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CHAPTER-5

In Vivo Hepatoprotective Activity

of Nigella sativa L. Seed in

Various Germination Phases

“One thing I have learned in a long life: that all our science,

measured against reality, is primitive and childlike -- and yet

it is the most precious thing we have.”

~Albert Einstein

http://www.goodreads.com/author/show/9810.Albert_Einstein

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Integral University, Lucknow 166

5.1 Rationale

Largest and complex organ in the body is liver which deals multiple and

diverse functions to safeguard internal environment of the body. Liver is involved in

synthesizing number of various plasma proteins such as albumin, fibrinogen and the

intermediate metabolism of protein, fat and carbohydrates. It is also involved in

synthesis of clotting factors and also produces a number of enzyme, formation and

excretion of bile. It acts as a storage depot for proteins, glycogen, various vitamins

and metals. It also has a role in the regulation of blood volume by transferring the

blood from portal to systemic circulation and its reticule endothelial system and

participates in immune mechanism. It plays a central role in detoxification and

excretion of many endogenous and exogenous compounds. Hence, any injury to it or

impairment of its function has grave implication for the health of the affected person.

Liver diseases are affecting humans of all ages and one of the major causes of

morbidity and mortality in public. About 20,000 deaths occur every year due to liver

disorder. Hepatocellular carcinoma is one of the ten most common tumors in the

world with over 2,50,000 new cases each year (Gupta and Misra, 2006). According

to WHO estimates, globally 170 million people are chronically infected with

hepatitis C alone and every year 3–4 millions are newly added into the list. Also,

there are more than 2 billion people infected by hepatitis B virus (Negi et al., 2008).

Every year about 18,000 people are reported to die due to liver cirrhosis caused by

hepatitis (Handa, 1991).

Usually liver fibrosis is initiated by hepatocyte damage and various biologic

factors such as hepatitis virus, bile duct obstruction, overload of cholesterol,

schistosomiasis or chemical factors such as CCl4 administration, alcohol intake etc.

were known to contribute to liver fibrosis. Hepatic fibrosis is a major feature of wide

range of chronic liver injuries including metabolic, viral, cholestatic and genetic

diseases. The failure of bile salt excretion in cholestatic leads to retention of

hydrophobic bile salts within the hepatocytes and causes apoptosis and/or necrosis

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(Miyoshi et al., 1999). Oxidative stress has been implicated in the pathogenesis of

various liver diseases including alcoholic liver disease, nonalcoholic fatty liver

disease, and chronic hepatitis C (Seki et al., 2005; Kitase et al., 2005). The liver

diseases are classified as acute or chronic depending on the duration of the disease.

Acute viral hepatitis and drug reactions account for the majority of cases of acute

liver diseases. Chronic liver damage is a worldwide common pathology

characterized by inflammation and fibrosis that can lead to chronic hepatitis,

cirrhosis and cancer (Tessitore and Bollito, 2006; Kohle et al., 2008). Chronic

hepatitis or long term intoxification can severely injure hepatic cells.

Drug/chemical-mediated hepatic injury is the common sign of drug toxicity

(Lee, 2003) and accounts for greater than 50% of acute liver failure cases. Hepatic

damage is the largest obstacle to the development of drugs and is the major reason

for withdrawal of drugs from the market (Cullen and Miller, 2006). Drug-induced

liver disease can be predictable (high incidence and dose-related) or unpredictable

(low incidence and may or may not be dose-related). Conventional drugs used in the

treatment of liver diseases are sometimes inadequate and can have serious adverse

effects. Herbal medicines are in great demand in the developed as well as developing

countries for primary healthcare because of their wide biological and medicinal

activities, higher safety margins and lesser costs (Chattopadhyay et al., 2007).

Presently a few hepatoprotective drugs and that too from natural sources are

available for the treatment of liver disorders. Hence, people are looking at the

traditional systems of medicine for remedies to hepatic disorders.

The present study is aimed to evaluate the hepatoprotective and antioxidant

activity of methanolic extract of the Nigella sativa L. from different germination

stages of seed against CCl4-induced hepatotoxicity in rats as hepatotoxin to prove its

claim in folklore practice against liver disorders with no side effects.

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5.2 REVIEW OF LITRARURE

5.2.1 Hepatotoxicity (causes and pattern of liver damage)

As the major drug metabolizing and detoxifying organ in the body, the liver

is subjected to potential damage from an enormous array of

pharmaceutical and environmental chemicals. More than 900 drugs have been

implicated in causing liver injury (Keeffe et al., 2004) and it is the most common

reason for a drug to be withdrawn from the market. Chemicals often cause

subclinical injury to liver which manifests only as abnormal liver enzyme tests.

Drug-induced liver injury is responsible for 5% of all hospital admissions and 50%

of all acute liver failures (Ostapowicz et al., 2002). Liver is susceptible to injury

from drugs and other substances. 75% of blood coming to the liver arrives directly

from gastrointestinal organs and then spleen via portal veins which bring drugs and

xenobiotics in near-undiluted form. There are many mechanisms behind liver

damage, many chemicals damage mitochondria and its dysfunction releases

excessive amount of oxidants which in turn injured hepatic cells. Activation of some

enzymes in the cytochrome P-450 system such as CYP2E1 also leads to oxidative

stress (Jaeschke H et al., 2002). Injury to hepatocyte and bile duct cells lead to

accumulation of bile acid inside liver that promotes further liver damage (Patel T et

al., 1998).

Drugs/chemicals produce a wide variety of clinical and pathological hepatic

injury. Biochemical markers (e.g. alanine transferase, alkaline phosphatase and

bilirubin) are often used to indicate liver damage. Liver injury is defined as a rise in

either ALT level more than three times of upper limit of normal, ALP level more

than twice upper limit of normal or total bilirubin level more than twice upper limit

of normal when associated with increased ALT or ALP (Mumoli N et al., 2006).

Three important characteristics of the P450 system have role in drug induced

toxicity (Figure 5.1).

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a. Direct toxicity

b. Hepatic conversion of xenobiotics to an active toxin

c. The immune mechanism usually by a drug or a metabolite acting as hepatic to

convert cellular protein into an immunogen.

Figure 5.1: Drug and toxin induced hepatic injury.

Exposure to various organic compounds including a number of

environmental pollutants and drugs can cause cellular damages through metabolic

activation of those compounds to highly reactive substances such as reactive oxygen

species (ROS). Drugs or toxins that have a pharmacological hepatotoxicity are those

that have predictable dose-response curves (higher concentrations cause more liver

damage) and well characterized mechanisms of toxicity such as directly damaging

liver tissue or blocking a metabolic process. Idiosyncratic injury occurs without

warning when agents cause non-predictable hepatotoxicity in susceptible individuals

which is not related to dose and has a variable latency period (Zimmerman, 1978).

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Idiosyncratic hepatotoxicity has led to the withdrawal of several drugs from market

even after rigorous clinical testing as part of the FDA approval process.

5.2.2 Carbon tetrachloride: A most commonly used hepatotoxin

Chemical toxicity comprises an important source of reactive oxygen species

(ROS), which may occur through processes such as inhibition of

mitochondrial electron transport chain and subsequent accumulation of

intermediates inactivation of antioxidant enzymes and deletion of radical

scavengers (Mates, 2000). Carbon tetrachloride (CCl4) is a well known hepatotoxin

and exposure to this chemical is known to induce oxidative stress and causes liver

injury by the formation of free radicals (Sil et al., 2007). Carbon tetrachloride is

one of the most commonly used hepatotoxin in the experimental study of liver

disease (Johnston et al., 1998).

