In vivo anti-diabetic activity of derivatives of isoliquiritigenin and liquiritigenin

Article abstract of DOI:10.1016/j.phymed.2013.10.015

Isoliquiritigenin (ISL), a chalcone and liquiritigenin (LTG), a flavonoid found in licorice roots and several other plants. ISL displays antioxidant, anti-inflammatory, antitumor and hepatoprotective activities whereas LTG is an estrogenic compound, acts as an agonist selective for the β-subtype of the oestrogen receptor. Both the phenolics were isolated from the rhizomes of Glycyrrhiza glabra. Five derivatives from ISL and four derivatives from LTG were synthesized. All the compounds were established by extensive spectroscopic analyses and screened through oral glucose tolerance test to gain preliminary information regarding the antihyperglycemic effect in normal Swiss albino male mice. ISL (1), ISL derivatives 3, 4, 5, 7 and LTG derivatives 9 and 10 showed significant blood glucose lowering effect. The structure-activity relationship indicated that the presence of ether and ester groups in ISL and LTG analogues are important for exhibiting the activity. Compounds 1, 4 and 10 were selected for in vivo antidiabetic activity and found to be potential candidates for treatment of diabetes. It is the first report on antidiabetic activity of ISL derivative 4 and LTG derivative 10.

Full text of DOI:10.1016/j.phymed.2013.10.015

Phytomedicine 21 (2014) 415–422
Contents lists available at ScienceDirect
Phytomedicine
journal homepage: www.elsevier.de/phymed
In vivo anti-diabetic activity of derivatives of isoliquiritigenin and
liquiritigenin
Rashmi Gaura,1, Kuldeep Singh Yadavb,1, Ram Kishor Vermac, Narayan Prasad Yadavb,∗
,
Rajendra Singh Bhakunia,∗
a Medicinal Plant Chemistry Division, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India
b Herbal Medicinal Product Department, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India
c Analytical Chemistry Division, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoliquiritigenin (ISL), a chalcone and liquiritigenin (LTG), a flavonoid found in licorice roots and sev-
eral other plants. ISL displays antioxidant, anti-inflammatory, antitumor and hepatoprotective activities
whereas LTG is an estrogenic compound, acts as an agonist selective for the ␤-subtype of the oestrogen
receptor. Both the phenolics were isolated from the rhizomes of Glycyrrhiza glabra. Five derivatives from
ISL and four derivatives from LTG were synthesized. All the compounds were established by extensive
spectroscopic analyses and screened through oral glucose tolerance test to gain preliminary information
regarding the antihyperglycemic effect in normal Swiss albino male mice. ISL (1), ISL derivatives 3, 4, 5,
7 and LTG derivatives 9 and 10 showed significant blood glucose lowering effect. The structure–activity
relationship indicated that the presence of ether and ester groups in ISL and LTG analogues are important
for exhibiting the activity. Compounds 1, 4 and 10 were selected for in vivo antidiabetic activity and found
to be potential candidates for treatment of diabetes. It is the first report on antidiabetic activity of ISL
derivative 4 and LTG derivative 10.
Received 12 August 2013
Received in revised form 5 September 2013
Accepted 5 October 2013
Keywords:
Glycyrrhiza glabra
Isoliquiritigenin
Liquiritigenin
Derivative
Antidiabetic activity
© 2013 Elsevier GmbH. All rights reserved.
Introduction
of diabetes throughout the world and popularized as nutraceuti-
cals. In addition, many of the currently available drugs have been
Diabetes is a metabolic disease which has become a serious
problem of the society due to the severe long term health compli-
cations associated with it. Type 2 diabetes mellitus (T2DM) is the
most encountered form of diabetes, accounting for more than 80%
of the total cases (Mlinar et al. 2007) and is expected to increase
by 5.4% in 2025 (Kim et al. 2006). Diabetes mellitus (DM) is a
chronic metabolic disorder characterized by high levels of glucose
in the blood due to the non-secretion of insulin (ADA 2007). The
plants have been used since ancient times to treat diabetes and
prevent conditions associated with diabetes (Soumyanath 2006).
The roots of Glycyrrhiza spp. is one of the oldest and most fre-
quently used crude drugs in traditional Chinese medicines for its
extensive pharmacological effects (Songpei et al. 2010). Medicinal
plants and their bioactive constituents are used for the treatment
derived directly or indirectly from plant source. The discovery of
the widely used hypoglycemic drug metformin also came from
the traditional approach of using Galega officinalis (Akpan et al.
2007).
Isoliquiritigenin (1, ISL), a chalcone and liquiritigenin (2, LTG),
a flavonoid found in licorice root and several other plants. ISL
displays antioxidant, anti-inflammatory and antitumor activities
as well as hepatoprotection against steatosis-induced oxidative
stress (Gaur et al. 2010). LTG is an estrogenic compound, acts as
an agonist selective for the ␤-subtype of the oestrogen recep-
tor (Mersereau et al. 2008). Chalcones possess a broad spectrum
of biological activities and have been shown the promising com-
pounds for the prevention or treatment of diabetic complications
(Hsieh et al. 2012). The mechanism is most often not completely
understood, but more and more studies are being conducted to
elucidate the mechanisms of action of different plants and natural
compounds. Biosynthetically and structurally, 1 is the precursor
and an isomer of 2 (Jayaprakasam et al. 2009). During the early
stages of the biosynthesis of these flavonoids, chalcone isomerase
(CHI) catalyzes the intramolecular cyclization of chalcone 1 into
flavanone 2 (Liu and Dixon 2001). Both the phenolics were isolated
from the rhizomes of Glycyrrhiza glabra. The present study was
undertaken to investigate the antidiabetic effect of derivatives of
Abbreviations: DEPT, distortionless enhancement by polarizationtransfer; COSY,
correlation spectroscopy; bw, body weight; AST, alkaline phosphatase; ALT, alanine
transaminase; AST, aspartate transaminase; T BIL, total bilirubin; TP, total protein;
TG, triglycerides; HDL, high density lipoprotein; LDL, low density lipoprotein.
