Two acylated flavone glucosides from Andrographis serpyllifolia

Article abstract of DOI:10.1016/S0031-9422(99)00113-2

Two new acylated flavone glucosides, skullcapflavone I 2'-O-β-D-(3_-E- cinnamoyl)glucopyranoside and skullcapflavone I 2'-Oβ-D-(2_-E- cinnamoyl)glucopyranoside, together with skullcapflavone I 2'-O-β-D- glucopyranoside and andrographidine C have been isolated from the whole plant of Andrographis serpyllifolia. Structural elucidation of the glycosides was achieved by various NMR techniques including ¹H-¹H COSY, HMQC, HMBC and ROESY experiments, FAB-mass spectrometry, acid hydrolysis and saponification.

Full text of DOI:10.1016/S0031-9422(99)00113-2

Phytochemistry 52 (1999) 147±151
Two acylated ¯avone glucosides from Andrographis serpyllifolia
b
a
A.G. Damu , B. Jayaprakasam , D. Gunasekar *, A. Blond , B. Bodo
a
a,
b
a
Natural Products Division, Department of Chemistry, Sri Venkateswara University, Tirupati, 517 502, India
Laboratoire de Chimie des Substances Naturelles, URA 401 CNRS, Museum National d'Histoire Naturelle, 63 rue Bu€on, 75005, Paris, France
b
Received 8 January 1999; accepted 10 February 1999
Abstract
Two new acylated ¯avone glucosides, skullcap¯avone I 2'-O-b-D-(30-E-cinnamoyl)glucopyranoside and skullcap¯avone I 2'-O-
b-D-(20-E-cinnamoyl)glucopyranoside, together with skullcap¯avone I 2'-O-b-D-glucopyranoside and andrographidine C have
been isolated from the whole plant of Andrographis serpyllifolia. Structural elucidation of the glycosides was achieved by various
1
1
NMR techniques including H± H COSY, HMQC, HMBC and ROESY experiments, FAB-mass spectrometry, acid hydrolysis
and saponi®cation. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Andrographis serpyllifolia; Acanthaceae; Whole plant; Acylated ¯avone glucosides; Skullcap¯avone I 2'-(30-E-cinnamoyl)glucoside;
Skullcap¯avone I 2'-(20-E-cinnamoyl)glucoside
1
. Introduction
spectrum of 1, which showed signals for all the 32 car-
bons of the molecule. The UV spectrum exhibited
absorption maxima at 273, 321 (sh) and 352 (sh) nm,
which was typical of ¯avones with 5,7,8-trioxygenation
Andrographis serpyllifolia Wt.Ic (Acanthaceae) is a
trailing and rooting procumbent herb widely distribu-
ted throughout Deccan and Carnatic regions of South
India (Gamble, 1956). Previous chemical examination
of the leaves, stems and roots of A. serpyllifolia
a€orded serpyllin, apigenin 7,4'-dimethyl ether and
(
Govindachari et al., 1968; Gupta, Taneja, Dhar &
Atal, 1983; Giessman, 1962). Addition of sodium acet-
ate did not cause any change in the band II absorption
maximum indicating the absence of a free hydroxyl at
C-7. The IR spectrum of 1 apart from hydroxyl
tectochrysin (Govindachari, Parthasarathy, Pai
&
Kalyanaraman, 1968). In the present communication
we describe the isolation and structure elucidation of
two new acylated ¯avone glucosides 1 and 2 in ad-
dition to skullcap¯avone I 2'-glucoside (3) and andro-
graphidine C (4).
� 1 � 1
(
bands, showed an additional carbonyl absorption band
3393 cm ) and carbonyl (1616 cm ) absorption
� 1
at 1678 cm indicating the presence of an ester group
conjugated with a double bond (Karl, Muller &
Pedersen, 1976; Aritomi, 1963).
