Three new iridoid glycosides from the fruit of gardenia jasminoides var. radicans

Article abstract of DOI:10.1248/cpb.c13-00262

Three new iridoid glycosides, 6″-O-trans-feruloylgenipin gentiobioside (1), 2'-O-trans-p-coumaroylgardoside (2), 2'-O-trans- feruloylgardoside (3), were isolated from the fruit of Gardenia jasminoides var. radicans MAKINO (Rubiaceae). The structures of th

Full text of DOI:10.1248/cpb.c13-00262

October 2013
Note
Chem. Pharm. Bull. 61(10) 1071–1074 (2013)
1071
Three New Iridoid Glycosides from the Fruit of Gardenia jasminoides
var. radicans
Fang-min Qin, Ling-jie Meng, Hui-liang Zou, and Guang-xiong Zhou*
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and
New Drugs Research, Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan
University; Guangzhou 510632, China.
Received April 4, 2013; accepted July 22, 2013
Three new iridoid glycosides, 6ꢀ-O-trans-feruloylgenipin gentiobioside (1), 2ꢁ-O-trans-p-coumaroylgardo-
side (2), 2ꢁ-O-trans-feruloylgardoside (3), were isolated from the fruit of Gardenia jasminoides var. radicans
MAKINO (Rubiaceae). The structures of these compounds were elucidated on the basis of MS, NMR spectra
analysis, glycoside hydrolysis, and sugar derivatization coupled with HPLC analysis.
Key words Rubiaceae; Gardenia jasminoides var. radicans; iridoid glycoside; 6″-O-trans-feruloylgenipin
gentiobioside; 2′-O-trans-p-coumaroylgardoside; 2′-O-trans-feruloylgardoside
Gardenia jasminoides var. radicans (THUNB.) MAKINO 147.2 (C-3‴), and 6.41 (d, J=15.9Hz, H-2‴)/115.2 (C-2‴). Aro-
(
Rubiaceae), is extensively distributed in Guangxi, Sichuan, matic proton signals at δ 7.08 (dd, J=8.1, 1.3Hz, H-9‴), 6.81
H
Jiangxi, Hubei provinces in China. Its fruit was used as a (d, J=8.1Hz, H-8‴), and 7.22 (d, J=1.3Hz, H-5‴) and proton
common folk medicine, or dye, and food additives for it con- signal at δ 3.90 (brs, 6‴-OCH ), together with carbon signals
H
3
1
,2)
tains high content of yellow pigments. As a variant of the at δC 111.7 (C-5‴), 116.5 (C-8‴), 124.4 (C-9‴), 127.6 (C-4‴),
Gardenia jasminoides ELLIS, it was reported that there were no 149.3 (C-7‴), and 150.5 (C-6‴), suggested the presence of a
3)
significant genetic diversity between it and G. jasminoides.
1,3,4-trisubstituted benzene moiety. Furthermore, the hetero-
Moreover, it has the higher content of the main medicinal nuclear multiple bond connectivity (HMBC) (Fig. 2) proton/
4)
iridoid ingredients than G. jasminoides. Recently, some of carbons correlations from δH 7.64 (H-3‴) to δ 127.6 (C-4‴),
C
iridoid ingredients isolated from the G. jasminoides were 169.2 (C-1‴), and from δH 7.22 (H-5‴) to δC 149.3 (C-7‴),
reported to be of a potential antagonistic effect against the 147.2 (C-3‴), from δ 7.08 (H-9‴) to δ 111.7 (C-5‴) and 149.3
H
C
5
)
Alzheimer’s disease. However, there are few reports on the (C-7‴), from δ 6.81 (H-8‴) to 127.6 (C-4‴) and 150.6 (C-6‴),
H
chemical constituents and pharmacological activity about the from δ 6.41 (H-2‴) to δ 127.6 (C-4‴), 169.2 (C-1‴), and from
H
C
2
,6)
variant. Still now, only five coumarins, three iridoid ingre- δ 3.90 (6‴-OCH ) to δ 150.5 (C-6‴) revealed the presence of
dients, four diterpenes, and other small molecule compounds a trans-feruloyl moiety.
