ORIGINAL RESEARCH

Lipid-lowering effects of methanolic extract

of Vernonia amygdalina leaves in rats fed on high
cholesterol diet

Oluwatosin A Adaramoye Abstract: We investigated the lipid-lowering effects of methanolic extract of Vernonia
Olajumoke Akintayo amygdalina (VA) leaves in rats fed an high cholesterol diet, and compared with a standard hypo-
Jonah Achem lipidemic drug, Questran (Qu). The effects of VA on the lipid profile were assessed by measuring
Michael A Fafunso the levels of total cholesterol, triglyceride, low-density lipoprotein (LDL) cholesterol, high-density
lipoprotein (HDL) cholesterol, lipid peroxidation (LPO), phospholipid, and glutathione (GSH) in
Department of Biochemistry, Faculty
of Basic Medical Sciences, College of the plasma and liver of the rats. Administration of cholesterol at a dose of 30 mg/0.3 ml, five times
Medicine, University of Ibadan, Ibadan, in a week for nine consecutive weeks resulted in a significant increase (p
0.05) in plasma and
Nigeria post mitochondrial fraction (PMF) cholesterol levels by 33% and 55%, respectively. However,
treatment with extract of VA at doses of 100 and 200 mg/kg caused a dose dependent reduction in
the plasma and PMF cholesterol by 20%, 23% and 23%, 29%, respectively. Similar reduction in
cholesterol levels was obtained in Qu-treated rats. Furthermore, VA at 200 mg/kg decreased the
plasma and PMF LDL-cholesterol levels by 23% and 49%, and also decreased plasma and PMF
triglyceride levels by 29% and 28%, respectively. Also, VA at 100 and 200 mg/kg caused a dose-
dependent increase in plasma HDL-cholesterol levels by 41% and 59%, respectively. However,
there were no significant differences (p  0.05) in the PMF HDL-cholesterol and phospholipid
levels of the treated rats when compared to hypercholesterolemic rats. There were significant
decreases (p
0.05) in the LPO levels of extract-treated rats. Precisely, VA at 100 and 200
mg/kg decreased the levels of plasma and PMF LPO by 38%, 42% and 35%, 45%, respectively.
In addition, VA augmented the cholesterol-induced decrease in PMF glutathione levels of the
rats. Taken together, these results suggest the lipid-lowering effects of VA and, probably serve
as a new potential natural product for the treatment of hyperlipidemia.
Keywords: hypercholesterolemia, lipid-lowering effect, Vernonia amygdalina, cholesterol

Introduction
It is known that hypercholesterolemia is a risk factor for cardiovascular diseases (CVD)
such as atherosclerosis and myocardial infarction, which is a common cause of mortal-
ity and morbidity (Wald and Law 1995; Krieger 1998). Although several factors, such
as life style, a diet rich in cholesterol, age and hypertension, have been reported to
cause heart failure (Schaefer et al 1995), high levels of cholesterol, particularly LDL
cholesterol, are mainly responsible for hypercholesterolemia (Krieger 1998). Recently,
hypercholesterolemia has been associated with enhanced oxidative stress related to
increase lipid peroxidation (Adaramoye et al 2005). Increased generation of oxidized
Correspondence: Oluwatosin A LDL is a major factor in the vascular damage associated with high cholesterol levels
Adaramoye
Department of Biochemistry, University (Pritchard et al 1995). Hence, the inhibition of oxidative stress under hypercholester-
of Ibadan, Ibadan, Nigeria olemic conditions is considered to be an important therapeutic approach and efforts
Tel +234 802 883 5954
Fax +234 2 810 3043
have been made to identify the antioxidative functions of various medicinal plants (Hu
Email aoadaramoye@yahoo.com et al 2006; Tomotake et al 2006; Visavadiya and Narasimhacharya 2007).

