J Basic Clin Physiol Pharmacol 2016; 27(2): 121–129

Shiv Vardan Singh, Atul Shrivastava, Jyotshna, Upma Chaturvedi, Subhash Chandra Singh,
Karuna Shanker, Jitendra K. Saxena, Gitika Bhatia and Anirban Pal*

A mechanism-based pharmacological evaluation

of efficacy of Flacourtia indica in management
of dyslipidemia and oxidative stress in
hyperlipidemic rats
DOI 10.1515/jbcpp-2015-0017 hyperlipidemic rats. In addition, the F. indica extract
Received February 19, 2015; accepted July 20, 2015; previously showed significant in vitro antioxidant and anti-adipo-
published online October 21, 2015
genic activity. HPLC analysis indicates the presence of fla-
Abstract vanones and flavones in the extract, and the extract was
found to be non-toxic up to a dose of 2000 mg/kg body
Background: Flacourtia indica (Burm. f.) Merr. is a medici- weight in the acute oral toxicity study.
nal plant indigenous to India and is broadly used world- Conclusions: These finding suggest that F. indica holds
wide for the treatment of a variety of health ailments. significant potential in preventing clinical deterioration
The present study was experimented on hyperlipidemic induced by dyslipidemia along with oxidative stress.
Charles Foster rats with the aim to explore the possible
Keywords: acute toxicity study; dyslipidemia; Flacourtia
mechanism responsible for the antidyslipidemic activity
indica; lipid lowering; oxidative stress.
of the hydromethanolic extract from F. indica leaves (FIL).
Methods: Hyperlipidemia was induced by a single intra-
peritoneal dose of Triton WR-1339 in Charles Foster rats.
The plasma lipid levels were estimated in control and
Introduction
treated groups. The antioxidant potential of F. indica was
Cardiovascular diseases, including atherosclerosis, are the
assessed in both enzymatic and non-enzymatic systems.
leading cause of death worldwide, with hyperlipidemia
An acute toxicity study of high-performance liquid chro-
being one of the central risk factors implicated in the devel-
matography (HPLC)-fingerprinted extract was carried out
opment of lesions and atherosclerosis progression [1]. Fur-
in Swiss albino mice.
thermore, disorders of lipid metabolism are also associated
Results: The F. indica extract at a dose of 150 mg/kg signif-
with overproduction of reactive oxygen species (ROS), thus
icantly lowers the plasma level of total cholesterol (17%),
enhancing oxidative stress [2]. Hydroxyl free radicals (OH●)
triglycerides (13%), and phospholipids (16%) by increas-
are potentially involved in the initiation and progression of
ing post-heparin lipolytic activity (19%) and lecithin-cho-
atherosclerosis in hyperlipidemic individuals via the per-
lesterol-acyltransferase activity (20%) in Triton-induced
oxidative damage of lipoproteins present in the blood [3].
*Corresponding author: Dr. Anirban Pal, Central Institute of In addition, enhanced oxidative stress in such individuals
Medicinal and Aromatic Plants, Council of Scientific and Industrial further increases the risk of cardiovascular diseases, and
Research (CSIR), Lucknow 226015, India, Phone: +91 52 227 186 44, thus, the management of dyslipidemia along with oxidative
Fax: +91 52 223 42666, stress might aid in reducing these cardiovascular events
E-mail: a.pal@cimap.res.in, drapaul@gmail.com
efficiently [4]. Modern pharmacological therapies and
Shiv Vardan Singh: Central Institute of Medicinal and Aromatic
Plants, Council of Scientific and Industrial Research (CSIR), available lipid-lowering drugs, viz. fibrates, statins, and
Lucknow, India; and Central Drug Research Institute, Council of bile acid sequestrants, are effective but are associated with
