Download Anti cancer effect of Gingerol and Ginger extract on Ethylene

ANTICANCER EFFECT OF GINGEROL AND CRUDE GINGER
EXTRACT ON ETHYLENE THIOUREA INDUCED THYROID & LIVER
CANCER IN ALBINO RATS- A COMPARISION
A PROJECT REPORT SUBMITTED TO THE TAMILNADU STATE COUNCIL FOR
HIGHER EDUCATION IN PARTIAL FULFILMENT OF THE MINI PROJECT
SUBMITTED BY
R. DHANYA, B.Sc.,
(REG.NO. 14MZO303)
GUIDED BY
Dr. K. SAKTHI SHREE, M.Sc., M.Phil., Ph.D.,
OCTOBER, 2016
PG AND RESEARCH DEPARTMENT OF ZOOLOGY
GOVERNMENT ARTS COLLEGE(AUTONOMOUS)
(ACCREDITED WITH ‗A‘ GRADE BY NAAC, THIRD CYCLE)
COIMBATORE-641018
TAMILNADU
INDIA
Certificate
DECLARATION
Acknowledgement
ACKNOWLEDGEMENT
I express my deepest and sincere thanks to Dr. K. Sakthi Shree, M.Sc.,
M.Phil., Ph.D., Associate Professor, PG and Research Department of Zoology,
Government Arts College (Autonomous), Coimbatore, for her inspiring and
valuable Guidance, constant support and encouragement brought my project to a
successful completion.
I am also thankful to Dr. P. Jayalakshmi, Principal, Government Arts
College, Coimbatore for permitting me to carry out the research work in the
college.
My sincere thanks to Dr. P. Esther Joice, Head, Department of Zoology,
Government Arts College, Coimbatore, for providing all the facilities necessary for
the project work.
I extend my deep regards to the lab technicians S. Velumani, K.
Ravichandran and V. Maheswari for providing necessary lab equipment.
My heartfelt thanks to Mrs. Sumaiya and Ms. Kiruba, Department of
Zoology, Government Arts College, Coimbatore for spending her valuable time,
providing physical and moral support in carrying out the experiments.
I take an opportunity to thank my parents Mr. G. Ravikumar, Mrs. R. Renuka
Devi and my sister Ms. R. Udhaya for supporting and helping me in doing the
project work successfully.
Abbreviations
ABBREVIATIONS
ALP
-
Alkaline Phosphatase
ALT
-
Alanine Amino Transanine
AST
-
Aspartate Amino Transaminase
ATP
-
Adenosine Triphosphate
bFGF
-
Beta Fibroblast Growth Factor
CYP
-
Cytochrome P
EPA
-
European Prosthodontic Association
ETU
-
Ethylene Thiourea
GRAS
-
Gleason Research Associates Corporated
GSH
-
Growth Stimulating Hormone
GST
-
Glutathione- s- transferase
HCT
-
Human Colorectal Carcinoma Cell Line
HDL
-
Human Derived Lactobacili
HIV
-
Human immuno deficiency Virus
KC
-
Kupffer Cell
LDH
-
Lactate Dehydrogenase
LTA4H
-
Leukotrine A4 Hydrolase
NAC
-
N - Acetyl - cystenine
NAG
-
N- Acetyl Glutcosamine
NAPQI
-
N – Acetyl- p- benzoquinoneimine
NFκB
-
Nuclear Factor B
NK
-
Natural killer cells
NKT
-
Natural killer T cell
NTP
-
National toxicology program
OTC
-
Over the counter
T3
-
Triiodothyronine
T4
-
Thyroxin
TCM
-
Traditional Chinese Medicine
TNF
-
Tumor necrotic factor
TRH
-
Thyrotropin releasing hormone
TSH
-
Thyroid stimulating hormone
UDP
-
Uridine diphosphate
VEGF
-
Vascular endothelial growth factor
Contents
CONTENTS
1. INTRODUCTION
1
2. REVIEW OF LITERATURE
2.1. THYROID
2.2. LIVER
2.3 GINGER
2.4. GINGEROL
2.5. ETHYLENETHIOUREA
4
3. SCOPE OF THE STUDY
17
4. MATERIALS AND METHODS
4.1. SELECTION OF THE ANIMAL MODEL:
4.2. PREPARATION OF GINGER EXTRACT
4.3. PREPARATION OF ETHYLENE THIOUREA AND (6) GINGEROL
4.4. EXPERIMENTAL GROUPS
4.5. ASSAY OF BIOCHEMICAL PARAMETERS
4.5.1. ESTIMATION OF THYROXINE
4.5.2. ESTIMATION OF TRIIODOTHYRONINE
4.5.3. ESTIMATION OF THYROID STIMULATING HORMONE
4.5.4. ESTIMATION OF TOTAL PROTEIN
4.5.5. ESTIMATION OF SGOT
4.5.6. ESTIMATION OF SGPT
4.5.7. ESTIMATION OF ALP
4.6. HISTOLOGICAL STUDIES
4.7. STATISTICAL ANALYSIS
18
5. RESULTS
5.1 EFFECT ON BODY WEIGHT
5.2 EFFECT ON THYROID HORMONES
5.3 EFFECT ON THYROID HISTOLOGY
5.4 EFFECT ON SERUM PROTEIN
5.5 EFFECTS OF LIVER MARKER ENZYMES
5.6 EFFECT ON LIVER HISTOLOGY
24
6. DISCUSSION
6.1 EFFECT ON BODY WEIGHT
6.2 EFFECT ON THYROID
6.3. EFFECT OF LIVER
26
7. CONCLUSION
31
8. REFERENCES
32
9. WORKSHOPS AND SEMINARS ATTENDED
introduction
1. INTRODUCTION
Herbal prescriptions and natural remedies are commonly employed in the developing
countries for the treatment of various diseases. The use of medical herbs have increased over
the past few years and research interest has focussed on various herbs that possess anti-tumor or
immune-stimulating properties that may be useful adjuncts in helping to reduce the risk of
cancer (Craigh, 1999). Most chemical carcinogens have to be metabolically activated in the
body before exerting their carcinogenicity (Miller, 1970)
The research on herbs have increased all over the world and a large body of evidence can
be collected to show the immense potential of medicinal plants, used in various traditional
system (Dhanukar, 2000). Herbal medicines are cheaper and available based on the promise
that plants contain natural substances. Numerous herbs have been employed for treatment for
cancer.
