Hepatoprotective amide constituents from the fruit of Piper chaba: Structural requirements, mode of action, and new amides

Article abstract of DOI:10.1016/j.bmc.2009.08.050

The 80% aqueous acetone extract from the fruit of Piper chaba (Piperaceae) was found to have hepatoprotective effects on d-galactosamine (d-GalN)/lipopolysaccharide-induced liver injury in mice. From the ethyl acetate-soluble fraction, three new amides, p

Full text of DOI:10.1016/j.bmc.2009.08.050

Bioorganic & Medicinal Chemistry 17 (2009) 7313–7323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Hepatoprotective amide constituents from the fruit of Piper chaba:
Structural requirements, mode of action, and new amides
Hisashi Matsuda a, Kiyofumi Ninomiya a,b, Toshio Morikawa a,b, Daisuke Yasuda a, Itadaki Yamaguchi a,
Masayuki Yoshikawa a,
*
a Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
b Pharmaceutical Research and Technology Institute, Kinki University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2009
Revised 18 August 2009
Accepted 19 August 2009
Available online 29 August 2009
The 80% aqueous acetone extract from the fruit of Piper chaba (Piperaceae) was found to have hepatopro-
tective effects on
ethyl acetate-soluble fraction, three new amides, piperchabamides E, G, and H, 33 amides, and four aro-
matic constituents were isolated. Among the isolates, several amide constituents inhibited -GalN/tumor
necrosis factor- (TNF- )-induced death of hepatocytes, and the following structural requirements were
D-galactosamine (D-GalN)/lipopolysaccharide-induced liver injury in mice. From the
D
a
a
suggested: (i) the amide moiety is essential for potent activity; and (ii) the 1,9-decadiene structure
between the benzene ring and the amide moiety tended to enhance the activity. Moreover, a principal
constituent, piperine, exhibited strong in vivo hepatoprotective effects at doses of 5 and 10 mg/kg, po
Keywords:
Hepatoprotective activity
Piperine
Piper chaba
Piperaceae
and its mode of action was suggested to depend on the reduced sensitivity of hepatocytes to TNF-a.
Ó 2009 Elsevier Ltd. All rights reserved.
Piperchabamide
Anti-TNF-a activity
1. Introduction
chaba as well as the hepatoprotective effects and structural
requirements of the amide constituents for the reduced sensitivity
Piper chaba HUNTER (syn. P. retrofractum VAHL., Piperaceae) is
widely distributed in Southeast Asia. The fruit of this plant is com-
monly called ‘Dee Plee’ in Thailand and has been used as an anti-
flatulent, expectorant, antitussive, antifungal, uterus-contracting
agent, sedative-hypnotic, appetizer, and counter-irritant in the tra-
ditional medicine of Thailand.1 In the course of our characteriza-
tion studies on this natural medicine,2–4 we previously reported
that 40 constituents were isolated from the 80% aqueous acetone
extract of the fruit of P. chaba. The aqueous acetone extract and
the isolates were found to show protective effects on ethanol- or
indomethacin-induced gastric lesions in rats,2 inhibitory effects
on the increase in serum aspartate aminotransaminase (sAST)
to TNF-a 3,5
.
2. Results and discussion
2.1. Protective effects of the aqueous acetone extract from the
fruit of P. chaba on liver injury induced by D-GalN/LPS in mice
The dried fruit of P. chaba (purchased in Thailand) was extracted
with 80% (v/v) aqueous acetone at room temperature to yield an
aqueous acetone extract (19.7% from the dried fruit).2 As shown
in Table 1, the 80% aqueous acetone extract from the fruit of P. cha-
ba showed inhibitory effects on the increase in sAST and sALT, as
and alanine aminotransaminase (sALT) levels induced by
tosamine ( -GalN)/lipopolysaccharide (LPS)-induced liver injury
in mice and on cell death induced by -GalN/tumor necrosis fac-
tor- (TNF-
) in primary cultured mouse hepatocytes,3 and pro-
D-galac-
markers of liver injury, induced by
of 25–50 mg/kg, po.
D-GalN/LPS in mice at doses
D
D
a
a
moting effects on adipogenesis of 3T3-L1 cells.4 As a continuing
study of the constituents from the fruit of P. chaba, we additionally
isolated three new amide constituents named piperchabamides E
(1), G (2), and H (3). This paper deals with the isolation and struc-
tural elucidation of the three new amides (1–3) from the fruit of P.
2.2. Effects of the aqueous acetone extract from the fruit of
P. chaba on -GalN or -GalN/TNF- -induced cytotoxicity in
primary cultured mouse hepatocytes
D
D
a
Infection with hepatitis C virus and chronic consumption of
alcohol are major causes of liver injury, cirrhosis, and hepatocellu-
lar carcinoma worldwide. TNF-
forms of organ injury through its induction of cellular apoptosis. In
a is known to mediate a number of
* Corresponding author. Tel.: +81 75 595 4633; fax: +81 75 595 4768.
E-mail address: myoshika@mb.kyoto-phu.ac.jp (M. Yoshikawa).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.050

7314
H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
Table 1
Inhibitory effects of the 80% aqueous acetone extract from the fruit of P. chaba on D-GalN/LPS-induced liver injury in mice
Treatment
Dose (mg/kg, po)
n
sAST
sALT
(Karmen unit)
Inhibition (%)
(Karmen unit)
Inhibition (%)
Normal (vehicle)
—
—
25
50
100
7
9
8
8
8
86 5a
—
—
49.0
72.9
90.0
28 6a
—
—
42.3
77.6
95.7
Control (D-GalN/LPS)
5016 560
2598 616a
1422 437a
577 35a
6373 928
3692 1301
1448 524a
304 12a
80% Aqueous acetone ext.
Each value represents the mean SEM.
Significantly different from the control.
a
p <0.01.
the liver, the biological effects of TNF-
a
have been implicated in
toxicity in hepatocytes [inhibition (%) at 30
lg/mL: 94 2 (p
hepatic injury induced by hepatic toxins, ischemia/reperfusion, vir-
<0.01) and 70 6 (p <0.01), respectively]. The EtOAc-soluble fraction
was subjected to SiO2 and ODS column chromatographies and finally
to HPLC (ODS column, eluted with MeOH–H2O or CH3CN–H2O sol-
vent system) to furnish three new amide constituents, piperchaba-
mides E3 (1, 0.0046%), G (2, 0.0064%), and H (3, 0.0085%) together
with four aromatic compounds, benzenepropanoic acid32 (0.13%),
3,4-methylendioxycinnamaldehyde33 (0.0019%), piperonal3,32 (4,
0.0047%), and methyl piperate2,34,35 (5, 0.11%), and 33 amide com-
pounds, piperchabamide A2 (6, 0.0029%), N-cinnamoylpiperidine36
(7, 0.0016%), ilepcimide37 (8, 0.0048%), Da,b-dihydropiperine [= pip-
eranine2,38,39 (9, 0.42%)], piperine2,32,35,40,41 (10, 2.95%), isopiper-
ine41 (11, 0.056%), isochavicine41 (12, 0.039%), chavicine41 (13,
0.0042%), piperoleine B3,40 (14, 0.0060%), pipernonaline2,42 (15,
0.47%), piperchabamide B2 (16, 0.0041%), dehydropipernonaline2,43
(17, 0.056%), piperundecalidine3,42 (18, 0.017%), piperchabamide C2
(19, 0.0032%), fragaramide44 (20, 0.0006%), 5,6-dihydropiperlon-
guminine3,39 (21, 0.015%), piperlonguminine2,35 (22, 0.072%), retro-
fractamide C3,45 (23, 0.036%), piperchabamide D2 (24, 0.0037%),
dihydroguineensine46 (25, 0.0015%), retrofractamide A3,40,47 (26,
0.0075%), pipercide [= retrofractamide B2,40,42,47 (27, 0.049%)], guin-
eensine2,35,40,42,47 (28, 0.081%), brachystamide B3,48 (29, 0.012%),
N-isobutyl-(2E,4E)-deca-2,4-dienamide3,49,50 (30, 0.018%), N-isobu-
tyl-(2E,4E)-dodeca-2,4-dienamide3,49,50 (31, 0.0034%), N-isobutyl-
(2E,4E)-tetradeca-2,4-dienamide50 (32, 0.0044%), N-isobutyl-(2E,
4E)-hexadeca-2,4-dienamide51 (33, 0.011%), N-isobutyl-(2E,4E)-
octadeca-2,4-dienamide2,51 (34, 0.23%), N-isobutyl-(2E,4E,8Z)-tet-
radeca-2,4,8-trienamide52 (35, 0.0017%), N-isobutyl-(2E,4E,12Z)-
octadeca-2,4,12-trienamide53 (36, 0.0057%), N-isobutyl-(2E,4E,
14Z)-eicosa-2,4,14-trienamide2,53 (37, 0.18%), and piperic acid iso-
pentyl amide54 (38, 0.0011%).
al hepatitis, and alcohol.6–8 Therefore, TNF-
a
is considered to be an
important target in research to discover anti-inflammatory and
hepatoprotective agents.
