Zhi-Qi Yin*a,
Lei Wangab,
Cheng-Hua Lia,
Dong-Mei Zhangb,
Wei Zhangab,
Ying Wangb,
Ming Zhaoc,
Chun-Tao Chec and
Wen-Cai Yeb
aDepartment of Natural Medicinal Chemistry & State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China. E-mail: cpu-yzq@cpu.edu.cn; Tel: +86-025-86185371
bInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China
cDepartment of Medicinal Chemistry and Pharmacognosy, WHO Collaborating Center for Tradition Medicine, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA
First published on 15th May 2017
Six new oleanane-type triterpene saponins, androsides A–F (1–6), along with two known compounds (7–8), were isolated from the aerial parts of Androsace umbellata. The structures were determined on the basis of spectroscopic data and acid hydrolysis. The aglycones in 1–5 are novel structures. Cytotoxicity assays using HepG2, HepG2/ADM, MCF-7, MCF-7/ADR and MDA-MB-231 cell lines indicated that 7 and 8, both bearing a 13β,28-epoxy group, were active. Compound 8 was shown to induce apoptosis in the HepG2/ADM cells.
In the search for bioactive constituents from A. umbellata, we have reported several triterpene and phenolic glycosides from this plant.7,8 Continuing phytochemical studies now led to the isolation of six new triterpenesaponins, androsides A–F (1–6) (Fig. 1), along with two known saponins (7–8).
Androside A (1) was obtained as an amorphous powder. Positive results from both Liebermann–Burchard and Molisch reactions indicated a triterpene glycoside structure. The molecular formula of 1 was determined to be C36H56O11 by the quasi-molecular ion [M + Na]+at m/z 687.3709 (calcd for C36H56O11Na: 687.3714) in HR-ESI-MS. The IR spectrum showed characteristic absorptions for hydroxyl (3442 cm−1), carboxyl (1726 cm−1) and olefinic bonds (1646 cm−1). Acid hydrolysis of 1 afforded D-glucose, which were detected by derivatization and HPLC analysis.
The 1H NMR spectrum of 1 (Table 1) displayed six tertiary methyl signals at δH 0.85, 0.85, 0.86, 1.29, 1.38, and 1.80 (each 3H, s), an olefinic proton at δH 5.62 (1H, br s), as well as an anomeric protons at δH 6.46 (1H, d, J = 8.0 Hz). The 13C NMR and DEPT spectra (Table 1) exhibited 36 carbon signals, 30 of which could be attributed to the aglycone. The 13C NMR resonances at δC 122.5 and 144.7 further suggested the presence of a Δ12,13 double bond on an oleanane skeleton.10 When the 13C NMR spectrum of 1 was compared with that of ardisicrenoside C,11 signals for carbons of rings B–E were almost identical (Table 1). On the other hand, a ketone carbon signal resonating at δC 211.1 was observed, which was assigned to C-2 by the HMBC correlations between δC 211.1 and δH 2.23 and 2.48 (H-1), as well as between δC 211.1 and δH 4.19 (H-3) (Fig. 2). Thus, the aglycone of 1 was determined to be 2-oxo-3β,16α, 28-trihydroxy-olean-12-en-30-oic acid, which was further confirmed by comparison of the NMR data for ring A of 1 with those of dillenic acid B.10