1
The H NMR spectrum of 1 showed a down®eld
signal at d 12.68, exchangeable with D O, which could
2
2
. Results and discussion
be attributed to a chelated C-5 hydroxyl group. A
sharp singlet at d 6.59 was assigned to H-6 as it
showed correlation with C-5 (156.6 ppm) in its HMBC
spectrum (Fig. 1). It also exhibited two methoxyl sig-
nals at d 3.91 and 3.80, and the former methoxyl
group was assigned to C-7 as it showed long range
correlation with H-6 in its ROESY spectrum (Fig. 1).
The other methoxyl group at d 3.80 was placed at C-8
as it resonated at 61.1 ppm, a chemical shift character-
Compound 1, obtained as a yellow amorphous pow-
+
der, showed [M+H]
HRFABMS corresponding to the molecular formula
C H O . This was corroborated by the C NMR
3
at m/z 607.1810 in its
13
2
30 12
*
7499.
Corresponding author. Tel.: +91-8574-50666; fax: +91-8574-
2
0
031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00113-2

1
48
A.G. Damu et al. / Phytochemistry 52 (1999) 147±151
down®eld shift (Markham & Geiger, 1994) of 1.78
ppm observed for H-30 (d 5.08, dd, J = 9.2, 9.2 Hz)
compared with that of 3 (d 3.30, dd, J = 9.0, 9.0 Hz).
The attachment of cinnamoyl moiety at C-30 in 1 was
further supported by the presence of a cross peak
between H-30 (d 5.08) of the glucose and carbonyl car-
bon (165.7 ppm) of the cinnamoyl residue in its
HMBC spectrum (Fig. 1) and two strong NOEs
observed for H-30 (d 5.08) with H-10 (d 5.35) and H-50
(
d 3.55) in its ROESY spectrum (Fig. 1). On the basis
of the foregoing studies, the structure of 1 was estab-
lished as skullcap¯avone I 2'-O-b-D-(30-E-cinnamoyl)-
glucopyranoside (1).
Compound 2, obtained as yellow amorphous pow-
+
der, showed a [M+H] peak at m/z 607.1807 in its
HRFABMS consistent with the molecular formula
C H O . The UV absorption maxima of 2 (268, 300
32
30 12
(sh), 348 (sh) nm) resembled that of 1 indicating the
presence of an identical ¯avone skeleton to that of 1.
The IR spectrum was characterised by a strong carbo-
2
-3
Fig. 1. Signi®cant HMBC (
lations for compound 1.
J
C11) (4) and ROESY (
) corre-
�
1
nyl ester band at 1710 cm , in addition to the usual
istic of a di-ortho substituted methoxyl group (Iinuma,
Matsuura & Kusuda, 1980). The presence of a sharp
� 1
carbonyl band of ¯avone at 1609 cm and hydroxyl
� 1
absorption band at 3457 cm . The acid and alkaline
1
3
1
singlet at d 7.01 was assigned to H-3 based on C- H
HMQC evidence. It also displayed the characteristic
pattern of a 2'-oxygenated ring B (Kuroyanagi, Sato,
Ueno & Nishi, 1987) with signals at d 7.87 (dd,
J = 7.9, 1.7 Hz, 1H), 7.56 (ddd, J = 8.5, 8.0, 1.7 Hz,
Table 1
13
C NMR spectral data (ppm) of compounds 1±3 (75 MHz, DMSO-
6
d )
C
1
2
3
1
1
H), 7.42 (m, 1H) and 7.26 (ddd, J = 8.0, 7.9, 0.9 Hz,
H), which were assigned to the 6', 4', 3' and 5' pro-
2
3
4
161.0
110.2
182.3
103.9
156.6
95.9
160.8
110.4
181.7
103.8
156.5
95.7
160.8
110.1
182.2
103.8
156.5
95.8
158.3
128.3
149.0
120.1
155.4
115.4
132.9
122.0
128.7
56.3
61.0
99.9
73.1
76.5
69.4
77.0
60.4
±
tons, respectively.