∗
Corresponding authors. Tel.: +91 5222718622; fax: +91 5222342666.
E-mail addresses: npyadav@gmail.com (N.P. Yadav), bhakunirs2000@yahoo.com
(R.S. Bhakuni).
1
These authors contributed equally to this study.
0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.phymed.2013.10.015

416
R. Gaur et al. / Phytomedicine 21 (2014) 415–422
OH
R
3
OH
R
2
OH
R
HO
O
a
1
HO
O
O
O
4
R
R
2
1
c/d
Isoliquiritigenin
Liquiritigenin
CH
3
R
3
Fig. 1. Structures of isoliquiritigenin (1) and liquiritigenin (2).
O
R
R
2
1
+
isoliquiritigenin (ISL) and liquiritigenin (LTG) (Fig. 1) on STZ-
induced diabetic mice.
R
3
O
5/6
R
3
OHC
Materials and methods
R
R
2
1
b
Plant material
7
O
Rhizomes of Glycyrrhiza glabra L. (Fabaceae) were collected from
the research farm of CSIR-Central Institute of Medicinal and Aro-
matic Plants (CIMAP), Lucknow, India in March 2011. The plant was
identified by a taxonomist in Botany and Pharmacognosy Depart-
ment, CSIR-CIMAP, Lucknow and a voucher specimen #7401was
deposited.
4, R1= R2= OMe, R3=OH; 5, R1= R2= OMe, R3=OAc; 6, R1= R2= OMe, R3=OBz
7, R1= R2= OMe, R3=H;
Reagents/conditions: (a) SOCl2-EtOH, RT, 2h, 87%; (b) NaOH-EtOH, RT, 2-4h,
58-93%; (c) Ac2O-C5H5N, RT, 0.5h, 91%; (d) PhCOCl-C5H5N, RT, 0.5h, 96%
Extraction and isolation
Fig. 3. Preparation of ISL (1) analogues 4–7.
Fresh rhizomes were cut into small pieces without peeling the
bark, dried at 35 ◦C in oven and powdered. The powder (625 g)
was extracted with hot acetone (1.5 l) for eight hours. The ace-
tone extract, after concentration, was fractionated with n-hexane
(3× 400 ml, 12.6 g) and ethyl acetate (4× 400 ml, 45.1 g). The
ethyl acetate extract (40 g) was column chromatographed (sil-
ica gel, 60–120 mesh, 6.5 cm × 120 cm), eluting with CHCl3 and
CHCl3:MeOH 99:1, 97:3, and 94:6, 4 l each. Similar CHCl3:MeOH
(97:3) fractions, 250 ml of each, monitored by TLC, provided LTG,
2, 1.02 g (0.18%, acetone-n-hexane). The mother liquor (1.4 g) upon
flash chromatography using CHCl3, with a flow rate of 2.5 ml/min
and a 2 min per tube collection time, yielded ISL, 1 (122 mg, 0.022%),
and LTG (605 mg, 0.11%). The purity of isolates were found to be
94–97%, detected by their HPLC analysis (chromatographic condi-
tions are shown below).
Synthesis of derivatives of isoliquiritigenin (Figs. 2 and 3)
4,4ꢀ-Diacetoxy-2ꢀ-hydroxy chalcone (3)
To a solution of ISL (300 mg, 1.17 mmol) in pyridine (10 ml)
acetic anhydride (1 ml, 10.53 mmol) was added drop wise over a
period of 10 min at 0 ◦C. The reaction mixture was allowed to stir
for 4 h at room temperature and monitored by TLC. After comple-
tion of the reaction, the reaction mixture was poured into ice cold
10% HCl solution (25 ml). The mixture was extracted with ethyl
acetate (3× 25 ml). The organic phase was washed with solution of
sodium bicarbonate (20 ml), brine (20 ml), dried over Na2SO4 and
concentrated under reduced pressure to give an oily residue which
was crystallized in methanol to afford yellow crystals in 65% (w/w)
yield, mp 88–90 ◦C (Figs. 2 and 3).
HPLC analysis
2ꢀ,4ꢀ-Dimethoxy-4-hydroxychalcone (4)
HPLC analysis was performed using a Shimadzu LC-10AD liq-
uid chromatography equipped with two LC-10A pumps controlled
by a CBM-10 interface module, SPD-M10A VP diode array detector,
and a SIL-10ADVP auto injector. Data were collected and analyzed
using a class LC-10 Work Station. The samples were analyzed by
using reverse phase chromatography on waters spherisorb ODS2
(250 × 4.6 mm i.d., 10 mm) column using binary gradient elution
with acetonitrile and water containing 0.1% TFA mobile phase
(30:70) at a flow rate of 0.6 ml/min, a column temperature of 25 ◦C
and UV detection at ꢀ 254 nm.
2ꢀ,4ꢀ-Dimethoxyacetophenone (1.80 g, 0.01 mol) and 4-
hydroxybenzaldehyde (0.01 mol) were dissolved in ethanol
(35 ml) and 0.05 ml SOCl2 was added at RT with constant stirring.
