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The Potential Hepatoprotective and Antioxidant Activities of Astragalus davisii against Paracetamol Induced Liver Damage in Rats

The current study aimed to estimate in vitro antioxidant effect of Astragalus davisii (A. davisii) extract. Further, the possible protective effect of A. davisii against paracetamol (PCM)-induced liver injury was assessed in rats. A. davisii was tested for its antioxidant activity using DPPH radical scavenging assay. The hepatoprotective potential of the extract was assessed in rats following oral Original Research Article Foudah et al.; JPRI, 32(23): 102-114, 2020; Article no.JPRI.61125 103 administration for 7 days. Liver injury was induced in rats following oral administration of PCM overdose. Hepatic biomarkers; alanine-aminotransferase, aspartate-aminotransferase, alkalinephosphatase, γ-glutamyl transferase and bilirubin were increased, while total protein and albumin were reduced in PCM control animals. Additionally, the activities of superoxide dismutase (SOD), catalase and glutathione-peroxidase (GPx) and the levels of glutathione were significantly declined, while levels of hepatic malondialdehyde (MDA) were significantly elevated in PCM alone treated rats. Oral administration of A. davisii (400 mg/kg) prior to PCM inhibited the elevation in the levels of liver damage markers in serum and protected against oxidative stress. Histopathological remarks confirmed the hepatoprotective potential of the extract. The results suggest that A. davisii extract at 400 mg/kg protects liver against injury induced by PCM overdose.


INTRODUCTION
The liver is a complex organ that is necessary for survival due to its role in the coordination of the metabolic activities, including glucose homeostasis, xenobiotic metabolism and detoxification. Industrial toxins, drugs, free radicals, food additives and alcohol are the risk factors for developing liver diseases [1]. PCM, (also known as acetaminophen, N-acetyl-p-aminophenol or APAP) is a commonly used mild analgesic and antipyretic available in numerous prescription and over-the-counter formulations [2]. PCM is widely used as a safe analgesic and antipyretic medication. However, its high dosages can lead to serious hepatic damage. Toxicity of PCM-overdose is attributed to the deleterious effect of its reactive metabolite known as N-acetyl-p-benzoquinoneimine (NAPQI). Elevated level of NAPQI-mediates oxidative damage, and therefore promotes tissue injury and organ dysfunction; including liver [3]. Currently available synthetic medications indicated for the management of liver disorders are inadequate and known for several adverse effects. Hinson et al., 2010 suggested that hepatotoxicity induced by paracetamol is mediated by oxidative damage [4].
Further, previous studies reported that, the use of plants with antioxidant activities minimizes the danger of liver disorders [5]. The genus Astragalus is the largest in the Leguminosae family and is cosmopolite, with 2000-3000 species grouped in 100 subgenera [6]. Some species of Astragalus has a history of being widely used in traditional medicine as antiperspirants, diuretics, and tonics for a wide array of diseases such as nephritis, diabetes mellitus and hypertension [7]. Others are a source of the economically important natural product, gum tragacanth. It is reported that some of Astragalus plants are well-known for their hepatoprotective, antiviral and immunostimulant activities [8]. Previous reports showed Astragalus plants to be a great source of saponins, flavonoids, and polysaccharides [9]. Total flavonoids, obtained from Astragalus demonstrated potent antioxidant effect and blocked the lipid peroxidation caused by O 2 and H 2 O 2 [10]. Growing evidence indicates that PCM enhances the excessive production of reactiveoxygen species (ROS), which resulted in massive lipid peroxidation and subsequent hepatic damage. Therefore, a substance with antioxidant effect may successfully prevent the development of liver damage due to PCMoverdose. Hence, this study aimed to assess the potential hepatoprotective potential of A. davisii extract against PCM-induced liver injury in rats.

Plant Extraction
The collected roots were shade dried, grinded to fine powders and extracted with 80% aqueous ethanol (2×3 L) under reflux. Ethanol extract of A. davisii was subjected to rotary evaporation (70±2°C) and lyophilized using freeze drier to yield 20.4 g and 15 g, respectively [11].

Estimation of in vitro Antioxidant Effect
In an attempt to estimate the antioxidant potential of A. davisii extract, the DPPH free-radical scavenging assay was used [12]. A solution of 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) in 0.004% methanol solution was prepared. Different concentrations of the extract were prepared in methanol. In a 96-well plate, 20 µl of each extract concentration was added to180 µl DPPH solution. The resultant mixture was incubated for 30 min at room temperature and the absorbance was estimated at 520 nm. DPPH solution was used as a control while methanol was served as a blank. In addition, pyrogallol was used as a positive control. The scavenging activities were calculated according to the equation: Where A control is absorbance of control (without the extract or Standard), and A sample is absorbance of sample. A plot between % inhibition verses concentration was used for the calculation of EC 50 of samples or standards.

