New furanoflavanoids, intestinal α-glucosidase inhibitory and free-radical (DPPH) scavenging, activity from antihyperglycemic root extract of Derris indica (Lam.)

Article abstract of DOI:10.1016/j.bmc.2009.05.051

A bioassay-guided fractionation and chemical examination of antihyperglycemic root extract of Derris indica resulted in isolation and characterization of two new furanoflavanoids (1, 2) along with thirteen known compounds (3-15). Their structures were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. All the compounds were tested in vitro for intestinal α-glucosidase inhibitory and DPPH radical activity. New compounds (1, 2) displayed moderate intestinal α-glucosidase inhibitory as well as free radical scavenging activity. Other compounds also displayed varying degrees of moderate intestinal α-glucosidase inhibitory activity. Pongamol (6) displayed potent intestinal α-glucosidase inhibition.

Full text of DOI:10.1016/j.bmc.2009.05.051

Bioorganic & Medicinal Chemistry 17 (2009) 5170–5175
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New furanoflavanoids, intestinal
a-glucosidase inhibitory and free-radical
(DPPH) scavenging, activity from antihyperglycemic root extract
of Derris indica (Lam.)
R. Ranga Rao a, Ashok K. Tiwari b, P. Prabhakar Reddy a, K. Suresh Babu a, Amtul Z. Ali b,
K. Madhusudana b, J. Madhusudana Rao a,
*
a Natural Products Laboratory, Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500607, India
b Pharmacology Division, Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A bioassay-guided fractionation and chemical examination of antihyperglycemic root extract of Derris
indica resulted in isolation and characterization of two new furanoflavanoids (1, 2) along with thirteen
known compounds (3–15). Their structures were determined on the basis of extensive spectroscopic
(IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. All the compounds
Received 30 March 2009
Revised 20 May 2009
Accepted 21 May 2009
Available online 28 May 2009
were tested in vitro for intestinal
(1, 2) displayed moderate intestinal
Other compounds also displayed varying degrees of moderate intestinal
Pongamol (6) displayed potent intestinal -glucosidase inhibition.
a
-glucosidase inhibitory and DPPH radical activity. New compounds
-glucosidase inhibitory as well as free radical scavenging activity.
-glucosidase inhibitory activity.
a
Keywords:
a
Derris indica
Leguminosae
Furanoflavonoids
a
Ó 2009 Published by Elsevier Ltd.
a-Glucosidase inhibition
1,1-Diphenyl-2-picrylhydrazyl radical
scavenging
Starch tolerance test
Antihyperglycemic activity
1. Introduction
mia, hyperinsulinemea, and burden of PPOS4,5 along with reducing
the increased risk of cardiovascular diseases.6 Natural agents that
delay the digestion and absorption of carbohydrates have been in
practice of traditional medical system world over. Evaluation of
such natural resources for their active principles is now becoming
one of the practical and logical strategy7,8 both in providing scien-
tific footage for such medical practices and development of newer
pharmacological agents for prevention and/or treatment of disor-
ders like PPHG and PPOS. In the course of our search for antihyper-
glycemic agents from traditional medicinal plants, we found
presence of these activities in DCM/MeOH extract of the root of
Derris indica.
D. indica (Lam.) Bennet [synonyms, Pongamia pinnata (L.) Pierre,
P. pinnata (L.) Merr, Pongamia glabra Vent. and Cytisus pinnaus (L.)]
is a medium sized, fast growing tree belonging to the Leguminosae
family. It is native to Western Ghats, and tidal forests of India.9 Dif-
ferent parts of the plant have been used in folk medicine for the
treatment of bronchitis, whooping cough, rheumatic joints and
have been advocated to quench dyspepsia in diabetes.10 Recently,
antidiabetic activities in fruits11 and flowers12 of this plant has
been reported. However, detail analysis of phytochemicals respon-
sible for antidiabetic activity is lacking. Bioassay guided fraction-
ation of antihyperglycemic DCM/MeOH extract of the roots of D.
Postprandial hyperglycemic excursion has been implicated as
an independent risk factor for cardiovascular disease; not only
among diabetes patients, but also among subjects in general pop-
ulation with mildly elevated postprandial glucose levels.1,2 Post-
prandial hyperglycemia (PPHG) is an exaggerated rise in blood
sugar following a meal with rich in carbohydrates and/or the lipids.
