Hepatoprotective and Antioxidant Potential of Phenolics-Enriched Fraction of Anogeissus acuminata Leaf against Alcohol-Induced Hepatotoxicity in Rats

Anogeissus acuminata is used to treat wounds, diarrhoea, dysentery, and skin ailments. However, its hepatoprotective effect against ethanol-induced liver damage is yet to be reported. The phenolic-enriched ethyl acetate fraction of Anogeissus acuminata (AAE) was evaluated for hepatoprotective activity against ethanol-induced liver toxicity in rats. The intoxicated animals were treated with a phenolic-rich fraction of Anogeissus acuminata (AAE) (100 and 200 mg/kg) and silymarin (100 mg/kg). The antioxidant activity of AAE was analysed. Biochemical markers (ALT, AST, ALP, GGT, and TBL) for liver injury in ethanol-administered animals resulted in higher levels of key serum biochemical injury markers, as evidenced by increased levels of ALT (127.24 ± 3.95), AST (189.54 ± 7.56), ALP (263.88 ± 12.96), GGT (91.65 ± 3.96), and TBL (2.85 ± 0.12) compared to Group I ALT (38.67 ± 3.84), AST (64.45 ± 5.97), GGT (38.67 ± 3.84), and TBL (0.53 ± 064) (p < 0.05). AAE administration decreased serum biochemical liver injury markers as manifested in Group III animals’ ALT (79.56 ± 5.16), AST (151.76 ± 6.16), ALP (184.67 ± 10.12), GGT (68.24 ± 4.05), TBL (1.66 ± 0.082) (p < 0.05), and Group IV ALT (55.54 ± 4.35), AST (78.79 ± 4.88), ALP (81.96 ± 9.43), GGT (47.32 ± 2.95), TBL (0.74 ± 0.075) (p < 0.05). Group IV exhibited the most significant reduction in serum biochemical markers as compared to Group III (p < 0.05) and close to silymarin-treated Group V ALT (44.42 ± 3.15), AST (74.45 ± 5.75), ALP (67.32 ± 9.14), GGT (42.43 ± 2.54), TBL (0.634 ± 0.077). Gene expression indices and histoarchitecture were evaluated to demonstrate the potential of AAE. The bioactive fraction of Anogeissus acuminata was rich in phenolics and flavonoid content. GC–MS analysis identified gallic acid, palmitic acid, cis-10-heptadecenoic acid, 9-octadecenoic acid, epigallocatechin, 2,5-dihydroxyacetophenone, and catechin. Oral administration of AAE (100 and 200 mg/kg) lowered the elevated levels of the biochemical markers and interleukin, and enhanced the level of enzymatic antioxidant. It also downregulated the expression level of proapoptotic genes and upregulated the expression level of the antiapoptotic gene along with improved liver histopathology.


Introduction
Alcohol is the second most psychoactive or recreational substance consumed after caffeine. The long-term consumption of alcohol leads to an increased risk of major health problems such as injuries, violence, liver diseases, and cancer. According to the World Health Organisation, alcohol consumption is more harmful to hypercholesterolemia and hypertension than cigarette smoking is [1]. Alcohol causes oxidative stress in the liver cells, which leads to metabolic abnormalities such as the accumulation of acetaldehyde, high vacuum and low temperature. The dried extract was further fractionated with solvent of different polarity indices (hexane, chloroform, and ethyl acetate). Among all fractions, the bioactive ethyl acetate (AAE) fraction was dried and stored for hepatoprotective investigation. Total phenolic content (TPC) was analysed by using the Ragazzi and Veronese (1973) method [11], and is expressed as mg gallic acid-equivalent (GAE)/gram AAE. Total flavonoid content (TFC) was investigated by the Oyaizu (1986) method [12] and expressed as mg quercetin-equivalent (QE)/gram AAE. The phytochemical characterisation of the bioactive fraction of Anogeissus acuminata leaf extract (AAE) was conducted via GC-MS. Sample (AAE) was derivatised by using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The derivatised fraction was evaluated on the GC-MS instrument comprising a gas chromatograph (Thermo Trace GC Ultra) and mass spectrometers (Thermo Fisher DSQ II). Data were recorded by mass selective detector operating in the electron impact (EI) mode with 70 eV ionisation energy at an ionisation current of 2.0 mA and mass range of 50-800 m/z. The resultant chromatographic and mass data were acquired using Xcalibur software. The software depicts the investigation of the m/z ratio values of each metabolite fragments detected in mass spectra using GC-MS spectral library databases such as WILLY and NIST. The relative concentration of detected metabolites was calculated as a percent peak area [13].