Carbon tetrachloride is used in the synthesis of chlorinated organic

compounds, including chlorofluorocarbon refrigerants. It is also used as an

agricultural fumigant and as a solvent in the production of semiconductors, in the

processing of fats, oils and rubber and in laboratory applications (Lewis, 1993;

Kauppinen et al., 1998). CCl4 continues to provide an important service today as a

model substance to elucidate the mechanisms of action of hepatotoxic effects such as

fatty degeneration, fibrosis, hepatocellular death, and carcinogenicity. CCl4 has been

extensively used to study liver injury induced by ROS in the mouse model, which is

closely analogous to hepatotoxicity in humans.

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Mechanism of CCl4 Toxicity:

CCl4

sER

CCl3•

Lipid radicals Lipid peroxidation

(Autocatalytic spread along

microsomal membrane)

Membrane damage Release of products of

to rER lipid peroxidation Polysome Damage to plasma

Detachment membrane

Permeability to Na+,

H2O, Ca2+

Apo protein synthesis Cell swelling

Fatty liver Massive influx of Ca2+

Inactivation of

Mitochondria, cell enzymes,

Denaturation of proteins

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The hepatotoxic effect of CCl4 are largely due to its active metabolites,

CCl3 (trichloro methyl radicals) CCl3•-

(tri chloro methyl per oxy radicals) is

most reactive species and causes damage to biological macromolecules by

combining with them there by causing covalent modification and setting the

chain reactions of lipid peroxidation and ultimately cell death (Benedetti et al.,

1987). Among various mechanism involved in hepatotoxic effect of carbon

tetrachloride, one is oxidative damage through free radicals generation (Deleve et

al., 1995). The hepatotoxicity of CCl4 is attributed to the formation of

trichloromethyl and trichloromethyl peroxyl radicals, initiating lipid peroxidation

and resulting in fibrosis and cell necrosis (Kadiiska et al., 2000).

At the molecular level CCl4 activates tumor necrosis factor TNF-alpha, nitric

oxide (NO), transforming growth factors, TGF-alpha and TGF-beta in the cell

processes that appear to direct the cell primarily toward self destruction or fibrosis.

TNF-alpha pushes toward apoptosis whereas the TGFs appear to direct toward

fibrosis. CCl4 intoxication also leads to hypomethylation of cellular components; in

the case of RNA the outcome is thought to be inhibition of protein synthesis, in the

case of phospholipids it plays a role in the inhibition of lipoprotein secretion (Weber

et al., 2003). The over production of ROS and therefore oxidative stress can be

initiated by a variety of factors including exposure to xenobiotics, such as

acetaminophen and carbon tetrachloride (Ronsein et al., 2005). Of concern here is

the adverse effect of carbon tetrachloride to the liver, having visualized the

prominent functions of the liver for survival. According to Liu et al., (1995),

ingestion of carbon tetrachloride can lead to marked hepatotoxicity.

5.2.3 Free radicals and oxidative damage in liver

A free radical can be defined as any species which is capable of

independent existence and contains one or more unpaired electrons (Halliwell and

Gutteridge, 1989). In excess, free radicals can damage cell membranes and cause

cell necrosis; they can cause damage to all macromolecules, lipids, proteins

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mitochondrial and nuclear DNA molecules of the cells causing inflammation or

lesion on various organs (Beckman et al., 1990; Halliwell and Gutteridge, 1984). It

is increasingly being realized that majority of disorders/diseases is mainly due to

the imbalance between pro-oxidant and antioxidant homeostatic phenomenon in the

body. Pro-oxidants conditions either dominate due to increase in the generation of

free radicals and/or their inadequate quenching or scavenging in the body (Tiwari,

2001). The reactive oxygen species play an important role related to the degenerative

or pathological processes of various serious diseases such as aging (Burns et al.,

2001), cancer, coronary heart disease, Alzheimer’s disease (Smith et al., 1996; Diaz

et al., 1997), neurodegenerative disorders, atherosclerosis, cataracts and

inflammation (Aruoma, 1998).

ROS such as superoxide radical, hydroxyl radical, peroxyl radical and nitric

oxide radical attack biological molecules such as lipids, proteins, enzymes, DNA

and RNA. This may be lead to cell or tissue injury associated with aging,

atherosclerosis, carcinogenesis (Keli Chen et al., 2005) and development of

chronic diseases related to the cardio and cerebrovascular systems. More

than one hundred disorders in humans including atherosclerosis, arthritis, ischemia,

reperfusion injury of many tissues, central nervous system injury, gastritis, cancer

and AIDS associate with reactive oxygen species (Cook and Samman, 1996).

ROS play an important role in fibrogenesis throughout increasing platelet-

derived growth factor. Most hepatocellular carcinomas occur in cirrhotic livers and

the common mechanism for hepatocarcinogenesis is chronic inflammation associated

with severe oxidative stress; other risk factors are dietary aflatoxin B1 consumption,

cigarette smoking and heavy drinking. Ischemia–reperfusion injury affects directly

on hepatocyte viability particularly during transplantation and hepatic surgery.

Ischemia activates Kupffer cells which are the main source of ROS during the

reperfusion period (Pablo Muriel, 2009). Hepatocellular carcinoma is one of the most

malignant and frequent worldwide spreading diseases. It is the third most common

cause of cancer deaths (Pisani et al., 1999; Parkin et al., 2005). Most Hepatocellular

http://www.ncbi.nlm.nih.gov/pubmed/?term=Muriel%20P%5Bauth%5D

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carcinoma occurs in cirrhotic livers and the common mechanism for

hepatocarcinogenesis is chronic inflammation associated with severe oxidative stress

(Seitz et al., 2006). There is a large body of evidence indicating that ROS play a

pathogenesis role in carcinogenesis in liver (Marx et al., 2004).

5.2.4 Antioxidant

Almost all organisms are well protected against free radical damage by anti-

oxidative enzymes such as superoxide dismutase (SOD) and catalase (CAT) or

chemical compounds such as α-tocopherol, ascorbic acid, carotenoids, polyphenol

compounds and glutathione. When the mechanism of antioxidant protection

becomes unbalanced by factors such as ageing, deterioration of physiological

functions may occur resulting in diseases and accelerated ageing. However,

antioxidant supplements or antioxidant containing foods may be used to help

the human body to reduce oxidative damage (Mau et al., 2001; Gulcin et al.,

2002).

