Gastroprotective flavonoid constituents from Oroxylum indicum Vent.

Article abstract of DOI:10.1016/j.bmcl.2009.11.024

Chemical investigation of the stem bark of Oroxylum indicum resulted in the isolation and characterization of two new flavonoid glycosides (1, 2), along with seven known compounds (3-9). Their structures were established on the basis of extensive spectroscopic (IR, MS, 2D NMR) data analysis and by the comparison with spectroscopic data reported in the literature. In addition, all the compounds were tested for their ulcer protective effects against various gastric ulceritis inducing models in rats.

Full text of DOI:10.1016/j.bmcl.2009.11.024

Bioorganic & Medicinal Chemistry Letters 20 (2010) 117–120
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Gastroprotective flavonoid constituents from Oroxylum indicum Vent.
a
b
a
a
c
T. Hari Babu , K. Manjulatha , G. Suresh Kumar , A. Hymavathi , Ashok K. Tiwari ,
b
a
a,
*
Muraleedhar Purohit , J. Madhusudana Rao , K. Suresh Babu
Natural Products Laboratory, Organic Division-I, Indian Institute of Chemical Technology, Hyderabad 500 607, India
Department of Pharmaceutical Chemistry, University of Gulbarga, Gulbarga, India
a
b
c
Division of Pharmacology, Indian Institute of Chemical Technology, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical investigation of the stem bark of Oroxylum indicum resulted in the isolation and characteriza-
tion of two new flavonoid glycosides (1, 2), along with seven known compounds (3–9). Their structures
were established on the basis of extensive spectroscopic (IR, MS, 2D NMR) data analysis and by the com-
parison with spectroscopic data reported in the literature. In addition, all the compounds were tested for
their ulcer protective effects against various gastric ulceritis inducing models in rats.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 27 August 2009
Revised 3 November 2009
Accepted 7 November 2009
Available online 12 November 2009
Keywords:
Oroxylum indicum
Bignoniaceae
Flavonoid glycosides
Anti gastric ulcer activity
Gastric or peptic ulcer constitutes major ailment that affects hu-
man gastrointestinal tract and presents major global health prob-
lem both in terms of morbidity and mortality. Consumption of
‘Syonaka’, has been used for centuries for the treatment of various
gastric disorders. Herein, we report the isolation and structure
elucidation of two new flavonoid glycosides (1,2), along with the
8
1
alcohol and analgesics have been identified as major risk factors
responsible for acute gastric mucosal injury in humans and pre-
sents life-threatening hemorrhages that require surgical interven-
seven known compounds, dihydroiso-
a
-lapachone (3), 7-O-meth-
0
ylchrysin (4), 5-hydroxy- 4 ,7-di methoxy flavone (5), dihydro
oroxylin A (6) oroxylin A (7), chrysin (8), and baicalein (9) as con-
stituents from hexane and acetone extracts of O. indicum and re-
port for the first time gastroprotective properties of these
extracts and isolates in rats under various ulcer inducing condi-
2
,3
tion.
Several other factors like heredity, smoking, elevated
calcium level, usage of corticosteroids in high dosage etc., are also
responsible for elevated risk of gastric ulceration.4
9
Presently prophylactic options for patients suffering from gas-
trointestinal ulceration include antacids, sucralfate, histamine-2-
tions like aspirin induced gastric ulceritis (AIU) , ethanol induced
1
0
11
ulcers (EIU) , cold restrain induced (CRU) gastric ulcer and pylo-
1
2
receptor antagonists (H
antagonists) and proton-pump inhibitors. However, it has been ob-
served that long-term use of H -RAs and proton-pump inhibitors
2
RAs), prostaglandins, muscarinic (M
1
-
rus ligation induced gastric lesions (PLU). This report further pro-
vides scientific basis for the use of O. indicum in traditional Indian
medicinal preparations meant for antigastric ulcer activities. Ex-
cept compounds7, 8, 9 all other compounds 3–6 are being reported
for the first time from this plant (Fig. 1).
