INTRODUCTION:

Contaminations of heavy metals in the environment are ecologically and globally major public health concern. One of the earliest heavy metals discovered by human is Lead. Low melting point, softness, malleability, ductility, resistance to corrosion - all these unique properties of lead have contributed to its widespread usage in different industries such as ceramics, automobiles, paint, plastics, etc. Chronic lead exposure causes life threatening detrimental effects on brain, liver, and other major organs like heart, kidney, bones, reproductive organs etc.¹

The liver is composed of highly active metabolic tissue containing a huge complement of detoxification machinery referred to as phase I and phase II enzyme systems that ideally serve to guard other physiological systems from the toxic effects of xenobiotic compounds. Liver is exposed to nutrients and other xenobiotics through the portal vein. In different studies it has been observed that acute and chronic intoxication of lead is responsible for alterations in hepatic xenobiotic metabolism, cholesterol metabolism, liver cell proliferation and DNA synthesis. It has been reported that after lead exposure a huge amount approximately 33% accumulated in soft tissues like liver. Lead exposure is responsible for detrimental effect on hepatic microsomal cytochrome P-450 and associated enzymatic activities in rat liver.²

Now a days, herbal medicines are popular throughout the world due to their potency and apparent safety profile. Polyphenolic compounds have many phenolic groups, are widely present in different plants, fruits and vegetables. Their beneficial effect against many diseased condition affecting different major organs already has been well established in heavy metal toxicity.^3,4

Polyphenols are reported for detoxification and removal of heavy metals.⁴ The reason by which it may show the protection is probably by scavenging reactive oxygen species generated by lead and other heavy metals. It has been found that they are also responsible for removal of accumulated heavy metals from major organs and detoxification. Polyphenols are also reported to attenuate ROS-mediated inflammatory cytokines secretion through ERK/JNK/p38 pathways resulting protection against lead induced inflammatory reactions.^5,6

Ellagic acid (EA), a polyphenolic phyto-chemical and an important component of fruits, nuts and vegetables, is well known for many medicinal properties.⁷ Pomegranates, raspberries, blackberries, strawberries, red guava, black raspberries, white guava, beefsteak fungus, Cranberries, walnuts and almonds are rich in EA.^8,9It is proved to be potential antioxidant, with hepato-protective, Neuro protective, Antiulcer, Anti inflammatory, Cardioprotective, Anticancer, Antiviral and Anti-cataract activities.^10-19 The present study has been planned to evaluate protective effect of Ellagic acid against lead induced hepatotoxicity.

MATERIAL AND METHODS:

Chemicals and Phyto-chemicals:

All chemicals were of analytical grade and purchased from standard and reputed companies. Lead acetate was purchased from Loba Chemicals, Mumbai. Biochemical kits were purchased from Crest Biosystems (Goa, India).

Ellagic acid samples were procured from Yucca Enterprises (Mumbai, India).

Experimental Animals

Healthy adult male wistar albino rats of approximately 170-200g were maintained under standardized condition (12 h L:D cycles, 25°±5°C) with paddy husk bedding in polypropylene cages, at the Central Animal House, Soniya Education Trust’s College of pharmacy, Dharwad. The animals were provided with standard pellet food and had free access to purified drinking water. The guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India were followed with prior permission from the Institutional Animal Ethics Committee for conducting the study.

Dose selection of Ellagic acid:

Rat dose of ellagic acid was selected based on earlier literature as 50mg/kg and 25mg/kg through oral route and termed as high and low dose respectively.²⁰

Experimental design:

After one week of acclimatization, the animals were randomly divided into 4 groups of 8 animals in each. Group I Served as normal control and received normal saline 2ml/kg body weight through oral route. Group II Served as toxic control and animals were treated with lead acetate (60mg/kg) p.o. for 28 days. Group III and IV animals were given Ellagic acid 50 and 25 mg/kg p.o. respectively for 28 days along with that lead acetate was administered as in group II.¹

