New iridoid glycoside from Gratiola officinalis

Article abstract of DOI:10.1080/10286020.2012.734299

A new iridoid glycoside, 1β,6β-di-O-trans-cinnamoyl-9-O-β-D- glucopyranosyl-3-iridene-5β-ol (1), along with four known compounds loliolide (2), β-sitosterol-3-O-β-D-glucopyranoside (3), betulinic acid (4), and β-amyrin (5), was isolated from the aerial parts of Gratiola officinalis L. The structure of the new compound was deduced on the basis of 1D ¹H and ¹³C NMR and 2D HMQC, HMBC, and COSY experiments, and mass spectrometric techniques (EI-MS and HR-EI-MS). The relative configuration of 1 was assigned by comparative analysis of the NMR spectral data with known analogs, together with NOESY experiments.

Full text of DOI:10.1080/10286020.2012.734299

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New iridoid glycoside from Gratiola
officinalis
Liaqat Ali a b , Tania Shamim Rizvi a , Manzoor Ahmad c & Farzana
Shaheen a
a HEJ Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi , Karachi ,
75270 , Pakistan
b Department of Biological Sciences and Chemistry , College of
Arts and Sciences, University of Nizwa , Nizwa , 616 , Oman
c Department of Chemistry , University of Malakand , Chakdara ,
Dir (L) , Pakistan
Published online: 08 Nov 2012.
To cite this article: Liaqat Ali , Tania Shamim Rizvi , Manzoor Ahmad & Farzana Shaheen (2012)
New iridoid glycoside from Gratiola officinalis , Journal of Asian Natural Products Research, 14:12,
1191-1195, DOI: 10.1080/10286020.2012.734299
To link to this article: http://dx.doi.org/10.1080/10286020.2012.734299
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Journal of Asian Natural Products Research
Vol. 14, No. 12, December 2012, 1191–1195
NOTE
New iridoid glycoside from Gratiola officinalis
Liaqat Aliab*, Tania Shamim Rizvia, Manzoor Ahmadc and Farzana Shaheena*
aHEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Pakistan; Department of Biological Sciences and
b
c
Chemistry, College of Arts and Sciences, University of Nizwa, Nizwa 616, Oman; Department of
Chemistry, University of Malakand, Chakdara, Dir (L), Pakistan
(Received 23 May 2012; final version received 18 September 2012)
A new iridoid glycoside, 1b,6b-di-O-trans-cinnamoyl-9-O-b-D-glucopyranosyl-3-
iridene-5b-ol (1), along with four known compounds loliolide (2), b-sitosterol-3-O-b-
D-glucopyranoside (3), betulinic acid (4), and b-amyrin (5), was isolated from the
aerial parts of Gratiola officinalis L. The structure of the new compound was deduced
on the basis of 1D 1H and 13C NMR and 2D HMQC, HMBC, and COSY experiments,
and mass spectrometric techniques (EI-MS and HR-EI-MS). The relative configuration
of 1 was assigned by comparative analysis of the NMR spectral data with known
analogs, together with NOESY experiments.
Keywords: Scrophulariaceae; Gratiola officinalis; iridoid
1. Introduction
phytochemical investigations of medicinal
plants from Pakistan and Turkey [7–11], we
investigated G. officinalis and obtained one
new iridoid glycoside, 1b,6b-di-O-trans-
cinnamoyl-9-O-b-D-glucopyranosyl-3-iri-
dene-5b-ol (1), along with four known
compounds 2–5.
Gratiola officinalis L., belonging to the
family Scrophulariaceae, is a small herb
which usually grows in wet habitats such as
river valleys and has become much rarer in
the last decades because of drainage [1,2]. In
Europe, theaerialpartsofthisplanthavebeen
used for a wide variety of ailments, including
menstrual disorders as well as skin and liver
diseases [2]. For centuries, it was a popular
medicinal plant and is still being used in
homeopathic and in folk medicine as an
anthelmintic [2]. The herb apparently tastes
very bitter, and the bitter principles were
thought to be cardenolides and glycosides of
cucurbitacine [1]. Two groups of Russian and
Austrian workers reported the presence of a
number of flavon C-monoglycosides, cucur-
bitacine glycosides, and scutellarein deriva-
tives in G. officinalis [3–6]. In the course of
2. Results and discussion
The water suspension of the crude extract,
obtained from the aerial parts of the plant,
was partitioned against n-hexane, dichlor-
omethane (DCM), ethyl acetate, and n-
butanol fractions. By repeated column
chromatography of the DCM and ethyl
acetate fractions, five compounds were
obtained: 1b,6b-di-O-trans-cinnamoyl-9-
O-b-D-glucopyranosyl-3-iridene-5b-ol (1),
loliolide (2), b-sitosterol-3-O-b-D-gluco-
pyranoside (3), betulinic acid (4), and
*Corresponding authors. Email: malikhejric@gmail.com; afnan.iccs@gmail.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.734299
http://www.tandfonline.com