The reaction was completed in 2 h and the solid obtained was
separated by filtration, dried and crystallized in ethyl acetate
hexane to provide chalcone 4, (2.47 g, 87% yield), mp 91–92 ◦C,
identified as 2,4-dimethoxy-4ꢀ-hydroxychalcone by comparison of
its mp, IR, 1H, 13C NMR and ESIMS with the reported data (Guantai
et al. 2011).
OH
CH
O
3
OH
O
HO
H C
OH
O
O
3
a
O
O
1
3
Reagents and conditions: a) pyridine, acetic anhydride, 0oC, 4hrs, 65%
Fig. 2. Preparation of 4,4ꢀ-Diacetoxy-2ꢀ-hydroxy chalcone (3).

R. Gaur et al. / Phytomedicine 21 (2014) 415–422
417
OH
Oral glucose tolerance test in normal mice
OH
O
HO
O
HO
The blood glucose level of each animal was checked by glu-
cometer using oxidase-peroxidase glucose strips (Breeze2, Bayer
India) after 18 h starvation. Animals showing blood glucose levels
between 60 and 80 mg/dl were divided into groups of five animals
in each (n = 5). Animals of the experimental group were adminis-
tered suspension of the desired test compound orally (made in 3%
cremophor EL and 0.3% carboxymethyl cellulose mixture) at a sin-
gle dose of 100 mg/kg bw. Animals of the glucose control group
were given an equal amount of 3% cremophor EL and 0.3% car-
boxymethyl cellulose mixture. An oral glucose load (2.0 g/kg bw)
was given to each animal exactly after 30 min per oral adminis-
tration of the test sample/vehicle. A blood glucose profile of each
mouse was again determined at 15, 30, 60, 90 and 120 min post
administration of glucose using glucometer (Beguinot and Nigro
2012).
a
NOH
O
11
1
Reagents and conditions: a) NH2OH.HCl, CH3COONa, EtOH, reflux
Fig. 4. Preparation of LTG-oxime (11).
4-Acetoxy-2ꢀ,4ꢀ-dimethoxychalcone (5)
Acetylation of compound 4 (0.198 g, 0.70 mmol) with acetic
anhydride (0.5 ml) in pyridine (2 ml) afforded light orange crystals,
0.207 g, 91% yield, mp 70–71 ◦C, identified as 4ꢀ-acetoxy-2,4-
dimethoxychalcone by comparison of its mp, IR, 1H, 13C NMR and
ESIMS with the reported data (Bianco et al. 2003).
Dose optimization
4-Benzoyloxy-2ꢀ,4ꢀ-dimethoxychalcone (6)
Screening of the test compounds for hypoglycaemic activity was
performed administering single dose (100 mg/kg bw) in normal
healthy mice with the oral glucose tolerance test (OGTT). The same
protocol was used to determine the most effective dose of selected
compounds at doses of 50, 100 and 200 mg/kg bw. The minimum
dose that improved glucose tolerance significantly was selected for
further studies (Genta et al. 2010).
Compound 4 (0.198 g, 0.70 mmol) and benzoyl chloride (0.5 ml)
were stirred in pyridine (2 ml) at room temperature. After com-
pletion of benzoylation, the reaction mixture was worked-up as
usual which afforded a residue. The residue upon crystallization in
ethyl acetate-hexane furnished dark orange crystals of derivative
6, 0.372 g, 96% yield, mp 90–91 ◦C.
Streptozotocin-nicotinamide induced antidiabetic activity in mice
2ꢀ,4ꢀ-Dimethoxychalcone (7)
2ꢀ,4ꢀ-Dimethoxyacetophenone (1.80 g, 0.01 mol) and benzalde-
hyde (0.01 mol) were dissolved in ethanol (35 ml) and 10% NaOH
in ethanol (5 ml) was added drop wise at RT with constant stirring.
After completion of the reaction it was poured on ice cooled water
(100 ml) and neutralized with dilute hydrochloric acid. The yellow
precipitate was obtained, filtered, washed with water (200 ml) and
crystallized to yield chalcone 7 as a yellow solid, 2.49 g, 93% yield,
mp 65–66 ◦C, identified as 2ꢀ,4ꢀ-dimethoxychalcone by comparison
of its IR, 1H, 13C NMR and ESIMS with the reported data (Bianco et al.
2004).
Swiss albino male mice (outbred strain) of 25–30 g body weight
were acclimatized in groups of five in controlled environmen-
tal conditions (23 2 ◦C, 55 10% RH and 12 h day/night cycle),
7 days before induction of diabetes. Overnight fasted animals
were administered nicotinamide (110 mg/kg bw, i.p.) dissolved in
normal saline followed by streptozotocin (200 mg/kg bw, i.p.) dis-
solved in 100 mM citrate buffer (pH 4.5) after 15 min. Animals
were fed with 5% glucose solution for 12 h to avoid hypoglycaemia.
Hyperglycaemia was confirmed after 3 days. After 10 days, hyper-
glycaemia was reached to steady state. The blood glucose level
was determined using a glucometer. Mice having blood glucose
≥300 mg/dl were considered as diabetic and selected for the study
(Badole and Bodhankar 2010; Yadav et al. 2013). Diabetic animals
were divided into five groups containing ten animals each (n = 10).
Animals of test groups were administered orally with 1, 4 and 10 at a
dose of 200, 50 and 50 mg/kg bw respectively, suspended in 3% cre-
mophor EL and 0.3% carboxymethyl cellulose mixture for 14 days.
