Diabetes mellitus, a chronic disease with complex etiologies (Robert
et al., 2002; Champe and Harvey, 1997) is a
disease that has created serious cause for concern all over the world. The World
Health Organization (WHO, 2002) reported that the number
of diabetic patients is expected to increase from 150 million (King
et al., 1998) in the year 2000 to 300 million or more by the year
2025. This has necessitated the screening of plants for anti-diabetic potentials.
The search for the cure for diabetes mellitus continues along traditional and
alternative medicine because many herbal supplements that have been used for
the control of the disease are either not easily reachable (Wild
et al., 2004) or they lack scientific evidence to support their effectiveness
(Morelli and Zoorob, 2000). The use of synthetic agents
such as sulphonylureas, on the other hand, has its limitations as it has been
associated with side effects such as weight gain, hypoglycaemia (Aldhahi
and Hamdy, 2003), cost effectiveness as well as correcting the fundamental
biochemical lesion and diabetic complications.
Mistletoe (Viscum album) is a plant that is indigenous to the South-Eastern
part of Nigeria where its used by the traditional medicine practitioners for
the management of many metabolic diseases such as hypertension and diabetes
mellitus (Obatomi et al., 1994). Mistletoe teas
have been used for the prevention and management of stokes in parts of Nigeria
and it is also believed to improve the circulatory system and heart function
in traditional medicine (Deeni and Sadiq, 2002).
Although, at present, there are no reports of toxicity arising from the usage of this plant in diabetics, there is need to document such data. This forms the basis of this study aimed at investigating the effect of mistletoe extract on the liver enzymes and electrolyte balance in alloxan induced diabetic rats.
MATERIALS AND METHODS
Preparation of plant materials: The mistletoe plant used for the experiment was harvested in 2010 and identified by a Taxanomist in Michael Okpara University of Agriculture, Umudike, Nigeria. The plant was pulverized and dried in an oven at 60oC. It was then ground to flour and stored in an air tight container for further analysis.
Chemicals: Alloxan, glucose oxidase, aspartate transaminase, alanine transaminase and electrolyte reagent kits used were obtained from Sigma and Aldrich Chemical company, Uk. All other chemicals used for the animal experiments were purchased locally from Associated Laboratories in Aba, Abia State, Nigeria and were of analytical grade.
Selection of animals and their care: Twenty male albino white rats
(Wistar strain) weighing between 90-120 g used for this experiment were purchased
from the University of Nigeria, Nsukka in 2010 and were kept in the animal house
in the Federal University of Technology Owerri, Imo State, Nigeria. The animals
were acclimatized for a period of 7 days to the laboratory conditions prior
to the experiment in line with the Universitys ethics of animal experiments.
Rats were housed in well ventilated colony cages with 2 rats per cage at room
temperature (27-30°C) with 12 h of light and dark cycle and had free access
to drinking water and their diets (ad libithium). The rats were fed with
commercial rat feed, obtained from Chukwuma Ventures Ltd, Owerri, Imo State,
Induction of diabetes: The rats were fasted for 24 h before injection of a freshly prepared solution of alloxan intraperitonially at a dosage of 100 mg kg-1 b.wt. This single dose of alloxan produced Type I diabetes having fasting blood sugar level of 204.37±1.24 mg dL-1 after five days of injection of alloxan.
Experimental procedure: The rats were divided into five groups with four animals in each group:
Group 1: Rats served as the normal (control) and they received oral administration of 60% aqueous extracts of the commercial rat feed and water for a period of four weeks at a dose of 100 mg kg-1 b.wt.
Alloxan was injected into the animals of group 2 at a single dose of 100 mg kg-1 b.wt. Those with fasting blood glucose above 180 mg dL-1 were categorized into groups 2-5:
Group 2: Diabetic control: The animals of this group received oral administration of 60% aqueous extract of the commercial rat feed and water for a period of four weeks at a dosage of 100 mg kg-1 b.wt.
