Effect of Artemisinin with Folic Acid on the Activities of Aspartate Amino Transferase, Alanine Amino Transferase and Alkaline Phosphatase in Rat
Edoho J. Edoho,
Emmanuel I. Etim
Studies on the effect of artemisinin alone and artemisinin with folic acid on the activities of aspartate amino transferase (ASAT), alanine amino transferase (ALAT) and alkaline phosphatase (ALP) in the serum of male Wistar rats were carried out. Different groups of rats (8 group-1) were orally given 0.75, 1.50, 3.00 and 6.00 mg kg-1 b.wt. of artemisinin. Each of these doses was also administered concurrently with 1.50 mg kg-1 of folic acid, respectively. Artemisinin only elevated the activities of serum ASAT, ALAT and ALP significantly at the four dose levels. When 1.50 mg kg-1 of folic acid was concurrently administered with artemisinin, the elevated serum level of ASAT, ALAT and ALP was significantly reversed almost completely at low dose of 0.75 and 1.50 mg kg-l artemisinin. Folic acid only reversed the elevated activity of ASAT, ALAT and ALP by artemisinin partially when the dose of artemisinin was high (i.e., 3.00 and 6.00 mg kg-1 of artemisinin). These results suggest that folic acid offers complete relief to metabolic disorders at low artemisinin concentration while the relief is partial at high concentrations.
to cite this article:
Aniefiok Udobre, Edoho J. Edoho, Olorunfemi Eseyin and Emmanuel I. Etim, 2009. Effect of Artemisinin with Folic Acid on the Activities of Aspartate Amino Transferase, Alanine Amino Transferase and Alkaline Phosphatase in Rat. Asian Journal of Biochemistry, 4: 55-59.
Malaria is transmitted to human by plasmodium through mosquito bite. Despite
increasing research and control efforts malaria still remains major health problem
malaria is the most important vector born disease (Curtis,
1993). About 400 million people experience clinical episodes and 2 million
deaths occur annually with about 90% mortality found in tropical Africa and
Southeast Asia where the disease is prevalent (WHO, 1993;
Coker et al., 2001).
These regions of the world are characterized by optimum conditions of temperature,
heavy rainfall and dense vegetation which promote the survival of the Plasmodium
species (Curtis, 1993).
The characteristics of malaria disease include shaking, chills and relapsing
fever (Maegraith and Fletcher, 1992). It is associated
with pulmonary edema renal failure, tropical splenomegaly syndrome and nephritic
syndrome (Guyton and Hall, 1996).
During the First World War, the first antimalarial drug, Dichloro-Diphenyl
Tricholorethane (DDT) was used (Alvin et al., 1978).
It was latter discovered to be a major environmental pollutants and was later
banned (Peters, 1990). Chloroquine is the cheapest and
the most widely available first line drug. The spread and intensity of resistance
by the malarial parasite to chloroquine and other antimalarials such as mefloquine
including combination drugs (Molta
et al., 1993; Olliaro and Trigg, 1995; Sowunmi
et al., 1996) necessitated the search for new drugs. This efforts led
to the choice of artesnuate which is obtained from the plant; Artemisia annua
(Woedenbag et al., 1994) and from which the antimalarial
principal (artemisinin) was discovered in 1971 (Liy and
Artemisinin works by destroying the cell membrane of the parasite slows down
protein synthesis disorganizes the ribosome, dilate the nuclear envelope and
disintegrate the food vacuoles. In the mosquito life cycle it inhibits the development
of the trophozoites, kills the schizont of the parasite and prevents progression
of the disease (Meshnik et al., 1996). Clinical
findings up to date have not revealed any pattern of resistance to artemisinin.
It is considered as an effective alternative drug for a more virulent falciparum
malaria (Baradell and Fitton, 1995; Karbwang
et al., 1994).
Artermisinin is commonly administered with folic acid. The objective of this study is to evaluate the effect of artemisinin and the role of folic acid on the activities of alanine aminotransferases, aspartate aminotransferases and alkaline phosphatases.
