Effect of MethanolicExtract of Justicia flava Leaves on Biochemical Markers in Male Wistar Rats Fed Crude Oil Contaminated Feed

: The medicinal potentials of plants have been documented. This study evaluated the capacity of the leaf of Justicia flava methanolic extract (JFME) to alter the biochemical distortions initiated by feeding on diet containing crude oil. Male Wistar albino rats, thirty six, were constituted into nine groups. Each group had six rats. Group 1 had untreated feed. Groups 2 to 4 had untreated feed but were given 100 mg, 200 mg and 300 mg/ kg b.wt of JFME, respectively. Group 5 had untreated feed and given 200 mg/kg b.wt of ascorbic acid as standard. Group 6 was fed with diet containing crude oil (4ml/100g v/w). Groups 7 to 9 were given contaminated feed and 100 mg, 200 mg and 300 mg/ kg b.wt of JFME, respectively. The rats were maintained on these treatments for thirty days and had water ad libitum. Thereafter exposure period, lipid profile, hematological and inflammatory markers in the blood were analyzed using standard methods. Petroleum in feed altered the lipid profile, hematological and inflammatory markers compared to values in positive control rats. However, treatment of the rats with JFME had a positive reversal of these markers close to values in control rats; which compared favorably with ascorbic acid, used as standard. This investigation discovered JFME as a candidate for managing crude oil- imposed health issues.

Petroleum is of economic importance due to the contribution to many a nation gross domestic product (GDP) through its export to earn foreign exchange and creation of employment (Usman et al., 2015). It is equally a source of numerous chemicals/ solvents that are produced via fractional distillation (Eneh, 2011). These are commodity chemicals required by industries for production of other goods and for domestic uses (Achuba and Okoh, 2014). However, this economic benefit does not come without negative implications on the health of humans in areas of production (Ordinioha and Brisibe, 2013). Hydrocarbons toxicities are preceded by oxidative stress due to petroleum-induced generation of free radicals. The free radical generated stimulates the peroxidation of membrane lipids (Achuba, 2010). Lipid peroxidation, though the outcome of oxidative insult on the biomembrane, is important in the assessment of the functional state of the cell (Achuba, 2002). It is applied in conjunction with antioxidant enzymes in measuring physiological regions of cells (Achuba and Okoro, 2010). These biomarkers are also utilized in environmental monitoring and assessment ; hydrocarbon-induced physiological as well as histological distortion in animals (Achuba, 2018a). Most importantly, the responses of animals to the protective influence of plant-derived products are catching the attention of so many scientists Ichipi-Ifukor et al., 2019;Achuba and Ichipi-Ifukor, 2020;Onakurhefe et al., 2020;Mordi et al., 2021). Similarly, the usefulness of organic substances in mitigating chemically-induced toxicity has been published: honey and coconut water (Mordi et al., 2015;Akintola et al., 2018), Hibiscus sabdariffa (Dahiru et al., 2003) and Moringa oil (Ezedom, 2018).On this premise, it is evident, therefore, that the importance of organic-derived substances in the prevention of chemically-induced cellular and metabolic distortions cannot be overemphasized. The present investigation examined the function of JFME against physiological changes in Wistar albino rats induced by petroleum in diet. Experimental Animals.Fifty-four male Wistar rats (weight: 150-170 g) used were inbreed from the animal house unit of Anatomy Department, Delta State University, Abraka, Nigeria. They were housed in plastic cages and fed with fish feed and water ad libitum for one month in order to allow for acclimatization to the new environment and feed. The rats were maintained at 12-12 h light/dark of normal day/night cycle. The rats were handled based on the care of experimental animals as outlined by the national research council on the use and care of experimental animals (NRC, 2011).

Plant extract preparation.
A sizeable quantity of the leaves was collected and dried at laboratory condition (28 ± 2ºC) for two weeks and four days until constant weight was obtained. The leaves were chopped from the stalk, broken into coarse sizes with hand and ground by means of a warring blender into fine powder form. It was then subjected to extraction using 70% methanol by cold maceration technique. The extract was concentrated with a rotary evaporator which produced brownish-green slurry at 40ºC.The slurry obtained was then dried in an open water bath at 40ºC.The yield was determined and the extract kept in a sample container at 4ºC until needed.

Treatment of animals.Male
Wistar albino rats, thirty six were constituted into nine groups. Each group had six rats. Group 1 had untreated feed. Groups 2 to 4 had untreated feed but were given 100 mg, 200 mg and 300 mg/ kg b.wt of JFME, respectively. Group 5 had untreated feed and given 200 mg/kg b.wt of ascorbic acid as standard. Group 6 was fed with feed that contained crude oil (4ml/100g v/w). Groups 7 to 9 were fed with contaminated feed and were given 100 mg, 200 mg and 300 mg/ kg b.wt of JFME, respectively. The rats were maintained on these treatments for thirty days and had water ad libitum.
Blood collection and determination of inflammatory markers. The rats were subjected to overnight fasting after thirty days of treatment and sacrificed under chloroform anesthesia. Blood samples were collected by cardiac puncture into EDTA containers and plain containers, respectively. The blood in plain sample containers were allowed to stand for 2 h to enable it clot and subjected to centrifugation at 4000rpm for 10 min to allow for collection of various sera of each treatment. The collected sera were stored at −4˚C and used within 12 h for assay. The serum lipid profile (triglyceride, total cholesterol, HDL-Cholesterol and LDL-Cholesterol concentrations in serum) were determined using Randox, UK, assay kits. The methods of Westegren and Singer (1957) were adopted for the determination of ESR and C-reactive protein (C-RP), respectively. While ceruloplasmin and creatinine concentrations were determined using the methods of Sunderman and Nomoto(1970) and Lustgarten and Wenk (1972), respectively. The bloods collected in EDTA-containers were used for hematological analysis using automated Mindray Hematology analyzer, model, BC-2300.
Statistical Analysis: All data were analyzed using Analysis of variance (ANOVA) and expressed as means ± SD. Significant difference between the control and treatment means were set at P < 0.05 confidence.

