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International Journal of Pharmacognosy and Phytochemical Research 2016; 8(8); 1372-1376

ISSN: 0975-4873
Research Article
 
Evaluation of Anti-Cholesterol Activity of Ethyl Acetate and N-
Hexane Extracts of Tetracarpidium conophorum (Mull. Arg.) Hutch
and Dalziel (African Walnut) Towards Hypercholesterolemic Rats
Ezealisiji K M1*, Stanley C N2, Ekanem E S1
1
Department of Pharmaceutical and Medicinal Chemistry, University of Port Harcourt, Nigeria.
2
Department of Pharmaceutical Microbiology and Biotechnology University of Port Harcourt Nigeria

Available Online:10th August, 2016

ABSTRACT
The main risk factor of atherosclerosis which is directly related to coronary heart disease is Hypercholesterolemia and this
is the most common cause of death in the developing world. Ethno-medical claim on Tetracarpidum conophorum is that it
is used in the treatment of hypertensive patients. Also, previous studies have shown that the seed of Tetracarpidum
conophorum has antioxidant properties. The present research was aimed at determining the anticholesterol activity of n-
hexane extract and ethyl acetate fractions of Tetracarpidum conophorum compared to atorvastatin in decreasing the plasma
level of total cholesterol, LDL and triglyceride and increasing the HDL level on hypercholesterolemic rats. Twenty-nine
male Wistar rats were divided into nine groups. Group I was fed with standard diet as negative control group and all the
other groups were fed with high-fat diet and were also given fructose solution. High-fat diet and fructose solution increased
the level of total cholesterol, LDL-cholesterol and triglyceride and decreased the level of HDL-cholesterol significantly
compared to the negative control group. The treatment groups were given 3 various doses of (0.5, 1.0, 2.0 ml/kgBW) of
crude extract, ethyl acetate and n-hexane fractions respectively and artovastatin 2.7 mg/kgBW daily for ten days. The data
were analyzed by one way Analysis of Variance (ANOVA) and Duncan’s Post Hoc Test. The best results were shown by
2.0 ml/kgBW of n-hexane extract fraction in decreasing total cholesterol, LDL-cholesterol and triglyceride as well as
increasing HDL-cholesterol when compared with the standard cholesterol lowering agent (artovastatin). Ethyl acetate
fraction (2.0 ml/kg) showed significant effect in increasing HDL- cholesterol and decreasing LDL- cholesterol.

Keywords: Anticholesterol; hypercholesterolemia; Tetracarpidium conophorum; African walnut.

INTRODUCTION damage, hence the degree of atherosclerosis was directly

Cardiovascular diseases are the second largest cause of proportional to the prevalence of CHD and stroke, and that
mortality worldwide. Progression of atherosclerosis, lipid levels were directly related to plaque damage. The
which is usually accompanied by the production of free higher the serum cholesterol, the greater the formation of
radicals are shown to be caused by elevated levels of plague1. Drugs such as clofibrate, niacin, cholestyramine,
cholesterol. The development of coronary heart disease statin and gemfibrozil are among the various drugs
(CHD) have been identified to be associated with the blood employed in lowering blood lipids and were successful in
lipids such as total cholesterol, HDL-cholesterol, LDL- reducing serum cholesterol levels, they produced
cholesterol and triglycerides, since these risk factors play unpleasant and distressing side effects. This was
an important role in determining atherogenesis and the established from the etiological preeminence of
subsequent pace of atherosclerosis. According to National hyperlipidemia in CHD1. The previous study which
Heart, Lung, and Blood Institute (NIH), CHD is a disease examined the antioxidant activity of crude extract and
in which a waxy substance called Plague builds up inside fractions of African walnut in vitro, shows that crude
the coronary arteries. These arteries supply oxygen-rich extract of the dried leaves had the best antioxidant activity;
blood to your heart muscle. This plague when build up in the broad range of this extract shows the potential of the
the arteries, results in a condition called atherosclerosis. plant as a source of natural antioxidants or nutraceuticals
Hyperlipidemia is the primary precondition for with potential application to reduce oxidative stress and
atherosclerosis manifested in premature cardiovascular consequent health benefits2. In vitro studies demonstrated
disability and death as confirmed from epidemiological that flavonoids are good free radical scavengers. About
and clinicopathological studies. Hyperlipidemia is caused half of the ingested flavonoids are absorbed into the
by elevated levels of fat in diets, especially saturated fat bloodstream through the gastrointestinal tract lining and
and cholesterol. The International Atherosclerosis Project, half are metabolized to other compounds by
discovered that CHD and stroke are related to plague gastrointestinal microflora. However, there is variation

*Author for Correspondence: [email protected].

