36
CHAPTER 4
4.1 HEPATOPROTECTIVE ACTIVITY
4.1.1 Introduction
Liver is the most important organ of metabolism and excretion of
drugs and food. About 20,000 deaths occur every year due to liver diseases.
Hepatocellular carcinoma is one of the ten most common tumors in the world
with over 2, 50,000 new cases each year (Hikino and Kiso 1988). Although
viruses are the main cause of liver diseases, environmental pollutants,
xenobiotics, hepatotoxins, excessive drug therapy and chronic alcohol
ingestions can also cause severe liver injury (Subrata et al., 1993). Liver injury
induced by chemicals has been recognised as a toxicological problem for more
than 100 years. During the late 1800s, scientists were concerned about the
mechanisms involved in the hepatic deposition of lipids following exposure to
yellow phosphorus. Hepatic lesions produced by arsphenamine, carbon-
tetrachloride and chloroform were also studied in laboratory animals during the
first 40 years of the 20th century. During the same period the correlation
between hepatic cirrhosis and excessive ethanol consumption was recognized.
(Plaa and Charbonneau, 2001).
4.1.2 Experimental model
4.1.2.1 CCl4 induced hepatotoxicity
Carbontetrachloride is a potent hepatotoxin producing
centilobular necrosis which causes liver injury. CCl4 an extensively studied
liver toxicant and its metabolite such as trichloro methyl peroxy radical (•CCl3
O2) is involved in the pathogenesis of liver damage (Al-Shabanah et al., 2000).
The toxicity of CCl4 depends on the cleavage of a carbon-chlorine bond to
37
generate a trichloromethyl free radical(•CCl3); this free radical reacts rapidly
with oxygen to form a trichloromethyl peroxy radical (•CCl3 O2), which may
contribute to the toxicity (Cheeseman et al., 1985, Mitchell et al., 1984,
Recknagel et al., 1973 & 1977), demonstrates that the cleavage occurs in the
endoplasmic reticulum and is mediated by the cytochrome P450 mixed function
oxidase system; the product of the cleavage can bind irreversersibly to hepatic
proteins and lipids ; and the CCl4 derived free radical(s) can initiate a process of
autocatalytic lipid peroxidation by attacking the methylene bridges of
unsaturated fatty acid side chains of microsomal lipids.
The peroxidative process initiated by the •CCl3 radical, for
example, is thought to result in early morphological alteration of the
endoplasmic reticulum, loss of activity of the cytochrome P450 xenobiotic
metabolizing system, loss of glucose-6-phosphatase activity, loss of protein
synthesis, loss of the capacity of the liver to form and excrete VLDL and
eventually, through as yet unidentified pathways, to cell death. Dingell and
Heimburg, (1968), Lal et al., (1970), Jaegar et al., (1973) and Anderson et al.,
(1979) have made use of barbiturate sleeping time to assess chemically induced
hepatotoxicity.
Normal cellular metabolism can result in the production of the
reactive oxygen species (superoxide, hydrogen peroxide, singlet oxygen,
hydroxyl radical) and all cells contain defense systems to prevent or limit
damage, glutathione is the major component of this system, but α-tocopherol
and Vitamin C play an important role (Liebler and Reed, 1997). The imbalance
between the prooxidants and antioxidants is known as oxidative stress (Reed,
1998). Toxicants are associated with the induction of lipid peroxidation or
38
oxidative stress in liver cells (Comporti, 1998). The CCl4 biotransformed
reactive free radicals promote membrane lipid peroxidation directly.
CCl4 intoxication disrupts the Ca2+ homeostasis within 2 to 4 hrs
and the calcium content of liver, mitochondria is also doubled (Reynolds et al.,
1962). Associated with this abnormal movement of calcium into the liver cells,
there are other disturbances in electrolyte distribution and swelling of liver
cells (Mclean et al., 1965). The lysosomes become disrupted between 5th and
10th hrs (Dianzani, 1963). Intracellular enzymes appear in the plasma due to
leakage (Rees et al., 1962). Focal necrosis is evident as early as 6 hrs after
poisoning and at first is midzonal. By 12 hrs the centrilobular cells exhibit
prenecrotic changes and ballon cells are prominent in the midzonal region. By
24 hrs, there is marked centrilobular necrosis affecting upto half of the lobule
(Recknagel, 1967).
In the present study, CCl4 has been selected, as its immense use
in industry leads to severe exposure to mankind, resulting in acute liver
diseases.
