1. INTRODUCTION:

The term hepatotoxicity is characterized by damage to the hepatic cells brought on by exposure to foreign chemicals such as drugs, high-fat diet, binge eating, consumption of alcoholic beverages, etc.¹ The substances which harm the hepatic tissues are called hepatotoxins².

It has different effects depending on the body’s health as well as their quality, place of entry, and rate of discrimination³. Hepatotoxins like ethanol and carbon tetrachloride (CCl₄) can undermine the liver's functional integrity, resulting in hepatocellular damage and dysfunction. Serious clinical diseases, such as fatty liver disease, cirrhosis, and even hepatocellular cancer, can result from this disruption^4,5.

Hepatotoxicity refers to the toxic effect of drugs, chemicals, or other substances on the liver. It can result in liver damage and can manifest in various ways, including elevated liver enzymes, jaundice, abdominal pain, and other symptoms. Hepatotoxicity can be caused by a wide range of medications, including over-the-counter (OTC) drugs, prescription medicines, and herbal enhancements.⁶ Particularly drug-induced hepatotoxicity is a big issue since it has the potential to alter therapy regimens and force the removal of potentially helpful drugs. The drug-inducedhepatotoxicity process is intricate and multifaceted, and it may involve direct toxicity, and idiosyncratic⁷. Numerous studies have been conducted to clarify the underlying processes of hepatotoxicity as a result of the clinical significance and potential for serious consequences that have been recognized⁸. Hepatotoxicity's pathophysiology is frequently associated with oxidative stress, mitochondrial dysfunction, cellular homeostasis disruption, and inflammation. The prediction and treatment of hepatotoxic reactions are further complicated by genetic predisposition, interindividual variability, and pre-existing liver disorders⁹.

According to a WHO report from 2022, excessive alcohol use is a major worldwide health issue that has severe social, economic, and clinical ramifications. It is estimated that 13.5% of all deaths are related to alcohol^10,11. Alcohol use has long been acknowledged for its dual nature: it may provide moments of enjoyment and relaxation while also having the potential to have negative health effects. Alcohol use is firmly ingrained in social and cultural practices around the world¹². ALD refers to a group of liver illnesses caused by prolonged and severe alcohol intake. These disorders vary from mild steatosis (fatty liver) to more serious indications including alcoholic hepatitis, liver fibrosis, cirrhosis, and hepatocellular cancer^13,14. The development of small droplets of fat beneath liver cells as they move toward the portal tracts is known as hepatic steatosis, the early stage of alcoholic liver disease.¹⁵ ALD is caused by a complex interplay of oxidative stress, inflammation, lipid metabolism dysregulation, and cellular death, which results in variable degrees of liver damage¹⁶. The oxidative metabolites of ethanol are principally responsible for the alterations in oxidation-reduction (redox) status that are the primary cause of ALD toxicity¹⁷. In particular, ethanol generates acetaldehyde, which is then converted to acetate, and nicotinamide adenine dinucleotide (NAD), which in liver cells decreases to its reduced form (NADH). Additionally, large amounts of acetaldehyde and NADH created by the oxidation of ethanol would promote lipogenesis and lead to hypertoxicity¹⁸.

The perennial herbaceous plant Alpinia galanga Willd. Linn, sometimes referred to as galangal, is a member of the Zingiberaceae family. It is indigenous to Southeast Asia and is extensively grown throughout the world's tropical and subtropical climates¹⁹. It includes a wide variety of phytochemicals, such as flavonoids, terpenoids, polyphenols and essential oils²⁰. Sesquiterpenes and monoterpenes including 1,8-cineole, camphene, -pinene, and -pinene dominate the essential oil makeup. These substances give the plant its scent and provide it its therapeutic effects²¹. A. galanga is a multipurpose plant with uses in aromatherapy, cooking, and medicine. Its rhizomes are rich in phytochemicals that give it a distinct scent and may have positive impacts on your health, including as antioxidant, anti-inflammatory, and antibacterial properties^22,23.

In view of these findings, the motive of this study is to evaluate the curative and preventive effects of aqueous extract of Alpinia galanga’s rhizome against ethanol and CCl4-induced liver injury in rat models. We hope to unravel the mechanisms underlying Alpinia galanga's protective effects on hepatocellular integrity by using a variety of biochemical and histological investigations. The findings of this study have the potential to not only broaden our understanding of herbal therapies for liver protection but also to open up new paths for therapeutic techniques in hepatology.

