1. INTRODUCTION:

The use of herbal medicines is gaining their importance and faith day by day. One of such plant used from ancient history of Indian subcontinent is Sesbania grandiflora (SG) (family Leguminosae). According to the current study, the fruits are used to treat anaemia, bronchitis, fever, tumours, as well as pain and thirst.

The various authenticated pharmacological activities reported are anxiolytic and anti-convulsive¹, anti-urolithiatic and antioxidant^2,3, erythromycin estolate-induced hepatotoxicity⁴, antiulcer, anticancer⁵, chemo preventive, antimicrobial⁶, analgesic and antipyretic⁷, hypolipidemic activity⁸, and cardio-protective⁹. The SG fruits contain varied concentration of phenols, flavonoids, and tannins. Specific compounds to these phytochemical groups were also identified in TLC, among which chief are gallic acid, caffeic acid, Kaemferol-3- rutinoside, quercetin, rutin¹⁰. Since the liver conducts a range of activities in metabolism, including detoxification, glycogen storage, vitamin A, D, and B₁₂ creation, the formation of several coagulation factors, cell proliferation (IGF-1), hormones (angiotensinogen), and biochemical substances required for digestion (bile). Widely known factors such as xenobiotic, oxidative stress, etc. in hepatic damage may contribute to several tasks becoming distorted¹¹. As per WHO, hepatotoxicity is a worldwide issue among developing countries where poor sanitary conditions and hygienic practices push the inhabitants into the darkness of sever health issues¹². Herbals drugs claims to possess hepatoprotective potential and the plentiful availability of Sesbania grandiflora along with its phytochemical and other pharmacological activities in it, has encouraged to appraise the hepatoprotective efficacy of the Sesbania grandiflora fruit’s petroleum ether concentrate against thioacetamide-induced hepatotoxicity in rats.

2. MATERIALS AND METHODS:

2.1 Herbal extracts:

Fruits of Sesbania grandiflora were harvested from the nearby fields of Raichur, located in Karnataka, India. The plant material was carefully identified and authenticated by Prof. Veda Vyas from the Department of Botany at L.V.D. College, Raichur. A voucher specimen of the collected sample was deposited in the department's herbarium for future reference.

To ensure the cleanliness of the fruits, they were thoroughly washed with filtered water and subsequently dried in the shade. The dried materials were then powdered and sieved through a 10-mesh sieve. The coarsely powdered material was separated using Soxhlet's column, employing petroleum ether as the solvent for a 24-hour extraction period.

The resulting solution was concentrated using a rotary flash evaporator, and the extract was subsequently dried and stored in an airtight container. For further pharmacological investigations, various concentrations were prepared through dilution of the extract.

2.2 Experimental animals:

Mature albino rats, weighing between 150-200grams, and comprising both males and females, were sourced from the central animal facility at N.E.T. Pharmacy College, located in Raichur, Karnataka, India. These rats were allowed a 7-day period for acclimatization to the laboratory conditions. Throughout this acclimatization period and the subsequent study, the rats were provided with a standard diet and had access to water ad libitum, all while being maintained under strict hygienic conditions.

The study protocol underwent thorough review and received approval by IAEC (576/2002/bc/IAEC/CPCSEA). Throughout the research, strict adherence to the CPCSEA.

2.3 Determination of acute toxicity (LD₅₀):

The experimental scheme was executed as per the OECD guideline no. 425¹³. Here, nonparous female albino mice (20-25g) were employed for protocol. The animals were deprived of food with free access to water for 3-4hour pre and 1-2 hour post administration of extract. The extract was administered at increasing dose from 5 to 500mg/kg to ensure any type of sign and symptoms of toxicity. The final validation of extract was done at amount of 2000mg/kg/p.o. Here, the extract did not created any sign about mortality or toxic effect. Thus, 1/5^th, 1/10^th, 1/20^th of maximum amount was considered as high, medium and low dose correspondingly.

2.4 Experimental design¹⁴:

Thirty-six albino Wistar rats of varying sexes, with weights falling in the range of 150 to 200grams, were divided into six groups, each consisting of six animals.

1. Group-I served as the normal control, receiving only Tween 80.

2. Group-II, the toxicant group, was administered thioacetamide (100mg/kg, s.c.) once every 72 hours.

3. Group-III received a daily dose of Silymarin (25 mg/kg, p.o.) alongside thioacetamide (100mg/kg, s.c.).

4. Groups IV, V, and VI were administered petroleum ether extract at doses of 100mg/kg, 200mg/kg, and 400mg/kg, respectively, along with thioacetamide.