All living organisms have endogenous defense systems against oxidative

damage such as lipid peroxidation DNA damage (Lee et al., 2005) and inhibition

of cell communication due to reactive oxygen species (ROS). Antioxidants protect

against chemotherapy toxicity and local toxic effects of tumors or surrounding

tissues. The protection of cells against damage from oxygen and its metabolites can

be accomplished through enzymatic and non-enzymatic means. Superoxide

dismutase, catalase and glutathione peroxides (GPx) are considered to be the

primary antioxidant enzymes, since they are involved in the direct elimination of

reactive oxygen species. Glutathione-S-transferase (GST), glutathione reductase

(GR) and glucose- 6-phosphate dehydrogenase (G6PD) are secondary antioxidant

enzymes which help in the detoxification of reactive oxygen species by

decreasing peroxide levels by GST or by maintaining a steady supply of metabolic

intermediates like glutathione as by GR and NADPH by, G6PD for the primary

antioxidant enzymes. The non-enzymatic small molecular antioxidants include

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sulfhydryl compounds such as glutathione (GSH) and thiols, NADPH, ascorbate, α-

tocopherol etc., (Halliwell and Gutteridge 1984). Phytochemical components such

as polyphenols, ascorbic acid, and carotenoids also serve as antioxidants (Rice-

Evans et al., 1997). Oxygen is essential for aerobic life process. However, cells

under aerobic condition are threatened with the insult of reactive oxygen molecules

that are efficiently taken care of by the powerful antioxidant system in human body.

5.2.5 Hepatoprotective activities of Nigella sativa L.

A large number of medicinal plants have been tested and found to contain

active principles with curative properties against a variety of diseases (Lewis,

1977). Liver protective plants contain a variety of chemical constituents like

phenols, coumarins, lignans, essential oil, monoterpenes, carotinoids, glycosides,

flavanoids, organic acids, lipids, alkaloids and xanthenes. Recent experience has

shown that plant drugs are relatively non-toxic, safe and even free from serious

side effects (Momin, 1987).

Nigella sativa L. is one of the most revered medicinal seed in history. A large

number of in vitro and in vivo studies have been conducted on laboratory animals

and humans in order to investigate its pharmacological properties like

immunostimulation, anti-inflammatory, hypoglycemic, antihypertensive,

antiasthmatic, antimicrobial, antiparasitic, antioxidant as well as anticancer

properties (Randhawa and Alghamdi, 2002). Acute and chronic toxicity studies on

laboratory animals have reported that N. sativa, its oil and thymoquinone the most

widely studied active principle are quite safe particularly when given orally (Badary

et al., 1997; Mansour et al., 2001; Ali et al., 2003).

It has been reported that N. sativa oil possesses hepatoprotective effects in

some models of liver toxicity. However, it is N. sativa seed that is used in the

treatment of liver ailments in folk medicine rather than its oil. Clinical and

experimental investigations have shown that N. sativa has a protective effect against

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oxidative damage in isolated rat hepatocytes (Daba et al., 1998). It was found that

the fixed oil of N. sativa has both antioxidant and anti-eicosanoid effects greater than

thymoquinone which is its active constituent (Houghton et al., 1995). Furthermore,

N. sativa has antioxidant activity by suppressing the chemiluminescence in

phagocytes (Haq et al., 1995).

Administration of oil extract of N. sativa to CCl4 intoxicated animals showed

significant hepatoprotective activity by restoring the hepatocellular activity. It has

been reported that thymoquinone (the active component of black cumin) presents in

high level in N. sativa possesses antioxidant properties (Hesham et al., 2002).

Thymoquinone prevents the formation of toxin stable complex by the combination of

CCl3O2 free radical and the glycolipid component of cell membrane and therefore

restores cellular architecture and prevent the leakage of its enzymes (Ali BH et al.,

2003). Significant hepatoprotective effects of N. sativa in carbon tetrachloride

(Mastour and Al-Ghamdi et al., 2003), D-galactosamine and turpentine oil- induced

liver damage (Subodh et al., 2011) were noted.

This was also reported that N. sativa has a significant hepatoprotective effect

in CCl4 intoxicated rabbits. N. sativa protects liver against fibrosis possibly through

immunomodulator and antioxidant activities. N. sativa seed extract reduced total

bilirubin, serum enzymes level in CCl4 treated animals and also prevent liver fibrosis

and cirrhosis, suggested that N. sativa protects liver against fibrosis possibly through

immunomodulator and antioxidant activities. (Turkdogan et al., 2003). N. sativa

seeds appeared to be safe and possibly protective against CCL4-induced

hepatotoxicity (Mastour and Al-Ghamdi, 2003).

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5.3 MATERIALS AND METHODS

5.3.1 Collection of N. sativa seeds

Seeds of N. sativa were procured in September, 2010 from a herbal

shop in Lucknow, India and authenticated by a botanist at National Botanical

Research Institute, Lucknow. A voucher specimen of the seeds was kept in the

museum of the Department for future reference.

5.3.2 Germination of N. sativa seeds

Germination was done according to the method of Ahmad et al., (2010). Seed

lots used for different experiments showed germination capacities ranging from 80 to

98%. The seeds were surface sterilized with 0.1% HgCl2 for 3 min. They were rinsed

thoroughly with double distilled water and soaked in de-ionized water for 30 min.

For germination of seeds, they were placed on four folds of damp filter paper at 25°C

and incubated in dark till the initiation of sprouting after which they were placed at a

light intensity of 100 µmol m-2

s-1

(that was measured by LI-190SA quantum Sensor,

Li-COR Co., USA) and a 14/10 h (day/night) photoperiod till the complete plantlet

with two leaves were obtained. The complete germination took eleven days with

emergence of epicotyl, hypocotyl, roots and green leaves. Germination, defined as 1

mm radicle emergence, was followed for 11days. No contamination by

microorganisms was observed during this time period.

5.3.3 Harvest of germinated seeds

The germinated seeds of different days were harvested with sterilized forceps

and were kept on blotting sheet to remove excess water. The germinated seeds

collected for different experiments were used immediately for preparing extracts.

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Seeds were considered to be germinated after the radicle emerged from the testa. All

the samples were stored at -80oC in a deep freezer until used further.

5.3.4 Preparation of distilled extracts

The samples of seed and germinated phases 5th, 7

th and 11

th day were shade-

dried and ground to a fine powder. The powder (20gm) was extracted with 200 ml

methanol solvent for 48 h in order to extract bioactive compounds using soxhlet

apparatus (AOAC method 1980). The extracts were filtered using Whatman filter

paper (No.1) and methanol was evaporated using rotary distillation apparatus to

obtained pure extract. Oily fraction of extracts was stored at 40C until use.

5.3.5 Animals

Male Wistar rats, weighing 150 - 200 g, were purchased from Central Drug

And Research Institute (CDRI), Lucknow, India and housed in a temperature

controlled room (22±2°C) with a 12 hour light-12 hour dark cycle and allowed free

access to a standard rat chow and filtered tap water for 7 days for acclimatization.

The study received the approval of the Institutional Animal Ethics Committee

(IAEC) of Era’s Lucknow Medical College & Hospital. Animals were cared for in

accordance with the internationally accepted principles for laboratory animal use and

care and the procedures followed were in accordance with the standards set forth in

the Guide for the Care and Use of Laboratory Animals (published by the National

Academy of Science, National Academy Press, Washington, D.C.). They were

housed under controlled conditions of temperature of 23±20C, relative humidity of

30–70% and 12 h light–12 h dark cycle. The animals were housed individually in

polypropylene cages containing sterile paddy husk (procured locally) as bedding

throughout the experiment. All animals were fed with sterile commercial pelleted rat

chow supplied by Hindustan Lever Ltd. (Mumbai, India) and had free access to

water. Animals were kept under fasting for overnight and weighed before the

experiment.