2. MATERIAL AND METHODS:

2.1 Selection of animals for the experiment:

All male Wistar rats weighing 150–200g were purchased from M/S Chakraborty Enterprise in Kolkata, a CCSEA-registered laboratory animal supplier. Before the trial, all rats were moved to a quarantine area to acclimatize to the animal house for 2 weeks. The animal was kept in cages made of polypropylene that have a 12hour light/dark cycle maintained at 25ºC under regular conditions. The Institutional Animal Ethics Committee (IAEC) gave the experimental protocol approval and assigned it the approval number (REG. No: UIP/IAEC/March/-2023/17). The laboratory and animal care were conducted in accordance with CCSEA regulations.

1. Animal Model: Wistar rats were used in the experiment.

2. Grouping: The rats were divided into five groups, with each group containing six animals. This results in a total of 30 rats (5 groups x 6 rats/group).

3. Duration: The experimental procedure was designed to last for up to eight weeks.

4. Administration of Alcohol: The rats were administered an alcohol bottle that contained 4% (v/v) ethanol. This suggests that the rats had access to a solution containing 4% ethanol throughout the experiment. This was likely done to induce hepatotoxicity, which is liver damage caused by excessive alcohol consumption.

5. CCl4 Administration: The rats also received oral administration of a mixture of carbon tetrachloride (CCl4) and olive oil. This mixture was given twice weekly.

I. Dosage: The rats received 0.2ml/kg body weight of the CCl4 and olive oil mixture.

II. CCl4 Concentration: The CCl4 was administered in a 1:1 combination with olive oil, meaning equal parts CCl4 and olive oil.

III. Olive Oil Concentration: The olive oil concentration in the mixture was 0.01% (v/v).

2.2 Treatment regimen of experimental animals:

Rats were fed a commercial diet, and water was available at all times. The water bottle of experimental animals was replaced by 4% (v/v) ethanol in the whole experiment,²⁴and 0.2ml/kg body weight dose of CCl₄dissolved in a 1:1 combination of olive oil (0.01%,v/v) orally twice a week for a total of 8 weeks²⁵. For eight weeks, distilled water was consumed orally by the normal group, which served as the control group. Alcohol and CCl₄ were administered orally to the disease-induced group of rats for a total of 8 weeks. Rats in the standard drug treatment group received 25mg/kg of silymarin, alcohol, and CCl₄ orally for eight weeks. The Curative group of rats received 500mg/kg of AEAG, alcohol, and CCl₄ orally for 8 weeks. Rats in the preventive group received 500mg/kg of AEAG, alcohol, and CCl₄ orally for 8 weeks, however, AEAG was administered one week before alcohol and CCl₄ were. After eight weeks of the study, withdraw alcohol 72 hours ago, before collecting blood and tissue samples. Animals from all five groups were slaughtered to obtain liver samples for biochemical and histological analyses, and the blood samples were taken using heart puncture.

2.3 Sample Preparation:

2.3.1. Blood Sample Collection:

Blood samples were taken from each animal through a technique known as retrobulbar. This is likely referring to retrobulbar blood sampling, which involves collecting blood from the vessels behind the eye.

2.3.2. Biochemical Parameters:

The collected blood samples were used to determine various biochemical parameters. These parameters include:

Serum, Total protein, Total cholesterol, SGPT (Serum Glutamic Pyruvic Transaminase) enzyme activity, SGOT (Serum Glutamic Oxaloacetic Transaminase) enzyme activity

2.3.3. Euthanasia:

After the blood samples were collected, the animals were euthanized. In this case, cervical dislocation was used as the method for euthanasia. It's a procedure in which the cervical spine is dislocated, causing rapid death.

2.3.4. Liver Removal:

Following euthanasia, the livers of the animals were quickly removed.

2.3.5. Liver Cleaning:

The removed livers were cleaned with a saline solution to remove any contaminants or blood.

2.3.6. Formalin Preservation:

Some liver lobes were stored in airtight containers with formalin (a 10% v/v formalin solution) for histological examinations. This means that these liver samples were preserved for later examination of their tissue structure.

2.4 In-vivo antioxidant analysis (Estimation of Oxidative Stress):

SOD, CAT, GPx and GSH activities carried out the in-vivo antioxidant functions of homogenized liver tissue. The SOD activity was determined extinction coefficient of H₂O₂ at 560 nm, CAT activity was carried out at a wavelength of 240nm, GSH activity was carried out at 412nm and GPx activity was examined at 412nm^29,30,31,32.

2.6 Histopathological study:

Pieces of the liver were fixed in 10% formalin for the histopathological analysis, and hydrate tissue sections were stained with hematoxylin and eosin at a 5µm thickness. Under a light microscope, the segment was thereafter examined and scored³³.

