Screening of DHB against Hepatorenal toxicity.

Paracetamol is a commonly used analgesic and antipyretic drug, but at a high dose level, it leads
to deleterious side effects. The need to investigate new hepatoprotective drugs is driven by the
lack of safety and efficiency of existing medications. A newly synthesized compound 2′, 3′-
dihydroxybenzylidene (DHB) was evaluated in the present study for its antioxidant, hepatoprotective and nephroprotective potential compared to ascorbic acid in paracetamol intoxicated rats. DHB and ascorbic acid were evaluated against 2, 2-diphenyl-1-picrylhydrazyl (DPPH)
for assessment of the antioxidant potential. Male albino rats (n = 20) were categorized into 5
groups with 4 rats each and the test compounds were administered for 14 days consecutively. On
day 15th, the rats were anesthetized, and blood was collected through heart puncture for the
evaluation of hematological and serological parameters. Subsequently, the rats were dissected for
the histopathology of liver and kidney. Alanine Transaminase (ALT), Alkaline Phosphatase (ALP),
Serum Bilirubin (SBR), Cholesterol level and Renal Function Tests (RFTs) showed a substantial
increase in the paracetamol treated group. Healing in liver and kidney tissues was observed in the
DHB treated groups compared to paracetamol intoxicated group. The hemoglobin (HB), mean
corpuscular hemoglobin (MCH), RBCs and mean corpuscular hemoglobin concentration (MCHC)
were found to be significantly elevated, while the total leukocyte count (TLC), platelets (PLT) and
neutrophils significantly decreased in the DHB treated group compared to the paracetamol
intoxicated group. It is concluded that DHB possesses antioxidant, hepatoprotective, neuroprotective, and anti-inflammatory potential against paracetamol induced hepatotoxicity and
nephrotoxicity in rats.

1. SUPERVISOR SUBMITTED BY Dr. Pradeep Kumar Samal Gulshan Athbhaiya Assistant Professor M. Pharm 3 rd Sem JOURNAL CLUB PRESENTATION ON “ Evaluation of Hepatorenal protective activity of Plant extract . in Paracetamol intoxicated Rats ” SUPERVISOR SUBMITTED BY Dr. Pradeep Kumar Samal Gulshan Athbhaiya Assistant Professor Roll no. 22084103 ( Pharmacology ) Regn.no. GGV/22/06303 Department of Pharmacy GURU GHASIDAS VISHWAVIDYALAYA ( A Central University established by the Central Universities Act.2009 No. 25 of 2009 )

2. Title, Author & Affiliations The research article has been selected from the Heliyon a Cell Press Journal, an Elsevier publication. Journal has an impact factor of 4.0 in 2023. Author: Mohammad Attaullah Aziz Ullah Muhammad Hussain Muhammad Zahoor Riaz Ullah Essam A. Ali Ateeq Ur. Rahman Arif Jan

3. INTRODUCTION:- Paracetamol, also known as Acetaminophen, is commonly used as an analgesic and antipyretic drug, but an overdose of 4g causes major hepatotoxicity and is recently known for nephrotoxicity. Paracetamol damages the liver and increases liver biomarkers. The liver is a functional organ for digestion, metabolism, detoxification & removal of waste. Drug metabolism generates ROS. The ROS needs to be cleared by antioxidants to reduce the oxidative stress. Ascorbic acid, a water-soluble vitamin well known as a strong antioxidant. It has a vital role in wound healing and protection against UV radiation. The compound N-(2, 4-Dinitrophenyl)-N’-(2′, 3′-dihydroxybenzylidene) hydrazone having common name 2′, 3′-dihydroxybenzylidene abbreviated as DHB. AIM- To evaluate the newly synthesized compound 2’,3’-dihydroxybenzylidene for its antioxidant, hepatoprotective & nephroprotective potential compared to ascorbic acid in paracetamol intoxicated rats. Fig. Structure of DHB

4. EXPERIMENTAL DESIGN:- S.NO TREATMENT GROUP NO. of Animals Dose & route 1 Normal Saline 4 2ml/kg, p.o. for 14 days. 2 Negative control ( Paracetamol) 4 650 mg/kg, p.o. 3 Positive control (DHB) 4 200mg/ kg,p.o . 4 Experimental group 4 200mg/kg of DHB + 650mg/kg of PCM 5 Experimental standard group 4 200mg/kg of Ascorbic acid + 650mg/kg of PCM The dose of DHB was prepared in 10 % DMSO (90 % water and 10 % DMSO). Paracetamol and ascorbic acid were prepared in a 5 % normal saline solution. After 14 days of dosage, the rats were fasted for 24 hours and then provided free access to tap water till the next morning on day 15.

