INTRODUCTION:

The quest for novel drug leads to combat the infectious diseases that are resistance to existing pharmaceuticals is a trend of the medicinal chemistry research. Traditionally, plants and terrestrial microorganisms are remains the major focus of the search for new drug leads. The novelty in their chemical structure associated with such natural products offer wide scope. In this context, marine organisms such as bacteria, fungi, algae, sponges, soft corals, tunicates, molluscs and bryozoans are potential sources of novel bio-active compounds.¹Among them, the seaweeds could produce a wide range of secondary metabolites with broad spectrum of bioactivity, have immense biomedical potential,^2,3and has been used in folk medicine for a variety of remedial purposes such as in eczema, gallstone, gout, scrofula, cooling agent for fever, menstrual trouble, renal problems, scabies.⁴A wide range of antibiotic, anti-HIV, anticoagulant, anticonvulsant, anti-inflammatory,⁵ antineoplastic, wound healing, antiulcer⁶hepatoprotective⁷and antitumor activities for seaweed derived compounds have been reported.^8-10Apart from the above biological activities, seaweeds are considered as the rich sources of varies antioxidant compounds.¹¹The lower prevalence of breast and prostate cancer in Japan and China has been linked directly to seaweeds that are consumed in appreciable quantities in these countries than in North America and Europe.¹²The distinctive seaweed resources of the Gulf of Mannar and Southeast coast of India are mainly utilized for the production of phycocolloids. The biomedical and nutritional potential of the seaweeds has not received much attention. The physiological processes such as metabolism, detoxification, secretion, and storage functions are done by liver and it’s injury is mainly associated with disruption of these functions.¹³ With many other factors involved in progression of liver diseases, production of large amounts of reactive oxygen species (ROS) has also been shown a strong connection to hepatotoxicity.¹⁴Efficient radical scavengers may prevent or alleviate many diseases associated with free radicals generation. The present study was evaluated the hepatoprotective activities of brown alga, L. variegate (J. V. Lamouroux) Womersley ex E.C. Oliveir (Dictyoace) against Cr(VI) induced oxidative damage and liver fibrosis in Wistarrats.

MATERIALS AND METHODS:

Chemicals:

Thiobarbituric acid (TBA; Research-Lab fine chemical industries, Mumbai, India), Nitro blue tertazolium chloride (NBT; Himedia laboratories Pvt. Ltd, Mumbai, India). 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB; Alfa Aesar, A Johnson Matthey Chemicals, Chennai, India). Bovine serum albumin (Spectrochem Pvt. Ltd. Mumbai, India), carboxymethyl cellulose (Research Lab Fine Chem Industry, Mumbai, India) and ethyl alcohol were used. All the chemicals used were of analytical grade and purchased from standard manufactures.

Collection of Algae Sample and preparation of extracts:

The marine brown alga Lobophora variegata (J.V. Lamouroux) Womersleyex E. C. Oliveir was collected from Mandapam, Ramanathapuram District, South East Coast of Tamilnadu, India. Seaweeds were collected by swimming and diving from the Islands of Mandapam by trained personnel of Central Salt Marine Research Institute, Mandapam. Details regarding the ecology of the Mandapam shore type (latitude 8°14'23.10" N, longitude 77°20'04.02"E), exposure of the substratum, tide level and mode of collection have been described earlier.^15-17The coastal region of Mandapam consists of discontinuous and irregular occurrence of the coral like stones which supports the marine algal vegetation, when the sea is rough; a large number of deep sea forms are uprooted and washed ashore in the Mandapam. Mandapam area enjoys a tropical climate with summer temperature ranging 31.5°C and winter temperature from 28 to 31°C. The experimental alga L. variegata was identified by the standard manual.¹⁸

The annual rainfall is about 77 mm. The voucher specimens (PCCACL05) were deposited as Herbarium in the PG and Research Department of Botany, Pachaiyappa’s College, Chennai-30. The collected materials were washed with sea water tore move impurities such as sand, rocks, epiphytes and epifauna. The washed samples were transported in a ice box containing slush ice to the laboratory. The samples were washed, in the laboratory, thoroughly in running tap water (to remove salt). They were shade dried for 48 hrs and pulverized to fine powder, packed in airtight container and stored at room temperature. The finely powdered sample was soaked in methanol (1:3 w/v) overnight and filtered to collect the methanol fraction. The residue was extracted two more times and the filtrates were combined and concentrated to obtain the crude extract. All the fractions were concentrated by a rotary evaporator and the dried extract was used for antioxidant activity studies.

