New antimicrobial pregnane glycosides from the stem of Ecdysanthera rosea

Article abstract of DOI:10.1016/j.fitote.2014.10.008

Phytochemical investigation on the stem of Ecdysanthera rosea led to the isolation of eight new C-21 pregnane glycoside ecdysosides A-H (1-8), together with one known pregnane glycoside ecdysantheroside A (9). Their structures were elucidated based on extensive spectroscopic data (MS, IR, 1D and 2D NMR) analysis, as well as comparison with the reported literature data. Antimicrobial activities of all the compounds were evaluated against bacteria and yeasts. Compounds 1, 9, 3 and 5 exhibited moderate antibacterial activities against respective Enterococcus faecalis and Providensia smartii, with MIC value of 12.5 μg/mL. Compound 8 showed significant anti-yeast activity against Cryptococcus neoformans with MIC value of 12.5 μg/mL.

Full text of DOI:10.1016/j.fitote.2014.10.008

Fitoterapia 99 (2014) 267–275
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
New antimicrobial pregnane glycosides from the stem of
Ecdysanthera rosea
Chang-Wei Song a,b, Paul-Keilah Lungaa,c, Xu-Jie Qin a,b, Gui-Guang Cheng a,b, Jian-Long Gu a,b
,
a,
Ya-Ping Liu a, , Xiao-Dong Luo
⁎
⁎
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic
of China
b
University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China
Department of Biochemistry, Laboratory of Phytobiochemistry and Medicinal Plants Study, Faculty of Science, University of Yaoundé 1, Yaoundé P.O. Box 812, Cameroon
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytochemical investigation on the stem of Ecdysanthera rosea led to the isolation of eight new
C-21 pregnane glycoside ecdysosides A–H (1–8), together with one known pregnane glycoside
ecdysantheroside A (9). Their structures were elucidated based on extensive spectroscopic data (MS,
IR, 1D and 2D NMR) analysis, as well as comparison with the reported literature data. Antimicrobial
activities of all the compounds were evaluated against bacteria and yeasts. Compounds 1, 9, 3 and 5
exhibited moderate antibacterial activities against respective Enterococcus faecalis and Providensia
smartii, with MIC value of 12.5 μg/mL. Compound 8 showed significant anti-yeast activity against
Cryptococcus neoformans with MIC value of 12.5 μg/mL.
Received 5 September 2014
Accepted in revised form 9 October 2014
Available online 16 October 2014
Keywords:
Ecdysanthera rosea
Apocynaceae
Pregnane glycosides
Antimicrobial activity
© 2014 Published by Elsevier B.V.
1. Introduction
(1–9) were isolated from this species, of these compounds 1–5
possessed a different aglycone from the previously reported
Ecdysanthera rosea Hook. et Arn. (Apocynaceae) is mainly
distributed in the southern region of China. As a Chinese folk
medicine, its whole plant has been used for the treatment of
traumatic injury, sore throat, and chronic nephritis [1]. Previous
phytochemical investigation reported the isolation of terpenoids,
flavonoids, phenolic glycosides, steroid and their glycosides from
this natural medicine [2–12].
The E. rosea has been used as a medicinal plant with definite
therapeutic effects. However, only a few steroid glycosides from
this plant have been previously reported with cytotoxic activity,
which is not related to its medicinal use [12], and this activated
our interest to search for more compounds like them from this
medicinal plant and conduct a further bioactive research. In our
current phytochemical investigation, nine pregnane glycosides
compound 9 [12]. Considering the fact that many diseases like
traumatic injury and sore throat could be caused by bacteria
and fungi, we evaluated their antibacterial activities against
three bacterial strains and antifungal activities against three
yeast species by using broth micro-dilution method. The results
demonstrated that compounds 1, 3, 5 and 9 showed moderate
activities against the corresponding bacterial strain, while
compound 8 showed significant anti-yeast activity against
Cryptococcus neoformans, comparable to nystatin, the positive
control. In this paper, we report the isolation, structure
elucidation and antimicrobial activities of these pregnane
glycosides.
2. Experimental
2.1. General methods
⁎
Corresponding authors. Tel.: +86 871 6522 3188; fax: +86 871 6522
3245.
E-mail addresses: xdluo@mail.kib.ac.cn (X.-D. Luo), liuyaping@mail.kib.ac.cn
Optical rotations were measured with a JASCO P-1020
digital polarimeter. UV spectra were obtained using a Shimadzu
(Y.-P. Liu).
http://dx.doi.org/10.1016/j.fitote.2014.10.008
0367-326X/© 2014 Published by Elsevier B.V.

268
C.-W. Song et al. / Fitoterapia 99 (2014) 267–275
UV-2401 PC spectrophotometer. IR spectra were recorded on a
Bruker Tensor 27 infrared spectrophotometer using KBr pellets.
1D and 2D NMR spectra were recorded on a Bruker AM-400, a
DRX-500, and an Avance III 600 or AV 600 MHz spectrometer
with TMS as internal standard. ESI-MS spectra were recorded on
Bruker HTC/Esquire and Agilent G6230 spectrometer. HREI-MS
was recorded on a Waters Auto Spec Premier P776 spectrom-
eter. HRESI-MS was recorded on an Agilent G6230 spectrome-
ter. Column chromatography was performed on silica gel (200–
300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China),
Rp-18 (40-63 μm, Merk), and Sephadex LH-20 (GE Healthcare,
Sweden). Fractions were monitored by TLC (GF254, Qingdao
Marine Chemical Ltd., Qingdao, China), and by heating silica gel
plates sprayed with 10% H2SO4 in ethanol. GC analysis was
performed on Agilent 7890A gas chromatograph equipped with
a H2 flame ionization detector.
(KBr) νmax 3441, 2971, 1760, 1630, 1451, 1381, 1368, 1332,
1196, and 1002 cm−1; positive-ion ESI-MS m/z 797 [M + Na]+;
positive-ion HRESI-MS m/z 797.4082 [M + Na]+ (calcd. for
C42H62O13Na+, 797.4088). 1H and 13C NMR: see Tables 1 and 2.