Group 3: Diabetic rats with 20% concentration of mistletoe flour extract: The animals of this group received oral administration of 20% concentration of aqueous extract of mistletoe flour for a period of four weeks at a dosage of 100 mg kg-1 b.wt.
Group 4: Diabetic rats with 40% concentration of mistletoe flour extract: The rats of this group received oral administration of 40% concentration of aqueous extract of mistletoe flour for a period of four weeks at a dosage of 100 mg kg-1 b.wt.
Group 5: Diabetic rats with 60% concentration of mistletoe flour extracts: The animals of this group received oral administration of 60% concentration of aqueous extract of mistletoe flour for a period of four weeks. At the end of four weeks, the animals were starved overnight, stunned by blow and killed by decapitation and their blood was collected intraveneously from the heart using a 10 mL syringe. Their fasting blood glucose, transaminase, electrolytes and phosphatase activities were determined using their respective kits while the changes in their body weights were recorded twice in a week throughout the duration of the experiment.
Similarly, the initial and final body weights were measured with an electronic weighing balance. From these, Fasting Blood Glucose (FBG %) reduction and % weight change were calculated using the formula:
The percentage growth rate was calculated as:
The percentage change in fasting blood glucose was calculated as:
Determination of glucose: The serum glucose was determined using the
glucose oxidase method as described by Cooper (1973).
The principle was based on the fact that β-D-glucose is oxidized by glucose
oxidase to produce D-glucoronic acid and hydrogen peroxide. The hydrogen peroxide
is oxidatively coupled with 4-aminoantipyrine and phenol substitute, p-HBS,
in the presence of peroxidase to yield a red quinone imine dye. The amount of
colored complex formed is proportional to glucose concentration and can be read
spectophotometrically at 500 nm against a reagent blank.
The concentration of glucose in the unknown samples was calculated from the equation:
Determination of aspartate transaminase activity (AST or SGOT): The
method of Henry et al. (1960) was used in the
analysis of the serum transaminase activity of the rats. This is measured by
monitoring the concentration of oxaloacetate hydrazone formed with 2-4 dinitrophenyl
hydrazine. The colored solution formed was measured spectrophotometrically at
546 nm against a reagent blank.
Determination of alanine transaminase (ALT or SGPT): Alanine transaminase
was measured using the method of Henry et al. (1960).
The principle is based on the reaction of α-ketoglutarate and L-alanine
to form L-glutamate and pyruvate. The enzyme activity was measured by monitoring
the concentration of pyruvate hydrazone formed with 2,-4 dinitrophenyl hydrazine
using a UV spectrophotometer at 546 nm.
Determination of chlorides (Cl¯): The chlorides were analyzed using
the method of (Tietz, 1976). The principle is based on
the fact that chloride ions form a soluble non-ionized compound with mercuric
ions and will displace thiocyanate ions from non-ionized mercuric thiocyanate.
The released thiocyanate ions react with ferric ions to form a red color complex
that absorbs light at 480 nm. The intensity of the color produced is directly
proportional to the chloride concentration. The concentration of Cl¯ released
was calculated from the equation:
Determination of sodium (Na+): The method of Henry
(1974) was adopted for the assay. The principle is based on the fact that
sodium is precipitated as the triple salt, sodium magnesium uranyl acetate with
the excess uranium then being reacted with ferrocyanide, producing a chromophore
whose absorbance varies inversely with the concentration of sodium in the test
specimen. The absorbance of the released Na+ was read using a UV
spectrophotometer at 550 nm and the concentration of Na+ in the sample
was derived from the standard using the equation:
Determination of bicarbonates (HCO3¯): This was measured
using the method of Henry (1974). The principle is based
on the fact that phosphoenol pyruvate carboxylase catalyzes the reaction between
phosphoenol pyruvate and carbon dioxide (bicarbonate) to form oxaloacetate and
phosphate ion. Oxaloacetate is reduced to Malate with simultaneous oxidation
of an equimolar amount of reduced nicotinamide adenine dinucleotide (NADH) to
NAD+. This reaction is catalyzed by Malate dehydrogenase. This results
in a decrease in absorbance at 340 nm that is directly proportional to HCO3¯
concentration in the sample. The concentration of HCO3¯ in the
unknown sample was quantified from the equation:
Determination of potassium (K+): The serum K+
components of the samples was determined using the method of Henry
(1974). This is determined by using the sodium tetraphenyl boron in a specifically
prepared mixture to produce a colloidal suspension, the turbidity of which is
proportional to the K+ concentration in the range of 2-7 mEq L-1
and which was read spectrophotometrically in a uv spectrophotometer at 500 nm
against a reagent blank.
