Two new natural products from the fruits of Alpinia oxyphylla with inhibitory effects on nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophage cells

Article abstract of DOI:10.1007/s12272-012-1211-7

Chemical investigation of the fruits of Alpinia oxyphylla led to an isolation of the two new natural products, 9-hydroxy epinootkatol (1) and (S)-2-pentanol-2-O-β-D-glucopyranoside (2), in addition to the nine known compounds, pinocembrin (3), tectochrysin (4), izalpinin (5), nookatone (6), yakuchinone A (7), protocatechuic acid (8), β-sitosterol (9), daucosterol (10) and β-sitosterol palmitate (11). Their structures were elucidated on the basis of physicochemical constants and NMR spectral data analysis. The effects of the isolated components on nitric oxide production in LPS-induced RAW 264.7 macrophages were examined. The two new natural compounds showed inhibitory activities with IC₅₀ values of 21.8 and 32.8 μg/mL, respectively.

Full text of DOI:10.1007/s12272-012-1211-7

Arch Pharm Res Vol 35, No 12, 2143-2146, 2012
DOI 10.1007/s12272-012-1211-7
Two New Natural Products from the Fruits of Alpinia oxyphylla with
Inhibitory Effects on Nitric Oxide Production in Lipopolysaccharide-
Activated RAW264.7 Macrophage Cells
Zhang Jun Qing1,2,3,4, Wang Yong2,3,4, Li Yong Hui2,3,4, Lai Wei Yong2,3,4, Li Hai Long2,3,4, Duan Jin Ao , and Pei Li Xia
1
5
1
Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China,
School of Pharmaceutical Sciences, Hainan Medical University, Haikou 571199, China, Hainan Provincial Key Labora-
tory of R&D on Tropical Medicinal Plants, Haikou 571199, China, Haikou Municipal Key Laboratory of Li Nationality
Medicines, Haikou 571199, China, and Pharmacology Laboratory of Traditional Chinese Medicine, Longhua Hospital,
2
3
4
5
Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
(Received May 8, 2012/Revised August 23, 2012/Accepted September 3, 2012)
Chemical investigation of the fruits of Alpinia oxyphylla led to an isolation of the two new nat-
ural products, 9-hydroxy epinootkatol ( ) and ( )-2-pentanol-2- -glucopyranoside ( ), in
addition to the nine known compounds, pinocembrin ( ), tectochrysin ( ), izalpinin ( ), nooka-
tone ( ), yakuchinone A ( ), protocatechuic acid ( ), -sitosterol ( ), daucosterol (10) and -sito-
1
S
O-β-D
2
3
4
5
6
7
8
β
9
β
sterol palmitate (11). Their structures were elucidated on the basis of physicochemical constants
and NMR spectral data analysis. The effects of the isolated components on nitric oxide produc-
tion in LPS-induced RAW 264.7 macrophages were examined. The two new natural compounds
showed inhibitory activities with IC50 values of 21.8 and 32.8 µg/mL, respectively.
Key words: Zingerberaceae, Alpinia oxyphylla, Sesquiterpenoids, Structure elucidation, Anti-
inflammatory effects
INTRODUCTION
together with nine known compounds. We reported
herein isolation, structure elucidation and inhibi-
The Zingerberaceae plant Alpinia oxyphylla Miq. is tory effects on NO production of the new natural pro-
widely cultivated and distributed in South China, of ducts 9-hydroxy epinootkatol (
which Hainan and Guangdong are the two main pro- O- -D-glucopyranoside ( ), and the nine known com-
ducing regions. Its fruits have been used as a tradi- pounds pinocembrin ( ), tectochrysin ( ), izalpinin ( ),
tional Chinese medicine for the treatment of intestinal nookatone ( ), yakuchinone A ( ), protocatechuic acid
disorders, urosis, diuresis, ulceration and dementia ), -sitosterol ( ), daucosterol (10), and -sitosterol
1) and (S)-2-pentanol-2-
β
2
3
4
5
6
7
(8
β
9
β
since ancient times (Liu, 2008). Sesquiterpenes, flavo- palmitate (11).