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5.3.6 Study design

Hepatoprotective effects of N. sativa methanolic extracts of different

germination stages were studied against carbon tetrachloride (CCl4) induced

hepatotoxicity in Wistar rats. Dose of N. sativa extracts was selected as per study of

Mehta et al., (2009). Silymarin (50 mg/kg b.w.) was used as standard

hepatoprotective drug (Nayak et al., 2012, Palanivel et al., 2008).

5.3.6.1 Carbon tetrachloride induced hepatotoxicity

Carbon tetrachloride (CCl4) toxicity was induced according to the method of

Asif Mir et al., 2010. Acute CCl4 toxicity was induced by treating rats for 24 hrs by

oral administration of CCl4 mixed with olive oil as vehicle in 1:1 ratio (3 ml/kg

b.w.).

5.3.6.2 Hepatoprotective effects of N. sativa

To study hepatoprotective effects of N. sativa during different phases of its

germination in carbon tetrachloride induced (CCl4) induced hepatotoxicity, animals

were divided in following groups containing 6 rats each.

Group-I (control) Received single dose of distilled water orally from day 1 to day 7

followed by olive oil (1.5ml/kg) on day 7.

Group-II (CCL4) Received distilled water for six days followed by CCl4 with olive

oil (3 ml/kg) orally on day 7 (Oral administration of CCl4 mixed

with olive oil as vehicle in 1:1 ratio (3 ml/kg b.w.)

Group-III Received Silymarin (50 mg/kg) orally from day 1 to day 7 followed by

CCl4 with olive oil (3ml/kg) on day 7 after 2 hrs.

Group- IV (Seed extract) Received N. sativa seed extract 1 g/kg orally from day 1

to day 7 followed by CCl4 with olive oil (3ml/kg) on day 7 after 2 hrs.

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Group-V (5th

day extract) Received N. sativa extract of 5th

day of germination

phase (1 g/kg) orally from day 1 to day 7 followed by CCl4 with olive

oil (3ml/kg) on day 7 after 2 hrs.

Group-VI (7th

day extract) Received N. sativa extract of 7th

day of germination

phase (1 g/kg) orally from day 1 to day 7 followed by CCl4 with olive

oil (3ml/kg) on day 7 after 2 hrs.

Group-VII (11th

day extract) Received N. sativa extract of 11th day of germination

phase (1 g/kg) orally from day 1 to day 7 followed by CCl4 with olive oil

(3ml/kg) on day 7 after 2 hrs.

Figure 5.2: Study design to investigate hepatoprotective effect of N. sativa extracts

of different germination phases in CCl4-induced hepatotoxicity.

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8

Collection of blood and liver samples

Grp-I. (control) Distilled water

Grp-II. Distilled water

Grp-III. Silymarin (50 mg/kg)

Grp- IV. Seed extract (1 g/kg)

Grp-V. 5th day extract (1 g/kg)

Grp-VI. 7th day extract (1 g/kg)

Grp-VII. 11th day extract (1 g/kg)

Grp-I. (control) Olive oil (1.5ml/kg)

Grp-II. CCl4 with olive oil (3ml/kg)

Grp- III. Silymarin (50 mg/kg) + CCl4 with olive

oil (3ml/kg)

Grp-IV. Seed extract (1 g/kg) + CCl4 with olive

oil (3ml/kg)

Grp-V. 5th day extract (1 g/kg) + CCl4 with olive

oil (3ml/kg)

Grp-VI. 7th day extract (1 g/kg) + CCl4 with

olive oil (3ml/kg)

Grp-VII. 11th day extract (1 g/kg) + CCl4 with

olive oil (3ml/kg)

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5.3.7 Blood sampling

Cardiac puncture (Diaphragmatic approach): General anesthesia was

administered and the animal placed on solid surface with its ventrum exposed

thexyphoid process was palpated at the caudal aspects of the animal sternum. A

notch was present on both side of this process. A 1.5O, 22 gauge needle attached to a

5 ml syringe was inserted into either notch and directed towards the heart as

determined by palpating the apex beat. Negative pressure was applied by placing

slight backward pull of the plunger, once it has been inserted beneath the skin.

Reflux of blood was apparent once the needle penetrated the heart.

Plate 5.1: Blood sampling by cardiac puncture and collection of liver by dissection.

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5.3.8 Biochemical estimation

5.3.8.1 Liver enzymes

Estimation of serum levels of glutamate oxaloacetate transaminase (SGOT),

glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP). Total

bilirubin was measured by standard method using semi-auto analyzer.

5.3.8.2 Tissue Homogenate Preparation

10% (w/v) homogenate of rat liver was prepared with the aid of York’s

homogenizer fitted with Teflon plunger in 0.1 M phosphate buffer (pH 7.5). The

whole homogenate was first centrifuged at 2500xg for 10 minutes in a refrigerated

centrifuge. The pellet consisting of nuclear fraction and cell debris was discarded.

The supernatant was further centrifuged at 11,000xg for 15 min and mitochondrial

fraction was separated. The clear supernatant was further centrifuged at 100,000xg

for 90 min and the resulted supernatant was used for enzyme activities.

5.3.8.3 Lipid peroxide level (Ohkawa et al., 1979)

Procedure: 0.2 ml of each sub-cellular fraction having 3 to 10 mg protein,

was mixed with 1.0 ml of 20% acetic acid followed by the addition of 0.2 ml of 8%

aqueous SDS. The mixture was adjusted to pH 4 by addition of concentrated NaOH

solution if needed. After adjusting the pH of reaction mixture, 1.5 ml of 0.8% TBA

solution and sufficient amount of distilled water was also added to achieve the final

volume of 4 ml. The reaction mixture was incubated in a boiling water bath for

1hour. After cooling to room temperature, 3 ml of n-butanol was mixed. The reaction

mixture was centrifuged at 10,000 x g for 15 min. A clear butanol fraction obtained

from centrifugation was used for measuring the absorbance at 532 nm.

Calculation: An appropriate standard made up of malondialdehyde (MDA)

2.5 nmol was run simultaneously. Result was expressed in nmol MDA/gm.

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5.3.8.4 Superoxide dismutase (McCord Fridovich, 1969)

Procedure: 2 ml of homogenate was dispended in centrifuge tube. The tube

was placed in a refrigerated centrifuge and spin at 10,000x g for 15 min. To the

supernatant for each sample, 313mg/ml ammonium sulphate was added to the final

concentration of 50%. The tube was shaken thoroughly and kept for 4 hours in cold

(40C). Thereafter, the tube was centrifuged at 14,000x g for 30 min at 4

0C. The

supernatant sample was dialyzed against cold tripled distilled water with 3 changes,

each change after 3 hrs interval. The content of dialysis bags were subsequently used

as enzyme source. Experimental tubes was contain 0.3 ml of 1.5mM

nitrobluetetrazolium (NBT), 0.2 ml of 0.93 mM phenazinemethosulphate, 1 ml of

20.4mM pyrophosphate buffer (pH 9.2), 1 ml triple distilled water and 0.2ml enzyme

source. The second setup reference tubes received the above reagents except the

enzyme source. The reaction was started simultaneously in two sets by the addition

of 0.1 ml 2.34mM NADH. After an interval of 90 sec, 1 ml glacial acetic acid was

added to each tube for checking the reaction then 0.2 ml enzyme source was added

for checking the reaction followed by addition of 0.2 ml enzyme source in the

reference tubes. The absorbance of these tubes was read at 560 nm in a

spectrophotometer against blank (NBT+PMS+Buffer+TDW).

