iv – results and disscusion -...

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IV – RESULTS AND DISSCUSION

4.1. PHYSICO-CHEMICAL ANALYSIS OF SEAWEEEDS EXTRACTS

The following seaweeds are used for the present study named as Seaweed 1

to Seaweed 4. They are 1. Sargassum wightii-(SW), 2. Gracilaria edulis-(GE),

3. Kappaphycus alvarezi-(KA) and 4. Gelidium acerosa - (GA).

4.1.1. Qualitative Analysis of seaweeds extracts

The results of the preliminary screening test using three different solvents

like methanol, ethanol and acetone of selected seaweeds are summarized in Table-

4.1. The phytochemical compositions (or) the secondary metabolite are varied

from species to species.

It reveals that all extracts (methanol, ethanol and acetone) showed the

presence for phytochemical constituents. Among the three solvents ethanolic

extract showed the presence of maximum number of phytochemicals, when

compared to methanol and acetone extracts (Dastmalachi and Dorman 2009). The

secondary metabolites identified in the selected seaweed are alkaloids,

carbohydrates, saponins, glycosides, proteins, aminoacids, phytosterols, phenolic

compounds, flavonoids, terpenoids and tannins. Most of the phytoconstituens are

well known for its medicinal property as well as exhibits various physiological

activities.

The presence of flavonoids, terpenoids and tannins is in agreement with the

earlier reports. In this study the ethanolic extracts of Sargassum Wightii (SW),

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Graciliaria Edulis (GE), Kappaphycus Ariezus (KA) and Gelidium Arosa (GA)

showed a maximum compounds (7 out of 10) and other extracts like acetone and

methanol exhibits the less number of phyto chemical constituents. For the further

examination only ethanolic extracts are used.

4.1.2. Quantitative analysis of phytochemicals in Ethanolic extract of selected

seaweeds

The phytochemical constituents of Ethanolic extracts of seaweeds are given

in Table - 4.2.

All the ethanolic extracts of all the selected exhibited the presence of

phytoconstituents and they are varied from species to species. On quantitative

analysis, the estimation of total phenolic content (mg/g), reveales that all the

selected seaweeds has phenolic content in ethanolic extracts. Higher values of

total phenolic content are also observed in Gracilaria Edulis (5.3 mg/g), followed

by Sargassum Wightii (4.1mg/g), Gelidium Acerosa (3.14 mg/g ) and

Kappaphycus Alvarezii (2.5 mg/g). The total phenolic compounds has high chain

breaking antioxidant properties with the additional ability to scavenge ROS, inhibit

lipid peroxidation and chelate metal ions (Shahid and Wanasundara, 1992; Rice-

Evans et al., 1995; Kahkonen et al., 1999).

The β-carotenes and other related compounds exhibited some antioxidant

properties. All the selected seaweeds has a significant amount of β-carotene in

ethanolic extracts and Sargassum wightii showed the maximum content

(36.3µg/ml) followed by the Gracilaria Edulis (34.1µg/ml) and other species


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Kappaphycus alvarezii (26.1 µg/ml) and Gelidium acerosa (23.14 µg/ml ) shows

the least.

Ascorbic acid (vitamin C) is considered as a potent antioxidant and found in

Marine Algae. Among the selected seaweeds Gracilaria Edulis shows the

maximum concentration (612.0mg / 100g) followed by Kappaphycus alvarezii

(521.8mg / 100g), Gelidium acerosa (415.0mg / 100g) and Sargassum wightii

(363.0mg / 100g).

Thiamine is one of the important vitamin belongs to B complex. Many

researchers reported that the seaweeds are the principle source of vitamins

(Buiminh et al., 2005). The ethanolic extracts of all the selected seaweeds exhibit

the presence of thiamine and Sargassum wightii showed the higher concentration

of Thamine content (0.22µg / 100g) followed by Gracilaria Edulis (0.16µg /

100g).Gelidium acerosa (0.13µg / 100g ) and Kappaphycus alvarezii (0.11µg /

100g ).

Flavonoids helps in protection of diseases caused by free radicals. It is most

commonly used as synthetic antioxidants, the flavonoids content of the seaweeds

balance the bodily antioxidant defence mechanism (Wong, 2000). Flavonoids are

hepato protection (Seevola et al., 1984; Wegner and Fintelmann, 1999). The total

content of flavenoids are higher in Sargassum wightii (19.2mg / g) followed by

Gracilaria Edulis (17.1mg / g), Kappaphycus alvarezii (15.5mg / g) and Gelidium

acerosa (14.2mg / g).


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The total ash content (g/100g) and minerals like K, Na, Ca are quantified in

the present study (mg/100g). The results showed that seaweeds provide the high

concentration of salt combined with sodium, potassium, calcium and the

concentration is higher than that of terrestrial plants (Buimin et al., 2005).

In this sargassum wightii showed the high content of total ash (4.6g/100g)

followed by Gelidium acerosa (4.5 g/100g), Kappaphycus alvarezii (4.1 g/100g)

and Gracilaria Edulis (3.8 g/100g).

Potassium is higher in Sargassum wightii (555.mg/100g) followed by

Gracilaria Edulis (512.2 mg/100g), Kappaphycus alvarezii (432.84mg/100g) and

Gelidium acerosa (405.2mg/100g). It is the principal element of the intracellular

fluid it is mainly concerned with the homeostasis and influence of the muscle. The

presence of potassium may therefore enchance the tissue damage of the liver.

Sodium is higher in Gracilaria Edulis (649.8 mg/100g) followed by

Sargassum wightii (549.8 mg/100g), Kappaphycus alvarezii (520.7 mg/100g) and

Gelidium acerosa (505.0 mg/100g). Sodium is involved in extra cellular and

intracellular fluid balance and maintenance the viscosity of the blood. The

Maximum content of the sodium content in the seaweed extract may help to

relieve antioxidant stress and control the complication of liver damage.

The calcium content is high in Gelidium acerosa (529.8 mg/100g) followed

by sargassum wightii (453.2 mg/100g), Gracilaria Edulis (432.8 mg/100g) and


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Kappaphycus alvarezii (420.8 mg/100g) showed the least (Muthuraman and

Ranganathan, 2004; Venkatesalu et al., 2004; Topping, 1973).

Based on the analysis ethanolic extracts were used for the further analysis.

4.1.3. Antimicrobial Assay

The antimicrobial activities of all the selected seaweeds in the ethanolic

extracts agains pathogenic bacteria and fungi are depicted in Table - 4.3.

The ethanolic extracts of the selected seaweeds exhibited the inhibitory

activity against all the strains (Samy and Ignacimuthu, 2000). The ethanolic

extracts of selected seaweeds were tested against six different bacterial strains and

5 fungi which are selected randomly.

In this study, special attention has been reported for anti bacterial and

antifungal activities related to marine algae against selected seaweeds are exhibited

(Borowitzka, 1992).

The ethanolic extracts of various algae have been shown to have

antibacterial activity against gram positive, gram negative bacteria and fungi. The

antimicrobial activity was determined by diffusion technique. The antimicrobial

activities of seaweeds also varied with species to species.

In this study the zone of inhibition ranged between 5 to 10mm and the

maximum activity (10mm) was recorded from the ethanolic extract of SW against

P.aeruginosa in bacteria and aspergillus fumigates in fungi and minimum (5mm).


