INTRODUCTION:

Acetaminophen (APAP), generally known as paracetamol, is among the most commonly utilised analgesic and antipyretic medications available over the counter globally¹. Statistics indicate that over 60 million Americans consume APAP weekly. It is noteworthy that APAP is utilised in conjunction with other medications, especially opioids and diphenhydramine, without public awareness².

Within the therapeutic dose range, adverse medication responses in healthy individuals are extremely infrequent. The British National Formulary states that adults weighing 50 kg or more should take 4 grammes of paracetamol daily, divided into two doses³. People who are at a high risk of hepatotoxicity, such as those who have a body weight less than 50 kg, are chronically alcoholic, are chronically dehydrated, are chronically malnourished, have hepatocellular insufficiency, and/or use P450 liver enzyme inducers simultaneously (such as antituberculosis drugs, antiepileptic drugs, and herbs/dietary supplements like St. John's wort), require special precautions. To this day, the exact molecular process by which APAP causes liver damage remains a mystery⁴.

The antipyretic effects result from the suppression of prostaglandin synthesis. Nonetheless, it exhibits no anti-inflammatory properties (cf. non-steroidal anti-inflammatory medicines (NSAIDs)), indicating that its action is confined to central mechanisms rather than peripheral ones⁵. At therapeutic dosages, the liver metabolises about 60%-90% of APAP via glucuronidation and sulfation, with the cytochrome P450 route accounting for a tiny portion, about 5%-15%⁶.

Paracetamol primarily causes liver damage through the action of cytochrome P450 enzymes, particularly CYP2E1 and CYP3A4, which transform it into a reactive metabolite known as NAPQI⁷. Under typical treatment settings, NAPQI is synthesised in minute quantities and safely eliminated from the body through glutathione conjugation⁸. On the other hand, when glutathione levels drop too low, NAPQI can bind to proteins in the cell, particularly at cysteine residues, and create protein adducts⁹. Mitochondria are particularly vulnerable to this damage, as NAPQI-induced oxidative stress activates the JNK pathway, resulting in mitochondrial dysfunction, ATP depletion, membrane rupture, and ultimately cellular necrosis¹⁰. This mitochondrial involvement has been extensively studied in the context of paracetamol-induced liver injury¹¹.

Severe hepatotoxicity impairs both synthetic and metabolic liver functions, causing coagulopathy, hypoglycemia, encephalopathy, and lactic acidosis. Clinical symptoms typically manifest with a delay, peaking two to three days post-overdose. Despite therapy, hepatotoxicity occurs in 12–13% of acute overdoses, with 2-5% developing liver failure and 0.2–0.5% dying¹².

In addition to liver damage, paracetamol overdose can cause acute kidney injury, which may occur independently of liver failure. This nephrotoxicity may result from direct tubular necrosis due to renal NAPQI formation or reflect hepatorenal syndrome¹³. Furthermore, at extremely high concentrations, paracetamol acts as a direct mitochondrial toxin, potentially leading to central nervous system depression and coma—even in the absence of liver damage or other CNS depressants—posing a risk for delayed diagnosis and treatment¹⁴.

Milk thistle (Silybum marianum [L.] Gaertn.) seeds contain a combination of flavonolignans known as silymarin, which has a long history of usage as a treatment for liver disorders, both acute and chronic¹⁵. Treatment with silymarin reduces the liver glutathione (GSH) depletion caused by hepatotoxicants such APAP, diethylnitrosamine, and ethanol, according to multiple studies¹⁶.

β-Asarone, a volatile ether found in plants like Acorus and Asarum, is known for its antimicrobial properties, particularly in combating fungal pests and bacteria. It is a propenylbenzene and serves as a major bioactive compound in Acorus calamus¹⁷. Despite its potential therapeutic application in managing cognitive impairments such as Alzheimer’s disease¹⁸, the development of β-asarone-based anthelmintics and insecticides is limited due to its toxicity and carcinogenicity¹⁹. Regulatory bodies, including the Council of Europe Committee of Experts on Flavouring Substances, have recognized its carcinogenic nature and recommended restrictions on its concentration in flavoring products like bitters derived from Acorus calamus.

The present study is to screening the pharmacological activity of β-Asarone against Pcm induced hepatotoxicity.

