Anti-hyperlipidemic Activity of Methanolic Extract of Impatiens
Balsamina L. in Hypercholestrolemic Induced Sprague Dawley Rats
Santosh Fattepur
1*
, Kiran Chanabasappa Nilugal
1
, Thurga Devi Balan
1
, , May Florence Dela Cruz
Bacayo
1
, Wong Charng Choon
1
, Mohamed Rasny Mohamed Razik
1
, Fadli Asmani
1
, Eddy Yusuf
2
1
School of Pharmacy, Management and Science University, 40100 Shah Alam, Selangor, Malaysia;
2
International Center for Halal Studies, Management and Science University, 40100 Shah Alam, Selangor, Malaysia
Keywords: Anti-cholesterol, Anti-hyperlipidemia, Impatiens Balsamina L., Simvastatin.
Abstract: Background & Aim: The occurrence of hyperlipidemia, is currently increasing at a remarkable rate
throughout the world. Hyperlipidemia graded as one of the greatest risk factors that contributes to the
prevalence and severity of life threatening coronary heart diseases. Medicinal plants and their products are
safer than their synthetic counterparts, including those involved in the anti-hyperlipidemic drugs statins.
Impatiens Balsamina (IB) is used medicinally for various ailments. No study was carried out on the anti-
cholesterol activity of IB. Objective: To study anti-hyperlipidemic activity of methanolic extracts of IB
(MEIB) in hypercholesterolemia induced Sprague Dawley rats. Materials & Method: MEIB leaves were
prepared using maceration method. Toxicity study was carried out using OECD guidelines.
Hypercholesterolemia in rats was induced by using 6% of lard oil, 2% of cheese and egg yolk. Two different
doses 200 and 400mg/kg of MEIB were used to study for anti-hyperlipidemic activity. Histopathological
study was carried out in rats. Results: No mortality was observed even up to 2g/kg. Only 400mg/kg of
MEIB statistically decreased in total cholesterol (P<0.05), LDL-cholesterol (P<0.05) and an increase in
HDL-cholesterol (P<0.05) as compared to the positive control. Histopathology study revealed that 400mg/kg
MEIB leaves administered group have mild steatosis and no inflammation as compared to control group.
Conclusion: MEIB could be a potential herbal medicine as adjuvant with existing therapy for the treatment
of hyperlipidemia.
1 INTRODUCTION
The occurrence of hyperlipidemia, is currently
increasing at a remarkable rate throughout the world
and there is a close connection between the
hyperlipidemia and cardiovascular diseases (CVD)
which has been well documented (Suanarunsawat et
al 2010). Hyperlipidemia represents a spectrum of
metabolic disorders that can be found in many
humans today. The manifestation of the
hyperlipidemia is defined as an abnormal increase in
one or more of the serum lipids profile such as either
total cholesterol (TC), low-density lipoprotein-
cholesterol (LDL-C) and triglycerides (TG) (Bencze
et al., 2012). Hyperlipidemia need to be given special
attention since it has been one of the greatest risk
factors that contributes to the prevalence and severity
of coronary heart diseases (Sudha et al 2011).
Statins reducing the risk of coronary events, has been
shown in large scale studies of both primary and
secondary intervention to reduce coronary artery
disease. These drugs will correct the modified blood
lipid profile by inhibiting the biosynthesis of
cholesterol and also by improving the clearance of
triglycerides rich lipoproteins (Girija et al 2011). The
use of synthetic hypolipidemic drugs may lead a
person to hyperuricemia, diarrhea, nausea, myositis,
gastric irritation, flushing, dry skin, and also
abnormal liver function (Subramaniam et al 2011).
Hence, there is a need to find other essential
materials from natural source that can give less
toxicity or side effects with better safety and efficacy
on long term usage. The cost of the therapy also
should be taken into consideration. Natural products
Fattepur, S., Nilugal, K., Balan, T., Bacayo, M., Choon, W., Razik, M., Asmani, F. and Yusuf, E.
Anti-hyperlipidemic Activity of Methanolic Extract of Impatiens Balsamina L. in Hypercholestrolemic Induced Sprague Dawley Rats.
