Protective effect of Saccharum officinarum Linn juice in Paracetamol Induced Acute Hepatotoxicity in Albino Rats
Rohtash Singh*, Rahul Shukla
School of Pharmaceutical Sciences, Shri Venkateshwara University, Gajraula, Amroha - 244236 (U.P) India.
*Corresponding Author E-mail: rohtasrajput@gmail.com
ABSTRACT:
Saccharum officinarum L (Poaceae) is popular in the native system of medicines like Ayurveda, Siddha, Unani and Homoeopathy. In the traditional system of medicine various plant parts are used as anti-diuretic, refreshment, diarrhea, jaundice and various liver disorders. Objective: This study aimed to investigate if Saccharum officinarum L. Juice had protective effect in acute liver toxicity with paracetamol. Material and Methods: This was a laboratory based experimental study. A total number of 24 Wister Albino rats were divided into four groups as the positive control group (1.5mL distilled water), negative control group treated with paracetamol (3gm/kg), standard group treated with Clofibrate (50mg/kg), test group treated with fresh juice (20 ml/kg) were administered for eight days. Paracetamol was administered on day 8th. In addition, liver tissues were evaluated histological (in terms of increased vein inlet, connective tissue, granular degeneration, necrotic cells and vascular congestion). Results: Experimental findings revealed that the fresh juice at dose level of 20 ml/kg b.w. showed dose dependant hepatoprotective effect against PCM induced hepatotoxicity by significantly restoring the levels of serum enzymes to normal that was comparable to that of Clofibrate, but the fresh juice at dose level of 20 ml/kg was found to be potency when compared to that of 50 mg/kg standard. Besides, the result of the histopathological studies also support the study. Conclusion: The results were concluded that fresh juice of Saccharum officinarum L afforded significant protection against PCM induced hepatotoxicity in rats.
KEYWORDS: Saccharum officinarum L, Paracetamol, Clofibrate, Hepatotoxicity, histopathology.
INTRODUCTION:
Paracetamol also known as acetaminophen (IUPAC name: N-(4-hydroxyphenyl) ethanamide), is the most commonly used antipyretic and pain reliever and since 1955 has been available over the-counter as a single formulation1. Although many side effects following drug use had been registered since the approved use in the 50s, its hepatotoxicity was not significantly recognized until 1980. Cases of fatal drug-related hepatotoxicity dubbed “therapeutic misadventures” and the association of paracetamol poisoning with alcohol were firstly reported during the mid 1980s2. While PCM is described as relatively nontoxic when administered in therapeutic doses3, it is known to cause toxicity when taken in a single or repeated high dose, or after chronic ingestion. The usual dosing of immediate-release oral preparations in adults is 325-750mg every 4-6 hours or 1g every 4-6 hours as necessary, without exceeding 4g per day4. Conversely, in children, the recommended dose is 10-15 mg/kg every 4-6 hours up to a maximum daily dose of 50-75mg/ kg5. Adverse events typically associated with paracetamol intoxication are represented by acute liver failure, centrilobular hepatic necrosis, renal tubular necrosis and hypoglycemic coma6.
Hepatotoxicity of Paracetamol and Related Fatalities:
After the administration of an oral dose, paracetamol is rapidly absorbed by the intestine, because of its weak acidity and lipid-solubility. Then, an amount between 50 and 60% is converted to its main and pharmacologically inactive glucuronidated and sulfated conjugates eliminated in urine. In liver microsomes, a small percentage of paracetamol (5-10%) is converted by cytochrome P450 isoforms (CYP2E1, CYP2A6) into a reactive metabolite, N-acetyl-para-benzo-quinoneimine that is primarily related to paracetamol hepatotoxicity7. About 2% of paracetamol is excreted in urine unchanged (Fig.1).