Scientific and Industrial Research, Lucknow, India various side effects [5]. Moreover, some recent studies have
Atul Shrivastava, Upma Chaturvedi, Jitendra K. Saxena and Gitika also shown that long-term use of cholesterol biosynthesis
Bhatia: Central Drug Research Institute, Council of Scientific and
inhibitors has an adverse effect on brain neurotransmis-
Industrial Research (CSIR), Lucknow, India
Jyotshna, Subhash Chandra Singh and Karuna Shanker: Central
sion [6]. Therefore, the search for effective bioactive sub-
Institute of Medicinal and Aromatic Plants, Council of Scientific and stances that could efficiently metabolize the lipids along
Industrial Research (CSIR), Lucknow, India with normalizing the oxidative stress is imperative.
122      Singh et al.: Antidyslipidemic and antioxidant activity of F. indica
Natural products are the most promising source of
effective bioactive substances in the treatment of various
health ailments. Flacourtia indica (Burm. f.) Merr. (family
Flacourtiaceae) is a small bushy tree native to India and
possesses worldwide traditional medicinal values [7] in
the treatment of various health disorders viz., jaundice,
enlarged spleen, cholera, diabetes, and malaria [8]. In
recent years, the phytochemical studies of F. indica led
to the isolation of phenolic glycosides [9], butyrolactone
lignan, sterols, poliothrysoside, coumarins, flavonoids,
and condensed tannins [10].
Dyslipidemia is a common incidence generally seen
in type II diabetes and contributes a major risk for car-
diovascular diseases. Recently, ethanolic extract from the
leaves of Flacourtia indica has been reported to possess
significant antidiabetic potential [11]. Thus, the present
study was designed to evaluate (a) the mechanism-
based antidyslipidemic and antioxidant activities of the
hydromethanolic extract of F. indica leaves (FIL), (b) acute
oral toxicity of the bioactive extract, and (c) chemical fin-
gerprinting analysis of the extract by reverse-phase HPLC.
Materials and methods
Preparation of plant extract
Fresh leaves of F. indica were collected in March 2013 from the Kukrail
forest near Lucknow, India. The specimen was identified by Dr. S.C.
Singh (taxonomist) and deposited at the institutional (CSIR-Central
Institute of Medicinal and Aromatic Plants) herbarium with voucher
no. 13689. Dry powdered leaves of F. indica (100 g) were extracted with
500  mL of hydromethanolic solvent (1:1), and the solvent was dried
under reduced pressure at 1034.21  kPa and 40 °C. The process was
repeated thrice for optimum recovery of the crude extract (19.5 g).
Phytochemical analysis of plant extract
Standard phytochemical tests were performed to determine the pres-
ence of alkaloids, flavonoids, tannins, saponins, glycosides, terpe-
noids, and steroids in the extract [12]. The free radical scavenging
activity of the extract studied by using the stable radical 1,1-diphenyl-
2-picrylhydrazyl (DPPH) method and obtaining the total phenolic
content using the Folin-Ciocalteau reagent. The flavonoid content
was estimated through the aluminum chloride method as reported
earlier [12].
Drugs and chemicals
All the chemicals were procured from Sigma Chemical Company
(St Louis, MO, USA), and the standard pellet diet was purchased from
Lipton India Limited (Bangalore, India).
Animals
Adult male rats of Charles Foster strain (age 2–4 weeks old, weight
100–150 g) were bred and maintained in the animal house of the
institute and used for the experiment after approval from the Institu-
tional Animal Ethics Committee (IAEC/2010/149). The animals were
kept in controlled conditions of temperature (25 °C–26 °C), relative
humidity (60%–80%), and 12/12-h light/dark cycle (light from 8:00