MATERIAL AND METHODS:

Drugs and Chemicals:

Analytical grade chemical was used in this study. β-asarone drug was obtained from Genentech biotech Pvt Ltd, Delhi 1gm. Drugs like paracetamol (Karnataka Antibiotic and Pharmaceuticals ltd, Bangalore) and Silymarin (Micro labs, Banglore) was purchased from local market. Chemical like ethanol (CDH, Mumbai), Anesthetic ether (CDH, Mumbai) and petroleum ether (Ranbaxy Fine Chemicals Ltd., New Delhi) were provided in institute.

Biochemical kits:

Sayoni Enterpriser in Cuttack, Orissa was the source of the biochemical kits that were used for the estimation of biochemical parameters. These kits included AST, ALT, total protein, direct bilirubin, and total bilirubin. These kits were obtained from Span Diagnostics Ltd. in Surat, India.

Animal:

For the purposes of conducting experiments, Wistar albino rats weighing between 150 and 200 grammes were kept in the animal house of the department of science and technology in Kolkata. After that, every single animal was acclimatised for a period of seven days under conventional husbandry settings, which included a room temperature of 25±1 degrees Celsius, relative humidity of 45-55%, and a light/dark cycle of 12 hours and 12 minutes. The animals were provided with unrestricted access to standard (Pranav Agro Industries Ltd, Vadodara, India), and they were provided with water on an ad libitum basis while being held to stringent hygiene standards. A distinct group of animals was utilised for each of the experimental groups, and every precaution was made to ensure that the animals that were used for one response were not used for any other purpose. A period of forty-eight hours previous to the experimental protocol, the animals were used to the conditions of the laboratory in order to reduce the likelihood of any non-specific stress. A prior approval from the Animal Ethical Committee (IAEC) of the SLT Institute of Pharmaceutical Sciences in Bilaspur (Chhattisgarh) was obtained before the tests were carried out. All of the protocols and experiments were carried out in a manner that was in complete accordance with the guidelines and recommendations of the Institutional Animal Ethical Committee (Reference No.IAEC/Pharmacy/2012/51) that were provided by the Committee for the Purpose of Control and Supervision of Experiments on Aminals (CPCSEA) (Approval No. 994/a/GO/06/CPCSEA).

Experimental design:

Animals were divided into 5 groups of 6 rats each and treated orally for 10 days.

Group I (Normal control): Served as normal control received distilled water. Group II (Toxic control): Administered with Paracetamol 2000mg/kg body wt. in distilled water. Group III (Standard): Paracetamol as in group II + Silymarin 100mg/kg body wt. Group IV (β-asarone test 1): Paracetamol as in group II +β-asarone 25mg/kg body wt. Group V (β-asarone test 2) Paracetamol as in group II + β-asarone 50mg/kg body wt. 24hours following the final treatment, the animals were put to death by being slaughtered while under light ether anaesthesia. Following the removal of the liver, which was then rinsed in cold saline and blotted with filter paper and weight, blood was taken through a heart puncture and placed in simple tubes.

RESULTS:

Acute Oral Toxicity:

Various doses of β-asarone were evaluated for acute oral toxicity in albino rats. No mortality was seen up to a dosage of 221mg/kg via intraperitoneal administration; therefore, the dosages for the animal study were established at 25mg/kg and 50mg/kg via intraperitoneal administration. Administration of paracetamol (2000 mg/kg, in two divided doses orally) significantly enhanced biochemical markers such as AST, ALT, direct bilirubin, and total bilirubin levels, whereas total protein levels were lowered compared to the normal group. Pretreatment with silymarin and β-asarone effectively mitigated the metabolic alterations generated by paracetamol. The hepatoprotective effect of β-asarone (50mg/kg, i.p.) was substantially superior to that of EEBM (25mg/kg, p.o.) and the reference group (silymarin 100mg/kg, i.p.).

Fig 1: Effect of β-asarone on AST level in PCM induced hepatotoxic rats

Fig 2: Effect of β-asarone on ALT level in PCM induced hepatotoxic rats

Fig. 3: Effect of β-asarone on Direct bilirubin level in PCM induced hepatotoxic rats

Fig. 4: Effect of β-asarone on total bilirubin in PCM induced hepatotoxic rat

Fig 5: Effect of β-asarone on Total protein level in PCM induced hepatotoxic rats

Fig 6 : A Section of liver of normal rat showing portal triad with hepatocytes (Group I Normal control)

Fig 7: A Section of liver of rat treated with paracetamol induces hepatotoxicity showing portal triad with hepatocytes. (Group II Toxic control

Fig 8: A Section of liver of rat treated with silymarine showing portal triad with hepatocytes (Group III Standard)

Fig 9 : A Section of liver of rat treated with β-asarone showing portal triad with hepatocytes (Group IV Test 1).