4.1.3 Herbal drugs
In spite of the tremendous advances made in allopathic medicine,
no effective hepatoprotective medicine is available. Herbal drugs are known to
play a vital role in the management of liver disorders. There are numerous
plants and polyherbal formulations claimed to have hepatoprotective activities.
Nearly 150 phyto constituents from 101 plants have been claimed to possess
liver protecting activity (Doreswamy and Sharma, 1995, Handa et al., 1989).
Some of the plants possessing hepatoprotective activities (Subrata et al., 1993)
are Silybum marianum, Withania somnifera, Ocimum sanctum, Tinospora
39
cordifolia, Picorrhiza kurroa, Andrographis paniculata, Phyllanthus embelica,
Phyllanthus amarus, Boerhaavia diffusa and Curcuma longa etc (Scott
Treadway, 1998).
4.2 Materials and Methods
4.2.1 Animals
Wistar albino mice of either sex and Wistar albino rats of either
sex were used for the experimental study as given in the section 3.1.2.1
4.2.2 Hepatoprotective evaluation
4.2.2.1 Pentobarbital induced sleeping time in mice
Pentobarbital induced sleeping time in mice model was evaluated
by the method Montilla et al., (1990).
4.2.2.1.1 Experimental protocol
The effect of plant extracts on pentobarbital induced sleeping
time and CCl4 induced prolongation of pentobarbital induced sleeping time was
studied in mice. The animals were divided into seven groups 10 in each group
and received the following regime of treatment.
Group I Animals received 1% carboxy methyl cellulose (10
ml/kg/po) and pentobarbital (45 mg/kg/ip).
Group II Animals received CCl4 mixture (1.5 ml/kg/po in 50%
v/v olive oil) followed by pentobarbital (45 mg/kg/ip).
Group III RCA (500 mg/kg/po) 4 doses of extract at 12 hr
40
intervals and CCl4 mixture (1.5 ml/kg /po in 50% v/v
olive oil ) 1 hour post treatment of the last dose of
extract, followed by pentobarbital (45 mg/kg/ip).
Group IV RCM (500 mg/kg/po) 4 doses of extract at 12 hr
intervals and CCl4 mixture (1.5 ml/kg /po in 50% v/v
olive oil ) 1 hour post treatment of the last dose of
extract, followed by pentobarbital (45 mg/kg/ip).
Group V CFA (500 mg/kg/po) 4 doses of extract at 12 hr
intervals and CCl4 mixture (1.5 ml/kg /po in 50% v/v
olive oil ) 1 hour post treatment of the last dose of
extract, followed by pentobarbital (45 mg/kg/ip).
Group VI CFM (500 mg/kg/po) 4 doses of extract at 12 hr
intervals and CCl4 mixture (1.5 ml/kg/po in 50% v/v
olive oil ) 1 hour post treatment of the last dose of
extract, followed by pentobarbital (45 mg/kg/ip).
Group VII Silymarin (25 mg/kg/po) 4 doses of extract at 12 hr
intervals and CCl4 mixture (1.5 ml/kg /po in 50% v/v
olive oil ) 1 hour post treatment of the last dose of
extract, followed by pentobarbital (45 mg/kg/ip).
After the administration of pentobarbital injection the animals
were placed on their back on a table and sleeping time was noted. The time
between loss of righting reflex and its recovery was taken as duration of
pentobarbitone induced sleeping time.
41
4.2.2.2 CCl4 induced liver injury model
CCl4 induced liver injury model was used to evaluate the
hepatoprotective activity by the method of Zhao et al., (2001).
4.2.2.2.1 Experimental protocol
The animals were divided into eleven groups of six animals each
Group I - Control animals received 1% CMC (10 ml/kg/po) for 7
days
Group II - CCl4 treated animal
Group III - RCA (250 mg/kg/po) for 7 days
Group IV - RCA (500 mg/kg/po) for 7 days
Group V - RCM (250 mg/kg/po) for 7 days
Group VI - RCM (500 mg/kg/po) for 7 days
Group VII - CFA (250 mg/kg/po) for 7 days
Group VIII - CFA (500 mg/kg/po) for 7 days
Group IX - CFM (250 mg/kg/po) for 7 days
Group X - CFM (500 mg/kg/po) for 7 days
Group XI - Silymarin (25 mg/kg/po) for 7 days
All the animal in Groups (II – XI) received single dose of equal
mixture of CCl4 and olive oil (50% v/v 5 ml/kg/po) on 7th day except normal
control animals (Group-I). All the animals were sacrificed by cervical
decapitation under light ether anesthesia on the eight day. Blood was collected
from jugular veins and centrifuged (3000 rpm for 10 min) to obtain serum. The
serum was used for marker enzyme estimation. Immediately after sacrifice, the
liver was dissected out, washed in the ice cold saline and the homogenate was
42
prepared in 0.1 ml Tris –HCl buffer (pH 7.4).The homogenate was centrifuged
and the supernatant was used for the biochemical analysis. Small pieces of liver
tissue were collected and preserved in 10 % formalin solution for
histopathological studies.