1192
L. Ali et al.
b-amyrin (5). The known compounds 2–5
were identified on the basis of comparison
with the published data [12–17].
skeleton [19] with cinnamoyl and glucosyl
moieties. It showed the signals for H-1 at d
5.18 as a broad singlet and the methylene
protons (H-7) resonating at d 2.79 as a
multiplet. Another doublet at d 1.09 (3H,
J ¼ 6.0 Hz) was assigned to the methyl
group, H-10. The presence of two olefinic
methine protons (H-3 and H-4) was also
indicated by two doublets at d 6.43 (1H, d,
J ¼ 6.6 Hz) and 5.73 (1H, d, J ¼ 6.6 Hz),
respectively, in the 1H NMR spectrum.
The two cinnamoyl moieties were inferred
from the signals of 10 aromatic protons at
d 7.05–6.55, whereas the trans olefinic
protons at d 7.58 and 6.26 appeared as
doublets with coupling constant 15.9 Hz.
The anomeric proton of the sugar moiety at
d 4.37 as a doublet (J ¼ 7.2 Hz) indicated
the b-linked sugar. The remaining protons
of the sugar moiety resonated between d
4.09 and 3.52. Furthermore, the methyle-
nic carbon C-60 at d 62.4 and four methine
carbons (C-20 to C-50) at d 76.1–70.4 also
supported the presence of b-linked sugar
in compound 1.
The new compound 1 was isolated as a
pale yellow gummy material from the ethyl
acetate soluble fraction by repeated column
chromatography over a silica gel column,
and thestructure was deducedonthe basisof
the spectroscopic methods. The IR spectrum
of compound 1 showed absorption bands for
hydroxyl and ester groups (3415 and
1708cm21) and aromatic moiety. The UV
spectrum showed absorption maxima at
203, 216, and 273 nm, characteristics of the
iridoid-enol ether system with a cinnamoyl
chromophore [18].
The molecular ion was established by
FAB-MS(-) atm/z623,correspondingtothe
formula, C33H35O12, for the deprotonated
ion. The molecular formula of compound 1,
C33H36O12, was further confirmed through
HR-FAB-MS showing the quasimolecular
ion at m/z 623.2187 [M-1]þ. The other
prominent ions in the EI-MS were observed
at m/z 316, 154, 137, and 85, which further
supportedthepresenceofaniridoidskeleton
with hydroxyl, glucosyl, and the cinnamoyl
moieties (Figure 1).
On acid hydrolysis with 10% H2SO4,
compound 1 gave the aglycone along with
two cinnamic acid molecules and a sugar
moiety that was identified as D-glucose by
Co-TLC with the authentic sample.
The 1H NMR spectrum of 1 also
indicated the presence of an iridoid
OH
R2O
Loss of OH
and two cinnamoyl
O
O
OGlu
H3C
OR3
H3C
OR1
R , R = cinnamoyl
= glucosyl
1
3
2
C15H24O7 (m/z = 316)
R
C33H36O12
(m/z = 624)
Loss of
deoxy
glucose
Loss of OH
O
O
OH
H3C
H3C
C9H13O (m/z = 137)
C9H14O2 (m/z = 154)
Figure 1. Key mass fragmentations of compound 1.