Twenty four hours after the last treatments, the animals were anesthetised with light ether anesthesia and blood was withdrawn by retro-orbital puncture. Serum was separated by centrifugation for the estimation of enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkanine phosphate (ALP), total bilirubin and lipid levels such as total cholesterol (TC), trigycerides (TG), low density lipoprotein (LDL), high density lipoprotein (HDL) levels by semi-autoanalyser (Robonik, Mumbai). Thereafter the animals were sacrificed; livers were used for the preparation of homogenate to estimate antioxidants like super oxide dismutase (SOD), catalase, thio-barbituric acid reactive substances (TBARS) and reduced glutathione (GSH). Remaining livers were embedded in formaline in saline solution (10%) for histological examination.^21,22

Preparation of Liver Tissue Homogenate:

The livers removed were gently rinsed with ice cold physiological saline solution (0.9% NaCl) to remove blood, mucus and other debris adhering them. The sliced liver was immediately homogenized in ice-cold o.1 M sodium phosphate buffer (pH 7.4) at 1-4̊ C to give a 10% (w/v) homogenate. The homogenates were centrifuged twice at 10000 rpm for 15 minutes at 4°C. The supernatants were used for the estimation of SOD, Catalase, GSH and TBARS.²³

Histological analysis

Liver sections were prepared from the remaining half of the liver samples in each group, stained with Hematoxylin and Eosin (HandE) and changes in histology were observed.

Statistical analysis:

Results were expressed as mean+/- SEM. Statistical significance was assessed using One-way Analysis of variance (ANOVA) followed by Tukey-Karmer multiple comparison tests. P<0.05 was considered significant.

RESULTS:

Effect on serum enzymes:

Effect on AST, ALT, ALP and Bilirubin (Table no. 1)

Toxic control (only lead acetate treated) group revealed extremely significant (P <0.001) increase in serum AST, ALT, ALP and bilirubin levels compared to normal control.

Treatment groups such as EA 50, EA 25 showed extremely significant (P <0.001) decrease in AST, ALT, ALP and bilirubin levels compared to toxic control.

Effect on TC, TG, HDL, LDL (Table no. 2):

Toxic control (only lead acetate treated) group demonstrated extremely significant (P <0.001) increase in serum TC, TG, LDL values compared to normal control, whereas extremely significant (P <0.001) decrease in HDL values compared to normal control.

Treatment groups such as EA 50, EA 25 showed extremely significant (P <0.001) decrease in serum TC, TG, LDL values, compared to toxic control. All the treatment groups showed extremely significant (P <0.001) increase in serum HDL values.

Effect on antioxidants in Liver tissue homogenate (HTH):

Effect on SOD, Catalase and GSH (Table no. 3):

Toxic control group reported extremely significant (P <0.001) decrease in SOD, Catalase and GSH activity compared to normal control.

Experimental groups EA 50 demonstrated extremely significant (P <0.001) where as for EA 25 treated groups it was found to be moderately significant increase in SOD, Catalase and GSH values compared to toxic control group.

Effect on TBARS (Table No. 3)

Toxic control group demonstrated extremely significant (P <0.001) increase in TBARS activity compared to normal control.

Treatment groups such as EA 50 showed extremely significant (P <0.001) where as for EA 25 treated group it was found to be moderately significant (P <0.01) decrease in TBARS activity compared to toxic control group.

Table no. 1: Effect on serum enzymes against lead acetate induced hepatotoxicity

Treatment	Blood serum level (U/L)
Treatment	AST	ALT	ALP	Bilirubin
Normal control	69.63±0.28	32.66±0.56	93.64±0.68	1.34±0.34
Toxic control	129.64±0.34^***	97.32±0.46^***	179.73±0.72^***	2.03±0.64^***
EA 50	76.45±0.65^###	43.45±0.63^###	113.42±0.65^###	1.44±0.52^###
EA 25	79.52±0.21^###	54.27±0.24^###	128.51±0.19^###	1.47±0.25^###

All values are mean ± SEM, n=8, ^***P <0.01 when compared to normal control; ^###P <0.001, compared to Toxic control group.

