Verticillosides A-M: Polyoxygenated pregnane glycosides from Asclepias verticillata L.

Article abstract of DOI:10.1016/j.phytochem.2012.02.019

As part of our ongoing effort to explore the chemical diversity of plants of the United States Midwest region, the isolation and identification of 13 pregnane glycosides named verticillosides A-M from Asclepias verticillata L. are reported. The structures of these compounds were elucidated by various spectroscopic techniques, including 1D and 2D NMR, IR, UV, and HRMS. The cytotoxicity of the isolates was evaluated against paired breast cell lines Hs578T (cancer) and Hs578Bst (normal), however, no significant growth inhibition was observed.

Full text of DOI:10.1016/j.phytochem.2012.02.019

Phytochemistry 78 (2012) 179–189
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Verticillosides A–M: Polyoxygenated pregnane glycosides from
Asclepias verticillata L.
Juan J. Araya a, Franklin Binns b, Kelly Kindscher c, Barbara N. Timmermann a,
⇑
a Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045, United States
b Facultad de Farmacia, Programa de Posgrado en Química, Universidad de Costa Rica, San Jose, Costa Rica
c Kansas Biological Survey, University of Kansas, KS 66047, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of our ongoing effort to explore the chemical diversity of plants of the United States Midwest
region, the isolation and identification of 13 pregnane glycosides named verticillosides A–M from
Asclepias verticillata L. are reported. The structures of these compounds were elucidated by various
spectroscopic techniques, including 1D and 2D NMR, IR, UV, and HRMS. The cytotoxicity of the isolates
was evaluated against paired breast cell lines Hs578T (cancer) and Hs578Bst (normal), however, no
significant growth inhibition was observed.
Received 3 November 2011
Received in revised form 11 January 2012
Available online 23 March 2012
Keywords:
Asclepias verticillata
Pregnane glycosides
Verticillosides
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
and in a stronger concentration, as an external and internal snake-
bite remedy (Campbell et al., 1951).
Pregnane glycosides are C21 steroidal compounds conjugated
with sugars that commonly occur in the family Asclepiadaceae.
They comprise a structurally diverse group that combines an agly-
cone skeleton and a variable number and type of sugar units, typ-
ically attached at the 3-position (Panda et al., 2006). Anticancer
and immunomodulating activities have been reported for this class
of compounds (Cao et al., 2006; Chen et al., 2010; Kim et al., 2005;
Kuroda et al., 2010; Li et al., 2006; Peng et al., 2008; Ye et al., 2005).
Members of the Asclepias genus, commonly known as ‘‘milk-
weeds’’, are widely distributed across the United States. They are
especially abundant in the Midwest, with over 130 plant species
and subspecies reported, many of them endemic to this geograph-
ical region. Although Asclepias is a rich source of cardiac and preg-
nane glycosides, it has been poorly investigated chemically and
biologically. For instance, Asclepias verticillata has not been investi-
gated phytochemically despite its traditional use as medicine and
food. The leaves and young shoots were boiled with meat and ea-
ten by the Hopi (Fewkes, 1896). The most common medicinal use
was the consumption by the Lakota and Hopi of a tea made from
the whole plant to increase the production of mother’s milk
(Fewkes, 1896; Rodgers, 1980). This may have been a Doctrine of
Signatures type use as the milky sap might indicate its value for
this purpose (Kindscher, 1992). The Choctaw used the root as a dia-
phoretic to promote sweating, and the root tea as both a stimulant,
As part of the ongoing effort to explore the biodiversity of the
American Great Plains, reported herein is the isolation and identi-
fication of 13 new pregnane glycosides named verticillosides A–M
(Fig. 1) from A. verticillata L. This report represents the first phyto-
chemical investigation of this species.
2. Results and discussion
Dried aerial parts of A. verticillata were extracted and subjected
to a series of purification steps, including a variety of chromato-
graphic techniques, to afford 13 new pregnane glycosides: verticil-
losides A (1; 38 mg), B (2; 42 mg), C (3; 10 mg), D (4; 36 mg), E (5;
27 mg), F (6; 37 mg), G (7; 11 mg), H (8; 19 mg), I (9; 10 mg), J (10;
13 mg), K (11; 12 mg), L (12; 9 mg), and M (13; 14 mg).
Verticilloside A (1) was obtained as a white, amorphous pow-
der. The HRMS displayed a [M+Na]+ ion at m/z 1055.5276 consis-
tent with a molecular formula of C50H80NaO22 (calcd 1055.5039).
The 1H NMR spectrum showed four singlet signals (d 2.49, 2.08,
1.94, 1.35) and an olefinic proton (d 4.99) that indicated the pres-
ence of an acetylated pregn-5-en-20-one skeleton (Table 2). The
proposed carbon skeleton was supported by the HMBC correlations
(Fig. 2) between the proton signal d 1.94 (Me-18) and the carbon
resonances d 58.3 (C-13), 73.9 (C-12), 89.8 (C-14), and 92.8 (C-
17); and proton signal d 1.35 (Me-19) and carbon resonances of d
39.2 (C-1), 139.6 (C-5), 44.8 (C-9), and 38.0 (C-10). Also, the HMBC
correlations between the methyl signal of the acetyl group (d 2.49)
and C-12 (d 73.9) clarified the attachment position of the methyl
⇑
Corresponding author. Tel.: +1 785 864 4844; fax: +1 785 864 5326.
E-mail address: btimmer@ku.edu (B.N. Timmermann).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2012.02.019

180
J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
Fig. 1. Structure of compounds 1–13.
NMR spectroscopic data analysis and comparison with previously
reported values. The connectivity of the sugars was established
by the following key HMBC correlations: cymarose anomeric pro-
ton H-10 (d 5.29) and C-3 (d 77.9); oleandrose anomeric proton
H-100 (d 4.70) and C-40 (d 83.6); thevetose anomeric proton H-1000
(d 4.90) and C-400 (d 83.6), and glucose anomeric proton H-1000 (d
5.16) and C-400 (d 83.8) (Fig. 2). The b-linkages of the four sugars
were established by the large coupling constants (J = 7.8–9.7) ob-
served for the anomeric protons. Finally, after acid hydrolysis of
compound 1, the optical rotation of the purified monomeric sugars
was measured in aqueous solution after 24 h equilibration period
and compared with previously reported values showing that the
sugars present in this compound had D-configuration. Therefore,
Fig. 2. Selected HMBC correlations observed in compound 1.
ester functionality. Finally, 1H,1H DQFCOSY, HSQC and HMBC
spectra allowed for the full assignment of the 1H and 13C signals
(Tables 1 and 2), and the aglycone moiety was deduced to be meta-
plexigenin (1a). The NMR spectroscopic data were in good agree-
ment with previously reported values for metaplexigenin
(Warashina and Noro, 2000; Ye et al., 2004). The initially proposed
stereochemistry was based on dipolar interactions observed in the
ROESY spectrum (Fig. 3) and further confirmed by X-ray crystallog-
raphy of the aglycone obtained by acid hydrolysis of the glycoside
(Fig. 4). In addition, four anomeric protons were observed in the 1H
NMR spectrum (d 5.29, 5.16, 4.90 and 4.70) suggesting that the
same number of sugars were attached at the 3-position. Further-
more, the presence of three methyl doublets in the aliphatic region
of the 1H NMR spectrum (d 1.78, 1.68 and 1.44) and three methoxy
groups (d 3.96, 3.56 and 3.49), indicated the presence of 6-deoxy-
3-methoxy sugars, commonly found in the Asclepias glycosides.
Using 1H,1H DQFCOSY, 1H, 1H-TOCSY, and HSQC-TOCSY spectra,
the proton spin systems and the carbon resonances of each sugar
were fully assigned (Tables 1 and 2). The sugar units were then
identified as cymarose, oleandrose, thevetose, and glucose by
the structure of 1 was determined to be metaplexigenin 3-O-b-
glucopyranosyl-(1 ? 4)-b- -thevetopyranosyl-(1 ? 4)-b- -olean-
dropyranosyl-(1 ? 4)-b- -cymaropyranose. To the best of our
D-
D
D
D
knowledge, compound 1 represents a previously unreported meta-
plexigenin glycoside that we named verticilloside A.
Verticilloside B (2), an amorphous white powder, showed a
HRMS [M+Na]+ ion at m/z 1052.5271, suggesting the same molec-
ular formula of C50H80NaO22 (calcd 1055.5039) as for compound 1.