Experimental Animals
In the current study, Wistar rats (both male and female, 180-200 g) were used. Animals were bred in Lab Animal-Care Unit, College of pharmacy, Prince Sattam-bin Abdulaziz University, KSA. Animals were kept under standard situation (at, 23 ± 2°C and 12-h lightdark cycle). Rats were fed standard-pellets and water ad libitum.

Acute Toxicity Study in Rats
To test the acute toxicity of A. davisii extract, two groups of healthy Wistar-rats (female) were used (n = 3). The study was carried out as per OECD guidelines-423, acute toxic class method OECD [13]. The extract was suspended in the vehicle (3% v/v Tween 80). Animals of the 1st group received the ethanol extract of A. davisii at oral dose of 2000 mg/kg (1 mL/100 g). Rats of the 2 nd group (control) medicated with the vehicle and kept under the same situations. The rats were observed for signs of toxicity, mortality, and morbidity for 24 h. They were also examined for the changes in neurological, autonomic, and behavioral profile. These rats were further observed for a period of 14 days.

Experimental design
Thirty mature male Wistar rats (180-200 g body weight) were divided into five equal groups as follows: Group I -Normal control (NC) rats received the vehicle at 5 mL/kg b.wt.
Group II -PCM control rats received the vehicle at 5 mL/kg b.wt.
Group III -Rats received silymarin (reference drug) at 100 mg/kg b.wt.
Group IV -Rats received A. davisii at 200 mg/kg b.wt.
Group V -Rats received A. davisii at 400 mg/kg b.wt.
The vehicle and the test extract were administered for 7 days using oral tube. Liver injury was produced on the 5 th day via oral dosing of PCM-overdose (2 g/kg) to all groups except rats in Group I.
At day-7, two h after last dosing, blood was collected from each animal through retro-orbital sinus plexus. Sera were taken out after centrifugation of blood samples at 4000 rpm for 15 min and maintained at −20°C until further biochemical evaluation.

Assessment of oxidative stress-related markers
Liver specimens were homogenized using Heidolph Diax 900 homogenizer, Germany, in 0.1 M phosphate buffer (25 mM, pH 7.4) and centrifuged at 18,000 rpm for 30 min. The supernatants were taken and stored at −80 • C till analysis. The levels of superoxide-dismutase (SOD), glutathione peroxide (GPx), catalase (CAT), reduced glutathione (GSH) and malondialdehyde (MDA) were evaluated via the assay kits obtained from Cayman Chemical Company (Michigan, USA) according to steps described by the manufacturer's guidelines.

Histopathological evaluation
Liver samples were kept in 10% buffered formalin, embedded in paraffin, and then serial sections of 5 μm thickness were cut. The sections of the livers were stained with hematoxylin and eosin (H & E) and evaluated for the pathological changes of hepatotoxicity [14].

Statistical Analysis
The results are expressed as mean ± standard error of six observations in each group. Data were analysed via one-way analysis of variance (ANOVA) followed by Dunnett's test using SPSS ver. 14.0. P-value of <0.05 was considered as the desired level of significance.

Estimation of in vitro Antioxidant Effect
The antioxidant effect of A. davisii extract was illustrated in Fig. 1

Acute Toxicity Study
Two out of three animals were showed good tolerance to the ethanol extract of A. davisii at 2000 mg/kg. For these animals, the dose of 2000 mg/kg was found to be non-lethal and did not show any noticeable signs of toxicity and mortality up to 14 days of observation. In addition, one of the animals exposed to 2000 mg/kg of A. davisii extract showed piloerection, lethargy and diarrhea and died 7 days after dosing. According to OECD-423 guidelines, LD50 value of A. davisii was calculated to be > 2000 -5000 mg/kg (category 5).

