Labdane diterpenoid glycosides from Alpinia densespicata and their nitric oxide inhibitory activities in macrophages

Article abstract of DOI:10.1021/np900019n

Four new labdane diterpene glycosides (1-4) named alpindenosides A, B, C, and D, two new norditerpene glycosides (5, 6) named noralpindenosides A and B, and three known flavonoid glycosides (7-9) have been isolated from the stems of Alpinia densespicata. Structural elucidation of compounds 1-6 was based on spectroscopic data. Compounds 1-9 all exhibited moderate NO inhibitory activities, whereas they were noncytotoxic at 20 μ M against several human tumor cell lines.

Full text of DOI:10.1021/np900019n

J. Nat. Prod. 2009, 72, 1097–1101
1097
Labdane Diterpenoid Glycosides from Alpinia densespicata and Their Nitric Oxide Inhibitory
Activities in Macrophages
Yu-Jen Kuo,†,‡ Ping-Chun Hsiao,† Li-Jie Zhang,† Ming-Der Wu,§ Yu-Han Liang,† Hsiu-O Ho,*,‡ and Yao-Haur Kuo*,†,⊥
National Research Institute of Chinese Medicine, Taipei 112, Taiwan, Republic of China, College of Pharmacy, Taipei Medical UniVersity,
Taipei 110, Taiwan, Republic of China, Food Industry Research and DeVelopment Institute, Hsinchu 300, Taiwan, Republic of China, and
Graduate Institute of Integrated Medicine, China Medical UniVersity, Taichuang 404, Taiwan, Republic of China
ReceiVed January 15, 2009
Four new labdane diterpene glycosides (1-4) named alpindenosides A, B, C, and D, two new norditerpene glycosides
(5, 6) named noralpindenosides A and B, and three known flavonoid glycosides (7-9) have been isolated from the
stems of Alpinia densespicata. Structural elucidation of compounds 1-6 was based on spectroscopic data. Compounds
1-9 all exhibited moderate NO inhibitory activities, whereas they were noncytotoxic at 20 µM against several human
tumor cell lines.
The plant family Zingiberaceae has about 1400 species, and most
of them grow or are cultivated in tropical and subtropical Asia.
The plants typically have large rhizomes that are used for food,
spice, or traditional medicines. Plants of this family have been
reported to possess antioxidant,1,2 anti-inflammatory,2 cytotoxic,3
and hepatoprotective activities.4 Some Alpinia species have been
used as Chinese herbal drugs for antiemetic and stomachic
purposes.5 Alpinia species have also been reported to have
antitumor, pungency, antibacterial, antiulcer, antifungal, and in-
secticidal properties.6-8 We report herein the separation of an EtOH
extract of Alpina densespicata Hayata (Zingiberaceae), by column
chromatography and HPLC, to yield new labdane diterpene and
norditerpene lactone glycosides (1-6), along with the known
flavonoid glycosides kaempferol-3-O-R-L-rhamnosyl-(1f2)-O-L-
rhamnoside (7), quercetin-3-O-R-L-rhamnosyl-(1f2)-O-R-L-rham-
noside (8), and morin-7-O-ꢀ-D-glucopyranoside (9). Structural
elucidation of compounds 1-6 was based on spectroscopic analysis,
mainly using HRESIMS, IR, and 1D and 2D NMR experiments.
Compounds 1-9 were then evaluated for anti-inflammatory and
antioxidant activities and inhibition of human tumor cell lines.
Results and Discussion
Compound 1 was obtained as a white powder and was determined
to have the molecular formula C32H48O13 on the basis of HRESIMS;
[M + Na]+ at m/z 663.3038. The IR spectrum of 1 showed
absorption bands at 1746 and 1656 cm-1 ascribable to lactone ring
and CdC functions, respectively, and strong absorption bands at
3387 and 1042 cm-1 suggestive of an oligoglycoside.9 Two
anomeric carbon signals (δ 105.6, 101.8) and signals characteristic
for three methyl groups and one oxygenated methylene group were
present in the 1H and 13C NMR and DEPT spectra (Table 1). Other
and H-12. The HMBC spectrum (Figure 1) showed correlations
between two protons (H-12, H-17) and olefinic quaternary carbons
(C-8, C-9) and between proton H-17 and secondary carbon C-7,
suggesting that two six-membered rings were fused to a six-
membered cyclic ether moiety. In addition, long-range correlations
occurred between H-12 and olefinic carbons C-9 and C-13, as well
as between three singlet methyl groups and quaternary carbons C-4
and C-10. These data suggested that 1 was a substituted labdane-
type diterpene.10
functional groups including a lactone ring were identified by the
presence of characteristic resonances at δ 175.9 (C-15), 73.94
(C-16), and 135.0, 149.0 (C-13, -14), and the HMBC spectrum
showed that H-16 was correlated with carbonyl (C-15), olefinic
(C-14), and oxygenated (C-12) carbons. Thus, the lactone ring was
linked at C-12.
