Buchholzia Coriacea Exhibited Antinecrotic, Antioxidant and Anti-Inflammatory Potentials in the Sub-Acute Dose of Paracetamol Induced Toxicity

and Anti-Inflammatory Potentials in the Sub-Acute Dose of Paracetamol Induced Toxicity. Abstract This study was designed to investigate the potential ameliorative effect of the ethanolic seed extract of Buchholzia coriacea in paracetamol-induced toxicities in male wistar rats using N-acetyl cysteine as a standard drug. Thirty-two male wistar rats were used for the study. The animals were divided into eight groups according to their weights, with four rats in each group. Group 1, which is the control, received only normal diet. Group 2 (negative control) received 14.28 mg/kg b.w of paracetamol daily. Groups 3 and 4 received 14.28 mg/kg b.w of paracetamol each followed by 200 and 400 mg/kg b.w of extract after 6 hours of paracetamol administration respectively. Groups 5 and 6 each received 14.28mg/kg b.w of paracetamol followed by 70 and 150 mg/kg b.w of N-acetyl cysteine respectively after 6 hours of paracetamol administration. Groups 7 and 8 received 200 and 400 mg/kg b.w of extract respectively once daily. These treatments were given orally for 28 days. The animals were sacrificed and the organs (liver and kidney) were excised, homogenized and analyzed for gene expression using quantitative polymerase chain reaction and gel electrophoresis. The results showed that the plant extract elicited upregulation


Introduction
Liver is a very important organ of the body which is responsible for majority of metabolism needed by the body for good health.
It makes many of the chemicals required by the body to function normally, it breaks down and detoxifies various metabolites, synthesizes proteins, and produces bio-chemicals necessary for digestion, and it also acts as a storage unit Kaplowitz [1]. The liver is highly specialized tissue consisting of mostly hepatocytes, regulates a wide variety of high-volume biochemical reactions Maton, et al. [2]. Furthermore, the kidney is another important organ of the body which is responsible for maintaining homeostasis in the body and is susceptible to certain drugs, environmental toxicants and protein overload Wasung, et al. [3]. The kidney also receives input from the parasympathetic nervous system, by way of the renal branches of the vagus nerve, although, the function of this is yet unclear Bard, et al. [4,5]. Nephrotoxicity is toxicity in the kidneys, it is a poisonous effect of some substances both toxic chemicals and medications on renal function. There are various forms that some drugs may affect renal function in more than one-way Galley [6].
RNA snap kit reagent across the groups. Tissues were homogenized and then stored in a refrigerator.

Gene Expression Profiling
Gene expression analysis: Total tissue RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's protocol.
RNA pellets were resuspended in diethyl/pyrocarbonate-treated deionized water. RNA samples were analyzed by agarose gel electrophoresis and integrity was confirmed by visualization of intact 18S and 28S rRNA under UV light. Spectrophotometric study (NanoDrop, Thermo Scientific 2000c) was used to confirm the purity of total RNA and then its concentration was determined.
The 1-2 μg RNA was reverse transcribed to cDNA at 42ºC for 60 min. After enzyme inactivation (95ºC, 10 min) cDNA fragments were amplified for 35 cycles using gene-specific primers for the genes of interest. PCR products were resolved on 2% agarose gels and visualized using Uvitec gel documentation systems (Uvitec ArminTeb, Iran). Gene expression study was carried out by statistical analysis using Uvitec Fire Reader Software (Cambridge) to compare the groups.

