INTRODUCTION:
The liver is the
major organ responsible for metabolism, detoxification, and secretory functions
in the body. Hence, it regulates various important metabolic functions in
mammalian systems. Hepatic damage is associated with the distortion of these
metabolic functions. The liver tissue is reported to be one of the tissues with
a high regenerative capacity.
Hepatocytes
exhibit a very good regenerative response to several stimuli, including massive
destruction of hepatic tissue by toxins, viral agents, or surgical extraction.
Regeneration of the liver tissues is a result of an organized and controlled
response of the liver toward tissue damage induced by toxic agents, trauma,
infections, or postsurgery resection. Different chemical agents, including
gasoline vapor constituents, are known to be hepatotoxic (1).
Chenopodium album L. (chenopodiaceae) is native of Western Asia. It is a summer
annual weed and attains a height upto 1 meter. The entire plant is covered with
varying amounts of a waxy substance giving the plant a light green appearance
it is commonly used for food and medicinal values and it grows in waste places
and as weed in wheat or other crops in almost all part is used in Sag and
Bathua Roti and Bathua Parantha [2].
MATERIALS AND
METHODS:
Extraction of Chenopodium album Linn:
The leaves plant
of Chenopodium album Linn.
was dried under shade and then powdered with a mechanical grinder to obtain a
coarse powder (500 gm) the fine powder of whole plant was packed in high
quality filter paper, which was then subjected to successive extraction in a
soxhlet apparatus using 50% ethanol for about 72 hour, solvent was recovered.
Extractive yield of Chenopodium
album Linn. was 17 %. After vacuum evaporation the crude extract
was dissolved in distilled water freshly as required.
Animals:
Albino rats (Wistar strain) weighing 125 - 150 gm of either sex were
used for the present study. The animals were housed in polypropylene cages at
control temperature (26 ± 2° C) relative humidity (60 ± 5%) and light. Rats
were fed with standard laboratory diet and drinking water was given through
drinking bottle throughout the experiment. The animals were maintained as per
CPCSEA regulation and cleared by IAEC at Bhupal Nobles College of Pharmacy,
Udaipur (Rajasthan), India.
Fig. 1 and 2: Effect of Chenopodium album Linn. on SGPT and SGOT
Values
are mean ± S.D (n = 6); P values: *** 0.0001 when compared with
control untreated rats; +++ 0.0001; ++ 0.001 when compared with
carbon tetrachloride treated rats.
Drug Formulation:
The extract of
plant fully dissolves in distilled water. The solution of the whole plant
extract (300 mg/ml) was freshly prepared in distilled water.
Experimental Induction of Hepatotoxicity:
All the animals,
except normal control group, will be received 40% ethanol (3.76 g/kg, p.o)
twice daily for a period of 25 days (3).
Fig. 3 and 4: Effect of Chenopodium album Linn. on ALP and Total
bilirubin
Values
are mean ± S.D (n = 6); P values: *** 0.0001 when compared with
control untreated rats; +++ 0.0001 when compared with carbon
tetrachloride treated rats.
Experimental Design:
Albino rats of
either sex weighing between 200 -250g were selected and divided into six groups
of six animals in each. The animals were fasted 24 hours prior to experiment.
Group I was maintained as normal control, which was given with distilled water
only. Group II received ethanol (3.76 gm/kg, twice daily, p.o) and animals in
Group III were treated with Silymarin (200 mg/kg, p.o) which served as
standard. Animals in Groups IV, V and VI were treated with three different
doses extract of Chenopodium album
Linn. (200mg/kg, 400mg/kg and 400mg/kg p.o.,) respectively. Group II,
III, IV, V were intoxicated with ethanol for 25 days. At the end of experimental period, all the
animals were sacrificed by cervical decapitation. Blood samples were collected,
allowed to clot. Serum was separated by centrifuging at 2500 rpm for 15 min and
analyzed for various biochemical parameters.
