Antidyslipidemic and antioxidant effects of novel lupeol-derived chalcones

Article abstract of DOI:10.1007/s11745-013-3824-0

A series of Lupeol-based chalcones have been synthesized aiming to enhance the therapeutic efficacy of parent compound, the novel compounds were evaluated for their antidyslipidemic activity in triton-WR 1339 induced hyperlipidemic rats. Among the ten synthesized chalcones, the most active K4, K8, and K9 reversed the plasma levels of TC by (24, 25, 27 %), phospholipid by (25, 26, 25 %) and triacylglycerol by (27, 24, 24 %) respectively. In addition, the compounds showed significant in vitro antioxidant activity. The lipid lowering activity of these compounds were mediated through lipoprotein lipase activation (12-21 %) and enhanced post-heparin lipolytic activity (15-16 %). The compounds also displayed noteworthy inhibitory effect on 3-hydroxy-3-methyl-glutaryl reductase activity (in vitro). The in vitro effect of the most active compounds on MDI-induced adipogenesis using 3T3-L1 preadipocytes at 10 and 20 μM concentrations showed significant inhibition (20-32 %) of adipogenesis.

Full text of DOI:10.1007/s11745-013-3824-0

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DOI 10.1007/s11745-013-3824-0
ORIGINAL ARTICLE
Antidyslipidemic and Antioxidant Effects of Novel
Lupeol-Derived Chalcones
•
Shishir Srivastava Ravi Sonkar Sunil Kumar Mishra
•
•
•
•
Avinash Tiwari Vishal Balramnavar Snober Mir
•
•
Gitika Bhatia Anil K. Saxena Vijai Lakshmi
•
Received: 14 March 2013 / Accepted: 29 July 2013
Ó AOCS 2013
Abstract A series of Lupeol-based chalcones have been
synthesized aiming to enhance the therapeutic efficacy of
parent compound, the novel compounds were evaluated for
their antidyslipidemic activity in triton-WR 1339 induced
hyperlipidemic rats. Among the ten synthesized chalcones,
the most active K4, K8, and K9 reversed the plasma levels
of TC by (24, 25, 27 %), phospholipid by (25, 26, 25 %)
and triacylglycerol by (27, 24, 24 %) respectively. In
addition, the compounds showed significant in vitro anti-
oxidant activity. The lipid lowering activity of these
compounds were mediated through lipoprotein lipase
activation (12–21 %) and enhanced post-heparin lipolytic
activity (15–16 %). The compounds also displayed note-
worthy inhibitory effect on 3-hydroxy-3-methyl-glutaryl
reductase activity (in vitro). The in vitro effect of the most
active compounds on MDI-induced adipogenesis using
3T3-L1 preadipocytes at 10 and 20 lM concentrations
showed significant inhibition (20–32 %) of adipogenesis.
Keywords Chalcones Á Lupeol Á Lupenone Á
Lipoprotein lipase (LPL) Á Antihyperlipidemic
activity
Abbreviations
PHLA Post-heparin lipolytic activity
LPL
TC
Lipoprotein lipase
Total cholesterol
TAG
PL
Triacylglycerol
Phospholipids
HDL
LDL
High density lipoprotein
Low density lipoprotein
VLDL Very low density lipoprotein
CVD
DCM
PCC
MHz
NMR
ESI
Cardiovascular diseases
Dichloromethane
Pyridinium chlorochromate
Mega hertz
Nuclear magnetic resonance
Electrospray ionization
Mass spectrometry
MS
S. Srivastava Á S. K. Mishra Á A. Tiwari Á V. Balramnavar Á
A. K. Saxena
Medicinal and Process Chemistry Division, Central Drug
Research Institute, Lucknow 226 001, India
Introduction
R. Sonkar Á G. Bhatia
Hypercholesterolemia is the main cause and concern for
development of atherosclerosis and other related fallouts
like myocardial ischemia and cardiac events [1]. Elevation
of blood cholesterol followed by the generation of reactive
oxygen species lead to endothelial dysfunction [2]. Cellular
damage to the endothelium is the basic cause for initiation
and maintenance of atherosclerosis. The cumulative effect
of hyperlipidemia and free radical species has a detrimental
effect on the body and begins a chain of events leading to
Department of Biochemistry, Central Drug Research Institute,
Lucknow 226 001, India
S. Mir
Department of Biotechnology, Integral University,
Lucknow 226 001, India
V. Lakshmi (&)
Department of Biochemistry, King George Medical University,
Lucknow 226 003, India
e-mail: vijlakshmius@yahoo.com
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coronary heart disease (CHD). Among the drug therapy
approaches for the disease, Statins have significantly
reduced the risk of CHD related deaths over the past two
decades [3]. Statins [4] generally focus on reducing the low
density lipoproteins (LDL). Although statins are effective
as a treatment but their drawback is that they induce certain
adverse coronary events as well as not possessing antiox-
idant properties. Fibrates represent another class of com-
pounds used for the treatment of hyperlipidemia but
requires higher doses for significant effects [5]. The com-
bination therapy is also used for these two classes resulting
in serious safety concerns. Therefore, there is an emergent
need for different classes of compounds to combat this
dreaded metabolic disorder. Natural products are the most
promising and consistent source of drug leads. As a part of
our drug discovery program on Indian medicinal plants
actively pursued by CDRI, we have been working on the
indigenous plant, Crataeva nurvala Buch–Ham (Capp-
aridaceae). The bark of the plant is a major source of lu-
peol, a molecule of interest, a pentacyclic triterpene with
diverse biological activities like antiurolithic [6], anti-
inflammatory [7], cytoprotective [8], antidiabetic, and an-
tidyslipidemic [9].