The concentration of the K+ was calculated thus:
Determination of alkaline phosphatase (ALP): Alkaline phosphatase was
measured using the method of Tietz (1976). The principle
is based on the fact that ALP acts upon the AMP-buffered sodium Thymolphthalein
monophosphate. The addition of an alkaline reagents stops enzyme activity and
simultaneously develops a blue chromogen which is measured photometrically at
590nm against a reagent blank.
The concentration of ALP in the unknown sample (IU L-1) was calculated using the equation:
Statistical analysis: Data was subjected to analysis of variance using the Statistical Package for Social Science (SPSS) 15.0 windows version. Results are presented as Mean±SD. Means with differences were separated using the Duncan Multiple Range Test and results were considered significant at p<0.05.
RESULTS AND DISCUSSION
Analysis of the nutritive constituents of the commercial feeds that were administered to the rats indicated that it contained 16% protein, 5% fat, 7% crude fibre, 1% calcium, 0.45% phosphorous, 0.75% lysine, 0.36% methionine and 0.3% salt (Table 1).
Alloxan is known to destroy the β-cells of the islet of the Langerhans
of the pancreas that function in the regulation of insulin secretion and thus
leads to an increase in the blood concentration of glucose and type I diabetes
mellitus (Eleazu et al., 2010). This accounts
for the increase in the blood glucose of the diabetic animals. The fundamental
mechanism underlying hyperglycemia in diabetes mellitus involves the over production
of glucose (excessive hepatic glycogenolysis and gluconeogenesis) and or decreased
utilization of glucose by the tissues (Latner, 1958; Dohi
et al., 1998).
However, findings from the study reveal that all concentrations of the extract administered significantly ameliorated the hyperglycemia that resulted from the diabetes (p<0.05) as shown in Table 2.
Diabetes mellitus brings about an increase in the plasma glucose leading to
cell dehydration and movement of K+ into the extra cellular fluid.
This leads to an increase in the activity of parietal cells of the distal and
cortical collecting tubules leading to increased renal excretion of K+
(Yared and Chiasson, 2003). In addition, glucosuria
as observed in diabetes also leads to excretion of excess water, Na+
and K+ in urine (Yared and Chiasson, 2003).
It is therefore possible that the electrolyte and water loss usually observed
in diabetes would lead to depletion of the Extracellular Fluid (ECF) electrolytes
and this could lead to the secretion of electrolytes by parietal and non-parietal
|| Nutrient composition of the feeds (growers feed)administered
to the rats
|| Effect of mistletoe extract on serum chemistry of diabetic
and non-diabetic rats
|Values in the same row with the same superscripts are not
significantly different at p>0.05, n = 4 animals per group
|| Effect of mistletoe extract on the enzyme activities of diabetic
and non-diabetic rats
|Values in the same row with different superscripts are significantly
different from each other at p<0.05, ALP: Alkaline phosphatase, AST:
Aspartate transaminase, ALT: Alanine transaminase
This may therefore account for the significant reduction in the Na+
and K+ of the diabetic rats compared with the control as observed
in Table 2.