In-Vitro Antioxidant Activity
Using the DPPH stable radical, the antioxidant potential of the bioactive fraction (AAE) was investigated [14]. The reducing potential of AAE was recorded at 515 nm with a calibration curve and determined by linear regression. Results obtained from AAE were compared with ascorbic acid, which is used as a standard antioxidant. DPPH radical inhibition was evaluated according to the equation.
DPPH˙radical inhibition = Control − Sample/Control × 100 Ferric reducing power (RP) evaluation was estimated by the ferric reducing power assay and denoted as mg ascorbic acid equivalents (ASE) per gram AAE [15]. Total antioxidant capacity was determined by using the spectrophotometric method [16]. Ascorbic acid was used as the standard, and total antioxidant capacity is expressed as mg ascorbic acid equivalents (ASE) per mg AAE.

Experimental Animals
Male Sprague Dawley rats were used in this study according to the regulations of the Institutional Animal Care Committee, CPCSEA, India (reg. no. 1732/GO/Re/s/13/CPCSEA). Acute toxicity was analysed according to OECD Guideline 423 [17]. The administration of AAE at doses of 300 mg/kg b.w. was a safe dose that exhibited no abnormal behaviour or mortality in tested rats. Twenty-five rats were divided into five groups (each group had five animals). All animals were orally intoxicated with ethanol (7 g/kg) for 28 days except Group I animals. Groups III and IV received AAE 100 and 200 (mg/kg, p.o.) once daily, while Group V received silymarin 100 (mg/kg, p.o.) for up to two weeks. Group II received 0.5% sodium carboxyl methyl cellulose [18]. After a two-week treatment period, the animals were sacrificed on an overnight fast by cervical dislocation. Blood was collected and centrifuged at 1000× g for 15 min to isolate serum for the investigation of biochemical markers. Collected vital organs were washed with phosphate buffer saline (PBS), fixed in formalin for histological studies, and the remaining tissue was stored at −80 • C for enzymatic antioxidants and molecular analysis.

Determination of Biochemical Parameters
Biochemical parameters were analysed by using Biochemical kits (Transasia Biomedicals Ltd.) from the collected blood serum (AST, ALT, ALP, GGT, and total bilirubin) with an autochemistry analyser (Csense 100).

Estimation of Interleukins and TNF-α in Hepatic Tissue
To estimate interleukins and TNF-α from liver tissue homogenate, an ELISA kit (Elabscience Biotech Co., Ltd., Wuhan, China) was used based on the principle of standard sandwich ELISA technology.

Gene Expression Analysis via Quantitative Real-Time PCR (qRT-PCR)
The TRizol reagent was used to isolate RNA. The NanoDropTm apparatus was used to measure the quality and concentration of RNA at 260/280 nm. Enhanced Avian HS RT-PCR kit was used to synthesise cDNA from isolated total RNA (Sigma-Aldrich, St. Louis, MO, USA). This cDNA was used as a template for qRT-PCR to assess total transcript levels in a StepOne real-time PCR system using SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA). The 2 −∆∆ct method was used to examine the quantitative real-time expression of the genes [26]. Table S1 shows the primer sequences developed for each gene.

Statistical Analysis
All estimated results are the mean of five replicates. Data were examined with Duncan's multiple-range test (DMRT) for the evaluation of the significant difference between means (p < 0.05). SD is depicted using the average of the five replicates.

Total Phenolic and Flavonoid Content
The total phenolic and flavonoid content of plant extracts depends on the type of solvent used for their extraction procedure. The extraction yield of a 70% ethanolic extract of A. acuminata was 13.71% w/w. The total phenolic content of AAE was 313 ± 17.23 (mg GA eq./gram AAE), and the total flavonoid content was 124 ± 7.19 (mg RE eq./gram AAE).

