New anthraquinone and iridoid glycosides from the stems of Hedyotis hedyotidea

Article abstract of DOI:10.1002/hlca.201000270

Five new anthraquinone glycosides, hedanthrosides A-E (1-5, resp.) and two new iridoid glycosides, hediridosides A and B (6 and 7, resp.), along with two known anthraquinones and four known iridoids, were isolated from the stems of Hedyotis hedyotidea (DC.) Merr. The structures of the new compounds were elucidated on the basis of 1D- and 2D-NMR, and HR-MS analysis and chemical methods. Copyright

Full text of DOI:10.1002/hlca.201000270

Helvetica Chimica Acta – Vol. 94 (2011)
675
New Anthraquinone and Iridoid Glycosides from the Stems of Hedyotis
hedyotidea
by Xiao-Ping Hu, Shu-Wei Zhang, Shan-Shan Liu, and Li-Jiang Xuan*
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai
201203, P. R. China (phone: þ 86-21-20231968; e-mail: ljxuan@mail.shcnc.ac.cn)
Five new anthraquinone glycosides, hedanthrosides A – E (1 – 5, resp.) and two new iridoid
glycosides, hediridosides A and B (6 and 7, resp.), along with two known anthraquinones and four known
iridoids, were isolated from the stems of Hedyotis hedyotidea (DC.) Merr. The structures of the new
compounds were elucidated on the basis of 1D- and 2D-NMR, and HR-MS analysis and chemical
methods.
Introduction. – The genus Hedyotis (Rubiaceae family) contains 69 species
distributed in China [1], some of which have been used as Traditional Chinese
Medicines (TCM), such as Hedyotis diffusa for treating cancer, and Hedyotis
chrysotricha for treating enteritis [2]. The characteristic chemical constituents of this
genus are anthraquinones, flavonoids, iridoids, and triterpenoids [3]. Plants of this
genus have been found to exhibit anti-inflammatory and anticancer activities [3].
Hedyotis hedyotidea (DC.) Merr. [4], a 3 – 5-m tall shrub growing in the south of
China [1], has been traditionally used for the treatment of cold, cough, gastro-enteritis,
heatstroke, Herpes zoster, rheumatoid arthritis, etc. [5]. Previously, some iridoids have
been identified from H. hedyotidea, and meanwhile the activity of the crude extract of
this herb against stress ulcer in mice was mentioned [6], but the bioactive components
were unknown. As part of our ongoing effort to discover new bioactive agents from
Chinese medicinal plants, the stems of H. hedyotidea were investigated. We report here
on the isolation and characterization of seven anthraquinones, including five new
glycosides, 1 – 5, together with six iridoids, which comprise two new glycosides, 6 and 7.
Inhibitory activities of compounds obtained to NF-kB induced by the NF-kB-activating
cytokine TNF-a (tumor necrosis factor a) was investigated, but all turned out to be
inactive.
Results and Discussion. – The stems of Hedyotis hedyotidea collected from Guangxi
were percolated with 70% aqueous acetone. After removal of the organic solvent under
reduced pressure, the extract was partitioned between CHCl3 and H2O. Compounds 1 –
7, along with 1,3-dihydroxy-2-(hydroxymethyl)-9,10-anthraquinone 3-O-b-d-glucopyr-
anoside [7], scandoside [8], borreriagenin [9], methyl deacetylasperulosidate [10], and
asperulosidic acid methyl ester [11] were obtained from the H2O fraction, and the
CHCl3-soluble fraction afforded rubiadin (8) [12].
The UV spectra of compounds 1 – 5 with the common absorbance maxima at 260
and 355 nm were typical of anthraquinone-type compounds. The IR spectra of them
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Helvetica Chimica Acta – Vol. 94 (2011)
also showed characteristic absorption bands for free OH groups in the range of 3363 –
3423 cmꢀ1, CO peaks at 1670 cmꢀ1, and aromatic group bands in the range of 1570 and
1670 cmꢀ1.
Compound 1, was obtained as a yellow amorphous powder, and analyzed for
C27H30O14 by means of HR-ESI-MS (m/z 601.1539 ([M þ Na]þ)). The aromatic region
1
of the H-NMR spectrum (Table 1) indicated the presence of five aromatic H-atoms.
Their multiplicity, an AABB system at d(H) 8.22 (dd, J ¼ 7.1, 2.0), d(H) 8.18 (dd, J ¼
6.3, 2.1), d(H) 7.90 – 7.95 (m, 2 H), and a 1-H singlet at d(H) 7.48, were suggestive of an
1
anthraquinone with one nonsubstituted and one trisubstituted ring. Other H-NMR
signals were a singlet of a Me group at d(H) 2.19, and characteristic signals of two b-
glucopyranosyl units with the anomeric H-atoms at d(H) 4.18 (d, J ¼ 7.5), and 5.18 (d,
J ¼ 6.6). The signals corresponding to a chelated CO group, a non-chelated CO group,
two O-bearing C-atoms, a Me group, along with signals of two sugar units in the
13C-NMR spectrum, supported the earlier assignments of the H-atoms. In the HMBC
spectrum, correlations from both of HꢀC(5) (d(H) 8.18) and the singlet aromatic H-
atom (d(H) 7.48) to the non-chelated C(10)¼O group (d(C) 181.6), confirmed that
C(4) is unsubstituted. Consequently, a free OH group should be located at C(1) to
chelate with C(9)¼O. Cross-peak from the Me (d(H) 2.19) to C(1) (d(C) 161.4) in the
HMBC supported the assignment of the Me group at C(2). Accordingly, the aglycone
of 1 was confirmed to be rubiadin (8), which was also found in H. hedyotidea.
