SUPPLEMENTARY MATERIAL
A new pregnane glycoside from Rubus phoenicolasius and its antiproliferative activity
Chao Liu a,b
, Zhi-Xin Liao a
*, Shi-Jun Liu a
, Jin-Yue Sun b
, Gui-Yang Yao c and
Heng-Shan Wang c a
Department of Pharmaceutical Engineering, School of Chemistry and Chemical
Engineering, Southeast University, Nanjing 211189, PR China; b Institute of Agro-Food
Science and Technology, Shandong Academy of Agricultural Sciences, 202 Gongye
North Road, Jinan 250100, PR China ;
c
State Key Laboratory Cultivation Base for the
Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal
University, Guilin 541004, PR China
*Corresponding author. E-mail: zxliao@seu.edu.com

Abstract
Chemical investigations of the whole plant ethanol extract of Rubus phoenicolasius led to the isolation and identification of a new pregnane glycoside, 3O β -glucopyranosyl-3 β ,15 β - dihydroxypregn-5-en-20-one ( 1 ), along with other nine known compounds ( 2
–
10 ). All the isolates were reported from this plant for the first time. The structure of Compound 1 was determined by detailed analysis of their spectral data including 1D and 2D NMR. In vitro anti-proliferative activities of Compounds 1 – 3 on MCF-7 and NCI-H460 tumor cell lines were evaluated, and
Compound 1 was active against the two cell lines with IC
50
values of 15.6 and 13.5 μM, respectively.
Keywords: Rubus phoenicolasius ; Rosaceae; pregnane glycoside; antiproliferative
1

1. Extraction and isolation
The dried plant (10.0 kg) was ground into powder and then extracted with 90% ethanol (100.0 L) at room temperature (4 × 7 days). Following filtration and vacuum-concentration of the combined solution yielded a crude extract (1069.0 g), which was then suspended in water (3000 mL) and extracted successively with petroleum ether (3 × 1000 mL), EtOAc (3 × 1000 mL) and n -BuOH (3 × 1000 mL) to afford three organic fractions.
The petroleum ether fraction (135.0 g) was submitted to column chromatography (CC) over silica gel, eluted with petroleum ether – ethyl acetate gradient to yield five subfractions (Fr.1 – Fr.4). The Fr. 2 – 4 were firstly subjected to
MCI gel CC (80% ethanol) to remove pigments. Fr. 2 was separated on silica gel CC eluted with petroleum ether – ethyl acetateto yield compounds 4 (638.7 mg) and 5
(154.4 mg). Fr. 3 was submitted to silica gel CC eluted with petroleum ether–ethyl acetate to yield 6 (545.3 mg). Purification of Fr. 4 by silica gel CC eluted with petroleum ether – ethyl acetate to afford compound 7 (22.2 mg) and 8 (12.2 mg).
The extract of EtOAc (86.5 g) was submitted to silica gel CC eluted gradiently with petroleum ether – ethyl acetate to produce fractions I – IV. Fraction II was first separated by silica gel CC eluted with petroleum ether – Me
2
CO, and then purified using Sephadex LH-20 to yield compound 2 (68.4 mg) and 9 (13.8 mg). Fr. III was fractionated by silica gel CC eluted with petroleum ether – ethyl acetate. The fraction was further separated over Sephadex LH-20 CC eluted with ethanol – water (7:3, 8:2,
9:1) to yield 3 (17.0 mg, 8:2) and 10 (12.0 mg, 9:1). Compound 1 (109.7 mg) was obtained from Fr. IV through silica gel CC eluted with CHCl
3
– MeOH.
2. Cytotoxicity activity experiments
The cytotoxic effects of compounds 1–3 were estimated in vitro against the MCF-7 and NCI-H460 cancer cell lines by MTT assay. Briefly, the cell suspensions (200 mL) at a density of 5 × 10 4 cells·mL
−1
were distributed into 96-well cell culture plates and cultured at 37°C in incubator with 5% CO
2
for 24 h. The solution of five different concentrations of the test compounds (2 mL in DMSO) was added to each well and
2