Fig 10: A Section of liver of rat treated with β-asarone showing portal triad with hepatocytes. (Group V Test)

Histology:

In healthy animals, liver slices exhibited normal hepatic cells characterised by well-preserved cytoplasm, a conspicuous nucleolus, and a central vein (Fig:6 ). In rats treated with paracetamol (2000mg/kg, administered in two divided doses orally), histological sections revealed a moderate extent of hepatic injury, characterised by periportal and lobular inflammation together with mild congestion. The alterations resulting by paracetamol are minimal (Fig: 7). In mice treated with silymarin (100mg/kg, p.o.), liver sections exhibited mild inflammation and moderate congestion (Fig: 8). In mice treated with β-asarone 1(25mg/kg, i.p.), liver sections exhibited mild inflammation and congestion (Fig: 9). In mice treated with β-asarone 2(50mg/kg, i.p.), liver sections exhibited minor inflammation and congestion, with the overall appearance resembling that of a normal liver (Fig: 10 )

DISCUSSION:

Numerous substances and medications have the potential to harm the liver. As a hepatotoxicant to produce liver damage, paracetamol was chosen for this study. Examining the hepatoprotective effects of various doses of β-asarone against paracetamol-induced hepatotoxicity is the main goal of this research. As an antipyretic and analgesic, paracetamol is a typical medication. Multiple investigations in both animals and humans have shown that greater doses induce liver damage or necrosis^20,21

The induction of hepatotoxicity by paracetamol has proven to be an effective strategy for the screening of hepatoprotective drugs. The kidneys are responsible for excreting paracetamol after it has been metabolised in the liver by conjugation with glucuronide and sulphate. A portion of paracetamol is converted to the very reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) by liver cytochrome P-450, which is thought to be the cause of paracetamol hepatotoxicity²². Increased lipid peroxidation from the abstraction of hydrogen from a polyunsaturated fatty acid and, finally, liver damage from higher dosages of paracetamol are outcomes of toxic metabolites (N-acetyl-p-benzoquinoneimine) that can alkylate and oxidise intracellular glutathione (GSH)²³. Initiating cell stress can be achieved by reactive metabolites by many pathways, such as reducing glutathione (GSH) levels or attaching to enzymes, lipids, nucleic acids, and other cellular components^24,25,26,27. A substantial decrease in AST, ALT, direct, and total bilirubin levels and an increase in total protein were observed in experimental rats that were pretreated with β-asarone and silymarin. Comparable to normal liver histopathology was observed in experimental mice that were pretreated with β-asarone and silymarin. Pretreatment with β-asarone considerably reduced the histopathological and biochemical changes caused by PCM in both blood and tissue, as shown by the present study's results. These changes included levels of total protein, direct bilirubin, and total bilirubin as well as serum AST and ALT. In comparison to β-asarone at a dose of 25mg/kg body weight, the highest effect is observed when the dosage is 50mg/kg body weight²⁸.

CONCLUSION:

The acute oral toxicity of β-asarone was evaluated in albino rats, revealing no mortality up to 221mg/kg i.p., leading to the selection of 25mg/kg and 50mg/kg i.p. doses for further studies. Paracetamol administration at 2000mg/kg (p.o.) significantly elevated liver enzymes (AST, ALT), bilirubin levels, and reduced total protein, indicating hepatic damage. Pretreatment with silymarin and β-asarone effectively mitigated these biochemical alterations, with β-asarone at 50mg/kg showing superior hepatoprotective effects compared to both silymarin and EEBM. Histological analysis supported these findings, as liver sections from β-asarone 2-treated rats displayed minimal inflammation and congestion, closely resembling normal hepatic architecture, whereas paracetamol-treated rats showed moderate liver damage. These results affirm the protective potential of β-asarone against paracetamol-induced hepatotoxicity.