H
3
C
6
,7)
have been isolated from this plant.
isolation and identification of three new iridoid glycosides 4.40 (d, J=7.7Hz, H-1″)/104.9 (C-1″) and other some proton
compounds 1–3, Fig. 1) by the combination of spectroscopic and carbon signals in δ /δ 4.52–3.20/77.9–64.7 revealed the
Herein, we report the
Signals at δ /δ 4.71 (d, J=7.8Hz, H-1′)/100.6 (C-1′), and
H C
(
H
C
analysis, acidic hydrolysis, and sugar derivatization. It is presence of two sugar moieties. On acid hydrolysis, compound
worth mentioning that the acyl moieties of iridoid glycosides 1 afforded D-glucose, the structure of which was confirmed
8
,9)
from the variant most were trans-feruloyl, which was different by HPLC method of Tanaka et al.
from the trans-sinapoyl moiety found in Gardenia jasminoi-
des.
The anomeric centers
6
)
Results and Discussion
The 90% EtOH extract of G. jasminoides var. radicans was
suspended in water and partitioned with petroleum ether and
ethyl acetate, respectively. The ethyl acetate extracts were
subjected to silica gel, polyamide, octadecyl silica gel (ODS),
Sephadex LH-20 column chromatography, and semiprepara-
tive reversed phase HPLC, affording three iridoid glycosides
(compounds 1–3).
Compound 1 was obtained as a colorless transparent
jelly. The molecular formula of C H O for this compound
33
42 18
was determined from the pseudo-molecular ion peak at m/z
+
7
49.2266 [M+Na] obtained by high-resolution electrospray
1
ionization mass spectrometry (HR-ESI-MS). The H- and
C-NMR spectra of compound 1 (Table 1) showed four ole-
13
finic protons and six olefinic carbon signals, including a set of
trans double bond signals at δ /δ 7.64 (d, J=15.9Hz, H-3‴)/
H
C
The authors declare no conflict of interest.
Fig. 1. The Structures of Compounds 1–3
*
To whom correspondence should be addressed. e-mail: guangxzh@sina.com
© 2013 The Pharmaceutical Society of Japan

1
072
Vol. 61, No. 10
Table 1. NMR Data (in Methanol-d ) of Compounds 1–3
4
1
2
3
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
a)
1
3
4
5
6
98.9
153.4
112.3
36.6
5.14 d (7.8)
96.4
152.9
111.9
30.7
5.53 d (2.8)
7.30 brs
96.6
152.8
112.0
30.9
5.54 d (3.0)
7.30 brs
7.47 brs
3.14 m
2.97 m
2.99 m
39.7
2.79 dd (15.9, 8.0)
39.5
2.18 m
39.8
2.20 m
2
.13 dd (15.9, 8.0)
1.79 m
1.80 m
7
8
9
0
129.1
144.6
46.8
5.83 brs
73.7
152.5
45.1
4.31 t (6.4)
73.8
152.5
45.3
4.31 t (6.5)
2.69 t (7.6)
4.22 m
3.02 brs
5.33 brs
5.28 brs
3.03 brs
5.33 brs
5.29 brs
1
1
61.5
112.2
112.2
4
.29 m
1
169.6
51.9
100.6
74.8
77.7
71.5
77.4
70.1
170.1
170.2
1
1-OCH3
3.67 brs
4.71 d (7.8)
3.28 m
b)
1
′
′
′
′
′
′
97.9
74.6
75.8
71.7
78.4
62.7
4.90 d (8.1)
98.2
74.8
76.0
71.9
78.5
62.9
4.89
b)
b)
2
3
4
5
6
4.86 dd
4.83
3.45 m
3.73 m
3.65 m
3.41 m
3.43 m
3.72 m
3.95 m
3.40 m
3.47 m
3.57 m
3.48 m
b)
4.10
3.95 d (11.7)
3.68 m
3
.76 m
1
2
3
4
5
6
″
″
″
″
″
″
104.9
75.0
77.9
71.5
75.3
64.7
4.40 d (7.7)
3.26 m
3.42 m
3.39 m
3.57 m
4.52 dd (11.7, 1.2)
b)
4
.32
1
2
3
4
5
6
7
8
9
6
‴
‴
‴
‴
‴
‴
‴
‴
169.2
115.2
147.2
127.6
111.7
150.5
149.3
116.5
124.4
56.6
168.3
114.9
146.9
127.4
131.4
116.9
160.9
116.9
131.4
168.3
115.4
147.3
128.1
112.2
150.6
149.4
116.5
124.2
56.6
6.41 d (15.9)
7.64 d (15.9)
6.26 d (15.9)
7.60 d (15.9)
6.29 d (15.9)
7.59 d (15.9)
7.22 d (1.3)
7.45 d (8.3)
6.81 d (8.3)
7.17 d (1.7)
6.81 d (8.1)
7.08 dd (8.1, 1.3)
3.90 brs
6.81 brd (8.3)
7.45 brd (8.3)
6.79 d (8.4)
7.06 dd (8.4, 1.7)
3.91 brs
‴
‴-OCH3
Assignments were performed by means of DEPT, COSY, HSQC, HMBC experiments. a) Multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet. b) Signals were over-
lapped.