 Ezealisiji et al. / Evaluation of Anti-Cholesterol… 

The plasma total cholesterol in hypercholesterolemic rat treated with n-hexane and ethyl acetate fraction of
Tetracarpidium conophorum. Result are expressed as mean ± SD of three animals per group, Duncan’s post Hoc tests
are showed by letter a,b,c are significantly different among treatment groups at confidence internal 95%

The plasma triglyceride level in hypercholesterolemic rat treated with n-hexane and ethyl acetate fraction of
Tetracarpidium conophorum. Result are expressed as mean ± SD of three animals per group, Duncan’s post Hoc tests
are showed by letter a,b,c are significantly different among treatment groups at confidence internal 95%

The plasma HDL-cholesterol level in hypercholesterolemic rat treated with n-hexane and ethyl acetate fraction of
Tetracarpidium conophorum. Result are expressed as mean ± SD of three animals per group, Duncan’s post Hoc tests
are showed by letter a,b,c are significantly different among treatment groups at confidence internal 95%

 
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The plasma LDL-cholesterol level in hypercholesterolemic rat treated with n-hexane and ethyl acetate fraction of
Tetracarpidium conophorum. Result are expressed as mean ± SD of three animals per group, Duncan’s post Hoc tests
are showed by letter a,b,c are significantly different among treatment groups at confidence internal 95%.

depending on the flavonoid3,4. Free radicals are chemical PortHarcourt were housed in standard cages and provided
species that have a singly occupied orbital. They possess with food and water ad libitum. The rats were adapted for
unpaired valence electrons, hence are highly reactive 7 days until the body weight were 125-175 g.
towards other substances as well as themselves. Formation Hypercholesterolemia were induced with high fat diet
of lipid peroxidation within plasma and organellar (5000 g of standard diet mixed with 550 g duck egg yolk,
membranes is as a result of free radicals in the presence of 500 g palm oil, 1250 g beef liver, 750 g beef brain and hot
oxygen. Initiation of free radicals could be blocked by water). The difference between the standard diet and high
antioxidants and terminate radical damage and inhibit lipid fat diet is shown in Table 1. In spite of high fat diet, the
peroxidation which was one of the factors implicated in rats were given fructose liquid 60% (100 g/400 ml
atherosclerosis. Natural antioxidants are safe, causes less aquadest) 1 mL/rat/day. The high fat diet and fructose
adverse reactions and widely used4,5. Since the clinical liquid were given for 2 weeks until the body weight were
efficacies of Tetracapidium conophorum in lowering the 150 – 200 g. The rats were divided into 9 groups (n=3) for
lipids profile is not established, it is important to study the different treatments. The first group of animals was given
anticholesterol activity of n-hexane, ethyl acetate fractions standard diet (negative control). The second group of rats
of Tetracapidium conophorum compared to atorvastatin in was given high fat diet (positive control). The third, fourth
decreasing the plasma level of cholesterol total, LDL and and fifth groups of rats were treated with high fat diet plus
triglyceride and increasing the HDL level on n-hexane fraction 0.5 ml/kgBW, 1.0 ml/kgBW and 2.0
hypercholesterolemic rats. ml/kgBW daily. The sixth, seventh and eighth groups of
rats were treated with high fat diet plus crude extract 0.5
MATERIALS AND METHODS ml/kgBW, 1.0 ml/kgBW and 2.0 ml/kgBW daily. Group
Phytoconstituent Extraction nine was treated with high fat diet plus atorvastatin 2.5
The unshelled seeds of Tetracarpidium conophorum were mg/kgBW daily. All the treatments were given for 10 days.
cut into smaller pieces to enhance drying. Drying was done Sample Preparation for Lipid Profile Test
under shade for one week before the seeds were pulverized After the treatments, 1.5 mL blood from the orbital vein
using a mechanical grinder. 600 g of the pulverized seeds were collected in tubes containing EDTA (heparinized
was defatted using 2.5 L of n-Hexane for 72h and filtered. tubes). The samples were centrifuged at 3000 rpm for 10
To the marc, 1.5 L of ethyl acetate was added and min and the plasma were used for measuring the total
macerated for 72 h. This was filtered using a sieve and cholesterol, LDL-cholesterol, HDL-cholesterol and
further separated by means of a separating funnel. The triglyceride level. The total plasma cholesterol and
different extracts (ethyl acetate and n-Hexane) were triglyceride were measured according to the instruction
concentrated using rotor evaporated and were further manual accompanying the diagnostic kits from Lively
subjected to evaporation using a water bath. A 23.115g Stones Medical Diagnostic Laboratories. High density
(equivalent to 25ml) of ethyl acetate extract and 120 ml lipoprotein (HDL) cholesterol, low density lipoprotein
(equivalent to 110.952g) of n-Hexane oil extract were (LDL) cholesterol were measured according to the
recovered and their extractive values were calculated using instruction manuals accompanying the diagnostic kits
the following formula: from Lively Stones Medical Diagnostic Laboratories.
Statistical Analysis
x 100 (1)
Mean and standard deviation (M ± SD) were calculated
Animals and Diets and 95% confidence interval (CI) of means was used. To
Twenty-nine adult male Wistar rats obtained from Animal compare between groups, analysis of variance (ANOVA)
house, Department of Agriculture, Choba, University of were calculated. P-values of less than 0.05 were considered