2.3.3. Ecdysoside C (3)
White amorphous powder; [α]2D1 +7.5 (c 0.06, MeOH); UV
(MeOH) λmax (log ε): 201 (3.8), 228 (3.8), and 245 (3.8) nm; IR
(KBr) νmax 3439, 2970, 2853, 1758, 1709, 1631, 1452, 1368,
1196, and 1004 cm−1; positive-ion ESI-MS m/z 959 [M + Na]+;
positive-ion HRESI-MS m/z 959.4617 [M + Na]+ (calcd. for
C48H72O18Na+, 959.4611). 1H and 13C NMR: see Tables 1 and 2.
2.3.4. Ecdysoside D (4)
White amorphous powder; [α]2D1 +30.7 (c 0.07, MeOH); UV
(MeOH) λmax (log ε): 200 (4.0), 222 (4.2), and 245 (4.0) nm; IR
(KBr) νmax 3442, 2972, 1760, 1604, 1450, 1381, 1266, 1196, and
1001 cm−1; positive-ion ESI-MS m/z 941 [M + Na]+; HREI-MS
2.2. Plant material
m/z 918.4970 [M]+ (calcd. for C49H74O1+6, 918.4977). 1H and 13
C
The stem of E. rosea was collected from Xishuangbanna
Autonomous Prefecture, Yunnan Province, People's Republic of
China, and identified by Jingyun Cui of Xishuangbanna Botanic
Garden. A voucher specimen (Cui 200811-03) has been
deposited at the Herbarium of Kunming Institute of Botany,
Chinese Academy of Sciences.
NMR: see Tables 1 and 2.
2.3.5. Ecdysoside E (5)
White amorphous powder; [α]2D1 +8.2 (c 0.1, MeOH); UV
(MeOH) λmax (log ε): 200 (4.2), 222 (4.2), and 247 (4.1) nm; IR
(KBr) νmax 3442, 2972, 2906, 1756, 1708, 1631, 1451, 1404,
1382, and 1002 cm−1
; positive-ion ESI-MS m/z 1121
2.3. Extraction and isolation
[M+Na]+; positive-ion HRESI-MS m/z 1121.5150 [M+Na]+
(calcd. For C54H82O23Na+, 1121.5145). 1H and 13C NMR: see
Tables 1 and 2.
The air dried and crushed stems of E. rosea (10 kg) were
extracted with MeOH (70 L, 24 h) three times at room
temperature. After filtration, all the solvent was evaporated
under reduced pressure at 50 °C to yield the extract (500 g).
This crude extract was exhaustively partitioned between water
and EtOAc to obtain the EtOAc fraction (120 g). The EtOAc
fraction was then subjected to column chromatography over
silica gel and eluted with chloroform–acetone (100:1→1:1) to
give 6 fractions [Fr.A (38.5 g), Fr.B (13 g), Fr.C (14 g), Fr.D (9 g),
Fr.E (18 g), Fr.F (26 g)] based on the TLC plate analysis. Fraction
C (14 g) was isolated by Sephadex LH-20 (MeOH–H2O 50:50)
and repeated silica gel column with chloroform–methanol to
yield compounds 1 (12 mg), 2 (4 mg) and 9 (22 mg). Fraction D
(9 g) was chromatographed over Sephadex LH-20 (MeOH–H2O
50:50), RP-C18 gel (MeOH–H2O 10:90→80:20) and further
purified by HPLC (MeCN–H2O 40:60) to provide compounds 3
(3 mg), 4 (7 mg), 7 (11 mg) and 8 (5 mg). Fraction E (18 g)
was separated over Sephadex LH-20, eluted with MeOH–H2O
(1:1) and chromatographed over RP-C18 gel (MeOH–H2O
10:90→70:30), followed by HPLC (MeCN–H2O 35:65) to give
compounds 5 (6 mg), and 6 (5 mg).
2.3.6. Ecdysoside F (6)
White amorphous powder; [α]2D1 −6.4 (c 0.05, MeOH); IR
(KBr) νmax 3442, 2969, 2853, 1755, 1631, 1452, 1381, 1367,
1267, and 1003 cm−1; positive-ion ESI-MS m/z 801 [M + Na]+;
positive-ion HRESI-MS m/z 801.4406 [M+Na]+ (calcd. for
C42H66O13Na+, 801.4401). 1H and 13C NMR: see Tables 3 and 4.
2.3.7. Ecdysoside G (7)
White amorphous powder; [α]2D1 −4.4 (c 0.05, MeOH); IR
(KBr) νmax 3439, 3432, 2970, 2872, 1756, 1631, 1454, 1381,
1190, and 1002 cm−1; positive-ion ESI-MS m/z 963 [M + Na]+;
positive-ion HRESI-MS m/z 963.4935 [M + Na]+ (calcd. for
C48H76O18Na+, 963.4929). 1H and 13C NMR: see Tables 3 and 4.
2.3.8. Ecdysoside H (8)
White amorphous powder; [α]2D1 −29.5 (c 0.04, MeOH); IR
(KBr) νmax 3443, 2969, 2855, 1759, 1634, 1451, 1382, 1192,
1097, and 1005 cm−1
; positive-ion ESI-MS m/z 1251
[M+Na]+; negative HRESI-MS m/z 1227.6518 [M − H]−
(calcd. for C62H99O−24, 1227.6526). 1H and 13C NMR: see
Tables 3 and 4.
2.3.1. Ecdysoside A (1)
White amorphous powder; [α]2D1 +36.6 (c 0.05, MeOH); UV
(MeOH) λmax (log ε): 201 (3.9), 228 (3.9), and 245 (4.0) nm; IR
(KBr) νmax 3442, 2971, 2876, 1757, 1709, 1631, 1455, 1383,
1195, and 1001 cm−1; positive-ion ESI-MS m/z 509 [M + Na]+;
HREI-MS m/z 486.2617 [M]+ (calcd. for C28H38O+7 , 486.2618).
1H and 13C NMR: see Tables 1 and 2.