the sugar chain was glucopyranosyl-(1→6)-glucopyranoside,
i.e., a gentiobiosyl moiety.
1
13
The remaining H- and C-NMR signals were similar to
5,10)
those of genipin by comparing with the reported data.
It
suggested that 1 should be an iridoid glycoside with a trans-
feruloyl, and gentiobiosyl moieties, which were confirmed by
1
1
H– H correlation spectroscopy (COSY), heteronuclear single
quantum coherence (HSQC), and HMBC spectra. The nuclear
Overhauser effect spectroscopy (NOESY) spectrum showed
the correlations between proton at δH 2.69 (H-9) and proton
at δH 2.13 (H-6β), and between two protons at δH 3.14 (H-5)
Fig. 2. Key COSY (in Bold Lines) and HMBC (H→C) Correlations of 1 and at δ 2.69 (H-9), confirming that the β-orientation of H-5
H
and H-9. There are no any nuclear Overhauser effect (NOE)
of the two glucopyranosyl moieties were determined to be correlations between protons at δ 5.14 (H-1) and 3.14 (H-5) or
H
β-configuration from the large coupling constant value at 2.69 (H-9), confirming the α-orientation of H-1. The gentiobio-
δH 4.71 (d, J=7.8Hz, H-1′), and 4.40 (d, J=7.7Hz, H-1″). syl moiety was attached at C-1 of genipin due to the HMBC
The HMBC correlations of protons/ carbons at δ /δ 4.40 correlations from proton at δ 5.14 (H-1) to carbon at δ 100.6
H
C
H
C
(
H-1″)/70.1 (C-6′), and 3.76 (H-6′b)/104.9 (C-1″) suggested that (C-1′), and proton at δ 4.71 (H-1′) to carbon at δ 98.9 (C-1).
H
C

October 2013
1073
bon at δ 97.9 (C-1′), and proton at δ 4.90 (H-1′) to carbon at
C
H
δC 96.4 (C-1). Furthermore, the linkage of the trans-p-couma-
royl-moiety to the gardoside group was located at C-2′ by the
HMBC correlations from proton at δ 4.86 (H-2′) to carbon at
H
δC 168.3 (C-1‴). Therefore, the structure of 2 was elucidated
as 2′-O-trans-p-coumaroylgardoside.
Compound 3 was also obtained as a colorless transparent
jelly. Its molecular formula C H O was determined from
2
6
30 13
+
the pseudo-molecular ion peak at m/z 573.1590 [M+Na] in
Fig. 3. Key COSY (in Bold Lines) and HMBC (H→C) Correlations of 2 HR-ESI-MS. The H- and C-NMR data of 3 were analogous
to those of 2, but showing the presence of one extra methoxyl
1
13
Furthermore, the correlation from proton at δ 4.32 (H-6″b) to groups (δ 3.91 and δ 56.6). The methoxyl group were locat-
H
H
C
carbon at δ 169.2 (C-1‴) in HMBC spectrum suggested that ed at C-6‴ on the basis of aromatic proton signals at δH 7.06
C
the linkage of the feruloyl to the gentiobiosyl group be estab- (dd, J=8.4, 1.7Hz, H-9‴), 6.79 (d, J=8.4Hz, H-8‴), and 7.17
lished to be at C-6″. Thus, the structure of 1 was deduced as (d, J=1.7Hz, H-5‴) as well as all comparable aromatic carbon
6
″-O-trans-feruloylgenipin gentiobioside.
