ABSTRACT

This study was carried out to assess the 
antioxidant  status of rats fed
diet  incorporating catfish contaminated
with crude petroleum  oil treated
with  Monodora
myrstica extracts. Thirty albino rats of weight 180 to 200 g were used for
the experiment and they were divided into six 
groups of five rats each.  The
grouping were as follows, group  1:  control, group 2: rats were fed crude
petroleum oil contaminated catfish diet (CPO-CCD) only, group 3: CPO-CCD plus
tween 80, group 4, 5, and 6 were given CPO-CCD 
and treated with  M. myristica water extract  (MWE), M.
myristica ethanol extract  (MEE) and M. myristica diethyl ether  extract 
(MDEE). The experiment lasted four weeks. The results showed significant
(p<0.05) decreased in  blood reduced glutathione (GSH), blood oxidised glutathione (GSSG), superoxide dismutase (SOD), catalase (CAT) and increase  malondialdehyde (MDA) level in the liver, kidney and brain rats fed CPO-CCD only and CPO-CCD + tween 80 when compared to the control. Administration of MWE, MEE and MDEE to  the rats fed CPO-CCD significantly (p<0.05) increase the level of  blood GSH, blood  GSSG, SOD, CAT and decrease MDA level in the liver, kidney and brain  when compared with the CPO-CCD only and CPO-CCD + Tween 80.  No significant difference was observed in the  blood GSH:GSSG ratio and brain GSH level  in all the experimental groups. In conclusion, M. myristica  extracts exhibited  beneficial effect by improvement  of the antioxidant status and showed to evade the oxidative insult elicited by the CPO-CCD intoxication and  in the various tissues.  Key words: Petroleum, Diet,  Antioxidants Indices,  Monodora Myrstica INTRODUCTION Exposure to crude oil pollution  leads to formation of free radicals (Won et al., 2016). Free radical is the most common reactive oxygen species in human (Georgewill and Nwankwoala, 2008). Once polyaromantic hydrocarbons enter the body of a living organism, each are metabolized to form highly reactive molecules such as diol epoxides that are polyromantic hydrocarbons intermediate metabolites that causes oxidative stress (Ekperusi and  Aigbodion, 2015; Penning, 2014). Reduced glutathione is a multifunctional intracellular non-enzymatic antioxidant which is well known to be the major thiol-disulphide redox buffer of the cell (Swaran, 2009). Oxidized glutathione is accumulated inside the cells and the ratio of GSH/GSSG is a good measure of oxidative stress of an organisms. Superoxide dismutase is an enzyme that alternately catalyzes the dismutation of the superoxide radical into either ordinary molecular oxygen or hydrogen peroxide (Hayyan et al., 2016). Catalase is also an enzymes. The name catalase was given to the enzyme owing to its catalatic action on the hydrogen peroxide. Antioxidant enzymes can be inactivated by lipid peroxides (Alantary et al., 2014; Hamza and Al-Harbi, 2015). The subchronic exposure of rats  to crude oil, decreased tissues catalases activities (Nwaogu et al., 2011). Several  studies have shown that, direct exposure to crude oil can disrupt antioxidant status in serum, brain, liver, and kidney (Adedara  et al.,2012; Ebokaiwe and Farombi, 2016) Monodora myristica  is a spice commonly consumed in the Niger Delta part of Nigeria especially by the Itsekiri, Urhobo and Ndokwa  people of Delta State. The seeds of  M. myristica  have  attractive small,  possess antioxidant properties and can be used in pharmaceutical industries (Talalaji, 1999).  The present study aimed to assess the effect of M. myristica  extracts in rats given  diet  incorporating catfish  polluted with crude petroleum  oil by evaluating  some antioxidant  status such as  reduced and oxidised GSH, CAT, SOD and lipid peroxidation  level.    MATERIALS AND METHODS Preparation of the Spice (Monodora myristica) Extracts M. myristica was obtained  from Obiaruku  main market, Ukwuani Local Government Area (LGA), Delta State  and then later identified at the Department of Botany, Delta State  University, Abraka, Delta State. The spice was briefly sun-dried to constant weight for two weeks and then crushed into fine particles using blender for at high speed. One hundred grams of the powdered spice was extracted with 500 ml of the respective solvent (hot water (60?C), ethanol (95 % v/v), and diethyl ether, 95 % v/v) and allowed to stand for 48 hrs. The mixture was then filtered using a clean muslin cloth. The  filtrate was evaporated to dryness using rotary evaporator attached to a vacuum pump.  