Biologically active iridoids from Hedyotis diffusa

Article abstract of DOI:10.1002/hlca.201000129

Two new iridoids, 10-O-benzoyl-6′-O-α-L-arabino(1→6)- β-D-glucopyranosylgeniposidic acid (1), deacetyl-6-ethoxyasperulosidic acid methyl ester (2), along with 14 other known iridoids, were isolated from the whole plant of Hedyotis diffusa Willd. Their structures were determined on the basis of spectroscopic analysis. The short-term-memory-enhancement activities of compounds 1, 7-9, 11, and 13 were evaluated on an Aβ transgenic drosophila model.

Full text of DOI:10.1002/hlca.201000129

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Helvetica Chimica Acta – Vol. 93 (2010)
Biologically Active Iridoids from Hedyotis diffusa
a
b
1
c
d
1
a
b
a
b
e
c
d
c
by Bo Ding ) ) ), Wei-Wei Ma ) ) ), Yi Dai ) ), Hao Gao ) ), Yang Yu ), Ye Tao ) ), Yi Zhong ), and
a
b e
Xin-Sheng Yao* ) ) )
a) Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University,
Guangzhou 510632, P. R. China
(
phone: þ 86-20-85225849; fax: þ 86-20-85221559; e-mail: tyaoxs@jnu.edu.cn)
b) Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs
Research, Guangzhou 510632, P. R. China
c) Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084,
P. R. China
d) Joekai Biotech. Co., Ltd., Beijing 100020, P. R. China
e) Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016,
P. R. China
Two new iridoids, 10-O-benzoyl-6’-O-a-l-arabino(1 ! 6)-b-d-glucopyranosylgeniposidic acid (1),
deacetyl-6-ethoxyasperulosidic acid methyl ester (2), along with 14 other known iridoids, were isolated
from the whole plant of Hedyotis diffusa Willd. Their structures were determined on the basis of
spectroscopic analysis. The short-term-memory-enhancement activities of compounds 1, 7 – 9, 11, and 13
were evaluated on an Ab transgenic drosophila model.
Introduction. – Hedyotis diffusa Willd. (Rubiaceae), an annual herbaceous plant
distributed in southern regions of China [1], is known as a folk medicine for the
treatment of sphagitis, bronchitis, dysentery, mastitis, pneumonia, appendicitis, pelvitis,
and some tumors [2]. In addition, the extract of this whole plant showed a series of
bioactivities in the modern pharmacological investigations, such as anticancer [3 – 4],
antimicrobial [5], anti-inflammatory [5], and immunoloregulation [6]. Previous
phytochemical studies revealed the presence of iridoid glycosides [7][8], flavones
[
9], anthraquinones [10], and phenylpropanoids [11]. Here, we report the isolation and
structural elucidation of two new iridoid glycosides, along with 14 known iridoids from
this plant. Compounds 1, 7 – 9, 11, and 13 showed potent activities in the short-term-
memory-enhancement assay on an Ab transgenic drosophila model.
Results and Discussion. – Structure Elucidation. The 60% EtOH/H O extract of the
2
whole plant of H. diffusa was chromatographed over macroporous resin HP-20 column,
eluted with EtOH/H O in gradient. The 50% EtOH/H O eluted fraction, through
2
2
further isolation by a series of silica gel, ODS, Toyopearl HW-40, Sephadex LH-20
columns as well as preparative HPLC, yielded two new iridoid glycosides, 1 and 2, and
14 known iridoids, 3 – 16. To the best of our knowledge, compounds 3 – 6 were isolated
from Hedyotis species for the first time.
1)
These authors contributed equally to this work.