Total Phenolic and Flavonoid Content
The total phenolic and flavonoid content of plant extracts depends on the type of solvent used for their extraction procedure. The extraction yield of a 70% ethanolic extract of A. acuminata was 13.71% w/w. The total phenolic content of AAE was 313 ± 17.23 (mg GA eq./gram AAE), and the total flavonoid content was 124 ± 7.19 (mg RE eq./gram AAE).

In Vitro Antioxidant Studies
DPPH is a free radical that can scavenge by accepting an electron from antioxidants and converting it into a stable diamagnetic molecule. The free radical scavenging effect of the active fraction on DPPH radicals increases with increasing concentration. At 5, 10, 15, 20, and 25 µg/mL, the scavenging activities of AAE on DPPH radical were 21%, 34%, 46%, 65% and 84%, respectively. The calculated IC 50 was 16 µg/mL. The reducing power of AAE exhibits the capability of Fe 3+ to Fe 2+ reduction by antioxidant. The analysed reducing power of AAE was 341.21 ± 2.43 mg ascorbic acid equivalents/gram AAE. Total antioxidant capacity was 232 ± 2.89 mg ascorbic acid equivalent/gram of AAE.

In Vitro Antioxidant Studies
DPPH is a free radical that can scavenge by accepting an electron from antioxidants and converting it into a stable diamagnetic molecule. The free radical scavenging effect of the active fraction on DPPH radicals increases with increasing concentration. At 5, 10, 15, 20, and 25 µg/mL, the scavenging activities of AAE on DPPH radical were 21%, 34%, 46%, 65% and 84%, respectively. The calculated IC50 was 16 µg/mL. The reducing power of AAE exhibits the capability of Fe 3+ to Fe 2+ reduction by antioxidant. The analysed reducing power of AAE was 341.21 ± 2.43 mg ascorbic acid equivalents/gram AAE. Total antioxidant capacity was 232 ± 2.89 mg ascorbic acid equivalent/gram of AAE.

Effect of AAE on Biochemical Parameters
Ethanol administered to Group II animals resulted in higher levels of serum biochemical injury markers, as evidenced by increased levels of ALT (127. 24

AAE Treatment Improves Antioxidant Enzymes Activity and Lowers Lipid Peroxidation
Ethanol administration (7 g/kg) reduced antioxidant activity and concomitantly increased level of stress marker (MDA  (Figure 3).
(DMRT) was used for the analysis of a significant difference between the means (p < 0.05) and compared each parameter. Bars with the same letters are not significantly different. All values are means of five replicates ± SD.

AAE Treatment Improves Antioxidant Enzymes Activity and Lowers Lipid Peroxidation
Ethanol administration (7 g/kg) reduced antioxidant activity and concomitantly increased level of stress marker (MDA  (Figure 3).
Med. Sci. 2022, 10, x FOR PEER REVIEW 8 of 14 highest lipid peroxidation. Duncan's multiple-range test (DMRT) was used for the analysis of a significant difference between the means (p < 0.05) and separately compared each parameter. Bars with the same letters are not significantly different. All values are means of five replicates ± SD.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Investigation
Ethanol administration promoted the expression of p53, Bax, caspase-3, and caspase-9, and significantly decreased the expression level of Bcl-2. AAE administration augmented the expression of p53, Bax, caspase-3, and caspase-9, and enhanced the expression level of Bcl-2 to protect ethanol intoxicated apoptosis and DNA damage in rat livers. The efficacy of AAE at 200 mg/kg was more significant than that at 100 mg/kg to improve altered gene expression, as in silymarin administration Group V. Gene-specific primers were used to investigate the relative expression of pro-and antiapoptotic genes. The GAPDH gene primer was taken as an endogenous control ( Figure 5).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Investigation
Ethanol administration promoted the expression of p53, Bax, caspase-3, and caspase-9, and significantly decreased the expression level of Bcl-2. AAE administration augmented the expression of p53, Bax, caspase-3, and caspase-9, and enhanced the expression level of Bcl-2 to protect ethanol intoxicated apoptosis and DNA damage in rat livers. The efficacy of AAE at 200 mg/kg was more significant than that at 100 mg/kg to improve altered gene expression, as in silymarin administration Group V. Gene-specific primers were used to investigate the relative expression of pro-and antiapoptotic genes. The GAPDH gene primer was taken as an endogenous control ( Figure 5). Duncan's multiple-range test (DMRT) was used for analysis of a significant difference between means (p < 0.05) and separately compared each parameter. Bars with the same letters were not significantly different. All values are means of five replicates ± SD.