2.3.9. Acid hydrolysis of 1–9 and GC analysis
Compounds 1–9 (2 mg) were dissolved with 2 M HCl (1,
4dioxane/H2O 1:1, 2 mL) and hydrolyzed on water bath at 90 °C
for 2 h. After cooling, the reaction products was partitioned
with CHCl3 (3 × 5 mL). The aqueous layer was evaporated to
dryness with MeOH until neutral. The dried residue was
dissolved in 1 mL anhydrous pyridine and treated with L-
cysteine methyl ester hydrochloride (1.5 mg) and stirred at
60 °C for 1 h. Trimethylsilylimidazole (1.5 ml) was added to the
2.3.2. Ecdysoside B (2)
White amorphous powder; [α]2D1 +32.8 (c 0.08, MeOH); UV
(MeOH) λmax (log ε): 201 (3.9), 228 (3.9), and 245 (4.0) nm; IR

C.-W. Song et al. / Fitoterapia 99 (2014) 267–275
269
Table 1
1H NMR data of compounds 1–5 (δ in ppm, J in Hz, C5D5N).
Position
1
2
3
4a
5
1
2
3
1.02 m, 1.73 m
1.74 m, 2.14 m
3.85 m
1.03 m, 1.73 m
1.70 m, 2.09 m
3.78 m
1.05 m, 1.75 m
1.72 m, 2.11 m
3.82 m
1.11 m, 1.90 m
1.61 m, 1.97 m
3.54 m
1.04 m, 1.74 m
1.70 m, 2.10 m
3.79 m
4
6
2.37 m, 2.58 m
5.36 m
2.32 m, 2.52 m
5.32 m
2.34 m, 2.55 m
5,36 m
2.24 m, 2.39 m
5.41 m
2.33 m, 2.53 m
5.33 m
7
8
2.00 m, 2.13 m
2.93 m
1.97 m, 2.10 m
2.90 m
2.00 m, 2.12 m
2.92 m
2.09 m, 2.27 m
2.92 m
1.98 m, 2.10 m
2.90 m
9
1.20 m
1.17 m
1.20 m
1.28 m
1.17 m
11
12
15
17
19
20
21
1′
1.60 m, 2.40 m
1.59 m, 2.40 m
5.94 d (1.5)
2.76 d (2.5)
1.11 s
1.58 m, 2.38 m
1.55 m, 2.35 m
5.90 d (1.7)
2.71 d (2.4)
1.08 s
1.60 m, 2.39 m
1.59 m, 2.40 m
5.94 d (1.7)
2.77 d (2.4)
1.10 s
1.72 m, 2.38 m
1.45 m, 2.32 m
5.86 d (1.8)
2.51 d (2.7)
1.14 s
1.58 m, 2.38 m
1.56 m, 2.36 m
5.90 d (1.5)
2.72 d (2.5)
1.07 s
4.85 m
4.81 m
4.85 m
4.68 m
4.81 m
1.38 d (6.4)
5.28 dd (9.6, 1.8)
1.89 m, 2.42 m
3.81 m
1.35 d (6.0)
5.27 dd (9.6, 1.6)
1.91 m, 2.32 m
4.10 m
1.39 d (6.4)
5.30 dd (9.6, 1.5)
1.94 m, 2.37 m
4.12 m
1.51 d (6.5)
4.84 dd (9.6, 1.6)
1.58 m, 2.09 m
3.79 m
1.36 d (6.4)
5.27 br d (9.6)
1.91 m, 2.33 m
4.09 m
2′
3′
4′
3.61 m
3.51 m
3.53 m
3.20 m
3.51 m
5′
4.19 m
4.20 m
4.24 m
3.83 m
4.21 m
6′
1.57 d (6.2)
3.51 s
1.36 d (6.0)
3.64 s
1.39 d (6.2)
3.65 s
1.20 d (6.1)
3.43 s
1.35 d (6.2)
3.62 s
OMe
1″
2″
3″
4″
5″
6″
OMe
1‴
2‴
3‴
4‴
5‴
6‴
OMe
1″″
2″″
3″″
4″″
5″″
6″″
5.12 dd (9.6, 1.6)
1.83 m, 2.32 m
4.06 m
3.49 m
4.19 m
1.41 d (6.2)
3.56 s
4.76 dd (9.8, 1.7)
1.72 m, 2.54 m
3.48 m
5.13 dd (9.7, 1.4)
1.81 m, 2.35 m
4.02 m
3.51 m
4.20 m
1.40 d (6.2)
3.42 s
4.68 br d (9.7)
2.21 m, 2.35 m
3.48 m
4.22 m
3.58 m
1.57 d (6.4)
3.41 s
5.18 d (7.8)
4.00 m
4.26 m
4.18 m
3.99 m
4.62 d (11.7)
4.38 dd (11.7, 5.8)
4.74 dd (9.7, 1.8)
1.63 m, 2.10 m
3.80 m
3.21 m
3.85 m
1.21 d (6.1)
3.43 s
4.44 dd (9.6, 1.7)
1.53 m, 2.30 m
3.70 m
5.14 br d (9.6)
1.80 m, 2.32 m
3.99 m
3.48 m
4.19 m
1.36 d (6.2)
3.40 s
4.61 br d (9.7)
2.15 m, 2.31 m
3.41 m
3.47 m
3.59 m
1.56 d (6.0)
3.45 s
3.15 m
3.45 m
4.32 m
3.54 m
1.28 d (6.0)
3.40 s
4.64 dd (9.7, 2.1)
1.64 m, 1.99 m
3.29 m
3.68 d (2.8)
3.30 m
1.35 d (6.2)
1.69 d (6.2)
3.36 s
5.09 d (7.8)
3.94 t (8.4)
4.14 m
4.00 m
4.09 m
4.30 m, 4.84 m
OMe
1″‴
2″‴
3″‴
4″‴
5″‴
6″‴
3.39 s
5.25 d (7.7)
4.06 m
4.24 m
4.23 m
4.25 m
4.54 d (11.4)
4.39 dd (11.4, 4.8)
a
Measured in CDCl3 at 600 MHz.
reaction mixtures, and they were kept at 60 °C for 30 min. The
supernatants (4 μL) were analyzed by GC, respectively, under
the following conditions: Agilent 7890A gas chromatograph
equipped with HP-5 (5% diphenyl polysiloxane) quartz capil-
lary column (30 m × 0.32 mm, film thickness 0.25 μm). The
oven temperature was programmed to rise from 100 to 230 at
the rate of 10 °C/min, and the carrier gas was N2 (2 mL/min);
FID temperature: 300; injector temperature: 250; and split
ratio: 1/5 [13,14]. The result showed that the standard
samples of D-glucose and D-cymarose derivatives gives a
peak at 10.86 and 11.43 min, respectively. Thus, the D-glucose in
compounds 3, 5, 7 and 8 and D-cymarose in compounds 1–9
were detected by comparing the retention times of the
corresponding derivatives with signals of the authentic D-
glucose and D-cymarose.
