Phenolic rich-extracts from Nauclea latifolia fruit restored Lead acetate-induced liver and kidney damaged in Wistar rats

Liver and kidney diseases are becoming order of the day in both developed and developing countries as a result of environmental pollutants such as lead. Restorative activities of methanol and methanol/acetone phenolic-rich extracts (MPR and MAPR, respectively) of the N. latifolia fruit (NLF) on lead acetate-induced liver and kidney damaged were assessed in Wistar rats. The antioxidant activities of both phenolic-rich extracts of NLF were also carried out using standard methods. Seven groups of Wistar rats comprising of 5 rats each were used for the study and 1000 mg/kg body weight (bw.) of lead acetate solution was administered orally to the 6 groups of animals to induce liver and kidney damage. The high and low dosages of 300 and of 150 mg/kg body weight (bw.) of both MPR and MAPR were administered orally to four groups for 14 days along positive (100 mg/kg bw. of silymarin), negative (treated with the placebo) and naïve control (non-induced). The percentage DPPH radical scavenging activities, ferric reducing antioxidant power and percentage inhibition of lipid peroxidation show high antioxidants activities dose-dependently. Furthermore, administration of lead acetate significantly (p > 0.05) reduces the weight gain and elevates the liver and kidney relative weight as well as their respective damage biomarkers with distortions in their histologies. However, treatment with MPR and MAPR resulted in significant (p < 0.05) improve in the percentage body weight gain, relative liver and kidney weight as well as restoration of the activities of the liver and kidney functions biomarkers of the treated animals. Likewise, lesser hepatic and renal cells injury were also observed in the treated groups with MAPR being more active at high dosage which significantly (p < 0.05) compared well with normal group. Hence, the phenolics content of the N. latifolia fruit can be exploited further for drug development for the management kidney and liver damage arise from lead-induced toxicity.

globally. According to the World Health Organization (WHO) global health estimate of 2015, chronic liver disease ranks as the 16th main cause of illness globally [6,7]. In 2017, the figure of mortality linked with chronic kidney diseases (CKD) or CKD-related complications was projected to be 1.2 million and this account for 4.6% of world-wide deaths [8]. Most of the chelating agents such as disodium edetate, dimercaprol, succimer among others used to curb lead induced toxicity exhibit various setbacks. These hinderances include toxicity, inability to chelate the accumulated lead in some tissues, prooxidant properties of some of the chelating agents as well as their high cost. For these reasons, it is justifiable to search for alternative drugs from natural sources.
Phenolic compounds are secondary metabolites that can be sourced from plant and they are one of the major sources of antioxidant which can protect the cells from oxidative impairment triggered by free-radicals [9]. Phenolics are known to serve as reducing agents, metal chelators, singlet oxygen quenchers, radical scavengers and hydrogen donors [10]. However, in order to extract maximum quantity of these secondary metabolites from several plants, selected solvents of different polarities must be used coupled with suitable method of phytoconstituent extraction [11].
Our previous study showed that phenolic-rich extracts of N. latifolia fruit ameliorates lead acetate-induced haematology and lung tissues toxicity in male Wistar rats [11]. Therefore, it is not out of place to evaluate the restorative effects of phenolics rich-extracts from N. latifolia fruit on lead acetate-induced liver and kidney damage in Wistar rats via changes in liver damaged markers, kidney damaged markers as well as the histology of both organs.

Plant collection
Fresh, matured fruits of N. latifolia were collected from Bosso Estate in Bosso Local Government area (Niger State, Nigeria) on Latitude: 9.6550611 and Longitude: 6.5138343. The fruit was identified by a Senior Botanist of the Biological Science Department of Federal University of Technology Minna with voucher number: FUT.-MIN/SLS/PB-019-016 and the specimen was deposited at the herbarium of the University.

Kits, chemicals and reagents
The biochemical kits used were obtained from AGAPE Laboratories, Hombrechtikon, Switzerland. The chemicals and reagents used were obtained from Sigma-Aldrich Chemical Company St. Louis, USA. The reagents include methanol, acetone, ethylacetate, Ferric chloride, DPPH, lead acetate, hematoxylin and eosin among others.