noids, diarylheptanoids and volatile oils are the main
components and have been found and reported pre- MATERIALS AND METHODS
viously (Morikawa et al., 2002; Liu et al., 2009; Xu et
al, 2009a, 2009b, 2010; Di et al., 2011; Lv et al., 2011; General
Yue et al., 2011). In our present research, we launch-
Melting points (m.p.) were determined on an XT-5
ed further study of its chemical constituents, which melting point apparatus and were uncorrected. Optical
led to an isolation of the two new natural products, rotations were carried out on a WZZ-2B automatic
polarimeter. IR spectra were recorded using JASCO
1
13
FT/IR-4100 spectrophotometer. H- and C-NMR spec-
tra were measured on a Bruker AM-400 or Bruker
DRX-300/500 instrument. Chemical shifts were report-
Correspondence to: Duan Jin Ao, Jiangsu Key Laboratory for
TCM Formulae Research, Nanjing University of Chinese Medi-
cine, Nanjing 210046, China
Tel: 86-25-8581-1116, Fax: 86-25-8581-1116
E-mail: dja@njutcm.edu.cn
ed on the
δ (ppm) scale with CDCl3 and CD3OD as
the solvents. Column chromatography was subjected
2143

2144
Z. J. Qing et al.
on silica gel (100-200 or 200-300 mesh) and silica gel MeOH to afford twelve fractions (Fr.1-12). Compound
H (60 m), which were obtained from Qingdao Marine 11 (6.3 mg) was obtained from Fr.1 by preparative
Chemical and Industrial Co. Lit., China. TLC was per- TLC with petroleum ether-EtOAC (10:1) as the devel-
formed on silica gel G or GF254. Sephadex LH-20 was oping solvent. Another 53.2 mg of compound was
µ
4
purchased from Pharmacia Biotech Sweden for the separated and purified by recrystallization from Fr.2.
purification of some compounds. RAW264.7 macrophage Fr.7 was further separated on silica gel column chro-
cells were purchased from Shanghai cell bank, Chinese matography to afford 8 (20.8 mg). The EtOAC extract
Academy of Sciences. DMEM and fetal bovine serum (500 g) was subjected on a silica gel column with a
were from Gibco Co. Indomethacin (Lot No: 100258- gradient elution system of CHCl3-CH3OH (1:0-0:1) to
200904) and was bought from National Institutes for afford 12 fractions (Fr.1-Fr.12). Fr.4 was separated on
Food and Drug Control, China.
silica gel column chromatography to afford 2 (22.4 mg).
Plant material
9-Hydroxy epinootkatol (1)
o
25
The fruits of Alpinia oxyphylla were collected from Colorless needles (CH COCH ); m.p. 180-181 C; [α]
3
3
D
3
Qiongzhong, Hainan province, China, in December, +31.0 (c 0.1, CHCl ); IR (film) max 3446, 2926, 1653,
-1
+
2
010. A voucher specimen was identified by vice pro- 1456 cm ; HR-ESI-MS m/z 259.1635 [M+Na ] (calcd
1
fessor Zeng Nian kai and deposited in Hainan provin- for C H O Na, 259.1669); H-NMR (CDCl , 300 MHz),
15
24
2
3
cial key laboratory of R&D on tropical medicinal plants,
Hainan Medical University.
δ 5.61 (1H, s, H-1), 4.72, 4.73 (1H each, both s, H-12a,
H-12b), 4.35 (1H, dd,
m, H-2), 2.72 (1H, ttt,
J
J
= 3.0, 3.1 Hz, H-9), 4.27 (1H,
= 12.9, 12.6, 5.7 Hz, H-7), 1.74
Extraction and isolation
3 3
(3H, s, 13-CH ), 1.23 (3H, s, 14-CH ), 0.88 (3H, d, J =
13
The fruits of A. oxyphylla (25 kg) were dried at a low 6.3 Hz, 15-CH3), C-NMR (CDCl , 75 MHz),
δ 149.8
3
o
temperature (48 C) for 24 h and grounded into a coarse
power (24.3 kg), then extracted twice with 95% ethanol
under reflux, each time 2 h. The solvent was removed
under reduced pressure to give a concentrated solution.
