Two anti-inflammatory steroidal saponins from Dracaena angustifolia Roxb.

Article abstract of DOI:10.3390/molecules18088752

Two new steroidal saponins, named drangustosides A-B (1-2), together with eight known compounds 3-10 were isolated and characterized from the MeOH extract of Dracaena angustifolia Roxb. The structures of compounds were assigned based on 1D and 2D NMR spectroscopic analyses, including HMQC, HMBC, and NOESY. Compounds 1 and 2 showed anti-inflammatory activity by superoxide generation and elastase release by human neutrophils in response to fMLP/CB.

Full text of DOI:10.3390/molecules18088752

Molecules 2013, 18, 8752-8763; doi:10.3390/molecules18088752
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Two Anti-inflammatory Steroidal Saponins from
Dracaena angustifolia Roxb.
1
1
2
3
Hui-Chi Huang , Ming-Kuem Lin , Syh-Yuan Hwang , Tsong-Long Hwang ,
4
,5
6
7
1,8,
Yao-Haur Kuo , Chi-I Chang , Chung-Yi Ou , Yueh-Hsiung Kuo
*
1
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources,
China Medical University, Taichung 404, Taiwan
2
3
4
Endemic Species Research Institute, Council of Agriculture, Nantou 552, Taiwan
Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan
Division of Herbal Drugs and Natural Products, National Research Institute of Chinese Medicine,
Taipei 211, Taiwan
5
6
Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University,
Taichung 404, Taiwan
Department of Biological Science and Technology, National Pingtung University of Science and
Technology, Pingtung 912, Taiwan
7
8
Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung 404, Taiwan
Tsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University,
Taichung 404, Taiwan
*
Author to whom correspondence should be addressed; E-Mail: kuoyh@mail.cmu.edu.tw;
Tel.: +886-4-2205-3366 (ext. 5701).
Received: 18 June 2013; in revised form: 11 July 2013 / Accepted: 21 July 2013 /
Published: 24 July 2013
Abstract: Two new steroidal saponins, named drangustosides A–B (1–2), together with
eight known compounds 3–10 were isolated and characterized from the MeOH extract of
Dracaena angustifolia Roxb. The structures of compounds were assigned based on 1D and
2
D NMR spectroscopic analyses, including HMQC, HMBC, and NOESY. Compounds 1
and 2 showed anti-inflammatory activity by superoxide generation and elastase release by
human neutrophils in response to fMLP/CB.
Keywords: Agavaceae; Dracaena angustifolia Roxb; steroidal saponins; drangustosides A–B;
anti-inflammatory activity

Molecules 2013, 18
. Introduction
The genus Dracaena (Agavaceae) includes more than 50 species found in tropical and subtropical
8753
1
regions of the eastern hemisphere. Dracaena angustifolia Roxb. is a native shrub or small tree in
southern Taiwan, widely planted in Australia, India, Malaysia, and Philippines [1]. The decoction of
the underground parts of this plant is used as a tonic and for the treatment of asthma, diarrhea, and
inflammation [2]. Previous phytochemical investigations on the genus Dracaena have reported the
presence of a variety of components, including steroidal saponins [3–7], flavonoids [5,8–10], and
phenolic compounds [8,11]. The pharmacological investigation indicated that the C27 steroidal
saponins present on the genus Dracaena showed broad biological activities, such as anti-inflammatory,
antifungal, antimicrobial, antiviral, analgesic, antioxidative, cytotoxic, and hypoglycemic
properties [3–5,11]. D. angustifolia Roxb. has been studied, and this revealed the presence of several
steroidal saponins with antifungal [4], antituberculosis [12], antiproferative [7], and cytotoxic
activities [13]. In our continuing phytochemical investigation on D. angustifolia Roxb., we have now
further identified two new steroidal saponins, drangustosides A–B (1–2), one known steroidal saponin,
alliospiroside A (3) [14], and seven benzenoids 4–10 in the MeOH extract of D. angustifolia Roxb.,
and report herein the structural determination of these substances using extensive spectroscopic
methods. Neutrophils play a significant role in the pathogenesis of several inflammatory diseases. The
production of vast amounts of superoxide anion and elastase by activated neutrophils can cause tissue
damage and contribute to the development of a wide spectrum of airway inflammatory diseases [15,16].