Table no. 2: Effect on serum enzymes against lead acetate induced hepatotoxicity

Treatment	Blood serum level (U/L)
Treatment	TC	TG	HDL	LDL
Normal control	120.4±8.34	39.6±6.31	49.6±8.81	71.8±5.93
Toxic control	165.3±10.41^***	74.9±8.21^***	24.8±6.49^***	126.7±9.38^***
EA 50	131.8±9.84^###	48.7±6.39^###	42.7±8.31^###	80.5±7.73^###
EA 25	133.7±11.59^###	49.2±7.75^###	41.5±4.73^###	83.5±5.17^###

All values are mean ± SEM, n=8, ^***P <0.01 when compared to normal control; ^###P <0.001 compared to Toxic control group.

Table No. 3: Effect on antioxidants in HTH and histological score against lead acetate induced hepatotoxicity

Treatment	Liver Tissue Homogenate (Units/mg of protein)
Treatment	SOD	CATALASE	TBARS	GSH
Normal control	13.83±0.53	44.34±0.76	29.83±0.38	9.53 ±0.31
Toxic control	03.43±0.67^**	19.78±0.39^**	56.32±0.59^**	2.19 ±0.62^***
EA 50	10.32±0.63^###	40.78±0.34^###	34.76±0.43^###	7.82 ±0.23^###
EA 25	08.54±0.49^##	33.85±0.28^##	39.62±0.52^##	5.76 ±0.41^##

All values are mean ± SEM, n=8, ^***P <0.001, ^**P <0.01 when compared to normal control; ^###P <0.001,^##P <0.01 compared to Toxic control group.

DISCUSSION:

The aim of the present study was to investigate the protective effect of Ellagic acid (EA) against lead acetate induced hepatotoxicity. Observed results suggested dose dependent beneficial effects for EA against lead acetate induced hepatotoxicity.

Lead exposure is responsible for generation of huge amount of free radicals which is responsible for development of oxidative stress. Enormous amount of oxidative stress associated with lead exposure is responsible for decline in intracellular ATP, oxidative DNA damage and the apoptosis of hepatocytes.²⁴

Reactive oxygen species (ROS) are effectively neutralized by the antioxidant defense system. But during patho-physiological conditions such as lead exposure results in enormous increased production of ROS and/or impaired antioxidant capacity, which culminate in oxidative stress.²⁵

Results of this study also witnessed extremely significant decrease in superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and increase in thio barbituric acid reactive substances (TBARS) in liver tissue. This may be due to excessive damage produced by lead exposure.

Four phenolic groups and two lactones present in EA can act as hydrogen-forming sides and electron acceptors delay, inhibit or prevent the oxidation of compounds, trapping free radicals and reduce oxidative stress. EA reported for inhibiting the generation of superoxide anions, hydroxyl radicals and prevent lipid peroxidation associated with damage of cell membrane. ^10,26

The antioxidant effect associated with EA may be due to direct action on free radical scavenging and indirect action through the induction of antioxidant enzymes. In addition, EA is able to inhibit PGE₂ produced and reduce the COX-2, thus, decreased TNF-α level.²⁷

In our present study also EA showed significant decrease in serum AST, ALT, ALP, total bilirubin levels which reflect protection against lead induced hepatotoxicity. Moreover EA treated groups showed significant increase in SOD, catalase, GSH and decrease in TBARS levels.

Lead intoxication is responsible for impaired hepatic cholesterol metabolism which is responsible for increase in both liver and serum total cholesterol levels.²⁸

In our present study also it has been evident that in the toxic group there is significant increase in TC, TG, LDL levels and significant decrease in beneficial HDL level. EA showed significant decrease in TC, TG, LDL levels and significant increase in HDL levels.

Only lead exposed group caused enormous changes in the liver cell associated with degeneration of liver tissue, focal necrosis with hepatocyte vacuolation, pykonotic nuclei, swelling, dilation of central vein and sinusoids, infiltration of inflammatory cells. Treatment with EA dose dependently inhibited lead induced damage by restoration of reticular fibres and inflammation.^29,30

It can be concluded from the present study that EA exhibited dose‑dependent protection against lead induced hepatotoxicity. Findings of the present study can be important for those who are chronically exposed to high level lead. Ellagic acid in the dietary source or in the form of formulation can keep their liver healthy and safe. Future studies can be carried out to establish the fact clinically.

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Received on 31.10.2019 Modified on 27.12.2019

Research J. Pharm. and Tech 2020; 13(9):4244-4248.

DOI: 10.5958/0974-360X.2020.00749.0