A preclinical antihyperlipidemic evaluation of Artemisia vulgaris root in diet induced hyperlipidemic animal model

Abstract

Hyperlipidemia is a major cause of atherosclerosis, coronary heart disease (CHD), ischemic cerebrovascular disease and peripheral vascular disease. The main objective of this study is to evaluate the hypolipidemic activity of aqueous root extract of Artemisia vulgaris in cholesterol diet induced hyperlipidemic rats. Rats were randomly divided into five groups each comprising six rats. The study was conducted for two months which included 30 days of feeding period and next 30 days of treatment period. Group I served as normal control, group II, III, IV & V were fed with high-fat diet for 30 days during the feeding period and then the high-fat diet was replaced by standard diet for the next 30 days of treatment period. Artemisia vulgaris extract showed significant serum lipid lowering effects in hyperlipidemic rats which brought down total cholesterol level (C) till 180 ± 9.48, triglycerides (TG) 147.2±1.28, LDL 126.3±9.54, VLDL 28.2±2.26, increased level of HDL 68±5.19 and Atherogenic Index (AI) 2.63±1.82 in comparison of diet-induced hyperlipidemic control, total cholesterol 282.23 ±15.15, triglycerides 243.2 ±9.52, LDL 209.16 ±18.36, VLDL 47.56 ±1.90 , HDL 34.17±2.312 and Atherogenic Index (AI) 8.2 ± 0.72 at 30th day and hypolipidemic activity of Artemisia vulgaris was compared with rosuvostatin in diet induced hyperlipidemic rats.

Full text

International Journal of Pharmacological Research www.ssjournals.com
ISSN: 2277-3312 Journal DOI:10.7439/ijpr
IJPR Volume 5 Issue 4 (2015) 110
A preclinical antihyperlipidemic evaluation of Artemisia vulgaris
root in diet induced hyperlipidemic animal model
K. Abedulla Khan*
Department of Pharmacology, Sultan ul Uloom College of Pharmacy, Banjara Hills, Hyderabad-34, Telangana, India.
Corresponding author*
Dr. K. Abedulla Khan
Associate Professor,
Sultan Ul Uloom College of Pharmacy
Banjara Hills, Road no 3, Hyderabad-500034.
A.P (State), India.
E-mail: abidulla.k@gmail.com
1. Introduction
Hyperlipidemia is a major cause of atherosclerosis, coronary heart disease (CHD), ischemic cerebrovascular
disease and peripheral vascular disease. Lipoproteins play an essential role in the absorption of dietary cholesterol,
long-chain fatty acids, and fat-soluble vitamins transport of triglycerides, cholesterol, and fat-soluble vitamins from the
liver to peripheral tissues and the transport of cholesterol from peripheral tissues to the liver. The plasma
lipoproteins are divided into five major classes based on their relative density they are as chylomicrons, very low
density lipoproteins (VLDLs), intermediate-density lipoproteins (IDLs), low-density lipoproteins (LDLs) and high-density
lipoproteins (HDLs) [1]. The two major clinical sequelae of hyperlipidemias are acute pancreatitis and atherosclerosis.
Atherosclerosis is a systemic disease process in which fatty deposits cause inflammation of cells and scar the tissue build up
within the walls of arteries, it is the underlying cause of the majority of clinical cardiovascular events. The metabolic
disorders that involve elevations in lipoprotein are termed as hyperlipoproteinemias or hyperlipidemias. Artemisia vulgaris
is a tall herbaceous perennial plant growing 1-2 m (rarely 2.5 m) tall with a woody root. The leaves are 5-20 cm long, dark
green, pinnate, with dense white tomentose hairs on the underside. The erect stem often has a red-purplish tinge [2,3]. The
aim of this study is to evaluate the hypolipidemic activity o f aqueous root extract of Artemisia vulgaris in cholesterol
diet-induced hyperlipidemic rats and compare with rosuvastatin in cholesterol diet- induced hyperlipidemic rats.