The solution was partitioned with petroleum ether,
methylene dichloride, ethyl acetate and n-butanol to
afford 429 g of petroleum ether extract, CH Cl (536
2
2
g), EtOAc (526 g) and n-BuOH (376 g) extracts, respec-
tively.
The petroleum ether extract (250 g) was then sub-
jected to silica gel, eluting with petroleum ether-EtOAc
(
1:0-0:1) to give ten fractions (Fr.1-10). Fr.1 and Fr.2
were divided into four subfractions (Fr.1.1-1.4) and
five subfractions (Fr.2.1-2.5), respectively. Compound
1
(7.2 mg) was isolated from Fr.1.1 by repeated column
chromatography. Compound
3 (9.8 mg) was obtained
from Fr.1.3 by column chromatography with petroleum
ether-acetone (1:0-0:1) as eluent. Compound (10.0
mg) was obtained from Fr.1.4 by using repeated silica
gel column chromatography. Compound (289.2 mg)
was obtained from Fr.1.5 by recrystallization with
acetone and gel column chromatography. Compound
1.2 g) was obtained from Fr.2.4 by recrystallization
with methanol. Fr.2.5 was further isolated and purified
by column chromatography to yield compound (10
mg) and compound (20 mg). Fr.10 was subjected on
silica gel H with a gradient elution of CH Cl -EtOAc
5:1, 2:1, 1:1) to yield compound 10 (31.9 mg).
The CH Cl extract (505 g) was separated by silica
6
4
9
(
7
5
2
2
(
2
2
gel column chromatography, eluted with a gradient
solvent system of petroleum ether-EtOAc followed by Fig. 1. Structures of compounds 1-11.

Two New Natural Products from A. oxyphylla
2145
(C-11), 146.8 (C-10), 129.6 (C-1), 108.8 (C-12), 74.5 (C- cumulation of nitrite in the culture medium using
9), 67.9 (C-2), 44.3 (C-6), 39.9 (C-4), 38.6 (C-3), 37.7 (C- Griess reagent. Cytotoxicity was determined by 3-(4,5-
5), 37.0(C-8), 34.6 (C-7), 21.0 (C-13), 19.9 (C-14), 15.1 dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
(
C-15).
mide (MTT) colorimetric assay. Briefly, after 24 h in-
cubation with test compounds, MTT (10 L, 5 mg/mL
in PBS) solution was added to the wells. After 4 h of
µ
(
S)-2-Pentanol-2-
O
-
β
-
D
-glucopyranoside (2)
o
29
Colorless needles (MeOH); m.p. 138-139 C; [
α]
D
−
34.1 culturing, the medium was removed and 2-propanol
+
(c 0.044, MeOH); HR-ESI-MS m/z 273.1295 [M+Na ] containing 0.04 M HCl was then added to dissolve the
1
(
4
(
calcd for C11
00 MHz): 4.35 (1H, d,
2H, m, H-6'a, H-5'), 3.69 (1H, dd,
H
22
O
6
Na, 273.1309); H-NMR (CD
= 7.8 Hz, H-1'), 3.83-3.90 the formazan solution was measured with a microplate
= 5.2, 11.8 Hz, H- reader at 570 nm (reference, 655 nm). Indomethacin
'b), 3.25-3.39 (3H, m, H-2, 3', 4'), 3.17 (1H, dd, = 8.0, was used as a reference compound. Each test compound
.8 Hz, H-2'), 1.63 (1H, m, H-3a), 1.41-1.47 (3H, m, H- was dissolved in dimethyl sulfoxide (DMSO), and the
b, H-4a, H-4b), 1.25 (3H, d, = 6.3 Hz, 1-CH3), 0.96 solution was added to the medium (final DMSO concen-
). C-NMR (CD OD, 100 MHz): tration was 0.5%). Inhibition (%) was calculated by
104.0 (C-1'), 78.2 (C-2), 77.9 (C-3'), 77.5 (C-5'), 75.4 the following formula, and the IC50 was determined
3
OD, formazan produced in the cells. The optical density of
δ
J
J
6
8
3
J
J
13
(3H, t,
J
= 7.2 Hz, 5-CH
3
3
δ
(
C-2'), 71.8 (C-4'), 62.9 (C-6'), 40.0 (C-3), 22.0 (C-1), graphically (N =4):
1
9.6 (C-4), 14.6 (C-5).
