Hepatoprotective Effects of the Leaves of Agauria salicifolia against Acetaminophen-Induced Liver Injury in Mice

Agauria salicifolia (Ericaceae) is a medicinal plant traditionally used for the treatment of liver ailments. The present study investigates the hepatoprotective effect of the residual aqueous fraction (RAF) of Agauria salicifolia on acetaminophen (APAP)-induced liver damage. The ethanol extract obtained as maceration of the dried leaves, was fractionated into hexane, ethyl acetate and residual aqueous fractions. Adult mice of both sexes were pre-treated with the reference drug silymarin (50 mg/kg) or RAF (100 and 200 mg/kg) during 6 days followed by a single administration of APAP (500 mg/kg) on day 7. The hepatoprotective effect and the contribution of antioxidant activities were evaluated by determining the level of transaminases in serum samples, the levels of proteins, nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase in liver homogenates. Histological analyses of liver slides were also performed. RAF at the tivities that might be mediated at least partially by its antioxidant effects.


Introduction
The liver is the largest organ in human beings, accounting for approximately 2% of his total weight [1]. Its key position in between the digestive tract and the systemic circulation makes it one of the major regulatory organs and one of the most exposed organs to various attacks by xenobiotic, pathogens and exogenous antigens. As such, the liver is subjected to numerous diseases among which, hepatitis is the most prominent. Two categories of hepatitis are described: infectious hepatitis which can be caused by viruses, parasites and bacteria [2], and non-infectious hepatitis which can be caused by excessive and chronic alcohol consumption (alcoholic hepatitis), metabolic diseases (metabolic syndrome, obesity, diabetes, and hypertriglyceridemia) which may lead to non-alcoholic fatty liver disease (NAFLD), autoimmune dysfunction (autoimmune hepatitis), genetic disorder (alpha-1-antitrypsin deficiency, hemochromatosis) or by toxins and medications.
Concerning the medication, acetaminophen (APAP) also called paracetamol, known for its analgesic and antipyretic properties, is of primary concern [3].
During the metabolism of APAP, it is converted by P450 enzymes into a highly reactive intermediate metabolite called N-acetyl-p-benzoquinone imine (NAPQI).
Under normal circumstances, NAPQI is rapidly converted and detoxified to a nontoxic metabolite by glutathione (GSH). However, in case of overdose or inappropriate use of APAP, part of NAPQI is detoxified after being conjugated with GSH and the remaining part attacks the hepatocytes and induces cell damages [4]. Pathways leading to these destructions comprise oxidative stress-induced molecules modification and enzymes inactivation, mitochondrial dysfunction and apoptosis, which will ultimately lead to cell death [5]. During the process of oxidative stress, lipids of the cell membranes are peroxidized, leading to membrane disruption. This lipid peroxidation is accompanied by the production of reactive lipid aldehydes, such as malondialdehyde. Oxidative stress is also known to induce inflammation by stimulating the production of inflammatory cytokines and increasing the level of NO synthesis. Therefore, it can be hypothesized that substances with antioxidant properties could have hepatoprotective effects against APAP-induced hepatocellular injury and cell death. In this prospect, many herbal formulations have been made available to ensure hepatocyte regeneration and protection against drugs-induced damages [6] [7]. The effectiveness of such remedy is underpinned by their content in secondary metabolites such as polyphenols and particularly flavonoids M. K. Tsague et al. Journal of Biosciences and Medicines which represent the most important and potent source of antioxidants. As an example, silymarin, a mixture of flavonoids from the herbal origin, is commercialized for its hepatoprotective activity.
In West Cameroon Region, Agauria salicifolia (Ericaceae) is used in folk medicine for the treatment of various diseases including liver ailments. Our previous studies showed that Agauria salicifolia contains phenolic compounds, possesses antioxidant properties and hepatoprotective effects against CCl 4 -induced hepatotoxicity, with the aqueous residual fraction (RAF) being the most efficient [8]. However, nothing is known about the effects of Agauria salicifolia against drug-induced liver damage. The present study aimed at investigating the protective effects of RAF against APAP-induced hepatotoxicity.