1
The H-1H COSY spectrum of 1 showed cross-peaks between
H-1, H-2, and H-3, between H-5, H-6, and H-7, and between H-11
The two sugar moieties were identified as glucose (Glc) and
rhamnose (Rha) on the basis of characteristic 13C NMR data, δ
105.6, 79.4, 78.9, 77.6, 72.0, 62.9 for glucose and δ 101.8, 74.0,
* To whom correspondence should be addressed. Tel: 886-02-28201999,
ext. 7051, and 886-02-27361661, ext. 6126. E-mail: kuoyh@nricm.edu.tw;
hsiuoho@tmu.edu.tw.
† National Research Institute of Chinese Medicine.
‡ Taipei Medical University.
72.1, 72.1, 70.0, 18.0 for rhamnose.11 In the H NMR spectrum,
1
§ Food Industry Research and Development Institute.
⊥ China Medical University.
anomeric protons at δ 4.42 (d, 7.6) and 5.37 (s) and a typical
rhamnose methyl signal (δ 1.21, d, 6.0) were observed. The glucose
10.1021/np900019n CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/08/2009

1098 Journal of Natural Products, 2009, Vol. 72, No. 6
Kuo et al.
Table 1. 1H NMR Data (δ, ppm) of 1-6 (MeOD, 600 and 500 MHz)
no.
1
1a
2b
3b
4b
5b
6b
1.32, td (11.4, 3.6)
1.71, d (12.0)
1.72, d (13.2)
2.08, (dd, 13.2, 3.2)
3.20, dd (12.0, 4.2)
1.24, m
1.77, dd (12.6, 6.6)
1.58, ddd (19.2, 12.0, 6.0)
1.90, d (7.2)
1.20, m
1.16, dt (14.0, 3.0)
1.62, dd (13.0,3.0)
1.64, m
1.10, m
1.64, m
1.65, m
1.93, m
1.10, m
1.45, m
1.66, m
1.10, m
1.45, m
1.66, m
1.66, d (12.5)
1.67, d (12.5)
2.04, dd (14.0, 3.5)
3.18, dd (12.0, 4.0)
1.15, d (12.0)
1.79, td (6.0, 13.5)
55, m
2
2.01, m
1.97, dd (13.0, 4.0)
3.17, dd (11.5, 4.0)
0.84, m
1.97 dd (13.0, 4.0)
3.17 dd (11.5, 4.0)
0.85, m
3
5
6
3.25, dd (11.5, 4.0)
0.94, dd (13.5, 1.5)
1.67, m
1.38, td (13.0, 4.0)
2.07, m
3.14, dd (11.5, 3.5)
0.94, d (12.0)
1.67, m
1.37, td (4.0, 13.0)
2.05, m
1.67, m
1.30, m
1.67, m
1.30, m
7
1.92, brd (6.0)
2.09, dd (12.4, 2.8)
0.98, m
1.43, m (ꢀ)
1.12, d (9.5, ꢀ)
1.75, dd, (2.5, 12.0, ꢀ)
1.41, m (R)
2.09, dd (12.4, 2.4)
0.97, dd (12.5, 4.0)
1.43, m (ꢀ)
0.99, m
0.99, m
8
9
11
1.31, m (ꢀ)
1.31, m (ꢀ)
1.10, m (ꢀ)
1.80, brd (9.0, ꢀ)
1.09, m (R)
4.23, d (8.0, ꢀ)
1.11, d (10.0, ꢀ)
91, brd (9.0, ꢀ)
1.09, m, (R)
1.06, m (ꢀ)
2.42, brd (15.6, R)
2.03, m (ꢀ)
2.31, brd, (16.0, ꢀ)
1.96, m (R)
1.66, m (ꢀ)
1.36, m (R)
12
13
14
15
4.33, brd (7.2,R)
4.15, brd (10.5, ꢀ)
4.23, brd (9.0,ꢀ)
2.34, m (ꢀ)
3.46, t (9.0, ꢀ)
2.22, m (ꢀ)
3 0.42, m (ꢀ)
7.54, s
4.88, s
7.54, s
4.88, s
7.50, s
7.56, s
4.93, s
5.04, s
4.84, s
4.74, s
1.72, s
16
17
4.90, s
4.85, m
4.05, s (2H)
4.06, d (16.2, R)
3.93, m (ꢀ)
1.08, s (R)
1.02, s (ꢀ)
4.06, d (15.0, R)
3.98, m (ꢀ)
1.08, s (R)
1.02, s (ꢀ)
4.34, d (8.0, ꢀ)
4.35, d (8.0, ꢀ)
1.04, s (R)
1.04, s (R)
18
19
20
G1
G2
G3
G4
G5
G6
1.08, s (R)
0.87, s (ꢀ)
0.85, s (ꢀ)
4.41, d (7.5)
3.41, d (7.5)
3.45, t (8.0)
3.27, m
1.05, s (R)
0.87 s (ꢀ)
0.87, s (ꢀ)
0.86, s (ꢀ)
0.87, s (ꢀ)
0.86, s (ꢀ)
0.90, s (ꢀ)
0.90, s (ꢀ)
0.87, s (ꢀ)
4.41, d (7.2)
3.41, d (7.6)
3.45, d (8.4)
3.25, t (8.8)
3.28, m
4.42, d (7.8)
3.42, d (8.0)
3.45, t (8.5)
3.27, m
4.42, d (7.5)
3.42, d (7.0)
3.45, t (8.5)
3.30, m
4.40, d (7.6)
3.42, m
4.40, d (7.6)
3.43, m
