Phenolic glycosides from the Chinese liverwort reboulia hemisphaerica

Article abstract of DOI:10.1002/hlca.201000423

Four new phenolic glycosides, named rebouosides A-D (1-4, resp.), along with three known ones 2-(3,4-dihydroxyphenyl)ethyl 2-O-α-L- rhamnopyranosyl-β-D-allopyranoside (5), 2-(3,4-dihydroxyphenyl)ethyl β-D-allopyranoside (6), 2-(3,4-dihydroxyphenyl)ethyl β-D- glucopyranoside (7), and a nucleoside, inosine (8), were isolated from Chinese liverwort Reboulia hemisphaerica. Their structures were elucidated by acidic hydrolysis and extensive spectroscopic methods, including 2D-NMR techniques.

Full text of DOI:10.1002/hlca.201000423

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Helvetica Chimica Acta – Vol. 94 (2011)
Phenolic Glycosides from the Chinese Liverwort Reboulia hemisphaerica
a
a
a
a
a b
by Li-Ning Wang ), Dong-Xiao Guo ), Shu-Qi Wang ), Chang-Sheng Wu ), Mujeeb Ur Rehman ) ),
a
and Hong-Xiang Lou* )
a) Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University,
No. 44 West Wenhua Road, Jinan 250012, P. R. China
(
phone: þ 86-531-88382012; fax: þ 86-531-88382019; e-mail: louhongxiang@sdu.edu.cn)
b) H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Science,
University of Karachi, Karachi 75270, Pakistan
Four new phenolic glycosides, named rebouosides A – D (1 – 4, resp.), along with three known ones
-(3,4-dihydroxyphenyl)ethyl 2-O-a-l-rhamnopyranosyl-b-d-allopyranoside (5), 2-(3,4-dihydroxyphe-
2
nyl)ethyl b-d-allopyranoside (6), 2-(3,4-dihydroxyphenyl)ethyl b-d-glucopyranoside (7), and a nucleo-
side, inosine (8), were isolated from Chinese liverwort Reboulia hemisphaerica. Their structures were
elucidated by acidic hydrolysis and extensive spectroscopic methods, including 2D-NMR techniques.
Introduction. – Reboulia hemisphaerica (L.) Raddi. is a thalloid liverwort of the
Aytoniaceae, widely distributed in moist areas of low altitude around the world [1]. It is
traditionally used in China as a folk medicine for the treatment of skin ulcer, burns,
pain, and swelling from injuries, external bleeding, and related diseases [2]. Samples of
R. hemisphaerica from various origins have been chemically investigated previously
[
1][3 – 14], and a series of sesquiterpenoids and bis-bibenzyls have been isolated from
its lipophilic fractions [3][4]. In our continuing effort to search for new biologically
active natural products from liverworts [15 – 17], the phytochemical investigation of the
H O-soluble portion of R. hemisphaerica led to the isolation and identification of four
2
new phenolic glycosides, named rebouosides A – D (1 – 4), together with the four
known compounds 5 – 8 (Fig. 1). This is the third report on the isolation and structural
elucidation of H O-soluble phenylethanoid glycosides (¼2-arylethyl glycosides) from
2
liverworts. Similar compounds were isolated from Marchantia polymorpha and
Ricciocarpos natans [18][19].
Results and Discussion. – Dried plant material of R. hemisphaerica was extracted
with 95% EtOH and then partitioned with Et O and H O. The H O extract was
2
2
2
repeatedly chromatographed on silica gel and Sephadex LH-20 and then further
separated by HPLC on a reversed-phase column to yield compounds 1 – 8.
Compound 1 was obtained as a colorless, amorphous powder. Its molecular formula
was established as C H O by the quasimolecular-ion peak at m/z 471.1465 ([M þ
1
9
28 12
þ
Na] ) in the positive-ion-mode HR-ESI-MS. The IR spectrum (KBr) showed
absorption bands of OH groups (3424 cm ) and aromatic rings (1610, 1523, and
446 cm ). The H-NMR spectrum of 1 (Table 1) exhibited characteristic signals
ꢀ1
ꢀ1
1
1
attributable to a 2-(3,4-dihydroxyphenyl)ethoxy moiety, i.e., three aromatic H-atoms
ꢀ
2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 94 (2011)
1147
Fig. 1. Compounds 1 – 8, isolated from Reboulia hemisphaerica
(
ABX systems), a CH (b) group at d(H) 2.71 – 2.68 (m), and two nonequivalent H-
2
atoms of CH (a) at d(H) 3.97 – 3.93 and 3.63 – 3.59 (2m) derived from the aglycon.
2
Compound 1 thus belonged to the phenylethanoid class of natural products. Addi-
tionally, two anomeric H-atom resonances appeared at d(H) 4.70 (d, J ¼ 7.8 Hz,
HꢀC(1’)) of an allose (All) unit and at d(H) 4.39 (d, J ¼ 7.2 Hz, HꢀC(1’’)) of a xylose
13
(
(
Xyl) unit, indicating an O-glycosylglycoside structure of 1. The C-NMR data
Table 2) confirmed the O-glycosylglycoside sugar chain of 1 by exhibiting two
anomeric-C-atom resonances at d(C) 100.3 and 102.5, which showed HSQC cross-
peaks with the anomeric H-atoms of the All and Xyl unit, respectively. In the HMBC
spectrum of 1, the cross-peaks (Fig. 2) between the anomeric H-atom of the All unit at
d(H) 4.70 (HꢀC(1’)) and the C-atom at d(C) 71.9 (C(a) of the arylethyl moiety), in
combination with those between the H-atom at d(H) 3.51 (HꢀC(2’) of All) and the
anomeric C-atom of the Xyl unit at d(C) 102.5 (C(1’’)) determined the 2-O-
glycosylglycoside chain linkage. Accordingly, the structure of 1 was established as 2-
1
(
3,4-dihydroxyphenyl)ethyl 2-O-b-d-xylopyranosyl-b-d-allopyranoside ), for which
the trivial name rebouoside A is proposed.
Compound 2 was isolated as a colorless, amorphous powder, with the molecular
formula C H O , as determined from the HR-ESI-MS quasimolecular-ion peak at
1
9
28 12
þ
m/z 471.1466 ([M þ Na] ) together with analysis of NMR data (Table 1 and 2).
1
13
Analysis of the H- and C-NMR spectra indicated that 2 also possessed a 2-(3,4-
dihydroxyphenyl)ethoxy moiety and two sugar units, while acid hydrolysis afforded
xylose and glucose (Glc). Moreover, the clear correlations (Fig. 2) C(a)/HꢀC(l’) and
1)
The absolute configurations d or l of the sugar moieties are tentative.