hexane–benzene, and benzene. The fraction collected with
benzene gave a single component, lupeol (yield 0.7 %), mp
215 °C, which was identified by spectroscopic techniques
[10].
Preparation of Lupenone (I) from Lupeol (S)
Lupeol (1) (10 g, 23.37 mmol) dissolved in dichloro-
methane (50 ml) was stirred at room temperature with
pyridinium chlorochromate (35.2 mmol, 1.5 equivalent)
for 24 h. The mixture was filtered through Celite with
ether, concentrated on a rotavapor to give the product 2
(8 g, 80 %), mp 166–168 °C [11].
Representative Procedure for the Synthesis of Target
Compounds (K1–K10)
To
a
magnetically stirred solution of lupenone
(I) (0.424 g, 1.0 mmol) in ethanol (20 ml), different
substituted benzaldehydes (1.0 mmol) and NaOH
(1.0 mmol) were added. The reaction mixture was stirred
for 24 h at room temperature. After completion of the
reaction (TLC monitoring) ethanol was evaporated under
reduced pressure. The compound was extracted with
chloroform. The combined organic extract was washed
with water, brine solution, dried (Na2SO4) and solvent
was removed under reduced pressure. The crude product
obtained was column chromatographed (60–120 mesh,
SiO2) using hexane and chloroform in different concen-
trations (ranging from 10 to 80 % chloroform in hexane)
to give the target compounds. (Scheme 1). These chal-
cone derivatives were synthesized in moderate to good
yield (Table 1).
The present study deals with (1) the synthesis of
various lupeol derived chalcones to enhance the thera-
peutic efficacy of the parent compound against dyslipi-
demia, (2) estimation of the plasma lipid profile and
determination of the possible mechanism of action, (3)
evaluation of the antioxidant potential of the derivatives,
and (4) effects of the most active compounds on
3-hydroxy, 3-methyl-glutaryl CoA (HMG-CoA) reductase
activity and adipogenesis (3T3L-1 cell line) in a dose-
dependent manner.
Biology
Materials and Methods
Chemistry
Animals
Male adult rats of the Charles Forest (CF) strain (body
weight 100–150 g) were kept in animal houses with con-
trolled temperature (25–26 °C), humidity (60–80 %) and
12/12 h light/dark cycle, under hygienic conditions. Before
starting the experiment, the animals were acclimatized for
1 week and had free access to the normal diet and water
ad libitum. Experimental procedures were approved by
ethical committee of our institute.
Plant Material
The stem bark of C. nurvala was purchased from a local
market and identified by the Botany Department of CDRI,
Lucknow, India. A voucher specimen (no. 3664) has been
preserved for future reference in our institution.
Isolation of Lupeol
Lipid Lowering Activity in Triton-Induced Hyperlipidemic
Rats
Lupeol was isolated from the stem bark of C. nurvala [5].
C. nurvala stem bark powder (1 kg) was extracted with
petroleum ether by cold percolation and the resulting
extract was subjected to column chromatography on silica
gel (60–120 mesh), eluting successively with hexane,
The rats were divided into 15 groups [group 1: control,
group 2: triton-induced hyperlipidemic rats, group 3–14:
triton-induced hyperlipidemic rats with compounds
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Scheme 1 Synthesis of lupeol
derived chalcone. Reagents and
conditions: (a) pyridinium
chlorochromate (pcc), DCM,
24 h. (b) Different aromatic
aldehyde (RCHO), NaOH,
ethanol, 24 h
earlier [21, 22]. Total cholesterol (TC), phospholipid
(PL), triacylglycerol (TAG), and apo-lipo-protein contents
of VLDL, LDL and HDL were analyzed by standard
procedures [23]. The free radical scavenging potential of
these compounds against formation of superoxide anions
(O2-) and hydroxyl free radicals (OHÁ) as well as their
effect on microsomal lipid peroxidation were measured
(in vitro) in the absence or presence of the compounds
(100 lg and 200 lg/ml) using the method reported earlier
[24].