3.46, m
3.25, m
3.24, m
3.67, m
3.45, m
3.24, m
3.22, m
3.27, m
3.26, m
3.24, d (2.0)
3.66, dd (11.6, 5.2)
3.85, dd (11.6, 2.0)
5.36, s
3.92, d (3.2)
3.75, d (3.2)
3.38, m
3.64, dd (12, 5.5)
3.84, d (11.5)
5.37, s
3.66, dd (12, 5.5)
3.83, d (11.5)
5.36, s
3.92, d (3.2)
3.75, dd (9.0, 3.0)
3.39, m
3.64, dd (12, 5.5)
3.84, d (11.5)
5.36, s
3.94, m
3.75, dd (9.0, 3.0)
3.39, m
4.22, dd (9.6, 4.4)
4.36, m
3.83, dd (12.0, 2.0)
5.36, s
R1
R2
R3
R4
R5
R6
5.37, s
3.95, m
3.72, d (3.2)
3.75, d (3.2)
3.39, d (9.6)
3.96, m
3.92, m
3.75, dd (9.0, 3.0)
3.40, t (9.2)
3.98, m
3.75, d (3.6)
3.38, m
3.95, m
1.22, d (6.4)
3. 97, m
1.20, d (6.0)
3.97, m
3.95, m
1.21, d (6.0)
1.20, d (6.0)
1.21, d (6.0)
1.21, d (6.0)
a 600 MHz. b 500 MHz. G ) glucose, R ) rhamnose.
had a ꢀ-configuration and rhamnose had an R-configuration on the
basis of the magnitude of the coupling constants of the anomeric
protons, J ) 7.6 and 0 Hz, respectively. Due to the HMBC cross-
peaks of Rha-H-1 with Glc-C-2, and of Glc-H-1 with C-3 of the
diterpene, their positions were assigned as Rha-(1f2)-O-ꢀ-Glc
connecting with C-3 of the aglycone. Major cross-peaks of H-3R/
Me-18, H-1R, H-5R, H-1R/H-11R, H-11R/H-12R, Me-20/H-1ꢀ, and
Me-19/H-1ꢀ, H-11ꢀ were observed in the NOESY spectrum of 1
(Figure 2). Thus, the structure and relative configuration of 1 was
assigned to be as indicated, and it was named alpindenoside A.
The molecular formula of 2 (alpindenoside B) was assigned as
C32H48O13 by HRESIMS (quasimolecular ion peak [M + Na]+ at
m/z 663.3048). The IR spectrum of 2 was essentially identical to
that of 1. The chemical shift values of C-l to C-20 and the signals
ring and oligoglycosidic moieties. The quisamolecular ion [M + Na]+
at m/z 681.3195 (HRESIMS) indicated that the formula of 3 was
C32H50O14. These data, together with signals consistent with three
tertiary methyl, one dioxymethine, two glycosidic, and one R,ꢀ-
unsaturated-γ-lactone group in the 1H and 13C NMR spectra (Tables
1 and 2), revealed that 3 was a labdane-type diterpene attached to a
glucose and a rhamnose. The 1H NMR and 13C NMR data of 3 were
very similar to those of curcumanggoside, except for the presence of
the rhamnose signals, and the signal of glucose C-2 in 3 was shifted
downfield to δ 79.0.12 Furthermore, long-range correlation between
H-1 of rhamnose and C-2 of glucose was observed in the HMBC
spectrum. These findings suggested that the rhamnose was linked to
C-2 of the glucose. The relative configuration of C-12 was determined
from the NOESY spectrum of 3, which showed cross-peaks of H-11
(2.31, brd, J ) 16.0 Hz)/Me-20, H-12. Thus, H-11 (2.31, brd, J )
16.0 Hz), H-12 should have a ꢀ-orientation, because Me-20 is
ꢀ-oriented. Thus, the structure of 3 was elucidated, and it was named
alpindenoside C.
1
of glucose and a rhamnose in the H NMR and 13C NMR spectra
of 2 were also nearly identical to those of 1, except for the sig-
nals of H-11 and H-12. There were obvious changes of H-11 and
1
H-12 signals between 1 and 2 in their H NMR spectra (Table 1).