The anti-inflammatory activity of new compounds was also evaluated as inhibitory activities against
formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion production and
elastase release in human neutrophils.
2
. Results and Discussion
The MeOH extracts of the whole plant of D. angustifolia Roxb. was extracted successively with
EtOAc and n-BuOH. Compounds 1–10 (Figure 1) were obtained from the EtOAc fraction by using a
series of chromatographic techniques on silica gel, Sephadex LH-20, and RP-HPLC. The structures of
the two new steroidal saponins 1–2 were elucidated as follows:
Compound 1 was obtained as a white amorphous solid. Its molecular formula was established as
+
C
45
H
72
O
17 based on an [M+Na] ion peak at m/z 907.4667 in its HRESIMS. The IR spectrum of 1
indicated the existence of hydroxyl groups (3,389) and the characteristic absorption bands of a
−1
1
(
25S)-spiroketal at 987, 918, 898, and 843 (intensity 918 > 898) cm [14]. The H-NMR spectrum
Table 1) of 1 showed signals due to two tertiary methyl groups (δ 1.34 and 0.87), four secondary
1.06 (d, J = 7.0 Hz), 1.08 (d, J = 7.0 Hz), 1.62 (d, J = 6.2 Hz), and 1.68 (d, J = 6.2 Hz)],
one olefinic proton [δ 5.53 (d, J = 5.1 Hz)], and three monosaccharide anomeric protons [δ 4.91 (d,
(
H
methyl groups [δ
H
H
H
J = 7.7 Hz), 5.73 (br s), and 5.82 (br s)], two of which were considered to be L-rhamnosides. Based on
the chemical shifts and coupling constants, the three monosaccharides were considered as a
1
3
combination of one D-glucoside and two L-rhamnosides. The C-NMR spectrum (Table 1) exhibited
5 carbon atoms, 27 of which belonged to the aglycone carbons while the remaining were due to three
4
1
3
hexose sugar units. The C-NMR and DEPT spectra revealed C27 signals including four methyl, nine

Molecules 2013, 18
8754
methylene, ten methine, and four quaternary carbons. Among the four quaternary carbon signals, the
signal at δ 110.0 was identified as an acetal carbon (C-22) and the signal at δ 139.3 was assigned as
C
C
a diakyl substituted olefinic carbon (C-5). The aforementioned data suggested that 1 is possibly a
spirostanol glycoside with the aglycone being a 27-carbons skeletonal aglycone along with three sugar
moieties [4].
Figure 1. Chemical structure of compounds 1–10 from D. angustifolia Roxb.

Molecules 2013, 18
8755
1
13
Table 1. H-NMR and C-NMR spectroscopic data (in ppm, J in Hz) of compounds 1–3 in CD OD.