Table 1 Percentage yield of differently substituted chalcone deriv-
atives of lupeol
Compound no. R1 R2
R3
R4
R5 Yield %
K1
K2
K3
K4
K5
K6
K7
K8
K9
K10
H
H
H
H
H
H
H
H
H
H
OCH3 OCH3
OCH3 OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
63
61
67
62
54
55
60
49
53
46
H
H
C2H6N
CH3O
H
H
OCH3 C6H5OCH2
H
NO2
H
H
H
Cl
Br
NO2
F
H
H
H
In-Vitro HMG-CoA Reductase Inhibitory Activity
H
H
H
H
The HMG-CoA reductase assay was performed using the
HMG-CoA reductase assay kit from Sigma-Aldrich (St.
Louis, MO, USA). HMG-CoA (substrate), NADPH, assay
buffer and enzyme HMGR were supplied with the assay
kit.
(S = lupeol, I = lupenone, K1–K10), and group 15: tri-
ton-induced hyperlipidemic rats treated with the standard
drug Gemfibrozil (100 mg/kg)] containing six rats in each
group. In this 18-h acute experiment, hyperlipidemia was
induced by the administration of triton WR-1339 [12–15]
(Sigma chemical company, St. Louis, MO, USA) intra-
peritoneally at a dose of 400 mg/kg body weight to rats of
all the groups except the control group. The compounds
were macerated with gum acacia suspended in water
(0.2 % w/v) and fed orally simultaneously with triton at a
dose of 100 mg/kg [16, 17] and the diet was withdrawn.
Rats of the control and triton-treated groups were given
the same amount of the gum acacia suspension (vehicle).
After 18 h of treatment, the rats were anaesthetized with
thiopentone solution (50 mg/kg body weight) prepared in
normal saline and then 1.0 ml blood was withdrawn from
the retro to orbital plexus using a glass capillary and put
into EDTA coated tubes (3.0 mg/ml blood). The blood
was centrifuged at 2,5009g for 10 min at 4 °C and the
plasma was separated and used for analysis of total
cholesterol (TC), phospholipids (PL), triacylglycerol
(TAG) by standard enzymatic methods [18–20]. Lipo-
protein lipase (LPL) and post-heparin lipolytic activity
(PHLA) were determined as per the method reported
Anti-Adipogenic Assay
The 3T3-L1 mouse embryo fibroblast cells were obtained
from the American Type Culture Collection. Cells were
seeded in 24-well plates (50,000 cells/well) and incubated
at 37 °C and 5 % CO2 in Dulbecco’s Modified Eagle
Medium (DMEM) containing 10 % heat-inactivated FBS
(v/v) and antibiotic amphotericin B and gentamycin, after
48 h of incubation, the culture media was replaced with
adipogenesis media, i.e., MDI ? ve (insulin 5 lg/ml,
dexamethasone 1 lM and isobutylmethylxanthine (IBMX)
0.5 mM). Two days after the induction medium treatment,
the cells were treated with insulin alone (5 lg/ml in
DMEM containing 10 % FBS). The culture medium was
replaced every 48 h until the preadipocytes differentiated
into mature adipocytes. The most active compounds (K4,
K8, and K9) were assessed for their effect on 3T3-L1
adipocyte differentiation. These compounds were added at
concentrations of 10 and 20 lM throughout the differen-
tiation. The differentiated cells were fixed in 4 % para-
formaldehyde w/v for 20 min, washed with phosphate
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buffered saline (PBS) and stained with 0.34 % Oil Red O
in 60 % isopropanol for 20 min. Then they were washed
with PBS two times and the stain was extracted with 100 %
isopropanol by keeping it at room temperature for 10 min
on an orbital shaker. An OD of the extracted dye was taken
at 520 nm [25].
Table 2 Antihyperlipidemic activity of lupeol based chalcone
derivatives (K1–K10) in Triton-induced hyperlipidemic model at a
dose of 100 mg/kg
S.
no.
