CHAPTER V
SUMMARY AND CONCLUSION
Diethyl phthalate is used ubiquitously as solvents and plasticisers worldwide.
The present study was undertaken to evaluate diethyl phthalate – induced toxic effects
on vital organs of mice (liver and kidney) with sub chronic exposure. Toxic effects of
diethyl phthalate on mice liver and kidney homogenates were also studied to get better
understanding of the mechanism involved in diethyl phthalate – induced toxicity.
Nigella sativa is an important therapeutic agent in the Indian traditional system of
medicine like Unani and Ayurveda. Nigella sativa seed extract was used in the study
to mitigate diethyl phthalate exerted toxicity in various in vitro and in vivo conditions.
PART I: To study the toxic effects of diethyl phthalate both in vitro
and in vivo conditions
In vitro study
Diethyl phthalate – induced lipid peroxidation (LPO) was measured in liver
and kidney homogenates by quantifying thiobarbituric acid reactive substances
(TBARS) in vitro. Results revealed that H2O2 – induced LPO was significantly
increased as the concentration of diethyl phthalate was increased in liver and kidney
homogenates. Maximum increase was found with 40 µg/mL concentration of diethyl
phthalate.
Similarly, effect of DEP treatment on protein content was evaluated in vitro
condition in liver and kidney homogenates. Results revealed that protein content
significantly decreased as the concentration of DEP was increased in liver and kidney
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homogenates. Maximum reduction in protein content was observed at 40 µg/mL
concentration of DEP. This might be due to increased LPO, which may be due to
DEP- induced generation of free radicals and oxidative stress.
In vivo study
In vivo study was designed to evaluate hepatic and renal damage caused by
administration of diethyl phthalate in mice. Summary of the diethyl phthalate –
induced toxicity is as follows:
Diethyl phthalate treatment for 30 days caused dullness and lethargy in treated
mice. However, no clinical signs were observed in untreated, as well as vehicle
control animals.
Oral administration of all three doses of diethyl phthalate resulted in
significant, dose-dependent reduction in body weight the animals as compared to
vehicle control. No significant changes in the body weight were noted between
different controls (Groups 1 and 2).
LIVER:
In groups 1 and 2 animals no significant differences in absolute and relative
liver weight were found. However, oral administration of diethyl phthalate for 30 days
caused significant, dose-dependent increase in absolute and relative liver weights
(Groups 3, 4 and 5) in mice as compared to vehicle control.
Groups 1 and 2 show no significant alteration in protein, lipid, cholesterol and
glycogen contents. When diethyl phthalate was administrated orally, there was
significant, dose-dependent decrease in protein content as well as increase in
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glycogen, lipid and cholesterol contents in liver of Groups 3, 4 and 5 animals when
compared with vehicle control.
Alteration in macromolecule contents of liver such as DNA and RNA was
found to be the prominent feature of diethyl phthalate toxaemia. DNA and RNA were
found to reduce significantly and dose-dependently with diethyl phthalate oral
administration. No significant change was observed between untreated and vehicle
control.
No significant changes were observed in the activities of succinic
dehydrogenase, adenosine triphosphatase and phosphorylase in liver of different
control groups (Group 1 and 2). However, oral administration of DEP caused dose-
dependent, significant reductions in the activities of succinic dehydrogenase,
adenosine triphosphatase and phosphorylase as compared to vehicle control.
Oxidative stress markers such as non – enzymatic (glutathione and ascorbic
acid contents) as well as enzymatic (glutathione peroxidase, glutathione reductase,
glutathione transferase, catalase and superoxide dismutase activities) antioxidants
along with lipid peroxidation were significantly altered in the liver of DEP -treated
mice. Level of lipid peroxidation was increased dose – dependently in DEP – treated
animals as compared to control which could be due to the reduction in non –
enzymatic and enzymatic antioxidants.
Oral administration of DEP for 30 days caused severe fat deposition, intra –
cellular vacuolation, necrosis and loss of histo - architecture as compared to controls
in liver of mice. No significant changes were observed in all groups of control
animals.
