Abstract: This research was undertaken to determine the effects of the extract of Vernonia amygdalina on some biochemical parameters in rat. The ethanol extract of leaf of Vernonia amygdalina was administered daily orally to four groups of rats at a dose of 100, 250, 500 and 1000 mg kg-1 weight, respectively, for 28 days. On the 29th day blood was obtained from the hearts of the rats. The blood samples were centrifuged to obtain the blood serum.Serum levels of the following enzymes and biomolecules were evaluated: alanine and aspartate transaminases, alkaline phosphatase, glucose, creatinine, proteins, total and conjugated bilirubin, total cholesterol, triglycerides, high density lipoproteins, low density lipoproteins and very low density lipoproteins. Weights of the animals were taken weekly. The results of this study show that the extract reduced significantly the levels of glucose by 15.6, 23.4, 19.5 and 11.7% at 100, 250, 500 and 1000 mg kg-1 doses, respectively. While only 250 mg kg-1 of the extract also decreased the value of triglycerides (1.0331 mmol L-1) compared to control (1.687 mmol L-1). It can be concluded from these results that the leaf extract of Vernonia amygdalina is safe for use in ethnomedicine, especially in ethnotherapy of diabetes mellitus.
INTRODUCTION
Vernonia amygdalina Del (Compositae) is a shrub or small tree that grows throughout tropical Africa. It is popularly called bitter leaf because of its abundant bitter principles. It is cultivated in Nigeria mainly for its nutritional value (Igile et al., 1995a). The plant (especially the leaf) has been found useful in the ethnotherapy of diabetes (Akah and Okafor et al., 1992; Uhegbu and Ogbuehi, 2004; Nwajo, 2005 ), asthma, schistosomiasis, malaria (Masaba, 2000), measles, diarrhoea, tuberculosis, abnominal pain and fevers, cough (Akinpelu, 1999). The fresh leaves of the plant contains 82%, 5.3 , 0.4, 10.0, 1.5 and 1.7 g of moisture, proteins, fat, carbohydrate, fibre and ash, respectively. It also contains 145 mg, 5.0 g and 51 mg of Ca, P, Fe and Vitamin C, respectively (Anne, 1979).
Phytochemicals contained in Vernonia amygdalina include saponins, sesquiterpenes, lactones and flavonoids. Steroid glucosides such as Vernoniosides A1, A2, A3, A4, B1, B2, B3, D and E have been isolated from the plant (Ohigashi, 1994; Aregbeore et al., 1997; Igile et al., 1995b). But it is yet to be ascertained which of these are responsible for some observed biological effects of the plant.
In view of the wide use of the plant in both human nutrition and ethnomedicine, the present research was undertaken to evaluate the effect of daily, prolonged consumption of the plant on some important biochemical parameters. This will enable us ascertain the safety or otherwise of the plant.
MATERIALS AND METHODS
Plant Collection
The fresh leaves of Vernonia amygdalina were collected in March 2005
from the medicinal garden of Faculty of Pharmacy, University of Uyo, Nigeria.
They were identified in the Pharmacognosy Department of the same Faculty by
Dr Kolawole Ajibesin (a pharmacognosist). The leaves were air dried before extraction.
Preparation of Extract
The leaves (662.5 g) were macerated with 4 L ethanol (95%) for 72 h. The
extract obtained was filtered, concentrated in vacuo in a rotary evaporator
and dried in a dessicator containing Silica gel (self indicating). 14.5 g of
extract was obtained , giving a yield of 2.18%.
Animals
Wistar rats of both sexes obtained from the animal house of the University
of Uyo, Nigeria, were used. They were bred and kept under standard laboratory
conditions. They had free access to both food and water. They were kept in the
care of experienced animal technicians. Animal work conformed to approved ethical
standards of the University.
Administration of Extract
Twenty five rats were divided into five equal groups A, B,C, D, and E. One
mL-1 of the extract of the plant was administered orally and once
daily in the dose of 100, 250, 500 and 1000 mg kg-1, to groups A,
B, C and D, respectively. Group E served as control and received 1 mL of the
vehicle (i.e., water only).
Collection of Blood
Blood sample was obtained on the 29th day from the hearts of the overnight
fasted animals under chloroform anaesthesia. Blood samples were allowed to clot
after which they were centrifuged at 7000 rpm for 10 min to obtain serum. Serum
was stored in a refrigerator at-4°C until used for analyses.
Biochemical Parameters
Appropriate commercial kits (Randox Laboratories) were used to evaluate
the following enzymes and biomolecules.
Glucose
This was evaluated using the glucose oxidase method (Trinder, 1969).
Alanine Transaminase (ALAT)
The method involves the monitoring of the concentration of pyruvate hydrazone
formed with 2,4-dinitrophenylhydrazine (Rietman and Frankel, 1957).
Aspartate aminotransferase (ASAT)
The principle of the method used involved monitoring the concentration of
oxaloacetate hydrazone formed with 2, 4,-dinitrophenyl hydrazine (Rietman and
Frankel, 1957).