A–B
A–C
inhibition rate (%) = -- - -- -- - -- -- ×100
Acid hydrolysis of 2
Compound (3.2 mg) was dissolved in THF (1 mL)
and 3 M hydrochloric acid (1 mL). After being stirred
2
A
−
C
B
: NO−
: LPS (+), sample (+);
2
concentration (
µ
g/mL) [
A: LPS (+), sample
(−
);
C: LPS (
−), sample (-)].
o
for 12 h at 90 C, the solution was evaporated. The
residue was chromatographed over ODS (Cosmosil RESULTS AND DISCUSSION
1
40C -OPN (Nacalai, Japan)) to obtain D-glucose (0.7
18
29
mg) ([
α]
D
+39.0 (c. 0.035, H O)).
Compound
1
was obtained as colorless needles, and
2
24 2
its molecular formula was assigned to C15H O from
NO production from RAW 264.7 macrophages HR-ESI-MS (observed
stimulated by LPS
showed the presence of hydroxyl (3446 cm ) and dou-
RAW 264.7 macrophages cells (2 × 10 cells/well) were ble bond (1653 cm ) groups. The H-NMR spectrum of
suspended in 200 L of DMEM supplemented with compound showed signals assignable to three meth-
0% fetal bovine serum, penicillin (100 units/mL) and yls [ 0.88( = 6.3 Hz,15-CH ), 1.23, 1.74 (both s, 14-
streptomycin (100 g/mL), and precultured in 96-well CH , 13-CH
microplates at 37 C in 5% CO in air for 1 h, and then bearing the oxygen function [
L of 20 g/mL LPS were added, and test compounds = 3.0, 3.1 Hz, H-9)], an exo-methylene [
6.25-100 g/mL) were cultured for 24 h. NO produc- both s] and an olefin [ 5.61 (s, H-1)]. The C-NMR
tion in each well was assessed by measuring the ac- spectrum data were identical to those of 9-hydroxy
m/z
236.1772).The IR spectrum
−1
5
−1
1
µ
1
d, J
1
δ
3
µ
3
3
)], a methine [
δ
2.72 (m, H-7)], two methines
4.27 (m, H-2), 4.35 (dd,
4.72, 4.73,
o
2
δ
1
(
0
µ
µ
µ
J
δ
1
3
δ
Table I. Inhibitory activities of compounds
1-5,
8,
10 and 11 from the fruits of Alpinia oxyphylla on NO production in
LPS-induced RAW 264.7 macrophagesa
Inhibition rate (%)
Compound
0
µg/mL
6.25
µg/mL
12.5
µg/mL
25
µg/mL
50
µg/mL
100
µg/mL
IC50 (µg/mL)
16.4 ± 2.4*b
12.3 ± 1.9*b
29.2 ± 2.5**
26.7 ± 2.8**
21.0 ± 1.4*b
29.1 ± 2.6**
23.1 ± 1.8**
59.9 ± 2.0**
31.7 ± 2.5**
48.2 ± 2.0**
32.6 ± 1.8**
25.9 ± 2.2**
40.7 ± 2.0**
43.7 ± 1.8**
53.5 ± 3.2** 64.5 ± 2.5**
46.7 ± 2.5*
b
66.0 ± 2.0**
31.5 ± 1.3**
67.6 ± 1.1**
63.1 ± 2.4**
53.7 ± 0.6**
69.4 ± 1.2**
68.2 ± 1.5**
70.4 ± 0.9**
75.1 ± 0.3**
b,c
b,c
b,c
b,c
b,c
b,c
b,c
b,c
b,c
21.8
32.8
17.9
14.7
13.1
18.9
18.2
16.3
12.9
1
2
3
4
5
8
0.0 ± 2.2
0.0 ± 1.5
0.0 ± 2.8
0.0 ± 3.1
0.0 ± 1.9
0.0 ± 0.9
0.0 ± 1.3
0.0 ± 1.8
0.0 ± 1.1
b
69.2 ± 1.5**
b,c
b,c
b,c
68.5 ± 3.5** 68.7 ± 1.2**
70.7 ± 4.0** 76.1 ± 3.2**
57.6 ± 2.4** 67.5 ± 2.4**
54.6 ± 2.3** 68.7 ± 0.4**
58.9 ± 1.5** 64.8 ± 1.0**
