Abstract: The sub chronic esculetin (6,7-dihydroxy-coumarin)-induced response on malondialdehyde concentration and some haematological and lipid metabolism parameters was studied in normal male Wistar rats to further understand the underlying esculetin metabolism and effects. Five groups (n = 6) of rats, A-E were respectively exposed to 6.0, 12.0 and 24.0 mg kg–1 b.wt. of esculetin, 0.2 mL of the vehicle control (10% dimethylsulfoxide (DMSO)) and 0.2 mL of the normal control (Distilled Water (DW)) per orally and daily for 28 days. Compared with the controls, the haematological parameters change in the esculetin-exposed rats was not significant (p>0.05) but decreased numerically with increasing dose viz: hemoglobin (99.83±5.04 g L–1-91±7.04 g L–1), packed cell volume (30±1.41%-27.33±2.16%), white blood cell count (9.63±0.91×109 L–1-7.95±0.58×109 L–1), neutrophil (1.93±0.51×109 L–1-1.72±0.34×109 L–1) and lymphocyte count (7.64±1.04×109 L–1-6.18±0.63×109 L–1). The reduction in the serum cholesterol (3.39±0.23 mmol L–1-3.01±0.78 mmol L–1) and triacylglyceride (0.49±0.45 mmol L–1-0.15±0.05 mmol L–1) concentrations in the esculetin-dosed rats was significant (p<0.05) and followed the numerical decreasing trend. The serum MDA concentration (1.90±0.53 mmol L–1-2.82±1.50 mmol L–1) decreased (p<0.05) but increased numerically with increasing dose. Thus, the sub-chronic esculetin-induced alteration was inversely related to esculetin dose and may not have resulted to significant pathologies or dysfunctional states in the rats.
INTRODUCTION
Esculetin (6,7-dihydroxycoumarin) is a coumarin (group of non-nutrient phenolic phytochemicals) derivative that is present in several plants, including Citrus limonia, Aesculus hippocastanum, Euphobia lathyris, Fraxinus rhynchophylla and Artemisia capillaris (Masamoto et al., 2003; Tien et al., 2011). Esculetin, like other coumarins, possesses diverse biological and pharmaceutical properties (Lacy and O’Kennedy, 2004) and is useful in drug synthesis (Kostova, 2005). It is common in foodstuffs, including vegetables, fruits, nuts, coffee, tea and wine and it is widely used as flavour-enhancing agents for variety of foods (Leung et al., 2005). The average western diet contains approximately 1.0 g day–1 of mixed coumarins, including esculetin (Lacy and O’Kennedy, 2004) and in the sub Saharan African, including Nigeria, plants generally serve as source for foods and herbs. Thus, the dietary and therapeutic exposure of animals to esculetin may be quite significant. Recent reports of beneficial effects of esculetin in experimentally induced diseased state in animals (Kadakol et al., 2015; Kim and Lee, 2015; Prabakaran and Ashokkumar, 2012, 2013) may increase its dietary and pharmacologic use with possible adverse physiologic influence, warranting this study aimed at ascertaining the sub chronic esculetin (6,7-dihydroxy-coumarin)-induced response on malondialdehyde concentration and some haematological and lipid metabolism parameters in normal male Wistar rats. Agent-induced influence in animals has been reported (Egbuonu et al., 2010; Egbuonu and Ezeanyika, 2013; Egbuonu and Osakwe, 2011) even at a low dose (Egbuonu et al., 2009).
MATERIALS AND METHODS
Thirty male Wistar rats (10-12 weeks old and an average body weight of 114.8 g) were used in this study. The rats were purchased from the animal house of the Faculty of Biological Sciences, University of Nigeria, Nsukka, Nigeria.
Chemicals: Chemicals used in this research were products of reputable companies, including May and Baker, England; Merck, Germany; BDH Chemicals, UK and Sigma Chemical Compound, USA. Reagents used were commercial kits and are products of Randox Laboratories Ltd., UK. The effective study was conducted between June, 2007 and June, 2008.