GINGER
Ginger (Zingiber officinale roscoe) is a flowering plant in the family Zingiberaceae
whose rhizome, ginger root or simply ginger, is widely used as a spice or a folk medicine. It is a
herbaceous perennial which grows annual stems about a meter tall bearing narrow green leaves
and yellow flowers.
Ginger has been traditionally used from the time immemorial for varied human ailments
in different parts of the globe to aid digestion and treat stomach upset, diarrhoea and nausea.
Some pungent constituents present in ginger and other zingiberaceous plants have potent antioxidant and anti-inflammatory activities and some of them exhibit cancer preventive activity in
experimental carcinogenesis. The anti-cancer properties of ginger are attributed to the presence
of certain pungent vallinoids, viz., (6)gingerol and (6) paradol, as well as some other constituents
like shogaols, zingerone etc. A number of mechanisms that may be involved in the
chemopreventive effects of ginger and its components have been reported from the laboratory
studies in a wide range of experimental models.
GINGEROL:
The oleoresin from rhizomes of ginger contains (6) gingerol [1-(4-hydroxyl-3methoxyphenyl)-5-hydroxy-3-decanone] and its homologs which are pungent ingredients that
have been found to possess many interesting pharmacological and physiological activities such
as anti-inflammatory, anti-hepatotoxic and cardiotonic effects. (6) gingerol inhibits cell adhesion,
invasion, motility and activities of MMp-2 and MMP-9 in MDA-MB-231 human reast cancer
cells. (6) gingerol, a pungent ingredient of ginger, has anti-bacterial, anti-inflammatory and antitumor promoting activities. Invitro, (6) gingerol inhibited both the VEGF- and bFGF- induced
proliferation of human endothelial cells and caused cell cycle arrest in the GI phase. It also
blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly
inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the
mouse cornea in response to VEGF.
ETHYLENE THIOUREA
Ethylene thiourea is a white crystalline solid used extensively in the rubber industry as an
accelerator in the vulcanization of elastomers. It is also a trace contaminant and metabolic
degradation product of a widely used class of ethylene bisdithiocarbamate fungicides. Ethylene
thiourea is known to produce thyroid neoplasms in rats and liver neoplasms in mice following
long-term administration; thus, it was chosen by the National Toxicology Program in an
investigation of the potential value of perinatal exposures in assessing chemical carcinogenicity.
Chronic toxicity and carcinogenicity studies of ethylene thiourea, 99% pure, were
conducted in F344/N rats and B6C3F1 mice of each sex. The studies were designed to determine
1) the effects of ethylene thiourea in rats and mice receiving adult exposure only (a typical
carcinogenicity study), 2) the toxic and carcinogenic effects of ethylene thiourea on rats and
mice receiving perinatal expo- sure only (dietary exposure of dams prior to breeding and
throughout gestation and lactation), and 3) the effects of combined perinatal and adult exposure
to ethylene thiourea.
It is an organosulphur compound , an example of an N-Substituted thiourea, this
compound is synthesised by treating ethylenediamine with carbon disulphide. Ethylene thiourea
is used in the rubber industry and in the production of some fungicides. Ethylene thiourea has
been shown to be a potent teratogen, in rats orally or dermally exposed. A study of female
workers occupationally exposed to thiourea did not report an increased incidents of thyroid
cancer. Increased incidence of carcinoma and hepatomas (liver tumors) have been observed in
rats and mice orally exposed to ethylene thiourea. In study by the NTP, an increase in incidence
of thyroid tumors in rats, and thyroid, liver and pituitary gland tumors in mice exposed to
ethylene thiourea were noted. EPA has classified ethylene thiourea as a group B2 probable
human carcinogen.
CANCER:
Cancer, known medically as a malignant neoplasm, is a broad group of varied diseases,
all involving cell growth. In cancer, cells divide and grow uncontrollably forming malignant
tumors, and invade near-by parts of the body. The cancer may also spread to moredistant part of
the body thgrough the lymphatics or blood stream. Not all tumors are cancerous. Benign tumors
do not grow uncontrollably, do not invade neighbouring tissue and do not spread throughout the
body. There are over 200 different known cancers that affect humans. Determining what causes
cancer is complex. Many things are known to increase the risk of cancer, including tobacco use,
certain infections, radiation, lack of physical activity, obesity and environmental pollution
(Anand et al., 2008).
LIVER:
The liver is the second largest organ in the human body after skin. And is the largest
internal organ. Most liver has 50,000 to 1,00,000 lobules that consist of the vein surrounded by
tiny liver cells, called hepatocytes. These cells purify the blood, remove wastes, toxins and
poisons and store healthy nutrient for the body to use when needed. The liver reacts with 8
different types of responses to injury towards variety of metabolic, toxic, microbial, circulation
and neoplastic insults (Cotran and Collins 1999).
Liver cancer is the second most common cancers in the world and most common in Asia,
Africa and Southern Europe. Liver cancer or hepatic cancer (from the Greek Hepar, meaning
liver) is a cancer that originates in the liver, and are malignant tumors that grow on the surface or
inside the liver. Liver tumors are discovered on medical imaging equipment or present
themselves symptomatically as an abdominal mass, abdominal pain, jaundice, nausea, or liver
dysfunction.
THYROID:
Thyroid is a large ductless gland in the neck which secretes hormones, that regulate
growth and development through the rate of metabolism. Thyroid cancers result in the
production of abnormal amount of thyroid hormones, upsetting the body‘s chemical balance, as
well as other physiological disturbances.
Review of literature
2. REVIEW OF LITERATURE
2.1. THYROID
The thyroid gland is a butterfly-shaped organ located in the base of your neck. It releases
hormones that control metabolism—the way your body uses energy. The thyroid hormones
regulate vital body functions, including:

Breathing,

Heart rate,

Central and peripheral nervous systems,

Body weight,

Muscle strength,

Menstrual cycles,

Body temperature and

Cholesterol levels.
The thyroid gland is about 2-inches long and lies in front of the throat below the
prominence of thyroid cartilage, sometimes called the Adam's apple. The thyroid has two sides
called lobes that lie on either side of the windpipe, and is usually connected by a strip of thyroid
tissue known as an isthmus. The thyroid is part of the endocrine system, which is made up of
glands that produce, store, and release hormones into the bloodstream so the hormones can reach
the body's cells. The thyroid gland uses iodine from the foods to make two main hormones:

Triiodothyronine (T3)

Thyroxine (T4)
Two glands in the brain—the hypothalamus and the pituitary communicate to maintain
T3 and T4 balance.
