Iridoid glycosides from Gardeniae Fructus for treatment of ankle sprain

Article abstract of DOI:10.1016/j.phytochem.2009.03.008

The iridoid glycosides, genipin 1-O-β-d-isomaltoside (1) and genipin 1,10-di-O-β-d-glucopyranoside (2), together with six known iridoid glycosides, genipin 1-O-β-d-gentiobioside (3), geniposide (4), scandoside methyl ester (5), deacetylasperulosidic acid

Full text of DOI:10.1016/j.phytochem.2009.03.008

Phytochemistry 70 (2009) 779–784
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Iridoid glycosides from Gardeniae Fructus for treatment of ankle sprain
a,b
a
a
a
a
c
Quan Cheng Chen , Wei Yun Zhang , UiJoung Youn , HongJin Kim , IkSoo Lee , Hyun-Ju Jung ,
d
e
a,
*
MinKyun Na , Byung-Sun Min , KiHwan Bae
College of Pharmacy, Chungnam National University, Daejeon 305–764, Republic of Korea
Institute for Biomedical Research, Xiamen University, Xiamen 361005, China
Department of Oriental Pharmacy, Wonkwang University, Jeonbuk 570-749, Korea
College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk 712–749, Republic of Korea
College of Pharmacy, Catholic University of Daegu, Gyeongbuk 712–702, Republic of Korea
a
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 December 2007
Received in revised form 15 January 2009
The iridoid glycosides, genipin 1-O-b-
together with six known iridoid glycosides, genipin 1-O-b-
D
-isomaltoside (1) and genipin 1,10-di-O-b-
D-glucopyranoside (2),
D-gentiobioside (3), geniposide (4), scandoside
methyl ester (5), deacetylasperulosidic acid methyl ester (6), 6-O-methyldeacetylasperulosidic acid
methyl ester (7), and gardenoside (8) were isolated from an EtOH extract of Gardeniae Fructus. The struc-
tures and relative stereochemistries of the metabolites were elucidated on the basis of 1D- and 2D-NMR
spectroscopic techniques, high-resolution mass spectrometry, and chemical evidence. Geniposide (4),
one of the main compounds of Gardeniae Fructus, was tested for treatment of ankle sprain using an ankle
sprain model in rats. From the second to fifth day, the geniposide (4) (100 mg/ml) treated group exhibited
significant differences (p < 0.01) with ꢀ21–34% reduction in swelling ratio compared with those of the
vehicle treated control group. This indicated the potential effect of geniposide (4) for the treatment of dis-
orders such as ankle sprain.
Keywords:
Gardenia jasminoides
Rubiaceae
Gardeniae Fructus
Ankle sprain treatment
Iridoid glycosides
Genipin 1-O-b-
D-isomaltoside
Genipin 1,10-di-O-b-
D
-glucopyranoside
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
Ankle sprain is a partial or complete tear of ligaments that sup-
hundreds of years in Korea, Japan, and China. However, their
clinical efficacy and pharmacological effect need to be further
studied.
Gardeniae Fructus, the dried ripe fruit of Gardenia jasminoides
port the ankle (Bosien et al., 1955), with symptoms like pain, swell-
ing, and bruising around the ankle in the acute stage. They may be
caused by sudden twisting of the ankle, such as stepping on an un-
even surface or in a hole, by taking an awkward step when running,
jumping, or stepping up or down, or by inversion of the foot, which
causes ankle to ‘‘roll over” when playing sports or exercising. Ankle
sprain is one of the most common injuries in athletes, particularly
in sports such as basketball, soccer, football, and volleyball
Ellis (Rubiaceae), is widely used in traditional medicine for its cho-
lagogue, sedative, diuretic, antiphlogistic, and antipyretic effects
(Guo et al., 1997a). Additionally, it is an externally used drug with
a long heritage and tradition in the treatment of sprain (Yao et al.,
1991). Many iridoid glycosides were isolated from Gardeniae Fruc-
tus in previous investigations (Junko et al., 1991). Some of them,
e.g. geniposide (4) (see Fig. 1), possess diverse biological activities
such as anti-inflammatory, antithrombotic, neuritogenic, and cho-
lagogue activities (Koo et al., 2006; Masaki et al., 1980; Suzuki
et al., 2001; Yamazaki et al., 1996). However, there are few chem-
ical and pharmacological studies related to Gardeniae Fructus for
treating sprain (Yao et al., 1991). The specific aim of the present
study was to investigate both the chemical constituents of Garde-
niae Fructus and their potential for treatment of ankle sprain in a
rat model.