RESULTS AND DISCUSSION
The consumption of feed containing crude oil altered the lipid profile of rats (Table 1). There are increases in total cholesterol and LDL-Cholesterol but a significant (P<0.05) decrease in HDL-cholesterol and triglyceride in rats fed crude contaminated feed against rats fed normal feed. On the contrary, the administration of JFME reversed these parameters close to the values in control rats and rats given the extracts but fed untreated feed. Lipid profile distortion is a major contributor in the initiation of cardiovascular disease (Einarson et al., 2018). The higher lipid level in plasma of rats fed diet containing crude oil (Table 1) noted here is in agreement with previous study . The abnormal high concentration of serum lipids in crude oil exposed rats is due to petroleum stimulated hypoglycemia . And in a bid to meet energy requirement there is increase in the mobilization of free fatty acids from the fatty depots in the body and hormone-activated lipolysis (Achubaet al., 2005). The trend observed in the plasma lipid profile in the crude oil exposed rats was consistent as the plasma LDL-C was significantly higher relative to control (Table 1). This finding is not unexpected as a high LDL-C has been reported to occur in blood of crude oil exposed rats (Achuba, 2005). The lowering of the plasma lipid profile in the control rats and rats treated with the extract is an indication of a hypolipidemic effect of the plant extract. The increase in plasma HDL-C in control rats treated with the extract is noteworthy as it did benefit crude oil intoxicated rats. This observation agrees with Achuba, (2005). Earlier publication indicated that high HDL-C protects against cardiovascular disease (Ganjali et al., 2017).This is predicated on phytochemicals that are shown to decrease blood lipid levels (Pires et al., 2018;Onakurhefe et al., 2019).This explains the positive modulation of the lipid profile by the plant extract. The consequence of consuming feed containing crude oil on hematological indices and the impact of the administration of various doses of methanolic extracts of Justicia flava in male Wistar albino rats are indicated in Table 2 Achuba, 2018c). The decrease in hematological profile, which is in tandem with previous study, has been attributed to a number of factors (Achuba, 2018c). One of the factors responsible for the reduced hemoglobin concentration, packed cell volume and red blood cell count is hydrocarbon-mediated decreased cell blood cell synthesis and increased hemolysis (Achuba, 2018c). However, the administration of Justicia flava leaves extract conferred protection against crude oilmediated hematotoxic imports of crude oil. Hematoprotective propensity of Justicia flava is no surprise as plant materials with antioxidant potentials have been reported to protect animals from the hematotoxicity of crude oil (Achuba, 2019). Each datum is the mean of six determinations and expressed as mean± SD. Different superscripts in a column signifies significant difference at 5% confidence limit The crude oil in rat feed altered the inflammatory indicators (Table 3). This was exhibited by the significant (P<0.05) increases in erythrocytes sedimentation rate (ESR), and the concentrations of Creactive protein (CRP), creatinine and ceruloplasmin. The administration of various doses of Justicia flavastimulated decreases in ESR as well as the concentrations of CRP, creatinine and ceruloplasmin compared to the values in rats fed diet containing crude oil only. These modulations of these inflammatory indicators are comparable to those of standard ascorbic acid given concurrently. Some important indices of inflammation in animals include erythrocyte sedimentation rate, C-reactive protein, ceruloplamine and creatinine, and the relationship between inflammatory markers and crude oil intoxication had been documented (Achuba and Obaremi, 2018). The health promoting potentials of Justicia flava extract is expressed in the positive alterations of petroleum-induced changes in inflammatory markers. Similarly, the medicinal capability of Justicia flava leaves was reported earlier (Baforet al., 2019). The anti-inflammatory disposition of Justicia flava leaves extract is not out of place since induction of free radical production is one mechanism that accounts for the initiation of petroleum-stimulated tissue damages (Achuba, 2018d;Achuba, 2019). The generation of free radical is quenched by the plant extract due to its richness in antioxidants (Bafor et al., 2019). This explains the anti-inflammatory propensity of Justicia flavaleaves extract because disease progression is associated with free radical generation cum oxidative stress.
Conclusion:This investigation revealed that JFME as a natural product for managing crude oil contaminated feed-imposed health issues. Thus, inhabitants of crude oil bearing areas of the world can take advantage of this plant that is readily available in the tropics.