Assessment of liver damage markers in serum
The effects of A. davisii extract on the activities of ALT, AST, ALP and γ-GT in serum of rats with PCM-induced hepatotoxicity are summarized in Table 1 and their % changes are depicted in Fig 15.95% and 17.41% decreases in the serum activities of ALT, AST, ALP and γ-GT, respectively in comparison with the PCM control group. Moreover, pretreatment of the PCM-exposed rats with A. davisii extract at 400 mg/kg significantly (P<0.05) inhibited the elevations in the activities of liver enzymes in which 30.62%, 38.57%, 35.38% and 31.78% reductions were found in the serum activities of ALT, AST, ALP and γ-GT, respectively.
The effects of A. davisii extract on the serum levels of BRN, TP and ALB in rats with PCMinduced hepatotoxicity are depicted in Table 2 and Fig. 3. PCM-control rats demonstrated marked elevation in serum level of BRN and decrease in total protein and albumin values in comparison with normal group. Fig. 3 depicts that pretreatment of PCM-exposed rats with silymarin or A. davisii extract at 400 mg/kg exhibited 38.73% and 32.39% reduction in the PCMinduced increase in the level of BRN, respectively. In addition, they markedly increased the level of TP and ALB, resulting in significant restoration towards their control values. BRN, TP and ALB were insignificantly (P<0.05) changed in the group of rats given the low dose of A. davisii extract.

Assessment of oxidative stress-related markers
The effect of A. davisii extract on the oxidative stress-related markers of PCM-exposed rats was investigated via assessment of the levels of SOD, GPx, CAT and GSH in their liver tissue (

Histopathological study
The histopathological study added a supportive confirmation for biochemical analysis is depicted in Fig. 5. Sections from liver of the normal rats manifested typical lobular architecture and common hepatocytes with well-preserved cytoplasm (Fig. 5-A). additionally, PCM-control rats revealed congestion, severe necrotic changes, inflammatory cell infiltration in many areas (Fig. 5-B). Treatment with A. davisii extract at 400 mg/kg showed marked liver protection against PCM hepatotoxicity, which is manifested by the presence of normal liver cells and reduced inflammatory infiltration and necrosis (Fig. 5-C).

Estimation of in vitro Antioxidant Effect
Many antioxidants from natural origin oftentimes have potent free radical scavenging activities. The protection against the diseases by antioxidants is considered to be due to their free radical scavenging capabilities in the biological systems. The DPPH free radical scavenging ability has exceedingly gained importance to establish the antioxidant potential of plant extracts. In our study, A. davisii extract showed in vitro antioxidant effect. In similar earlier reports, Astragalus squarrosus showed free radical scavenging effect that might be due to its phenolic and flavonoid constituents [15]. Moreover, Astragalus sinicus showed antioxidant capacity measured by DPPH assay, and is correlated to its total phenolic content [16]. The antioxidant effect of phenolic compounds is predominantly due to their redox characteristics [17]. Accordingly, the present study indicated that the antioxidant effect of A. davisii extract is due to the presence of phenolic and flavonoid contents. It has earlier been reported that among most important constituent of the genus Astragalus are phenolic and flavonoid compounds [18].
In vitro antioxidant potential of a compound is inversely proportional to its EC50 value. In this regard, Lee et al., 2008 reported that EC50 value of <10 mg/mL suggests that A. davisii has effective antioxidant activity [19]. In our study, the EC50 values of A. davisii extract was found to be less than 10 mg/mL that confirm the effectiveness of the extract as antioxidant.

Acute Toxicity Study
In this study, A. davisii extract at 2000 mg/kg had no adverse effect on 2 out of 3 rats during 14 days of observation. At 2000 mg/kg, there was a case of mortality observed in only one rat throughout the experimental period. According OECD-423 guidelines, A. davisii extract was related to category 5 with LD50 > 2000 -5000 mg/kg. In a previous study, ethyl acetate extract from the aerial parts of Astragalus corniculatus, containing flavonoids, was found to be practically nontoxic as its acute oral toxicity value was > 5 g/kg in mice [6].

Hepatoprotective Activity
Liver is among the most important organs involved in metabolic activities. Hepatocellular injury induced by PCM overdose has been documented by a number of studies [19][20]. In this study, the potential hepatoprotective effect of