3
1
δH
2
3
δH
δC
δC
δH
δC
1
2
3.68 (dd, 11.6, 3.9)
78.1
3.70 (dd, 12.4, 4.2)
2.63 (br d, 12.4),
2.36 (q, 11.9)
4.06 (m)
78.2
3.86 (dd, 12.1, 3.7)
2.72 (br d, 12.4),
2.36 (q, 11.8)
3.86 (m)
83.9
37.7
2
.58 (br d, 12.5),
41.9
41.1
2
.28 (q, 11.8)
4.02 (m)
3
4
5
6
75.0
39.4
75.1
39.7
68.5
44.1
2.77 (m), 2.75 (m)
2.84 (m), 2.82 (m)
-
2.61, 2.57
-
-
139.3
125.5
139.4
125.4
139.9
125.0
5.53 (d, 5.1)
5.54 (d, 6.2)
5.57 (d, 5.2)
1
.90 (m), 1.54 (br d,
.6)
7
32.7
1.92 (m), 1.57 (m)
32.6
1.89 (m), 1.55 (m)
32.3
9
8
9
1.58 (m)
1.32 (m)
33.2
51.4
44.0
1.59 (m)
1.36 (m)
33.2
51.5
44.1
1.57 (m)
1.50 (m)
33.4
50.7
43.2
1
0
-
-
-
2
.83 (dd, 11.3, 3.5),
2.85 (m)
2.91 (m)
1
1
24.4
24.4
24.3
1
.70 (m)
1.75 (m)
1.63 (m)
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
1.72 (m), 1.21 (m)
-
40.7
40.4
57.1
32.5
81.6
63.2
16.8
14.0
42.7
15.1
110.0
26.7
26.5
27.8
1.73 (m), 1.23 (m)
-
40.8
40.5
57.1
32.7
81.4
63.3
16.8
14.0
42.8
15.1
110.0
26.7
26.5
27.8
1.54 (m), 1.28 (m)
-
40.6
40.5
57.1
32.7
81.5
63.1
17.0
15.3
42.7
15.1
110.0
26.7
26.5
27.8
1.13 (m)
1.13 (m)
1.13 (m)
2.05 (m), 1.47 (m)
4.51 (dd, 14.5, 7.9)
1.77 (t, 7.9)
0.87 (s)
2.07 (m), 1.49 (m)
4.51 (dd, 14.7, 8.0)
1.78 (t, 8.0)
0.90 (s)
2.01 (m), 1.39 (m)
4.50 (dd, 14.5, 7.5)
1.71 (t, 7.5)
0.84 (s)
1.34 (s)
1.37 (s)
1.44 (s)
1.87 (m)
1.88 (m)
1.84 (m)
1.08 (d, 7.0)
-
1.11 (d, 6.9)
-
1.10 (d, 7.0)
-
1.88 (m), 1.43 (m)
2.11 (m), 1.36 (m)
1.58 (m)
1.91 (m), 1.45 (m)
2.15 (m), 1.37 (m)
1.61 (m)
1.86 (m), 1.40 (m)
2.11 (m), 1.34 (m)
1.55 (m)
4
.05 (br d, 11.5),
.34 (d, 11.5)
1.06 (d, 7.0)
4.09 (br d, 10.8),
3.36 (d, 10.8)
1.07 (d, 7.0)
3-O-β-D-Glc
5.06 (d, 7.3)
4.23 (m)
4.06 (br d, 10.8),
3.35 (d, 10.8)
1.06 (d, 7.1)
3-O-α-L-Ara
4.72 d (7.0)
4.59 (m)
2
2
6
7
65.3
16.5
65.3
16.6
65.3
16.6
3
3
-O-β-D-Glc
1
'
'
'
'
4.91 (d, 7.7)
100.2
78.3
87.6
70.2
100.7
78.0
79.9
72.9
100.7
75.4
76.2
70.4
2
3.99 (dd, 8.0, 7.7)
4.14 (t, 8.0)
4.03 (m)
3
4
4.26 (m)
4.08 (m)
4.16 (t, 8.6)
4.06 (m)
4
.25 (m)
5
6
'
'
3.78 (m)
78.3
62.4
3.88 (m)
78.5
62.8
67.6
3
.66 (d, 11.0)
4
.38 (br d, 11.5),
4.46 (dd, 11.8, 1.9),
4.31 (m)
-
4
.31 (dd, 11.5, 5.9)
α-L-RhaI
α-L-Rha
α-L-Rha
1
2
''
''
5.82 (br s)
104.1
72.9
6.36 (br s)
4.80 (m)
102.3
72.8
6.31 (br s)
101.9
72.9
4.73 (m)
4.70 (br d, 3.5)

Molecules 2013, 18
8756
Table 1. Cont.