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In this present study higher activity is recorded from SW followed by GE

and other showed the moderate effects. There was no inhibitory effects was seen

in the ethanolic extract against staphyloccous aerus. The zone of inhibition

against staphyloccous aerus is 6mm in SW, 5mm in KA and 7mm in GA

respectively.

GE and KA did not show any inhibitory effect against the test bacteria

bacillus cereus 7mm for SW and 6mm for GA is noticed. There was no inhibitory

effect noticed in ethanolic extract of SW and KA against test bacteria. In

klebsiella pneumonia 6mm for GE and 5mm for GA is noticed.

No inhibitory effect was seen against E.coli and A.famigucus ethanolic

extract of GA. 10mm for GA, 7mm for SW, 5mm for KA is noticed for

A.famigucus and for E.Coli the zone of inhibition is 7mm for SW, 8mm for GE

and 6mm for KA respectively.

There was no inhibitory effect seen in GE and KA extracts against

Aspergillus niger and candida tropicals. 8mm for GA, 8mm and 7mm for SW,

5mm for KA and 7mm for GA respectively against Aspergillus niger and Candida

tropicals. GE and SW were the two seaweeds active against all the tested

pathogens. This may be due to active compounds which are present in seaweed

extracts.


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As suggested by (Schwarz and Noble, 1999) the bacterial and fungal strains

may have some kind of resistance mechanism like enzymatic inactivation. Target

sites modifications and decrease intracellular drug accumulation or the

concentration of the compound used may not be sufficient.

The alkaloids are commonly found in seaweeds in pocesses to have

antimicrobial properties (Neli Loko Pfoz et al., 2011) presence of alkaloids in all

the extracts and hence exerting a remarkable antibacterial activity against the

tested micro organism. Sodium salts are shown to processes antibacterial activity

against S.aereus and E.Coli (Lee et al., 2002) and on treatment to produce

significant reduction of microbial constituents.

The findings of the phytochemical analysis reveal the presence of the

following. The phenolic compound exhibits the antibacterial and antifungal

activity (Conner, 1993; Didry et al., 1993; Karapinar and Tung, 1987).

The antimicrobial activity may be attributed to the presence of terpenoid

(Konig and Wright, 1997; Bansemir et al., 2004) are in agreement with the

antimicrobial activity found in the present study for selected seaweeds against test

bacteria and fungi. Phlorotannins, phenolic compounds, terpenoids are reported to

be produced by seaweeds exhibited antibacterial (Kubo et al., 1992; Alam et al.,

1994; Nagayama et al., 2002) and antifungal activity (Bhargava et al., 1998;

Vairappan et al., 2004).


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4.1.4. In Vitro Antioxidant studies in Ethanolic extracts of seaweeds

Several studies have investigated the activity of natural products in marine

and fresh water algae (Fujimoto and Kaneda, 1984; Matsukawa et al., 1997; Lim et

al., 2002; Xue et al., 2004). In this study, all the selected algae, exhibited the most

valuable antioxidant activities and are summerised in Table - 4.4. The ethanolic

extracts of seaweeds displayed high antioxidant potential (Duffy and Power, 2001)

thus act as a potential natural antioxidants.

To find out the presence of different antioxidant components in biological

samples, several assay methods have been developed and applied in recent years to

screen and evaluate the total antioxidant activity. The scavenging activity of

ethanolic extracts of seaweeds on DPPH radicals increased with increasing

concentrations IC50 value (the amount of antioxidant material required to scavenge

50% of free radicals in the assay system). DPPH is a stable free radical that

possesses a characteristic absorption maximum at 517 nm, which is diminished in

the presence of a compound capable of reducing it to its hydrazine form by

hydrogen/ electron donation. Free radical scavenging is one of the known

mechanisms by which antioxidants inhibit lipid oxidation. In this assay, results are

expressed as the ratio percentage of the absorbance decrease of DPPH radical

solution in the presence of extract at 517 nm to the absorbance of DPPH radical

solution at the same wavelength (Rastianzahra et al., 2007).

The antioxidant activity was determined by free radical scavenging effect

(DPPH decolourization method) (Sinha and Shana, 2007). The scavenging activity


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was assesed and expressed as % reduction of the initial DPPH absorption by the

tested compound.

Kappaphycus alvarezii (61.8%) displayed significantly higher Scavenging

activity followed by Sargassum wightii (66.8%) Gracilaria edulis (61.8%) and

Gelidium acerosa (59.0%). Natural antioxidants are found in the seaweeds.

Superoxide dismutase reduces the half–life of the superoxide anion is

expressed in µg/minute/mg of Proteins. Ethanolic extract of Gracilaria Edulis

exhibited the high activity of superoxide dismutase (574.0 µgm/minute/mg of

protein) (Pedersenn et al., 1996; Weinberger et al., 2001) followed by sargassum

wightii (512.0µgm/minute/mg of protein), Gelidium acerosa (47.5 µgm/ minute

/mg of protein). Superoxide dismutases (SOD) are a class of closely related

enzymes that catalyze the breakdown of the superoxide anion into oxygen and

hydrogen peroxide. SOD enzymes are present in almost all aerobic cells and in

extracellular fluids.

Reduced form of Glutathione is present in high concentration (mg/g) in

ethanolic extract of Sargassum wightii (76.8mg/g) and significantly decreased

amount is noticed in Gracilaria edulus (71.8mg/g) and Kappaphycus alvarezii

(69.0 mg/g) and Gelidium acerosa (53.2 mg/g). It is one of the water soluble

natural antioxidant, Glutathione has antioxidant properties since the thiol group in

its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced


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it rejuvenates immune system, and detoxifies the blood and protects the liver

(Pawlik et al ., 2007).

Catalase is the enzyme converts the hydrogen peroxide in to simple water

molecule using either an iron or manganese cofactor and has high potential of free

radical scavenging activity is found in all the selected seaweeds. Sargassum

wightii showed highest activity (61.7 µg/mol/min/mg of protein) followed by

Gracilaria edulis (56.2 µg/mol/min/g of protein), Kappaphycus alvarezii (55.7

µg/mol/min/g of protein) and Gelidium acerosa (45.9 µg/mol/min/g of protein)

(Toshiki Nakano et al., 1995).

4.1.5. Bio chemical parameters analysed in Ethanolic extracts

Seaweeds are potentially good source of biochemical factors like

carbohydrates, protein, fat, total amino acid content, proline, and all these factor

are quantified and summerized in Table - 4.5.

Based on the results obtained, some variations in biochemical compositions

are noticed. The carbohydrate content was maximum in SW (11.2g/100g) and the

lowest value is recorded in GA (9.6g/100g) than KA (10.5g/100g) and GE

(10.1g/100g) (Dawes et al., 1993; Norzian and Chiag 2002; Sancez-Machado, et

al., 2002).


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The total proteins of seaweeds have remarkable variation and high content

of protein is noticed in ethanolic extract of GE, when compared to other sea weeds

(Rao et al., 1983; Ochiai et al., 1987).

The total amino acid and proline content are analyzed. In this study it was

found that among the four selected seaweeds, ethanolic extracts of SW exhibits the

high amino acid content and proline in large amount followed by GE and the KA,

GA shows the least (Manivannan et al ., 2009). The proline detected in seaweed

molecule indicates the presence of all essential amino acids in considerable

quantities. This shows that seaweed proteins are nutritionally superior to the

terrestrial plant proteins (Rashidagasim, 1991).

