Rhizophora Mucronata Lam. (Mangrove) Bark Extract Prevents Ethanol-induced Liver Injury, Oxidative Stress and Apoptosis in Swiss Albino Mice


 The bark extract of Rhizophora mucronata (BERM) was recently reported for its prominent in vitro protective effects against liver cell line toxicity caused by various toxicants, including ethanol. Here, we aimed to verify the in vivo hepatoprotective effects of BERM against ethanol intoxication. An oral administration of different concentrations (100, 200, and 400 mg/kg) of BERM prior to high-dose ethanol via intraperitoneal injection was performed in mice. On the 7th day, liver and kidney sections were dissected out for histopathological examination. The ethanol intoxication caused large areas of liver necrosis while the kidneys were not affected. Pre-BERM administration decreased ethanol-induced liver injury, as compared to the mice treated with ethanol alone. In addition, the pre-BERM administration resulted in a decrement in the level of ethanol-induced oxidative stress, revealed by a concomitant increase of GSH and a decrease of MDA hepatic levels. The BERM extract also reversed the ethanol-induced liver injury and hepatotoxicity, characterized by the low detection of TNF-α gene expression level and fragmented DNA, respectively. Altogether, BERM extract exerts antioxidative activities and present promising hepatoprotective effects against ethanol intoxication. The identification of the related bioactive compounds will be of interest for future use at physiological concentrations in ethanol-intoxicated individuals.


Introduction
Ethanol, also called ethyl alcohol or alcohol, is considered one of the potent hepatotoxins capable of causing chronic liver damage (Julien et al., 2020). Liver diseases including alcoholic liver disorder (ALD) have been associated to chronic alcohol abuse leading to highest morbidity and mortality worldwide (Asrani et al., 2019;Crabb et al., 2020). The time and dosage contingent intake of alcohol increase the risk of ALD (Marugame et al., 2007). ALD progression is revealed by a series of liver diseases, which begins from fatty liver to swelling and noxious cells such as steatohepatitis, cholecystitis, and cirrhosis to ultimately develop into hepatocellular carcinoma (HCC) (Morgan et al., 2004). To overcome any side effects caused by toxicants or conventional chemotherapeutic drugs causing liver cell damage Ethanol consumption followed by its metabolism results in high toxic levels of acetaldehyde via alcohol dehydrogenase, which generates oxidative stress (Yin et al., 1999;Zima et al., 2001;Zhou et al., 2003).
Through its highly reactive nature, acetaldehyde interacts with cellular proteins, lipids, and deoxyribonucleic acid (DNA) leading to the production of adducts and reactive species, which subsequently cause elevated hepatotoxicity and severe liver injury. Acetaldehydes mainly cause the formation of protein adducts that are toxic and highly immunogenic (Niemelä, 2001). Consequently, acetaldehyde-adducted proteins and alcohol-induced oxidative stress increase the synthesis and release of tumor necrosis factor-alpha (TNF-α), an in ammatory cytokine mainly secreted by the macrophages and demonstrated to contribute to liver injury and damage (Sapkota et al., 2016;). In addition, one of the factors playing major roles in the alcohol toxicity is the oxidative stress caused by excessive generation of reactive oxygen species (ROS) (Bailey and Cunningham, 1998). Under normal physiological situations, the liver oxidative stress is regulated by the hepatic enzymatic (i.e., glutathione reductase) and nonenzymatic (i.e., reduced glutathione (GSH) and malondialdehyde (MDA)) antioxidant systems to maintain the cellular redox homeostasis. An excessive consumption of alcohol impairs the hepatic antioxidant system and results in lipid peroxidation, indicated by MDA, and in GSH de ciency (Chen et al., 2016;Pérez-Hernández et al., 2017).
Consumption of ethanol leads to another major consequence leading to cell fate towards programmed cell death (i.e., apoptosis), a complex process characterized by DNA fragmentation, which is occurred not only in the liver (Nanji, 1998) but also in other tissues including brain , salivary gland (Zhang et al., 1998) and gastric mucosa . Furthermore, ethanol toxicity also interferes with the electron transport chain that provokes mitochondrial dysfunction, apoptosis, cellular damage, and ultimately necrosis, a form of a premature cell death caused by autolysis and occurring in response to injury (Hoek et  Based on a previous report, using human hepatocarcinoma cell line HepG2, the plant parts of Rhizophora mucronata Lam. (R. mucronata), also known as Mangrove, including leaves, roots, owers, bark and fruits were shown to have promising therapeutic values and to be capable of neutralizing various toxicants, including ethanol intoxication (Jairaman et al., 2019). However, no studies have explored the in vivo biological protective impact of the bark extract of R. mucronata (BERM) against ethanol-induced liver injury and hepatotoxicity. Thus, in this present study, BERM was tested for its hepatoprotective properties against ethanol intoxication in Swiss albino mice. This in vivo assessment was made in an attempt to nd a novel and safe hepatoprotective drug against ethanol-induced liver injury. This study primarily focused on the evaluation of liver injury biomarkers, including the measurements of nonenzymatic antioxidant components, MDA and GSH, using oxidative stress-related biochemical assays; the monitoring of TNF-α gene expression level using real time-polymerase chain reaction (RT-PCR) technique; and the evaluation of cell damage based on apoptotic status using terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay. Hepatoprotective avonoid milk thistle seeds-derived Silybum marianum (Silymarin), well-known for its anti-oxidative properties, was used as a positive control throughout this study.