The linkage sites of the two glucopyranosyl units were deduced from the HMBC
correlations HꢀC(1’) (d(H) 5.18)/C(3) (d(C) 161.2), and HꢀC(1’’) (d(H) 4.18)/C(6’)
(d(C) 68.0). Furthermore, in the ROESY spectrum (Fig. 1), correlation HꢀC(1’)/
HꢀC(4) confirmed that the glucopyranosyl units were located at C(3). Enzymatic
hydrolysis of 1 afforded glucose, which was identified by TLC comparison with
authentic sample. The glucose isolated from the hydrolysate gave optical rotation
[a]2D0 ¼ þ42.3 (c ¼ 0.90, H2O), indicating it as d-glucose. Therefore, 1 was determined
to be 1,3-dihydroxy-2-methyl-9,10-anthraquinone 3-O-b-d-glucopyranosyl-(1 ! 6)-b-
d-glucopyranoside1), named hedanthroside A.
1
)
Arbitrary atom numbering. For systematic names, see Exper. Part.

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677
Table 1. 1H- and 13C-NMR Data ((D6)DMSO) for 1, 2, and 51). d in ppm, J in Hz.
1
2
5
d(H)
d(C) d(H)
d(C) d(H)
d(C)
C(1)
C(2)
C(3)
HꢀC(4)
HꢀC(5)
HꢀC(6)
HꢀC(7)
HꢀC(8)
C(9)
C(10)
C(11)
C(12)
C(13)
161.4
120.7
161.2
105.9 7.47 (s)
161.2
123.6
162.0
106.4 7.50 (s)
159.6
117.0
159.8
105.6
127.0
134.9
134.7
126.5
186.8
181.2
132.9
132.7
111.1
135.3
7.48 (s)
8.18 (dd, J ¼ 6.3, 2.1) 126.8 8.17 (dd, J ¼ 6.7, 2.1) 127.0 8.18 – 8.22 (m)
7.90 – 7.95 (m)
7.90 – 7.95 (m)
134.7 7.90 – 7.95 (m)
134.6 7.90 – 7.95 (m)
134.8 7.92 – 7.99 (m)
134.9 7.92 – 7.99 (m)
8.22 (dd, J ¼ 7.1, 2.0) 126.4 8.17 (dd, J ¼ 7.0, 2.0) 126.6 8.22 – 8.27 (m)
187.1
181.6
132.4
133.0
110.9
131.9
187.1
181.5
132.9
132.8
111.4
133.8
50.9
C(14)
MeꢀC(2) or
HOCH2ꢀC(2)
MeOOCꢀC(2)
Glc’
2.19 (s)
8.4 4.65 (d, J ¼ 11.4),
4.56 (d, J ¼ 11.4)
3.86 (s)
52.6, 163.7
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
CH2(6’)
5.18 (d, J ¼ 6.6)
3.36 – 3.38 (m)
3.62 – 3.65 (m)
3.36 – 3.38 (m)
3.36 – 3.40 (m)
4.00 (d, J ¼ 9.6),
3.65 (d, J ¼ 9.6)
100.2 5.15 (d, J ¼ 6.8)
76.0 3.77 – 3.82 (m)
75.6 3.30 – 3.50 (m)
73.1 3.00 – 3.20 (m)
69.0 3.30 – 3.50 (m)
68.0 4.02 (d, J ¼ 9.8),
3.79 (d, J ¼ 9.8)
100.8 5.30 (d, J ¼ 7.6)
75.6 3.36 – 3.38 (m)
69.8 3.66 – 3.69 (m)
73.5 3.22 – 3.27 (m)
68.9 3.33 – 3.39 (m)
68.0 3.99 (d, J ¼ 9.5),
3.65 (d, J ¼ 9.5)
99.9
76.2
75.6
72.9
68.9
67.9
Glc’’
HꢀC(1’’)
HꢀC(2’’)
HꢀC(3’’)
HꢀC(4’’)
HꢀC(5’’)
CH2(6’’)
4.18 (d, J ¼ 7.5)
2.98 – 3.00 (m)
3.03 – 3.07 (m)
2.94 – 2.98 (m)
3.02 – 3.06 (m)
3.60 (d, J ¼ 10.7),
3.42 (d, J ¼ 10.7)
103.4 4.19 (d, J ¼ 7.4)
76.8 3.00 – 3.20 (m)
76.6 3.00 – 3.20 (m)
73.4 3.00 – 3.20 (m)
69.9 3.30 – 3.50 (m)
60.9 3.60 (d, J ¼ 11.6),
3.38 (d, J ¼ 11.6)
103.5 4.15 (d, J ¼ 7.6) 103.4
76.9 3.07 (m)
73.4 3.07 (m)
76.5 2.98 (m)
73.2 3.08 (m)
76.8
76.5
73.4
69.9
61.0 3.62 (d, J ¼ 10.3), 60.9
3.44 (d, J ¼ 10.3)
Compound 2, an orange amorphous powder, displayed a sodiated molecular-ion
peak at m/z 617.1482 ([M þ Na]þ) in its HR-ESI-MS, corresponding to the molecular
formula C27H30O15, which was suggestive of an oxidized form of 1. Comparison of the
NMR data (Table 1) of 2 with those of 1 indicated the presence of the CH2 H-atoms
represented by two doublets (d(H) 4.56 (d, J ¼ 11.4), and d(H) 4.65 (d, J ¼ 11.4)), with
the disappearance of the singlet peak of Me H-atoms. Correlations of the CH2 to C(1)
and C(3), confirmed that the Me at C(2) in compound 1 was hydroxylated in
compound 2. The configuration of other functional groups were determined by HMBC
experiment (Fig. 1), which revealed that compound 2 exhibited the same diglucosyl
unit as 1, including the location and the interglycosidic linkage. From the above results,
compound 2 was characterized as 1,3-dihydroxy-2-(hydroxymethyl)-9,10-anthraqui-
none 3-O-b-d-glucopyranosyl-(1 ! 6)-b-d-glucopyranoside1), named hedanthroside B.

