Humans and animals are constantly exposed to
crude petroleum contaminated diets in petroleum producing areas of the world.
As a result, researches are on-going to find simple ameliorative agent against
crude petroleum contaminated diet toxicity. The aim of this study was to
evaluate the protective effect Monodora
myristica on some biochemical parameters of rats fed with crude petroleum
oil contaminated catfish (Clarias
gariepinus) meal. Thirty male albino rats were separated into six groups of five rats and were maintained as follows: group 1: control, group 2: rats were fed crude petroleum
oil contaminated catfish diet (CPO-CCD) only, group 3: CPO-CCD plus 1 ml/kg of
1 % tween 80, group 4: CPO-CCD plus M.
myristica water extract (MWE), group
5: CPO-CCD plus M. myristica ethanol extract
(MEE) and group 6: CPO-CCD plus M.
myristica diethyl ether extract (MDEE). The feeding of the rats with CPO-CCD
and administration of extracts using cannula lasted for 28 days. The results
showed significant (p<0.05) decrease in total protein, albumin, and globulin in the serum and liver of rats fed CPO-CCD only when compare with control. Significant increases were observed in aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) in the serum, liver, kidney and brain of rats fed CPO-CCD only when compare with control. Significant increase were observed in total protein, albumin, and globulin in the serum and liver and decrease in AST, ALT, ALP activities in the serum and tissues when compare with CPO-CCD only and CPO-CCD plus tween 80 after treatments with MWE, MEE and MDEE. However, it could be concluded that MDEE revealed a strong effect when compare with the MEE and MWE. Key words: Petroleum, Catfish, extracts, Monodora myristica, Diet. *Corresponding author: okpo[email protected] 1. Introduction Crude petroleum oil (CPO) contains high amount of toxic chemicals, which can cause a wide range of health effects in people and wildlife, depending on the level of exposure and susceptibility 1.The constituents of crude petroleum oil are very complex. It contains aliphatic, alicyclic, polycyclic aromatic hydrocarbons, oxygen, sulphur and nitrogen containing substances 2. The polycyclic aromatic hydrocarbon content of CPO consists of fused aromatic benzene 3. Exposure of human and animals to these chemicals depends on different stage of CPO usage and environmental level 4. Crude petroleum oil has been reported as a mediator in oxidative stress 5, which may lead to various dreadful diseases like cancer 6, dementia, atherosclerosis, multiple sclerosis, cardiac dysfunction, blood disorders, hepatic morphological abnormalities 6, nephrotoxic effects 7 etc. The use of antioxidant in ameliorating the deleterious effect of free radicals has been the subject of previous investigations 8,9,10,11. Hence, use of spice with antioxidants property is necessary to suppress oxidative stress in a healthier way 12. Spice and herbs have been studied for their antioxidant activities. The study was carried out to examine the protective effect of M. myristica extracts on crude petroleum oil contaminated catfish (Clarias gariepinus) diet stimulated toxicity in rats. 2. Materials and Methods Chemicals and reagents Dichloromethane, ethanol and diethyl ether were all purchased from BDH Chemical Laboratory England, United Kingdom. Anhydrous sodium sulphate was purchased from Sigma Chemical Company, London, United Kingdom. Kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin and total protein were obtained from Randox laboratories limited (Antrim, United Kingdom). All other reagents used were of analytical grade 2.1 Spice (Monodora myristica) The spice M. myristica was purchased from the local market in Obiaruku, Delta State, Nigeria. The spice was identified at the Department of Botany, Delta State University, Abraka, Delta State, Nigeria 2.2. Preparation of the M. myristica Extracts The hot water (60?C), ethanol (95 % v/v), and diethyl ether (95 % v/v) extracts of M. myristica was obtained using the previous method stated by George et al. 12. 2.3 Stimulation of Crude Petroleum Oil Pollution The crude petroleum oil (CPO) was gotten from the Nigerian National Petroleum Cooperation (NNPC), refinery, and reported to have been extracted from Warri (Warri Excarvos light crude oil) in Delta State, Nigeria. The fish used for this study was collected from a commercial fish farm in Obiaruku, Nigeria and allowed to acclimatize for 7 days and then divided into two groups. Group 1: control: the catfish was cultured in plastic aquaria with 30 L borehole water for four weeks. Group 2: the catfish was cultured in plastic aquaria with 30 L borehole water and then polluted with crude petroleum oil ( LD 50 toxicity in catfish, 823.3 µl/L) as described by Ikeogu et al. 14 for four weeks. The water was changed and re-polluted every 24 hours. 