2.4. Antimicrobial assays
The microorganisms used in antimicrobial assays were
obtained from the American Type Culture Collection (ATCC),
“centre Pasteur” of Yaounde Cameroon and “Institut Pasteur de
Paris” (IP). They included three bacterial strains: Enterococcus
faecalis ATCC 10541, Staphylococcus aureus ATCC 25922 and
Providensia smartii ATCC29916; three yeasts: Candida albicans
ATCC 2091, C. neoformans IP 90526 and Candida guilimondis.
The MIC values of the isolated compounds were determined by

270
C.-W. Song et al. / Fitoterapia 99 (2014) 267–275
Table 2
Table 3
13C NMR data of compounds 1–5 in C5D5N (δ in ppm, J in Hz, C5D5N).
1H NMR data of compounds 6–9 (δ in ppm, J in Hz, C5D5N).
Position
1
2
3
4a
5
Position
6
7
8
1
2
3
4
5
6
7
8
37.7
30.6
77.4
39.5
37.0
29.9
76.9
38.8
37.7
30.6
77.5
39.5
37.1
37.7
30.6
77.5
39.5
1
2
3
4
6
7
8
9
11
12
15
16
17
19
20
21
1′
2′
3′
4′
5′
1.14 m, 1.80 m
1.27 m, 2.11 m
3.81 m
2.29 m, 2.50 m
5.38 m
2.00 m, 2.61 m
2.51 m
1.22 m
1.50 m, 2.42 m
1.28 m, 1.96 m
1.86 m, 1.99 m
2.03 m
2.15 m
1.36 s
1.10 m, 1.80 m
1.69 m, 2.09 m
3.81 m
2.49 m, 2.89 m
5.38 m
2.00 m, 2.60 m
2.47 m
1.20 m
1.47 m, 2.40 m
1.25 m, 1.93 m
1.88 m, 2.59 m
2.01 m
2.13 m
1.02 s
1.13 m, 1.76 m
1.72 m, 2.11 m
3.85 m
2.32 m, 2.51 m
5.40 m
2.03 m, 2.63 m
2.52 m
1.23 m
1.50 m, 2.43 m
1.27 m, 1.96 m
1.89 m, 1,97 m
2.04 m
2.16 m
1.05 s
29.4
77.3
38.6
140.5
119.6
29.0
33.8
49.6
37.8
19.9
35.4
54.5
183.7
126.4
205.4
60.8
174.3
19.2
75.7
23.9
95.9
35.4
76.9
82.4
68.4
18.2
57.9
99.6
35.4
77.2
82.5
68.2
18.2
58.1
101.2
36.3
79.2
82.3
70.5
18.3
56.8
100.6
31.9
70.8
66.7
77.8
16.8
55.7
141.0
120.7
29.8
34.3
50.1
38.4
20.6
35.5
55.1
183.2
127.5
205.9
61.3
175.4
19.7
76.6
24.1
96.7
36.6
79.4
74.7
71.4
19.6
58.5
140.3
120.0
29.1
33.7
49.5
37.8
20.0
34.9
54.4
182.5
126.7
205.1
60.7
174.6
19.0
75.8
23.4
96.1
37.0
77.8
83.1
68.8
18.3
58.6
100.2
36.7
77.6
82.9
68.7
18.3
58.6
101.9
37.0
81.1
75.9
72.7
18.4
56.8
140.9
120.7
29.8
34.3
50.1
38.4
20.6
35.5
55.1
183.2
127.5
205.9
61.3
175.4
19.7
76.6
24.1
96.7
37.6
78. 4
83.8
69.5
19.0
59.2
100.9
36.9
77.9
83.5
69.4
19.0
58.7
103.1
33.3
80.3
73.9
71.4
18.4
56.6
105.3
76.3
78.9
72.3
79.1
63.6
141.0
120.8
29.8
34.3
50.1
38.4
20.6
35.5
55.1
183.2
127.5
205.9
61.4
175.4
19.7
76.6
24.1
96.7
37.6
78.4
83.8
69.5
19.0
59.2
100.9
36.8
77.9
83.6
69.4
19.0
58.7
103.2
33.3
80.3
73.5
71.5
18.6
56.7
105.2
76.0
78.8
72.3
78.2
70.9
9
10
11
12
13
14
15
16
17
18
19
20
21
1′
2′
3′
4′
5′
6′
OMe
1″
2″'
3″'
4″'
5″'
6″'
OMe
1‴
2‴
3‴
4‴
5‴
6‴
OMe
1″″
2″″
3″″
4″″
5″″
6″″
OMe
1″‴
2″‴
3″‴
4″‴
5″‴
6″‴
4.65 m
4.66 m
4.69 m
1.24 d (6.2)
5.25 br d (9.2)
1.88 m, 2.29 m
3.41 m
3.49 m
4.18 m
1.1.36 d (6.1)
3.38 s
5.10 br d (9.3)
1.79 m, 2.30 m
4.07 m
1.23 d (6.2)
5.25 br d (9.2)
1.87 m, 2.29 m
4.06 m
3.49 m
4.20 m
1.37 d (6.5)
3.59 s
5.09 br d (9.7)
1.76 m, 2.29 m
3.98 m
1.26 d (6.1)
5.29 br d (9.5)
1.92 m, 2.33 m
4.10 m
3.49 m
4.23 m
1.40 d (5.7)
3.63 s
5.14 br d (9.2)
1.86 m, 2.35 m
3.98 m
6′
OMe
1″
2″
3″
4″
5″
6″
OMe
1‴
2‴
3‴
4‴
5‴
6‴
OMe
1″″
2″″
3″″
4″″
5″″
6″″
3.51 m
4.19 m
3.46 m
4.15 m
3.48 m
4.19 m
1.39 d (6.2)
3.61 s
4.68 br d (9.2)
2.15 m, 2.27 m
4.06 m
3.88 m
3.56 m
1.53 d (6.3)
3.50 s
1.35 d (6.6)
3.38 s
4.63 br d (9.7)
2.17 m, 2.30 m
3.43 m
4.14 m
3.52 m
1.53 d (6.3)
3.36 s
5.12 d (7.7)
3.94 m
4.21 m
4.12 m
3.93 m
4.56 d (11.2)
4.34 dd (11.4, 5.7)
1.40 d (6.6)
3.59 s
4.72 br d (9.6)
1.76 m, 2.51 m
3.56 m
3.49 m
3.52 m
1.41 d (5.3)
3.41 s
4.92 d (9.6)
1.75 m, 2.52 m
3.62 m
3.52 m
3.54 m
1.46 m
OMe
1″‴
2″‴
3″‴
4″‴
5″‴
6″‴
OMe
1‴‴
2‴‴
3‴‴
4‴‴
5‴‴
6‴‴
3.51 s
4.90 d (9.7)
2.01 m, 2.31 m
3.46 m
4.20 m
3.55 m
1.56 d (6.2)
3.48 s
5.19 d (7.6)
4.01 m
4.04 m
4.21 m
106.1
75.6
79.0
72.1
79.1
63.1
a
Measured in CDCl3 at 125 MHz.