Position | 1 | 2 | 4 | |||
---|---|---|---|---|---|---|
δH | δC | δH | δC | δH | δC | |
a 1H NMR Spectrum was measured at 500 MHz; 13C NMR Spectrum was measured at 125 MHz.b Overlapping signals were reported without designating multiplicity. | ||||||
1 | 2.23, br d (12.0) 2.48, m | 53.8, CH2 | 2.21, d (12.5) 2.47, m | 53.8, CH2 | 6.52, s | 129.5, CH |
2 | — | 211.1, C | — | 211.1, C | — | 146.3, C |
3 | 4.19, s | 83.3, CH | 4.17, s | 83.3, CH | — | 201.1, C |
4 | — | 45.7, C | — | 45.7, C | — | 44.6, C |
5 | 1.55, br d (10.8) | 54.8, CH | 1.56, br d (11.0) | 54.8, CH | 1.65, br d (9.2) | 54.1, CH |
6 | 1.36.1.54, m | 18.9, CH2 | 1.36, 1.56, m | 18.9, CH2 | — | 19.1, CH2 |
7 | 1.35, 1.68, m | 32.8, CH2 | 1.34, 1.69, m | 32.8, CH2 | 1.34, 1.68, m | 33.0, CH2 |
8 | — | 40.3, C | — | 40.3, C | — | 40.6, C |
9 | 2.02, m | 46.7, CH | 2.02, m | 46.7, CH | 2.04, m | 42.8, CH |
10 | — | 43.2, C | — | 43.6, C | — | 38.4, C |
11 | 1.70, 2.01, m | 23.6, CH2 | 1.76, m | 23.7, CH2 | 1.90, 2.05, m | 23.8, CH2 |
12 | 5.62, s | 122.5, CH | 5.79, br s | 122.4, CH | 5.74, br s | 122.6, CH |
13 | — | 144.7, C | — | 144.8, C | — | 145.0, C |
14 | — | 41.9, C | — | 42.0, C | — | 42.2, C |
15 | 1.63, m 2.20, br d (13.4) | 34.9, CH2 | 1.63, m 2.19, dd (6.8.10.8) | 34.8, CH2 | 1.60, m 2.21, br d (11.1) | 34.8, CH2 |
16 | 4.66, s | 74.0, CH | 4.72, br s | 74.0, CH | 4.72, br s | 74.1, CH |
17 | — | 40.4, C | — | 40.4, C | — | 40.6, C |
18 | 2.78, m | 43.4, CH | 2.65, m | 43.4, CH | 2.86, dd (10.8, 11.6) | 43.8, CH |
19 | 2.35, br d (12.7) 2.85, d (12.2) | 44.7, CH2 | 2.41, m 2.88, t (13.0) | 44.2, CH2 | 2.42, 2.88, m | 44.1, CH2 |
20 | — | 44.6, C | — | 44.6, C | — | 44.6, C |
21 | 2.48, m | 33.6, CH2 | 2.53, m | 33.4, CH2 | 2.50, m | 33.9, CH2 |
22 | 2.35, br d (12.7) 2.57, m | 31.8, CH2 | 2.46, m | 31.8, CH2 | 2.43, m | 31.8, CH2 |
23 | 1.29, s | 29.4, CH3 | 1.28, s | 29.4, CH3 | 1.19, s | 27.8, CH3 |
24 | 0.85, s | 17.3, CH3 | 0.84, s | 17.2, CH3 | 1.11, s | 20.2, CH3 |
25 | 0.85, s | 16.6, CH3 | 0.85, s | 16.6, CH3 | 1.10, s | 22.1, CH3 |
26 | 0.86, s | 16.5, CH3 | 0.85, s | 16.5, CH3 | 0.92, s | 17.4, CH3 |
27 | 1.80, s | 27.3, CH3 | 1.80, s | 27.2, CH3 | 1.76, s | 27.1, CH3 |
28 | 3.65, dd (8.2, 2.0) | 70.0, CH2 | 3.52, 3.68, dd (10.8) | 70.1, CH2 | 3.53, 3.70, dd (9.5) | 70.1, CH2 |
29 | 1.38, s | 28.6, CH3 | 1.48 s | 28.5, CH3 | 1.49, s | 28.4, CH3 |
30 | — | 177.1, C | — | 177.1, C | — | 177.2, C |
Glc1′ | 6.46, d (8.0) | 95.9, CH | 6.38, d (8.0) | 93.7, CH | 6.39, d (8.0) | 93.7, CH |
2′ | 4.21, t (8.1) | 74.5, CH | 4.46, d (8.7) | 81.3, CH | 4.46, t (8.5) | 81.3, CH |