The seaweeds are also rich in total fat content. The SW showed a high

content of fat (g/100g) next to GE, KA and GA showed an equal amount of fat

content (Dawer et al., 1974; Idler and Wiseman, 1970).

Out of four seaweeds the SW and GE are possessing many phytochemical

constituents and exhibited high biochemical parameters, antioxidant properties and

antimicrobial activity. So for the further studies only the two seaweeds GE and SW

are selected for GC-MS analysis, animal study and cell line analysis and

comparision among the seaweeds are reported.


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4.1.6. GC-MS SPECTRA OF THE PURIFIED FRACTION OF SELECTED

SEAWEEDS (SW and GE)

GC-MS Chromotogram analysis of the Ethanolic extracts of SW and GE

showed peaks which indicate the presence of many phytochemical constituents.

On comparison of the mass spectra of the constituent with NIST library the phyto

compounds were identified. The various phytochemicals which contributes to the

medicinal activities of the seaweeds are identified. The mass spectra of all the

phytochemicals identified in the Ethanolic extracts of SW and GE were presented

below.

4.1.6.1. Phytocomponents identified in Ethanolic extracts of GE and SW

Constituents of the extract were identified by comparison of their mass

spectral pattern and retention time with those of standard samples. The

components identified from the extract, their retention time and percentage

compositions are summarized in Table - 4.6.1. and Table - 4.6.2.

The compounds were identified from the GC-MS spectra which accounted

for 99.80% of the extract composition. The extract was predominated by alkaloid.

Majority of the compounds identified in the seaweeds are reported to have

pharmacological and toxicological Importance in humans. Thus SW and GE could

be harnessed as a source of raw materials in drug development.

In addition to antimicrobial activity, it was also reported to have anticancer,

anti-oxidant, chemo-preventive, pesticide, anti-tumor, and sunscreen properties

and anti-inflammatory properties. The phenolic compound and that the terpenes


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are found in SW/GE of some plants and physiological function of these

compounds are generally believed to be a chemical in defense against certain

pathogens causing human and animal diseases.

The presence of various bioactive compounds in the GE and SW justifies

the use of Seaweeds for various ailments by traditional practitioners. The isolation

of individual phytochemical constituents and subjecting it to the biological activity

will definitely give fruitful results.

4.2. EFFECT OF SARGASSUM WITGHTII AND GRACILARIA EDULIS

ON HAEMATOLOGY PARAMETERS BY CARBON TETRA

CHLORIDE / CHROMIUM INDUCED HEPATOTOXICITY IN

RATS

The haemostatic defects in the liver disease are complex and multifactorial,

which are often unpredictable, and their mechanisms are still elusive. The

screening of various hematological parameters depicts its significance and

diagnosis to estimate the degree of hepatocellular function (Lee and Baglin, 1994).

4.2.1. Effect of Ethanolic extracts of Gracilaria Edulis and Sargassum Wightii

on Hematological parmeters in Carbon tetra chloride induced Rats

Liver injury due to carbon tetrachloride in rats was first reported in 1936

and has been widely and successfully used by many investigators. Carbon

tetrachloride is metabolized by cytochrome P-450 in endoplasmic reticulum and

mitochondria with the formation of CCl3O-, a reactive oxidative free radical, which

initiates lipid peroxidation. Hepato toxicity are also induced by CCl4 (carbon tetra


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chloride) an organic compound, which are widely used as fire extinguishers, and a

precursor of cleaning agent, It is a colorless liquid with a sweet, small that can be

detected at low levels.

Carbon tetra chloride is a volatile organic alkyl halogen that is present in

the environment largely because of release from manmade sources (Grigg, 2004).

Carbon tetra chloride is readily absorbed from the Gastro Intestinal tract and

the lungs. CCl4 is distributed preferentially to fat tissue and is found in highest

concentration in bone marrow, brain, liver, kidney and blood carbon tetrachloride

is metabolized in cell microsomal membrane to a highly toxic trichloromethyl

radical that initiates lipid per oxidation, lipids, and protein in various cell

membranes, especially the hepatic endoplasmic reticulum (Friedman et al., 2003;

Ostapowic et al., 2002).

Carbon tetrachloride is metabolized by cytochrome p-450 in endoplasmic

reticulum and mitochondria with the formation of CCl3- a reactive oxidative free

radical, which initiates lipid metabolism. (McNally and Peter, 2006).

Hematological parameters like WBC, RBC, HB, PCV and PT are analysed

with CCl4 induced rats with the treatment of GE and SW are summarized in Table

- 4.7. and Table - 4.8. In this study group II to V is compared with group I with

CCl4 induced groups. In this analysis, the results demonstrates that, when CCl4 is

induced to the rats there is significant decrease in all the parameters (p<0.01) than


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control. And when the CCl4 rats are treated with ethanolic extracts of seaweeds in

various concentrations (100 mg/kg of bw, 200 mg/kg of bw/ 300 mg/kg of bw),

there is a noticeable changes are observed. All the Blood parameters are restoring

to that of control. Among the three concentrations 300mg/kg of bw showed the

maximum restorations of the parameters, and bring back the parameters to almost

to that of control groups. This study shows indicated that ethanolic extract of GE

and SW at a quantity of 100mg/ kg of bw and 200mg/ of bw was the minimal

effective dose and 300mg/of bw was the maximum effective dose. From the result,

it was inferred that The GE and SW was able to increase the blood parameters

levels considerably in dose dependent manner. The effectiveness of GE and SW

treatment was compared with control.

The decrease in the RBC count may be due to the destruction of

erythrocytes or the adverse effect of CCl4 on the erythropiotic tissue namely bone

marrow. Further the reduction of RBC correlates well with the decreased Hb

content (Meral, 2003).

In this present analysis Sargassum Wightii shows the significance

restoration of the changes observed in Carbon tetra chloride (CCl4) induced rats

than Gracilaria Edulis.


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4.2.2. Effect of Ethanolic extracts of Gracilaria Edulis /Sargassum wightii on

Hematological parmeters in Cr induced Rats

Chromium is an essential mineral found in very low concentration. Even

though chromium has beneficial effect, chromium compounds Cr (VI) are

powerful oxidizing agents and thus tend to be irritating and corrosive. Chromium

is absorbed by the lungs and gastrointestinal tract and even to certain extent by

skin. The reduction of Cr (VI) is considered to serve as detoxification process.

If Cr (VI) is reduced to Cr (III) extracellularly this form of the metal is not

readily transported into cells, so the toxicity is not observed. The balance that

exist between extra cellular Cr (VI) and Intracellular Cr (IV) enter the cell and

impact its toxic effect.

Cr (IV) enters many types of cells and under physiological condition, it

reduced to hydrogen peroxide (H2O2), Glutathione (GSH) reductase, ascorbic acid

and GSH to produce reactive intermediate, including Cr (V), Cr (VI) thiyiracucals

and hydroxyl radicals (Salnikow and Zhitovich, 2008).

The Cr3+ become toxic only at extremely high doses; or acts as a toxic

element gastric irritants rather than as that adversely affects physiology and

metabolism (Lukazski, 1999; Mosinger et al.,1954).

The hematological parameters are analysed with Cr induction followed by

the treatment with GE and SW are summarized in the Table - 4.9. and Table -

4.10.


