Ecdysteroids and a sucrose phenylpropanoid ester from Froelichia floridana

Article abstract of DOI:10.1016/j.phytochem.2009.01.017

Phytoecdysteroid glycosides (1-5) and a phenylpropanoid ester of sucrose (6) were isolated from the whole plant of Froelichia floridana, along with eight known compounds including three ecdysteroids (7-9), four flavonoids (10-13), and one phenolic compoun

Full text of DOI:10.1016/j.phytochem.2009.01.017

Phytochemistry 70 (2009) 430–436
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Ecdysteroids and a sucrose phenylpropanoid ester from Froelichia floridana
*
Ping Wang, Shiyou Li , Stacy Ownby, Zhizhen Zhang, Wei Yuan, Wanli Zhang, R. Scott Beasley
National Center for Pharmaceutical Crops, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University, Nacogdoches, TX 75962, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 April 2008
Received in revised form 12 December 2008
Available online 28 February 2009
Phytoecdysteroid glycosides (1–5) and a phenylpropanoid ester of sucrose (6) were isolated from the
whole plant of Froelichia floridana, along with eight known compounds including three ecdysteroids
(
7–9), four flavonoids (10–13), and one phenolic compound (14). Structures were determined using a
combination of spectroscopic techniques. Compounds 1, 2 and 6–14 were tested in vitro for their activity
against human DNA topoisomerase I. Compound 13 (diosmetin) showed marginal inhibition against
Keywords:
Froelichia floridana
Amaranthaceae
topoisomerase I with IC50 of 130
lM in conjunction with low intercalation ability.
Ó 2009 Elsevier Ltd. All rights reserved.
Ecdysteroid glycosides
Phytoecdysteroids
Sucrose phenylpropanoid ester
DNA topoisomerase I
1
. Introduction
attempted discovery of novel bioactive agents from native Texas
plants, the isolation and structure elucidation of 14 compounds
are reported, including five new ecdysteroid glycosides and one
new phenylpropanoid ester of sucrose from F. floridana. All com-
pounds except 3–5 were tested in vitro for the activity against hu-
man DNA topoisomerase I (Topo I).
Froelichia Moench (cottonweed or snake-cotton) is a genus of
the family Amaranthaceae with 16 species of annuals, perennials,
and shrubs native to the Western Hemisphere (McCauley, 2004).
Although the genus has a wide range from the southern extremes
of Canada to Northern Argentina and Uruguay (McCauley, 2004),
there is no report available on any medicinal uses of cottonweeds.
The Froelichia genus is probably best known as a roadside weed, as
thrives in environments with little competition (Flora of North
America Editorial Committee, 1993). To date, there is only one re-
port on chemical investigations of the genus, with two ecdyster-
oids (insect molting hormones) identified (Sarker et al., 1998).
Froelichia floridana (Nuttall) Moquin-Tandon is an annual herba-
ceous species, commonly known as ‘‘Florida snake-cotton” or
2
. Results and discussion
The EtOH extract of the whole plant of F. floridana was repeatedly
subjected to silica gel, Sephadex LH-20, or ODS column chromato-
graphic purification steps, followed by preparative HPLC and TLC
to afford compounds 1–14 (Fig. 1). Compounds 1–6 were new,
whereas compounds 7–14 were known previously. All were identi-
fied by analysis of their physical and spectroscopic data and compar-
ison with literature values. The known compounds included three
phytoecdysteroids: 20-hydroxyecdysone (7) (Zhu et al., 2001),
blechnoside B (8) (Suksamrarn et al., 1986), achyranthesterone A
‘
1
‘plains snake-cotton” (Flora of North America Editorial Committee,
993). This species is native to North America and to the West Indies
of the Caribbean and has also been naturalized in Australia (USDA
ARS, 2008). It can be found on open sand prairies, edges of wood-
lands in sandy soils, roadsides, and railroad rights-of-way (Flora of
North America Editorial Committee, 1993). Interestingly, the species
has been listed as an endangered species in the Ohio Rive area of
southeastern Ohio (McCauley and Ungar, 2002), but as invasive in
both Nebraska and the Great Plains according to authoritative
sources (USDA NRCS, 2008). From the seeds of F. floridana, Sarker
et al. (1998) isolated 20-hydroxyecdysone and 2-dehydro-3-epi-
(
9) (Meng et al., 2005), four flavonoids: laricitin 3-O-b-
ranoside (10) (Braca et al., 2001; Zhao et al., 2007), kaempferol 3-
O-b- -(6-O-p-E-coumaroyl)-glucopyranoside (11) (Tsukamoto
et al., 2004), daidzin (12) (Kinjo et al., 1987), and diosmetin (13)
Timmermann et al., 1979; Yu and Yang, 1999) and one phenylprop-
D-glucopy-
D
(
anoid: 3,4-dihydroxyphenyl caffeate (14) (Zhang et al., 2008).
Compound 1 was obtained as a colorless amorphous powder
+
and had a [M + Li] ion peak at m/z 617.3881 in the HRESIMS, cor-
2
0-hydroxyecdysone. As part of our continuing research on the
responding to a molecular formula of C33
tion spectrum gave an unusually broad peak with a maximum
absorption at 246 nm, which was characteristic of an , b-unsatu-
rated ketone (Ikekawa et al., 1980; Sadikov et al., 2000; Jayasinghe
54
H O10. The UV absorp-
a
*
Corresponding author. Tel.: +1 936 468 2071; fax: +1 936 468 7058.