ꢀ
2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 93 (2010)
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Compound 1 was obtained as a yellow amorphous powder. HR-ESI-MS gave a
ꢀ
quasi-molecular-ion peak at m/z 609.1821 ([M ꢀ H] ), corresponding to the molecular
1
formula C H O . In the H-NMR spectrum of 1 (Table 1), the H-atom signals at
28
34 15
d(H) 7.53 (d, J ¼ 0.9, 1 H) and 5.20 (d, J ¼ 8.0, 1 H) were characteristic for HꢀC(3) and
HꢀC(1) of iridoids. In addition, another olefinic H-atom (d(H) 5.98 (br. s, HꢀC(7))
and four olefinic C-atom signals at d(C) 153.5 (C(3)), 112.4 (C(4)), 131.8 (C(7)), and
139.8 (C(8)) were assigned to the iridoid moiety. The H-Atom signals at d(H) 8.05 (dd,
J ¼ 7.8, 1.3, HꢀC(2’’’,6’’’)), 7.60 (tt, J ¼ 7.4, 1.3, HꢀC(4’’’)), and 7.48 (t, J ¼ 7.8,
HꢀC(3’’’,5’’’)), together with the C-atom signals at d(C) 131.5 (C(1’’’)), 130.6
(C(2’’’,6’’’)), 129.7 (C(3’’’,5’’’)), and 134.3 (C(4’’’)) indicated the presence of a
monosubstituted benzene ring. Furthermore, the HMBC correlations observed for

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HꢀC(2’’’,6’’’) (d(H) 8.05)/C(7’’’) (d(C) 167.8) revealed the presence of a benzoyl (Bz)
13
moiety. The C-NMR spectrum of 1 exhibited 28 C-atom signals, with ten from the
iridoid moiety and seven from the Bz group. Of the remaining eleven signals, six could
be assigned to a glucopyranosyl moiety, and another set of five signals could be assigned
1
1
to an arabinopyranosyl moiety on the basis of the analysis of the H, H-COSY and
TOCSY experiments. After acid hydrolysis and derivatization of 1 by the method of
Hara et al. [12][13], GC Analysis revealed the presence of d-glucose and l-arabinose.
Table 1. 1H- and 13C-NMR Data (400 MHz for H, CD OD) of Compound 1. d in ppm, J in Hz. Arbitrary
1
3
atom numbering.
d(H)
d(C)
HꢀC(1)
HꢀC(3)
C(4)
5.20 (d, J ¼ 8.0)
98.7
153.5
112.4
36.8
7.53 (d, J ¼ 0.9)
–
HꢀC(5)
3.22 (br. t, J ¼ 7.7)
CH (6)
2.89 (dd, J ¼ 16.6, 8.9), 2.24 (dd, J ¼ 16.6, 8.9)
40.0
2
HꢀC(7)
5.98 (br. s)
131.8
139.8
47.4
C(8)
–
HꢀC(9)
2.80 (t, J ¼ 8.0)
CH (10)
5.08 (br. d, J ¼ 13.9), 5.02 (br. d, J ¼ 14.0)
64.4
2
C(11)
–
170.8
100.7
74.8
77.9
71.8
77.6
69.8
105.4
72.4
74.2
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
4.72 (d, J ¼ 7.8)
3.25 (overlapped)
3.37 (overlapped)
3.28 (overlapped)
3.49 (overlapped)
4.05 (dd, J ¼ 11.8, 1.9), 3.66 (dd, J ¼ 11.9, 6.9)
4.26 (d, J ¼ 6.8)
CH (6’)
2
HꢀC(1’’)
HꢀC(2’’)
HꢀC(3’’)
HꢀC(4’’)
3.51 (overlapped)
3.44 (overlapped)
3.73 (dd, J ¼ 3.2, 1.2)
3.76 (dd, J ¼ 12.2, 3.9), 3.42 (m)
–
69.4
66.7
CH (5’’)
2
C(1’’’)
131.5
130.6
129.7
134.3
167.8
HꢀC(2’’’,6’’’)
HꢀC(3’’’,5’’’)
HꢀC(4’’’)
C(7’’’)
8.05 (dd, J ¼ 7.8, 1.3)
7.48 (t, J ¼ 7.8)
7.60 (tt, J ¼ 7.4, 1.3)
–
Interpretation of the HSQC and HMBC spectra of 1 (Fig. 1) revealed the
1
13
substitution pattern, and allowed the assignment of all H- and C-NMR signals
(