Compound 2 was obtained as a colorless transparent jelly. C-2′, same as compound 1. Therefore, the structure of 3 was
Its molecular formula C H O was determined from the elucidated as 2′-O-trans-feruloylgardoside.
signals, suggesting that 3 had a trans-feruloyl substituent at
2
5
28 12
+
pseudo-molecular ion peak at m/z 543.1475 [M+Na] in HR-
ESI-MS. The H- and C-NMR spectra of 2 (Table 1) showed Experimental
1
13
five olefinic protons and six olefinic carbons signals, including
General Experimental Procedures Melting points were
a set of trans double bond signals at δH 7.60 (d, J=15.9Hz, determined on an X-5 micro-melting point apparatus (Beijing
H-3‴), and 6.26 (d, J=15.9Hz, H-2‴). Proton/carbon signals at Tech Instrument Co., Ltd., Beijing, China). Optical rotations
δ /δ 6.81 (2H, brd, J=8.3Hz, H-6‴, 8″)/116.9 (C-6‴,8‴), and were carried out using a JASCO P-1020 automatic digital
H
C
7.45 (2H, brd, J=8.3Hz, H-5‴, 9‴)/131.4 (C-5‴,9‴), together polarimeter (JASCO Corporation, Tokyo, Japan). UV spec-
with carbon signals at δ 127.4 (C-4‴), and 160.9 (C-7‴), sug- tra were recorded on a JASCO V-550 UV/VIS spectrometer
C
gested the presence of a symmetrical 1,4-disubstituted ben- (JASCO Corporation, Tokyo, Japan). IR spectra were obtained
zene ring. Furthermore, the HMBC correlations (Fig. 3) ob- using a Fourier Transform infrared spectrometer (Bruker
served between protons and carbons at δ /δ 7.60 (H-3‴)/168.3 Instrument, Inc., German). 1D- and 2D-NMR spectra were re-
H
C
(
C-1‴), 7.45 (H-5‴, 9‴)/146.9 (C-3‴), 6.81 (H-6‴, 8‴)/127.4 corded in CD OD using Bruker AV-300 spectrometer (Bruker
3
(C-4‴), 6.26 (H-2‴)/127.4 (C-4‴) and 6.26 (H-2‴)/168.3 (C-1‴), Instrument, Inc., German) with tetramethylsilane (TMS)
revealed the presence of a trans-p-coumaroyl-moiety. Sig- as the internal standard, and the chemical shifts were ex-
nals at δ /δC 4.90 (d, J=8.1Hz, H-1′)/97.9 (C-1′) and other pressed in δ values (ppm). Analytical reversed-phase HPLCs
H
oxygenated methine signals revealed the presence of sugar were performed on an Ultimate™ XB-C18 column (5µm,
residual. After acid hydrolysis and derivatization of 2 by 4.6×250mm, Welch, Potamac, MA, U.S.A.) and Materi-
8
,9)
the method of Tanaka et al.,
in HPLC analysis. The β-configuration was established due purification. Open column chromatography was performed
to the coupling constant of the anomeric proton signal at δ
by silica gel (300–400 mesh, Qingdao, Haiyang Chemical
.90 (d, J=8.1Hz, H-1′). The remaining C-NMR signals of Group Corporation, Qingdao, China), and ODS (50µm, YMC,
compound 2 displayed the characteristic carbons at δC 96.4 Tokyo, Japan). All the reagents were purchased from Tianjin
the D-glucose was detected als XB-C18 (5µm, 10×250mm) for semipreparative HPLC
H
13
4
(
C-1), 111.9 (C-4), 152.9 (C-3), and 170.1 (C-11) of the iridoid Damao Chemical Company (Tianjin, China). Thin-layer chro-
1
nucleus. The H-NMR spectrum of 2 showed the presence of matography was performed by precoated aluminum gel plate
exocyclic methylene protons at δH 5.33 (brs, H-10a) and 5.28 (aluminum gel HSAF254, 1mm, Yantan, China). The spraying
(
brs, H-10b), and a signal at δH 4.31 (t, J=6.4Hz, H-7) at- reagent used for TLC was 10% H SO in EtOH. Standard sug-
2 4
tributable to an allylic hydroxymethine proton. In the HMBC ars D-glycose and L-glycose, and L-cysteine methyl ester were
spectrum of 2, the correlations of signals from δ 5.33 (H-10a) purchased from Adamas-beta Company (Basel, Switzerland).