further incubated for 48 h under the same conditions. The MTT solution (20mL) was then added to each well and incubated for 4 h. Finally, the supernatant was discarded and limited DMSO was added to each well to dissolve the blue-violet crystals completely, the optical density (OD) values were then read on the microplate reader at
570 nm. All tests and analyses were carried out in triplicate. Dose-response curves were generated and the IC
50
values were defined as the concentration of compound required to inhibit cell proliferation by 50%. Cis -platinum, an approved agent for the treatment of many tumors, was applied as the positive control.
3. The chemical data for nine known compounds 2-10
3
β
-hydroxy-cholest-5-en-7-one ( 2 ): mp 168~169
℃, 1
H-NMR (500 MHz, CDCl
3
)
δ
(ppm): 1.12 (3H, s), 0.88 (3H, s), 0.70 (3H, s), 0.89 (3H, d, J = 6.6 Hz), 0.94 (3H, d, J
= 6.4 Hz);
13
C-NMR (125 MHz, CDCl
3
) δ (ppm): 36.1 (C-1), 31.2 (C-2), 70.5 (C-3),
41.8 (C-4), 165.1 (C-5), 126.1 (C-6), 203.3 (C-7), 45.1 (C-8), 49.9 (C-9), 38.3 (C-10),
21.2 (C-11), 38.7 (C-12), 41.8 (C-13), 49.9 (C-14), 26.3 (C-15), 28.5 (C-16), 54.7
(C-17), 12.0 (C-18), 17.3 (C-19), 35.7 (C-20), 18.9 (C-21), 36.2 (C-22), 23.8 (C-23),
39.5 (C-24), 28.0 (C-25), 22.6 (C-26), 22.8 (C-27).
7-oxoβ -sitosterol ( 3 ): 1 H-NMR (400 MHz, CDCl
3
) δ (ppm): 1.20 (3H, s), 0.87 (3H, s), 0.68 (3H, s), 0.82 (3H, d, J = 7.0 Hz), 0.94 (3H, d, J = 7.0 Hz); 13 C-NMR (100
MHz, CDCl
3
) δ (ppm): 36.3 (C-1), 31.2 (C-2), 70.5 (C-3), 41.8 (C-4), 165.2 (C-5),
126.1 (C-6), 202.4 (C-7), 45.4 (C-8), 49.9 (C-9), 38.3 (C-l0), 21.2 (C-11), 38.7 (C-12),
43.1 (C-13), 49.9 (C-14), 26.3 (C-15), 28.5 (C-16), 54.7 (C-17), 12.0 (C-18), 17.3
(C-19), 36.1 (C-20), 18.9 (C-21), 33.9 (C-22), 26.0 (C-23), 45.8 (C-24), 29.1 (C-25),
18.6 (C-26), 19.8 (C-27), 23.0 (C-28), 12.0 (C-29).
Stigmasterol ( 4 ): IR (KBr,
 max
cm -1 ): 3400, 3100, 1677 。 EI-MS m/z : 412 [M + H] + ,
397, 369, 340, 301, 273.
1
H-NMR (300 MHz, CDCl
3
)
δ
(ppm): 0.66 (3H, s, H-18),
0.78 (3H, d, J = 6.0 Hz, H-27), 0.81 (3H, d, J = 6.2 Hz, H-26), 0.82 (3H, t, J = 7.5 Hz,
H-29), 0.90 (3H, d, J = 6.5 Hz, H-21), 0.98 (3H, s, H-19), 5.32 (1H, brs, H-6), 3.52
(1H, m, H-3a), 5.17 (1H, dd, J = 14.6, 8.0 Hz, H-22), 5.03 (1H, dd, J = 14.6, 8.0 Hz,
3