(Kofotolis et al., 2007; Liu and Nguyen, 1999). Non-steroidal anti-
inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen,
naproxen, and piroxicam, have been used immediately post-injury
and considered to be the best drug treatments due to their analge-
sic and anti-inflammatory effects (Buckwalter, 1995; Elder et al.,
2
001). However, taking such NSAIDs causes adverse effects, and
is somewhat controversial regarding long-term healing (Stanley
and Weaver, 1998; Stovitz and Johnson, 2003). For the treatment
of soft tissue injuries, traditional drugs, such as Gardeniae Fructus
(
Guo et al., 1997a), Notoginseng Panax (Guo et al., 1997b), and
2
. Results and discussion
Carthami Flos (Guo et al., 1997c) etc., have also been used for
Two new iridoid glycosides 1 and 2 were isolated from the
n-BuOH-soluble fraction of the EtOH extract of Gardeniae Fructus,
*
Corresponding author. Tel.: +82 42 821 5925; fax: +82 42 823 6566.
E-mail addresses: baekh@cnu.ac.kr, khbae@hotmail.com (K. Bae).
together with six known iridoid glycosides (Fig. 1). The known
0
031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.03.008

7
80
Q.C. Chen et al. / Phytochemistry 70 (2009) 779–784
0
0
00
1
COOCH3
COOCH3
or between H-1 and H-5 (Fig. 2). The H NMR spectroscopic data
of an octaacetyl derivative (1a) (Table 1), peracetylated from 1,
provided better resolution and this enabled facile measurement
of coupling constants for the proton signals of the sugar units
R4
H
H
H
R3
3
5
9
O
O
1
HO
H
OR1
OR
0
which were hitherto partly overlapping, i.e., d 3.24 (H-2 ), d 3.26
H-4 ), d 3.32 (H-4 ), d 3.36 (H-2 ), d 3.59 (H-3 ), and d 3.61
(H-5 ). Carbon signals at d 73.81, 75.3, 72.0, 73.85, and 62.9 were
assigned to C-2 , C-3 , C-4 , C-5 , and C-6 and this was further
confirmed by analysis of the HMQC spectrum. The HMBC long-
range correlations from H-6 [d 3.76 (1H, dd, J = 10.8, 1.8 Hz) and
d 3.86 (1H, dd, J = 10.8, 6.0 Hz)] to C-1 (d 100.2), and H-1 to
C-6 (d 68.2) suggested the connection of an
a b-glucose at the 6 -OH position (Fig. 2). Therefore, the sugar
moiety was a disaccharide, and identified as b-isomaltosyl. The
aglycone ‘genipin’ was identified following hydrolysis of 1 and
comparison of its spectroscopic data with that previously reported
(Drewes et al., 1996). The anomeric carbon (d 100.7, C-1 ) of the
b-isomaltosyl group showed an HMBC correlation with d 5.17
R
2
O
OH
0
0
0
00
00
(
1
1
2
2
3
4
5
6
7
R
= Z, R2 = R3 = R4 = H;
8 R = X
00
1
a R = Z(Ac) , R =Ac, R3 = R4 = H;
1
7
2
00
00
00
00
00
R
= R = X, R = R4 = H;
2 3
1
a R = R = R = R = H;
1
2
3
4
0
R
= Y, R = R = R = H;
1
2
3
4
R1 = X, R2 = R3 = R4 = H;
00
00
R
R
= X, R = R = H, R3 = OH;
1
1
2 4
0
a-glucose moiety with
= X, R = R = H, R4 = OH;
OH
2
3
0
R1 = X, R2 = R3 = H, R4 = OCH3
OH
6"
O
HO
HO
6"
1"
O
OH
HO
HO
O
OH
O
6'
6'
6
'
1"
OH
0
O
O
O
HO
HO
HO
HO
HO
HO
OH 1'
OH 1'
OH 1'
(
1H, d, J = 7.8 Hz, H-1) as well as that of d 98.8 (C-1) with the
anomeric proton d 4.81 (1H, d, J = 7.8 Hz, H-1 ). This established
β
0
the connectivity of the glycosylation at the 1-OH position of
Fig. 1. Chemical structures of isolated compounds from Gardeniae Fructus.
genipin. Thus, compound 1 was genipin 1-O-b-D-isomaltoside.
