Hepatoprotective Activity of Lathyrus sativus Leaf Extract in Paracetamol-Induced Hepatotoxic Rats

Dipendra Nirmalkar, Aarti Tiwari, Gulshan Athbhaiya, Pradeep Kumar Samal*

Department of Pharmacy, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, 495009, India.

*Corresponding Author E-mail: samalpharmacology@rediffmail.com

ABSTRACT:

Aim: - The purpose of this study was to evaluate the hepatoprotective properties of Lathyrus sativus leaves against liver damage caused by paracetamol in Wistar rats. Methods: -Rats with hepatotoxicity from paracetamol were utilised to test the hepatoprotective benefits of a methanol extract made from Lathyrus sativus leaves. To cause hepatotoxicity, albino Wistar rats were fed paracetamol (2 g/kg) orally for 14 days. For 14 days, oral administration of the Lathyrus sativus leaf methanol extract at 200 and 400 mg/kg/day was conducted. The quantities of albumin, total bilirubin, ALT, AST, ALP, and protein were found using a serum analysis. To measure the amounts of glutathione, malondialdehyde, and superoxide dismutase, the liver was removed and ground up. Furthermore, investigations related to histology were performed on the liver samples. Results: The groups who received extract treatment were able to reduce the negative effects of paracetamol while restoring blood biochemical parameters to levels that were nearly normal. Conclusions: - According to the study, Lathyrus sativus leaves have significant hepatoprotective effects.

KEYWORDS: Paracetamol, Hepatoprotective activity, Lathyrus sativus, Oxidative stress, Antioxidant.

1. INTRODUCTION: 

The liver is the principal organ that regulates the metabolic and physiological equilibrium of the body at various phases. It plays a role in synthesising essential biomolecules that fight infections and eliminate toxic chemicals from the body^1,2. Liver disease results in over 2 million deaths per year worldwide. The causal factors encompass illnesses that impair liver function, drugs including ethanol, CCl4, thioacetamide, and D-galactosamine, environmental contaminants, and pharmaceuticals such as paracetamol. Paracetamol, commonly referred to as PCM, is a prevalent analgesic and antipyretic agent. Acute liver failure and hepatotoxicity are frequently linked to this specific risk factor^4,5. Medicinal herbs have been utilised for generations to mitigate various human ailments. Numerous natural phytochemicals, such as silymarin, curcumin, quercetin, and resveratrol, frequently serve as preventive agents against hepatic injury^6,7,8,9.

The Fabaceae family plant Lathyrus sativus L. has long been used as a traditional medicine to cure a variety of ailments, such as allergies, eczema, and scabies. Starch, cane sugar, legumelin, terpenes, gum resin, oleo-resin, alkaloids, carbohydrates, flavonoids, phenols, tannins, vitamin C, riboflavin, beta-carotene, carotenoids, proteins, and amino acids were among the constituents of the material. The material demonstrated a number of pharmacological characteristics, such as cardioprotective, antidiabetic, analgesic, neuroprotective, and antioxidant¹⁰.  More research on the hepatoprotective properties of Lathyrus sativus leaf extract against paracetamol-induced liver damage is required. Thus, using rats as an experimental model, the current work attempts to evaluate the hepatoprotective benefits of Lathyrus sativus against liver damage caused by paracetamol.

2. MATERIALS AND METHODS:

2.1 Drugs and Chemicals:

Paracetamol was acquired from Shreya Chemicals in Bilaspur, whereas Silymarin (Silybin 140, Micro Labs Limited) was obtained from the local market in Bilaspur. All the other compounds included in the investigation were of analytical grade.

2.2 Collection and authentication of plant material:

The fresh leaves of Lathyrus sativus L. were obtained at the local market in Arang, Raipur district, Chhattisgarh, India, in January 2023. After being washed with tap water, the leaves were air-dried at room temperature. The plant sample was verified by a botanist from the Department of Botany at Guru Ghasidas Vishwavvidyalaya, located in Koni, Bilaspur (C.G.), with the postal code 495009. The specimen voucher, with the identification number (Bot/GGV/2023/72), was submitted to the department for deposit.

2.3 Plant extract’s preparation:

After being shaded for two weeks, the leaves of Lathyrus sativus were allowed to dry naturally at room temperature. A mechanical grinder was used to finely grind the dried plant material until it became black. Then, methanol was extracted using a soxhlet method. A greenish-colored solvent was observed in the syphon tube once the extraction procedure was finished. Following filtration, the solvent extract was subjected to a Rotavapor with temperature control and reduced pressure until it was completely dry. A value for extraction was calculated.

