Preparation of plant sample
The leaves of Carica papaya were plucked and washed with distilled water to be free from dirt or debris and other contaminants while the seeds were gotten from ripe C. papaya fruits which were cut into two halves so as to obtain the seeds easily. The leaves and seeds were air-dried separately for 2 weeks and blended into powdered form respectively. The blended leaves and seeds weighed 2492g and 1005g respectively. The blended leaves and seeds of C. papaya were soaked in 1500ml of distilled water for 24hours in an air-tight container and were kept in a room temperature (250C). The extracts were filtered, separating the residue from the solvent using a funnel and whatman No. 1 filter paper into separate well-labeled beakers. The solvents from both the leaves and seeds were concentrated in a water bath at 500 C for three consecutive days and slurry formed from the leaves and seeds samples were obtained and were preserved in a refrigerator at 40c to avoid spoilage until use.
Compliance with ethical standards
The animal study was conducted in accordance with the Ethical Guidelines for the Use of Animals in Research National Committee for Research Ethics in Science and Technology (NENT) (2018).
Experimental design and animal grouping
Male albino rats weighing between 174g to 196g were obtained from the animal house of the Department of Science Technology, Biochemistry unit, Akwa Ibom State Polytechnic, Ikot Osurua. Animals were housed and maintained in wooden cages (47 by 35.5 by 33cm) with wire mesh covers on the cages at 25ºC under a 12:12 light/dark cycle, with free access to food and water. Experiments were carried out during the normal light/dark cycle and always started at the same hour (10 am).
Thirty adult male albino rats were randomly allocated to six groups of five rats each:
Group 1 (control): Animals in this group served as control and received only 0.2ml of distilled water.
Group 2 (Pb(NO3)2): Animals in this group received 0.2ml lead nitrate at a dose of 50mg/kg body weight for 31 days by oral gavaging. The chosen dose fell within the ranges of doses applied in previous studies (11, 12).
Group 3 (CPL): Animals in this group received 0.2ml aqueous extract of C. papaya leaves at a dose of 500mg/kg body weight for 21 days by oral gavaging.
Group 4 (CPS) : Animals in this group received 0.2ml aqueous extract of C. papaya seeds at a dose of 500mg/kg body weight for 21 days by oral gavaging.
Group 5 (CPL + Pb(NO3)2): This group of animals received 0.2ml aqueous extract of C. papaya leaves (500mg/kg body weight) beginning eleven (11) days after the start of lead nitrate treatment (13). An hour after the treatment with C. papaya leaves, the animals receive 0.2ml of lead nitrate (50mg/kg) (14).
Group 6 (CPS + Pb(NO3)2): This group of animals received 0.2ml aqueous extract of C. papaya seeds (500mg/kg body weight) beginning eleven days after the start of lead nitrate treatment. An hour after the treatment with C. papaya seeds, the animals receive 0.2ml of lead nitrate (50mg/kg).
All experimental groups received treatment once daily for 3 weeks following 1 week treatment with lead nitrate for the lead treated groups. No group received above 1ml of extract or lead nitrate.
Collection of blood sample
After 24hours fast, the animals were anesthesized with chloroform on the morning of the 4th week. The unconscious animal was pinned down on the dissecting board with the help of a tag pin and was dissected medioventially and whole blood was collected from the aorta with the use of a syringe and needle using cardiac puncture method. The blood was immediately transferred into an EDTA bottle which contained anticoagulant to prevent blood clotting.
Determination of body weights of experimental animals
Body weight of animals in each group was determined at the beginning and end of the experiment using a weighing balance. Change in body weight was calculated as: Final body weight- Initial body weight
Determination of organ weights of experimental animals
The weights of the heart, liver, spleen, lung and kidney, liver were carefully removed after the animals were sacrificed using a sensitive weighing balance and filter paper.
Determination of Metal Concentration in Liver
Metal levels in liver and kidney were estimated according to the method of Ballantine and Barrford (15). To 0.2g of the homogenates of liver and kidney, 10ml of nitric acid was added, followed by 20ml of perchloric acid. The sample was then digested over a sand bath until the solution became clear and yellow in colour. The digest was made up to known volume with deionized water. Aliquots of this were used to estimate the metals by atomic absorption spectrophotometer. The estimation of lead concentration was performed with hollow cathode lamps at wavelengths: 283.3 with air/acetylene by using atomic absorption spectrophotometer model GBC SENSES AA. Result of accumulated heavy metal were measured as (mean + SE) and expressed as the percentage of the total dose. Statistical evaluations were based on 5 rats per group.
Statistical analysis
Results were analyzed using the Statistical Package for Social Sciences (SPSS) version 25 and expressed as Standard Error of Mean (SEM). Multiple Comparison test was made between control and experimental groups using ANOVA test. Significant differences among treatments were detected using LSD test and values of P < 0.05 was considered statistically significant.