D
-GalN/LPS-induced liver injury is recognized to develop via
immunological responses.9 This type of liver injury occurs in two
forms. First, depletion of uridine triphosphate and increased sensi-
tivity to TNF-
pro-inflammatory mediators, such as TNF-
LPS-activated macrophages (Kupffer’s cells). Apoptosis of hepato-
cytes by TNF- is reported to have an important role in -GalN/
LPS-induced liver injury.10
a
in hepatocytes is induced by
D-GalN. Second,
a
, are released from
a
D
In our previous study on hepatoprotective compounds from nat-
ural medicines, we reported that several constituents from Hovenia
dulcis,11 Bupleurum scorzonerifolium,12,13 Curcuma zedoaria,14–16
Angelica furcijuga,17,18 Betula platyphylla var. japonica,19 Pisum sati-
vum,20 Salacia reticulata,21 Tilia argentea,22 Anastatica hierochunti-
ca,23 Panax notoginseng,24 Cyperus longus,25 Erycibe expansa,26
Camellia sinensis,27 Sedum sarmentosum,28,29 Sinocrassula indica,30
and Hedychium coronarium31 showed hepatoprotective effects on li-
ver injury induced by D-GalN/LPS in mice and/or D-GalN-induced
cytotoxicity in primary cultured hepatocytes. Since the aqueous ace-
tone extract from the fruit of P. chaba showed hepatoprotective ef-
fects on
inhibitory effects on
in primary cultured mouse hepatocytes were examined using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. As shown in Table 2, the aqueous acetone extract of this
herbal medicine inhibited the cytotoxicity induced by both -GalN
and -GalN/TNF- in hepatocytes (IC50 value = 18 and 11 g/mL,
respectively).
D
-GalN/LPS-induced liver injury in mice (vide ante), the
D
-GalN or -GalN/TNF- -induced cytotoxicity
D
a
D
D
a
l
2.4. Structures of piperchabamides E (1), G (2), and H (3)
2.3. Chemical constituents from the fruit of P. chaba
Piperchabamide E (1) was obtained as colorless oil with positive
The 80% aqueous acetone extract from the fruit of P. chaba was
partitioned between EtOAc–H2O (1:1, v/v) to give an EtOAc-soluble
fraction (9.7%) and an aqueous phase. The EtOAc-soluble fraction
optical rotation (½a D24
ꢀ
+18.7 in MeOH). Its IR spectrum showed
absorption bands at 2963, 1646, 1545, 1506, 1489, 1446, 1257,
and 1238 cmꢁ1 ascribable to methylene, an unsaturated amide
group, and an aromatic ring. The EIMS of 1 showed a molecular
significantly inhibited the D-GalN and D-GalN/TNF-a-induced cyto-
Table 2
Inhibitory effects of the 80% aqueous acetone extract from the fruit of P. chaba on D-GalN or D-GalN/TNF-a-induced cytotoxicity in primary cultured mouse hepatocytes
Inhibition (%)
IC50 (lg/mL)
0
0
0
l
g/mL
3
7
l
g/mL
10
l
g/mL
30
l
g/mL
100 lg/mL
D
-GalN-induced (hepatocytes)
80% Aqueous acetone ext.
2
1
3
17 1a
57 4a
76 2a
66 4a
ꢁ22 5b
18
11
D-GalN/TNF- -induced (hepatocytes)
a
80% Aqueous acetone ext.
22 2a
105 9a
Each value represents the mean SEM (N = 4).
Significantly different from the control.
a
p <0.01.
Cytotoxic effect was observed.
b

H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
7315
ion peak at m/z 287 (M+), and the molecular formula was deter-
mined to be C17H21NO3 by high-resolution EIMS measurement.
The 1H and 13C NMR spectra of 1 (CDCl3, Tables 3 and 4) showed
signals assignable to two methyls [d 0.91 (3H, dd, J = 6.9, 7.6 Hz,
H3-40), 0.91 (3H, d, J = 6.7 Hz, H3-50)], two methylenes [d 1.17,
1.41 (1H each, both m, H2-30), 3.15, 3.29 (1H each, both m, H2-
10)], a methine [d 1.59 (1H, m, H-20)], a 3,4-methylenedioxyphenyl
group [d 5.97 (2H, s, H2-12), 6.75 (1H, d, J = 8.2 Hz, H-10), 6.88 (1H,
dd, J = 1.8, 8.2 Hz, H-11), 6.96 (1H, d, J = 1.8 Hz, H-7)], two trans-
olefinic proton pairs [d 5.93 (1H, d, J = 15.0 Hz, H-2), 6.65 (1H, dd,
J = 10.7, 17.7 Hz, H-4), 6.78 (1H, d, J = 17.7 Hz, H-5), 7.35 (1H, dd,
J = 10.7, 15.0 Hz, H-3)], and a conjugated amide group [dC 166.2
(C-1)]. The planar structure of 1 was constructed on the basis of
various NMR experiments.55 Specifically, the 1H–1H COSY experi-
ments on 1 indicated the presence of three partials written in bold
lines, while in the HMBC experiments, long-range correlations
were observed between the following proton and carbon pairs:
H-2 and C-1; H-5 and C-6; and H2-10 and C-1 (Fig. 2). Finally, acid
both br d, J = ca. 13 Hz), H-1000,5000], 3.61, 3.62 (2H each, both br s,
H-20,40)}, two 3,4-methylenedioxyphenyl groups [d 5.87, 5.90 (2H
each, both br s, H2-12, 1200), 6.56 (1H, dd, J = 1.5, 7.9 Hz, H-1100),
6.60 (1H, d, J = 1.5 Hz, H-700), 6.68 (2H, br s, H-7, 11), 6.74 (1H, d,
J = 8.2 Hz, H-10), 6.88 (1H, br s, H-1000)], a cis-olefin pair [d 5.71
(1H, ddd, J = 2.1, 5.1, 9.8 Hz, H-3), 5.84 (1H, d, J = 9.8 Hz, H-2)],
and two amide groups [dC 170.4 (C-1), 173.2 (C-100)]. As shown in
Figure 1, the 1H–1H COSY experiment on 2 indicated the presence
of partial structures written in bold lines. In the HMBC experiment
on 2, long-range correlations were observed between the following
protons and carbons (H-2, H2-10,50 and C-1; H-4, 5 and C-6, 7, 11;
H-7, 11 and C-5; H-200, H2-1000, 5000 and C-100; H2-500 and C-600, 700,
1100; H-700, 1100 and C-500), as shown in Figure 2. Thus the connectiv-
ities of quaternary carbons (C-1, 6, 100, 600) in 2 were clarified, and
the planar structure of 2 was elucidated.
Piperchabamide H (3) was isolated as an amorphous powder. Its
molecular formula, C38H46N2O6, was determined by EIMS and HRE-
IMS measurements. The 1H and 13C NMR spectra of 3 (CDCl3, Tables
3 and 4) showed signals assignable to four methylenes {d [1.26 (1H,
m), 1.44 (1H, br s), H2-400], [1.36 (1H, m), 1.56 (1H, br s), H2-500],
1.56 (2H, br s, H2-600), 2.13 (2H, m, H2-700)}, four methines [d
2.13, 3.54 (1H each, both br s, H-300, 4), 3.67 (1H, br d, J = ca.
11 Hz, H-5), 4.02 (1H, dd, J = 10.8, 11.0 Hz, H-200)], two piperidine
rings {d 1.20, 1.26 (2H each, both m, H2-2000,4000), 1.44 (4H, br s,
H2-30,3000), 1.44, 1.56 (2H each both br s, H2-20,40), [3.07 (2H, dd-
like), 3.43 (1H, br d, J = ca. 13 Hz), 3.90 (1H, d-like), H2-1000,5000],
3.54 (4H, br s, H2-10,50)}, two 3,4-methylenedioxyphenyl groups
[d 5.87, 5.93 (2H each, both br s, H2-12, 1600), 6.68 (1H, br s, H-
11), 6.73 (1H, d, J = 1.2 Hz, H-7), 6.74 (2H, br d, J = ca. 8 Hz, H-10,
1500), 6.87 (1H, br s, H-1100), 6.88 (1H, br s, H-1400)], a cis-olefin pair
[d 5.70 (1H, m, H-3), 5.83 (1H, d, J = 9.8 Hz, H-2)], and two amide
groups [dC 170.5 (C-1), 173.5 (C-100)], which were superimposable
on those of 2, except for the signals due to two additional methy-
hydrolysis56 of
1 with 6 M HCl liberated (R)-2-methylbutyl-
amine,57 which was identified by HPLC analysis using the refrac-
tive index and optical rotation detectors. On the basis of the
above evidence, the stereostructure of 1 was elucidated as shown.