Vascular Health and Risk Management 2008:4(1) 235–241 235

© 2008 Adaramoye et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access
article which permits unrestricted noncommercial use, provided the original work is properly cited.
Adaramoye et al

Drugs that lower cholesterol such as fibrates and bile Preparation of plant extract
acid sequestrants were used for several decades, but the high Fresh leaves of Vernonia amygdalina were obtained from
prevalence of their adverse effects led to the introduction of a local market, Sasa in Ibadan, Nigeria. The botanical
statins (HMG CoA inhibitors) (Miller 2001). Although the identification and authentication was confirmed at the
adverse effect of statins is relatively low, one rare effect called Department of Botany, University of Ibadan, Nigeria, where
rhabdomyolysis can be very serious (Miller 2001). This effect voucher specimen was kept at the herbarium (Voucher no.
is very pronounced when a statin is taken with another type UI-02567). The leaves were air-dried at room temperature
of cholesterol lowering drug, in particular fibrates (Miller and then powdered. The powdered sample (1 kg) was de-
2001). Rhabdomyolysis led to the voluntary withdrawal of fatted with n-hexane (2.5 litres) and then extracted with
cerivastatin (Baycol) by its manufacturer. In view of the 75% methanol (2.5 litres) overnight in a soxhlet extractor
adverse effects associated with synthetic lipid-lowering drugs, (Sri Rudran Instrument Co, Chennai, India). The methanolic
the quest for natural products with lipid-lowering potential extract was concentrated and evaporated to dryness at 50 oC
and with minimal or no side effect is warranted. with a rotary evaporator under reduced pressure, and the
Plants have been the companions of man since time imme- yield of the preparation was 6.8%. The extract was dissolved
morial and formed the basis of useful drugs since they are in water at a concentration of 4 g/100 ml, and aliquots of
less toxic than synthetic drugs. Screening of medicinal plants different concentrations were given orally to the animals
presents an avenue for the discovery of new drugs. Vernonia with a gavage needle.
amygdalina (VA) is a shrub that grows predominantly in the
tropical Africa. Leaves from this plant serve as food vegetable Grouping of animals and study design
and culinary herb in soup (Argheore et al 1998). In Nigerian Forty-two male albino rats (Wistar strain) weighing between
herbal homes, extracts of the plant are used as tonic, in the 120–130 g were used as experimental animals. The rats
control of tick and treatment of cough, feverish condition, were kept in cages in a room maintained at 26–29 °C with
constipation and hypertension (Regassa 2000; Kambizi and a 12-hour light-dark cycle for 4-weeks to acclimatize, and
Afolayan 2001; Amira and Okubadejo 2007). Phytochemical were allowed free access to food and water ad libitum. The
screening of VA revealed the presence of saponins, sesqui- protocol conforms to the guidelines of the National Institute
terpene, and flavonoids (Igile et al 1994). Strong antioxidant of Health (NIH) (NIH publication 85–23, 1985) for laboratory
activities have been reported for flavonoids from VA and, its animal care and use. Animals were randomly distributed into
saponins have been reported to elicit antitumoral activities seven groups of six animals each. Group A served as control
in leukemia cells (Jisaka et al 1993). Peptides from VA are (received drug-vehicle, water), rats in groups B and C were
known to be potent inhibitor of mitogen-activated proteins administered VA at doses 100 and 200 mg/kg body weight,
kinases, which are crucial for breast tumor growth and also respectively in addition to oral administration of cholesterol.
represents a key regulatory point for the tumour (Izevbigie Rats in group D were intubated with cholesterol-only
2003; Izevbigie and Ernest 2005). However, there is dearth (hypercholesterolemic animals). Rats in group E served as
of scientific data to support the folkloric use of this plant in positive control and were given VA at a dose of 100 mg/kg
the treatment of hypertension or related vascular diseases body weight, while rats in group F were intubated with
in Nigerian herbal homes. Therefore, the present work was cholesterol and Questran. Rats in group G were treated orally
designed to provide scientific proof of the use of VA in the with Questran-only. Cholesterol was orally administered at
treatment of vascular diseases. a dose of 30 mg/0.3 ml/ animal, while Questran was given
at a therapeutic dose of 0.26 g/kg body weight (Adaramoye
Methods et al 2005). Extract of VA was administered at doses 100 and
Chemicals 200 mg/kg body weight (Iwalokun et al 2006). VA, Questran,
Cholesterol and thiobarbituric acid (TBA) were procured and cholesterol were administered five times in a week for a
from Aldrich Chemical Co. (Milwaukee, WI, USA). Questran period of nine weeks.
(Bristol-Myers Squibb, Hounslow, UK) was purchased
from a local chemist in Ibadan, Nigeria. Diagnostic kits for Sample collection
cholesterol and triglyceride were procured from Boehringer After the last dose of drugs, rats were fasted overnight and
Mannheim GmbH Diagnostica, Germany. Other reagents sacrificed by cervical dislocation. Blood was collected
were of analytical grade and the purest quality available. from the heart into EDTA tubes. Plasma was prepared