The hypothalamus produces TSH Releasing Hormone (TRH) that signals the pituitary to
tell the thyroid gland to produce more or less of T3 and T4 by either increasing or decreasing the
release of a hormone called thyroid stimulating hormone (TSH).

When T3 and T4 levels are low in the blood, the pituitary gland releases more TSH to stimulate
the thyroid gland to produce more thyroid hormones.

If T3 and T4 levels are high, the pituitary gland releases less TSH to the thyroid gland to slow
production of these hormones. The hormones regulate the speed with which the cells/metabolism
works.
The thyroid gland is capable of meeting physiologic demands for T4 and T3 up
to a point. However, beyond that point, continuous stimulation of the thyroid may result in
changes that could eventually lead to disease, including neoplasia. Persistent elevation of TSH
levels stimulates the thyroid gland to deplete its existing stores of thyroid hormone. When the
thyroid is not able to keep up with the demand, the follicular cells hypertrophy and cells divide,
leading to hyperplasia and nodular hyperplasia. Generally, effects are reversible upon removal of
the stimulus, at least early in the process. However, if the stimulus continues, benign and then
malignant neoplasms can result. In some cases, chronic stimulation also results in pituitary
hyperplasia or tumors involving the cells that produce TSH. There are many ways chemicals
produce antithyroid effects (i.e., perturb thyroid-pituitary homeostasis) that reduce circulating
thyroid hormone, increase TSH, and increase thyroid cancer potential in rodents (Capen and
Martin 1989). In the thyroid, these include: 1) inhibition of the active transport of inorganic
iodide into the follicular cell (iodide pump); 2) inhibition of thyroid peroxidase that converts
inorganic iodide into organic iodide and couples iodinated tyrosyl moieties into thyroid hormone;
3) damage to follicular cells; and 4) inhibition of thyroid hormone release into the blood. Outside
the thyroid, chemicals can cause 5) inhibition of the conversion of T4 to T3 by 5'monodeiodinase at various sites in the body and 6) enhancement of the metabolism and excretion
of thyroid hormone by the liver, largely through the action of uridine diphosphate (UDP)
glucuronosyltransferase.
2.2. LIVER
The liver is among the most complex and important organs in the human body. Its primary
function is to control the flow and safety of substances absorbed from the digestive system
before distribution of these substances to the systemic circulatory system. It lies below the
diaphragm in the pelvic region of the abdomen. The liver is a reddish brown organ with four
lobes of unequal size and shape. It is both the largest internal organ and the largest gland in the
human body. It is connected to two large blood vessels, one called the hepatic artery and one called
the portal vein. It constitutes about 2.5% of an adult‘s body weight. During rest, it receives 25%
of the cardiac output via the hepatic portal vein and hepatic artery. The hepatic portal vein
carries the absorbed nutrients from the GI tract to the liver, which takes up, stores, and
distributes nutrients and vitamins. It produces bile, an alkaline compound which aids in
digestion via the emulsification of lipids. Two major types of cells populate the liver lobes:
parenchymal and non-parenchymal cells. 80% of the liver volume is occupied by parenchymal
cells commonly referred to as hepatocytes. Non-parenchymal cells constitute 40% of the total
number of liver cells but only 6.5% of its volume. Sinusoidal endothelial cells, kupffer cells and
hepatic stellate cells are some of the non-parenchymal cells that line the hepatic sinusoid (Kmiec
2001).
The major aspects of hepatic structure include: The hepatic vascular system, which has
several unique characteristics relative to the organ, the biliary tree, which is a system of ducts
that transport bile out of the liver into small intestine, the three dimensional arrangement of the
liver cells, or hepatocytes and their association with the vascular and biliary system.
The liver is covered with covered with connective tissue capsules that branches and extend
throughout the substance of the liver as septae. This connective tissue is the highway through
which afferent blood vessels, lymphatic vessels and bile duct traverse the liver. Additionally, the
sheets of connective tissue divide the parenchyma of the liver into very small units called
lobules.
2.2.1. FUNCTION OF LIVER
The liver has well over 500 functions and is known as the laboratory of the human body.
The liver is tied to all bodily processes because it is responsible for filtration of all incoming
foods and fluids. The body relies upon the liver to remove toxins so that nutrients supplied to the
body are pure and capable of providing nourishment. Many scientists believe the liver is
connected to, or at least aware, of every disease or dysfunction that is happening inside the body.
Carbohydrate metabolism
Liver maintains the normal blood glucose level. It converts glucose to glycogen
(glycogenesis) when blood sugar level is high and break down glycogen to glucose
(glycogenolysis) when blood sugar level is low. Also liver can convert amino acid and lactic
acid to glucose (gluconeogenesis) when blood sugar level is low.
Lipid metabolism
Liver stores some triglycerides (neutral fat) and breaks down fatty acids into acetyl
coenzyme-A. This process is called as oxidation and converts excess acetyl coenzyme A into
ketone bodies (ketogenesis). It synthesizes lipoproteins. Hepatic cells synthesize cholesterol and
use cholesterol to make bile salts.
Protein metabolism
The liver deaminates (remove the amino group, NH2) from amino acids so that they can
be used for ATP production. It converts the resulting toxic ammonia (NH3) into the much less
toxic urea for excretion in urine. Hepatic cells synthesize plasma protein such as alpha and beta
globulin, albumin, prothrombin and fibrinogen.
Haematological function:
The liver produces coagulation factors I (fibrinogen), II (prothrombin), V, VII, IX, X and
XI, as well as protein C, protein S and antithrombin. In the first trimester foetus, the liver is the
main site of red blood cell production. By the 32nd week of gestation, the bone marrow almost
completely takes over that task.
Secretion and excretion of bile:
Bile is partially an excretory product and partially a digestive secretion. Each day the
hepatic cells secrete 800-1000ml of bile, a yellow or olive green liquid. It has pH of 7.6- 8.6.
Bile mainly consists of water, bile salts, cholesterol, a phospholipid called lecithin, bile
pigments and several ions. The principle bile pigment is bilirubin. When worn out red blood
cells break down, iron, globin‘s and bilirubin (derived from heam) are released.
Breakdown:
The breakdown of insulin and other hormones. The liver glucoronidates bilirubin,
facilitating its excretion into bile. The liver breaks down or modifies toxic substances
(e.g.,methylation) and most medicinal products in a process called drug metabolism. This
sometimes results in toxication, when the metabolite is more toxic than its precursor.