Silymarin was employed both as a positive control and for assessing its hepatoprotective potential in comparison to the various doses of Sesbania grandiflora (SG) fruit extract.

On the 7th day of the study, the animals were sacrificed, and blood samples were collected via retro-orbital puncture for subsequent biochemical analysis. Liver tissues were dissected, sections were prepared, and various parameters such as cell necrosis, fatty changes, hyaline generation, and ballooning generation were observed.

2.5 Pentobarbitone Induced Sleeping Time¹⁵:

The animal grouping was conducted as previously described. All animals were deprived of food but had access to water ad libitum during the study.

1. Group I served as the normal control and received 5 ml/kg of normal saline orally (p.o.).

2. All animals in groups II to VI were administered thioacetamide (100mg/kg; s.c.). Group II animals were designated as the thioacetamide control group and did not receive any drug treatment.

3. Groups III, IV, and V received petroleum ether extract at doses of 100mg/kg, 200mg/kg, and 400 mg/kg orally (p.o.), respectively.

4. Group VI animals were treated with Silymarin (25 mg/kg, p.o.) along with thioacetamide (100mg/kg, s.c.), serving as the standard group.

To assess the liver's protection against damage caused by thioacetamide, the reduction in sleep time was measured. On the first day, all animals were administered their respective doses, and two hours later, thioacetamide was administered to both groups. On the second day, the same doses were given, and Pentobarbitone sodium (40mg/kg i.p.) was administered one hour after treatment. The onset of action and duration of sleep were observed for evaluation.

2.6 Determination of biochemical parameters^16,17,18

All the samples were observed and microscopically photographed. The samples of blood obtained were left to coagulate at room temperature for 45 minutes. At 4000rpm for 20min, the serum was alienated by centrifugation. Serum aminotransferases activities including SGOT and SGPT SALP, direct and total Bilirubin, total protein albumin, cholesterol and triglyceride values were tartan using the semi autoanalyser (Erba Mannheim Chem. 5 plus V₂).

3. RESULTS:

Administration of thioacetamide led to a significant increase in various biochemical parameters, including SGOT, SGPT, ALP, direct and total bilirubin, cholesterol, and triglycerides, along with a decrease in protein and albumin levels when compared to the control group.

Specifically, there was a notable increase in SGPT levels, reaching 120.99IU/L in the thioacetamide-treated group. However, treatment with the SG extract at doses of 200mg/kg and 400mg/kg reversed SGPT levels to near-normal values, i.e., 87.25IU/L and 71.40IU/L, respectively. The standard drug, Silymarin at 100 mg/kg, significantly restored SGPT levels to 42.43 IU/L.

Similarly, the rise in ALP serum levels due to thioacetamide challenge was significant, reaching 210.23 IU/L. However, treatment with the SG extract at doses of 200 mg/kg and 400mg/kg brought ALP levels back to near-normal, i.e., 156.47 IU/L and 137.80 IU/L, respectively. Standard Silymarin at 100 mg/kg also effectively restored ALP levels to 116.02 IU/L.

Serum SGOT levels were elevated in the thioacetamide-treated group, reaching 299.23 IU/L. Treatment with standard Silymarin at 100 mg/kg returned SGOT levels to near-normal, i.e., 112.50 IU/L. The SG extract, administered at doses of 200mg/kg and 400mg/kg, also significantly reduced SGOT levels to 155.59 IU/L and 145.22 IU/L, respectively.

Regarding total and direct bilirubin, there was a significant increase, with levels reaching 2.751mg/dl and 2.29mg/dl, respectively, in the serum due to thioacetamide treatment. Treatment with SG extract at doses of 200mg/kg and 400mg/kg reversed total and direct bilirubin serum levels to 1.76mg/dl and 1.05 mg/dl, and 1.22mg/dl and 0.83mg/dl, respectively. These changes were statistically significant compared to the thioacetamide-treated group. Standard Silymarin at 100mg/kg also led to significant reductions, with levels of 0.30mg/dl for total bilirubin and 0.22mg/dl for direct bilirubin.

Total protein levels showed a significant reduction in the thioacetamide-treated group, reaching 6.15. However, groups treated with Silymarin and SG extract at doses of 200mg/kg and 400mg/kg showed significant increases in total protein levels, reaching 11.95, 8.44, and 11.24, respectively.

