Hypocholesterolaemic Effect of Aqueous Extract of Acalypha wilkesiana ‘Godseffiana’ Muell Arg on Rats Fed Egg Yolk Supplemented Diet: Implications for Cardiovascular Risk Management.
Ikewuchi Jude C.* and Ikewuchi Catherine C.
Department of Biochemistry, Faculty of Science, University of Port Harcourt, P.M.B. 5323, Choba, Nigeria
The effect of aqueous extract of Acalypha wilkesiana on the mean daily weight gain, lipid profile and atherogenic indices of rats fed egg yolk supplemented diets was investigated. The control group was given normal feed while other three groups received 50g egg yolk/kg feed. The extract was orally administered daily at 200 and 250mg/kg body weight; while the test control and control groups received appropriate volumes of water by the same route. The mean daily weight gain of the test groups were each significantly lower than the control, but not significantly different from the test control. The plasma total, LDL and non-HDL cholesterol levels of the test groups were each significantly less than the control and test control. The cardiac risk ratio and atherogenic coefficient of the test groups were each significantly (P<0.05) lower than those of the test control, but not different from the control. These results indicate a hypocholesterolaemic effect of the extract, thus suggesting a likely protective role of the extract against the development of cardiovascular diseases.
Dyslipidemia is one of the major risk factors for the development of cardiovascular disease, and is often found, associated with hypertension, diabetes mellitus and obesity1-4. Dyslipidemia usually involve elevated plasma levels of triglycerides, total, LDL and VLDL cholesterol and a low level of HDL cholesterol1-4. Therefore, any nutritional and pharmacologic intervention that improves or normalizes abnormal lipid metabolism may be useful for reducing the risk of cardiovascular diseases1,4. Several drugs are at present, available for the management of dyslipidemia. However, there is renewed interest in the use of herbal products.
Acalypha wilkesiana Muell Arg (family Euphorbiaceae) is a popular outdoor plant that provides color throughout the year, although it is also grown indoors as a container plant. Many cultivars are available with different leaf forms and colors: A. wilkesiana ‘Godseffiana’ has narrow, drooping, green leaves with creamy-white margins, 'Marginata' has coppery-green leaves with pink or crimson margins, 'Macrophylla' has larger leaves, variegated with bronze, cream, yellow and red, while 'Musaica' has green leaves that are mottled with orange and red5,6. In traditional medicine, the leaves of this plant are eaten as vegetables in the management of hypertension, consequent upon which we undertook a study of the effect of the plant leaves on: plasma sodium and potassium levels of normal rabbits7; urinary and plasma chemistry8, blood pressure and aorta contractility9, tissue profiles of ATPase activity10 and enzymes of energy metabolism11, of salt-loaded rats.
In the present study, the effect of aqueous extract of the leaves on weight, plasma lipid profile and atherogenic indices was investigated in rats fed egg yolk supplemented diet, with a view to unveiling any likely cardioprotective potential.
MATERIAL AND METHODS:
Collection of Animals and Preparation of Plant Extract: Albino rats were collected from the animal house of the Department of Physiology, University of Nigeria, Enugu Campus, Enugu, Nigeria. Samples of the fresh Acalypha wilkesiana plants were collected from within the Choba and Abuja Campuses of University of Port Harcourt, Port Harcourt, Nigeria. After due identification at the University of Port Harcourt Herbarium, Port Harcourt, Nigeria, they were rid of dirt and the leaves were removed, oven dried at 550C and ground into powder. The resultant powder was soaked in boiled distilled water for 12h, after which the resultant mixture was filtered and the filtrate, hereinafter referred to as the aqueous extract was stored for subsequent use. A known volume of this extract was evaporated to dryness, and the weight of the residue used to determine the concentration of the filtrate, which was in turn used to determine the dose of administration of the extract to the test animals.
Experimental Design: Studies were conducted in compliance with applicable laws and regulations. The rats were randomly sorted into four groups of five animals each, so that the average weight difference was ±1.4g. The animals were housed in plastic metabolic cages. After a one-week acclimatization period on guinea growers mash (Port Harcourt Flour Mills, Port Harcourt, Nigeria), the treatment commenced and lasted for two-weeks. The control group was given normal feed while the three test groups received 50g egg yolk/kg feed. The first test group (Test 1) received daily by intra-gastric gavages 200mg/kg body weight of the Acalypha wilkesiana extract; the second group (Test 2) received 250mg/kg body weight of the Acalypha wilkesiana extract; while the third group (test control) and the control group received appropriate volumes of water by the same route. The two weeks egg yolk supplementation is a modification of the two weeks 5% loading reported by Agbafor and Akubugwo12. The animals were allowed food and water ad libitum. They were exposed to 12 hour light-dark cycle and were handled according to standard protocol. At the end of the treatment period the rats were weighed, fasted overnight and anaesthetized by exposure to chloroform. While under anesthesia, they were painlessly sacrificed and blood was collected from each rat into heparin sample bottles. Whole blood was immediately used to determine the triglyceride levels (using test strips), while the heparin anti-coagulated blood samples were centrifuged at 1000g for 10min, after which their plasma was collected and stored for subsequent analysis.