The extracts were stored in refrigerator (- 4 oC) until required. Crude petroleum oil pollution and diet preparation  The crude petroleum oil (CPO) was got from the Nigerian National Petroleum Cooperation (NNPC), refinery, Warri Delta State, Nigeria. Fifty  catfish (with length between 20-25 cm  and weight between  250-300 g) was got  from commercial farm, then acclimatized for 7 days  for  the experiment. The catfish was divided into two groups; group 1 : control : contains twenty-five catfish which was cultured in plastic aquaria with 30 L borehole water for four weeks. Group 2:  also contain twenty-five which was cultured in plastic aquaria with 30 L borehole water and then  polluted with crude petroleum oil, 823.3 µl/L as described by Ikeogu et al. (2013) for four weeks.   At the end of the experimental period, the catfish was harvested and the used in the preparation of  diet for the experimental rats  following the method described by Sunmonu  and Oloyede (2007).  The  catfish  were oven dried at 40°C and  used as a source of  protein. The diet for each group were  prepared by mixing known quantities of sources of each food class comprising: protein (25 %), corn  starch (52 %), groundnut oil (4 %), maize  cob (4 %), granulated  refined sugar (10 %) and vitamin/mineral mixture (5 %). The food components  were  mixed together and then made into pellets which was  feed rats. Experimental Rats Thirty male albino Wistar rats were  used for the study. The rats were allowed to acclimatized for two weeks to suite the  laboratory condition. They  had free access to water and standard growers mash diet. The rats  used for the study were in accordance to the  guide for care and use of laboratory animals (NIH, 1985). They were divided into six  groups of  five  rats; group 1:  control, group 2: CPO-CCD only, group 3:  CPO-CCD plus  1 ml/kg b. wt. of 1 % tween 80, group 4: CPO-CCD plus 200 mg/kg b. wt. of MWE, Group 5:  CPO-CCD plus 200 mg/kg b. wt. of MEE and group 6: CPO-CCD plus 200 mg/kg b. wt. of MDEE. Rats in group 1 to 6 received tap water daily throughout the experiment. The  administration of the CPO-CCD and extracts orally was allowed  for four weeks.  Blood Collection and Preparation of Tissue Homogenate The rats were  sacrified after 24 hours fast on the last day. The blood was collected by cardiac puncture using hypodermic syringe and needle and then transferred to an anticoagulant tube and organs were harvested. One gram of various tissue (liver, kidney and brain) were  homogenized in 10 ml of normal saline and then centrifuged at 2,500 revolution per minutes for 15 minutes to obtained the supernatant  which was immediately used for biochemical analysis.   BIOCHEMICAL ANALYSIS Estimation of blood GSH/GSSG (Reduced/Oxidized glutathione) ratio. Blood GSH/GSSG  was  estimated using the enzymatic method described by Tietze (1976). GSSG Sample preparation Thirty microliters (30 ?L) of thiol-scavenging reagent (50 mg 1- methyl pyridinium trifluoromethane sulphonate)  to a micro-centrifuge tube and 100 ?L of whole blood was carefully added to the  bottom of the centrifuge tube and mixed gently. GSSG sample 130 ?L was incubated at room temperature for 5-10 minutes. Thereafter 270 ?L of ice-cold 5 % MPA was added  to the tube  and centrifuged at 1000 x g and 4°C for 10 minutes. The supernatant (50 ?L) was added to 700 ?L of assay buffer (phosphate buffer, 2M, pH 8) in a new micro-centrifuge tube, this was placed the  on ice until used.  GSH Sample preparation Fifty microliter (50 ?L) of whole blood  was added to the bottom of a micro-centrifuge tube and  then mixed. Thereafter 350 ?L of ice-cold 5% MPA  was added to the micro-centrifuge tubes and centrifuged at 1000 x g  in  4°C for 10 minutes. Then, 25 ?L of the supernatant was added to 1.5 mL of assay buffer in a new micro-centrifuge tubes and this was placed on ice until required. Procedure Two hundred microliters (200 ?L)  of  samples  and blank were  added to 200 ?L of the DTNB solution in  respective test-tubes, then  200 ?L of the reductase solution (recombinant glutathione reductase)  was added immediately then mixed. These were allowed to  incubate at room temperature for 5 minutes, then 200 ?L of  2 mg/mL NADPH solution  was added  to the test tubes.  