The dried stem-bark (5 kg) were ground, extracted three times
with hexane and acetone in a soxhlet apparatus for 72 h. The
resulting extracts were evaporated to dryness under reduced pres-
sure, affording syrupy residues. The hexane extract was subjected
to column chromatography over a silica gel column (60–120 mesh,
150 ꢀ 15 cm) and eluted with a step wise gradient of chloroform:
MeOH, (100, 99.5:0.5, 99:1, 98:2 by volume) to afford a total of
30 fractions of 50 ml each. Column fractions were analyzed by
TLC (Silica Gel 60 F254, chloroform/MeOH, 92:8), and fractions
with similar TLC patterns were combined to give two major frac-
2
induces hyperplasia in enterochromal-like (ECL) cells, which may
lead to further relapse of ulcer disease and induction of gastric can-
5
cer. Moreover, most of these therapeutics are largely empirical
6
and have several other adverse side-effects also. Therefore, there
is a need of more effective and safer antigastric ulcer agents with
lesser side effects.
Plants have been used in traditional medicine systems all over
the world since the dawn of human civilization. Also, plant derived
metabolites have provided important basis for discovery and
development of modern therapeutics.7
In Indian traditional medicine, the roots as well as stem bark of
Oroxylum indicum (Family: Bignoniaceae), commonly known as
tions (F
1
and
F
2
). Flash chromatography of fraction
F
1
(
331.88 mg) by elution with hexane/EtOAc (80:20), yielded dehy-
*
Corresponding author. Tel.: +91 40 27191881; fax: +91 40 27160512.
E-mail address: suresh@iict.res.in (K.S. Babu).
1
3
2
droiso-a lapachone (3) (0.120 g). Fraction F was purified on
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.024

1
18
T. Hari Babu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 117–120
OMe
O
O
COOMe
O
OMe
O
HO
HO
O
O
O
OH
OH
O
HO
O
OH
OH
O
OMe
OH
O
OH
Dihydro iso lapacone (3)
1
2
R2
OH
R1
O
R1
OH
O
O
O
OMe
OH
O
OH
OH
O
1
1
2
7 . R = OMe ( Oroxylin A )
4
5
. R = OMe, R = H
Dihydro oroxylin A (6)
1
1
2
8 . R = H ( Chrysin )
. R = OMe, R = OMe
1
9
. R = OH ( Baicalein )
Figure 1. Compounds (1–9) isolated from Oroxylum indicum.
silica gel column chromatography (100–200 mesh) eluted with
1
4
0
(
.5% MeOH in chloroform to afford 7-O-methyl chrysin(4)
0
15
COOMe
O
0.7 g), 5-hydroxy-4 ,7-dimethoxy-flavonoid (5) (0.12 g), dihydro
1
6
HO
HO
O
O
O
oroxylin A (6) (0.08 g) and eluted with 2% MeOH in chloroform to
1
7
17
18
afford oroxylin A (7) (3 g), chrysin (8) (2 g) and baicalein (9)
HO
(
(
(
5 g). Similarly, the dark yellow colored crude acetone extract
1 g) was subjected to column chromatography over silica gel
100–200 mesh, 300 g), and eluted with chloroform/MeOH in the
MeO
OH
Figure 2. Key HMBC correlations of compound 1.
increasing order of polarities to afford the three fractions. Succes-
sive flash chromatography of fraction I by elution with 4% MeOH
in chloroform to get the dihydrooroxylin 7-O-glucuronide-6 -methl
0
ester (1) (0.02 g) and fraction II, elution with 6% MeOH in chloro-
form yielded flavonoid derivative (2) (0.34 g).