An anomeric proton signal at d 5.35 (d, J = 7.9 Hz)
suggested the presence of a sugar residue with b-con-
4a
5
®
guration. The remaining signals at d 6.67 (d,
6
J = 16 Hz, 1H), 7.68 (d, J = 16 Hz, 1H), 7.42 (m, 3H)
and 7.73 (m, 2H) indicated a trans-cinnamoyl moiety
in 1 and its presence was further evidenced by the for-
mation of trans-cinnamic acid, D-glucose and an agly-
cone, skullcap¯avone I (1a) (Jalal, Overton & Rycroft,
7
158.5
128.5
149.1
120.4
155.2
115.6
133.1
122.3
129.0
56.5
158.1
128.2
149.0
121.5
155.1
116.3
132.9
122.8
129.1
56.1
8
8a
1'
2
3
4
5
'
'
'
'
1
979) when 1 was subjected to acid hydrolysis. The
glucose residue in 1 was found to be linked to C-2'
since a NOE was observed between H-10 and H-3' in
its ROESY spectrum and a cross peak between H-10
and C-2' was seen in its HMBC spectrum (Fig. 1).
Alkaline hydrolysis of 1 gave trans-cinnamic acid
and skullcap¯avone I 2'-O-b-D-glucoside (3) (Gupta,
Taneja & Dhar, 1996) indicating that the cinnamoyl
moiety was attached to the glucosyl residue. The cin-
namoyl residue in 1 was found to be linked to the C-
6'
7
8
1
2
-OMe
-OMe
61.1
99.6
60.6
99.5
0
0
a
71.4
77.9
73.2
73.6
30
40
a
67.4
76.8
69.8
77.5
5
6
1
2
0
0
1
60.2
60.5
134.2
128.3
129.0
130.3
144.2
118.7
165.7
133.4
128.0
128.5
130.2
144.5
117.0
164.8
3
shifted down®eld (Dorman Roberts, 1970;
0 hydroxyl of the glucose as this carbon signal was
1, 61
±
&
31, 51
41
±
Kamerling, De Bie & Vliegenthart, 1972; Yamasaki et
al., 1977; Agrawal & Bansal, 1989) by 1.40 ppm, while
the C-20 and C-40 signals were shifted up®eld by 1.70
and 2.0 ppm, respectively compared with 3 (Table 1).
The site of esteri®cation in 1 was con®rmed by a
±
71
81
9
1
±
±
±
a
Assignments exchangeable.

A.G. Damu et al. / Phytochemistry 52 (1999) 147±151
149
hydrolysis products of 2 were found to be identical
with those obtained from 1. From the molecular for-
mula and from the nature of the products obtained in
acid and alkaline hydrolysis, 2 should be an isomeric
glycoside of 1 di€ering in the point of attachment of
the cinnamoyl moiety to the glucose unit.
of acylated ¯avone glycosides in the genus
Andrographis.
3
. Experimental
1
3
3.1. General
The C NMR spectrum of 2 was similar to 1 (Table
) except in the carbon resonances of the glucose unit.
1
Mps were uncorr. IR spectra were recorded in KBr.
A close comparison of the sugar carbon signals in 2
with that of 3 (Table 1) showed that the cinnamoyl
moiety in 2 was located at C-20 as this carbon signal
was deshielded by 0.1 ppm whereas the C-10 and C-30
signals had moved up®eld by 0.4 and 2.9 ppm, respect-
1
13
H and C NMR spectra were determined on a
Bruker AC 300 spectrometer operating at 300.13 and
5.43 MHz, respectively using DMSO-d with TMS as
7
6
1
1
int. standard. H± H COSY, HMQC, HMBC and the
phase-sensitive ROESY (with 150 ms mixing time)
spectra were recorded using the standard pulse
sequences. FAB and HRFAB mass spectra were
obtained on a 700 JEOL mass spectrometer in thiogly-
cerol matrix. CC was performed on Acme silica gel
®ner than 200 mesh (0.08 mm). PC was carried out on
1
ively. Also, in the H NMR spectrum, the chemical
shift for H-20 (d 4.94, dd, J = 9.5, 8.1 Hz) was indica-
tive of acylation at C-20 of the glucose moiety
(
Birkofer, Kaiser, Hillges & Becker, 1969). The site of
acylation was further supported by the HMBC corre-
lation between the carbonyl carbon (164.8 ppm) of the
cinnamoyl moiety and H-20, and a strong NOE
observed for H-20 (d 4.94) with H-40 (d 3.31) in its
ROESY spectrum (Fig. 2). Thus, 2 was characterised
as skullcap¯avone I 2'-O-b-D-(20-E-cinnamoyl)gluco-
pyranoside.