236 Vascular Health and Risk Management 2008:4(1)

 Lipid-lowering effects of Vernonia amygdalina

by centrifugation at 3,000 g for 15 minutes in an MSC bench Determination of lipid peroxidation (LPO) and
centrifuge (Beckman and Hirsch, Burlington, IO, USA). The reduced glutathione (GSH) levels
plasma was used in the estimation of lipid profile. Liver from Lipid peroxidation and reduced glutathione levels were
the animals were rinsed in ice- cold 1.15% KCl, dried and assayed by the methods of Walls and colleagues (1976) and
weighed. It was homogenized in 4 volumes of ice-cold 5 mM Moron and colleagues (1979), respectively. Briefly, GSH
phosphate buffer, pH 7.4, and centrifuged at 10,000 g for level was assayed by measuring the rate of formation of
20 min to obtain post mitochondrial fraction (PMF). Organs chromphoric product in a reaction between DTNB (5, 51-
such as kidneys, lungs, liver and heart were removed from dinitro bis (2-nitrobenzoic acid) and free sulphydryl groups
the animals and weighed. (such as GSH) at 412 nm, while the extent of LPO was
estimated by the measuring the intensity of pink precipitate
Determination of triglyceride and cholesterol levels formed in a reaction between malondialdehyde (MDA,
Triglyceride and cholesterol levels were assayed using end-product of lipid peroxidation) and thiobarbituric acid at
Boehringer Mannheim diagnostic kits. 535 nm spectrophotometrically.

Determination of protein and phospholipid levels

Protein level was determined by the method of Lowry
Statistical analysis
Results were expressed as the mean ± S.D. (n = 6). A one-
and colleagues (1951). Briefly, the method involved the
way analysis of variance (ANOVA) was used for the data
reduction of phospho-18 molybdic tungstic complex
analysis, using SPSS (Statistical Package for Social Sciences)
by phenolic groups present in amino acids to blue
(10.0) software. Significant differences between groups were
precipitate at alkaline pH. The precipitate was read
detected in the ANOVA using Duncan’s Multiple Range Test
spectrophotometrically at 720 nm. The level of phospholipid
at p values less than 0.05 and 0.001.
was assayed by the method of Fiske and SubbaRow (1925).
The method involves the release of inorganic phosphate
from phospholipid in presence of an acceptor in aqueous Results
medium. The inorganic phosphate reacts with ammonium Table 1 depicts the effect of Vernonia amygdalina on body
molybdate and ascorbate to form a blue precipitate which weight, organs weight and relative weight of visceral organs of
was read at 660 nm spectrophotometrically. hypercholesterolemic rats. Extract of VA at 100 and 200 mg/kg
did not affect the body weight gain during this study. However,
Determination of HDL and LDL cholesterol levels there was a significant increase (p
0.001) in the body weight
The lipoproteins, VLDL and LDL were precipitated using gain of hypercholesterolemic rats when compared with the
phosphotungstic acid and magnesium chloride. After cen- control. In Table 2, there were significant (p
0.001) increases
trifugation, the supernatant contained the HDL fraction, in the plasma HDL cholesterol levels of animals treated with
which was assayed for cholesterol with diagnostic kit. LDL VA when compared with the hypercholesterolemic rats. Fur-
cholesterol was calculated using the formula of Friedewald thermore, administration of cholesterol caused a significant
and colleagues (1972). increase (p
0.05) in the plasma and PMF total cholesterol