A rich meal also causes increased susceptibility of the organism to-
ward oxidative damage, known as postprandial oxidative stress
(PPOS). PPOS is characterized by overt generation of free radicals
and increased susceptibility of the organism towards oxidative
damage. Furthermore, PPOS is associated with higher risk for ath-
erosclerosis, diabetes, and obesity.2,3 Therefore, combination of
agents that reduce PPHG and PPOS may become therapeutics of
interest in combating these multiple disorders.
Slowing down of the digestion and absorption of dietary carbo-
hydrates either by means of dietary manipulations or by intestinal
a-glucosidase inhibitory drugs like acarbose, Voglibose or the
Miglitol, have shown promise in reducing postprandial hyperglyce-
* Corresponding author. Tel.: +91 40 27193166; fax: +91 40 27160512.
E-mail address: janaswamy@iict.res.in (J.M. Rao).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.05.051

R. Ranga Rao et al. / Bioorg. Med. Chem. 17 (2009) 5170–5175
5171
Indica in our study led to the isolation of two new furanoflavonoids
(1, 2) along with thirteen known compounds (3–15). We report
herein isolation, structure elucidation and synthesis of compound
the presence of 8 methine carbons and 9 quaternary carbons
including carbonyl at d 177.2 ppm. A signal at d 177.2 indicated
the presence of a,b-unsaturated carbonyl group. Where as a signal
1 along with their biological activity against intestinal
dase inhibition and free radical scavenging activity for all the iso-
lated fifteen compounds mentioned in Figure 1.
a
-glucosi-
at 105.4 is attributed to an olefinic carbon. A signal at d 104.6 and d
147.7 indicated the presence of a furan ring. The DEPT experiment
revealed presence of eight methines and nine quaternary carbons.
The explanation was further supported by several diagnostic corre-
lations (H-3/C-2, C-4, C-10; H-5/C-7, C-8, C-9, C-10; H-6/C-5, C-7, C-
8; H-20/C-2, C-10, C-30, C-40; H-50/C-10, C-40, C-60; H-60/C-2, C-10, C-40,
C-50; H-200/C-7, C-8, C-9, C-300; H-300/C-7, C-8, C-9, C-200) and 1H–1H
COSY (H-5/H-6; H-50/H-60; H-200/H-300). The HMBC correlations of
H-200 with C-7 and C-8 indicated that furan ring should be fused
at C-7 (oxygenated) and C-8 of the aromatic ring. Hence, the struc-
ture of compound 1 was identified as 30,40-dihydroxy-4H-furo[2,3-
h]chromen-4-one (Fig. 2).
Compound 2 was isolated as a pale yellow amorphous solid, mp
320 °C. The molecular formula was established as C17H10O6 using
HRESIMS. It is spectral data was similar to that of compound 1.
The IR spectrum displayed absorption bands at 3283, 1692 cmꢀ1
suggesting presence of a hydroxyl group and a carbonyl group
The significant difference in the 1H NMR spectrum (Table 1) was
the absence of a peak at d 6.80 (1H, s, H-3) in compound 2. Increase
in molecular weight 16 amu as compared with that of compound 1,
suggested presence of another hydroxyl group at C-3 position. This
explanation was further supported by the disappearance of three
signals at d 8.85, d 9.15 and d 9.36 in deuterium exchange condi-
tions indicated the presence of three hydroxyl groups. In the 13C
NMR spectrum of compound 2 absence of a signal at d 105.4 (C-
3) suggested that the presence of a hydroxyl group at (C-3) posi-
tion. 13C and DEPT-135 spectrum clearly demonstrated seven
methines and 10 quaternary carbon atoms (including carbonyl).
Thus, the structure of compound 2 was conformed as 3,30,40-trihy-
droxy-4H-furo[2,3-h]chromen-4-one (Fig. 3).