First, the aglycone present in 2 was determined to be metaplexige-
nin and it was identified as described before. Second, the 1H NMR
spectroscopic also showed the presence of four anomeric protons
(d 5.27, 5.26, 5.00 and 4.67); however, only the chemical shift of
H-1000 changed significantly when compared with the anomeric
protons of 1. After analysis of the spin systems of each sugar aided
by analyses of 1H,1H DQFCOSY, and 1H, 1H-TOCSY and HSQC-TOCSY
spectra, the 1H and 13C signals were completely assigned and the
four sugars were elucidated as cymarose, oleandrose, 6-deoxy-3-
O-methyl allopyranose, and glucose. The HMBC correlations unam-
biguously established the connectivity of the sugars: anomeric
proton of cymarose H-10 (d 5.26) with C-3 (d 78.1); anomeric

J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
181
Table 1
13C NMR (125 MHz, C5D5N) spectroscopic data for compounds 1–13.a,b
Atom
1
2
3
4
5
6
7
8
9
10
11
12
13
1
2
3
4
5
6
7
8
39.2 t
30.2 t
77.9 d
39.5 t
139.6 s
119.6 d
35.0 t
74.6 s
44.83 d
38.0 s
25.2 t
73.9 d
58.3 s
89.8 s
34.1 t
33.2 t
92.8 s
10.8 q
18.5 q
210.6 s
28.0 q
39.4 t
30.4 t
78.1 d
39.7 t
139.8 s
119.7 d
35.2 t
74.1 s
45.0 d
38.2 s
25.4 t
74.8 d
58.4 s
90.0 s
34.3 t
33.4 t
92.0 s
11.0 q
18.7 q
210.8 s
28.2 q
39.3 t
30.2 t
78.0 d
39.6 t
139.6 s
119.5 d
35.0 t
74.6 s
44.8 d
37.7 s
25.2 t
73.9 d
58.3 s
89.8 s
34.1 t
33.2 t
92.8 s
10.8 q
18.5 q
210.6 s
28.0 q
39.3 t
30.2 t
78.0 d
39.6 t
139.6 s
119.5 d
35.0 t
74.6 s
44.8 d
37.7 s
25.2 t
73.9 d
58.2 s
89.8 s
34.1 t
33.2 t
92.8 s
10.8 q
18.5 q
210.6 s
28.0 q
39.4 t
30.3 t
78.1d
39.4 t
30.3 t
78.1d
39.7 t
139.5 s
120.2 d
35.7 t
74.4 s
44.9 d
37.7 s
29.5 t
71.1 d
59.2 s
89.2 s
34.9 t
34.6 t
89.3 s
11.8 q
18.7 q
73.5 d
18.2 q
39.3 t
30.3 t
78.1d
39.7 t
139.5 s
119.9 d
35.4 t
74.6 s
45.3 d
37.7 s
29.8 t
69.2 d
60.7 s
89.7 s
34.6 t
33.1 t
92.9 s
9.8 q
39.3 t
30.3 t
78.1d
39.7 t
139.5 s
120.2 d
35.4 t
74.6 s
45.3 d
37.7 s
29.8 t
69.3 d
60.7 s
89.7 s
34.6 t
33.1 t
92.9 s
9.8 q
39.3 t
30.3 t
78.1 d
39.7 t
139.7 s
119.9 d
35.4 t
74.1 s
44.9 d
37.7 s
29.8 t
69.2 d
60.7 s
89.7 s
34.6 t
33.2 t
92.9 s
9.8 q
39.4 t
30.3 t
78.1 d
39.7 t
139.5 s
120.1 d
35.6 t
74.1 s
44.9 d
37.7 s
29.5 t
71.1 d
58.4 s
89.2 s
34.9 t
34.6 t
89.4 s
11.8 q
18.7 q
73.5 d
18.2 q
39.2 t
30.3 t
78.0 d
39.6 t
139.5 s
120.0 d
35.4 t
74.6 s
44.5 d
37.7 s
26.0 t
75.7 d
57.8 s
89.0 s
34.6 t
33.2 t
89.2 s
12.2 q
18.5 q
71.3 d
19.8 q
39.2 t
30.3 t
78.0 d
39.6 t
139.5 s
120.0 d
35.4 t
74.6 s
44.4 d
37.6 s
26.0 t
75.7 d
57.8 s
89.0 s
34.6 t
33.2 t
89.2 s
12.1 q
18.5 q
71.3 d
19.8 q
39.2 t
30.3 t
78.1 d
39.6 t
139.5 s
39.7 t
139.5 s
120.2 d
35.7 t
74.4 s
44.9 d
37.7 s
29.5 t
71.1 d
59.2 s
89.2 s
34.9 t
34.6 t
89.4 s
11.8 q
18.6 q
73.5 d
18.2 q
120.0 d
35.4 t
74.6 s
44.5 d
37.6 s
26.0 t
75.7 d
57.8 s
89.0 s
34.6 t
33.2 t
89.2 s
12.1 q
18.5 q
71.3 d
19.8 q
9
10
11
12
13
14
15
16
17
18
19
20
21
18.7 q
209.9 s
28.2 q
18.7 q
209.9 s
28.2 q
18.8 q
209.9 s
28.2 q
12-Ac
12-Ac
12-Ac
12-Ac
12-Bz
12-Bz
12-Bz
1
2
3
4
5
170.3 s
21.1 q
170.5 s
21.3 q
170.3 s
21.1 q
170.3 s
21.1 q
167.0 s
132.1 s
130.8 d
129.2 d
133.6 d
167.0 s
132.1 s
130.8 d
129.2 d
133.6 d
167.0 s
132.1 s
130.8 d
129.2 d
133.6 d
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D-Cym
10
96.7 d
37.6 t
78.2 d
83.6 d
69.3 d
19.0 q
59.2 q
96.9 d
37.7 t
78.4 d
84.0 d
69.2 d
19.0 q
59.4 q
96.7 d
37.3 t
78.4 d
83.7 d
69.4 d
18.8 q
59.3 q
96.7 d
37.6 t
78.4 d
83.7 d
69.4 d
18.8 q
59.3 q
96.7 d
37.6 t
78.2 d
83.6 d
69.3 d
19.0 q
59.2 q
96.7 d
37.6 t
78.7 d
83.8 d
69.3 d
19.0 q
59.0 q
96.7 d
37.6 t
78.2 d
83.9 d
69.3 d
19.0 q
59.2 q
96.7 d
37.5 t
78.2 d
83.8 d
69.2 d
19.0 q
59.0 q
96.7 d
37.6 t
78.4 d
83.7 d
69.4 d
18.7 q
59.2 q
96.7 d
37.5 t
78.4 d
83.7 d
69.4 d
18.8 q
59.2 q
96.7 d
37.6 t
78.2 d
83.5 d
69.3 d
19.0 q
59.2 q
96.7 d
37.6 t
78.2 d
83.8 d
69.3 d
19.0 q
59.2 q
96.7 d
37.6 t
78.4 d
83.8 d
69.4 d
18.9 q
59.2 q
20
30
40
50
60
30-OMe
D
-Ole
D
-Ole
D
-Cym
D
-Cym
D
-Ole
D
-Ole
D
-Ole
D
-Ole
D
-Cym
D
-Cym
D
-Ole
D
-Ole
D-Cym
100
102.3 d
37.7 t
79.6 d
83.6 d
72.4 d
19.1 d
57.8 q
102.5 d
37.9 t
79.8 d
83.7 d
72.4 d
19.4 d
58.0 q
100.8 d
37.3 t
78.1 d
83.5 d
69.2 d
18.9 q
59.2 q
100.8 d
37.3 t
78.0 d
83.5 d
69.2 d
18.9 q
59.2 q
102.3 d
37.7 t
79.6 d
83.6 d
72.4 d
19.1 d
57.8 q
102.3 d
38.0 t
79.6 d
83.3 d
72.2 d
19.2 d
57.8 q
102.3 d
38.0 t
79.6 d
83.6 d
72.3 d
19.1 d
57.8 q
102.3 d
38.0 t
79.6 d
83.3 d
72.3 d
19.2 d
57.8 q
100.8 d
37.3 t
78.7 d
83.5 d
69.2 d
18.8 q
59.3 q
100.8 d
37.3 t
78.7 d
83.4 d
69.2 d
18.9 q
59.2 q
102.3 d
38.0 t
79.6 d
83.6 d
72.3 d
19.1 q
57.5 q
102.3 d
38.0 t
79.6 d
83.3 d
72.3 d
19.2 q
57.5 q
100.8 d
37.3 t
78.0 d
83.5 d
69.2 d
18.8 q
59.3 q
200
300
400
500
600
300-OMe
D
-Thv
D
-Allme
D
-Ole
D
-Ole
D
-Thv
D
-Allme
D
-Thv
D
-Allme
D
-Ole
D
-Ole
D
-Thv
D
-Allme
D-Ole
1000
104.4 d
75.3 d
86.7 d
83.8 d
72.3 d
19.1 q
61.0 q
102.4 d
73.2 d
83.7 d
83.8 d
70.1 d
18.8 q
62.3 q
102.3 d
37.6 t
79.6 d
83.6 d
72.3 d
19.0 q
57.8 q
102.2 d
38.0 t
79.6 d
83.3 d
72.3 d
19.2 q
57.8 q
104.4 d
75.3 d
86.7 d
83.8 d
72.3 d
19.1 q
61.0 q
102.2 d
73.0 d
83.5 d
83.6 d
70.0 d
18.6 q
62.1 q
104.4 d
75.3 d
86.7 d
83.6 d
72.3 d
19.1 q
61.0 q
102.2 d
73.0 d
83.5 d
83.6 d