These data, together with NOESY cross-peaks of H-3R/H-1R, H-5R,
Me-18, H-1ꢀ/H-11ꢀ, H-1R/H-11R, and H-11ꢀ/H-12ꢀ, Me-20,
revealed that 2 was the H-12 ꢀ-epimer of 1. Comparing 13C NMR
spectra of 1 and 2, the signal of C-12 for 2 shifted to lower field
and was consistent with the structure and relative configuration
indicated for 2. Acid hydrolysis of 1 and 2 with 2 N HCl afforded
aglycones 1a and 2a, respectively, along with D-glucose and
L-rhamnose in a ratio of 1:1, both of which were identified by chiral
GC.
1
Compounds 4 (alpindenoside D) and 3 had similar IR and H
and 13C NMR spectra (Table 2), revealing that 4 was a labdane
diterpene containing glucose and rhamnose. The HRESIMS spec-
trum of 4 showed the quasimolecular ion [M + Na]+ at m/z
723.3280, indicating a molecular formula of C34H52O15. Thus, 4
differed from 3 by an additional C2H2O unit. The 13C NMR
spectrum of 4 showed signals of an OAc group (δ 172.7, 20.8),
and the signal of C-6 of glucose in 3 (δ 62.8) was shifted to lower
field in 4 (δ 64.8), suggesting that the OAc group in 4 was linked
to C-6 of Glc. Like 3, H-12 of 4 was ꢀ-oriented mainly on the
Compound 3 was obtained as a white powder, and its IR spectrum
with absorption bands at 1743, 3380, and 1046 cm-1 indicated lactone

Labdane Diterpenoid Glycosides from Alpinia densespicata
Journal of Natural Products, 2009, Vol. 72, No. 6 1099
Table 2. 13C NMR Data (δ, ppm) of 1-6 (MeOD, 100 MHz)
no.a
1
2
3
4
5
6
1
2
3
4
5
6
7
8
36.6, t
27.7, t
89.9, d
40.4, s
52.8, d
18.9, t
28.2, t
126.3, s 126.2, s
135.9, s 136.2, s
37.8, s
27.6, t
70.2, d
135.0, s 135.5, s 135.4, s 135.3, s
149.0, d 148.7, d 148.7, d 148.8 d
175.9, s 174.9, s 174.9, s 174.9, s
73.9, t
69.2, t
28.4, q
20.6, q
17.1, q
35.3, t
27.5, t
90.1, d
40.2, s
52.8, d
19.0, t
27.8, t
38.4, t
27.5, t
90.1, d
40.3, s
56.0, d
21.4, t
29.6, t
42.6, d
53.1, d
36.8, s
30.6, t
71.4, d
38.4, t
27.4, t
90.4, d
40.4, s
56.0, d
21.7, t
29.7, t
42.6, d
53.0, d
36.8, s
30.8, t
71.4, d
38.5, t
37.5, t
90.4, d
40.3, s
56.9, d
22.3, t
31.4, t
47.7, d
55.3, d
36.9, s
27.5, t
50.3, d
83.5, d
153.3, s 148.5, s
108.1, t 110.3, t
65.7, t
28.6, q
17.0, q
14.0, q
38.5, t
27.5, t
90.4, d
40.3, s
56.9, d
22.4, t
31.5, t
47.5, d
55.3, d
36.9, s
28.7, t
54.5, d
82.0, d
9
10
11
12
13
14
15
16
17
18
19
20
G1
G2
G3
G4
G5
G6
R1
R2
R3
R4
R5
R6
OAc
37.9, s
28.9, t
71.1, d
72.5, t
72.4, t
72.3, t
20.4, q
28.6, q
17.0, q
13.9, q
69.9, t 101.9, d 101.8, d
28.4, q
19.5, q
17.1, q
28.5, q
18.0, q
14.7, q
28.5, q
18.0, q
14.7, q
105.6, d 105.6, d 105.6, d 105.5, d
105.6, d 105.6, d
78.9, d
79.4, d
72.0, d
77.6, d
62.7, t
78.9, d
79.4, d
72.1, d
77.6, d
62.7, t
79.0, d
79.5, d
72.2, d
77.6, d
62.8, t
78.7, d
79.3, d
72.3, d
74.9, d
64.8, t
79.0, d
79.5, d
72.1, d
77.6, d
62.8, t
79.0, d
79.5, d
72.1, d
77.6, d
62.7, t
101.8, d 101.8, d 101.8, d 101.9, d
101.9, d 101.8, d
72.1, d
72.1, d
74.0, d
70.0, d
18.0, q
72.0, d
72.0, d
74.0, d
70.0, d
18.0, q
72.1, d
72.1, d
74.0, d
70.0, d
18.0, q
72.0, d
72.0, d
74.0, d
70.0, d
18.0, q
72.1, d
72.0, d
74.0, d
70.0, d
18.0, q
72.0, d
72.0, d
73.9, d
70.0, d
18.0, q
Figure 1. Major HMBC correlations of compounds 1, 3, and 5.
172.7, 20.8
a G ) glucose, R ) rhamnose.
postulated biogenetic pathway to 1-4 is shown in Scheme 1
(Supporting Information).