4.2.3 Estimation of Tissue and Serum enzymes
4.2.3.1 Aspartate aminotransferase (EC: 2.6.1.1 AST)
Aspartate aminotransferase was estimated by the method of King
(1965b) under the section 3.1.4.3.7
4.2.3.2 Alanine aminotransferase (EC: 2.6.1.2 ALT)
Alanine aminotransferase was assayed by the method of King
(1965b) under the section 3.1.4.3.8
4.2.3.3 Alkaline phosphatase (EC 3.1.3.1 ALP)
Alkaline phosphatase was assayed by the method of King (1965 a)
under the section 3.1.4.3.9
4.2.3.4 Estimation of total bilirubin
Total bilirubin was estimated by the method of Malloy and
Evelyn (1937) under the section 3.1.4.3.6
4.2.3.5 Estimation of protein
Protein was estimated by the method of Lowry et al., (1951) under
the section 3.1.4.3.5
43
4.2.4 Estimation of Enzymic Antioxidants
4.2.4.1 Superoxide dismutase (EC: 1.15.1.1, SOD)
The enzyme was assayed according to the method of Marklund and
Marklund (1974).
Reagents
1. Tris-HCl buffer 0.1 M, pH 8.2: 1.21 g of Tris was dissolved in 90
ml of distilled water, the pH was adjusted to 8.2 with 3 N HCl and
the volume was made up to 100 ml with distilled water.
2. Tris-HCl buffer 0.5 M, pH 7.4: 605 mg of Tris was dissolved in 90
ml of distilled water, the pH was adjusted with 3 N HCl and the
volume was made up to 100 ml with distilled water.
3. Pyrogallol solution 2 mM: 2.52 mg of pyrogallol was dissolved in
10 ml of 0.05 M Tris-HCl buffer in an aluminium foil wrapped
stoppered test tube.
4. Absolute ethanol (AR).
5. Chloroform (AR).
Procedure
To 1 ml of the sample, 0.25 ml of absolute ethanol and 0.15 ml of
chloroform were added. After 15 min of shaking in a mechanical shaker, the
suspension was centrifuged and the supernatant obtained constituted the
enzyme extract. The reaction mixture for autoxidation consisted of 2 ml of the
buffer (Tris-HCl, pH 8.2), 0.5 ml of 2 mM pyrogallol and 1.5 ml water.
44
Initially, the rate of autoxidation of pyrogallol was noted at an interval of one
minute for 3 min.
The assay mixture for the enzyme contained 2 ml of 0.1 M
Tris-HCl buffer, 0.5 ml of pyrogallol, aliquots of the enzyme preparation and
water to give a final volume of 4 ml. The rate of inhibition of pyrogallol
autoxidation after the addition of the enzyme was noted.
The enzyme activity is expressed in terms of units/min/mg
protein in which one unit corresponds to the amount of enzyme required to
bring about 50% inhibition of pyrogallol autoxidation.
4.2.4.2 Catalase (EC: 1.11.1.6)
The catalase activity was assayed by the method of Sinha (1972).
Reagents
1. Dichromate–acetic acid reagent: 5% potassium dichromate in water
was mixed with acetic acid in the ratio 1:3 (v/v). The solution was
further diluted to 1:5 with distilled water.
2. Phosphate buffer 0.01 M, pH 7.0 : 173 mg of disodium hydrogen
phosphate and 122 mg of sodium dihydrogen phosphate were
dissolved in 61 ml and 39 ml of distilled water, respectively.
3. Hydrogen peroxide 0.2 M : 2.27 ml of hydrogen peroxide was
made up to 100 ml with distilled water.