2.7 Statistical Analysis:

Six rats from each group were used to determine the means±SD using GraphPad Prism 9. The results were analyzed using one-way and two-way ANOVA. The test groups were contrasted with the control, untreated, and treated groups.

3. RESULTS:

3.1 BIOCHEMICAL PARAMETERS:

3.1.1 Assessment of Liver Function Test:

Liver diseases result in elevated levels of serum enzymes such as SGPT, SGOT, ALT, albumin, and bilirubin. Serum enzymes reflecting liver illness considerably rise in the disease-induced group, when it compares normal to disease-induced to test group and standard to test groups are shown in Table 1.

Table 2: Effect of Silymarin and AEAG on lipid profile in the experimental animals

Group’s Name	LIPID PROFILE
Group’s Name	Triglyceride (mg/dL)	T.Cholesterol (mg/dL)	HDL (mg/dL)	LDL (mg/dL)	VLDL (mg/dL)
Normal	86.33 ± 2.28	81.33 ± 2.73	49.33 ± 1.97	45.5± 2.62	28.83 ± 2.03
Disease induced	155.5 ± 2.75^a	172.8 ± 2.85^a	24.5 ± 2.92^x	82.5 ± 2.62^a	32.83± 2.54^a
Std. Treated	88.66 ± 3.39^b	72 ± 1.82^b	33.66±2.28^b	55.5 ± 2.75^b	16.67± 2.13^b
Curative	85.33± 1.69^b,c	64.66± 2.35^b,c	27.33±1.49^c	51.33± 2.42^b,c	24.83± 2.67^b,c
Preventive	78.33±1.97^b,c,d	70.66±1.59^b,c,d	32 ± 1.63^c,d	44.33±1.97^b,c,d	19 ± 2^b,c,d

The data show as mean±std. deviation of six rats in each group. The data show as mean±std. deviation of six rats in each group. ^ap<0.0001 as compare to the normal group. ^bp<0.0001 as compared to the disease-inducedgroup. ^cp<0.001 as compare to the standard group. ^dp<0.05 as compared to the curative group. ^x p<0.001 as comparedto the normal group.

Table 3: Effect of A. galanga rhizome extract on antioxidant activity in liver

Group	Antioxidant activity in Liver
Group	SOD	CAT	GPx	GSH
Normal	20.68±2.30	67.08 ± 3.94	15.66±1.17	55.08±2.04
Disease-induced	7.33±1.30^a	37.58 ± 1.59^a	7.58±1.23^a	25.68±1.76^a
Std. treated	17.89±1.45^b	61.83 ± 1.26^b	13.16±1.34^b	48.58±3.8^b
Curative	11.10±1.20^b,c	52.34 ± 2.86^b,c	10.31±2.0^b,c	39.16±1.49^b,c
Preventive	14.30±1.62^b,c,d	57 ± 2.88^b,c,d	11.61±1.97^b,c,d	43.76±2.3^b,c,d

The data show as mean±std. deviation of six rats in each group. ^ap<0.0001 as compare to the normal group.^b p<0.0001 as compared to the disease-inducedgroup. ^cp<0.001 as compare to the standard group. ^dp<0.05 as compared to the curative group.

4. DISCUSSION:

The primary objective of the study is to assess whether the Alpinia galanga rhizome extract can protect the liver from the damaging effects of ethanol and CCl4. The evaluation likely involves measuring various liver parameters such as liver enzymes (e.g., alanine transaminase, aspartate transaminase), histological examination of liver tissue, and other relevant biochemical markers. These assessments help determine the extent of liver damage and the effectiveness of the extract in preventing or reducing it.

The results of this investigation can further our knowledge of the potential hepatoprotective qualities of Alpinia galanga rhizome extract and may have implications for creating medicinal remedies or dietary supplements to lessen alcohol- and toxin-induced liver disorders.