2. Results and discussion
2.1. Chemistry
2.1.1. Isolation and structure elucidation
Compound 1 was obtained as a pale yellow amorphous solid,
mp 284 °C and the molecular formula was analyzed as C17H10O5
by HRESIMS. IR spectrum displayed absorption bands at 3303,
1694 cmꢀ1 that suggested presence of a hydroxyl group and
a,b-
unsaturated carbonyl group. The 300 MHz 1H NMR spectrum in
DMSO-d6 (Table 1) displayed 10 signals. A singlet at d 6.80 inte-
grating for one proton was attributed to an olefinic proton, which
was adjacent to the carbonyl group, two doublets at d 6.93
(J = 8.4 Hz, H-50), 7.51 (J = 2.4 Hz, H-20) and a double doublet at d
7.55 (J = 8.4 Hz, 2.4 Hz, H-60) each integrating for one proton sug-
gesting the presence of a 1,3,4-tri substituted aromatic ring. A dou-
blet at d 8.22 (J = 2.0 Hz) and a doublet at d 7.47 (J = 2.0 Hz)
integrating for one proton each indicated the H-200, H-300 of the fur-
an ring. Two doublets at d 7.88 (J = 8.8 Hz, H-5), 7.67 (d, J = 8.8 Hz,
H-6), each integrating for one proton indicated the presence of a
tetra substituted aromatic ring. Two signals at d 9.54 and d 9.97
showed no connectivity with any carbon nucleus in the HSQC plot.
This suggests presence of two aromatic hydroxyl groups. This
explanation is further supported by the disappearance of two
peaks at d 9.54 and 9.97 in a heavy water exchange condition.
The 13C NMR spectrum in DMSO-d6 of compound 1 exhibits the
presence of 17 carbon atoms whose multiplicity is explained using
DEPT 135 and HMBC correlations. 13C and DEPT spectra indicated
In addition to the above compounds, thirteen known com-
pounds were also isolated from the DCM + MeOH (1:1) extract
O
R3
O
2''
R1
3''
R2
2'
3'
4'
5'
3'
O
OMe
R2
4
O
O
O
1'
O1''
7
O
5
9
8
2
1
2
6'
9
1'
3
5'
7
6
8
OMe
4
10
3
R1
OH
O
5
O
1. R1 = H, R2 = R3 = OH
2. R1 = R2 = R3 = OH
3. R1 = OMe, R2 = R3 = H
8
6. R1 = R2 = H
7. R1 = R2 = -OCH2O-
4
. R = OMe, R = R = -OCH2O-
5. R11 = H, R2 =2R3 =3-OCH2O-
COOH
OMe
OMe
O
3'
O
MeO
O
O
R4
7
1'
O
R2
9 O
1
5' R5
O
OMe
O
OMe
13
3
5
R1
10
15
14
R3
O
9.
R1 = OMe, R2 = OMe, R3 = H, R4 = R5 = OMe
10. R1 = OMe, R2 = OH, R3 = H, R4= R5 = -OCH2O-
11. R1 = R4 = R5 = H, R2 = OMe, R3 = OH
12. R1 = R5 = H, R2 = R4 = OMe, R3 = OH
Figure 1. Isolated compounds from DCM/MeOH (1:1) extract of D. indica.

5172
R. Ranga Rao et al. / Bioorg. Med. Chem. 17 (2009) 5170–5175
Table 1
1H NMR (300 MHz) and 13C NMR (75 MHz) spectral data for 1 and 2
Position
Compound 1
1H, d (mult, J, Hz)
Compound 2
1H, d (mult, J, Hz)
dc
dc
1
—
—
—
—
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
100
200
300
163.0
105.4
177.2
121.3
110.4
157.9
119.1
149.9
117.3
122.3
113.7
150.3
146.2
119.2
116.59
—
—
146.0
136.9
171.3
119.6
108.0
156.0
115.5
147.9
115.4
121.2
113.6
143.9
143.7
114.2
118.6
—
—
6.80 (1H, s)
—
7.88 (1H, d, J = 8.8)
7.67 (1H, d, J = 8.8)
—
—
—
—
9.36 (br s) (OH)
—
8.07 (1H, d, J = 8.6)
7.58 (1H, d, J = 8.6)
—
—
—
—
—
—
7.51 (1H, d, J = 2.4)
9.54 (br s) (OH)
9.97 (br s) (OH)
6.93 (1H, d, J = 8.4)
7.55 (1H, dd, J = 8.4, 2.4)
—
7.87 (1H, d, J = 2.1)
8.85 (br s) (OH)
9.15 (br s) (OH)
6.95 (1H, d, J = 8.4)
7.70 (1H, dd, J = 8.4, 2.1)
—
147.7
104.6
8.22 (1H, d, J = 2.0)
7.47 (1H, d, J = 2.0)
144.7
102.8
7.96 (1H, d, J = 2.0)
7.32 (1H, d, J = 2.0)
2.1.2. Synthesis
Confirmation of the structure of compound 1 was done through
OH
its synthesis (Scheme 1). By following earlier synthetic protocol,24
the resorcinol was treated with acetic anhydride in the presence of
polyphosphoric acid under reflux conditions to afford resacetoph-
enone(16). Resacetophenone was then treated with bromoacetal-
dehyde diethyl acetal in the presence of potassium carbonate in
anhydrous DMF to give compound 17, which on treatment with
acidic Amberlyst 15 in toluene at 90 °C yielded 4-hydroxy-5-acetyl
coumarone (18) that was condensed with aldehyde 19 in the pres-
ence of KOH in anhydrous ethanol to give chalcone(20). This chal-
cone was cyclized using I2 in trigol25,24c at 120 °C 30,40-dihydroxy-
4H-furo[2,3-h]chromen-4-one (1).