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All the Test Groups are compared with the control Groups. A significant

decrease in Hb, RBC, WBC and PCV was noted with Cr treated rats when

compare to normal (p<0.01) (Knoll and Mohanty, 2009). RBC, WBC, Hb, PCV

and Platelets level in both 100mg/kg of bw and 200mg/of bw of Ethanolic

extracts of and GE and SW was significantly decreased (p<0.01). 300mg/of bw of

Ethanolic extracts of GE and SW treated groups are highly significant similar to

the normal (p<0.01).

The hematological parameters have diagnostic significances and plays a

role in providing information concerning hematological changes caused by Cr.

Most phytochemical constituents affect the immune system and hematological

parameters. The increase in PVC and increased level of haemoglobin on

treatment with GE and SW may due to the presence of Haematinic factors in GE

and SW such as iron which plays an important role which play in iron metabolism

that increase the level of PVC and synthesis of haemoglobin (Alda, 2000). The

Phyto-constituents of GE includes flavenoids and phytosterols are possible

candidates that increase white blood cells (Iweala et al., 2009).

4.3. EFFECT OF ETHANOLIC EXTRACTS OF GRACILARIA EDULIS

AND SARGSSUM WIGHTII IN CARBON TETRA CHLORIDE /

CHROMIUM INDUCED RATS [LIVER FUNCTION TEST (LFT)]

The liver has a great capacity to regenerate the damage tissue. The liver

produces symptoms after extensive damage. CCl4 and Cr damages mitochondria


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the power house of the cell which in turn release excessive amount of oxidants that

are capable for injuring hepatic cells.

Injury of hepatocytes and bile duct cells leads to accumulation of bile acid

inside the liver, and promotes liver damage (Patel et al., 1998) In the present study

the animal treated with CCl4 and Cr showed a significant liver damage. Elicited by

the decreased activities of hepatic marker enzymes.

4.3.1. Liver function test

The effect of ethanolic extract of GE and SW in CCl4 induced rat liver are

(LFT) shown in Table - 4.11. and Table - 4.12.

Decline in the activities of hepatic enzymes indicate the liver damage. The

reversal of increased serum enzymes in CCl4 induced liver damage by the extracts

may be due to the prevention of the leakage of intracellular enzymes by its

membrane stabilizing activity. This is in agreement with the accepted view that

transaminase level in the serum returns to the normal with the healing of hepatic

parenchyma and the regeneration of hepatocytes (Thabrew and Joice, 1987).

Administration of extracts of SW and GE to the respective rats at the dose

of 100µg/ml, 200 µg/ml and 300 100µg/ml for 15 days. There was a significant

(P<0.01) restoration of enzyme levels on administration of the ethanolic extracts of

seaweeds in dose dependent manner.


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The effective control of ALT, SGOT, Bilirubin and total protein levels

points towards an early improvement in the secretary mechanism of the hepatic

cells.

The carbon tetra chloride (CCl4) when induced to the test rats cause the

liver damage and produces significant change in the intracellular marker enzymes.

The SGOT, ALT, Bilirubin and protein are the most effective markers employed in

diagnosis of hepatic damage. The increase in activities of the enzymes subsequent

fall in the tissue might due to the leakage of the cytosolic enzymes in to the

circulatory system, resulting from hepatocellular damage due to the liver

dysfunction and disturbance of biosynthesis of these enzymes with alteration in the

permeability of liver membrane. The ethanolic extraction of GE and SW restored

the elevated activities of liver marker enzymes against liver damage induced by

carbon tetra chloride (CCl4) in male albino rats (Nevin and Vijammal, 2005). The

level of ALT are decreased in CCl4 induced rats (11.2±0.71) when compare to the

control rats (38.0±0.71). Increased level of Bilurubin is noticed in the CCl4

induced rats (0.97±0.001), when compare to control groups (0.51±0.005). The

level of SGOT is also noticeably decreased upon hepatotoxicity induction with

CCl4 (141.5±0.80) than control groups (370.0±0.5).

Upon treatment with GE and SW in the concentration of 100 100µg/ml,

200µg/ml and 300µg/ml showed a significant restoration of the enzyme levels. Out

of the three different concentrations of seaweed treatments the highest restoration

is observed in 300µg/ml.


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The ALT level become normal in higher concentration 300mg/kg of bw

(p<0.01). GOT and on treatment with GE and SW in the concentration of

100µg/ml and 200µg/ml showed the significant increase in the concentration of

bilurubin. The level become normal in treatment with 300 µg/ml (p<0.01). This

shows treatment of GE and SW on higher concentration reverse the toxicity of

liver induced by CCl4. In this present analysis SW showed the significance

restoration of the changes observed in Carbon tetra chloride induced rats than GE.

The present study revealed that CCl4 administration (3mg/kg of bw) for 15 days

caused a significant increase in serum ALT, SGOT, bilirubin and protein. This

increase may be due to liver damage induced by CCl4 intoxication (Manna, 2006).

The decreased activity of the enzymes was considered as an indicator for

the improvement of the functional status of the liver cells, which may due to the

action free radical scavenging activities and antioxidant properties of the

amomoacid content. The results are in agreement with other researchers even the

design of the experiment was changed (Ningappa et al., 2008; Mohammed et al.,

2009).

4.3.1. Effect of Ethanolic extracts of Gracilaria Edulis and Sargassum Wightii

in Cr induced Rats (LFT)

The level of Alanine transaminase (ALT), Glutamate oxaloacetate

transaminase (GOT), Bilirubin and Protein were used as biochemical markers to

assess hepatic damage.


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The chromium toxicity and detoxification of GE and SW are summarized in

Table - 4.13. and Table - 4.14.

The liver is the largest organ in the vertebrate body and is the major site of

xenobiotic metabolism and excretion. Liver injury can be caused by toxic

chemicals, drugs and virus infiltration from ingestion or infection. The toxins

absorbed from the intestinal tract gain access first to the liver resulting in a variety

of liver ailments. Thus liver diseases remain one of the major health problems

(Karan et al., 1999).

The serum level of hepatic enzymes ALT, GOT and total bilirubin levels

were increased and reflected the hepatocellular damage in the Cr induced

hepatotoxicity animal model. This is indicative of cellular leakage and loss of

functional integrity of cell membrane in liver.

The Cr when induced to the test rats cause the liver damage and causes the

significant change in the intracellular enzymes. The level of ALT are decreased in

Cr induced rats, and on treatment with GE and SW in the concentration of

100mg/ml and 200mg/ml showed the significant increase in the enzyme level

(P<0.01), the ALT level become normal in higher concentration 300mg/kg of bw

(p<0.01).

GOT, Blirubin level are increased when the rats are treated with Cr, and on

treatment with GE and SW in the concentration of 100 mg/kg of bw and 200


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mg/kg of bw showed the significant increase in the enzyme level (p< 0.01).

Bilirubin, an endogenous organic anion, binds reversibly to albumin and is

transported to the liver, where it is conjugated to glucuronic acid and excreted in

the bile. It is derived primarily from catabolism of red blood cells, heme and to a

lesser extent from degradation of myoglobulin, cytochrome, catalase and

peroxidase.

The ALT level become normal in 300mg/kg of bw (p<0.01). This shows

than on treatment of GE and SW on higher concentration reverse the toxicity of

liver induced by Cr. Increase activity of liver enzyme such as Alanine

Transaminase, SGOT, Bilirubin and protein are roughly proportional to the extent

of liver damage.

