G.F. Chi, et al.
Steroids163(2020)108724
Table 2
1H (600 MHz) and 13C (150 MHz) NMR data for sugar units of 1–4.
Position
δH (J in Hz)
δC
37.3
δH (J in Hz)
δC
37.1
δH (J in Hz)
δC
37.1
δH (J in Hz)
δC
37.1
1
1.80, m0.97, m
1.72, m0.92, m
1.75, m1.38, m
3.44, m
1.88, m
1.90, m
2
1.75, m1.40, m
29.5
76.4
38.0
140.7
121.7
31.9
31.4
50.0
36.9
20.8
38.0
40.5
56.2
31.4
80.6
62.2
16.5
19.4
41.5
15.2
108.9
31.9
28.9
30.3
66.4
17.6
29.3
77.7
38.0
140.4
121.2
31.4
31.3
50.2
36.2
20.5
39.4
40.2
56.3
31.3
81.0
63.6
15.4
18.4
39.7
14.7
112.5
31.7
27.6
33.5
74.5
15.8
1.74, m1.30, m
3.59, m
29.3
77.9
38.0
140.4
121.1
31.3
29.9
50.2
36.6
20.7
39.7
40.4
56.3
31.7
81.0
63.6
15.4
18.4
40.0
14.7
112.5
31.3
27.5
33.5
74.8
15.9
1.93, m1.62, m
3.65, m
29.3
77.7
38.0
140.5
121.2
31.3
33.5
50.2
36.6
20.5
39.7
40.4
56.3
31.7
81.0
63.6
15.4
18.4
40.4
14.7
112.5
31.3
27.5
33.5
74.5
15.9
46.2
3
3.48, m
4
2.16, t (11.7)2.42, dd (11.7, 4.6)
2.32, m2.09, m
2.28, m2.43, m
2.33, m2.46, m
5
6
5.34, br d (4.7)
1.56, m1.30, m
1.62, m
5.28, br s
1.82, m1.07, m
1.47, m
5.40, br s
1.98, m1.28, m
1.92, m
5.40, d (3.0)
1.68, m1.32, m
1.77, m
7
8
9
0.89, m
0.82, m
0.98, m
0.97, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
22-OCH3
1.57, m
1.41, m
1.56, m
1.58, m
2.15, t (12.4)1.80, m
1.63, m1.06, m
1.78, m1.18, m
2.19, t (6.6)1.80, m
1.15, m
2.01, m
4.27, m
1.67, m
0.73, s
1.01, m
1.88, m
4.38, m
1.61, m
0.67, s
1.13, m
1.98, m
4.39, m
1.74, m
0.85, s
1.11, m
1.58, m
4.39, dd (6.0, 1.8)
1.75, m
0.85, s
0.96, s
0.89, s
1.06, s
1.07, s
1.82, m
0.91, d (6.8)
1.89, m
0.85, d (6.6)
2.18, m
1.03, d (6.8)
2.19, m
1.03, d (7.2)
1.93, m
1.88, m
1.65, m
1.68, m
1.23, m
1,43, m
1.14, m
1.20, m
1.75, m
1.58, m
1.76, m
1.77, m
3.41, m3.18, m
0.75, d (6.4)
3.48, m3.25, m
0.76, d (6.3)
3.72, m3.42, m
0.85, d (4.4)
3.75, m3.41, m
0.96, d (7.2)
3.16, s
a
b
Recorded in DMSO‑d6.