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Fig. 1. Key HMBC (H ! C) and ROESY (H $ H) correlations of 1 – 5
Compound 3, isolated as a yellow amorphous powder, had the molecular formula
C28H32O15 on the basis of the pseudo-molecular ion at m/z 631.1637 ([M þ Na]þ) in the

Helvetica Chimica Acta – Vol. 94 (2011)
679
HR-ESI-MS. Together with the MS analysis, NMR data led to the conclusion that 3 was
1
an 1-O-Me derivative of 2. The H- and 13C-NMR spectra (Table 2) displayed MeO
signals at d(H) 3.68 (s) and d(C) 63.0. The 3J correlation (Fig. 1) observed between the
MeO H-atoms (d(H) 3.68) and C(1) (d(C) 160.4) supported the location of the MeO
moiety at C(1). Other HMBCs similar to those of 2 as shown in Fig. 1 established 3 as
3-hydroxy-2-(hydroxymethyl)-1-methoxy-9,10-anthraquinone 3-O-b-d-glucopyrano-
syl-(1 ! 6)-b-d-glucopyranoside1), named hedanthroside C.
Compound 4 was obtained also as a yellow amorphous powder. The molecular
formula, C29H34O16, was determined from the HR-ESI-MS signal at m/z 661.1749
1
([M þ Na]þ). The aromatic region of the H-NMR spectrum (Table 2) indicated the
presence of four aromatic H-atoms. Their multiplicity, an ABC system at d(H) 7.66 (dd,
Table 2. 1H- and 13C-NMR Data for Compounds 3 and 41). d in ppm, J in Hz.
3a)
4b)
d(H)
d(C) d(H)
d(C)
C(1)
C(2)
C(3)
HꢀC(4)
HꢀC(5)
HꢀC(6)
HꢀC(7)
C(8)
160.4
130.6
160.4
109.3 7.64 (s)
161.7
132.4
161.5
110.0
120.6
136.6
120.6
160.9
183.8
184.7
135.2
123.2
124.0
136.5
64.6
6.96 (s)
7.20 – 7.30 (m)
7.20 – 7.30 (m)
7.28 – 7.33 (m)
7.38 – 7.48 (m)
126.4 7.62 (d, J ¼ 6.8)
134.2 7.66 (dd, J ¼ 7.6, 6.8)
135.4 7.42 (d, J ¼ 7.6)
126.9
C(9)
181.4
182.3
130.9
133.0
120.1
135.2
C(10)
C(11)
C(12)
C(13)
C(14)
MeOꢀC(1) 3.68 (s)
63.0 3.84 (s)
52.5 4.54 (d, J ¼ 11.4), 4.56 (d, J ¼ 11.4)
CH2ꢀC(2)
MeOꢀC(8)
Glc’
4.65 (d, J ¼ 11.4), 4.56 (d, J ¼ 11.4)
54.0
57.5
3.82 (s)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
CH2(6’)
Glc’’
5.06 (d, J ¼ 6.7)
100.1 5.28 (d, J ¼ 6.7)
76.3 3.38 – 3.44 (m)
75.6 3.65 – 3.70 (m)
73.5 3.64 – 3.68 (m)
69.1 3.84 – 3.90 (m)
101.9
77.6
74.6
70.7
77.1
69.7
3.28 – 3.32 (m)
3.50 – 3.55 (m)
3.50 – 3.55 (m)
3.50 – 3.55 (m)
4.04 (d, J ¼ 9.5), 4.32 (d, J ¼ 9.5)
68.2 4.30 (d, J ¼ 10.1), 3.97 (d, J ¼ 10.1)
HꢀC(1’’)
HꢀC(2’’)
HꢀC(3’’)
HꢀC(4’’)
HꢀC(5’’)
CH2(6’’)
4.52 (d, J ¼ 6.4)
103.2 4.46 (d, J ¼ 7.6)
76.1 3.34 – 3.38 (m)
75.7 3.62 – 3.70 (m)
73.0 3.34 – 3.42 (m)
70.0 3.30 – 3.34 (m)
104.6
77.8
77.3
71.4
75.0
62.5
3.50 – 3.55 (m)
3.50 – 3.55 (m)
3.30 – 3.55 (m)
3.30 – 3.55 (m)
3.75 (d, J ¼ 11.3), 3.90 (d, J ¼ 11.3)
61.2 3.92 (d, J ¼ 10.5), 3.71 (d, J ¼ 10.5)
a) Recorded in D2O. b) Recorded in CD3OD and D2O.