Compound 2 was purified as a white amorphous powder. The
HR–FAB–MS spectrum of 2 showed a quasi-molecular ion at m/z
+
compounds were identified as genipin 1-O-b-
D
-gentiobioside (3),
573.1812 [M+Na] , corresponding to a molecular formula of
geniposide (4) (Endo and Taguchi, 1973), scandoside methyl ester
23 34
C H O15. The structure of the compound was established from
1
13
(
(
5) (Guvenalp et al., 2006), deacetylasperulosidic acid methyl ester
6) (Damtoft et al., 1981; Kim et al., 2005), 6-O-methyldeacetylas-
analyzing its H and C NMR spectroscopic data (Table 1) which
were compared to those of 3, as well as the 2D-NMR spectra
including HMQC, HMBC, COSY, and ROESY. Compound 2 showed
perulosidic acid methyl ester (7) (Machida et al., 2003), and gar-
denoside (8) (Farid et al., 2002) by comparing their physical and
spectroscopic data with those of published literature.
1
13
similar UV, IR, and H and C NMR spectra to 3. Two glucopyran-
1
13
osyl signals were shown in the H and C NMR spectra of 2. A sig-
1
Compound 1 was obtained as a white amorphous powder. The
nificant difference was observed in the H NMR spectrum, namely
HR–FAB–MS spectrum of 1 showed a quasi-molecular ion at m/z
that the signals of H-10 [d 4.60 (1H, d, J = 12.6 Hz) and d 4.27 (1H, d,
J = 12.6 Hz)] of 2 were more downfield than those of 3 [d 4.32 (1H,
d, J = 14.7 Hz) and d 4.19 (1H, d, J = 14.7 Hz)]. This suggested that
one glucose moiety was connected to the 10-OH group of the agly-
cone. The HMBC experiment further supported this assignment as
evidenced by the long-range correlations from H-10 to that of the
+
5
73.1757 [M+Na] , with a molecular formula of C23
34
H O
15. Its UV
spectrum exhibited an absorption maximum similar to that of
geniposide (4) with a maximum at 239 nm, suggesting the pres-
ence of
a,b-unsaturated ester carbonyl groups. The IR spectrum
ꢁ1
indicated the presence of hydroxyl (3360 cm
)
and ester
1700 cm ) groups. The 1H and C NMR spectroscopic data of 1
ꢁ1
13
anomeric carbon [d 104.7 (C-1 )] of one glucose, and the anomeric
00
(
0
0
were almost identical to those of genipin 1-O-b-
D
-gentiobioside
proton H-1 [d 4.34 (1H, d, J = 7.8 Hz)] to d 69.1 (C-10). In addition,
the proton signal at d 5.26 (1H, d, J = 7.8 Hz, H-1) showed a long-
range correlation with the anomeric carbon of the other glucose
(3) (Table 1). Sugar unit analyses using the GC method for the
hydrolysis product of 1 established the presence of
D
1
-glucose (Hara
13
0
et al., 1987). However, careful examination of the H and C NMR
spectra of both compounds showed considerable differences in the
signals of one of the sugar units. In the HMQC spectrum, a correla-
tion between the proton resonance at d 4.81 (1H, d, J = 3.6 Hz, H-
at d 100.3 (C-1 ); this indicated linkage between the glucose and
the 1-OH group of the aglycone, with the same pattern of 4
(Fig. 2). The configurations of the glycosidic linkage for two gluco-
pyranosyl units were determined to be b on the basis of examina-
tion of the J1,2 values for the anomeric protons at 7.5 Hz (H-1’) and
0
0
00
00
1
) and the carbon signal at d 100.2 (C-1 ) suggested that H-1
0
0
00
was an anomeric proton of a sugar moiety. However, H-1 dis-
played a more downfield shift and a smaller coupling constant
7.8 Hz (H-1 ). The aglycone was also identified as genipin by anal-
ysis of the spectroscopic data of its hydrolysis product as well as
from a ROESY experiment (Fig. 2). Therefore, compound 2 was
determined to be genipin 1,10-di-O-b-D-glucopyranoside.