The location of the glycosyl linkage was determined to be at C-7
since the anomeric proton signal at d 5.02 showed a HMBC correla-
tion with an oxygenated quaternary carbon at d 156.44, assignable
to C-7. The ‘D’ configuration of glucuronic acid was determined
after acid hydrolysis of 1 by isolating the sugar using preparative
HPLC and measuring the purified sugar’s optical rotation. Absolute
configuration of 1was deduced from the aglycone (dihydrooroxylin
A) moiety, which is known from the literature. Based on these
results, the structure of 1 was confirmed as methyl-3,4,5-trihy-
droxy-6-(5-hydroxy-6-methoxy-4-oxo-2-phenylchroman-7-yloxy)-
tetrahydro-2H-pyran-2-carboxylate and has been trivially named
as dihydrooroxylin A-7-O-methyl glucuronide.
25
Compound (1) was isolated as pale yellow solid with ½
a
ꢁ
ꢂ46.4
D
(
c 0.05 MeOH) and mp 165 °C. The IR spectrum indicated the pres-
ꢂ1
ꢂ1
ence of OH (3395 cm ), carbonyl (1618 cm ), ester carbonyl
ꢂ1
(
1735 cm ) and aromatic functional groups. UV absorption max-
ima at kmax 295 and 320 (sh) nm were indicative of a flavanone
1
9
structure. The molecular formula was determined as C23
24 11
H O
2
1
by HRESIMS, which provided a pseudomolecular ion peak at m/z
+
13
4
2
99.1535 (M +Na), in conjunction with its C NMR, displaying
3 resonances. A DEPT NMR experiment permitted differentiation
of 23 carbons into twelve methine, one methylene, two methoxyls,
two carbonyls and, six quaternary carbons, of which sixteen car-
bons were attributed to a flavonoid skeleton and six carbons to su-
gar moiety. Regarding the aglycone moiety, signals at d 5.46 (1H,
dd, J = 12.0, 3 Hz, H-2), d 3.14 (1H, dd, J = 17.5, 12.0 Hz, H-3ax),
Compound 2 was obtained as a white, amorphous powder with
24
the molecular formula C23H O11, determined on the basis of NMR
+
and HRESIMS data (m/z 499.1211 [M+Na] ). The IR spectrum dis-
ꢂ1
played absorption bands diagnostic of hydroxyl (3329 cm ), con-
1
ꢂ1
and d 2.82 (1H, dd, J = 17.5 3.0 Hz, H-3eq) in the H NMR spectrum
jugated ketone (1633 cm ) and aromatic functional groups,
1
13
(
in MeOH-d
4
+ CDCl
3
) together with the presence of carbonyl reso-
respectively. Examination of H and C NMR data of 2 indicated
that molecule consisted of a flavone and sugar moiety. The 1
NMR spectrum of 2 (MeOH-d + CDCl ) showed a pair of one-pro-
nance at d 198.78 confirmed the flavonone skeleton. Two multi-
plets at d 7.57–7.48 (2H), d 7.45–7.39 (3H), and correlations of H-
H
4
3
0
0
0
0
0
0
0
0
0
0
2
/C-1 , C-3 , C-2; H-3 /C-4 , C-5 , H-4 /C-5 , C-3 , C-6 in the HMBC
ton doublets d 6.42 (d, J = 2 Hz) and d 6.34 (d, J = 2 Hz), were as-
spectrum supported that 1 having unsubstituted ring B. Further
more, H NMR spectrum indicated a three-proton singlet at d
signed to H-6 and H-8, corresponding with the HMBC cross
1
H C
peaks between d 6.42/d 161.05 (C-5), 165.39 (C-7), 92.48 (C-8)
3
.85, assigned to aromatic methoxyl group at C-6. On the basis of
and d 6.34/d 98.04 (C-6), 158.76 (C-9), indicating a presence of
H
C
the above analyses of spectroscopic data, the flvanone aglycone
was suggested as 5,7-di hydroxyl-6-methoxyflavanone, which
was identical to 6, isolated from the same plant. Acid hydrolysis
a flavone with substitutions at C-5 and C-7. It also exhibited a sin-
glet at d 6.41 (1H, s), which was assigned to H-3. Further, the sig-
2
0
0
nals for two doublets at d 6.80 (1H, d, J = 8 Hz, H-3 ), d 6.70 (1H, d,
0
0
1
of 1 yielded glucuronic acid and dihydrooroxylin A (6), as deter-
mined by the TLC comparison with standards. The 1H NMR spec-
trum also revealed one three-proton singlet at d 3.75 assigned to
ester methoxyl group, based on the HMBC correlation to C-
J = 7.8 Hz, H-5 ) and a triplet at d 7.40 (1H, J = 8 Hz, H-4 ) in the H
NMR spectrum suggested the 2,6-disubstituted pattern in ring B.