45
Procedure
0.1 ml of the homogenate was taken to which 1 ml of phosphate
buffer and 0.5 ml of hydrogen peroxide were added. The reaction was arrested
by the addition of 2 ml dichromate-acetic acid reagent. Standard hydrogen
peroxide in the range of 4 to 20 μmoles were taken and treated similarly. The
tubes were heated in a boiling water bath for 10 min. The green colour
developed was read at 570 nm.
Catalase activity in tissue homogenates is expressed as μmoles of
H2O2 consumed /min/mg protein at 37°C.
4.2.4.3 Glutathione peroxidase (EC: 1.11.1.9, GPx)
The activity of glutathione peroxidase was assayed by the method
of Rotruck et al., (1973).
Reagents
1. Sodium phosphate buffer 0.32 M, pH 7: 6.96 g of disodium
hydrogen phosphate and 3.89 g of sodium dihydrogen phosphate
were dissolved in 61 ml and 39 ml distilled water, respectively.
2. Ethylene diamine tetra acetic acid (EDTA) 0.8 mM : 233 mg of
EDTA was dissolved in 100 ml of distilled water.
3. Sodium azide 10 mM : 165 mg of sodium azide was dissolved in
100 ml of distilled water.
4. Reduced glutathione (GSH) 4 mM : 123 mg of GSH was dissolved
in 100 ml of distilled water.
46
5. Hydrogen peroxide 2.5 mM: 0.03 ml of hydrogen peroxide solution
was made up to 100 ml with water.
6. 10% TCA
7. Disodium hydrogen phosphate 0.3 M : 5.33 g of disodium
hydrogen phosphate was dissolved in 100 ml of distilled water.
8. 5,5’-dithio bis (2-nitrobenzonic acid) (DTNB) : 40 mg of DTNB
was dissolved in 100 ml of 1% Tri sodium citrate.
9. Glutathione standard: 10 mg of reduced glutathione was dissolved
in 100 ml of distilled water.
Procedure
0.2 ml each of EDTA, sodium azide, glutathione (reduced),
hydrogen peroxide, and 0.4 ml of buffer and 0.1 ml of homogenate were mixed
and incubated at 37°C for 10 min. The reaction was arrested by the addition of
0.5 ml of TCA and the tubes were centrifuged. To 0.5 ml of supernatant, 4 ml
of disodium hydrogen phosphate and 0.5 ml of DTNB were added and the
colour developed was read at 420 nm immediately. Standards were also treated
similarly.
Glutathione peroxidase activity is expressed as μg of glutathione
utilized/minute/mg protein at 37°C.
4.2.4.4 Estimation of glutathione-S-transferases
Glutathione-S-transferase of liver homogenate was assayed
according to the method (Habig et al., 1974).
47
Reagents
1. 0.3 mM Phosphate buffer pH 6.5
2. 30 mM 1-Chloro –2,4- dinitrobenzene (CDNB)
3. 30 mM Reduced Glutathione
Procedure
The reaction mixture (3 ml) contained 1.0 ml of 0.3 mM
phosphate buffer (pH 6.5), 0.1 ml of 30 mM 1-chloro-2, 4-dinitrobenzene
(CDNB) and 1.7 ml of distilled water. After pre-incubating the reaction
mixture at 37°C for 5 min, the reaction was started by the addition of 0.1 ml of
tissue homogenate and 0.1 ml of glutathione as substrate. The absorbance was
followed for 5 min at 340 nm. Reaction mixture without the enzyme was used
as blank.
The activity of GST is expressed as μ moles of GSH-CDNB
conjugate formed/min/mg protein.
4.2.5 Estimation of Non-Enzymic Antioxidants
4.2.5.1 Reduced glutathione
Reduced glutathione was determined by the method of Moron
et al., (1979).
Reagents
1. Phosphate solution: 5.33 g of disodium hydrogen phosphate was
dissolved in 100 ml of distilled water.
2. 10% TCA
48
3. DTNB 0.6 mM: 400 mg of DTNB was dissolved in 100 ml of 1%
trisodium citrate solution.
4. Standard: 10 mg of reduced glutathione was dissolved in 100 ml
of distilled water.
Procedure
One ml of tissue homogenate was precipitated with 1 ml of 10%
TCA. The precipitate was removed by centrifugation. To an aliquot of the
supernatant was added 4 ml of phosphate solution and 0.5 ml of DTNB
reagent. The colour developed was read at 420 nm.
The amount of glutathione in tissue is expressed as μg/mg
protein.