Calculation: The unit of enzyme activity was defined as the amount of

enzyme required to inhibit the reduction in optical density of nitro blue tetrazolium

up to 50%, in 1 min at 560 nm under the assay condition. Results were expressed as

unit/mg protein

5.3.8.5 Catalase (Aebi, 1974)

Procedure: 3.0 ml of H2O2 phosphate buffer was pipette into the cuvette

followed by addition of required amount of tissue supernatant (cytosolic fraction) as

enzyme source and the content was mixed thoroughly. The decrease in absorbance at

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240 nm recorded after every 30 sec for 3 min using UV spectrophotometer. Result

was expressed as unit/mg protein.

5.3.8.6 Reduced glutathione (Eliman et al., 1959)

Procedure: 1 ml liver (10%) homogenate was deprotenized by adding 1 ml

of 10% TCA and centrifuged at 6000x g for 5 min. 0.5 ml aliquots for supernatant

was mixed with 0.5 ml of double distilled water thereafter 2 ml of 0.4 M Tris buffer

and 0.1 ml DTNB were added to it with proper stirring. The absorbance was read at

412 nm within 5 min of the addition of DNTB.

Calculation: GSH in the sample was calculated using the standard curve and

results were expressed as µmol/g tissue.

5.3.9 Histopathological study

For histopathology half of liver tissue of each rat was stored in 10% formalin

solution for 48 hours or until processing. For block preparation, liver were processed

using a graded ethanol series and embedded in paraffin. 4 µm paraffin sections were

cut and stained with Haematoxylin and eosin. The study was conducted in

consultation with the trained pathologist under light microscope with 10x, 40x, and

100x magnifications.

Tissue fixation, embedding & sectioning

Tissue Fixation

The rats were sacrificed after the completion of the period of the dose and the

organs were collected in normal saline. The tissue was cut in small pieces

(L.Section/T.Section, approx 2.3 mm in size). The tissue was fixed in 10% formalin

for 24 hours and washed in running tap-water after that tissue was kept in distilled

CHAPTER-5


water for 10-15 minutes. Tissue was dehydrated in following graded series of

alcohol.

30% alcohol for 45 minutes.





Then, this tissue was transferred in absolute alcohol + xylene (1:1) for 35 minutes

and then after in pure xylene for 30 minutes.

Following chemicals are transferred in incubator at 60°C

Xylene + wax in 1:1 ratio for 45 minutes and pure wax for 45 minutes.

Tissue Embedding

The processed tissues were then transfer to the molten wax poured into the L-

block at 60°C, allowed to solidify for 10-15 minutes and removed from the L-block.

The casted wax blocks were left overnight at room-temperature to uniform

solidification. (Precautions were taken to minimize the appearance of air bubbles in

the blocks and to maintain the orientation of the tissue for transverse section (TS) or

longitudinal section (LS) while placing it into the molten wax in L-molds).

Section Cutting

With the help of microtome the section of the tissue has been cut (2-5 µm in

size). The thin section has been taken in clean slide coated with a mixture of egg

albumin + glycerol (1:1). Then it was unfolded with slight warm distilled water, then

heat fixed and the slides were kept for 12 hours.

CHAPTER-5


Slide Staining

The tissue was immersed in xylene for 10 min.

Immersed in 100% alcohol for 10 min.



Immersed in 50% alcohol for 5-8 min.


Immersed in distilled water for 5 min.

Immersed in Haematoxylin for 2-3 min.

Immersed in tap water for 10-15 min.

Immersed in acid water 3-5 min.

Immersed in distilled water for 5 min.




Immersed in acid for 3-5 min.



Immersed in xylene for 5-10 min.

Then mounted on the slide with DPX.

Then left for 12 hours & seen under microscope.

CHAPTER-5


5.3.10 Statistical analysis

Statistical significance was determined by One Way Analysis of Variance

(ANOVA) followed by Dunnet’s t-test to compare group means. The level of

significance was P < 0.001.

5.4 RESULTS AND DISCUSSION

5.4.1 Hepatoprotective effect of N. sativa extracts of different germination

phases against CCl4 induced hepatotoxicity.

Administration of carbon tetrachloride CCl4 caused a significant increase in

liver enzymes and total bilirubin level which was attenuated by oral administration

of N. sativa extracts from different phases of seed germination.

Table 5.1: Effect of N. sativa extracts of different germination phases on liver

enzymes in CCl4 induced hepatotoxicity.

*Group-I (control); Group-II (CCL4); Group-III (silymarin); Group- IV (Seed

extract); Group-V (5th day extract); Group-VI (7

th day extract); Group-VII (11

th day

extract). *Values in Mean ±SEM, N=6.* ap <0.001 compared with Group I (control)

and bp<0.001 compared with Group II (CCl4 treated).

Groups ALP(U/L) SGOT (U/L) SGPT (U/L) Total bilirubin

(mg/dl)

Group I 74.87±5.09 44.77±3.37 39.57±5.04 0.19±0.01

Group-II 267.22±18.4a 1217.98±156.38

a 434.89±71.37

a 0.668±0.08

a

Group-III 75.2±5.9b 45.1±6.12

b 49.14±4.32

b 0.2±0.04

b

Group- IV 110.5±6.95b 63.96±7.67

b 100.22±7.1

b 0.285±0.01

b

Group-V 74.74±5.7b 45.42±3.33

b 49.33±3.54

b 0.206±0.02

b

Group-VI 86.77±3.44b 45.88±4.47

b 59.1±5.9

b 0.208±0.02

b

Group-VII 100.94±3.4b 64.44±7.8

b 81.33±4.2

b 0.2±0.022

b

CHAPTER-5


5.4.1.1 ALP

Results of present study showed that level of ALP in control group was

74.87±5.09 U/L serum. There was a significant (P<0.001) increase in mean ALP

level in rats administered with CCl4 (3ml/kg) for 24 hrs (267.22±18.4 U/L serum).

Oral administration of N. sativa seed extract (non-germinated) and extracts of

different germination phases (5th day, 7

th day and 11

th day) ameliorated this increase

in ALP level. In all the four groups receiving N. sativa extracts rise in ALP level was

prevented (Table 5.1 and Figure 5.3). Among all extracts 5th

day germination extract

showed significant protective effect and reduced serum ALP level (74.74±5.7 U/L

serum) which was equal to control group (74.87±5.09 U/L serum) and the standard

drug silymarin (75.2±5.9 U/L serum). Extract of 7th day germination was on second

position (86.77±3.44 U/L serum) followed by 11th day extract (100.94±3.4 U/L

serum) and seed extract (110.5±6.95 U/L serum).

Figure 5.3: Effect of N. sativa extracts of different germination phases on ALP level

against CCL4-induced toxicity.




th day

extract).