Whatman No 1 using BAW (n-BuOH±HOAc±H O,
2
4:1:5).
3
.2. Plant material
The whole plant of A. serpyllifolia Wt.Ic was col-
lected in March 1996 at Talakona Hills, Andhra
Pradesh, India. A voucher specimen (DG-198) has
been deposited in the Herbarium of the Department of
Botany, Sri Venkateswara University, Tirupati.
Compounds 3 and 4 were identi®ed as skullcap¯a-
vone I 2'-O-b-D-glucopyranoside and andrographidine
C, respectively by acid hydrolytic studies and by direct
comparison of their spectral data with those earlier
reported from A. paniculata (Kuroyanagi et al., 1987;
Gupta et al., 1996).
3
.3. Extraction and isolation
The occurrence of 1 and 2 constitutes the ®rst report
Dried and powdered whole plant (2.5 kg) was suc-
cessively extracted with hexane, Me CO and MeOH.
2
The Me CO extract was concentrated in vacuo to give
2
a brown viscous residue. It was defatted with n-hexane
and C H . The residual brown solid (66 g) was sub-
6
6
jected to CC on silica gel and eluted with CHCl3,
CHCl ±EtOAc and EtOAc±MeOH mixtures. CHCl ±
3
3
EtOAc 1:1 and 2:8 frs yielded 1 (30 mg) and 2 (40 mg),
respectively. EtOAc±MeOH 9:1 and 8:2 frs gave 3
(
45 mg) and 4 (50 mg), respectively.
3.3.1. Skullcap¯avone I 2'-O-b-D-(20-E-
cinnamoyl)glucopyranoside (1)
Yellow amorphous powder (MeOH); mp 236±2378;
2
5
[
mode) m/z: 607.1810 ([M+H] ; C H O +H
a] � 0.098 (MeOH, c 5.0); HRFABMS (positive
D
+
32
30 12
requires 607.1815); FABMS (positive mode) m/z
+
+
(
(
l
rel. int): 607 [M+H] (32), 477 [M+H� cinnamoyl]
MeOH
+
2), 315 [M+H� cinnamoyl glucosyl]
(53); UV
nm (log e): 273 (4.84), 321 sh (4.52), 352 sh
4.36); (NaOMe) 275, 385; (NaOAc) 272, 320 sh, 352
max
(
sh; (NaOAc+H BO ) 272, 320 sh, 352 sh; (AlCl ) 280,
3
3
3
KBr
max
2-3
J
Fig. 2. Signi®cant HMBC (
lations for compound 2.
C11) (4) and ROESY (
) corre-
335, 392 sh; (AlCl +HCl) 273, 330 sh, 360; IR n
3
�
1
cm : 3393 (OH), 1678 (C1O), 1616 (C1O), 1570,

1
50
A.G. Damu et al. / Phytochemistry 52 (1999) 147±151
1
1
7
2
520; H NMR (DMSO-d ): d 12.68 (1H, s, OH-5),
acid was identi®ed by co-PC (BAW). The sugar in the
aq. layer was identi®ed as D-glucose by co-PC (BAW).