Experimental design: Approval for this animal study was sought and obtained from the Ethical Committee, Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Nigeria. The rats were housed in separate cages, acclimatized for 7 days and then randomly assigned to five groups (A, B, C, D and E) of 6 rats each. The rats in group A, B and C were exposed to esculetin solution at a dose of 6, 12 and 24 mg kg–1 b.wt., respectively.
The rats in group D, the vehicle control were exposed to 0.2 mL of 10% dimethylsulfoxide (DMSO) whereas those in group E, the normal control were exposed to 0.2 mL of Distilled Water (DW). The administration was per oral for 28 consecutive days. All the animals were handled humanely and fed ad libitum with pelletised growers feed manufactured by Grand Cereals and Oil Mills Limited, Jos, Nigeria. The rats were scarificed on day 29 to obtain the samples (whole blood which shared to two and one part processed to obtain the serum).
Methods: Hemoglobin was determined by the method described by Cheesbrough (2000) based on the principle that haemoglobin converts to stable haemiglobincyanide (HiCN) following haemolysis of whole blood cells on dilution with modified Drabkin’s solution (potassium ferricyanide and potassium cyanide). The absorbance of the coloured HiCN solution was read with a spectrophotometer at 540 nm and compared with that of a reference HiCN standard solution. The intensity of the colour produced is directly proportional to the amount of haemoglobin present. The differential white blood cell count was determined by counting the different white cell types in a Leishman stain using a microscope as described in Cheesbrough (2000).
The Packed Cell Volume (PVC) was measured by the method of Cheesbrough (2000) as the proportion of whole blood occupied by red cells, expressed as a ratio while the WBC count was determined by diluting whole blood with a fluid that cause haemolysis of the erythrocytes but had no effect on leukocytes, which can then be counted microscopically using a haemocytometer as the number of white blood cells per liter of blood calculated (Cheesbrough, 2000).
The serum MDA concentration was determined by the method of Wallin et al. (1993) based on the principle as in Egbuonu and Ezeanyika (2012). The serum concentration of total cholesterol and triacylglyceride were determined with Randox enzyme kit based on the method of Trinder (1969) and on the principle of colorimetric estimation and quantification of quinoneimine formed after the enzymatic hydrolysis and oxidation of either cholesterol or triacylglyceride.
Statistical analysis: The data were analyzed by the Least Significant Difference (LSD) and significant difference in means accepted at p<0.05, using one way analysis of variance (ANOVA). The results were expressed as mean value±standard deviation of the measured variables in each group.
RESULTS
On comparison with the controls (D and E), the esculetin-induced alteration on the haematological parameters was not significant (p>0.05) but decreased numerically from the lowest to highest esculetin-exposed group viz: hemoglobin (99.83±5.04 g L–1-91±7.04 g L–1), packed cell volume (30±1.41%-27.33±2.16%), white blood cell count (9.63±0.91×109 L–1-7.95±0.58×109 L–1), neutrophil (1.93±0.51×109 L–1-1.72±0.34×109 L–1) and lymphocyte count (7.64±1.04×109 L–1-6.18±0.63×109 L–1) (Table 1).
The reduction in the serum concentration of cholesterol and triacylglyceride in the esculetin-dosed rats was significant (p<0.05) and followed the numerical decreasing trend in the concentration of cholesterol (3.39±0.23 mmol L–1-3.01±0.78 mmol L–1) and triacylglyceride (0.49±0.45 mmol L–1-0.15±0.05 mmol L–1). Lipid peroxidation products (MDA) concentration decreased significantly (p<0.05) but the numerical decreasing trend was reversed (1.90±0.53 mmol L–1-2.82±1.50 mmol L–1) (Table 2).