2.4 Determination of total phenolic content (TPC):

In order to determine the total phenolic content (TPC), the Folin-Ciocalteu reagent assay was used to evaluate the methanolic extract of Lathyrus sativus (LSME). The phenolic content of the extract was measured in milligrammes of gallic acid equivalent per gramme (mg GAE/g). Add 0.5ml of an aqueous extract solution to 2.5ml of a 10% Folin-Ciocalteu reagent (v/v) and 2.0ml of a 7.5% Na₂CO₃ solution to quantify the total phenolic content. A Shimadzu Spec-1800 spectrophotometer was used to measure the absorbance at a wavelength of 765nm after incubating the reaction mixture at 45^◦C for 40 minutes.

2.5 Total flavonoid content (TFC) Determination:

An aluminium-flavonoid complex constituted the basis for the TFC determination. The sample was mixed with an equal volume of a 2% aluminium chloride solution, with a volume of 1millilitre. After 15 minutes of incubation at room temperature, the absorbance at 306 nm was recorded from the reaction mixture. There was a total of milligrams of quercetin equivalent per gramme (mg QE/g) of flavonoids in the extract.

2.6 GC-MS analysis:

The LSME was examined by gas chromatography–massage spectrometry (G.C.–M. Son at the Central Instrumentation Laboratory (CIL), Central University of Punjab, Bathinda, Punjab, India.

2.7 Phytochemical analysis of the extract:

A preliminary phytochemical analysis was conducted on the leaf extract of Lathyrus sativus to determine the presence of carbohydrates, proteins, alkaloids, steroids, glycosides, free amino acids, phenolics, flavonoids, and other compounds.

2.8 Experimental animals:

Wistar albino rats of both sexes weighing between 100-160g were obtained from Chakraborty companies. The animal home of GGV, Bilaspur, maintains the 3D Girish Vidyaratna lane, Narkeldanga, Kolkata-700011, for experimental purposes. Subsequently, the animals had a seven-day acclimatization period by typical husbandry settings, which included a room temperature of 25±1°C, a relative humidity range of 45-55%, and a 12:12 hour light/dark cycle. The animals were provided unrestricted access to a standard rat pellet (Kapila Posuahar) and water, fed ad libitum in a spotless and sanitary environment. The tests were conducted following the rules of CCSEA. IAEC (07/IAEC/Pharmacy/2023) granted ethical approval for the study conducted at Guru Ghasidas Vishwavvidyalaya, Koni, Bilaspur, India.

2.9 Experimental design:

Both male and female Wister strain rats, weighing between 100 and 160grams, were randomly assigned to 5 groups, each including six animals. The Group I rats were designated as the Normal control group and received a single dosage of distilled water (1ml/kg body weight, p.o.) for 14 days. The Group II rats were designated as the negative control (Toxic) group and were administered a single dose of distilled water (1ml/kg body weight, p.o.) for 14 days. The positive control group of rats, classified as Group III, were administered a single oral dosage of silymarin at a concentration of 100mg/kg body weight for 14 days. Group IV and V rats were administered Lathyrus sativus methanolic extract (LSME) at 200mg/kg and 400mg/kg body weight orally, as Test I and II treatments. The dose of paracetamol was preceded by 12hours of food withdrawal in order to increase the severity of acute liver damage. Rats from Groups II, III, IV, and V were supplied paracetamol (2gm/kg, p.o.) diluted with distilled water on the 14th day, 1 hour after silymarin and LSME administration, respectively. The rats were then euthanized 4 hours after the administration of paracetamol.

2.10 Serum biochemical analysis:

After collecting blood samples through heart puncture, they were spun at 12,000rpm for ten minutes in a centrifuge. Biological testing on the serum was subsequently performed. Biochemical diagnostic kits were obtained from Beacon Diagnostics Pvt. Ltd., Gujrat, India, to evaluate the levels of total protein, total bilirubin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

2.11 Tissue homogenate preparation:

A surgical knife was used to finely cut the liver that was retrieved from the deceased animal. Then, it was mixed with phosphate-buffered saline¹¹. Centrifugation was used on the homogenate of tissues at 4 degrees Celsius for 15 minutes at 8000 revolutions per minute. For biochemical investigation, the recovered supernatant liquid confirmed the maximum enzyme release.