Our findings point to a significant hepatoprotective effect that is explained by decreased liver enzyme levels, lipid profiles, and oxidative stress as a result of ethanol and carbon tetrachloride-induced free radical production in rats, one of the primary mechanisms by which hepatocellular damage is induced in this model. Previous research has demonstrated that persistent alcohol consumption affects liver cells by causing oxidative stress, mitochondrial malfunction, and changes in respiratory enzymes³⁴. The most significant biomarkers of ALD include liver enzymes and proteins including ALT, AST, and bilirubin, among others. The liver-toxicant (EtOH+CCl4)-induced increase in the test drug's (A. galanga) level in experimental rats was decreased. The modulation of the glutathione pathway by A. galanga's antioxidant activities against alcohol and CCl4 is well understood, and it has been demonstrated that the optimal balance between oxidant and antioxidant is a crucial component in the efficient operation of the liver's metabolism. In rats exposed with hepatotoxicants, A. galanga boosted the activity of antioxidant indices as SOD, CAT, GSH, and GPx that stimulate antioxidant defenses in the liver. In rats used in experiments, A. galanga accelerated the transition from oxidant to antioxidant. These studies have demonstrated the ability of A. galanga to activate antioxidant enzymes and scavenge free radicals in rat liver cells subjected to hepatotoxicants. Additionally, a microscopical analysis of the disease-induced (EtOH+CCl4) group revealed several hepatocellular damages. A vast area of fatty alteration, necrosis, with only a few possessing Mallory's hyaline, and ballooning degeneration were confirmed by histological investigation. In the groups treated with the standard and test drugs, hepatotoxicant-induced microscopic and histological alterations were mitigated. There was no evidence of liver injury in the normal group. A small amount of hepatic cell distribution was seen in the standard drug-treated group. It had small lipid vacuoles that were disrupted, while the curative test group also had small fat vacuoles with nuclei that were shifted to the periphery. While the hepatocytes in the test drug-treated preventative group seemed normal, a few little fat vacuoles with peripherally displaced nuclei were visible. According to this study, there were normal liver function tests (SGPT, SGOT, ALP, albumin, and bilirubin), lipid profiles (triglycerides, T. cholesterol, HDL, VLDL, and LDL), antioxidant parameters, and histopathological analyses before the disease was induced. However, after the disease was induced, the test values for all of these parameters were significantly higher than they were for the normal group. These values were significantly lower after treatment with conventional, curative, and preventative medications when compared to the disease-induced group. Standard medication significantly outperforms curative, preventative, and disease-induced groups in terms of results. While less significant than the standard group, the preventive group significantly outperformed the curative group in terms of results. In comparison to the standard and preventative groups, the results for the curative group are marginally better.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

6. ACKNOWLEDGMENTS:

I would like to thank prof. Dr. Alok Mukerjee, Principal UIP, for their priceless effort and provision all through the research progression. Their understandings and proficiency were contributory in influential the direction of this project. The accomplishment of this study scheme would not have been imaginable without the contributions and backing of many entities and organizations. I’m deeply obliged to all individuals who engage in recreation a role in the accomplishment of this project.

7. LIST OF ABBREVIATIONS:

Abbreviation	Full name
EtOH	Ethanol
WHO	World Health organization
ALD	Alcoholic liver disease
HCC	Hepatocellular carcinoma
CCl₄	Carbon tetrachloride
AEAG	Alcoholic extract of Alpinia galanga
NADH	Nicotinamide adenine dinucleotide
H₂O₂	Hydrogen peroxide
TCA	Tricarboxylic acid
SOD	Superoxide dismutase activity
LFT	Liver function test
ROS	Reactive oxygen species
CPCSEA	committee for the purpose of control and supervision of experiments on animals
w/v	Weight/volume
v/v	Volume/volume
NBT	Nito blue tetrazolium
PMS	Phenazine methosulfate
Req.	Require
EtOH	Ethanol
CHCl₃	Chloroform
Amt.	Amount

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Received on 02.09.2023 Modified on 28.11.2023

Res. J. Pharmacology and Pharmacodynamics.2024;16(1):52-57.

DOI: 10.52711/2321-5836.2024.00010

Group’s Name	Liver Function Test
	SGPT (IU/L)	SGOT (IU/L)	ALP (U/L)	Albumin (mg/dl)	Bilirubin (mg/dl)
Normal	52.37 ± 2.81	62.5 ± 2.45	137.83± 2.57	7.93±0.43	0.35 ± 0.10
Disease induced	105.83± 3.64^a	115.67 ± 2.30^a	203.67± 3.78^a	1.25±0.50^a,x	2.32 ± 0.50^a,x
Std. Treated	59.83± 2.80^b	55 ± 2.88^b	155.67± 2.89^b	5.11±0.47^b	0.85 ± 0.10^b
Curative	67.67 ± 2.56^b,c,	64.5 ± 1.20^b,c	168.83± 3.71^b,c	3.85±0.57^b,c	0.91 ± 0.23^b,c
Preventive	61 ± 2.37^b,c,d	58.17± 1.97^b,c,d	160.5± 3.59^b,c,d	3.95±0.71^b,c,d	0.89± 0.19^b,c,d