Discussion
Liver and kidney damage induced by drugs are world wide's health problem. It is a major health issue that challenges not only health care professionals but also the pharmaceutical industry and drug regulatory agencies James, et al. [13]. Liver is one of the most important organs in the biotransformation of food, drugs, endogenous and exogenous substances. Profuse supply of blood and the presence of many redox systems (e.g., cytochromes and various enzymes) enable liver to convert these substances into different kinds of inactive, active or even toxic metabolites. The burden of metabolism and exposure to dangerous chemicals make liver vulnerable to a variety of disorders, such as acute or chronic inflammation, toxin or drug-induced hepatitis, cirrhosis and hepatitis after viral infection Sherlock and Dooley [14].
Kidney is another crucial organ. It functions in regulating blood and urine homeostasis in the body. It receives messages from the parasympathetic nervous system, meaning, it connects to the brain and the lower part of the spinal cord to affect its function Bard, et al. [4]. Kidney can also be exposed to poisonous substances that result in nephrotoxicity, such substances can either be chemicals or medications on renal function.
Acetaminophen (N-acetyl-p-aminophenol, APAP; paracetamol) is a commonly prescribed analgesic and antipyretic and its overdose can cause overproduction of ROS during formation of N-acetylp-benzoquinone imine (NAPQI) by cytochrome P450 leading to hepatic toxicity and nephrotoxicity Jaeschke, et al. [15]. As such, the United States Food and Drug Administration recommends N-acetyl cysteine (NAC), a known antioxidant, as the only therapeutic option for APAP-overdosed patients Green, et al. [16]. However, this medication has its limitations including adverse effects and narrow therapeutic window Du, et al. [17]. Hence, the development of new drugs that are superior to NAC, in terms of effectiveness and therapeutic time frame, is clearly needed. In recent years, there have been intensive studies demonstrating the antioxidant and hepatoprotective effects of natural products against APAPinduced hepatotoxicity in experimental animals Ang-lee, et al. [18]. Therefore, we present this research work to demonstrate the significant effect of B. coriacea extract as dose dependent protective measure against paracetamol induced hepatoxicity and nephrotoxicity.
Glutathione Peroxidase-1 (GP x -1) GP X -1 is ubiquitously expressed in many tissues, where it protects cells from oxidative stress Brigelius-Flohé, et al. [19]. GP X -1 gene produces a protein that protects us from the damaging effects of free radicals which can lead to tissue damage and speed up aging processes Higashi, et al. [20]. Within cells, it is concentrated on a particular spot of the cytoplasm and mitochondria Gouaze, et al. [21]. Glutathione peroxidase, GP X -1, functions in the detoxification of hydrogen peroxide to water Straif, et al. [22]. The glutathione peroxidase also catalyzes the reduction of other organic hydroperoxides. GP X -1 overexpression delays cell growth and protects them from H 2 O 2 toxicity. It drains the cellular reduced GSH pool and therefore, has protective effect on endothelial cell lining Faucher, et al. [23]. From the result in (Figure 1), no significant difference was observed in the relative gene expression of GP X -1 when all the groups were compared to one another. But figuratively, when paracetamol group (negative control) was compared to the basal control, there was a repression in the level of GP X -1.  P<0.05 (95% confidence interval), Turkey post hoc test, 95.00% CL *p<0.033, **p<0.002, ***p<0.001 Since phytochemicals (flavonoid) from plants have been described as free radicals' scavengers Murni, et al. [25], therefore, the consumption of Buchholzia coriacea extract at 400mg/kg will be effective as an hepato-protective agent. In GPX-1 relative expression in the kidney as shown in (Figure 2), no significant difference was observed when the groups were all compared with each other. But figuratively, when paracetamol group was compared to the basal control, there was also a down-regulation in the level coriacea, if taken as food/drug supplement, could be more effective for preventing nephrotoxicity that can result from paracetamol overdose.

Cytochrome P450 1A2 (CYP1A2)
Cytochrome P450 1A2 (abbreviated CYP1A2 ), a member of the cytochrome P450 mixed-function oxidase system, is involved in the metabolism of xenobiotics in the body. In humans, the CYP1A2 enzyme is encoded by the CYP1A2 gene Nelson, et al. [26]. Of more than 300 identified P450 isozymes, CYP1A2 in chemical carcinogenesis play a critical role, and is classified as 'toxic' isoenzyme Borba, et al. [27]. The content of CYP1A2 in the liver is relatively high, carcinogens such as amino acids, aflatoxin, toxins and aromatic compounds are metabolized by CYP1A2 and eventually produce carcinogenic substances Chaudhry, et al. [28].