a.m. to 8:00 p.m.) and provided with standard pellet diet and water
ad libitum. After the end of experiments, the animals were sacrificed
with an overdose of anesthetic ether.
Induction of hyperlipidemia
The animals were divided into six groups with six animals each:
group 1, control animals; group 2, Triton-treated animals; group 3,
Triton+FIL (50 mg/kg body weight); group 4, Triton+FIL (100 mg/kg
body weight); group 5, Triton+FIL (150 mg/kg body weight); group
6, Triton+standard drug gemfibrozil (50 mg/kg body weight).
Hyperlipidemia in rats was induced by an intraperitoneal injection
of Triton WR-1339 at 400 mg/kg body weight, prepared in normal
saline, which was administered to all the groups except the con-
trol group [13]. Simultaneously, the F. indica extract and gemfibro-
zil were prepared (macerated) with 0.2% w/w aqueous gum acacia
and administered orally at their respective doses. The control and
Triton group animals received equal volume of vehicle (gum acacia
suspension). Pellet diet was withdrawn after dosing, and the rats
were fasted for next 18 h, followed by anesthesia with sodium pen-
tothal solution (50 mg/kg i.p.), prepared in normal saline. Blood
was collected from the retro-orbital plexus using glass capillary in
EDTA-coated tubes (3 mg/mL blood). The blood was centrifuged at
2500 g for 10 min at 4 °C to harvest the plasma for further biochemi-
cal analysis.
Plasma lipids and lipoproteins
The levels of total cholesterol (TC), triglycerides (TG), phospho-
lipids (PL), and high-density lipoproteins (HDLs) were estimated
according to the methods reported earlier [14, 15]. Very-low-density
lipoprotein (VLDL) and low-density lipoprotein (LDL) were also
evaluated according to the following formulas: VLDL = TG/5 and
LDL = TC−HDL−TG/5.
Plasma lipolytic enzymes
Lecithin-cholesterol acyltransferase (LCAT) activity and post-heparin
lipolytic activity (PHLA) in plasma were measured according to meth-
ods reported earlier [16].
Risk of atherogenicity
The risk for the development of atherosclerosis was expressed in
terms of atherogenic index [(TC−HDL)/HDL] and the HDL/LDL ratio.
 Singh et al.: Antidyslipidemic and antioxidant activity of F. indica      123
In vitro antioxidant activity
The free radical scavenging efficacy of the F. indica extract (50–
150  μg/mL) was determined against the generation of superoxide
anions (O2−) and hydroxyl free radicals (OH●) in both enzymatic and
non-enzymatic systems by a method reported earlier [17].
Cell viability assay
The MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay was used to determine cell viability [18]. Briefly, the 3T3-
L1 cells (1 × 104/well) suspended in Dulbecco’s modified Eagle medium
(DMEM) containing 10% FBS were seeded into a 96-well culture plate
and incubated for 24 h under 5% CO2 with or without FIL at 5-, 10-, 25-,
and 50-μg/mL concentrations. The following day, 10 μL (5  mg/mL)
of MTT was added and incubated for 4 h, and the media was replaced
with 150 μL dimethyl sulfoxide (DMSO). The absorbance at 550 nm
was measured using spectroscopic plate (ELISA) Reader (Synergy
HT, SN. 253580, Biotech Instrument). All the samples were assayed in
triplicate to minimize the error.
Anti-adipogenic assay
Two days post-confluency, 3T3-L1 cells were treated with the induc-
tion media (10% calf serum/DMEM containing 1 μg/mL insulin, 1 μM
dexamethasone, and 500 μM IBMX). After the induction of medium
treatment (day 2), the cells were treated with insulin alone (10% calf
serum/DMEM containing 1 μg/mL insulin). Complete differentiation
was normally achieved after 8 days. To test the effect of FIL on the
differentiation of 3T3-L1 preadipocytes to adipocytes, 10- to 50-μg/