Assessment of liver function Biochemical parameters i.e.,
aspartate amino transferase (AST or SGOT) [4], alanine amino transferase (ALT
or SGPT) [4], alkaline phosphatase (ALP) [5], total bilirubin [6], total
cholesterol [7] and total protein [8], were analyzed according to the reported
methods. The liver was removed, morphological changes were observed. A 10% of
liver homogenate was used for antioxidant studies such as lipid peroxidation
(LPO) [9], superoxide dismutase (SOD) [10], Catalase [11] and glutathione [12].
A portion of liver was fixed in 10% formalin for histopathological studies.
Fig.
5 and6: Effect of Chenopodium album
Linn. on SOD and GSH
Values
are mean ± S.D (n = 6); P values: *** 0.0001 when compared with
control untreated rats; +++ 0.0001 when compared with carbon
tetrachloride treated rats.
Histopathology:
The tissues of liver were removed from animals, washed with normal
saline to remove blood, fixed in 10% formalin and embedded in paraffin wax.
Sections of 5 μm thickness were made using rotary microtome and stained
with haematoxylin-eosin and histological observations were made under light
microscope[13,14].
Statistical Analyses:
The experimental results were expressed as the Mean ± S.D for six
animals in each group. Statistical analyses were performed using the unpaired t
test. A p value of 0.05 or less was considered to indicate a significant
difference between groups [15].
RESULTS:
Effect of Chenopodium
Album Linn. on Serum Biochemical Markers:
As showed in
figures, the levels of SGPT, SGOT ALP, total bilirubin, total protein and total
cholesterol in serum are all common biomarkers of liver damage, compared to the
normal group, were significantly elevated by ethanol administration to rats (p
< 0.0001, 0.0001, 0.0001, 0.0001,0.0001, 0.0001 respectively),
indicated liver cell damage and the alcoholic liver injury model had been
established successfully. However, Chenopodium album Linn. treatment for 25 days significantly
decreased the levels of the serum biochemical indicators by alcohol-induced
hepatic damages. Notably, Chenopodium album Linn. administration of at different doses (200 to
600 mg/kg) recovered the impaired liver functions to varying degrees resulting
from alcohol-induced toxicity (p < 0.01).
Interestingly, treatment groups had a significant dose-dependent behavior
antagonizing acute alcoholic liver injury.
Fig.
7 and 8: Effect of Chenopodium album Linn. on CATALASE and LPO
Values
are mean ± S.D (n = 6); P values: *** 0.0001 when compared with
control untreated rats; +++ 0.0001; ++ 0.001 when compared with
carbon tetrachloride treated rats.
Lipid
peroxidation: Malondialdehyde (MDA) is the product of lipid peroxidation and is
a common marker of lipid peroxidation. The content of MDA was significantly
(p<0.0001) increased in the liver of alcohol treated rats (5.71 ± 0.698 nm
MDA/mg tissue) as compared with the normal group (1.52± 0.094 nm MDA/mg
tissue). Treatment with Chenopodium album Linn. at the doses 200, 400 and
600 mg/kg significantly (p<0.001) suppressed the formation of TBARS in the
liver, suggestive of less oxidative damage of liver. SOD activity: The effect
of Chenopodium album Linn. on hepatic SOD activity is shown in graph . SOD
activity of the ethanol treated control group (2.90 ± 0.261 U/mg tissue) was
found to be significantly lower than the normal group (11.65 ± 0.602 U/mg
tissue). However, a significant dose dependent reversal of the SOD level was
observed Chenopodium album Linn. treated groups. Glutathione
content: Figure 6 shows the effect of Chenopodium
album Linn. on the content of GSH in
ethanol induced hepatotoxicity in rats. The administration of ethanol alone
significantly decreases the total glutathione content of the liver homogenate.
Ethanol administration with the tested compounds significantly (p<0.0001)
inhibited the depletion of GSH compared to the control group. Catalase
activity: Catalase activity in the liver homogenate is shown in figure 8. CAT
activity of the Ethanol treated control group (3.79 ±0.323 U/mg tissue) was
significantly (p<0.0001) lower than the normal group (14.02± 0.685 U/mg
tissue). The CAT activities in Chenopodium
album Linn. and silymarin treated
rats were significantly (p<0.0001) higher compared to Ethanol treated group.