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KIDNEY:
There were no significant effects observed in absolute and relative kidney
weights in different control groups (Group 1 and 2). Treatment of DEP for 30 days
caused significant, dose-dependent increase in absolute and relative kidney weights of
mice (Group 3, 4 and 5) as compared to vehicle control.
No significant alterations in protein, lipid, cholesterol contents were observed
in different control groups (Group 1 and 2). DEP treatment caused significant
decrease in protein content whereas lipid and cholesterol contents were significantly
increased (Group 3, 4 and 5) as compared with vehicle control (Group 2).
In kidney, no significant changes in DNA and RNA contents were observed
between different control groups (Group 1 and 2). However, DEP treatment caused,
significant dose-dependent decrease in DNA and RNA contents (Group 3, 4 and 5), as
compared to vehicle control.
There were no significant changes observed in the activities of succinic
dehydrogenase and adenosine triphosphatase between different control groups (Group
1 and 2). However, oral administration of DEP caused dose-dependent, significant
reductions in the activities of succinic dehydrogenase and adenosine triphosphatase in
kidney of mice as compared to the controls.
Results revealed that oral administration of DEP for 30 days caused significant
increase in lipid peroxidation in kidney of mice. This could be due to significant dose-
dependent reduction in non-enzymatic (glutathione and ascorbic acid contents) and
enzymatic (glutathione peroxidase, glutathione reductase, glutathione transferase,
catalase and superoxide dismutase activities) antioxidants in kidney of groups 3, 4 and
5 animals as compared to vehicle control. However, there were no significant
alterations found in different control groups in above mentioned parameters.
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The transverse section of kidney of vehicle and untreated control mice showed
normal Bowman’s capsule with glomerulus as well as proximal and distal convoluted
tubules (Group 1 and 2). Oral administration of DEP for 30 days caused distortion of
the tubules, increased vacuolization, necrosis, atrophy of the glomerulus and increased
space between glomerulus and capsule wall (Groups 3, 4 and 5). The effect was more
prominent in high dose DEP -treated animals than that of low dose.
No significant difference was observed between different controls (Groups 1
and 2). The oral administrations of DEP for 30 days caused significant increase in
activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST),
alkaline phosphatase (ALP), acid phosphatase (ACP) as well as creatinine and glucose
contents in serum of mice. However, total protein, cholesterol contents were
significantly reduced in DEP-treated mice. The effect was more prominent in high
dose DEP- treated animals than that of low dose.
PART II: Phytochemical analysis of the Nigella sativa seed extract
Phytochemicals are the secondary metabolites present in plants in minute
concentrations and principally responsible for the medicinal value of the same.
Various polyphenol content of hydro-alcoholic extract of Nigella sativa seed was
quantitatively estimated in the present study. The extract showed presence of
phenolics, flavonoids, tannins and ascorbic acid in considerably high amount
indicationg its good antioxidative potency as phytochemicals are known reductant.
Various chemical antioxidant assay systems were used to evaluate free radical
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scavenging activity of Nigella sativa seed extract. Hydro – alcoholic extract of Nigella
sativa seed effectively scavenged superoxide, hydroxyl, nitrous oxide and DPPH
radicals in in vitro condition. Results indicate that hydro – alcoholic extract of Nigella
sativa seed can effectively reduce the oxidized compounds. Free radical scavenging
assay as well as reducing ability showed that hydro- alcoholic extract was potent due
to higher amount of phytochemicals present in Nigella sativa seed extract.
Nigella sativa seed extract was used to study their possible mitigatory effects
against diethyl phthalate toxicity in in vitro conditions. Results revealed that
concurrent addition of Nigella sativa seed extract (0-200 µg/mL) along with DEP (50
µg/mL) caused concentration-dependent significant decrease in lipid peroxidation as
well as increase in protein content in liver and kidney homogenates. Therefore,
Nigella sativa seed extract was selected to study its possible amelioration against
DEP- induced toxicity in in vivo conditions.