DOI: 10.5220/0008357600690076
In Proceedings of BROMO Conference (BROMO 2018), pages 69-76
ISBN: 978-989-758-347-6
Copyright
c
2018 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
69
that come from a plant are used for centuries in order
to cure various ailments (Desu, 2013). Besides that,
in many cases, medicinal plants and its active
ingredients have shown the ability to cure or improve
diseases.
Various research studies have been conducted from
many natural sources having anti-hyperlipidemic
effects from the dietary fiber, plant sterols, herbal
extracts, and some yeast extracts (Yoon et al., 2008).
Unlike conventional medicines or treatments, herbal
treatments are less expensive, more effective in
certain chronic conditions, reduced occurrence of
adverse effects as well as widespread availability or
in other words, it is easily obtained. Lot of
pharmacological work have been carried out on the
Impatiens Balsamina L. extracts. The leaves were
selected to investigate the anti hyperlipidemic
activity of methanolic extract of Impatiens
Balsamina (MEIB). However there is lack of
scientific report, on the anti hyperlipidemic activity
of MEIB.
2 METHODOLOGY
2.1 Preparation of Plant Materials
The fresh leaves of Impatiens Balsamina L. were
obtained from Temerloh, Pahang, Malaysia and were
authenticated by Forest Research Institute Malaysia.
Fresh plant materials were washed, shade dried for
about 7 days and subjected for coarse powder. Then,
these dry leaves were powdered using a mechanical
grinder. The coarse powder was subjected for
methanolic extraction using soxhlet apparatus and
extracts were concentrated using rotary evaporator
under reduced pressure. The final MEIB was stored
in umber colored bottle and kept refrigerator at 4,
until use for the pharmacological and phytochemical
screening. (Abdelwahab et al 2011).
2.2 Animals Maintenance
Healthy male Sprague Dawley rats weight ranging
between (150 to 175gm) were selected for the study.
The animals were brought form the local vender and
are kept in animals for 1 week to acclimatized to the
laboratory condition. During the study, they were
kept at temperature (25 ±1)
O
C, with relative
humidity (50 ± 15) % in 12 h light - dark cycles.
They were maintained in standard diet and water ad-
libitum. Institutional animal ethics clearance (IAEC)
was obtained from management and science
university, Malaysia before the study.
2.3 Preparation of High Cholesterol Diet
Eggs and high cholesterol cheese were procured
from local market at Klang Valley, Malaysia. The
lard oil was prepared by rendering method where
mutton fats was bought from Masai, Johor Market
and chopped to fine cubes. These fat cubes were
placed in a large stockpot and heated slowly,
delicately over medium heat. The fat was stirred at
regular interval for 30min. The high cholesterol diet
was prepared by mixing 60ml of melted lard oil, with
2 egg yolk and 20ml of melted cheese. These
ingredients were mixed at mild heat water bath to
prevent solidification of oil and cheese at room
temperature. This high cholesterol diet was fed to the
SD rats for 4 weeks according to their body weight
by using gavage needle. Each rat was weighed and
received 20ml/kg of this semi-solid high cholesterol
diet twice daily (Morning and evening) according to
table 3 in order to induce cholesterol
(Balasubramanian et al 2008).
2.4 Toxicity Study
Toxicity study was carried out using the OECD
guidelines. Single administration of MEIB extracts of
500, 1000 and 2000 mg/kg of the extract was given
by intra-gastric intubation by gavage needle to SD
rats (n=3) respectively. The rats were observed for
mortality and toxicity signs for 14 days. Animals
were observed individually at least once during the
first 30 min, periodically during the first 4 h, and
daily thereafter, for a total of 14 days.
2.5 Experimental Design
The rats were randomly divided into five groups
(n=8). Tail marker was used for identification and all
the animals were housed in cage according to their
group at ambient temperature. The feeding and drug
administration schedule for the five groups was
presented in Table 1.
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
70
Table 1: The feeding and drug administration schedule for the 5 groups of rats.
Group Schedule
Group 1
Normal control rats were fed with the normal diet for 4 weeks.
Group 2
High cholesterol rats fed with high cholesterol diet for 4 weeks.
Group 3
Rats fed with high cholesterol diet for 4 weeks. During the last 2 weeks, daily
administration of 200 mg/kg of MEIB extracts by intra-gastric intubation.