The absorbance were read and recorded at 412 nm. Standard curve After preparing 1mM stock solution of  GSH /GSSG. Different concentrations  were prepared and from each of the GSH/GSSG dilution, 200?l was taken and added into 2300 ?l of 0.2 M phosphate buffer pH 7.6 then 500?l of 1mM DTNB was added, these five mixtures were well shaken and incubated for five minutes. After the  incubation  period, absorbance of each mixture was recorded at  412 nm. 5,5-dithiobis-2-nitrobenzoic acid (DTNB) blank was prepared by adding 500 ?l  DTNB to 2500 ?l phosphate buffer pH 7.6. Absorbance of DTNB was also taken at 412 nm. The real absorbance of each mixture was obtained by subtracting  absorbance of DTBN blank from absorbance of each of the mixture. The concentration of GSSG is much lower in the reaction mixture compared to GSH,standard calibration  curve was  plotted separately,  0, 0.50, 0.75, 1.0, and 1.50 ?M  GSSG,  and 0, 1.0,1.5, 2.0, and 3.0 ?M GSH Concentration of GSH/GSSG ratio (units/ml)  =   GSH-2GSSG     GSSG   Estimation of tissue  reduced glutathione The  reduced glutathione  concentration in the  liver, kidney and brain were  estimated using the method of Ellman (1959). Procedure: To 0.5 ml of tissue homogenate was added 2 ml 10% trichloroacetic acid and centrifuged. One milliliters (1ml) of supernatant was treated with 0.5 ml of Ellman's reagent and 3 ml of phosphate buffer. The colour developed was read at 412 nm. A series of standard were treated in similar manner along with a blank containing 3.5 ml of buffer. Determination of superoxide dismutase activity The activity of  SOD  in the  liver, kidney and brain were assayed using the method of Misra and Fridovich (1972). Procedure: The assay was carried out by adding 0.2 ml of the supernatant to 2.5 ml of 0.05 M carbonate buffer, pH 10.2. The reaction was started by addition of 0.3 ml freshly prepared epinephrine as the substrate to the buffer supernatant mixture and was quickly mixed by inversion. The reference cuvette contained 2.5 ml of the buffer; 0.3 ml of the substrate and 0.2 ml of distilled water. The increase in absorbance at 480 nm due to the adrenochrome formed was monitored every 30 seconds for 120 seconds. Determination of Catalase Activity The method of Kaplan et al. (1972) was adopted for the assay of liver, kidney and brain catalase activity. Procedure: Two milliliter (2 ml) of H2O2 was added to 1ml of sample  in the reaction cuvette. Absorbance was read at 360 nm for 70 seconds. The reference cuvette contained 2 ml H2O2 and 1ml of water. The disappearance of hydrogen peroxide was calculated using the Molar extinction  co- efficient, ? = 39.4 M-1 cm-1. Determination of Lipid Peroxidation Lipid peroxidation in form of malondialdehyde (MDA) were determined in the  liver, kidney and brain by using the method of Buege and Aust (1978). Procedure: One millilitre of the sample  was added to 2 ml of  TCA-TBA-HCL reagent 0.37% Thioarbituric acid (TBA), 15% Tricarcoxylic acid (TCA) and 0.24 N Hydrochloric acid (HCl) (1:1:1 ratio). The tube was stoppered  loosely and immersed in boiling water for 15 minutes  and swirled slightly at intervals. The mixture was cooled and centrifuged for 10 minutes at 5000 g. The absorbance was read at 532 nm using the reagent blank. Lipid peroxidation in units/g of wet tissue was calculated  with a molar extinction co-efficient of 1.56 x 105M-1 STATISTICAL ANALYSIS The  data obtained  and results were expressed as mean ±SD. The significant differences between groups were analyzed using  one way analysis of variance (ANOVA) and least significant difference  (LSD). The SPSS-PC programme package (version 17.0) were used for statistical analysis. A significant threshold of p< 0.05 was regarded statistically significance between the test and control group for the analysis.   