0
0
than that of 3 at d 4.37 (1H, d, J = 7.5 Hz, H-1 ). In the COSY spec-
trum, the proton resonance at d 3.36 (1H, dd, J = 9.6, 3.6 Hz)
0
0
showed a cross-peak with H-1 , indicating that the proton signal
Gardeniae Fructus, as a traditional medicine, is often externally
used for the treatment of soft tissue injuries. Geniposide (4) is one
of the main components for Gardeniae Fructus. Because it is a nat-
urally occurring, biodegradable molecule with low cytotoxicity, it
has recently been used in many biological investigations (Huang
et al., 1998). In this study, the EtOH extract of Gardeniae Fructus
and geniposide (4) isolated from the n-BuOH-soluble fraction of
the EtOH extract were investigated for treatment of ankle sprain
using a rat model. This model was first established in the research
of acupuncture analgesia for ankle sprain (Koo et al., 2002), and lat-
eral ankle sprain is also a common source of persistent pain in hu-
mans. To model this condition, the rat’s right hind ankle was bent
repeatedly, overextending lateral ligaments, for 4 min under ethyl
ether anesthesia. The rat subsequently showed swelling of the
ankle for the next several days. To estimate the degree of edema
0
0
00
of H-2 appeared at d 3.36. Similarly, H-3 at d 3.59 (1H, dd,
0
0
00
J = 9.6, 9.0 Hz), H-4 at d 3.26 (1H, t, J = 9.0 Hz), H-5 at d 3.61
1H, m), H-6 a at d 3.81 (1H, dd, J = 11.4, 1.8 Hz), and H-6 b at d
.63 (1H, dd, J = 11.4, 5.4 Hz) could be assigned by analysis of the
0
0
00
(
3
COSY spectrum (Fig. 2). The coupling constant of J
Hꢁ100;Hꢁ200
3.6 Hz
1
in the H NMR spectrum of 1 suggested the presence of an axial-
0
0
00
equatorial form between H-1 and H-2 , while the large coupling
0
0
constant of J
Hꢁ200;Hꢁ300
9.6 Hz indicated that the H-2 was axial
according to the vicinal Karplus correlation (Karplus, 1959). There-
0
0
fore, H-1 was a proton in the equatorial position. The above data
thus suggested that the sugar unit was an -glucosyl moiety, and
this was supported further by the H– H ROESY correlation peak
a-D
1
1
0
0
00
between the anomeric proton H-1 and that of H-2 . There was,
0
0
00
however, no correlation observed between either H-1 and H-3 ,

Q.C. Chen et al. / Phytochemistry 70 (2009) 779–784
781
Table 1
NMR spectroscopic data of compounds 1–3.
No.
1
1a
2
3
dC,
mult.
dH (J in Hz)a
5.13, d (7.8)
HMBC
dH (J in Hz)b
dC,
mult.
dH (J in Hz)a
5.26, d (7.8)
HMBC
dC,
mult.
dH (J in Hz)c
5.15, d (7.8)
0
0
1
3
4
5
6
98.8, d
153.4, d 7.51, d (0.9)
112.6, s
3, 5, 8, 9, 1
1, 4, 5, 11
5.16, d (7.5)
7.41, s
97.8, d
153.4, d 7.50, d (0.9)
112.8, s
3, 5, 8, 9, 1
1, 4, 5, 11
98.9, d
153.5, d 7.51, d (1.2)
112.5, s
36.7, d
40.0, t
3.18, br dd (8.4, 7.8)
2.81, br dd (8.4, 8.4)
1, 3, 4, 6, 9
7, 8
3.20, q (7.0)
2.87, m
2.27, m
36.1, d
39.8, t
3.18, br dd (8.4, 7.5)
2.83, br dd (8.4, 8.4)
2.12, ddt (8.4, 8.4, 2.1) 4, 7, 8
1, 3, 4, 6, 7, 8, 9 36.8, d
4, 7, 8 39.8, t
3.17, br dd (8.4, 7.8)
2.83, br dd (8.4, 8.4)
2.17, ddt (8.4, 8.4, 2.1)
2.14, ddt (8.4, 8.4, 2.1) 7, 8
7
8
9
129.2, d 5.85, br s
144.7, s
5, 6, 9, 10
5.86, br s
130.5, d 5.87, br s
141.6, s
5, 6, 9, 10