Animals
Mice (25 to 30 g) of both sexes were obtained from the animal house of the Department of Animal Biology, University of Dschang, Cameroon. The animals were housed in plastic cages and maintained at room temperature and in a natural light/dark cycle. They were fed with standard laboratory food and water ad libitum. Animals were maintained and used in accordance with the internationally accepted standard ethical guidelines for laboratory animal use and care as described in the European Community guidelines (EEC Directive of 2010; 10/609/EEC).

Plant Material and Extracts
The fresh leaves of A. salicifolia were harvested in the South West Region of

Acetaminophen-Induced Acute Hepatotoxicity in Mice
For the experimental design, 30 mice were randomly divided into five groups (I -V) of six animals each (3 males and 3 females) and treated orally as follows: Group I (naive) was used as normal control and they were given distilled water orally for seven days.
Group II (disease control) received daily oral administration of distilled water for six days and acetaminophen on the seventh day.
Group III (reference control) received silymarin (50 mg/kg) orally daily for six days and acetaminophen on the seventh day.
Group IV and V (test groups) received dally administration of RAF (100 and 200 mg/kg) for six days and acetaminophen on the seventh day.
Acetaminophen suspension was administered orally to animals belonging to groups II to V at the dose of 500 mg/kg, b.w. Six hours post-administration of APAP, blood samples were obtained via retro-orbital sinus plexus and the mice were later sacrificed. Blood was left to clot at room temperature and the serum was obtained by centrifugation at 3000 rpm for 10 min and was immediately used for alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) assays. The assays were performed using commercial kits from INMESCO, according to the manufacturer's instructions. The liver was rapidly collected after sacrifice, weighted and divided into two parts. The biggest liver lobe was fixed in 10% buffered formalin while the rest was used for the different biochemical measurements.

Measurement of Lipid Peroxidation
Lipid peroxidation assay was performed by determining spectrophotometrically the level of malondialdehyde (MDA) in liver homogenates as thiobarbituric acid reactive substances (TBARS) according to the methodology described by Fofie et al. [9]. Briefly, 100 μl of a sample, 500 μl of orthophosphoric acid (1%), 500 μl of thiobarbituric acid prepared in 1% trichloroacetic acid were added in a test tube. The mixture was kept at 100˚C in a water bath for 15 minutes and then cooled in iced water. After centrifugation at 3000 rpm for 15 min, the absorbance of the supernatant was read at 532 nm against a blank.

Measurement of Nitric Oxide (NO) Production and Proteins
The NO level was determined in the supernatant of liver tissue homogenates by the method of Griess as described by Nguelefack-Mbuyo et al. [10]. Briefly, 250 μl of the tissue sample was mixed with 250 μl of 1% sulfanilamide prepared in 5% orthophosphoric acid. After 5 minutes incubation in the dark, 250 μl of 0.1% naphthyl ethylenediamine was added, and then all incubated in the dark for an additional 5 minutes. The optical density was read at 530 nm. The quantity of nitric oxide was calculated from sodium nitrite's standard curve. Tissue protein

Catalase Activity Determination
Catalase activity was measured in liver homogenates according to the following protocol: to 50 μl of a sample, was added 500 μl phosphate buffer (200 mM) and 100 μl of H 2 O 2 (100 mM). The obtained solution was mixed with vortex and incubated at room temperature for 3 minutes. Then, 1000 μl of potassium dichromate solution (prepared by mixing one volume of 5% potassium dichromate with 3 volumes of glacial acetic acid) was added to the mixture. After that, tubes were incubated for 10 minutes in boiling water, cooled with tape water and centrifuged at 2000 rpm for 5 minutes to remove precipitated proteins. The supernatant was read at 570 nm against the reagent blank.

Superoxide Dismutase (SOD) Activity Determination
Superoxide dismutase (SOD) activity was measured in the liver homogenates as described by Wandji et al. [12]. The sample (70 μl) was mixed with 830 μl of carbonate buffer (pH 10.2). The reaction was initiated by introducing 100 μl of epinephrine (0.3 mM). The absorbance was read at 60 and 120 seconds after epinephrine was introduced, at 480 nm using a spectrophotometer (Helios Epsilon). The SOD activity was expressed as units/mg of liver protein. One unit was defined as the enzyme activity that inhibited the auto-oxidation of adrenaline by 50%.