mL concentrations were used. For the assessment of adipogenesis,
the differentiated cells were fixed in 4% w/v paraformaldehyde for 20
min, washed with 1 ×  phosphate-buffered saline (PBS), and stained
with 0.34% Oil Red O in 60% isopropanol for 15 min. The cells were
washed thrice with 1 ×  PBS, and the stain was extracted with 80%
isopropanol by keeping it at room temperature for 30  min on an
orbital shaker. The optical density (OD) of the extracted dye was read
at 520 nm [19].
Acute oral toxicity
The acute oral toxicity of F. indica was carried out in Swiss albino mice
to explore its safety profile in accordance with the OECD test guide-
line no. 423 (1987). Mice were divided into three groups of six mice in
each of both sexes (group 1, vehicle control; group 2, FIL at 1000 mg/
kg body weight; group 3, FIL at 2000 mg/kg body weight). The extract
was suspended in 0.7% carboxymethylcellulose (CMC in water) and
orally administered in a single dose. In parallel, the control animals
received only the vehicle (CMC). The animals were monitored every
hour for any abnormal symptoms on the day of administration and
checked for mortality thereafter until the end of the experiment (day
7). The animals were sacrificed on the seventh day after treatment,
and blood and serum samples were collected from all the animals for
hematological and biochemical investigations.
HPLC analysis and chromatographic conditions
The chemical fingerprint of the hydromethanolic extract of F. indica
was developed by reverse-phase HPLC using a monolith column
(Merck 150 × 4.6 mm i.d.) and (A) acidified water, 0.1% AcOH, and (B)
acetonitrile-methanol, 50:50 v/v, as gradient elution. The flow rate of
mobile composition was 1.0 mL/min, and the column temperature
was maintained at 30 °C. The elution conditions were as follows: 0.01
min, 5% B; 10 min, 10% B; 15 min, 22% B; 30 min, 23% B; 40 min,
35% B; 45 min, 40% B; 50 min, 45% B 55 min, 50% B; 60 min, 60% B.
The injection volume was 10 μL, and data acquisition was performed
in the range of 200–400 nm to monitor the column eluent. The PDA
detector was set at 280  nm for the quantitative analysis of the tar-
geted compounds in the extract. A representative chromatogram
(3D-HPLC fingerprint) of the extract is depicted in Figure 4.
Statistical analysis
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 (α = 2) probability,
p < 0.05, was considered statistically significant. The number of inde-
pendent determinations for in vivo experiments was n = 6 and for in
vitro experiments was n = 3.
Results
Phytochemical analysis of plant extract
The results of the phytochemical analysis revealed the
presence of the alkaloids, flavonoids, tannins, saponins,
glycosides, terpenoids, and steroids in the active extract.
RS50 for the DPPH radical scavenging was found to be
43.34±2.65 μg/mL, whereas the total phenolic content was
found to be 12.20±1.2  mg gallic acid equivalent/g of dry
plant extract. The total flavonoid content was quantified
as 2.35±0.2 mg quercetin equivalent/g of dry plant extract.
Triton-induced hyperlipidemia
Plasma lipids and lipoproteins
Acute administration of Triton WR-1339 caused a marked
increase in plasma levels of TC (3.1-fold), TG (3.4-fold), PL
(2.9-fold), LDL (6.1-fold), and VLDL (3.4-fold) and a sig-
nificant decrease in the plasma level of HDL (−42.4%).
Treatment with F. indica hydromethanolic extract (FIL) of
hyperlipidemic rats at 150 mg/kg body weight dose signifi-
cantly lowered the plasma levels of TC (−17%), TG (−13%),
PL (−16%), LDL (−22%), and VLDL (−13%) and increased
the plasma HDL level (15%) (Figure 1).
124      Singh et al.: Antidyslipidemic and antioxidant activity of F. indica