3′ | 4.28, t (8.1) | 78.6, CH | 4.20, t (8.9) | 78.5, CH | 4.20, t (8.9) | 79.7, CH |
4′ | 4.30, br d (9.1) | 71.3, CH | 4.29, m | 71.0, CH | 4.29, t (9.0) | 71.0, CH |
5′ | 4.01, m | 79.3, CH | 3.94, m | 79.0, CH | 3.94, m | 79.0, CH |
6′ | 4.34, dd (4.5, 11.6) 4.42, d (9.8) | 62.4, CH2 | 4.30, dd (5.3, 9.5) 4.41, dd (4.5.11.6) | 62.3, CH2 | 4.30, br d (9.0) 4.41, br d (10.8) | 62.3, CH2 |
Glc1′′ | 5.53 d (8.0) | 105.6, CH | 5.55 d (8.0) | 105.6, CH | ||
2′′ | 4.73, br s | 76.4, CH | 4.72, br s | 76.4, CH | ||
3′′ | 4.33, t (8.7) | 78.2, CH | 4.33, t (9.0) | 78.2, CH | ||
4′′ | 4.32, t (8.7) | 71.4, CH | 4.32, t (9.0) | 71.4, CH | ||
5′′ | 3.89, m | 78.4, CH | 3.89, m | 78.4, CH | ||
6′′ | 4.30, 4.41, m | 62.6, CH2 | 4.30, 4.41, m | 62.6, CH2 |
For the glycone part, the anomeric carbon signal at δC 95.9 showed correlation with proton at δH 6.46 (d, J = 8.0 Hz) in the HSQC spectrum. Detailed analysis of the HSQC, HMBC and ROESY data of 1 led to the assignment of a glucopyranosyl unit (Table 1). The β-configuration of the anomeric proton was confirmed by the large 3JH1-H2 coupling constant. The linkage position of the sugar unit was determined with the aid of HMBC data (Fig. 2), in which correlation between H-1 of glucose (δH 6.46) and C-30 of the aglycone (δC 177.1) was clearly observed. In conclusion, all available evidence suggested the structure of 2-oxo-3β, 16α, 28-trihydroxy-olean-12-en-30-oic acid 30-O-β-D-glucopyranosyl ester for 1. It is noteworthy that the aglycone of 1 is reported for the first time.
Androside B (2) was isolated as an amorphous powder. The high resolution ESI-MS spectrum showed a quasi-molecular ion at m/z 849.4259 [M + H]+ (calcd for C42H67O16: 849.4243), consistent with the molecular formula C42H66O16. The 1H NMR spectrum of 2 (Table 1) displayed six tertiary methyl signals at δH 0.84, 0.85, 0.85, 1.28, 1.48, and 1.80 (each s), an olefinic proton at δH 5.79 (br s), as well as two anomeric protons at 6.38 (d, J = 8.0 Hz) and 5.53 (d, J = 8.0 Hz). The 13C NMR data (Table 1) were similar to those of 1 except for the appearance of an additional glucopyranosyl unit (δC 105.6, 76.4, 78.2, 71.4, 78.4, 62.6). Acid hydrolysis of 2 afforded D-glucose only, which was identified by derivatization and HPLC analysis. Comparison of the NMR data of 2 with those of 1 revealed the downfield shift (+6.8 ppm) for C-2′ of glucose, suggesting an additional glucopyranosyl residue at C-2′. This conclusion was supported by the HMBC correlation between H-1′′ (δH 5.53) of the terminal glucose and C-2′ (δC 81.3) of the inner glucose. Based on the above evidence, 2 was determined to be 2-oxo-3β,16α,28-thihydroxy-olean-12-en-30-oic acid 30-O-β-D-glucopyranosyl(1→2)-glucopyranosyl ester.
