Pentacyclic Triterpenoids from Sabia discolor Dunn and Their α-Glycosidase Inhibitory Activities

Four new pentacyclic triterpenoids named Sabiadiscolor A–D (1 and 7–9) together with eleven known ones were isolated by repeated column chromatography. Their structures were identified and characterized by NMR and MS spectral data as 6 oleanane-type pentacyclic triterpenoids (1–6), 7 ursane-type ones (7–13), and 2 lupanane-type ones (14–15). Except for compound 15, all other compounds were isolated from Sabia discolor Dunn for the first time. Their α-glycosidase inhibitory activities were evaluated, which showed that compounds 1, 3, 8, 9, 13, and 15 implied remarkable activities with IC50 values ranging from 0.09 to 0.27 μM, and the preliminary structure–activity relationship was discussed.


Introduction
Diabetes mellitus (DM) is one of the most prevalent metabolic diseases worldwide. This disease is a chronic metabolic disease mainly characterized by hyperglycemia caused by a variety of factors, among which type 2 diabetes mellitus (T2DM) is the most common, accounting for 90% of the total number of diabetic patients. T2DM is a type of diabetes that is associated with an imbalance in glucagon/insulin homeostasis that leads to the formation of amyloid deposits in the brain, in pancreatic islet cells, and possibly in the kidney glomerulus. With increasing human material resources and improvements in living standards, the incidence of T2DM is increasing, which seriously affects human health and quality of life. When diet and exercise fail to control hyperglycemia, patients are forced to start therapy with antidiabetic agents. Currently, long-term medication remains an important tool for T2DM control and treatment, and these drugs are characterized by low bioavailability and immediate drug release, resulting in the need to increase the frequency of administration to achieve therapeutic goals. It is inconvenient for the patient [1]. Therefore, there is no ideal drug for the treatment of this disease and it is still urgent and necessary to develop new candidates with improved clinical therapeutic effects. Natural products, particularly those derived from plants, have been proven to exert anti-diabetic effects via diverse mechanisms [2,3]. However, these drugs present several drawbacks that can affect the course of treatment. α-Glucosidase inhibitors are an important class of drugs that can be used for the treatment of T2DM and widely exist in fruits, leaves, seeds, and other tissues and organs of plants. In the past 30 years, research on α-GI from Chinese herbal medicine has become active at home and abroad, and has gradually become a hot spot in the prevention and treatment of diabetes [4]. Based on this situation, in our continuous discovery of structurally interesting and biologically active triterpenes from medicinal plants [5][6][7], four new pentacyclic triterpenes (1 and 7-9), including one new oleanane and three new ursane-type triterpenes, as well as eleven known triterpenes, were isolated from the dried stems of S. discolor Dunn. Furthermore, the α-glycosidase inhibitory activities of these fifteen triterpenoids were screened by an enzyme-inhibitor model using maltose as a substrate. Herein, we mainly describe the structural elucidation of four new pentacyclic triterpenes (1 and 7-9) and the α-glycosidase inhibitory activities of all triterpenoids obtained from S. discolor Dunn. It will be of great significance to provide a scientific basis for the utilization and development of plant resources of genus Sabia.
Molecules 2022, 26, x FOR PEER REVIEW 2 of 13 spot in the prevention and treatment of diabetes [4]. Based on this situation, in our continuous discovery of structurally interesting and biologically active triterpenes from medicinal plants [5][6][7], four new pentacyclic triterpenes (1 and 7-9), including one new oleanane and three new ursane-type triterpenes, as well as eleven known triterpenes, were isolated from the dried stems of S. discolor Dunn. Furthermore, the α-glycosidase inhibitory activities of these fifteen triterpenoids were screened by an enzyme-inhibitor model using maltose as a substrate. Herein, we mainly describe the structural elucidation of four new pentacyclic triterpenes (1 and 7-9) and the α-glycosidase inhibitory activities of all triterpenoids obtained from S. discolor Dunn. It will be of great significance to provide a scientific basis for the utilization and development of plant resources of genus Sabia.