OH
O
O
O
2.2. Biological activity
Figure 2. Key HMBC correlations of compound 1.
Plant medicines have been used successfully to treat various
disorders world over even before the dawn of modern medications.
Though several herbal medicines used in diabetes have been found
to possess acarbose like activity,8,26 very few are studied for the
identification of phytochemicals responsible for biological activi-
ties. Identification of bioactive phytochemicals help standardiza-
tion of traditional plant based therapeutics for treatment of
specific disorders.27 DCM/MeOH extract from the root of D. indica
displayed antihyperglycemic activity in starch-induced-postpran-
dial hyperglycemia in normal rat model in our study (Fig. 4). Dose
titration disclosed that pretreatment of rats with 100 mg/kg body
weight dose could significantly decrease the rise in blood glucose
level post starch feeding.
OH
OH
O
O
OH
Phytochemical analysis of this extract led us isolate fifteen mol-
ecules of various classes (Fig. 1) including compounds 1 and 2 that
O
exhibited intestinal a-glucosidase inhibition and DPPH radical scav-
Figure 3. Key HMBC correlations of compound 2.
enging activity (Table 2). It is evident from the table that none of the
compound could display enzyme inhibition more than that of the
extract at primary screening concentration. It appears therefore that
greater enzyme inhibitory activity in extract may be due to the po-
sitive synergetic effect of individual compounds. Though majority of
(Fig. 1). By comparison of their physical and spectroscopic data
with the literature, they were characterized as Karanjin (3),13 Pon-
gapin (4),13 Pongaglabrone (5),14 Pongamol (6),15 Ovalitenone
(7),16 Pongachromene (8),17 Fisetin tetramethyl ether (9),18 3-
methoxy-7-hydroxy-30,40-methylenedioxyflavone (10),19 7-O-
methylchrysin (11),20 7,40-dimethoxy-5-hydroxyflavone (12),21
Pinnatin (13),19 Pongapinone-B (14),22 Piperonylic acid (15).23 To
the best of our knowledge, this is the first report of the isolation
of compounds 10, 11, 12 and 15 from D. indica.
the isolated compounds displayed varying degrees of
inhibition, only 1, 2 and 10 could display DPPH radical scavenging
activity with SC50 values of 84.6 M, 21 M and 39 M, respectively
and 6 potent -glucosidase inhibition (IC50 = 103.5 M, Table 2).
a-glucosidase
l
l
l
a
l
Recently Karanjin (3) and Pongamol (6) have been reported to
possess antidiabetic activity in various animal models by virtue of

R. Ranga Rao et al. / Bioorg. Med. Chem. 17 (2009) 5170–5175
5173
O
OH
O
HO
OH
HO
OH
BrCH2CH(OC2H5)2
Amberlyst 15
(Ac2O)O
dry DMF/K2CO3,
800C, 90 %
Toluene, 900C, 40%
Polyphosporic acid
ref lux, 68 %
CH3
CH3
EtO
OR
OEt
17
16
O
OH
OR
OR
OH
OR
19
O
O
O
O
OH
OR
O
OHC
I2/Trigol
EtOH, 40% NaOH
75%
1200C, 2h, 50%
CH3
R = CH2Ph
O
1
O
20
18
R = CH2Ph
Scheme 1. Synthesis of compound 1.