2. Material and methods
2.1. Drugs and Chemicals
Cholesterol :(Himedia Pvt Ltd, Mumbai); Deoxycholic Acid:( Sigma-Aldrich Pvt Ltd, Mumbai); Rosuvastatin:
(Reddy labs Pvt Ltd, Hyderabad); Serum Total cholesterol diagnostic kit:(RFCL Diagnova, Dehradun); Serum Triglyceride
diagnostic kit :( RFCL Diagnova, Dehradun); Serum HDL cholesterol diagnostic kit: (RFCL Diagnova, Dehradun)
Other chemicals and reagents were of analytical grade.
2.2. Equipments
Oral feeding tube, Oral feeding needle, Microphage tubes (centrifuge tubes, 1.5 ml), Micro Pipettes (10µl, 100 µl,
Abstract
Hyperlipidemia is a major cause of atherosclerosis, coronary heart disease (CHD), ischemic
cerebrovascular disease and peripheral vascular disease. The main objective of this study is to evaluate the
hypolipidemic activity of aqueous root extract of Artemisia vulgaris in cholesterol diet induced hyperlipidemic rats.
Rats were randomly divided into five groups each comprising six rats. The study was conducted for two months which
included 30 days of feeding period and next 30 days of treatment period. Group I served as normal control, group II, III,
IV & V were fed with high-fat diet for 30 days during the feeding period and then the high-fat diet was replaced by
standard diet for the next 30 days of treatment period. Artemisia vulgaris extract showed significant serum lipid
lowering effects in hyperlipidemic rats which brought down total cholesterol level (C) till 180 ± 9.48, triglycerides (TG)
147.2±1.28, LDL 126.3±9.54, VLDL 28.2±2.26, increased level of HDL 68±5.19 and Atherogenic Index (AI) 2.63±1.82
in comparison of diet-induced hyperlipidemic control, total cholesterol 282.23 ±15.15, triglycerides 243.2 ±9.52, LDL
209.16 ±18.36, VLDL 47.56 ±1.90 , HDL 34.17±2.312 and Atherogenic Index (AI) 8.2 ± 0.72 at 30th day and
hypolipidemic activity of Artemisia vulgaris was compared with rosuvostatin in diet induced hyperlipidemic rats.
Keywords: Hypolipidemic activity, Artemisia vulgaris, extraction & Atherogenic Index

K. Abedulla Khan Research Article
IJPR Volume 5 Issue 4 (2015) 111
1000 µl), Tuberculin syringe, Remi Centrifuge, UV spectroscopy (Single Monochromator UV-2600 company shimadzu).
2.3 Preparation of extract
The air-dried stem of Artemisia vulgaris were subjected to hydrodistillation for four hour using a clevenger-type
apparatus [4,5].
2.4 Experimental Animals
Animals were procured from Sainath agencies, Hyderabad. Healthy albino rats of wistar strain weighing between
180-200g were used in this study. Thirty male albino rats were randomized into treatment, standard and control groups. All
rats were allowed one-week acclimatization period to become accustomed to the laboratory conditions. The study
protocol was approved by our Institutional Animal Ethical Committee (IAEC/SUCP/08/2012).
Rats were randomly divided into five groups, each comprising six rats. The study was conducted for two months
which included 30 days of feeding period and next 30 days of treatment period.
Group I served as normal control was fed with standard rat chow throughout the study. Group II, III, IV & V
were fed with high-fat diet for 30 days during the feeding period and then the high-fat diet was replaced by standard
diet for the next 30 days of treatment period. Rats were supplied food and water ad libitum.
Group I served as normal control (N) & received normal saline (5ml/kg, per oral by oral feeding needle with tuberculin
syringe) daily for 30 days.
Group II served as hyperlipidemic control (H) & received normal saline (5ml/kg, per oral by oral feeding needle with
tuberculin syringe) daily for 30 days.
Group III served as standard drug control (R) hyperlipidemic control (H) & received Rosuvastatin (10 mg/kg, per oral by
oral feeding needle with tuberculin syringe) daily for 30 days.
Group IV served as test group - A test drug extract (25 mg/kg per oral by oral feeding needle with tuberculin syringe) daily
for 30 days.
Group V served as test group - B (test drug extract (50 mg/kg per oral by oral feeding needle with tuberculin syringe) daily
for 30 days.