Structural Analysis of New Compounds
The crude petroleum ether extract of S. discolor Dunn was isolated and purified by various column chromatography techniques, including MCI gel, Sephadex LH-20, silica gel, RP-C18 silica gel, and a semipreparative HPLC column, allowing for the isolation of four new pentacyclic triterpenes, namely Sabiadiscolor A-D (1 and 7-9), along with eleven known compounds. Their structures are shown in Figure 1. Compared with the literature, eleven of these structures were known compounds based on their NMR and MS data, and were identified as ursolic acid (2) [8], juglangenin A (3) [9], 3β, 28-dihydroxy-12-oleanenel-one (4) [10], 3-hydroxyolean-12-en-1-one (5) [11], 1α, 2α, 3β-trihydroxyl-olean-12-en-28oic acid (6) [12], dandelion alkan-3β, 20β-diol (10) [13], olean-12-ene-1, 3-diol (11) [14], 3oxo-20S-hydroxytaraxastane (12) [15], ψ-taraxasterone (13) [16], betulinic acid (14) [17], and birch ester alcohol (15) [18].  Compound 1 was obtained as a white solid and its molecular formula was inferred to be C 30 Figure S1), 13 C NMR (Table 2 and Figure S2), and DEPT ( Figure S3) spectral data revealed the presence of eight quaternary carbons, five methine groups, ten methylene groups, and seven methyl groups, including a carbonyl group (δ C 214.6) and a trisubstituted olefinic unit (δ C 122.9 (CH) and δ H 5.20 (dd, J = 4.5, 2.9 Hz, 1H); δ C 144.1 (C)). According to 1 H NMR (Table 1) [10] in Tables 1 and 2 showed that the two compounds should share the same basic skeleton and that both were very similar. According to further HMBC ( Figure S5) correlations in Figure 2A, H-3 (δ H 3.88, m, 1H) was correlated with C-1 (δ C 214.6), C-5 (δ C 51.3), and C-24 (δ C 22.3), and H-28 (δ H 3.30 and 3.21, 2H) was correlated with C-16 (δ C 21.9), C-17 (δ C 36.9), and C-18 (δ C 42.5). Based on this data, it was predicted that the two hydroxyl groups should be at positions C-3 and C-28. Therefore, the planar structure of compound 1 was the same as that of compound 4, as shown in Figure 2A. The relative configuration of compound 1 was further determined according to the NOESY ( Figure S6) correlation spectrum in Figure 2B. The correlation signals between H-23, H-25, and H-3 indicate that H-3 is in the β configuration and the 3-substituted hydroxyl group has an α configuration. The relative configuration of compound 4 was determined according to the NOESY ( Figure S7) correlation spectrum in Figure 2C. The correlation signals between H-24 and H-3 indicated that H-3 was the α configuration and the 3-substituted hydroxyl group has a β configuration. Therefore, it was confirmed that compound 1 and compound 4 are isomers, and compound 1 is 3α, 28-dihydroxy-12-oleanene-l-one, named Sabiadiscolor A.   Figure S1), 13 C NMR (Table 2 and Figure  S2), and DEPT ( Figure S3) spectral data revealed the presence of eight quaternary carbons, five methine groups, ten methylene groups, and seven methyl groups, including a carbonyl group (δC 214.6) and a trisubstituted olefinic unit (δC 122.9 (CH) and δH 5.20 (dd, J = 4.5, 2.9 Hz, 1H); δC 144.1 (C)). According to 1 H NMR (Table 1) [10] in Tables 1  and 2 showed that the two compounds should share the same basic skeleton and that both were very similar. According to further HMBC ( Figure S5) correlations in Figure  Based on this data, it was predicted that the two hydroxyl groups should be at positions C-3 and C-28. Therefore, the planar structure of compound 1 was the same as that of compound 4, as shown in Figure 2A. The relative configuration of compound 1 was further determined according to the NOESY ( Figure S6) correlation spectrum in Figure  2B. The correlation signals between H-23, H-25, and H-3 indicate that H-3 is in the β configuration and the 3-substituted hydroxyl group has an α configuration. The relative configuration of compound 4 was determined according to the NOESY ( Figure S7) correlation spectrum in Figure 2C. The correlation signals between H-24 and H-3 indicated that H-3 was the α configuration and the 3-substituted hydroxyl group has a β configuration. Therefore, it was confirmed that compound 1 and compound 4 are isomers, and compound 1 is 3α, 28-dihydroxy-12-oleanene-l-one, named Sabiadiscolor A.  