0

50

100

150

200

250

300

Group I Group II Group III Group IV Group V Group VI Group VII

AL

P (

U/L

ser

um

)

CHAPTER-5


5.4.1.2 SGOT

Table 5.1 and Figure 5.4 showed that administration of CCl4 (3ml/kg) for 24

hrs significantly (P<0.001) increase SGOT level as compared to control. The SGOT

value of CCl4 group (1217.98±156.38 U/L serum) was very high than control group

(44.77±3.37 U/L serum). Administration of N. sativa seed extract (non-germinated)

and extracts from different germination phases of seed (5th day, 7

th day and 11

th day)

ameliorated the increase in SGOT level due to CCl4 toxicity. Among all extracts 5th

day and 7th day germination extract showed significant protective effect against

CCl4-induced toxicity and reduced serum SGOT level (45.42±3.33 U/L serum and

45.88±4.47 U/L serum respectively) which was significantly (P<0.001) equal to

control group (44.77±3.37 U/L serum) as well as standard group (45.1±6.12 U/L

serum) followed by 11th

day extract (64.44±7.8 U/L serum) and seed extract

(63.96±7.67 U/L serum).

Figure 5.4: Effect of N. sativa extracts of different germination phases on SGOT

level against CCL4-induced toxicity.




th day

extract).

0

200

400

600

800

1000

1200

1400

1600


SG

OT

(U

/L s

eru

m)

CHAPTER-5


5.4.1.3 SGPT

Results showed SGPT level in control group was 39.57±5.04 U/L serum.

There was a significant (P<0.001) increase in mean SGPT level in rats administered

with CCl4 (3ml/kg) for 24 hrs (434.89±71.37U/L serum). Oral administration of N.

sativa non-germinated seed extract and extracts from different germination phases of

seed (5th day, 7

th day and 11

th day) ameliorated the increase in SGPT level due to

CCl4 toxicity. In all the four groups, receiving N. sativa extracts rise in SGPT level

was prevented (Table 5.1 and Figure 5.5). Among all extracts 5th day germination

extract showed significantly protective effect against CCl4-induced toxicity and

reduced serum SGPT level (49.33±3.54 U/L serum) which was approximately equal

to control group (39.57±5.04 U/L serum) in comparison to the toxic group. Level of

SGPT in group which received silymarin was (49.14±4.32 U/L serum) which was equal

to 5th day germination extract. 7

th day extract was on second position (59.1±5.9 U/L

serum) followed by 11th

day extract (81.33±4.2 U/L serum) and seed extract

(100.22±7.1 U/L serum).

Figure 5.5: Effect of N. sativa extracts of different germination phases on SGPT





th day

extract).

0

100

200

300

400

500

600


SG

PT

(U

/L s

eru

m)

SGPT/ALT

CHAPTER-5


5.4.1.4 Total bilirubin

Mean value of total bilirubin in control group was 0.19±0.01 (mg/dl serum).

There was a significant (P<0.001) increase in value of total bilirubin level in rats

administered with CCl4 (3ml/kg) for 24 hrs (0.668±0.08 mg/dl serum).

Administration of N. sativa seed extract (non-germinated) and extracts from different

germination phases (5th day, 7

th day and 11

th day) ameliorated the increase in total

bilirubin level due to CCl4 toxicity. In all the four groups receiving N. sativa extracts

rise in total bilirubin level was prevented (Table 5.1 and Figure 5.6). 7th day extract

was on second position followed by 11th

day extract and seed extract to ameliorate

the level of bilitubin.

Figure 5.6: Effect of N. sativa extracts of different germination phases on bilirubin





th day

extract).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


Bil

iru

bin

(m

g/d

l se

rum

)

BILIRUBIN(TOTAL)

CHAPTER-5


5.4.2 Effect of N. sativa extracts of different germination phases on antioxidant

enzymes level against CCL4-induced hepatotoxicity.

Carbon tetrachloride (CCl4) administration caused a significant oxidative stress

as evident by increase in MDA level and decrease in SOD, CAT and GSH levels.

This altered redox status was attenuated by administration of N. sativa extracts of

different phases of seed germination.

Table 5.2: Effect of N. sativa extracts of different germination phases on antioxidant

level against CCl4-induced hepatotoxicity.




th day

extract).*Values in Mean ±SEM, N=6.*p<0.001 compared with Group I (control)

and bp< 0.001 compared with Group II (CCl4 treated).

5.4.2.1 MDA LEVEL

Level of MDA in control group was 289.4±19.23 nmol MDA/gm tissue.

There was a significant (P<0.001) increase in mean MDA level in rats administered

with CCl4 (3ml/kg) for 24 hrs (363.22±56 nmol MDA/gm tissue). This effect of CCl4

Groups nmol MDA/gm

tissue

SOD

U/mg protein

CAT

U/mg protein

GSH

µmol/g protein

Group I 289.4±19.23 12.5±0.56 9.5±0.23 38.12±0.52

Group-II 363.22±56.12a 4.56±0.48

a 3.46±0.25

a 25.39±0.52

a

Group-III 295.2±7.42b 11.02±0.61

b 8.91±0.45

b 37.1±0.21

b

Group- IV 321.01±6.67b 8.24±0.99

b 6.24±0.25

b 35.1±0.31

b

Group-V 299.32±10.33b 11±0.56

b 8.9±0.56

b 36.99±0.4

b

Group-VI 303.12±6.47b 10.19±0.88

b 8.1±0.61

b 36±0.12

b

Group-VII 316.1±8.8b 9.97±0.88

b 6.97±0.64

b 35.89±0.52

b

CHAPTER-5


toxicity was ameliorated in rats which received N. sativa extracts from different

germination phases of seed. In all the four groups receiving N. sativa extracts rise in

MDA level was prevented (Table 5.2 and Figure 5.7). Among all extracts 5th day

germination extract showed significant protective effect against CCl4-induced

toxicity and reduced MDA level as control (299.32±10.33 nmol MDA/gm tissue).

Silymarin also reduced MDA level (295.2±7.42 nmol MDA/gm tissue) significantly.

7th day, 11

th day and seed extract (non-germinated) also reduced the MDA level.

Figure 5.7: Effect of N. sativa extracts of different germination phases on MDA





th day

extract).

5.4.2.2 Superoxide dismutase

There was a significant (P<0.001) decrease in SOD level observed in rats

administered with CCl4 (4.56±0.48 U/mg) in comparison to control group (12.5±0.56

U/mg). Extracts of N. sativa raise the level of superoxide dismutase enzyme and

maintain the level of catalase as in control group. The mean value of 5th day, 7

th day,

11th day and seed extract was 11±0.568 U/mg, 10.19±0.88 U/mg, 9.97±0.88 U/mg

0

50

100

150

200

250

300

350

400

450


MD

A (

nm

ol/

gm

tis

sue)

CHAPTER-5


and 8.24±0.99 U/mg respectively (Table 5.2 and Figure 5.8). Results showed that 5th

day germinated extract significantly increased the level of SOD. Extract of 7th

day

germination phase was on second position followed by 11th

and seed extract (non-

germinated).

Figure 5.8: Effect of N. sativa extracts of different germination phases on SOD level





th day

extract).

5.4.2.3 Catalase

There was a significant (P<0.001) decrease in CAT enzyme level in rats

administered with CCl4 (3ml/kg) for 24 hrs (3.46±0.25 U/mg) in comparison to

control group (9.5±0.23 U/mg). Extracts of N. sativa from germination phases of

seed raise the level of catalase and maintain the level of catalase as was in control

group. The mean value of silymarin, 5th

day, 7th

day, 11th day and seed extract (non-

germinated) was 8.91±0.45 U/mg, 8.9±0.56 U/mg, 8.1±0.61 U/mg, 6.97±0.64 U/mg

0

2

4

6

8

10

12

14


SO

D (

U/m

g p

rote

in)

CHAPTER-5


and 6.24±0.25 U/mg respectively (Table 5.2 and Figure 5.9). Extract of 5th day

germination phase increased catalase activity as in control group.