4.2.5.2 Vitamin C (Ascorbic acid)
Ascorbic acid was estimated by the method of Omaye et al.,
(1979).
Reagents
1. 5% TCA
2. 65% H2SO4
3. 2,4 dinitrophenyl hydrazine (DNPH), thiourea, copper sulphate
(CuSO4) reagent (DTC): 3 g DNPH, 0.4 g thiourea and 0.05 g
CuSO4 were dissolved in 9 N H2SO4 and made up to 100 ml.
4. Standard: Standards of ascorbic acid were made in 5% TCA in
the range of 0 to 20 µg/ml.
49
Procedure
Aliquots of homogenate were precipitated with 5% ice-cold TCA
and centrifuged for 20 min at 3500 xg. 1ml of the supernatant was mixed with
0.2 ml of DTC and incubated for 3 hr at 37o C. Then 1.5 ml of ice cold 65%
H2SO4 was added, mixed well and the solutions were allowed to stand at room
temperature for an additional 30 min. Absorbance was determined at 520 nm.
Ascorbic acid values were expressed at µg/mg protein.
4.2.5.3 Vitamin E
Vitamin E was estimated by the method of Desai (1984).
Reagents
1. Absolute ethanol: Analytical grade ethanol redistilled in glass
apparatus after adding pellets of KOH and crystals of KMNO4.
2. Hexane: Analytical grade purified by glass distillation.
3. Bathophenanthroline reagent: 0.2% solution of 4,7-diphenyl-1,10
phenanthroline in purified absolute ethanol.
4. Ferric chloride reagent: 0.001 M ferric chloride solution in
purified absolute ethanol. This reagent was prepared fresh and
was kept in amber coloured bottle.
5. Orthophosphoric acid reagent: 0.001 M orthophosphoric acid
solution in purified absolute ethanol.
6. Vitamin E standard: α-tocopherol standards in the range of 1-10
µg/ml of purified absolute ethanol were prepared and treated in
the same manner as test samples.
50
Procedure
a. Saponification and extraction
To 500 mg of the tissue, 5 ml of isotonic KCl was added and
homogenised. To 1.5 ml of homogenate, add 1 ml of ethanol, 0.5 ml of 25%
ascorbate and preincubate at 70oC for 5 min in glass-stoppered tubes. To this 1
ml of saturated KOH was added and mixed again. The mixture was further
incubated at 70oC for 30 min. The tubes were immediately cooled in an ice bath
and 1 ml of distilled water and 4 ml of purified hexane were added. The tubes
were shaken vigorously for 2 min and centrifuged at 1500 rpm for 10 min to
separate the phases.
b. Estimation
3 ml aliquots of hexane extract were pipetted out into suitable
reaction tubes and evaporated to dryness under nitrogen. The residue was then
carefully dissolved in 1 ml of purified ethanol. Tubes containing α-tocopherol
standards were treated in the same way as test samples. To all the tubes,
including a reagent blank, 0.2 ml of 0.2% bathophenanthroline reagent was
added and the contents of the tubes were thoroughly mixed. The assay
proceeded very rapidly from this point and care was taken to reduce
unnecessary exposure to direct sunlight. 0.2 ml of ferric chloride reagent was
added and the tubes were mixed by vortexing. After 1 min, 0.2 ml of
orthophosphoric acid was added and the tubes were thoroughly mixed again.
The absorbance was read at 536 nm. Vitamin E value is expressed as mg/g
tissue.
51
4.2.6 Estimation of lipid peroxidation
4.2.6.1 Tissue lipid peroxidation
Lipid peroxidation was estimated by the method of Ohkawa et
al., (1979).
Reagents
1. 0.8% TBA
2. 8.5% Sodium dodecyl sulphate
3. 20% Glacial acetic acid
4. Distilled water
Procedure
1.5 ml of TBA, 0.2 ml of sodium dodecyl sulphate, 1.5 ml of
glacial acetic acid were added to test tubes containing 0.1 ml of samples. The
test tubes were heated in water bath for 1hr. The test tubes were then cooled
and 1 ml of distilled water was added. The optical density was determined at
532 nm using a reagent blank. Standard malondialdehyde was also processed in
a similar fashion.