CONFLICTS OF INTEREST:

All authors declared that there is no conflict of interest

Authors Contribution and credit:

# indicated authors considered as 1^st author and given equal contribution:

Nivedita Singh: Writing original manuscript, Aarti Tiwari: Data curation and plagiarism checked, Satyabrata Rout: Re Editing of the manuscript, Chandan Sahu: Data Collection, Prasanjit Deep: formal analysis Gulshan Athbhaiya: Editing of the manuscript, Pradeep Kumar Samal: Supervision and guiding

ACKNOWLEDGEMENTS:

We acknowledge the Department of Pharmacy, Guru Ghasidas Vishwavidyalaya, Bilaspur for their kind support

REFERENCES:

1. Ayoub SS. Paracetamol (acetaminophen): A familiar drug with an unexplained mechanism of action. Temperature. Routledge. 2021; 8(4): 351-371. doi:10.1080/23328940.2021.1886392

2. Liao J, Lu Q, Li Z, Li J, Zhao Q, Li J. Acetaminophen-induced liver injury: Molecular mechanism and treatments from natural products. Front Pharmacol. 2023; 14. https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2023.1122632

3. Tsang WH, Chan CK, Tse ML. Paracetamol-induced hepatotoxicity after normal therapeutic doses in the Hong Kong Chinese population. Hong Kong Medical Journal. 2024; 30(5): 355-361. doi:10.12809/hkmj2311194

4. Samal PK, Dangi JS. Isolation, preliminary characterization and hepatoprotective activity of polysaccharides from Tamarindus indica L. Carbohydr Polym. 2014; 102(1): 1-7. doi:10.1016/j.carbpol.2013.10.059

5. Gunaydin C, Bilge SS. Effects of nonsteroidal anti-inflammatory drugs at the molecular level. Eurasian Journal of Medicine. AVES İbrahim KARA. 2018; 50(2): 116-121. doi:10.5152/eurasianjmed.2018.0010

6. Rotundo L, Pyrsopoulos N. Liver injury induced by paracetamol and challenges associated with intentional and unintentional use. World J Hepatol. 2020; 12(4): 125-136. doi:10.4254/wjh.v12.i4.125

7. Prakash Patel O, Tiwari A, Athbhaiya G, Vaishnaw B, Choudhary R, Kumar Samal P. Hepatoprotective Activity of Methanolic Extract of Amaranthus cruentus in Paracetamol Intoxicated Rats. Journal of Chemical Health Risks. www.jchr.org

8. Samal PK. Evaluation of Antioxidant activity of Ardisia solanacea in albino rats. Research J Pharm and Tech. 2013; 6(5). www.rjptonline.org

9. Samal PK, Vaishnaw B, Ahirwar B, et al. Evaluation of the Hepatoprotective potential and Antioxidant activity of 4–Hydroxybenzyl alcohol on Carbon tetrachloride Induced liver damage in Experimental animals. Res J Pharm Technol. 2023; 16(12): 6022-6027. doi:10.52711/0974-360X.2023.00977

10. Sahu P, Tiwari A, Athbhaiya G, Mandal S, Samal PK. Pharmacological Screening of Hepatoprotective Activity of Vanillylacetone in Paracetamol Induced Hepatotoxic Rats. Res J Pharm Technol. 2024; 17(10): 5085-5090. doi:10.52711/0974-360X.2024.00782

11. Chidiac AS, Buckley NA, Noghrehchi F, Cairns R. Paracetamol (acetaminophen) overdose and hepatotoxicity: mechanism, treatment, prevention measures, and estimates of burden of disease. Expert Opin Drug Metab Toxicol. 2023; 19(5): 297-317. doi:10.1080/17425255.2023.2223959

12. Fontana RJ. Acute Liver Failure Including Acetaminophen Overdose. Medical Clinics of North America. 2008; 92(4): 761-794. doi:https://doi.org/10.1016/j.mcna.2008.03.005

13. Kumar Samal P, Dangi J, Pd Meena K, Beck N, Maheshwari G, Patel A. Hepatoprotective Activity of Butea monosperma bark on Liver Damage Caused by Paracetamol in Rats. Research J Pharm and Tech. 2011; 4(5). www.rjptonline.org

14. Saad M, Flament J. Paracetamol overdose causing acute kidney injury without hepatotoxicity: a case report. Int J Emerg Med. 2024; 17(1): 81. doi:10.1186/s12245-024-00662-w

15. Akhtar MN, Saeed R, Saeed F, et al. Silymarin: a review on paving the way towards promising pharmacological agent. Int J Food Prop. 2023; 26(1): 2256-2272. doi:10.1080/10942912.2023.2244685