2.12 In vivo assessment of antioxidant enzymes:

An analysis was conducted on the liver tissue homogenate to determine the levels of antioxidant enzymes, including nitric oxide (NO), glutathione (GSH), lipid peroxidation (LPO), and superoxide dismutase (SOD). using the modified methods described by Ohkawa et al., Kakkar et al., and Ellman^12,13,14,15, respectively.

2.13 Histopathology of liver tissue:

Liver tissue from the experimental animals underwent histological testing. The tissues were preserved in a solution of 10% formalin and then dried using a series of alcohol solutions with increasing concentration. After the material was stained with haematoxylin and eosin, it was embedded in paraffin blocks. Under a microscope, the abnormal changes in the liver were studied to identify their symptoms.

2.14 Statistical analysis:

The data were shown using group means±SEM (standard error of the mean). All tests were conducted with a significance threshold of p<0.05. All graphical representations were generated using GraphPad Prisms® version 5.0 software. The research used one-way analysis of variance (ANOVA) followed by post hoc Tukey multiple comparison testing to determine the disparities across the therapy groups.

3. RESULTS:

3.1 Phytochemical screening:

During the early qualitative screening of Lathyrus sativus, it was discovered that the plant included amino acids, carbohydrates, alkaloids, flavonoids, and phenols.

3.2 Total phenolic and flavonoid content:

The total phenolics and flavonoids identified in LSME were reported to have a concentration of 4.18 and 99.94 mg GAE/g and mg QE/g of extract, respectively.

3.3 GC-MS analysis of methanolic extract of Lathyrus sativus leaves:

The component discovered using GC-MS analysis of the methanolic extract of Lathyrus sativus (LSME) is shown in (Figure 1 and Figure 2).

Figure 1. GC-MS spectrum of methanolic extract of Lathyrus sativus leaves

Figure 2. GC-MS spectral analysis of methanolic extract of Lathyrus sativus leaves

3.4 Effect of LSME on biochemical markers in paracetamol induced hepatotoxicity in rats:

The oral administration of PCM at a dosage of 2 grammes per kilogramme resulted in substantial (p< 0.05) increases in serum biochemical marker activity, including elevated levels of ALP, ALT, and AST, as well as elevated levels of total bilirubin (T.B.), when compared to the control groups that were considered to be expected. When compared to rats that were healthy and normal, the levels of albumin (ALB), globulin (GLB), and total protein (T.P.) in the negative control group were considerably lower. In contrast, rats that were administered LSME at doses of 200 and 400 mg/kg exhibited noteworthy reductions in biochemical marker levels (p < 0.05) and a noticeable increase in ALB, globulin, and T.P. in rats with paracetamol hepatotoxicity as compared to the group that served as the disease control. A considerable normalization of the abnormal biochemical markers was also seen in the group treated with silymarin (Figures 3 and 4).

3.5 Antioxidant activity:

Oral paracetamol administration significantly increased liver levels of malondialdehyde (MDA) and nitric oxide (NO) compared to group I, the control group. The levels of MDA and NO were significantly reduced in rats when given 200 and 400 mg/kg of LSME and silymarin, respectively, at a p‚0.005 level of significance. After being subjected to paracetamol-induced hepatotoxicity, rats showed a significant drop in glutathione and superoxide dismutase (SOD) levels, in contrast to the healthy animals. These levels were significantly higher (p‚0.005) in rats given dosages of LSME (200 mg/kg) and silymarin (100 mg/kg) compared to animals in Group II.

This is seen in Figure 5.

Figure 3. Effect of LSME on ALT, AST and ALP level in paracetamol induced hepatotoxicity in rats. Fig. 3A ALT, Fig. 3B AST, Fig. 3C ALP. Each value is expressed as the mean ± SEM (n = 6) in each group and was estimated using one-way ANOVA followed by post hoc Tukey multiple comparison tests. A significantly different from group I at p < 0.05. ^b Significantly different from group II at p < 0.05. ^c Significantly different from group III at p < 0.05. ^d Significantly different from group IV at p < 0.05.