H
to δC 73.7 (C-7) and 45.1 (C-9), from δH 5.28 (H-10b) to δC o-Tolyl isothiocyanate was purchased from Sigma Company
7
3.7 (C-7) and 45.1 (C-9), from δ 7.30 (H-3) to δ 30.7 (C-5), (Santa Clara, CA, U.S.A.).
H C
9
6.4 (C-1), and 170.1 (C-11), and from δ 5.53 (H-1) to δ 30.7
Plant Material The fruit material of G. jasminoides var.
H
C
(C-5) and 152.9 (C-3) further supported the structural elucida- radicans was purchased in Bozhou, Anhui province, China,
tions. Thus, the structure of this iridoid moiety was assigned and identified by Professor GX Zhou, college of pharmacy,
11)
as a gardoside nucleus moiety. The NOESY spectrum of Jinan University. A specimen was deposited in the Pharma-
compound 2 showed the correlations between protons at δH cognosy Department of the college.
4.31 (H-7) and 2.18 (H-6α), and between protons at δH 3.02
Extraction and Isolation The dried fruit of G. jasminoi-
(
(
H-9) and 1.79 (H-6β), as well as between protons at δH 2.97 des var. radicans were grounded, and extracted three times
H-5) and 1.79 (H-6β). However, there are no any NOE corre- with 85–95% alcohol under soaking at room temperature for
lations between proton at δ 5.57 (H-1) and protons at δ 2.97 24h. The extract solution were combined and concentrated
H
H
(
H-5) or δ 3.02 (H-9), confirming that the H-5 and H-9 were under reduced pressure to about 3.5kg and then partitioned
H
β-orientational, and H-1 and H-7 were α-orientational. The successively with petroleum ether, ethyl acetate, and n-buta-
glucopyranosyl moiety was attached at C-1 of gardoside due nol, to afford 500g, 270g, and 950g of extracts, respectively,
to the HMBC correlations from proton at δ 5.53 (H-1) to car- and 840g water-soluble residue. The ethyl acetate extract
H

1
074
Vol. 61, No. 10
(270g) was subjected to silica gel chromatography eluting isothiocyanate (10µL) was added to the mixture, which was
with gradient CHCl –MeOH systems (1:0→20:1→18:1→ heated at 60°C for 1h. The reaction mixture was directly
3
1
5:1→12:1→10:1→8:1 →5:1→5:2→1:1→0:1) to afford 24 analyzed by reversed-phase HPLC. Analytical HPLC was
fractions (Fr. A–W) after combination of fractions by TLC performed on the column at 28°C with isocratic elution of
pattern. Fraction U (17.8g) was further separated by silica gel 25% CH CN containing 0.08% formic acid for 40min and
3
chromatography eluting with CHCl –MeOH systems (20:1→ subsequent washing the column with MeOH at a flow rate of
3
1
5:1→10:1→5:1→5:2→1:1→0:1) to obtain 9 fractions (Fr. 1mL/min. Peaks were detected by UV detector at 250nm. The
1–9) by TLC. Fraction 7 (1.2g) was subjected to sephadex peak of the derivative of 1 was observed at tR 15.95 (D-Glu)
LH-20 column eluting with MeOH to obtain compound 1 min. The standard sugars D-glycose was subjected to the same
(
180mg). Fraction 8 was subjected to ODS column chromatog- method. The peak of the standard was at t 16.05min. Follow-
R
raphy eluting with MeOH–H O (20→30→40→60→80→100%) ing the above procedure, the derivatives of 2 gave peaks at tR
2
to afford 3 subfractions (SFr. 8-1–3). Subfraction 8-1 (580mg) 16.13 (D-Glu) min.
was purified on sephadex LH-20 column eluting with MeOH
to obtain compounds 2 (120mg) and 3 (15mg).