The disease control group was given 3% cremophor EL and 0.3%
carboxymethyl cellulose mixture, orally at the same volume. The
standard control group was treated with metformin (200 mg/kg bw,
p.o.). The normal control group was constituted using non-diabetic
animals and 3% cremophor EL and 0.3% carboxymethyl cellulose
mixture (vehicle) at same volume was given to them with the help
of oral gavage. All animals had free access to pathogen free water
and normal rodent chow (Dayal Industries, Lucknow, India) during
the experimental period. Blood glucose levels were measured at
1, 7 and 14 days of the study. On the 15 day, mice were weighed
and blood was collected from each mouse by heart puncture under
deep ether anaesthesia. Animals were sacrificed by cervical dis-
location and liver, kidney and spleen were collected from each
animal and weighed. Serum was separated from the blood and
used for estimation of alkaline phosphatase (AST), alanine transam-
inase (ALT), aspartate transaminase (AST), total bilirubin (T BIL),
total protein (TP), triglycerides (TG), high density lipoprotein (HDL),
low density lipoprotein (LDL), uric acid and creatinine using com-
mercially available kits (Randox Laboratories Ltd., Co. Antrim, UK).
Synthesis of derivatives of liquiritigenin
LTG derivatives (8–10)
LTG was acetylated and benzoylated as per methodology
reported previously by us (Gaur et al. 2010), to LTG 7,4ꢀ-diacetate
(8), LTG 4ꢀ-acetate (9) and LTG 7,4ꢀ-dibenzoate (10).
LTG-oxime (11) (Fig. 4)
It was prepared by treating (0.256 g, 0.001 mol) LTG with
hydroxylamine hydrochloride (0.069 g, 0.01 mol) in ethanol and
sodium acetate trihydrate (0.136 g, 0.001 mol) in water. The solu-
tion was heated on a water bath for 4 h with constant stirring. Upon
cooling and addition of water, a creamy precipitate was obtained.
It was filtered, washed with water, and dried. The product was
crystallized from ethanol to yield 0.235 g (91.7%), mp 125–126 ◦C.
Experimental animals
Swiss albino male mice (body weight 25–30 g) were selected
for this study. They were bred in the Animal House of the CSIR-
Central Institute of Medicinal and Aromatic Plants, Lucknow, India
and maintained on a standard rodent pellets diet and water ad libi-
tum. Permission and approval for animal studies was obtained from
CPCSEA (Reg. No. 400/01/AB/CPCSEA, AH-2012-08), Government of
India through the Institutional Animal Ethics Committee.

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R. Gaur et al. / Phytomedicine 21 (2014) 415–422
Statistical comparison is made by one way ANOVA followed by
Tukey’s multiple comparison tests using GraphPad PRISM version
5.01 (GraphPad software, USA). The value of p < 0.05 is considered
statistically significant.
Results and discussion
Chemistry
ISL (1) and LTG (2) were isolated from the chloroform/ethyl
acetate fraction of rhizomes as the procedure described earlier
and identified by the analysis of their IR, 1H, 13C NMR, DEPT-
135 and electron spray mass spectroscopy and compared with
the data reported in literature (Gaur et al. 2010). Because of
low quantity of ISL, the chalcone 1 was further modified to sev-
eral ether and ester derivatives by directly synthesizing them.
2ꢀ,4ꢀ-Dimethoxy-4-hydroxychalcone (4) was synthesized via aldol
condensation by adding thionyl chloride to a mixture of 2ꢀ,4ꢀ-
dimethoxyacetophenone and 4-hydroxybenzaldehyde in ethanol
at room temperature (Jaypal et al. 2010). The resulted chalcone
4 was further acetylated to 4-acetoxy-2ꢀ,4ꢀ-dimethoxychalcone
(5) and benzoylated to a new derivative, 4-benzoyloxy-2ꢀ,4ꢀ-
dimethoxychalcone (6).
The natural LTG upon acetylation provided LTG 7,4ꢀ-diacetate
(8) at room temperature and by refluxing the reaction mixture for
3 h, the diacetate was partially hydrolyzed to LTG 4ꢀ-acetate (9).
LTG was further benzoylated to LTG 7,4ꢀ-dibenzoate (10).
Biology
Biological screening
The test compounds were screened for antihyperglycemic
effect. The antihyperglycemic activity was evaluated in artificially
enhanced blood glucose in albino mice called oral glucose tolerance
test. ISL (1) and its derivatives 3, 4, 5, 7 and LTG derivative 9 and
10 were found to be effective at 100 mg/kg bw in OGTT. All these
compounds showed ∼30.82, 25.59, 36.07, 31.74, 24.31, 40.22 and
23.44% change in blood glucose level after 120 min during OGTT,
respectively. Therefore, compounds 1, 3, 4, 5, 7, 9 and 10 were iden-
tified as potential blood glucose-lowering agents. The rest of the
molecules were inefficient in the lowering of blood glucose levels.
These inspiring results were further verified in streptozotocin-
nicotinamide induced diabetic model in mice (Table 1).
Dose optimization
The data so far collected reveals that the compounds 1, 3, 4,
5, 7, 9 and 10 exhibited significant antihyperglycemic activity but
due to insufficient quantity of compounds 3, 5, 7 and 9 only 1, 4
and 10 were selected for antidiabetic study in in vivo model using
mice and also evaluated through OGTT to ascertain the lowest dose
required for antidiabetic effect. After 15, 30, 60, 90 and 120 min
post-administration of ISL (1) (200 mg/kg bw), ∼12.53, 40.42, 48.85,
46.81, and 36.13% improvement was noticed in glucose tolerance,
respectively (Fig. 5a). Compound 4 (50 mg/kg bw) exhibited ∼25.79,
34.65, 20.81, 14.87, and 27.05% improvement in glucose tolerance,
after 15, 30, 60, 90 and 120 min respectively (Fig. 5b), whereas Com-
pound 10 (50 mg/kg bw) displayed∼12.42, 43.43, 38.60, 29.22 and
23.92% improvement in glucose tolerance, respectively (Fig. 5c).