129.1, d 5.85, br s
144.9, s
47.4, d
61.5, t
2.70, br t (7.8)
4.32, br d (14.4)
.17, br d (14.4)
1, 5, 6, 7, 8
7, 8
7, 8
2.82, t (7.5)
4.75, br d (14.0)
4.69, br d (14.0)
47.1, d
69.1, t
2.95, br t (7.5)
4.60, br d (12.6)
4.27, br d (12.6)
1, 5, 6, 7, 8
47.1, d
61.6, t
2.71, br t (7.8)
4.32, br d (14.7)
4.19, br d (14.7)
0
0
1
0
7, 8, 9, 1
00
1
4
1
1
2
3
4
5
6
1
169.7, s
100.7, d 4.71, d (7.8)
169.7, s
169.7, s
100.7, d 4.71, d (7.8)
0
0
0
0
0
0
1, 2 , 3
4.87, d (8.0)
4.95, dd (9.0, 8.0)
5.24, t (9.0)
5.04, t (9.0)
3.75, m
3.76, m
4.05, m
5.13, d (3.0)
4.80, dd (10.0, 3.0)
5.43, dd (10.0, 9.5)
5.02, t (9.5)
100.3, d 4.71, d (7.5)
75.0, d 3.24, m
78.13, d 3.37, m
71.8, d
78.6, d
62.92, t 3.86, br d (12.0)
3.65, dd (12.0, 5.4)
104.7, d 4.34, d (7.8)
1, 2 , 3 , 5
0
0
0
0
0
0
0
0
0
0
0
0
0
74.9, d
78.0, d
71.9, d
76.9, d
68.2, t
3.24, dd (9.0, 7.8)
3.39, t (9.0)
3.32, m
1 , 3
2 , 4
3 , 6
1 , 3
2 , 4 , 5
3 , 5 , 6
3 , 4 , 6
4 , 5
4 , 5
10, 3 , 5
1 , 3
2 , 4 , 5
3 , 5 , 6
3 , 4 , 6
4 , 5
74.9, d
78.1, d
71.7, d
77.9, d
69.9, t
3.16, m
3.40, m
3.37, m
3.51, m
3.87, br d (12.0)
3.65, dd (12.0, 5.4)
0
0
0
0
0
0
0
0
0
0
0
3.28, m
3.29, m
0
0
3.52, ddd (9.0, 6.0, 1.8) 1 , 3 , 4 , 6
0
0
00
0
0
0
0
0
3.76, dd (10.8, 1.8)
.86, dd (10.8, 6.0)
4 , 1
4 , 5 , 1
6 , 2 , 3 , 5
00
3
0
0
0
0
0
0
0
0
00
00
00
00
00
1
2
3
4
5
6
100.2, d 4.81, d (3.6)
73.81, d 3.36, dd (9.6, 3.6)
75.3, d
72.0, d
73.85, d 3.61, m
104.9, d 4.37, d (7.5)
0
0
0
0
0
00
00
00
00
00
00
3
75.4, d
78.0, d
71.7, d
3.20, m
3.37, m
3.29, m
75.3, d
78.0, d
71.8, d
77.8, d
62.7, t
3.23, m
3.39, m
3.31, m
3.27, m
3.87, br d (11.4)
3.65, dd (11.4, 5.4)
3.71, s
00
00
00
00
00
3.59, dd (9.6, 9.0)
3.26, t (9.0)
4 , 5
3 , 5 , 6
00
00
00
00
00
00
00
4
3.75, m
78.10, d 3.28, m
62.90, t 3.87, br d (12.0)
3.69, dd (12.0, 5.4)
00
00
00
00
00
62.9, t
3.81, dd (11.4, 1.8)
.63, dd (11.4, 5.4)
3.71, s
4 ,
4.06, br d (12.0)
4.22, dd (12.0, 4.5)
3.72, s
0
0
00
3
4 , 5
11
4 , 5
–
OCH
3
51.8, q
51.8, q
3.71, s
11
51.8, q
Ac
1.99, 2.01, 2.02, 2.03,
2
.07, 2.09, 2.10, 2.11
a
3
Spectrum recorded at 600 MHz in CD OD.
b
c
5
3
00 MHz in CDCl
3
.
00 MHz in CD OD.
3
COOCH3
COOCH3
through fifth days with ꢀ20–38% reduction in the swelling ratio.
The EtOH ex. (100 mg/ml) treated group also showed significant
differences (p < 0.05) on the fourth and fifth days with ꢀ13 and
18% reduction, respectively. The geniposide (4) (10 mg/ml) treated
group showed significant differences (p < 0.01) on the third
through fifth days with ꢀ20–23% reduction, whereas at higher dos-
age (100 mg/ml), the treated group through the second and fifth
days had ꢀ21–34% reduction (Fig. 4). These findings suggest that
external use of Gardeniae Fructus was helpful in the treatment of
soft tissue injuries, and that geniposide, one of the main com-
pounds of Gardeniae Fructus, might be applied as topical drug
for the treatment of ankle sprain.
H
H
H
H
H
H
5
9
5
9
3
3
OH
1
O
H
1
O
H
7
7
O
H
H
HO
HO
H
O
O 10
O
OH
O
OH 1"
H
H
1"
HO
O
O
O
OH
1'
OH
HO
1'
H
H
OH
6
'
H
H
HOHO
HOHO
HOHO
HMBC
H
H
C
ROESY
H
1
2
COSY HC
CH
Fig. 2. Selected HMBC, ROESY, and correlations of 1 and 2.