Compound 7 was obtained as a white solid and its molecular formula was inferred to be C 30 H 50 O 2 with 6 degrees of unsaturation by HR-ESI-MS with 465.3701 [M + Na] + (calc. 465.3703). The IR spectrum of this compound revealed the presence of a hydroxyl group (3368 cm −1 ). As shown in Tables 1 and 2, its 1 H NMR ( Figure S11), 13 C NMR (Figure S12), and DEPT ( Figure S13) spectral data showed that compound 7 contained thirty carbons, including six quaternary carbons, eight methine groups, eight methylene groups, and eight methyl groups. 1 H NMR (Table 1)  showed seven methyl groups at the sp 3 quaternary carbons and one methyl group at the sp 3 tertiary carbon. According to the above data, compound 7 was inferred to be a five-membered ring triterpene with one olefinic unit (δ C 140.0 (C); δ C 119.3 (CH) and δ H 5.33 (dd, J = 6.7, 2.0 Hz, 1H)) and two oxygen-substituted methine groups (δ C 79.7 (CH) and δ H 3.80 (dd, J = 11.1, 4.2 Hz, 1H); δ C 75.5 (CH) and δ H 3.63 (dd, J = 12.0, 4.4 Hz, 1H)). Compared with the chemical structure of the pentacyclic triterpenes isolated from the genus Sabia, all spectral data showed that the compound was an ursane-type pentacyclic triterpene and was structurally similar to the known compound, which is 20-taraxastene-3β, 22α-diol [19]. By further analyzing the HSQC ( Figure S14) and HMBC ( Figure S15) correlation signals of compound 7 in Figure 3A, it was shown that H-3 (δ H 3.63, dd, J = 12.0, 4.4 Hz) was correlated with C-2 (δ C 39.5) and C-24 (δ C 16.2), while H-1 (δ H 3.63, dd, J = 12.0, 4.4 Hz) was correlated with C-3 (δ C 75.5), C-5 (δ C 52.1), and C-25 (δ C 13.2). Based on the above-mentioned data, it was predicted that the two hydroxyl groups should be at positions C-1 and C-3. According to the 1 H-1 H COSY ( Figure S16) signal in Figure 3A, both H-3 (δ H 3.63) and H-1 (δ H 3.80) are correlated with H-2 (δ H 2.36), as shown in Figure 3A. The relative configuration of compound 7 was further determined according to the NOESY ( Figure S17) correlation spectrum in Figures 3B and S16. The correlation signals between H-3 and H-24 indicate that H-3 has an α configuration. The correlation signals between H-3 and H-1 indicate that H-1 has an α configuration. Both substituted hydroxyl groups at C-1 and C-3 had a β configuration. Therefore, the structure of the compound can be determined as 20-taraxastene-1β, 3β -diol, named Sabiadiscolor B. should be at positions C-1 and C-3. According to the 1 H-1 H COSY ( Figure S16) signal in Figure 3A, both H-3 (δH 3.63) and H-1 (δH 3.80) are correlated with H-2 (δH 2.36), as shown in Figure 3A. The relative configuration of compound 7 was further determined according to the NOESY ( Figure S17) correlation spectrum in Figure 3B and Figure S16. The correlation signals between H-3 and H-24 indicate that H-3 has an α configuration. The correlation signals between H-3 and H-1 indicate that H-1 has an α configuration. Both substituted hydroxyl groups at C-1 and C-3 had a β configuration. Therefore, the structure of the compound can be determined as 20-taraxastene-1β, 3β -diol, named Sabiadiscolor B.  1H); δC 139.9 (C)) existed in compound 8. According to NMR spectral data, compound 8 was found to be very similar to the known compound pseudotaraxasterol [20]. By comparing their NMR data, it was found that the main difference lies in the chemical shift values of C-6, C-7, C-23, and C-25. Therefore, it was speculated that another hydroxyl group might be at C-6 or C-7. The HSQC ( Figure S24) and HMBC ( Figure S25) spectra of compound 8 showed that H-3 (δH 3.50) was correlated with C-1 (δC 38.1) and C-23 (δC 12.0). H-6 (δH 3.29) is associated with C-7 (δC 17.9), C-24 (δC 27.8), and C-26 (δC 16.3). The relative configuration of compound 8 was determined according to its NOESY ( Figure S27) correlation spectrum, as shown in Figure 4B. The strong correlations of Me-23/H-3/H-5 and H-6/Me-23 indicated that these protons or methyl groups were α-orientations. Thus, Me-23, H-3, H-5, and H-6 were arbitrarily assigned