69.9 d
18.6 q
62.1 q
102.2 d
38.0 t
79.6 d
83.3 d
72.3 d
19.2 d
57.8 q
102.2 d
38.0 t
79.6 d
83.3 d
72.3 d
19.2 d
57.8 q
104.4 d
75.3 t
86.7 d
83.6 d
72.4 d
19.1 d
60.9 q
102.2 d
73.0 d
83.5 d
83.6 d
70.0 d
18.7 q
62.1 q
102.2 d
38.0 t
79.6 d
83.3 d
72.3 d
19.2 q
57.5 q
2000
3000
4000
5000
6000
3000-OMe
D
-Glc
D
-Glc
D
-Thv
D
-Allme
D
-Glc
D
-Glc
D
-Glc
D
-Glc
D
-Allme
D
-Allme
D
-Glc
D
-Glc
D-Allme
10000
105.2 d
76.2 d
79.0 d
72.3 d
78.5 d
63.3 t
107.2 d
76.2 d
78.9 d
72.5 d
78.5 d
63.5 t
104.4 d
75.3 d
86.7 d
83.6 d
72.3 d
19.1 q
61.0 q
102.3 d
73.0 d
83.5 d
83.6 d
69.9 d
18.2 q
62.1 q
105.2 d
76.2 d
79.0 d
72.3 d
78.5 d
63.3 t
107.0 d
75.8 d
78.7 d
72.2 d
78.2 d
63.3 t
105.2 d
76.2 d
79.0 d
72.4 d
78.5 d
63.4 t
107.0 d
75.8 d
78.8 d
72.2 d
78.7 d
63.3 t
102.3 d
73.0 d
83.4 d
83.6 d
69.8 d
18.6 q
62.0 q
102.3 d
73.0 d
83.5 d
83.6 d
69.8 d
18.2 q
62.0 q
105.3 d
76.2 d
79.1 d
72.3 d
78.5 d
63.5 t
107.0 d
76.0 d
78.8 d
72.2 d
78.8 d
63.3 t
102.3 d
73.0 d
83.5 d
83.6 d
69.9 d
18.7 q
62.1 q
20000
30000
40000
50000
60000
30000-OMe
D
-Glc
D
-Glc
D
-Glc
D
-Glc
D-Glc
100000
200000
300000
400000
500000
600000
105.0 d
76.2 d
79.1 d
72.4 d
78.5 d
63.5 t
107.0 d
75.8 d
78.8 d
72.2 d
78.7 d
63.3 t
107.0 d
75.8 d
78.8 d
69.8 d
78.8 d
63.5 t
107.0 d
75.8 d
78.8 d
72.2 d
78.8 d
63.3 t
107.0 d
75.9 d
78.8 d
72.2 d
78.7 d
63.3 t
a
Ac: Acetyl; Bz: Benzoyl;
-Glucopyranose.
s: singlet; d: doublet; t: triple;: q: quartet.
D-Cym: D-Cymaropyranose; D-Ole: D-Oleandropyranose; D-Thv: D-Thevetopyranose; D-Allme: 6-deoxy-3-O-methyl-D-allopyranose; D-Glc:
D
b

182
J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
Table 2
1H NMR (500 MHz, C5D5N) spectroscopic data for compounds 1–5.a,b
Atom
1
1
J (Hz)
2
J (Hz)
3
J (Hz)
4
J (Hz)
5
J (Hz)
1.82 m⁄
1.12 ddd
2.10 m
1.82 m
3.86 m
2.56 br dd
2.45 m
5.31 m
2.47 m
2.33 m
1.72 m
2.24 ddd
2.10 m
4.99 dd
2.12 m
3.25 m
2.02 m
1.94 s
14.3, 13.4, 3.9
1.78 m
1.08 ddd
2.08 m
1.80 m
3.85 m
2.54 br dd
2.41 m
5.29 m
2.45 m
2.32 m
1.69 m
2.22 ddd
2.08 m
4.97 dd
2.10 m
3.23 m
2.01 m
1.94 s
14.2, 13.5, 3.8
1.79 m
1.09 ddd
2.08 m
1.81 m
3.85 m
2.56 br dd
2.44 m
5.28 m
2.47 m
2.32 m
1.70 m
2.24 m
2.09 m
4.98 m
2.10 m
3.23 m
2.03 m
1.94 s
13.9, 13.2, 3.9
1.80 m
1.09 ddd
2.08 m
1.80 m
3.85 m
2.54 br dd
2.43 m
5.29 m
2.47 m
2.32 m
1.71 m
2.23 ddd
2.09 m
4.98 dd
2.11m
13.8, 13.3, 3.9
1.94 m
1.19 ddd
2.13 m
1.86 m
3.91 m
2.60 br dd
2.50 m
5.40 m
2.50 m
2.38 m
1.66 m
2.54 m
2.01 m
3.92 m
2.06 m
2.01 m
13.3, 13.2, 3.7
2
3
4
12.8, 4.6
12.9, 4.6
12.5, 4.5
13.0, 4.6
12.7, 4.8
6
7
9
11
12.7, 12.3, 12.0
12.1, 4.1
12.9, 12.6, 12.4
11.6, 3.9
12.5, 12.3, 12.2
12.2, 4.2
12
15
16
3.23 m
2.02 m
1.94 s
18
19
20
21
1.98 s
1.43 s
1.35 s
1.34 s
1.34 s
1.34 s
2.08 s
12-Ac
2.49 s
2.08 s
12-Ac
2.48 s
2.48 s
12-Ac
2.08 s
2.48 s
12-Ac
2.08 s
1.54 d
6.4
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D-Cym
10
20
5.29 br d
2.32 m
1.90 ddd
4.05 m
3.49 dd
4.23 m
1.44 d
9.1
5.26 br d
2.28 m
1.88 ddd
4.02 m
3.47 dd
4.22 m
1.43 d
9.4
5.29 m
2.32 m
1.92 m
4.10 m
3.52 m
4.24 m
1.40 d
3.58 s
5.29 m
2.31 m
1.92 m
4.10 m
3.52 m
4.23 m
1.38 d
3.62 s
5.29 dd
2.32 m
1.90 m
4.05 m
3.49 dd
4.25 m
1.44 d
3.56 s
9.5, 1.7
13.2, 9.8, 2.6
9.6, 2.7
6.1
13.5, 9.6, 2.4
9.5, 2.7
6.2
30
40
9.5, 2.7
6.0
50
60
6.0
6.2
30-OMe
3.56 s
3.55 s
D
-Ole
D
-Ole
D
-Cym
D
-Cym
D-Ole
100
200
4.70 dd
2.50 m
1.78 m
3.59 m
3.62 dd
3.56 m
1.68 d
3.49 s
9.7, 1.6
4.67 dd
2.46 m
1.73 m
3.56 m
3.56 m
3.54 m
1.61 d
3.53 s
9.6, 1.7
5.13 dd
2.32 m
1.82 m
4.02 m
3.44 dd
4.20 m
1.39 d
3.63 s
9.7, 1.6
5.12 dd
2.31 m
1.81 m
4.00 m
3.42 dd
4.17 m
1.37 d
3.55 s
9.6, 1.5
4.70 dd
2.50 m
1.78 m
3.59 m
3.62 m
3.56 m
1.68 d
3.49 s
9.7, 1.7
300
400
9.0, 8.8
6.1
9.6, 2.6
6.0
9.6, 2.6
6.2
500
600
5.4
8.1
6.0
300-OMe
D
-Thv
D
-Allme
D
-Ole
D
-Ole
D-Thv
1000
2000
4.90 d
3.93 dd
7.9
9.0, 7.9
5.27 d
3.83 m
4.68 dd
2.49 m
1.77 m
3.56 m
3.62 m
3.57 m
1.68 d
3.52 s
9.7, 1.5
4.67 dd
2.48 m
1.73 m
3.57 m
3.57 m
3.55 m
1.61 d
3.53 s
9.7, 1.4
4.90 d
3.93 m
7.9
3000
3.71 dd
3.89 dd
3.77 dq
1.78 d
9.2, 9.0
9.3, 9.2
9.3, 6.1
6.1
4.50 dd
3.76 dd
4.29 m
1.66 d
3.84 s
2.6, 2.5
9.5, 2.2
3.71 dd
3.89 m
3.77 m
1.78 d
3.96 s
8.9, 8.9
4000
5000
9.4, 6.2
6.1
6000
6.2
6.0
5.5
3000-OMe
3.96 s
D
-Glc
D
-Glc
D
-Thv
D
-Allme
D-Glc
10000
20000
30000
40000
50000
60000
5.16 d
7.8
5.00 d
8.2
8.6, 8.5
4.89 d
3.93 m
3.71 dd
3.89 m
3.76 m
1.79 d
7.8
5.28 m
3.85 m
4.50 dd
3.77 dd
4.30 m
1.67 d
5.16 d
7.8
4.07 m
4.26 m
4.26 m
4.00 ddd
4.56 dd
4.38 dd
4.05 dd
4.28 m
4.24 m
4.02 m
4.57 dd
4.41 dd
4.07 m
4.26 m
4.26 m
4.00 ddd
4.56 dd
4.38 dd
9.0, 8.8
2.7, 2.6
9.5, 2.4
8.6, 5.4, 2.9
11.5, 2.1
11.5, 5.3
8.6, 5.4, 3.0
11.5, 2.7
11.5, 5.6
11.5, 2.0
11.5, 5.3
6.1
7.8
6.2
30000-OMe
3.96 s
3.84 s
D
-Glc
D-Glc
100000
200000
300000
400000
500000
600000
5.16 d
5.01 d
7.9
4.06 m
4.26 m
4.25 m
4.01 m
4.56 br d
4.40 br d
4.06 m
4.28 m
4.25 m
4.02 m
4.58 br d
4.40 br d
11.7
11.7
11.5
11.5
a
Ac: Acetyl;
D
-Cym:
D
-Cymaropyranose;
D
-Ole:
D
-Oleandropyranose;
D
-Thv:
D
-Thevetopyranose;
D
-Allme: 6-deoxy-3-O-methyl-
D-allopyranose;
D
-Glc:
D
-Glucopyranose.
b
s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet; br: broad.

J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
183