Sample Collection and Extract Preparation
During January 2018, the barks of R. mucronata Lam. were collected from Pichavaram Mangrove forest and were authenticated by Prof. P. Jayaraman with the specimen no: PARC/2018/3854 at Plant Anatomy Research Centre, West Tambaram, Chennai, India for future reference. The barks were dried in shade for 15 days, roughly powdered and kept in containers, which were impermeable to air and later used for further study.
The pre-weighed 500 g of powdered bark of R. mucronata were brought in a tight glass container with lid and soaked with ethanol: water (3:1 v/v) weighing about 1500 mL. The container was sealed and kept for a period of 2 weeks with sporadic mixing and agitation. The extract was then ltered through Grade I Whatmann lter paper. In order to get the crude bark extract of R. mucronata (BERM), the ltrate was evaporated at room temperature and stored in refrigeration at 4°C for further use.

Animal Procurement and Maintenance
The animals were procured from Biogen Laboratory Animal Facility (Bangalore, Karnataka, India). For the present study, healthy male Swiss C57/BL/6 Albino mouse strains (n = 36) aged between 8 and 10 weeks, weighing approximately 25 to 30 g, were obtained. The animal experiments were conducted in accordance with the ethical norms and guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (New Delhi, India) and was also approved by the Institutional Animal Ethical Committee (IAEC) of Saveetha Medical College (SU/CLAR/RD/002/2018).
The mice were shifted 10 days before the start of the experiment to the laboratory conditions for acclimation. The mice were kept in plastic cages and were marked on the tail for the identi cation of each individual. Throughout the experiment, the mice were fed with ADILAID Hemster pellets vegetable (Mumbai, India) and drank potable water ad libitum, except during the short fasting period where the supply of food was still in ad-lib but potable water was not supplied 2 h before the treatment.

Animal Study Design and Sample Preparation
The experiment was designed as per the previously published protocol (Padmanabhan and Jangle, 2014). The 36 Swiss Albino mice were sorted into 6 groups consisting of 6 animals in each group. Group 1: standard control group. For 6 days, the mice were orally administered with distilled water (5 mL/kg body weight (b.w.)). Group 2: ethanol-induced liver injury group. The mice were administered with ethanol (cat. #64-17-1, Sigma-Aldrich Corp.) alone. Group 3: silymarin (cat. #S0292, Sigma-Aldrich Corp.) + ethanol group. The mice were orally administered with a single dose of silymarin (50 mg/kg b.w.) prior to ethanol administration. Group 4: 100 BERM + ethanol group. The mice were orally administered with a single dose of 100 mg/kg b.w. BERM prior to ethanol administration. Group 5: 200 BERM + ethanol group. The mice were orally administered with a single dose of 200 mg/kg b.w. BERM prior to ethanol administration. Group 6: 400 BERM + ethanol group. The mice were orally administered with a single dose of 400 mg/kg b.w. BERM prior to ethanol administration. Except the untreated mice in Group 1, all the treated mice were given ethanol (5 mL/kg b.w. of 25% v/w ethanol), via intraperitoneal (i.p.) injection for 6 days, after half-hour oral administration of the plant extract.
On day 7, the mice were euthanized by applying pressure to the neck and dislocating the spinal cord. Kidneys and liver were removed, thoroughly rinsed with regular brine, then dried with tissue paper. The upper left lobe of the liver was cut with sterile scissors and wrapped in the aluminum foil, and kept at -70°C before processing RT-PCR for TNF-α gene expression level monitoring and TUNEL assay. The remaining part of the liver was mixed evenly to get the homogenate, which was dissolved in 0.2 M phosphate buffer (pH 7.4). By using a tissue homogenizer (MC Dalal & Co., Chennai, India), 10% homogenized liver tissue was made. After centrifugation at 2075 ×g for 15 min, the supernatant was utilized for the detection of MDA and GSH.