E-mail address: lis@sfasu.edu (S. Li).
0
031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.01.017

P. Wang et al. / Phytochemistry 70 (2009) 430–436
R4
431
2
1
1
R6
R3
2
6
23
24
25
27
2
2
1
R =H; R =H-β; R =OH; R =H; R =Glc; R =CH
1 2 3 4 5 6 3
2
0
8
2
1
OR5
2 R =H; R =H-β; R =H; R =H; R =Glc -Glc ; R =CH
3 R =H; R =H-β; R =H; R =H; R =Glc; R =CH
1 2 3 4 5 6 3
4 R =H; R =H-α; R =H; R =H; R =Glc -Glc ; R =CH
1 2 3 4 5 6
5 R =H; R =OH-β; R =H; R =H; R =Glc -Glc ; R =CH
1 2 3 4 5 6 3
1
2
3
4
5
6
3
3
1
2
1
7
1
1
1
3
1
9
1
6
2
1
1
4
R1
15
9
2
1
1
4
2
10
8
OH
7 R =OH; R =H-β; R =OH; R =OH; R =H; R =CH
1 2 3 4 5 6 3
8 R =H; R =H-β; R =H; R =OH; R =Glc; R =CH
1 2 3 4 5 6 3
3
5
7
6
HO
R2
9 R1=OH; R2=H-β; R3=OH; R4=OH; R5=H; R6=CH2OH
O
R2
OH
OH
6'
4'
O
HO
O
O
R O
5'
2'
OR1
5
R3
HO
1'
1
3
'
CH OR
2
1
OR4
O
O
OH
4
O
OH
OH
2
5
3
OH
HO
O
CH OR
2
3
6
OR2
1
0 R =H; R =OCH ; R =OH;
1 2 3 3
6
R =R =Ac; R =R =R =
p
-
E
-coumaroyl
11 R1=
p
-
E
-coumaroyl; R =H; R =H
1
5
2
3
4
2
3
13
OCH3
OH
1
2
O
O
O
HO
O
O
O
OH
HO
OH
OH
OH
O
OH
O
14
OH
OH
OH
OH
Fig. 1. Compound structures from Froelichia floridana.
et al., 2003; Hunyadi et al., 2004; Liktor-Busa et al., 2007). By ana-
lyzing the H and C NMR spectroscopic data, it was determined
supported by the chemical shift of C-3 at d
C
62.8 ppm, whereas a
1
13
3a-OH configuration usually gives a downfielded signal that is
that 1 was an ecdysone analog with a monosaccharide moiety (Ta-
shifted 3–5 ppm (Lafont et al., 2008). Moreover, a NOE for Me-18
to Me-21 was observed, in agreement with a 20R configuration
(Fig. 3) (Pongrácz et al., 2003). Thus, the aglycone of 1 was assigned
as 2,22-dideoxy-20-hydroxyecdysone. Acid hydrolysis of 1 yielded
a sugar, identified as D-glucose by co-TLC analysis and by measure-
ment of optical rotation (Zhang et al., 2006). The monosaccharide
ble 1). Five methyls observed at d
.13 (Me-21), 1.15 (Me-26 and Me-27) in the H NMR spectrum
of 1 were unambiguously assigned by HMBC correlations (Fig. 2).
H
0.73 (Me-18), 0.85 (Me-19),
1
1
1
3
The C NMR spectrum of 1 had one oxymethine at d
and three oxyquaternary carbons at d 85.0 (C-14), 73.6 (C-20),
and 76.5 (C-25), which were also assigned by HMBC correlations
Fig. 2). The Me-19/H -11, Me-19/H -1, and Me-19/H-5 correla-
tions in the NOESY spectrum were indicative of a cis-A/B ring junc-
tion, whereas the H -11/Me-18, H -15/Me-18, and H -12/H-17
cross-peaks indicated a trans-C/D ring junction; at the same time
it was verified that H-17 was in the -position (Fig. 3). In addition,
C
62.8 (C-3)
C
moiety was deduced as glucopyranose with a b-anomeric configu-
1
3
(
b
b
ration from its C NMR spectroscopic data and the anomeric pro-
ton coupling constant (J = 7.7 Hz, see Table 1) (Maria et al., 2005).
As observed in the HMBC spectrum of 1, a key HMBC correlation
b
b
a
of the anomeric proton of sugar at d
25 (d 76.5) of the aglycone confirmed that the glucose was at-
tached to the C-25 position (Fig. 2). Accordingly, compound 1
H
4.26 (d, J = 7.7 Hz) with C-
a
C
the coupling constants of the H-17 signal (t, J = 9.0 Hz) established
the b-configuration of the side-chain at C-17 according to previ-
ously reported (Girault et al., 1996; Suksamrarn and Yingyongnar-
was established as 2,22-dideoxy-20-hydroxyecdysone 25-O-b-D-
glucopyranoside.
ongkul, 1996). The NOESY correlation of H
small NOE for H -2 to H-3 indicated that H-9 was
OH-3 was b-oriented. The orientation of the 3b-OH group was also
a
-2 with H-9 and the
Compound 2, a colorless amorphous powder, was assigned a
molecular formula of C39 14 by HRESIMS, which exhibited a
[M + Li] ion peak at m/z 763.4463. The UV spectrum was consistent
a
a-oriented and
64
H O
+

4
32
P. Wang et al. / Phytochemistry 70 (2009) 430–436
Table 1
6 3 4
NMR spectroscopic data of 1ꢀ5 (1 and 2 in DMSO-d , 3–5 in CD OD-d ).