69.8 ± 1.8** 70.2 ± 1.1**
66.1 ± 2.0** 65.4 ± 0.9**
b
b
b
b
17.0 ± 0.8*
18.3 ± 1.2*b
30.1 ± 1.5**
33.9 ± 1.6**
b
10
b,c
b,c
b
11
b,c
Indomethacin
a
c
N p < 0.05, **p < 0.01;
= 4); bSignificantly different from the control: *
Each value represents the mean ± S.E.M. (
Cytotoxic effect was observed.

2146
Z. J. Qing et al.
epinootkatol which has been reported in the literature (2011BA101B07), Hainan Special Plan for the Mor-
Morikawa et al., 2002). Thus compound was deter- dernization of Chinese Medicines (2010ZY012 and
mined as 9-hydroxy epinootkatol. It should be noted 2011ZY004), the foundation of Shanghai Municipal
(
1
that this is the first report of an isolation of
new natural product.
1
as the Health Bureau (20114060), natural science foundation
of Hainan province (812189) and research develop-
Compound
2 was also obtained as colorless needles. ment fund supported by Hainan Medical University
13
The C-NMR spectrum indicated the presence of a (HY2012-006 and HY2012-013).
glucopyranosyl moiety and the remaining five carbon
signals consisted of two methyls, whose protons ap- REFERENCES
peared as a doublet [
a triplet [ 0.96 (3H, d
in the H-NMR spectrum, two methylenes and a sec-
ondary alcohol. The coupling constant = 7.8 Hz indi-
cated the -configuration of the sugar residue (Ahmed
et al., 1992). Acid hydrolysis of yielded a free sugar
that was identified as -glucose by measurement of
δ
1.25 (3H, d
J = 7.2 Hz, H-1)] and
Ahmed, W., Ahmad, Z., and Malik, A., Stigmasteryl galacto-
side from Rhynchosia minima Phytochemistry, 31, 4038-
039 (1992).
Chen, Y. K., Xie, B., Liu, X. Y., Kong, N. C., and Li, C., Cry-
stal structure of nootkatone and its application as tobacco
flavorant. Fine Chemicals, 23, 980-982 (2006).
Di, L., Wang, Z. Y., Wang, Z., Li, N., and Wang, K. J., Chemi-
cal constituents in Alpinia oxyphylla seed. J. Plant
Resour. Environ., 20, 94-96 (2011).
δ
J
= 7.2 Hz, H-5)], respectively,
1
.
4
J
β
2
D
2
9
the optical rotation ([
fore, the planer structure of compound
mined to be
configuration of the chiral center in aglycone portion
of compound is according to the chemical shifts in
the C-NMR spectrum [ 22.0 (C-1), 78.2 (C-2), 104.0
α]
+39.0 (c. 0.035, H O)). There-
D 2
2
was deter-
β-glucoside of 2-pentanol. The absolute Hidehiko, K., Hideaki, O., Toshinori, I., Choei, O., Eui, H.,
Anki, T., and Yoshio, T., Glycosides of megastigmane and
2
S
of the simple alcohols from Alangium premnifolium
.
13
δ
Phytochemistry, 42, 723-727 (1996).
Liu, M. S., Introduction of Li nationality medicine. People’s
Medical Publishing House, pp. 123-126, (2008).