Histopathological Studies
Histopathological examination of the liver section was observed with a microscope at 40X. H&E-stained liver section of Group I animals showed normal cell morphology with clear cellular boundaries, nucleus, and nucleolus with well-preserved granulated cytoplasm. No steatosis or inflammatory cells were observed compared to ethanolintoxicated Group II animals' liver sections. Ethanol-intoxicated animals in Group II exhibited a disarrangement of hepatocytes and number of inflammatory cells with cellular degeneration having centrilobular necrosis ( Figure 6B). AAE-administered Groups III and IV showed improved hepatocyte architecture, reduction in inflammatory cells, and necrosis with clear nucleus and nucleolus, as in silymarin-treated Group V ( Figure 6).

Histopathological Studies
Histopathological examination of the liver section was observed with a microscope at 40X. H&E-stained liver section of Group I animals showed normal cell morphology with clear cellular boundaries, nucleus, and nucleolus with well-preserved granulated cytoplasm. No steatosis or inflammatory cells were observed compared to ethanol-intoxicated Group II animals' liver sections. Ethanol-intoxicated animals in Group II exhibited a disarrangement of hepatocytes and number of inflammatory cells with cellular degeneration having centrilobular necrosis ( Figure 6B). AAE-administered Groups III and IV showed improved hepatocyte architecture, reduction in inflammatory cells, and necrosis with clear nucleus and nucleolus, as in silymarin-treated Group V ( Figure 6).