Preferably, the toxins are conjugated to avail excretion in bile or urine. The liver converts
ammonia to urea (urea cycle).
Other functions
The liver stores a multitude of substances, including glucose (in the form of glycogen),
vitamin (1-2years' supply), vitamin D (1-4 months' supply), vitamin B12 (1-3 years' supply),
iron, and copper. The liver is responsible for immunological effects—the reticulo endothelial
system of the liver contains many immunologically active cells, acting as a 'sieve' for antigens
carried to it via the portal system. The liver produces albumin, the major osmolar component
of blood serum. The liver synthesizes angiotensinogen, a hormone that is responsible for
raising the blood pressure when activated by renin, an enzyme that is released when the kidney
senses low blood pressure. The liver also produces insulin-like growth factor 1 (IGF-1), a
polypeptide protein hormone that plays an important role in childhood growth and continues to
have anabolic effects in adults. The liver is a major site of thrombopoietin production.
Thrombopoietin is a glycoprotein hormone that regulates the production of platelets by the
bone marrow.
The liver is one of the most important organs that performs high activity in metabolism
and has a chief role in detoxification process and withdrawal of many toxic substances which
enter the body (Yamazuki and LaRusso, 1988).
Liver plays a pivotal role in regulating various physiological processes. It is also involved
in several vital function, such as metabolism, secretion and storage. It has great capacity to
detoxicate toxic substances and synthesize useful principle. It helps in the maintenance,
performance and regulation of homeostasis of the body. It is involved with almost all the
biochemical pathways to growth, fight against disease, nutrient supply, energy provision and
reproduction. It aids in metabolism of carbohydrate, protein and fat, detoxification, secretion of
bile and storage of vitamins
The role played by this organ in the removal of substances from the portal circulation makes
itsusceptible to first and persistent attack by offending foreign compounds, culminating in liver
dysfunction. These hepatotoxic agents activate some enzyme activity in the cytochrome p-450
system such as CYP2E1 leading to oxidative stress. Injury to hepatocyte and bile duct cells lead
to accumulation of bile acid inside liver. This promotes further liver damage. Liver is also the
major reticulo endothelial organ in the body and as such has important immune function in
maintaining body veracity. Damaging hepatocyte results in the activation of innate immune
system like kupffer cells (KC), natural killer (NK) cells, and natural killer T(NKT) cells and
result in producing proinflammatory
mediators such as tumor necrosis factor-α (TNF),
interferon-ᵞ (IFN), and interleukin-β (IL) which produced liver injury. Many agents damage
mitochondria, an intracellular organelle that produces energy. In mitochondria, hepatocellular
death is a direct result of drugs acting on these organelles (e.g., drug accumulation, inhibition of
electron transport and fatty acid oxidation, or depletion of anti-oxidant defenses). An indirect
result ensuing from mitochondrial participation is program of cell death. These programs lead to
necrosis or apoptosis; they are mediated through signalling mechanisms arising at the cell
membrane (e.g., death receptors) or in subcellular compartments (e.g., the endoplasmic reticulum
or cell nucleus).Its dysfunction releases excessive amount of oxidants which, in turn, injure
hepatic cells. Nonparenchymal cells such as Kupffer cells, fat storing stellate cells, and
leukocytes (i.e. neutrophil and monocyte) also have role in the mechanism of hepatotoxicity.
Thus liver is an important organ which is actively involved in many metabolic functions
and is the frequent target for a number of toxicants (Meyer 2001) Hepatic damage is associated
with distortion of these metabolic functions (Wolf and Clin, 1999). Liver disease is still a
worldwide health problem. Unfortunately, conventional or synthetic drugs used in the treatment
of liver diseases are inadequate and sometimes can have serious side effects (Guntupalli et
al.,2006) In view of severe undesirable side effects of synthetic agents, there is growing need to
utilize abundant plant resources available and to evaluate scientific basis for the medicinal plants
that are claimed to possess hepatoprotective activity.
The hepaticlobule is the structured unit of the liver. It consists of roughly hexagonal
arrangement of plates of hepatocytes radiating outward from a central vein in the cancer. At the
vertices of the lobule are regularly distributed portal vein (Ledezma et al., 1999).
2.2.2. LIVER TOXICITY
Hepatotoxicity implies chemical-driven liver damage. Certain medicinal agents, when taken
in overdoses and sometimes even when introduced within therapeutic ranges, may injure the
organ. Other chemical agents, such as those used in laboratories (e.g. CCl4, paracetamol) and
industries (e.g. lead, arsenic), natural chemicals (e.g.microcystins, aflatoxins) and herbal
remedies (Cascara sagrada, Ephedra) can also induce hepatotoxicity. Chemicals that cause liver
injury are called hepatotoxins. These agents convert chemically reactive metabolites in liver,
which have the ability to interconnect with cellular macromolecules such as protein, lipids and
nucleic acids, leading to protein dysfunction, lipid per oxidation, DNA damage and oxidative stress.
This damage of cellular function can result in cell death and likely liver failure. More than 900
drugs have been implicated in causing liver injury 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. More than 75 percent of cases of
idiosyncratic drug reactions result in liver transplantation or death.
Some of the liver injuries are caused by the use and abuse of drugs. Conventional or
synthetic drugs such as steroids, vaccines, antivirals, and other medications can cause serious
side effects, even toxic effects on the liver, especially when used for prolonged periods of time
(Ali & Fahmy 2009). Paracetamol, a widely used over-the-counter (OTC) analgesic and
antipyretic, is one of the best known experimental models of hepatotoxicity (Bailey et al., 2003).
It is safe at therapeutic doses, but causes a fatal hepatic necrosis and hepatic failure in overdose
(Bessems, et al., 2001). It was found that induction of CYP2E1, CYP1A2, CYP3A4, depletion of
intracellular GSH, and oxidative stress are the major mechanisms involved in the pathogenesis of
paracetamol induced liver injury (Bhandari et al., 2005). Paracetamol at therapeutic doses is
rapidly metabolized in the liver principally through glucuronidation and sulfation and only a
small portion is oxidized by cytochrome P-450 2E1, to generate a highly reactive and cytotoxic
intermediate, N-acetyl -P- benzoquinoneimine (NAPQI), which is quickly conjugated by hepatic
glutathione to yield a harmless water soluble product, mercapturic acid (Cheng et al., 1999).