4.3 Results and Discussion
4.3.1 Hepatoprotective activity
4.3.1.1 Pentobarbital induced sleeping time
Figure 1 shows sleeping time of CCl4 treated animals (Group II)
was significantly (P<0.001) increased when compared with control animals
(Group I). The extracts RCA, RCM, CFA and CFM at the dose level of 500
52
mg/kg/po, significantly decreased (P<0.05, P<0.001) the sleeping time when
compared with CCl4 treated animals. Silymarin (25 mg/kg/po) treated animals
also showed significant decrease (P<0.001) in sleeping time when compared
with CCl4 treated animals.
Plaa et al.,(1958), demonstrated that prolongation of
pentobarbital sleeping time could be used to quantify the relative hepatotoxicity
of haloalkanes. Pentobarbital sleeping is directly dependent on the ability of the
liver to biotransform the barbiturate. Hepatocellular injury can lead to
decreased activity of hepatic drug metabolizing enzymes and therefore a
prolongation of pentobarbital hypnosis.
This method is used to screen anti - CCl4 toxicity of drugs in
animals (Burger, 1968). The extracts RCA, RCM, CFA, CFM were tested
against pentobarbital induced sleeping time. Hepatotoxic chemicals like CCl4
reduce the levels of drug metabolizing enzymes in liver. Therefore metabolism
of pentobarbitone is reduced resulting in prolongation of pentobarbitone
induced sleeping time. The extracts RCA, RCM, CFA, CFM reduced the CCl4
induced prolongation of sleeping time hence the extracts (RCA, RCM, CFA,
CFM) can be considered hepatoprotective against CCl4 toxicity.
4.3.1.2 CCl4 induced hepatotoxicity
4.3.1.2.1 Tissue and serum enzymes
Effects of RCA, RCM, CFA and CFM at dose levels (250, 500
mg/kg/po) on marker enzymes of serum, total bilirubin, total protein in CCl4
induced hepatotoxicity are shown in (Table 6 & 7). Liver damage induced by
CCl4 caused significant increase in marker enzymes AST, ALT, ALP in serum
53
and liver homogenate. Oral administration of the extracts RCA, RCM, CFA,
and CFM significantly decreased the level of marker enzymes AST, ALT, ALP
in both serum and liver. The total bilirubin level was significantly increased in
CCl4 treated animals. The extracts treated animals showed a lower total
bilirubin level in serum. The total protein level in serum and liver was
considerably reduced in CCl4 toxicity. The extracts RCA, RCM, CFA, and
CFM treated animals showed significant increase in the total protein level in
both serum and liver.
Since the changes associated with CCl4 induced liver damage is
similar to that of acute viral hepatitis (Suja et al., 2004). CCl4 induced liver
toxicity was chosen as the experimental model. The ability of the liver
protective drugs to reduce the injurious effects or to preserve the normal
hepatic physiological mechanisms, which have been disturbed by a
hepatotoxin, is the index of its protective effect (Yadav and Dixit, 2003). The
liver damage induced by CCl4 is due to its metabolite •CCl3, a free radical that
alkylates cellular proteins and other macromolecules with a simultaneous
attack on polyunsaturated fatty acids, in the presence of oxygen, to produce
lipid peroxides, leading to liver damage (Bishayee et al., 1995). Hepatocellular
necrosis leads to elevation of the marker enzymes which are released from the
liver into blood (Ashok Shenoy et al., 2002). The increased levels of AST,
ALT, ALP and serum bilirubin are conventional indicators of liver injury
(Achliya et al., 2004). The present study revealed a significant increase in the
marker enzymes like AST, ALT, ALP and serum bilirubin levels,on exposure
to CCl4, indicating considerable hepatocellular injury, administration of RCA,
RCM, CFA, CFM at two different dose levels attenuated the increased levels of
the marker enzymes produced by CCl4 and caused a subsequent recovery
54
towards normalization almost like that of standard silymarin treatment. The
decreased total protein level observed in the rats treated with CCl4 may be due
to the decrease in the number of hepatocytes which in turn may result in
decreased hepatic capacity to synthesis protein (Bhandarkhar and Khan, 2004).
On administration of extracts RCA, RCM, CFA, CFM showed significant
increase in total protein level, which indicates the increase in hepatocyte levels,
accounting for its hepatoprotective effect. The results show that the extracts
RCA, RCM, CFA, CFM at different dose levels offer hepatoprotection. But the
extracts (RCA, RCM, CFA, and CFM) at 500 mg/kg/po is more effective than
250 mg/kg/po treated groups.