Determination of the Plasma Lipid Profiles/Indices: Plasma triglyceride concentration (TG) was determined using multiCareinTM triglyceride strips and glucometer (Biochemical Systems International, Arezzo, Italy). The test is based on lipase/glycerol kinase/glycerol phosphate oxidase/peroxidase/chromogen reaction. The intensity of the colour developed from the reaction is proportional to the concentration of triglycerides in the blood. Plasma total (TC) and high density lipoprotein (HDLC) cholesterol concentration were assayed enzymatically with Randox commercial test kits (Randox Laboratories, Crumlin, England). In the presence of magnesium ions, low density lipoproteins (LDL and VLDL) and chylomicrons fractions are precipitated quantitatively by the addition of phosphotungstic acid. After centrifugation, the cholesterol concentration of the high density lipoprotein (HDL) fraction, which remains in the supernatant, can be determined, as in total cholesterol. The cholesterol released by enzymatic hydrolysis is oxidized with the concomitant release of hydrogen peroxide, whose breakdown leads to the conversion of 4-aminoantipyridine to quinoneimine (the indicator) whose concentration can be determined spectrophotometrically at 500nm.
Plasma VLDL- and LDL-cholesterol (LDLC and VLDLC) concentrations was calculated using the Friedewald equation13 as follows:
· [LDL cholesterol] (mg/dL) = [Total cholesterol] – [HDL cholesterol] – [Triglyceride]
· [VLDL cholesterol] (mg/dL) = [Triglyceride]
While the plasma non-HDL cholesterol concentration was determined as reported by Brunzell et al.14:
· [Non-HDL cholesterol] = [Total cholesterol] – [HDL cholesterol]
The atherogenic indices were calculated as earlier reported by Ikewuchi and Ikewuchi15 using the following formulae:
· Cardiac Risk Ratio (CRR) = [Total Cholesterol]
· Atherogenic Coefficient (AC) =
[Total Cholesterol] - [HDL Cholesterol]
· Atherogenic Index of Plasma (AIP) =
Statistical Analysis of Data: All values are quoted as the mean ± SEM. The values of the various parameters for all the groups were analyzed for statistical significant differences using the student’s t-test, with the help of SPSS Statistics 17.0 package. P<0.05 was considered to significant.
Table 1 Effects of aqueous extract of Acalypha wilkesiana on the daily weight gain and plasma lipid profile of rats fed egg yolk supplemented diet
Mean daily weight gain (g/day)
Total cholesterol (mg/dL)
HDL cholesterol (mg/dL)
VLDL cholesterol (mg/dL)
LDL cholesterol (mg/dL)
Non-HDL cholesterol (mg/dL)
Values are mean ± SEM, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.
Table2: Effect of aqueous extract of Acalypha wilkesiana on atherogenic indices of rats fed egg yolk supplemented diet.
Cardiac risk ratio
Atherogenic index of plasma
Values are mean ± SEM, n=5, per group. Values in the same row with the different superscripts are significantly different at P<0.05.
The effects of aqueous extract of Acalypha wilkesiana on the daily weight gain and plasma lipid profile of rats fed egg yolk supplemented diet is given in Table 1. The mean daily weight gain of the test groups were each significantly lower than the control, but not significantly different from the test control. The plasma triglyceride and VLDL cholesterol levels of the test groups were not significantly (P<0.05) different from the control and test control. The plasma total, LDL and non-HDL cholesterol levels of the test groups were each significantly less than the control and test control. The effects seem to be dose dependent, with the 200mg/kg body weight dose being more effective.
Table 2 shows the effect of aqueous extract of A. wilkesiana on the atherogenic indices of rats fed egg yolk supplemented diet. The cardiac risk ratio and atherogenic coefficient of the test groups were each significantly (P<0.05) lower than those of the test control, but not different from the control. There were no significant differences in the atherogenic index of plasma of the three groups.
The effects of the extract was dose dependent, with the 200mg/kg dose being more effective (Tables 1 and 2). The extract produced low plasma total cholesterol levels in the treated rats. This may be cardioprotective, since elevated plasma total cholesterol level is a recognized and well-established risk factor for developing atherosclerosis and other cardiovascular diseases16. This hypocholesterolaemic activity of the extract may be due to its high saponin content17. In human nutrition, saponins are reported to assist in the prevention of cardiovascular diseases18 by lowering plasma cholesterol concentrations through the excretion of cholesterol directly or indirectly as bile acids19-21.
Decreases in plasma LDL cholesterol have been considered to reduce risk of coronary heart disease3,4. In this study, a significantly lower plasma LDL cholesterol level was produced by the extract, indicating its likely cardio-protective potential. Several studies have shown that non-HDL cholesterol is a better predictor of cardiovascular disease risk than is LDL cholesterol14,22,23. Therefore, the significantly lower plasma non HDL cholesterols observed in the test groups indicate the ability of the extract, to reduce cardiovascular risk.
The atherogenic indices are strong indicators of the risk of heart disease: the risk of developing cardiovascular disease increases with increases in the values of these indices, and vice versa24-27. Low atherogenic indices are protective against coronary heart disease26. In this study, the extract produced significantly lower cardiac risk ratio and atherogenic coefficient.
Finally, these results indicate a dose dependent hypocholesterolaemic effect of the extract, thus suggesting a likely protective role of the extract against the development of cardiovascular diseases.
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