1
2
δH
3
δC
δH
δC
δC
δH
3
4
5
6
''
''
''
''
4.52 (m)
4.46 (m)
73.0
74.0
70.1
18.6
4.64 (dd, 9.2, 3.1)
4.34 (m)
73.1
74.4
69.7
18.9
4.63 (dd, 9.3, 3.5)
4.31 (dd, 9.3, 9.3)
4.85 (dq, 9.3, 6.2)
1.73 (d, 6.2)
72.8
74.8
69.5
19.3
4.76 (dq, 8.9, 6.2)
1.62 (d, 6.2)
α-L-RhaII
4.99 (m)
1.71 (d, 6.2)
1
2
3
4
5
6
'''
'''
'''
'''
'''
'''
5.73 (br s)
102.8
72.7
72.8
73.8
70.9
18.9
4.81 (br s)
4.46 (m)
4.29 (m)
4.86 (dq, 9.1, 6.2)
1.68 (d, 6.2)
1
13
Comparison of the H and C-NMR signals (Table 1) of the aglycone moiety of 1 with those of
alliospiroside A (3) [14], indicated that the structures of the aglycone parts of 1 and 3 were almost
13
superimposable. The only significant differences were seen in the C-NMR signals of C-1 and C-3 in
1
3
the ring A portion. Generally, a carbon attached to an -O-glycoside group exhibits the higher C NMR
shift than that of a carbon attached to a hydroxyl group. The O-glycoside group in compound 3 was
assigned at C-1 [δC-1 83.9; δH-1 3.86 (dd, J = 12.1, 3.7 Hz) and δC-3 68.5, δH-3 3.86 (m)]. The NMR
peaks of C-1 were at δ
in compound 1, while the anomeric proton H-1′ [δ
correlation with δ 75.0 (C-3). These results lead to the conclusion that the -O-glycoside is
C
78.1 [δH-1 3.68 (dd, J = 11.6, 3.9 Hz)] and C-3 at δ
C
75.0 [δH-3 4.02 (m)] were
H
4.91 (d, J = 7.7 Hz)] exhibited an HMBC
C
unambiguously located at C-3 in compound 1. Comparing the NMR data of C-1 and C-3 between 1
and 3, the -O-glycosidation at C-3 in compound 1 and at C-3 in compound 3 agrees with the general
knowledge. The coupling constant of H-1 (dd, J = 11.6, 3.9 Hz) can be assigned to an α-axial orientation.
H-1 and H-3 have a NOESY correlation, therefore revealing that H-3 was located with the same α-axial
orientation. In general, the difference in chemical shifts between axial and equatorial protons among
on H
difference in chemical shift (Δea = δe − δa) for H
configurations whereas it is < 0.20 ppm in 25R compounds. Therefore, the axially oriented H
and S-configuration of C-25 were deduced based on the presence of the following different chemical
shift data: Δea = 0.45 for H -23, Δea = 0.75 for H -24, Δea = 0.71 for H -26. The 22R stereochemistry
was deduced based on the presence of the two proton signals at the C-26 of spirostanol appeared as
separate signals at δ 4.05 (H-26eq) and 3.34 (H-26ax)] and the spirostanol had a normal type F ring
Figure 1) [18]. The correlations from H-26ax (δ 3.34) to H-25 (δ 1.58) and from H-23ax (δ 1.43)
to H-20 (δ 1.87) and H -27 (δ 1.06) in the NOESY spectrum (Figure 2) were consistent with the
2
-23, H
2
-24, and H
2
-26 can be used to resolve the absolute configuration of C-25 [17,18]. The
-23, -24, -26 are usually > 0.35 for 25S
-27 (1.06)
2
3
2
2
2
H
(
H
H
H
H
3
H
C-22R and C-25S configurations. The stereostructure of 1 was confirmed by NOESY analysis, as
shown in Figure 2.

Molecules 2013, 18
Figure 2. Key NOESY correlations of the aglycone moiety of 1–2.
8757
D-Glucose and L-rhamnose in a 1:2 ratio were obtained upon acidic hydrolysis of 1 with HCl in
1
,4-dioxane [19], as indicated by chiral HPLC methodology [20]. Analysis of the NMR data indicated
the presence of a trisaccharide unit connected to an aglycone moiety, with three anomeric protons [δ
.91 (d, J = 7.7 Hz, H-1' of β-D-glucopyranosyl), 5.73 (br s, H-1''' of α-L-rhamnopyranosyl), and 5.82
br s, H-1'' of α-L-rhamnopyranosyl)] which correlated to carbon signals at δ 100.2, 102.8, and 104.1,
respectively [21]. In the TOCSY spectrum of 1, the anomeric proton that was ascribed to D-glucoside.