2.5 Induction of hyperlipidemia
High cholesterol diet (HCD) comprised the following ingredients: cholesterol 5g, deoxycholic acid 5g, coconut
oil 300 ml (300 g), and standard rat chow 700g. Deoxycholic acid was mixed thoroughly with of powdered rat
chow diet, Simultaneously cholesterol was dissolved in 300 ml of warm coconut oil. This oil solution of cholesterol
was added slowly into the powdered mixture and thoroughly mixed to obtain soft homogenous cakes. These cakes
were daily supplied to rats in each cage in sufficient quantities [4-6].
Body weights of all rats were checked on the day before the start of feeding period and on day 1, 15 & 30
o f the treatment period.
Calculation of weight gain: Total weight gain on day 30 = Final body weight – Initial body weight
Daily single dosage of Artemisia vulgaris extract (dissolved in normal saline 5 ml/kg) were given orally for
30 days in the treatment period to the test groups through oral by oral feeding needle with tuberculin syringe. The
control groups received normal saline alone. The standard group received Rosuvastatin 10 mg/kg/day orally dissolved
in 5 ml/kg normal saline. Doses of Artemisia vulgaris extract and rosuvastatin were selected based on the reports
in previous study which had hypolipidemic activity. All doses were administered between 10-11am.
All blood samples were collected within one hour period between 8:00am and 9:00 am. Twelve hours fasted
blood samples were collected under light ether anaesthesia by retro orbital puncture. Blood samples were collected
on the day before the start of feeding period and on day 1, 15 & 30 of the treatment period. These blood samples were
used for serum lipid analysis.
Serum lipid profile were analyzed for all rats on the day before the start of feeding period and on day 1, 15 &
30 of the treatment period. Blood samples were allowed to clot for 30 minutes and serum was separated by
centrifugation at 3000 revolutions per minute for 5 minutes in Remi- centrifuge and transferred to sterile 1.5mL
centrifuge tubes. Serum total cholesterol (TC), triglycerides ( TG) and high density lipoprotein (HDL), low density
lipoproteins (LDL) ,very low density lipoproteins (VLDL) were determined by endpoint colorimetric analysis using
commercial kits and UV Spectroscopy(Single Monochromator UV-2600 company shimadzu) using with biochemical
diagnostic kit (RFCL Diagnova, Dehradun) according to the manufacturer directions[17-18].
Results were expressed as mean + standard deviation (SD) of six values (n=6) for each group. Statistical
differences between the controls and the treatment groups were evaluated by using student’s paired t-test wherever
applicable using prism-5 software package. Percentage change from initial values (day 1 of treatment period) of
serum lipid levels were calculated on day 15 and day 30 of treatment period.

K. Abedulla Khan Research Article
IJPR Volume 5 Issue 4 (2015) 112
Calculation of Percentage Change in serum parameters
Day 1 serum levels – Day 30 serum levels
Percentage Change (%) on day 30 = ----------------------------------------------------------- X 100
Day 1serum levels
Day 1 serum levels – Day 15 serum levels
Percentage Change (%) on day 15 = ----------------------------------------------------------- X 100
Day 1 serum levels
Calculation of Atherogenic Index (AI)
Total serum cholesterol
Atherogenic Index = ----------------------------------
Total serum HDL-C
Calculation of Percentage Protection
AI of Control – AI of Treated group
Protection (%) = -------------------------------------------------- X 100
AI of Control
3. Results and discussion
Artemisia vulgaris extract showed significant serum lipid lowering effects in hyperlipidemic rats which brought
down total cholesterol level (C) till 180 ± 9.48, triglycerides (TG) 147.2 ± 1.28, LDL 126.3 ± 9.54, VLDL 28.2 ± 2.26,
increased level of HDL 68 ± 5.19 and Atherogenic Index (AI) 2.63 ± 1.82 in comparison of diet-induced hyperlipidemic
control, total cholesterol 282.23 ± 15.15, triglycerides 243.2 ± 9.52, LDL 209.16 ± 18.36, VLDL 47.56 ± 1.90 , HDL
34.17 ± 2.312 and Atherogenic Index (AI) 8.2 ± 0.72 at 30th day [Table 1].