3.99 m
4.61 d (11.2)
4.39 dd (11.2, 5.4)
the broth microdilution method in 96-well microtitre. The 96-
well plates were prepared by dispensing into each well 100 μL
of Mueller Hinton broth for bacteria and Sabouraud dextrose
broth for fungi. The compounds were initially prepared in
10% DMSO in broth medium at 400 μg/mL for compounds or
50 μg/mL for the reference antibiotics. A volume of 100 μL of
each test sample was added into the first wells of the microtitre
plate (whose wells were previously loaded with 100 μL of broth
medium). Serial two-fold dilutions of the test samples were
made and 100 μL of inoculum standardized at 106 CFU/mL for
bacteria, and 2.5 × 105 CFU/mL for yeasts (at 600 nm, Jenway
6105 UV/Vis spectrophotometer 50 Hz/60 Hz). This gave final
concentration ranges from 100 to 0.781 μg/mL for the com-
pounds and 12.5 to 0.097 μg/mL for reference substances. The
plates were sealed with parafilm, then agitated with a plate

C.-W. Song et al. / Fitoterapia 99 (2014) 267–275
271
Table 4
Sigma-Aldrich, South Africa). Viable bacteria reduced the yellow
dye to a pink color. For yeasts, MICs were determined by
visualizing the turbidity of the wells. The MIC corresponded to
the lowest well concentration where no color turbidity change
was observed, indicating no growth of microorganism. All tests
were performed in triplicates. Gentamycin for bacteria, and
nystatin for yeast were used as positive controls, and the MIC
value was defined as the lowest concentration that inhibited
visible growth.
13C NMR data of compounds 6–9 (δ in ppm, J in Hz, C5D5N).
Position
6
7
8
1
2
3
4
5
6
7
8
38.8
31.5
78.5
40.4
37.3
30.0
77.0
38.9
37.8
30.5
77.5
39.5
141.1
123.0
28.7
40.1
47.4
38.0
22.7
34.8
61.1
86.3
27.0
36.6
58.3
180.0
21.0
84.6
22.6
97.5
38.4
80.1
84.3
70.2
19.9
56.6
101.7
38.0
79.2
84.4
70.3
19.8
60.0
104.0
34.0
79.0
67.9
72.9
18.8
59.9
139.7
121.6
27.2
38.6
45.9
37.3
21.2
33.3
59.6
84.8
25.6
36.9
56.8
178.5
21.0
82.8
21.0
96.0
36.9
77.7
83.1
68.8
18.3
58.5
100.2
36.2
77.2
82.9
68.7
18.3
58.1
102.4
32.6
79.5
73.4
70.7
17.6
55.9
104.7
75.6
78.2
71.6
78.3
62.9
140.1
122.1
27.8
39.2
46.4
37.9
21.7
33.8
60.1
85.3
26.1
35.7
57.3
179.2
20.1
83.4
21.6
96.5
37.4
78.3
83.4
69.3
18.9
59.1
100.8
37.2
78.0
83.4
69.2
18.8
59.2
102.2
37.8
79.2
83.0
71.2
18.9
56.5
100.4
37.7
79.7
83.3
71.9
19.0
57.6
101.5
33.4
80.3
73.7
71.9
18.2
57.2
105.1
76.3
78.7
72.1
78.9
63.4
9
3. Results and discussion
10
11
12
13
14
15
16
17
18
19
20
21
1′
2′
3′
4′
5′
6′
OMe
1″
2″
3″
The MeOH extracts of E. rosea was partitioned between
EtOAc and water. The EtOAc fraction was subjected to silica gel,
Sephadex LH-20 and ODS column chromatography and finally
by semi-preparative HPLC to yield eight new pregnane
glycosides 1–8 and a known compound 9 [12]. The aglycone
of compounds 1–5 was identified with the same skeleton of
pregna-5,14-dien-18-oic acid,3,20-dihydroxy-16-oxo-,γ-lactone,
(3β,20R), and compounds 6–9 with the same aglycone of pregn-
5-en-18-oic acid,3,14,20-trihydroxy-,γ-lactone,(3β,14β,20R) by
detailed analysis of their NMR spectra data in association with
literature information. The D-configuration of glucose in the
compounds was identified by GC analysis of acid hydrolysis
products. For the deoxysugars, since only the D-glucose and D-
cymarose could be obtained, the L-configuration of cymarose in
the compounds was determined mainly by comparison of their
13C NMR chemical shift values with reported data. By analyzing
the reported literature data, it was confirmed that the chemical
shift of C-2 is at 35.0–38.0 ppm in β-D-cymaropyranosyl and
α-D-cymaropyranosyl. On the other hand, this values are
between 31.0 and 34.0 ppm in β-L-cymaropyranosyl and α-L-
cymaropyranosyl [12–20]. Therefore, compounds 1–8 were
identified as ecdysosides A–H and the known compound 9 was
determined to be ecdysantheroside A.
