The study was carried out in the Laboratory of Biochemistry Department, Faculty of Basic Medical Sciences University of Calabar, Calabar-Nigeria

Introduction

Macromolecules are complex bio-molecules found in the cells and tissues of higher animals and these include; deoxyribonucleic acid, ribonucleic acid, polysaccharides, protein and lipids ₁ . In the brain and spinal cord, lipids and proteins are important since they make up the bulk of the neuronal membrane, cytosol and organelles of the neurons, neuroglia and other connective tissues including myelin sheath.

Proteins and lipids are very necessary for proper functioning of each brain cell. Lipid functions in the replacement of damaged and worn membranes and as electrical insulators allow rapid propagation of depolarization waves along myelinated nerve ₂,₃ . The most abundant lipids being fatty acids and cholesterol (CH). Fatty acids are stored in the body as triacylglycerol (TAG) which constitutes the body's main caloric reserve, with CH being the precursor of all other steroids ₃ .

Substances taken into the body affect the brain bio-molecules in different ways. This includes increase in cholesterol and triacylglycerol ₄,₅,₆,₇ , and decrease in lipids due to neurodegeneration ₈ . Some of these effects may result from peroxidation of these macromolecules due to the presence of free radicals ₉,₁₀ .

These free radicals are generated by both amodiaquine and artesunate (AQ and AS) ₉,₁₀ , and these are antimalarial drugs manufactured in the form of a combination called Larimal (AQ+AS). These drug functions in the body as two different drugs having high efficacy ₁₁,₁₂,₁₃,₁₄ .

AQ is a 4-aminoquinoline and AS is a water soluble hemi-succinate derivative of artemisinin. AQ acts by accumulating in the lysosomes of the parasites bringing loss of its function, and also binds to their nucleoproteins inhibiting the DNA and RNA polymerase ₁₅ , which end up generating free radicals ₉ . AS act by heme-mediated decomposition of its endoperoxide bond to produce carbon-centered free radicals ₁₀ .

The actions of these drugs may affect the bio-molecules of the brain, hence we studied its effect on some brain bio-molecules of Wistar rats.

Materials And Methods

Twenty-four adult Wistar albino rats weighing between 150-210g were divided into four groups of six animals each. Group A served as the control and the animals received distilled water, while groups B, C and D served as the experimental groups. Each packet of Larimal contains twelve blister tablets each of AQ Hydrochloride USP equivalent to AQ base (153.1mg) and AS (50mg). The drugs were administered in milligram per kilogram body weight (mg/kg) twice a day, three days for groups 2 and 3 animals, and six days for group 4 animals. These are as shown in Table 1. Twelve hours after the last administrations the animals were weighed before sacrificing by anaesthesia with chloroform and their brains removed, blotted dry on filter papers and weighed using a Mettler p163 balance. The brain of all the animals were homogenised in cold 0.25m STKM buffer. The homogenates were used to estimate; total proteins by Biuret kit method, cholesterol by CHOP-PAP kit method and triacylglycerol by GPO-PAP kit method.

Statistical analysis was carried out using one way analysis of variance (ANOVA), thereafter post-hoc test was carried out to find the level of significance at p<0.05. All the results are expressed as mean standard error of mean.

Figure 1

Table 1: Drugs administration

Results

Anthropometric Measurements

Bio-Molecules Estimations

Cholesterol (CH)

The control group had significantly (p<0.001) higher level than the groups treated with 17.50±5.71mg/kg of AQ+AS (C) and 8.75±2.86mg/kg of AQ+AS (D) for three and six days respectively, but not significantly different from the group treated with 8.75±2.86mg/kg of AQ+AS for three days (B). These are as seen in Fig. 4.

Triacylglycerol (TAG)

The control was significantly (p<0.05) higher than the group treated with 8.75±2.86mg/kg of AQ+AS for six days (B), but not different from the groups treated with 8.75±2.86mg/kg of AQ+AS (D) and 17.50±5.71mg/kg of AQ+AS (C) for three days. These are as seen in Fig. 5.