Group 4
Rats fed with high cholesterol diet for 4 weeks. During the last 2 weeks, daily
administration of 400 mg/kg of MEIB extracts by intra-gastric intubation.
Group 5
Rats fed with high cholesterol diet for 4 weeks. During the last 2 weeks, the
reference drug Simvastatin at dose of 10 mg/kg was administered by intra-gastric
intubation.
2.6 Collection of Blood and Biochemical
Analysis
Blood was collected by retro-orbital sinus puncture
which is a good choice when less quantity aseptic
sample is needed on 1
st
, 14
th
and 31
st
day of the test
under mild ether anesthesia Isoflurane 300µL by
using drop jar method where the cotton with
anesthesia will be placed inside the jar with the SD
rats.
These blood samples were tested using the blood
cholesterol testing kit in order to check Total
cholesterol, LDL-Cholesterol and HDL-Cholesterol
for each rat. Diagnostic kits to screen for cholesterol
and LDL-Cholesterol was procured from Mercury
Pharmacy, Temerloh, Malaysia (Dhulasavant et al
2010).
2.7 Histopathological Assessment
The liver sections of the SD rats were fixed in 10%
formaldehyde, dehydrated in gradual ethanol (50% to
100%), cleared in xylene and embedded in paraffin.
Sections (4 to 5 μm thick) were prepared and stained
with hematoxylin and eosin (HE) dye and observed
under a microscope. (Arsad et al 2014)
2.8 Statistical Analysis
The results were evaluated for statistical significant
difference by one-way ANOVA followed by post
hoc Dunnett’s test using SPSS software version 24.
Significant difference was accepted at the level of P
< 0.05.
3. RESULTS
3.1 Toxicity Study
There were no toxicity symptoms nor mortality were
observed in all the SD rats fed with 500mg/kg, 1 and
2g/kg of the MEIB extracts.
3.2 Biochemical Analysis
3.2.1 Effect On Body Weight
Table 2: Average body weight of experimental rats (n=6 per group) are expressed as mean ± S.E.M
Group
Average Body Weight of Experimental Rats (g)
Week 0
Week 4
I
124.83 ± 3.21
173.72 ± 2.98
II
119.04 ± 2.49
210.75 ± 1.59
III
129.61 ± 1.08
190.66 ± 2.53
IV
131.43 ± 3.13
187.39 ± 1.88
V
128.32 ± 1.84
185.78 ± 1.42
Anti-hyperlipidemic Activity of Methanolic Extract of Impatiens Balsamina L. in Hypercholestrolemic Induced Sprague Dawley Rats
71
3.2.2 Effect On Total Cholesterol (TC)
Table 3: Anti-hyperlipidemic effect of MEIB extracts 200mg/kg and 400mg/kg in hyperlipidemia induced rats. The values of
Total Cholesterol (TC) are expressed as mean ± S.E.M of six rats per group,
a
P < 0.05;
b
P > 0.05 compared with positive
control.
Figure 1: Graph shows the effect of MEIB extracts on Total Cholesterol (TC) of the hypercholesterolemia rats according to
different time.
3.2.3 Effect on Low-Density Lipoprotein (LDL)
Table 4: Anti-hyperlipidemic effect of MEIB extracts 200mg/kg and 400mg/kg in hyperlipidemia induced rats. The values of
Low-Density Lipoprotein (LDL) are expressed as mean ± S.E.M of six rats per group,
a
P < 0.05 and
b
P > 0.05 compared with
positive control.