The HREI-MS spectra gave a single peak at m/z 486.2617
[M]+ (calcd. for 486.2618) which indicated that the molecular
formula of compound 1 was C28H38O7 with 10° of unsaturation.
The IR spectrum gave characteristic signals at 3442, 1709 cm−1
for hydroxyl and carbonyl groups, respectively. The 13C NMR
and DEPT spectrum of 1 (Table 2) exhibited twenty eight carbon
signals for four methyls (one oxygenated), seven methylenes,
eleven methines, and six quaternary carbons, of which 7 carbon
signals were assigned to the sugar part, and 21 carbon signals
were assigned to a C21-steroidal aglycone. In the 1H NMR
spectra, a characteristic anomeric proton at δH 5.28 (dd, J = 9.6,
1.8 Hz, H-1′) indicated the presence of a sugar unit with β
configuration based on its large coupling constant value (9.6 Hz)
and supported by the ROESY experiment. The terminal methyl
group at δH 1.57 (d, J = 6.2 Hz, Me-6′), methoxy methyl group at
4″
5″
6″
OMe
1‴
2‴
3‴
4‴
5‴
6‴
OMe
1″″
2″″
3″″
4″″
5″″
6″″
OMe
1″‴
2″‴
3″‴
4″‴
5″‴
6″‴
1‴‴
2‴‴
3‴‴
4‴‴
5‴‴
6‴‴
δH 3.51 (s, OMe-3′) and methylene at δH 2.42 (m, H-2′a) could
be assigned to the sugar moiety by the correlations from δH 5.28
(H-1′) to δC 36.6 (C-2′) and δC 79.4 (C-3′); δH 1.57(Me-6′) to δC
71.4 (C-5′) and δC 74.7 (C-4′); and δH 3.51 (OMe-3′) to δC 79.4
(C-3′) in the HMBC spectrum. Furthermore, the detailed COSY
correlations between δH 2.42 (m, H-2′a), δH 5.28 (H-1′) and δH
3.81 (H-3′); δH 3.81 (m, H-3′) and δH 3.61 (m, H-4′); and δH 4.19
(m, H-5′), δH 3.61 (H-4′) and δH 1.57 (H-6′) revealed the
presence of a 2, 6-dideoxysugar. The sugar unit was further
determined to be β-D-cymaropyranose based on its chemical
shift of δC 36.6 (C-2′) which was very close to the reported data
shaker to mix their contents and incubated at 35 °C for 24 h for
bacteria, 48 h for yeast.
For bacteria, MICs were determined upon addition of
50 μL (0.2 mg/mL) p-iodonitrotetrazolium chloride (INT,

272
C.-W. Song et al. / Fitoterapia 99 (2014) 267–275
of 36.7 ppm in the same solvent (pyridine), and N35.0 ppm
[13,14,16], this was also supported by the GC analysis of the
sugar derivative from compound 1 after acid hydrolysis. Besides
the sugar unit, the aglycone should include two carbonyl groups
and two double bonds which accounted for 4° of unsaturation,
the remaining 5° of unsaturation suggested that the aglycone
had a pentacyclic skeleton. The literature data suggested that
the aglycone in compound 1 was a known C-21 pregnane [2]. As
the methyl groups at C-18 and C-19 in pregnane were typical
methyl groups with β-configuration [21] in the ROESY spectral
data, the correlation between δH 1.11 (H-19) and δH 2.93 (H-
8) suggested the β-configuration of H-8; the correlation
between δH 1.11 (H-19) and δH 2.37 (H-4a) suggested the α-
configuration of H-4b, therefore, the correlation from δH 2.58
(H-4b) to δH 3.85 (H-3) indicated the β-configuration of the
hydroxyl group at C-3. Likewise, the correlation between δH
2.93 (H-8) and δH 2.40 (H-12a) suggested the β-configuration
of H-12a which revealed the α-configuration of H-12b, thus,
the observation of the correlation between δH 1.59 (H-12b) and
(calcd. for 959.4611). Detailed analysis of the1H and 13C NMR
data of compound 3 with those of 2 indicated that 3 had the
same aglycone as 2 but with a different sugar moiety. The
anomeric proton signals at δH 5.30 (dd, J = 9.6, 1.5 Hz, H-1′), δH
4.68 (dd, J = 9.7, 1.5 Hz, H-1″), δH 5.18 (d, J = 7.8 Hz, H-1‴), δH
5.13 (dd, J = 9.7, 1.4 Hz, H-1″″) in 1H NMR spectrum implied
the presence of four sugar units with a β-configuration. The 1D
and 2D NMR spectral data supported the presence of one β-D-
glucose, and two β-D-cymarose residues, together with another
β-L-cymarose unit, which were further confirmed by their
characteristic chemical shifts of δC 37.6 (C-2′) [13,14], δC 36.9
(C-2″) [16], δC 33.3 (C-2‴) [17]. In the HMBC spectrum, the key
correlations from δH 5.30 (H-1′) to δC 77.5 (C-3), from δH 4.68
(H-1″) to δC 83.8 (C-4′), from δH 5.18 (H-1‴) to δC 83.5 (C-4″)
and from δH 5.13 (H-1″″) to δC 80.3 (C-4‴) suggested that the
sugar moiety was also a straight chain and its linkage position
was at the C-3 hydroxyl group of the aglycone. As a result, the
above data allowed the structural assignment of 3 as shown in
Fig. 1, and named ecdysoside C.
δ
H 1.20 (H-9) suggested the α-configuration of H-9. In addition,
The molecular formula C49H74O16 of compound 4 could be
determined according to its HREI-MS m/z 918.4970 (calcd. for
918.4977). The 13C and DEPT NMR spectra revealed the same
aglycone unit as in compound 3 except for the sugar moiety. In
the 1H NMR spectra, it gave out four anomeric protons at δH
4.84 (dd, J = 9.6, 1.6 Hz, H-1′), δH 4.64 (dd, 9.7 2.1 Hz, H-1″), δH
4.74 (dd, 9.7 1.8 Hz, H-1‴) and δH 4.44 (dd, 9.6 1.7 Hz, H-1″″).