Piperchabamide G (2) was isolated as an amorphous powder
and the molecular formula, C34H40N2O6, was determined by EIMS
and HREIMS measurements. The 1H and 13C NMR spectroscopic
properties (CDCl3, Tables 3 and 4) of 2 showed signals assignable
to two methylenes [d 1.58, 1.67 (1H each, both m, H2-400), 2.40,
2.50 (1H each, both m, H2-500)], four methines [d 2.22 (1H, m, H-
300), 3.60 (1H, br s, H-4), 3.68 (1H, ddd, J = 2.1, 2.1, 9.7 Hz, H-5),
4.05 (1H, dd, J = 9.7, 10.0 Hz, H-200)], two piperidine rings {d [1.15
(1H, m), 1.25 (2H, m), 1.43 (1H, m), H-2000,4000], 1.43, 1.67 (2H each,
both m, H-30,3000), 1.58 (4H, m, H-20,40), [2.95 (1H, ddd, J = 2.4, 12.5,
12.5 Hz), 3.01 (1H, ddd, J = 3.1, 13.2, 13.2 Hz), 3.48, 3.98 (1H each,
Table 3
1H NMR (500 MHz, CDCl3) data on piperchabamides E (1), G (2), and H (3)
Position
1
Position
2
Position
3
dH (J Hz)
dH (J Hz)
dH (J Hz)
2
3
4
5
5.93 (1H, d, 15.0)
7.35 (1H, dd, 10.7, 15.0)
6.65 (1H, dd, 10.7, 17.7)
6.78 (d, 17.7)
6.96 (d, 1.8)
6.75 (d, 8.2)
6.88 (dd, 1.8, 8.2)
5.97 (2H, s)
3.15 (1H, m)
3.29 (1H, m)
1.59 (1H, m)
1.17 (1H, m)
1.41 (1H, m)
0.91 (3H, dd, 6.9, 7.6)
0.91 (3H, d, 6.7)
5.66 (1H br s)
2
3
4
5
5.84 (1H, d, 9.8)
5.71 (1H, ddd, 2.1, 5.1, 9.8)
3.60 (1H, br s)
3.68 (1H, ddd, 2.1, 2.1, 9.7)
6.68 (1H, br s)
6.74 (1H, d, 8.2)
6.68 (1H, br s)
5.87 (2H, br s)
3.61 (2H, br s)
3.62 (2H, br s)
1.58 (4H, m)
1.43 (1H, m)
1.67 (1H, m)
4.05 (1H, dd, 9.7, 10.0)
2.22 (1H, m)
1.58 (1H, m)
1.67 (1H, m)
2.40 (1H, m)
2.50 (1H, m)
6.60 (1H, d, 1.5)
6.88 (1H, br s)
6.56 (1H, dd, 1.5, 7.9)
5.90 (2H, br s)
2
3
4
5
5.83 (1H, d, 9.8)
5.70 (1H, m)
3.54 (1H, br s)
3.67 (1H, br d, ca. 11)
6.73 (1H, d, 1.2)
6.74 (1H, br d, ca. 8)
6.68 (1H, br s)
5.87 (2H, br s)
3.54 (4H, br s)
1.44 (2H, br s)
1.56 (2H, br s)
1.44 (2H, br s)
4.02 (dd, 10.8, 11.0)
2.13 (1H, br s)
1.26 (1H, m)
1.44 (1H, br s)
1.36 (1H, m)
1.56 (1H, br s)
1.56 (2H, br s)
2.13 (2H, m)
5.99 (1H, dt, 15.5, 7.0)
6.25 (1H, d, 15.5)
6.87 (1H, br s)
6.88 (1H, br s)
6.74 (1H, br d, ca. 8)
5.93 (2H, br s)
3.07 (2H, dd-like)
3.43 (1H, br d, ca. 13)
3.90 (1H, d-like)
1.20 (2H, m)
7
7
7
10
11
12
10
10
11
12
10,50
10
11
12
10,50
20,40
20
30
20,40
30
30
200
300
400
40
50
CONH
200
300
400
500
500
600
700
700
1000
1100
1200
800
900
1100
1400
1500
1600
1
2
000,5000
2.95 (1H, ddd, 2.4, 12.5, 12.5)
3.01 (1H, ddd, 3.1, 13.2, 13.2)
3.48 (1H, br d, ca. 13)
3.98 (1H, br d, ca. 13)
1.15 (1H, m)
1.25 (2H, m)
1.43 (1H, m)
1.43 (1H, m)
1.67 (1H, m)
1
000,5000
000,4000
2
000,4000
3000
1.26 (2H, m)
1.44 (2H, br s)
3000

7316
H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
Table 4
mide B (16, inhibition: 63% at 3
retrofractamide C (23, 51%), piperchabamide D (24, 57%), and pip-
ercide (= retrofractamide B, 27, 54%) showed potent effects at 3
lM), piperlonguminine (22, 50%),
13C NMR (125 MHz, CDCl3) data on piperchabamides E (1), G (2), and H (3)
lM
Position
1
Position
2
Position
3
dC
dC
dC
with more than 50% inhibition. However, several amide constitu-
ents such as 23, 24, and 27 exhibited no concentration-dependent
inhibition at 3–30 lM, suggesting that they would show cytotoxic
effects at higher concentrations. With regard to the structural
requirements of the amide constituents for the activity, the amide
structure was important [methyl piperate (5, inhibition: 21% at
30 lM) < piperine (10, 68%)], and 16 and 24, which have the 1,9-
decadiene structure between the benzene ring and amide moiety,
tended to show more potent activity.
1
2
3
4
5
6
7
8
166.2
123.3
140.9
124.7
138.8
130.9
105.7
148.2
148.2
108.5
122.6
101.3
45.3
1
2
3
4
5
6
7
8
170.4
134.0
123.2
38.3
1
2
3
4
5
6
7
8
170.5
133.9
123.4
37.9
46.6
46.6
137.8
108.1
147.5
146.0
108.9
121.4
100.7
42.9
47.1
25.9
27.1
24.6
173.2
45.0
39.3
31.9
33.5
136.0
108.7
147.5
145.5
108.1
120.9
100.7
42.9
137.9
108.2
147.5
146.0
108.9
121.4
100.7
42.8
47.0
25.8
25.9
24.6
173.5
45.1
39.6
29.8
29.4
9
9
9
10
11
12
10
20
30
40
50
10
11
12
10,50
10
11
12
10,50
2.6. Effects of the chemical constituents on NO production in
LPS-activated mouse peritoneal macrophages
35.1
27.1
11.3
17.2
20,40
20,40
Next, the effects of the aqueous acetone extract from the fruit of
P. chaba and its constituents on NO production, as a marker of mac-
rophage activation, in LPS-activated mouse peritoneal macro-
phages were examined. As shown in Table 7, piperchabamides G
(2, IC50 value = 28 lM), A (6, 20 lM), and C (19, 42 lM) showed
inhibitory effects without cytotoxic effects in the MTT assay, and
30
30
100
100
200
200
300
300
400
400
500
500
600
600
27.0
32.9
their inhibitory activities were equivalent to or stronger than that
of an NO synthase inhibitor [NG-monomethyl-
L-arginine (L-
700
700
800
800
129.2
129.5
132.4
105.3
147.9
146.5
108.0
120.2
100.9
42.8
NMMA), 36 l
M].60,61
900
900
These in vitro findings led us to suggest the following possible
mechanisms of action for the hepatoprotective effects of the fruit
1000
1100
1200
1000
1100
1200
1300
1400
1500
1600
1000,5000
of P. chaba: (i) protection of hepatocytes on
toxity (14, 31, and 37), (ii) inhibitory effects on
D
-GalN-induced cyto-
-GalN/TNF- -in-
1
000,5000
D
a
47.0
26.0
27.1
24.6
duced cell death in hepatocytes (16, 22–24, 27, etc.), and (iii)
inhibitory effects on activation of macrophages induced by LPS
(2, 6, and 19).
2
000,4000
3000
47.0
26.1
2
000,4000
2.7. Effects of the chemical constituents on TNF-
cytotoxicity in L929 cells
a-induced
26.8
24.6
3000
In order to clarify the effects of the constituents on the sensitiv-
ity of hepatocytes to TNF- , the effects on the TNF- -induced de-
crease in cell viability of L929 cells, a TNF-
-sensitive cell line,62
were examined using the MTT assay. After incubation of L929 cells
with 20 pg/mL TNF- and 0.5 g/mL actinomycin D for 20 h, the
viability of L929 cells without test sample was reduced to ca.