Position
1
2
3
4
5
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
d
H
(J in Hz)
d
C
H
d (J in Hz)
1
1
2
2
3
a
b
a
b
b
29.1
1.49 m
1.22 m
1.63 m
1.32 m
3.84 br. s
28.8
1.42 m
1.26 m
1.56 m
1.43 m
3.78 br. s
29.9
1.62 m
1.43 m
1.80 m
1.65 m
3.98 br. s
(W1/2 = 15.8)
1.31 m
38.0
1.85 m
1.39 m
2.10 m
1.39 m
3.61 m
25.5
1.83 m
1.45 m
1.96 m
1.77 m
4.09 br.s
(W1/2 = 16.2)
1.61 m
28.2
62.8
32.1
27.7
62.0
32.2
29.0
65.6
33.4
31.0
71.8
31.4
29.2
67.1
36.7
(
W1/2 = 15.6)
(W1/2 = 15.7)
1.32 m
4
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
a
1.37 m
1.78 m
b
1.57 m
2.42 dd (12.1, 4.0)
2.11 m
2.36 dd (12.3, 3.5)
2.03 m
50.8
2.27 dd (12.1, 3.7)
5.63 d (3.0)
3.07 m
50.7
205
120.4
167.0
31.7
2.19 dd (12.2, 3.3)
5.59 s
52.5
207
54.7
81.0
203
120.4
167
38.1
43.4
22.4
a
203.4
120.3
165.8
32.1
36.1
20.1
203.4
123.3
166.7
47.6
39.7
21.8
121.8
168.6
37.7
43.4
21.8
5.81 s
5.85 d (2.7)
2.75 m
5.86 s
3.07 m
3.34 m
3.38 m
0
1
1b
2
2b
3
4
5
5b
6
6b
7
8
9
0
1
2a
2b
3a
3b
4a
4b
5
36.5
20.6
a
1.47 m
1.59 m
1.99 dd (11.8, 7.8)
1.66 m
1.37 m
1.51 m
1.86 m
1.53 m
1.53 m
1.74 m
2.08 m
1.77 m
1.58 m
1.77 m
2.07 m
1.73 m
1.55 m
1.70 m
2.09 m
1.76 m
a
30.7
30.9
32.4
32.2
32.3
48.6
85.0
29.9
49.1
84.0
30.8
48.3
86.1
31.9
47.8
85.8
32.0
47.9
86.0
32.0
a
a
1.50 m
1.79 m
1.82 m
1.67 m
2.17 t (9.0)
0.73 s
1.42 m
1.73 m
1.86 m
1.19 m
1.79 t (9.0)
0.50 s
1.58 m
1.95 m
2.06 m
1.42 m
1.97 t (9.5)
0.71 s
1.58 m
1.92 m
2.03 m
1.40 m
1.92 m
0.72 s
1.58 m
1.93 m
2.01 m
1.40 m
1.94 m
0.73 s
a
20.7
26.9
28.0
28.0
27.9
51.2
17.7
23.8
73.6
26.6
44.5
50.4
15.8
24.1
35.4
19.2
36.7
52.0
16.4
24.5
37.0
19.6
38.0
52.0
16.5
13.4
37.1
19.8
38.0
52.1
16.4
17.2
37.0
19.6
38.0
0.85 s
0.78 s
0.97 s
0.86 s
0.89 s
1.28 m
0.79 d (6.7)
1.25 m
0.87 m
1.99 m
1.25 m
1.37 m
1.25 m
1.50 m
0.96 d (6.4)
1.45 m
1.02 m
1.62 m
1.47 m
1.56 m
1.46 m
1.47 m
0.97 d (6.5)
1.46 m
1.10 m
1.56 m
1.32 m
1.53 m
1.42 m
1.48 m
0.97 d (6.5)
1.42 m
1.05 m
1.45 m
1.30 m
1.55 m
1.44 m
1.13 s
1.33 m
1.26 m
1.38 m
1.27 m
1.38 m
18.2
42.1
20.6
42.3
21.8
43.7
21.8
43.9
21.8
43.8
76.5
26.3
26.2
97.1
73.3
77.0
70.3
76.5
61.2
78.0
25.7
26.9
95.9
82.1
76.7
70.4
77.3
61.3
79.2
26.9
27.1
98.8
75.4
78.4
71.9
77.7
63.0
79.9
26.4
27.3
97.4
82.7
78.4
71.3
78.7
63.1
79.8
26.4
27.2
97.3
82.6
78.3
71.7
78.5
63.1
6
7
1.15 s
1.15 s
4.26 d (7.7)
2.90 m
3.13 m
3.01 m
3.04 m
3.76 d (11.0)
1.08 s
1.08 s
4.38 d (7.4)
3.14 d (8.4)
3.34 d (8.3)
3.04 m
3.07 m
3.60 d (11.6)
3.43 m
4.33 d (7.7)
2.96 t (8.4)
3.13 m
1.25 s
1.25 s
4.45 d (7.8)
3.13 m
3.36 m
3.30 m
3.24 m
3.82 dd (11.7, 2.3)
3.65 m
1.27 s
1.27 s
4.59 d (8.5)
3.41 m
3.53 m
3.54 m
3.27 m
3.84 d (11.5)
3.69 m
4.58 d (8.1)
3.22 m
3.38 m
3.31 m
3.24 m
3.81 d (11.8)
3.64 m
1.27 s
1.29 s
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
4.59 d (8.1)
3.42 m
3.55 m
3.63 m
3.27 m
3.85 m
3.68 m
4.58 d (8.1)
3.24 m
3.39 m
3.28 m
3.26 m
3.83 m
3.66 m
3.63 m
0
0
0
0
0
0
Glc -1
Glc -2
Glc -3
Glc -4
Glc -5
Glc -6
104.3
75.4
76.3
70.2
76.3
61.4
105.5
76.5
77.7
71.6
77.9
62.9
105.4
76.4
77.6
71.7
77.8
62.0
3.07 m
3.06 m
3.55 d (11.6)
3
.37 m
a
Assignments were based on COSY, HMQC, and HMBC experiments.