Hence, minimum effective dose of compounds 1, 4 and 10 was
calculated as 200, 50 and 50 mg/kg bw, respectively.
Fig. 5. Dose optimization of compound 1 (Isoliquiritigenin), compound 4 (2ꢀ, 4ꢀ-
Dimethoxy-4-hydroxychalcone) and compound 10 (liquiritigenin 7,4ꢀ-dibenzoate)
by oral glucose tolerance test (OGTT) in normal 18 h fasted mice, (a) mice admin-
istered with compound 1: 50 mg/kg bw/10 ml; 100 mg/kg bw/10 ml; 200 mg/kg
bw/10 ml, (b) mice administered with compound 4: 50 mg/kg bw/10 ml; 100 mg/kg
bw/10 ml; 200 mg/kg bw/10 ml, and (c) mice administered with compound 10:
50 mg/kg bw/10 ml; 100 mg/kg bw/10 ml; 200 mg/kg bw/10 ml, before 30 min of glu-
cose load (2 g/kg bw/10 ml). All the treatments were given orally. Data is expressed
as percent reduction in blood glucose level. n = 5 mice/group.
∼73.24% (10) towards normal levels after 14 days of treatment. The
standard drug metformin also significantly (p < 0.001) recovered
random blood glucose level by ∼32.93% (Fig. 6).
Effect on biochemical parameters
Antidiabetic activity in Swiss albino mice
Compounds 1, 4 and 10 were administered to hyperglycaemic
albino mice once daily for 14 days at a dose of 200, 50 and 50 mg/kg
bw, respectively. All the tested compounds recovered (p < 0.001)
from the hypoglycaemic state by ∼53.20% (1), ∼54.56% (4) and
The effects of compounds 1, 4 and 10 on liver glycogen contents
as well as serum biochemical parameters of liver, lipid and kidney
were also scrutinized in experimental mice to see further ben-
eficial effect of the compounds. The liver glycogen content is in

R. Gaur et al. / Phytomedicine 21 (2014) 415–422
419
Table 1
Percent change in blood glucose level of normal mice after single oral administration of test compounds (100 mg/kg bw) during glucose tolerance test.
Test compound number Test compound name
Structure of compound
Percent change in blood glucose level after
15 min 30 min 60 min 90 min 120 min
Metformin
38.13
41.80
50.55
52.09
55.12
OH
OH
O
HO
1
Isoliquiritigenin
24.72
40.72
32.93
35.73
30.82
OH
O
HO
2
3
Liquiritigenin
21.43
10.55
−5.56
−4.07
1.09
O
OCOCH
3
OH
CH COO
3
4,4ꢀ-Diacetoxy-2ꢀ-hydroxy chalcone
26.66
50.22
41.12
38.97
25.59
O
OH
OCH
H CO
3
3
4
2ꢀ,4ꢀ-Dimethoxy-4-hydroxychalcone
22.89
49.94
41.73
49.74
36.07
O
OCOCH
3
OCH
O
H CO
3
3
5
6
4-Acetoxy-2ꢀ,4ꢀ-dimethoxychalcone
27.32
44.88
47.70
34.02
31.74
OCOC H
6
5
OCH
3
H CO
3
4-Benzoyloxy-2ꢀ,4ꢀ-dimethoxychalcone
26.69
11.59
15.58
8.38
6.85
O
OCH
O
H CO
3
3
7
2ꢀ,4ꢀ-Dimethoxychalcone
47.36
30.84
35.04
25.07
24.31
OCOCH
3
O
CH COO
3
8
Liquiritigenin-7,4ꢀ-diacetate
6.80
−1.20
−8.19
−4.24
1.96
O

420
R. Gaur et al. / Phytomedicine 21 (2014) 415–422
Table 1 (Continued)
Test compound number
Test compound name
Structure of compound
Percent change in blood glucose level after
15 min
30 min
60 min
90 min
120 min
40.22
OCOCH
3
O
HO
9
Liquiritigenin-4ꢀ-acetate
7.35
34.06
35.56
47.29
O
OCOC
H
5
6
O
H
C OCO
6
5
10
Liquiritigenin-7,4ꢀ-dibenzoate
14.17
26.07
45.31
24.12
34.83
28.25
29.40
23.44
12.42
O
OH
O
HO
11
Liquiritigenin-oxime
2.44
NOH
Table 2
Effect of test compounds on serum biochemical parameters in STZ-induced diabetic mice.
Normal control
347.83 20.21
Diabetic control
Metformin
Compound 1
Compound 4
Compound 10
Liver glycogen (mg/100 mg)
Liver parameters
ALP (IU/L)
ALT (IU/L)
AST (IU/L)
172.53 15.39###
244.78 28.72
260.04 8.04*
217.60 21.02
222.82 12.91
257.09 12.54
57.35 6.17
136.89 14.78
0.27 0.04
356.86 9.14###
190.66 8.60###
273.16 13.84###
1.16 0.07###
4.45 0.21
281.19 10.07***
88.98 5.08***
160.10 12.77***
0.83 0.04*
296.23 12.38**
92.10 7.17***
195.73 13.81**
0.81 0.07**
289.35 10.79**
101.48 6.42***
234.20 11.42
0.78 0.07**
299.05 11.26*
124.98 5.06***
233.70 10.19
0.80 0.07**
T BIL (mg%)
TP (g%)
5.13 0.18
4.91 0.19
4.77 0.24
4.95 0.35
4.55 0.18
Lipid parameters
TG (mg%)
HDL (mg%)
LDL (mg%)
98.10 13.88
32.95 2.10
35.96 1.72
177.33 14.06###
19.37 2.47#
31.33 3.27
108.01 12.06**
31.69 4.23
32.18 1.37
109.22 7.32**
30.15 3.02
33.14 1.98
111.11 8.62**
28.67 3.29
32.66 1.33
105.75 9.17**
31.33 1.83
34.51 2.55
Kidney parameter
Uric acid (mg%)
Creatinine (mg%)
2.92 0.28
0.37 0.04
3.81 0.28
0.46 0.03
3.01 0.31
0.36 0.03
3.45 0.37
0.42 0.08
3.66 0.42
0.36 0.04
3.37 0.36
0.33 0.02
Each value presents mean S.E.M., n = 10.