Fig. 1 Paracetamol metabolism
Other Side Effects of Paracetamol:
Paracetamol-induced liver necrosis has been studied extensively, but the extra hepatic manifestations of paracetamol toxicity are currently not well described in the literature. Renal insufficiency occurs in approximately 1-2% paracetamol users following overdose8. Limited data in a retrospective case series of pediatric patients with paracetamol poisoning suggests that associated nephrotoxicity may be more common in children and adolescents9.
S. officinarum L, commonly known as sugar cane is a tall grass native to tropical regions of Asia10. It has been used as a folk remedy for arthritis, bedsores, boils, cancer, colds, cough, diarrhoea, dysentery, fever, hiccups, inflammation, laryngitis, sore throat and wounds for centuries. Its main properties being that of an antiseptic and bactericide, it has also been used as a cardiotonic, diuretic, and laxative11. It has been reported to possess antioxidant12, anti-cancerous and anti-proliferative properties13-14. Different extracts of sugarcane are reported to have cholesterol lowering and antiplatelet effects15 along with prevention of injury in brain16. Sugarcane juice is a rich source of antioxidant compounds, i.e., polyphenolics predominantly falvonoids17. The present work was, therefore, designed to evaluate the effect of Saccharum officinarum L. on paracetamol induced hepatotoxicity.
MATERIAL AND METHODS:
Collection and Authentication of plant:
The plant of S.officinarum was selected after the literature survey and collected from Gajraula, Amroha (U.P). The plant of S.officinarum was authenticated by the senior botanist Dr D.C Kasana, Head of the Department of Botany, I.P College of Science, Bulandshahr (U.P). Specification – IP College of Science - SOP- BVSO/09/1753
Preparation of Juice:
The sugarcane (S.officinarum) is washed well and peeled to remove the outer layer with a suitable knife and then as small pieces pressed between tow metal rollers. The peel extract is collected in a big container and strained using a muslin cloth. It is then stored at room temperature (12 to 20ºc) in well-closed glass container for future use.
Evaluation of Experimental Animals:
Healthy adult Male Wister albino rats of Sprague dawley strain, weighing 150-200gm was selected for the study. They were fed with a standard pellet diet and water ad libitum. All animal protocols were approved by the Institutional Animal Ethical committee (IAEC) of the organization (Reg. The Institutional Animal Ethical Committee of Janta College of Pharmacy Butana, (Sonepat) Haryana, India (CPCSEA-667/02/c/CPCSEA) approved the studies.). All animals were maintained under standard conditions of humidity (50±10%), temperature (22±200c) and light (12 hours light and 12 hours dark). The animals were acclimatized to laboratory conditions (RT-25°C) for 4 days and given pelleted animal feed (Hindustan Lever) and drinking water.
Acute toxicity studies:
The WHO has set guidelines for toxicity studies of herbal medicine. It supports appropriate usage of herbal medicines and encourages the remedies, which are proved to safe and efficacy.18 Acute toxicity study was performed for water extract according to the acute toxic classic method as per OECD guidelines19, albino rats were used for acute toxicity study. The animals were kept fasting for overnight providing only water, after which the fresh juice was administered orally at the dose of 1, 5, 10, 20 and 40ml/kg and observed for 8th days. If mortality was observed in 2 out of 3 animals, then the dose administered was assigned as toxic dose. If the mortality was observed in 1 animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher dose i.e., 20ml/kg.
Paracetamol induced Hepatotoxicity:
Experimental animals: 24 male (n=6) Wister albino rats which were just weaned with a weight of 150-200gm (4-5 weeks of age) were used in the study20.