Table 2
In vitro intestinal
compounds 1–15
200
150
100
50
a-glucosidase inhibitory and DPPH scavenging activity of
Control
Extract
Acarbose
%
a
-Glucosidase inhibition
g/mL)
M)
% DPPH Scavenging activity
Extract
1
2
3
4
5
6
7
63.2 1.4 (19.2
25.6 0.341 (335
37.9 2.6
l
25.6 0.7 (49.8
55.1 1.2 (84.6
l
l
g/mL)
M)
l
64.9 0.8 (21
lM)
26.3 1.8
NA
21.4 0.7
NA
8.6 0.1
NA
58.2 0.2 (103.5
29.7 0.5
22.8 5.5
19.7 0.3
36 1.8
lM)
11.2 2.3
NA
NA
NA
c
c
b
a
c
8
9
c
c
10
11
12
13
14
15
68.3 1.6 (39
lM)
c
28.7 1.8
4.4 0.1
NA
NA
36.5 5.9
1.2 0.2
NA
NA
18.4 2.2
2.4 1.6
Primarily DPPH scavenging activity was determined at 25
rat intestinal -Glucosidase inhibition at 50 g/mL. Scavenging concentration for
50% of DPPH free radical (SC50) was calculated from logarithmic regression equation
lg/mL concentration and
a
l
0 min
30 min
60 min
90 min
120 min
obtained from the values of at least five dilutions of the primary concentration. The
Time in minutes
50%
a-Glucosidase enzyme inhibitory concentrations (IC50) were also obtained
similarly. Acarbose was taken as standard
Values represent mean standard deviations, n = 3; in parentheses is SC50, IC50
value of respective sample. NA; not active.
a
-Glucosidase inhibitor (IC50 = 24 M).
l
Figure 4. Antihyperglycemic activity of D. indica extractin rats. Plasma glucose
levels of rats after starch tolerance test in various groups of animals at different
time points. One way ANOVA analysis followed by Bonferroni’s multiple compar-
ison tests was applied to find difference between the groups. Values represent
mean SD, n = 5, a, b, and c represent statistical comparisons between control and
test groups. ap < 0.05, bp < 0.01, cp < 0.001. Extract dose was given at 100 mg/kg
body weight and acarbose 10 mg/kg body weight.
enedioxy moiety (7) in chalcones (6, 7) decreased enzyme inhib-
itory potential. Pongamol (6) was found to be the most potent
a-
glucosidase inhibitor (IC50 = 103.5 M) in this study. However,
l
presence of other active molecules in the extract can not be ruled
out.
their Protein Tyrosine Phosphatase-1b (PTP-1b) inhibitory activ-
ity.11 PTPase inhibitors increase insulin sensitivity by blocking
the PTPase-mediated negative insulin signaling pathway and are
also one of the attractive target in type 2 diabetes and related
complications.11 Our study is the first of its kinds that report anti-
hyperglycemic activity in root of D. indica identifies and assign
3. Conclusion
Two new compounds (1, 2) including thirteen known com-
pounds were isolated from D. indica. The newly isolated compound
1 was also synthesized involving methodology of benzyl deprotec-
tion. I2 in trigol was demonstrated to be a simple and convenient
reagent to cleave benzyl ethers. The advantage of this method is
that it is inexpensive, simple reaction conditions, high selectivity,
and retention of unsaturation. All the compounds displayed mod-
intestinal
activity to the compounds present in this extract. Both, the new
furanoflavanoids 1 and 2 displayed moderate -glucosidase inhi-
a-glucosidase inhibitory and DPPH radical scavenging
a
bition and potent DPPH radical scavenging activity. Due to very
few numbers of compounds in each class, structure–activity rela-
tionship could not be discussed properly. However, it is evident
that presence of 3,30,40-hydroxyls (2) were responsible for in-
erate intestinal
showed potent DPPH radical scavenging activity. Compound 6
was identified as potent -glucosidase inhibitor. Compounds 10,
11, 12 and 15 are being reported for the first time from D. indica
root. This is the first report identifying presence of intestinal -glu-
cosidase inhibitory and DPPH scavenging activity in D. indica.