Two signals at d 3.87 and d 3.82 suggested the presence of two aro-
matic methoxyl groups. These assignments are clearly supported
0
0
1
6
(170.75) (see Fig. 2).
by the H–1H COSY and HSQC experiments. Moreover, three

T. Hari Babu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 117–120
119
all the isolates displayed varying degrees of gastroprotective
potentials. Among them, chrysin (8) displayed better activity in
PLU and CRU models. Further, fractionation of active acetone ex-
tract led to the isolation of two new compounds (1,2) with the bet-
ter gastroprotective potential in various ulcer inducing models
(Fig. 4). Compound 1 displayed more potent activity than com-
pound 2. Though it is difficult to discuss the structure activity rela-
tionship criteria responsible for gastroprotective activities in this
set of compounds, presence of free phenyl ring (ring B) 6, 7, 8 ap-
pears important in imparting gastroprotective activity when com-
pared with 5. However, absence of methoxyl group at 6th position
in compound 8 may responsible for better activity in PLU and CRU
animal models (Fig. 4A and B). Additionally, presence of hydroxyl
group at 7th position in gastroprotection cannot be ruled out, as
its absence in compounds 4 and 2 drastically decreased gastropro-
tective activities. However, methoxy group at 7th position appears
to improve gastroprotection in EIU and AIU models (Fig. 4C and D)
as is observed with the compounds 4, 5 and 2. Furthermore, glycos-
MeO
O
MeO
OH
OH
O
O
OH
OH
OH
O
Figure 3. HMBC correlations of the compound 2.
multiplets at d 3.92–3.70, d 3.55–3.40, and d 3.10–2.90, together
with a characteristic signal at d 4.95 (1H, d, J = 7.8 Hz,) in
1
H
NMR spectrum confirmed the glycoside linkage. Acid hydrolysis
of 2 produced aglycon and -glucose as the sole sugar, as deter-
D
mined by TLC, GC and comparison with the authentic sugar sam-
ple. Finally, the HMBC correlation between the anomeric proton
at d 4.95 (1H, d, J = 7.8 Hz) and C-2 (d 158.37) indicated the pres-
-glucopyranosyl moiety, attached to C-2 of the agly-
con moiety through the anomeric carbon (Fig. 3).
0
0
ence of a b-
D
0
idation at 2 -position in compound 2 was observed to enhance the
In the HMBC spectrum of 2, the carbon skeleton suggested by
several diagnostic correlations (H-3/C-2, C-4; H-6/C-5, C-7, C-8;
activity than 5. It is important to note that glucuronization of hy-
droxyl group at 7th position in compound 6 significantly improved
the gastroprotective properties in all the ulcer induced models as
evident in the case of compound 1.
0
0
0
0
0
0
0
0
00
0
H-8/C-6, C-9; H-3 /C-1 , C-4 , C-5 ; H-4 /C-2 , C-3 , C-6 ; H-1 /C-2 ;
0
0
00
00
1
0
0
0
0
00
H-2 /C-1 , C-3 ; and H–1H COSY (H-3 /H-4 ; H-4 /H-5 ; H-1 /
0
0
00
00
22
H-2 ; H-5 /H-6 ). Based on the above data, compound 2 was
identified as 5- hydroxyl-7-methoxy-2-(2-methoxy-6-(3,4,5-trihy-
droxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yoloxy)phenyl)-
This communication observed that compound 1 with free phe-
nyl ring (ring B) together with glucuronide linkage is the most po-
tent compound with gastroprotective activities among all the
isolates and animal models studied, however, gastroprotective
contribution of other molecules cannot be ruled out taking in to ac-
count the holistic therapeutic approach of traditional medicinal
formulations. This is the first scientific report explaining gastropro-
tective properties in traditional Indian medicinal plant O. indicum
based on the chemical composition and isolation of new com-
pounds 1 and 2.
