Loniceroside C, an antiinflammatory saponin from Lonicera japonica

Article abstract of DOI:10.1248/cpb.51.333

A new triterpenoid saponin, loniceroside C was isolated from the aerial parts of Lonicera japonica. Its structure was established to be 3-O-β-D-glucopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl (1→2)-[β-D-xylopyranosyl(1→6)]-β-D-glucopyranosyl ester by spectroscopic techniques and chemical transformations. Loniceroside C showed in vivo antiinflammatory activity against mouse ear edema provoked by croton oil.

Full text of DOI:10.1248/cpb.51.333

March 2003
Notes
Chem. Pharm. Bull. 51(3) 333—335 (2003)
333
Loniceroside C, an Antiinflammatory Saponin from Lonicera japonica
Wie Jong KWAK,a Chang Kyun HAN,a Hyeun Wook CHANG,b Hyun Pyo KIM,c Sam Sik KANG,d and
,e
*
Kun Ho SON
a SK Chemicals Ltd.; Suwon 440–745, Korea: b College of Pharmacy, Yeungnam University; Kyongsan 712–749, Korea:
d
c College of Pharmacy, Kangwon National University; Chunchen 200–701, Korea: Natural Products Research Institute,
e
Seoul National University; Seoul 110–460, Korea: and Department of Food and Nutrition, Andong National University;
Andong 760–749, Korea. Received October 7, 2002; accepted November 29, 2002
A new triterpenoid saponin, loniceroside C was isolated from the aerial parts of Lonicera japonica. Its struc-
ture was established to be 3-O-b-D-glucopyranosyl hederagenin 28-O-a-L-rhamnopyranosyl (1®2)-[b-D-xylopy-
ranosyl(1®6)]-b-D-glucopyranosyl ester by spectroscopic techniques and chemical transformations. Loniceroside
C showed in vivo antiinflammatory activity against mouse ear edema provoked by croton oil.
Key words Lonicera japonica; Caprifoliaceae; triterpenoid saponin; loniceroside C; antiinflammatory activity
a)
Table 1. 13C-NMR Spectral Data for Compound 1 in Pyridine-d5
Lonicera japonica (THUNB.) (Caprifoliaceae) is a twining
shrub that has been used as an antidote and to treat urinary
Carbon no.
1
Carbon no.
1
Carbon no.
1
disorders, fever, and headache.1) It has been known as an an-
tiinflammatory agent in Korea from ancient times and is used
widely for treating upper respiratory tract infections, diabetes
mellitus, and rheumatoid arthritis.2) In a previous paper, we
elucidated the structures of flavonoid derivatives3) as well as
two new hederagenin glycosides, lonicerosides A and B.4)
Furthermore, we reported that loniceroside A showed in vivo
antiinflammatory activity in a croton-oil induced ear edema
model and antiarthritic activity.5,6) In our continuing research
on L. japonica, we isolated a new triterpenoid saponin,
loniceroside C (1). This paper describes the structure eluci-
dation and antiinflammatory activity of this compound.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
38.8
25.8
82.5
43.4
47.8
18.3
32.4
40.0
48.2
37.0
23.9
20
21
22
23
24
25
26
27
28
29
30
30.7 28-O-Sugar moieties
34.0
33.0
64.9
13.6
16.3
17.6
25.9
176.6
33.1
23.9
Glc 1
94.8
75.5
79.6
71.2
78.0
69.0
101.6
72.2
72.6
73.9
69.8
18.8
105.7
74.8
77.6
71.1
67.0
2
3
4
5
6
Rha 1
(®2Glc) 2
3
4
5
6
122.8 3-O-Sugar moiety
144.2
42.3
28.7
23.4
47.2
42.0
46.4
Glc 1
105.9
75.9
78.7
71.6
78.3
62.8
Repeated silica gel and RP-18 chromatography of the
BuOH fraction afforded a saponin (1). Compound 1 was pos-
itive in the Liebermann–Burchard and Molisch tests for
saponins and its molecular formula was found to be
C53H86O22 by high-resolution FAB-MS [(MϩNa)ϩ at m/z
1097.5508, calcd., 1097.5509]. Acid hydrolysis of 1 yielded
xylose, rhamnose, glucose, and an aglycone, hederagenin,
2
3
4
5
6
Xyl 1
(®6Glc) 2
3
4
5
a) Assignments based upon 1H–1H COSY, 13C–1H COSY, and HMBC experiments.
identified by direct comparison with an authentic sample.