Plasma lipolytic enzymes Risk of atherogenicity

The acute administration of Triton also caused the inhi-
bition of PHLA (−28.1%) and LCAT (−47.3%) activities,
whereas the treatment with FIL at 150 mg/kg body weight
restored PHLA (19%) and LCAT (20%) activities, which
was found comparable to standard drug gemfibrozil
(20%) (Figure 2A).
Triton-treated animals exhibited higher atherogenic index
(5.7-fold) and lower HDL/LDL ratio (87%), which indicates
a higher risk for development of atherosclerosis. Treat-
ment with FIL at 150 mg/kg body weight significantly
reduced the atherogenic index by 26% and increased the
HDL/LDL ratio by 19% (Figure 2B).

Total cholestrol Triglycerides

300
***
ns ns 300
**

200
*** 200
mg/dL

mg/dL
100
100

0 0
FIL FIL FIL FIL FIL FIL
Control Triton Gemfibrozil Control Triton Gemfibrozil
50 mg/kg 100 mg/kg 150 mg/kg 50 mg/kg 100 mg/kg 150 mg/kg

Plasma lipids High density lipoproteins

250 50
***
ns
ns 40
200 **

*** 30 ***
150
mg/dL

mg/dL

ns *
*** ns

100 20

50 10

0 0
FIL FIL FIL FIL FIL FIL
Control Triton Gemfibrozil Control Triton Gemfibrozil
50 mg/kg 100 mg/kg 150 mg/kg 50 mg/kg 100 mg/kg 150 mg/kg

Low density lipoproteins Very low density lipoproteins

200
*** 75
ns
* *** ns ns
150 *** *
50
mg/dL

mg/dL

***
100 ***

25
50

0 0
FIL FIL FIL Control Triton FIL FIL FIL
Control Triton Gemfibrozil Gemfibrozil
50 mg/kg 100 mg/kg 150 mg/kg 50 mg/kg 100 mg/kg 150 mg/kg

Figure 1: Effect of the F. indica extract on plasma lipids and lipoproteins.

Data are presented as mean±SE of six animals. The Triton-treated group was compared with control; FIL 50 mg/kg, FIL 100 mg/kg,
FIL 150 mg/kg, and gemfibrozil 50 mg/kg. ***p < 0.001, **p < 0.01, * p < 0.05, and p > 0.05 (ns).
 Singh et al.: Antidyslipidemic and antioxidant activity of F. indica      125

Cell viability and adipogenesis manner, exhibiting 23.2% inhibition at 50-μg/mL concen-
tration (Figure 2C).
The results of the MTT assay showed that FIL treatment
at concentrations between 5 and 50 μg/mL had no sig-
nificant cytotoxic effect on 3T3-L1 preadipocytes. As FIL In vitro antioxidant activity
did not show any cytotoxic effect on the proliferation of
preadipocytes, we then assessed the effect of FIL on adipo- The generation of superoxide anions (16% and 22%)
cytes differentiation. FIL treatment significantly inhibited and hydroxyl free radicals (14% and 17%) in enzymatic
the differentiation of preadipocytes in a dose-dependent systems were significantly inhibited by FIL at 100- and

A
Lecithin cholestrol acyltransferase Post heparin lipolytic activity
n mole Cholestrol released/h/L

80

n mole free fatty acid released/h/L

20 ***
ns **
60 ns
15 ***
***
ns ns **
40 *** 10

20 5

0 0
FIL FIL FIL FIL FIL FIL
Control Triton Gemfibrozil Control Triton Gemfibrozil
50 mg/kg 100 mg/kg 150 mg/kg 50 mg/kg 100 mg/kg 150 mg/kg

B
15 Atherogenic index HDL/LDL ratio
1.5
***
ns
10 *
1.0
**

5 ***
0.5
***
ns ns *
***
0 0.0
Control Triton FIL FIL FIL Control Triton FIL FIL FIL
50 mg/kg 100 mg/kg 150 mg/kg Gemfibrozil 50 mg/kg 100 mg/kg 150 mg/kg Gemfibrozil

Adipogensis MTT
C
Adipocytes formation percentage

ns ns
100 100 ns ns ns

*
% Viable cell

**
50 50

0 0
Control FIL FIL FIL FIL
Control FIL FIL FIL
5 µg/mL 10 µg/mL 25 µg/mL 50 µg/mL
10 µg/mL 25 µg/mL 50 µg/mL