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Helvetica Chimica Acta – Vol. 94 (2011)

Helvetica Chimica Acta – Vol. 94 (2011)
1149
Fig. 2. Key HMBCs (H ! C) of 1 – 4
C(2’)/HꢀC(1’’), indicated that the Xyl unit was attached to C(2’) of the Glc unit and the
latter to C(a) of the arylethyl moiety [20]. Therefore, the structure of compound 2
(
rebouoside B) was established as 2-(3,4-dihydroxyphenyl)ethyl 2-O-b-d-xylopyrano-
1
syl-b-d-glucopyranoside ).
Compound 3 was obtained as a colorless, amorphous powder. The molecular
formula was determined as C H O from the HR-ESI-MS quasimolecular-ion peak
2
0
30 12
þ
1
13
at m/z 485.1617 ([M þ Na] ). Analysis of the H- and C-NMR spectra (Table 1 and
2
) and the acid hydrolysis experiment suggested the presence of three fragments,
including a 2-(3,4-dihydroxyphenyl)ethoxy, a b-glucose, and an a-rhamnose (Rha)
unit. The sugar moieties were assigned according to the coupling-constant values of the
13
sugar H-atoms and their C-NMR data (Table 2) [21]. In the HMBC spectrum of 3,
the correlations HꢀC(1’) (Glc)/C(a), HꢀC(1’’) (Rha)/C(2’) (Glc) were observed
(
Fig. 2). On the basis of these results, 3 was established as 2-(3,4-dihydroxyphenyl)-
1
ethyl 2-O-a-l-rhamnopyranosyl-b-d-glucopyranoside ), and named rebouoside C.
Compound 4 was obtained as a colorless, amorphous powder. Its molecular formula
þ
C H O was deduced from the [M þ Na] peak at m/z 593.1840 in the HR-ESI-MS.
26
34 14
ꢀ1
The IR spectrum showed absorptions of OH groups (3355 cm ) and aromatic rings
ꢀ
1
1
13
(
1614, 1516, and 1456 cm ). The H- and C-NMR, COSY, and HMQC data (Tables 1
and 2) of 4 showed the presence of two ABX systems at d(H) 7.05 (d, J ¼ 8.9 Hz,