Histopathological Analysis
The liver and kidneys were rst xed in 10% formalin then they were dehydrated using gradual ethanol (50-100%), rinsed in xylene, and were impregnated in para n wax. The tissue sections (5-6 µm thickness) were generated using rotary microtome and later stained with haematoxylin and eosin (HE) dye for histopathological examination.
2.6. Oxidative Stress-Related Non-Enzymatic Assays 2.6.1. Estimation of Reduced Glutathione Reduced GSH was measured in the homogenized supernatant as described in (Moron et al., 1979). Brie y, in order to precipitate the proteins, 125 µL of 25% of trichloroacetic acid (TCA) were added to 0.5 mL of supernatant. The test tubes were cooled on ice for 5 min and the supernatant was then diluted with 0.6 mL of 5% TCA and centrifuged for 10 min at 9000 ×g. To the 0.3 mL of the aliquot, 0.7 mL of 0.2 M sodium phosphate buffer (pH 8.0) were added to make it up to 1 mL. Further, to the tubes freshly prepared, the 5-5'-dithio-bis(2-nitrobenzoic acid) (DTNB) solution (2.0 mL) was added. After 10 min, the formed yellow color produced by the presence of 2-nitro-5-thiobenzoic acid, generated from the reduced glutathione GSH and DTNB reaction, was read at 412 nm using a spectrophotometer (Lovibond, ACD Company, New Delhi, India). Similarly, standards were also included to measure the content of GSH.

Estimation of Malondialdehyde
The measurement of MDA detected in the homogenized supernatant was carried out as described in (Högberg et al., 1974). Brie y, in a total volume of 2 mL, 0.2 mL of supernatant, 0.03 M Tris-HCl buffer (pH 7.4) and 0.2 mM sodium pyrophosphate were added. The mixture was incubated for 20 min at 37°C. The reaction was stopped by the addition of 1 mL of 10% TCA, after which 1.5 mL of the organic compound 2-thiobarbituric acid (TBA) were added and the mixture was heated. The pink-colored product formed revealing the presence of MDA due to the oxidation of fatty acids, was measured using spectrophotometer at an absorption of 535 nm.

RNA Extraction and RT-PCR
Total ribonucleic acid (RNA) was isolated utilizing ONE STEP-RNA Reagent (Biobasic Inc.) from untreated and treated liver tissue homogenates. Concentration and quality of RNA samples were assessed using ultra-violet (UV) spectrophotometry. Easy Script Plus™ Reverse Transcriptase (Tinzyme, New Delhi, Indian) was used for the reverse transcription of high-quality RNA extracts. Brie y, 0.5 µg total RNA, 2 µL oligo dT and 0.5 µg/mL random hexamer primers in diethyl pyrocarbonate (DEPC)-treated water were inoculated for 5 min at 65°C and instantly cooled down on ice. After the addition of 4 µL dithiothreitol (10 mM), 2 µL dNTP (10 mM) and 8 µL First Strand buffer, the temperature of the solution was lowered to 55°C and completed with 200 U Superscript II® (MiRXES, Heal Force Company, Shanghai, China). The solution was then incubated at 55°C for 60 min then at 85°C for 15 min thus generating complementary DNA (cDNA).
TNF-α and internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were ampli ed by PCR using selected primer pairs of sequences as follows: TNF-a, 5′-CCGAGGCAGTCAGATCATCTT-3′ (forward), 5′-AGCTGCCCCTCAGCTTGA-3′ (reverse); GAPDH, 5′-GCAAGTTCAACGGCACAGTCAAG-3′ (forward), 5′-ACATACTCAGCACCAGCATCACC-3′ (reverse). PCRs were performed in duplicate for each sample. To verify whether the results were con ned to one single ampli ed product, all the reactions were assessed through dissociation curve analysis. Each of the amplicons had different melting temperatures, which were 83°C and 84°C for TNF-α and GAPDH, respectively. Pfa 's mathematical model was used to calculate the relative quanti cation of TNF-α transcripts (Pfa , 2001). In a total volume of 25 mL, 1.5% agarose and 1X Tris-acetate-EDTA (TAE) were prepared and cascaded onto a gel tray. The loading dye was blended with the PCR product. Along with the 1 kilobase pair (kbp) DNA ladder used as a reference; the mixture sample was loaded to each well. The gel was run at 50 volt (V) for 90 min and then PCR products were visualized by ethidium bromide staining and analyzed using Gel Pro Analyzer software (version 4.0.). The quantity of TNF-α transcript was related to GAPDH.