Sleeping time induced by pentobarbitone was significantly longer in thioacetamide-treated rats compared to normal control rats. However, pretreatment with SG extract resulted in a substantial reduction in sleeping time, approaching near-normal values, which were comparable to the values observed in the standard drug (Silymarin)-treated group of animals.

Table 1: Effect of FESG on diverse biochemical parameters in thioacetamide induced hepatotoxicity in rats.

	Serum marker enzyme levels
Treatment	SGPT	SGOT	SALP	Direct bilirubin	Total bilirubin	Cholesterol	Triglyceride	Total protein	Albumin
Normal control (10ml/kg)	40.28 ± 4.11	107.23 ±15.35	107.22 ± 7.39	0.20 ± 0.02	0.29± 0.02	133.69 ± 2.36	30.53 ± 1.45	13.56 ± 0.57	4.40 ± 0.16
Toxicant control (100mg/kg)	120.99 ± 1.52	229.23 ± 3.44	210.23 ± 1.20	2.29 ± 0.07	2.75 ± 0.06	210.28 ± 2.49	117.91 ± 1.87	6.51 ± 0.33	3.35 ± 0.11
Silymarin (100mg/kg)+ thioacetamide	42.43 ±1.21**	112.50 ± 1.40**	116.02 ± 0.74**	0.22 ±0.008**	0.30 ±0.009**	147.57 ± 1.45**	41.89 ± 1.47**	11.95 ±0.15**	4.19 ±0.03**
Low dose of FESG (100mg/kg.) + thioacetamide	114.65±2.11^ns	205.58± 1.51^ns	181.70±1.76**	2.02± 0.02**	2.34± 0.03**	200.71 ±1.43*	89.45 ±3.41**	7.67± 0.08^ns	3.44± 0.02^ns
Medium dose of FESG (200mg/kg) +thioacetamide	87.25 ±1.64**	155.59 ± 7.63**	156.47 ± 2.80**	1.22 ± 0.02**	1.76 ±0.028**	180.38 ± 3.67**	73.78 ±1.75**	8.44 ± 0.07**	3.67 ± 0.03*
High dose of FESG (400mg/kg) +thioacetamide	71.40± 1.05**	145.22 ± 2.14**	137.80± 1.74**	0.83 ± 0.01**	1.05 ± 0.03**	153.90 ± 1.62**	63.14 ±2.38**	11.24 ±0.51**	4.00 ±0.02**

“Values are mean ± SEM (n=6) one way ANOVA followed by Dunnett’s ‘t’ test. Where, * represents significant at p<0.05, ** represents highly significant at p< 0.01, *** represents very significant at p<0.001and ns represents non significant.”

Table 2: Effects of of FESG on pentobarbitone induced sleeping time in thioacetamide induced hepatotoxicity in rats.

Group	Onset of time	Duration of sleep
Normal control: Pentobarbitone (40mg/kg, i.p.)	16.38 ±1.45	71.05±2.56
Toxicant Control Group Thioacetamide (100mg/kg, s/c) +Pentobarbitone (40mg/kg, i.p.)	7±1.10	200.43±7.29
Low dose of FESG: 100mg/kg of SG+ Thioacetamide (100mg/kg, sc) + Pentobarbitone (40mg/kg, i.p.)	6±0.13	187.56±5.89
Medium dose of FESG :200mg/kg + Thioacetamide (100mg/kg, sc)+ Pentobarbitone (40mg/kg, i.p)	10±1.12	145.73±6.33**
High dose of FESG : 400mg/kg+ Thioacetamide (100mg/kg, sc)+ Pentobarbitone (40mg/kg, i.p)	12.6±1.68	121.15±.5.12**
Standard Group Silymarin (25 mg/kg p.o.) +Thioacetamide (100mg/kg, sc)+ Pentobarbitone (40mg/kg, i.p)	15.18±1.39	89.52±4.68**
“Values are mean ± SEM (n=6) one way ANOVA followed by Dunnett’s‘t’ test. Where, * represents significant at p<0.05, represents highly significant at p< 0.01, * represents very significant at p<0.001and ns represents non significant”

Histopathological study:

Figure 1: Control Group - In this group, hepatocytes are arranged in single cords, displaying the characteristic lobular structure of the liver. A centrally positioned nucleus is observed, along with occasional binucleate cells. Additionally, sinusoidal cells are present, with the nuclei of Kupffer cells in close proximity.

