ORYCTES RHINOCEROS LARVA OIL SUPPLEMENTATION IMPROVES TISSUE ANTIOXIDANT STATUS IN CHOLESTEROL-FED RATS

Experimental evidence from previous study has demonstrated the hypolipidemic effects of Oryctes rhinoceros oil (ORO) when fed as a supplement to a cholesterol-based diet. Due to renew interest in the consumption of insect derived oil, the present study was designed to elucidate the effect of Oryctes rhinoceros oil (ORO) supplementation in comparison to vitamin E on oxidative status in some tissues of rats fed a cholesterol-based diet. Forty (40) Swiss albino rats were divided into 4 groups (n = 10) and maintained on a basal diet (cholesterol free as control), a cholesterol-based diet (5% cholesterol as cholesterol), a cholesterol-based diet supplemented with ORO (cholesterol + ORO) and a cholesterol-based diet supplemented with vitamin E (Cholesterol + vit E) for 10 weeks. Animals in the cholesterol group had a significantly (p <0.05) higher malondialdehyde (MDA), conjugated diene and nitric oxide concentrations in the serum, liver, heart, kidney and lung compared to control, cholesterol + ORO and cholesterol + vit E groups. Tissue glutathione (GSH) concentration was significantly (p <0.05) higher in rats fed cholesterol-based diet supplemented with ORO and vitamin E compared to those fed cholesterol-based diet alone. Xanthine oxidase activity was significantly (p <0.05) reduced in tissues of rats fed ORO and vitamin E supplemented diets compared to cholesterol rat group. In addition, catalase and superoxide dismutase activities in the various tissues examined were significantly (p <0.05) higher in both ORO and vitamin E supplemented groups compared to the cholesterol group. No significant difference was observed between animals fed ORO and vitamin E supplemented diets. These results showed that Oryctes rhinoceros larva oil exhibited similar protective effects to vitamin E against diet-induced oxidative stress in rats. In addition, data from this study showed that Oryctes rhinoceros larva oil possessed antioxidant property. Overall, the potential nutritional benefit of Oryctes rhincoceros larva oil consumption on cardiovascular health could possibly involve its ability to upregulation of cellular antioxidant defense mechanisms.


INTRODUCTION
Palm oil remains the most traded vegetable oil globally as it constitutes over 50% packaged products in supermarkets. However, in order to sustain the increase demand for palm oil or palm oil-based products several hectares of land are cleared yearly for palm plantations. The world population is estimated to exceed 9 billion by 2050 (Teoh, 2010). This no doubt portends a serious challenge in terms of food security and environmental issues. Specifically, an astronomical increase in the demand for palm oil or palm-based product is expected. Thus, there is an urgent need to evaluate other nutrient sources for feed production. The use of edible insects for feed is widely viewed as a healthier and more sustainable potential solutions to overcoming the challenge of food security (Van Huis, 2013). Insects are rich sources of valuable nutrients such proteins, fat, vitamins, minerals and energy. Compared to oil palm trees insects have better yield per hectare due to their high rate of proliferation and short life cycle, requires less space and can be reared on agro-waste stream.
Thus far, research on the utilization of edible insects for animal feed production has focused on their protein content. Nevertheless, insects contain between 5% to 40% (% dry matter, DM) oil with a better fatty acid profile compared to palm oil (Womeni et al., 2009). In Europe, the use of animal-based protein with the exception of fish for animal feed production is restricted. However, there exist no prohibition on the use insect oil. Research on the use of insect oil in animal feeding trial is scanty. A recent study by Belghit et al. (2019) demonstrated that liver triacylglycerol level was reduced in freshwater Atlantic salmon fed insect meal and insect oil compared fish fed the control diet. Similarly, Oluba et al. (2008a) reported that the supplementation of O. rhinoceros oil with a cholesterol-based diet in rats resulted in improvement in serum lipid profile and reduced susceptibility to atherosclerosis. No. 1/2019 The highly lipid soluble radical, nitric oxide (NO -) diffuses readily through cellular membranes, interacting with other radicals including superoxide, peroxide etc thus potentiating their actions (Pacher, Beckman and Liaudet, 2007;Birben et al., 2012). Nitric oxide radical is also capable of simultaneously reacting with superoxide radical to form peroxinitrite radical (Pacher, Beckman and Liaudet, 2007). Peroxinitrite radical is a very reactive 1thiolesters group of cysteine and methionine residues in peptides and proteins are potential oxidizing targets for peroxinitrite. Unregulated generation of free radicals in the body has been implicated in the pathogenesis of tissue damage in several diseased conditions such as ageing, diabetes, cardiovascular diseases, etc (Uttara et al., 2009;Valko et al., 2007). Dietary fats in the form of cholesterol are considered to be important in the initiation of free radical production in these clinical disorders (Nevin and Rajamohan, 2006). These free radicals subsequently attack and breakdown membrane phospholipids and thus trigger lipid peroxidation (Sevanian and Hochstein, 1985; Thomas et al., 1990 The consumption of Oryctes rhinoceros (palm beetle) larva as a delicacy is a common practice in Nigeria especially in the Southern part of the country where palms are cultivated on a commercial scale. O. rhinoceros larvae feeds on decaying organic matter (palm logs, manure and rubbish dumps). These larvae are either eaten raw, boiled, fried or roasted and are sometimes used as meat substitute in the preparation of stews and soups. Reports on the proximate composition of O. rhinoceros larva from Nigeria by several investigators have shown that it contains as much as 38% oil (by dry weight). with high level (65%) of unsaturated fatty acids. The oil being majorly composed of unsaturated fatty acids has been shown to be hypercholestrolemic in action (Oluba, Josiah and Fagbohunka, 2014). Presently, insect oil is gaining prominence in the scientific field as well as increased acceptability among consumers. Hence, there is urgent need to intensify scientific efforts on the possible nutritional and health benefits of insect oil. Therefore, this study was carried out to investigate the effects of Oryctes rhinoceros oil (ORO) supplementation in comparison to vitamin E on tissue oxidative status in rats fed a cholesterol-based diet.