Compound 1, 4 and 10 refers to Isoliquiritigenin, 2ꢀ,4ꢀ-Dimethoxy-4-hydroxychalcone) and liquiritigenin-7,4ꢀ-dibenzoate, respectively.
*
Statistical significance vs. diabetic control (p < 0.05).
Statistical significance vs. diabetic control (p < 0.01).
Statistical significance vs. diabetic control (p < 0.001).
Statistical significance vs. normal control (p < 0.05).
**
***
#
###
Statistical significance vs. normal control (p < 0.001).
proportion to insulin deficiency which is the primary intracellular
storable form of glucose (Chandramohan et al. 2008). Only ISL (1)
was able to modify liver tissue glycogen level significantly (p < 0.05)
by ∼49.92%.
compound 1 group by ∼56.82%. Significant recovery of T BIL levels
were observed in all treatment groups (Compound 1, 4 and 10) by
∼39.33, 42.70 and 40.45%, respectively.
Enhanced proteolysis and lowered protein synthesis occurs due
to distinct metabolic renal alterations in experimental diabetes
(Tuvemo et al. 1997) but no significant differences were noticed for
TP levels, uric acid and creatinine levels during experimentation in
any group.
The disturbances in carbohydrate, lipid and protein
metabolisms together with oxidative stress under hypergly-
caemic condition possibly affect hepatic and renal functions. An
increase in liver marker enzymes activities reflects active liver
damage (Yadav et al. 2010). Increased liver enzymes level under
insulin deficiency has been related with increased gluconeogen-
esis and ketogenesis during diabetes (Nabi et al. 2013). The oral
administration of test compounds 1 (200 mg/kg bw), 4 (50 mg/kg
bw) and 10 (50 mg/kg bw) for the period of 14 consecutive days
recovered (p < 0.05) the serum ALP levels by ∼60.77, 67.67 and
57.94%, respectively. Significant (p < 0.001) recoveries were also
observed in ALT levels by ∼73.93, 66.90 and 49.27%, respectively.
The levels of AST was significantly (p < 0.01) recovered only in the
STZ-induced diabetes also developed hyperlipidaemia which
represents a risk factor for coronary heart diseases. Due to insulin
deficiency or its resistance, a variety of alterations in metabolic
and regulatory mechanisms occur, which are responsible for the
accumulation of lipids (Sancheti et al. 2013). In this experiment, it
was observed that compound 1, 4 and 10 significantly (p < 0.01)
recovered the TG levels by ∼85.96, 83.58 and 90.34% respec-
tively. No significant changes were perceived in HDL and LDL
levels.

R. Gaur et al. / Phytomedicine 21 (2014) 415–422
421
Fig. 6. Effect of compound 1 (Isoliquiritigenin), compound 4 (2ꢀ,4ꢀ-Dimethoxy-4-
hydroxychalcone) and compound 10 (liquiritigenin-7,4ꢀ-dibenzoate) on random
Fig. 7. Effect of compound 1, compound 4 and compound 10 on organ coefficient
blood glucose level of STZ induced diabetic mice.
of diabetic mice.
The starting synthones for our synthesis, ISL and LTG were iso-
lated from ethyl acetate extract of fresh rhizomes of G. glabra as per
process reported by us (Gaur et al. 2010).
The antihyperglycemic activity of compound 1, 4 and 10 was
compared with metformin which also significantly (p < 0.05) recov-
ered the ALP, ALT, AST, T BIL and TG by ∼75. 84, 76.27, 82.97, 37.08
and 87.49% respectively, towards normal levels (Table 2).
Ethyl acetate extract of G. uralensis containing LTG is also
reported to have antidiabetic activity (Kuroda et al. 2003). ISL
and LTG were also previously reported as anti-inflammatory
(Kobayashi et al. 1995), hepatoprotective (Gaur et al. 2010) antimy-
cobacterial and antitumor agents (Jaypal et al. 2010). However, this
is the first report on in vivo antidiabetic activity of their derivatives.
The recent findings in literature reveal that antidiabetic activ-
ity of chalcone derivatives is mediated via stimulation of PPAR-␥
(Hsieh et al. 2012) and flavonoids, which are based on 2-
phenylchromone or 2-phenyl benzopyrone, have shown beneficial
effects in the treatment of hyperglycemic disease probably through
changes in the activity of intracellular enzymes, such as glucosidase
enzyme (Zhang et al. 2012). Therefore it is likely that isoliquiriti-
genin (ISL), a chalcone and liquiritigenin (LTG), a flavonoid and their
derivatives may act by above means to exert its antidiabetic effect.
The present study has shown that the ISL type chalcones as
well as LTG derived flavonoids could be used as possible leads for
the control of blood glucose level/diabetes. Therefore, plants are
excellent source or information centre of diverse molecules to be
modified or synthesized for the discovery of new therapeutics.