2.1. Concluding remarks
Two new genipin glycosides (1 and 2) were isolated from Gar-
produced by ankle sprain, changes in the volume of the foot were
measured. As shown in Fig. 3, the edema increased to peak swell-
ing at around 12 h and then gradually recovered afterward. Addi-
tionally, the rats were greatly inactive during high swelling and
then gradually recovered to normal activity as swelling lessened.
Inflammation is a necessary component of the healing process.
Current evidence suggests that reducing the inflammatory re-
sponse too early following injury may actually delay acute healing,
slow muscle regeneration and compromise long-term healing
deniae Fructus. In the structure of 1, the sugar moiety was identi-
fied as b-isomaltose. To our knowledge, the structure combining
with such a disaccharide is rare in plant secondary metabolites
to date. The EtOH extract of Gardeniae Fructus and geniposide
(4), one of the main components for Gardeniae Fructus, exhibited
a potential effect on treating ankle sprain in rat. Although Garde-
niae Fructus has been externally used for the treatment of soft tis-
sue injuries in traditional medicine for a long time, only few
related chemical and pharmacological studies have been reported
(Yao et al., 1991). Our findings provided a better understanding
for the traditional use, as well as will help for further insight into
the pharmacological action and mechanism. Furthermore, whether
the EtOH extract of Gardeniae Fructus and geniposide (4) affect lig-
ament cells proliferation and collagen synthesis is under investiga-
tion in our research group.
(Almekinders, 1999; Hertel, 1997; Stovitz and Johnson, 2003).
Thus, the ankle swelling in rats was treated with 200 l of a drug
l
every 24 h from the twelfth hour after induction of ankle sprain.
Compared with those of the vehicle treated control group, a posi-
tive control (commercial diclofenac 12.5 mg/ml in gel) showed
good effect with significant differences (p < 0.01) on the first

7
82
Q.C. Chen et al. / Phytochemistry 70 (2009) 779–784
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
3.2. Plant material
Gardeniae Fructus was purchased from the pharmacy store in
Daejeon, Korea in July 2006, and identified by Prof. KiHwan Bae.
A voucher specimen (CNU 1516-3) has been deposited at the
Herbarium in the College of Pharmacy, Chungnam National
University.
0
0.5
1.5
Time (day)
2.5
3.5
4.5
5.5
3.3. Extraction and isolation
The dried Gardeniae Fructus slice (3.0 kg) was extracted with
Fig. 3. Changes in foot swelling volume after ankle sprain. Swelling volumes were
expressed as the differences of foot volume at various times before and after the
induction of ankle sprain. Data were presented as mean ± SE.
hot EtOH (5 l ꢂ three times) for two days. The EtOH extracts were
filtered, combined, and concentrated in vacuo to give a rufous
2
gummy residue (980 g). To this was then added H O (2 l) to give
a suspension that was partitioned consecutively with n-hexane,
EtOAc, and n-BuOH (each 2 l), then exhaustively concentrated to
yield an n-hexane-soluble fraction (95.0 g), an EtOAc-soluble
fraction (36.5 g), and an n-BuOH-soluble fraction (760.0 g),
respectively.
3
. Experimental
3.1. General experimental procedures
An aliquot of the n-BuOH-soluble fraction (700 g) was dissolved
FT–IR and UV spectra were obtained using a Jasco Report-100
in a minimal amount of MeOH–H
plied to a Diaion HP 20 column (100 ꢂ 20 cm, I.D.) eluting with a
step gradient of H O–MeOH (1:0, 4:1, 1:1, 1:4, and 0:1, v/v) to give
fractions GB1ꢀ5. GB1 was next subjected to an ODS CC
O (1:2, v/v) as eluent to give five
2
O (1:1, v/v). This was then ap-
infrared spectrometer and a Beckman Du-650 spectrophotometer,
respectively, whereas optical rotations were measured on a JASCO
DIP-370 polarimeter. FT–NMR spectra were recorded on a Bruker
2
1
13
DRX-300 spectrometer ( H NMR, 300 MHz; C NMR, 75 MHz)
using CD OD as solvent and tetramethylsilane (TMS) as an internal
(
50 ꢂ 2.5 cm, I.D.) using MeOH–H
2
3
sub-fractions. The second sub-fraction was then applied to a semi-
preparative RP HPLC [Gilson trilution system; Optimapak OP C18
standard. Chemical shifts (d) were expressed in ppm with reference
to TMS signals. Two-dimensional (2D) NMR (HMQC, HMBC, COSY,
and ROESY) experiments were acquired on a Bruker Avance 500
spectrometer. HR–FAB–MS were measured with a JEOL JMS-700
MStation mass spectrometer. Semipreparative HPLC were con-
ducted using a Gilson instrument with TRILUTION LC control soft-
ware, Gilson 321 pumps, UV/VIS 151 detector and GX-271 liquid
(
2
(
250 ꢂ 10 mm, I.D.) column; MeOH–H
40 nm] to afford (5.9 mg), (7.0 mg),
132.0 mg), 6 (60.0 mg), and 7 (40.0 mg). GB4 was applied to sil-
2
O (2:8, v/v); UV detection,
1
2
3
(164.5 mg),
5
ica-gel column (100 ꢂ 10 cm, I.D.) with EtOAc–MeOH (20:1, v/v)
eluent to give 4 (5.0 g) and 8 (160.0 mg).