16. Kim YC, Na JD, Kwon DY, Park JH. Silymarin prevents acetaminophen-induced hepatotoxicity via up-regulation of the glutathione conjugation capacity in mice. J Funct Foods. 2018; 49: 235-240. doi:https://doi.org/10.1016/j.jff.2018.08.025

17. Wang A, Zhou Y, Fu X, et al. Structural Derivatives of β-Asarone from Acorus calamus Linn. as Insecticide Candidates and the Insecticidal Mechanism Against Small Brown Planthopper. Agronomy. 2024; 14(10). doi:10.3390/agronomy14102420

18. Balakrishnan R, Cho DY, Kim IS, Seol SH, Choi DK. Molecular Mechanisms and Therapeutic Potential of α-and β-Asarone in the Treatment of Neurological Disorders. Antioxidants. MDPI. 2022; 11(2). doi:10.3390/antiox11020281

19. Mcgaw L, Jäger A, Staden J, Eloff J. Isolation of β-asarone, an antibacterial and anthelmintic compound, from Acorus calamus in South Africa. South African Journal of Botany. 2002; 68: 31-35. doi:10.1016/S0254-6299(16)30450-1

20. Yan M, Huo Y, Yin S, Hu H. Mechanisms of acetaminophen-induced liver injury and its implications for therapeutic interventions. Redox Biol. 2018; 17: 274-283. doi:https://doi.org/10.1016/j.redox.2018.04.019

21. Samal PK. Investigation of Antioxidant activity of Butea monosperm barks. Research J Pharm and Tech. 2013; 6(6). www.rjptonline.org

22. Ulicna O, Greksák M, Vancova O, et al. Hepatoprotective effect of Rooibos tea (Aspalathus linearis) on CCl4 –induced liver damage in rats. Physiological research / Academia Scientiarum Bohemoslovaca. 2003; 52: 461-466. doi:10.33549/physiolres.930340

23. Moore M, Thor H, Moore G, Nelson S, Moldéus P, Orrenius S. The toxicity of acetaminophen and N-acetyl-p-benzoquinone imine in isolated hepatocytes is associated with thiol depletion and increased cytosolic Ca2+. Journal of Biological Chemistry. 1985; 260(24): 13035-13040. doi:10.1016/s0021-9258(17)38834-8

24. Lushchak VI. Glutathione Homeostasis and Functions: Potential Targets for Medical Interventions. J Amino Acids. 2012; 2012(1): 736837. doi:https://doi.org/10.1155/2012/736837

25. Nirmalkar D, Tiwari A, Athbhaiya G, Samal PK. Hepatoprotective Activity of Lathyrus sativus Leaf Extract in Paracetamol-Induced Hepatotoxic Rats. Res J Pharm Technol. 2025; 18(2): 625-631. doi:10.52711/0974-360X.2025.00093

26. Ekka NR, Namdeo KP, Samal PK: Standardisation strategies for herbal drugs- an overview. Res J Pharmcol Technol. 2008; 1(4): 310–312.

27. Beck, N.R.; Samal, P. Traditional medicinal plants used by the tribes and rural people of Bilaspur district, Chhattisgarh (India). Res. J. Pharm. Technol. 2012; 5: 1281–1282.

28. Athbhaiya, G., Tiwari, A., Choudhary, R., & Samal, P. K. (2026). Pharmacological and toxicological effects of paracetamol: current knowledge and a review. Drug and Chemical Toxicology, 1–24. https://doi.org/10.1080/01480545.2025.2604674.

Received on 04.11.2025 Revised on 19.03.2026

Accepted on 23.05.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3053-3058.

DOI: 10.52711/0974-360X.2026.00434

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group	SGOT	SGPT	Direct Bilirubin	Total Bilirubin	Total Protein
Normal control	42.48±5.300	58.81±5.501	0.3233±0.06652	0.8067±0.1380	7.140±0.5697
Toxic (paracetamol)	156.8±9.097^c	156.8±7.728^c	0.7283±0.05741^c	2.312±0.3605^b	3.003±0.4293^b
Standard	51.31±6.286^f	62.15±5.220^f	0.3883±0.04679^e	0.9383±0.1860^e	7.272±0.6234^e
β-asarone 1	70.79±6.147^f	71.57±6.386^f	0.4733±0.05566^d	1.140±0.3111^d	5.807±1.154
β-asarone 2	54.51±6.763^f	62.84±6.382^f	0.4138±0.05029^f	0.9200±0.1432^f	6.587±0.6145^d