60% compared with that of TNF- -untreated cells. As shown in Ta-
ble 8, piperchabamide A (6, IC50 value = 13 M), piperine (10,
42 M), piperleine B (14, 64 M), piperchabamides B (16, 33 M)
and C (19, 42 M), and N-isobutyl-(2E,4E,14Z)-eicoca-2,4,14-tri-
enamide (37, 25 M) inhibited the decrease in the cell viability.
a
a
lene groups. The connectivities of the quaternary carbons in 3 were
elucidated on the basis of the 1H–1H COSY and HMBC experiments,
as shown in Figure 2. Next, the stereostructures of 2 and 3 were
characterized in nuclear Overhauser enhancement spectroscopy
(NOESY) experiments. NOE correlations in 2 and 3 were observed
between the following proton pairs (H-2 and H-3; H-3 and H-5;
H-5 and H-300; and H-200 and H-7, 11) as shown in Figure 3. Conse-
quently, the stereostructures of 2 and 3 were determined to be as
shown.
a
a
l
a
l
l
l
l
l
l
2.5. Effects of the chemical constituents on
TNF- -induced cytotoxicity in primary cultured mouse
hepatocytes
D-GalN or D-GalN/
2.8. Protective effects of piperine (10) on liver injury induced
by -GalN/LPS in mice and its mode of action
a
D
Finally, we examined the effects of the principal constituent
piperine (10) on -GalN/LPS-induced liver injury in mice. As
shown in Table 9 and 10 significantly inhibited the increase in
sAST and sALT induced by -GalN/LPS in mice at doses of 5 mg/
kg, po. To clarify the mode of action of 10, its effect on the in-
crease in serum TNF- levels induced by -GalN/LPS in mice
was examined. As a result, 10 at 5–20 mg/kg, po did not inhibit
the increase in serum TNF- levels apparently different from
hydrocortisone (Table 10). Although compound 10 inhibited the
death of hepatocytes induced by -GalN/TNF- , it could not pro-
tect the cells against -GalN. On the other hand, 10 at 10–100
significantly inhibited the death of L929 cells caused by TNF-
indicating a decrease in the sensitivity of L929 cells to TNF-
Since the aqueous acetone extract of P. chaba was found to show
protective effects on both -GalN and -GalN/TNF- -induced cyto-
toxicity in primary cultured mouse hepatocytes (Table 2), the cyto-
protective effects of the constituents from this herbal medicine
were also examined. As shown in Table 5, piperoleine B (14, IC50
D
D
D
a
D
a
D
value = 2.9 lM), N-isobutyl-(2E,4E)-dodeca-2,4-dienamide (31,
9.3
lM), and N-isobutyl-(2E,4E,14Z)-eicoca-2,4,14-trienamide (37,
a
6.4
lM) showed strong hepatoprotective activity against cytotox-
icity induced by D-GalN. Their activities were greater than that of
D
a
commercial silybin (39 l
M),23–31 which is well known to show po-
D
lM
tent hepatoprotective effects.58,59 On the other hand, most of the
a,
amide constituents except for brachystamide B (29) and 37 signif-
a
icantly inhibited the cell death induced by
D-GalN/TNF-a in the
(vide ante). These findings suggest a reduction in the sensitivity
effect of 10.3 Many compounds that inhibit cell death induced
hepatocytes at 1–30 M, as shown in Table 6. Notably, piperchaba-
l

H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
7317
O
9''O
13'' O
16''
10''
6''
12''
14''
15''
7
3'
4'
1'
8
9
6
2'
5'
O
3
1
11''
5
8'' O
7''
O
N
H
4
2
12
5'''
12''
4'''
O
2''
11
11''
10''
4'' 5''
O
8''
3'''
10
N1''
5'''
9''
2'
4'''
3''
2'
4'
O
1'
7''
O
1'''
7
5'' 6''
4''
3
2'''
3'''
piperchabamide E (1)
4
N1''
3'
2''
5
N
1
3''
5
6
1'
O
1'''
2'''
2
3
5'
4
8
3'
N
6
1
O
11
7
O
2
4'
12
5'
8
9
10
CHO
O
O
O
O
11
OCH3
12
O 9
10
O
O
piperchabamide G (2)
piperonal (4)
methyl piperate (5)
piperchabamide H (3)
O
O
O
O
O
O
O
O
O
N
N
N
N
,
piperchabamide A (6)
N-cinnamoylpiperidine (7)
ilepcimide (8)
α β-dihydropiperine
Δ
(= piperanine, 9)
O
O
O
O
O
O
O
O
O
N
N
O
N
N
O
O
piperine (10)
isopiperine (11)
isochavicine (12)
chavicine (13)
O
O
O
O
N
(CH2)n
N
N
(CH2)n
O
O
O
O
O
piperoleine B (14)
n = 4: pipernonaline (15)
n = 6: piperchabamide B (16)
n = 2: dehydropipernonaline (17)
n = 4: piperundecalidine (18)
n = 6: piperchabamide C (19)
O
O
O
O
O
O
O
O
O
O
N
H
N
H
N
H
(CH2)n
N
H
O
O
fragaramide (20)
5,6-dihydropiperlonguminine (21) piperlonguminine (22)
n = 4: retrofractamide C (23)
n = 6: piperchabamide D (24)
n = 8: dihydroguineensine (25)
O
O
N
H
N
H
(CH2)n
(CH2)n
O
O
n = 2: retrofractamide A (26)
n = 4: N-isobutyl-(2E,4E)-deca-2,4-dienamide (30)
n = 6: N-isobutyl-(2E,4E)-dodeca-2,4-dienamide (31)
n = 8: N-isobutyl-(2E,4E)-tetradeca-2,4-dienamide (32)
n = 10: N-isobutyl-(2E,4E)-hexadeca-2,4-dienamide (33)
n = 12: N-isobutyl-(2E,4E)-octadeca-2,4-dienamide (34)
n = 4: pipercide ( = retrofractamide B, 27)
n = 6: guineensine (28)
n = 8: brachystamide B (29)
O
O
O
O
N
H
N
H
(CH2)n
piperic acid isopentyl amide (38)
n = 2: N-isobutyl-(2E,4E,8Z)-tetradeca-2,4,8-trienamide (35)
n = 6: N-isobutyl-(2E,4E,12Z)-octadeca-2,4,12-trienamide (36)
n = 8: N-isobutyl-(2E,4E,14Z)-eicosa-2,4,14-trienamide (37)
Figure 1. Chemical structures of compounds 1–38 from the fruit of Piper chaba.
by
there are few reports on compounds that selectively reduce the
sensitivity of hepatocytes to TNF-a 63,64
D
-GalN or production of TNF-
a
have been reported,14,16,22 but
diene structure between the benzene ring and amide moiety
were suggested to be important for potent inhibition of the
D-GalN/TNF-a-induced death of hepatocytes. Furthermore, a
.
In conclusion, three new amide constituents, piperchaba-
mides E (1), G (2), and H (3), together with 33 known amide
constituents and four aromatic constituents, were isolated from
the fruit of P. chaba. Among the isolates, several constituents
were found to show hepatoprotective activities. With regard to
the structural requirements, the amide moiety and the 1,9-deca-
principal amide constituent, piperine (10), dose-dependently
inhibited the increase in sAST and sALT at doses of 5 and
10 mg/kg po in
fect was suggested to depend on the reduced sensitivity of
hepatocytes to TNF- . The mechanisms of action of the amide
constituents should be studied further.
D-GalN/LPS-treated mice, and this inhibitory ef-
a

7318
H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
O
1
O
O
12''
16''
13''
14''
9''
10''
11''
5
1'
7
4
O
O
8
9
15''
9''
O
12'' O
N
H
6
12
2
8''
5'''
O
6''
7''
11''
11
10''
8''
1'
10
N
1''
5'''
5''O
2'' 3''
5
1'
O
1
1'''
8
1
N
4
1''
N
3''
2''
5
7
O 1
6
1'''
8
3
2
5'
4
N
11
O
12
7
9
6
3
2
5'
9
10
1H—1H COSY :
HMBC :
11
O
O
12
2
10
O
3
Figure 2. 1H–1H COSY and HMBC correlations of 1–3.
O
eter (l = 5 cm); UV spectra, Shimadzu UV-1600 spectrometer; IR
spectra, Shimadzu FTIR-8100 spectrometer; 1H and 13C NMR spec-
tra, JEOL JNM-LA500 (500 and 125 MHz) and JEOL EX-270 (270 and
68 MHz) spectrometers with tetramethylsilane as an internal stan-
dard; EIMS and high-resolution EIMS, JEOL JMS-GCMATE mass
spectrometer; HPLC detector, Shimadzu RID-10A refractive index,
Shimadzu SPD-10A UV–vis, and Shodex OR-2 optical rotation
detectors. Cosmosil 5C18-MS-II (Nacalai Tesque Inc., 250 ꢂ 4.6
mm i.d.) and (250 ꢂ 20 mm i.d.) columns were used for analytical
and preparative HPLC, respectively.
The following experimental conditions were used for chroma-
tography: normal-phase silica gel column chromatography (CC),
silica gel BW-200 (Fuji Silysia Chemical, Ltd, 150–350 mesh); re-
versed-phase silica gel CC, Chromatorex ODS DM1020T (Fuji Silysia
Chemical, Ltd, 100–200 mesh); normal-phase TLC, pre-coated TLC
plates with silica gel 60F254 (Merck, 0.25 mm); reversed-phase
TLC, pre-coated TLC plates with silica gel RP-18 F254S (Merck,
0.25 mm); and reversed-phase HPTLC, pre-coated TLC plates with
N
H
3''
2''
H
O
5
H
N
4
H
3
7
2
O
11
O
2, 3
NOE :
Figure 3. NOESY experiments on 2 and 3.