MATERIALS AND METHODS
Adult male Wistar rats weighing between 150 and 180 g were used. They were
fed with growers marsh (Vital Feeds Limited, Lagos) and water ad
libitum and kept in the animal house of the Faculty of Pharmacy, University
of Uyo under standard laboratory conditions.
Artemisinin (manufactured by Mekopharm Chemical Company, Vietnam but purchased
in Uyo), folic acid (Unique Pharmaceuticals, Lagos, Nigeria) and freshly prepared
Administration of Drugs/Chemicals on the Animals
Seventy overnight fasted rats were divided into 9 equal groups (each containing
8 rats). The drugs were administered orally to the rats once daily for 5 days
as shown below:
||Received 0.75 mg kg-l artemisinin only
||Received 0.75 mg kg-l artemisinin and 1.50 mg kg-l
||Received 1.50 mg kg-l artemisinin only
||Received 0.50 mg kg-l artemisinin and 1.50 mg kg-l
||Received 3.00 mg kg-l artemisinin only
||Received 3.00 mg kg-l artemisinin and 1.50 mg kg-l
||Rerceived 6.00 mg kg-l artemisinin only
||Received 6.00 mg kg-l artemisinin and 1.50 mg kg-l
||Saline water only (control)
Collection of Blood and Preparation of Serum
Overnight fasted rats were sacrificed on the 6th day and blood collected
by passing a needle on syringe through the ventricle of the heart.
The serum was prepared from clotted blood by gently decanting the serum from the cells into iron-free centrifuge tubes and the tubes were spun at 10,000 rpm for 5 min in a centrifuge machine (MSE England). The serum was then decanted into another set of clean tubes and stored at 15°C for a period not exceeding 24 h.
Determination of Enzymes
The prepared serum samples obtained from the blood of the sacrificed animals
were analyzed. Aspartate amino transferase and alanine amino transferase were
estimated using end point calorimetric method as explained in Randox enzyme
kits (Reitman and Frankel, 1957). The absorbance of
the test solution was measured using an UV-Vis-NIR spectrophotometer with 540
nm at 37°C.
The activity of alkaline phosphatase in the serum was determined using phenolphthalein
monophosphate method (Nashar, 1974; Ngaha
and Plumer, 1977). The absorbance of the test solution was measured with
540 nm at 37°C. Enzyme activity is given in IU L-l.
The data obtained were expressed as Mean±l;SD. Students t-test
was used for the analysis the level of significance and p<0.05 was taken
to be statistically significant.
RESULTS AND DISCUSSION
The results in Table 1 shows that the activity of ASAT in
the serum increases as the dose of the drug increases. At a dose of 1.5 mg kg-l
artemisinin, the activity of ASAT (51.26±l;2.79) doubled that of the corresponding
control value (20.75±l;4.10). As the dose was increased to 3.0 and 6.0
mg kg-l artemisinin, the ASAT correspondingly tripled (75.36±l;5.75)
and quadrupled (86.00±l;1.31) that of the corresponding control value,
respectively. This increase was statistically significant (p<0.05). This
elevated activity is an indication of liver dysfunction (Murray
et al., 1990). Concurrent administration of 0.75 mg kg-l
artemisinin and 1.50 mg kg-l folic acid reduced the ASAT activity
from 46.563.52 to 34.50±l;4.84. This combination did not reduce the ASAT
activity to the control value of (20.75±l;4.10). This shows that folic
acid can reverse metabolic disorder associated with low concentration of artemisinin.
Concurrent administration of 6.0 mg kg-l artemisinin with 1.50 mg
kg-l of folic acid and that of 3.00 mg kg-l artemisinin
with 1.50 mg kg-l folic acid did not reduce the ASAT level significantly.
It is probable that the 1.50 mg kg-l of folic acid administered was
inadequate and perhaps a higher dose could have reversed this metabolic disorder.