H
4
(
C
[
δ
H
4.91 (d, J = 7.7 Hz, H-1')] showed connectivity with four consecutive methines as follows: δ
H-2'), 4.14 (H-3'), 4.03 (H-4'), and 3.78 (H-5'), and then two methylene protons [δ 4.38 (H-6a'), and
.31 (H-6b′)]. The TOCSY spectrum gave a result that showed two rhamnoside units connected on the
H
3.99
(
H
4
3
glucoside unit. In the HMBC spectrum, a J correlation between H-1'' (δ
H
5.82) of rhamnoside and C-2'
87.6) of glucoside
(
δ
C
78.3) of glucoside, and between H-1''' (δ 5.73) of rhamnoside and C-3' (δ
H
C
confirmed that two rhamnoside units located at C-2' and C-3' of glucoside units, respectively (Figure 3).
Consequently, the connectivity of the three monosaccharide in 1 was identified as 3-O-{α-L-
rhamnopyranoside(1→2)-O-[α-L-rhamnopyranoside(1→3)]-β-D-glucopyranosyl}. Therefore, the
structure 1 was ultimately identified as 3-O-{α-L-rhamnopyranoside(1→2)-O-[α-L-rhamno-
pyranoside(1→3)]-β-D-glucopyranosyl} (1,3,22R,25S)-spirost-5-ene-1β,3β-diol, to which the trivial
name drangustoside A was given.
Figure 3. The COSY and key HMBC correlations of compounds 1–2.
27
21
O
25
18
17
22
24
OH
OH
O
3
1
9
O
1
HO
HO
OH
O
3
O
5
HO
O
O
O
HO
O
O
HO
OH
HMBC 1H
COSY
1H
13C
1H
HO
OH
HO
O
HO
OH
2
1

Molecules 2013, 18
Compound 2 was obtained as an amorphous solid with a molecular formula of C H O
62 13
8758
3
9
+
determined by the HRESIMS data (m/z 761.4085 [M+Na] ). The IR spectrum of 2 indicated the
existence of hydroxyl groups (3,389) and the characteristic absorption bands of (25S)-spiroketal at 987,
−1
1
13
9
20, 897, and 840 (intensity 920 > 897) cm [14]. The H- and C-NMR spectroscopic features of the
aglycone moiety of 2 (Table 1) were very similar to those of 1, which was suggested that compounds 2
1
13
and 1 possessed the same (1,3,22R,25S)-spirost-5-ene-1,3-diol aglycone. The H- and C-NMR
spectra of 2 exhibited two anomeric protons signals at δ 6.36 (br s) and 5.06 (d, J = 7.3 Hz), as well
H
C
as the corresponding anomeric carbon resonances at δ 102.3 and 100.7, respectively, that established
1
13
the existence of two sugar units. The differences in the H and C-NMR spectra of 1 and 2 showed
that 2 had one less rhamnoside unit than 1. In addition, the molecular ion mass spectrum of 2 was 146
H
atomic units lower than that of 1, and only two anomeric proton signals were observed at δ 5.06 (d,
J = 7.3 Hz) and 6.36 (br s). Acid hydrolysis of 2 yielded a L-rhamnose and a D-glucose in 1:1 ratio.
The TOCSY spectrum of 2 showed that the proton at δ 5.06 (H-1′) was coupled to the signals at δH
H
4
.23 (H-2′), 4.26 (H-3′), 4.16 (H-4′), 3.88 (H-5′), 4.31 (H-6′) and 4.46 (H-6′), successively. The carbon
signals at δ 100.7, 78.0, 79.9, 72.9, 78.5, and 62.8 were assigned to the D-glucoside C-1′, C-2′, C-3′,
C
C-4′, C-5′, and C-6′, respectively, from the HSQC analysis. The proton and carbon signals of
L-rhamnoside were fully assigned by the same method. Moreover, the α-anomeric configurations of
the L-rhamnoside units [δ
H
6.36 (br s)] and the β-anomeric configurations of the D-glucoside units
[δ
H
5.06 (d, J = 7.3 Hz)] were confirmed by their coupling constants and chemical shifts [19]. In the
3
HMBC spectrum, J-correlations were observed between H-1′′ (δ
H C
6.36) of rhamnoside and C-2′ (δ 78.0)
of glucoside and H-1′ (δ 5.06) of glucoside and C-3 (δ 75.1) of aglycone. These findings indicated
H
C
that the terminal rhamnose was linked at C-2′ of the inner glucose and the sugar chain was attached to
C-3 of the aglycone (Figure 3). Consequently, compound 2 was identified as (1,3,22R,25S)-spirost-5-
ene-1,3-diol 3-O-[α-L-rhamnopyranoside (1→2)-β-D-glucopyranosyl] and named drangustoside B.