3. Experimental
3.1. General
The following instruments were used to obtain spectral and
physical data: specific rotations, Horiba SEPA-300 digital polarim-
Table 5
Inhibitory effects of the chemical constituents from the fruit of P. chaba on D-GalN-induced cytotoxicity in primary cultured mouse hepatocytes
Inhibition (%)
IC50 (lM)
0
lM
3
l
M
10
lM
30
lM
100 lM
Piperonal (4)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
4
2
0
0
0
0
3
1
4
8
0
3
3
0
2
ꢁ3
ꢁ2
5
2
0
4
2
2
4
5
3
2
11 1b
19 3b
11 2b
Methyl piperate (5)
2
2
6
3
2
2
3
3
1
8
4
6
1
6
1
Da,b-Dihydropiperine (= piperanine, 9)
Piperine (10)
14 2b
14 3b
6
9
ꢁ6
ꢁ12
7
5
2
2
3
2
ꢁ2
ꢁ5
3
2
0
ꢁ9
ꢁ17
ꢁ21
5
ꢁ14
ꢁ18
1
2
3
2
3
2
2
1
Pipernonaline (15)
Piperundecalidine (18)
5,6-Dihydropiperlonguminine (21)
Piperlonguminine (22)
Retrofractamide C (23)
Retrofractamide A (26)
Pipercide (= retrofractamide B, 27)
Guineensine (28)
ꢁ4
ꢁ9
ꢁ4
7
1
3
15 4a
13 3a
1
7
10
0
2
3
4
1
3
6
5
1
2
1
5
3
3
0
5
ꢁ6
ꢁ19
12
10
5
7
6
ꢁ20 4c
ꢁ22 4c
Brachystamide B (29)
18
2
ꢁ17
4
N-Isobutyl-(2E,4E)-deca-2,4-dienamide (30)
N-Isobutyl-(2E,4E)-dodeca-2,4-dienamide (31)
N-Isobutyl-(2E,4E)-octadeca-2,4-dienamide (34)
N-Isobutyl-(2E,4E14Z)-eicosa-2,4,14-trienamide (37)
Silybind
6
1
17
2
41 14b
82 6b
75 9b
9.3
1
1
ꢁ1
2
3
2
32 6b
53 2b
66 4b
45 9b
89 10b
77 6b
6.4
39
5
1
8
1
0
0
l
M
1
l
M
3
l
M
10
l
M
30
lM
Piperleine B (14)
3
21
4
64 5b
73 8b
92 3b
2.9
Each value represents the mean SEM (N = 4).
Significantly different from the control.
a
p <0.05.
p <0.01.
b
c
Cytotoxic effect was observed.
Commercial silybin was purchased from Funakoshi Co, Ltd. (Tokyo, Japan).
d

H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
7319
M)
Table 6
Inhibitory effects of the chemical constituents from the fruit of P. chaba on D-GalN/TNF-
a
-induced cytotoxicity in primary cultured mouse hepatocytes
Inhibition (%)
IC50
4.9
ca. 4
ca. 11
(l
0
lM
1
lM
3
lM
10
l
M
30 lM
98 6b
Piperchabamide E (1)
Piperchabamide G (2)
Piperchabamide H (3)
Piperonal (4)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
5
4
2
4
2
3
10
4
2
2
3
6
6
3
1
3
3
2
3
7
3
5
4
34 2b
43 6b
24 3b
20 1b
71 2b
104 4b
42 3b
23 5b
29 2b
32 2b
50 2b
46 4b
33 1b
31 3b
74 3b
49 8b
52 1b
43 5b
54 6b
31 6b
77 3b
35 5b
51 3b
33 3b
11 2a
25 3b
31 6b
41 1b
19 12
118 4b
39 2b
Methyl piperate (5)
18
3
21
5
Piperchabamide A (6)
29 1b
31 5a
23 5b
29 3b
70 7b
53 2b
68 3b
64 7b
48 2b
78 5b
58 5b
68 5b
66 6b
66 3b
37 2b
37 4b
60 5b
26 2b,c
12 1b,c
14
17
12
17
> 30
<3
11
6.7
8.2
ca. 3
ca. 3
<3
Da,b-Dihydropiperine (= piperanine, 9)
Piperine (10)
Piperoleine B (14)
Pipernonaline (15)
Piperchabamide B (16)
Piperundecalidine (18)
Piperchabamide C (19)
5,6-Dihydropiperlonguminine (21)
Piperlonguminine (22)
Retrofractamide C (23)
Piperchabamide D (24)
20 3a
17
6
63 6b
40 3b
42 5b
43 9a
50 6b
51 2b
57 3b
32 5b
54 4b
26 2b
22 3b
19 3a
21 3b
37 2b
27 6a
34 12
27
4
30 13
Retrofractamide A (26)
Pipercide (= retrofractamide B, 27)
Guineensine (28)
21
2.6
32
12
5
2
Brachystamide B (29)
-6
2
N-Isobutyl-(2E,4E)-deca-2,4-dienamide (30)
N-Isobutyl-(2E,4E)-dodeca-2,4-dienamide (31)
N-Isobutyl-(2E,4E)-octadeca-2,4-dienamide (34)
N-Isobutyl-(2E,4E14Z)-eicosa-2,4,14-trienamide (37)
42 3b
44 4b
43 5b
15 10
10
5
3
2
27
6
Silybind
0
2
11
9
19
6
37 5b
93 4b
15
Each value represents the mean SEM (N = 4).
Significantly different from the control.
a
p <0.05.
p <0.01.
b
c
Cytotoxic effect was observed.
Commercial silybin was purchased from Funakoshi Co., Ltd (Tokyo, Japan).
d
silica gel RP-18 WF254S (Merck, 0.25 mm). Detection was achieved
by spraying with 1% Ce(SO4)2–10% aqueous H2SO4, followed by
heating.
3.2. Plant material
This item was described in a previous report.2
Table 7
Inhibitory effects of the aqueous acetone extract from the fruit of P. chaba and its chemical constituents on NO production in LPS-activated mouse peritoneal macrophages
Inhibition (%)
10 g/mL
10 3b
IC50
(
l
g/mL)
0
0
0
l
l
g/mL
2
3
3
l
g/mL
l
30
34 2b
30
l
g/mL
100
90 1b
100
l
g/mL
80% Aqueous acetone ext.
4
1
44
M
lM
10
lM
l
M
l
M
IC50
(
lM)
Piperchabamide E (1)
Piperchabamide G (2)
Piperchabamide H (3)
Piperonal (4)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
2
2
2
3
2
1
2
3
5
2
8
4
9
4
5
1
ꢁ2
ꢁ2
1
2
3
4
2
1
1
3
1
1
2
2
1
6
10
3
1
3
2
1
2
16 1b
75 1b
16 2b
46 2b
81 1b
21 1b
28
20
2
5
2
8
4
1
7
1
Methyl piperate (5)
14 2b
100 1b,c
20 1b
Piperchabamide A (6)
Da,b-Dihydropiperine (= piperanine, 9)
Piperine (10)
27 1b
98 1b
2
2
3
2
7
3
3
1
7
11
2
5
49 4b
Pipernonaline (15)
-3
5
47 1b
24 1b
28 7b
46 3b
23 1b
99 1b,c
99 1b,c
101 1b,c
97 1b,c
86 1b
Piperchabamide B (16)
Dehydropipernonaline (17)
Piperundecalidine (18)
Piperchabamide C (19)
5,6-Dihydropiperlonguminine (21)
Piperchabamide D (24)
Retrofractamide A (26)
11 3a
25 5b
23 4b
6
9
ꢁ13
ꢁ1
4
10
2
8
2
4
3
6
10 2a
42
ꢁ1
4
1
3
ꢁ4
5
25 3a
7
10 2a
22 1b
ꢁ12
20 3b
44 3b
ꢁ8
4
ꢁ14
7
L
-NMMA60,61
0
0
3
2
1
6
3
5
43 2b
86 1b
71 2b
36
11
CAPE60,61
100 1b,c
Each value represents the mean SEM (N = 4).
Significantly different from the control.
a
p <0.05.
p <0.01.
Cytotoxic effect was observed.
b
c

7320
H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
Table 8
Inhibitory effects of the aqueous acetone extract from the fruit of P. chaba and its chemical constituents on TNF-a-induced cytotoxicity in L929 cells
Inhibition (%)
10 g/mL
19 1b
IC50
(
l
l
g/mL)
M)
0
0
0
l
g/mL
1
3
8
3
l
l
g/mL
3
l
30
85 2b
30
ꢁ1
20 6a
l
g/mL
100
100
l
g/mL
M
80% Aqueous acetone ext.