H-23)
； 13
C-NMR (75 MHz, CDCl
3
)
δ
(ppm): 31.6 (C-1), 31.9 (C-2), 71.8 (C-3), 39.7
(C-4), 140.7 (C-5), 121.6 (C-6), 31.9 (C-7), 31.6 (C-8), 50.2 (C-9), 36.5 (C-10), 21.6
(C-11), 39.6 (C-12), 42.8 (C-13), 56.6 (C-14), 24.3 (C-15), 28.0 (C-16), 56.6 (C-17),
11.9 (C-18), 19.4 (C-19), 40.5 (C-20), 21.6 (C-21), 138.5 (C-22), 129.5 (C-23), 51.6
(C-24), 31.6 (C-25), 18.9 (C-26), 21.6 (C-27).
Ursolicacid ( 5 ) ： EI-MS m/z : 456 [M]
+
, 423, 438, 410, 248, 207, 203, 189.
1
H-NMR
(300 MHz, DMSOd
6
)
δ
(ppm): 5.13 (1H, t, C
12
-H), 3.01 (1H, m, C
3
-H), 0.82 (3H, d,
J = 6.40 Hz, C
29
-H), 0.92 (3H, d, J = 7.60 Hz, C
30
-H), 1.04, 0.90, 0.87, 0.75, 0.68
(each 3H, s), 11.91 (1H, brs, COOH). 13 C-NMR (75 MHz, DMSOd
6
) δ (ppm): 38.4
(C-1), 26.9 (C-2), 76.8 (C-3), 38.4 (C-4), 54.7 (C-5), 17.9 (C-6), 32.7 (C-7), 38.7
(C-8), 46.8 (C-9), 36.3 (C-10), 22.8 (C-11), 124.5 (C-12), 138.1 (C- 13), 41.6 (C-14),
28.2 (C-15), 23.8 (C-16), 52.3 (C-17), 47.0 (C-18), 38.3 (C-19), 38.2 (C-20), 30.1
(C-21), 36.5 (C-22), 27.5 (C-23), 15.2(C-24), 16.0 (C-25), 16.9 (C-26), 23.2 (C-27),
178.2 (C-28), 16.9 (C-29), 21.0 (C-30).
Oleanolic acid ( 6 ) : ESI-MS m/z : 457 [M + H]
+
, 455 [M - H]
-
;
1
H-NMR (300 MHz,
CDCl
3
)
δ
H
(ppm): 0.72 (3H, s), 0.76 (3H, s), 0.86 (3H, s), 0.92 (3H, s), 0.99 (3H, s),
1.08 (3H, s), 1.13 (3H, s), 3.48 (1H, m), 5.27 (1H, m);
13
C-NMR (75 MHz, CDCl
3
)
δ
C
(ppm): 38.4 (C-1), 27.0 (C-2), 79.2 (C-3), 38.8 (C-4), 55.3 (C-5), 18.4 (C-6), 32.8
(C-7), 39.6 (C-8), 47.9 (C-9), 37.3 (C-10), 23.2 (C-11), 122.8 (C-12), 143.5 (C-13),
41.8 (C-14), 27.7 (C-15), 23.8 (C-16), 46.6 (C-17), 40.2 (C-18), 45.9 (C-19), 30.9
(C-20), 33.9 (C-21), 32.9 (C-22), 28.1 (C-23), 15.7 (C-24), 15.5 (C-25), 17.3 (C-26),
25.9 (C-27), 182.4 (C-28), 33.3 (C-29), 23.7 (C-30).
19
α
-hydroxy-3-acetylursolic acid ( 7 ): mp 171 ～ 173; ESI-MS m/z : 515 [M + H]
+
;
1
H-NMR (500 MHz, CDCl
3
)
δ
(ppm): 5.34 (1H, brs, H-12), 4.50 (1H, dd, J = 11.2,
6.3 Hz, H-3), l.24 (3H, s, H-27), 1.23 (3H, s, H-25), 0.95 (3H, s, H-26), 0.84 (3H, s,
H-24), 0.77 (3H, s, H-23), 0.96 (3H, d, J = 6.4 Hz, H-30), 2.05 (3H, s, H-32);
13
C-NMR (125 MHz, CDCl
3
)
δ
(ppm): 38.1 (C-1), 23.6 (C-2), 80.9 (C-3), 37.7 (C-4),
52.8 (C-5), 18.3 (C-6), 32.6 (C-7), 40.0 (C-8), 47.1 (C-9), 36.9 (C-10), 23.5 (C-11),
4