Figure 5.9: Effect of N. sativa extracts of different germination phases on CAT level





th day

extract).

5.4.2.4 Reduced glutathione GSH

There was a significant (P<0.001) decrease in GSH level in rats administered

with CCl4 (3ml/kg) (25.39±0.52 µmol/gm protein) in comparison to control group

(38.12±0.52 µmol/g protein). Extracts of N. sativa raise the level of GSH. All the

extract showed protective effect in CCl4-induced toxicity and increased the level of

GSH significantly. Extract of 5th

day germination phase increased the level of GSH

significantly (36.99±0.4 µmol/gm protein), this was higher than non-germinated seed

extract (35.1±0.31 µmol/gm protein).

0

2

4

6

8

10

12


Cata

lase

(U

/mg p

rote

in)

CHAPTER-5


Figure 5.10: Effect of N. sativa extracts of different germination phases on GSH





th day

extract).

5.4.3 Histopathology studies

In control group animals showed normal liver parenchyma and normal

architecture of hepatocytes, vasculature, distinct hepatic cords and central vein (Plate

5.2). However, animals treated with carbon tetrachloride showed extensive necrosis,

fraying of cell margins, and portal triditis with mononuclear lymphoplasmocytic

inflammatory infiltration (Plate 5.3). This effect was significantly decreased in

animals pretreated with N. sativa extracts from different germination phases of seed.

Plate 5.2: Photomicrograph from control (Group-I) rat showing normal liver

parenchyma and normal architecture of hepatocytes and vasculature,

distinct hepatic cords and central vein. Haematoxylin and eosin stained

(H and E) 5 micron thick section (H&E 40X).

0

5

10

15

20

25

30

35

40

45


GS

H (

µm

ol/

g p

rote

in)

CHAPTER-5


Hepatic cord

Central vein

Plate 5.3: Photomicrograph of CCl4 treated rats (Group-II) showing extensive

necrosis, fraying of cell margins, portal triditis with mononuclear

lymphoplasmocytic inflammatory infiltration, strip focal necrosis, feathery

degeneration (in the mid zone) involving several hepatocytes, necrosis of several

hepatocyte extended in band like fashion in the mid zonal area and occasional

apoptotic bodies.

CHAPTER-5


Plate 5.4: Liver section from rat orally treated with seed extract (non-germinated

1g/kg) showing a slight protective effect but shows some

lymphoplasmocytic inflammatory infiltration.

Plate 5.5: Liver section from rat orally treated with 5th

day germination extract (1

g/kg), showing highly protective effect without lymphoplasmocytic

inflammatory infiltration

CHAPTER-5




g/kg) showing slight protective effect with some inflammatory cells.



g/kg) showing protective effect with reduced necrosis.

CHAPTER-5


A number of chemicals including various environmental toxicants and

clinically useful drugs can cause severe cellular damages in different organs of our

body through the metabolic activation to highly reactive substances such as free

radicals. CCl4 is one of such widely studied environmental toxicant (Sil et al., 2006).

Up to the present time the etiology and treatment of most liver diseases are not

known. The liver is the commonest site affected during the toxic manifestation of

many drugs. Toxicity in liver due to CCl4 and other chemicals is attributed to the

toxic metabolites formed which are responsible for the initiation of CCl4 dependent

lipid peroxidation (Arulkumaran et al., 2007). In the liver CCl4 is metabolized by the

cytochrome P450-dependent monooxygenase systems followed by its conversion to

more chemically active form namely trichloromethyl radical (CCl3•-

) that bind

covalently to the macromolecules and induce peroxidative degradation of membrane

lipids of endoplasmic reticulum rich in polyunsaturated fatty acids which leads to the

formation of lipid peroxides. This lipid peroxidative degradation of biomembranes is

one of the principle causes of hepatotoxicity of CCl4 (Kaplowitz et al., 1986). CCL4

also causes depletion of glutathione content and reduced the level of SOD and CAT

enzymes (Kamiyama et al., 1993).

In the assessment of liver damage by CCl4 the determination of enzyme

levels such as SGOT (AST), SGPT (ALT) and bilirubin is largely used. Necrosis or

membrane smash up releases the enzyme into circulation and hence they can be

measured in the serum. Increase levels of SGOT indicate liver damage in the same

manner caused by viral hepatitis as well as cardiac infarction and muscle injury. This

enzyme catalyses the conversion of alanine to pyruvate and glutamate and is released

in a similar manner. Therefore SGPT is more specific to the liver and is a better

parameter for detecting liver injury. Elevated levels of serum enzymes are indicative

of cellular leakage and loss of functional integrity of cell membrane in liver

(Drotman et al., 1978). On the other hand serum ALP, bilirubin and total protein

levels are related to the function of hepatic cell. Increase level of ALP in serum is

due to increased synthesis in presence of increasing biliary pressure (Muriel et al.,

1992).

CHAPTER-5


As the results showed that oral administration of CCl4 caused a significant

(P<0.001) elevation of enzyme levels such as ALP, SGPT, SGOT, total bilirubin and

decrease in antioxidant system increased in MDA level and depletion of glutathione

content, SOD and CAT activity when compared to control group. There was a

significant (P<0.001) restoration of these enzyme levels on oral administration of the

N. sativa extracts from different germination phases in day-dependant manner and

also by silymarin at a dose of 50 mg/kg. Among all tested extracts 5th

day

germination extract showed significant protective effect like standard drug silymarin.

Extract of 7th

day germination was on second position followed by 11th

day and seed

extract. The reversal of increased serum enzymes in CCl4-induced liver damage by

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

its membrane stabilizing activity. This is in agreement with the commonly accepted

view that serum levels of transaminases returns to normal with the healing of hepatic

parenchyma and the regeneration of hepatocytes (Thabrew et al., 1987). Effective

control of ALP and bilirubin levels point towards an early improvement in the

secretary mechanism of the hepatic cells.

The worth of any hepatoprotective drug is reliant on its capability of either

reducing the harmful effect or restoring the normal hepatic physiology that has been

disturbed by a hepatotoxin. Silymarin, the standard hepatoprotective drug used in the

present study exerted marked protective effects against CCl4-induced liver injury. It

has been reported in several in vitro and in vivo studies (Letteron et al., 1990). The

crucial protective mechanism of silymarin is an inhibition of lipid peroxidation by its

free radical scavenging properties (Farghali et al., 2000). Silymarin prevented CCl4

induced lipid peroxidation and hepatotoxicity in mice by decreasing the metabolic

activation of CCl4 and by acting as a chain-breaking antioxidant (Letteron et al.,

1990). Both silymarin and the N. sativa extracts of germination phases (specially of

5th day germination extract) decreased CCl4 induced elevated enzyme levels in tested

groups indicating the protection of structural integrity of hepatocyte cell membrane

or regeneration of damaged liver cells as they showed in histopathology results. The

increase in MDA (LPO) level in liver induced by CCl4 suggests enhanced lipid

CHAPTER-5


peroxidation leading to tissue damage and failure of antioxidant defense mechanism

to prevent formation of excessive free radicals. Treatment with N. sativa

significantly reverses these changes. Hence it is likely that the mechanism of

hepatoprotection of N. sativa is due to its antioxidant effect. N. sativa reduced lipid

peroxidation significantly during germination phases by scavenging free hydroxyl

radical was reported in our previous research (Kamal et al., 2010).