Fig. 5. Photomicrographs of rat liver (H & E stain, X400), (A) Normal control group showing normal histological structure of hepatic lobule; (B) PCM-hepatotoxic group showing necrosis and dissociation of centrolobular hepatocytes; (C) A. davisii (400 mg/kg) + PCM showing no histopathological changes
A. davisii extract was evaluated in PCMoverdose rat model and compared with the reference drug; silymarin. The serum levels of some remarkable biochemical parameters are used as characteristic markers of liver damage. One of the most diagnostic signs of liver damage is the liberation of intracellular enzymes, such as ALT, AST, ALP and γ-GT. The increased activities of liver marker enzymes are suggestive of cellular infiltration and the loss of the functional integrity of hepatocyte membranes that are constantly associated with hepatonecrosis [21].
In our study, the oral administration of PCMoverdose showed drastic elevation in the level of serum marker enzymes when compared with normal rats, indicating PCM mediated hepatic damages. Prescott et al., 1977 described the biochemical changes of PCM hepatotoxicity to be a considerable elevation in serum ALT and AST levels [22]. The elevation of these markers in serum following overdose of PCM could be referred to the damaged hepatocytes as these enzymes are located in cytoplasm and are released into circulation after hepatocyte injuries. The pretreatment of PCM-exposed rats with silymarin or A. davisii extract at 400 mg/kg exhibited inhibition of PCM-induced increase in the serum levels of ALT, AST, ALP and γ-GT enzymes, resulting in significant restoration towards their control values. In this investigation, the activity of A. davisii extract at 400 mg/kg was comparable with that of the standard drug silymarin. In addition, the reduction in the activities of liver marker enzymes in serum by the pre-treatment of PCM-exposed rats with A.
davisii extract (400 mg/kg) is a marker of repair of hepatic tissue damage caused by PCM.
The protective potential of A. davisii extract at 400 mg/kg might be a result of the improvement of oxidative stress and preservation of the antioxidant capacity conferred by the extract. This suppresses the leakage of ALT, AST, ALP and γ-GT enzymes into blood circulation. Moreover, the mechanism by which A. davisii extract reduced serum activities of liver enzymes may be referred to their capacity to inhibit the cytochrome P450 so decrease the generation of the reactive metabolites of PCM.
Serum BRN is considered as one of the important tests of liver functions. The increased concentrations of serum BRN are a sign of biliary obstruction, hepatic injury and alterations in binding, conjugation and excretory functions of hepatocytes [22]. The increased serum concentration of BRN in alone PCM-intoxicated rats suggests PCM-induced liver injury.
The liver is the main source of most of blood proteins as the liver cells are responsible for production of fibrinogen, albumin and most of the α-and β-globulins [23]. Therefore, the marked decrease in serum levels of total protein and albumin in PCM-control rats indicates cellular damage produced. In the current study, the marked decrease in serum levels of albumin and total protein in PCM-control animals might be a result of a reduction in the number of liver cells responsible for the albumin synthesis through necrosis or might be due to the functional failure of endoplasmic reticulum of hepatocytes [24]. Besides, reduction of serum albumin in PCM treated group may be due to binding of the reactive metabolite of PCM (NAPQI) to the amino acid cysteine in proteins, forming PCM protein adducts [25]. Pretreatment with A. davisii extract at 400 mg/kg reduced the increased serum level of BRN in rats due to PCM overdose, suggests the stabilization of biliary function as well as repair of damaged liver tissue of rats exposed to PCM [26]. The significant elevation in serum levels of total protein and albumin in A. davisiimedicated rats indicated the stabilization of the ER (Endoplasmic reticulum) that is responsible for the synthesis of protein. This assures the hepatoprotective activity of A. davisii extract against PCM-hepatotoxicity.
PCM hepatotoxicity is characterized by an extensive oxidative stress [27]. A key mechanism of the toxicity is cytochrome P450-catalyzed metabolic activation of PCM, which generates the highly reactive metabolite; NAPQI [28]. This reactive metabolite is responsible for oxidative stress and glutathione depletion and liver damage [29]. However, several cellular mechanisms defend the cells against the toxic effects of ROS generated by PCM and other toxicants. In this respect, hepatocytes have possess an antioxidant defense system including enzymatic and non-enzymatic antioxidants to protect themselves against free radicals and oxidative stress. Physiologically, the levels of free radicals are maintained within normal concentrations by the enzymatic antioxidants, mainly SOD, GPx and CAT and non-enzymatic antioxidants such as GSH [30]. Oxidative stress leads to toxicity when there is an increased free radicals generation than the cell's capacity for their removal. Monitoring of the levels of the cellular antioxidants in biological samples is commonly used to determine the state of oxidative stress.