0
50
100
150
200
GROUP I GROUP II GROUP III GROUP IV GROUP V
Total Cholesterol (TC),
mmol/L
week 0
week 2
week4
Groups
Treatment
Total Cholesterol (TC), mmol/L
Week 0
Week 2
I
Control with normal diet
106.88 ± 2.75
109.76 ± 6.01
II
Control with high cholesterol diet
105.75 ± 4.97
139.49 ± 5.53
III
High Cholesterol diet treated with
200mg/kg of MEIB extracts
103.61 ± 3.28
137.09 ± 5.53
IV
High Cholesterol diet treated with
400mg/kg of MEIB extracts
110.47 ± 2.49
135.52 ± 9.60
V
High cholesterol diet treated with
Simvastatin 10mg/kg
106.02 ± 4.77
140.80 ± 5.72
Groups
Treatment
Low-Density-Lipoprotein Cholesterol (LDL) mmol/L
Week 0
Week 2
Week 4
I
Control with normal diet
79.51 ± 1.65
82.52 ± 2.39
82.70 ± 1.51
II
Control with high cholesterol diet
80.88 ± 4.72
106.95 ± 4.48
112.91 ± 4.37
III
High Cholesterol diet treated with
200mg/kg of MEIB extracts
75.36 ± 3.43
101.72 ± 5.21
103.50 ± 6.03
b
IV
High Cholesterol diet treated with
400mg/kg of MEIB extracts
82.28 ± 3.02
105.98 ± 7.33
100.71 ± 6.20
a
V
High cholesterol diet treated with
Simvastatin 10mg/kg
79.80 ± 3.97
101.94 ± 6.81
79.37 ± 11.55
a
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
72
Figure 2: Graph shows the effect of MEIB extracts on Low Density Lipoprotein Cholesterol (LDL-C) of the
hypercholesterolemia rats according to different time.
3.2.4 Effect on High-Density Lipoprotein (HDL)
Table 5: Anti-hyperlipidemic effect of MEIB extracts 200mg/kg and 400mg/kg in hyperlipidemia induced rats. The values of
High-Density Lipoprotein (HDL) are expressed as mean ± S.E.M of six rats per group,
a
P < 0.05; and
b
P > 0.05 compared with
positive control.
Figure 3: Graph shows the effect of MEIB extracts. leaves on High Density Lipoprotein Cholesterol (HDL-C) of the
hypercholesterolemia rats according to different time.
0
50
100
150
GROUP I GROUP II GROUP III GROUP IV GROUP V
(LDL) mmol/L
week 0
week 2
week4
0
5
10
15
20
GROUP I GROUP II GROUP III GROUP IV GROUP V
(HDL) mmol/L
week 0
week 2
week4
Groups
Treatment
High-Density-Lipoprotein Cholesterol (HDL) mmol/L
Week 0
Week 2
Week 4
I
Control with normal diet
12.26 ± 0.97
12.10 ± 0.99
12.27 ± 0.87
II
Control with high cholesterol diet
12.65 ± 1.21
11.20 ± 0.72
12.65 ± 1.04
III
High Cholesterol diet treated with
200mg/kg of MEIB extracts
13.41 ± 0.82
12.64 ± 1.19
13.91 ± 1.16
b
IV
High Cholesterol diet treated with
400mg/kg of MEIB extracts
12.30 ± 1.12
10.72 ± 1.79
14.50 ± 1.17
a
V
High cholesterol diet treated with
Simvastatin 10mg/kg
12.85 ± 1.30
11.71 ± 1.57
15.73 ± 0.69
a
Anti-hyperlipidemic Activity of Methanolic Extract of Impatiens Balsamina L. in Hypercholestrolemic Induced Sprague Dawley Rats
73
Table 6: Anti-hyperlipidemic effect of MEIB extracts 200mg/kg and 400mg/kg in hyperlipidemia induced rats on 4
th
week (28
th
day). The increase (+) or decrease (-) value of Total Cholesterol, Low-Density Lipoprotein and High-Density Lipoprotein (HDL)
are expressed as percentage.
Groups
Treatment
Percentage increase (+) or decrease (-) of lipid values at week 4
(28
th
Day), mmol/L (%)
Total
Cholesterol
Low Density
Lipoprotein
Cholesterol
High Density
Lipoprotein
Cholesterol
I
Control with normal diet
111.47 ± 4.71
82.70 ± 1.51
12.27 ± 0.87
II
Control with high cholesterol
diet
143.51 ± 4.55
112.91 ± 4.37
12.65 ± 1.04
III
High Cholesterol diet treated
with 200mg/kg of MEIB
extracts
142.27 ± 2.98
(-0.86%)
103.50 ± 6.03
(-8.32%)
13.91 ± 1.16
c
(+9.96%)
IV
High Cholesterol diet treated
with 400mg/kg of MEIB
extracts
133.12 ± 8.44
(-7.24%)
100.71 ± 6.20
(-10.80%)
14.50 ± 1.17
a
(+14.62)
V
High cholesterol diet treated
with Simvastatin 10mg/kg
107.44 ± 8.09
(-25.13%)
79.37 ± 11.55
(-29.70)
15.73 ± 0.69
b
(+24.35)
3.3 Histopathological Result
High cholesterol diet rat (severe steatosis, and
inflammation found)
Hypercholestrolemia rats treated with 200mg/kg of MEIB
extracts (severe steatosis and inflammation found),
Hypercholestrolemia rats treated with
400mg/kg of MEIB extracts (mild steatosis and
no inflammation found),
Hypercholestrolemia rats treated with 10mg/kg of
Simvastatin (very mild steatosis and no inflammation
found).