4H-chromen-4-one.
The Gastroprotective activities were investigated for crude ex-
tracts and the isolates against various gastric ulceration models
2
3,24
in Wistar rats.
At two random dosage of 100 mg and 250 mg/
kg body weights, hexane and acetone extracts displayed mild to
moderate gastroprotective activity (Fig 4). Acetone extract dis-
played better activity than hexane extract. As shown in Figure 4,
1
00
100
75
50
25
0
7
5
2
5
0
5
0
1
00
100
75
50
25
0
7
5
2
5
0
5
0
Figure 4. Ulcer protective potentials of the compounds isolated from O. indicum. (A) Pylorus ligation induced ulcer (PLU), (B) cold-restrain induced ulceritis (CRU), (C) ethanol
induced ulceritis (EIU) and (D) aspirin induced ulceration (AIU) models in rats. Values represent% protection against individual models (mean ± SD). HE1, AE1 and HE2, AE2
represent hexane and acetone extracts at the dose of 250 and 100 mg/kg body weight, respectively. Compounds were dosed at 25-mg/kg-body weight each. Percentage of
protection with standard drugs Omiprazole, Ranitidine Sucralfate and Diazepam were studied at the dose of 30 mg, 50 mg, 400 mg and 1 mg/kg body weight, respectively.

1
20
T. Hari Babu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 117–120
2
5
Sigma) (t
R
: 4.1 min) and measurement of their optical rotations (½
aꢁ
+26.4, c
Acknowledgment
D
0
.05 H O).
2
2
5
ꢂ46.4 (c 0.05 MeOH). 1H
2
1. Spectral data for 1: pale yellow solid, mp 165 °C; ½
aꢁ
D
Authors gratefully acknowledge keen interest of Dr. J. S. Yadav,
Director, IICT, Hyderabad, India.
NMR (300 MHz, CDCl3 + MeOH-d ): d 5.46 (1H, 12.0, 3.0 Hz, H-2), 3.14, (1H, dd,
4
J = 17.5, 12.0 Hz, H-3ax), 2.82 (1H, dd, J = 17.5 3.0 Hz, H-3eq), 3.85 (3H, s, OMe-
0
0
0
0
0
5
5
), 6.28 (1H, s, H-8), 7.57–7.48 (2H, m, H-2 , 6 ), 7.45–7.39 (3H, m, H-3 , 4 , 5 ),
0
0
00
00
00
.02 (1H, d, J = 7.8 Hz, H-1 ), 3.70–3.52 (3H, m, H-2 , 3 , 4 ), 4.02 (1H, d,
References and notes
00
00 13
J = 4.0 Hz, H-5 ), 3.78 (3H, s, OMe-6 ). C NMR (75 MHz, CDCl
3 4
+ MeOH-d ): d
8
0.89 (C-2), 44.37 (C-3), 198.78 (C-4), 159.26 (C-5), 132.13 (C-6), 156.44 (C-7),
0 0 0
96.15 (C-8), 159.88 (C-9), 105.37 (C-10), 139.98 (C-1 ), 127.48 (C-2 ,6 ), 129.85
1
2
.
.
Laine, L. Rev. Gastroenterol. Disord. 2004, 4, S33.
Roth, A. L. J. In Gasteroenterolgy; Bockers, H. L., Ed.; Saunders: Philadelphia,
0 0 0 00 00 00 00
(C-3 , 4 , 5 ), 101.52 (C-1 ), 77.00 (C-2 ), 76.75 (C-3 ), 74.33 (C-4 ), 72.77 (C-
+
00 00
5 ), 170.75 (C-6 ), 61.75 (OMe), 53.15 (OMe). ESIMS: m/z 499 [M+Na] .