RESULTS   Table 1: Blood GSH, GSSG and GSH:GSSG ratio  of rats fed CPO-CCD   treated with extracts of M. myristica Groups Blood     GSH (units/ml) Blood             GSSG (units/ml) Blood GSH:GSSG ratio 1: Control  1.81±0.04 a 0.72±0.03 a 0.99±0.02 a 2: CPO-CCD only  0.42±0.02 b 0.11±0.07 b 0.80±0.05 a 3: CPO-CCD + Tween 80 0.42±0.06 b 0.12±0.05 b 0.83±0.04 a 4: CPO-CCD + MWE 1.09±0.05 a 0.53±0.01 a 1.00±0.01 a 5: CPO-CCD + MEE 1.41±0.32 a 0.56±0.01 a 1.01±0.02 a 6: CPO-CCD + MDEE 1.63±0.10 a 0.61±0.02 a 1.00±0.01 a Values are given in mean ± SD. n=5. Mean values with different superscript letter in the same column differ significantly at p<0.05.   Table 2:  GSH level in the liver,  kidney and brain  of rats fed CPO-CCD   treated with extracts of M. myristica   GSH  (units/g wet tissue) Groups Liver Kidney Brain 1: Control  7.19±0.99 a 6.54±0.05 a 3.33±0.47 a 2: CPO-CCD only  3.36±0.94 b 2.24±0.79 b 1.01±0.05 a 3: CPO-CCD + Tween 80 2.34±0.73 b 1.76±0.57 b 2.81±0.08 a 4: CPO-CCD + MWE 3.46±0.54 b 3.31±0.66 b 2.36±0.16 a 5: CPO-CCD + MEE 3.96±0.09 b 4.29±0.19 b 2.20±0.16 a 6: CPO-CCD + MDEE 6.25±0.58 a 5.31±0.29 a, b 2.64±0.08 a Values are given in mean ± SD. n=5. Mean values with different superscript letter in the same column differ significantly at p<0.05.   Table 3:  Changes in superoxide dismutase activity in the liver, kidney and brain of rats fed CPO-CCD   treated with different extracts of M. myristica   SOD (units/g wet tissue) Groups Liver Kidney Brain 1: Control  89.41±19.16 a 86.41±14.28 a 73.50±6.52 a 2: CPO-CCD only  57.36±6.12 b 55.07±8.00 b 40.28±3.15 b 3: CPO-CCD + Tween 80 56.07±14.40 b 54.46±3.03 b 40.90±2.67 b 4: CPO-CCD + MWE 66.43±6.98 c 65.29±6.29 c 50.48±4.33 c 5: CPO-CCD + MEE 78.11±6.77 d 74.50±3.38 d 67.32±2.90 d 6: CPO-CCD + MDEE 85.47±3.58 a 82.41±1.42 a 70.23±5.21 a Values are given in mean ± SD. n=5. Mean values with different superscript letter in the same column differ significantly at p<0.05.   Table 4:  Changes in catalase activity in the liver, kidney and brain of rats fed CPO-CCD   treated with extracts of M. myristica.   CAT  (units/g wet tissue) Groups Liver Kidney Brain 1: Control  74.28±10.19 a 70.18±8.65 a 64.26±5.94 a 2: CPO-CCD only  46.53±12.62 b 40.31±7.62 b 35.71±3.92 b 3: CPO-CCD + Tween 80 46.16±512 b 41.15±2.95 b 36.27±4.37 b 4: CPO-CCD + MWE 55.31±10.19 c 52.28±3.47 c 45.49±3.64 c 5: CPO-CCD + MEE 61.47±11.53 d 60.74±5.42 d 50.39±5.99 d 6: CPO-CCD + MDEE 72.20±5.65 a 68.32±3.57 a 61.17±2.38 a Values are given in mean ± SD. n=5. Mean values with different superscript letter in the same column differ significantly at p<0.05.   Figure 1:  MDA  level in the liver,  kidney and brain  of rats fed CPO-CCD treated with different extracts of M. myristica. Bars represent mean values from five rats in each group. For each organs, bars with different superscript letter in the same column differ significantly at p<0.05.   RESULT AND DISCUSSION   Alterations of blood GSH, GSSG and GSH:GSSG ratio of rats fed CPO-CCD   treated with different extracts of M. myristica are shown in Table 1. Significant (p<0.05) decrease level of blood GSH and GSSG  were observed in rats fed CPO-CCD only and CPO-CCD + tween 80 when compare with control. Treatment of rats fed CPO-CCD with MWE, MEE and MDEE CPO-CCD significantly (p<0.05) increase the level of blood GSH and GSSG.  The reduction in  GSH and GSSG levels  could be a compensatory mechanism by which the rats fed the formulated feed mixed with crude oil contaminated catfish to overcome the effect of the oxidant stress caused by free radicals produced by crude petroleum oil. The is in line with the findings of Shang et al. (2016), indicating that the decreased GSH to GSSG ratio in the blood showed  that oxidative stress occurred in the distant organs and systemically upon crude petroleum induced nephrotoxicity. No significant difference were observed in the blood GSH, GSSG and GSH : GSSG ratio levels when rats fed CPO-CCD only were compared rats  with fed CPO-CCD + tween 80, these results may be considered as  pathological evidences to confirm the nontoxic effect  of tween 80 as previously reported (Rowe, 2009).   