Discussion
The liver is the essential vital organ that metabolises various toxins and nutrients to keep the healthy human body in normal biochemical and physiological action; it should always function properly. GC-MS analysis of A. acuminanata revealed valuable pharmacologically active phytocompounds. The identified compounds were gallic acid (4.66%), palmitic acid (19.78%), cis-10-heptadecenoic acid (5.38%), 9-octadecenoic acid (22.39%), epigallocatechin (6.34%), 2,5-dihydroxyacetophenone (1.27%), and catechin (0.41%) ( Table 1). Gallic acid and epigallocatechin have hepatoprotective properties in rat livers, as evidenced by a significant reduction in liver injury markers with CYP2E1, and increased antioxidant enzyme activity [2,27], indicating a strong therapeutic potential of gallic acid in this experimental liver disorder due to its potent antioxidant properties, DPPH scavenging activity, and reducing power assay. Palmitic acid and its derivatives are hepatoprotective and have anticancer activity [9]. 9-Octadecenoic acid showed antiandrogenic, 5-alpha reductase-inhibiting, cancer-preventive, anti-inflammatory, anaemiagenic, insectifuge, dermatitigenic, and hypocholesterolemic activity [28]. Thus, these pharmacologically active phytocompounds may exhibit potent restorations of altered biochemical injury markers in ethanol-intoxicated animals. The hepatoprotective potential of AAE was due to its valuable antioxidant properties, or phytocompound like phenolics and flavonoids, which reduced oxidative stress and its complications, such as ethanol-induced hepatic damage and inflammation [29][30][31][32]. DPPH (DPPH•) is a persistent radical that can accept an electron or hydrogen radical to produce a stable diamagnetic compound, leading to a change in colour. The percentage of DPPH colour change is widely used to estimate radical scavenging efficiency [33,34]. The existence of reducing agents (i.e., antioxidants) enables the Fe 3+ /ferricyanide complex to be reduced to ferrous form. As a result, evaluating the production of Perl's Prussian blue at 700 nm can measure Fe 2+ concentration. AAE's reducing ability was enhanced with concentrations [35]. Ethanol is a unique substance with a rapid action owing to its solubility in both water and lipids, which is absorbed from the stomach and intestine and then rapidly diffuses into the blood circulation, where it is dispersed throughout the body. Hepatocellular necrosis is caused by alcohol use, resulting in an increase in serum marker enzymes released into the bloodstream [2]. Elevated ALT, AST, ALP, GGT, and TBL levels are prominent biomarkers of hepatic damage [9]. It also increases the production of reactive oxygen species inside the living system, since ethanol is extensively metabolised by the microsomal oxidising system to acetaldehyde and ultimately to acetate through cytochrome P450 [31]. Steatosis is the most significant alteration that happens in the liver following alcohol use. As a result of redox state imbalance, lipid peroxidation occurs [36]. In organisms, GSH is an essential antioxidant capable of reducing damage produced by reactive oxygen species. MDA is produced by lipid peroxidation and is a sign of oxidative stress. Free radicals and oxidative stress caused by ethanol promote MDA overproduction and GSH depletion [37]. Excessive free radicals are likely to elicit Kupffer cells, which can regulate the inflammatory process in the liver by releasing TNF-alpha and other pro-inflammatory cytokines [38]. Increased proinflammatory mediators and cytokines (e.g., TNF-α, IL-1 β, and IL-6) aggravated in ethanol-induced groups may have been through NF-κB activation [39]. The potent effect of AAE reduced free radicals and apoptotic properties by the enhanced antioxidant capacity against oxidative stress induced by alcohol for hepatoprotection. Histopathological examination revealed the hepatoprotective properties of AAE. AAE at a dosage of 200 mg/kg reflected more effective results, as in silymarin at 100 mg/kg. In the current study, we examined the gene expression levels of Bax, Bcl2, caspases3, caspases 9, and Bcl2 to evaluate apoptosis. The Bcl-2 family is associated with the initiation of the mitochondrial apoptotic pathway [40]. Bax, a proapoptotic protein, translocates to mitochondria and forms complexes with Bcl2. Increasing the Bax/Bcl2 ratio induces mitochondrial defects, resulting in the release of cytochrome C [41]. Caspases are proteolytic enzymes that cleave specific proteins in the nucleus and cytoplasm to damage the cell. When mitochondrial cytochrome c is released into the cytoplasm and activates caspase 9, which eventually activates caspase 3 via intrinsic apoptotic pathways. Cell membrane death receptors can activate initiator caspases, which leads to caspase-3 activation via extrinsic apoptosis pathways [4]. On the basis of real-time PCR, increased mRNA levels of the Bax/Bcl2 ration, and levels of caspases 3 and 9 were observed in the livers of ethanol-intoxicated rats, indicating that ethanol might reduce the level of Bcl2 while improving the level of Bax, which was consistent with our findings [42]. AAE might decrease mRNA expression levels of the Bax/Bcl2 ratio in the livers of intoxicated rats. As a result, AAE was highly hepatoprotective against ethanol toxicity, as was silymarin. All of the mentioned positive attributes confirmed the protective effects of AAE on ethanol toxicity symptoms via antilipid peroxidation, and antiapoptotic and antioxidant capabilities in our present study. The phenolics-enriched fraction of Anogeissus acuminata plays a significant role in hepatoprotective functions. As a result, in future studies, the potent phytocompounds will be isolated from the bioactive fraction, and their activities and mechanisms related to apoptosis will be explored.

Conclusions
According to the findings of this study, Anogeissus acuminata (AAE) may be useful in the treatment of alcohol-induced hepatotoxicity and oxidative stress. AAE showed significant hepatoprotective activity according to the results of various biomarkers. These findings contribute to the significance of the bioactive fraction's constituents in hepatoprotective activity. Antioxidant, anti-inflammatory, and antiapoptotic properties of AAE may justify its beneficial impacts on alcoholic liver injury. Before AAE can be used in humans, extensive clinical studies must be carried out to demonstrate its safety and effectiveness. This study demonstrates the plant's hepatoprotective properties, which could lead to the development of new hepatoprotective herbal medicines to treat alcohol-induced liver injury.