Experimental animals
Male Wistar rats (125.00 ± 3.16) g were obtained from the animal facility of Federal University of Technology Minna (FUT.MINNA), Niger State, Nigeria. The animals were housed in polypropylene cages under a controlled environment with 12 h light/dark cycles, temperature of 28 ± 3°C and relative humidity of 45-50%. The animals were fed on pelleted diet (Vital Feeds, Jos Nigeria) with supply of water ad libitum. The experiment was conducted according to protocol review (1997) of Canadian Council on Animal Care and use guidelines. Ethical clearance number 0000012EAU was given by FUT.-MINNA/Nigeria Ethical Review Committee.

Sample preparations
Matured Fruits of N. latifolia were washed, sliced and dried at room temperature (28 ± 2)°C. The dried fruits were pulverised using electric blender (Binatone BLG 450, United Kingdom). The milled sample was kept in an air tight plastic vessel at room temperature (28 ± 2)°C until further use.

Phenolics extraction from the N. latifolia fruits
Fifty grams (50 g) of the powdered sample was positioned in a round bottom flask and steeped with 400 mL of 70% methanol and another 50 g of the fruits sample was steeped with 40% acetone and 60% methanol mixture at temperature of (28 ± 2)°C for 72 h with occasional mixing. The extracts were filtered with Whatman filter paper after which the methanol or methanol/acetone solvent were evaporated at low pressure in a rotary vacuum evaporator. The filtrate of N. latifolia fruit gotten were freeze-dried and phenolics rich extract obtained was refrigerated at − 4°C.
In vitro antioxidant activities of phenolics from N. latifolia fruits The method of Oyaizu [30] was used for the determinations of scavenging activity of 1, 1-diphenyl-2-picryl hydrazyl (DPPH) and the ferric reducing antioxidant power of the phenolics from N. latifolia fruits while Halliwell et al. [31] method was used to determine percentage inhibition of lipid peroxidation of the extract using a modified thiobarbituric acid reactive substances (TBARS) assay as all reported by Ibrahim et al. [32].
Animal grouping for lead acetate-induced hepato-renal toxicity study The Wister rats were grouped into seven of five rats each as shown below; Group A (Naïve Control): 0.5 mL/kg bw. of Normal saline.
The 1000 mg/kg bw. of lead acetate was orally administered to induce lead toxicity in six groups for 14 days after weighing the animals. Each extract of 150 mg/kg bw. and 300 mg/kg bw. were administered to four of the groups after one hour of lead acetate administration. One of the remaining two groups of the lead induced toxic group was treated with 100 mg/kg bw. of silymarin (Standard drug) while the other one (Untreated) was given 2 mL/kg bw. of 0.9% saline solution. The animals were weighed and euthanized by cardiac puncture at 15th day after an overnight fasting while blood, liver and kidney were collected for further analysis.

Serum, liver and kidney preparations
Serum was prepared by using the procedure described by Yakubu et al. [33]. Briefly, the rats were euthanized by cervical dislocation at day 15th and the blood samples were collected by cardiac puncture from the inferior vena cava of the heart into serum sample bottles. The blood samples were allowed to clot at room temperature and serum was obtained by centrifuging at 3000 rpm for 10 min. The liver and kidney were collected, washed blotted and weighed as described by Busari et al. [34].

Histologies of the liver and kidney
A section of liver and kidney tissues was quickly fixed in 10% neutral buffered formalin solution prior to at least 24 h of examination. The fixed specimen was processed by using conventional paraffin embedding technique (dehydration via ascending grades of C 2 H 5 OH, clearing using chloroform and embedding with paraffin wax at 60°C. Paraffin blocks was prepared from which 3-4 μm thick sections was gotten and stained with haematoxylin and eosin (H&E). The histology observations were then made under light microscope at Magnification × 40.

Data analysis
The analysis of variance (ANOVA) followed by Post hoc Duncan multiple comparisons test (Statistical Package for Social Sciences, version 22.0, SPSS Inc., Chicago, IL, USA) at 95% confidence interval was used on the data obtained with p-value less than 0.05 was considered significant difference. The data were expressed as mean ± standard error mean of five replicates.

Results
Percentage of scavenging activity of methanol and methanol/acetone phenolic-rich extracts (MPR and MAPR) of NLF against 1, 1-Diphenyl-2-Picryl Hydrazyl radical (DPPH) The % DPPH Scavenging activities of methanol and methanol/acetone phenolic-rich extracts of the NLF are shown in Table 1. Both extracts and the standard (vitamin C) exhibit dose dependent % DPPH Scavenging activities. The % DPPH Scavenging activities are in following order; methanol extract < methanol/acetone extract < vitamin C with the highest at 500 μg/mL (42.32 < 62.32 < 91. 35).
Values are Means of five replicates ± standard error mean. Values with different superscripts and on the same column are significantly different (p < 0.05).