Recorded in MeOD.
cells treated with various concentrations of test samples were incubated
at 37 °C in a humidified atmosphere and 5% CO2 for 72 h. The fluor-
escence was then measured with an Infinite M2000 ProTM plate reader
(Tecan, Crailsheim, Germany) using an excitation wavelength of
544 nm and an emission wavelength of 590 nm. The IC50 values were
calculated from a calibration curve by linear regression using Microsoft
Excel 2013. Doxorubicin was used as a positive control.
germinal protons H-26a (δ 3.41, m) and H-26b (δ 3.18, m)
(Δδab = 0.23 ppm): Δδab < 0.48 ppm for 25R and Δδab > 0.57 ppm
for 25S [28]. The Δ5-spirostane type steroidal tetracyclic skeleton was
verified by key HMBC correlations (Fig. 2) of H-18 (δH 0.73) with C-13
(δHC 40.2), C-14 (δC 56.2), C-17 (δC 62.2), and the correlation of H-19
(δH 0.91) with C-1 (δC 37.2), C-5 (δC 140.9), C-9 (δC 50.0), C10 (δHC
36.8). The 1H NMR spectrum showed the presence of two anomeric
proton signals at δH 4.36 (1H, d, J = 8.0) and 5.04 (1H, br s) correlating
in the HSQC spectrum with two anomeric carbon signals at δC 98.5 and
100.5, respectively, indicating the existence of two sugar moieties in 1.
Acid hydrolysis of compound 1 with 2 M HCl liberated D-glucose and L-
rhamnose on the basis of comparative TLC analysis using authentic
samples of sugars as reference and also by comparing their 13C NMR
data, coupling constants with literature. The coupling constant of the
anomeric protons (3J1,2 > 7.0 Hz) revealed the β-configuration of
glucose, and coupling constant (3J1,2 < 7.0 Hz) implied the α-config-
uration of rhamnose. The sequence and linkage sites of the sugar chain
were deduced from the glycosylation-shifted 13C resonance for C-3 (δC
76.7), as well as from the HMBC data. The key HMBC correlations
(Fig. 2) between Glc-H-1 (δH 4.36) and C-3 (δHC 76.7) and Rha-H-1 (δC
5.04) and Glc-C-3 (δC 77.0) further suggested a sequence of the dis-
accharide chain at C-3. Furthermore, the NMR data of compound 1
showed it to be nearly identical to the known compound progenin III.
The most substantial differences between progenin III and compound 1
were revealed from the NOESY correlation between C-21 protons of a
methyl (δH 0.91) and C-23 protons of a methylene (δH 1.85), indicating
that methyl and methylene protons are α-oriented in compound 1 but β-
oriented in progenin III. Thus, the structure of compound 1 was de-
termined to be (25R)-spirost-5-ene-3β,22β-3-O-β-D-glucopyranosyl(1 →
2)-O-α-L-rhamnopyranoside.
3. Results and discussion
The air-dried fruits of R. vinifera (1.23 kg) were extracted twice with
CH3Cl/MeOH (1:1) for 48 h by percolation at room temperature and the
resulting extract was partitioned successively between n-hexane and
EtOAc. Repeated silica gel column chromatography led to the isolation
of four new secondary metabolites (1–4) and six previously isolated
compounds (6–10). The structures of these known compounds were
identified as, diosgenin (5) [18,19] diosgenin-3-O-β-D-glucopyranoside
viously as trillin (6) [20] deltonin (7) [21] progenin III (8) [22] 26-O-β-
molecular formula C39H62O12 based on its positive mode HRESIMS
analysis ([M + Na]+ m/z 745.4131, calcd 745.4139) and 13C NMR
typical angular methyl proton signals at δH 0.73 (s, CH3-18) and 0.96 (s,
CH3-19), two methyl doublets at δH 0.91 (d, J = 6.8 Hz, CH3-21) and
0.75 (d, J = 6.4 Hz, CH3-27), two olefinic carbon signals at δC 140.9 (C-
5) and 121.4 (C-6), and five characteristic carbon signals of an F-ring in
a (25R) form at δC 108.8 (C-22), 31.9 (C-23), 28.9 (C-24), 30.2 (C-25),
66.3 (C-26) and 17.5 (C-27), indicating the presence of a steroidal
aglycone of the (25R)-spirost-5-ene type [25–27]. The 25R configura-
Compound 2 was obtained as a cream powder. The HRESIMS ana-
lysis (m/z 925.4756, calcd 925.4773), together with its NMR data
1
3