The level of GSH, SOD and CAT activity in the liver, kidney and brain of rats fed CPO-CCD treated with extracts of M. myristica are shown in Table 2, 3 and 4 respectively. Rats fed with CPO-CCD only  and CPO-CCD + Tween 80  showed significant (p<0.05) decreased in SOD and CAT activity  in the liver, kidney and brain when compared to the control. Administration of MWE, MEE and MDEE to CPO-CCD rats  significantly (p<0.05) increased level of SOD and CAT in the liver, kidney and brain when compared with the CPO-CCD only and CPO-CCD + Tween 80 respectively.  No significant difference was seen in GSH level in the brain of all the experimental groups.  The decrease kidney and liver GSH level in the rats fed CPO-CCD may be due to the decrease in the activity of the hepatic glutamate-cysteine ligase (a key enzyme responsible for glutathione synthesis). The depletion in brain GSH in CPO-CCD induced oxidative stress may leads to increased productions of superoxide, hydroxyl radicals, and H2O2, because there is no known enzymatic defense against hydroxyl radicals (Dringen, 2000), making GSH the only compound capable of scavenging these radicals in the brain.   These findings are in line with the study of Aoyama et al. (2008) which states that, when comparing the brain with other organs, the brain is especially vulnerable to oxidative stress. This is because it has lower SOD, CAT, and glutathione peroxidase; GPx activities, while it contains an abundance of lipids with unsaturated fatty acids that are targets of lipid peroxidation (Dringen, 2000). Furthermore, the brain GSH concentration is lower than those of the liver and kidney (Aoyama et al., 2008). The detoxification mechanisms promoted by enhanced glutathione production indicates the protective effects of MDEE, MEE and MWE. This also might be the reason for the restoration of other antioxidant enzymes (SOD and CAT).    The marked reduction in SOD activity of rats fed CPO-CCD  and the enhanced SOD activity  when M. myristica extracts was administered were in agreement with other studies (Nwaogu et al., 2011; Sunmonu and Oloyede, 2007). The inhibition of CAT activity during CPO-CCD induced toxicity  may be due to the increased generation of reactive free radicals, which can lead to oxidative stress in the cells. The administration of MDEE, MEE and MWE inversed the catalase activity in the liver, kidney and brain tissues and thus enhance the antioxidant defense against  ROS. This findings are  in collaboration with Oyinloye et al.(2016) who reported that M. myristica aqueous extract prevent lipid peroxidation and replenish hepatic antioxidant enzymes against cadmium induced  liver tissue damage.   Figure 1, showed the level of MDA; malondialdehyde (end product of membrane lipid peroxidation) in the  tissues (liver, kidney and brain)of rats fed CPO-CCD treated with extracts of M. myristica. MDA level in  the respective tissues were significantly (p<0.05) increased in rats fed  CPO-CCD only and CPO-CCD + tween 80 when compared with the control rats.   However, treatment of rats fed CPO-CCD with the different extracts significantly decreased the level of MDA as compare with that of the control in the respective tissues. The excessive ROS generated during crude  petroleum  oil toxicity rapidly react with lipid membranes and thus initiates the lipid peroxidation chain reaction, resulting in lipid peroxyl radicals' formation (Ita  and Edagha, 2016; Ujowundu et al., 2012; Nwaogu et al., 2011). The elevation of lipid peroxidation caused by rats administered crude petroleum oil has been previously reported (Sunmonu and Oloyede, 2007), which is in line with the results obtained in this study.  In the present study, the lower MDA levels  in  the  tissues of rats fed CPO-CCD plus M. myristica  extracts, apparently indicating the anti-oxidative protective role of  M. myristica  extracts against CPO-CCD induced damage on cell membranes. Moreover, MDEE revealed a strong inhibitory ability towards lipid peroxidationas compared with MEE and MWE.    CONCLUSION The results of this study showed that M. myristica  extracts  rescued the CPO-CCD induced tissues damage/lipid peroxidation and improvement of  antioxidant status owing to its free radical scavenging properties.

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