The molecular formula of androside C (3) was determined to be C42H66O15 by HR-ESI-MS (833.4288 [M + Na]+; calcd for C42H66O15Na: 833.4293). The 1H and 13C NMR spectral features of the aglycone (Table 2) were similar to those of 2, suggesting the same aglycone structure. For the glycone part of 3, the presence of two sugar units was implied by the observation of two anomeric proton signals at δH 6.32 (d, J = 8.0 Hz) and 6.56 (br s), as well as two anomeric carbon signals at δC 101.5 and 94.5. Indeed, acid hydrolysis of 3 yielded L-rhamnose and D-glucose. The anomeric configurations of D-glucose and L-rhamnose were determined to be β and α, respectively, based on the coupling constants of the anomeric protons and 13C NMR data.12
Position | 3 | 5 | 6 | |||
---|---|---|---|---|---|---|
δH | δC | δH | δC | δH | δC | |
a 1H NMR Spectrum was measured at 500 MHz; 13C NMR Spectrum was measured at 125 MHz.b Overlapping signals were reported without designating multiplicity. | ||||||
1 | 2.17, 2.46, m | 53.8, CH2 | 6.54, s | 129.5 d | 0.88, 1.41, m | 38.9, CH2 |
2 | — | 211.1, C | — | 146.3, C | 2.18, 1.78, m | 26.6, CH2 |
3 | 4.18, s | 83.3, CH | — | 201.1, C | 3.27, dd (8.0, 2.0) | 88.9, CH |
4 | — | 45.7, C | — | 44.6, C | — | 39.5, C |
5 | 1.56, m | 54.8, CH | 1.65, m | 54.1, CH | 0.71, d (11.7) | 55.8, CH |
6 | — | 18.9, CH2 | — | 19.1, CH2 | 1.42, 1.29, m | 18.4, CH2 |
7 | 1.34, 1.68, m | 32.8, CH2 | 1.34, 1.68, m | 33.0, CH2 | 1.56, 1.27, m | 33.2, CH2 |
8 | — | 40.3, C | — | 40.6, C | — | 40.0, C |
9 | 2.02, t (9.8) | 46.8, CH | 2.04, m | 42.8, CH | 1.66, t (8.9) | 47.0, CH |
10 | — | 43.4, C | — | 38.4, C | — | 36.8, C |
11 | 1.76, m | 23.7, CH2 | 1.9, 2.05, m | 23.8, CH2 | 1.78, m | 23.7, CH2 |
12 | 5.79, br s | 122.4, CH | 5.84, br s | 122.6, CH | 5.64, br s | 123.1, CH |
13 | — | 144.8, C | — | 145.0, C | — | 144.7, C |
14 | — | 41.9, C | — | 42.2, C | — | 41.7, C |
15 | 1.63, br d (14.2) 2.18, br d (12.2) | 34.9, CH2 | 1.60, 2.18, m | 34.8, CH2 | 1.61, 2.19, | 34.9, CH2 |
16 | 4.62, br s | 74.0, CH | 4.62, br s | 74.1, CH | 4.66, br s | 74.2, CH |
17 | 40.5, C | — | 40.6, C | — | 40.4, C | |
18 | 2.88, m | 42.8, CH | 2.88, m | 43.0, CH | 2.74, m | 43.2, CH |
19 | 2.49, 2.91, m | 44.1, CH2 | 2.45, 2.91, m | 44.0 t | 2.34, 2.78, m | 44.7, CH2 |
20 | — | 44.7, C | — | 44.6, C | — | 44.7, C |
21 | 2.50, m | 33.9, CH2 | 2.50, m | 33.9, CH2 | 2.49, 1.58, m | 33.6, CH2 |
22 | 2.35, 2.43, m | 31.8, CH2 | 2.35, 2.43, m | 31.8, CH2 | 2.58, 2.34, m | 31.8, CH2 |