Terminal deoxynucleotidyl transferase (TdT)-Mediated dUTP Nick-End Labelling (TUNEL) Assay
DNA fragmentation analyses were carried out in para n-impregnated liver tissues using a terminal deoxynucleotidyl TUNEL reaction conforming to manufacturer's instructions. TUNEL reaction mixture (250 µL) was formulated using: TdT (25 µL) diluted in the nucleotide mixture (225 µL). Nucleotide solution excluding TdT was considered as negative control in all experiments. After lysing the cells and the DNA strands were decondensed, the slides containing the para n-impregnated liver tissues were washed twice with PBS. The TUNEL reaction mixture (25 µL) was added to each slide and a coverslip was placed on the top for mounting. Later, the slides were incubated in a dark and highly moist chamber for 60 min at 37°C. The coverslips were then taken off and the slides were rinsed three times with PBS. Slides were developed with diaminobenzidine substrate, counterstained with HE dye, and scrutinized for con rmation of programmed cell death (i.e., apoptosis), revealed by DNA fragmentation. The count of brown apoptotic cells was normalized to total cells as visualized by HE staining. The apoptotic index was calculated by dividing the number of apoptotic cells by the total number of cells in random elds.

Statistical Analysis
Sigma Plot-13 software (version 14.0, Systat Softwarem, CA, USA) was used to carry out statistical analysis. The results are expressed as the mean ± standard error of mean (SEM). One-way ANOVA with Dunnett's comparison post-hoc test was used for evaluating the signi cance of difference. If p < 0.001, the data were considered statistically signi cant.

BERM Prevents Ethanol Intoxication-Induced Liver Injury
Histopathological observations were made in gross liver and kidney tissues, dissected from all the studied mouse groups. In the group treated with ethanol alone, spotty necrosis was visualized in the liver tissue while the sections of kidney appeared to be normal, as seen in the Figure 1. The mouse groups, which received treatment with either silymarin (the positive control) or BERM extract (100 mg and 200 mg) prior to ethanol administration showed a gradual improvement towards normal tissue architecture indicated by the absence of spotty necrosis induced by ethanol, as compared to the control (Group 1), the untreated tissues (Figure 1). The oral treatment with BERM prior to the administration of the toxic ethanol for 7 consecutive days showed an obvious decrease in toxicant liver tissue injury, as compared with ethanol-induced liver injury (Figure 1).
Based on histopathological microsection examination of the liver and kidney tissues collected from the 6 groups of mice, different tissue characteristics were observed, however both tissues showed necrotic cells and brosis due to ethanol intoxication, as seen in the Figure 2. The liver and kidney tissues collected from ethanol-administered groups pretreated with BERM presented normal tissue architecture, as compared with the normal untreated group, the negative control ( Figure 2).

BERM Decreases Ethanol-Induced MDA Levels and Enhances Ethanol-decreased GSH Levels in Liver Tissues
As a biomarker of ethanol-induced oxidative stress and of liperoxidation (Galicia-Moreno et al., 2016), the content of MDA was measured in the supernatants of tissue homogenates. The administration of alcohol signi cantly increased the level of MDA produced in liver tissues, as seen in the Figure 3. The oral pretreatment with silymarin followed by the administration of ethanol resulted in the signi cant decrease in ethanol-induced MDA production, as compared with hepatic tissue MDA levels detected in the ethanol study group (Figure 3). A signi cant gradual reduction in ethanol-induced MDA production was observed by the increased concentrations of the oral pre-treatment with BERM extracts (100, 200 and 400 mg), as compared to the ethanol group ( Figure 3). The decrease in ethanol-induced MDA production by the pretreatment with 400 mg BERM was similar to the decrease in ethanol-induced MDA production in liver tissues caused by the pre-treatment with silymarin ( Figure 3). GSH levels was found to be the least measured in the toxic ethanol study group and the highest in the silymarin study group, as seen in the Figure 3. A signi cant gradual increase in ethanol-induced GSH production was observed by the increased concentrations of the oral pre-treatment with BERM extracts (100, 200 and 400 mg), as compared to the ethanol group (Figure 3). A signi cant difference was still observed between the increased level of GSH content detected in ethanol-induced liver injury pre-treated with 400 mg BERM and the increased level of GSH detected in ethanol-induced liver injury pre-treated with silymarin ( Figure 3).