Fas Ligand
Fas (also called CD95 or APO-1 or TNFRSF6) is a type I transmembrane protein Ashkenazi, et al. [30], containing a death domain (DD) in its cytoplasmic region, which is essential for the induction of apoptosis Fesik [31]. The induction of apoptosis is triggered by the interaction of Fas with its ligand (FasL), a 40-kDa membrane protein Tanaka, et al. [32] allowing recruitment of the adaptor protein Fas-associated death domain (FADD) Peter, et al. when compared to basal control was not as high as that of negative control. In the study carried out by Jin [38], it was discovered that the antioxidant effect of N-acetylcysteine enables it to inhibit inflammation and apoptotic processes in human conjunctional epithelial cells in a high glucose environment. Based on our study, the extract was able to inhibit inflammation and apoptosis.

Cyclooxygenase -2 (COX-2)
COX-2 is an enzyme in humans that is encoded by the PTGS2 gene Hla, et al. [39]. In humans it is one of two cyclooxygenases. It is involved in the conversion of arachidonic acid to prostaglandin H2, an important precursor of prostacyclin, which is expressed in inflammation Rouzer, et al. [40]. COX-2 enzyme is also necessary for inflammation, a normal, healthy attempt by the body to heal itself.
However, when inflammation gets out of control (such as in the case of arthritis, or other chronic inflammatory disorders) ongoing pain and discomfort is the result. That's where botanical COX-2 inhibitors can help. Botanical COX-2 inhibitors block the action of the COX-2 enzyme, which helps reduce inflammation and pain. Rouzer, et al. [40]. From the relative expression of COX-2 gene as shown in (Figure 7), statistically, there was a significant difference at p<0.05 in paracetamol group (negative control) when it was compared to the basal control. There was also a significant difference at p<0.033 in the group treated with 14.28mg/kg paracetamol and 70mg/kg NAC when it was compared to the negative control group.
This explained that the extract at 200mg/kg and 400mg/kg respectively are able to downregulate the inflammatory property of paracetamol. There was a repression in the level of COX-2 gene when the groups treated with 14.28mg/kg paracetamol and 70mg/kg NAC, 14.28mg/kg paracetamol and 150mg/kg NAC were compared to both basal control and negative control, although the expression of the gene in the group treated with 14.28mg/kg paracetamol and 150mg/kg NAC was more repressed. Recent clinical trials suggest that N-acetyl cysteine replenishes glutathione stores, scavenges hydroxyl free radicals and also has anti-inflammatory properties Dimari , et al. [46] and this confirms the study carried out by Erica, et al. [47] in which the anti-inflammatory property of NAC enables it to enhance the down-regulation of COX-2 gene induced by paracetamol toxicity. Likewise, in the treatment with 14.28mg/ kg paracetamol and 150mg/kg of NAC, NAC was able to repress the inflammatory property of paracetamol. Therefore, the result suggested that the extract was able to serve as an analgesic drug and also prevent inflammation caused by paracetamol toxicity.

Glutathione S-Transferase (GST)
The glutathione S-transferase (GST) gene family encodes genes that are critical for certain life processes, as well as for detoxication and toxification mechanisms, via conjugation of reduced glutathione (GSH) with numerous substrates such as pharmaceuticals and environmental pollutant Franco, et al. [48].
GSTs have multiple biological roles, including cell protection against oxidative stress and several toxic molecules, and are involved in the synthesis and modification of leukotrienes and prostaglandins Hayes, et al. [49]. As an example, GSTs protect cellular DNA against oxidative damage that can lead to an increase of DNA mutations or induce DNA damage promoting carcinogenesis Li, et al. [50]. GSTs are able to conjugate glutathione (γ-l-glutamyl-l-cysteinyl-glycine, GSH) to a wide range of hydrophobic and electrophilic molecules including many carcinogens, therapeutic drugs, and many products of oxidative metabolism, making them less toxic and predisposed to further modification for discharge from the cell Hayes, et al. [49]. An analysis of the GST gene family in the human genome organizationsponsored human gene nomenclature committee database showed 21 putatively functional genes.
Its primary function is to detoxify xenobiotics by catalyzing the nucleophilic attack by GSH on electrophilic carbon, sulfur, or nitrogen atoms of specific nonpolar xenobiotic substrates, thereby preventing their interaction with crucial cellular proteins and nucleic acids Josephy [51,49]. The GST genes are upregulated in response to oxidative stress Bae [52]. From the relative expression of the GST gene in the liver as shown in (Figure 8  P<0.05 (95% confidence interval), Turkey post hoc test, 95.00% CL *p<0.033, **p<0.002, ***p<0.001

