Investigation of The Biochemical, and Histopathological Effects of Alpinia Ocinarum Rhizome Extract Against Cisplatin-Induced Hepatotoxicity in Rats

This study aimed to evaluate the effect of Alpinia ocinarum rhizome extract (AORE) on cisplatin (CP)-induced hepatotoxicity in rats. In this regard, 44 male rats were divided into six groups including the control group, AORE control group, CP control group, and three groups of CP (7 mg/kg dose, on the tenth day) with AORE (at concentrations of 100, 200 and 400 mg/kg, daily for 14 days). After 14 days, the rats' livers were removed and their liver function was assessed using biochemical marker enzymes. Oxidative stress was assessed by evaluating malondialdehyde concentration and hepatic superoxide dismutase activity and a histopathological test was also conducted. According to the results, the group treated with CP had severe liver damage and the group treated with AORE had no harm compared with the control group. Among the groups receiving CP and AORE (at different concentrations), the group with 200 mg/kg AORE showed the lowest liver damage compared to the other two groups.


Introduction
It is known that cancer is one of the most important health issues around the world ( . Although this substance plays an important role in different cancer treatments, several negative impacts have been reported such as hepatotoxicity, ototoxicity, renal dysfunction, nausea, and vomiting (Poon 2009). It has also signi cant side effects on the kidney, however, it is metabolized by the liver (Townsend 2009; Yimit 2019 ; Ghosh S 2019). According to histopathological investigations, severe damages can be induced to the liver by CP such as degeneration of hepatocytes and it can moderate the sinusoid dilatation ( Lynch 2005;Miyamoto 2007). To alleviate the adverse effects of CP, antioxidant treatment has been used. Nephrotoxicity and hepatotoxicity have been shown to stem from oxidative stress produced by CP (Chirino 2009 ;Sezen 2008 ;Yüce 2007).
The medicinal plant of Alpinia o cinarum mostly grows in the tropical and subtropical regions of Southeast Asia. This is a kind of Chinese medicinal material, which is consumed as a food additive. Alpinia o cinarum Hance is obtained from the rhizome of ginger family, Zingibriaceae, and has a pleasant and pungent odor. Due to its high safety, it has been registered by the National Health and  ( Pillai 2018;Lin 2015).Previous studies have identi ed bioactive compounds of Alpinia o cinarum rhizome extract (AORE). Higher content of the compounds exists in the rhizomatous extract of Alpinia o cinarum compared to those found in the aerial parts. These bioactive compounds include linear, dimeric, and cyclic diarylheptanoids, volatile oils, avonoids, lignin, phenylpropanoid, and diterpenoids (Zhang 2016 Although there are several studies about the effect of AORE on preventing hepatotoxicity of CP, the biochemical mechanism or cellular response by which AORE protects the liver against CP-induced damage has not been investigated. We hypothesized that AORE prevents CP-induced hepatotoxicity due to having inherent antioxidant potential and improves metabolism and antioxidant defense mechanisms of the liver. The aim of this study was to evaluate the biochemical, and histopathological effects of AORE in rats against CP-induced hepatotoxicity.

Materials And Methods
Preparation of AORE Alpinia o cinarum rhizome was procured from the local market in December 2019 (Bushehr, Iran). The plant was washed twice with distilled water to remove any pollution and air-dried at room temperature for 24 h. Then, the freshly dried rhizomes were ground to obtain a ne powder. For the preparation of hydroalcoholic extraction, dried rhizomes were mixed with ethanol (95% v/v). The mixture was ltered by a Whatman lter paper (No. 4). The hydro-alcoholic extract was concentrated using a rotary evaporator under vacuum at 50 °C for 10 min. AORE was prepared in different concentrations of 100, 200, and 400 mg/kg. The extract was stored in a refrigerator at 4 °C prior to experimental trials (Lee 2009 ;Mazaheri 2014).