1
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Helvetica Chimica Acta – Vol. 94 (2011)
Table 2. 13C-NMR Data (150 MHz) of Compounds 1 – 4. d in ppm.
1a
)
2a)
3 )
b
4c)
134.4
152.5
117.9
116.2
154.4
120.0
36.9
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(a)
C(b)
131.9
117.4
144.9
146.3
116.3
121.6
71.9
131.7
131.0
117.3
144.8
146.3
116.4
121.4
72.3
116.5
143.8
142.2
116.2
121.0
70.6
36.7
36.8
34.4
33.9
All:
100.3
77.8
Glc:
103.2
83.7
Glc:
100.8
78.7
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
135.4
116.5
144.3
145.9
121.2
117.1
Glc:
103.2
75.3
78.3
71.5
78.1
62.6
70.3
68.8
75.5
63.2
Xyl:
102.5
74.7
77.6
70.3
77.9
71.4
78.0
62.7
Xyl:
106.1
75.9
77.5
71.3
75.6
69.4
75.6
60.6
Rha:
100.9
70.0
69.4
71.8
C(1’’)
C(2’’)
C(3’’)
C(4’’)
C(5’’)
C(6’’)
C(1’’’)
C(2’’’)
C(3’’’)
C(4’’’)
C(5’’’)
C(6’’’)
67.1
67.3
68.9
16.5
103.2
75.1
78.5
71.6
78.1
62.6
a) Recorded in CD OD. ) Recorded in D O. ) Recorded in (D )DMSO.
b
c
3
2
6
HꢀC(3)), 6.83 (dd, J ¼ 8.9, 2.4 Hz, HꢀC(4)), and 6.75 (d, J ¼ 2.4 Hz, HꢀC(6)) for a 2,5-
disubstituted phenyl moiety (ring A), and at d(H) 6.60 (d, J ¼ 1.8 Hz, HꢀC(2’)), 6.59
(
dd, J ¼ 1.8, 7.8 Hz, HꢀC(6’)), and 6.45 (d, J ¼ 7.8 Hz, HꢀC(5’)) for a 3,4-dihydroxy-
phenyl moiety (ring B). The NMR data of 4 were similar to those reported for a,b-
dihydrostilbene-2,4’,5-triol 2,5-di(b-d-glucopyranoside) [18]. Rings A and B of 4 were
connected via the fragment CH (a)CH (b), based on the HMBCs (Fig. 2) of CH (a) at
2
2
2
d(H) 2.92 – 2.87 and 2.85 – 2.80 with C(1), C(2), and C(6) at d(C) 134.4, 152.5, and
20.0, respectively, and correlations of CH (b) at d(H) 2.74 – 2.65 with C(1’), C(2’), and
1
2
C(6’) at d(C) 135.4, 116.5, and 117.1, respectively. Thus, the aglycone of 4 was deduced
to be bibenzyl-2,3’,4’,5-tetrol (¼4-[2-(2,5-dihydroxyphenyl)ethyl]benzene-1,2-diol).
The acid hydrolysis afforded only glucose. Two anomeric H-atoms at d(H) 4.61
(
1
HꢀC(1’’)) and 4.76 (HꢀC(1’’’)) displayed long-range correlations with C(2) (d(C)
52.5) and C(5) (d(C) 154.4), respectively, in the HMBC plot, which determined that
the glucose units were connected with the bibenzyl moiety at C(2) and C(5). Finally, 4