23 | 1.29, s | 29.4, CH3 | 1.20, s | 27.8, CH3 | 1.25, s | 28.1, CH3 |
24 | 0.84, s | 17.3, CH3 | 1.10, s | 20.2, CH3 | 1.09, s | 16.9, CH3 |
25 | 0.85, s | 16.6, CH3 | 1.12, s | 22.1, CH3 | 0.82, s | 15.7, CH3 |
26 | 0.86, s | 16.5, CH3 | 0.93, s | 17.4, CH3 | 0.87, s | 16.8, CH3 |
27 | 1.81, s | 27.3, CH3 | 1.77, s | 27.1, CH3 | 1.81, s | 27.4, CH3 |
28 | 3.62, dd (8.2, 2.0) | 69.8, CH2 | 3.62, dd (8.2, 2.0) | 70.1, CH2 | 3.65, dd (8.2, 2.0) | 70.0, CH2 |
29 | 1.54, s | 28.4, CH3 | 1.54, s | 28.4, CH3 | 1.37, s | 28.6, CH3 |
30 | — | 177.3, C | — | 177.2, C | — | 177.1, C |
Glc1′ | 6.32, d (8.0) | 94.5, CH | 6.34, d (8.0) | 94.5, CH | 4.88, d (7.5) | 105.0, CH |
2′ | 4.51, t (8.8) | 76.1, CH | 4.53, m | 76.2, CH | 4.21, dd (2.8.8.8) | 83.4, CH |
3′ | 4.32, t (9.0) | 78.6, CH | 4.33, m | 79.7, CH | 4.25, dd (4.0.9.0) | 77.9, CH |
4′ | 4.23, m | 71.7, CH | 4.24, m | 71.8, CH | 4.11, t (9.3) | 71.7, CH |
5′ | 3.96, m | 79.7, CH | 3.97, m | 78.6, CH | 3.88, m | 78.3, CH |
6′ | 4.26, m 4.36, dd (2.5.11.8) | 62.4, CH2 | 4.26, br d (9.4) 4.36, br d (11.4) | 62.6, CH2 | 4.42, br d (10.0) 4.51, dd (2.3.11.7) | 62.7, CH2 |
Glc1′′ | 5.34, d (7.5) | 106.0, CH | ||||
2′′ | 4.09, t (9.0) | 77.1, CH | ||||
3′′ | 4.21, dd (2.8.8.8) | 78.2, CH | ||||
4′′ | 4.11, t (9.3) | 71.6, CH | ||||
5′′ | 3.88, m | 77.9, CH | ||||
6′′ | 4.42, br d (10.0) 4.45, dd (3.2.11.5) | 62.8, CH2 | ||||
Rha 1′′ | 6.56, s | 101.5, CH | 6.56, br s | 101.5, CH | ||
2′′ | 4.73, m | 72.5, CH | 4.74, m | 72.5, CH | ||
3′′ | 4.52, t (8.8) | 72.5, CH | 4.53, m | 72.5, CH | ||
4′′ | 4.28, m | 74.1, CH | 4.28, br d (7.9) | 73.9, CH | ||
5′′ | 4.66, dd (6.2, 9.4) | 70.1, CH | 4.66, dd (6.2, 9.9) | 69.8, CH | ||
6′′ | 1.73, d (6.1) | 18.8, CH3 | 1.73, d (6.1) | 18.8, CH3 | ||
Glc 1′′′ | 6.45, d (8.0) | 95.9, CH | ||||
2′′′ | 4.21, dd (2.8.8.8) | 74.4, CH | ||||
3′′′ | 4.29, t (7.5) | 78.6, CH | ||||
4′′′ | 4.30, t (6.9) | 71.2, CH | ||||
5′′′ | 3.99, m | 79.3, CH | ||||
6′′′ | 4.33, dd (4.8.11.8) 4.42, br d (10.0) | 62.4, CH2 |
The sequence and linkage of the sugars were then determined by 2D NMR analysis. Thus, HMBC cross peaks between H-1′′ (δH 6.56) of rhamnose and C-2′ (δC 76.1) of glucose, and between H-1′ of glucose (δH 6.32) and C-30 of the aglycone (δC 177.3), were clearly observed. The above findings led to the assignment of 3 as 2-oxo-3β, 16α,28-thihydroxy-olean-12-en-30-oic acid 30-O-α-L-rhamnopyrano syl(1→2)-β-D-glucopyranosyl ester.