Selection and maintenance of Animals:

Adult male albino wistar rats were obtained from K.M. College of Pharmacy, Madurai, India. Animals weighing between 180 and 220 gm were used for the study. Animals were housed in large spacious cages and ad libitum fed with commercial pellets (Amrut, Bangalore) and water. Animals were well acclimatized for a week before the commencement of the experiment and standard environmental condition of temperature (22 ± 5⁰C), humidity (55 ± 5%) and 12 hrs light/dark cycles throughout the experimental period.

Antioxidant study:

In order to assess the toxic effects and tolerance limit and to determine a safe dose an acute oral toxicity study was carried out as per the CPCSEA guidelines. The rats were fasted for 3-4 hours before administration of extracts. All the procedures described in this study were reviewed and approved by the Institute Animal Ethical Committee (IAEC) No: IAEC/KMCP/152/FT/ 4379/2013-2014.

Grouping of animals for biological studies

The normal and treatment rats were divided into four groups, comprising of minimum six rats in each group.¹⁹

Table. 1: Animal groups

Group	Type	Treatment	Dose (per Kg of body weight)*
I	Control	0.9% saline	10 mL
II	Toxic control	Chromium dissolved (30% v/v, 1mL/100 kg)	30 mg (Ping et al., 2006)
III	Positive or standard control	Vitamin C	5 mg (Amany et al., 2009)
IV	Treatment control	Methanol extract of L. variegata (intraperitoneally)	10 mL

*Experiment conducted for 28 days

Sample Collection and Processing:

On the 29^thday of the experiment, rats were anesthetized and the blood was withdrawn through intra-cardiac puncture of rats. The animals were fasted for 12 hrs before the collection of blood. After the blood was collected, the rats were sacrificed by cardiac dislocation and their liver excised, rinsed in ice-cold normal saline and stored in refrigerator. A 10% w/v tissue homogenate was prepared in 0.9% saline (NaCl) and centrifuged at 5000 rpm for 10mins at 5^οC. The supernatant was taken for the estimation of various biochemical parameters. A small piece of liver also preserved in 10% buffered formalin solution was further processed and they were embedded in paraffin and sectioned at 5 µm thickness, then they were stained with haematoxylin and eosin for his to pathological examination. All the bio chemical assays were carried out within 48 hrs after sacrifice of the animals.

Biochemical Studies:

Lipid per Oxidation Assay (LPO):

Malondialdehyde (MDA), a secondary product of lipid per oxidation reacts with thiobarbituric acid at pH 3.5. The red pigment produced was extracted in n-butanol-pyridine mixture and estimated by measuring the absorbance at 532 nm.

Catalase Activity (CAT):

Catalase activity was assessed by the method of Luck (1974), where the breakdown of H₂O₂ was measured at 240 nm. Briefly, the assay mixture consisted of 3 mL of H₂O₂ phosphate buffer (0.0125 M; H₂O₂) and 0.05 mL of supernatant of liver homogenate and the change in the absorbance was measured at 240nm. The enzyme activity was calculated using the millimolar extension coefficient of H₂O₂ (0.07). The results were expressed as micromole of H₂O₂ decomposed per min per milligram of protein.

Estimation of Reduced Glutathione:

Reduced glutathione (GSH) in the liver was assayed according to the method of Ellman (1959).²⁰ 0.75mL of the homogenate was precipitated with 0.75 mL of 4% sulphosalicyclic acid and centrifuged at 1200 rpm for 15 mins at 4^oC. The assay mixture contained 0.5mL of supernatant and 4.5mL of 0.01 M, DTNB (5-5’-dithiobis (2-nitro benzoic acid) in 0.1 M, phosphate buffer (pH 8.0). The yellow colour developed was read immediately at 412 nm. The results were expressed as micromole of GSH per milligram of proteins.

Determination of AST (Aspartate aminotransferase)

AST (Aspartate aminotransferase) from the liver in the blood serum was assayed according to the method of Reitman and Frankel (1957).²¹ L-Aspartate react with a-Ketoglutarate to form oxaloacetate. The formed oxaloacetate is coupled with 2, 4-dinitrophenyl hydrazine (2, 4-DNP) to give the corresponding hydrazone derivative, which gives a brown colour in alkaline medium and this is measured colorimetric ally.