Effect on body weight of mice
STZ-induced diabetes is characterized by a severe loss in body
weight. The decrease in body weight is due to the increased mus-
cle destruction or degradation of structural proteins (Stephen et al.
2012). Repeated administration of test compounds 1, 4 and 10 for
14 consecutive days at oral dose of 200, 50 and 50 mg/kg bw, respec-
tively showed significant increase in animal body weight by ∼28.76,
24.23 and 24.38% respectively, compared to the toxin treated group.
Metformin treatment also increased the body weight of mice signif-
icantly by ∼23.08% as compared to diabetic control group (Table 3).
The toxicity of some drugs may only result in animal organ
weight or organ coefficient changes during the process of non-
clinical drug safety evaluation (Piao et al. 2013). Liver, kidney
and spleen coefficient were measured in this study but no signif-
icant change was observed in any treatment groups. Only liver
coefficient of diabetic control group was significantly changed as
compared to a normal control group (Fig. 7).
Acknowledgements
The antidiabetic activity of G. glabra extracts have also been
reported earlier (Gupta et al. 2011; Sharma et al. 2007). The efforts
to obtain antidiabetic compounds, structural modifications at C-4,
C-2ꢀ and, 4ꢀ positions on ISL and C-7, C-4ꢀ position on LTG were
focused.
The authors are grateful to the Director, CSIR-CIMAP for pro-
viding necessary facilities for this work. Financial support from
the National Medicinal Plant Board (NMPB), New Delhi, is duly
acknowledged.
References
Table 3
Effect of test compounds on body weight of diabetic mice.
Akpan, H.B., Adefule, A.K., Fakoya, F.A., Caxton-Martins, E.A., 2007. Evaluation of
LDH and G6-PDH activities in auditory relay centers of streptozotocin-induced
diabetic wistar rats. Journal of Analytical Sciences 1, 21–25.
American Diabetes Association (ADA), 2007. Diagnosis and classification of diabetes
mellitus. Diabetes Care 30, S42–S47.
Badole, S.L., Bodhankar, S.L., 2010. Antidiabetic activity of cycloart-23-ene-3beta,
25-diol (B2) isolated from Pongamia pinnata (L. Pierre) in streptozotocin-
nicotinamide induced diabetic mice. European Journal of Pharmacology 632,
103–109.
Beguinot, F., Nigro, C., 2012. Measurement of glucose homeostasis in vivo: glu-
cose and insulin tolerance tests. Methods in Molecular Biology (Clifton, NJ) 933,
219–228.
Bianco, A., Cavarischia, C., Farina, A., Guiso, M., Marra, C., 2003. A new synthesis of
flavonoids via heck reaction. Tetrahedron Letters 44, 9107–9109.
Bianco, A., Cabarichia, C., Guiso, M., 2004. The heck coupling reaction using aryl
vinyl ketones: synthesis of flavonoids. European Journal of Organic Chemistry,
2894–2898.
Groups
Day 1
Day 15
Normal Control
Diabetic Control
Metformin
Compound 1
Compound 4
Compound 10
26.63 1.53
28.18 1.27
25.96 1.33
27.70 1.12
26.10 1.53
27.24 0.51
27.60 1.60
19.89 0.61##
24.48 0.82*
25.61 1.02**
24.71 1.33*
24.74 0.86*
Each value presents mean S.E.M., n = 10.
Compound 1,
4
and 10 refers to Isoliquiritigenin, 2ꢀ,4ꢀ-Dimethoxy-4-hydroxy-
chalcone) and liquiritigenin-7,4ꢀ-dibenzoate, respectively.
##
Statistical significance vs. normal control (p < 0.01).
Statistical significance vs. diabetic control (p < 0.05).
*
**
Statistical significance vs. diabetic control (p < 0.01).

422
R. Gaur et al. / Phytomedicine 21 (2014) 415–422
Chandramohan, G., Ignacimuthu, S., Pugalendi, K.V., 2008. A novel compound from
Casearia esculenta (Roxb.) root and its effect on carbohydrate metabolism in
streptozotocin-diabetic rats. European Journal of Pharmacology 590, 437–443.
Gupta, N., Belemkar, S., Gupta, P.K., Jain, A., 2011. Study of Glycyrrhiza glabra on
glucose uptake mechanism in rats. International Journal of Drug Discovery and
Herbal Research 1 (2), 50–51.
Gaur, R., Kumar, S., Trivedi, P., Bhakuni, R.S., Bawankule, D.U., Pal, A., Shanker,
K., 2010. Liquiritigenin derivatives and their hepatoprotective activity. Natural
Product Communications 5, 1243–1246.
Genta, S., Cabrera, W., Mercado, M., Grau, A., Catalán, C., Sánchez, S., 2010.
Hypoglycemic activity of leaf organic extracts from Smallanthus sonchifolius:
Constituents of the most active fractions. Chemico-Biological Interactions 185,
143–152.
Mersereau, J.E., Levy, N., Staub, R.E., Baggett, S., Zogric, T., Chow, S., Ricke,
W.A., Tagliaferri, M., 2008. Liquiritigenin is plant-derived highly selec-
a
tive estrogen receptor ␤-agonist. Molecular and Cellular Endocrinology 283,
49–57.
Mlinar, B., Marc, J., Janez, A., Pfeifer, M., 2007. Molecular mechanisms of insulin
resistance and associated diseases. Clinica Chimica Acta 375, 20–35.
Nabi, S.A., Kasetti, R.B., Sirasanagandla, S., Tilak, T.K., Kumar, M.V.J., Rao, C.A.,
2013. Antidiabetic and antihyperlipidemic activity of Piper longum root aque-
ous extract in STZ induced diabetic rats. BMC Complementary and Alternative
Medicine 13, 37.