The protein levels are reflects major functional changes in liver function

(Pachathundikandi and Varghese, 2006). The protein level in the hepatotoxic

animal could be due to stabilization in protein synthesis secondary to a decreased

amount and availability of mRNA in the liver and this could indicate liver

dysfunction (Orchue et al., 2005). In this present analysis SW Showed the

significance restoration of the changes observed in Chromium (Cr) induced rats

than GE.


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4.4. EFFECT OF ETHANOLIC EXTRACTS OF GRACILARIA EDULIS

AND SARGSSUM WIGHTII ON ANTIOXIDANT PARAMETERS IN

CARBON TETRA CHLORIDE AND CHROMIUM INDUCED RATS

Rats when treated with CCl4 and Cr decreases the level of antioxidant

parameters like GPx, GSH, Vitamin-E, SOD, catalase, and increase the

concentration of vitamin-C. The ethanolic extracts of SW and GE restores the

antioxidant parameters which was altered by CCl4 and Cr induced in test groups,

indicating the protection of structural integrity of hepatocytic cell membrane or

regeneration of damaged liver cells.

The treatment with ethanolic extracts of seaweeds significantly reverse the

change, hence it is likely that the mechanism of due to antioxidant effects.

Antioxidant assay in CCl4 induced hepatotoxicity in rats and treatment with

ethanolic extracts of GE and SW are depicted in Table - 4.15. and Table - 4.16.

Decreased Plasma ALT are noticed. The treated groups showed the

reversal of the impaired hepatic SOD activity and increased in total GSH level.

Supplement of GE/SW a significantly improved hepatic antioxidant status and

therefore the supplementation of GE/SW protects against CCL4 induced liver

injury by alternating the free radical formation and enhancing antioxidant defense.


120

Antioxidant status was significantly lowered in CCL4 treated animals (p<

0.01) significantly lower level of glutathione (GSH) and lowered activities of

SOD, CAT and GPX.

The protective effect of GE/SW are evident from lowering levels of marker

enzymes and in serum and maintenance of antioxidant status in the liver as from

increased level of GSH and increase activity of SOD, CAT and GPx. The result

suggested that the antioxidant property of extract (Shajahan, 2005). Polyphenolic

natural flavonoids pocesses antioxidant and anticancer activity.

Vitamin C is the major ubiquitous non-enzymatic antioxidant has a crucial

role in scavenging several reactive O2 species. At physiological concentration.

Vitamin C is a potent free radical scavenger in the serum, protecting cell against

oxidative damage caused by ROS.

In the present study the levels of Vitamin-C decreased on exposed to the

CCl4 which was effectively improved by the SW/GE. Exposure to the CCl4

significantly depleted Vitamin-C which was counteracted effectively by the oral

Administration of SW and GE.

Vitamin E is one of the major chain breaking lipophitic antioxidant, with in

the cell membrane, where it protects membrane fattyacids from per oxidation,

Vitamin E has been effective in blocking per oxyl mediated chain reaction and in


121

combination with ascorbate, it scavenges SO- in the lipid membrane (Manikandan

and Devi 2005).

A significant reduction in the level of Vitamin-E was observed when it is

exposed to CCl4, which were improved on exposed to GE and SW. Thus from the

present study that the study there is increase in Vitamin E level on the

administration of extracts proves it is an antioxidant potential.

GSH is an intracellular defense against damage by ROS. The reduced form

of glutathione is necessary to maintain the normal reduces state of cells so as to

alleviate all the injurious effects of oxidative stress (Ahmad et al., 2009). In the

present investigation ,a marked depletion in the levels of GSH was observed when

the rats are exposed to CCl4. The levels improved when the rats are co-operated

with the ethanolic extracts of GE and SW in dose-dependent manners.

Thus, the results obtained revealed that the ethanolic extracts of GE and SW

influences favorably the antioxidant status of CCl4 induced rats.

Rats when treated carbon tetra chloride (CCl4) induce hepatocellular

damage and results in the notiacble change in enzymatic and non enzymatic

Antioxidants parameters Gpx, SOD, CAT, GSH, Vitamin E show a decrease level

when compare to the normal and Vitamin C level significantly increased than the

control groups. When the groups of rats are administered respective dosage of GE

and SW along with the fixed dose of Ccl4, in the respective concentration of


122

100mg/kg of bw, 200 mg/kg of bw and 300 mg/kg of bw showed the significant

restoration of the enzymatic and non enzymatic levels both the groups. 100mg/kg

of bw, 200 mg/kg of bw reverse the antioxidants only to the certain level and

significant restoration are noticed in the higher concentration 300mg/kg of bw in

the both the groups. In the antioxidant analysis SW showed significant higher

restoration than the GE (P <0.01) There was a stabilization in the level of

antioxidant profiles in the control and GE and SW fed rats which could be

attributed to the presence of antioxidants phytochemicals including Phenolic

compounds, flavonoids which favour the reaction with free radicals and Reactive

Oxygen Species (ROS).

4.4.2. Effect of Ethanolic extracts of Gracilaria edulis and Sargssum wightii

on antioxidant parameters in Chromium induced rats

The liver plays a key role in accumulation and detoxification of toxic

chemicals and heavy metals. According to Roch and McCarter (1984), the

bioaccumulation of trace elements in the Liver tissue reaches a proportion in which

the function of the liver is impeded, thus resulting in gradual degeneration of the

liver cells syncytial arrangement. This cirrhosis, the outcome of prolonged

hepatocellular injury is manifested by fibrosis of hepatic cords.


123

Assay of antioxidant parameters in Cr induced rats and treatment with GE

and SW extracts are shown in Table - 4.17. and Table - 4.18.

Rats when treated Chromium (Cr) induce hepatocellular damage and results

in the notiacble change in enzymatic and non enzymatic Antioxidants parameters.

Gpx, SOD, CAT, GSH, Vitamin E showed a decrease level when compare to the

normal and Vitamin C level significantly increased than the control groups. When

the groups of rats are administered respective dosage of GE and S w along with

fixed dosage of chromium in the respective concentration of 100mg/kg of bw, 200

mg/kg of bw, 300 mg/kg of bw shows the significant restoration of the enzymatic

and non enzymatic levels both the groups. 100µg/ml, 200 µg/ml reverse the

antioxidants only to the certain level and significant restorations are noticed in the

higher concentration 300µg/ml in the both the groups .In the antioxidant analysis

SW showed significant higher restorations than the GE (P <0.01) (Patiolla et al.,

2008).

Glutathione is one of the most abundant antioxidant in the reduced form

removes the free radical and maintain removes the free radical and maintain

membrane protein thiols (Prakash et al., 2001). Administration of Ethanolic

extracts of seaweeds significant (P<0.01) increases Gpx level.

Glutathione peroxide acts on the substrate glutathione also has the

antioxidant activity vitamin C and Vitamin E are non enzymatic substance with

free radical scavenging effects (Halliwell, 1986). The selected seaweeds increases


124

Vitamin E and Vitamin C increase antioxidant status. It is well established that

physiological antioxidant Vitamin-E (α-tocopherol) is located on the biological

membranes and play an important antioxidant role against oxidative damage of the

membrane (Dean, 1987; Summerfield and Tappel, 1984). Vitamins are water

soluble physiological anti oxidants in aqueous compartment of cells. Vitamin –C

act as a prooxidant, producing H2O2, and free radicals (Shamberger, 1984). This

ascorbic acid is employed as an antichrom agent for the prevention of and

protection against chromium (VI) toxicity in animals and in humans. The

modification of chromium induced damages by vitamin-C may be due to its ability

to reduce chromium (VI) to (III) with in the cells.