When paracetamol is dosed at higher dose levels in animals or humans, metabolism of
paracetamol through glucuronidation and sulfation is saturated and NAPQI is synthesized in
enough amounts to cause acute hepatotoxicity (Chrubasik et al., 2005). Many research efforts are
directed to the discovery and development of agents, which might protect cells from oxidative
reactions with potential antioxidant and hepatoprotectiveeffects (Sharaky et al., 2009). The most
popular antioxidant for paracetamol hepatotoxicity is N-acetyl-L-cysteine (NAC) (Farag et al.,
2010).
2.5 GINGER
Medicinal plants play a key role in the human health care system. Pharmacological
medicinal plants play a key role in the evaluation of these plants and their taxonomical health
care system. Herbal medicines are in great demand. Various chemicals and drugs have been
developed for primary health care because of their efficacy, safety, lesser side effects and narrow
therapeutic window. Therefore, the use of herbal drugs is much safer than synthetic products
available in the market. Herbal remedies support natural healing phenomena through blocking of
the progression of the degenerative pathological processes. Modern medicine offers limited
success in providing effective cure and there is a severe need to develop new drug capable of
healing toxic liver damages. In traditional system of medicine, plant were claimed to be effective
and used successfully to alleviate multiple liver disorders. Ayurveda has a clinical speciality
called rasayana, which prevent disease and counteracts the aging process by means of
optimization of homeostasis. It has been reported that rasayanas are rejuvenators, nutritional
supplements and possess strong antioxidant activity. Hepatic injury leads to disturbances in
transport, function of hepatocytes, resulting in leakage of plasma membrane, thereby causing an
increased enzyme level in serum. All the hepatotoxins induced ROS production in the body,
leading to depletion of antioxidant status of hepatic tissue, inducing lipid peroxidation and
degradation of bio membrane. Administration of antioxidant, which can scavenge the free
radicals, could reduce the hepatic injury.
There are many herbal medicinal plants having
antioxidant properties which show hepatoprotective activity.
Ginger (Zingiber officinale Rosc) (Family: Zingiberaceae) is a herbaceous perennial, the
rhizomes of which are used as a spice. India is a leading producer of ginger in the world and
during 2006-07 the country produced 3.70 lakh tones of the spice from an area of 1.06 lakh
hectares. Ginger is cultivated in most of the states in India (Tapsell et al.,2006). However, states
namely Kerala, Meghalaya, Arunachal Pradesh, Mizoram, Sikkim, Nagaland and Orissa together
contribute 70 per cent to the country‘s total production. In India the average daily consumption is
8-10 gms of fresh ginger root. Warm and humid and is cultivated from sea level to an altitude of
1500 m about sea level. It can be grown both under rain fed and irrigated conditions (Sasikumar,
et al.,2008).
Ginger (Zingiber officinale Roscoe) is a widely used herb and food-flavouring agent. Its
neutraceutical properties have long been of interest to the food processing and pharmaceutical
industries. The volatile essential oils contributing to the characteristic flavour of ginger, varies
from 1.0-3.0%. While the oleoresin, responsible for the pungent flavour of ginger, varies from
4.0-7.5% and also possesses substantial antioxidant activity (Balachandran et al., 2006). [6]gingerol is the most abundant constituent of fresh ginger but it decreases during post-harvest
storage and processing, especially thermal processing (He et al., 1998).
The Chinese have used ginger for at least 2500 years as a digestive aid and antinausea
remedy and to treat bleeding disorders and rheumatism; it was also used to treat baldness,
toothache, snake bite and respiratory conditions (Duke et al.,1985). In Traditional Chinese
Medicine (TCM), ginger is considered as a pungent, dry, warming, yang herb to be used for
ailments triggered by cold, damp weather.
In Malaysia and Indonesia, ginger soup is given to new mother for 30 days after their
delivery to help warm them and to help them sweat out impurities. In Arabian medicine, ginger
is considered an aphrodisiac (Qureshi et al.,1989). Nowadays ginger is extensively cultivated
from Asia to Africa and the Caribbean and is used worldwide as a nausea remedy, as an antispasmodic and to promote warming in case of chills (Kapil et al.,1990). The use of plant to treat
various diseases in India dates back to the times of Rigveda. Later, the monumental Ayurvedic
works like Charaksamhita and Sushrutasamhita followed by other Ayurveda and Siddha treatises
have incorporated nearly 700 plant drugs entering in to several medicine preparation used in the
management of the health care. In fact this system has been in practice even in remote areas of
our country for centuries (Yoganarasimhan, 1996). The ginger family is a tropical group
especially abundant in Indo- Malaysia, consisting of more 1200 plant species in 53 genera. The
genus Gingiber includes about 85 species of aromatic herbs from East Asia and tropical
Australia. The name of the genus, Zingiber, derived from a Sanskrit word denoting ―hornshaped,‖ in reference to the protrusions on the rhizome (Awang, 1992).Ginger has been noted to
treat migraine headaches without side effects (Mustafa, and Srivastava 1990).
The volatile oil components in ginger consist mainly of sesquiterpene hydrocarbons,
predominantly zingeberene (35%), curcumene (18%) and farnesene (10%), with lesser amounts
of bisabolene, mono terpenoid hydrocarbons, 1, 8-cineole, linalool, borneol, neral, and geraniol
and bsesquiphellandrene. Many of these volatile oil constituents contribute to the distinct aroma
and taste of ginger. Non-volatile pungent compounds contain biologically active constituents
including the non-volatile pungent principles, such as the gingerols, shogaols, paradols and
zingerone that produce a ‗‗hot‘‘ sensation in the mouth. The gingerols, a series of chemical
homolog differentiated by the length of their unbranched alkyl chains, were identified as the
major active components in the fresh rhizome (Connell, & Sutherland,1969). Other constituents
are oleoresins, fats, waxes, carbohydrates, vitamins and minerals. Ginger rhizomes also contain a
potent proteolytic enzyme called zingibain (Govindarajan, 1982).
Thus, Ginger is one of the most important natural medicinal plant which is used for the
various traditional and medicinal purposes in India as well as China, Caribbean, Africa and
African countries. This natural gift is consumed worldwide as a spice and flavouring agent from
the ancient time. Ginger has been used for the centuries to support various digestive imbalances
including, indigestion, nausea, motion sickness, vomiting, diarrhoea, coughing, heartburn and
many other uses. Ginger bears an enormous number of pharmacological activities among those,
neuro-protective activity and activity against colon cancer have facilitated the extent of further
research for finding out less toxic and more potent drugs for the better treatment of those
diseases.