Determination of ALT (Alanine aminotransferase):

ALT (Alanine aminotransferase) from the liver in the blood serum was assayed according to the method of Reitman and Frankel (1957).²¹ALT react with a-Ketoglutarate to form pyruvate ester. The ester has ketone functional group and it coupled with 2, 4-dinitrophenylhydrazine (2, 4-DNP) to give the corresponding hydrazone, which gives a brown colour in alkaline medium and this can be measured colorimetric ally.

Determination of ALP (Alanine phosphatase):

Estimation of Serum ALP Activity This method is used for in vitro quantitative determination of alkaline phosphatase (ALP) in serum and plasma.ALP catalyzes the hydrolysis of p-Nitrophenyl phosphate (pNPP) to pnitrophenol. The hydrolysis of pNPP resulted in the conversion of colourless pNPP to p-Nitrophenol which has a strong absorbance at 405 nm.

His to pathological examination of liver:

Liver was cleaned and fixed at 10% neutral buffered formalin solution. After dehydration in graded ethanol solutions and in toluene, they were embedded in paraffin. Tissue sections of 3–5 μm thickness were stained with haematoxylin and eosin (H.E.) for histopathological examination. Other sections were stained with (Periodic acid staining) and PAS Stain for histopathological examination of glycogen.

RESULTS:

In vivoantioxidant activity in animals under chromium (VI) stress:

Effect of body weight of normal and experimental animals:

Effect of the treatment of methanol extract of L. variegata to animals against chromium induced oxidative stress on body weight, vide Materials and Methods are given in Table.1 Administration of chromium did not cause any significant change in the food and water intake. Nevertheless, chromium feeding resulted in a 4% decrease in the body weight with the duration of 28 days of treatment. However, animals fed with the methanol extract of L. variegata and those treated with vitamin-C did not cause an appreciable change in the body weight of the animals under chromium stress (Table.2).

Table. 2: Efficiency of methanol extract of L. variegata on body weight of normal and experimental animals

Groups	Initial Body Weight	Final Body Weight
Normal control (G I)	225.6 ± 7.58	219.90 ± 6.15
Negative control (Chromium (IV) (G II))	217.5 ± 7.32	175.40 ± 4.32^(a*)
Positive control (Vitamin C, (G III))	225.9 ± 7.30^(a*)	226.90 ± 6.35
Treatment control (Extract of L. variegate (G IV))	228.4 ± 6.50	215.38 ± 5.28

Units: gram

Values are expressed as mean ± SEM.

No. of animals in each group (n) = 6

Values were found out by using One Way ANOVA followed by Newman Keul’s multiple range test.

(a*) values were significantly different from Initial Body Weight of G_II at (P<0.01).

Effect on Glucose and Peroxidases:

The levels of glucose, catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidase (LPO) were determined and the results are presented in Table 3. The administration of Chromium (VI) resulted in liver injury and extensive oxidative damage in the experimental animals as manifested by a significant increase in the levels of LPO and glucose (p<0.01) coupled with a drastic decrease in CAT and GPx. The level of LPO increased by 19.40%, whereas the level of GPx and CAT decreased by 27.50% and 54.55% respectively as compared to control. Pretreatment with vitamin-C increased the levels of glucose while those of CAT and GPx registered a decrease. A similar condition comparable to that observed for positive controls (vitamin-c treated) was exhibited by the animals that received L. variegata extracts as a pretreatment.

Effect on Total Serum Enzymes:

The animals are treated with chromium (VI) showed significant changes in the levels of the serum protein, albumin and the enzymes AST (Aspartate aminotransferase), ALT (Alanine aminotransferase) and ALP (Alanine phosphatase) (Table 4). The animals under chromium (VI) stress resulted in a significant (p<0.01) decrease in the total protein and total albumin contents and a marked increase in the levels of AST, ALT and ASP (Table4). The animals which received a pre-treatment with vitamin-C (Group III), the enzymes ASP and ALT alone showed an increase 19% and 72% increase as compared with control (Group 1). In group IV animals which were received a pretreatment with the algal extract, the enzyme levels were elevated significantly as compared to control animals. Nevertheless, the levels of these enzymes decreased with the pretreatment as compared to chromium (VI) stressed animals to a marginal extent unlike the treatment with vitamin-C.