1
The H-NMR spectra of compound 1 indicated the presence of terminal rhamnose (d 6.49) and C-2 of glucose (d 75.5),
of four anomeric proton signals at d 4.84 (1H, d, Jϭ7.4 Hz), and H-1 of terminal xylose (d 4.84) and C-6 of glucose (d
5.06 (1H, d, Jϭ7.8 Hz), 6.09 (1H, d, Jϭ8.1 Hz), and 6.49 69.0) clearly demonstrated the presence of the 28-O-
(1H, s), which were in agreement with the 13C-NMR signals rhamnopyranosyl (1®2)-[xylopyranosyl (1®6)]glucopyra-
for anomeric carbons at d 105.7, 105.9, 94.8, and 101.6 by nosyl moiety in compound 1 (Fig. 1). Furthermore, the 13C-
1H–1H shift correlation spectroscopy (1H–1H COSY) and NMR signals of the three sugars were identical to those of
13C–1H shift correlation spectroscopy (13C–1H COSY) (Table the C-28 acyl moiety in lonicerosides A and B. Accordingly,
1). In the 13C-NMR spectra, two signals at d 94.8 and 82.5 the structure of compound 1 was determined to be 3-O-b-D-
due to C-1 of inner glucose attached to C-28 of the hedera- glucopyranosyl hederagenin 28-O-a-L-rhamnopyranosyl
genin and C-3 of the hederagenin moiety, respectively, (1®2)-[b-D-xylopyranosyl (1®6)]-b-D-glucopyranosyl ester.
strongly indicated that compound 1 is a bisdesmosidic glyco-
The new compound, loniceroside C (1), possesses in vivo
side with an ester function at C-28. Alkaline hydrolysis of antiinflammatory activity against mouse ear edema provoked
compound 1 gave a prosapogenin (compound 2) which was by croton oil (Table 2). It inhibited ear edema (15.0—31.0%
identified as hederagenin 3-O-b-D-glucopyranoside by com- inhibition at 50—200 mg/kg). Reference compound, pred-
parison of its NMR data with those previously reported for nisolone, showed potent inhibition (57.9% inhibition at 10
hederoside B.7) The results showed that the acyl moiety at C- mg/kg), while aspirin weakly inhibited ear edema (15.0% in-
28 is comprised of three sugars, i.e., glucose, rhamnose, and hibition at 100 mg/kg). It is significant to note that 1 at the
xylose. The sugar sequence of the acyl moiety was deter- dose of 100 mg/kg showed antiinflammatory activity with
mined by two-dimensional (2D) NMR analysis. In the 1H-de- potency similar to that of loniceroside A, previously reported
tected heteronuclear multiple-bond correlation (HMBC) to be one of active components.5) Therefore, 1 also con-
spectrum, the long-range correlation between the H-1 of tributes to the in vivo antiinflammatory activity of L. japon-
inner glucose (d 6.09) and C-28 of aglycone (d 176.6), H-1 ica.
* To whom correspondence should be addressed. e-mail: sonkh@andong.ac.kr
© 2003 Pharmaceutical Society of Japan

334
Vol. 51, No. 3
residue (1.08 kg) that was suspended in H2O and partitioned successively
with hexane (41 g), CHCl3 (5 g), EtOAc (16 g), and then with BuOH (45 g).
The BuOH-soluble fraction (45 g) was subjected to column chromatography
(CC) over silica gel with CHCl3–MeOH–H2O (8 : 2 : 0.5, lower layer, and
then 52 : 28 : 8, lower layer) to give nine subfractions. The eighth sub-frac-
tion was further subjected to repeated CC over silica gel using EtOAc satu-
rated with H2O–MeOH (0—7%, gradient) and RP-18 with MeOH–H2O (6 :
4) to obtain 1 (350 mg).