2.4 Formulation of Diet The formulation of diet was carried out by the method described by Sunmonu and Oloyede (2007). The catfish were used as a source of protein to formulate diet for the rats. 2.5. Experimental Procedure Male albino rats were used for the study to avoid oestrous cycle complication. They were allowed to acclimatize for two weeks and had free access to water. The rats were supplied with standard growers mash diet gotten from Lagos State (Bio-Ingredients Limited Ikeja, Lagos). They were maintained in accordance with the National Institutes of Health (NIH) guide lines and then placed in six groups, were, n =5. Group 1: Control Group 2: Crude petroleum oil contaminated catfish diet (CPO-CCD) control Group 3: CPO-CCD plus tween 80 Group 4: CPO-CCD plus 200 mg/kg b. wt. of M. myristica water extract (MWE) Group 5: CPO-CCD plus 200 mg/kg b. wt. of M. myristica ethanol extract (MEE) Group 6: CPO-CCD plus 200 mg/kg b. wt. of M. myristica diethyl ether extract of (MDEE) Rats in group 1 to 6 received tap water daily throughout the experiment and then sacrificed after 24 hours fast on the last day of the study. The blood was collected into an anticoagulant free test tube. One gram (1 g) of various tissues were homogenized in ten milliliters of normal saline. After wards the clotted blood and the tissue homogenate, were centrifuged at two thousand five hundred revolution per minutes (RPM) for fifteen minutes to separate the serum and tissues supernatants which was stored in the refrigerator (-4? C), for further biochemical analysis. 2.6. Biochemical Analysis The total hydrocarbon content in the catfish sample was determined using a modified method of Soley et al. 16. The method of Kaplan and Righetti 17 was used in the assay of alkaline phosphatase activity. The method of Reitman and Frankel 18 was adopted for the assay of aspartate aminotransferase and alanine aminotransferase activities. Albumin level was analysed using the method of Doumans et al. 19. The total protein level was estimated using the method of Tietz 20. Globulin levels was calculated; Globulin = total protein – albumin. The liver histology was carried out according to method of Drury and Wallington 21. 2.7. Statistical Analysis Descriptive statistics was carried on the data obtained. Results were expressed as mean bars and 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 22.0) was used for statistical analysis. A significant threshold of p< 0.05 was regarded statistically significance between the test and control group for the analysis 3. Results Effect of crude petroleum oil pollution on catfish total hydrocarbon content level The mean value of total petroleum hydrocarbon content obtained in crude petroleum oil polluted catfish was significantly (p<0.05) higher when compared with DPR (Department of Petroleum Resource) limit (Figure 1). Effect of M. myristica extracts on AST, ALT and ALP activities of rats fed with CPO-CCD. Changes in AST activity in the serum, liver, kidney and brain (tissues) of rats fed CPO-CCD treated with extracts of M. myristica are shown in Table 1. The activity of ALT in the serum and tissues of rats fed CPO-CCD treated with extracts of M. myristica are presented in Table 2. The effect of M. myristica on ALP activity in the serum and tissues of rats fed CPO-CCD are presented in Table 3. The AST, ALT and ALP activities increased significantly (p<0.05) in rats fed CPO-CCD only (group 2) and CPO-CCD plus tween 80 (group 3) in the serum, liver, kidney, and brain when compere with the control. However treatment with MWE, MEE and MDEE (group 4, group 5 and group 6) showed significant decrease in AST, ALT and ALP activities in the serum and various tissues as compared with CPO-CCD only. Effect of M. myristica extracts treatment on albumin, total protein and globulin levels of rats fed with CPO-CCD. Significant decrease level of albumin in the serum and liver were observed in rats fed CPO-CCD only and CPO-CCD + tween 80 when compared with control. Treatment with the extracts of M. myristica significantly (p<0.05) increase the level of albumin in the serum and liver when compared with rats fed CPO-CCD only and CPO-CCD plus tween 80 (Figure 2). The total protein level in the serum and liver were significantly (p<0.05) lower in rats fed CPO-CCD only and CPO-CCD + tween 80 when compared with control. However the altered total protein levels in the serum and liver were significantly (p<0.05) normalized after treatment with MWE, MEE and MDEE (Figure 3). Figure 4 indicated significant (p<0.05) decreased level of globulin in the serum and liver in rats fed CPO-CCD only and CPO-CCD + tween 80 when compared with control. Upon treatment with different extracts of M. myristica (MWE and MEE) significant (p<0.05) elevated levels of globulin in the serum and liver. No significant (p>0.05) difference were observed in the level of
globulin in the serum and liver after treatment with MDEE when compared with
of M. myristica extracts on the liver histology of rats fed CPO-CCD.