PART III: To study the ameliorative effect of Nigella sativa seed
extract on toxicity induced by diethyl phthalate
Efficiency of Nigella sativa seed extract in reducing diethyl phthalate exerted
toxicity in mice was evaluated using two different doses. Cotreatment of Nigella
sativa with diethyl phthalate in mice resulted in significant mitigation of diethyl
phthalate- induced toxicity in liver and kidney which are as follows:
Cotreatment of Nigella sativa seed extract and DEP significantly mitigated
DEP- induced clinical symptoms such as lethargy and dullness. No treatment related
mortality was observed in any of the experimental groups.
No significant difference in body weight was observed between different
control groups (Group 1 and 2). Oral administration of DEP (HD) caused significant
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reduction in body weight. However, oral administration of 150 and 300 mg/kg body
weight/ day of Nigella sativa seed extract along with HD of DEP caused significant
mitigation in retardation in body weight, as compared to HD of DEP alone treated
animals (Group 3) in a dose – dependent manner.
LIVER:
No significant difference in the absolute and relative liver weights was found
between different control group animals (Group 1 and 2). As compared to the vehicle
control (Group 2), oral administration of DEP (HD) for 30 days caused significant
increase in absolute and relative liver weights of mice. Nigella sativa seed extract
treatment along with HD of DEP significantly reduced absolute and relative weights
of liver which was significantly increased with HD of DEP – treatment (Group 3).
Animals of group 1 and 2 show no significant alterations in protein, lipid,
cholesterol and glycogen contents. Conversely, as compared to vehicle control (Group
2) oral administration of DEP in animals of group 3 caused significant decrease in
protein content, whereas significant rise was observed on glycogen, lipid and
cholesterol contents. Concurrent administration of Nigella sativa seed extract and HD
of DEP leads to significant amelioration in all parameters in liver of Group 4 and 5
animals, when it was compared with groups treated with HD of DEP alone (Group 3).
There were no significant changes in DNA and RNA contents in liver of all
control group animals (Group 1 and 2). Significant elevation in DNA and RNA
contents were noted in liver of mice treated with DEP (HD) along with Nigella sativa
seed extract while were reduced in case of diethyl phthalate intoxication.
No significant changes were observed in the activities of succinic
dehydrogenase and adenosine triphosphatase in liver of different control group
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(Group 1 and 2) animals. Cotreatment of Nigella sativa seed extract along with HD of
DEP for 30 days caused significant amelioration in all parameters as compared to HD
of DEP alone treated groups.
Nigella sativa seed extract is known to possess strong antioxidative potency
and was used to combat DEP - induced oxidative stress in this study. In mice liver, no
significant changes were found in non-enzymatic (glutathione and ascorbic acid
contents) as well as in enzymatic (glutathione peroxidase, glutathione reductase,
glutathione transferase, catalase and superoxide dismutase activities) antioxidants in
between different control groups (Group 1 and 2). There was no significant change in
lipid peroxidation in all control groups. Treatment with Nigella sativa seed extract
along with HD of DEP shows significant amelioration in all above parameters
(Groups 4 and 5) as compared to HD of DEP alone treated group (Group 3).
The transverse section of liver of vehicle control and antidote control mice
showed normal hepatocytes and sinusoids. Oral administration of DEP (HD) for 30
days caused severe fat deposition, intra – cellular vacuolation, necrosis and loss of
histo - architecture as compared to controls in liver of mice (Group 3). Treatment with
Nigella sativa seed extract for 30 days along with HD of DEP caused amelioration in
DEP - induced changes as compared to only HD of DEP treated group (Group 3).
Amelioration was more in high dose Nigella sativa seed extract treated animals than
that of low dose.
KIDNEY:
There were no significant effects found in absolute and relative kidney weights in
different control groups (Group 1 and 2). Results revealed that DEP (HD) treatment
for 30 days caused significant, increase in absolute and relative kidney weights as
compared to vehicle control (Group 2). Oral administration of Nigella sativa seed
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extract along with HD of DEP significantly ameliorates DEP – induced changes in
absolute and relative kidney weights (Groups 4 and 5)
No significant alterations in protein, total lipid and cholesterol contents were
observed between different control groups (Group 1 and 2). Oral administration of
DEP (HD) in animals of Group 3 caused significant increase in lipid and cholesterol
contents whereas protein content reduced significantly. However, concurrent
administration of Nigella sativa seed extract and DEP leads to significant
amelioration in all parameters in Group 4 and 5, when it was compared with DEP
alone treated group (Group 3).