|
S. No |
Groups |
Treatments |
|
1. |
Positive Control |
1.5mL/day distilled water was given orally for eight days. |
|
2. |
Negative Control |
1.5mL/day distilled water was given by gavage for eight days, paracetamol was given orally at a dose of 3000 mg/kg on the 8th day |
|
3. |
Standard (Clofibrate) |
Drug control animal: Paracetamol induced hepatotoxicity animals treated with Clofibrate (50mg/kg-bw) for 8th days. |
|
4. |
S. officinarum L (Fresh Juice) |
S. officinarum L+ Paracetamol: 20 ml/kg sugarcane juice was given by gavage for eight day; paracetamol was given orally at a dose of 3000 mg/kg on the 8th day. |
Anesthesia, obtaining tissue and blood samples: On the 9th day, anesthesia was achieved by i.p (intraperitoneal) route ketamine (80mg/kg) and xylazine (10mg/kg) before sacrifice and collect the blood sample (3-6mL) was obtained from the aorta with sterile injector. The blood samples were centrifuged at 5000 RPM for 5 minutes and their sera were separated. At the end of the study, the abdomen was cut in the midline and the right lobe of the liver was removed for histological examination21.
Histopathological examination:
The tissues were fixed in 10% neutral formaldehyde solution and paraffin blocks were prepared. Five-micron-thick sections were obtained from the paraffin blocks prepared. The samples were stained with hematoxylin-eosine for histological evaluation22.
The results were expressed as mean±standard error mean. The statistical significance was assessed using one-way analysis of variance, followed by Bonferroni's Multiple Comparison test.
RESULTS:
Acute toxicity:
The fresh juice was found to be safe in the dose used and no mortality up to a dose of 40ml/kg, b.w. for fresh juice was observed. Hence, 20ml/kg b.w. p.o. were selected for the activity
The administration of PCM resulted in a marked increase in serum SGOT, SGPT, total and direct bilirubin and triglycerides. The protective actions of fresh juice at dose level of 20 ml/kg b.w on hepatotoxicity induced by PCM are summarized in table and figures (Table 1 and 2-6). Pretreatment of the rats with fresh juice before PCM administration caused a significant reduction in the values of serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), direct bilirubin, total bilirubin, and triglycerides.
Table1. Effect of Saccharum officinarum L fresh Juice on different biochemical parameters in paracetamol-induced hepatotoxicity in rats
|
Groups |
Treatment |
SGPT (IU/L) |
SGOT (IU/L) |
Direct Bilirubin (mg/dl) |
Total Bilirubin (mg/dl) |
Triglycerides (mg/dl) |
|
I |
Control (Saline, 1ml/kg) |
63.06±0.13** |
129.5±0.42** |
0.32±0.041** |
0.60±0.03* |
88.56±1.06* |
|
II |
Positive Control (PCM, 3g/Kg) |
115.34±0.37 |
231.27±0.63 |
1.21±0.18 |
1.17±0.02 |
171.12±0.53 |
|
III
|
Standard (Clofibrate), 50 mg/kg) + PCM |
73.00±2.45* |
147.39±2.13* |
0.49±0.05** |
0.76±0.02** |
120.79±1.56* |
|
IV
|
Fresh Juice (20ml/kg) + PCM |
88.16±0.83** |
170.71±0.81** |
0.83±0.02* |
0.86±0.06* |
136.91±4.39** |
Values are mean ± SEM (n=6) one way ANOVA. Where, * represents significant at p<0.05, ** represents highly significant at p< 0.01
|
Fig 2: Representing the effect of fresh juice of S. officinarum on SGPT levels in PCM- induced hepatotoxic rats. |
Fig 3: Representing the effect of fresh juice of S. officinarum on SGOT levels in PCM- induced hepatotoxic rats. |
|
|
|
Fig 4: Representing the effect of fresh juice of S. officinarum on direct bilirubin in paracetamol induced hepatotoxic rats. |
Fig 5: Representing the effect of fresh juice of S. officinarum on total bilirubin in paracetamol induced hepatotoxic rats. |
|||
Fig 6: Representing the effect of fresh juice of S. officinarum on triglyceride in paracetamol induced hepatotoxic rats.