Interleukin-6 (IL-6)
Interleukin-6 is an interleukin that act as a pro-inflammatory cytokine and in human; it is encoded by the interleukin-6 gene Ferguson-smith, et al. [53]. After IL-6 is synthesized in a local lesion in the initial stage of inflammation, it moves to the liver through the blood stream, followed by the rapid induction of an extensive range of acute phase proteins such as C-reactive proteins (CRP), serum ameloid A (SAA), fibronogen, haptoglobin and α1-antichymotrypsin (Heinrich, et al. [54]. When high concentration of SAA persists for a long time, it leads to a serious complication of several chronic inflammatory diseases through the generation of amyloid A amyloidosis and this results in amyloid fibril deposition which causes progressive deterioration in various organs Gillmore, et al. [55]. IL-6 is involved in the regulation of serum iron and zinc levels via control of their transporters. As for serum iron, IL-6 induces hepcidin production which blocks the action of iron transporter ferroportin1 on gut and thus reduces serum iron level Nemeth, et al. [56]. This means that, the IL-6 hepcidin access is responsible for hypoferrinia and anaemia associated with chronic inflammation. P<0.05 (95% confidence interval), Turkey post hoc test, 95.00% CL *p<0.033, **p<0.002, ***p<0.001

IL-6 also enhances zinc importer (ZIP14) expression on hepatocytes and so induces hypozincemia seen in inflammation
Luizzi, et al. [57]. From the relative expression of IL-6 gene as shown in (Figure 10), there was no statistical difference when all the groups were compared to each other. But figuratively, there was an upregulation in the expression of IL-6 in the group treated with paracetamol (negative control) when compared to basal control.
While paracetamol is classified as an antipyretic drug, it does not have any significant anti-inflammatory activity McKay, et al. [42] which explains the upregulation in the level of gene in group treated with paracetamol Hochhauser [41]. A down-regulation in the level of the gene was also observed in the groups treated with 14.28mg/ kg paracetamol and 200mg/kg extract, 14.28mg/kg paracetamol and 70mg/kg NAC, 14.28mg/kg paracetamol and 150mg/kg NAC when compared to basal control and negative control respectively.
Nevertheless, the group treated with 14.28mg/kg paracetamol and 70mg/kg NAC showed more repression of the gene.
There was an up-regulation in the level of the gene in the gene treated with 14.28mg/kg paracetamol and 400mg/kg when compared to basal and negative control. In the group treated with 400mg/kg extract alone, a down-regulation was observed in the level of the gene when compared with negative control but there was no difference when compared with basal control. In the group treated with 200mg/kg extracted, a little upregulation was observed in the level of the gene when compared to basal control, but a down-regulation was observed for negative control. Based on the result from the experiment carried out by Ibiam [58], ethanolic extract of B. coriacea was proven to have anti-inflammatory potential which could be attributed to phytochemical components acting individually or collectively as seen in some of the groups treated with extract. But NAC has been scientifically proven to have a higher rate of antioxidant and anti-inflammatory properties Mata, et al. [59], which is in accordance with the previous report on the study carried out by Laura [60]. where pre-treatment and posttreatment with NAC in paracetamol administration diminished the elevation of interleukin 6 gene. This explains the downregulation in the group treated with paracetamol and 70mg/kg NAC. Though NAC is good as an anti-inflammatory drug, it can be suggested that treatment with extracts of B. coriacea would also serve as a potent anti-inflammatory agent and could be a good reparative drug in treating kidney injury.