Piao, Y., Liu, Y., Xie, X., 2013. Change trends of organ weight background data
in Sprague Dawley rats at different ages. Journal of Toxicologic Pathology 26,
29–34.
Guantai, E.M., Ncokazi, K., Egain, T.J., Gut, J., Rosenthal, P.J., Bhampidipati,
R., Kopinathan, A., Smith, P.J., Chibali, K., 2011. Enone- and chalcone-
chloroquinoline hybrid analogues: in silico guided design, synthesis, antiplas-
modial activity, in vitro metabolism, and mechanistic studies. Journal of
Medicinal Chemistry 54, 3637–3649.
Hsieh, C.T., Hsieh, T.J., El-Shazly, M., Chuang, D.W., Tsai, Y.H., Yen, C.T., Wu, S.F.,
Wu, Y.C., Chang, F.R., 2012. Synthesis of chalcone derivatives as potential anti-
diabetic agents. Bioorganic & Medicinal Chemistry Letters 22 (12), 3912–3915.
Jaypal, M.R., Sreenivasa, K., Sreedhar, N.V., 2010. Synthesis and characterization of
2,4 dihydroxy substituted chalcones using aldol condensation by SOCl2/EtOH.
Journal of Chemistry and Pharmaceutical Research 2, 127–132.
Jayaprakasam, B., Doddaga, S., Wang, R., Holmes, D., Goldfarb, J., Li, X.M., 2009.
Licorice flavonoids inhibit eotaxin-1 secretion by human fetal lung fibroblast
in vitro. Journal of Agriculture and Food Chemistry 57, 820–825.
Kim, D., Choi, S.S., Kim, S., Yun, B.S., Yoo, I.D., Reddy, N.R., Yoon, H.S., Kim, K.T., 2006.
Isoliquiritigenin selectivity inhibits H(2) histamine receptor signaling. Molecu-
lar Pharmacology 70 (2), 493–500.
Kobayashi, S., Miyamoto, T., Kimura, I., Kimura, M., 1995. Inhibitory effect of
isoliquiritigenin, a compound in licorice root, on angiogenesis in vivo and tube
formation in vitro. Biological & Pharmaceutical Bulletin (Japan) 18, 1382–1386.
Kuroda, M., Mimaki, Y., Sashida, Y., Mae, T., Kishida, H., Nishiyama, T., Tsukagawa, M.,
Konishi, E., Takahashi, K., Kawada, T., Nakagawa, K., Kitahara, M., 2003. Phenolics
with PPAR-ligand-binding activity obtained from licorice (Glycyrrhiza uralensis
roots) and ameliorative effects of glycyrin on genetically diabetic KK-A(y) mice.
Bioorganic & Medicinal Chemistry Letters 13, 4267–4272.
Liu, C.J., Dixon, R.A., 2001. Elicitor-induced association of isoflavone o-
methyltransferase with endomembranes prevents the formation and 7-o-
methylation of daidzein during isoflavonoid phytoalexin biosynthesis. Plant Cell
13, 2643–2658.
Sancheti, S., Sancheti, S., Seo, S.Y., 2013. Antidiabetic and antiacetylcholinesterase
effects of ethyl acetate fraction of Chaenomeles sinensis (Thouin) Koehne fruits
in streptozotocin-induced diabetic rats. Experimental and Toxicologic Pathology
65, 55–60.
Sharma, A., Rastogi, T., Bhartiya, M., Shasany, A.K., Khanuja, S.P.S., 2007. Type 2
diabetes mellitus: phylogenetic motifs for predicting protein functional sites.
Journal of Biosciences 32 (5), 999–1004.
Songpei, L., Wei, L., Yinghua, W., Yoshihisa, A., Kazuo, K., 2010. Prenylflavonoids
from Glycyrrhiza uralensis and their protein tyrosine phosphatase-1B inhibitory
activities. Bioorganic & Medicinal Chemistry Letters 20 (18), 5398–5401.
Soumyanath, A., 2006. Traditional Medicines for Modern Times – Antidiabetic Plants.
CRC Press, Boca Raton/London/New York.
Stephen, I.S., Sunil, C., Duraipandiyan, V., Ignacimuthu, S., 2012. Antidiabetic and
antioxidant activities of Toddalia asiatica (L.) Lam. leaves in streptozotocin
induced diabetic rats. Journal of Ethnopharmacology 143, 515–523.
Tuvemo, T., Ewald, U., Kobbah, M., Proos, L.A., 1997. Serum magnesium and pro-
tein concentrations during the first five years of insulin-dependent diabetes in
children. Acta Paediatrica (Oslo, Norway: 1992) Supplement 418, 7–10.
Yadav, K.S., Yadav, N.P., Shanker, K., Thomas, S.C., Srivastav, S., Srivastava, S., Rai,
V.K., Mishra, N., Sinha, P., 2013. Assessment of antidiabetic potential of Cissam-
pelos pareira leaf extract in streptozotocine-nicotinamide induced diabetic mice.
Journal of Pharmacy Research 6, 874–878.
Yadav, N.P., Chanda, D., Chattopadhyay, S.K., Gupta, A.K., Pal, A., 2010. Hep-
atoprotective effects and safety evaluation of coumarinolignoids isolated
from Cleome viscosa seeds. Indian Journal of Pharmaceutical Sciences 72,
759–765.
Zhang, B., Chen, T., Chen, Z., Wang, M., Zheng, D., Wu, J., Jiang, X., Li, X., 2012. Syn-
thesis and anti-hyperglycemic activity of hesperidin derivatives. Bioorganic &
Medicinal Chemistry Letters 22, 7194–7197.