Standard antihyperlipidemic agent Rosuvostatin 10 mg/kg body weight also able to reduce the elevated serum
lipid level toward the normal. It brought down total cholesterol 183.3±5.74, triglycerides 139.2±1.08, LDL 102.36 ±5.13,
VLDL 20.1±2.22, increased level of HDL 68.2±2.53 and Atherogenic Index (AI) 2.68 ±2.26 when compared to diet-
induced hyperlipidemic control, total cholesterol 282.23 ±15.15, triglycerides 243.2 ±9.52, LDL 209.16 ±18.36, VLDL
47.56 ±1.90, HDL 34.17±2.312 and Atherogenic Index (AI) 8.2 ± 0.72 at 30th day.
Hyperlipidemia is a well known risk factor for cardiovascular disease, especially atherosclerotic coronary
artery disease (CAD) [7-9]. It is one of the major causes of premature death globally and is expected to be the most
important cause of mortality in India by the year 2010.
From the results obtained it was observed that keeping the animal on High cholesterol diet (HCD)
significantly increased the total cholesterol (TC), TG, LDL-C level in serum as compared to rats on normal diet.
With administratio n o f Artemisia vulgaris root extracts, the elevated levels of TC, TG and LDL showed a
considerable decline as compared to High cholesterol diet rats, thus indicating the efficacy of Artemisia vulgaris
extract in decreasing levels of various components of lipid profile under experimental condition of diet-induced
hyperlipidemia. Rosuvastatin which was used as positive control in this study is a HMG-CoA reductase inhibitor.
Rats treated with rosuvastatin showed marked reduction in all serum lipoproteins and increase in HDL level as
compared to High cholesterol diet (HCD) group. Though both rosuvastatin and Artemisia vulgaris showed significant
TC, TG, LDL lowering activity and significant HDL increasing activity, the percentage reduction of TC, TG, LDL
and percentage increase in HDL was lesser with Artemisia vulgaris than with rosuvastatin. Also 50mg/kg of
Artemisia vulgaris showed greater response than 25mg/kg of Artemisia vulgaris.
An ideal drug is one which raises HDL along with the lowering of LDL. In the present study, Artemisia vulgaris
has markedly decreased the triglycerides level. Studies have shown the presence of flavonoids, saponins, tannins,
triterpenoids, steroids, and polyphenolics in root extracts of Artemisia vulgaris. Flavonoids are reported to increase
HDL concentration and decrease LDL and VLDL levels in hypercholesteremic rats. Several studies show that plant
saponins and steroids are known to possess both hypolipidemic and antihyperlipidemic activities [10-12].
The aqueous extract of Artemisia vulgaris root has anti-oxidant property[5,14]. This property could be another
factor that contributes to hypolipidemic & anti-atherosclerotic effect. Further investigation is required to elucidate this
mechanism. Atherogenic index (AI) is a marker of atherogenecity, showed significant decline with both Artemisia vulgaris
and rosuvastatin. During the experimentation rats did not show any mortality or any other adverse effects. This indicates that
the Artemisia vulgaris has a good margin of safety. The result shows that the Artemisia vulgaris has a definite
hypolipidemic activity hence it may have cardioprotective and antiatherosclerotic activity[15-16]. There is also a valid
scientific basis for consuming it in the treatment of coronary artery diseases in India. Hence, the present study
helps to support the traditionally claimed cardioprotective and cardiotonic activity of Artemisia vulgaris. However
further studies are necessary to support these findings. Also an extensive case-control study is required to document its
therapeutic application in human beings.

K. Abedulla Khan Research Article
IJPR Volume 5 Issue 4 (2015) 113
Figure 1: Serum lipid levels, Atherogenic Index (AI) of different groups on 30th day of treatment.
.
Table 1: Serum lipid levels, Atherogenic Index (AI) of different groups on the 1st day, 15th day and 30th day.