In this investigation, a marked decrease in activities of the antioxidant enzymes in liver homogenate was induced by PCM overdose. The results suggested that administration of A. davisii extract at 400 mg/kg had successfully restored the activities of the hepatic antioxidant enzymes of PCM-overdosed rats. Improvement in the actions of the antioxidant enzymes does provide evidence to an improvement in the endogenous antioxidant defense system [31]. Accordingly, the beneficial effect of the extract could relate to its ability to maintain the activities of the hepatic antioxidant enzymes of PCM-overdosed animals. Following treatment of PCMexposed rats with A. davisii extract at 400 mg/kg, the hepatic levels of SOD, GPx and CAT were significantly restored to established values that were not different from silymarin-treated group, suggesting the capability of the extract to scavenge and neutralize PCM induced oxidative stress.
GSH is the main non-enzymatic antioxidant that is responsible as the major defense mechanism during oxidative stress. At non-toxic dose, the reactive metabolite N-acetyl-pbenzoquinoneimine (NAPQI) of PCM was detoxified by the SH-group (sulfhydryl-group) of GSH forming paracetamol-GSH conjugate. In case of PCM-overdose, NAPQI depleted the stores of GSH in hepatocytes by as much as 80-90% and thereafter bound to cellular proteins leading to necrosis of hepatocytes [32] in this investigation, a marked reduction in hepatic GSH contents in PCM-exposed animals is an indication of tissue injury produced by free radicals. The depletion of cellular-GSH leaves the cell vulnerable to oxidative-stress following PCM-overdose. In our study, A. davisii extract (400 mg/kg) preserved the hepatic GSH contents of PCM-overdosed rats. This indicates the antioxidant potential of A. davisii extract against PCM-induced oxidative stress in rat liver. Therefore, we can predict that the tested extract may stimulate the synthesis of endogenous GSH that antagonizes the oxidative damage induced by PCM-overdose.
The significantly raised level of MDA, a byproduct of lipid peroxidation, generated by PCM overdose indicated free radical stress induced LPO [33]. In this investigation, MDA levels increased markedly in liver homogenate of PCM control rats in comparison with normal control. Subsequent treatment with A. davisii extract at 400 mg/kg led to a significant decrease in MDA levels in PCM-exposed rats. LPO is one of the hallmark properties of oxidative stress in PCMinduced liver injury. According to Luqman and Rizvi [34] LPO is known to injure the cells by inactivation of membrane enzymes, decrease fluidity of the membrane and resolve into cytotoxic aldehydes such as MDA. Kanbur et al [35] have reported MDA, a product of LPO, to increase in the liver-tissues of PCM-induced liver injury in mice.
In this investigation, LPO induced by PCMoverdose was confirmed by the increase in hepatic level of MDA. The increased liver contents of MDA has been regarded as a measure for cellular injury and suggests disability of the antioxidant defense system to protect against the generation of excessive free radicals.  [20]. Plants of the genus Astragalus are well known to contain biologically active compounds such as polyphenolics, triterpenoid saponins and polysaccharides. Flavonoids represent the largest group of polyphenolic compounds occurring in Astragalus species [6]. It is recognized that flavonoids can balance various classes of oxidizing species such as hydroxyl radical, superoxide anion or peroxy radicals. Further, flavonoids may act as quenchers of singlet oxygen [36]. Additionally, the flavonoid mixture and polysaccharides of some Astragalus plants are documented to have strong antioxidant effect [37]. Therefore, we believe that the possible mechanism of hepatoprotection offered by A. davisii extract is due to its phytocomponents as flavonoids and polysaccharides.
The histopathological findings supported the biochemical results. As demonstrated in this study, PCM-control animals revealed serious histological injury that might be due to the generation of free radicals because of PCMoverdose and subsequent lipid peroxidation (LPO). Many studies demonstrated the induction of necrosis of hepatic cells by high doses of PCM in animals [38]. The histologic alteration were ameliorated in rats that were pre-treated with A. davisii extract suggesting marked protection of liver tissues against damage induced by PCMoverdose. It can be referred to the antioxidant effect of the extract that markedly decreased the oxidative stress and thereby reduced the histopathological alterations of the liver.

CONCLUSION
In conclusion, this study demonstrates that A. davisii had a protective effect against liver damage induced by PCM-overdose in rats. The hepatoprotective potential of A. davisii may be explained by increasing levels of antioxidant enzymes and decreasing the lipid peroxidation and liver enzyme on PCM-induced damage in rats. Additional phytochemical studies are in progress to separate and characterize the active compound(s) responsible for the antioxidant and hepatoprotective activities.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not approval.

ETHICAL APPROVAL
Experimental procedures were done in accordance with international regulations on animal protection, and/or the Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the Institutional Animal Ethics Committee.