a-glucosidase inhibition. Compounds 1, 2 and 10
creased DPPH radical scavenging and
a-glucosidase inhibitory
activity for angular furanoflavanoids (1–5). On the other hand
a
40,50-methylenedioxy moiety (10) may be held responsible for
inhibition of
a-glucosidase and presence of 7-hydroxy (10) for
a
the DPPH scavenging activity (9–12). Nevertheless, 30,40-methyl-

5174
R. Ranga Rao et al. / Bioorg. Med. Chem. 17 (2009) 5170–5175
4.4. Animal experiment
4. Experimental
4.1. General
Antihyperglycemic activity study was done according to the
method reported earlier.28 Wistar rats of either sex weighing be-
tween 195 and 215 g were obtained from National Institute of Nutri-
tion (CPCSEA Reg. No. 154, Government of India), Hyderabad. The
animals were housed in standard polyvinyl cages. The room temper-
ature was maintained at 22 1 °C with an alternating 12 h light dark
cycle. Food and water were provided ad libitum. Experiments were
performed as per the Institutional Animal Ethical Committee norms.
The rats were divided into various groups’ viz. control, extracts
group and a group with standard drug acarbose (10 mg/kg body
weight). Five rats in each group were taken. All the animals were
kept for overnight fasting. Next day forenoon blood was collected
from retro orbital plexus in EDTA containing tubes, and plasma glu-
cose levels for basal (‘0’ h) value were measured by glucose-oxidase
test method using auto blood analyzer instrument (Bayer EXPRESS
PLUS). Test sample was suspended in normal saline and adminis-
tered orally through gastric intubation in the dose of 100 mg/kg-
body weight. The control group of animals was treated sham with
normal saline. Fifteen minutes after test sample treatment, animals
were fed with soluble-starch dissolved in normal saline at a dose of
2 g/kg-body weight. Thereafter, blood was collected at intervals of
30, 60, 90 and 120th min post starch feeding. Plasma was separated
out for glucose measurement as described above.
Optical rotations were recorded on a JASCO DIP 300 digital
polarimeter at 25 °C. IR spectra were recorded on Nicolet-740 spec-
trometer with NaCl optics. The 1H and 13C NMR, COSY, HMQC,
HMBC and NOESY spectra were recorded on
a Bruker FT-
300 MHz spectrometer at 300 MHz for 1H and 75 MHz for 13C,
respectively, using TMS as internal standard. The chemical shifts
are expressed as d values in parts per million (ppm) and the cou-
pling constants (J) are given in hertz (Hz). HRESIMS were measured
on LC-MSD-Trap-SL instrument. Column chromatography was car-
ried out using silica gel 60–120 mesh (Qingdao Marine Chemical,
China) and precoated silicagel plates (Merck, 60 F254) were used
for preparative TLC. Rat intestinal acetone powder as a source of
intestinal
a-glucosidase, p-nitrophenyl a-D-glucopyranoside (p-
NPG)as substrate for enzyme, soluble potato starch, DPPH radical,
and EDTA were purchased from Sigma Chemical Co., St Louis,
MO, USA. Other chemicals of analytical grade were procured from
indigenous manufacturers.
4.2. Plant material
Roots of D. indica was collected from the forest of Tirumala hills
in Chitoor Dist. (Andhra Pradesh, India) in the month of January
2005 and identification was made by Professor Dr. K. Madhava
Chetty, Department of Botany, Sri Venkateswara University, Tiru-
pati. A voucher specimen No. 589 of the plant is deposited at the
herbarium of the S. V. University, Tirupati.
4.5.
a-Glucosidase inhibitory assay
a
-Glucosidase inhibitory activities were determined as per ear-
lier reported methods.27b Rat intestinal acetone powder in normal
saline (100:1; w/v) was sonicated properly and the supernatant
was used as a source of crude intestinal
gation. In brief, 10 L of test samples (5 mg/mL DMSO solution)
were reconstituted in 100 L of 100 mM-phosphate buffer (pH
6.8) in 96-well microplate and incubated with 50 L of crude intes-
tinal -glucosidase for 5 min before 50 L substrate (5 mM, p-nitro-
phenyl- -glucopyranoside prepared in same buffer) was added.