6
.87 (1H, dd, J = 7.9, 1.7 Hz, H-6') 7.73 (2H, m, H-
1, 61), 7.68 (1H, d, J = 16.0 Hz, H-71), 7.56 (1H,
ddd, J = 8.5, 8.0, 1.7 Hz, H-4'), 7.42 (4H, m, H-3', 31,
3.5. Alkaline hydrolysis of compounds 1 and 2
4
(
(
1, 51), 7.26 (1H, ddd, J = 8.0, 7.9, 0.9 Hz, H-5') 7.01
A soln of the glycoside (5 mg) in 1% KOH (5 ml)
was re¯uxed for 2 h. The reaction mixt was acidi®ed
with 1N HCl and extracted with Et O followed by n-
1H, s, H-3), 6.67 (1H, d, J = 16.0 Hz, H-81) 6.59
1H, s, H-6), 5.35 (1H, d, J = 7.9 Hz, H-10), 5.08 (1H,
2
dd, J = 9.2, 9.2 Hz, H-30), 3.91 (3H, s, OMe-7), 3.80
BuOH. The Et O extract was washed with H O, evapd
2
2
(
2
3H, s, OMe-8), 3.71 (1H, m, H-60a), 3.55 (4H, m, H-
to dryness, redissolved in MeOH (0.5 ml) and sub-
mitted to PC when trans-cinnamic acid was identi®ed,
while the residue obtained from the n-BuOH extract
was cryst. from MeOH to yield skullcap¯avone I 2'-O-
b-D-glucoside (4 mg), mp. 260±2628.
1
3
0, 40, 50, 60b); C NMR in Table 1.
3
.3.2. Skullcap¯avone I 2'-O-b-D-(20-E-
cinnamoyl)glucopyranoside (2)
Yellow amorphous powder (MeOH); mp 196±1978,
[a] � 0.158 (MeOH, c 4.0); HRFABMS (positive
mode) m/z: 607.1807 ([M+H] ; C H O +H
2
5
D
+
3
2
30 12
Acknowledgements
requires, 607.1815); FABMS (positive mode) m/z
(
+
rel. int): 607 [M+H] (100), 477 (1), 315 (44); UV
MeOH
max
BJ acknowledges Council of Scienti®c and Industrial
Research, New Delhi for ®nancial assistance. We are
grateful to Mr J. P. Brouard, Laboratoire de Chimie
des Substances Naturelles, MNHN, Paris and Michel
Â
Becchi, Centre de Spectrometrie de masse du, CNRS,
Lyon, France for providing mass spectral data.
l
(
(
nm (log e): 268 (4.45), 300 sh (3.94), 348 sh
3.51); (NaOMe) 278, 380; (NaOAc) 268, 300 sh, 348;
NaOAc+H BO ) 268, 300 sh, 348; (AlCl ) 275, 325
3
sh, 362; (AlCl +HCl) 265, 270, 305, 360; IR n
cm : 3457 (OH), 1710 (C1O ester), 1609 (C1O),
3
3
KBr
max
3
�
1
1
1
586, 1511, 1448; H NMR (DMSO-d ): d 12.68 (1H,
6
s, OH-5), 7.66 (1H, dd, J = 7.5, 1.8 Hz, H-6'), 7.57
1H, ddd, J = 8.5, 7.5, 1.8 Hz, H-4'), 7.41 (1H, dd,
J = 8.5, 0.9 Hz, H-3'), 7.38 (2H, m, H-21, 61), 7.35
1H, m, H-41), 7.31 (2H, m, H-31, 51), 7.23 (1H, ddd,
(
References
(
J = 7.5, 7.5, 0.9 Hz, H-5'), 7.21 (1H, d, J = 16.0 Hz,
H-71), 6.41 (2H, s, H-3, 6), 6.32 (1H, d, J = 16.0 Hz,
H-81), 5.21 (1H, d, J = 8.1 Hz, H-10), 4.94 (1H, dd,
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3
6
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+
(
[
+
13
M+H� glucosyl] (100); C NMR in Table 1; MS,
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&
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+
(
[
polia: isolation and structure of serpyllin,
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a new ¯avone.
+
1
M+H� glucosyl] (100); MS, UV, IR and H and
1
3
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(
13
3.4. Acid hydrolysis of compounds 1 and 2
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(
and recryst. from MeOH to give pale yellow needles of
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& Nishi, K. (1987).
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1
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(
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