CH3
HO
CH3
OH
H
OH
H
O
CH3
O
H
H
H
HO
H
OH
H
H
H
OH
O
OH
H
H
H
OH
H
HO
H
H
H
H
HO
C
H
H
H
O
Fig. 3. Relative stereochemistry of 1. Arrows show characteristic NOE steric
Fig. 2. Key HMBC correlations of 1.
proximities.

P. Wang et al. / Phytochemistry 70 (2009) 430–436
433
with the presence of a 7-en-6-one chromophore in ecdysteroids
indicated that the major difference was a methine at d
in 2 being replaced by an oxyquaternary at d
correlations between H-7 and Me-19 with the signal at d
established the presence of an OH substituent at C-5. Thus com-
pound 5 was assigned as the 5-hydroxy derivative of 2. The 5b-
OH configuration was indicated by the upfield resonance for the
C
50.7 (C-5)
with a maximum value at 248 nm. The 13C NMR spectrum of com-
pound 2 showed 39 signals, of which 27 were assigned to the agly-
cone, the remaining 12 to two hexose units. Acid hydrolysis of 2
81.0 in 5. The HMBC
80.1
C
C
yielded D-glucose by co-TLC analysis with an authentic sample. De-
tailed NMR spectroscopic analysis showed that the aglycone of 2
was different from 1 only at the C-20 position. As shown in the
C
Me-19 at d 17.2 ppm (Jayasinghe et al., 2003; Tóth et al., 2008; La-
1
13
H and C NMR spectra, the oxyquaternary carbon at d
C
73.6 (C-
font et al., 2008). The similar NMR spectrum of rings C and D, the
side chain, and the disaccharide chain in 5 and 2 suggested a similar
structure and stereochemistry for these moieties (Tóth et al., 2008).
Compound 5 was thus elucidated as 2,22-dideoxy-5b-hydroxyec-
2
0) in 1 was replaced by a methine at d 35.4 in 2, indicating that
C
the aglycone of 2 was a 20-deoxy-derivative of 2,22-dideoxy-20-
hydroxyecdysone. The methyl group at C-20 in 2 which appeared
a doublet peak at d
H
0.79 (J = 6.7 Hz) also supported this conclusion
dysone 25-O-b-D-glucopyranosyl-(1?2)-b-D-glucopyranoside.
(
Girault et al., 1990). The cis A/B and trans B/C and C/D ring junc-
The HRESIMS of compound 6, a colorless amorphous powder,
exhibited a [M + Li] ion peak at m/z 871.2642 consisted with a
molecular formula C43H O19. Alkaline hydrolysis of 6 yielded su-
44
crose and p-coumaric acid, which were identified by co-HPLC and
+
tions and C-20 (R) configuration were deduced from NOESY corre-
lations, and comparison with NMR spectroscopic data of related
compounds (Lafont et al., 2008). Therefore, the aglycone of 2 was
defined as 2,22-dideoxyecdysone. The presence of two hexose units
was indicated by two anomeric protons at d
4
served in the HMQC spectrum. Both sugars were assigned as b-glu-
copyranose based on their 13C NMR spectroscopic data and two
anomeric proton coupling constants (Table 1). The linkage and
the sequence of the disaccharide moiety were deduced from HMBC
correlations. The long-range correlation of d
inner glucose (Glc) with d 78.0 (C-25) of the ecdysteroid skeleton
suggested that Glc was located at C-25 position, while the HMBC
TLC with authentic samples. The presence of a sucrose moiety
1
3
H
4.38 (d, J = 7.4 Hz) and
95.9, 104.3) ob-
could be confirmed by its typical C NMR spectroscopic data with
eight oxymethines at d 76.6 (C-3), 79.7 (C-4), 72.7 (C-5), 91.0
.33 (d, J = 7.7 Hz) and two anomeric carbons (d
C
C
0
0
0
0
0
(C-1 ), 72.3 (C-2 ), 68.9 (C-3 ), 68.8 (C-4 ), 67.8 (C-5 ), three oxym-
0
ethylenes at d
ternary carbon at d
p-coumaroyl moieties in 6 were easily recognized by three pairs
of trans-olefins at d 6.32 (d, J = 15.9 Hz) and 7.50 (d, J = 15.9 Hz),
C
62.9 (C-1), 64.6 (C-6), 62.7 (C-6 ), and one oxyqua-
C
103.9 (C-2) (Takasaki et al., 2001). Three
H
4.38 (Glc H-1) of the
H
C
6.44 (d, J = 15.8 Hz) and 7.66 (d, J = 15.8 Hz), and 6.44 (d,
J = 15.8 Hz) and 7.61 (d, J = 15.8 Hz), and three 1,4-disubstituted
aromatic rings. In addition, the typical NMR signals of two acetyl
0
0
correlations d
with d
H
4.33 (Glc H-1) of the terminal glucosyl group (Glc )
0
C
82.1 (Glc C-2), and d
H
0
3.14 (Glc H-2) with d
C
104.3 (Glc C-1)
H C
groups at d 1.84 and 2.05, and d 20.3, 20.5, 169.4, and 170.2 were
demonstrated that the Glc was assigned the position. Thus, the
structure of 2 was formulated as 2,22-dideoxyecdysone 25-O-b-
glucopyranosyl-(1?2)-b- -glucopyranoside.