Loniceroside C (1) was obtained as amorphous powder from MeOH. mp
227—230 °C. [a]D27 Ϫ26.7° (cϭ0.12, pyridine). IR (KBr) cmϪ1: 3410 (OH),
2922 (C–H), 1741 (CϭO), 1078 (glycosidic C–O). Positive-ion FAB-MS
m/z: 1097.5508 [MϩNa]ϩ (Calcd for C53H86O22Na: 1097.5509) 1H-NMR
(pyridine-d5) d: 0.79 (3H, s, H3-29), 0.86 (3H, s, H3-30), 0.90 (3H, s, H3-24),
0.93 (3H, s, H3-25), 1.06 (3H, s, H3-26), 1.15 (3H, s, H3-27), 1.71 (3H, d,
Jϭ6.2 Hz, H3-6 of rhamnose), 4.84 (1H, d, Jϭ7.4 Hz, H-1 of xylose), 5.06
(1H, d, Jϭ7.8 Hz, H-1 of glucose attached to C-3 of aglycone), 5.37 (1H,
br s, H-12), 6.09 (1H, d, Jϭ8.1 Hz, H-1 of glucose attached to C-28 of agly-
cone), 6.49 (1H, s, H-1 of rhamnose). 13C-NMR: Table 1.
Acid Hydrolysis of Compound 1 Compound 1 (20 mg) was refluxed
with 4% H2SO4 (10 ml) in MeOH for 1 h. The reaction mixture was then
concentrated under reduced pressure to remove MeOH, diluted with H2O,
and filtered. The precipitate was purified by recrystallization from MeOH to
afford an aglycone, hederagenin (6 mg), as needles, which was identified by
direct comparison with an authentic sample. The filtrate was adjusted to pH
7 with BaCO3 and filtered. The filtrate was concentrated and examined by
cellulose TLC (pyridine–EtOAc–HOAc–H2Oϭ36 : 36 : 7 : 21). Xylose (Rf
0.42), rhamnose (Rf 0.49), and glucose (Rf 0.28) were identified by compari-
son with authentic samples.
Fig. 1. Significant Long-Range Correlation of Loniceroside C (1) in the
HMBC Spectrum
Table 2. Antiinflammatory Activity of Lonicerosides A and C (1) against
Alkaline Hydrolysis of Compound 1 Compound 1 (100 mg) in 3%
KOH (in MeOH) was refluxed for 30 min. The reaction mixture was neutral-
ized with 0.05 M H2SO4 and then extracted with BuOH. The BuOH layer was
subjected to CC over silica gel to give the prosapogenin 2 (30 mg). Com-
pound 2 was recrystallized from MeOH as an amorphous powder, mp 245—
247 °C; [a]D27 ϩ44.5° (cϭ0.1, pyridine). 13C-NMR (pyridine-d5) d 82.1 (C-
3), 64.3 (C-23), 180.1 (C-28), 105.9 (C-1Ј), 76.1 (C-2Ј), 78.8 (C-3Ј), 71.9
(C-4Ј), 78.3 (C-5Ј), 62.8 (C-6Ј). Compound 2 was identified as hederagenin
3-O-b-D-glucopyranoside by comparison of its NMR data with those previ-
ously reported for hederoside B.7)
Mouse Ear Edema Induced by Croton Oil
Dose
(mg/kg, p.o.)