Table 1). The Bz group is located at C(10), due to the HMBC correlations of
H ꢀC(10) (d(H) 5.08) and H ꢀC(10) (5.02) with C(7’’’) (d(C) 167.8). The d-
a
b
glucopyranosyl moiety is obviously attached to C(1) of the aglycone, due to the HMBC
correlations HꢀC(1’) (d(H) 4.72)/C(1) (d(C) 98.7), and HꢀC(1) (d(H) 5.20)/C(1’)
(d(C) 100.7). The l-arabinopyranosyl moiety is attached to C(6’) of d-glucopyranosyl
moiety, according to the HMBC correlations HꢀC(1’’) (d(H) 4.26)/C(6’) (d(C) 69.8)
and of H ꢀC(6’) (d(H) 4.05)/C(1’’) (d(C) 105.4). The b-configuration for d-
a
glucopyranosyl unit and the a-configuration for l-arabinopyranosyl unit were

Helvetica Chimica Acta – Vol. 93 (2010)
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established due to the large J(HꢀC(1’),HꢀC(2’)) and J(HꢀC(1’’),HꢀC(2’’)) coupling
constants (J ¼ 7.8 and J ¼ 6.8 Hz). Therefore, the structure of compound 1 was deduced
as 10-O-benzoyl-6’-O-a-l-arabino(1 ! 6)-b-d-glucopyranosylgeniposidic acid.
Fig. 1. Structures and significant HMBC (H ! C) and COSY (—) correlations of 1 and 2
Compound 2 was obtained as a yellowish-brown amorphous powder with a
1
13
molecular formula of C H O . The H- and C-NMR data of 2 were identical to
19
28 11
those of deacetylasperulosidic acid methyl ester [14], except for additional signals, i.e.,
at d(H) 3.49 (dd, J ¼ 13.0, 7.0, H ꢀC(1’’)), 3.38 (dd, J ¼ 12.8, 6.7, H ꢀC(1’’)), 1.04 (t, J ¼
a
b
7.0, Me(2’’)), and at d(C) 66.1 (C(1’’)), 15.9 (C(2’’)), arising from an EtO group in 2.
Interpretation of the HSQC and HMBC spectra of 2 (Fig. 1) allowed the
1
13
assignment of all H- and C-NMR signals (Table 2). The EtO group is located at C(6),
due to the HMBC correlations between HꢀC(6) (d(H) 4.46) and C(1’’) (d(C) 66.1).
Furthermore, the linkage could be confirmed by the significant obvious low-field shift
of C(6) (d(C) 83.3) of 2 compared to that of deacetylasperulosidic acid methyl ester
(
d(C) 75.5). The relative configuration of HꢀC(6) was established as b from the
correlation HꢀC(6)/HꢀC(9) in the ROESY experiment. The H-atom signals of
HꢀC(6) (d(H) 4.46 (d, J ¼ 7.8)) further confirmed the above inference, which is
consistent with the values found for 6a-butoxygeniposide [15]. The presence of d-
glucose was revealed by acid hydrolysis, derivatization, and GC analysis. The b-
configuration for d-glucopyranosyl was established due to its large J(HꢀC(1’),
HꢀC(2’)) coupling constant (J ¼ 7.8 Hz ) . Thus, 2 was deduced as deacetyl-6-ethoxy-
asperulosidic acid methyl ester.
To find out whether compound 2 was an artefact of isolation, dried plant material
(
12 g) was refluxed twice with CH Cl (2 ꢁ 250 ml) for 2 h each time. After evaporation
2
2
under reduced pressure, the residue and compound 2 were analyzed by HPLC,
respectively, eluted with 30% MeOH/H O. The absence of peak of compound 2 (t
2
R
1
3.8 min) in the HPLC chromatogram of CH Cl extract indicated that compound 2 is
2 2
an artefact.