Compound 3, a colorless amorphous powder, was established
also observed. The attachment of the three p-coumaroyl moieties
0
D
-
to C-3, C-6 and C-2 was determined by HMBC correlations of H-
D
H
3 (d 5.53) of fructose with the ester carbonyl carbon of one p-cou-
maroyl group (d
the second p-coumaroyl group, and H-2 (d
C
165.5), H-6 (d
H
4.50) of fructose with d
C
166.5 of
+
0
by HRMS (m/z 617.3250 [M + Na] ) as C33
H
54
O
9
with a maximal
H
5.27) of glucose with
UV absorption at 247 nm. Detailed comparison of the NMR spec-
troscopic data of 3 with those of 2 indicated that both compounds
had the same aglycone of a 2,22-dideoxyecdysone moiety, but dif-
fered in the sugar part (Table 1). Compared to 2, the NMR spectra of
C
d 166.0 of the third p-coumaroyl group. In the same way, the
HMBC correlations of d
bonyl carbon signal at d
H
4.13 (H-1) of fructose with one acetyl car-
0
C
170.2 and d
H
4.89 (H-4 ) of glucose with d
C
169.4 of the other acetyl group established that the two acetyl
0
3
displayed only one monosaccharide, which was assigned as b-
glucopyranose by its NMR data (Table 1). Acid hydrolysis of 3 also
afforded -glucose, as determined by co-TLC analysis and by mea-
surement of optical rotation. A key HMBC correlation between H-1
4.45, d, J = 7.8 Hz) of glucose and C-25 (d 79.2) established the
groups were linked to C-1 and C-4 of sucrose, respectively. On
the basis of above evidence, compound 6 was determined to
D
be b-
D
-(1-O-acetyl-3,6-O-p-E-dicoumaroyl)-fructofuranosyl-
a-
D-
0
0
(4 -O-acetyl-2 -O-p-E-coumaroyl)-glucopyranoside.
(
d
H
Compounds 1, 2 and 6–14 isolated from F. floridana were
screened for activity against human DNA Topo I. Compound 13
1
3
1
position of glycosilation at C-25. Full assignments of the C and H
NMR signals were achieved by a combination of 1H, C, COSY,
HMQC, and HMBC spectroscopic analysis. Consequently, 3 was elu-
13
showed activity with IC50 value of 130
pounds proved inactive (IC50 > 500 M). This compound was ob-
served to completely inhibit Topo I relaxation activity at the
concentration of 285 M in conjunction with intercalation ability
at 312 M. Its activity was similar to luteolin (the positive control,
IC50: 108 M), but much less than camptothecin, a well known
lM, while the other com-
l
cidated as 2,22-dideoxyecdysone 25-O-b-
D-glucopyranoside.
Compound 4 had the molecular formula of C39
H
64
O
14 as 2.
l
Extensive NMR analysis indicated that compounds 4 and 2 were
structurally different in terms of the orientation of H-5 (Table 1).
The configuration of H-5 of 4 could be determined from the chem-
ical shift of C-19 (Kim et al., 1988; Russell et al., 1981). The orien-
l
l
Topo I poison (Webb and Ebeler, 2003). Our data showed that 13
inhibited Topo I catalytic activity by interacting directly with the
free enzyme and preventing formation of the DNA-Topo I complex,
but not stabilizing the intermediate DNA-Topo I covalent complex
as camptothecin. Diosmetin (13) was previously found to poison
human Topo II (Bandele and Osheroff, 2007), and here we report
its inhibition on Topo I for the first time. Thus, 13 is a dual topoi-
somerase inhibitor as luteolin (Webb and Ebeler, 2004).
tation of 5
3.4), while the C-19 with a 5b-H configuration was usually down-
field shifted 10–11 ppm (Kim et al., 1988; Russell et al., 1981). The
stereochemistry of 5 -H of 4 was further confirmed by H -9/H-5
and H -1/H-5 correlations in the NOESY spectrum. The C NMR
a-H for 4 was suggested by the chemical shift of C-19 (d
1
a
a
1
3
a
spectroscopic data also allowed for differentiation of 4 and 2 as
the chemical shifts of ring A and B displayed significant differences
(
(
Table 1) (Lafont et al., 2008). Structure 4 was thus elucidated as
)-2,22-dideoxyecdysone 25-O-b- -glucopyranosyl-(1?2)-b-
5a
D
D
-
3. Conclusion
glucopyranoside.