4 DISCUSSION
Natural products that come from a plant are used for
centuries in order to cure various ailments. Plants
have been the found to be companions to man and
formed the basis for various drugs synthesis since
they are less toxic than synthetic drugs (Srujana et al
2012). Screening of medicinal plants presents basic
path for the discovery of new drugs. From very long
ancient times medicinal plants are considered to be
important source for drug discovery having with
potential therapeutic effect and safer entity. Plant
species that have been traditionally used as an anti-
cholesterol folk medicine should be seen as strategy
in research for new anti-cholesterol drugs with better
therapeutic effect and less side effects. Many drugs
used for the treatment are found to be associated with
side effects when used. These may leads to
hyperuricemia, diarrhea, nausea, myositis, gastric
irritation, flushing, dry skin, and also abnormal liver
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
74
function (Katzung et al 2012) . Hence there is an
increase in demand to produce anti hypolipidemic
activity from the natural source.
According to the toxicity test result, MEIB extracts
produced non-toxic symptoms or mortality up to
dose level of 2000mg/kg body weight orally in rats
and hence the drug was considered safe for further
pharmacological screening. Phytochemical analysis
of the MEIB extracts revealed the presence of
alkaloid, flavonoid, terpenoid and tannins.
In the present study, the anti-cholesterol activity of
the test extract was measured by inducing
hypercholesterolemia in rats model. MEIB extracts
was used to evaluate the anti-cholesterol in this
hypercholesterolemia induced rats. The doses of the
test extract used for this study are 200mg/kg and
400mg/kg body weight respectively. The result
obtained from this study has been showed in lipid
profile of rats where 200mg/kg of MEIB extracts
shown to have non-significant anti-cholesterol effect
(P>0.05), whereas 400mg/kg of MEIB extracts has
significant anti-cholesterol effect (P<0.05) when
compared to the positive control. The results are
statistically analyzed using One Way ANOVA.
From previous studies, it showed that flavonoids
have the ability to reduce LDL-C and increase HDL-
C in hypercholesterolemia induced rats. The
antihyperlipidemic activity may also be attributed to
some of its active principles. The hypolipidemic
activity of natural products can be correlated to the
presence of flavonoids due to their properties of
inhibiting cholesterol biosynthesis and absorption
and modifying the activity of lipogenic and lipolytic
enzymes, leading to reduced lipid metabolism
(Borradaile et al 2003). So in this study, Flavonoids
presence in BPR extract might be the reason for
reducing TC, LDL-C and increasing HDL-C in
400mg/kg treated rats. Further experiments are
required to prove the exact mechanism and active
ingredients involved in the hypolipidemic activity.
5 CONCLUSION
The findings of the study revealed that MEIB extracts
are having anti-cholesterol properties with the dose
of 400mg/kg, and the anti-cholesterol effect is not
significant with 200mg/kg. This was proven
statistically with that P> 0.05.
Therefore, MEIB extracts could be a potential herbal
medicine as adjuvant with existing therapy for the
treatment of hyperlipidemia. Further studies to
isolate, identify, and characterize the active
principle(s) present in the extract have to be done.
ACKNOWLEDGEMENTS
The author is thankful to Professor Tan Sri Dato'
Wira Dr Mohd Shukri Ab Yajid, President,
Management & Science University, Malaysia, for
providing financial help and providing facilities to
complete the research.
CONFLICT OF INTERESTS
Authors shows no conflict of interests.
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