DISCUSSION:

Experimental evidence suggests that free radicals (FR) and reactive oxygen species (ROS) can be involved in a high number of diseases.²²The antioxidants works against the molecules that form free radicals, destroying them before they can begin the domino effect that leads to oxidative damage. The present study revealed that the L. variegata contain pharmacologically active substances such as alkaloids, glycosides, saponins, tannins, flavonoids and phenolic compounds, which are responsible for the antioxidant activity. The present study was undertaken to evaluate the antioxidant activity against Cr(VI) induced oxidative stress in male albino wistar rats. Chromium induces a broad spectrum of toxicological effects and biochemical dysfunctions constituting serious hazards to health.²³ recent studies have reported that oxygen free radicals are considered to be important mediators of Cr(VI) induced acute toxicity.²⁴Cr(VI) compounds are easily taken up by cells and are subsequently reduced to Cr(III) species. This reduction generates free radicals, which play a major role in the adverse biological effect of these compounds.²⁵The main approaches used to ameliorate Cr(VI) induced toxicity is the use of agents with powerful antioxidant properties. In the present study, involves the antioxidant efficiencies of the methanol extract of L. variegata against Cr(VI) induced liver-cell injury. Redox disturbances are known to have a negative impact on the body system through ROS generation, which destroy proteins, lipids and DNA by oxidation.²⁶Although chromium itself does not directly generate free radicals, it indirectly generates various radicals such as superoxide, nitrogen species like peroxynitrite, nitric oxide and hydroxyl causing damage consistent with oxidative stress.²⁷ These radicals attack the cell membrane and lead to the destabilization and the disintegration of the cell membrane as a result of lipid peroxidation.²⁸The body has endogenous antioxidant enzymes, such as SOD, CAT, GPX and GST. When the balance between ROS production and antioxidant defenses is lost, oxidative stress results, which through a series of events deregulates the cellular functions leading to various pathological conditions.²⁹Any compounds, natural or synthetic, with antioxidant properties may contribute towards the partial or total allevation of this type of damage. In the present study, decline in the level of antioxidant enzymes like CAT, GPx observed in chromium treated rat is a clear manifestation of excessive formation of free radicals and activation of the lipid peroxidation system resulting in tissue damage. The significant increase (p<0.01) in the concentration of these constituents in tissues of a chromium and methanol extract of L. variegata and standard vitamin-C treated animals indicate the antioxidant effect of methanol extract of L. variegata. It was reported that Dunaliella salina, a green marine algae having the ability to protect against oxidative stress in vivo using animal models.³⁰ MDA (Malondialdehyde) is the major oxidation product of peroxidized poly-unsaturated fatty acids and the increased MDA content is an important indicator of lipid peroxidation.³¹In the present study, we observed exposure marked elevation in liver MDA after Cr(VI), is an index of lipid peroxidation. Pre-treatment with the methanol extract of L. variegata and Vitamin-C in the prevention of oxidative damage induced by Cr(VI), objectified by a significant decrease in pancreatic MDA. Reduced glutathione (GSH) acts as a ROS scavenger within the cell. Increasing the synthesis of GSH would improve the antioxidant/ prooxidant ratio within the cell. Antioxidant enzymes are considered to be the body’s primary defense, which prevents biological macromolecules from oxidative injury and removes peroxides, free radicals and superoxide anion generated inside the cell. These enzymes work independently, cooperatively and synergistically to maintain the integrity of the organ tissues. The level of such enzymes is well maintained under normal physiological conditions. However, increase or decreases in the amount of such enzymes are marked through their modification in gene expression, decreased uptake or when cells are overloaded with oxidants.^{32, 33} On the other hand, reactive oxygen radicals have the ability to sufficiently modify a protein, leading to altered enzyme activity.³⁴ In the present study, statistically significant depletion in the activity of the antioxidative enzymes relates to the high ROS levels in the liver tissues. Such type of correlation is already reported in a wide variety of tissues treated with heavy metals including Cr(VI).^35,36Most of the antioxidant enzymes become inactive after Cr(VI) exposure either due to the direct binding of heavy metal to enzyme active sites if it contains SH groups or to the displacement of metal cofactors from active sites.³⁷ In standard drug, vitamin-C and Cr(VI) treated rats, the activities of GPx and CAT reached almost the control value levels, indicating that the methanol extract of L. variegata eliminated the toxic effects of Cr(VI) significantly. Furthermore, the methanol extract of L. variegata prevented the rise in blood glucose level/hyperglycemia in rats treated with acute doses of Cr(VI). The antioxidant enzymes and glutathione form the first line of defense against free radical-induced damage, offer protection against free radicals, and thereby maintain lower levels of lipid peroxide.³⁸ Glucose oxidation is believed to be the main source of free radicals. In its enediol form, glucose is oxidized in a transition-metal dependent reaction to an enediol radical anion that is converted into reactive ketoaldehydes and to superoxide anion radicals. The superoxide anion radicals undergo dismutation to hydrogen peroxide, which, if not degraded by catalase or glutathione peroxidase, and in the presence of transition metals, can lead to the production of extremely reactive hydroxyl radicals.³⁹ Superoxide anion radicals can also react with nitric oxide to form reactive peroxynitrite radicals.²⁶Hyperglycemia is also found to promote lipid peroxidation of low density lipoprotein (LDL) by a superoxide-dependent pathway resulting in the generation of free radicals.⁴⁰ The methanol extract of L. variegata brings about its anti-hyperglycemic effect through insulin secretion from the remnant β-cells and insulin sensitivity.^41,42