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Helvetica Chimica Acta – Vol. 94 (2011)
J ¼ 7.6, 6.8), 7.62 (d, J ¼ 6.8), and 7.42 (d, J ¼ 7.6), and a 1-H singlet at d(H) 7.64,
suggested that ring A is monosubstituted with a trisubstituted ring C. Presence of two
MeO groups was evident from signals at d(H) 3.84 (s, HꢀC(1a)) and 3.82 (s, HꢀC(8a)).
The HMBCs from HꢀC(4) (d(H) 7.64) and HꢀC(5) (d(H) 7.62) to the C(10)¼O group
(d(C) 184.7), as well as the presence of the cross-peaks between both of the two MeO
groups and the C(9)¼O group (d(C) 183.8) confirmed that two of the MeO groups were
located at C(1) and C(8), respectively. Other HMBCs as shown in Fig. 1 led to the
structure 3-hydroxy-2-(hydroxymethyl)-1,8-dimethoxy-9,10-anthraquinone 3-O-b-d-
glucopyranosyl-(1 ! 6)-b-d-glucopyranoside1), named hedanthroside D, for 4.
Compound 5, obtained as a yellow amorphous solid, was analyzed for C28H30O16 by
means of HR-ESI-MS (m/z 645.1443 ([M þ Na]þ)). The aromatic region of the
1H-NMR spectrum (Table 1) was similar to that of 1, revealing a non-substituted ring A
1
and a trisubstituted ring C. Comparison of the H- and 13C-NMR chemical shifts of 5
with those of 1 further revealed the presence of an ester CO group (d(C) 163.7) and a
MeO group (d(H) 3.86) in 5 instead of a Me group in 1. The HMBC spectrum showed
correlations from the the MeO H-atoms to ester CO (d(C) 163.7), and from HꢀC(4)
(d(C) 7.50) to C(2) (d(C) 117.0), C(13) (d(C) 111.1), C(10) (d(C) 181.2), and C(3)
(d(C) 159.8). Comparing 1H- and 13C-NMR data (Table 1) and key HMBCs of
compound 5 with those of 1, the only difference was that the substituent at C(2) was a
COOMe group instead of a Me group in 1. Thus, compound 5 was determined as 1,3-
dihydroxy-2-(methoxycarbonyl)-9,10-anthraquinone 3-O-b-d-glucopyranosyl(1 ! 6)-
b-d-glucopyranoside1), named hedanthroside E.
Optically active compound 6, [a]D20 ¼ ꢀ12.7 (c ¼ 0.15, H2O), was isolated as an
almost white amorphous powder, with the molecular formula C21H24O13 determined by
means of HR-ESI-MS (m/z 507.1104 ([M þ Na]þ)). The IR spectrum exhibited
absorption bands due to OH (3444 cmꢀ1), ester CO (1641 cmꢀ1), and alkene groups
(1384 and 1078 cmꢀ1). The 1H- and 13C-NMR spectra of 6 (Table 3) showed signals of a
furan-2-carboxy group (d(H) 6.74 (dd, J ¼ 3.5, 0.8), 7.48 (d, J ¼ 3.5), and 7.85 (d, J ¼
0.8); d(C) 146.1, 122.2, 115.1, 150.6, and 162.8), which were similar to those of synthetic
compounds with a furan-2-carboxy group reported in [13]. The 1H,1H-COSY
correlations (Fig. 2) from HꢀC(5’’) through HꢀC(4’’) to HꢀC(3’’), in combination
Table 3. 1H- and 13C-NMR Data (D2O) of Compound 61). d in ppm, J in Hz.