In addition, 14 known iridoids were identified as monotropein methyl ester
(
galioside; 3) [14], gardenoside (4) [16], 6-O-methyldeacetylasperulosidic acid methyl
ester (5) [17], 4-epiborreriagenin (6) [18], (E)-6-O-p-coumaroylscandoside methyl

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Helvetica Chimica Acta – Vol. 93 (2010)
Table 2. 1H- and 13C-NMR Data (400 MHz for H, CD OD) of Compound 2. d in ppm, J in Hz. Arbitrary
1
3
atom numbering.
d(H)
d(C)
HꢀC(1)
HꢀC(3)
C(4)
HꢀC(5)
HꢀC(6)
HꢀC(7)
C(8)
5.01 (d, J ¼ 8.8)
101.7
154.9
108.5
42.1
7.60 (d, J ¼ 1.2)
–
3.07 (td, J ¼ 6.7, 1.4)
4.46 (d, J ¼ 7.8)
83.3
6.11 (d, J ¼ 1.8)
128.3
152.1
46.0
–
HꢀC(9)
2.53 (t, J ¼ 8.2)
CH (10)
4.45 (d, J ¼ 14.8), 4.19 (d, J ¼ 15.7)
61.7
2
C(11)
–
169.5
100.7
75.0
78.3
71.4
77.9
62.6
51.8
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
4.71 (d, J ¼ 7.8)
3.23 (t, J ¼ 8.9)
3.40 – 3.34 (m)
3.40 – 3.34 (m)
3.40 – 3.34 (m)
CH (6’)
3.81 (dd, J ¼ 12.1, 2.1), 3.66 (dd, J ¼ 12.1, 5.2)
2
MeO
3.73 (s)
CH (1’’)
Me(2’’)
3.49 (dd, J ¼ 13.0, 7.0), 3.38 (dd, J ¼ 12.8, 6.7)
1.04 (t, J ¼ 7.0)
66.1
15.9
2
ester (7) [19], (E)-6-O-feruloylscandoside methyl ester (8) [19], (Z)-6-O-p-coumar-
oylscandoside methyl ester (9) [7], (Z)-6-O-feruloylscandoside methyl ester (10) [7],
deacetylasperulosidic acid methyl ester (11) [14], deacetylasperuloside (12) [20],
asperulosidic acid (13) [21], scandoside methyl ester (14) [14], asperuloside (15), and
[
22], asperulosidic acid methyl ester (16) [22] by direct comparison with authentic
samples, and/or by comparison of various spectral and chemical data with those
reported in the literature.
Biological Studies. Compounds 1, 7 – 9, 11, and 13 were evaluated for the short-
term-memory-enhancement activities by using the human Ab42 transgenic fly AD
model. The activities were indicated by the performance index (PI), as shown in Fig. 2.
Fig. 2. Performance index (PI) of AD flies fed with compounds. N ¼ 8 for all groups. Each value is the
mean ꢂ SE (SE refers to standard error), *: P < 0.05, and **: P < 0.01, compared with AD flies without
drug group.

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All six compounds showed potent activities, with compound 7 being the most active.
Combined with our recent research on Gardenia jasminoides [23], this suggests that the
iridoid glycosides could improve the short-term-memory capacity of Ab42 transgenic
flies, and might have a potential antagonism effect against Alzheimerꢂs disease.
This work was supported by grants from the National Natural Science Foundation of P. R. China (No.
3
0701047), the Key Project of the Chinese Ministry of Education (No. 208173), the National Basic
Research Projects (973 Program) of the Ministry of Science and Technology of China (2006CB500806 and
2
8
009CB941301), the Team Project of Natural Science Foundation of Guangdong Province (No.
351063201000003), the Tsinghua-Yue-Yuen Medical Sciences Fund, and the Project of Beijing Municipal
Science & Technology Plan (Z07000200540705).