Superoxide dismutase (SOD) is a sensitive index in hepatocellular damage

is decreased is one of the most important enzymes in enzymatic antioxidant

defense system (Curtis and Mortiz, 1972). The selected seaweeds causes

significant increase in hepatic SOD activity and thus reduces reactive free radicals

induced oxidative damage to liver.

Catalase (CAT) is also an enzymatic antioxidant highly distributed in red

cells and liver. It decomposes in red cells and liver. It decomposed hydrogen

peroxides and protects the liver tissues from highly reactive hydroxyl radicals

(Chance and Greenstein 1992). A higher dose (300 mg/kg of bw) increases the

level of CAT as control


125

4.5. EFFECT OF GRACILARIA EDULIS and SARGASSUM WIGHTII ON

CCl4/Cr INDUCED HEPATOTOXICITY MEDIATED

HISTOPATHOLOGICAL CHANGES

The histopathological changes observed in liver of CCl4/Cr induced rats (6

groups) were almost recovered after treatment with Gracilaria Edulis and

Sargassum wightii. A detailed necroscopy was then conducted and sliced tissues

are viewed under microscope and the hepatocellular degeneration in liver was

graded and following observation was noticed.

Slight (degree: 1): Mild hepatocellular swelling due to hydropic degeneration and

fatty changes only in centrilobular areas (fatty infiltration)

Moderate (degree 2): Clear hepatocellular swelling in both centrilobular and

midzonal areas.

Severe (degree3): Diffuse and hepatocellular swelling, cytoplasmic paleness and

rupture (this grade was not seen in any treatment).


126

4.5.1. Effect of Gracilaria Edulis and Sargassum Wightii on CCl4 Induced

Hepatotoxicity Mediated Histopathological Changes

Fig 4.1: Control Rat liver

Control Rat liver show trabacule, central vein and normal hepatocytes with

one or two nuclei. The liver of control rats showed a normal structure (Fig 4.1). It

is composed of hexagonal or pentagonal lobules with central veins and peripheral

hepatic triads or tetrads embedded in connective tissue. Hepatocytes are arranged

in trabecules running radiantly from the central vein and are separated by sinusoids

containing kupper cells. The kupffer cells are fixed macrophages, which belongs to

the mononuclear phagocytic system. They are associated with endothelial cells and

possess cellular processes, which extended via the spaces between endothelial cells

in to the leumen of the sinusoid.


127

Fig 4.2: liver induced by carbon tetra chloride (CCl4)

Liver of a rat exposed to single dose of 3 mg of CCl4 on 15th day. The

trabecular structure of the lobules is blurred in some places. The cytoplasm of

some hepatocytes is enlarged, light with vacuoles. Fatty cells are observed in most

parts of the liver. Accumulation of mononuclear cells are seen in the vicinity of

sinusoids. The sinsoid walls showed many kupffer cells.

Hepatocytes are regular and contain a large spheroidal nucleus with a

distinctly marked nucleolus and peripheral chromatin distribution. Some cells have

two nuclei each. Lipocytes or Ito cells are typically found in the space and have the

ability to accumulate lipid droplets. They are the main source of vitamin A storage

in the body and also play a role in wound healing (hepatic fibrogenesis). Following

exposure to CCl4 and Cr single dose (3mg and 500 mg), the trabecular structure of

the lobules was blurred. Cytoplasm of hepatocytes contained empty vacuole like

spaces and were enlarged. Some sinusoids were overfilled with erythrocytes and


128

the walls of most sinusoids showed numerous kupffer cells. Locally mononuclear

cell infiltrations were observed frequently in the hepatocytes of zone I. (Fig 4.2).

The inflammatory cell infiltrates afore mentioned were observed more

frequently. However increased numbers of activated Kupffer cells characterized

by sinusoidal walls with increased amounts of cytoplasm and vacuolated nuclei as

well as small loci of extramedullary hematopoiesis (EMH) were also present. The

walls of the sinusoids in both zones showed numerous kupffer cells. In zone I

hepatocytic necrotic changes were evident, a small pycnotic cellular nucleus with

condensed chromatin, lack of nucleolus and strongly acidophilic cytoplasm were

observed. Mononuclear cell infiltration were also noted in zone I hepatocytes.


129

Fig 4.3: Liver induced with CCl4 + Gracilaria Edulis

Liver of rat exposed to Ethanolic extract of Gracilaria Edulis (200mg) and

CCl4 (3mg). Moderate affected hepatocytes and infiltration of mono nuclear cells.

Rats treated with Ethanolic extract of GE (200mg/kg of bw) and single dose of

CCl4 (3mg) and Cr (500mg) (groupIII) regained nearly normal structure of liver.

Fatty cells between hepatocytes disappeared, assumed their normal

histoarchitecture (Fig 4.3). Kupffer cells number in the walls of sinusoids

decreased significantly. In the rats pretreated with Ethanolic extracts of GE

(200mg/kg) and single dose of CCl4 (3ml/kg) and Cr (500 mg) (Group IV), nearly

normal structure of liver was observed.


130

Fig 4.4: Liver induced with CCl4 + Sargassum Wightii

Liver of rat exposed to Ethanolic extracts of Sargassum Wightii

(200mg/kg) and CCl4 (3ml/kg). Repaired Hepatocytes similar to normal size.

Granular or vacuolated degeneration and necrosis of the liver cells, sinusoidal and

central vein dilatation, bile duct proliferation, enlargement of periportal areas with

mononuclear cell inflammatory infiltration and mild degree fibrous tissue

proliferation were Partially get repaired. The similar progressive changes were

observed in rats pretreated with SW (200mg/ kg of bw) (Group V, VI) (Fig 4.4).

This investigation proved that Ethanolic extract of SW showed an effective

hepato protective effect than GE in rat liver induced with CCl4 and Cr.


131

4.5.2. Effect of Gracilaria Edulis and Sargassum Wightii on Cr Induced

Hepatotoxicity Mediated Histopathological Changes

The hepatotoxicity of liver is induced by Cr and histopathological changes

are observed. The following noticeable changes are seen.

Fig 4.5: Liver induced with Cr (chromium)

Liver of a rat exposed to single dose of 5 mg of Cr as potassium di

chromate on 15th day. The trabecular structure of the lobules is blurred in some

places. The cytoplasm of some hepatocytes is enlarged, light with vacuoles. Fatty

cells are observed in most parts of the liver. Accumulation of mononuclear cells

are seen in the vicinity of sinusoids. The sinsoid walls showed many kupffer cells.

Hepatocytes are regular and contain a large spheroidal nucleus with a

distinctly marked nucleolus and peripheral chromatin distribution. Some cells have

two nuclei each. Lipocytes or Ito cells are typically found in the space and have


132

the ability to accumulate lipid droplets. They are the main source of vitamin A

storage in the body and also play a role in wound healing (hepatic fibrogenesis).

Following exposure to single dose of Cr (5mg), the trabecular structure of the

lobules was blurred. Cytoplasm of hepatocytes contained empty vacuole like

spaces and were enlarged. Some sinusoids were overfilled with erythrocytes and

the walls of most sinusoids showed numerous kupffer cells. Locally mononuclear

cell infiltrations were observed frequently in the hepatocytes of zone I. (Fig 4.5).