Determination of Glutathione
The determination of GSH in liver samples was done using the Ellman's reagent and according to the method of [13]. One milliliter of Na 2 HPO 4 · 2 H 2 O (0.3 M) and 1 mL of dithiobisnitrobenzoate (0.4 mg/mL prepare in 1% of sodium citrate) were successively added to 250 μl of a sample. The absorbance of the mixture was measured spectrophotometrically at 412 nm.

Liver Histopathological Analysis
Fragments of liver tissues in each group were collected in 10% buffered formalin for fixation at room temperature. These tissues were further embedded in paraffin. Sections of 5 -6 μm in thickness were made and stained with hematoxylin and eosin (H&E). These sections were microscopically examined for morphology and necrosis.

Statistical Analysis
Data are expressed as mean ± SEM for each group. Results were statistically analyzed using one-way analysis of the variance (ANOVA) followed by Tukey's post-test. Differences between groups were considered significant when the probability p was less than 0.05. Statistical analyses were performed using GraphPad Prism software (version 5.0).

Effect of the Residual Aqueous Fraction of A. salicifolia on the Body and Liver Relative Weights
The bodyweight of mice treated solely with APAP was not significantly different from that of healthy mice; none of the treatment administered also affected this parameter (Figure 2(a)). A significant (p < 0.01) increase of 26% of the liver weight was observed in disease mice when compared to the naive group. Only silymarin was able to significantly (p < 0.01) prevent this liver overweight by 25% (Figure 2(b)).

Effect of the Residual Aqueous Fraction of A. salicifolia on the Levels of Proteins and Nitric Oxide
Treatment of mice with APAP increased the liver's protein content by 36% as compared to the naive mice (Figure 3(a)). Administration of silymarin or RAF at both doses significantly reduced the increased protein content induced by APAP. APAP administration also significantly (p < 0.01) increased the nitric oxide level by 44% in the mice liver. Silymarin and ARF at the dose of 100 mg/kg, significantly reversed the NO increase (Figure 3(b)).

Effect of the Residual Aqueous Fraction of A. salicifolia on Catalase, Superoxide Dismutase, Glutathione and Lipid Peroxidation Levels in APAP Treated Mice
Although the APAP administration did not significantly reduce catalase activity in the liver, RAF at the dose of 100 mg/kg significantly (p < 0.05) increased it by 71.60% as compared to the control group (Figure 4(a)). None of the treatments administered significantly affected SOD activity (Figure 4(b)). APAP significantly (p < 0.001) and drastically reduced (85.5%) the level of GSH in the liver. Extract or silymarin administrations significantly reversed this reduction. Silymarin antagonized the reduction by 54% while extract prevented it by 39% and 43% at respective doses of 100 and 200 mg/kg (Figure 4(c)). As shown in Figure  4     control. Silymarin administered at the dose of 50 mg/kg inhibited the MDA production by 54% while RAF showed the best effect with inhibition percentages of 98% and 85% when administered at the respective doses of 100 and 200 mg/kg.

Effects of the Residual Aqueous Fraction of A. salicifolia on the Histology of the Liver of APAP-Treated Mice
Liver slices were examined at the level of the central lobular vein and the portal hepatic artery. APAP administration induced severe injuries when compared to the healthy liver from the naive mice ( Figure 5(a) and Figure 6(a)). These damages were characterized by features typical of drug-induced diseases, including alteration of hepatic architecture with trabecular collapse and disorder, infiltration of leucocytes, presence of ballooned cells and important necrosis as depicted by the hepatocellular degeneration ( Figure 5(b) and Figure 6(b)). Besides, in the liver of disease mice, a vascular congestion was also observed (Figure 6(b)).
These harmful effects of APAP were significantly attenuated by pre-treating the mice with silymarin ( Figure 5

