Oryctes rhinoceros oil protect rat tissue against lipid peroxidation.

Oryctes rhinoceros oil possesses antioxidant activity.

Live Oryctes rhinoceros larvae were collected from decaying palm trees at Igoba village near Akure (Nigeria). The larvae were transported to the laboratory in an open plate within 2 h of collection. They were authenticated and identified at the Department of by a zoologist at the Department of Biology, University of Benin (Nigeria). The larvae were rinsed with distilled water before being anaesthetized by freezing. The frozen larvae were thaw at 37 °C and oven dried at 50 °C for 72 h. The dried larvae sample was powdered using a mechanical grinder and the powdered sample kept in an air-tight container at 4 °C for further analysis.

Oil from the powdered larvae sample (100 g) was extracted using Soxhlet apparatus using hexane as solvent. The extracted oil was dried and stored in a dried dark airtight container and refrigerated until required for further analysis.

This study was approved by the Animal Ethics Committee of the Department of Chemical Sciences, Joseph Ayo Babalola University, Ikeji-Arakeji, Nigeria and was conducted in compliance to NIH guidelines for the care and use of laboratory animals (ILAR, 1985). Forty (40) male Swiss albino rats (weighing between 50.5 – 55.1 g) aged 6 weeks were purchased from the Department of Biochemistry, University of Ibadan (Nigeria) and used for the study. The animals were housed in wooden cages with raised wire-gauze floors at a temperature of 25.7 ±2.3 °C and a relative humidity of 45% – 60%, with 12 h light/dark cycles.

Diet formulation and preparation followed the prescription of the American Institute of Nutrition. The formulated diets were designated: control, cholesterol, cholesterol + ORO and cholesterol + vit E. The composition of the respective diet is as shown in Table 1. Prior to the feeding experiment animals were conditioned to the laboratory environment for a period of 2 weeks. All through the 10 weeks feeding trial, animals in each group were given food and water ad libitum. Food intake and body weight were recorded daily. At the end of the 10-week feeding experiment, the animals were fasted overnight, weighed and euthanized with chloroform and sacrificed by cervical dislocation. The blood from the rats was rapidly collected by direct heart puncture into plain sample bottles, and the serum was prepared and stored at -4 °C until it was required for analysis. The liver, heart, kidneys and lung were quickly excised, washed with icecold phosphate buffered saline, freed of fat, weighed and stored separately at -4 °C until required for further analysis.

Weighed portions of liver, heart, kidney and lung were separately minced with scissors and homogenized in solution (1:2 w/v) containing 0.15 M KCl and 3 mM EDTA, pH 7.4 in ice. The homogenates were diluted 4-folds and centrifuged at 10,000 x g at 4 °C for 15 min. The supernatant was decanted and used for the various analyses. Lipid peroxidation was determined by the method described by Buege and Aust (1978) while conjugated diene level was determined spectrophotometrically following the method of Recknagel and Glende Jr. (1984). Nitric oxide (NO) concentration was estimated in terms of its stable metabolic product, nitrite, using Griess reaction as described by Kang, Bansal and Mehta (1998). Xanthine oxidase (XOD) was determined according to the method described by Litwack et al. (1953). Reduced glutathione (GSH) level was estimated according to the method of Moron, Depierre and Mannervik (1979). Catalase (CAT) activity was assayed according to the methods described by Aebi (1984). Superoxide dismutase (SOD) activity was determined following the method of McCord and Fridovich (1969). The method of Gornall, Bardawill and David (1949) was adopted in protein determination.

Results are mean ± SEM of 10 determinations. Statistical comparison of means was by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using SPSS version 20. p <0.05 was considered significant.

Rats fed cholesterol-based diet consumed significantly (p <0.05) higher amount of food compared to control rats fed normal rat diet (Table 2). Mean weekly body weight gain was significantly (p <0.05) in rats fed cholesterolbased diet only compared to control and those fed cholesterol-based diet supplemented with ORO and vit E. No observable significant (p >0.05) difference was seen in rats fed ORO supplemented diet and vit E supplemented diet in terms of their mean weekly body weight gain (Table 2).

Lipid peroxide concentration as determined by tissue malondialdehyde concentration was significantly (p <0.05) elevated following feeding with cholesterol. However, when rats were fed cholesterol + ORO and cholesterol + Vit E diets, serum, liver, heart, kidney and lung MDA concentrations were significantly (p <0.05) reduced compared to its level in rats fed cholesterol diet only. Serum, heart, kidney and lung MDA concentrations in cholesterol + ORO was not significantly (p >0.05) different from that in cholesterol + vit E fed rats (Figure 1a). Conjugated dienes concentration in the serum, liver, heart, kidney and lung in rats fed cholesterol diet was significantly (p <0.05) higher compared to rats fed cholesterol + ORO and cholesterol + vitamin E diets (Figure 1b). Nitric oxide (NO) level in the serum and lung of rats fed cholesterol diet supplemented with ORO and vitamin E was significantly (p <0.05) lower compared to the observed level in rats fed cholesterol diet alone (Figure 1c).