Figure 4. Effect of LSME on T.B., T.P., ALB and GLB level in paracetamol induced hepatotoxicity in rats. Fig. 4A TB, Fig. 4B TP, Fig. 4C ALB and Fig. 4D GLB.  Each value is expressed as the mean±SEM (n = 6) in each group and was estimated using one-way ANOVA followed by post hoc Tukey multiple comparison tests. ^a significantly different from group I at p<0.05.^b Significantly different from group II at p<0.05. ^c Significantly different from group III at p<0.05. ^d Significantly different from group IV at p < 0.05.

Figure 5. Effect of LSME on SOD, GSH, MDA and Nitrite level in paracetamol induced hepatotoxicity in rats. Fig. 5A SOD, Fig. 5B GSH, Fig. 5C MDA and Fig. 5D NO.  Each value is expressed as the mean±SEM (n = 6) in each group and was estimated using one-way ANOVA followed by post hoc Tukey multiple comparison tests. ^a significantly different from group I at p<0.05. ^b Significantly different from group II at p<0.05. ^c Significantly different from group III at p<0.05. ^d Significantly different from group IV at p<0.05.

3.5 Histopathological observations:

The various groups of rats were examined using histopathological examination, shown in Figure 6(A-E). The purpose of this study was to investigate the cellular architecture of the liver tissue. Histopathological examinations offered information that agreed with the findings of the biochemical study. In the photomicrograph of the G-I animals' livers, the hepatic cells' architecture was typical, with transparent cytoplasm and slightly dilated central veins. Additionally, the kupffer cells were normal, and all of the cells had big nuclei that were considered to be expected (Fig. 6A). Figure 6B demonstrates that the liver tissue in the group that was exclusively treated with PCM had a deformed architecture, along with a large region of necrosis and haemorrhage. In addition, the group that was treated with silymarin (G-III) had a lower level of inflammation and necrosis in the liver cells (Fig. 6C). A greater degree of typical architecture of the liver tissue was seen in G-IV and G-V animals that were treated with plant extracts at doses of 200 and 400 mg/kg, respectively (Fig. 6D, 6E). Necrosis and inflammation rates were also reduced. Histological findings confirm the induction of hepatotoxicity by PCM and the hepatoprotective effect of LSME. This is obvious from the levels of blood and tissue biochemical parameters of the samples.

Figure 6. 6A Histopathology of G-I; group treated with distilled water (1ml/kg) only, 6B: Histopathology of G-II; group treated with paracetamol (2g/kg) only, 6C: Histopathology of G-III; group treated with silymarin (100mg/kg) paracetamol (2g/kg), 6D: Histopathology of G-IV; group treated with LSME (200mg/kg) paracetamol (2g/kg), 6E: Histopathology of G-V; group treated with LSME (400mg/kg) paracetamol (2g/kg).

4. DISCUSSION:

The various forms that plant medicines can take make them very useful in treating a broad variety of illnesses. Some of their potentials have been studied and confirmed by scientific studies. With the ultimate aim of converting these leaves into safe-to-use natural medicine candidates, we designed these studies to explore the hepatoprotective characteristics of a methanolic extract of leaves from Lathrus sativus. Many people turn to paracetamol, an antipyretic and analgesic, when they're sick with a fever, headache, or other pain. Additionally, a valid prescription is not required to obtain it. At toxic concentrations, it becomes a potent hepatotoxin that kills test subjects and humans alike through fulminated hepatic and renal tubular necrosis^16,17,18. To form N-acetyl-para-benzoquinone-immine (NAPQI), a reactive metabolite, the liver isoform enzymes cytochrome P450 CYP2E1 and CYP2A6 transform paracetamol^19,20. Increased levels of NAPQI cause cellular damage and oxidative stress in the liver. Hepatocyte membranes are vulnerable to reactive oxygen species (ROS) accumulation, which in turn causes lipid peroxidation and, eventually, liver necrosis^21,22,23. Most studies to date have utilised blood levels of transaminase enzymes as a proxy for liver injury severity^24,25. Serum ALT and AST values are useful indicators to monitor for hepatic necrosis. Enzymes ALT and AST are responsible for the reductive transfer of amino acids from alanine and aspartate to alpha-ketoglutarate, respectively, producing pyruvate and oxaloacetate. A variety of substances, including ALT and AST, can be released into the extracellular space below by injured hepatocytes. Accordingly, damage to the liver is indicated by an elevated level of these enzymes^26,27,28. Although ALT is present in the brain, skeletal muscle, and heart, the liver contains the highest concentration of the compound.