2.6. GINGEROL
Ginger contains essential oils especially gingerol and gingiberene. It also contains
pungent principles such as ginger one, gingerol and shogaol (Yamahara, 1985). Gingerol can be
seen in two forms depending upon its structure (the position of the methoxy group) known as
[6]-gingerol and [10]-Gingerol. Both are the active constituent of fresh ginger which is
chemically related with capsaicin and piperine. It is normally found as pungent yellow oil, but
also can form a low-melting crystalline solid (McGee.,2004). In the fresh ginger rhizome, the
gingerols were identified as the major active components and [6] gingerol [5-hydroxy-1-(4hydroxy-3-methoxy phenyl) decan-3-one is the most abundant constituent in the gingerol series.
In dried ginger powder, shaogaol, a dehydrated product of gingerol, is a predominant pungent
constituent of biosynthesis (Mustafa et al., 1993).
Chul et al., (2009)have studied that [6]-gingerol, the major pharmacologically active
component of ginger which have antioxidant and anti-inflammatory properties and exert
substantial anticarcinogenic and antimutagenic activities.
Compound [6]-gingerol was most effective at lower doses in inhibiting endothelial cell
tube formation. The in vitro studies show that [6]-gingerol has two types of antitumor effects: 1)
direct colon cancer cell growth suppression, and 2) inhibition of the blood supply of the tumor
via angiogenesis. Further research is warranted to test [6]-gingerol in animal studies as a
potential anticancer plant bioactive in the complementary treatment of cancer.
Kim et al.,(2005) have performed that [6]-Gingerol has anti-tumor-promoting activities.
They reported its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol
inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused
cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells
in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and
formation of new blood vessel in the mouse cornea in response to VEGF. The results
demonstrate that [6]- gingerol inhibits angiogenesis and may be useful in the treatment of tumors
and other angiogenesis-dependent diseases. [6]-gingerol stimulates apoptosis through up
regulation of NAG-1 and G1 cell cycle arrest through down regulation of cyclin D1 (Seong, et
al., 2008).
The leukotrienes are structurally related to paracrine hormones derived from the
oxidative metabolism of arachidonic acid. Leukotriene can provoke human cancer and chronic
inflammation (Funk, 2001). Leukotrienes are found at high levels in most inflammatory lesions
and are involved in the physiologic changes that are characteristic of the inflammatory process
(Fabre et al., 2002) Leukotrienes, such as leukotriene B4 (LTB4), a potent chemo attractant that
induces a forceful inflammatory response, are implicated in cancer development (Gunning et
al.,2002). Because LTB4 have a role in carcinogenesis, where leukotriene A4 hydrolase
(LTA4H) act as an attractive target for chemoprevention and cancer therapy (Chen et al., 2004).
LTA4H is a bifunctional zinc enzyme that catalyzes the final rate-limiting step in the
biosynthesis of LTB4. Besides catalyzing the production of LTB4, LTA4H also possesses
aminopeptidase activity (Orning et al.,,1991). LTA4H was shown to exhibit high levels of
protein expression in certain types of cancers, and its inhibition leads to reduced cancer
incidence in animal models (Arguello et al., 2006). LTA4H caused carcinoma in the HCT116
cell in the colonrectal cancer. [6]-Gingerol directly binds with LTA4H, [6]-gingerol inhibits
LTA4H enzyme activity. The secreted LTB4 levels in HCT and HT cells. [6]-gingerol
suppresses LTB4 production in both cell lines. The inhibitory effect of [6]-gingerol against
aminopeptidase activity was further evaluated in vitro by using a p-nitroanilide derivative of
alanine (Ala-p-NA) as substrate. The aminopeptidase activity of LTA4H was also potently
suppressedby [6]-gingerol. [6]-gingerol suppresses tumor growth of HCT116 cells implanted in
nude mice by inhibiting the enzymatic activity of LTA4H. Results showed that dietary intake of
ginger does not significantly changes the proliferative or apoptosis indexes of the colonic crypt
cells (Dias et al., 2006). It also contains acrid resinous substances 5-8%19. Ginger contains up to
3% of a fragrant essential oil whose main constituents are sesquiterpenoids, with (-)-gingeberene
as the main components. Ginger also contains amadaldehyde, paradols, gingerdiols,
gingerdiacetates, gingerenones, 6-gingersulfonic acid, diterpenes, gingerglycolipids A, B and C.
The other minor compounds are methylegingediol, methylegingediacetates and C20- dialdehyde.
The oleoresin from rhizomes of ginger contains (6) gingerol [1-(4-hydroxy -3-methoxy
phenyl)-5-hydroxy-3-decanone] and its homologs which are pungent ingredients that have been
found to possess many interesting pharmacological and physiological activities, such as antiinflammatory, anti-hepatotoxic and cardiotonic effects. (6)gingerol inhibits cell adhesion,
invasion, motility and activities of MMP-2 and MMP-9 in MDA-MB-231 human breast cancer
cells. (6) Gingerol, a pungent ingredients of ginger (Zingiber officinale roscoe, Zingiberaceae),
has anti-bacterial, anti-inflammatory and anti-tumour promoting activities . Invitro, (6)gingerol
inhibited both the VEGF-and bFGF induced proliferation of human endothelial cells and caused
cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells
in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and
formation of new blood vessel in the mouse cornea in response to VEGF.
2.7. ETHYLENETHIOUREA
Ethylene thiourea occurs as white-to-pale-green needle-like crystals with a faint amine
odour. It is very soluble in hot water; slightly soluble in cold water, methanol, ethanol, ethylene
glycol, pyridine, acetic acid, and naphtha; and insoluble in acetone, ether, chloroform, and
benzene. When heated to decomposition, ethylene thiourea emits toxic fumes of nitrogen oxides
(NOx) and sulfur oxides (SOx). Ethylene thiourea is available in the United States as crystals, as a
powder, as an 80% dispersion of the powder in oil, or encapsulated in a matrix of compatible
elastomers. Ethylene thiourea is used primarily as an accelerator for vulcanizing polychloroprene
(neoprene) and polyacrylate rubbers. Neoprene rubbers are used almost exclusively in industrial
applications, e.g., for mechanical and automotive products, in wire and cable production, in
construction, and in adhesives. Polyacrylate rubbers are used in products such as seals, o-rings,
and gaskets for automotive and aircraft applications. Ethylene thiourea is used in the
manufacture of ethylenebisdithiocarbamate pesticides, such as amobam, maneb, mancozeb,
metiram, nabam, and zineb. Ethylene thiourea is also used in electroplating baths, as an
intermediate in antioxidant production, in dyes, pharmaceuticals, and synthetic resins. There is
sufficient evidence for the carcinogenicity of ethylene thiourea in experimental animals. When
administered in the diet, ethylene thiourea induced thyroid follicular cell carcinomas in rats of
both sexes. When administered by gavage, ethylene thiourea induced hepatomas in mice of both
sexes.