BERM Reduces Ethanol-Induced TNF-α Gene Expression Level
Chronic ethanol consumption also leads to the increase in the gene expression level of TNF-α, a proin ammatory cytokine used as a biomarker of liver injury (Yin et al., 1999). Ampli ed by RT-PCR then visualized using gel electrophoresis, the liver treatment with ethanol signi cantly up-regulated TNF-α mRNA expression level, as compared to TNF-α mRNA expression level monitored in the untreated control liver tissue, as seen in the Figure 4. The pre-treatment with either BERM or silymarin signi cantly decreased ethanol-induced TNF-α mRNA expression level, as compared to TNF-α mRNA expression level monitored in the toxic alcoholic liver tissue (Figure 4).

BERM Inhibits Apoptosis Due to Ethanol-Induced Liver Injury
Ethanol intoxication-induced liver injury leads to premature and programmed cell death, including apoptosis (Wang et al., 2016). Known as a hallmark of apoptosis, nuclear DNA fragmentation was assessed using TUNEL assay under light microscope. Mainly observed in the toxic ethanolic liver tissue, the shrunken cells of which nuclei was stained brown were identi ed as TUNEL-positive apoptotic cells, as compared with the healthy cells that were observed in the untreated control liver tissue, as seen in the Figure 5A. Fewer TUNEL-positive apoptotic cells were spotted in the ethanol-induced liver injury after pretreatment with either BERM or silymarin, as compared to TUNEL-positive apoptotic cell number observed in the toxic ethanolic liver tissue ( Figure 5A). The apoptotic index was signi cantly decreased in ethanolinduced liver injury in the mice group pre-treated with BERM, as compared with the mice treated with ethanol alone, as seen in the Figure 5B. The pre-treatment with silymarin decreased at a higher extent the apoptotic index induced by ethanolic intoxication alone than the one determined following to pretreatment with BERM ( Figure 5B).

Discussion
Liver and kidneys are both vital organs that are crucial for xenobiotic and drug elimination from our body We previously evaluated the in vitro hepatoprotective activities of BERM based on the reduction of the cytotoxicity in HepG 2 cell line exposed to the combined BERM and toxicants (i.e., CCl 4 , ethanol and paracetamol) treatment (Padmanabhan and Jangle, 2014). In this present study, we demonstrated the hepatoprotective activities of BERM in ethanol-intoxicated mice associated with a decrease of ethanolinduced MDA production, enhancement of ethanol-decreased GSH production, and with the concomitant reduction of ethanol-induced hepatotoxicity, revealed by the down-regulation of TNF-α gene expression level and by the quasi-disappearance of brosis and apoptotic hepatocytes.
Oxidative stress has been mainly associated with the pathological process of ethanol-induced liver injury (Phaniendra et al., 2015). The production of MDA, commonly known as oxidative stress marker and as a marker of lipid peroxidation, was considerably enhanced in the ethanol study group in comparison with the untreated control group. The MDA overproduction due to ethanol-induced liver damage aligned with previous research studies (Chang et al., 2021). Another study reported that oxidative stress in brain due to ethanol consumption also elevated MDA levels (Das et al., 2007).
A crucial non-enzymatic antioxidant pertaining to oxidative stress is GSH, which removes H 2 O 2 radicals and reacts directly with certain ROS (e.g., the hydroxyl radical) and nullify its toxic effects. In the present study, the ethanol toxicity group exhibited decreased levels of GSH in comparison with the control and treated groups, resulted in reduced synthesis of GSH, as previously reported (Husain et al., 2001).
Observed in rats subjected to alcohol and tobacco smoke exposure, the generation of oxidative stress was also stated to decrease GSH levels in liver (Ignatowicz et al., 2013), which agreed with our present ndings.
Oxygen radicals generated by the ethanol intoxication-induced injury play an important role in the stimulation of in ammation through up-regulation of in ammatory cytokines such as TNF-α ( Gutierrez-Ruiz et al., 2001). TNF-α is a key pro-in ammatory cytokine which induces the secretion of enzymes and other cytokines in various cells and tissues. In this present study, the prolonged exposure to ethanol leads to increased level of TNF-α gene in the toxic study group. Similar results were obtained by Nowak and Relja who demonstrated that NF-κB signaling pathway was activated during alcoholic liver disease, which resulted in the increased of gene expression levels of pro-in ammatory cytokines and chemokines (Nowak and Relja, 2020). In humans, chronic alcohol consumption is associated with increase in the production of serum pro-in ammatory cytokines (e.g., TNF-α, IL-1, IL-6, IL-8) Cohen, 1989, McClain et al., 1999). Thus, the correlation between oxidative stress and in ammation within the course of alcoholic liver injury is indisputable. Moreover, improper metabolism of ROS ends up in the expression of hypoxia-inducible factor-1 alpha that may also increase TNF-α secretion, resulting in associate immune reaction that intensi es the liver injury (Wilson et al., 2014). In addition to play a major role in in ammation, TNF-α bound to its receptor, which initiates programmed death pathways such as apoptosis through activation of downstream kinases and proteases, including caspases (Fouad et al., 2019). A deeper investigation of the reverse effect of ethanol intoxication inducing apoptosis-related molecular mechanisms, including caspase-dependent (extrinsic) and mitochondria-dependent (intrinsic) pathways, contributing to hepatoprotective activities of BERM would be of interest.
The TUNEL assay was carried out for the detection of apoptotic cells that undergo massive DNA fragmentation during the nal stages of apoptosis. The DNA damage may be incurred due to ethanolinduced oxidative stress exposed to the hepatocytes causing production of ROS and of TNF-α-induced cell death, which in turn lead to hepatic injury (Wang et al., 2016). The current study showed that ethanol intoxication towards mouse hepatocytes increased the number of apoptotic cells that was determined using TUNEL assay and observed with light microscopy, which agrees with previous studies using human alcoholic hepatitis specimens (Zhao et al., 1997;Natori et al., 2001). However, in this present study when pre-treated with either silymarin or BERM, a considerable decrease in the number of ethanol-induced apoptotic cells was noticed, con rming the in vivo hepatoprotective effect of BERM from alcohol intoxication.