14
lM
M
10
l
M
lM
l
IC50
(
Piperchabamide E (1)
Piperchabamide G (2)
Piperchabamide H (3)
Piperonal (4)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
3
2
4
1
4
1
2
2
3
4
1
4
1
4
4
2
1
3
5
2
3
0
8
3
1
9
5
2
3
8
2
ꢁ4
12
ꢁ2
ꢁ4
10
2
4
1
6
2
3
22 3b
18 6a
ꢁ4
1
8
5
10
3
7
0
Methyl piperate (5)
13 2b
15 3b
90 5b
Piperchabamide A (6)
23 5b
12
10
7
35 3b
71 1b
13
Da,b-Dihydropiperine (= piperanine, 9)
Piperine (10)
4
3
3
1
7
3
3
2
4
3
1
1
1
5
2
11
2
2
1
ꢁ1
2
17 5b
22 1b
82 2b
42
64
Piperoleine B (14)
Pipernonaline (15)
Piperchabamide B (16)
Piperundecalidine (18)
Piperchabamide C (19)
Piperlonguminine (22)
Piperchabamide D (24)
Pipercide (= retrofractamide B, 27)
Guineensine (28)
18 1b
25 1b
66 4b
8
4
5
10
12
ꢁ1
2
3
3
9
7
ꢁ5
1
ꢁ10
2
19 3a
24 1b
25 4b
25 1b
94 4b
29 3b
92 5b
23 2b
33
42
ꢁ2
16
8
25 2b
ꢁ3
3
2
1
1
ꢁ23
2
2
7
1
ꢁ20
3
3
2
1
1
9
4
4
0
5
5
1
ꢁ9
0
ꢁ18
ꢁ15
2
1
Brachystamide B (29)
ꢁ11
N-Isobutyl-(2E,4E)-deca-2,4-dienamide (30)
N-Isobutyl-(2E,4E)-dodeca-2,4-dienamide (31)
N-Isobutyl-(2E,4E)-octadeca-2,4-dienamide (34)
N-Isobutyl-(2E,4E14Z)-eicosa-2,4,14-trienamide (37)
10 1a
18 2b
19 3b
34 6b
ꢁ2
5
1
ꢁ3
4
3
ꢁ2
ꢁ4
ꢁ3
2
14 1b
25 2b
32 1b
47 4b
45 2b
52 4b
82 2b
25
20
Curcumin14–16
0
2
Each value represents the mean SEM (N = 4).
Significantly different from the control.
a
p<0.05.
p<0.01.
b
Table 9
Inhibitory effects of piperine (10) on D-GalN/LPS-induced liver injury in mice
Treatment
Dose (mg/kg, po)
n
sAST
sALT
(Karmen unit)
Inhibition (%)
(Karmen unit)
Inhibition (%)
Normal (vehicle)
—
—
5
8
95 5b
9126 1477
—
—
19 1b
9830 1605
—
—
Control (D-GalN/LPS)
Piperine (10)
2.5
5
10
4
7
8
9098 1195
4545 1259a
450 145b
0.3
50.7
96.1
7555 796
4817 1489a
575 196b
23.2
51.1
94.3
Hydrocortisone
10
7
627 262b
94.2
247 123b
97.7
Each value represents the mean SEM.
Significantly different from the control.
a
p <0.05.
p <0.01.
b
Table 10
3.3. Extraction and isolation
Inhibitory effects of piperine (10) on the increase in serum TNF-
GalN/LPS in mice
a
levels induced by D-
Inhibition (%)
The dried fruit of P. chaba (4.0 kg) was finely cut and extracted
four times with 80% (v/v) aqueous acetone at room temperature for
one day. Evaporation of the solvent under reduced pressure pro-
vided an 80% aqueous acetone extract (788 g, 19.7% from the dried
fruit). The 80% aqueous acetone extract (463 g) was partitioned be-
tween EtOAc–H2O (1:1, v/v) and removal of the solvent under re-
duced pressure yielded an EtOAc-soluble fraction (227 g, 9.7%)
and an aqueous phase. The EtOAc-soluble fraction (118 g) was sub-
jected to normal-phase silica gel CC [3.0 kg, n-hexane–EtOAc
(15:1?10:1?5:1?2:1?1:1, v/v)?MeOH] to give nine fractions
[Fr. 1 (7.3 g), 2 (4.0 g), 3 (3.1 g), 4 (22.0 g), 5 (3.8 g), 6 (1.8 g), 7
(11.1 g), 8 (44.2 g), and 9 (20.7 g)] as described previously.2
Dose
n
TNF-a
(mg/kg, po)
(pg/mL)
Normal (vehicle)
Control ( -GalN/LPS)
—
—
4
6
4
3a
—
—
D
233 46
Piperine (10)
5
10
20
4
4
4
197 52
212 27
238 71
15.7
9.2
ꢁ2.2
Hydrocortisone
10
4
22 2a
92.1
Each value represents the mean SEM.
Significantly different from the control.
a
p <0.01.

H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
7321
The fraction 2 (3.2 g) was separated by reversed-phase silica gel
CC [120 g, MeOH–H2O (70:30?90:10, v/v)?MeOH] to give piper-
onal (4, 45.9 mg, 0.0047%) together with methyl piperate (5,
1.0 g, 0.11%). The fraction 3 (3.1 g) was separated by reversed-
phase silica gel CC [630 g, MeOH–H2O (60:40?70:30?
80:20?90:10?95:5, v/v)?MeOH] and finally HPLC [CH3CN–H2O
(35:65, v/v)] to give 3,4-methylenedioxycinnamaldehyde (23 mg,
0.0019%). The fraction 4 (20.0 g) was separated by reversed-phase
silica gel CC [700 g, MeOH–H2O (60:40?70:30?80:20?90:10?
95:5, v/v)?MeOH] to give eight fractions [Fr. 4-1 (1.2 g), 4-2
(0.21 g), 4-3 (0.29 g), 4-4 (1.1 g), 4-5 (7.3 g), 4-6 (4.6 g), 4-7
(3.9 g), and 4-8 (1.4 g)]. The fraction 4-1 (1.2 g) was purified by
HPLC [CH3CN–H2O (35:65, v/v)] to give benzenepropanoic acid
(980 mg, 0.090%). The fraction 4-2 (0.21 g) was subjected to pre-
parative HPLC [CH3CN–H2O (50:50, v/v)] to give piperchabamide
A (6, 27 mg, 0.0029%) and N-isobutyl-(2E,4E)-deca-2,4-dienamide
(30, 27 mg, 0.0025%). The fraction 4-3 (0.29 g) was subjected to
preparative HPLC [CH3CN–H2O (65:35, v/v)] to give 30 (80 mg,
0.0074%), N-isobutyl-(2E,4E)-dodeca-2,4-dienamide (31, 37 mg,
0.0034%), and N-isobutyl-(2E,4E,8Z)-tetradeca-2,4,8-trienamide
(35, 18 mg, 0.0017%). The fraction 4-4 (1.1 g) was purified by HPLC
[CH3CN–H2O (83:17, v/v)] to give N-isobutyl-(2E,4E)-tetradeca-
2,4-dienamide (32, 49 mg, 0.0044%). The fraction 4-5 (0.91 g) was
purified by HPLC [MeOH–H2O (90:10, v/v)] to give N-isobutyl-
(2E,4E)-hexadeca-2,4-dienamide (33, 15 mg, 0.011%). The fraction
4-6 (0.80 g) was purified by HPLC [MeOH–H2O (90:10, v/v)] to give
N-isobutyl-(2E,4E)-octadeca-2,4-dienamide (34, 185 mg, 0.18%),
N-isobutyl-(2E,4E,12Z)-octadeca-2,4,12-trienamide (36, 12 mg,
0.0057%), and N-isobutyl-(2E,4E,14Z)-eicosa-2,4,14-trienamide
(37, 362 mg, 0.18%). The fraction 4-7 (0.91 g) was purified by HPLC
[MeOH–H2O (90:10, v/v)] to give 34 (127 mg, 0.048%). The fraction
5 (3.8 g) was subjected to reversed-phase silica gel CC [200 g,
MeOH–H2O (60:40?70:30?80:20?90:10, v/v)?MeOH] to fur-
nish four fractions [Fr. 5-1 (1.2 g), 5-2 (0.62 g), 5-3 (1.3 g), and 5-
[CH3CN–H2O (45:55, v/v)] to furnish piperchabamide E (1, 27 mg,
0.0046%), piperine (10, 43 mg, 0.0075%), isopiperine (11, 157 mg,
0.027%), isochavicine (12, 111 mg, 0.020%), chavicine (13, 24 mg,
0.0042%), piperlonguminine (22, 76 mg, 0.013%), and piperic acid
isopentyl amide (38, 7 mg, 0.0011%). The fraction 7-6 (0.58 g)
was purified by HPLC [CH3CN–H2O (67:33, v/v)] to give pipernon-
aline (15, 36 mg, 0.0063%), 17 (145 mg, 0.0025%), 23 (107 mg,
0.019%), and 26 (25 mg, 0.0043%). The fraction 7-7 (1.0 g) was fur-
ther purified by HPLC [CH3CN–H2O (67:33, v/v)] to give 15
(874 mg, 0.40%), 17 (64 mg, 0.029%), and 23 (18 mg, 0.0081%).