α-orientations, while two hydroxyl groups on the third and sixth carbons were β-oriented. Therefore, the structure of compound 8 can be determined to be 6β-pseudotaraxasterol, named Sabiadiscolor C.  1H); δ C 139.9 (C)) existed in compound 8. According to NMR spectral data, compound 8 was found to be very similar to the known compound pseudotaraxasterol [20]. By comparing their NMR data, it was found that the main difference lies in the chemical shift values of C-6, C-7, C-23, and C-25. Therefore, it was speculated that another hydroxyl group might be at C-6 or C-7. The HSQC ( Figure S24) and HMBC ( Figure S25) spectra of compound 8 showed that H-3 (δ H 3.50) was correlated with C-1 (δ C 38.1) and C-23 (δ C 12.0). H-6 (δ H 3.29) is associated with C-7 (δ C 17.9), C-24 (δ C 27.8), and C-26 (δ C 16.3). The relative configuration of compound 8 was determined according to its NOESY ( Figure S27) correlation spectrum, as shown in Figure 4B. The strong correlations of Me-23/H-3/H-5 and H-6/Me-23 indicated that these protons or methyl groups were α-orientations. Thus, Me-23, H-3, H-5, and H-6 were arbitrarily assigned α-orientations, while two hydroxyl groups on the third and sixth carbons were β-oriented. Therefore, the structure of compound 8 can be determined to be 6β-pseudotaraxasterol, named Sabiadiscolor C.  Figure S33) spectral data could be classified into six sp 3 quaternary carbons, eight sp 3 methine groups, eight sp 3 methylene groups, and eight methyl groups, as shown in Tables 1 and 2. Among them, one sp 3 methine (δC 77.9 (CH) and δH 3.49 (dd, J = 10.6, 5.6 Hz, 1H)) and one sp 3 quaternary carbon(δC 73.8 (C)) were ascribed as bearing oxygen atoms. According to 1 H NMR data (Table 1) 3H, s, H-27). The comparison of the NMR data of compound 9 with the known compound ursan-3β, which is 5α-diol [21], suggested that compound 9 possessed an ursane-type pentacyclic triterpene skeleton. The HSQC ( Figure S34) and HMBC ( Figure S35) spectra of compound 9 showed that C-13 (δC 73.8) was correlated with H-14 (δH 1.42) and H-18 (δH 1.33), which revealed that the hydroxyl group should be assigned at C-13. The relative configuration of compound 9 was determined according to its NOESY ( Figure S37) correlation spectrum, as shown in Figure 5B. The strong correlations of Me-24/H-3/H-5 indicated that Me-24, H-3, and H-5 were α-orientations, while the hydroxyl group on the third carbon was β-oriented. Therefore, the structure of compound 9 can be determined as ursan-3β, 13β-diol, named Sabiadiscolor D.

α-Glycosidase Inhibitory Activities
All compounds (1-15) isolated from S. discolor Dunn were evaluated for their α-glycosidase inhibitory activity. As shown in Table 3, compounds 1, 3, 8, 9, 13, and 15 showed remarkable activities with IC50 values from 0.09 to 0.27 μM, while compound 7 showed weak activity with an IC50 value of 0.56 ± 0.0331 μM. The other compounds had low inhibitory activity against α-glycosidase and are not listed in Table 3.  Figure S33) spectral data could be classified into six sp 3 quaternary carbons, eight sp 3 methine groups, eight sp 3 methylene groups, and eight methyl groups, as shown in Tables 1 and 2. Among them, one sp 3 methine (δ C 77.9 (CH) and δ H 3.49 (dd, J = 10.6, 5.6 Hz, 1H)) and one sp 3 quaternary carbon(δ C 73.8 (C)) were ascribed as bearing oxygen atoms. According to 1 H NMR data (Table 1) The comparison of the NMR data of compound 9 with the known compound ursan-3β, which is 5α-diol [21], suggested that compound 9 possessed an ursane-type pentacyclic triterpene skeleton. The HSQC ( Figure S34) and HMBC ( Figure S35) spectra of compound 9 showed that C-13 (δ C 73.8) was correlated with H-14 (δ H 1.42) and H-18 (δ H 1.33), which revealed that the hydroxyl group should be assigned at C-13. The relative configuration of compound 9 was determined according to its NOESY ( Figure S37) correlation spectrum, as shown in Figure 5B. The strong correlations of Me-24/H-3/H-5 indicated that Me-24, H-3, and H-5 were α-orientations, while the hydroxyl group on the third carbon was β-oriented. Therefore, the structure of compound 9 can be determined as ursan-3β, 13β-diol, named Sabiadiscolor D.  