Figure 2: Antidyslipidemic and antiatherogenic effects of F. indica extract; effect on lipolytic enzymes of plasma (A) and risk of
­atherogenicity (B).
Data are presented as mean±SE of six animals. Triton-treated group was compared with control; FIL 50 mg/kg, FIL 100 mg/kg, FIL 150 mg/kg, and
gemfibrozil were compared with Triton-treated animals. Effect of the F. indica extract on cell viability and adipogenesis (C). Data are presented as
mean±SE of triplicate experiments (n = 3). All groups were compared with control. ***p < 0.001, **p < 0.01, *p < 0.05, and p > 0.05 (ns).
 126      Singh et al.: Antidyslipidemic and antioxidant activity of F. indica

150-μg/mL concentrations, respectively. FIL also inhibited HPLC analysis and chromatographic
the non-enzymatic generation of superoxide anions (19%) conditions
and hydroxyl free radicals (21%) at 150 μg/mL concentra-
tion (Figure 3). Fourteen major peaks representing phenolics, viz., 7.584,
15.601, 20.07, 23.511, 24.227, 25.073, 26.757, 30.767, 34.661,
49.713, 50.499, 51.526, 52.546, and 54.499 min, were
Acute oral toxicity observed in the hydromethanolic extract of F. indica,
using monolithic-HPLC methodology. Each of the flavo-
No observational changes, morbidity, or mortality was noid peaks was well resolved from the neighboring peaks,
observed throughout the experimental period up to the displaying excellent peak symmetry and separation effi-
dose of 2000 mg/kg body weight. Blood (serum) samples ciency (Figure 4).
upon analysis showed non-significant changes in all the
parameters like total hemoglobin, red blood cell (RBC)
count, white blood cell (WBC) count, serum glutamic Discussion
pyruvic transaminase (SGPT), alkaline phosphatase
(ALKP), creatinine, TGs, cholesterol, albumin, and serum Hyperlipidemia, along with oxidative stress, has been con-
protein (Table 1). No significant changes were found in the sidered as a more prominent causative factor for the devel-
relative organ weight of the experimental animals. opment of cardiovascular diseases such as atherosclerosis,

Superoxide anions (Enzymatic) Hydroxyl free radicals (Enzymatic)

75
ns ns
300
n mole Melondealdehyde formed

** *
**
n mole Formazone formed

***
50
*** ***
200

25
100

0 0
Control FIL FIL FIL Standard Control FIL FIL FIL Standard
50 µg/mL 100 µg/mL 150 µg/mL 50 µg/mL 100 µg/mL 150 µg/mL

Hydroxyl free radicals (Non-enzymatic)

Superoxide anions (Non-enzymatic)
300
200
ns
n mole Melondealdehyde formed

*
n mole Formazone formed

** 150 ns
200 *
**
***
***
100

100

50

0
Control FIL FIL FIL Standard 0
Control FIL FIL FIL Standard
50 µg/mL 100 µg/mL 150 µg/mL
50 µg/mL 100 µg/mL 150 µg/mL

Figure 3: Effect of F. indica extract on in vitro generation of superoxide anions and hydroxyl free radicals in enzymatic and non-enzymatic
systems.
Data are presented as mean±SE of triplicate experiments (n = 3). All groups were compared with control. ***p < 0.001, **p < 0.01, *p < 0.05,
and p > 0.05 (ns).
 Singh et al.: Antidyslipidemic and antioxidant activity of F. indica      127

Table 1: Effect of FIL hydromethanolic extract on different hematological and biochemical parameters in Swiss albino mice.