By further analyzing these anomeric proton signals with their
large coupling constant they were assigned to four sugar units
with a β-linkage. In the HSQC, HMBC and COSY spectra, the
β-D-configuration of three cymarose residues and the β-L-
configuration of the remaining cymarose residues were deter-
mined and further supported by comparison of the characteristic
chemical shift values of δC 35.4 (C-2′) [14], δC 35.4 (C-2″) [13], δC
36.3 (C-2‴) [13], and δC 31.9 (C-2″″) [14,17] with reported data.
In addition, the sequence of the sugar chain could be determined
by the HMBC correlations from δH 4.84 (H-1′) to δC 77.3 (C-3),
from δH 4.74 (H-1″) to δC 82.4 (C-4′), from δH 4.44 (H-1‴) to δC
82.5 (C-4″), and from δH 4.64 (H-1″″) to δC 82.3 (C-4‴).
Therefore, the structure of 4 was elucidated as shown in Fig. 1,
and named ecdysoside D.
The positive HRESI-MS peak at m/z 1121.5150 (calcd. For
1121.5145) suggested the molecular formula of compound 5 to
be C54H82O23 which was further supported by its 13C and DEPT
NMR spectra. The evidences from NMR spectra indicated that
the aglycone of 5 was the same as that of compound 4. Their
major differences appeared on the sugar moiety as the 1H NMR
spectra showed five anomeric proton signals at δH 5.27 (br d,
J = 9.6 Hz, H-1′), δH 5.14 (br d, J = 9.6 Hz, H-1″), δH 4.61 (br d,
J = 9.7 Hz, H-1‴), δH 5.09 (d, J = 7.8 Hz, H-1″″) and δH 5.25
(d, J = 7.7 Hz, H-1″‴), supporting the presence of five sugar
units with β-configuration. Based on the detailed correlations
of the 2D spectra data, the sugar moiety was deduced to be a
straight chain consisting of two D-glucose, together with two D-
cymarose and one L-cymarose which had similar chemical shift
values of 37.6 (C-2′) [13,14], δC 36.8 (C-2″) [14,16], and δC 33.3
(C-2‴) [17] as those in literature. The unambiguous HMBC
correlations from δH 5.27 (H-1′) to δC 77.5 (C-3), from δH 5.14
(H-1″) to δC 83.8 (C-4′), from δH 4.61 (H-1‴) to δC 83.6 (C-4″),
from δH 5.09 (H-1″″) to δC 73.5 (C-4‴), and from δH 5.25 (H-1″‴)
to δC 70.9 (C-6″″), revealed the sequence of the sugar chain and
it connected to the C-3 of aglycone in compound 5. Therefore,
as the correlation between δH 1.38 (H-21) and δH 2.76 (H-17)
with δH 1.59 (H-12b) could be observed, it suggested the
α-configuration of C-21 and H-17. The above observation
associated with literature data showed that the aglycone had a
skeleton of ecdysantherin [2]. Moreover, the linkage position of
the sugar moiety was at the C-3 hydroxyl group of the aglycone
by the unambiguous correlation from δH 5.28 (H-1′) to δC 77.4
(C-3) in the HMBC spectra. Thus, the structure of compound 1
was elucidated as shown in Fig. 1, and named ecdysoside A.
Compound 2 had the molecular formula of C42H62O13
,
which was identified by its positive HRESI-MS at m/z 797.4082
[M+Na]+ (calcd. for 797.4088). The similar signals to those of
compound 1 in NMR spectra suggested that compound 2
contained the same aglycone skeleton as 1 except for the sugar
moiety. In 1H NMR spectrum of compound 2, the characteristic
anomeric protons at δH 5.27 (dd, J = 9.6, 1.6 Hz, H-1′), δH 5.12
(dd, 9.6 1.6 Hz, H-1″), 4.76 (dd, J = 9.8 1.7 Hz, H-1‴) assumed
the presence of three sugar units and this could be confirmed
by its molecular formula and acid hydrolysates of compound 2.
All the three sugar units were identified with a β-configuration
on the basis of their large coupling constant (N9.0 Hz). The 13C
and DEPT NMR spectrum of 2 showed three methylene groups
at δC 37.0 (C-2′), 36.7 (C-2″), and 37.0 (C-2‴), three methyl
groups at δC 18.3 (C-6′), 18.3 (C-6″), and 18.4 (C-6‴), and three
methoxyl groups at δC 58.6 (−OMe), 58.6 (−OMe), and 56.8
(−OMe) which were assigned to the sugar moiety. These
observations further suggested the presence of three 2, 6-
dideoxysugar units. According to the detailed 2D NMR spectral
data together with acid hydrolysis experiment, all the sugar
signals were assigned to three cymaropyranoses. The sugar
units were determined to be with D-configuration by compar-
ing their corresponding 13C NMR chemical shifts at δC 37.0 (C-
2′) [14], δC 36.7 (C-2″) [13,14,16], and δC 37.0 (C-2‴) [14] which
were N35.0 ppm with literature data. Furthermore, the straight
sugar chain and its sequence were revealed by the correlations
from δH 5.27 (H-1′) to δC 76.9 (C-3), from δH 5.12 (H-1″) to δC
83.1 (C-4′) and from δH 4.76 (H-1‴) to δC 82.9 (C-4″) in HMBC
experiment. Thus, the structure of compound 2 was elucidated
as shown in Fig. 1, and named ecdysoside B.
The molecular formula of compound 3 was deduced to be
C48H72O18 on the basis of its HREI-MS m/z 959.4617 [M+Na]+

C.-W. Song et al. / Fitoterapia 99 (2014) 267–275
273
Fig. 1. Structures of pregnane glycosides 1–9 from Ecdysanthera rosea.
compound 5 was elucidated as shown in Fig. 1, and named
ecdysoside E.
indicated that the sugar moiety consisted of three 2, 6-dideoxy
sugar units. Furthermore, the presence of two β-D-cymarose
and one β-L-cymarose was determined on the basis of their
coupling constant in 1H NMR spectrum and characteristic
chemical shift values of 38.4 (C-2′) [16], δC 38.0 (C-2″) [13,14],
δC 34.0 (C-2‴) [19] which were in good agreement with
previously reported data. Moreover, the HMBC correlations
observed from δH 5.25 (H-1′) to δC 78.5 (C-3), from δH 5.10 (H-
1″) to δC 84.3 (C-4′), and from δH 4.68 (H-1‴) to δC 84.4 (C-4″)
indicated the structure of a straight sugar chain, which was
placed at C-3 of the aglycone. Thus, compound 6 was elucidated
as shown in Fig. 1, and named ecdysoside F.