Compound
TCa
PLa
TAGa
PHLAb
1
2
3
4
5
6
S (Lupeol)
-11*
-13*
-13*
-15*
-15*
-
-12*
-14*
-13*
-16*
-11*
-
-11*
-13*
-11*
-14*
-11*
-
?3
?4
I (Lupenone)
K1
K2
K3
K4
?8
Statistical Analysis:
?10
?7
All groups were compared by one way analysis of variance
(ANOVA) and the significance of the mean difference
between different groups was done by Tukey’s post hoc
test. A two-tailed (a = 2) probability p \ 0.05 was con-
sidered statistically significant (*p \ 0.05, **p \ 0.01,
***p \ 0.001, p [ 0.05 (ns = not significant).
?15
24***
25***
27***
7
K5
K6
K7
K8
-10*
-6ns
-9ns
-
-8ns
-9ns
-10*
-
-9ns
-5ns
-12*
-
?5
?3
8
9
?5
10
?15
25***
26***
24***
11
K9
-
-
-
?16
27***
-9ns
25***
-6ns
24***
-8ns
Results
12
13
K10
?4
Gemfibrozil
(Standard)
-
31***
-
33***
-
33***
?19
The acute administration of triton WR-1339 caused a
marked increase in plasma levels of TC (2.00 fold), PL
(1.59 fold), and TAG (2.83 fold) and inhibition of plasma
PHLA (19 %). A significant decrease was noticed in TC,
PL, and TAG after treatment with lupeol derivatives in
comparison to the parent compound. However, gemfibrozil
exerted a greater lipid lowering effect than these com-
pounds The results shows that lupeol derivatives K4, K8,
and K9 significantly decreased the levels of TC by 24, 25,
and 27 %; PL by (25, 26, and 25 % and TAG by 27, 24,
and 24 % respectively. Other compounds were mildly
active. The lupeol derivatives partially reactivated PHLA
activity in hyperlipidemic rats. K4, K8, and K9 were found
to be more active and enhanced the PHLA by 15, 15, and
16 % respectively (Table 2). Other compounds were
moderately active.
Values are means SD of six rats
Units of chalcone series compounds: amg/dl, bn mol of free fatty acids
formed/h/ml of plasma
TC Total cholesterol, PL phospholipid, TAG triacylglycerol, ns
insignificant starting, I intermediate
*** p \ 0.001; ** p \ 0.01; *p \ 0.05
Triton administration inhibited the LPL activity by 35 %
while treatment with K4, K8, and K9 reactivated the LPL
activity by 12.6 %, 16.1, and 20.8 % respectively (Fig. 2).
The results are significant and explain the possible mech-
anism of action.
The inhibitory activity of K4, K8, and K9 were evaluated
at five different concentrations ranging from 5 to 80 lM for
HMG-CoA reductase activity. Compounds exhibited max-
imum inhibition at a dose of 80 lM (69.5 64.44, and
70.30 %) (Fig. 3), results are comparable to those of lova-
statin which showed an inhibition of 73.13 % at this dose.
It is well established that an excess of reactive oxygen
species induces lipid peroxidation as well as protein
modification, which in turn results in cardiovascular com-
plications. In view of the above facts, we evaluated anti-
oxidant activity of the lupeol derivatives in vitro.
Compounds K2, K4, K8, and K9 significantly decreased
superoxide anions (17, 28, 23, and 23 % at 100 lg/ml and
27, 39, 39, and 25 % at 200 lg/ml), hydroxyl radicals (18,
15, 17, and 18 % at 100 lg/ml and 25, 24, 26, and 20 % at
200 lg/ml) and microsomal lipid peroxidation (17, 16, 18,
and 12 % at 100 lg/ml and 28, 25, 26, and 20 % at 200 lg/
ml) respectively. Other compounds were moderately
active. The standard drug Allopurinol, Mannitol and
The most active compounds were further analyzed for
their effect on the lipid profile of lipoproteins. The triton
administration in rats significantly increased the TC, PL,
TAG and apo-protein level of VLDL by 2.8, 1.8, 22, and
2.3 folds, LDL by 2.4, 3.1, 3.0 and 2.0 folds and followed
by decrease in HDL by 23, 26, 25, and 23 % respectively.
The results shows that lupeol derivatives K4, K8, and K9
significantly decreased the levels of VLDL–TC by 22, 24,
and 24 %; VLDL–PL by 20, 23, and 25 %; VLDL–TAG
by 20, 16, and 27 % and VLDL–apoprotein by 23, 26, and
25 % respectively. Similarly LDL–TC decreased by 25, 26,
and 23 %; LDL–PL by 27, 25, and 26 %; LDL–TAG by
23, 27, and 25 % and LDL–apoprotein by 26, 23, and 25 %
followed by increase in HDL–TC by 25, 26, and 22 %,
HDL–PL by 23, 22, and 24 %, HDL–TAG by 22, 24, and
25 % and HDL–apoprotein by 25, 23, and 24 % respec-
tively (Fig. 1).
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