In the present study, SOD and GPx activities decreased slightly, but in most organs, these changes have no statistical signiﬁcance. It is likely that, the decrease in the activity of SOD and GPxare not the main factors in lipid peroxidative damage. To cope with the oxidative stress, there was a significant decrease in reduced glutathione (GSH) and catalase level in the liver tissue. Catalase and GSH-Px activities in the marine brown alga L. variegata, supplementation group were higher than in controls. However, SOD activity was not affected by seaweed supplementation. These results suggest that marine brown alga L. variegata supplementation decreases oxidative stress in vivo due to a combination of improved antioxidant activities and decreased peroxidation processes. No significant change in the SOD activity was observed in the chromium-treated animals and our results were confirmed by earlier studies.⁴³. At the end of the experimental duration, the total protein and total albumin levels were decreased in group II rats due to the Cr(VI) toxicity. Co-administration of the methanol extract of the L. variegata at 10 mg/kg body weight alters the total protein and total albumin to an almost normal level. The total protein was raised suggesting the stabilization of endoplasmic reticulum leading to protein synthesis. Besides activating the oxidative stress.

Cr(VI) has caused a marked increase in AST, ALT and ALP levels, suggesting that the Cr(VI) caused severe hepatic damage. The significant (p<0.01) increase was observed in the levels of diagnostic marker enzymes (ALT, AST and ALP) in plasma of Group III vitamin-C injected rats as compared to that of Group I control rats. This is an indicative of the cellular leakages and loss of functional integrity of cell membranes in the liver. The present observation is in agreement with earlier reported studies, which have shown that the amount of diagnostic marker enzymes present in plasma is directly proportional to the number of necrotic cells present in the liver tissue.^44,45On the other hand, pre-treatment with L. variegate significantly (p<0.01) improved the levels of these diagnostic marker enzymes in plasma of Group IV animals as compared to Group III vitamin-C-injected rats, demonstrating the cytoprotective activity of L. variegata. These findings indicated that the methanol extract of L. variegate have an ability to rebuild the structural integrity of the hepatocellular membrane and reduce damage caused by Cr (VI). Previous studies have shown that natural antioxidant molecules impart stabilization to cell membranes in relation to the degree of their free radical scavenging ability^46,
47 hence; it is possible that likewise L. variegata may also prolong the viability of liver cell membranes from vitamin-C-induced necrotic damage by its membrane stabilizing action. Many researchers have also demonstrated the hepato-toxic effect of chromium (VI), which is mainly due to the lipid peroxidation.^{48, 49} these adverse effects of Chromium (VI) could be significantly curtailed by pre-treating the animals with the methanol extract of L. variegata. This result implies that excessive administration of crude extract will decrease their antioxidant ability with unknown reasons.

Histopathology:

Chromium has been extensively studied as a liver toxicant. Liver and kidney are the two important vital organs mostly affected by drugs and xenobiotics. The massive generation of free radicals in the chromium induced liver damage provokes a sharp increase of lipid peroxidation in liver.³⁰ Chromium induces fatty liver and cell necrosis are plays a significant role in inducing depletion of reduced glutathione, increased lipid peroxidation, membrane damage and depression of protein synthesis and loss of enzyme activity. Hepatocytes make up 70–80% of the cytoplasmic mass of the liver. These cells are involved in protein synthesis, protein storage and transformation of carbohydrates. Chronic liver disease is characterized by the excessive deposition of collagen and other extracellular matrix (ECM) proteins within the liver. It is thought that activated hepatic stellate cells in the perisinusolidal space are the main contributors to the fibrotic process.⁵⁰Diagnosis of liver fibrosis is based on histological examination of chronic liver damage for lobular architecture, the degree of hepatocyte damage, inflammatory infiltration, firbrous deposition, and regeneration and nodular formation.