Androside D (4) was shown to possess a molecular formula C42H64O16 from its HR-ESI-MS data, m/z 847.4088 [M + Na]+ (calcd for C42H64O16Na: 847.4092). The IR spectrum displayed characteristic absorptions of hydroxyl (3424 cm−1), carboxyl (1728 cm−1) and olefinic bonds (1642 cm−1). In the UV spectrum, an absorption maximum at 270 nm indicated the presence of an α, β-unsaturated ketone. The 1H NMR spectrum of 4 (Table 1) displayed signals for six tertiary methyl groups [δH 0.92, 1.10, 1.11, 1.19, 1.49 and 1.76 (each s)], two olefinic protons [δH 6.52 (s) and 5.74 (br s)], and two anomeric protons [δH 6.39 (d, J = 8.0 Hz) and 5.55 (d, J = 8.0 Hz)]. In the 13C NMR and DEPT spectra (Table 1), 42 carbon signals (Table 1) were observed, of which 30 signals could be assigned to the aglycone. Comparison of the NMR data of 4 with those of 2 revealed the signals were almost identical, except for differences in ring A. The HMBC correlations between C-3 (δC 201.1) and H-1 (δH 6.52)/H-23 (δH 1.19)/H-24 (δH 1.11) led to the assignment of a carbonyl at the C-3 position (Fig. 2). Furthermore, HMBC cross peaks between H-1 (δH 6.52) and C-2 (δC 146.3)/C-5 (δC 54.1)/C-9 (δC 42.8)/C-25 (δC 22.1) revealed a double bond between C-1 and C-2, as well as a hydroxyl group at C-2 (Fig. 2). These conclusions could be further confirmed by the ROESY correlations between H-1(δH 6.52) and H-9 (δH 2.04), and between H-1 and H-25 (δH 1.10). Therefore, the aglycone of 4 was identified to be 3-oxo-2, 16α, 28-trihydroxy-olean-1, 12-dien-30-oic acid.
For the glycone part, D-glucose was identified by acid hydrolysis and HPLC analysis. The NMR data for the sugar chain of 4 were identical to those of 2, indicating the presence of 30-O-β-D-glucopyranosyl(1→2)-glucopyranosyl ester, which could be further verified by an HMBC experiment (Fig. 2). Thus, HMBC correlations between H-1 (δH 6.52) of the outer glucose and C-2 (δC 81.3) of the inner glucose, as well as between H-1 (δH 6.39) of the inner glucose and C-30 (δC 177.2) of aglycone, were observed. Based on the above evidence, the structure of 4 was established to be 3-oxo-2,16α,28-trihydroxy-olean-1,12-dien-30-oic acid 30-O-β-D-glucopyranosyl(1→2)-glucopyranosyl ester.
The molecular formula of androside E (5) was assigned as C42H64O15 on the basis of its HR-ESI-MS data (m/z 831.4143 [M + Na]+; calcd for C42H64O15Na: 831.4137). The 1H and 13C NMR spectra (Table 2) indicated that 5 was also a triterpene saponin bearing two sugar units. The NMR data of 5 were similar to those of 4, except the signals of glucopyranosyl in 4 (δC 105.6, 76.4, 78.2, 71.4, 78.4 and 62.5) were now replaced by those of a rhamnopyranosyl unit (101.5, 72.5, 72.5, 73.9, 69.8 and 18.8). Acid hydrolysis of 5 yielded D-glucose and L-rhamnose. The chemical shifts and J values of the anomeric protons indicated the β-configuration of glucopyranosyl and α-configuration of rhamnopyranosyl units. In the HMBC spectrum, cross-peaks were observed between H-1 (δH 6.58) of rhamnose and C-2 (δC 76.2) of glucose, as well as between H-1 (δH 6.34) of glucose and C-30 (δC 177.2) of aglycone. Thus, the structure of 5 was determined to be 3-oxo-2, 16α,28-trihydroxy-olean-1,12-dien-30-oic acid 30-O-α-L-rhamnopy ranosyl(1→2)-β-D-glucopyranosyl ester. It is noteworthy that the new aglycone structure in 4 and 5 is reported for the first time.