d(H)
d(C)
d(H)
d(C)
HꢀC(1) 5.29 (d, J ¼ 6.7)
HꢀC(3) 7.28 (s)
C(4)
99.6 HꢀC(1’) 4.85 (d, J ¼ 6.8)
150.9 HꢀC(2’) 3.32 – 3.38 (m)
117.4 HꢀC(3’) 3.53 – 3.57 (m)
101.3
75.4
78.3
72.2
78.8
HꢀC(5) 3.14 (dd, J ¼ 4.7, 6.6) 47.5 HꢀC(4’) 3.42 – 3.46 (m)
HꢀC(6) 4.67 (d, J ¼ 4.6)
HꢀC(7) 6.09 (s)
C(8)
83.5 HꢀC(5’) 3.44 – 3.48 (m)
134.7 CH2(6’) 3.89 (dd, J ¼ 12.0, 1.4), 3.72 (dd, J ¼ 12.0, 5.6) 63.4
143.0 C(2’’)
146.1
122.2
115.1
150.6
162.8
HꢀC(9) 3.23 (dd, J ¼ 7.1, 6.7) 48.9 HꢀC(3’’) 7.48 (d, J ¼ 3.5)
CH2(10) 5.13 (s)
65.6 HꢀC(4’’) 6.74 (dd, J ¼ 3.5, 0.8)
178.3 HꢀC(5’’) 7.85 (d, J ¼ 0.8)
C¼O
C(11)

Helvetica Chimica Acta – Vol. 94 (2011)
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Fig. 2. Key HMBC (H ! C) and 1H,1H-COSY (—) correlations of 6 and 7
with HMBCs (Fig. 2) from HꢀC(5’’) to C(3’’) and C(2’’), and from HꢀC(4’’) to C(2’’),
further confirmed the presence of a furan-2-carboxy moiety in 6.
The remaining 16 13C-NMR signals were attributed to the iridoid glycoside skeleton
of scandoside [8]. The scandoside skeleton was further confirmed by the NOESY
spectrum (Fig. 3) in which correlations were observed between HꢀC(5) and HꢀC(9),
and between HꢀC(6) and HꢀC(1). The glucopyranosyl moiety was at C(1), established
by means of HMBC between HꢀC(1’) and C(1) (d(C) 99.6). The furan-2-carboxy
group was at C(10), corroborated by means of the HMBC between CH2(10) (d(H)
5.13) and C¼O (d(C) 162.8). From these data, the structure of 6 was elucidated as rel-
(1R,4aR,5S,7aR)-7-[(furan-2-yl-carboxy)methyl]-1-(b-d-glucopyranosyloxy)-5-hydroxy-
1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylic acid, named hediridoside A.
Fig. 3. Selected ROESY correlations of 6 and 7
Compound 7 was obtained as a white, amorphous powder, and its molecular
formula was deduced as C23H32O15 by HR-ESI-MS (m/z 571.1660 ([M þ Na]þ)). The
IR spectrum exhibited absorption bands for OH (3446 cmꢀ1) and CO (1633 cmꢀ1)
groups. The 1H- and 13C-NMR spectra (Table 4) of 7 not only showed signals
characteristic for an iridoid skeleton at d(H) 5.74 (d, J ¼ 3.6, HꢀC(1)), 7.59 (s,
HꢀC(3)), and 6.55 (d, J ¼ 1.5, HꢀC(7)); and at d(C) 98.0 (C(1)), 155.0 (C(3)), and
114.7 (C(4)), but also displayed additional glucopyranosyl and rhamnopyanosyl
resonances (d(H) 5.05 (HꢀC(1’)), 4.85 (HꢀC(1’’)), 1.36 (d, J ¼ 6.2, HꢀC(6’’)); d(C)
101.0 (C(1’)), 103.7 (C(1’’)), 19.3 (C(6’’))). Different from most iridoid glycosides

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Helvetica Chimica Acta – Vol. 94 (2011)
Table 4. 1H- and 13C-NMR Data (D2O) of Compound 71). d in ppm, J in Hz.
d(H)
d(C)
d(H)
d(C)
HꢀC(1)
HꢀC(3)
C(4)
5.74 (d, J ¼ 3.6)
7.59 (s)
98.0
155.0
114.7
35.1
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
5.05 (overlapped)
3.36 (dd, J ¼ 9.2, 8.4)
3.53 (dd, J ¼ 9.2, 9.2)
3.84 (dd, J ¼ 9.2, 9.2)
3.40 – 3.44 (m)
101.0
75.1
78.1
72.8
72.6
HꢀC(5)
3.35 – 3.39 (m)
CH2(6)
2.48 (ddd, J ¼ 18.2, 7.5, 2.2, Ha),
2.88 (ddd, J ¼ 18.2, 7.5, 2.2, Hb)
6.55 (d, J ¼ 1.5)
40.8
HꢀC(7)
142.8
CH2(6’)
4.06 (br. d, J ¼ 10.8),
3.78 (dd, J ¼ 10.8, 5.6)
4.85 (overlapped)
4.00 – 4.04 (m)
3.62 – 3.68 (m)
3.44 – 3.50 (m)
70.0
C(8)
142.0
49.4
176.1
172.7
54.4
HꢀC(1’’)
HꢀC(2’’)
HꢀC(3’’)
HꢀC(4’’)
HꢀC(5’’)
Me(6’’)
103.7
72.7
77.8
74.7
71.4
19.3
HꢀC(9)
C(10)
C(11)
MeO
3.30 – 3.34 (m)
3.80 (s)
3.76 – 3.80 (m)
1.36 (d, J ¼ 6.2)
obtained from the Hedyotis genus, compound 7 had a C(10)OOH (d(C) 176.1), and not
a HOꢀCH2(10) group.