Fig 4.6: Liver induced with Cr (Chromium) + Gracilaria Edulis

Liver of rat exposed to Ethanolic extract of Gracilaria Edulis (200mg) and

Cr (5mg). Moderately affected hepatocytes and infiltration of mono nuclear cells.

Rats treated with ethanolic extract of Gracilaria Edulis (200mg/kg of bw) and

single dose of Cr (3mg) (groupIII) regained nearly normal structure of liver. Fatty

cells between hepatocytes disappeared, assumed their normal histoarchitecture


133

(Fig 4.6). Kupffer cells number in the walls of sinusoids decreased significantly.

In the rats pretreated with Ethanolic extracts of Gracilaria Edulis (200mg/kg) and

single dose of Cr (5ml/kg) (Group IV) nearly normal structure of liver was

observed

Fig 4.7: Liver induced with Cr (Chromium) + Sargassum Wightii

Liver of rat exposed to Ethanolic extracts of Sargassum Wightii

(200mg/kg) and Cr (5ml/kg). Repaired Hepatocytes similar to normal size.

Granular or vacuolated degeneration and necrosis of the liver cells, sinusoidal and

central vein dilatation, bile duct proliferation, enlargement of periportal areas with

mononuclear cell inflammatory infiltration and mild degree fibrous tissue

proliferation were Partially get repaired. The similar progressive changes were

observed in rats pretreated with Sargassum Wightii (200mg/ kg of bw).

This investigation proved that Ethanolic extract of Sargassum Wightii

showed an effective hepato protective effect than Gracilaria Edulis in rat liver

induced with Cr (Fig 4.7).


134

4.6. EFFECT OF THE GRACILARIA EDULIS / SARGASSUM WIGHTII ON

TOXICITY INDUCED BY CARBON TETRA CHLORIDE (CCl4) AND

CHROMIUM IN HepG2 CELL LINES 4.6.1. Effect of ethanolic extract of GE on the cell viability by MTT assay

Cytotoxicity was measured based on the alteration of plasma membrane

permeability. MTT is cleaved in the tetrazolium ring by the succinate tetrazolium

reductase in active mitochondria. This yellow MTT is cleaved by all living,

metabolically active cells and not by the dead cells thereby forming formazon,

which form purple crystals and is directly proportional to cell number (Mosmann,

1983).

Table – 4.19. depicts the effect of Ethanolic extract of GE on the

cytotoxicity of Hepatocellular carcinoma cell lines as determined by MTT assay.

Treatment with CCl4/Cr (10 mM) caused significant loss of viability of cells

as measured by this assay. Cell lines pretreated with Ethanolic extract GE,

(100µg/ml, 200µg/ml and 300 µg/ml) and CCl4/Cr caused significant increment in

viability of cells in a dose dependent manner. The GE extract treatments along

with CCl4/Cr significantly increased cell viability by MTT assay. The highest

concentration of GE (300 µg/ml) (Group 6) was most effective when compared to

other concentrations of the extract. The efficacies of different solvent extracts of

GE/SW were also tested. The ethanol extracst of GE showed most efficiency in

protecting the cells against CCl4/Cr toxicity.


135

There was no significant difference in cell viability between cells incubated

with seaweed extracts (300 µg/ml) for 72 h and control cells, which indicates that

the extracts GE had no toxic effect up to 300 µg/ml. Thus the result indicates that

moderate to good protection is offered by the extracts of GE. The highest

protection is observed in 300 µg/ml concentration of ethanolic extract of GE.

From these results, it is proved that 300 µg/ml concentration of ethanolic

extract of GE had the most efficient effect against CCl4/Cr induced cytotoxicity.

Hence the ethanolic extract of GE was used for the in vivo studies.

4.6.2. Effect of ethanolic extract of SW on the cell viability by MTT assay

Cytotoxicity was measured based on the alteration of plasma membrane

permeability. MTT is cleaved in the tetrazolium ring by the succinate tetrazolium

reductase in active mitochondria. This yellow MTT is cleaved by all living,

metabolically active cells and not by the dead cells thereby forming formazon,

which form purple crystals and is directly proportional to cell number (Mosmann,

1983).

Table – 4.20. depicts the effect of Ethanolic extract of GE on the

cytotoxicity of Hepatocellular carcinoma cell lines as determined by MTT assay.

Treatment with CCl4/Cr (10 mM) caused significant loss of viability of cells

as measured by this assay. Pretreated with SW extracts (100 µg/ml, 200µg/ml,

300µl/ml) and CCl4/Cr caused significant increment in viability of cells in a dose


136

dependent manner. SW extract treatments along with CCl4 significantly increased

cell viability by MTT assay. The highest concentration of SW (300 µg/ml) (Group

6) was most effective when compared to other concentrations of the extract. The

efficacies of different solvent extracts of SW were also tested. Of these the ethanol

extract of SW showed most efficiency in protecting the cells against CCl4 toxicity.

There was no significant difference in cell viability between cells incubated

with Ethanolic t extracts of SW (300 µg/ml) for 72 h and control cells, which

indicates that the extract SW had no toxic effect up to 300 µg/ml. Thus the result

indicates that moderate to good protection is offered by the extracts of SW. The

highest protection is observed in 300 µg/ml concentration of ethanolic extract of

GE.

From these results, it is proved that 300 µg/ml concentration of ethanolic

extract of SW had the most efficient effect against CCl4/Cr induced cytotoxicity.

Hence the ethanolic extract of SW was used for the in vivo studies.

4.7. Effect of Ethanolic extracts of Gracilaria Edulis / Sargassum wightii on

Antioxidant parameters Carbon tetra chloride (CCl4) /Cr induced

hepato in toxicity in HepG2 cell lines

4.7.1. Effect of Ethanolic extracts of Gracilaria Edulis / Sargassum wightii on Antioxidant parameters (CCl4) induced hepato in toxicity in HepG2 cell lines

The results of antioxidant parameters are depicted in Table 4.21. and Table

4.22.


137

All the results were expressed in g/ml of protein. When the HepG2 cell

lines are treated with ccl4, the antioxidant parameters like GPx, GSH, SOD, CAT

are showa a significant decrease when compare to control. On treatment with GE

and SW significant increase the antioxidant parameters when compare to control

cell lines. out of various concentrations 300µg/ml of the Ethanolic extracts of

seaweed display the maximum restorations of antioxidants parameters than

100µg/ml and 200µg/ml concentrations. The ethanolic extracts of the seaweed GE

and SW stabilizes and increases all the components of defence system that were

examined in this study Viz., GSH, CAT, SOD and GPX. These factors protects

cells from ROS damage in CCl4 induced hepatocellular carcinogenis, as GE and

SW abolishes the the oxidative state induced by the initiator CCl4, and does so by

interacting directly by activating the antioxidant defense system. In this analysis

SW exhibited the maximum antioxidant potentials than GE. Thus the ethanolic

extract of GE and SW counteracts free radicals induced hepatocacinogenis and

promote the enzymatic and non – enzymatic antioxidant defense system and

potential chemo prevention might be due to synergistic effect of the

phytomolecules present extract.