Animals
Forty-eight Wistar albino male rats (250-300 g) were procured from the animal house of the School of Medicine. The rats were maintained under a standard laboratory condition with thermally controlled temperature (20-25 °C), automatically illuminated room (12 h light: 12 h darkness) for one week. They were kept under proper diet and good ventilation during the experiment and they had free access to food and water. All experimental protocols were approved by the Institutional Animal Care and Ethics Committee of the Medical science of Jundishapur University (Ahvaz, Iran).

Experimental design
The rats were randomly divided into 6 groups (n=6 for Group I and Group II and n=8 for other groups): Group I (control); rats received normal saline by gavage for 14 days and received a single intraperitoneal injection of normal saline on the 10th day. Group II (CP -/AORE + ); rats received just AORE (400 mg/kg) daily by oral administration for 14 days without any injection of CP. Group III (CP + /AORE -); rats received normal saline by gavage for 14 days and received a single intraperitoneal injection of CP (7 mg/kg) on the 10th day. Group IV (CP + /AORE + 100); rats received AORE (100 mg/kg) for 14 days and CP (7 mg/kg) on the 10th day. Group V (CP + /AORE + 200); rats received AORE (200 mg/kg) for 14 days and CP (7 mg/kg) on the 10th day. Group VI (CP + /AORE + 400); rats received AORE (400 mg/kg) for 14 days and CP (7 mg/kg) on the 10th day. In groups that received both CP and AORE, AORE was administered one hour before CP treatment. On the 14 th day, biochemical, histopathological, and immunohistochemical assays were carried out. The entry and exit criteria were male rats, without receiving any medication before the study initiation and the use of healthy rats without anatomical abnormality.

Specimen collection
Xylazine (5 mg/kg) and ketamine (50 mg/kg) were utilized to anesthetize the rats after 14 days. Blood samples were taken from the heart of each rat. To evaluate serum liver enzymes, serum was separated from the blood. Thereafter, the rats' livers were removed immediately after sacri cing the rats. A part of the liver was washed using PBS and after weighing was freshly used for biochemical evaluations. The remaining tissue was xed in 10% buffer formalin for immunohistochemical and histological assays (Amiri 2018).

Evaluation of liver function
The biochemical markers of liver damage, including serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), albumin, total protein, and total bilirubin (T. bilirubin) levels were determined using Pars Azmoon kits (Tehran, Iran) according to the manufacturer's instructions.

Evaluation of oxidative markers
Malondialdehyde (MDA) concentration in liver homogenate was measured based on the reaction of thiobarbituric acid with MDA to form thiobarbituric acid reactive species (TBARs). The measurements were conducted colorimetrically at 534 nm and relative to MDA content similar to previous studies (Puntel 2007;Nelson 1993;Ohkawa 1979). using ZellBio kits (Germany).

Evaluation of antioxidant activity
Hepatic superoxide dismutase (SOD) activity was determined based on the ability of the enzyme to inhibit phenazine methosulfate-mediated reaction of nitroblue tetrazolium. The measurement was carried out colorimetrically at 560 nm according to previous studies (Lee 2009 ;Mazaheri 2014) using ZellBio kits (Germany).

Histopathological analysis of liver tissue
For histopathological assay and to determine the effect of CP and AORE on the liver, samples were xed in 10% (w/v) buffer formalin for 24 hours. The standard protocol was used to process and embed the tissue in para n and prepare sections with a thickness of 5 μm. The sections were then stained with eosin and hematoxylin and evaluated using a light microscope with a 40× magni cation by a histologist who was blinded to the groups and the corresponding treatments. Histological photomicrographs converted to quantitative analysis using a scoring system (Amiri 2018(. Based on the extent of cytoplasmic vacuolization, in ammatory cell in ltration, sinusoidal dilatation, degeneration, and congestion, the results scored as 0, 1, 2, and 3 representing normal, mild, moderate, and severe damage, respectively. Statistical analysis SPSS 19 (Chicago, USA) was employed for statistical data analysis. The measured data were reported as the mean value ± SD. The comparison among the groups was performed by one-way ANOVA followed by the Post hoc Tukey test and statistical signi cance was considered to be P < 0.05.