 
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Table 1: The nutrient value of standard and high fat diet. occurring compounds found in plant cell membranes.
Item High fat diet Standard diet However, because phytosterols are structurally similar to
Water (%) 14.20 6.20 the body’s cholesterol, when they are consumed they
Ash (%) 7.18 4.86 compete with cholesterol for absorption in the digestive
Crude protein (%) 20.08 24.12 system. As a result, cholesterol absorption is blocked, and
Crude fiber (%) 10.02 14.02 blood cholesterol levels reduced. The most common
Crude fat (%) 30.50 8.09 phyosterols in human diet are campesterol, sitosterol and
Carbohydrate (%) 42.42 60.04 stigmasterol. It is known that phytosterols have a greater
affinity for micelles than cholesterol because of their
as statistically significant. Furthermore, to know the best greater hydrophobicity, thereby reducing intestinal
treatment, Duncan’s post-Hoc test 95% confidence cholesterol absorption, and consequently reduce hepatic
interval was used. Statistical analysis was carried out using and plasma cholesterol concentrations. Saponins are also
SPSS 16.0 program. capable of precipitating cholesterol from micelles and
interfering with enterohepatic circulation of bile acids
RESULTS AND DISCUSSION making it unavailable for intestinal absorption and hence
The present research showed that high fat diet and fructose reduce plasma cholesterol levels. The anticholesterolemia
liquid significantly increased the plasma total cholesterol, effect of ethyl acetate and n-hexane extract of
LDL-cholesterol and triglyceride, however, the HDL- Tetracarpidium conophorum could be due to its high
cholesterol was decreased compared to basal diet. After content of flavonoid and high antioxidant activity.
treatment with various dose of ethyl acetate and n-hexane Isoflavones could protect the circulating and membrane
fractions of Tetracarpidium conophorum, and 2.7 lipids by sparing endogenous antioxidant6. An antioxidants
mg/kgBW atorvastatin for 10 days, the effect on plasma such as flavonoid either blocks the initiation of free radical
total cholesterol is as shown in Figure 13. Statistical formation or inactivate (e.g. scavenge) free radicals and
analysis with ANOVA showed that the treated groups were terminates radical damage5. Lipid peroxidation may occur
significantly different (p = 0.000) and Duncan post-Hoc due to free radicals presence in oxygen. The lipid-free
test showed that 2.0ml/kgBW/day of n-Hexane fraction of radical interactions yield peroxides, which are unstable and
Tetracarpidium conophorum, gave the best result in reactive which resulting in extensive membrane,
decreasing the plasma total cholesterol. This is better organellar and cellular damage. Free radical could be
compared to atorvastatin (patent anticholesterol agent). captured by a scavenger, such as flavonoid, a potent free
The effect of treatment observed for LDL-cholesterol is radical scavengers, even greater than vitamin E4. For
shown in Figure 2. The statistical analysis showed that example, superoxide is unstable and decays (dismutates)
there was significant difference (p = 0.000) and 2.0ml/ spontaneously into oxygen and hydrogen peroxide in the
kgBW/day of n-hexane fraction of Tetracarpidium presence of water. However, several non-enzymatic and
conophorum gave the best result in decreasing the LDL- enzymatic systems contribute to inactivation of free radical
cholesterol compared to atorvastatin. Ethyl acetate fraction reactions. A series of enzymes act as free-radical
at concentration of 2.0ml/kgBW/day showed similar scavenging systems and break down hydrogen peroxide
activity to atorvastatin. The effects of the treatments on and superoxide anion. Superoxide dismutase (SOD),
HDL-cholesterol is shown in Figure 3. There were which is found in many cell types, can convert superoxide
significant differences with p=0.000 and 2.0ml/kgBW/day to hydrogen peroxide. Flavonoid as an antioxidant in the
n-hexane extract of Tetracarpidium conophorum increased ethyl acetate and n-hexane fraction of African walnut
the HDL-cholesterol when compared to atorvastatin and could increase SOD activity in hypercholesterolemic rats.
other treatments. Ethyl acetate fractions at concentration of Walnuts are rich in linoleic and alpha linolenic acids (ω-3
2.0ml/kgBW/day showed similar activity in increasing the fatty acids) and in other health-related compounds such as
HDL-cholesterol level when compared to artovastatin. high-biological-value proteins (e.g. arginine) fibre,
Figure 4 shows the effect of treatments on triglyceride vitamins, tannins, folates and polyphenols which may
concentration. Statistical analysis with ANOVA showed a provide additional antiatherogenic properties7. The omega-
highly significant (p = 0.000) difference and Duncan’s 3 fatty acids are believed to lower lipids by inhibiting the
post-Hoc test showed that n-hexane fraction 2.0 ml/ synthesis of vLDL in the liver. This results in smaller, less-
kgBW/day of Tetracarpidium conophorum had similar dense vLDL and LDL particles and therefore, a reduced
activity in decreasing the triglyceride concentration when plague producing lipid profile. In 2001, the Adult
compared to atorvastatin. The seeds of Tetracarpidium Treatment Panel (ATP111) of the National Cholesterol
conophorum are high in protein, carbohydrates, lipids, Education Program recommended that omega-3 fatty acids
fiber and minerals. They are also rich in novel alkaloids, be used as adjunct to pharmacological therapy for lowering
saponins, and sterols. The seeds of Tetracarpidium triglycerides. Walnuts contain polyunsaturated fatty acids,
conophorum contains essential fatty acids such as alpha which may protect against cardio vascular disease (CVD)
linolenic acid (ω-3 fatty acids), fatty acids such as n- and may enhance tocopherol absorption8. n-Hexane and
Hexadecanoic acid (palmitic acid) and vitamins such as ethyl acetate fraction of Tetracarpidium conophorum
methyl nicotinate, which all play vital role in lowering significantly decreased the total cholesterol, LDL
cholesterol levels. Phytosterols, which are usually referred cholesterol and triglycerides and increased the HDL
to as plant sterol and stanol esters, are a group of naturally cholesterol in hypercholesterolemic rats.