In fig. 5, the control
group showed normal histological structure of hepatic lobule, hepatic cell
(HC), and central vein (CV). Rat liver section of CPO-CCD control group
indicated congestion of central vein (CCV), perivascular mononuclear cells
infiltration (MCI), and ballooned hepatocyte (BH). Treatment of CPO-CCD with
MWE the rat liver sections showed CCV, and HC moderate regeneration. Treatment
of CPO-CCD with MEE group showed slight CCV, and regeneration of HC. No
necrosis and ballooning formation were also observed. However, treatment of
rats fed CPO-CCD with MDEE showed normal histological structure of hepatic
lobule with moderate CV, and HC.
Crude petroleum oil polluted catfish had high
content of total petroleum hydrocarbon (PHC) that higher than the Department of
petroleum resource (DPR) exposure limits 22. This observation suggests that consumption
of crude oil contaminated diet exposes man and animals to hydrocarbon toxicity. This finding is in line with the study
conducted by Ubong et al. 23, who hinted that presence of petroleum hydrocarbon in the aquaculture environment is
of global importance because fishes bioaccumulation and transfer toxic and carcinogenic chemicals to humans.
That the consumption of
petroleum contaminated diet is injurious to animal health is indicated by the
increase in serum liver function enzymes (Table 1 and 2).This is consistent
with earlier studies (Achuba and Ogwumu, 2014; Achuba and Nwokogba 2015). The
increase in serum liver enzymes has been attributed to damage to the liver cell
membrane and cellular leakage of these enzymes into general circulation. This
explains why a positive correlation was observed between AST and ALT (r
=0.946). Exposure to crude petroleum contaminated diet induced organ/tissue
damages is further expressed by the increase in alkaline phosphatase activities
(Table 3). This finding is also in accordance with the studies of Adeyemi et
al. 25; Momoh and Oshin 26 which indicated that crude petroleum oil induced
renal toxicity, damage to the plasma membranes and vascular endothelium of the
brain. However, the activities of AST
and ALT in various tissues (liver, brain and kidney) in rats fed CPO-CCD
treated with MDEE, MEE and MWE were restored close to control values. These
observations therefore portray the protective effect of M. myristica extracts against damage to the plasma membranes of the
tissues. The protective influence of plant materials against chemical toxicity
is previously reported (Achuba et al 2016; Kadiri, 2017)
In addition, other markers
of organ/tissue damage were affected by consumption crude oil contaminated diet.
This is indicated by decreases in albumin, globulin and total protein levels in
the serum of rats fed with CPO-CCD only and CPO-CCD when compared to control (figure
3 and 4). Generally, that the liver is damaged by the contaminated diet is
further highlighted by histological study. The hepatic histoarchitechure of
rats fed CPO-CCD resulted in severe hepatic necrosis (HN), congestion of central
vein (CCV), perivascular mononuclear cells infiltration (MCI), and ballooned
hepatocyte (BH) (Figure 5; group 2 and
3) when compared with the control ( group 1). The distortion of architectural integrity
of damaged organs and tissues by petroleum was reported previously (Achuba and
The damage produced result in failure of the
endoplasmic reticulum, which leads to decrease in protein synthesis 27. The
results is also in line with previous study conducted by
Sunmonu and Oloyede 15, who
stated that crude petroleum oil contaminated diet may lead to reduced dietary
intake and hence a reduction in serum albumin levels. Similar to liver function
enzymes treatment with MDEE, MEE and MWE were restored close to control values.
The extracts may have stabilized the endoplasmic reticulum leading to protein
synthesis which is in line with the report of Sureshkumar and Mishra 27.
The protective potency
of polysorbates (tween 80), a known food additive, against crude petroleum oil
contaminated diet induced liver toxicity was also evaluated. No change was showed in AST and ALT activities when
rats fed CPO-CCD only were compared
with rats fed CPO-CCD + tween 80, thus confirming the nontoxic effect of tween 80 with respect to liver function.
This is in agreement with the statements of Rowe et al. 24, who reported that
polysorbates (tween 80) are widely used in food products, and topical
pharmaceutical formulations, and are regarded as nontoxic materials. Similar to M. myristica extracts, tween 80 protected the exposed rats
from liver damage. The use of commercially available chemical against crude oil
toxicity is previously documented (Achuba and Ahwin 2009; Uboh et al 2009; Ita
et al 20016)
It is pertinent to suggest
that the CPO-CCD triggered hepatotoxicity and disturbance in the levels of
hepatic enzymes activities and other tissues (kidney and brain) as shown in
this study. However, administration of M. myristica extracts (MDEE, MEE and
MWE) significantly rescued the CPO-CCD induced tissue damage and structural
alterations. Moreover, MDEE showed a strong effect as compared with MEE and MWE
(MDEE > MEE > MWE).Thus this study was able to establish that M. myristica extracts compares
favourably with synthetic protective agents.