Hyperglycemia as seen in uncontrolled diabetes is known to result in metabolic
acidosis (Stoner, 2005) which causes an increase in
respiratory activities and thus leads to an increase in the loss of CO2
from the blood. The loss of CO2 from the blood results in a reduction
in bicarbonates concentration of the ECF. This loss of bicarbonates from the
ECF could be responsible for the significant reduction in the bicarbonate levels
of the diabetic animals.
In the ECF, an inverse relationship is known to exist between the bicarbonates and chlorides in order to keep the anion concentration constant. Thus a decrease in the bicarbonate concentration in the ECF will likely cause an increase in the concentration of chlorides. This increase in ECF chloride concentration may therefore account for the difference in the chloride concentration observed in the diabetic animals as observed in Table 2. However, none of the concentrations of the extract administered significantly ameliorated the altered K+ and Na+ levels of the diabetic animals compared with the diabetic control (p>0.05), 40 and 60% of the extract were most effective in ameliorating the altered bicarbonate levels of the diabetic with respect to the diabetic control while all the concentrations of the extract administered significantly ameliorated the altered chloride levels of the diabetic animals compared with the diabetic control and non diabetic (p<0.05).
The liver is the most vital organ for the metabolism of drugs and other toxicants.
The destruction of the liver cell results in the impairment of the liver cell
membrane permeability which results in the leakage of tissue contents into the
blood stream (Saeed et al., 2008). In addition,
physical trauma or disease process can cause lyses, resulting in the release
of these intracellular enzymes into the blood. It has been reported that the
liver is necrotized in diabetic rats which leads to increased activities of
AST, ALT and Alkaline Phosphatase enzymes as they leak from the liver cytosol
into the blood stream (Saeed et al., 2008) and
this is also an indicator of the hepatotoxicity of alloxan. Therefore, the increase
in the activities of AST, ALT and ALP in serum of the diabetic animals as observed
in Table 3, is mainly due to the leakage of these enzymes
from the liver cytosol into the blood stream (Mansour et
al., 2002; Whitehead et al., 1999).
|| Effect of mistletoe extract on the body weight of diabetic
and non-diabetic rats (g)
|n = 4 animals per group
|| Percentage change in glucose, weight and growth rate
Phosphatase activity is normally high in diseased states and is often used
as a tool in clinical investigations (Bull et al.,
2000). Data generated from this study indicates that all concentrations
of the extract administered could significantly ameliorate the altered phosphatase
and transaminase activities of the diabetic rats (p<0.05) (Table
The loss in weight in the diabetic groups as observed in Table
4, is attributed to the alloxan that was injected into the animals. Alloxan
is known to destroy the β-cells of the islets of the langerhams of the
pancreas that function in insulin regulation, producing type 1 diabetes (Eleazu
et al., 2010). The destruction of the pancreas results in the utilization
of non-carbohydrate moieties such as protein for the synthesis of glucose. The
loss of structural proteins in increased gluconeogenesis together with increased
lipolysis and increased synthesis of ketone bodies results in severe weight
loss. All the concentration of the extracts that were administered ameliorated
the weight loss observed in the diabetic animals compared with the control.
There was a 12.23±3.87, 8.47±2.69 and 2.95±1.44% increase in weights of the diabetic rats respectively, after administration of 20, 40 and 60% extracts of mistletoe to the diabetic rats. Similarly, we recorded a 44.97±6.82, 32.57±2.73 and 10.68±3.54% increase in growth rates after administration of 20, 40 and 60% extracts of mistletoe respectively to the diabetic rats while we recorded a 42.88±0.52, 21.47±0.23 and 16.54±3.37% decrease in fasting blood glucose levels after administration of 20, 40 and 60% extracts of mistletoe respectively to the diabetic animals (Table 5). The study shows that 20% of the aqueous extract of mistletoe flour was most effective in ameliorating the hyperglycemic status of the diabetic rats with a corresponding increase in body weights and growth rates.
The result of the study carried out reveals that mistletoe has anti-diabetic potentials and could be useful in improving the altered electrolytes that arise from diabetes. Finally, the dietary supplement could protect the liver cells from free radical damage that arises from diabetes.