Figure 2: Control (toxicant): A fatty modification and degeneration with loss of nuclear architecture were shown by the liver hepatocytes

Figure 3: Standard- No centrilobular necrosis and no hydropic degeneration were observed in this sample, suggesting the prevention of thioacetamide-induced hepatic damage.

Figure 4: Low dose (100mg/kg): Minimal variations such as centrilobular necrosis were found in parts of the liver

Figure 5: Medium dose (200mg/kg): The sections of the liver showed mild improvements in hydropic degeneration and centrilobular necrosis.

Figure 6: High dose (400mg/kg): With preservation of normal lobular architecture, the liver parts displayed limited centrilobular necrosis, which confirms hepatoprotective function.

4. DISCUSSIONS:

Liver is engrossed in detoxification of xenobiotics and contaminants¹⁹. Being time, hasty oxidative radicals are also created at the time of detoxification. Due to the overdose of drugs or toxins or use of drugs for extensive period, there is strong possibility of generation of free radicals resulting in oxidative stress which may dent liver. The oxidative stress to liver imposes stern trouble, ultimately leading to compromised function^19,20. There is no perfect therapy to protect the liver from oxidative injuries. Thus, it is very much important to get and acknowledge various therapies to protect and heal liver from various contributory factors. The chemical used here was thioacetamide which states to be a compelling hepatotoxicant which get metabolized by Cytp-450 enzymes and get renovated into thioacetamide S-oxide which pushes the hepatic cells towards oxidative stress^21,22. Here, this free radical species should be considered as culprit as it alter cellular permeability, augmented Ca⁺⁺intracellularly, disturbed nuclei and its volume along with diminished mitochondrial bustle. These all transformations push the hepatic cells towards darkness of death²³.

Injury to hepatic cells often manifests as elevated enzyme levels in the bloodstream, detectable through various techniques²⁴. In this study, the significant improvement in parameters such as SGOT, SGPT, SALP, direct and total bilirubin, cholesterol, and triglycerides, coupled with a decrease in protein and albumin levels in the thioacetamide-treated group, serves as an indication of liver damage. Therefore, the use of herbal extracts from the Sesbania grandiflora plant at different dosage levels demonstrated a reduction in these elevated parameters, establishing its efficacy against hepatotoxic agents like thioacetamide²⁵.

Elevated serum total bilirubin levels, resulting from impaired bile excretion by the liver, signify a loss of liver functionality and necrosis. This condition can be related to the rate of erythrocyte breakdown. The extract notably reduced total bilirubin levels at doses of 200mg/kg and 400mg/kg, supporting the hepatoprotective potential of the extract²⁶.

Disturbances in total protein levels are often due to disruptions in polyribosomes on the endoplasmic reticulum, resulting in faulty protein synthesis. Thioacetamide leads to a decrease in protein levels, while the Sesbania grandiflora extract significantly increased protein levels at doses of 200mg/kg and 400 mg/kg, indicating its protective effect on the liver against thioacetamide-induced hepatotoxicity.

A damaged liver may struggle to metabolize drugs efficiently, potentially leading to prolonged drug action or toxic effects. Additionally, the reduction in pentobarbitone-induced sleeping time in the animal groups clearly indicates that the SG extracts offers protection to the liver.

Furthermore, histopathological examination corroborated these findings. Thioacetamide induced focal fatty changes and degenerative necrosis in liver cells through the production of free radicals during its metabolism. In contrast, the high-dose SG fruit extract showed minimal centrilobular necrosis while retaining a normal lobular architecture, confirming its hepatoprotective activity. Both serum markers and histopathological evaluation affirmed the extract's potential in mitigating hepatic cell damage induced by thioacetamide.

The presence of herbal polyphenolic compounds in the fruit extracts, which act as antioxidants, contributed to hepatoprotection through enzymatic and non-enzymatic reactions^27-29.

5. CONCLUSION:

The substantial hepatoprotective activity of the petroleum ether extract derived from Sesbania grandiflora fruit, particularly at a high dose of 400mg/kg, was established based on enhancements in serum enzyme markers, physical parameters, histopathological findings, and the presence of phytoconstituents. This conclusion strongly supports the traditional application of the extract (p<0.001).

6. ACKNOWLEDGEMENT:

The authors wish to express their gratitude to the Dr. Bheemachari, Akshaya Institute of Pharmacy, Tumkur (K.A.) for their valuable support throughout the work.

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Received on 12.01.2022 Modified on 20.03.2023

Research J. Pharm. and Tech 2023; 16(10):4929-4934.

DOI: 10.52711/0974-360X.2023.00799