Alanine amino transferase (ALAT) in the serum decreased with increasing dose of artemisinin. At a dose of 0.75, 1.50, 3.0 and 6.0 mg kg-l artemisinin the serum ALAT was 24.67±l;8.39, 22.69, 22.33 and 18.40 IU L-l. The decrease in serum ALAT was dose dependent. Concurrent administration of these doses with 1.50 mg kg-l of folic acid increased ALAT level to control levels in the first three doses (i.e., 0.75, 1.50 and 3.0 mg kg-l of artemisinin). However, at the highest dose of 6.0 mg kg-l of artemisinin the increase in the level of ALAT was lowest. The results of this study showed that artemisinin might cause some metabolic disorders and that folic acid could reverse, partially or completely, this effect.
The Mean serum alkaline phosphatase (ALP) activity of samples treated with
0.75, 1.50, 3.0 and 6.0 mg of artemisinin was 170.23±l;22.15, 181.01±l;13.59,
189.76±l;6.27 and 200.40±l;16.77 IU L-l, respectively.
These values showed a significant dose dependent increase of ALP activity over
the control (121.33±l;20.91). While the concurrent administration of 1.50
mg kg-l folic acid with 0.75 or 1.50 mg kg-l artemisinin
significantly reduced the elevated value of ALP that of 1.50 mg kg-l
of folic acid with 3.0 and 6.0 mg kg-l artemisinin did not significantly
reduce the elevated ALP activity.
||The effect of different doses of artemisinin (artesunate)
alone and artemisinin with folic acid on serum aspartate amino transferase
(ASAT), alanine amino-transferase (ALAT) and alkaline phosphatase (ALP)
|ART: Artemisinin, FA: Folic acid, Control: Physiological saline.
The results are expressed as Mean±l;SD. n = 8, *Statistical significance
Some enzymes act as markers and indicators of disease states. Enzyme levels
in the intracellular fluids such as blood form an integral part of diagnosis.
Increase in the activities of enzymes in serum is indicative of cell damage.
Some disease states also decrease enzyme synthesis leading to reduced enzyme
concentration in the serum. ALAT, ASAT and ALP are often present in high concentrations
in the liver. ASAT (EC 18.104.22.168) is found primarily in heart, liver, skeletal
muscle and kidney whereas ALAT (EC 22.214.171.124) is found primarily in liver and
kidney, with lesser amounts in heart and skeletal muscle (Giannini
et al., 2005). Liver disease is the most important cause of increased
ALAT activity and a common cause of increased ASAT activity. In hepatocellular
injury or necrosis they leak into the circulation and raise the serum level
of the enzymes. In most types of liver disease, ALAT activity is higher than
that of ASAT. Progressive increase in the ASAT/ALAT ratio is correlated with
a concomitant decrease in liver function. An ASAT/ALAT ratio of 1 or higher
has a good specificity for the diagnosis of cirrhosis (Giannini
et al., 2003). The major diagnostic use of ASAT is in myocardial
infarction and ALAT in viral hepatitis and acute pancreatitis (Ellis
et al., 1978; Reichling and Kaplan, 1988).
In inflammatory conditions, there is usually a leakage of cytoplasmic enzymes
into the circulation. As such, ALAT is observed to be higher than that of ASAT.
In gross necrosis, the level of ASAT may rise above that of ALAT (Giannini
et al., 2005).
From the results obtained in this study, it can be inferred that the administration of different doses and most especially, 6.00 mg kg-l artemisinin to male wistar rats caused significant increase in serum aspartate amino transferase, alkaline phosphatase and alanine amino transferase activities. These effects are indicative of liver problems. Folic acid gave complete relief to the metabolic disorders only at low doses of artemisinin while it effected partial or no relief at much higher doses. The administration of folic acid alongside that of artemisinin in the treatment of malaria is therefore beneficial and advisable.
Authors acknowledged the kind assistance of the staff of Animal House, Faculty of Pharmacy, University of Uyo, Uyo in the course of this study and also the Department of Pharmaceutical and Medicinal Chemistry for materials provided.
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