Fig.
9 and 10: Effect of Chenopodium
album Linn. on total protein and total cholestrol
Values
are mean ± S.D (n = 6); P values: *** 0.0001 when compared with
control untreated rats; +++ 0.0001; ++ 0.001; +<0.01 when
compared with carbon tetrachloride treated rats.
Fig.
1: Histopathalogical monograph of extract and standard. a: Control; b: Ethanol
(3.76 g/kg) alone; c: Ethanol+ Chenopodium album ( 3.76 g/kg +200 mg/kg); d: Ethanol + Chenopodium album ( 3.76 g/kg+400 mg/kg);
e: Ethanol+ Chenopodium album
(3.76 g/kg+600 mg/kg); f Ethanol+Silymarin (
3.76 g/kg+ 200 mg/kg).
DISCUSSION:
Ethanol is a
natural product that has been available for human consumption for thousands of
years. It has well characterized psychophysical and mood-altering effects. It
is also a common cause for the generation of reactive oxygen species (ROS),
which can damage cellular lipids, proteins, and DNA leading to oxidative stress
and induce liver injury. Although the pathophysiological mechanism of chemical
induced hepatotoxicity is not yet fully figured out, it is mostly associated with
the metabolic conversion of xenobiotics into ROS, which induce oxidative stress
and damage the cellular macromolecules. Disturbances of the delicate balance of
endogenous antioxidant defense system of the organism causes oxidative stress,
which is associated with various liver disorders, therefore leading to
alcoholic liver damage, non-alcoholic fatty liver disease, and drug-induced
liver injury . Therefore, more attention has been paid to the research and
development of effective therapy for alcoholic liver disease (ALD) and agents
for protecting alcohol-induced liver injury. It has been recognized that
generation of free radicals and oxidative stress play a critical role in the
development of ALD. Recently, accumulating evidence has revealed that dietary antioxidant
supplementation may contribute to keeping this balance, finally inhibiting the
hepatotoxicity.
Administration
of ethanol significantly (P<0.0001) increased the serum intracellular
enzymes such asalanine aminotransferase (AST) and aspartate aminotransferase
(ALT) compared to the control group while administration of Chenopodium album Linn. decrease significantly (P<0.001) these enzyme
levels. The reversal of elevated serum intracellular enzyme levels by Chenopodium album Linn. extract after ethanol administration may be attributed
to the stabilizing ability of the cell membrane preventing enzymes leakages. In
addition, it was reported that the reversal of increased levels of
transaminases to nearly normal predicts the restoration of hepatocytes and
regeneration of hepatic parenchyma [16]. In
addition, the antioxidant enzymes and lipid peroxidation levels can be used to
predict the severity of ethanol induced liver damage. Antioxidants enzymes such
as SOD, CAT, GSH dependently act in the metabolic pathways that involve free
radicals. Therefore, SOD, CAT, and GSH levels decrease in liver suggest the
toxic effects of ethanol on liver functions but the administration of Chenopodium album Linn. can counter the efficacy of ethanol on liver cells
thereby blocking the decrease antioxidants levels. Since it was proved that the
significance of GSH in the detoxification of chemically reactive metabolite in
drug induced toxicity after decrease in GSH [16] then we can deduce that
increased oxidation and decrease synthesis of GSH causes decrease in GSH
levels. Therefore increase in antioxidant enzyme activities levels (SOD, CAT,)
after extracts administration might contribute to the ameliorating effects of
oxidative stress. MDA is a known biomarker of lipid peroxidation and oxidative
stress, the increase in MDA level signifies the toxic effects of ethanol on
liver [17] but the counteractions of Chenopodium album Linn. reducing MDA level suggest the potential attributes of
in the restoration of damaged liver tissues after ethanol administration.
Therefore, the antioxidant potential of Chenopodium album Linn. improves the
liver functions by promoting antioxidant enzyme activities, thus can be
recommended as a therapeutic agent for heavy alcohol drinkers in alcohol related
liver damage. Therefore we can deduce from our findings that Chenopodium album Linn. tentatively
mitigates the effects of alcohol on the liver of rats.
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