Groups
C(mg/dl)
Mean ± SD
TG(mg/dl)
Mean ± SD
HDL(mg/dl)
Mean ± SD
VLDL(mg/dl)
Mean ± SD
LDL(mg/dl)
Mean ± SD
AI
Mean ± SD
1st day
15th day
30th day
1st day
15th day
30th day
1st day
15th day
30th day
1st day
15th day
30th day
1st day
15th day
30th day
1st day
15th day
30th day
N
104.93±14.18
103.0±3.59
102.3±8.94
80.32±8.13
79.00±8.71
78.75±6.63
41.83±2.91
41.0±2.1
40.3±4.48
16.6±1.6
15.89±1.74
15.3±1.21
49.43±16.1
48.00±1.65
47.6±6.55
2.60±0.53
2.50±1.64
2.53±0.42
H
283.80±18.89
272.0±18.9
282.23±15.15
263.1±15.9
251.9±11.3
243.2±9.52
40.77±8.23
36.7±2.1
34.17±2.31
49.6±3.1
46.39±2.27
47.56±1.90
216.4±19.1
210.6±21.0
209.16±18.3
6.96±0.95
7.39±0.99
8.2±0.72
R
292.28±13.88
196.5±15.67
183.3±5.74***
215.5±10.3
158.2±16.0
139.2±1.08***
42.29±4.98
60.2±5.4
68.2±2.53***
43.1±2.0
31.65±3.20
20.1±2.22***
206.8±12.8
109.6±15.0
102.36±5.13***
6.99±0.83***
3.26±0.39
2.68±2.26***
T-A
285.67±16.41
242.2±13.2
203.1±2.93
204.3±12.5
196.8±13.8
187.2±5.34
42.1±7.17
48.4±4.8
64.3±2.60
48.2±2.5
37.36±2.77
31.2±3.07
212.7±19.6
186.4±14.6
143.2±4.40
6.78±1.24
5.40±0.53
3.15±1.12
T-B
292.0±14.26
205.0±19.9
180.31±9.48***
206.4±13.2
170.0±16.1
147.2±1.28***
42.5±4.31
52.4±2.4
68.5±5.19***
47.2±2.6
34.01±3.23
28.2±2.26***
210.8±14.19
158.5±19.0
126.3±9.54***
6.87±0.96
4.68±0.49
2.63±1.82***
Data expressed as Mean + SD of ( n=6) ; N=Normal, H= Hyperlipidemic control, R=Rosuvostatin group, T-A= Test group-A,
T-B= Test group-B, Atherogenic Index (AI).; *** Significant at P<0.001; ** Significant at P< 0.01; *Significant at P<0.05 compared to
diet-induced hyperlipidemic control.
0
50
100
150
200
250
300
Cholesterol
Triglycerides
HDL
VLDL
LDL
AI
mg/dl
HDL-High-density lipoprotein; VLDL-Very-low-density lipoprotein; LDL-Low-density lipoprotein; AI-Atherogenic
Index
Normal Group
Hyperlipidemic control Group
Rosuvostatin Group
Test group-A
Test group-B

K. Abedulla Khan Research Article
IJPR Volume 5 Issue 4 (2015) 114
Table 2: Weight Gain Different between 30th day to 1st day in various groups .
Groups
Weight Gain(gm)
Mean ± SD
N
6.66 + 2.58
H
17.3 + 3.76
R
7.5 + 2.73
T-A
10.0 + 4.47
T-B
8.16 + 3.76
Data expressed as Mean + SD of (n=6); N=Normal, H= Hyperlipidemic control, R=Rosuvostatin group, T-A= Test group-A,
T-B= Test group-B, Atherogenic Index (AI).
4. Conclusion
The aqueous extract of roots of Artemisia vulgaris has a definite hypolipidemic effect in cholesterol diet-induced
hyper lipidemia in rats. The aqueous extract of root of Artemisia vulgaris has hypolipidemic activity same as rosuvastatin.
These findings provide some biochemical basis for the use of aqueous root extract of Artemisia vulgaris as hypolipidemic
agent in hyperlipidemia. Further studies are required to gain more insight into the possible mechanism of action.
Acknowledgment
The authors are thankful to the management of Sultan ul Uloom College of Pharmacy, Hyderabad, for providing
the laboratory facilities and providing financial assistance.
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