Compound 5 was obtained as a white powder, and the molecular
formula was assigned to be C31H52O12 by HRESIMS, which showed
the molecular ion [M]+ at m/z 616.3438. The IR spectrum of 5 had
peaks at 3362 and 1038 cm-1, suggestive of an oligoglycosidic
moiety. In the 1H and 13C NMR and DEPT spectra, signals
indicating three methyl groups, seven methylene groups (one
olefinic methylene and one oxymethylene), six methines (two
oxymethines δC 90.4, 83.5), four quaternary carbons (one olefinic
carbon), and 12 carbon signals for two hexose moieties were found.
Valence bond calculations revealed that the unsaturation degree of
compound 5 was 6, indicating one double bond, two hexose
moieties, and three rings.
1
The H-1H COSY spectrum of 5 showed cross-peaks of H-8/
H-9/H-11/H-12/H-13, together with partial long-range correlations
(C-3/H-G1, C-11/H-9, C-13/H-8) present in the HMBC spectrum,
suggesting that 5 contained a 6-6-5 ring structure. On the basis
of long-range correlations, OH groups were attached to C-13 (δC
83.5) and C-16 (δC 65.7), and an isopropenyl unit was located at
C-12 (δC 50.3); a Rha-(1f2)-O-ꢀ-Glc was also attached at C-3.
These data, together with the absence of a carbon atom compared
with 3, indicated 5 to be a norditerpene glycoside. On the basis of
NOE correlations in the NOESY spectrum of 5, the relative
configuration of 5 was determined. The cross-peaks of H-3R/Me-
17R, Me-18ꢀ/Me-19ꢀ, Me-19ꢀ/H-9ꢀ, H-9ꢀ/H-8ꢀ, H-8ꢀ/H-12ꢀ, and
H-12ꢀ/H-13ꢀ and R-orientations were assigned to the oxygenated
isopropenyl group at C-12 and to the OH group at C-13 in 5. Thus,
the structure of 5 was determined, and it was named noralpinde-
noside A.
Figure 2. Major NOESY connectivities of compounds 1, 3, and 5.
basis of the characteristic NOE correlations and carbon chemical
shift of C-12 (δ 71.4). Thus, the structure of 4 was determined to
be as shown.
Compounds 5 and 6 had similar IR and 1H and 13C NMR
1
spectra. Detailed comparisons of the H and 13C NMR spectra
Isolated labdane diterpenes with a six-membered cyclic ether
moiety (such as 1-4) are rarely reported, except for curcumang-
of 5 with 6 indicated that the CH2OH group (δC 65.7) at C-15
in 5 was replaced by a CH3 group (δC 20.4) in 6. Comparison
goside, which was isolated from another zingiberaceous plant.12
A

1100 Journal of Natural Products, 2009, Vol. 72, No. 6
Kuo et al.
NMR spectra were recorded on a Varian NMR spectrometer (Unity
Plus 400 MHz) using CD3OD or pyridine-d5 as solvents. The chemical
shifts are given in δ (ppm) and coupling constants in Hz. Low-resolution
ESIMS were recorded on a VG Quattro 5022 mass spectrometer. High-
resolution ESIMS were measured on a MAT-95XL high-resolution mass
spectrometer. Sephadex LH-20 (Pharmacia) and silica gel (Merck
70-230 mesh and 230-400 mesh) were used for column chromatog-
raphy, and precoated silica gel (Merck 60 F-254) plates were used for
TLC. The spots on TLC were detected by spraying with 5% H2SO4
and then heating at 110 °C. HPLC separations were performed on a
Waters 600 series apparatus with a Waters 996 photodiode array
detector, equipped with a 250 × 10 mm i.d. preparative Cosmosil AR-
II column (Nacalai, Tesque).
Table 3. Effects of 1-9, 1a, and 2a on LPS-Induced NO
Production in RAW264.7 Macrophagesa
compound
IC50 (µM)
1
2
3
4
5
6
7
8
34.2
49.3
38.0
30.00
31.8
44.3
40.1
32.9
39.1
12.2
44.6
9.6
9
1a
2a
quercetin
Plant Material. Alpinia densespicata was collected from YangMing
Mountain of Taiwan in July 2003. A voucher specimen (No. 2003-
07-066) has been deposited in the National Research Institute of Chinese
Medicine, Taipei, Taiwan.
a RAW264.7 cells were incubated with compounds in the presence of
LPS (1 µg/mL). The medium was harvested 24 h later and assayed for
nitrite production. NO released was measured using Griess reagent. Cell
viability was evaluated with the MTT assay. The results are presented as
a percentage of the control value obtained from nontreated cells. Values
are expressed as mean ( SD of three individual experiments, performed
in triplicate.