Ferric reducing antioxidant power (FRAP) of methanol and methanol/acetone phenolic-rich extracts of NLF
The ferric reducing antioxidant power of methanol and methanol/acetone phenolic-rich extracts of the NLF are shown in Table 2. Both extracts and the standard (vitamin C) also exhibit dose dependent FRAP as it was observed in % DPPH Scavenging activities. The FRAP of methanol and methanol/acetone phenolic-rich extracts of NLF are in following order; methanol extract < methanol/acetone extract < vitamin C with significant high value of FRAP at 500 μg/mL (0.82 < 0.88 < 2.50).
Values are Means of five replicates ± standard error mean. Values with different superscripts and on the same column are significantly different (p < 0.05).

Inhibition of lipid peroxidation of methanol and methanol/acetone phenolic-rich extracts of NLF
The Inhibition of lipid peroxidation of methanol and methanol/acetone phenolic-rich extracts of the NLF are shown in Table 3. No significant differences (p > 0.05) between the Inhibition of lipid peroxidation of methanol, methanol/acetone phenolic-rich extracts of the NLF and the standard (vitamin C) at 125 μg/mL. However, Inhibition of lipid peroxidation of methanol/acetone phenolicrich extracts of the NLF is significant higher (p < 0.05) when compared with both methanol and vitamin C at 500 μg/mL respectively (47.35 > 42.88 > 41.73). methanol/acetone phenolic-rich extract of the NLF is significant higher (p < 0.05) when compared with both methanol and vitamin C at 500 μg/mL respectively (47.35 > 42.88 > 41.73).
Values are Means of five replicates ± standard error mean. Values with different superscripts and on the same column are significantly different (p < 0.05).
Changes in body weight, relative liver and kidney weight of male Wistar rats administered with lead acetate and treated with methanol and methanol/acetone phenolicrich extracts of N. latifolia fruit The body weight changes along with relative weights of liver and kidney were shown in Tables 4 and 5 respectively. Oral administration of lead acetate to the experimental animals significantly decreases (p > 0.05) the body weight with significant increase in relative weight of liver and kidney of rats in negative control group when compared with other treated groups. In most cases animals treated with 300 mg/kg bw. MAPR NLF are significantly (p < 0.05) compared well with those in normal groups.
Values are Means of five replicates ± standard error mean. Values with different superscripts and on the same column are significantly different (p < 0.05).
Values are Means of five replicates ± standard error mean. Values with different superscripts and on the same column are significantly different (p < 0.05).
Liver and kidney damage biomarkers of male Wistar rats administered with lead acetate and treated with methanol and methanol/acetone phenolic-rich extracts of N. latifolia fruit The biomarkers for liver damage in Wistar rats administered with lead acetate and treated with methanol and methanol/acetone phenolic-rich extracts of N. latifolia fruit is presented in Table 6. Administration of lead  The biomarkers for kidney damage in Wistar rats administered with lead acetate and treated with methanol and methanol/acetone phenolic-rich extracts of N. latifolia fruit is presented in Table 7. Administration of lead acetate significantly increase (p > 0.05) the urea (98 mg/ dL), sodium (136.50 mmol/L), potassium (15.10 mmol/ L), chloride (99.50 mmol/L) enzymes as well as creatinine (4.00 mg/dL) when compared with control group. Treatment with lead acetate and treated with methanol and methanol/acetone phenolic-rich extracts of N. latifolia fruit however significantly (p > 0.05) restored the level of these kidney damage biomarkers at all dosages. Methanol/acetone phenolic-rich extracts of N. latifolia fruit at dosage 300 mg/kg bw. performed excellently well when compared with methanol phenolic-rich extracts and the standard drug (silymarin) at chosen dosages but not comparable to un-induced control group.
Values are Means of five replicates ± standard error mean. Values with different superscripts and on the same column are significantly different (p < 0.05).
Values are Means of five replicates ± standard error mean. Values with different superscripts and on the same column are significantly different (p < 0.05).
Histology of the liver and kidney of male Wistar rats administered with lead acetate and treated with methanol and methanol/ acetone phenolic-rich extract of N. latifolia fruits The administration of lead acetate caused marked hepatocytes necrosis, central vein congestion, vacuolar degeneration and scattered lymphocytes in between the hepatocytes and in the sinusoids. and in the histology of the liver tissue of the lead acetate treated group (Figure 1) while their kidney tissue showed capsular and tubular degeneration ( Figure 2). However, the methanol and methanol/ acetone phenolic-rich extract of N. latifolia fruits treated groups showed mild hepatocytes necrosis, mild central vein congestion, mild vacuolar degeneration and hepatocytes regeneration which is comparable to the reference drug (100 mg/kg bw silymarin) treated group.