Acknowledgment We gratefully acknowledge financial
6
″-O-trans-Feruloylgenipin Gentiobioside (1): Colorless support from the Chinese National S&T Special Project on
2
5
transparent jelly; mp 175–177°C; [α]D −20.1 (c=0.04, MeOH); Major New Drug Innovation (2011ZX09307-002-01).
UV λmax (MeOH): 288nm; IR ν (KBr): 3428, 2931, 1694,
632, 1083cm ; H- and C-NMR spectral data, see Table 1. References
HR-ESI-MS m/z: 749.2266 [M+Na] (Calcd for C H O Na,
49.2269).
max
−1
1
13
1
+
1) Liu S. J., Zhang X. T., Wang W. M., Qin M. J., Zhang L. H., Zhong
Cao Yao (Chinese Tranditional and Herbal Drugs), 43, 238–241
33
42 18
7
(2012).
2′-O-trans-p-Coumaroylgardoside (2): Colorless transpar-
2
5
2) Zhang L. H., Liu S. J., Zhao Z. D., Wang W. M., Zhang X. T., Hai
Xia Yao Xue (Strait Pharmaceutical Journal), 24, 39–40 (2012).
ent jelly; mp 179–180°C; [α]D −15.0 (c=0.02, MeOH); UV
λmax (MeOH): 312nm; IR ν (KBr): 3406, 1695, 1634, 1604,
173 cm ; H- and C-NMR spectral data, see Table 1. HR-
max
3
)
Huh M. K., J. Life Sci., 17, 24–30 (2007).
−1
1
13
1
4)
Xie Z. W., Zhong Yao Cai (Journal of Chinese Medicinal Materi-
als), 14, 45–47 (1991).
+
ESI-MS m/z: 543.1475 [M+Na] (Calcd for C H O Na,
2
5
28 12
5
43.1479).
′-O-trans-Feruloylgardoside (3): Colorless transparent
5
)
Yu Y., Xie Z. L., Gao H., Ma W. W., Dai Y., Wang Y., Zhong Y.,
Yao X. S., J. Nat. Prod., 72, 1459–1464 (2009).
2
2
5
jelly; mp 181–182°C; [α]D −45.2 (c=0.02, MeOH); UV λmax
MeOH): 232nm; IR ν
275 cm ; H- and C-NMR spectral data, see Table 1. HR-
ESI-MS m/z: 573.1590 [M+Na] (Calcd for C H O Na,
73.1484).
Acid Hydrolysis and HPLC Analysis The absolute
configuration of the sugar moieties in the structures was
determined by the method of Tanaka et al.
4mg) was hydrolyzed with 4mL of 2M HCl for 6h at 80°C.
6) Yu Y., Gao H., Dai Y., Yao X. S., Zhong Cao Yao (Chinese Trandi-
tional and Herbal Drugs), 41, 148–153 (2010).
(
1
(KBr): 3458, 1633, 1518, 1385,
max
−1
1
13
7
)
Moon H. I., Joa J. S., Kim J. S., Pei P. C., Zee O. P., Saenyak
Hakhoechi, 33, 1–4 (2002).
+
2
6
30 13
8)
Tanaka T., Nakashima T., Ueda T., Tomii K., Kouno I., Chem.
Pharm. Bull., 55, 899–901 (2007).
5
9)
Wei L. B., Chen J. M., Ye W. C., Yao X. S., Zhou G. X., J. Asian
Nat. Prod. Res., 14, 521–527 (2012).
8
,9)
Compound 1
1
0) Dinda B., Debnath S., Banik R., Chem. Pharm. Bull., 59, 803–833
2011).
1) Chrysoula G., Anastasia K., Jörg H., Helen S., Phytochemistry, 68,
1799–1804 (2007).
(
(
The mixture was evaporated to dryness under vacuum. The
residue was dissolved in pyridine (2mL) containing L-cysteine
methyl ester (2mg) and heated at 60°C for 1h. Then, o-tolyl
1