Experimental Part
General. Column chromatography (CC): Diaion HP-20 (Mitsubishi-Chemical, Japan), silica gel
SiO ; 100 – 200 mesh, Qingdao Marine Chemical Ltd., China), octadecylsilanized (ODS) SiO (YMC
(
2
2
Ltd., Japan), Toyopearl HW-40 (TOSOH Co., Japan), and Sephadex LH-20 (Amersham Pharmacia
Biotech, Sweden) columns. TLC: SiO GF (Yantai Chemical Inst., China) plates and visualized by
2
254
spraying with conc. H SO /vanillin soln., followed by heating. Optical rotations: JASCO P-1020 digital
2
4
polarimeter. UV Spectra: JASCO V-550 UV/VIS spectrometer; l (log e) in nm. IR Spectra: JASCO
max
ꢀ1
FT/IR-480 plus spectrometer; ˜n in cm . 1D and 2D NMR spectra: Bruker AV-400 spectrometer at r.t.;
CD OD solns.; d in ppm rel. to Me Si as internal standard, J in Hz. ESI-MS: Finnigan LCQ Advantage
3
4
MAX mass spectrometer; in m/z (rel. %). HR-ESI-MS: Micromass Q-TOF mass spectrometer; in m/z.
Plant Material. The plant material was purchased from the Qingping Market of Traditional Chinese
Medicine, Guangdong Province, China, in July 2008, and was identified as the whole plant of H. diffusa by
Prof. Danyan Zhang, Guangzhou University of Traditional Chinese Medicine. A voucher specimen
(
20080731 HEDI) was deposited with the Institute of Traditional Chinese Medicine & Natural Products,
College of Pharmacy, Jinan University, Guangzhou, China.
Extraction and Isolation. The whole plant (4.4 kg) of H. diffusa was refluxed twice with 60% aq.
EtOH (2 ꢁ 40 l) for 2 h each time. After filtration, the filtrate was concentrated under reduced pressure.
The concentrated soln. was suspended in H O, centrifuged, and passed through a macroporous resin
2
Diaion HP-20 column, successively eluted with H O, and EtOH/H O 1:1 and 95 :5, to afford 381.1, 102.5,
2
2
6
8.4 g of extracts, resp. The EtOH/H O 1:1 eluate (72.5 g) was passed through a SiO column, eluted with
2
2
a stepwise gradient mixture of CHCl
3
/MeOH 100 :0, 95 :5, 9 :1, 8 :2, 7:3, and 5 :5, and finally with MeOH
alone, to give ten fractions, Frs. 1 – 10. Fr. 5 (1.5 g; eluted with CHCl /MeOH 9 :1) was fractionated on
3
HW-40 CC, eluted with MeOH/H O in gradient, to yield four subfractions, Frs. 5.1 – 5.4. Fr. 5.2 (eluted
2
with 30% MeOH/H O) afforded compounds 2 (25.6 mg), 5 (17.3 mg), 6 (15.1 mg), 15 (529.4 mg), and 16
2
(
66.2 mg) after purification by prep. ODS HPLC with 27% MeOH/H O as mobile phase. Fr. 6 (8.2 g;
2
eluted with CHCl /MeOH 8 :2) was further fractionated by CC (ODS; MeOH/H O in gradient) to yield
3
2
eight subfractions, Frs. 6.1 – 6.8. Fr. 6.5 (eluted with 50% MeOH/H O) was subjected to CC (HW-40;
2
MeOH/H O in gradient) to yield five subfractions, Frs. 6.5.1 – 6.5.5. Subfr. 6.5.4 (eluted with 60% MeOH/
2
H O) yielded compound 7 (4.8 g). Subfr. 6.5.3 (eluted with 40% MeOH/H O) was purified by prep.