Helvetica Chimica Acta – Vol. 94 (2011)
1151
1
was determined as a,b-dihydrostilbene-2,3’,4’,5-tetrol 2,5-di(b-d-glucopyranoside) ),
which was given the trivial name rebouoside D.
The four known compounds were identified as 2-(3,4-dihydroxyphenyl)ethyl 2-O-
a-l-rhamnopyranosyl-b-d-allopyranoside (5) [18], 2-(3,4-dihydroxyphenyl)ethyl b-d-
glucopyranoside (6) [22], 2-(3,4-dihydroxyphenyl)ethyl b-d-allopyranoside (7) [23],
and inosine (8), based on comparison of their NMR and MS data with those reported.
They were all obtained for the first time from this plant species.
This investigation was supported by grants from the National Natural Science Foundation of China
(
Nos. 30925038 and 30730109).
Experimental Part
General. TLC: precoated silica gel GF254 plates (Qingdao Marine Chemical Industry); eluent CHCl3/
MeOH 2 :1. Column chromatography (CC): silica gel (SiO ; 200 – 300 mesh; Qingdao Marine Chemical
2
Industry), Sephadex LH-20 gel (Pharmacia Biotech), MCI gel (CHP20P, 75 – 150 mm; Mitsubishi
Chemical Industries Ltd.), silica gel 100 C -reversed phase (40 – 63 mm; SigmaꢀAldrich.). Semi-prep.
18
HPLC: YMC-Pack ODS-A column (250 ꢁ 20 mm, 10 mm (spherical), 12 nm), with Agilent-1100-G1310A
isopump and Agilent-1100-G1314 detector (210 nm); mobile phase MeOH/H O 15 :85, flow rate 1.8 ml/
2
min. Optical rotations: Perkin-Elmer-241MC polarimeter. UV Spectra: Shimadzu-UV-2450 spectropho-
ꢀ
1
tometer; l (log e) in nm. IR Spectra: Thermo-Nicolet-670 spectrophotometer; KBr disks; ˜n in cm
.
max
max
1
13
1
13
H- and C-NMR Spectra: Bruker-Avance-DRX-600 spectrometer; at 600 ( H) or 150 MHz ( C); in
CD OD, (D )DMSO, or D O; d in ppm rel. to Me Si as internal standard, J in Hz; 2D spectra recorded
3
6
2
4
with standard pulse programs and acquisition parameters. MS: API-4000 triple-stage quadrupole
DECA
instrument for electrospray ionization (ESI) and Finnigan LC-Q
m/z.
mass spectrometer for HR-ESI; in
Plant Material. R. hemisphaerica was collected from Libo, Guizhou Province, P. R. China, in August
008, and authenticated by Prof. Yuan-Xin Xiong (School of Agricultural Sciences, Guizhou University,
2
P. R. China). A voucher specimen has been deposited with the School of Pharmaceutical Sciences,
Shandong University (accession number: TX-18-200807-RH), Shandong, P. R. China.
Extraction and Isolation. The air-dried parts of R. hemisphaerica (380 g) were ground and extracted
four times exhaustively with (each 2.0 l) 95% aq. EtOH at r.t. The combined EtOH extract was
concentrated to yield a semi-solid (32.2 g). The residue was partitioned between Et O (5 ꢁ 200 ml) and
2
H O (200 ml). The aq. portion (10.3 g) was purified further by CC (MCI gel, H O ! MeOH) to give
2
2
fractions eluting with H O (6.4 g) and MeOH (3.5 g). A portion (3.4 g) of the MeOH fraction was
2
subjected to CC (SiO , gradient cyclohexane/acetone 100 :10 ! 0 :100): Fractions 1 – 6. Fr. 3 (0.23 g) was
2
subjected to CC (Sephadex LH-20, CHCl /MeOH 1:1): Frs. 3.1 – 3.6. Fr. 3.3 (25 mg) was further
3
fractionated by semi-prep. HPLC (see General): 6 (3.2 mg; tR 25.3 min), 7 (2.4 mg; tR 27.1 min), and 8
(
2.1 mg; tR 30.5 min). Fr. 4 (0.18 g) was applied to CC (Sephadex LH-20, CHCl /MeOH 1:1): Frs. 4.1 –
3
4
.5. Fr. 4.3 (24 mg) was further fractionated by semi-prep. HPLC (see General): 1 (2.1 mg; t 33.8 min), 2
R
(
2.4 mg; tR 38.6 min), 3 (5.6 mg; tR 40.1 min), and 5 (6.3 mg; tR 47.9 min). Fr. 5 (0.12 g) was subjected to
CC (Sephadex LH-20, CHCl /MeOH 1:1): Frs. 5.1 – 5.4. Fr. 5.2 (3.2 mg) was further fractionated by
3
semi-prep. HPLC (see General): 4 (1.3 mg).
1
Rebouoside A (¼2-(3,4-Dihydroxyphenyl)ethyl 2-O-b-d-Xylopyranosyl-b-d-allopyranoside ); 1):
2
0
Colorless, amorphous powder. [a] ¼ ꢀ36 (c ¼ 0.5, MeOH). UV (MeOH): 202 (3.84), 274 (3.17). IR
D
1
13
ꢀ
(
KBr): 3424, 1610, 1523, 1446. H- and C-NMR: Tables 1 and 2. ESI-MS (neg.): 447 ([M ꢀ H] ). HR-
þ þ
ESI-MS: 471.1465 ([M þ Na] , C H NaO ; calc. 471.1473).
19
28
12
1
Rebouoside B (¼2-(3,4-Dihydroxyphenyl)ethyl 2-O-b-d-Xylopyranosyl-b-d-glucopyranoside ); 2):
2
0
Colorless, amorphous powder. [a] ¼ ꢀ27 (c ¼ 0.9, MeOH). UV (MeOH): 202 (3.96), 274 (3.16). IR
D
1
13
ꢀ
(
KBr): 3423, 1610, 1523, 1446. H- and C-NMR: Tables 1 and 2. ESI-MS (neg.): 447 ([M ꢀ H] ). HR-
þ þ
ESI-MS: 471.1466 ([M þ Na] , C H NaO ; calc. 471.1473).
19
28
12