Alkaline Phosphatase (Phenolphthalein Monophoshate Method)
This method is based on the principle that serum alkaline phosphatase hydrolyzes
a colourless substrate of phenolphthalein that results in phosphoric acid and
phenolphthalein at alkaline PH.values. The pinkly coloured product is measured
colorimetrically at 550 nm.
Triglycerides
This involves the enzymatic colorimetric test of glycerol phosphate oxidase
method (Zoppi and Fenili, 1976).
Total Cholesterol
This was carried out by the enzymatic colorimetric chod-PAP method (Zoppi
and Fenili,1976).
HDL-Cholesterol
High Density Lipoprotein (HDL) separated from chylomicrons. Very low density
lipoproteins (VLDL) and Low Density Lipoproteins (LDL) by the addition of a
phosphotungstic and magnesium chloride (precipitating reagent) to the serum.
After centrifugation, the cholesterol content was determined by the enzymatic
colorimetric method (Zoppi and Fenili, 1976).
LDL and VLDL Cholesterol
These were calculated as recommended by Tietz (1999).
Total Protein
This was done using the Biuret method.
Creatinine
Modified Jaffe’s method was used. Creatinine which is a hydride of
creatine reacts with alkaline sodium picrate to form a red complex which can
be determined photometrically (Jaffe, 1886).
Total and Conjugated Bilirubin
This was based on colorimetric method of Jendrassik and Grof (1938).
Statistical Analysis
Data were expressed as Mean±SEM and were analysed by one way ANOVA
and Scheffe’s post test. p<0.05 was taken as significant.
RESULTS AND DISCUSSION
Only the serum concentration of glucose (39.0, 35.4, 37.2 and 40.8 mg dL-1) was significantly different from the control (46.2 mg dL-1) at dose levels of 100, 250, 500 and 1000 mg dL-1, respectively (Table 1). The percentage reduction is 15.6, 23.4, 19.5 and 11.7%, respectively. While the extract reduced the levels of Triglycerides at all dose levels to some extent, only the 250 mg kg-1 dose significantly reduced the concentration (1.0331 mmol L-1) at p<0.05 compared to control (1.687 mmol L-1). All the other biomolecules did not statistically differ at p<0.05 from the control.
However, the observed hypoglycemic activity of the extract was not dose dependent. 250 mg kg-1 doses gave the highest percentage reduction.
The hypoglycaemic effect of vernonia amygdalina is well known. The hypoglycemic
effect of all doses of the plant extract as observed in this work is therefore
not surprising, it confirms the effectiveness of the leaves of V. amygdalina
in the ethnotherapy of diabetes mellitus (Akah and Okafor, 1992). Alanine and
aspartate transaminases and alkaline phosphatases are often present in high
concentrations in the liver. They leak into the circulation when there is necrosis
or hepatocellular injury. They are therefore employed in clinical diagnosis.
Principally ASAT level is of use in diagnosis of myocardial infarction.
| Table 1: | Effect of Vernonia amygdalina on some biochemical parameters in rats |
Mean±SEM, n = 5, *p<0.05, n is
the number of animals per group p is 95% significance level |
|
ALAT in viral hepatitis. Hyperbilirubinemia which may lead to jaundice is caused by over production of bilirubin, liver damage or obstruction to the excretory ducts of the liver. The values of both conjugated and total bilirubin , which were not significantly different from control in this study, further show that the extract did no harm to the liver. The results of this study therefore indicate that the extract did not injure either the heart or liver of the rats. The extract also did not generate any inflammatory conditions.
Lipid profile (which involves levels of total cholesterol, HDL, LDL and VLDL) serve as diagnostic indices in conditions such as chronic obstructive jaundice, hepatitis, coronary heart disease and atherosclerosis. Hyperlipidaemia is one of the risk factors for coronary heart disease while cholesterol is the major lipid constituent of atherosclerotic plague. Extract of v. amygdalina did not affect lipid profile significantly. indicating that the use of the plant will not likely contribute to any disease associated with hyperlipidaemia. 250 mg kg-1 of the extract significantly reduced the level of Triglycerides from 1.687 mmol L-1 (control) to 1.0331 mmol L-1. It is not clear why the reduction in the level of Triglycerides at other dose levels were not significant. The plant extract also did not seem to have adverse effect on the kidney, since the creatinine levels were not significantly altered.
The present research show that there were no changes in most of the investigated parameters following the administration of the ethanolic extract of V. amygdalina. It confirms that V. amygdalina is useful in the ethnotherapy of diabetes mellitus. It could also be concluded that the plant is also safe for consumption as food or medicine, since there were no indication of toxicity judging from the values of the biomolecules evaluated. This research also seems to show that the dose of 250 mg kg-1 of the extract is the optimal dose for the hypoglycemic and hypotriglyceridemic effects of the extract.