Liver biochemical marker enzymes
The levels of the biochemical marker enzymes for liver damage, including AST, ALT, ALP, and LDH for different groups are shown in Fig. 1. According to the results, the CP -/AORE + group showed no signi cant differences with the control group, whereas the level of the markers signi cantly enhanced (P <0.001) in the CP + /AOREgroup in comparison to the control group. The level of biochemical enzymes in the CP + /AORE + 100 group showed a signi cant increase compared to the control group (P <0.001). The level of AST and ALP enzymes in this group exhibited a signi cant decrease in comparison with the group treated with CP + /AORE -(P <0.001 and P <0.05, respectively).
Levels of chemical enzymes in the CP + /AORE + 200 group showed a signi cant reduction compared to the CP + /AOREand CP + /AORE + 100 groups (P <0.001). This group showed a signi cant enhancement in ALT enzyme level compared to the control group (P <0.001) but did not differ from the control group in other enzyme levels. The level of the enzymes in the CP + /AORE + 400 group showed a signi cant decrease in comparison with the CP + /AOREand CP + /AORE + 100 groups (P <0.001). In this group, a signi cant rise in the level of ALT (P <0.001), ALP (P <0.001), as well as AST (P <0.05) was observed compared to the control group. Even the ALT enzyme level was signi cantly enhanced compared to the CP + /AORE + 200 group (P <0.001).
Data represented in Table 1 show the serum total bilirubin, protein, and albumin levels. According to the results, the level of bilirubin in the CP + /AOREgroup increased compared to the control group, while the level of protein and albumin was reduced signi cantly compared. There was a statistically signi cant difference between the results of this group and the control group. The level of these serums in the CP -/AORE + group did not change noticeably compared to the control group and the difference between the results of the two groups was not signi cant.
Serum bilirubin level in the CP + /AORE + 100 group enhanced signi cantly compared to the control group and there was a signi cant difference in the results, but compared to the CP + /AORE -, CP + /AORE + 200, and CP + /AORE + 400 groups, signi cant changes were not observed and there was no statistical difference between their results. The serum protein level in the CP + /AORE + 100 group was reduced compared to the control group and there was a statistically signi cant difference between the results, while compared to the CP + /AORE -, CP + /AORE + 200, and CP + /AORE + 400 groups, there was no signi cant difference between the results. The results of serum albumin levels in this group showed similar results to total serum protein, however, the serum albumin level of group CP + /AORE + 200 was higher than that of the CP + /AORE + 100 group and there was a signi cant difference between their results.
The level of serum bilirubin in the CP + /AORE + 200 group increased signi cantly compared to the control group and there was a signi cant difference in the statistical results, but compared to the CP + /AOREgroup, it decreased with a statistically signi cant difference. The serum protein level in group CP + /AORE + 200 did not change remarkably relative to the control group and there was no statistically signi cant difference between the results. The result of the serum albumin level was similar to that of the total serum protein.
The serum bilirubin level in the CP + /AORE + 400 group rised signi cantly compared to the control group, however, it showed a decrement compared to the CP + /AOREgroup. The differences were statistically signi cant. Furthermore, the level of serum protein in the CP + /AORE + 400 group decreased relative to the control group and there was a statistically signi cant difference between the results, but there was no signi cant change and no statistical difference between the results of this group and the CP + /AOREgroup. The results of the serum albumin level for this group were also similar to those of total serum protein.

Oxidative stress
As shown in Table 2, the CP + /AOREgroup showed a signi cant increment in the level of MDA. According to the result of the CP -/AORE + group, the MDA level was diminished relative to the control group. All groups showed a signi cant difference with the control group (P <0.05). Moreover, the CP + /AORE + 200 group showed a lower liver MDA level than did CP + /AORE + 100 and CP + /AORE + 400. The difference between the result of CP + /AORE + 100 and CP + /AORE + 400 was not signi cant.
The SOD activity of the CP + /AOREgroup was signi cantly reduced in the liver tissue compared to the control group. On the other hand, an increment in the SOD activity of the CP -/AORE + group was not signi cant in comparison with the control group. The CP + /AORE + 400 group demonstrated a lower SOD activity level compared to the CP + /AORE + 100 and CP + /AORE + 200 groups in the liver tissue. A signi cant enhancement of the SOD activity was exhibited in the CP + /AORE + 200 compared to the CP + /AOREgroup.