handler, equipped with a YMC-Pack Pro C18 column (10 lm,
2
50 ꢂ 20 mm, I.D.). Column chromatography (CC) was performed
3.4. Genipin 1-O-b-D-isomaltoside (1)
using silica-gel (Kieselgel 60, 70–230 mesh and 230–400 mesh,
Merck), whereas thin layer chromatography (TLC) used Merck
pre-coated Silica-gel 60 F254 and/or RP-18 F254s plates (0.25 mm);
compounds were observed under either UV 254 and 365 nm or
2
D
2
MeOH
White amorphous powder, ½
a +50.0 (c 0.3, MeOH); UV kmax
ꢃ
KBr
ꢁ1
nm (log
1080; for H NMR (600 MHz, CD
CD OD) spectroscopic assignments, see Table 1; HR–FAB–MS m/z
e): 240 (3.18); IR mmax cm : 3400, 2900, 1698, 1625,
1
13
3
OD) and C NMR (75 MHz,
were visualized by spraying the dried plates with 10% H
2
SO
4
fol-
3
+
lowed by heating at 180 °C.
34
573.1757 [M+Na] (calcd. for C23H O15Na, 573.1795).
1
00
Control
EtOH ex. 100 mg/ml
Geniposide 10 mg/ml
Geniposide 100 mg/ml
Diclofenac 12.5 mg/ml
80
60
40
20
0
**
**
**
*
*
*
*
**
*
*
**
**
5
**
**
**
**
1
2
3
4
Time (day)
Fig. 4. Effect of geniposide and EtOH extract of Gardeniae Fructus on ankle swelling in rats with ankle sprain. A commercial diclofenac in gel was used as positive control.
Swelling ratio was expressed as the ratio of swelling volume at various times before and after drug treatment. Data were presented as mean ± SE. *, p < 0.05; **, p < 0.01
compared with vehicle treated control group, one-way ANOVA, Dunnett’s post hoc test.

Q.C. Chen et al. / Phytochemistry 70 (2009) 779–784
783
3
.5. Acetylation of genipin 1-O-b-
D
-isomaltoside
in air. The right hind foot was repeatedly bent for 4 min in the
direction of simultaneous inversion and plantar flexion so that it
eventually reached a 180° inversion. Anesthesia was discontinued
and the rat recovered from anesthesia within 5–10 min.
A solution of 1 (2.0 mg) in Ac
:1, v/v) was stirred for 2 h at room temperature. Evaporation of
2
O and anhydrous pyridine (1 ml,
1
the reagents under reduced pressure afforded the octaacetyl deriv-
1
ative 1a. White amorphous powder; for H NMR (500 MHz, CDCl
assignments, see Table 1.
3
)
3.9.3. Drug treatment and measurement of foot volume change
Test samples were administered cutaneous (topical administra-
tion to the skin). Swollen ankles in rats were treated with EtOH–
3.6. Enzymatic and acidic hydrolysis of 1
2
H O (7:3, v/v) (control group), 100 mg/ml of EtOH extract of Garde-
niae Fructus (EtOH ex. 100 mg/ml group), geniposide (4) (genipo-
side 100 mg/ml group), geniposide (4) (geniposide 10 mg/ml
group), and VoltarenÒ Schmerzgel (diclofenac 12.5 mg/ml, positive
Naringinase (100.0 mg, 300 U/g) (Sigma–Aldrich, St. Louis, MO,
USA) was added to a suspension of 1 (2.0 mg) in 50 mM acetate
buffer (pH 5.5) and the mixture was stirred at 37 °C for 3 h. The
reaction mixture was then extracted with EtOAc (3 ꢂ 20 ml) and
evaporated to dryness. The resulting residue was then dissolved
in MeOH and subjected to reversed phase HPLC [Gilson trilution
system; YMC-pack Pro C18 (250 ꢂ 20 mm, I.D.) column; MeOH–
control group), 200
ll per day from the twelfth hour after induc-
tion of ankle sprain.