The HMBC spectrum showed cross-peaks between the anomeric H-atoms HꢀC(1’)
with C(1), and HꢀC(1’’) with C(6’). These interactions supported the presence of a
sugar moiety rha-(1 ! 6)-glc, and its linkage to C(1) of the iridoid aglycon. Acid
hydrolysis of 7 afforded glucose and rhamnose, which were identified by TLC
comparison with authentic samples. The glucose and rhamnose isolated from the
hydrolysate gave optical rotations [a]2D0 ¼ þ42.3 (c ¼ 0.90, H2O) and [a]D20 ¼ þ6.3 (c ¼
1.10, H2O), respectively, indicating that they were d-glucose and l-rhamnose.
In the ROESY NMR spectrum (Fig. 3) of 7, cross-peaks from HꢀC(5) to HbꢀC(6),
from HꢀC(5) to HꢀC(9), from HꢀC(9) to HbꢀC(6), and from HaꢀC(6) to HꢀC(1)
indicated the b-, b-, and a-orientations of HꢀC(5), HꢀC(9) and HꢀC(1), respectively.
Accordingly, the structure of compound 7 was determined as 1,4a,5,7a-tetrahydro-4-
(methoxycarbonyl)-1-[(6-O-a-l-rhamnopyanosyl-b-d-glucopyranosyl)oxy]cyclopenta-
[c]pyran-7-carboxylic acid, named hediridoside B.
Anthraquinones and iridoids are the characteristic chemical constituents of the
genus Hedyotis. The anthraquinone glycosides we obtained are common anthraqui-
nones with a substituent at C(2), which is biogenetically reasonable. Most iridoid
glycosides obtained from the Hedyotis genus contain the skeleton of scandoside,
geniposide, deacetyl asperulosidic acid, and deacetyl asperuloside [2], and several
derivatives have been isolated from the genus, such as the benzoylated geniposide
derivatives [14], 10-acetylscandoside methyl ester [2], etc. Furan-2-carboxy moiety is a
usual functional group in synthetic compounds, but rarely occurred in the plant
secondary metabolites. Hediridoside A (6) is the first iridoid glycoside with a furan-2-
carboxy moiety. Furthermore, hediridoside B (7) is the first iridoid glycoside with a
carboxy group at C(10), found in the genus Hedyotis.
Since the plants from the genus Hedyotis were reported to possess anti-
inflammatory and anticancer activities, we evaluated the effects of compounds 1 – 7
as well as of the six isolated known compounds on TNFa-induced transcriptional

Helvetica Chimica Acta – Vol. 94 (2011)
683
activity of NF-kB, using a luciferase reporter gene assay [15]. However, none of the
compounds exhibited any significant effect on basal NF-kB transcription.
We are grateful to the National Science and Technology Major Protect ꢂKey New Drug Creation and
Manufacturing Programꢃ, P. R. China (Number: 2009ZX09301-001), the State Key Laboratory
(SIMM0907 KF-04), and the National Natural Science Foundation of China (No. 3090185).
Experimental Part
General. Column chromatography (CC): silica gel H (200 – 300 mesh; Qingdao Marine Chemical
Ltd., Qingdao, P. R. China), Chromatorex C18 and C8 (20 – 45 mm; Fuji Silysia Chemical Ltd.), NH-silica
gel (20 – 45 mm; Fuji Silysia Chemical Ltd.), MCI gel CHP-20P (75 – 150 mm; Mitsubishi Chemical
Industries Co., Ltd.), TSK gel Toyopearl HW-40F (30 – 60 mm; Toso Co., Ltd.), Sephadex LH-20 (20 –
80 mm; Amersham Pharmacia Biotech AB), and Diaion HP20 (Mitsubishi Chemical Industries Co.,
Ltd.). Reversed-phase (RP) HPLC: Agilent 1100 series system equipped with YMC-Pack ODS-A
(250 ꢁ 20 mm, S-5 mm, 12 nm) column. TLC: Precoated SiO2 GF254 plates (Yantai Huiyou Inc., Yantai,
P. R. China). Optical rotations: Perkin-Elmer 341 polarimeter. UV and IR spectra: Shimadzu UV-2450
and Perkin-Elmer 577 spectrophotometer, resp. NMR Spectra: Varian Mercury NMR spectrometer, at
400 MHz for 1H and 100 MHz for 13C. LR- and HR-ESI-MS: Finnigan LCQ-DECA and Waters
Micromass Q-TOF Ultima Globe spectrometer, resp.
Plant Material. The stems of Hedyotis hedyotidea (Rubiaceae) were collected from Ningming, in
Guangxi Province, R. P. China, in September 2006, and authenticated by Prof. Heming Yang. A voucher
specimen (No. SIMMGX51) is deposited with the Herbarium of Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, P. R. China.
Extraction and Isolation. Air-dried stems of Hedyotis hedyotidea (1 kg) were collected and powered.