Liver histopathological study
The photomicrographs of the liver tissue in different groups are presented in Fig. 2. The control group (Fig. 2a) showed normal histoarchitecture of lobules in the liver (sinusoids, hepatocytes, and Kupffer cells). No change in the liver structure was observed in the rats treated with AORE (CP -/AORE + ) (Fig. 2b). Fig. 2c illustrates a liver tissue from the CP + /AOREgroup. Administration of CP resulted in disorganization in lobules such as dilation sinusoids, congestion, hemorrhage, periportal leukocyte in ltration, hepatocyte vacuolization, enlarged nuclei, and focal necrosis with pyknotic (Score 3). On the other hand, the groups treated with CP and AORE (CP + /AO + 100, CP + /AORE + 200, and CP + /AORE + 400) exhibited little hepatic damage (Score 1) in comparison with those treated with CP alone (Fig. 2d-2f). The damage in these groups was determined as slight hydropic degeneration of hepatocytes and congestion of blood sinusoid. However, lower hepatic damage was observed in the CP + /AORE + 200 group relative to the CP + /AORE + 100 and CP + /AORE + 400 groups. The damage severity in all groups have been listed in Table 3 based on the de ned scoring.

Discussion
CP-induced hepatotoxicity is commonly characterized by the changes in biochemical, molecular, and histological parameters (Ekinci 2020). Speci c liver enzymes can be measured to determine hepatotoxicity. As liver enzyme activities are much higher than those of serum (about 1000 times), the serum enzyme activity will be doubled in case only 1% of hepatocytes necrotize occurs (Mir 2015). In this study, the levels of AST, ALT, LDH, and ALP, as the biochemical marker enzymes of damaged liver tissue, were measured to study the effect of AORE in the prevention of CP-induced hepatotoxicity. An increment in the enzyme levels is the result of losing functional integrity of hepatocyte cell membrane in response to adverse effects of some drugs as well as pathological conditions as cirrhosis leading to the leakage of these enzymes (Kadir 2013). In this study, the levels of the biochemical marker enzymes were the highest in the group that received CP compared to other groups. CP has been found to be accumulated in the liver leading to some toxic activities and increasing the hepatic cell permeability or provoking cellular destruction (Mansouri 2017).
The measurement of other liver parameters exhibited that serum total albumin and protein levels of the CP-treated group decreased relative to the control and CP -/AORE + groups, while the total bilirubin level enhanced. The increment in the total bilirubin level has been reported to be due to defense mechanisms against oxidative injury induced by free radicals. These mechanisms involve the reduction of free radicals by increasing the level of electron donors such as bilirubin (Hozayen 2014). Poor health and worse consequences can be inferred from the low levels of total protein (Ibrahim 2008). The alteration of the serum levels of bilirubin, albumin, protein, and the hepatic enzymes indicated impaired liver function in the CP + /AOREgroup. Similar results have been reported in previous studies. It has been indicated that the level of ALP, LDH, ALT, AST, and serum total bilirubin increased and the albumin and protein levels decreased in CP-treated groups showing CP-induced hepatotoxicity (İşeri 2007; Al-Malki 2014).
The liver function indices improved in the groups administered by AORE only or combined with CP.
Similar results were obtained for the control group and the CP -/AORE + group and the best results were observed in the CP -/AORE + 200 compared to other doses of AORE. The hepatoprotective effect of AORE might be due to the presence of antioxidants, bioactive phytochemicals such as avone and avonoids (Abbas 2018). Rajesh et al (Rajesh 2013) reported an enhancement in the serum hepatic enzyme levels in ischemic reperfusion rats treated by 200 mg/kg and 400 mg/kg of AORE. In another study by Xia et al. (Xia 2010). the ALT activity was similar to that of the control group in rat models administered by AORE. It was also reported by Abbas et al. (Abbas 2018) that using 400 mg/kg of AORE led to the improvement of the liver function indices.
In this study, liver damage caused by CP was also determined by the increase in MDA besides the reduction of hepatic antioxidants such as SOD activity. Potential oxidative stress induction by CP injection which results in cell death in liver tissue was reported by previous studies (Ekinci 2020;Hassan 2020 ; Choi 2019 ;Soares 2013; Ekinci 2017 ). Oxidative stress is caused by the overproduction of reactive oxygen species and/or antioxidant mechanism failure (Karale 2017). In normal metabolism, produced reactive oxygen species are removed in the liver through speci c reactions in which some enzymes including catalase, SOD, peroxiredoxin, and glutathione peroxidase (GSH Pxs) are involved. In the case of oxidative stress exposure like CP-induced hepatotoxicity, a large volume of reactive oxygen species beyond the detoxi cation capacity is produced in liver cells. Therefore, hepatocyte injury is caused by DNA damage (reduced proliferation and promoted cell death), lipid peroxidation (radical formation and in ammation), protein oxidation (reduced enzymatic functions and growth factor inhibition), as well as a direct effect on signal pathways (reduced NO level, reduced angiogenesis, and transcription factor activation) (Bilgic 2018).
The treatment of the groups with AORE led to a noticeable decrement in the hepatic MDA content together with an increment in hepatic SOD activities. Similar performance was observed in the control and CP -/AORE + groups and the CP + /AORE + 200 exhibited the best results in liver function indices. The antioxidant effect of AORE is stemmed from avonoid antioxidant compounds (Sani 2019; Tungmunnithum 2020). The avonoid (galangin) has free-radical scavenging activities resulting in suppression of the genotoxicity of chemicals and modulation of enzyme activities (Swain 2020).
According to the histopathological studies, the liver structure of the CP -/AORE + group was similar to that of the control group, while hepatic damage was observed in the CP + /AOREgroup. Treatment with AORE led to the alleviation of liver injury in rats. The results are in good agreement with other histopathological investigations revealing the incidence of less damage to the liver tissue in AORE-treated rats in comparison with reperfusion/hepatic ischemia-induced damage in rats ( Rajesh 2013)