Changes in the volume of the foot were measured before and
after the sprain procedure, using the Archimedes’s principle as de-
scribed in the literature (Koo et al., 2002). Briefly, each rat was
marked at about 5 mm proximal to the ankle in the distal part of
the leg. Then, a small beaker, about half full of water, was placed
on a digital scale that has the capability to weigh at a resolution
of 0.01 g. The foot was immersed into the water up to the pre-
marked line. The change in the weight of the water was then re-
corded. The weight increase in each gram is equated to 1 ml of foot
volume.
H
2
O (3:7, v/v); UV detection, 240 nm] to give genipin (2a), identi-
fied by comparing physical and spectroscopic data with those of
published literature data (Drewes et al., 1996). The H O layer
2
was passed through a Sep-Pak C18 cartridge (Waters, Milford,
MA) and concentrated to dryness. The dried residue was dissolved
in 2 M HCl and dioxane (50 ml each) and heated to 100 °C for 1 h.
After dilution with H
Amberlite IRA-60E column. The eluate was concentrated to dry-
ness and stirred with -cysteine methyl ester hydrochloride, hex-
2
O, the solution was passed through an
D
3.10. Statistical analysis
amethyldisilazane, and trimethylsilyl chloride in pyridine using
the same procedures as in previous reports (Hara et al., 1987). After
the reactions, the supernatant was analyzed by GC analysis. GC
conditions: column, Supelco SPB-1 (30 m ꢂ 0.32 mm); detector,
FID; detector temp, 300 °C; injector temp, 270 °C; carrier gas, He;
The parameters included in the statistical evaluation were de-
fined as follow:
Swelling volume ðS
v
i
Þ ¼ V
i
ꢁ V
Þ ꢂ 100
column temperature, 190 °C; t
R
22.4 min (
D
-glucose), 20.6 min (
L
-
Swelling ratio ð%Þ ¼ ðS
v
i
=S
v
0
glucose). -Glucose was detected from 1.
D
i
where V was the foot volume at various times after induction of an-
kle sprain (i from 0 to 5 according to days of administration), and V
was the foot volume before induction of ankle sprain. Foot volumes
were tested before administration and measured as ml. Data were
expressed as the mean ± standard error of mean (SE) for the number
3
.7. Genipin 1,10-di-O-b-D-glucopyranoside (2)
2
D
2
MeOH
max
White amorphous powder, ½
a
ꢃ
+3.0 (c 0.3, MeOH); UV k
KBr
ꢁ1
nm (log
e
): 239 (3.19); IR
m
cm : 3360, 2920, 1700, 1640,
max
(n = 3–6) in each group. The statistical analysis accomplished using
1
13
1
(
440, 1300, 1080; for H NMR (600 MHz, CD
75 MHz, CD OD) spectroscopic data, see Table 1; HR–FAB–MS
m/z 573.1812 [M+Na] (calcd. for C23
3
OD) and C NMR
the SPSS 13.0 statistical software (SPSS ins., Chicago, IL, USA). Anal-
ysis of variance was performed using ANOVA procedures followed
by the Dunnett’s post hoc test. A p value <0.05 was considered to
be statistically significant.
3
+
34
H O15Na, 573.1795).
3.8. Enzymatic hydrolysis of 2
Acknowledgements
Enzymatic hydrolysis of 2 used the same method as for 1 above
to give genipin (2a), which was identified by comparing physical
and spectroscopic data with those of published literature data
This research was supported in part by the BK21 Program of the
Korean Government. W.Y. Zhang wishes to acknowledge the finan-
cial support of the Korea Research Foundation Grant KRF-2007-
2
(Drewes et al., 1996). The H O layer was passed through a Sep-
Pak C18 cartridge (Waters, Milford, MA). The eluate was concen-
trated and subjected to the same procedure described above.
2
11-E00002. M. Na was supported in part by the Korea Research
Foundation Grant funded by the Korean Government (MOEHRRD,
Basic Research Promotion Fund, KRF-2008-331-E00451). We are
grateful to Korea Basic Science Institute (KBSI) for supplying the
NMR spectra.
D
-Glucose was detected from 2.
3
3
.9. Biological assay
.9.1. Experimental animals
Male Wistar rats, eight-week-old, weight 200–250 g, were ob-
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