Release of p-nitrophenol was measured at 405 nm spectrophoto-
metrically (SpectraMaxÒ plus384), Molecular Devices Corporation,
Sunnyvale, CA, USA) 5 min after incubation with substrate. Individ-
ual blanks for test samples were prepared to correct background
a-glucosidase after centrifu-
l
4.3. Isolation procedures
l
l
The air dried and powdered roots of D. indica (5 kg) were ex-
tracted three times with DCM/MeOH (1:1) at room temperature
(25 °C) for 48 h. The combined extracts were concentrated under
vacuum. Portion of active DCM/MeOH (1:1) extract (50 g) was
subjected to column chromatography (silica gel, 100–200 mesh)
using step gradient of hexane, EtOAc, acetone, CHCl3, MeOH to
yield six major fractions (F1–F6). Fraction F1 was subjected to re-
peated silica gel (100–200 mesh) column chromatography (CC)
by eluting with EtOAc/hexane (10:90) to yield 1.28 g, 1.23 g,
1.48 g of compounds, respectively 3, 6 and 7. Fraction F2 was
subjected to silica gel column chromatography eluting with ace-
tone/hexane (8:92) to yield 0.9 g of compound 8. A portion of
fraction F3 was subjected to silica gel column chromatography
with EtOAc/CHCl3 (5:95) to yield 3.52 g, 1.20 g of compounds 4
and 5. Fraction F4 was subjected to silica gel column chromatog-
raphy with MeOH/CHCl3 (10:90) to yield 0.92 g, 0.12 g 0.22 g of
compounds, respectively 9, 10 and 13. Fraction F5 was subjected
to repeated column chromatography eluting with MeOH/CHCl3
(15:85) to yield 0.32 g, 0.045 g, 0.025 g of compounds, respec-
tively 11, 12 and 15. Similarly Fraction F6 was subjected to re-
peated column chromatography eluting with MeOH/CHCl3
(25:75) to yield 0.015 g, 0.022 g of compounds 1 and 2, respec-
tively (Fig. 1).
a
l
a-D
absorbance where substrate was replaced with 50
lL of buffer. Con-
trol sample contained 10 L DMSO in place of test samples. Percent-
l
age of enzyme inhibition was calculated as (1 ꢀ B/A) ꢁ 100 where
[A] represents absorbance of control without test samples, and [B]
represents absorbance in presence of test samples.
4.6. DPPH free radical scavenging activity
Assay for the scavenging of stable free radical 1,1-diphenyl-2-
picrylhydrazyl (DPPH) was done as reported earlier.27b Briefly, in
a 96-well micro plate, 25
lL of test sample dissolved in DMSO
(1 mg/mL), 125 L of 0.1 M tris–HCl buffer (pH 7.4) and 125
l
lL of
0.5 mM DPPH solution dissolved in absolute ethyl alcohol were
added. The reaction mixture was shaken well and incubated in
dark for 30 min and read at 517 nm spectrophotometrically (Spec-
traMaxÒ plus384, Molecular Devices Corporation, Sunnyvale, CA,
USA). Percentage of DPPH scavenging was calculated as (1 ꢀ B/
A) ꢁ 100 where A represents absorbance of control without test
samples, and B represents absorbance in presence of test samples.
Compound 1: Pale yellow amorphous solid, mp 284 sC; IR (KBr)
m
max: 3303, 1694, 1270, 1167, 775, 671 cmꢀ1. 1H NMR and 13C NMR
data in DMSO-d6 given in Table 1. HRESIMS: calcd for C17H11O5
295.0601; found 295.0604 [M+H].
Acknowledgements
Compound 2: Pale yellow amorphous solid, mp 320 sC; IR (KBr)
m
max: 3283, 1692, 1215, 1131, 764, 670 cmꢀ1 1H NMR and 13C NMR
The authors thank Dr. J. S. Yadav, Director, IICT, for his encour-
agement and support during the course of this work. RRR thank
CSIR, New Delhi for financial support.
.
data in DMSO-d6 given in Table 1. HRESIMS: calcd for C17H9O6
309.0405; found 309.0395[M+ꢀH].