Conclusions
To summarize, the positive impact of the AORE administration on the improvement of hepatotoxicity in rats induced by CP was demonstrated. This is conducted through a number of intracellular pathways such as improving liver function parameters, and suppressing oxidative stress, as well as modifying antioxidant defense mechanisms. The rise in the levels of ALT, AST, LDH, ALP, and serum total bilirubin as well as the reduction of serum total protein and albumin were the determining factors for hepatic injury, which was remarkably improved in AORE-treated groups. Moreover, AORE treatment resulted in a reduction in SOD and an increase in MDA. In the histopathological examination of AORE-treated groups, signi cantly fewer hepatocytes damage was observed. Overall, the strongest antioxidant properties were shown in the CP + /AORE + 200 group. The results may modify cancer treatment protocols to include phytoceuticals to render safer cancer treatments.

Declarations
Ethics approval and consent to participate All institutional and national guidelines for the care and use of laboratory animals were followed.

Consent for publication
Not applicable' for that speci c section.

Availability of data and materials
The data that support the ndings of this study are available on request from the rst author or corresponding author upon request.

Competing interests
The authors declare that they have no con ict of interest.

Authors' Contributions
FN performed the histological examination of the Liver, and was a major contributor in writing the manuscript. AA participated in animal interventions and drug therapy phase and was a major contributor in writing the manuscript. MA participated in conducting experiments and writing the article. NC analyzed and interpreted the data regarding the biochemical assay. All authors read and approved the nal manuscript. Tables   Table 1 The levels of serum total bilirubin, serum     the CP+/AORE-group; ***, p < 0.001 vs. the CP+/AORE+100 group; ααα, p < 0.001 vs. the CP+/AORE+ 200 group