| Table 1: | Haematological indices in controls and esculetin-exposed rats |
| *Statistically significant difference (p<0.05) between the control and esculetin-exposed groups. Results were expressed as Mean±SD for n = 6 rats, WBC: White blood cells | |
| Table 2: | Serum malondialdehyde, triacylglyceride and cholesterol concentration in controls and esculetin-exposed rats |
| *Statistically significant difference (p<0.05) between the control and esculetin-exposed groups. Results were expressed as Mean±SD for n = 6 rats | |
DISCUSSION
Esculetin (6,7-dihydroxycoumarin) is coumarin derivative found in various plants, plant products and average western diet, hence may be inadvertently consumed on daily basis, warranting this study to further understand the underlying esculetin metabolism and effects. On comparison with the controls (D and E), the esculetin-induced alteration on the haematological parameters was not significant (p>0.05), implying negligible esculetin-induced influence on blood cells production, circulation and functions in the rats. The observed numerical decrease in the haematological indices from the lowest to the highest esculetin-exposed group could be a pointer to apparent esculetin-induced inverse response with varying dose in the rats.
The change in the WBCs in the rats exposed to esculetin was not significant (p>0.05), suggesting that esculetin may not have induced significant toxic effect that could trigger an immune response in the rats. The WBCs are important in immune response (Guilhermino et al., 1998) and are generally released in response to toxic effect in animals. The packed cell volume range in esculetin-treated rats (30.00±1.41%-27.33±2.16%) was not significant (p>0.05) but within the normal range (25%-45%) as reported by Oleforuh-Okoleh et al. (2015), though in broiler chicks. The reduction in the serum concentration of cholesterol and triacylglyceride in the esculetin-dosed rats was significant (p<0.05) and dose dependent, indicating significant up-regulation in the utilization or down-regulation in the synthetic pathways of cholesterol and triglyceride catabolism which could occur regardless of esculetin dose.
The hydroxycoumarins, including esculetin are typical phenolic compounds with metal chelating free radical scavenging potentials (Lin et al., 2000; Nishiyama et al., 2001; Kaneko et al., 2003). As expected, the lipid peroxidation products (MDA) concentration decreased significantly (p<0.05) and dose dependently in the esculetin-exposed groups compared to the controls. Similar observations were reported earlier by Lin et al. (2000), Martin-Aragon et al. (1998) and Gilani et al. (1998), suggesting prevailing healthy antioxidant status, perhaps due to the absence of significant esculetin-induced pathologies or dysfunctional states and/or potential antioxidant activities of esculetin in the rats. Antioxidant properties of esculetin have been related with its radical scavenging activity (Kostova, 2005), inhibition of tyrosine kinases (Masamoto et al., 2003) and enhancement of glutathione status (Martin-Aragon et al., 1998). The observed decrease in the serum MDA concentration in the esculetin-exposed rats appears to support the observation on the other parameters in this study and in earlier studies which reported by Egbuonu et al. (2012) and Egbuonu et al. (2015) suggesting the absence of significant esculetin-induced adverse influence in the rats.
The numerical decreasing trend observed in the other studied parameters was however reversed in the serum MDA concentration (1.90±0.53 mmol L–1-2.82±1.50 mmol L–1), which could imply novel underlying mechanism in esculetin metabolism and actions. The esculetin-induced decreasing response with increasing dose for some parameters but an increasing response with increasing dose for MDA as observed in this study may be apparently pointing to esculetin potential to selectively elicit potentiated effect, either at low or high concentration, that could diminish and possibly vanish with increasing or decreasing concentration, respectively. This is a novel observation that could be indicating interplay of multiple catabolic routes in esculetin metabolism, hence underlying complex mechanisms in the biochemical and therapeutic actions of esculetin, warranting further studies.
CONCLUSION
The sub-chronic esculetin-induced alterations were inversely related to esculetin dose and may not have resulted to significant dysfunctional states in the rats. The noted inverse dose related response in either decreasing or increasing direction could be pointing to significant underlying complex mechanisms in esculetin metabolism and actions, warranting further investigations.