Prostaglandin Synthase
Prostaglandin synthase, also known as cyclooxygenases (COX-1 and COX-2) is an enzyme in humans that is encoded by prostaglandin synthase gene (PTGS gene) Funk, et al. [61]. Prostaglandins P<0.05 (95% confidence interval), Turkey post hoc test, 95.00% CL *p<0.033, **p<0.002, ***p<0.001 Although, the group treated with 14.28mg/kg of paracetamol and 150mg/kg of NAC showed more repression of the gene in comparison with negative control. Based on recent clinical trials that suggest that N-acetyl cysteine replenishes glutathione stores, scavenges hydroxyl free radicals and also has anti-inflammatory properties Dimari, et al. [46], which is confirmed in the study carried out by Ancha [66,67] where treatment with NAC suppressed prostaglandin synthase expression to control values in TNBSinduced colitis in rats. Also, the expression pattern of prostaglandin synthetase on the graph has shown that the B. coriacea extract was able to elicit physiological effect on diseased kidney. The expression of prostaglandin that was repressed confirmed the report by Mosly, et al. [68] on the use of pharmacological agents to ameliorate the symptoms of kidney inflammation. Therefore, I suggest that though NAC is good as an anti-inflammatory drug, treatment with extracts of B. coriacea can also serve as an alternate natural potent antiinflammatory agent and in combination as an adjuvant, could be a good reparative drug in treating kidney injury.

Kidney Injury Molecule-1 (KIM-1)
P<0.05 (95% confidence interval), Turkey post hoc test, 95.00% CL *p<0.033, **p<0.002, ***p<0.001 Kidney injury molecule -1, also known as T cell immunoglobulin and mucin-1 (TIM-1) and hepatitis A virus cellular receptor 1(Havcr1) is a type 1 cell membrane glycoprotein Yang, et al. [69]. KIM-1 plays critical roles in regulating immune cell activity especially regarding the host response to viral infection. KIM-1 is also involved in allergic response, asthma, and transplant tolerance Meyers, et al. [70]. Kim-1 mRNA levels are elevated more than any known gene in rodents and humans after the initiation of kidney injury Lim, et al. [71,72]. Urinary Kim-1 has been shown to be a sensitive and early diagnostic indicator of renal injury in a variety of acute and chronic rodent kidney injury models Vaidya, et al. [72] and it was subsequently shown that KIM-1 is also a sensitive marker for kidney injury in children undergoing cardiac surgery Han, et al. [73]. Sancho-Martnez , et al. [74] investigated the use of measuring the mRNA expression or protein levels of KIM-1 and other biomarkers to detect nephrotoxicity in vitro. The expression of KIM-1 mRNA was previously reported to be markedly induced in rats in response to renal injury caused by cisplatin Ichimura, et al. [75]. From the relative expression of KIM-1 gene as shown in implies that if 400mg/kg of extract is taken as food supplement or as drug, it can useful as a measure to reduce nephrotoxicity. In the groups treated with 14.28mg/kg paracetamol and 70mg/kg NAC, 14.28mg/kg paracetamol and 150mg/kg, a downregulation was observed in the level of KIM-1 gene expressed. When the group treated with 14.28mg/kg paracetamol and 400mg/kg extract was compared to the control group, there was an upregulation observed in the level of the gene but a repression in the gene level when compared to paracetamol (negative control). According to the experiment carried out by Sao [77,78] on the protective effect of N-acetylcysteine on kidney as a remote organ after skeletal muscle ischemia-reperfusion, it was concluded that administration of N-acetylcysteine treatment significantly decreased renal injury by skeletal muscles ischemia-reperfusion. Likewise, in this study, NAC was able to lessen the effect of paracetamol and therefore, I suggest that intake of 70mg/kg NAC may be effective in preventing nephropathy induced by paracetamol toxicity.

Conclusion
Based on the present studies, Buchholzia coriacea could be studied for its possible antigenotoxic and biosafety before being considered for clinical trials as potent cytoprotective agent in the treatment of paracetamol induced nephro-and hepatotoxicities.