IV – RESULTS AND DISSCUSION -...
Transcript of 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.
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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
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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
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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.
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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
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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
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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).
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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.
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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
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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.
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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.
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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.
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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
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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
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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).
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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.
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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
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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.
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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
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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.
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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
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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.
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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)
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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).
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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
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Fig 4.10: HepG2 cell line induced with CCl4 + Gracilaria Edulis
Fig 4.11: HepG2 Cell line induced with CCl4 + Sargassum Wightii
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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
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