Table 1 Effect of Vernonia amygdalina on body weight, organ weight, and relative weight of visceral organs of hypercholesterolemic
rats
Grouping Body weight (g) Lungs Kidneys Heart Liver Relative weight
(g) (g) (g) (g) (% body weight)
Initial Final Change Heart Liver
CONTROL 125.0 ± 5.2 171.0 ± 7.6 46.0 ± 8.6 1.6 ± 0.1 1.6 ± 0.1 0.80 ± 0.1 6.1 ± 0.5 0.47 ± 0.1 3.6 ± 0.4
VA1 + CH 120.2 ± 4.7 168.3 ± 5.7 48.1 ± 11.3 1.5 ± 0.4 1.5 ± 0.1 0.75 ± 0.1 5.8 ± 0.8 0.47 ± 0.1 3.7 ± 0.5
VA2 + CH 130.0 ± 4.5 174.0 ± 6.1 44.0 ± 5.5 1.6 ± 0.3 1.5 ± 0.3 0.82 ± 0.2 6.0 ± 0.6 0.47 ± 0.1 3.5 ± 0.3
CH ONLY 120.0 ± 3.0 178.3 ± 6.0 58.3 ± 6.0* 1.7 ± 0.3 1.8 ± 0.2 0.86 ± 0.2 6.2 ± 0.3 0.48 ± 0.1 3.5 ± 0.4
VA1 ONLY 120.3 ± 3.5 166.6 ± 4.3 46.3 ± 8.3 1.6 ± 0.3 1.8 ± 0.5 0.80 ± 0.2 6.1 ± 0.7 0.48 ± 0.0 3.7 ± 0.4
QU + CH 125.0 ± 4.3 170.0 ± 7.9 45.0 ± 7.2 1.5 ± 0.1 1.6 ± 0.2 0.82 ± 0.1 5.9 ± 0.5 0.48 ± 0.1 3.5 ± 0.5
QU ONLY 130.0 ± 5.8 176.0 ± 8.7 46.0 ± 8.8 1.7 ± 0.2 1.7 ± 0.4 0.79 ± 0.1 6.0 ± 0.6 0.45 ± 0.1 3.4 ± 0.5
Notes: Values are means ± S.D. of six animals; *Significantly different at p
0.001 from others.
Abbreviations: VA, Vernonia amygdalina; CH, cholesterol; QU, Questran; VA1, 100 mg/kg;VA2, 200 mg/kg.

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Adaramoye et al

Table 2 Effect of Vernonia amygdalina on protein, phospholipid, high-density lipoprotein (HDL), and low-density lipoprotein (LDL)
cholesterol levels of hypercholesterolemic rats
Grouping Protein content Phospholipid HDL-cholesterol LDL-cholesterol
Plasma PMF PMF Plasma PMF PMF
(g/ml) (mg/g liver) (mg/dL) (mmol/L) (mmol/g liver) (mmol/g liver)
CONTROL 7.0 ± 1.3 2.4 ± 0.3 542.8 ± 13.6 0.28 ± 0.07* 0.09 ± 0.02 2.9 ± 0.9**
VA1 + CH 6.8 ± 1.4 2.8 ± 0.8 532.5 ± 10.3 0.24 ± 0.04* 0.10 ± 0.02 4.8 ± 1.2
VA2 + CH 6.5 ± 1.8 2.1 ± 0.6 535.7 ± 16.7 0.27 ± 0.06* 0.10 ± 0.02 3.2 ± 1.2**
CH ONLY 6.1 ± 1.5 2.4 ± 0.3 545.2 ± 11.4 0.17 ± 0.03 0.09 ± 0.02 6.2 ± 0.8
VA1 ONLY 6.2 ± 0.6 2.5 ± 0.5 548.6 ± 11.9 0.28 ± 0.05* 0.09 ± 0.01 2.6 ± 0.8**
QU + CH 6.5 ± 1.4 2.6 ± 1.5 547.8 ± 14.6 0.19 ± 0.05 0.08 ± 0.02 5.0 ± 1.4
QU ONLY 6.0 ± 0.5 2.5 ± 0.5 536.5 ± 13.8 0.26 ± 0.05* 0.09 ± 0.01 3.2 ± 0.9**
Notes: Values are means ± S.D. of six animals; *Significantly different from CH group at p
0.001; **Significantly different from CH group at p
0.05.
Abbreviations: VA, Vernonia amygdalina; CH, cholesterol; QU, Questran; PMF, post mitochondrial fraction; VA1, 100 mg/kg;VA2, 200 mg/kg.