(
C-1')], while these data are 19.7 (C-1), 75.1 (C-2), and
1
02.1 (C-1') in its corresponding
R
configuration, res-
was
-gluco-pyranoside,
which is also a new natural compound isolated from
A. oxyphylla
The compounds (
son of reference or their physical and spectra data
Liu, N., Yu, X. Y., Zhao, H., and Zhao, Y. Q., Studies on the
pectively (Hidehiko et al., 1996). Thus, compound
deduced as ( )-2-pentanol-2-
2
chemical constituents from the fruits of Alpinia oxyphylla
.
S
O-β-D
China Tradit. Herb. Drugs, 40, 29-32 (2009).
Lv, X. Q., Luo, J. G., Wang, X. B., Wang, J. S., Luo, J., and
Kong, L. Y., Four new sesquiterpenoids from the fruits of
.
3-11) were identified by compari-
Alpinia oxyphylla. Chem. Pharm. Bull., 59, 402-406 (2011).
Morikawa, T., Mastuda, H., Toguchida, I., Ueda, K., and
Yoshokawa, M., Absolute stereostructures of three new
sesquiterpenes from the fruit of Alpinia oxyphylla with
inhibitory effects on nitric oxide production and degranu-
lation in RBL-2H3 cells. J. Nat. Prod., 65, 1468-1474 (2002).
Wang, X.B., Yang, C. S., Hua, S. Z., and Kong, L. Y., Chemi-
cal constituents from the seeds of Alpinia katsumadai
Hayata. Chin. J. Nat. Med., 8, 419-421 (2010).
1
13
(m.p., IR, H-NMR, C-NMR, MS) with reported litera-
ture values (Zhou et al., 1999; Chen et al., 2006; Wang
et al., 2010; Yue et al., 2011). Of the nine compounds,
compound
3 was isolated from the fruits of A. oxyphylla
for the first time.
The effects of all isolated constituents from A. oxy-
phylla on NO production from LPS-activated RAW264.7
macrophage cells were examined. The compounds were Xu, J. J., Tan, N. H., Zeng, G. Z., Han, H. J., Huang, H. Q.,
screened at a concentration of 50 g/mL. Then the can-
Ji, C. J., Zhu, M. J., and Zhang, Y. M., Studies on chemical
constituents in fruit of Alpinia oxyphylla China J. Chin.
µ
.
didate compounds with an inhibitory rate over 50%
Mater. Med., 34, 990-993 (2009a).
were examined at concentrate of 6.25 g/mL, 12.5 g/
mL, 25 g/mL, 50 g/mL, and 100 g/mL to calculate
their IC50 and the results are summarized in Table I.
From the result we can easily conclude that phenolic
µ
µ
Xu, J. J., Tan, N. H., Xiong, J., Adebayo, A. H., Han, H. J.,
Zeng, G. Z., Ji, C. J., Zhang, Y. M., and Zhu, M. J., Oxy-
phyllone A and B, novel sesquiterpenses with an unusual
µ
µ
µ
4
,5-secoeudesmane skeleton from Alpinia oxyphylla
. Chin.
acid derivatives (compounds
4, 5 and 8) have more
Chem. Lett., 20, 945-948 (2009b).
potent NO inhibitory activity than other compounds.
Maybe the phenolic acid compounds were the main
anti-inflammatory components in A. oxyphylla.
Xu, J. J., Tan, N. H., Zeng, G. Z., Han, H. J., and Peng, Y. F.,
Two new norsesquiterpenes from the fruits of Alpinia
oxyphylla. Chin. J. Nat. Med., 8, 6-8 (2010).
Yue, Y., Liu, Y. Q., Sun, L., and Cai, Q., Isolation and identi-
ACKNOWLEDGEMENTS
fication of chemical constituents from Alpinia oxyphylla
.
Asia-Pacific Tradit. Med., 7, 19-20 (2011).
We are grateful for the financial support from Zhou, L. D., Guo, J., and Yu, J. G., Flavonoids from Beijing
National 12th Five-Year Plan Regional Base Project
propolis. China J. Chin. Mater. Med., 24,162-164 (1999).