1
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Helvetica Chimica Acta – Vol. 94 (2011)
Rebouoside C (¼2-(3,4-Dihydroxyphenyl)ethyl 2-O-(6-Deoxy-a-l-mannopyranosyl)-b-d-glucopyr-
1
20
D
anoside ); 3): Colorless, amorphous powder. [a]
¼ ꢀ56 (c ¼ 0.9, MeOH). UV (MeOH): 202 (3.82), 274
1
13
(
3.14). IR (KBr): 3423, 1609, 1522, 1446. H- and C-NMR: Tables 1 and 2. ESI-MS (neg.): 461 ([M ꢀ
ꢀ
þ
þ
12
H] ). HR-ESI-MS: 485.1617 ([M þ Na] , C H NaO ; calc. 485.1629).
20
30
Rebouoside D (¼a,b-Dihydrostilbene-2,3’,4’,5-tetrol 2,5-Di(b-d-glucopyranoside) ¼ 2-[2-(3,4-Dihy-
1
20
D
droxyphenyl)ethyl]-1,4-phenylene Bis(b-d-glucopyranoside) ); 4): Colorless, amorphous powder. [a]
¼
1
ꢀ
37 (c ¼ 0.9, MeOH). UV (MeOH): 202 (3.94), 274 (3.74). IR (KBr): 3355, 1614, 1516, 1456. H- and
13
þ
þ
C-NMR: Tables 1 and 2. ESI-MS (pos.): 593 ([M þ Na] ). HR-ESI-MS: 593.1840 ([M þ Na] ,
þ
C H NaO ; calc. 593.1841).
26
34
14
Acid Hydrolysis of Compounds 1 – 4. Each compound 1 – 4 (each 0.5 mg) was hydrolyzed with 0.5n
HCl for 3 h at 1008. The mixture was then diluted with H O (10 ml) and extracted with CH Cl (3 ꢁ 5 ml).
2
2
2
The aq. layer was lyophilized to give a residue. The sugars were identified by co-TLC with a authentic
samples. TLC (CHCl /AcOH/H O 30 :35 :5): R value of glucose 0.26, of allose 0.33, of xylose 0.50, and of
3
2
f
rhamnose 0.75.
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Received August 31, 2010