 
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3. Koshy, A.S., Anila, L. & Vijayalaksmi, N.R. 2001.

CONCLUSION Flavonoids from Garcinia combagia lower lipid levels
The present study has shown that the extracts of T. in hypercholesterlemic rats. Food Chemistry 72: 289-
conophorum had significant anticholesterol activity which 294.
could be attributed to the oleic acids (ω-9 fatty acids) and 4. Boik J. 1996. Cancer and Natural Medicine. USA:
alpha- linolenic acids (ω-3 fatty acids) which are believed Oregon Medical Press. 150-156.
to lower lipid levels. 5. Kumar V., Abbas A.K. & Fausto N. 2005. Robbins and
Cotran Pathologic Basis of Disease, 7th ed.
ACKNOWLEDGEMENT Philadelphia: Elsevier saunders 16-18.
The Authors are grateful to Dr. Ukwueze S E, the Head of 6. Tsai, P.J. & Huang, P.C. 1999. Effects of isoflavones
Department of Pharmaceutical and Medicinal Chemistry, containing soy protein isolate compared with fish
University of Port Harcourt, Nigeria for using his protein on serum lipids and susceptibility of low
laboratory. density lipoprotein and liver lipids to in vitro oxidation
in hamsters. J.Nutr. Biochem, 10: 631-637.
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