Ethylene Thiourea and its derivatives have found extensive applications in the fields of
medicine, agriculture and analytical chemistry. They are known to exhibit a wide variety of
biological activities such as antiviral, antibacterial, antifungal, (Saeed et al.,2009) antitubercular,
herbicidal, insecticidal (Zhang 2004,) and to act as chelating agents, in catalysis, (Arslan et
al.,2009) in anion recognition and to play a role in some epoxy resin curing agents containing
amino functional groups. (Saeed et al., 2009).
It is an organosulfur compound, an example of an N-Substituted thiourea, this compound
is be synthesized by treating ethylene diamine with carbon disulfide. Ethylene thiourea is used in
the rubber industry and in the production of some fungicides. Ethylene thiourea has been shown
to be a potent teratogen in rats orally or dermally exposed. Increased incidence of thyroid
carcinoma and hepatomas (liver tumors) have been observed in rats and mice orally exposed to
ethylene thiourea.
Ethylene Thiourea are important organic compounds that possess high biological
activity, act as corrosion inhibitors and antioxidant, and are polymer component1.Thiourea and
urea derivatives show a broad spectrum of biological activities as anti-HIV, antiviral, HDLelevating, antibacterial and analgesic properties. Acyl thiourea derivatives are well known for
wide range of biological activities like bactericidal, fungicidal, herbicidal, insecticidal action and
regulating activity for plant growth. On the other hand, some thiourea derivatives have been used
in commercial fungicides. They are selective analytical reagents, especially for the determination
of metals in complex interfering materials.
Scope of the study
3. SCOPE OF THE STUDY
Plant derivatives have been used for medical purpose for centuries and are also being
used in our daily food intake. At present, it is estimated that about 80% of the world population
relies on botanical preparation as medicines to meet their health needs (Ajith et al., 2007).
Recently, considerable research has been carried out in the search for natural or synthetic
compounds as a means of chemically preventing cancer. Many of chemicals are capable of
generating oxidative stress in tissue also act as tumor promoters (Kensler and Taffe et al., 1986).
In the present study, the aqueous extract of ginger and gingerol are to be investigated for
their cancer therapeutic property by induction of ethylene thiourea in experimental rats. The
effect on thyroid hormones, serum transaminases, ALP and the histological studies of liver and
thyroid have been undertaken to ascertain the anticarcinogenic effect of ginger in the current
doses and duration.
Materials &methods
4. MATERIALS AND METHODS
4.1. Selection of the animal model:
Albino rats, which had tissue absorption, metabolism and excretion of test compound
comparable to that of human beings were selected for the present study. Wistar strain
albino rats weighing about 130-250 grams were to be selected, procured and acclimatized
to our laboratory condition for 2 weeks. The animal will be housed in a well-ventilated,
temperature and humidity controlled house with a light schedule of 14 hours and 10 hours
darkness. They were to be fed with standard diet and drinking water made available ad
libitum.
4.2. Preparation of ginger extract
To prepare an aqueous extract of ginger, ginger was washed with distilled water and
dried. 150 gm of ginger was taken and powdered in electric grinder to a fine powder and
passed through a 24- mesh sieve and stored in a plastic bag. 50 gm of ground powder was
soaked in 150 ml of hot water in a water bath for 6 hours, the filtrate were stored in dark
bottles, in the refrigerator at 45°C for future use.
4.3.Preparation of ethylene thiourea and (6) gingerol:
Ethylene thiourea and (6 ) gingerol were purchased from Sigma Chemical Co. Ethylene
thiourea and gingerol were dissolved in saline and given orally once a week at a dose of
5mg/100g BW for ethylene thiourea and 0.1ml/100g BW/day for 28 days for gingerol.
4.4.Experimental groups
Healthy male albino rats were divided into 6 groups of 4 animals and received the
following regimen of treatments
I.
GROUP I (Control)- Animals to receive normal saline 1ml/100mg BW/day for 28
days and used as control.
II.
GROUP II- Animals to receive ginger extract orally at 0.2ml/100g BW/day for 28
days.
III.
GROUP III- Animals to receive gingerol orally at 0.1ml/100g BW/day for 28 days.
IV.
GROUP VI- Animals to receive ethylene thiourea 5mg/100g BW/week for 28 days.
V.
GROUP V- Animals to receive both ethylene thiourea 5mg/100g BW/week and
gingerol 0.1ml/100g BW/day for 28 days.
VI.
GROUP VI- Animals to receive both ethylene thiourea 5mg/100g BW/week and
ginger extract 0.2ml/100g BW/day for 28 days.
All treatments were given between 9.30 to 10.00 hours in the morning.
After the
treatment protocol (28 days) animals were anesthetized with ether and sacrificed by cervical
decapitation. Blood was collected and allowed to clot for four hours at room temperature
centrifuged at 3000 rpm for 10 minutes to separate serum and stored at -20o until analysis. This
was then used to determine activities of liver marker enzymes such as AST, ALT, ASP as well as
protein and thyroid hormone levels. The thyroid and liver tissues were excised in 10 % neutral
buffered formalin for histopathological analysis.
4.5. ASSAY OF BIOCHEMICAL PARAMETERS:
4.5.1 ESTIMATION OF THYROXINE (T4)
The quantitative determination of thyroxine (T4) in serum is done using the ADVIA
Centaur CP System.
4.5.2. ESTIMATION OF TRIIODOTHYRONINE (T3)
The quantitative determination of triiodothyronine (T3) in serum is done using the
ADVIA Centaur CP System.
4.5.3. ESTIMATION OF THYROID-STIMULATING HORMONE (TSH)
The quantitative determination of thyroid-stimulating hormone (TSH, thyrotropin) in
serum is done using the ADVIA Centaur CP System
4.5.4. ESTIMATION OF TOTAL PROTEIN:
The protein present in the samples were determined using Ensure Biotech analyzer
instrument in the laboratory, (Kjeldahl, 1883).