Journal of Asian Natural Products Research
1193
The 13C NMR spectrum (BB and
DEPT) also supported the presence of
two cinnamoyl groups. The signals for two
ester carbonyl carbons (C-900, C-9000) were
resonated at d 168.3, whereas those for
olefinic and aromatic carbons skeleton
were observed in the downfield region (d
148.0–116.3) with double intensity of the
aromatic carbon signals (Table 1).
The direct one-bond 1H–13C connectiv-
ities were inferred from the HMQC
spectrum, whereas the COSY and HMBC
interactions (Figure 2) helped in construct-
ing the structure of compound 1 by
assigning the position of the individual
groups on the iridoid skeleton. The
anomeric proton (H-10) showed HMBC
interactions with C-9 (d 81.7), C-20 (d 72.3),
and C-50 (d 76.1), indicating the attachment
of the sugar moiety at C-9 position.
Similarly, H-1 and H-6 showed interactions
with C-900 (d 168.3) and C-9000 (d 168.3),
respectively. On the basis of these obser-
vations, the attachments of the two cinna-
moyl groups were assigned at C-1 and C-6
positions of the iridoid nucleus.
The multiplicities of carbons were
determined through the DEPT exper-
iments, whereas the relative stereochem-
istry of compound 1 was deduced on the
basis of the NOESY interactions. The
1H–1H NOESY correlations of Me-10
Table 1. 1H and 13C NMR spectral data for compound 1.
C. No.
13C (d)
1H (d)
Multiplicity
HMBC correlation
1
3
4
5
103.1
146.2
104.2
76.2
5.18, br s
6.43, d, J ¼ 6.6 Hz
CH
CH
CH
C
9, 900
4
5.73, d, J ¼ 6.6 Hz
3, 5
6
7
8
9
70.6
36.5
49.3
81.7
4.91, m
2.79, m
3.27, m
CH
CH2
CH
C
5, 9, 9000
9, 10
10
10
18.5
1.09, d, J ¼ 6.0 Hz
4.37, d, J ¼ 7.2 Hz
CH3
CH
CH
CH
CH
CH
CH2
C
CH
CH
CH
CH
CH
CH
CH
C
8
104.2
72.3
73.8
70.4
76.1
9, 20
20
30
3.90–3.52
Overlap H-20 to H-50
40
500
600
100
200
300
400
500
600
700
8
62.4
4.09, m; 3.98, m
131.5
121.3
123.2
127.5
123.2
121.3
148.0
116.3
168.3
131.5
121.3
123.2
127.5
123.2
121.3
148.0
117.1
168.3
7.05, dd, J ¼ 7.6, 2.0 Hz
6.75, dd, J ¼ 7.6, 6.9 Hz
6.55, t, J ¼ 6.9 Hz
6.75, dd, J ¼ 7.6, 6.9 Hz
7.05, dd, J ¼ 7.6, 2.0 Hz
7.58, d, J ¼ 15.9 Hz
100, 800
900, 700
6.26, d, J ¼ 15.9 Hz
900
1000
2000
3000
4000
5000
6000
7000
8000
9000
C
7.01, dd, J ¼ 7.6, 2.0 Hz
6.82, dd, J ¼ 7.6, 6.9 Hz
6.55, t, J ¼ 6.9 Hz
CH
CH
CH
CH
CH
CH
CH
C
6.82, dd, J ¼ 7.6, 6.9 Hz
7.01, dd, J ¼ 7.6, 2.0 Hz
7.58, d, J ¼ 15.9 Hz
1000, 8000
9000, 7000
6.26, d, J ¼ 15.9 Hz