Conclusion
There is growing interest in the discovery of new antioxidant plant-based bioactive compounds that can reverse the deleterious effects of toxicants, including ethanol overdose. In this present study, BERM at the concentration of 400 mg/kg showed the highest protective effect from ethanol intoxication-induced liver damage in mice and were revealed to be comparable with the hepatoprotective standard herbal drug, silymarin. The hepatic MDA levels were subsequently found to be low, indicating the decrease in the oxidative stress levels, and the increase in hepatic GSH levels clearly re ected the hepatoprotective effect of BERM due to the presence of antioxidants. Thus, the recently reported puri cation and isolation of unidenti ed bioactives compounds from BERM along with those characterized BERM-derived hepatoprotective agents, including daidzein, epicatechin, hesperidin, diosmin, and quercetin (Saha et al., 2019; Chitra et al., 2020), will pave the way for the development of an alternative and cost-effective hepatoprotective agents against toxic liver disorders.

Declarations Data availability
The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Con icts of interest
The authors do not have any con ict of interest to declare.
Funding statements 51. Saha P, Das Talukdar A, Nath R, Sarker SD, Nahar L, Sahu J, Choudhury MD. Role of natural phenolics in hepatoprotection: A mechanistic review and analysis of regulatory network of associated genes. Front Pharmacol. 2019; 10: 509. Figure 1 BERM pre-administration prevented ethanol-induced liver injury in mice. Representative gross sections of liver and kidney tissues of each mouse from 6 groups of mice after (5 mL of 25% ethanol/kg b.w.) ethanol treatment via i.p. injection following to either oral pre-administration with sylimarin tested at 50 mg (Group 4) or oral pre-administration with BERM tested as 100 mg (Group 3), 200 mg (Group 4) and 400 mg (group 5), as compared to healthy untreated control mice (Group 1) and mice administered with ethanol alone (Group 2). Spotty necrosis as indicator of tissue damage was only observed in the liver tissue of ethanol-treated mice.  Effect of BERM pre-administration on hepatic tissue GSH and MDA levels following to ethanol-induced liver injury. The bar graphs show hepatic tissue levels of GSH and MDA measured using colorimetric methods involving speci c substrates such as DNTB and TBA solutions, respectively. Refer to methods section for more information. Alphabets a, b and ab, clearly indicate that they are statistically signi cant with the control (untreated) mouse group.