The fraction 7-8 (0.30 g) was subjected to HPLC [CH3CN–H2O
(67:33, v/v)] to furnish 14 (8 mg, 0.0016%), 15 (198 mg, 0.039%),
17 (7 mg, 0.0014%), and 27 (14 mg, 0.0027%). The fraction 7-10
(0.17 g) was separated by HPLC [CH3CN–H2O (70:30, v/v)] to give
piperchabamide H (3, 12 mg, 0.0020%), 16 (13 mg, 0.0023%), and
18 (79 mg, 0.014%). The fraction 8 (44.2 g) was recrystallized in
MeOH to give 10 (30.8 g, 2.64%). The mother liquor fraction
(13.4 g) was subjected to reverse-phase silica gel CC [450 g,
MeOH–H2O (60:40?90:10, v/v)?MeOH] to give nine fractions
[Fr. 8-1 (0.29 g), 8-2 (0.32 g), 8-3 (6.6 g), 8-4 (4.8 g), 8-5 (0.39 g),
8-6 (0.74 g), 8-7 (0.17 g), 8-8 (0.32 g), and 8-9 (0.17 g)]. The frac-
tion 8-2 (0.32 g) was separated by HPLC [CH3CN–H2O (50:50, v/
v)] to give fragaramide (20, 7 mg, 0.0006%). The fraction 8-3
(2.0 g) was separated by normal-phase silica gel CC [500 g, n-hex-
ane–EtOAc (4:1?3:1?2.5:1?1:1, v/v)?MeOH] and finally HPLC
[CH3CN–H2O (55:45, v/v)] to give ilepcimide (8, 16 mg, 0.0048%),
Da,b-dihydropiperine [= piperanine (9, 1268 mg, 0.36%)], and 22
(554 mg, 0.15%). The fraction 8-4 (0.88 g) was further purified by
HPLC [CH3CN–H2O (45:55, v/v)] to give 9 (126 mg, 0.057%), 10
(483 mg, 0.22%), 11 (24 mg, 0.011%), 12 (32 mg, 0.015%), and 22
(130 mg, 0.059%). The fraction 8-5 (0.32 g) was subjected to HPLC
[CH3CN–H2O (45:55, v/v)] to give 10 (124 mg, 0.013%), 11
(18 mg, 0.0018%), and 12 (39 mg, 0.0041%). The fraction 8-6
(0.74 g) was further purified by HPLC [CH3CN–H2O (60:40, v/v)]
4
(0.68 g)]. The fraction 5-1 (1.2 g) was subjected to HPLC
to give piperchabamide G (2, 76 mg, 0.0064%), 15 (266 mg,
[CH3CN–H2O (35:65, v/v)] to give benzenepropanoic acid
(489 mg, 0.040%) and 30 (94 mg, 0.0085%). The fraction 5-2
(0.62 g) was subjected to HPLC [CH3CN–H2O (70:30, v/v)] to give
piperchabamide D (24, 20 mg, 0.0016%), pipercide [= retrofracta-
mide B (27, 100 mg, 0.0083%), and guineensine (28, 292 mg,
0.024%). The fraction 5-3 (1.3 g) was further purified by HPLC
[CH3CN–H2O (75:25, v/v)] to give dihydroguineensine (25, 18 mg,
0.0015%), 28 (690 mg, 0.057%), and brachystamide B (29, 83 mg,
0.0069%). The fraction 6 (1.6 g) was subjected to reversed-phase
silica gel CC [200 g, MeOH–H2O (60:40?70:30?80:20?
90:10?95:5, v/v)?MeOH] to give five fractions [Fr. 6-1 (0.14 g),
6-2 (0.07 g), 6-3 (0.80 g), 6-4 (0.39 g), and 6-5 (0.20 g)]. The frac-
tion 6-1 (0.14 g) was further purified by HPLC [CH3CN–H2O
(45:55, v/v)] to give N-cinnamoylpiperidine (7, 17 mg, 0.0016%).
The fraction 6-3 (0.80 g) was subjected to HPLC [CH3CN–H2O
(67:33, v/v)] to give piperoleine B (14, 47 mg, 0.0042%), dehydro-
0.022%), and 17 (165 mg, 0.014%). The fraction 8-7 (0.17 g) was
separated by HPLC [CH3CN–H2O (70:30, v/v)] to give 3 (51 mg,
0.0042%) and 15 (27 mg, 0.0023%). The fraction 8-8 (0.32 g) was
further purified by HPLC [CH3CN–H2O (60:40, v/v)] to give 3
(29 mg, 0.0023%).
3.3.1. Piperchabamide E (1)
Colorless oil, ½a D24
ꢀ
+18.7 (c 0.72, CHCl3). High-resolution EIMS:
Calcd for C17H21NO3 (M)+: 287.1521. Found: 287.1523. UV [EtOH,
nm (log e)]: 241 (4.2), 252 (4.1), 308 (4.2), 338 (4.3). IR (film):
2963, 1646, 1545, 1506, 1489, 1446, 1257, 1238, 1039, 991 cmꢁ1
.
1H NMR (500 MHz, CDCl3) d: given in Table 3. 13C NMR
(125 MHz, CDCl3) dC: given in Table 4. EIMS m/z: 287 (M+, 48),
201 (100).
3.3.2. Piperchabamide G (2)
pipernonaline (17, 97 mg, 0.0088%), retrofractamide
C (23,
An amorphous powder, ½a D28
ꢀ
0 (c 0.63, CHCl3). High-resolution
103 mg, 0.0093%), 24 (12 mg, 0.0011%), retrofractamide A (26,
36 mg, 0.0032%), and 27 (322 mg, 0.029%). The fraction 6-4
(0.39 g) was further purified by HPLC [CH3CN–H2O (67:33, v/v)]
EIMS: Calcd for C34H40N2O6 (M)+: 572.2886. Found: 572.2884. UV
[EtOH, nm (log e)]: 235 (3.9), 288 (3.8). IR (film): 2936, 1624,
1485, 1441, 1244, 1040, 928 cmꢁ1. 1H NMR (500 MHz, CDCl3) d: gi-
ven in Table 3. 13C NMR (125 MHz, CDCl3) dC: given in Table 4.
EIMS m/z: 572 (M+, 48), 112 (100).
to give 14 (3 mg, 0.0002%), piperchabamide
B (16, 19 mg,
0.0018%), piperundecalidine (18, 30 mg, 0.0028%), piperchabamide
C (19, 34 mg, 0.0032%), 24 (11 mg, 0.0010%), 27 (94 mg, 0.0089%),
and 28 (58 mg, 0.0055%). The fraction 7 (5.9 g) was subjected to re-
versed-phase silica gel CC [270 g, MeOH–H2O (60:40?70:30?
80:20?90:10, v/v)?MeOH] to yield 11 fractions [Fr. 7-1 (0.17 g),
7-2 (0.28 g), 7-3 (0.27 g), 7-4 (1.0 g), 7-5 (0.09 g), 7-6 (0.58 g), 7-
3.3.3. Piperchabamide H (3)
An amorphous powder, ½a D27
ꢀ
0 (c 0.38, CHCl3). High-resolution
EIMS: Calcd for C38H46N2O6 (M)+: 626.3355. Found: 626.3352. UV
[EtOH, nm (log e)]: 362 (4.2), 288 (4.0). IR (film): 2932, 1628,
7
(2.7 g), 7-8 (0.30 g), 7-9 (0.09 g), 7-10 (0.17 g), and 7-11
1487, 1443, 1248, 1040, 930 cmꢁ1. 1H NMR (500 MHz, CDCl3) d: gi-
ven in Table 3. 13C NMR (125 MHz, CDCl3) dC: given in Table 4.
EIMS m/z: 626 (M+, 48), 112 (100).
(0.25 g)]. The fraction 7-2 (0.28 g) was further purified by HPLC
[CH3CN–H2O (45:55, v/v)] to give 5,6-dihydropiperlonguminine
(21, 84 mg, 0.015%). The fraction 7-4 (1.0 g) was subjected to HPLC

7322
H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
3.4. Acid hydrolysis of 1
the fresh medium (100 lL) containing D-GalN (2 mM) and TNF-a
(40 ng/mL) with or without the test sample was added, and the
A solution of 1 (10 mg) in 6 M HCl (1.0 mL) was heated at 110 °C
overnight in a sealed tube. After cooling, the reaction mixture was
basified (pH 10) with aq NaOH and then the mixture was extracted
with n-hexane. The n-hexane phase was evaporated in vacuo to
give a residue, which was subjected to HPLC analysis [column: Cos-
mosil 5C18-MS-II, 250 ꢂ 4.6 mm i.d.; mobile phase: MeOH–H2O
(50:50, v/v); detection: RI and OR; flow rate: 1.0 mL/min] to iden-
tify (R)-2-methylbutylamine [tR 9.1 min (positive)].
hepatocytes were cultured for 20 h. The medium was then ex-
changed with 100 lL of fresh medium, and 10 lL of MTT [5 mg/
mL in phosphate buffered saline (PBS)] solution was added to the
medium. After 4 h of culture, the medium was removed, and
100 lL of isopropanol containing 0.04 M HCl was then added to
dissolve the formazan produced in the cells. The optical density
(OD) of the formazan solution was measured with a microplate
reader at 570 nm (reference: 655 nm). Inhibition (%) was obtained
using following formula.