and triglyceride levels of the rats (Figures 1 and 2). Precisely, Figure 4 show that cholesterol administration significantly
plasma and PMF levels of total cholesterol and triglyceride (p
0.05) elevated the plasma and PMF LDL cholesterol
were increased by 33%, 55% and 44%, 80%, respectively. The levels of hypercholesterolemic rats. Specifically, plasma and
hypercholesterolemia induced by the dietary cholesterol was PMF LDL cholesterol levels were elevated by 41% and 115%,
significantly attenuated (p
0.05) in animals treated with VA respectively. However, VA at 200 mg/kg significantly attenu-
and Questran. Furthermore, VA at a dose of 200 mg/kg was ated (p
0.05) the cholesterol-induced increase in plasma and
able to decrease the hypertriglyceridemia induced by dietary PMF LDL cholesterol by 23% and 49%, respectively. Similarly,
cholesterol (Figure 2). Administration of dietary cholesterol the cholesterol-induced decrease in PMF reduced glutathione
significantly (p
0.05) increased plasma and PMF LPO by levels of hypercholesterolemic rats were significantly (p
0.05)
58% and 90%, respectively (Figure 3). However, the choles- ameliorated in animals treated with VA (Figure 4).
terol-induced increase in plasma and PMF LPO was signifi-
cantly (p
0.05) attenuated by extract of VA. Specifically, Discussion
VA at 100 and 200 mg/kg decreased the plasma and PMF The presence of a high amount of cholesterol in the diet
LPO by 38%, 42% and 35%, 45%, respectively. Table 2 and has been demonstrated to elevate plasma cholesterol and

140
Plasma CH

120 PMF CH

100
Cholesterol levels (mmol/L)

* * * *
* *

80

60

40
* *
* * *
*
20

0
CONTROL VA1 +CH VA2 +CH CH ONLY VA1 ONLY QU+CH QU ONLY
Treatment

Figure 1 Effect of Vernonia amygdalina on plasma and post mitochondrial fraction (PMF) cholesterol levels of hypercholesterolemic rats.
Note: *Significantly different from CH group at p
0.05.
Abbreviations: CH, Cholesterol; VA,Vernonia amygdalina; QU, Questran; PMF, Post mitochondrial fraction; VA1, 100 mg/kg; VA2, 200 mg/kg.

238 Vascular Health and Risk Management 2008:4(1)

 Lipid-lowering effects of Vernonia amygdalina

Plasma triglyceride
45
PMF Triglyceride

40

35 *
Triglyceride levels (mmol/L)

30 * *
*
25

20

15

10 * *
* *

0
CONTROL VA1 + CH VA2 + CH CH ONLY VA1 ONLY QU + CH QU ONLY
Treatment

Figure 2 Effect of Vernonia amygdalina on plasma and post mitochondrial fraction (PMF) triglyceride levels of hypercholesterolemic rats.
Note: *Significantly different from CH group at p
0.05.
Abbreviations: CH, Cholesterol; VA,Vernonia amygdalina; QU, Questran; PMF, Post mitochondrial fraction; VA1, 100 mg/kg;VA2, 200 mg/kg.

may increase aortic atherosclerosis (LRCP 1984). Several therapeutic benefits of plant foods have been the focus
studies have indicated that diet treatment or drug therapy to of many extensive dietary studies (Yokozawa et al 2006;
regulate cholesterol can reduce subsequent CVD-associated Zhang et al 2007).
mortality and morbidity (Kwiterovich 1997). On the basis In the present study, we investigated the lipid-lowering
of this, great efforts have been made to reduce the risk effects of Vernonia amygdalina in rats fed on high cho-
of CVD through the regulation of cholesterol, thus the lesterol diet for a period of nine weeks. Notably, the rats

25 Plasma LPO
PMF LPO

20

15
LPO (nmol/mg protein )

*
* *

*
*
10

* * * * *
5

0
CONTROL VA1 + CH VA2 + CH CH ONLY VA1 ONLY QU + CH QU ONLY
Treatment

Figure 3 Effect of Vernonia amygdalina on plasma and post mitochondrial fraction (PMF) lipid peroxidation levels of hypercholesterolemic rats.
Note: *Significantly different from CH group at p
0.05.
Abbreviations: CH, Cholesterol;VA, Vernonia amygdalina; QU, Questran; LPO, Lipid peroxidation; VA1, 100 mg/kg;VA2, 200 mg/kg.