Discussions
Lead toxicity has proven to be one of the leading global problems associated with a number of pathological disorders which include neurotoxicity, inflammation, liver and kidneys damages, immunomodulation, hypertension, cardiovascular diseases, and anaemia [43]. Owing to the pharmacological properties alongside with little or no toxicity attributed to the compounds of natural origins possessing antioxidant activities, the search for such compounds is warranted [44].
A number of studies are available backing the implication of oxidative stress in several human and animal diseases. In human or animal body, free radicals are produced naturally from the metabolism of nutrients and indigenous compounds. These free radicals can be   [32]. According to Ibrahim et al. [32], when oxidative stress set in, free radicals cause oxidation of vital biomolecules such as lipids, proteins, carbohydrates and deoxyribonucleic acid (DNA) in healthy human cells leading to diverse physiological disorders. Growth has been considered a very important indicator of living organisms health status. Lead as a toxic metal has been reported to alter body weight and relative organ weight of animals as the ingestion of this toxic substances has been previously linked with poor growth rate [34]. In addition, ingestion of lead was reported to have lead to increased relative organ weights as a result of necrosis and apoptosis triggered by the accumulation of lipids in such organs [45,46]. Likewise, relative organs weight is one of the markers to indicate oedema, atrophy, or hypertrophy of some organs [34]. Hence, the reversion of body weight and relative organs weights of the experimental animals treated with phenolic extract when compared with negative control group in this study infers that the extracts were able to prevent hepatomegaly and kidney enlargement caused by leadinduced toxicity. The higher protective activities of the extracts at dose of 300 mg/kg bw could be due to increased concentrations of phenolic compounds at this dosage which in turn maximumly prevent, protect or repair the tissues from lead induced oxidative damage.
Natural antioxidant compounds such as phenolics possess free radicals-quenching power which in turn results in the prevention of organs damage emanating from the actions of free radicals. The antioxidant activities of the phenolic compounds have been shown to be through a number of mechanisms which are structure-dependent, and these include: inhibition of the activities of the enzymes involved in the production of reactive oxygen species, stabilizing produced free radicals or chelating metal ions, or up-regulation of genes involved in the production of antioxidant enzymes [47]. The ability of the extracts (methanol and methanol/acetone phenolic-rich extracts) to exhibit excellent antioxidant properties through percentage DPPH scavenging activities, ferric reducing antioxidant power and inhibition of lipids peroxidation might be as a result of their phenolics content. As such, higher antioxidant activity of methanol/acetone phenolic-rich extract could be due to its higher phenolics content. This result agrees with the report of Busari et al. [11] who reported higher phenolics content (phenols, flavonoids and tannins) of methanol/acetone extract of NLF than its methanol extract. The higher activities (in vivo) of MAPR actually corresponds to its antioxidant activities (in vitro) which could be through  stabilization of free radicals or metal chelation capacity (by donating hydrogen atom or electron to the free radical) which depend on the quantity of phenolic compounds present in the extract. Liver is considered the major target for numerous toxicants such as lead. Environmental and occupational exposure to lead can result in many changes in the architecture of the liver [48]. Autopsy reports of humans exposed to lead indicated that liver is the principal reservoir (33%) of lead, followed by kidneys. According to Adaramoye et al. [49], biochemical parameters such as total protein, albumin, bilirubin, AST, ALT, and ALP are major biomarkers for liver functions and integrity. In acute hepatotoxicity or mild hepato-cellular damage, there is upsurge levels of these biochemical markers, but are observed to decrease with long-time intoxication due to liver damage [34]. In this study, the oral administration of lead acetate resulted in significant increased levels ALP, AST, ALT and conjugated bilirubin, while significant decrease in the levels of total protein and albumins was concomitantly observed. Liver structural integrity damage has been associated with increased levels of AST and ALT resulting from toxicants such as lead. The function of ALP in maintaining integrity of plasma membrane for proper functioning of organs [50]. In our study, a decrease in total protein level was observed in the liver tissues, which agrees with the