Compound 5, a colorless amorphous powder, exhibited a
M + H] ion peak at m/z 773.3975 in the HRESIMS, 16 mass units
Eight ecdysteroids (compounds 1–5 and 7–9) were isolated from
F. floridana including five new phytoecdysteroid glycosides (1–5).
HPLC analysis showed that 1–5 and 8 were found in stems and
leaves, while 7 and 9 were detected in each part of the plant. All
compounds except for 4 and 5 were tested in vitro for their activity
+
[
higher than that of 2, implying that 5 possessed an additional oxy-
gen atom. Acid hydrolysis of 5 also afforded a -glucose. Compari-
son of the C NMR spectroscopic data between 5 and 2 (Table 1)
D
1
3

4
34
P. Wang et al. / Phytochemistry 70 (2009) 430–436
against DNA topoisomerase I. None of these ecdysteroids shows any
activity in the Topo I assays. Of the four flavanoids isolated from the
plant, only diosmetin (13) showed bioactivity against DNA Topo I.
Ecdysteroids are the steroid hormones found in animals and
plants. There is a growing interest in ecdysteroids due to their
important human pharmacological effects and insect-molting
activity, particularly their potential as lead compounds for insecti-
cide development (Dhadialla et al., 1998). Of 325 phytoecdyster-
oids isolated to date, 45 are ecdysteroid glycosides (Lafont et al.,
A voucher specimen (TX-Nac-20060914-#0001-wp) is deposited at
the National Center for Pharmaceutical Crops at Stephen F. Austin
State University, USA.
4.3. Extraction and isolation
Air-dried plant material (1 kg) was ground to a coarse powder
and extracted with 9 L of EtOH/H O (95:5, v/v) at room tempera-
2
ture. The combined EtOH extracts were concentrated to give a res-
idue (40 g) under reduced pressure. The residue was then
2
008). These ecdysteroid glycosides were isolated from Caryo-
phyllaceae (20 compounds), ferns (12), Amaranthaceae (3), Malva-
ceae (3), Limnanthaceae (3), Menispermaceae (3), Liliaceae (2),
Lamiaceae (1), and Melanthiaceae (1). The sugar moiety of the
ecdysteroid glycosides may primarily be monosaccharide residue
suspended in MeOH/H
sively with n-hexane (8.6 g) and EtOAc (17 g). The EtOAc extract
exhibited Topo I inhibition properties with IC50 value of 15 g/
mL, and thus was subjected to further purification. Silica gel cc
500 g) of the EtOAc extract with gradient CHCl /MeOH (30:1,
5:1, 7:1, 5:1, 3:1 and 1:1, v/v, each 3000 ml) afforded seven frac-
tions (1–7) on the basis of their TLC profiles. Fraction 2 was devel-
oped on preparative TLC (silical gel, CHCl /MeOH, 12:1) to give 13
7 mg). Fraction 3 was applied to a Sephadex LH-20 column eluting
with MeOH to afford four subfractions: Ia, Ib, Ic, and Id. Fraction Ia
afforded 12 (4 mg) after preparative HPLC separation (MeOH/H O,
3:57, v/v). By preparative TLC (silica gel, CHCl /MeOH, 6:1), 11
4 mg), 14 (3 mg) and 10 (3 mg) were purified from Ib, Ic, and Id,
2
O (500 ml, 1:1, v/v) and extracted succes-
l
(
glucose, galactose, xylose, or allose). Usually C-3 (31 compounds)
(
3
and C-22 (10) are the positions of glycosylation in the plants with
occasional C-25 (4), C-2 (2), and C-26 (1). Among these reported
compounds, rhamnose is restricted to the fern Polypodium (Jizba
et al., 1971; Kim et al., 1988; Kim and Kinghorn, 1989), xylose is
only found in Limnanthes (Limnanthaceae) (Sarker et al., 1997;
Meng et al., 2001), and allose is reported only in Lilium (Mimaki
et al., 1989). Thus, it is possible that ecdysteroid glycosides may
have taxonomic significance in some plant groups.
Froelichia is morphologically distinguished within the subfamily
Gomphrenoideae of Amaranthaceae (McCauley and Ballard, 2007).
It is the first report of ecdysteroid glycosides in the subfamily and
the second in the family after Pfaffia. Its ecdysteroid glycosides (1–
1
3
(
2
4
3
(
respectively. Compound 7 (400 mg) was obtained from fraction 4
by recrystallization in MeOH. After being combined, fractions 5
and 6 were further fractionated by ODS cc (100 g) eluting with
2
MeOH/H O (15:85, 30:70, 50:50, and 75:25, v/v, each 700 ml),
5
and 8) have sugar moieties attached at C-25. Ecdysteroid glyco-
and 800 ml of 100% MeOH. According to the HPLC profiles, the elu-
ents were combined to give four subfractions: IIa, IIb, IIc, and IId.