Total protein (TBP)

The control group had significantly (p<0.001, 0.05) higher level than the groups treated with 17.50±5.71mg/kg of AQ+AS (C) and 8.75±2.86mg/kg of AQ+AS (D) for three and six days respectively, but not significantly different from the group treated with 8.75±2.86mg/kg of AQ+AS for three days (B). These are as seen in Fig. 6.

Figure 2

Figure 1: Body weights

Data are presented as mean±standard error of mean ^NS Not significantly different from the control (A)

Figure 3

Figure 2: Brian weight

Data are presented as mean±standard error of mean
** Significantly different from control (A) at p<0.01
*** Significantly different from control (A) at p<0.001

Figure 4

Figure 3: Organ weight to body/brain weight ratio

Data are presented as mean±standard error of mean ^NS Not significantly different from the control (A)

Figure 5

Figure 4: Cholesterol (CH)

Data are presented as mean±standard error of mean *** Significantly different from the control (A) at p<0.001 ^NS Not significantly different from the control (A)

Figure 6

Figure 5: Triacylglycerol (TAG)

Data are presented as mean±standard error of mean * Significantly different from the control (A) at p<0.05 ^NS Not significantly different from the control (A)

Figure 7

Figure 6: Total Protein (TBP)

Data are presented as mean±standard error of mean * Significantly different from the control (A) at p<0.05 *** Significantly different from the control (A) at p<0.001 ^NS Not significantly different from the control (A)

Discussion

The effect of amodiaquine+artesunate (AQ+AS) combination on some macromolecules in the brain of Wistar rats was carried out. Anthropometric results revealed a higher body and brain weights in the group treated with 8.75±2.86mg/kg of AQ+AS for three days, but a lower body and brain weights in the groups treated with 17.50±5.71mg/kg of AQ+AS and 8.75±2.86mg/kg of AQ+AS for three and six days respectively.

Analysis of organ weight in toxicology studies is an important end point for identification of potentially harmful effects of chemicals. Differences in organ weights between treatments groups are often accompanied by differences in body weight between these groups, making interpretation of organ weight differences difficult. Bailey et al ₁₆ reported that analysis of organ weight and body/brain weight is predictive for evaluating the brain. In our study, the organ weight and body/brain weight ratio revealed no significant difference among the groups. This may be as result of the drug having no significant effect on the brain of the Wistar rats at these doses.

In the bio-molecules study, there was a lower level of brain cholesterol (CH), triacylglycerol (TAG) and total proteins (TBP) in the group treated with 8.75±2.86mg/kg of AQ+AS for six days, but a higher level of these bio-molecules in the group treated with 8.75±2.86mg/kg of AQ+AS for three days. This is consistent with the weight of the brain as discussed in this study. Increase in brain size also increases CH level which is an essential component of myelin in white matter ₁₇ . But the formation of CH involves specific proteins which may also increase thereby adding to the total brain proteins ₁₈ .

However brain TAG of the group treated with17.50±5.71mg/kg of AQ+AS for three days was not significantly different from the control, while CH and TBP where significantly (p<0.001, 0.5) lower. The lower levels of CH and TBP are consistent with the brain size in this study. The non consistency of TAG in the group treated with17.50±5.71mg/kg of AQ±AS for three days may be due to trauma caused by the treatment. Ikeda et al ₄ reported increased brain TAG on rats subjected to hypoxia which later decreased on recovery. The drug may also have affected adrenocorticotropin hormone level whose increase level stimulates the synthesis of brain TAG ₁₉ . Sun ₂₀ had earlier reported marked alterations in acyl group compositions of major phosphoglycerides from whole brain homogenates in rat maintained on fatty acid deficient diet.

The drugs AQ±AS combination releases free radical and this usually result in lipid and protein peroxidation ₉,₁₀ . This drug combination did not really show these effects and hence may not be neurotoxic at these dosage and time as the difference seen may be attributed to the rat's size. Thus, this result encourages the use of the drug in malarial treatment as recommended by the manufacturers.