The molecular formula of compound 6 was determined to
be C42H66O13 on the basis of the positive HRESI-MS m/z
801.4406 [M+Na]+ (calcd. for 801.4401). The similar IR and
NMR spectra data indicated that compound 6 possessed the
same aglycone but different sugar moiety to ecdysantheroside
A [12]. The anomeric protons at δH 5.25 (br d, J = 9.2 Hz, H-1′),
δH 5.10 (br d, J = 9.3 Hz, H-1″) and δH 4.68 (br d, J = 9.2 Hz, H-
1‴) suggested that there were three sugar units, and the
assumption was further confirmed by analyzing the derivatives
of its acid hydrolyzate. The 1D and 2D NMR spectral data

274
C.-W. Song et al. / Fitoterapia 99 (2014) 267–275
Table 5
Compound 7 with the molecular formula C48H76O18 was
Minimum inhibitory concentration (MIC, μg/mL) of compounds 1–9 against
bacteria and yeasts.
determined on the basis of its positive HRESI-MS m/z 963.4935
[M+Na]+ (calcd. for 963.4929). The same aglycone unit as 6
was confirmed by the 1H and 13C NMR spectra. For the sugar
moiety, the characteristic anomeric protons in 1H NMR spectrum
at δH 5.25 (br d, J = 9.2 Hz, H-1′), δH 5.09 (br d, J = 9.7 Hz, H-1″),
EF
SA
29916
CA
CN
CG
1
2
3
4
5
6
7
8
9
12.5
50
50
NA
50
NA
NA
25
50
NA
25
100
50
NA
NA
50
25
1
2
3
4
5
6
7
8
9
NA
NA
NA
NA
NA
100
100
50
50
100
25
100
25
50
NA
NA
100
100
NA
50
NA
12.5
50
12.5
NA
NA
25
δH 4.63 (br d, J = 9.7 Hz, H-1‴) and δH 5.12 (d, J = 7.7 Hz, H-1‴)
indicated the presence of four sugar units with β configuration.
Detailed analysis of its NMR spectroscopic data suggested
the presence of a straight sugar chain composed of one β-D-
glucose as well as two β-D-cymaropyranose and one β-L-
cymaropyranose which had characteristic chemical shift values
of 36.9 (C-2′) [16], δC 36.2 (C-2″) [15,16], δC 32.6 (C-2‴) [14,16]
in compound 7. The correlations from δH 5.25 (H-1′) to δC 77.0
(C-3), from δH 5.09 (H-1″) to δC 83.1 (C-4′), from δH 4.63 (H-1‴)
to δC 82.9 (C-4″), from δH 5.12 (H-1″″) to δC 73.4 (C-4‴) in HMBC
experiment suggested the sequence of the sugar moiety in
compound 7 and its location was similar to compound 6.
Therefore, the structure of 7 was elucidated as shown in Fig. 1,
and named ecdysoside G.
Compound 8 was found to possess a molecular formula of
C62H99O24 based on its positive HRESI-MS m/z 1227.6518 [M −
H]− (calcd. for 1227.6526). The 1H and 13C NMR spectra
showed the same aglycone part as 7. The 1H NMR data showed
six anomeric protons at δH 5.29 (br d, J = 9.5 Hz, H-1′), δH 5.14
(br d, J = 9.2 Hz, H-1″), δH 4.72 (br d, J = 9.6 Hz, H-1‴), δH 4.92
(d, J = 9.6 Hz, H-1″″), δH 4.90 (d, J = 9.7 Hz, H-1″‴) and δH 5.19
(d, J = 7.6 Hz, H-1‴‴), which indicated the presence of six sugar
units with β-configuration in compound 8. On the basis of
1D and 2D NMR spectral data, it was suggested that, besides
one β-D-glucose unit, the sugar moiety included four β-D-
cymaropyranose and one β-L-cymaropyranose which had
characteristic chemical shift values of 37.4 (C-2′), δC 37.2 (C-
2″), δC 37.8 (C-2‴), δC 37.7 (C-2″″) [13,14] and δC 33.4 (C-2″‴)
[17], corroborating reported literatures. The sequence of the
straight sugar chain could be further determined by the HMBC
correlations from δH 5.29 (H-1′) to δC 77.5 (C-3), δH 5.14 (H-1″)
to δC 83.4 (C-4′), δH 4.72 (H-1‴) to δC 83.4 (C-4″), δH 4.92 (H-1″″)
to δC 83.0 (C-4‴), δH 4.90 (H-1″‴) to δC 84.4 (C-4″″), and δH 5.19
(H-1‴‴) to δC 73.7 (C-4″‴), and it was deduced to be at C-3 of the
aglycone. Thus, the structure of 8 was elucidated as shown in
Fig. 1, and named ecdysoside H.
25
25
12.5 50
25 NA
12.5
50
25
NA
Gentamycin 1.562 0.195 0.195
Nystatin 12.5 12.5 3.125
NA: not active (MIC N 100 μg/mL).
Bacteria strains: EF: Enterococcus faecalis ATCC 10541, SA: Staphylococcus aureus
ATCC 25922, 29916: Providensia smartii ATCC29916.
Yeasts strains: CA: Candida albicans ATCC 2091, CN: Cryptococcus neoformans IP
90526, CG: Candida guilimondis.
research of these pregnane glycosides, some of them displayed
in vitro antimicrobial activities against specify microbes, which
might suggest that the pregnane glycosides are a kind of possible
active constituents in this plant.
Acknowledgments
This project was supported by the National Natural Science
Foundation of China (81225024), the National Science and
Technology Support Program of China (2013BAI11B02).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.fitote.2014.10.008.
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