+
1
974.
3
4
.
.
Salim, S. A. J. Pharm. Pharmacol. 1987, 39, 553.
Kumar, V.; Cotran, R.; Robbins, S., 6th ed.. In Pathogenic Basis of Disease; W.B.
Saunders, 1999; Vol. 18.
HRESIMS: m/z 499.1535 [M+Na] (calcd for C23
24
H O11, 499.1216).
22. Spectral data for 2: 1H NMR (300 MHz, CDCl
+ MeOH-d
): d 6.42 (1H, s, H-3),
3
4
0
6.42 (1H, d, J = 2 Hz, H-6), 6.34 (1H, d, J = 2 Hz, H-8), 6.82 (1H, d, J = 8 Hz, H-3 ),
0
0
5
6
.
.
Bays, E. D.; Finch, H. Nat. Prod. Rep. 1990, 409.
Peptic disorders. Peptic ulcer. The Merck Manual, 2nd Home ed.; Merck & Co.:
New Jersy, 2000; Chapter 14, p 13.
7.40 (1H, t, J = 8 Hz, H-4 ), 6.70 (1H, d, J = 8 Hz, H-5 ), 4.97 (1H, d, J = 7.8 Hz, H-
00 00 00 00
1 ), 3.89–3.70 (2H, m, H-2 , H-3 ), 3.10–2.90 (1H, m, H-4 ), 3.55–3.40 (2H, m,
13
0
0
00
H-5 , H-6 ), 3.95 (3H, s, OMe), 3.90 (3H, s, OMe).
CDCl + MeOH-d ): d 161.87 (C-2), 113.33 (C-3), 182.64 (C-4), 161.05 (C-5),
98.04 (C-6), 165.39 (C-7), 92.48 (C-8), 158.76 (C-9), 105.54 (C-10), 112.33 (C-
C NMR (75 MHz,
7
8
9
.
.
.
Maurya, R.; Gupta, P.; Gupta, C. M. In Perspective on Biodiversity—A Vision for
Megadiverse Countries; Verma, D. D., Arora, S., Rai, R. K., Eds.; Ministry of
Environment and Forest, Government of India: New Delhi, 2006; p 295.
Shastri, R, et al., Eds.; Charak Samhita of Agnivesha. Vidyabhavan Ayurveda
Granthamal 32. Chaukhambha Vidya Bhavan, Banaras (India) Ch. Chi. 13, 1962;
pp 170–171.
3
4
0
0
0
0
0
0
1 ), 158.37 (C-2 ), 105.73 (C-3 ), 132.56 (C-4 ), 108.35 (C-5 ), 156.25 (C-6 ),
101.46 (C-1 ), 73.21 (C-2 ), 76.60 (C-3 ), 69.93 (C-4 ), 76.80 (C-5 ), 61.81 (C-
6 ), 56.09 (OMe), 55.76 (OMe). ESIMS: m/z 499. HRESIMS: m/z 499.1211
24
[M+Na] (calcd for C23H O11, 499.1216).
0
0
00
00
00
00
0
0
+
Yetkin, G.; Celebi, N.; Ozer, C.; Gonul, B.; Ozogul, C. Int. J. Pharm. 2004, 277, 163.
23. Rao, J. M.; Babu, K. S.; Babu, T. H.; Manjulatha, T.; Purohit, M. J.; Srinivas. P. V.;
Yadav, J. S. US Patent 20070213281, 2007.
1
0. Jayaraj, A. P.; Lewin, F. I.; Tovey, M. E.; Clark, K. C. G. Eur. J. Pharmacol. 1988, 146,
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1. Ogle, C. W.; Cho, C. H.; Jorg, M. C.; Koo, M. W. L. Eur. J. Pharmacol. 1985, 112,
99.