4.3.1.2.2 Enzymic, non – enzymic antioxidants and tissue lipid
peroxidation
Decreased liver enzymic and non- enzymic antioxidant levels,
and enhanced activity of lipid peroxidation were seen in the CCl4 treated group
(Table 8), whereas the extracts (RCA, RCM, CFA, CFM) and standard drug
silymarin treated groups showed significant increase in antioxidant levels, with
reduction in lipid peroxidation level when compared with CCl4 treated animals.
It has been hypothesized that one of the principal causes of CCl4
induced liver injury is formation of lipid peroxides by free radical derivatives
of CCl4. Thus the antioxidant activity or the inhibition of the generation of free
radicals is important in the protection against CCl4 induced hepatotoxicity
(Castro et al., 1974). The body has an effective mechanism to prevent and
neutralize the free radicals induced damage. This is accomplished by a set of
endogenous antioxidant enzymes such as SOD, Catalase, GST and GPx. These
enzymes constitute a mutually supportive team of defense against ROS
55
(Venukumar and Latha, 2002). In CCl4 induced liver toxicity, the balance
between ROS production and antioxidant defenses may be lost and leads to
“oxidative stress”, which deregulates the cellular functions through a series of
events leading to hepatic necrosis. The reduced activities of SOD, Catalase,
GST and GPx observed, pointed out the hepatic damage in the animals treated
with CCl4, but the drug treated animals showed significant increase in the
levels of these enzymes, which indicates the antioxidant activity of the extracts
RCA, RCM, CFA, and CFM.
Regarding non-enzymic antioxidants, GSH is a critical
determinant of tissue susceptibility to oxidative damage and the depletion of
hepatic GSH has been shown to be associated with an enhanced toxicity to
chemicals, including CCl4 (Hewawasam et al., 2003). In the present study, a
decrease in hepatic tissue GSH level was observed in the CCl4 treated groups.
The increase in the hepatic GSH level in rats treated with RCA, RCM, CFA,
CFM may be due to the denovo GSH synthesis or GSH regeneration. There
was also depletion of other nonenzymic antioxidants like Vitamin C and
Vitamin E in the hepatic tissues of CCl4 treated groups, when compared with
the control group. Vitamin C is reported to be associated with better
scavenging activities invivo than the antioxidant enzymes, because they are
present both in intracellular as well as extracellular fluid (Chatterjee and Nandi,
1991). The antioxidant effect of Vitamin E has ability to quench both singlet
oxygen and peroxides (Karthikeyan and Rani, 2003). Within the membrane
tocopherol is the only protective agent that can act against the toxic effects of
oxygen free radicals (Suntress and Shek, 1995). The significant increase in
these nonenzymic antioxidant levels in the hepatic tissues of extracts (RCA,
56
RCM, CFA, CFM) treated groups indicates that the antioxidant effect of these
drugs exists both intracellularly and extracellularly.
LPO level is a measure of membrane damage and alterations in
structure and function of cellular membrane (Ilavarasan et al., 2003). In the
present study, elevation of lipid peroxidation level was observed in rats treated
with CCl4. Increase in MDA levels in liver suggests enhanced lipid
peroxidation leading to tissue damage and failure of antioxidant defense
mechanisms to prevent formation of excessive free radicals (Ashok Shenoy et
al., 2001). Administration of RCA, RCM, CFA, and CFM significantly
reversed these changes. Hence it is possible that the mechanisms of
hepatoprotection of RCA, RCM, CFA, and CFM may be due to its antioxidant
action. The results shows that RCA, RCM, CFA, CFM at the dose level of 500
mg/kg/po produced greater activity which is close to the values with standard
drug silymarin.
4.3.1.2.3 Histopathology of liver tissue (Plate 8)
Histopathology of the Group II (CCl4 treated) showed perivenular
inflammatory infiltration and hepatocytic fatty changes, diffused mild
hepatocellular vacuolation, where as extracts (RCA, RCM, CFA, CFM) treated
groups showed absence of cell necrosis, but minimal perivenular inflammation
and occasional hepatocytic fatty change. The extracts treated groups (RCA,
RCM, CFA, CFM) at 500 mg/kg/po dose level showed minimal inflammatory
conditions with near normal liver architecture possessing greater
hepatoprotective action.
From the above results, it can be concluded that the extracts
RCA, RCM, CFA and CFM at the dose level of 500 mg/kg/po, showed
significant protection against CCl4 induced liver injury in rats.