Extraction and Isolation. Dried stems and leaves of A. densespicata
(18 kg) were chipped, extracted with EtOH (80 L, three times) at 50
°C, and concentrated under reduced pressure. The EtOH extract (3.6
kg) was partitioned between n-hexane and H2O (1:1) to give the
n-hexane-soluble fraction (A, 350 g), CHCl3 and H2O (1:1) to give
the CHCl3-soluble fraction (B, 221 g), BuOH and H2O (1:1) to give
the BuOH-soluble fraction (C, 110 g), and the H2O fraction, respec-
tively. Fraction C (110 g) was chromatographed on a silica gel column
(22 × 9 cm i.d.) eluted with CHCl3-MeOH (1:0 f 1:1) to give 12
fractions (C-1 to C-12). Fraction C-11 (5 g, eluted with CHCl3-MeOH,
5:1) was further separated on an LH-20 column (94 × 4 cm i.d.) eluted
with CHCl3-MeOH (1:1) to obtain fractions C-111 to C-117. Fraction
C-113 (640 mg) was separated by HPLC (Cosmosil 5C18-AR II, 250
× 10.0 mm i.d., flow rate: 2.5 mL/min, 60% MeOH) to yield 1 (345
mg) and 2 (92 mg). Fraction C-114 (133 mg) was separated by HPLC
(Cosmosil C18-AR II, 250 × 10.0 mm i.d., flow rate: 2.5 mL/min, 60%
MeOH) to yield 3 (6.9 mg), 4 (6.0 mg), and 6 (13.5 mg). Fraction
C-12 (6 g, CHCl3-MeOH, 1:1) was repeatedly chromatographed on a
LH-20 column (70 × 6 cm i.d.) with CHCl3-MeOH (1:1) and then
purified using preparative TLC (CHCl3-MeOH (3:1)) to afford 5
(7.0 mg).
of the HRESIMS spectra of 5 and 6 indicated one less oxygen
atom in 6. HMBC experiments confirmed the OH group at C-13
and two sugars connected by Rha-(1f2)-O-ꢀ-Glc at C-3. These
data, together with the NOE correlations, confirmed the structure
of 6 (noralpindenoside B).
The other isolated compounds, 7-9, were identified as kaempferol-
3-O-R-L-rhamnopyranosyl-(1f2)-O-R-L-rhamnopyranoside (7),13
quercetin-3-O-R-L-rhamnopyranosyl-(1f2)-O-R-L-rhamnopyrano-
side (8),14 and morin-7-O-δ-D-glucopyranoside (9),15 respectively,
by comparison with literature data.
Compounds 1-9, 1a, and 2a were evaluated for cytotoxicity
against human tumor cell lines [HeLa (cervix epitheloid carcinoma),
KB (oral epidermoid carcinoma), Doay (medulloblastoma), and
WiDr (colon adenocarcinoma)] and anti-inflammatory activity, using
RAW264.7 cells supplemented with LPS, which induced cell
inflammation and caused nitrite accumulation in the medium. None
of the isolates (1-9) nor 1a and 2a were cytotoxic against the
above-mentioned tumor cells, and all of them had high cell viability
(>80%) for RAW264.7 cells. Except for 1a, all compounds showed
only moderate anti-NO effects, with their IC50 values about 3 to 5
times that of quercetin (IC50 9.6 µM). It was noted that 1a (IC50
12.2 µM) and 2a (IC50 44.6 µM) had significantly different
inhibitory effects against NO production. These data implied that
the configuration at C-12 of labdane diterpenoids plays a crucial
role for the NO production induced by LPS.
As shown in Table 3, quercetin (positive control) had greater
inhibition of LPS-induced NO production compared with flavonoid
glycosides 7-9. Moreover, the labdane diterpenoid 1a had an IC50
value close to that of quercetin and exhibited much higher inhibitory
activity than its glycoside, 1. Sugar substituents may decrease the
anti-inflammatory activity of flavonoid glycosides due to increased
steric hindrance by or the hydrophilic character of additional sugar
moieties, which may decrease the penetration and absorption of
constituents into the cell. Several reports are consistent with the
above conclusion.16,17 In addition, comparing with quercetin (ED50
18.0 µM), the isolated labdane diterpenoids (1-6) showed no
antioxidative activity (ED50 >200 µg/mL) when assayed by DPPH.
The active flavonoid glycosides 8 (ED50 16.6 µM) and 9 (ED50
22.2 µM) both have two OH groups in the B ring. Comparison of
the inactive flavonoid glycoside 7, together with a literature report,18
indicates that additional OH groups in flavonoids increase their
antioxidant activity.
Alpindenoside A (1): white, amorphous powder; mp 168 °C; [R]24
D
-61.0 (c 1.00, MeOH); UV (MeOH) λmax195.8 nm; IR (neat) νmax 3387,
1
2937, 1746, 1656, 1445, 1356, 1209, 1042 cm-1; H NMR (MeOD,
600 MHz), Table 1; 13C NMR (MeOD, 100 MHz), Table 2; HRESIMS
m/z 663.3038 [M + Na]+ (calcd for C32H48O13Na, 663.2993).