129.3 (C-12), 137.9 (C-13), 41.1 (C-14), 28.3 (C-15), 25.3 (C-16), 47.7 (C-17), 55.2
(C-18), 73.1 (C-19), 41.1 (C-20), 26.0 (C-21), 37.5 (C-22), 28.0 (C-23), 16.7 (C-24),
15.3 (C-25), 17.0 (C-26), 24.5 (C-27), 183.9 (28-COOH), 27.4 (C-29), 16.1 (C-30),
171.0 (C-31), 21.3 (C-32).
2-oxo-pomolic acid ( 8 ):
1
H-NMR (500 MHz, CDCl
3
)
δ
(ppm): 5.35 (1H, brs, H-12),
3.91 (1H, s, H-3), l.32 (3H, s), 1.26 (3H, s), 1.21 (3H, s), 0.88 (3H, s), 0.77 (3H, s),
0.96 (3H, d, J = 6.4 Hz, H-30), 0.69 (3H, s);
13
C-NMR (125 MHz, CDCl
3
)
δ
(ppm):
53.4 (C-1), 211 (C-2), 82.9 (C-3), 45.4 (C-4), 54.5 (C-5), 18.3 (C-6), 32.4 (C-7), 41.4
(C-8), 47.2 (C-9), 43.7 (C-10), 23.6 (C-11), 128.3 (C-12), 138.3 (C-13), 41.8 (C-14),
29.1 (C-15), 26.0 (C-16), 47.7 (C-17), 54.5 (C-18), 72.3 (C-19), 42.0 (C-20), 27.4
(C-21), 37.4 (C-22), 29.4 (C-23), 16.6 (C-24), 16.2 (C-25), 16.5 (C-26), 24.3 (C-27),
183 (C-28), 28.2 (C-29), 16.1 (C-30).
Euscaphic acid ( 9 ): HR-EI-MS m/z 488.3508 (C
30
H
48
O
5
);
1
H-NMR (400 MHz, CDCl
3
)
δ
(ppm): 5.16 (1H, s, H-12), 3.77 (1H, m, H-2
β
), 3.49 (1H, d, J = 2.5 Hz, H-3
α
), 2.73
(1H, dt, J = 13.5, 4.4 Hz, H-16
α
), 3.04 (1H, s, H-18), 0.66, 1.27, 1.22 (each 3H, s,
H-27, H-29, H-24), 1.09 (3H, d, J = 6.5 Hz, H-30), 0.91, 1.06 和 0.89 (each 3H, s,
H-26, H-25, H-23); 13 C-NMR (100 MHz, CDCl
3
) δ (ppm): 46.8 (C-1), 67.1 (C-2),
82.2 (C-3), 38.9 (C-4), 54.8 (C-5), 18.1 (C-6), 32.5 (C-7), 39.0 (C-8), 46.6 (C-9), 37.6
(C-10), 23.1 (C-11), 126.7 (C-12), 138.6 (C-13), 41.1 (C-14), 28.0 (C-15), 25.1
(C-16), 47.0 (C-17), 53.1 (C-18), 71.6 (C-19), 41.4 (C-20), 25.9 (C-21), 37.2 (C-22),
28.8 (C-23), 16.3 (C-24), 16.3 (C-25), 17.1 (C-26), 23.9 (C-27), 178.7 (C-28), 26.4
(C-29), 16.6 (C-30).
2 α ,3 α ,19 α -trihydroxyl-urso-12-en-28-oic acid ( 10 ): HR-EI-MS m/z 488.3508,
1
H-NMR (500 MHz, CDCl
3
)
δ
(ppm): 5.58 (1H, s, H-12), 4.30 (1H, ddd, J = 10.3, 4.1,
3.0 Hz, H-2 β ), 3.76 (1H, d, J = 2.5 Hz, H-3 β ), 3.10 (1H, dt, J = 13.5, 4.4 Hz, H-16 α ),
3.04 (1H, s, H-18), 2.33 (1H, dt, J = 13.5, 4.8 Hz, H-15 β ), 1.89 (1H, dd, J = 12.0, 4.8
Hz, H-1
β
), 1.75 (1H, t, J = 11.8 Hz, H-1
α
), 1.63, 1.40, 1.25 (each 3H, s, H-27, H-29,
5

H-24), 1.09 (3H, d, J = 6.5 Hz, H-30), 1.09, 0.97 and 0.89 (each 3H, s, H-26, H-25,
H-23);
13
C-NMR (125 MHz, CDCl
3
)
δ
(ppm): 41.4 (C-1), 65.2 (C-2), 78.3 (C-3), 37.8
(C-4), 47.7 (C-5), 17.7 (C-6), 32.5 (C-7), 39.6 (C-8), 46.6 (C-9), 37.7 (C-10), 23.1
(C-11), 127.0 (C-12), 138.9 (C-13), 41.8 (C-14), 28.2 (C-15), 25.4 (C-16), 47.3
(C-17), 53.6 (C-18), 71.8 (C-19), 41.2 (C-20), 25.9 (C-21), 37.5 (C-22), 28.4 (C-23),
21.4 (C-24), 15.8 (C-25), 16.3 (C-26), 23.7 (C-27), 179.7 (C-28), 26.2 (C-29), 15.7
(C-30).
Legends for Figures
Figure S1. 1 H-NMR spectrum of compound 1
Figure S2. 13 C-NMR spectrum of compound 1
Figure S3. HSQC spectrum of compound 1
Figure S4. HMBC spectrum of compound 1
Figure S5. ROESY spectrum of compound 1
Figure S6. HR-ESI-MS of compound 1
Figure S7. DEPT spectra of compound 1
Figure S8. ROESY-2 spectrum of compound 1
Figure S9. ROESY-3 spectrum of compound 1
6

Figure S1. 1 H-NMR Spectrum of compound 1
Figure S2. 13 C-NMR Spectrum of compound 1
7

Figure S3. HSQC Spectrum of compound 1
Figure S4. HMBC Spectrum of compound 1
8

Figure S5. ROESY Spectrum of compound 1
21
SZ-140429-03 49 (0.912)
100
517.2788
TOF MS ES+
1.09e3
%
518.2825
0
259.0919 336.2025
459.0853
515.2369
601.3289 645.3561 713.3056
m/z
250 300 350 400 450 500 550 600 650 700
Figure S6. HR-ESI-MS of compound 1
9

Figure S7. DEPT spectra of compound 1
Figure S8. ROESY-2 spectrum of compound 1
10

Figure S9. ROESY-3 spectrum of compound 1
11