The intensity of the degenerative and necrotic changes of hepatocytes in rats treated with L. variegata and Vitamin-C was mild when compared with that of chromium (VI) intoxicated rats. Moreover, the improvement of these changes in rats treated with methanol extract of L. variegata extract or vitamin-C after 28 days of chromium (VI) administrator. The level of Vitamin-C was significantly depleted in chromium (VI) intoxicated rats. This depletion may be due to the excessive utilization of non-enzyme (vitamin-C) involved in quenching the free radicals produced during chromium (VI) intoxification. The seaweed pre-treated rats showed an improvement in the levels of vitamin-C. This emphasizes that treatment with L. variegata considerably prevented the alterations in the liver cell structural integrity triggered by chromium (VI) and restored the induced histopathological abnormalities.

CONCLUSION:

The methanol extracts of seaweeds L. variegata showed hepatoprotective activities and it could be a potential candidate of biomedical importance. It appears that the hepatoprotection afforded by these compounds was mainly due to their free-radical scavenging activity that protected the cells from the free radicals generated by chromium (VI) strong oxidizing species induced hepatotoxicity. The hepatoprotective effects of seaweed L. variegata enhanced the structural integrity of the hepatocyte due to their antioxidant activity. The results were supported by histopathological examination of liver and the methanol extract of L. variegata treatment improved liver architecture damage caused by chromium(VI).

CONFLICT OF INTEREST STATEMENT:

We declare that we have no conflict of interest.

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Received on 23.04.2017 Modified on 04.05.2017

Res. J. Pharmacology & Pharmacodynamics.2017; 9(2): 61-69.

DOI: 10.5958/2321-5836.2017.00011.8

Groups	Glucose	Catalase (CAT)	Glutathione Peroxidase (GPx)	Lipid peroxidation (LPO)
Normal control (G I)	65.68 ± 3.90	68.50 ± 2.68	39.33 ± 2.38	15.38 ± 1.95
Negative control (Chromium (IV) (G II))	132.80 ± 5.56^(**a)	32.34 ± 1.87^(**a)	16.32 ± 1.48^(**a)	28.42 ± 2.46^(**a)
Positive control (Vitamin C, (G III))	91.78 ± 4.56^(**b)	61.54 ± 2.36^(**b)	28.70 ± 1.77^(**b)	23.40 ± 1.60^(**b)
Treatment control (Extract of L. variegata(G IV))	95.38 ± 3.70^(**b)	54.55 ± 2.30^(**b)	27.50 ± 1.55^(**b)	19.40 ± 1.62^(**b)

Groups	Total protein	Total Albumin	Aspartate Aminotransferase (AST)	Alanine Aminotransferase (ALT)	Alanine Phosphatase (ALP)
Normal control - (Group I)	7.00 ± 1.82	5.12 ± 1.25	152.00 ± 5.20	79.80 ± 2.00	124.50 ± 2.50
Toxic control - Chromium (VI) (Group II)	5.10 ± 1.30⁽*^a)	4.10 ± 2.50⁽*^a)	250.10 ± 5.20⁽*^a)	165.00 ± 4.10⁽*^a)	259.20 ± 5.10⁽*^a)
Positive control-Vitamin C (Group III)	7.10 ± 1.50⁽*^b)	5.30 ± 2.51⁽*^b)	181.30 ± 5.20⁽*^b)	89.10 ± 5.20⁽*^b)	215.20 ± 5.80⁽*^b)
Treatment control-Extract of S. asperum (Group IV)	8.48 ± 1.46⁽*^b)	5.36 ± 0.58⁽*^b)	215.20 ± 7.15⁽*^b)	98.65 ± 5.05⁽*^b)	225.45 ± 6.05⁽*^b)

KEYWORDS: Hepatotoxicity; Lobophora variegata, radical scavenger, chromium (VI), Liver fibrosis,

Oxidative stress.