Xanthine oxidase activity was significantly (p <0.05) lower in rats fed ORO and vitamin E supplemented diets compared to those fed cholesterol diet without supplementation (Figure 2a). Reduced glutathione (GSH) concentration reduced significantly (p <0.05) in tissues of rats fed cholesterol diet compared to rats fed normal diet. However, in rats fed cholesterol diet supplemented with Oryctes rhinoceros oil (Cholesterol + ORO) and vitamin E (Cholesterol + Vit E) significant improvement was observed in serum, liver, heart, kidney and lung GSH concentrations compared rats fed cholesterol diet only (Figure 2b). Catalase activity was observed to be significantly (p <0.05) lower in serum, liver, heart, kidney and lung of rats fed cholesterol diet compared to rats fed normal diet. On the other hand, in rats fed cholesterol diet supplemented with Oryctes rhinoceros oil (Cholesterol + ORO) and vitamin E (Cholesterol + Vit E) CAT activity was significant (p <0.05) higher in serum, liver, heart, kidney and lung compared rats fed cholesterol diet only (Figure 2c). Tissues (serum, liver, heart, kidney and lung) SOD activities were significantly (p <0.05) lower in rats fed cholesterol diet compared to rats fed normal diet. However, in rats fed cholesterol diet supplemented with Oryctes rhinoceros oil (Cholesterol + ORO) and vitamin E (Cholesterol + Vit E) SOD activity was significant higher in these tissues compared rats fed cholesterol diet only (Figure 2d).

Cholesterol-based diets have been implicated to be particularly damaging to the vascular membrane. Thus, hypercholesterolemia and related cardiovascular syndrome are predominant in regions of the world, where fat-rich foods constitute the bulk of their daily meals. A recent report from a study by Oluba, Josiah and Fagbohunka (2014) showed that consumption of a cholesterol-based diet led to imbalance in serum lipid profile and release of proinflammatory cytokines. The inflammation resulting from tissue damage that most often accompany high fat consumption is viewed as a consequence of oxidative stress.

In the present study, inclusion of ORO as a supplement to a cholesterol-based diet led to normalization of body weight. Rats fed cholesterol-based diet tend to be overweight which could have been a consequence of increased fat deposit in the body. The observed higher relative organ (liver, heart, kidney and lung) weight further gave credence to this assumption. The development of fatty liver has been reported in high-fat fed rats.

The hyperlipidemia that develops following the consumption of a high-fat diet has been shown to substantially contributes to the depletion of both nonenzymic (GSH) and enzymic (SOD, catalase and GPx) (Oluba et al., 2008b; Ojieh et al., 2009; Oluba et al., 2011). Findings from this study showed that increased dietary consumption of cholesterol could create of a prooxidant state and a depletion of cellular redox status. This observation agrees concur with the report of Oluba et al. (2008b) which showed a significant increase in plasma MDA concentration with a concomitant decrease in cellular antioxidant level in rats fed a high cholesterol diet.

The increased tissue MDA, conjugated dienes and nitric oxide concentrations in animals fed cholesterol-based diet alone in this study could be attributed to augmentation in the rate of cellular lipid peroxidation due to high fat (cholesterol) in the diet. This assertion is further reimbursed by the fact that xanthine oxidase activity was higher in rats fed cholesterol diet without supplementation compared to rats fed ORO and vitamin E supplemented diets. Xanthine oxidase has been demonstrated to be a primary source of superoxide radical (Gomez-Cabrera et al., 2005). MDA and conjugated dienes are established markers of lipid peroxidation while nitric oxide through its free radical activity is capable of interacting with cellular components such proteins, DNA, and lipids within the cell in a process known as nitrosative stress leading to cytotoxicity (Chang, Liao and Kuo, 2001). Thus, the reduction in tissue MDA, conjugated diene and nitric oxide levels in rats fed ORO and vitamin E supplemented diets in this study is beneficial and could give an indication of an anti-oxidative potential of ORO. The antioxidant activity of vitamin E is well established in literature (Niki et al., 1985; Traber and Atkinson, 2007). In this study, ORO demonstrated a similar effect on tissue MDA, conjugated diene and nitric oxide concentrations to vitamin E showing that its potential antioxidant effect could be comparable to that of vitamin E.

An important mechanism of action of oxidative stress is via enhanced generation of ROS and/or RNS, which most often form conjugates or adducts with cellular components such as DNA, membrane lipids, proteins and carbohydrates. Thus, the increased serum and lung xanthine oxidase activities observed in rats fed cholesterol diet without supplementation could provide a justification that the pro-oxidant effect of dietary cholesterol could involve its effect on xanthine oxidase activity. In the coronary artery of hypercholesterolemic individuals, NADPH oxidases and xanthine oxidase have been reported to be the major sources of superoxide radicals. However, these reactive oxygen species in the cells are neutralized by cellular antioxidant defense system including GSH, CAT, and SOD. Thus, oxidative stress is the resultant effect of a disequilibrium between cellular oxidants versus antioxidants (Oluba, 2019). Several studies have shown that alteration of antioxidant enzyme activities in different kinds of stress was associated with a depletion of GSH, CAT and SOD and an increase of lipid peroxidation, all of which can lead to oxidative stress and finally cell death (Bouayed and Bohn, 2010). Overexpression of the peroxisomal enzyme, catalase which catalyzes the reduction of hydrogen peroxide to water and molecular oxygen have been shown to reduce atherosclerosis in highfat fed rats (Yang et al., 2009). Results from the present study showed that ORO could act to normalize the attendant depletion of cellular antioxidant molecules when fed as a supplement to a cholesterol-based diet.