The other eight isolated compounds were identified as alliospiroside A (3) (ESIMS m/z 709
+
+
[
(
(
M+H] , C38
H
60
O
12) [14], p-hydroxybenzaldehyde (4) (ESIMS m/z 123 [M+H] , C
7
H
6
O
2
) [22],
+
E)-cinnamic acid (5) (ESIMS m/z 149 [M+H] , C H O ) [23], ethyl (E)-4-hydroxycinnamate (6)
9
8
2
+
+
ESIMS m/z 193 [M+H] , C11
H O
12 3
) [24], ethyl 4-hydroxybenzoate (7) (ESIMS m/z 167 [M+H] ,
+
C
H
9 10
O
3
) [25], benzoic acid (8) (ESIMS m/z 123 [M+H] , C
7
H
6
O
2
) [26], 4-hydroxybenzoic acid (9)
+
(
ESIMS m/z 139 [M+H] , C H O ,) [27], and methyl 2,3-dimethoxy-4-hydroxybenzoate (10) (ESIMS
7 6 3
+
12 5
m/z 213 [M+H] , C10H O ) [28], respectively, by comparison with the spectroscopic data reported in
the literature for these compounds.
Inhibition of superoxide generation and elastase release by human neutrophils in response to fMLP
were utilized to measure the anti-inflammatory activity of compounds 1 and 2 (Table 2). LY294002, a
phosphatidylinositol-3-kinase inhibitior, was used as a positive control for inhibition of superoxide
anion generation and elastase release with IC50 values of 2.00 ± 0.59 and 4.94 ± 1.69 μM, respectively.
Compound 1 showed the highest anti-inflammatory activity against superoxide generation
(
IC50= 18.55 ± 0.23 μM) and elastase (IC50= 1.74 ± 0.25 μM).

Molecules 2013, 18
8759
Table 2. Inhibitory effects of pure compounds on superoxide generation and elastase
release in fMLP/CB-induced human neutrophils.
Superoxide anion
Elastase release
Compound
a
a
IC (μM)
IC (μM)
5
0
50
1
2
26.39 ± 1.63
18.55 ± 0.23
2.00 ± 0.59
3.94 ± 0.19
1.74 ± 0.25
4.94 ± 1.69
LY294002
a
Concentration necessary for 50% inhibition (IC50). Results are presented as mean ± S.E.M. (n = 2 or 3).
3. Experimental
3.1. General Procedures
The infrared (IR) spectra were measured on a Mattson Genesis II spectrophotometer using a KBr
matrix. The optical rotations were measured on a JASCO P-1020 polarimeter equipped with a sodium
1
13
lamp (589 nm). H- and C-NMR spectra were recorded on a Bruker DRX-500 spectrometer with
CD OD as the solvent. The HRESIMS data were collected using a Finnigan MAT95S mass
3
spectrometer. The GC-MS was performed using a Thermo Finnigan TRACE GC Ultra instrument.
Sephadex LH-20, and silica gel (Merck 70–230 mesh and 230–400 mesh) were used for the column
chromatography. The preparative HPLC was performed using a reverse phase column (Cosmosil 5C18-AR-
II column, 5 μm particle size, 250 mm × 20 mm i.d.) and the stereoselective HPLC analysis was
performed using a normal phase column (Chiralpak AD-H column, 5 μm particle size, 250 mm× 10 mm
i.d.) on a Shimadzu LC-6AD series apparatus with a RID-10A Refractive Index detector. The MPLC
was performed using a reverse phase column (Buchi MPLC glass column, C18, 460 mm × 36 mm i.d.)
on a Buchi pump module C-601 series apparatus without detector.