In kidney, no significant changes were observed in DNA and RNA contents in
all control groups (Group 1 and 2). As compared to animals of vehicle control, DEP
treatment caused (Group 3) significant reduction in DNA and RNA contents.
Combined treatment of Nigella sativa seed extract plus DEP caused significant
amelioration in all parameters as compared to DEP alone treated groups.
No significant changes were observed in the activities of succinic
dehydrogenase and adenosine triphosphatase in kidney of different control groups
(Group 1 and 2). Oral administration of DEP (HD) caused significant reduction, in
these enzyme activities as compared to control. However, treatment with Nigella
sativa seed extract along with DEP caused significant amelioration in all parameters
as compared to DEP alone treated group.
Oral administration of DEP (HD) for 30 days caused significant increase in
lipid peroxidation in kidney of mice. This could be due to significant dose- dependent
reduction in non-enzymatic (glutathione and ascorbic acid contents) and enzymatic
(glutathione peroxidase, glutathione reductase, glutathione transferase, catalase and
superoxide dismutase activities) antioxidants in group 3 as compared to vehicle
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control. Simultaneous treatment of Nigella sativa seed extract and DEP (Group 4 and
5) caused significant amelioration, as compared to DEP alone treated groups, in all
parameters. No significant alterations were found in different control groups in above
mentioned parameters.
Oral administration of DEP for 30 days caused distortion of the tubules,
increased vacuolization, necrosis, atrophy of the glomerulus and increased space
between glomerulus and capsule wall (Groups 3). Cotreatment with Nigella sativa
seed extract (Group 4 and 5) along with DEP (HD) for 30 days showed almost
complete recovery in histopathological features; it was almost comparable to vehicle
control. No significant changes was observed between vehicle control and antidote
control groups (Group1 and 2).
No significant difference in activities of alanine aminotransferase (ALT),
aspartate aminotransferase (AST), alkaline phosphatase (ALP), acid phosphatase
(ACP) as well as total protein, creatinine, glucose and cholesterol contents in serum
were observed between different control groups ( Groups 1 and 2). Oral
administration of DEP significantly increased activities of ALT, AST, ACP and ALP
as well as creatinine and glucose contents as compared with vehicle control. However,
total protein and cholesterol contents were significantly decreased in DEP- treated
animals as compared to vehicle control. These changes were effectively mitigated by
the administration of Nigella sativa seed extract along with DEP in a dose - dependent
manner.
In conclusion, DEP leads to the formation of free radicals and thus it creates
oxidative stress which in turn damages the vital organs. DEP also disturbs the energy
metabolism by altering the activities of enzymes of mitochondrial function. These
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toxic effects of DEP had been mitigated by Nigella sativa seed extract because of its
antioxidative properties.
FUTURE PERSPECTIVES:
Present study was an attempt to establish a correlation between xenobiotic-
induced toxicities and its amelioration by medicinal plant. As this thesis provides
various biochemical and histopathological evidence for diethyl phthalate – induced
toxicity (in vitro and in vivo) and protective effect of the plant extract against it
following are the some of the perspectives for which the study can be extended:
1. Detailed and systematic investigation of mechanism of diethyl phthalate-
toxicity in various in vitro and in vivo models by advanced analytical
techniques and provide more scientific research base for the same.
2. Evaluation of diethyl phthalate exposure and toxicity on human beings
supported by surveys and clinical trials.
3. Comparative potency evaluation of Nigella sativa seed extract on various
chemical and animal models.
4. Isolation, purification and characterization of various active components
possessing physiological action from Nigella sativa seed extract which can be
tested further for the management of numerous diseases and disorders.
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