The material was extracted three times with aq. acetone (70%) at r.t. (10 l, 3 ꢁ , each for 1 week). Solvent
was removed under reduced pressure to obtain a crude extract (98 g), which was suspended in H2O and
partitioned with CHCl3. The H2O fraction (78 g) was subjected to CC (MCI gel; H2O, 10, 50, 70, and
100% MeOH gradiently) to give five fractions, Frs. A – E. Fr. A (27 g) was further submitted to CC
(Sephadex LH-20; MeOH/H2O 0 to 20%) to afford two fractions, Frs. A1 – A2. Fr. A1 (0.9 g) was
subjected to CC (C8; MeOH/H2O 5 to 10%) to give compound 6 (17 mg). Fr. A2 (1.3 g) was subjected to
CC (HW-40F; MeOH/H2O 5 to 15%) to afford scandoside (26 mg). Isolation of Fr. B (13 g) by repeated
CC (C8; MeOH/H2O 8 to 40%) afforded compound 7 (27 mg) and borreriagenin (18 mg). Fr. C (7.0 g)
which was loaded on a MCI gel column (MeOH/H2O, 15 to 50%) finally was separated by HW-40F
(MeOH/H2O 10 to 40%) to afford two fractions, Frs. C1 – C2. Fr. C1 (1.1 g) was purified by CC (C8;
MeOH/H2O 15 to 25%) to give methyl deacetylasperulosidate (34 mg). Fr. C2 (0.9 g) was passed
through a C18 column with MeOH/H2O (30 to 45%) to afford asperulosidic acid methyl ester (35 mg).
Fr. D (9.9 g) was submitted to CC (C8; MeOH/H2O 35 to 42%), followed by HW-40F CC (MeOH/H2O
32%), and further separation by RP-HPLC (YMC-Pack ODS-A (250 ꢁ 20 mm); MeOH/H2O 30%; flow
rate of 2.0 ml/min) to yield compound 3 (15 mg; tR 12.0 – 14.0 min ) and 4 (20 mg; tR 15.3 – 17.2 min).
Fr. E (13.0 g) was loaded on MCI gel column (MeOH/H2O 50%) to afford compound 2 (100 mg) and
Fr. E2, which was purified by RP-HPLC (MeOH/H2O 50%; flow rate of 2.0 ml/min) to yield compound 1
(27 mg; tR 4.1 – 5.9 min), 1,3-dihydroxy-2-(hydroxymethyl)-9,10-anthraquinone 3-O-b-d-glucopyrano-
side (13 mg; tR 9.0 – 11.0 min) and compound 5 (4 mg; tR 15.3 – 16.8 min). The CHCl3 fraction (18 g) was
subjected to CC (SiO2; petroleum ether (PE)/Me2CO 35 :1 ! 5 :1) to give three fractions, Frs. a – c). Fr. c
was concentrated and subjected to CC (SiO2; CHCl3/AcOEt 10 :1) and repeated prep. TLC (CHCl3/
Me2CO 50 :1) to yield rubiadin (8; 12 mg).
Hedanthroside A (¼ 9,10-Dihydro-4-hydroxy-3-methyl-9,10-dioxoanthracen-2-yl 6-O-b-d-Glucopy-
ranosyl-b-d-glucopyranoside; 1). Yellow amorphous powder. [a]2D3 ¼ ꢀ37.0 (c ¼ 0.20, MeOH). UV
1
(MeOH): 208 (4.00), 268 (4.05), 353 (3.13). IR (KBr): 3363, 1668, 1631, 1591. H- and 13C-NMR: see
Table 1. ESI-MS (pos.): 601.2 ([M þ Na]þ). HR-ESI-MS: 601.1539 ([M þ Na]þ, C27H30NaO1þ4 ; calc.
601.1533).

684
Helvetica Chimica Acta – Vol. 94 (2011)
Hedanthroside B (¼ 9,10-Dihydro-4-hydroxy-3-(hydroxymethyl)-9,10-dioxoanthracen-2-yl 6-O-b-d-
Glucopyranosyl-b-d-glucopyranoside; 2). Orange amorphous powder. [a]2D3 ¼ ꢀ33.0 (c ¼ 0.20, MeOH).
UV (MeOH): 203 (4.04), 256 (3.92), 357 (3.01). IR (KBr): 3403, 1660, 1631, 1591. 1H- and 13C-NMR: see
Table 1. ESI-MS (pos.): 617.3 ([M þ Na]þ). HR-ESI-MS: 617.1482 ([M þ Na]þ, C27H30NaO1þ5 ; calc.
617.1482).
Hedanthroside C (¼ 9,10-Dihydro-3-(hydroxymethyl)-4-methoxy-9,10-dioxoanthracen-2-yl 6-O-b-d-
Glucopyranosyl-b-d-glucopyranoside; 3). Yellow amorphous powder. [a]2D3 ¼ ꢀ35.0 (c ¼ 0.25, MeOH).
1
UV (MeOH): 210 (3.99), 269 (4.15), 360 (3.00). IR (KBr): 3403, 1672, 1577. H- and 13C-NMR: see
Table 2. ESI-MS (pos.): 631.2 ([M þ Na]þ). HR-ESI-MS: 631.1637 ([M þ Na]þ, C28H32NaO1þ5 ; calc.
631.1639).