3
.2. Plant Material
The leaves of D. angustifolia Roxb. used in this experiment was collected on the mountains of
Nantou County, Taiwan, in September 2009. The plant was identified by Dr. Shy-Yuan Hwang,
Endemic Species Research Institute. A voucher specimen of D. angustifolia Roxb (No. CMR200909DA)
was deposited in the Department of Chinese Pharmaceutical Sciences and Chinese Medicine
Resources, China Medical University, Taichung, Taiwan.
3.3. Extraction and Isolation
The leaves of D. angustifolia Roxb. (6.5 kg) were sectioned and extracted five times with MeOH
(20 L) for 72 hr each time. The MeOH extract was continuously dried under reduced pressure at 45 °C
to yield the brown syrup (ca. 972.4 g). The combined extracts were suspended on H O (3 L) and then
2
successively partitioned with EtOAc (3 L, three times) and n-BuOH (3 L, three times). The EtOAc
layer (57.7 g) was subjected to silica gel CC (6.5 × 43.5 cm, Merk 70–230 mesh), eluting with a
CHCl /MeOH gradient to give ten fractions (fr. 1–10). Fraction 3 (679.2 mg) was again subjected to
3
Sephadex LH-20 column chromatography eluting with MeOH to yield five subfractions (fr. 3.1–3.5).
Fr. 3.3 (141.8 mg) was subjected to silica gel CC (3 × 25 cm, Merk 70–230 mesh) with gradient

Molecules 2013, 18
8760
n-hexane-EtOAc to yield nine subfractions (fr. 3.3.1–3.3.9). Fr. 3.3.5 was chromatographed using
preparative RP-HPLC (Cosmosil 5C -AR-II column, flow rate: 5.0 min/mL) with isocratic
1
8
MeOH-H
2
O (65:35) to yield 7 (6.0 mg), 8 (3.6 mg), and 10 (3.5 mg). Using preparative RP-HPLC
(
Cosmosil 5C -AR-II column, flow rate: 5.0 min/mL) with isocratic MeOH-H O (60:40), 4 (8.4 mg),
1
8
2
5
(4.6 mg), 6 (5.5 mg), and 9 (2.1 mg) were obtained from fr. 3.3.7. Fraction 7 was subjected to
Sephadex LH-20 column (10 × 70 cm) chromatography eluting with MeOH to yield six subfractions
(
fr.7.1–7.6). Fr. 7.4 was subjected to preparative RP-HPLC (Cosmosil 5C -AR-II column, flow rate:
18
5
2
.0 min/mL), eluting with MeOH-H O (55:45) to yield 2 (3.6 mg). Fr. 8 was further separated by
chromatography on RP-MPLC (Buchi MPLC glass column, flow rate: 8.0 min/mL) with gradient
MeOH/H O (0%-100%) to yield 14 subfractions (fr.8.1–8.14). Fraction 8.4 was further separated by
2
chromatography on Sephadex LH-20 (10 × 70 cm) with MeOH to yield six subfractions
(
fr. 8.4.1–8.4.6). Fr. 8.4.4 was chromatographed using RP-HPLC with isocratic MeOH-H
2
O (70:30) to
yield 1 (5.7 mg) and 3 (5.4 mg).
3.4. Spectral Data
2
D
5
Drangustoside A (1). white amorphous powder; [α]
−54.74 (c 0.29, MeOH); IR (KBr) νmax 3,389
(
OH), 2,928, 1,681 (C=C), 1,367, 1,043 (C-O-C), 987, 918, 898 and 843 (intensity 918 > 898,
-1
1
13
spiroketal chain of the 25S series) cm ; for H- and C-NMR spectroscopic data, see Table 1;
+
HRESIMS m/z 907.4667 [M+Na] (calcd for C45
H
72
O17Na, 907.4662).