R. Ranga Rao et al. / Bioorg. Med. Chem. 17 (2009) 5170–5175
5175
16. Lee, Y. R.; Morehead, A. T., Jr. Tetrahedron 1995, 51, 4909.
17. Mukerjee, S. K.; Sarkar, S. C.; Seshadri, T. R. Tetrahedron 1968, 25, 1063.
References and notes
18. Mukerjee, S. K.; Sarkar, S. C.; Seshadri, T. R. Indian J. Chem. 1969, 7, 1275.
19. Das, B.; Chakravarty, A. K.; Masuda, K.; Suzuki, H.; Ageta, H. Phytochemistry
1994, 37, 1363.
20. Audier, H. Bull. Soc. Chim. Fr. 1966, 2892.
21. Benerji, A.; Das, R. Indian J. Chem. 1977, 15B, 495.
1. Bonora, E.; Muggeo, M. Diabetalogia 2001, 44, 2107.
2. Ceriello, A. Diabetes Vasc. Dis. Res. 2008, 5, 260.
3. Sies, H.; Stahl, W.; Sevanian, A. J. Nutr. 2005, 135, 969.
4. Maki, K. C. Am. J. Cardiol. 2004, 93, 12C.
5. Maki, K. C.; Carson, M. L.; Miller, M. P.; Turowski, M.; Bell, M.; Wildor, D. M.;
Reeves, M. S. Diabetes Care 2007, 30, 1039.
6. Delorme, S.; Chiasson, J. L. Curr. Opin. Pharmacol. 2005, 5, 184.
7. McCarty, M. F. Med. Hypotheses 2000, 65, 152.
8. Matsui, T.; Ogunwande, I. A.; Abesundona, K. J. M. Mini-Rev. Med. Chem. 2006, 6,
109.
9. Krishnamurthi, A.. In The Wealth of India; Publication and Information
Directorate, CSIR: New Delhi, India, 1969; Vol. VIII.
10. (a) Kritikar, K. R.; Basu, B.D.; Basu, L. M. Indian medicinal plants, 2nd ed.;
Allahabad, India, 1956; Vol. 1, pp 830–832.; (b) Yadav, P. P.; Ahmad, G.;
Maurya, R. Phytochemistry 2004, 65, 439.
11. Tamarakar, A. K.; Yadav, P. P.; Tiwari, P.; Maurya, R.; Srivastava, A. K. J.
Ethnopharmacol. 2008, 118, 435.
22. Kitagawa, I. Chem. Pharm. Bull. 1992, 40, 2041.
23. Dhar, K. L.; Atal, C. K.; Pelter, A. Planta Med. 1970, 18, 332.
24. (a) Goel, A.; Dixit, M. Indian pat. Appl. 2006, 17pp. IN2004DE01730,
A20060922. Application: In 2004, DE1730 20040914. Chem. Abstr. 147,
427141; (b) Goel, A.; Dixit, M. Synlett 2004, 1990; (c) Anuradha, V.; Srinivas,
P. V.; Rao, R. R.; Manjulatha, K.; Purohit, G. M.; Rao, J. M. Bioorg. Med. Chem.
2006, 14, 6820.
25. Miyake, H.; Takizawa, E.; Sasaki, M. Bull. Chem. Soc. Jpn. 2003, 76, 635.
26. Tiwari, A. K.; Rao, J. M. Curr. Sci. 2002, 83, 30.
27. (a) Pullela, P. V.; Tiwari, A. K.; Vanka, U. M. S.; Vemmenthula, A.; Tatipaka, H. B.;
Dasari, K. R.; Khan, I. A.; Janaswamy, M. R. J. Ethnopharmacol. 2006, 108, 445;
(b) Rao, S. A.; Srinivas, P. V.; Tiwari, A. K.; Vanka, U. M. S.; Rao, R. V. S.; Dasari, K.
R.; Rao, J. M. J. Chromatogr., B 2007, 855, 166.
12. Punitha, R.; Manoharan, S. J. Ethnopharmacol. 2006, 105, 39.
13. Siddiqui, Z. N.; Zaman, A. Indian J. Chem. 1998, 37B, 1081.
14. Khanna, R. N.; Seshadri, T. R. Tetrahedron 1963, 19, 219.
15. Virinder, S. P.; Jatendera, S. R.; Jain, R.; Henderson, D. A.; Malone, J. F.
Phytochemistry 1989, 28, 591.
28. Tiwari, A. K.; Kumbhare, R. M.; Agawane, S. B.; Ali, A. Z.; Kumar, K. V. Bioorg.
Med. Chem. Lett. 2008, 18, 4130.