Hedanthroside D (¼ 9,10-Dihydro-3-(hydroxymethyl)-4,5-dimethoxy-9,10-dioxoanthracen-2-yl 6-O-
b-d-Glucopyranosyl-b-d-glucopyranoside; 4). Yellow amorphous powder. [a]2D3 ¼ ꢀ34.0 (c ¼ 0.17,
1
MeOH). UV (MeOH): 206 (4.08), 258 (4.12), 357 (2.87). IR (KBr): 3423, 1627, 1690, 1585. H- and
13C-NMR: see Table 2. ESI-MS (pos.): 661.2 ([M þ Na]þ). HR-ESI-MS: 661.1749 ([M þ Na]þ,
C29H34NaOþ16 ; calc. 661.1745).
Hedanthroside E (¼ Methyl 3-[(6-O-b-d-Glucopyranosyl-b-d-glucopyranosyl)oxy]-9,10-dihydro-1-
hydroxy-9,10-dioxoanthracene-2-carboxylate; 5). Yellow amorphous solid. [a]2D3 ¼ ꢀ32.0 (c ¼ 0.20,
1
MeOH). UV (MeOH): 207 (3.83), 265 (4.15), 354 (2.69). IR (KBr): 3372, 1731, 1673, 1633, 1591. H-
and 13C-NMR: see Table 1. ESI-MS (pos.): 645.2 ([M þ Na]þ). ESI-MS (neg.): 621.9 ([M ꢀ H]ꢀ). HR-
ESI-MS: 645.1443 ([M þ Na]þ, C28H30NaO1þ6 ; calc. 645.1432).
Hediridoside A (¼ rel-(1R,4aR,5S,7aR)-7-{[(Furan-2-ylcarbonyl)oxy]methyl}-1-(b-d-glucopyrano-
syloxy)-1,4a,5,7a-tetrahydro-5-hydroxycyclopenta[c]pyran-4-carboxylic Acid; 6). White amorphous
powder. [a]2D0 ¼ ꢀ12.7 (c ¼ 0.15, H2O). UV (MeOH): 259 (3.91). IR (KBr): 3444, 1641, 1384, 1078.
1H- and 13C-NMR: see Table 3. ESI-MS (pos.): 507.1 ([M þ Na]þ). ESI-MS (neg.): 483.5 ([M ꢀ H]ꢀ).
HR-ESI-MS: 507.1104 ([M þ Na]þ, C21H24NaO1þ3 ; calc. 507.1115).
Hediridoside B (¼ rel-(1R,4aR,7aR)-1,4a,5,7a-Tetrahydro-4-(methoxycarbonyl)-1-[(6-O-a-l-rham-
nopyranosyl-b-d-glucopyranosyl)oxy]cyclopenta[c]pyran-7-carboxylic Acid; 7). White amorphous pow-
der. [a]2D0 ¼ ꢀ4.0 (c ¼ 0.125, MeOH), UV (MeOH): 238 (3.62). IR (KBr): 3446, 1633. 1H- and 13C-NMR:
see Table 4. ESI-MS (pos.): 571.2 ([M þ Na]þ). ESI-MS (neg.): 547.5 ([M ꢀ H]ꢀ). HR-ESI-MS:
571.1660 ([M þ Na]þ, C23H32NaO1þ5 ; calc. 571.1639).
Enzymatic Hydrolysis of 1. Compound 1 (15 mg) was dissolved in H2O (10 ml), b-cellulase (15 mg)
was added, and the soln. was kept at 378 for 2 d. The mixture was extracted with AcOEt, and the aq.
phase was compared with an authentic sugar sample by co-TLC (AcOEt/MeOH/H2O/AcOH 13 :3 :3 :4;
Rf 0.40 for glucose). Identification of d-glucose was carried out by comparing the optical rotation of the
liberated glucose with that of an authentic sample of d-glucose ([a]2D0 ¼ þ52).
Acidic Hydrolysis of 7. A soln. of compound 7 (8 mg) in 5% H2SO4/EtOH was refluxed for 3 h. The
mixture was neutralized and concentrated in vacuo to remove the alcohol, and extracted with AcOEt.
The aq. layer was evaporated and separated over a C8 and a NH-SiO2 column. Glucose and rhamnose
were separated, and compared with authentic samples by co-TLC (AcOEt/MeOH/H2O/AcOH
13 :3 :3 :4, Rf 0.44 for glucose and 0.62 for rhamnose). Identification of d-glucose and l-rhamnose
was carried out by comparing the optical rotations of the liberated glucose and rhamnose with those of
authentic samples of d-glucose ([a]2D0 ¼ þ52) and l-rhamnose ([a]D20 ¼ þ7.2 ) .
Bioassay. A HEK293 cell line was stably transfected with a kB-luciferase reporter gene. For the
luciferase assay, the cells were seeded into 24-well plate at ca. 90% confluency and were pretreated with
the tested compounds (10 mm) for 1 h. After stimulation of the cells by TNF-a (25 IU/ml) for 5 h, equal
cell numbers were collected for the assay, and the luciferase activity was measured by a luminometer
using a luciferase assay system (Promega, Shanghai, P. R. China).
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Received July 19, 2010