Fraction IIa and IIb afforded compounds 8 (8.9 mg) and 1 (7 mg),
sides with sugar moieties at C-25 have only been reported in Pfaffia
in the Amaranthaceae (Nishimoto et al., 1988), Silene in the Caryo-
phyllaceae (Girault et al., 1990), and the fern Blechnum of Blechn-
aceae (Suksamrarn et al., 1986). Three compounds (2, 4, and 5)
from Froelichia have unique disaccharide residues at C-25. The
compounds of ecdysteroid glycosides with a disaccharide residue
at C-3 were isolated from the fern Polypodium (Jizba et al., 1971;
Kim et al., 1988), Silene (Baltayev, 1998), and Limnathes (Meng
et al., 2001). To date, reported ecdysteroid glycosides show struc-
tural diversity in plants. With extensive ecdysteroid analysis in
more plants, the taxonomic implications of phytoecdysteroids will
be better understood.
respectively, by preparative HPLC (MeOH/H
isolation of IIc eluted with MeOH/H O (63:37, v/v) gave a mixture
of 2, 4, and 5. Further separation of this mixture was conducted on
2
O, 50:50, v/v). HPLC
2
3 2
analytical HPLC (CH CN/H O, 31:69, v/v), which afforded
2
(
70 mg), 4 (2.3 mg), and 5 (1.2 mg), respectively. Compound 3
(
2.6 mg) was isolated from IId (preparative HPLC, CH
3 2
CN/H O,
3
3:67, v/v). Compound 9 (22 mg) were purified by preparative
O, 35:75, v/v). The rest parts of
fraction 7 after removal of 9 gave 6 (3.6 mg) (preparative HPLC,
MeOH/H O, 32:68, v/v). All fractions were detected at 254 nm, ex-
HPLC from fraction 7 (MeOH/H
2
2
cept for compound 6 (360 nm). The distribution of compounds 1–5
and 7–9 was also investigated by HPLC analysis in different plant
parts (leaves, stems, flowers, and roots).
4
. Experimental
4
.1. General experimental procedures
4
.4. 2, 22-dideoxy-20-hydroxyecdysone 25-O-b-
D
-glucopyranoside (1)
a + 26(c 0.07, MeOH); UV
D
Optical rotation values were measured on a JASCO P-1010 polar-
2
1
Colorless amorphous powder. ½ ꢂ
imeter, whereas UV spectra were recorded in MeOH with a
l
Quant
MeOH
max
1
13
k
nm: 246; For H NMR (600 MHz, DMSO-d
150 MHz, DMSO-d ) spectroscopic data, see Table 1; HRESIMS
m/z 617.3881 (calcd for C33 10Li, 617.3877).
6
) and C NMR
spectrophotometer (Bio-Tek Instruments, Inc.). NMR experiments
were performed using a Bruker 600 MHz NMR instrument with data
reported in d ppm values and referenced to the solvent used, with
HRESIMS being measured on a PE SCIEX QSTAR LC/MS/MS spec-
trometer. Octadecyl-functionalized silica gel, silica gel, Sephadex
LH-20, and TLC plates were purchased from Aldrich Chemical Co.
HPLC analysis was performed on a Hewlett Packard Series 1100
with a HP 1100 diode array detector using a Hypersil ODS column
(
6
54
H O
4.5. 2,22-dideoxyecdysone 25-O-b-
glucopyranoside (2)
D-glucopyranosyl-(1?2)-b-D-
2
1
Colorless amorphous powder. ½
aꢂ
+ 48 (c 0.92, MeOH); UV
D
MeOH
max
1
13
(
(
150 ꢁ 4.6 mm, 5
lm, Supelco; flow rate, 1 ml/min; MeOH/H
2
O
k
6
nm: 248; For H NMR (600 MHz, DMSO-d ) and C NMR
v/v) linear gradient, 2:98–98:2 in 35 min). Preparative HPLC was
(150 MHz, DMSO-d ) spectroscopic data, see Table 1; HRESIMS
6
performed with an Acuflow Series III pump connected with an
m/z 763.4463 (calcd for C39H64O14Li, 763.4456).
Acutect 500 UV/VIS detector using an Econosil ODS column (250 ꢁ
2
2 mm, 10 lm, Alltech). D-Glucose was purchased from Aldrich.
4.6. 2,22-deoxyecdysone 25-O-b-D-glucopyranoside (3)
2
1
4
.2. Plant material
Colorless amorphous powder. ½
aꢂ
+ 14 (c 0.43, MeOH); UV
D
MeOH
max
1
13
k
nm: 247; For H NMR (600 MHz, CD
OD-d ) spectroscopic data, see Table 1; HRESIMS
m/z 617.3250 (calcd for C33 Na, 617.3666).
3 4
OD-d ) and C NMR
Whole plants of F. floridana were collected in Nacogdoches,
Texas, USA on September 14, 2006, and identified by Wanli Zhang.