proton H-1000 (d 4.68) and C-400 (d 83.5); thevetose anomeric proton
H-10000 (d 4.89) and C-4000 (d 83.6); and glucose anomeric proton
H-10000 (d 5.16) and C-4000 (d 83.6). In a comparable manner, the
HMBC of 4 showed correlations between cymarose I anomeric
proton H-10 (d 5.29) and C-3 (d 78.0); cymarose II anomeric proton
H-100 (d 5.12) and C-40 (d 83.7); oleandrose anomeric proton H-1000
(d 4.67) and C-400 (d 83.5); 6-deoxy-3-O-mehtyl allopyranose
anomeric proton H-10000 (d 5.28) and C-4000 (d 83.3); and glucose
anomeric proton H-10000 (d 5.01) and C-4000 (d 83.6). Hence, 3 was
determined to be metaplexigenin 3-O-b-
(1 ? 4)-b- -thevetopyranosyl-(1 ? 4)-b- -oleandropyranosyl-
(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)-b- -cymaropyranose. Com-
pound 4 was established to be metaplexigenin 3-O-b- -glucopyr-
anosyl-(1 ? 4)-b-(6-deoxy-3-O-methyl)- -allopyranosyl-(1 ? 4)-
b- -oleandropyranosyl-(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)-b-
cymaropyranose.
D-glucopyranosyl-
Fig. 3. Observed ROE couplings of compound 1.
D
D
D
D
D
D
D
D
Verticilloside E (5), isolated as a white amorphous powder,
showed a calculated molecular formula of C48H80NaO21 based on
the observed HRMS ion [M+Na]+ at m/z 1015.5098 (calcd
1015.5090). The observation of two singlet methyl groups
(d 1.98, 1.43), a methyl doublet (d 1.54), and an olefinic proton
(d 5.40) in the 1H NMR spectrum as well as two olefinic carbons
(d 139.5 s and 120.2 d) in the 13C NMR suggested the presence of
a 20-hydroxy-pregn-5-ene skeleton. Using 1H,1H COSY, HSQC and
HMBC spectra, 1H and 13C signals in the pregnane skeleton could
be assigned unambiguously (Tables 1 and 2) and allowed for the
identification of the aglycone as sarcostin (5a). Relative orientation
of the hydroxy group at position C-17 was determined as beta
based on the X-ray structure of the aglycone (5a) obtained by acid
hydrolysis of 5 (Fig. 5). The 1H and 13C NMR spectroscopic data
were in good agreement with previously reported data for sarco-
stin (Li et al., 2008). In addition, 5 showed four anomeric protons
in the 1H NMR spectrum (d 5.29, 5.16, 4.90, and 4.70) indicating
the presence of four sugars attached to the aglycone. The sugars
were found to be the same as those present in 1 based on their
1H and 13C NMR data (Tables 1 and 2). Consequently, 5 was
Fig. 4. X-ray structure of metaplexigenin (1a) obtained by hydrolysis of 1.
proton of oleandrose H-100 (d 4.67) and C-40 (d 84.0); anomeric pro-
ton of 6-deoxy-3-O-methyl allopyranose H-1000 (d 5.27) and C-400
(d 83.7); and anomeric proton of glucose H-1000 (d 5.00) and C-400
(d 83.8). As a result, the structure of 2 was assigned to be
elucidated as sarcostin 3-O-b-
D
-glucopyranosyl-(1 ? 4)-b-
D
-theve-
topyranosyl-(1 ? 4)-b- -oleandropyranosyl-(1 ? 4)-b-
D
D-cymar-
opyranose. Moreover, verticilloside F (6) presented the same
calculated molecular formula as 5 (C48H80NaO21) based on the
experimental HRMS ion [M+Na]+ at m/z 1015.5153 (calcd
1015.5090). The 1H and 13C NMR signals of the aglycone portion
of 5 and 6 were almost superimposable, indicating that 6 also
had sarcostin as the pregnane core. On the other hand, after full
assignment of the 1H and 13C NMR signals using 2D NMR spectra,
the four sugars present in 6 were found to be the same as
those present in compound 2 by means of careful comparison of
their NMR data (Tables 1–3). Consequently, 6 was defined to
metaplexigenin 3-O-b-
methyl)- -allopyranosyl-(1 ? 4)-b-
b- -cymaropyranose.
Verticillosides C (3) and D (4) were both isolated as white,
D-glucopyranosyl-(1 ? 4)-b-(6-deoxy-3-O-
D
D
-oleandropyranosyl-(1 ? 4)-
D
amorphous powders. The HRMS of 3 and 4 showed very similar
[M+Na]+ ions at m/z 1199.5857 and 1199.5845, respectively, sug-
gesting that both shared the same molecular formula C57H92NaO25
(calcd 1199.5825). The aglycone structure in the two compounds
was established to be metaplexigenin (vide supra). However, five
anomeric protons were identified in the 1H NMR spectroscopic
(3: d 5.29, 5.16, 5.13, 4.89 and 4.68; 4: d 5.29, 5.28, 5.12, 5.01
and 4.67) indicating that each compound contained five sugar
units. Only one proton signal corresponding to H-10000 (d 4.89 in 3,
and d 5.28 in 4) showed a significant difference when the 1H
NMR spectrum of 3 was compared to that of 4. The 1H,1H DQFCOSY
and 1H,1H-TOCSY and HSQC-TOCSY spectra assisted significantly
with the identification for the spin systems of each sugar as well
as with the assignment of all the 1H and 13C signals (Tables 1
and 2). Two units of cymarose and single units of oleandrose,
thevetose, and glucose were identified in 3. On the other hand,
the sugars present in 4 were found to consist of two units of cyma-
rose and single units of oleandrose, 6-deoxy-3-O-methyl allopyra-
nose, and glucose. As previously described, HMBC correlations
allowed for the establishment of the sugar sequence in 3: cymarose
I anomeric proton H-10 (d 5.29) and C-3 (d 78.0); cymarose II ano-
meric proton H-100 (d 5.13) and C-40 (d 83.7); oleandrose anomeric
be sarcostin 3-O-b-
methyl)- -allopyranosyl-(1 ? 4)-b-oleandropyranosyl-(1 ? 4)-b-
-cymaropyranose.