Alpindenoside B (2): white, amorphous powder; mp 162-165 °C;
[R]24 -31.7 (c 0.60, MeOH); UV (MeOH) λmax 195.8, 194.2 nm; IR
D
1
(neat) νmax 3373, 2933, 1739, 1630, 1380, 1205, 1042, 806 cm-1; H
NMR (MeOD, 500 MHz), Table 1; 13C NMR (MeOD, 100 MHz), Table
2; HRESIMS m/z 663.3048 [M + Na]+ (calcd for C32H48O13Na,
663.2993).
Alpindenoside C (3): white, amorphous powder; mp >295 °C; [R]24
D
-42.0 (c 0.69, MeOH); UV (MeOH) λmax 195.6 nm; IR (neat) νmax
3380, 2933, 1743, 1634, 1391, 1046, 813 cm-1; 1H NMR (MeOD, 500
MHz), Table 1; 13C NMR (MeOD, 100 MHz), Table 2; HRESIMS
m/z 681.3195 [M + Na]+ (calcd for C32H50O14 Na, 681.3200).
Alpindenoside D (4): white, amorphous powder; mp 240 °C; [R]24
D
-46.7 (c 0.60, MeOH); UV (MeOH) λmax 195.6 nm; IR (neat) νmax
3373, 2933, 1739, 1369, 1242, 1046, 810 cm-1; 1H NMR (MeOD, 500
MHz), Table 1; 13C NMR (MeOD, 100 MHz), Table 2; HRESIMS
m/z 723.3280 [M + Na]+ (calcd for C34H52O15 Na, 723.3204).
Noralpindenoside A (5): white, amorphous powder; mp 228 °C;
[R]24 -28.6 (c 1.40, MeOH); UV (MeOH) λmax 195.8 nm; IR (neat)
D
1
νmax 3362, 2930, 1568, 1402, 1038, 813 cm-1; H NMR (MeOD, 500
MHz), Table 1; 13C NMR (MeOD, 100 MHz), Table 2; HREIMS m/z
616.3438 [M]+ (calcd for C31H52O12, 616.3459).
Noralpindenoside B (6): white, amorphous powder; mp >295 °C;
[R]24 -34.8 (c 1.35, MeOH); UV (MeOH) λmax 195.6 nm; IR (neat)
D
ν
max 3380, 2933, 1391, 1042, 806 cm-1; 1H NMR (MeOD, 500 MHz),
Table 1; 13C NMR (MeOD, 100 MHz), Table 2; HRESIMS m/z
623.3476 [M + Na]+ (calcd for C31H52O11Na, 623.3407).
1a: [R]24 264.0 (c 0.5, MeOH); UV (MeOH) λmax 210.0 nm; IR
D
(neat) νmax 3457, 2926, 2847, 2360, 1746, 1651, 1457, 1069 cm-1; 1H
NMR (CDCl3) 0.80 (3 H, s, H-18), 0.97 (3 H, s, H-19), 1.00 (3 H, s,
H-20), 1.22 (1 H, m, H-5), 1.33 (1 H, m, H-1), 1.54 (1 H, ddd, J )
18.0, 12.6, 6.6, H-6), 1.63 (1 H, m, H-6), 1.71 (1 H, m, H-7), 1.76 (1
H, d, J ) 7.2 Hz, H-1), 1.84 (1 H, m, H-2),1.96 (1 H, m, H-2), 1.90
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on a JASCO P-1020 polarimeter. IR spectra were measured on a
Mattson Genesis II spectrophotometer (Thermo Nicolet, Madison, WI).

Labdane Diterpenoid Glycosides from Alpinia densespicata
Journal of Natural Products, 2009, Vol. 72, No. 6 1101
(1 H, m, H-11), 2.47 (1 H, d, J ) 14.4 Hz, H-11), 3.24 (1 H, dd, J )
4.2, 12.0 Hz, H-3), 3.95 (1 H, d, J ) 15.0 Hz, H-17), 4.10 (1 H, d, J
) 16.4 Hz, H-17), 4.34 (1 H, d, J ) 7.8 Hz, H-12), 4.81 (1 H, s,
H-16), 7.37 (1 H, s, H-14); ESIMS m/z 355.4 [M + Na]+ (calcd for
C20H28O4).
al.23 with minor modifications. An aliquot of each sample (120 µL,
200-3.125 µg/mL) or quercetin (25-3.125 µg/mL) was mixed with
30 µL of 0.75 mM DPPH methanol solution in a 96-well microplate.
The mixture was shaken vigorously on an orbital shaker in the dark at
room temperature for 30 min, and then the absorbance at 517 nm was
measured with an ELISA reader. The negative control was the
measurement using methanol to replace the sample in the reaction
solution. The DPPH radical scavenging activities of compounds 1-9
were compared to the negative control and to quercetin as a positive
control. The final results were reported as ED50, the concentration of
sample required to cause 50% reduction of DPPH radicals in solution.