Figure S33) spectral data could be classified into six sp 3 quaternary carbons, eight sp 3 methine groups, eight sp 3 methylene groups, and eight methyl groups, as shown in Tables 1 and 2. Among them, one sp 3 methine (δC 77.9 (CH) and δH 3.49 (dd, J = 10.6, 5.6 Hz, 1H)) and one sp 3 quaternary carbon(δC 73.8 (C)) were ascribed as bearing oxygen atoms. According to 1 H NMR data (Table 1) 3H, s, H-27). The comparison of the NMR data of compound 9 with the known compound ursan-3β, which is 5α-diol [21], suggested that compound 9 possessed an ursane-type pentacyclic triterpene skeleton. The HSQC ( Figure S34) and HMBC ( Figure S35) spectra of compound 9 showed that C-13 (δC 73.8) was correlated with H-14 (δH 1.42) and H-18 (δH 1.33), which revealed that the hydroxyl group should be assigned at C-13. The relative configuration of compound 9 was determined according to its NOESY ( Figure S37) correlation spectrum, as shown in Figure 5B. The strong correlations of Me-24/H-3/H-5 indicated that Me-24, H-3, and H-5 were α-orientations, while the hydroxyl group on the third carbon was β-oriented. Therefore, the structure of compound 9 can be determined as ursan-3β, 13β-diol, named Sabiadiscolor D.

α-Glycosidase Inhibitory Activities
All compounds (1-15) isolated from S. discolor Dunn were evaluated for their α-glycosidase inhibitory activity. As shown in Table 3, compounds 1, 3, 8, 9, 13, and 15 showed remarkable activities with IC50 values from 0.09 to 0.27 μM, while compound 7 showed weak activity with an IC50 value of 0.56 ± 0.0331 μM. The other compounds had low inhibitory activity against α-glycosidase and are not listed in Table 3.

Discussion
By modern natural medicinal chemistry experiments, fifteen natural pentacyclic triterpenoids (1-15) were obtained and identified from the traditional Chinese ethnic medicinal plant named S. discolor Dunn, collected from minority areas, and four of them (1 and 7-9) were new compounds. Their preliminary α-glycosidase inhibitory activities were evaluated. The results showed that six compounds (1, 3, 8, 9, 13, and 15) showed remarkable activities with IC 50 values of 0.27 ± 0.0499, 0.11 ± 0.0222, 0.23 ± 0.0135, 0.23 ± 0.0307, 0.26 ± 0.0383, and 0.09 ± 0.0045 µM, respectively. It was found that ursane-type pentacyclic triterpenes have better hypoglycemic activities and especially new compounds 1, 8, and 9 have more significant activity than the positive control (Acarbose), which revealed that they might be a class of potential α-glycosidase inhibitors. According to the structure and activity data of these pentacyclic triterpenoids with potential hypoglycemic activity, it is speculated that the number and location of hydroxyl groups as well as double-bond groups might contribute more greatly to the inhibition rate of α-glycosidase, and both oleanane-type pentacyclic triterpenoids and lupanane-type ones should be the potential α-glycosidase inhibitor. Due to limited quantity of the isolated compounds, it is not possible to systematically discuss the structure-activity relationship of such compounds, but compound 15 with the lupanane-type pentacyclic triterpenoid skeleton was implied to have the best activity.
S. discolor Dunn is one of the most important species in the genus Sabia, which is rich in resources in the minority areas of southwest China. The medical plant is used to treat rheumatism, bone pain, bruises, hepatitis, and other diseases in the folk [22]. The main types of chemical constituents in the genus Sabia included pentacyclic triterpenoids, alkaloids, benzene derivatives, and fatty acids [23], but there was less literature reporting on the chemical composition of S. discolor Dunn. As our research suggested its hypoglycemic activity for the first time, it provided an important basis for the comprehensive utilization of this plant resource.
If the inhibitory rate was close to or higher than that of acarbose, the compound was considered to have α-glycosidase inhibitory activity. The IC 50 of the potential compound was measured and calculated by the same method after 5-fold dilution.