|
|
|
|
Fig. 7: Histopathology of liver of healthy normal control animals showing normal histological activity, with central vein from which chords of hepatocytes were radiating with no damage. |
|
|
|
|
|
Fig. 8: Histopathology of liver of animals treated with PCM only showing severe damage in central vein, and changes of fatty degenerations as well as necrosis of hepatocytes. |
|
|
|
|
|
Fig. 9: Histopathology of liver of animal treated with standard drug (Clofibrate) showing central vein with mild to moderate diffuse granular degeneration and very mild necrosis in hepatocytes. |
|
|
|
|
|
Fig. 10: Histopathology of liver of animals treated with S. officinarum L fresh juice at 20ml/kg reduced damage due to PCM in hepatocytes showing regeneration of hepatocytes and reduced necrosis. |
|
The histological observations of control rats did not show any histological alterations in the hepatocytes. The liver sections showed normal architecture with no damage in the central vein and no change in sinusoids and hepatocytes architecture (Figure 7). In toxic control group, the liver sections showed hepatic cell necrosis along with severe damage associated with central vein due to PCM (Figure 8). In group treated with Clofibrate, the liver sections showed mild to moderate diffuse granular degeneration and necrosis in hepatocytes (Figure 9). In group treated with fresh juice (20ml/kg), the liver sections showed reduced severity of damage and regeneration of hepatocytes, bile duct, branch of hepatic portal vein and minimal necrosis caused by PCM intoxication (Figures 10).
DISCUSSION:
The present study investigated the protective effect of fresh juice of S. officinarum L whole plant compared with the protective effect of Clofibrate on PCM induced hepatotoxicity in albino rat model. PCM is a very common antipyretic agent that is safe in therapeutic doses, but can produce fatal hepatic necrosis in human being, mice and rats with toxic doses. PCM toxicity is due to the development of toxic metabolites when a part of it is metabolized by cytochrome P-450. Cytochrome P450 or depletion of hepatic glutathione is a condition for PCM- induced hepatotoxicity. Due to liver damage (in terms of increased vein inlet, connective tissue, granular degeneration, necrotic cells and vascular congestion).
The experimental findings of hepatoprotective activity of S. officinarum L Juice exhibited remarkable protective effect against the paracetamol induced hepatotoxicity in rats. Administration of paracetamol (3g/kg, b.w) to each group except control group caused elevation of serum enzyme levels (SGOT, SGPT), direct bilirubin, total bilirubin and triglycerides as compared to control group.
The fresh juice dose (20ml/kg, b.w) showed significant reduction in elevated levels of serum enzymes (SGOT, SGPT), direct bilirubin, total bilirubin and triglycerides.
The effect was comparable to that of standard drug Clofibrate (50mg/kg, b.w). The fresh juice at dose level (20ml/kg, b.w) was found to be more effective as compared to dose (1.5ml/kg, b.w).
The histopathological studies provided supportive evidence for biochemical parameters. Histology of liver section of control animals (group I) showed the normal histological architecture with well defined cytoplasm, nucleus and central vein (Fig.7), while the positive control group showed severe damage (Fig.8), hepatic necrosis and fatty degenerations of hepatocytes. Treatment with standard and fresh juice recovered the injured liver to quite normal (Fig.9-10).
CONCLUSION:
S. officinarum L plant fresh juice revealed significant hepatoprotective activity on paracetamol induced hepatotoxicity in rat model.
These investigations revealed that the plant juice had potent hepatoprotective activity. However, further research on detailed pharmacological screening, isolation of active phytoconstituents responsible for therapeutic activity and clinical study for evaluation of safety and efficacy of the drug, need to be assessed.
ACKNOWLEDGEMENT:
I would like to thank entire department of Pharmacy, Shri Venkateshwara University, Gajraula, Amroha, (U.P) for their support and cooperation during the research.
CONFLICT OF INTEREST:
We declare that we have no conflict of interest.
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Received on 18.08.2020 Revised on 12.09.2020
Accepted on 28.09.2020 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Res. 2021; 11(1):17-22.
DOI: 10.5958/2231-5691.2021.00005.8