Superoxide dismutase (SOD) has been reported as one of the most important

enzymes in the enzymatic antioxidant defense system which scavenges the

superoxide anion to form hydrogen peroxide and thus diminishing the toxic effect

caused by this radical. Decrease in enzyme activity of SOD is a sensitive index in

hepatocellular damage and is the most sensitive enzymatic index in live injury

(Curtis et al., 1972). Catalase (CAT) is an enzymatic antioxidant widely distributed

in all animal tissues and the highest activity is found in the red blood cells and liver.

CAT decomposes hydrogen peroxide and protects the tissues from highly reactive

hydroxyl radicals (Chance et al., 1992). N. sativa extracts from germination phases

cause a significant (P<0.001) increase in hepatic SOD and CAT activity and thus

reduces reactive free radical induced oxidative damage to liver. Previous research

has been proved that N. sativa shows significant high antioxidant activity during

germination phases as compared non-germinated seed (Kamal et al., 2010).

Glutathione is one of the most abundant tripeptide, non-enzymatic biological

antioxidant present in the liver. It removes free radical species such as hydrogen

peroxide, superoxide radicals and maintains membrane protein thiols. Also it is

substrate for glutathione peroxidase (Prakash et al., 2001). Decreased level of GSH

is associated with an enhanced lipid peroxidation in CCl4 treated rats. Administration

of N. sativa significantly (P<0.001) increased the level of GSH especially in 5th

day

germination extract treated group.

Many studies have demonstrated hepatoprotective effect of N. sativa seeds

and reported significant improvement in liver functional test. After giving N. sativa

oil significant improvement of deranged liver functional test parameters against

CHAPTER-5


various hepatotoxic compounds (Mohideen et al., 2003; Al-Ghamdi et al., 2003;

Kanter et al., 2005; El-Gharieb et al., 2010; Janakat et al., 2010). N. sativa

administration in several model of induced hepatotoxicity has been shown to exert

hepatoprotective effect along with restoration of reduced glutathione, superoxide

dismutase, lactate dehydrogenase, catalase and decrease in lipid per oxidation

reaction (Al-Ghamdi et al., 2003; Mahmoud et al., 2002; Kanter et al., 2005; El-

Gharieb et al., 2010). Present study is the first study to investigate hepatoprotective

effect of N. sativa during germination phases of seed. As results showed that N.

sativa possesses better hepatoprotective effect during germination phases (especially

during 5th day) than non-germinated seed.

Protective effect of N. sativa may be due to the presence of high amount of

bioactive compounds such as thymoquinone (TQ), thymol, carvacrol and other

phytochemicals like flavonoids, phenols and tannins. Both biochemical and

histopathological data showed that there was no significant difference in extract

treatment when compared to control group. Extract of 5th day germination phase was

highly protective among all the extracts due to presence of higher quantity of

secondary metabolites, TQ, thymol and other active constituents were higher in 5th

day germination extract (chapter 2).

TQ has been reported to have potent superoxide anion (O2−) scavenging

abilities and to inhibit iron-dependent microsomal lipid peroxidation (Badary et al.,

2003). TQ may protect against CCl4-induced hepatotoxicity by a combination of two

mechanisms, i.e. TQ antioxidant potential (Houghton et al., 1995) and a previous

report of the quinone to have in vitro and in vivo superoxide anion radical

scavenging ability (Nagi and Mansour, 2000). TQ provided good protection against

lipid peroxidation and the oxidative damage caused by several toxic agents, as in

cisplatin nephrotoxicity (Badary et al., 1997), doxorubicin cardiotoxicity (Al-

Shabanah et al., 1998) and benzo(a)pyrene-induced forestomach carcinogenesis

(Badary et al., 1999). Burits and Bucar (2000) found that TQ had a scavenger effect

against the OH˙ radical in vitro. Moreover, Houghton et al. (1995) reported that TQ

CHAPTER-5


had potent anti-inflammatory and inhibitory effects on non-enzymatic peroxidation

of brain phospholipid liposomes. This was also reported by Abdulrahman L. Al-

Malki (2010) that pretreatment with thymol ameliorates the deleterious effect of

CCl4. It was speculated that thymol exert their effects by decreasing lipid

peroxidation and enhancing the activities of antioxidant enzymes. For this reason,

thymol could be used as hepatoprotective agent with free medication side effects

(Abdulrahman L. Al-Malki, 2010). Carvacrol, another constituent of N. sativa

affords significant hepatoprotective and antioxidant effect against D-GalN-induced

hepatotoxicity rats (Baser et al., 2008; Aristatile et al., 2009). The protective effects

of alpha-Hederin (constituent of N. sativa) on carbon tetrachloride-induced

hepatotoxicity were also observed by Jeong et al., (1998). N. sativa seed have been

shown to significantly decrease TNF-α (tumor necrosis factor α), IFN-γ (interferon

γ), IL-β (interleukin β) in CCl4-induced hepatotoxicity (Al-Ghamdi, 2003). These

hepatoprotective effects were probably due to antioxidant activities of N. sativa and

their active constituent TQ.

The above observation shows that N. sativa produces hepatoprotective effect

by restoration of anti oxidation enzyme system of the liver, reduction of lipid

peroxidation and inhibition of neutrophil infiltration and release of cytokines from

the inflammatory cells. Our study is also in conformity with Mahmoud et al., (2002)

who reported that active components of N. sativa increased hepatic antioxidant

enzymes activities (superoxide dismutase, catalase, glutathione) and reduces the

hepatic lipid peroxidation.

http://www.ncbi.nlm.nih.gov/pubmed?term=Aristatile%20B%5BAuthor%5D&cauthor=true&cauthor_uid=19650854

CHAPTER-5


5.5 Conclusion

It may be concluded the N. sativa methanolic extracts from different

germination phases showed high protective activity in CCl4-induced hepatotoxicity.

The extracts of germination stages were more protective than non-germinated seed

extract. This may be due to the fact that during germination many phytoconstituents

are produced in high quantities like flavonoids, phenols, tannins, which increase the

level of antioxidant enzymes and reduce the toxicity of CCl4. In the present study 5th

day germination phase followed by 7th

day extracts possesses significant

hepatoprotective activity and reduces the CCl4-induced hepatotoxicity compared

than non-germinated seed extract. The activity seems to be due to active component

thymoquinone which increase the antioxidant enzyme level. Further study is required

in this area as preliminary results are very encouraging to investigate the mechanism

of action of the compound to minimize toxicity.

CHAPTER-5


5.6 REFERENCES

Abdulrahman, L., Al-Malki. (2010). Antioxidant Properties of Thymol and

Butylated Hydroxytoluene in Carbon Tetrachloride Induced Mice Liver

Injury. JKAU.AU. Sci., 22(1):239-248.

Aebi, H. (1974).Catalase In: Bergmeyer HV, editor.Methods in enzymatic an

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