The molecular formula of androside F (6) was determined to be C48H78O20 by its HR-ESI-MS spectrum (m/z 1009.4775 [M + Cl]−; calcd for C48H78O20Cl, 1009.4775). The 1H and 13C NMR spectroscopic data of the aglycone (Table 2) were in good agreement with those of ardisicrenoside C11 The β-configuration of 3-OH and α-configuration of 16-OH were deduced by the coupling constants of H-3 (dd, J = 8.0, 2.0 Hz) and H-16 (br s), respectively. Thus, the aglycone of 6 was determined to be jacquinic acid (3β, 16α, 28-trihydroxy-olean-12-en-30-oic acid). The NMR data for the sugar moiety and GC analysis of the derivatives of its acid hydrolysateled to the assignment of three β-D-glucopyranose units. Connectivity of the sugars was determined on the basis of HMBC data. Thus, correlations between H-1 of the inner glucose [δH 4.88 (1H, d, J = 7.5 Hz)] and C-3 of the aglycone (δC 88.9), between H-1 of the outer glucose [δH 5.34 (1H, d, J = 7.5 Hz)] and C-2 of the inner glucose (δC 83.4), as well as between H-1 of the third glucose [δH 6.45 (1H, d, J = 8.0 Hz)] and C-30 (δC 177.1) of the aglycone, were observed. These data revealed the structure of sugar chain as shown in Fig. 1, which was further confirmed by comparison of the NMR data with those of asteryunnanoside I.13 Consequently, the structure of 6 was elucidated to be 3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl 3β,16α,28-trihydroxy-olean-12-en-30-oic acid 30-O-β-D-glucopyranosyl ester.
Compounds | IC50 ( ± SD) μMb | ||||
---|---|---|---|---|---|
HepG2 | HepG2/ADM | MCF-7 | MCF-7/ADR | MDA-MB-231 | |
a Cytotoxic activities of compounds 1–8 were tested by using MTT assay. All data are presented as means ± standard deviation of at least three independent experiments.b IC50: concentration of the tested compound inhibiting 50% cell growth.c DOX: doxorubicin was used as positive control. | |||||
DOXc | 0.18 ± 0.03 | 143.62 ± 5.12 | 0.85 ± 0.24 | 37.86 ± 5.56 | 21.13 ± 0.15 |
1 | >200 | >200 | >200 | >200 | >200 |
2 | >200 | >200 | >200 | >200 | >200 |
3 | >200 | >200 | >200 | >200 | >200 |
4 | >200 | >200 | >200 | >200 | >200 |
5 | >200 | >200 | >200 | >200 | >200 |
6 | >200 | >200 | >200 | >200 | >200 |
7 | 40.34 ± 2.51 | 36.19 ± 2.57 | 42.57 ± 3.75 | 43.86 ± 2.50 | 51.86 ± 5.86 |
8 | 17.71 ± 0.62 | 9.97 ± 0.46 | 10.32 ± 0.13 | 10.52 ± 1.13 | 9.29 ± 0.32 |
In the presence of compound 8, an increase in sub-G1 DNA content was observed in HepG2/ADM cells after 72 h treatment in a dose-dependent manner (Fig. 3A). Apoptosis was analyzed by Annexin V-FITC/PI double staining assay and the population of apoptotic cells was quantified. As shown in Fig. 3B, the population of early and late apoptotic cells was increased from 19.1% to 44.5% in a dose-dependent manner. Compound 8 also induced PARP cleavage (Fig. 3C), a marker of apoptosis.14 Taken together, the findings suggested that compound 8 induced apoptosis in HepG2/ADM cells.
Footnote |
† Electronic supplementary information (ESI) available: Spectroscopic data and other relevant information for compounds 1–8. See DOI: 10.1039/c7ra03948d |
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