D-glucopyranosyl-(1 ? 4)-b-(6-deoxy-3-O-
D
D
Fig. 5. X-ray structure of sarcostin (5a) obtained by hydrolysis of 5.

184
J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
Table 3
1H NMR (500 MHz, C5D5N) spectroscopic data for compounds 6–10.a,b
.
Atom
1
6
J (Hz)
7
J (Hz)
8
J (Hz)
9
J (Hz)
10
J (Hz)
1.93 m
1.18 ddd
2.13 m
1.86 m
3.90 m
2.60 br dd
2.49 m
5.41 m
2.48 m
2.37 m
1.65 m
2.53 m
2.00 m
3.92 m
2.06 m
2.00 m
13.4, 13.2, 3.5
1.89 m
1.14 ddd
2.12 m
1.87 m
3.89 m
2.59 br d
2.48 m
5.38 m
2.49 m
2.37 m
1.64 m
2.54 m
1.90 m
3.95 m
2.13m
13.4, 13.2, 3.6
1.89 m
1.13 ddd
2.11 m
1.86 m
3.89 m
2.58 br dd
2.47 m
5.36 m
2.49 m
2.37 m
1.64
2.46 m
1.90 m
3.96 m
2.14 m
3.40 m
2.12 m
2.02 s
13.8, 13.7, 3.5
1.90 m
1.13 dd
2.12 m
1.85 m
3.89 m
2.59 br dd
2.49 m
5.36 m
2.50 m
2.38 m
1.65 m
2.47 m
1.92 m
3.96 m
2.15 m
3.41 dd
2.03 m
2.02 s
13.8, 13.7, 3.5
1.92 m
1.20 ddd
2.13 m
1.86 m
3.90 m
2.61 br dd
2.50 m
5.39 m
2.49 m
2.38 m
1.67 m
2.54 m
2.01 m
3.91 m
2.07 m
2.01 m
13.5, 13.1, 3.8
2
3
4
12.6, 4.5
12.6, 4.6
12.8, 4.7
12.8, 4.6
12.6, 3.6
6
7
9
11
12
15
16
3.39 m
2.13 m
2.02 s
9.8, 2.7
18
19
20
21
1.97 s
1.43 s
1.98 s
1.43 s
4.49 q
1.54 d
1.42 s
1.41 s
1.41 s
6.4
6.4
1.54 d
6.4
2.60 s
2.65 s
2.66 s
D
-Cym
D
-Cym
D
-Cym
D
-Cym
D-Cym
10
20
5.30 dd
2.30 m
1.90 m
4.03 m
3.48 dd
4.23 m
1.43 d
3.56 s
9.6, 1.7
5.30 dd
2.32 m
1.89 m
4.05 m
3.48 dd
4.23 m
1.42 d
3.58 s
9.5, 1.6
5.30 dd
2.30 m
1.89 m
4.03 m
3.48 dd
4.23 m
1.41 d
3.55 s
9.4, 1.6
5.30 dd
2.33 m
1.92 m
4.10 m
3.52 m
4.24 m
1.38 d
3.63 s
9.6, 1.6
5.32 dd
2.33 m
1.92 m
4.11 m
3.52 m
4.25 m
1.38 d
3.63 s
9.5, 1.60
30
40
9.6, 2.7
6.2
9.6, 2.7
6.2
9.5, 2.6
6.2
50
60
6.2
6.2
30-OMe
D
-Ole
D
-Ole
D
-Ole
D
-Cym
D-Cym
100
200
4.67 dd
2.47 m
1.74 m
3.56 m
3.56 m
3.53 m
1.60 d
3.53 s
9.7, 1.6
4.69 dd
2.48 m
1.75 m
3.57 m
3.60 m
3.76 dq
1.68 d
3.51 s
9.7, 1.6
4.68 dd
2.40 m
1.74 m
3.56 m
3.56 m
3.54 m
1.61 d
3.52 s
9.6, 1.6
5.12 dd
2.33 m
1.88 m
4.02 m
3.43 dd
4.18 dq
1.38 d
9.6, 1.5
5.11 dd
2.32 m
1.81 m
4.00 m
3.43 dd
4.17 dq
1.38 d
9.7, 1.5
300
400
9.8, 2.7
9.8, 6.2
6.2
9.6, 2.6
9.6, 6.2
6.2
500
9.4, 6.1
6.0
600
5.8
8.0
5.8
8.0
300-OMe
3.55 s
3.56 s
D
-Allme
D
-Thv
D
-Allme
D
-Ole
D-Ole
1000
2000
5.28 d
3.84 m
4.89 d
3.92 m
7.9
5.27 d
3.83 m
4.68 dd
2.48 m
1.74 m
3.58 m
3.57 m
3.55 m
1.62 d
3.53 s
9.8, 1.5
4.67 dd
2.48 m
1.74 m
3.57 m
3.58 m
3.54 m
1.60 d
3.53 s
9.6, 1.4
3000
4.50 dd
3.76 dd
4.29 m
1.67 d
3.85 s
2.7, 2.7
9.6, 2.4
3.71 dd
3.89 dd
3.55 m
1.78 d
3.95 s
9.0, 9.0
9.0, 8.8
4.50 dd
3.77 dd
4.29 m
1.66 d
3.84 s
2.7, 2.7
9.6, 2.4
4000
5000
6000
6.2
6.1
6.2
5.5
8.1
5.4
8.1
3000-OMe
D
-Glc
D
-Glc
D
-Glc
D
-Allme
D-Allme
10000
20000
30000
40000
50000
60000
5.01 d
7.7
8.8, 8.4
5.16 d
7.8
5.01 d
7.8
5.27 d
5.28 d
3.86 m
4.51 dd
3.77 dd
4.30 m
1.68 d
4.06 dd
4.28 m
4.25 m
4.00 m
4.58 dd
4.41 dd
4.05 m
4.26 m
4.25 m
3.99 m
4.55 dd
4.39 dd
4.06 m
4.27 m
4.25 m
4.04 ddd
4.58 br d
4.41 dd
3.85 m
4.51 dd
3.77 dd
4.26 m
1.67 d
2.7, 2.7
9.5, 2.5
2.7, 2.7
9.6, 2.4
8.9, 5.4, 2.4
11.8
11.8, 5.9
11.6, 2.4
11.6, 5.4
11.4, 2.4
11.4, 5.5
6.2
8.0
6.2
30000-OMe
3.85 s
3.86 s
D
-Glc
D-Glc
100000
200000
300000
400000
500000
600000
5.02 d
5.02 d
8.2
8.6, 8.2
4.07 m
4.29 m
4.30 m
4.05 m
4.58 dd
4.41 dd
4.06 dd
4.29 m
4.26 m
4.03 ddd
4.58 dd
4.42 dd
9.0, 5.6, 2.5
11.6, 2.5
11.6, 5.6
11.5, 5.4
11.5, 2.5
a
D
-Cym:
D-Cymaropyranose; D-Ole: D-Oleandropyranose; D-Thv: D-Thevetopyranose; D-Allme: 6-deoxy-3-O-methyl-D-allopyranose; D-Glc: D-Glucopyranose.
b
s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet; br: broad.

J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
185
Table 4
1H NMR (500 MHz, C5D5N) spectroscopic data for compounds 11–13.a,b
.