3.5. Bioassay
Inhibition ð%Þ ¼ ½ðODðsampleÞ ꢁ ODðcontrolÞÞ=ðODðnormalÞ
ꢁ ODðcontrolÞÞꢀ ꢂ 100
3.5.1. Reagents
LPS (from Salmonella enteritidis), minimum essential medium
(MEM), and William’s E medium were purchased from Sigma
Chemical (St. Louis, MO, USA); fetal calf serum (FCS) was from Life
Technologies (Rockville, MD, USA); and other chemicals were from
Wako Pure Chemical Industries (Osaka, Japan). 96-Well micro-
plates were purchased from Nalge Nunc International (Naperville,
IL, USA).
3.5.5. Effects on production of NO in LPS-stimulated mouse
peritoneal macrophages
The screening test for NO production using TGC-induced mouse
peritoneal macrophages was performed as described previ-
ously.60,61 Briefly, peritoneal exudate cells (5 ꢂ 105 cells/well) were
collected from the peritoneal cavities of male ddY mice, suspended
in 100
l
L of RPMI 1640 supplemented with 5% FCS, penicillin (100
3.5.2. Animals
units/mL), and streptomycin (100
lg/mL), and pre-cultured in 96-
Male ddY mice weighing about 25–30 g were purchased from
Kiwa Laboratory Animal Co., Ltd, (Wakayama, Japan). The animals
were housed at a constant temperature of 23 2 °C and were fed
standard laboratory chow (MF, Oriental Yeast Co., Ltd, Tokyo,
Japan). The animals were fasted for 20–24 h prior to the beginning
of the experiment, but were allowed free access to tap water. All of
the experiments were performed with conscious mice unless
otherwise noted. The experimental protocol was approved by the
Experimental Animal Research Committee of Kyoto Pharmaceuti-
cal University.
well microplates at 37 °C in 5% CO2 in air for 1 h. Nonadherent cells
were removed by washing with PBS, and the adherent cells were
cultured in 200 lL of fresh medium containing 10 lg/mL LPS and
various concentrations of test compound for 20 h. NO production
in each well was assessed by measuring the accumulation of nitrite
(NO2ꢁ) in the culture medium using Griess reagent. Cytotoxicity
was determined with the MTT colorimetric assay, after 20 h incu-
bation with test compounds. Each test compound was dissolved
in DMSO, and the solution was added to the medium (final DMSO
concentration 0.5%). NG-Monomethyl-
inhibitor) and caffeic acid phenethyl ester (CAPE, an inhibitor of
NF- B activation) were used as reference compounds. Inhibition
L-arginine (L-NMMA, an NOS
3.5.3. Protective effects on
mice
D-GalN/LPS-induced liver injury in
j
(%) was calculated using the following formula, and the IC50 value
was determined graphically (N = 4).
The method described by Tiegs et al.65 was modified and used
for this experiment. Briefly, male ddY mice weighing about 25–
Inhibition ð%Þ ¼ ½ðA ꢁ BÞ=ðA ꢁ CÞꢀ ꢂ 100
30 g were fasted for 20 h before the experiment.
kg) and LPS (10 g/kg) dissolved in saline were injected intraperi-
toneally to produce liver injury. Each test sample was given orally
1 h before -GalN/LPS was injected. Blood samples were collected
from the infraorbital venous plexus 10 h after -GalN/LPS injection.
D-GalN (350 mg/
ꢁ
l
A ꢁ C : NO2 concentration ð
lg=mL or
lMÞ
½A : LPS ðþÞ; sample ðꢁÞ; B : LPS ðþÞ; sample ðþÞ;
C : LPS ðꢁÞ; sample ðꢁÞꢀ
D
D
sAST and sALT levels were determined using the Reitman-Frankel
method (commercial kit, S.TA-Test Wako, Wako Pure Chemical
Industries Co. Ltd). Hydrocortisone was used as a reference com-
pound. Test samples were suspended with 5% Arabic gum solution,
and the solution was administered orally at 10 mL/kg in each
experiment, while the vehicle was given orally at 10 mL/kg in the
corresponding control group.
3.5.6. Inhibitory effects on TNF-
cells
L929cells(DainipponPharmaceuticals, Osaka, Japan)weremain-
tained in Eagle’s MEM (Sigma–Aldrich) containing 10% FCS, 1% MEM
Non-Essential Amino Acids (Invitrogen), penicillin G (100 units/mL),
a-induced cell death in L929
and streptomycin (100 lg/mL) at 37 °C under a 5% CO2 atmosphere.
The cells were inoculated in a 96-well tissue culture plate [3 ꢂ 104
cells/well in 100 lL/well in MEM]. After 20 h of incubation in the
3.5.4. Protective effects on cytotoxicity induced by
D-GalN or D-
medium containing actinomycin D (0.5 lg/mL) and TNF-a (20 pg/
GalN/TNF- in primary cultured mouse hepatocytes
a
mL) with or without the test sample, the viability of the cells was as-
sessed using the MTT colorimetric assay (vide ante). Each test com-
pound was dissolved in DMSO, and the solution was added to the
medium (final DMSO concentration 0.5%).
The hepatoprotective effects of the constituents were deter-
mined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) colorimetric assay of primary cultured mouse
hepatocytes.23–31 Hepatocytes were isolated from male ddY mice
(30–35 g) by collagenase perfusion method. A cell suspension of
4 ꢂ 104 cells in 100
lL William’s E medium containing FCS (10%),
3.5.7. Effects on increase in serum TNF-a levels induced by D-
penicillin G (100 units/mL), and streptomycin (100
l
g/mL) was
GalN/LPS in mice
inoculated in a 96-well microplate and precultured for 4 h at
37 °C under a 5% CO2 atmosphere. The fresh medium (100 L) con-
taining -GalN (2 mM) with or without the test sample was added,
and the hepatocytes were cultured for 44 h for the -GalN-induced
cytotoxicity test. For the -GalN/TNF- -induced cytotoxicity test,
Male ddY mice weighing about 25–30 g were fasted for 20 h be-
fore the experiment. -GalN (350 mg/kg) and LPS (10 g/kg) dis-
solved in saline were injected intraperitoneally to produce liver
injury. Each test sample was given orally 1 h before -GalN/LPS
was injected. Blood samples were collected from the infraorbital ve-
l
D
l
D
D
D
D
a

H. Matsuda et al. / Bioorg. Med. Chem. 17 (2009) 7313–7323
7323
24. Yoshikawa, M.; Morikawa, T.; Kashima, Y.; Ninomiya, K.; Matsuda, H. J. Nat.
Prod. 2003, 66, 922.
25. Xu, F.; Morikawa, T.; Matsuda, H.; Ninomiya, K.; Yoshikawa, M. J. Nat. Prod.
2004, 67, 569.
nous plexus 1.5 h after D-GalN/LPS injection. The serum TNF-a level
was determined using an ELISA kit (GE Healthcare Sciences).66
26. Matsuda, H.; Morikawa, T.; Xu, F.; Ninomiya, K.; Yoshikawa, M. Planta Med.
2004, 70, 1201.
3.6. Statistics
27. Li, N.; Morikawa, T.; Matsuda, H.; Ninomiya, K.; Li, X.; Yoshikawa, M.
Heterocycles 2007, 71, 1193.
28. Ninomiya, K.; Morikawa, T.; Zhang, Y.; Nakamura, S.; Matsuda, H.; Muraoka, O.;
Yoshikawa, M. Chem. Pharm. Bull. 2007, 55, 1185.
29. Zhang, Y.; Morikawa, T.; Nakamura, S.; Ninomiya, K.; Matsuda, H.; Muraoka, O.;
Yoshikawa, M. Heterocycles 2007, 71, 1565.
Values are expressed as means SEM. One-way analysis of var-
iance (ANOVA) followed by Dunnett’s test was used for statistical
analysis. Probability (p) values of less than 0.05 were considered
to represent a statistically significant difference.
30. Ninomiya, K.; Morikawa, T.; Xie, H.; Matsuda, H.; Yoshikawa, M. Heterocycles
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M.Y. and H.M. were supported by the 21st COE Program, Aca-
demic Frontier Project, and a Grant-in Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Tech-
nology (MEXT) of Japan. T.M. and K.N. were supported by a Grant-
in Aid for Scientific Research from the ‘High-tech Research Center’
Project for Private Universities: matching fund subsidy from a
Grant-in Aid for Scientific Research from MEXT, 2007–2011. T.M.
was also supported by Takeda Science Foundation.
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injection of
D
-GalN/LPS injection, and serum TNF-
a
levels were determined
using a commercial kit. The serum TNF-
a level was maximal 90 min after the
injection, as reported previously.22 Therefore, serum TNF-
the injection were compared.
a levels 90 min after