Scientific hypothesis
Oryctes rhinoceros oil protect rat tissue against lipid peroxidation.

MATERIAL AND METHODOLOGY Insect material
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 extraction
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.

Animal care and ethical consideration
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.

Experimental diet
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. No. 1/2019

Biochemical analyses
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.

Statistic analysis
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.

RESULTS AND DISCUSSION Food intake and body weight
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 peroxidation parameters
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).

Antioxidant parameters
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 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,  Note: ORO, Oryctes rhinoceros larva oil. a Mineral premix (AIN-76) composed of Ca as CaSO4 (5.2 g.kg -1 ), Ka as KCl (3.8 g.kg -1 ), Na as NaCl (1.1 g.kg -1 ), Mg as MgSO4 (0.5 g.kg -1 ), Fe as FeSO4 (34.25 mg.kg -1 ), Zn as ZnSO4 (36.75 mg.kg -1 ), Mn as MnSO4 (59.34 mg.kg -1 ), Cu as CuSO4 (6.73 mg.kg -1 ), Co as CoCl2 (0.03 mg.kg -1 ), and I as KI (0.21 mg.kg -1 ). b Vitamin premix (AIN-76) composed of vitamin A (4.0 IU.g -1 ), vitamin D3 (1.0 IU.g -1 ), α-tocopherol (64.24 IU.kg -1 ), thiamine (5.90 mg.kg -1 ), riboflavin (6.29 mg.kg -1 ), niacin (30.15 mg.kg -1 ), pantothenic acid (15.26 mg.kg -1 ), choline (1040.0 mg.kg -1 ), pyridoxine (7.12 mg.kg -1 ), folic acid (2.10 mg.kg -1 ), biotin (0.21 mg.kg -1 ), vitamin B12 (10.10 mg.kg -1 ), and vitamin K (0.50 mg.kg -1 ). 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.

CONCLUSION
In conclusion, this study which was carried out to evaluate the effect of Oryctes rhinoceros larva oil in comparison with vitamin E on tissue lipid peroxidation and antioxidant defense systems in rats fed a cholesterol-based diet showed that Oryctes rhinoceros larva oil supplementation decreased tissue lipid peroxidation and increased the antioxidant defense molecules in rats. These results showed that consumption of Oryctes rhinoceros larva oil extracted from Oryctes rhinoceros larva, exhibited similar protective effects to vitamin E against diet induced oxidative stress in rats. Overall, the potential nutritional benefit of Oryctes rhinoceros larva oil on cardiovascular health could possibly involve its ability to upregulation of cellular antioxidant defense mechanisms.