report of El-Zayat et al. [51], who observed decrease in hepatic total protein levels resulting from lead intoxication. Inhibition of protein synthesis caused by lead may be as a result of its ability to cause damage or mutation to DNA or RNA [52]. Lead causes DNA mutation via base pair mutation, oxygen radical attack on DNA or deletion. In addition, Pb 2+ disturbs homeostasis of Ca 2+ intracellularly, and causes damage of endoplasmic reticulum resulting in reduced protein synthesis. Reduced albumin level was observed in rats treated with Pb owing to the decreased hepatic protein synthesis. The hypoalbuminemia observed in this study is in agreement with the study of Lakshmi et al. [53]. Offor et al. [54], bilirubin has been implicated in the protection of cell membrane against oxidative damage induced by metals. Thus, increased bilirubin levels observed in rats treated with lead acetate may be due to excessive destruction of heme and blockage of biliary tract resulting in the inhibition of the conjugation reaction and the release unconjugated bilirubin from damaged hepatocytes [50].
However, oral administration of methanol and methanol/acetone phenolic-rich extracts of NLF ameliorated the liver damages by reversing abnormal increase or decrease in the levels of the liver biomarkers. This effect also reflected in the liver histology of the rats treated with the extracts. The rats treated with the extracts showed mild hepatocytes necrosis, central vein congestion, vacuolar degeneration as well a gradual hepatocytes regeneration. The excellent and most promising activity observed with methanol/acetone phenolic-rich extract of NLF was not surprising due to its excellent exhibition of in vitro antioxidant activity when compared to the methanol phenolic-rich extract of NLF. Phenolic compounds such as tannins, flavonoids and other phenolics possess ability to quench free radicals such reactive oxygen species (ROS), which in turn prevent oxidation of essential biomolecules [32]. The chelating properties attributed to phenolics could be a contributing factor to the prevention of DNA and RNA damage, which would have been inflicted by lead via its interaction with intracellular Ca 2+ signalling and the destruction of hepatic endoplasmic reticulum.
The increased levels of blood urea and creatinine in lead acetate-treated rats implies inability of the kidneys to excrete these by-products leading to their elevated levels in the blood and decreased excretion in urine [55]. Longtime exposure to lead resulted in the retention of electrolytes evidenced by upsurge levels of sodium, potassium and chloride since lead has been implicated in distortion of renal tubular transport mechanisms. The other possible way that might result in elevated levels of these electrolytes is the degeneration of functioning nephrons that stimulate a number of adaptive processes in the augmented rates of electrolytes reabsorption [56,57]. Nonetheless, treatment with the phenolic-rich extracts of NLF restored all the abnormalities in the levels of these biomarkers with methanol/acetone phenolic-rich extracts having higher activity and brought the levels of these parameters close to normal and can be well compared with standard drug, silymarin. The histology of the kidneys validates the reversion of the kidneys damage via manifestation of less capsular and tubular degeneration alongside decreased inflammatory cell infiltration. The ability of the phenolic-rich extracts to play roles of reducing agents, metal chelators, singlet oxygen quenchers, radical scavengers and hydrogen donors could be a collective factor that contributed to the reversal of liver and kidneys damage resulting from lead-induced toxicity, by normalizing the levels of their biomarkers.
Apart from the previously mentioned mechanisms through which phenolic compounds could elicit their actions; Barrera et al. [58] also suggests that phenolic compounds can also exhibit hepatoprotective effects by upregulating Nrf2 signalling, a known transcription factor that activates the transcription of genes involved in cytoprotection [59,60]. These antioxidant compounds were also observed to simultaneously downregulated NF-kB [61], a type of transcription factor that activates the transcription of inflammation-related genes [62]. Thus, hepato-renal protective effects of the phenolicrich extracts which resulted into amelioration of leadinduced hepato-renal injury in extracts treated rats in this research could be as a result of upregulation of Nrf2 and/or downregulation of NF-kB transcription factors as suggested by aforementioned researchers.

Conclusions
It can be concluded that phenolic-rich extracts from Nauclea latifolia fruit exhibit ameliorative effects on lead acetate-induced liver and kidney toxicity in male Wistar rats with the exhibition of higher activities in methanol/acetone phenolic-rich extracts of the NLF.