Thickness increase
(mm)
Group
Croton oil
% Inhibition
—
100
10
100
50
0.126Ϯ0.011a)
0.107Ϯ0.019
0.053Ϯ0.012*
0.088Ϯ0.018*
0.107Ϯ0.019
0.087Ϯ0.010*
0.092Ϯ0.016
—
Aspirin
15.0
57.9
30.2
15.0
31.0
28.7
Prednisolone
Loniceroside A
Loniceroside C (1)
Determination of the Absolute Configuration of Sugars of Compound
100
200
1
A sample of 1 (10 mg) was hydrolyzed with 1 M HCl (H2O–dioxane,
1 : 1; 5 ml) at 80 °C for 3 h. The reaction mixture was neutralized with a
small column of Amberlite IRA67 (OHϪ form), and the filtrate was concen-
trated to dryness in vacuo. The residue was dissolved in pyridine (0.1 ml),
and then the solution was added to a pyridine solution (0.1 ml) of L-cysteine
methyl ester hydrochloride (2 mg) and warmed at 60 °C for 1 h. The solvent
was evaporated under a N2 stream and dried in vacuo. Then trimethylsilylim-
idazole (0.1 ml) was added, and the mixture was heated at 60 °C for 1 h.
After the addition of hexane and water (1 ml each), the hexane layer was an-
alyzed by GC. The retention times of the peaks were 18.38 (D-xylose), 23.73
(L-rhamnose), and 37.87 min (D-glucose), respectively.
In Vivo Antiinflammatory Activity To measure antiinflammatory
(antiedema) activity in vivo, the croton oil-induced ear edema assay was car-
ried out according to the modified procedure of Kim et al.8) based on the
original procedure of Tonneli et al.9) In brief, the test compounds suspended
in 0.5% carboxymethylcellulose were administered orally. One hour later,
2.5% croton oil (Sigma Chemical) dissolved in acetone (25 ml/ear) was ap-
plied topically to the inner and outer surfaces of mouse ears. After 5 h, ear
thickness was measured using a spring-loaded dial thickness guage (Lux
Scientific Instruments, U.S.A.). The increase in thickness compared with
that before croton oil application was regarded as edematous inflammation.
The statistical significance of the difference in ear thickness was evaluated
using one-way analysis of variance (ANOVA).
a) Arithmetic meanϮS.D. (nϭ6), *: pϽ0.05, significantly different from the croton
oil-treated group.
Experimental
General Procedures Melting points were measured on a Mitamura-
Riken apparatus and are uncorrected. The optical rotations were determined
on a JASCO P-1020 polarimeter. The NMR spectra were measured on a
Bruker AMX-500 instrument (500 MHz for 1H-NMR and 125 MHz for 13C-
NMR), and the chemical shifts are referenced to tetramethylsilane (TMS).
FAB-MS was obtained in a 3-nitrobenzyl alcohol matrix in positive-ion
mode with a VG-VSEQ spectrometer. Gas chromatography (GC) analysis
was performed with a Hewlett Packard 5890 Series II gas chromatograph
equipped with a H2 flame ionization detector. The conditions were HP-5
capillary column (30 mϫ0.32 mmϫ0.25 mm), column temperature, 200 °C;
injector and detector temperature, 290 °C; and He flow rate, 1 ml/min. Col-
umn chromatography was carried out on Kieselgel 60 (Merck; 40—63 mm)
and LiChroprep RP-18 (Merck; 40—63 mm). TLC was performed on pre-
coated silica gel 60 F254 sheets (Merck), and detection was achieved by
spraying with 10% H2SO4 followed by heating. Sugars were run on pre-
coated cellulose plates (Merck) and detected using aniline phthalate.
Plant Material The aerial parts of L. japonica were collected in Kyung-
bug province, Korea, in the summer of 1998 and were authenticated by Dr.
Hyung Joon Chi of the Natural Products Research Institute, Seoul National
University. A voucher specimen is deposited in the Department of Food and
Nutrition, Andong National University.
Acknowledgments This investigation was financially supported by a re-
search grant from the Ministry of Health and Social Welfare, Republic of
Korea (HMP-01-PJ2-PG4-J201PT01-0009), which is gratefully acknowl-
edged.
Animals Male ICR mice (specific pathogen free, 18—22 g) were pur-
chased from Charles River, Japan. Animals had access to Purina lab. chow
and water ad libitum and were maintained in our animal facility for at least
7 d prior to experiments at 21Ϯ1 °C, 40—60% relative humidity, and a 12
h/12 h (light/dark) cycle.
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March 2003
335
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