Parameters   Control  1000 mg/kg  2000 mg/kg

Hematological      
 Body weight, g   36.03±2.89  31.09±2.98  30.19±2.12
 Total WBC count, thousand cells/mm3   4.28±0.45  5.56±1.41  5.59±0.85
 Total RBC count, million cells/mm3   8.17±1.13  7.69±0.74  7.32±.72
 Hemoglobin, g/dL   15.18±0.34  13.58±0.37  15.68±0.87

Biochemical      
 SGOT, U/L   32.40±3.84  32.66±2.50  32.50±1.53
 SGPT, U/L   11.07±1.84  11.23±1.310  12.60±1.26
 ALKP, U/L   310.23±19.87  311.92±46.86  314.04±11.7
 Creatinine, mg/dL   0.84±0.04  0.91±0.08  1.03±0.12
 Serum protein, mg/dL   1.02±0.08  0.98±0.05  1.03±0.07
 Serum bilirubin, mg/dL   0.6±0.05  0.71±0.023  0.69±0.021
 Serum TGs, mg/dL   101.01±21.40  118.17±17.29  129.56±20.65
 Total serum cholesterol, mg/dL   110.90±5.50  106.44±7.13  123.36±14.20

Data are the mean percentage±SD of six mice (both sexes) in each group. SGOT, Serum glutamic oxaloacetic transaminase.

acute myocardial infarction, hypertension, and coronary
heart diseases [20]. In the present study, hydromethanolic
extract from the leaves of F. indica was investigated for
its antihyperlipidemic activity in Triton-induced hyper-
lipidemic rats. Triton WR-1339 (tyloxapol) is non-ionic sur-
factant being widely used to investigate the possible mode
of action of lipid-lowering drugs/molecules [13]. Triton
also inhibits the lipases activity and obstructs the uptake
of lipoproteins from circulation by extra-hepatic tissues,
which results into higher level of circulatory lipids [21]. The
F. indica extract treatment significantly reduced the lipid
level in hyperlipidemic animals at a dose of 150 mg/kg
body weight. In fasting condition, the only source of serum
lipids is endogenous production; thus, the reduction in
plasma lipids level by the F. indica extract clearly indicates
that it has an effect on endogenous lipid metabolism.
Furthermore, to explore the possible mode of action of
the F. indica extract, LCAT activity and PHLA were analyzed
in experimental animals. The LCAT and PHLA activities
were found to be reduced in Triton-induced hyperlipidemic
animals, whereas they increased in the F.  indica-treated
groups. As LCAT converts cholesterol into a cholesteryl
ester (a more hydrophobic form of cholesterol), which is
then sequestered into the core of a lipoprotein particle
(HDL) [17], a reduced cholesterol and enhanced HDL level
was found in F. indica-treated animals. Administration of
heparin induces the release of various lipolytic lipases (viz.
TG lipase and lipoprotein lipase) located on the surface of
endothelial cells, which causes the breakdown of lipopro-
teins and lipids [22]. In our study, the enhanced activity of
PHLA in F. indica-treated animals may be responsible for
the reduction in the plasma levels of lipids (TG and PL)
and lipoproteins (LDL and VLDL). The HDL/LDL ratio and
the atherogenic index are the two common indicators that
reflect the risk of cardiovascular diseases in an individual.
In Triton-treated hyperlipidemic rats, the HDL/LDL ratio
was found to be much lower than normal, whereas the F.
indica extract enhanced the HDL/LDL ratio by reducing
the LDL level and increasing the HDL level in treated rats.
The atherogenic index is considered as a better indicator
of cardiovascular disease risk than individual lipoprotein
concentrations [23]. In our experiments, Triton increased
the atherogenic index in hyperlipidemic rats. F. indica,
meanwhile, significantly lowered the atherogenic index
and increased the HDL/LDL ratio, indicating that F. indica
significantly reduces the risk of cardiovascular diseases
including atherosclerosis.
Besides in vivo lipid-lowering activity, F. indica also
possesses potential antioxidant activities. The DPPH free
(stable) radical scavenging activity results revealed that
the F. indica extract has significant radical scavenging effi-
cacy. As DPPH radical scavenging activity is widely used as
a marker to evaluate the antioxidant potential of the plant
extracts/molecules, we further explored its antioxidant
potential in enzymatic and non-enzymatic systems. The
F. indica extract commendably inhibits the generation of
O2− and OH● in a concentration-dependent manner in both
enzymatic and non-enzymatic systems (in vitro). Inhibi-
tion of the enzymatic system suggests that the F.  indica
extract has inhibitory effect on the enzymes/agents
responsible for the endogenous production of O2− and OH●
radicals, whereas the results of the non-enzymatic system
128      Singh et al.: Antidyslipidemic and antioxidant activity of F. indica