In the present work a significant work decreased in the total protein in

serum rat was recorded after the 15 days of CCl4 adminstration as compared to the

control groups. This may be due to hepatopathy and inhibiton of protein synthesis

(Faroon et al., 1994; Manjunath et al., 2005). Upon the treatment with GE and

SW caused a significant increased in total protein content in serum.This increase

may be due to the stabilization of ER to improve the synthesis of protein.The


138

stimulisation of protein synthesis has been advanced as a contribytry

hepatoprotective mechanismof the studied protein which accelerates the

regeneration processes and protection of liver cells.

Regarding the activity of antioxidant enzymes in the tissue, There is a

significant in significant decrease in liver SOD and CAT activities of rats were

recorded after exposure to CCl4 as compare to control groups.

This decreases may be due to tissue damage and failure of antioxidant

defense mechanism to prevent the formarion of excessive freeradicals which cause

inactivation of the antioxidant enzyme (Escorbar, 1996).

Concerning the possible protective role of GE and SW significant increase

in hepatic SOD and CAT were observed in rats pretreated with SE and GE .The

increased SOD and CAT activities as a result of pre-treatment with GE and SW

may be due to the antioxidant activities of SW and GE which recover the damaged

caused by CCl4. The SW and GE have inhibiting the membrane damage and

enhancing the activities of endogenous antioxidants. GPx plays a common role in

cellular resistance to oxidative damage as a free radical scavenging and as a

protein bound glutathione and generation of ascorbic acid and tocopherol in liver.

The present study the rats adminstred with CCl4 alone exhibited a significant

decrease in hepatic GSH contents as compare to the control group.


139

A significant increase in hepatic GSH level was recorded in HepG2 Cell

lines treated with GE and SW to CCl4 administred groups.The results may due to

denova synthesis or regeneration of GSH as a result of pretreatment with GE and

SW and enhance antioxidant defence mechanism.

4.7.2. Effect of Ethanolic extracts of Gracilaria Edulis and Sargassum wightii

on Antioxidant parameters Chromium induced to in toxicity in HepG2

cell lines

Effect of GE and SW on chromium induced hepato cellular carcinoma cell

line are depicted in the Table - 4.23. and Table - 4.24. All the results were

expressed in g/ml of protein. When the HepG2 cell lines are treated with Cr, the

antioxidant parameters like GPx, GSH, SOD, CAT are show the significant

decrease when compare to control. On treatment with GE and Sw significant

increase the antioxidant parameters when compare to control cell lines. out of

various concentrations 300µg/ml of the ethanolic extracts of seaweed display the

maximum restorations of antioxidants parameters than 100µg/ml and 200µg/ml

concentrations.In this analysis Sw exhibited the maximum antioxidant potentials

than GE. The Glutathione which is present in the cells act as a potential

physiological antioxidant in biological system (Kettererer, 1988). Many

investigation have been made in to the reaction of glutathione with chromium (VI)

forma a Chromium (V)-glutathine complex and are produced by the reduction of

chromium (VI) by GSH (Goodgame Joy, 1986; Kawanishi et al., 1986; Kitagawa

et al 1988; Shi and Dalal, 1990). Glutathione is also known to form sssssstable

complexwith chromium III (Abdulla et al., 1985). The studies shows that the


140

glutathione depletes the free radicals formaton by DNA damage caused by

chromate, indicating the intracellular glutathione has a protective effects in Cr

induced hepatocytes. The studies showed that the ethanolic extracts of seaweeds of

HepG2 cells cause an increase in cellular content of Glutathione, resulting in

enhanced chromate induced DNA damage and act as a potential antioxidants.

The active oxygen species such as superoxide radical hydroxyl radical,

singlet oxygen and H2O2 have wide potential of cellular injury. The physiological

antioxidant such as superoxide dismutase (SOD) and catalase, which remove

superoxide radical and H2O2 respectively. and shown to prevent cell injury

mediated by oxygen radical under the variety of radical. H2O2 is not afreeradiacl

produce by superoxide radicals involment of active oxygen radical in chromium

produce DNA strand breaks and the chromate reacts with H2O2 invitro to form

tetra peroxochromate (V)[CrV(O2)4 3- ] lead to the oxy reactive species and cause

cellular damage. In addition of SOD to the chromate result in an inhibition of

hydroxyl radical production and indicating the hydroxyl radical generation and

result in DNA breakage. The treatment of chromium induced cell lines with

ethanolic extracts of GE and SW at the dose dependent manner reverse the

valuable physiological antioxidant potentials and proved as effective

Anticarcinogenic agent in HepG2 cell lines and in rats.


141

4.8. EFFECT OF ETHANOLIC EXTRACT GE AND SW ON THE CELL VIABILITY OF CCl4/Cr INDUCED HepG2 CELL LINES BY TRYPAN BLUE STAINING ASSAY.

4.8.1 .Effect of ethanolic extract GE and SW on the cell viability of carbon

tetra chloride (CCl4) induced HepG2 Cell lines by trypan blue staining assay.

Table - 4.25. indicates the effect of ethanolic extracts on GE and SW Cell

viability assay in CCl4 induced HepG2 cell line.

The cell viability by trypan blue assay depict of percentage of cell death.

The percentage of cell death significantly increased in ccl4 induced group

(Group2) when compared to control (Group I). The Ethanolic extracts of GE and

SW showed decrease in the cell death after induction with CCl4/Cr. The ethanol

extract of SW treatment showed better effect than ehtanolic extracts of GE. Of the

three concentrations of the ethanol extract, 300 µg/ml was found to be more

effective than other concentrations (100 µg/ml (Group IV) and200 µg/ml

(GroupV). Treatment with ethanolic extract of GE and SW alone (Group V) did

not change cell viability when compared with control (Group 1).

4.8.2. Effect of ethanolic extract GE and SW on the cell viability of

Chromium (Cr) induced HepG2 Cell lines by trypan blue staining

assay

Table-4.26. depicts the % of cell viability of HepG2 (Hepatocellular

Carcinoma cell lines) exposed to plant extract (Gracilaria Edulis and Sargassum

wightii)


142

The cell viability by trypan blue assay depict of percentage of cell death.

The percentage of cell death significantly increased in Cr induced group (Group2)

when compared to control.

(Group I). The Ethanolic extracts of GE/SW showed decrease in the cell

death after induction with Cr.The ethanol extract of SW treatment showed better

effect than ehtanolic extracts of GE. Of the three concentrations of the ethanol

extract, 300 µg/ml was found to be more effective than other concentrations (100

µg/ml (Group IV) and200 µg/ml (GroupV). Treatment with ethanolic extract of

GE/SW alone (Group V) did not change cell viability when compared with control

(Group I).


143

4.9. HISTOLOGICAL STUDIES

HepG2 cell line is used for the histological studies. The following slides

(Fig 4.8 - Fig 4.14) shows the effect of various toxic substances and recovery of

the same when it is treated with SW and GE. It confirms the effect of SW and GE

on controlling the Hepatocellular carcinoma in selected cell line also.

(Hepatocellular carcinoma cell line)

Fig 4.8: Control (HepG2 cell line)

Fig 4.9: HepG2 cell line induced with CCl4


144

Fig 4.10: HepG2 cell line induced with CCl4 + Gracilaria Edulis

Fig 4.11: HepG2 Cell line induced with CCl4 + Sargassum Wightii


145

Fig 4.12: HepG2 cell line induced with Cr (Chromium)

Fig 4.13: HepG2 cell line induced with Cr (Chromium) + Gracilaria

Edulis

Fig 4.14: HepG2 cell lines induced with Cr (Chromium) + Sargassum

Wightii


iv – results and disscusion -...

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