(150 MHz, CD
3
4
54 9
H O

P. Wang et al. / Phytochemistry 70 (2009) 430–436
435
4
.7. (5
a
)-2,22-dideoxyecdysone 25-O-b-
D
-glucopyranosyl-(1?2)-b-
D
-
(4.6 ꢁ 150 mm, 5
l
m) (mobile phase: 10% MeOH in 0.1% acetic
glucopyranoside (4)
acid solution, flow rate: 1 ml/min, UV detector: 280 nm). By com-
parison of its retention time at 7.8 min and DAD UV absorption
o-coumaric acid was identified (Yuan et al., 2007). Sucrose was
2
1
Colorless amorphous powder. ½
aꢂ
+ 32 (c 0.38, MeOH); UV
D
MeOH
max
1
13
k
nm: 248; For H NMR (600 MHz, CD
3
OD-d
4
) and C NMR
identified by TLC in CHCl
3 2
–MeOH–H O (6:4:1) using an authentic
(
150 MHz, CD
3
OD-d
4
) spectroscopic data, see Table 1; HRESIMS
14H, 757.4374).
sample (Takasaki et al., 2001; Li et al., 2002).
m/z 757.4299 (calcd for C39
64
H O
4.12. Topoisomerase I assay
4
.8. 2,22-dideoxy-5-hydroxyecdysone 25-O-b-
D
-glucopyranosyl-
(
1?2)-b- -glucopyranoside (5)
D
Compounds 1–2 and 6–14 were individually evaluated for their
ability to inhibit Topo I activity following the protocol described by
Webb and Ebeler (2003). Compounds were tested at concentra-
2
D
1
Colorless amorphous powder. ½
a
ꢂ
+ 46 (c 0.11, MeOH); UV
MeOH
1
13
k
(
nm: 248; For H NMR (600 MHz, CD
OD-d
m/z 773.3975 (calcd for C39
3
OD-d
4
) and C NMR
tions of 30, 63, 125, and 312
lM, respectively. Camptothecin
max
150 MHz, CD
3
4
) spectroscopic data, see Table 1; HRESIMS
15H, 773.4324).
(100 M), luteolin (312 M), and ethidium bromide (0.4 l
l
l
g/ml)
H
64
O
were used as positive controls for poisoning, inhibition and inter-
calation, respectively. All compounds were analyzed for DNA Topo
I inhibition, poisoning and intercalation activities using a gel elec-
trophoresis assay. IC50 values were estimated by analyses of en-
zyme activity versus inhibitor concentration. Three replicates
were used in the Topo I assay.
4
.9. b-
D
-(1-O-acetyl-3,6-O-p-E-dicoumaroyl)-fructofuranosyl-
4 -O-acetyl-2 -O-p-E-coumaroyl)-glucopyranoside (6)
a-
D-
0
0
(
2
1
Colorless amorphous powder. ½
aꢂ
+ 33 (c 0.34, MeOH); UV
D
MeOH
nm: 211, 231, 314; 1H NMR (600 MHz, DMSO-d
k
6
) sucrose
max
0
moiety: d
H
1.84 (3H, s, H-Ac-4 ), 2.05 (3H, s, H-Ac-1), 3.45 (2H, m,
Acknowledgments
0
0
H-1a and H-6 a), 3.71 (1H, br. d, J = 12.9 Hz, H-3 ), 4.09 (1H, dd,
0
J = 7.9, 8.1 Hz, H-4), 4.10 (1H, m, H-6 b), 4.13 (1H, m, H-1b), 4.29
This study was funded by the US CDC grant (R01 CI000315-01).
The authors would like to thank the Keck/IMD NMR Center, which
is funded by the W.M. Keck Foundation and the University of Hous-
ton; Dr. Youlin Xia for NMR analysis assistance; and Dr. Shane Ti-
chy of Texas A&M University for HRESI-MS analysis.
0
(
1H, m, H-5), 4.31 (1H, m, H-6a), 4.35 (1H, d, J = 9.8 Hz, H-5 ),
.50 (1H, d, J = 10.5 Hz, H-6b), 4.89 (1H, t, J = 9.7 Hz, H-4 ), 5.27
1H, t, J = 9.6 Hz, H-2 ), 5.41 (1H, s, H-1 ), 5.53 (1H, d, J = 7.9 Hz,
0
4
0
0
(
0
000
H-3); p-coumaroyl moiety: d
H
6.32 (1H, d, J = 15.9 Hz, H-8 ),
0
0
000
00
6
.44 (2H, d, J = 15.8 Hz, H-8 and H-8 ), 6.79ꢀ6.84 (6H, m, H-3
00 000 000 0000 0000
and H-5 , and H- 3 and H-5 , and H-3 and H-5 ), 7.50 (1H,
0
000
0000
0000
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0
00
000
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2
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0
0
00
000
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0
0
000
0000
00
0
0
000
000
0000
0000
00
0
00
0000
000
000
0000
1
6
1
25.4 (s, C-1 and C-1 ), 130.4 (d, C-2 and C-6 , C-2 and C-
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000
00
00
0000
000
0
0
000
0000
00
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0
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(
44
H O
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.10. Acid hydrolysis of 1-5
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CHCl . The organic solutions were analyzed using an Agilent
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100 HPLC system with an Agilent Eclipse XDB-C18 column

4
36
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