutea* was investigated and the results showed that compound **8** demonstrated the highest alpha-glucosidase inhibitory activity with IC50 value of 11.3 ± 1.0 μg/mL, followed by compounds **10** and **7** with IC50 values of 17.1 ± 1.0 μg/mL and 22.9 ± 2.0 μg/mL, as indicated in Table 3. The IC50 value of compound **8** is consistent with the previously reported value of 12.1 ± 1.0 μM [35]. The higher inhibitory activity demonstrated by compound **8** (compared to compound **7**) could be explained by the shift of the C-29 methyl group from C-20 to C-19, which has enhanced the inhibition of the alpha-glucosidase enzyme [35]. In addition, the lowest alpha-glucosidase inhibitory activity demonstrated by compound **10** among the tested triterpenes might be due to the absence of the carboxylic group in its chemical skeleton. Among all the tested abietane diterpenes, only compound **6** demonstrated moderate alpha-glucosidase inhibitory activity, with an IC50 value of 81.7 ± 2.1 μg/mL.

Remarkably, compound **7** demonstrated the strongest alpha-amylase inhibitory activity among the tested compounds with an IC50 value of 12.5 ± 0.7 μg/mL, followed by compounds **8** and **10** with IC50 values of 66.1 ± 2.0 μg/mL and 76.6 ± 2.1 μg/mL, respectively. None of the tested abietane diterpenes showed alpha amylase inhibitory activity, as shown in Table 3.


**Table 3.** Inhibitory activities of the isolated compounds on alpha-glucosidase and alpha-amylase.

Not active (NA) at the test concentrations. The results are expressed as mean ± SEM for *n* = 3.