Atom
1
11
J (Hz)
12
J (Hz)
13
J (Hz)
1.80 m
1.13 ddd
2.10 m
1.82 m
3.89 m
2.60 br dd
2.47 m
5.40 m
2.52 m
2.40 m
1.82 m
2.53 m
2.19 m
5.40 m
2.14 m
1.99 m
2.24 s
14.1, 13.8, 3.6
1.81 m
1.12 ddd
2.10 m
1.82 m
3.89 m
2.59 br dd
2.47 m
5.39 m
2.51 m
2.40 m
1.82 m
2.53 m
2.19 m
5.40 m
2.14 m
1.99 m
2.24 s
14.1, 13.8, 3.4
1.80 m
1.12 ddd
2.09 m
1.84 m
3.89 m
2.59 br dd
2.47 m
5.38 m
2.51 m
2.39 m
1.81 m
2.52 m
2.19 m
5.40 m
2.13 m
1.98 m
2.24 s
14.1, 13.5, 3.6
2
3
4
13.1, 4.4
13.0, 4.5
13.0, 4.3
6
7
9
11
12
15
16
18
19
20
21
1.37 s
4.12 dq
1.29 d
1.37 s
4.12 dq
1.29 d
1.36 s
4.12 m
1.29 d
6.1, 6.1, 6.1, 5.3
6.1
6.2, 6.2, 6.2, 5.6
6.2
6.1
12-Bz
12-Bz
12-Bz
8.59 br d
7.41 br dd
7.50 m
8.2
8.2, 7.6
8.59 br d
7.41 br dd
7.50 m
8.2
8.2, 7.6
8.59 br d
7.41 br dd
7.50 m
8.2
8.2, 7.6
D
-Cym
D
-Cym
D-Cym
10
20
5.31 dd
2.33 ddd
1.90 m
4.06 m
3.50 m
4.25 m
1.45 d
9.7, 1.5
13.5, 2.9, 1.5
5.30 dd
2.31 m
1.89 m
4.04 m
3.49 dd
4.24 m
1.44 d
3.56 s
9.7, 1.5
5.30 dd
2.32 m
1.90 m
4.10 m
3.53 m
4.24 m
1.38 d
3.63 s
9.6, 1.6
30
40
9.6, 2.4
6.2
50
60
6.2
6.2
30-OMe
3.59 s
D
-Ole
D
-Ole
D-Cym
100
200
4.70 dd
2.49 m
1.78 m
9.6, 1.6
4.68 dd
2.49 m
9.6, 1.6
5.12 dd
2.31 m
9.4, 1.3
1.75 m
3.57 m
3.56 m
3.54 m
1.62 d
3.54 s
1.81 m
4.00 m
3.41 dd
4.17 m
1.38 d
3.55 s
300
3.56 m
3.63 m
3.56 m
1.69 d
3.52 s
400
9.6, 2.5
6.2
500
600
300-OMe
6.0
7.9
5.9
D
-Thv
D
-Allme
D-Cym
1000
2000
4.89 d
3.92 m
5.28 d
3.85 m
7.9
4.67 dd
2.48 m
1.73 m
3.56 m
3.57 m
3.55 m
1.61 d
3.53 s
9.6, 1.2
3000
3.72 dd
3.89 dd
3.76 dq
1.67 d
9.0, 8.9
9.4, 9.0
9.4, 6.1, 6.1, 6.1
6.1
4.50 dd
3.76 m
4.31 m
1.68 d
3.85 s
2.7, 2.7
6.4
4000
5000
6000
5.3
8.0
3000-OMe
3.84 s
D
-Glc
D
-Glc
D-Allme
10000
20000
30000
40000
50000
60000
5.16 d
7.7
5.01 d
7.7
5.28 d
3.85 m
4.50 dd
3.76 dd
4.29 m
1.68 d
4.06 m
4.26 m
4.26 m
4.00 m
4.55 br d
4.40 br d
4.07 m
4.28 m
4.26 m
4.04 m
4.58 br d
4.41 br d
2.6, 2.4
9.6, 2.4
11.5
11.5
11.5
11.5
6.2
30000-OMe
3.85 s
(continued on next page)

186
J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
Table 4 (continued)
Atom
11
J (Hz)
12
J (Hz)
13
J (Hz)
D
-Glc
100000
200000
300000
400000
500000
600000
5.01 d
7.8
4.05 m
4.28 m
4.25 m
4.00 m
4.59 br d
4.42 m
11.5
a
Bz: Benzoyl;
D
-Cym:
D-Cymaropyranose; D-Ole: D-Oleandropyranose; D-Thv: D-Thevetopyranose; D-Allme: 6-deoxy-3-O-methyl-D-allopyranose; D-Glc: D-Glucopyranose.
b
s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet; br: broad.
Verticillosides G (7) and H (8) displayed a HRMS ion [M+Na]+ at
(C-13), 75.7 (C-12), 89.0 (C-14), and 89.2 (C-17); and between pro-
ton signal d 1.37 (Me-19) and carbon resonances of d 39.2 (C-1),
139.5 (C-5), 44.5 (C-9), and 37.7 (C-10). The experimental NMR
spectroscopic data (Tables 1 and 4) of the aglycone were in good
agreement with previously reported values (Gan et al., 2008).
The 1H NMR and 13C NMR values of the aglycone in compound
12 were almost identical to those just described for 11, indicating
that the two compounds shared the same aglycone (Tables 1 and
4). Furthermore, following full assignment of 1H and 13C NMR data
using 2D NMR spectra, close comparison of their spectra revealed
that the sugars present in 11 and 12 were identical to those present
in 1 and 2, respectively. Consequently, the structure of 11 was
m/z 1013.4913 and 1013.4921, respectively suggesting the same
molecular formula of C48H78NaO21 (calcd 1013.4933) for both com-
pounds. As in the case of compounds 1–4, three methyl singlets
were observed in the 1H NMR (7: d 2.60, 2.02, 1.42; 8: d 2.65,
2.02, 1.41); however, the acetyl 1H and 13C signals were missing
in 7 and 8. Following a similar analysis of the 1H, 13C and 2D
NMR spectroscopic data as described before, the aglycones present
in the two compounds were identified to be 12-O-deacylmetaplex-
igenin and the signal assignments were in good agreement with
previously reported values (Warashina and Noro, 2000). The 1H
and 13C signals were totally assigned for the sugar portion using
2D NMR spectra (Tables 1 and 3). While the sugars in 7 were found
to be the same as in 1, the sugars in 8 were identical to those pres-
ent in 2 based on their NMR data comparison. The structures of 7
and 8 were determined to be 12-O-deacylmetaplexigenin 3-O-
established as 12-O-benzoylsarcostin 3-O-b-
(1 ? 4)-b- -thevetopyranosyl-(1 ? 4)-b- -oleandropyranosyl-
(1 ? 4)-b- -cymaropyranose, and structure of 12 as sarcostin 3-O-
b- -glucopyranosyl-(1 ? 4)-b-(6-deoxy-3-O-methyl)- -allopyran-
osyl-(1 ? 4)-b-
D-glucopyranosyl-
D
D
D
D
D
b-
oleandropyranosyl-(1 ? 4)-b-
metaplexigenin 3-O-b- -glucopiranosyl-(1 ? 4)-b-
-oleandropyranosyl-(1 ? 4)-b- -cymaropyranose,
D
-glucopyranosyl-(1 ? 4)-b-
-cymaropyranose and 12-O-deacyl-
-thevetopyr-
D
-thevetopyranosyl-(1 ? 4)-b-
D
-
D-oleandropyranosyl-(1 ? 4)-b-D-cymaropyranose.
D
Finally, verticilloside M (13) had a HRMS [M+Na]+ ion at m/z
D
D
1263.6037 (calcd 1263.6138) suggesting a molecular formula of
C62H96NaO25. By closed comparison of the 1D and 2D NMR spectra,
anosyl-(1 ? 4)-b-
D
D
respectively.
the aglycone of 13 was identified to be 12-O-benzoylsarcostin, as
in compound 11 (Tables 1 and 4). The sugar moiety, however,
contained the same sequence of five units as those described for
compound 4. Hence, compound 13 was identified as 12-O-benzoyl-
Verticilloside I (9) showed a HRMS ion [M+Na]+ at m/z 1159.5904
indicating a molecular formula of C55H92NaO24, (calcd 1159.5876).
Complete assignment of 1H and 13C NMR signals using 2D NMR
spectroscopic showed that the aglycone corresponded to 12-O-dea-
cylmetaplexigenin. The five sugars present in 9 were identical to
those found in 4 and their 1H and 13C sugar signals were almost
identical (Tables 1 and 3). Hence, structure of 9 was confirmed to
sarcostin 3-O-b-
-allopyranosyl-(1 ? 4)-b-
opyranosyl-(1 ? 4)-b- -cymaropyranose.