Discussion
Acetaminophen (APAP) commonly called paracetamol is a well-known analgesic and antipyretic drug widely used, and often without medical prescription.
Meanwhile, overdose or improper use can cause significant morbidity and mortality. Indeed, APAP toxicity remains the most common cause of drug-induced liver failure [14]. In accordance with the literature, results from the present study showed an increase in serum transaminases, in liver weight, MDA and NO content, and reduction in GHS level that matched the histological alterations in APAP treated mice. These altered parameters were significantly corrected by the pre-treatment with RAF.  APAP is generally well metabolized in the liver. However, in the case of overdose, it is highly converted through cytochrome P 450 system into the toxic free-radical product N-acetyl-p-benzoquinone imine (NAPQI), which produces necrosis of hepatocytes located in central lobules [15]. AST and ALT enzymes then leak out from the liver into the bloodstream when these hepatocytes are disrupted [16] [17]. Serum levels of these transaminases reflect the degree of liver damage. It is, therefore, an important marker of the liver, routinely used in clinical diagnosis and experiments to appraise liver injury [18] [19]. To evidence this fact, histological analysis was performed. Results showed, in contrary to APAP treated mice, that liver slices from RAF or silymarin treated groups presented very low cell loss and leucocyte infiltration, as well as more or less normal liver architecture, confirming therefore, the hepatoprotective effect of RAF.
Besides the increase of ASAT and ALAT caused by APAP administration, an increase in liver weight and protein level was noted. It has been demonstrated that APAP intoxication is accompanied with hepatic congestion in humans and rodents [20] [21] that occurs early and before the appearance of necrosis. In mice, the congestion results from the accumulation of red blood cells within endocytic vacuoles and the Space of Disse with a collapse of the sinusoidal lumens [22]. This leads to an increase in liver weight and even liver proteins, reaching the maximum at about 6 h [23]. In the present study, RAF was unable to prevent the increase in liver weight. Besides, it was also noticed that mice treated with RAF still presented vascular congestion, strengthening the direct relationship between the increase in liver weight and liver congestion. Ito et al. [24] have attributed this liver congestion to the dysfunction of the sinusoidal endothelial cells. It could then be hypothesized that although RAF potently protects hepatocytes, it does not have the same effect on the function of endothelial cells.
APAP-induced liver disease is an inflammatory process and as such, many inflammatory mediators contribute to its development. One of these mediators is nitric oxide (NO) [25]. An increase in liver NO content in APAP treated animals is an obvious typical observation and the reduction in iNOS has been correlated with reduced serum ALT [26], although not very efficient for complete hepatoprotection. In this study, a significant increase in liver NO was indeed observed in APAP-treated mice and RAF pre-treatment significantly prevented it. This result suggests the inhibition of NO production as one of the mechanisms of RAF and further indicates its anti-inflammatory and/or antioxidant effect.
Oxidative stress in APAP liver intoxication highly involves NO which reacts with superoxide anion to form peroxynitrite, an oxidizing and nitrating agent. Peroxynitrite is normally detoxified by glutathione; but the latter is depleted by NAPQI in acetaminophen-induced hepatotoxicity [27] and therefore, enhancing the peroxidation. Results obtained in this study are in accordance with this mechanism and show that overdose of APAP in mice drastically depleted glutathione. Pre-treatment with RAF significantly prevented this depletion and may justify the hepatoprotective effect of this extract. It could then be speculated that antioxidant activity is one of the main mechanisms of action of RAF.
To further understand the RAF action mechanism, we focused on oxidative stress, given the paramount importance of this parameter in the development of APAP administration did not significantly affect the activity of both enzymes in the liver. Nevertheless, RAF significantly increased the catalase activity but did not significantly affect the SOD activity. These findings suggest that RAF has antioxidant properties either through a direct scavenging effect or by enhancing the endogenous antioxidant such as catalase and GSH.

Conclusion
The residual aqueous fraction of the ethanol extract of the leaves of Agauria salicifolia possesses hepatoprotective activities in acetaminophen treated mice. This hepatoprotective effect evidenced by histological and biochemical parameters is at least partially mediated by the antioxidant effects of the extract. This antioxidant effect of RAF might combine direct scavenging effect and enhancement of the activity of endogenous antioxidant enzymes.

Availability of Data and Materials
The data used and analyzed in this study are available from the corresponding author on reasonable request.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.