Vascular Health and Risk Management 2008:4(1) 239

Adaramoye et al

140 Plasma LDL-cholesterol

PMF GSH

120
LDL-cholesterol (mmol/L); GSH (mg/ 100g)

100 *
*
*

80 *

60
* *
* *
40 *

20

0
CONTROL VA1 + CH VA2 + CH CH ONLY VA1 ONLY QU + CH QU ONLY
Treatment
Figure 4 Effect of Vernonia amygdalina on plasma LDL-cholesterol and post mitochondrial fraction (PMF) reduced glutathione levels of hypercholesterolemic rats.
Note: *Significantly different from CH group at p
0.05.
Abbreviations: CH, Cholesterol; VA, Vernonia amygdalina; QU, Questran; LDL, Low-density lipoprotein; PMF, Post mitochondrial fraction; GSH, Reduced glutathione;
VA1, 100 mg/kg; VA2, 200 mg/kg.

intubated with dietary cholesterol showed higher plasma against CVD. The protective roles of HDL cholesterol from
and hepatic levels of total cholesterol and triglyceride than CVD have been suggested to occur in various ways (Nofer
the extract-treated animals. It is obvious that the reducing et al 2002). HDL exerts part of its anti-atherogenic effect
effect produced by Vernonia amygdalina on plasma and PMF by counteracting LDL oxidation and, recent studies also
cholesterol levels is dose dependent. Dietary cholesterol showed that HDL promotes the reverse cholesterol transport
has been shown to reduce fatty acid oxidation, which, in pathway, by inducing an efflux of excess accumulated cellular
turn, increases the levels of hepatic and plasma triglycerol cholesterol and prevents the generation of an oxidatively
(Fungwe et al 1993). The elevated levels of plasma and modified LDL (Yokozawa et al 2006). Furthermore, HDL
PMF triglyceride obtained in cholesterol-fed rats are in line inhibits the oxidation of LDL by transition metal ions, but
with the findings of Adaramoye and colleagues (2005) and also prevents 12-lipooxygenase-mediated formation of lipid
Yokozawa and colleagues (2006). The results suggest that VA hydroperoxides (Nofer et al 2002). On the basis of our results,
(200 mg/kg) reduced the hepatic triglyceride biosynthesis and VA may probably plays an anti-atherogenic role through the
favour the redistribution of cholesterol among the lipoprotein inhibition of lipids oxidation, (due to its anti-lipoperoxidative
molecules. This observation is consistent with the findings effect observed in this study) as well as the elevation of HDL
of Nwanjo (2005) in which VA significantly attenuated the cholesterol.
hepatic triglyceride and LDL cholesterol levels of strepto- LDL cholesterol is another primary target of CVD risk-
zotocin diabetic rats. reduction therapy (Kwiterovich 1997). In this study, VA
The crucial risk factor for CVD includes a low level administered at a dose of 200 mg/kg lowered LDL cholesterol
of HDL-cholesterol. The association between a low level levels of hypercholesterolemic rats. Excess LDL can be
of HDL-cholesterol and an increased risk of CVD has deposited in the blood vessel walls and becomes a major
been well established through epidemiological and clinical component of atherosclerotic plaque lesions. Therefore,
studies (Assmann and Gotto 2004). Since low level of HDL- plasma LDL cholesterol level may be used for monitoring
cholesterol plays a direct role in the atherogenic process, the treatment of patients with elevated blood cholesterol
therapeutic intervention to raise HDL-cholesterol together levels. In view of our results, VA elicited beneficial effects by
with other risk factors is widely encouraged. In this study, lowering the plasma and PMF levels of cholesterol including
treatment with VA led to significant elevation of plasma low-density lipoprotein of the treated rats. Oxidative stress,
HDL-cholesterol, indicating its promising protective role defined as a disruption of the balance between oxidative

240 Vascular Health and Risk Management 2008:4(1)

 Lipid-lowering effects of Vernonia amygdalina

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