2
24. Animal experiments: male Wister rats (170–180 g Body weight) were purchased
from National Institution of Nutrition, Hyderabad, India. All the experiments
were performed as per the norms and permission of institutional ethical
committee of University of Gulbarga, Gulbarga, India. In each group six animals
were taken. In brief, experiments were carried out as follows: (i) Pylorus
ligation test: 48-h food deprived animals were given test samples in respective
dosages. After 1 h of test sample administration (1% Twen-80 suspension, 1 mL
each rat), abdominal cavity was cut open under anesthesia and stomach was
ligated at pylorus end. Four hours after pylorus ligation, animals were
sacrificed and studied as reported.12 (ii) Cold restraint induced ulceritis:
experiments were performed as reported by Ogle et al.11 After oral
administration of test samples, animals were restrained to cold (4 °C) for 2 h.
Thereafter, abdominal cavity was cut open. Stomach was exposed along greater
curvature and severity of gastric ulcer was assessed in terms of mean ulcer
index. (iii) Ethanol induced ulceritis: This experiment was performed as
described by Jayaraj et al.1 Forty eight-hour fasting rats were given
respective dosages of the test sample and 1 h after oral test sample
administration, ethanol was given intragastrically. One hour after ethanol
challenge, stomach of rats was ligated at the pylorus end under anesthesia.
Four hours after pylorus ligation, animals were sacrificed for the study of ulce9r
indexing (iv) Aspirin induced ulceritis was produced according to Yetkin et al.
In this experiment, each group of animal received respective dosages test
samples for five days, followed by administration of 200 mg/kg body weight
Aspirin suspended in Tween-80 (1%). At the end of the drug administration
schedule, animals were fatsed for 24 h and sacrificed by cervical dislocation.
Their stomach was opened along the greater curvature, washed with luke
warm saline and examined under a dissecting microscope for ulcer index.
1
3
1
1
2. Alphino, R. S.; Ward, J. W. Eur. J. Pharmacol. 1969, 6, 61.
3. (a) Joshi, K. C.; Singh, P.; Paudasani, R. T. Planta Med. 1978, 34, 219; (b) Gafner,
S.; Wolfender, L. J.; Nianga, M.; Stoeckli-Evans, H.; Hostettmann, K.
Phytochemistry 1996, 42, 1315.
1
1
1
1
1
1
4. Benerji, A.; Das, R. Indian J. Chem., Sect B 1977, 15B, 495.
5. Dhar, K. L.; Atal, C. K.; Pelter, A. Planta Med. 1970, 18, 332.
6. Hansel, R.; Sauer, H. Planta Med. 1967, 15, 443.
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8. Buschi, C. A.; Pomilio, A. B.; Gros, E. G. Phytochemistry 1981, 20, 1178.
9. (a) Fang, W.; Ruan, J.; Wang, Z.; Zhao, Z.; Zou, J.; Zhou, D.; Cai, Y. J. Nat. Prod.
2
006, 69, 1641; (b) Harborne, J. B. Photochemical Methods: A Guide to Modern
Techniques of Plant Analysis; Chapman & Hall: London, 1972; (c) Somdej, K.;
Kwan Jai, K.; Komkrich, N.; Palangpon, K. J. Nat. Prod. 2004, 67, 968.
0
2
0. Acid hydrolysis of compound 1: compound 1 (10 mg) was refluxed with 7% HCl
(
10 mL) at 70 °C for 3 h. After cooling, the reaction mixture was diluted with
H
2
O and then extracted with CH Cl (2 mL each) four times. The combined
2
2
organic layers were evaporated to dryness to give residue (3 mg), which was
identified as dihydrooroxylin A (6) on the basis of TLC and spectral data (NMR
and MS).The aqueous phase was neutralized with Ag CO powder and then
2 3
filtered to remove the inorganic materials. The filtrate was concentrated to
dryness and purified by preparative HPLC (Column Phenomenex Luna (C18,
1
(
50 ꢀ 4.6 mm,
1:1); PDA detector) to yield 2 mg of sugar, respectively. The sugar
constituent of 1 was determined to be -glucuronic acid by comparison of
their HPLC retention times with that of an authentic sample (obtained from
5 lm); Solvent system: acetonitrile/water/0.1% acetic acid
D