2a: [R]24 296.0 (c 0.5, MeOH); UV (MeOH) λmax 210.0 nm; IR
D
(neat) νmax 3457, 2926, 2847, 2364, 1746, 1647, 1457, 1069 cm-1; 1H
NMR (CDCl3): 0.81 (3 H, s, H-18), 1.00 (3 H, s, H-19), 1.00 (3 H, s,
H-20), 1.13 (1 H, d, J ) 12.6 Hz, H-5), 1.18 (1 H, dd, J ) 12.6, 3.0
Hz, H-1), 1.58 (1 H, m, H-2), 1.67(1 H, dd, J ) 10.2, 4.8, H-1), 1.72
(1 H, m, H-6), 1.76 (1 H, dd, J ) 6.0, 10.2, H-6), 1.85 (1 H, brd, H-7),
1.88 (1 H, brd, H-11), 2.38 (1 H, d, J ) 16.8 Hz, H-11), 3.24 (1 H, dd,
J ) 3.0, 11.4 Hz, H-3), 3.99 (1 H, d, J ) 15.0 Hz, H-17), 4.03 (1 H,
d, J ) 15.6 Hz, H-17), 4.23 (1 H, d, J ) 10.2 Hz, H-12), 4.80 (1 H,
s, H-16), 7.37 (1 H, s, H-14); ESIMS m/z 355.4 [M + Na]+ (calcd for
C20H28O4).
Acknowledgment. The authors would like to thank Dr. M. J. Don,
National Research Institute of Chinese Medicine, for preliminary EIMS
measurements, and the Instrument Center of National Taiwan Univer-
sity, for the HRESIMS measurements. This work was supported by
grants from the National Science Council (NSC92-2323-B-077-003)
and the National Research Institute of Chinese Medicine (NRICM-96-
DHM-002), Taiwan, Republic of China.
Acid Hydrolysis. Solutions of compounds 1 and 2 (each 20 mg) in
2 N HCl (2 mL) were refluxed at 100 °C for 3 h, respectively. Each
reaction mixture was extracted with CH2Cl2 to yield the aglycone (1a
or 2a). After separating the organic layer, the aqueous phase was
neutralized with NaHCO3 and filtered. The filtrate was condensed, then
dissolved in MeOH (0.2 mL) and purified by TLC in n-BuOH-
MeOH-H2O (8:8:1) to obtain the glucose and rhamnose. The sugars’
specific optical rotations were compared with authentic samples. In
addition, part of the filtrate was evaporated to dryness; then 1-(trim-
ethylsilyl)imidazole and pyridine (0.2 mL) were added. After reacting
for 1 h, the mixture was dried by a stream of N2 and partitioned with
n-hexane and water. The n-hexane layer was analyzed by GC with the
following conditions: CP-Chirasil-L-Val column (25 m × 0.25 mm);
injection temperature 200 °C; column temperature 100-200 °C; rate
3 °C/min. Peaks of trimethylsilyl derivatives derived from D-glucose
(29.08 min) and L-rhamnose (16.74 min) were detected by comparison
with retention times of authentic samples treated with 1-(trimethylsilyl)-
imidazole.
NO Production and Cell Viability Assays. The murine macrophage
cell line RAW264.7 (BCRC 60001 ) ATCC TIB-71) was cultured in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL Life
Technologies, Inc.) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) and incubated at 37 °C in a humidified 5% CO2
atmosphere using a 96-well flat-bottomed culture plate. After 24 h, the
medium was replaced with fresh DMEM and FBS. Then compounds
1-9, 1a, and 2a (0, 1, 5, 10, or 20 µg/mL) were added, respectively,
in the presence of lipopolysaccharide (LPS, 1 µg/mL; Sigma, cat no.
L-2654) and incubated at the same condition for 24 h. The cultured
cells were then centrifuged, and the supernatants were used for NO
production measurement. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide] assay was used to determine cell
viability.
Supporting Information Available: NMR spectra of 1-6, including
HMBC and NOESY spectra, and the presumed biosynthetic pathway
of 1-4. This material is available free of charge via the Internet at
http://pubs.acs.org.
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The supernatant was mixed with an equal volume of the Griess
reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine
dihydrochloride in 2.5% phosphoric acid solution) and incubated
for 10 min at room temperature. Nitrite concentration was determined
by measuring the absorbance at 540 nm using an ELISA plate reader
(µ Quant).19
The MTT colorimetric assay was modified from that of Mosmann.20
The test is based upon the selective ability of living cells to reduce the
yellow soluble salt, MTT, to a purple-blue insoluble formazan. MTT
(Merck; dissolved in phosphate-buffered saline at 5 mg/mL) solution
was added to the attached cells mentioned above (10 µL per 100 µL
culture) and incubated at 37 °C for 4 h. Then, DMSO was added and
the amount of formazan formed was determined by absorbance at 550
nm. The optical density of formazan formed in the control (untreated)
cells was taken as 100% viability.
Scavenging Activity of 1,1-Diphenyl-2-picrylhydrazyl (DPPH)
Radical.21 The radical scavenging activity of 1-9 on DPPH free radical
was measured using the method of Rangkadilok et al.22 and Chung et
NP900019N