5. Results:- Weight (g) of rats before experiments and after 15 days of experimental work. Weight (g) (Mean± SD) Day 01 Day 15 Normal Control group 145 c ± 4.20 155 c ± 5.65 Negative control group 116.75 ± 4.64 97.25 ± 4.64 Positive control group 118 c ±4.83 127 c ± 3.16 Experimental group 143 c ± 5.03 159 c ± 4.96 Experimental standard group 128 c ± 13.96 146 c ± 11.35 C =(p<0.001). DPPH assays of DHB and ascorbic acid. Compound Concentration ( μg /mL) DPPH scavenging assay (%) DHB 1000 80.5 500 78.36 250 76.02 125 73.46 62.5 69.89 31.25 64.4 Ascorbic acid 1000 95.7 500 90.2 250 87.3 125 83.3 62.5 80.1 31.25 76.9

6. Lipid profile and blood sugar level in different experimental groups of rats. Parameters Normal control Negative control Positive control * Exp.group * Exp.standard Cholestrol (mg/dl) 85 c ± 5.6 105.6 ± 7.7 104.3 c ± 4.1 91.6 c ± 3.0 97.6 c ± 4.1 TG(mg/dl) 71.5 c ± 3.5 74 c ± 3.6 70 c ± 8.5 73 c ± 4 73 c ± 7.2 HDL(mg/dl) 15.5 a ± 2.1 16.3 ± 2.3 13.6 a ± 1.1 13.6 a ± 3.0 14 a ± 2 LDL(mg/dl) 52.2 c ± 2.1 76.6 ± 6.8 71.6 c ± 3.0 71.6 c ± 3.0 75 c ± 7.2 Total lipids (mg/dl) 229 c ± 4.2 269.3 ± 12.5 259.6 c ± 4.0 267.2 c ± 2.3 267.6 c ± 2.3 Blood sugar(mg/dl) 115 a ± 8.4 117 ± 5.2 118.66 c ± 9.5 106 c ± 8.1 99.3 c ± 7.5 a,c =a(p<0.05),c(p<0.001)

7. Histology- Liver tissue of normal control rats indicating normal hepatocytes, central vein and Kupffer cells. (a) image at low magnification 100X (b) image at high magnification 400X Liver tissue of negative control group indicating necrosis in hepatocytes, dilation in central vein and proliferation in Kupffer cells. Inflammation, fibrosis, and steatosis are found as indicated by the arrows. (a) image at low magnification 100X (b) image at high magnification 400X. Liver tissue of positive control group indicates dilation in central vein while other areas are normal with no proliferation in Kupffer cells and no inflammation in hepatocytes. (a) image at low magnification 100X (b) image at high magnification 400X Liver tissue of experimental group indicating recovery in hepatocytes, central vein dilated but recovered up to some extent as indicated by vascular degeneration. Inflammatory cells between the hepatocytes were recovered while most areas were found clear. (a) image at low magnification 100X (b) image at high magnification 400X

8. Liver tissue of experimental standard group indicating slight dilation in the central vein and recovery in the hepatocytes and Kupffer cells. No inflammation was seen. (a) image at low magnification 100X (b) image at high magnification 400X Kidney tissue of the negative control group (paracetamol administered rats). Necrosis and congestion in the glomerulus and tubules are evident. Inflammatory cells can clearly be seen in between the renal tubules indicated by the arrow heads. (a) image at low magnification 100X (b) image at high magnification 400X. Kidney tissue of the normal group of rats. The arrow heads indicate the normal structure of glomeruli and tubules. No infiltration or damage were seen. (a) image at low magnification 100X (b) image at high magnification 400X Kidney tissue of DHB administered rats. The arrow heads indicate the normal structure of glomeruli and tubules. No infiltration or damage were seen. (a) image at low magnification 100X (b) image at high magnification 400X.

9. Kidney tissue of the experimental group (DHB and paracetamol administered rats). The arrow heads indicate the normal structure of glomeruli and tubules. Slight infiltration or damage were seen. Damage was healed back in the inflammatory cells. (a) image at low magnification 100X (b) image at high magnification 400X Kidney tissue of the experimental standard group (ascorbic acid and paracetamol administered rats). The arrow heads indicate the normal structure of glomeruli and tubules. Slight infiltration or damage were seen. Damage was healed back in the inflammatory cells. (a) image at low magnification 100X (b) image at high magnification 400X Discussion- Antioxidant, hepatoprotective, and nephroprotective studies were evaluated in Paracetamol intoxicated rats. The DHB antioxidant activity as compared to Ascorbic acid against DPPH in rats possessed strong antioxidant potentials may be due to the presence of 2 hydroxyl groups. The weight in the Negative control group was significantly decreased as compared to the DHB & Ascorbic acid-treated group. The paracetamol-treated group displayed a markedly increase in the liver biomarkers with a decrease in ALT, ALP and SBR at a dose of 200mg/kg in DBH.Decrease in the cholesterol levels and LDL levels in DHB proves its hepatoprotective potential. Paracetamol causes tubular necrosis by damaging nephrons of kidney, marked by elevated levels of blood urea and creatinine level.DHB treatment reduced the levels as compared to ascorboic acid, which proving its nephroprotective potential. Reference- A. Adeneye , J. Olagunju , Protective effect of oral Ascorbic Acid (Vitamin C) against Acetaminophen-induced hepatic injury in rats, Afr. J. Biomed. Res. 11 (2008) 183–190, https://doi.org/10.4314/ajbr.v11i2.50704. O. Ozkaya , G. Genc , K. Bek , Y. Sullu , A case of acetaminophen (paracetamol) causing renal failure without liver damage in a child and review of literature, Ren. Fail. 32 (9) (2010) 1125–1127, https://doi.org/10.3109/0886022x.2010.509830 K. Okaiyeto , U. Nwodo , L. Mabinya , A. Okoh , A review on some medicinal plants with hepatoprotective effects, Pharmacogn . Rev. 12 (24) (2018) 186–199, https://doi.org/10.4103/phrev.phrev_52_17.

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