D
-glucopyranosyl-(1 ? 4)-b-(6-deoxy-3-O-methyl)-
D
D
-oleandropyranosyl-(1 ? 4)-b- -cymar-
D
D
Cytotoxicity of the isolates (1–13) was tested against the paired
breast cell lines Hs578T (cancer) and Hs578Bst (normal); however,
no toxicity was observed in the experimental concentration range
be 12-O-deacylmetaplexigenin 3-O-b-
b-(6-deoxy-3-O-methyl)- -allopyranosyl-(1 ? 4)-b-
pyranosyl-(1 ? 4)-b- -cymaropyranosyl-(1 ? 4)-b- -cymaropyra-
nose. Likewise, verticilloside J (10) had a molecular formula of
55H90NaO24 on the basis of the observed HRMS ion [M+Na]+ at
D
-glucopyranosyl-(1 ? 4)-
D
D
-oleandro-
D
D
of 0.2–50 lM (see Supplementary Data).
C
3. Conclusions
m/z 1057.5734 (calcd 1157.5720). Following the complete assign-
ment of 1H and 13C NMR signals aided by 2D NMR data, the aglycone
was found to be sarcostin and the sugars the same as those present
in 4 (Tables 1 and 3). Hence, 10 was determined as sarcostin 3-O-
The present work reports the isolation and chemical character-
ization of 13 new polyoxygenated glycosides from A. verticillata.
The structures of these compounds were established on the basis
of spectroscopic data, including 800 MHz 2D NMR experiments.
Furthermore, relative stereochemistry for two compounds (1 and
5) was confirmed by means of X-ray crystallography. Our investi-
gation shows the diversity of secondary metabolites present within
Asclepias, a genus widely distributed in the US Midwest flora.
Although the isolates did not show significant cytotoxicity against
the tested cell lines, evaluation against other biological targets is
underway and will be reported in due course.
b-
osyl-(1 ? 4)-b-
syl-(1 ? 4)-b-
D
-glucopyranosyl-(1 ? 4)-b-(6-deoxy-3-O-methyl)-
-oleandropyranosyl-(1 ? 4)-b- -cymaropyrano-
-cymaropyranose respectively.
D-allopyran-
D
D
D
Verticillosides K (11) and L (12) displayed very similar [M+Na]+
ions at m/z 1119.5337 and 1119.5364, respectively, thus sharing
the same calculated molecular formula of C55H84NaO22 (calcd
1119.5352). Unlike previously described compounds, 1H NMR
spectrum of 11 showed aromatic signals corresponding to a ben-
zoyl group (d 8.59, 7.50, and 7.41) and further confirmed by 13C
NMR resonances (d 167.0, 133.6, 132.1, 130.8, and 129.2). The ben-
zoyl group was found to be attached to position 12 by means of the
observed HMBC correlation between H-12 (d 5.40) and the benzoyl
carbonyl signal (d 167.0). The aglycone was established as 12-O-
benzoylsarcostin based on the HMBC correlations between the
proton signal d 2.24 (H-18) with carbon resonances of d 57.8
4. Experimental section
4.1. General experimental procedures
Melting points were recorded with an OptiMelt automatic
apparatus. IR spectra were obtained with a Thermo Nicolet Avatar

J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
187
380 FT-IR. UV–Vis spectra were acquired in a Varian Cary 50 Scan; 1H
NMR, 13C NMR and two dimensional spectra were acquired with a
Bruker Avance AV-III 500 with a dual carbon/proton cryoprobe.
for C50H80NaO22) for 1H and 13C NMR spectroscopic data see Tables
1 and 2.
Some NMR experiments were conducted in
a
Bruker Unity
4.6. Verticilloside C (3)
800 MHz NMR with direct detection probe. HRMS were conducted
with a LCT Premier Waters corp. (Milford, MA). Semi-preparative
HPLC was conducted using an Agilent 1200 HPLC system with a
Amorphous white powder; mp 169.0–170.5 °C; [a]
25 = +6.6 (c.
D
0.378, MeOH); IR mmax (film) cmꢁ1: 3389.7 (OH), 1708.7 (C@O),
1644.8 (C@O), 1156.2 (C–O), 1059.8 (C-O); UVmax (nm, MeOH):
206.9, 274.1; HRMS m/z: 1199.5857 [M+Na]+ (1199.5825 calcd
for C57H92NaO25); for 1H and 13C NMR spectroscopic data, see Ta-
bles 1 and 2.
Phenomenex Luna C18 column (5
4.5 mL/min (approx. 160 bar), injection volume of 50
l
m, 250 ꢀ 10 mm), flow rate of
l
L (ca. 10 mg
sample), and UV detection using diode array. Preparative HPLC
separations were done using an Agilent 1100 HPLC system with a
Phenomenex Luna C18 column (5
35 mL/min (aprox. 35 bar), injection volume of 800
l
m, 250 ꢀ 21 mm), flow rate of
l
L (ca. 100 mg
4.7. Verticilloside D (4)
sample), and UV detection using multi-wavelength detector.
Amorphous white powder; mp 171.1–175.2 °C; [a]
25 = +31.8
D
(c. 1.63, MeOH); IR mmax (film) cmꢁ1: 3388.7 (OH), 1703.8.0
(C@O), 1642.2 (C@O), 1148.3, 1054.7; UVmax (nm, MeOH): 207.0,
282.0; HRMS m/z: 1199.5845 [M+Na]+ (1199.5825 calcd for
C57H92NaO25) for 1H and 13C NMR spectroscopic data, see Tables
1 and 2.
4.2. Plant Material
Above-ground biomass of A. verticillata was collected in July
10th of 2009 by Loring and Long at the Dog Leg Prairie of the Nelson
Environmental Study Area, Lawrence, Kansas (latitude: 39.0550°;
longitude: 95.1967°). Botanical identification was performed by
Kelly Kindscher and a voucher specimen was deposited in the R.L.
McGregor Herbarium (Collection number H. Loring 3559).
4.8. Verticilloside E (5)
Amorphous white powder; mp 160.1–162.6 °C; [a]
25 = +17.5
D
(c. 0.479, MeOH); IR mmax (film) cmꢁ1: 3378.3 (OH), 1150.1 (C–O),
992.8 (C–O); UVmax (nm, MeOH): 207.0, 267.0; HRMS m/z:
1015.5098 [M+Na]+ (1015.5090 calcd for C48H80NaO21); 1H and
13C NMR spectroscopic data, see Tables 1 and 2.
4.3. Plant extraction and isolation
A. verticillata fresh biomass (5.5 kg) was left to dry at room tem-
perature. The dry material (1.2 kg) was then ground to a fine pow-
der and extracted four times with a mixture of MeOH and CH2Cl2
(1:1, v/v) at room temperature. The organic solvents (c.a. 20 L)
were removed under reduced pressure to afford the crude extract
(138 g) (11.5% w/w of dry weight). The organic extract was sus-
pended in MeOH:H2O 9:1 (1 L) and extracted with hexanes
(500 mL ꢀ 3) to give a hexanes fraction. Then, MeOH was removed
from the aqueous layer under reduced pressure, with the volume
adjusted to 500 mL with distilled H2O. This was then successively
extracted with CH2Cl2 (500 mL ꢀ 3) and n-BuOH (500 mL ꢀ 3) to
give CHCl2 and n-BuOH fractions respectively. The n-BuOH extract
(41 g) was suspended in H2O (500 mL) and adsorbed in a MCI gel
(500 g) column, washed with H2O (2 L), and then eluted with mix-
tures of H2O–MeOH (9:1 ? 0:100, v/v) starting with 10% step
increments (2 L each fraction) to afford a total of 10 fractions (1–
10). Fractions 6–9 were purified as follows: first Sephadex LH-20
(500 g) CC using MeOH as eluent, followed by silica gel CC using
CHCl3:MeOH 95:5 (v/v) or CHCl3:MeOH 90:10 (v/v) as mobile
phase, and finally semi-preparative or preparative HPLC chroma-
tography using mixtures of CH3CN–H2O for elution. A total of 13
pregnane glycosides were isolated and chemically elucidated using
1H NMR, 13C NMR, 2D NMR, IR, UV, and HRMS.
4.9. Verticilloside F (6)
Amorphous white powder; mp 165.8–167.6 °C; [a]
25 = +21.7
D
(c. 0.974, MeOH); IR mmax (film) cmꢁ1: 3385.3 (OH), 1152.8 (C–O),
996.3 (C–O); UVmax (nm, MeOH): 207.2, 272.5; HRMS m/z:
1015.5153 [M+Na]+ (1015.5090 calcd for C48H80NaO21); for 1H
and 13C NMR spectroscopic data, see Tables 1 and 3.