2
D
5
Drangustoside B (2). white amorphous powder; [α]
−38.25 (c 0.15, MeOH); IR (KBr) νmax 3,389
(
OH), 2,929, 1,681 (C=C), 1,368, 1,044 (C-O-C), 987, 920, 897 and 840 (intensity 920 > 897,
−1
1
13
spiroketal chain of the 25S series) cm ; for H- and C-NMR spectroscopic data, see Table 1;
+
HRESIMS m/z 761.4085 [M+Na] (calcd for C39
H
62
O13 Na, 761.4088).
3
.5. Acid Hydrolysis of 1–2
Compound 1 or 2 (2 mg each) were heated with 1N HCl (dioxane-H
and then evaporated under reduced pressure to give a residue. The residue was partitioned with CH
and H O three times. The aqueous (pH = 2.0) was neutralized with aqueous 1N NaOH and then evaporated
2
O, 1:1, 2 mL) at 90 °C for 4 h,
2
Cl
2
2
under reduced pressure. The aqueous layer (0.3 mg; pH = 7.0) was dissolved in MeOH (0.5 mL) and
then analyzed by stereoselective HPLC under the following conditions: column: Chiralpak AD-H;
solvent system: n-hexane-ethanol-TFA (7:3:0.1, v/v); flow rate: 0.5 mL/min; inject volume: 20 μL;
detector: refractive index. D-Glucose and L-rhamnose for compounds 1 (t = 14.31 and 15.80 min,
R
respectively) and 2 (t
R
= 14.36 and 15.89 min, respectively) were determined by comparison of the
= 14.33 min) and L-rhmanose (t =15.85 min) standards.
respective retention times of D-glucose (t
R
R
3.6. Superoxide Generation and Elastase Release by Human Neutrophils
Human neutrophils were obtained using dextran sedimentation and Ficoll centrifugation. Blood was
drawn from healthy human donors (20–32 years old) by venipuncture using a protocol approved by the
institutional review board at Chang Gung Memorial Hospital. Neutrophils were isolated using a

Molecules 2013, 18
8761
standard method as previously described [29,30]. Measurements of superoxide anion generation and
elastase release were carried out according to previously described procedures [29,30].
4
. Conclusions
Two new steroidal saponins 1–2, one known steroidal saponin 3, and seven known benzenoids 4–10
were isolated from the MeOH extract of D. angustifolia Roxb. Their structures were determined based
on extensive spectroscopic analyses, and the anti-inflammatory activity of 1 and 2 were evaluated by
superoxide anion generation and elastase assays, which revealed that the steroidal saponin
(
1,3,22R,25S)-spirost-5-ene-1,3-diol with a disaccharide, 2, had better anti-inflammatory activity
against superoxide generation than 1 with a trisaccharide. Another reported steroidal saponin with a
disaccharide, 3-O-β-chacotriosyl-25(S)-spirost-5-en-3β-ol [21], which was similar to 2, but missing an
hydroxyl group at C-1, also showed very potent anti-inflammatory activity against superoxide
generation. These results implied that the number of sugars in this type of steroidal saponins could be
important for the anti-inflammatory activity. Moreover, 3-O-β-chacotriosyl-25(S)-spirost-5-en-3-ol
(
IC = 4.65 ± 0.25 μM, reported data) had more potent anti-inflammatory activity against superoxide
50
generation than compound 2 (IC50 = 18.55 ± 0.23 μM); whereas, 2 (IC50 = 1.74 ± 0.25 μM) was more
active than 3-O-β-chacotriosyl-25(S)-spirost-5-en-3β-ol (IC50 = 4.65 ± 0.25 μM, reported data) in the
human neutrophil elastase release assay [21]. Thus, the functional group in C-1 for this type of
steroidal saponins seems to play a crucial role for the anti-inflammatory activity against superoxide
generation and human neutrophil elastase release.
Acknowledgements
The financial supports from the National Science Council (NSC 98–2320-B-039–026-MY3) to H.C
Huang and the Taiwan Department of Health Clinical Trial and Research Center of Excellence
(
DOH-102-TD-B-111-004) and the China Medical University (CMU98-N1-17, CMU98-CT-04, and
CMU99-N1-13-1) to Y.H. Kuo and H.C. Huang are appreciated.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–10 are available from the authors.
©
2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(