2
2
HPLC (ODS; 45% MeOH/H O) to yield compounds 8 (211.9 mg), 9 (88.4 mg), and 10 (16.2 mg). Fr. 7
2
(
5.8 g; eluted with CHCl /MeOH 8 :2) was fractionated by CC (ODS; MeOH/H O in gradient) to yield
3
2
nine fractions, Frs. 7.1 – 7.9. Fr. 7.2 (eluted with 30% MeOH/H O) was subjected to CC (HW-40; gradient
2
MeOH/H O) to yield seven subfractions, Frs. 7.2.1 – 7.2.7. Compounds 3 (31.2 mg), 4 (75.6 mg), 11
2
(
563.8 mg), 12 (188.7 mg), and 14 (94.3 mg) were obtained from Fr. 7.2.5 (eluted with 30% MeOH/H O)
2
after purification by prep. HPLC (ODS; 15% MeOH/H O); compound 13 (526.6 mg) was obtained from
2
Fr. 7.3 (eluted with 30% MeOH/H O) after purification by CC (Sephadex LH-20; with 50% MeOH/H O;
2
2
and compound 1 (262.0 mg) was obtained from Fr. 7.7 (eluted with 50% MeOH/H O) after purification
2
by CC (HW-40; 40% MeOH/H O.
2
1
0-O-Benzoyl-6’-O-a-l-arabino(1 ! 6)-b-d-glucopyranosylgeniposidic Acid ( ¼ (1S,4aS,7aS)-7-
[
(Benzoyloxy)methyl]-1,4a,5,7a-tetrahydro-1-(6-O-a-l-arabinopyranosyl-b-d-glucopyranosyloxy)cyclo-

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Helvetica Chimica Acta – Vol. 93 (2010)
1
8
penta[c]pyran-4-carboxylic Acid; 1). Yellow amorphous solid. [a] ¼ þ0.8 (c ¼ 0.5, MeOH). UV
D
1
13
(
MeOH): 230.8 (3.82). IR (KBr): 3330, 1707, 1277, 1075, 716. H- (400 MHz) and C-NMR (100 MHz):
þ
þ
ꢀ
see Table 1. ESI-MS (pos.): 1243 ([2M þ Na] ), 633 ([M þ Na] ). ESI-MS (neg.): 609 ([M ꢀ H] ). HR-
ꢀ
ESI-MS (neg.): 609.1821 ([M ꢀ H] ).
Deacetyl-6-ethoxyasperulosidic Acid Methyl Ester ( ¼ Methyl (1S,4aS,5S,7aS)-5-Ethoxy-1-(b-d-
glucopyranosyloxy)-1,4a,5,7a-tetrahydro-7-(hydroxymethyl)cyclopenta[c]pyran-4-carboxylate; 2). Yel-
1
8
lowish-brown amorphous solid. [a] ¼ þ13.5 (c ¼ 0.5, MeOH). UV (MeOH): 237.2 (3.42). IR (KBr):
D
1
13
3
381, 2925, 1703, 1632, 1439, 1384, 1158. H-NMR (400 MHz) and C-NMR (100 MHz): see Table 2.
þ
þ
ꢀ
ESI-MS (pos.): 887 ([2M þ Na] ), 455 ([M þ Na] ). HR-ESI-MS (neg.): 431.1574 ([M ꢀ H] ).
Acid Hydrolysis. Acid hydrolysis of 1 and 2 was performed according to the method of Hara et al.
[
2
12][13] to determine the absolute configuration of the monosaccharide. After hydrolysis with 1m HCl for
h at 808 and then further derivatization, the derivatives of 1 were analyzed by GC [13]. Two peaks were
observed at tR 15.43 (l-Ara) and 29.61 min (d-Glc), while the peaks of the mixed standard
monosaccharide derivatives were recorded at tR 15.81 (l-Ara), 19.63 (l-Rha), 29.67 (d-Glc), 32.21 (l-
Glc), and 32.88 min (d-Gal). In addition, the peak of derivatives of 2 was observed at tR 30.16 min (d-
Glc).
Determination of Short-Term-Memory-Enhancement Activity. The bioassays were performed by
using the method of Yu et al. [23]. The activities of compounds 1, 7 – 9, 11, and 13 were indicated by the
performance index (PI; see Fig. 2).
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Received March 29, 2010