4.10. Verticilloside G (7)
Amorphous white powder; mp 157.6–159.8 °C; [a]
25 = +13.6 (c.
D
0.366, MeOH); IR mmax (film) cmꢁ1: 3385.3 (OH), 1685.8 (C@O),
1152.8 (C–O), 996.3 (C-O); UVmax (nm, MeOH): 207.1, 282.0; HRMS
m/z: 1013.4913 [M+Na]+ (1013.4933 calcd for C48H78NaO21) for 1H
and 13C NMR spectroscopic data, see Tables 1 and 3.
4.11. Verticilloside H (8)
Amorphous white powder; mp 163.1–164.3 °C; [a]
25 = +12.3 (c.
D
0.674, MeOH); IR mmax (film) cmꢁ1: 3425.2 (OH), 1696.5 (C@O),
1152.1 (C–O), 997.4 (C–O); UVmax (nm, MeOH): 206.1, 280.7; HRMS
m/z: 1013.4921 [M+Na]+ (1013.4933 calcd for C48H78NaO21) for 1H
and 13C NMR spectroscopic data, see Tables 1 and 3.
4.4. Verticilloside A (1)
Amorphous white powder; mp 164.9–166.9 °C; [
a
]
25 = ꢁ14.3
D
4.12. Verticilloside I (9)
(c. 0.412, MeOH); IR mmax (film) cmꢁ1: 3366.6 (OH), 1706.0
(C@O), 1635.8 (C@O), 1158.4 (C–O), 1059.8 (C-O); UVmax 206.9,
277.5; HRMS m/z: 1055.5276 [M+Na]+ (1055.5039 calcd for
Amorphous white powder; mp 172.2–174.0 °C; [a]
25 = +25.2
D
(c. 0.447, MeOH); IR mmax (film) cmꢁ1: 3391.3 (OH), 1149.6 (C–O),
999.4 (C–O); UVmax (nm, MeOH): 206.1, 269.2; HRMS m/z:
1159.5904 [M+Na]+ (1159.5876 calcd for C55H92NaO24); for 1H
and 13C NMR spectroscopic data, see Tables 1 and 3.
C
50H80NaO22) for 1H and 13C NMR spectroscopic data see Tables 1
and 2.
4.5. Verticilloside B (2)
4.13. Verticilloside J (10)
Amorphous white powder; mp 169.8–172.7 °C; [
a
]
25 = ꢁ5.0 (c.
D
0.735, MeOH); IR mmax (film) cmꢁ1: 3394.8 (OH), 1708.6 (C@O),
1635.0 (C@O), 1058.7 (C–O), 1035.2 (C–O); UVmax (nm, MeOH):
208.3, 267.9; HRMS m/z: 1052.5271 [M+Na]+ (1055.5039 calcd
Amorphous white powder; mp 170.9–172.1 °C; [a]
25 = +13.1
D
(c. 0.288, MeOH); IR mmax (film) cmꢁ1: 3455.0 (OH), 1154.8 (C–O),
1051.3 (C–O); UVmax (nm, MeOH): 206.0, 268.9; HRMS m/z:

188
J.J. Araya et al. / Phytochemistry 78 (2012) 179–189
1057.5734 [M+Na]+ (1157.5720 calcd for C55H90NaO24); for 1H and
13C NMR spectroscopic data, see Tables 1 and 3.
336033; email: deposit@ccdc.cam.ac.uk). See also Supplementary
Information for crystal data and structure refinement.
4.14. Verticilloside K (11)
4.19. Cytotoxicity assay
Amorphous white powder; mp 173.1–174.8 °C; [a]
25 = +53.6
D
Breast cancer cell line Hs578T and one normal breast Hs578Bst
cell lines were seeded in separated 384-well plates (seeding den-
sity of 3000 cells per well, in a volume of 30 lL per well) and al-
lowed to attach and grow overnight in a cell incubator. Then,
compounds were added using a Lybcyte ECHO acoustic liquid han-
(c. 0.467, MeOH); IR mmax (film) cmꢁ1: 3389.0 (OH), 3032.3 (Ar–
H), 1732.5 (C@O), 1635.8 (C@O), 1155.5 (C–O), 977.7 (C–O); UVmax
(nm, MeOH): 204.1, 226.1, 274.1; HRMS m/z: 1119.5337 [M+Na]+
(1119.5352 calcd for C55H84NaO22) for 1H and 13C NMR spectro-
scopic data, see Tables 1 and 4.
dling instrument (eight concentrations in the range 0.2–50
and plates were incubated for 72 h. Next, cell viability was deter-
mined adding 10 L of CellTiter-Glo (CTG) reagent, shaking plates
lM)
4.15. Verticilloside L (12)
l
for 2 min followed by reading of luminescence after 15 min stabi-
lizing period. Each dose-response curve was determined by tripli-
cate. The data were normalized dividing by the median and IC50
calculation was done using GraphPad Prism software.
Amorphous white powder; mp 176.8–178.2 °C; [a]
25 = +25.2
D
(c. 0.254, MeOH); IR mmax (film) cmꢁ1: 3340.5 (OH), 3031.8
(Ar–H), 1698.8 (C@O), 1635.8 (C@O), 1153.4 (C–O), 978.5 (C–O);
UVmax (nm, MeOH): 204.0, 226.0, 274.1; HRMS m/z: 1119.5364
[M+Na]+ (1119.5352 calcd for C55H84NaO22) for 1H and 13C NMR
spectroscopic data, see Tables 1 and 4.
Acknowledgment
This study was supported by grant IND0061464 (awarded to
B.N.T and K.K.) from the Kansas Bioscience Authority (KBA) and
Center for Heartland Plant Innovations (HPI). The authors thank
Q. Long and H. Loring, botanists at the University of Kansas or at
the Kansas Biological Survey at the University of Kansas for assis-
tance in plant collections and identifications; Dr. Justin Douglas
(KU, Lawrence) for assistance in running the NMR experiments,
Dr. Victor Day for conducting the X-ray crystallographic analyses
(NSF grant CHE-0923449, to V. Day), and Dr. Huaping Zhang for
his critical reading of this manuscript. J.A. thanks LASPAU-Fulbright
program and the University of Costa Rica for financial support.
4.16. Verticilloside M (13)
Amorphous white powder; mp 176.8–178.2 °C; [a]
25 = +25.2
D
(c. 0.254, MeOH); IR mmax (film) cmꢁ1: 3340.5 (OH), 3031.8
(Ar–H), 1698.8 (C@O), 1635.8 (C@O), 1153.4 (C–O), 978.5 (C–O);
UVmax (nm, MeOH): 204.0, 226.0, 274.1; HRMS m/z: 1263.6037
[M+Na]+ (1263.6138 calcd for C62H96NaO25); for 1H and 13C NMR
spectroscopic data, see Tables 1 and 4.
4.17. Acid hydrolysis of glycosides
Hydrolysis of glycosides was conducted as described elsewhere
(Warashina and Noro, 2010). Acid hydrolysis of a mixture of 1–4
(50 mg) afforded the aglycone metaplexigenin (1a); mixtures of
5, 6 and 10 (50 mg) produced sarcostin (5a); a mixture of 7–9
(50 mg) gave 12-O-deacylmetaplexigenin; and a mixture of 11–
13 (50 mg) afforded 12-O-benzoylsarcostin (11a). Structure of the
aglycones were established by spectroscopic methods and com-
pared with previously reported data. From the combined aqueous
layers the following sugars were purified and identified by com-
parison of spectroscopic and optical rotation (after 24 h equilibra-
tion time) data reported in literature:
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2012.02.019.
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D
-Cymarose [
et al., 1986).
-Thevetoside [
et al., 2004).
a]
25 = +50.1 (c. 0.1 H2O); lit. + 51.6 (Tsukamoto
D
D
a
]
25 = +38.0 (c. 0.1 H2O); lit. + 42.3 (Hamed
D
D-Oleandrose [a]
25 = ꢁ9.8 (c. 0.1 H2O); lit. + 10.3 (Nakagawa
D
et al., 1983).
(6-Deoxy-3-O-methyl)-
D
-allose [
a]
25 = +3.9 (c. 0.1 H2O); lit. + 5
D
(Allgeier, 1968).
Glucose [
1944).
a]
25 = +50 (c. 0.1 H2O); lit. + 56 (Jacobs and Craig,
D
Jacobs, W.A., Craig, L.C., 1944. The veratrine alkaloids - xxii. On pseudojervine and
4.18. X-ray structure determination
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