Harvesting of the mushroom
Fresh fruiting bodies of wild P.tuber-regium were collected from a forest at the back of University of Nigeria Nsukka by a taxonomist working with the university. These fresh fruiting bodies were cleaned and air dried away from direct sunlight. The mushroom were ground and stored in a clean dry plastic container until use [10]. Little is known about bacterial contamination of the Nigerian harvested P.tuber-regium. P.tuber-regium were not tested for micriobiological contaminations
Animals husbandry
Thirty six male Sprague–Dawley rats with body weights 180–200 g acclimatized for two weeks were maintained under controlled conditions of temperature (23 ± 2 °C) and humidity (50 ± 5%) and a 12-h light–dark cycle, were used for the experiment. The animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding. The bedding of the cages was changed daily and the cages were cleaned as well. They had free access to standard rat
pellet diet and water ad libitum. All the experimental procedures were performed according to the committee for the purpose of control and supervision of experiments on animals, norms and approved by the University of Port Harcourt Animal Ethical Committee with an approval number UNIPORT/PHARM/0133.
Carbon tetrachloride (CCl4)
Thirty percent carbon tetrachloride (Sigma Aldrich) in Olive oil [11] was used to induce renal and hepatic damage at a dose of 10 ml/kg (i.p) [12].
Acute toxicity studies
Different concentrations of P.tuber-regium (50–5000 mg/kg body weight b.w.) were administered orally to male rats. These animals were observed daily for toxicological manifestations like behavioral changes, neural and autonomic toxicities, feeding pattern changes etc. There was no mortality recorded during this period even up to the dose of 5000 mg/kg [13].
Experimental design
The animals were divided into six groups with each group consisting of six animals each. The administration of CCl4 10 ml/kg body weight of 30% CCl4 in olive oil was given on days 0, 7, 14, and 21 concomitantly with the daily feeding of the mushroom.
The animals were treated for 4 weeks as follows
-
Group I – normal control received olive oil 10 ml/kg i.p. weekly in addition to standard food and water.
-
Group II – Positive control received CCl4 (30% CCl4 in olive oil) at a dose of 10 ml/kg weekly in addition to standard feed and water.
-
Group III – rats were treated orally with 100 mg/kg b.w. of P.tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly.
-
Group IV – rats were treated orally with 200 mg/Kg b.w. of P.tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly.
-
Group V – rats were treated orally with 500 mg/kg b.w. of P.tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly. In all CCl4 was given intraperitoneally.
-
Group VI – rats were treated orally with 500 mg/kg b.w. of P.tuber-regium in feed (33.3% w/w) only with standard feed and water.
Necropsy
Animals were sacrificed 24 h after the last treatment. Blood was collected by retero orbital sinus puncture and serum was separated by centrifugation at 3000 r.p.m for 10mins at 4 °C for assay of biochemical parameters. Rats were sacrificed under ether anesthesia; liver and kidney were excised, rinsed clean in saline, weighed and preserved in 10% formalin for histopathological study.
Determination of biochemical parameters
Commercial reagent kits for the determination of bilirubin, cholesterol, creatinine and urea concentrations and alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), were assayed using Quimica Clinica Aplicada (QCA) Spain. Fasting blood glucose was determined using commercial glucometer made by accu-chek.
Antioxidant assay
The liver and kidney were minced separately into small pieces and homogenzed with ice cold 0.05 M potassium phosphate buffer (pH 7.4) to make 10% homogenates. The homogenates were centrifuged at 6000 rpm for 15 mins at 4 °C. The supernatant was collected for the estimation superoxide dismutate (SOD) and malondialdehyde (MDA) assays. Superoxide dismutate (SOD) was assayed by the method described by Misra and Fridovich [14]. Lipid peroxidation was quantified as malondialdehyde (MDA) according to the method described by Ohkawa et al. [15]. and the MDA level was calculated according to the method of Todorova et al. [16] and expressed as μmol MDA/mg protein.
Histopathological studies
Portions of the liver and kidney from all the experimental groups were fixed in 10% formaldehyde, dehydrated in graded alcohol, cleared in xylene and then embedded in paraffin. Microtome sections (5 μm thick) were prepared from each liver and kidney sample and stained with heamtoxylin-eosin (H&E) dye. The sections were examined for the pathological findings.
Statistical analysis
Data obtained were analyzed using graph pad prism 5. The values represent means and their standard deviations. Differences between the means were determined using one way analysis of variance (one way ANOVA) followed by Bonferroni’s test. P values of 0.05 or less were considered statistically significant.