In contrast, AST is found in other organs, leading some to believe that it is less selective for inflammation in the liver. The liver-resident enzyme alkaline phosphatase (ALP) is another potential candidate for further study. The membranes lining the bile duct and canaliculi produce ALP, an enzyme that can be hydrolysed. When the hepatobiliary system is injured, it is typically found in elevated blood levels. which are cited as^29,30. The PCM group found that hepatotoxicity causes biliary obstruction, which makes it harder for the body to excrete ALP, which causes levels of ALP to rise. It is possible to gauge the liver's secretory and synthetic functions by measuring the quantities of serum albumin, globulin, and bilirubin, which are well-known markers. Further, the kinds of liver injury that have taken place can be identified using these concentrations^31,32,33.

The degree of cell dysfunction is indicated by a reduction in total protein level in chronic liver disease. Furthermore, it has been suggested that promoting protein synthesis, which in turn increases the generation of hepatic cells, can act as a liver protectant^34,35,36. Damage to DNA, proteins, and phospholipids can occur when reactive oxygen species (ROS) are present in excess. This can cause oxidative stress^14,37,38 by leading to lipid peroxidation and a decrease in antioxidant enzymes such as glutathione (GSH), superoxide dismutase (SOD), and GPx. Liver cells had a greater concentration of malondialdehyde, a byproduct of lipid peroxidation. Based on these results, it appears that ineffective antioxidant defence was the cause of the lipid peroxidation.

This study found that in an animal model, paracetamol causes cell death in the liver when administered at high doses. Also, in groups IV and V, rats that were inebriated on paracetamol had significantly lower levels of AST, ALT, ALP, TBL, and GLB after receiving 200 and 400mg/kg of LSME, respectively. In comparison to the control group of rats, the T.P. levels in Group II animals were much lower. The results showed that rats given plant extracts had much higher T.P. levels, which could mean that LSME sped up the development of hepatic cells and boosted protein manufacturing. Hepatotoxic control group II levels of antioxidant enzymes, especially hepatic GSH and SOD, were significantly reduced after administration of 2g/kg p.o. of paracetamol, as shown in the present study.

Hepatic MDA levels also increased relative to normal control group I, suggesting oxidative stress, as a result. On the other hand, the GPx level did not show any notable alterations. Results showed that hepatic GSH and SOD levels were significantly higher and MDA levels were lower in rats pre-treated with 200 and 400 mg/kg/day p.o of LSME before paracetamol than in the hepatotoxic group-II. When it came to bringing liver markers and antioxidant markers back to normal levels, the administration of LSME at a dosage of 200mg/kg was more effective than 400mg/kg. Two hundred milligrammes of LSME per kilogramme per day had the same hepatoprotective effect as one hundred milligrammes of the gold standard drug silymarin per kilogramme per day. These results suggest that LSME reduces paracetamol-induced liver damage in part by restoring liver function test (LFT) markers and reducing heightened liver injury. Bioactive chemicals like flavonoids and phenolics may limit the production of free radicals and scavenge them, which may help the body deal with many damaging processes ^[39,40,41]. One possible explanation for LSME's hepatoprotective effects is the presence of phytoconstituents, which have antioxidant capabilities. These phytoconstituents have anti-inflammatory properties that protect the liver from inflammation-induced damage, and they also help reduce oxidative stress that paracetamol and related analgesics generate.

5. CONCLUSION:

Based on the hepatoprotective study, it has been shown that Lathyrus sativus leaves can act as hepato-protectants by restoring liver function and oxidative stress indicators to optimal levels. Additional confirmation by examination of their histology micrographs demonstrates the reduction of liver damage. Nevertheless, more research using a broader range of experimental liver injury models is necessary to clarify the precise molecular and biochemical processes at play and demonstrate this compound's therapeutic potential as a hepatoprotective agent.

6. CONFLICT OF INTEREST:

We pronounce that we have no conflict of interest.

7. ACKNOWLEDGEMENTS:

The authors thank the Central Instrumentation Laboratory (CIL), Central University of Punjab, Bathinda, Punjab, India, for GC-MS analysis.

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Received on 21.01.2024      Revised on 08.06.2024 Accepted on 12.09.2024 Published on 28.01.2025 Available online from February 27, 2025 Research J. Pharmacy and Technology. 2025;18(2):625-631. DOI: 10.52711/0974-360X.2025.00093 © RJPT All right reserved
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