34.661
15.601

23.511

26.757

49.713
30.767

50.499
25.073
24.227
20.07
7.584

51.526

54.499
52.546
323.7

328.4

328.4
328.4
323.7

318.9
311.8

289.2
285.7

285.7
284.5

285.7
283.3

283.3
283.3
271.5

271.5

256.1
229.0
229.0

227.8

226.6
226.6

226.6
226.6

224.3
220.8
217.2
217.2

217.2
217.2
217.2
2.80
2.60
2.40
2.20
2.00
1.80
1.60

AU
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

250.00
300.00
350.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
Min

Figure 4: 3D-HPLC chromatogram of FIL hydromethanolic extract (100 mg/mL).

The upper plot comprises the characteristic UV-VIS spectrum of major peaks corresponds to the flavonoidal-glycosides group of compounds
present in the extract.

proved that it also has the efficacy to remove the radicals mobile phases, such as methanol, acetonitrile, tetrahydro-
from the circulation. These results collectively indicate furan, or acetic acid solutions. The chromophoric nature of
that F.  indica may reduce oxidative stress by inhibiting flavonoids makes them unique to identify based on their
endogenous generation and neutralization of preformed UV spectra [26]. The classes of flavonoids that characterize
free radicals. Moreover, F.  indica treatment significantly FIL hydromethanolic extract (flavanones, flavones, and, to
increases plasma levels of HDL, which is itself a powerful a lesser extent, flavonols/flavanols) have their maximum
antioxidant on its own [24]. Furthermore, the effect of the absorption at specific wavelength ranges: flavanones
F. indica extract on adipogenesis of 3T3-L1 preadipocytes (280–290 nm), flavones (304–350 nm), and flavonols (352–
was also studied because many known lipid-lowering 385  nm). A representative 3D-PDA HPLC chromatogram
drugs like niacin targets the adipogenesis and inhibits (200–400 nm) of F. indica extract (Figure 4) highlights the
lipid accumulation [25]. The F. indica extract significantly presence of flavanones and flavones with their characteris-
inhibited lipid accumulation in preadipocytes, without tic UV spectrum plot index. Presence of substantial amount
causing any effect on the viability of cells. This indicates of flavonoids in the active extract may be responsible for
that F. indica reduces adipogenesis without causing apop- the observed antidyslipidemic and antioxidant activities.
tosis, so there might be a possibility of another mechanism
behind its anti-adipogenic activity. In acute oral toxicity,
the F. indica extract was found to be well tolerable and did Conclusions
not create any sign of toxicity and is thus considered safe
up to a dose of 2000 mg/kg body weight. The results of the present study clearly indicate that FIL
Reverse-phase chromatography has been extensively have significant potential to lower plasma lipids level
employed for the separation of flavonoids on C8 or C18 in hyperlipidemic conditions and also possess potential
columns but rarely on monolithic columns with polar antioxidant activity. Thus, F. indica may be used as a good
 Singh et al.: Antidyslipidemic and antioxidant activity of F. indica      129
herbal candidate for the treatment of cardiovascular dis-
eases and related complications. This preliminary work
will be also helpful in the further characterization of active
extract to obtain effective phytomolecules (responsible for
the observed activity) with their possible mode of action.
Acknowledgments: The authors are grateful to the direc-
tor of CSIR-CDRI and CSIR-CIMAP for providing the neces-
sary research facilities to carry out this work. S.V.S. is also
thankful to the Indian Council of Medical Research (ICMR),
India, for the award of Senior Research Fellowship.
Author contributions: All the authors have accepted
responsibility for the entire content of this submitted
manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played
no role in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; or in the
decision to submit the report for publication.
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