1194
L. Ali et al.
O
O
OH
H
4
6
3
7
8
O
1
10
O
H3C
H
HO
O
H
HO
HO
O
O
1
HO
1H
13C HMBC
1H NOESY
1H
1H
1H
COSY
Figure 2. Key COSY, HMBC, and NOESY interactions for compound 1.
with H-1 and H-6 were helpful in assign-
ment of the relative stereochemistry of C-1
and C-6, whereas the remaining positions
were confirmed on the basis of biogenetic
considerations and by comparing the NMR
spectral data with the reported data for
iridoids [20,21]. Compound 1 was closely
related to the known compound, harpago-
side, earlier reported from Harpagophy-
tum procumbens [20,21], with the
difference of the number of cinnamoyl
moieties and the position of the sugar
group in the skeleton.
values are reported in ppm (d) units and
the coupling constants (J) are given in Hz.
For TLC, pre-coated aluminum sheets
(silica gel 60F-254, E. Merck) were used.
Visualization of the TLC plates was
achieved under UV at 254 and 366 nm
and by spraying with the ceric sulfate
reagent. MeOH:DCM 1–5% was used as
solvent system.
3.2 Plant material
The plant G. officinalis L. (Scrophular-
iaceae) was collected from Swat (Pakistan)
in 2003 and was identified by Mr Mehboob
ur Rahman, Assistant Professor, Depart-
ment of Botany, Govt Jehanzeb Post
Graduate College, Saidu Sharif, Swat,
K.P.K., Pakistan. A voucher specimen
(OG-14) has been deposited at the
herbarium of the department.
The above spectral studies thus fully
supported the structure of 1b,6b-di-O-
trans-cinnamoyl-9-O-b-D-glucopyrano-
syl-3-iridene-5b-ol for compound 1 as a
new constituent from G. officinalis.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a
JASCO DIP 360 polarimeter (Easton, MD,
USA). IR spectra were recorded on a
Bruker VECTOR 22 spectrophotometer.
EI-MS and HR-EI-MS were recorded on
mass spectrometers JEOL JMS HX 110
3.3 Extraction and isolation
The air dried and powdered aerial parts
(G. officinalis 7 kg) were extracted exhaus-
tively with 80% ethanol–water at room
temperature to get 400 g of the crude
extract. The residue was suspended in
water and partitioned in n-hexane (10 g),
DCM (25 g), ethyl acetate (20 g), and
n-butanol (120 g) extracts. The ethyl
1
(Akishima, Tokyo, Japan). H NMR and
13C NMR spectra were recorded on Bruker
NMR spectrometers operating at 300 MHz
(75 MHz for 13C). The chemical shifts

Journal of Asian Natural Products Research
1195
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subjected to further fractionation and
purification. On repeated silica gel column
chromatography using 1% MeOH:DCM,
the ethyl acetate extract yielded com-
pounds 1 (4.7 mg) and 2 (7.5 mg). The
DCM extract was subjected to column
chromatography on silica gel by using
various polarity solvent systems which
gave 16 sub-fractions.
The semi-pure fractions 7–12 were
again loaded on silica gel and eluted with
MeOH:DCM. Compounds 3 (12 mg) and 4
(3.5 mg) were purified from fraction 12 by
eluting with MeOH:DCM (2:98) and
(3:97), respectively, and compound 5
(4 mg) was purified by repeated silica gel
column chromatography eluted with
MeOH:DCM (5:95).
3.3.1 1b,6b-di-O-Trans-cinnamoyl-9-O-
b-D-glucopyranosyl-3-iridene-5b-ol (1)
30
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CH3OH); UV (MeOH) lmax (log 1): 203
(3.94), 216 (3.74), 273 (3.13) nm; IR
(KBr): nmax 3415, 2922, 1705, 1635, 1450,
1395 cm21. 1H NMR (300 MHz, CD3OD):
see Table 1; 13C NMR (CD3OD, 75 MHz):
see Table 1; FAB-MS: m/z 623 [M-1]þ;
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Acknowledgments
The authors are grateful to Mr Mehboob ur
Rahman, Assistantt Professor, Department of
Botany, Govt Jehanzeb Postgraduate College,
Saidu Sharif, Swat, K.P.K., Pakistan, for help in
the collection and identification of the plant and
to the Higher Education Commission (Pakistan)
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