Principle:
Total protein was first determined by the Kjeldahl‘s method. The peptide bond Of protein
reacts with copper II ions in Alkaline solution to form a Blue- violet
complex. The colour
formed is proportional to the protein concentration and is measured at 540 nm (520-560)
Procedure:
Clean dry test tubes were taken and labeled as B (Blank), S (Standard) and T (Test).
In the test tube B, 1.0ml of protein reagent and 10 µl distilled water was taken. In the test tube
S,1.0ml of protein reagent and 10µl of Standard solution were added. In the test tube T, 1.0ml of
protein reagent and 10µl of the sample were taken. The test tubes were mixed well and incubated
at room temperature for 5 minutes. Then the absorbance of the solution were read at 540 nm.
Using the absorbance the amount of protein present in the samples were calculated using the
formula,
Total protein conc =
(mg/100mg)
4.5.5.ESTIMATION OF SGOT
Estimation of serum glutamate oxaloacetate tansaminase was done by Reitman and
Frankel (1957) method.
Principle
SGOT catalyses the transfer of amino group from L-asparate to α ketoglutamate. The
oxaloacetate so formed were allowed to react with 2.4, dinitrophenylhydrazone derivative which
was brown color in alkaline medium. The absorbance of this hydrazone derivative was correlated
to SAT activity by plotting a calibration curve using Oxaloacetate.
Reagents
2.4 dinitrophenyl hydrazine (DNPH), 0.4N Sodium hydroxide, GOT Buffer substrate
(0.2M solution of Monobasic Sodium phosphate, 0.0M solution of Diabasic Sodium phosphate).
Procedure
Test tubes were marked as T and B corresponding to test and control. 0.5ml of buffer
substrate was added in the test tubes and incubated at 37˚C for three minutes. 0.1 ml of serum
was added to the test tube T alone and shaken well. Then the tubes were incubated at 37˚ C for
30 minutes. After 30 minutes, the tube were removed from the incubator and 0.5ml of DNPH
colour reagent was added and mixed well and allowed to stand at room temperature for 20
minutes to start the colour development. After 20 minutes 0.1ml of distilled water was added to
blank alone and 5ml of working Sodium hydroxide was added to both the test tubes. It was
mixed well and allowed to stand at room temperature for 10 minutes and both the tubes were
read at 505nm against water blank. The results were calculated.
SGOT level =
the sample
O.D of Sample×0.4×1000×µmOxaloacetate formed/mg
O.D of the standard× 0.266.
4.5.6. ESTIMATION OF SGPT
Estimation of serum glutamate puiruvate transaminase was done by Reitman and Frankel
(1957) method.
Principle
SGPT catlalyses the transfer of amino groups from L-alanine to alpha ketoglutarate with
formation of Pyruvate. The Pyruvate so formed was allowed to react with 2.4
dinitophenylhydrazone derivative which was brown coloued in acid medium. The aqbsorbance
of this hydrazone derivative was corelated to ALT activity by plotting a calibration curve using
Pyruvate standard.
Reagents
2.4 dinitrophenylhydrazone (DNPH), Sodium hydroxide, GOT buffer substrate (0.2M
solution of monobasic sodium phosphate, 0.2m solution of Dibasic sodium phosphate).
Procedure
Test tubes were marked as T and B corresponding to test and control. 0.2 buffer substrare
was added in the tubes and incubated at 30 C for three minutes. 0.1 ml of serum was added to the
test tube T alone and shaken well.Then the tubes were incubated at 37˚C for 30 minutes, the
tubes were removed from the incubator and 0.5 ml of DNPH colour reagent was added and
mixed well and allowed to start for room temperature for 20 minutes to start the colour
development. After 20 minutes 0.1 ml of distilled water was added to blank alone and 5ml of
working sodium hydroxide was added to both the test tubes. It was mixed well and allowed to
stand at room temperature for 10 minutes and both the tubes were read at 505 nm against water
blank. The results were calculated.
SGPT levels
=
O.D of the sample×0.4×1000µm pyruvate formed/mg
in the sample
O.D of the standard×0.266
4.5.7. ESTIMATION OF ALKALINE PHOSPHATASE
Alkaline phosphatase is determined by colorimetric method of Bowers and Mc Comb
(1988).
Principle
The substrate P-Nitrophenyl phosphate is hydrolyzed by Alkaline phosphatase from the
sample, in the presence of magnesium ions, to form P-Nitrophenyl which is yellow in colour and
can be read at 405nm. The intensity of colour produced is proportional to the alkaline
phosphatase activity in the sample.
P-Nitrophenyl phosphate + H20 →P
The reagent composition of the kit was follows
CONTENT
CONCENTRATIONS
R1. Buffer
0.35 mol/11,
2-Amino-2-methyl-propano Mg2+
PH 10.4
R2.Supstrate
2.0m mol
P-Nitrophenyl phosphate
10m mol/1
All the contents are ready for use. Values expressed as u/1.
4.6. HISTOPATHOLOGICAL STUDIES
Anotamy of the liver and thyroid tissues were to be studied immediately after sacrificing
the animals. A small portion was fixed in 10 % neutral buffered formalin as described by Luna
(1968). Thin sections of 4-5 µm were taken, stained with Haematotoxylin and Eosin and
histology was studied.
4.7. STATISTICAL ANALYSIS
The results were expressed in Mean±SEM, Student‘s ―t‖ test was used for statistical
significance between groups (Steele and Torrie, 1960).
results
5. RESULTS
5.1 EFFECT ON BODY WEIGHT (Table.1 Figure.1)
When compared to control, the body weight of all the treated groups showed a
comparative increase of above 10%, except for the ethylene thiourea induced group, where a
significant decrease can be observed. Individual administration of ginger extract and gingerol as
well as co-administration of both with ethylene thiourea were seen to cause a more or less similar
percentage of significant increase in body weight.
5.2 EFFECT ON THYROID HORMONES (Table.2 Figure.2)
The level of T3 and T4 are decreased significantly on administration of ginger extract as
well as gingerol alone and on co-administration of both along with ethylene thiourea. But the
level of TSH was insignificantly increased from control levels on treatment with ginger extract
only.
5.3 EFFECT ON THYROID HISTOLOGY (Plates 1-6)
The normal structure of thyroid with active follicles can be seen in control rats.
Administration of ginger extract brought about mild focal changes in the thyroid follicles, while
gingerol administration caused slight disturbances in follicular structure, with follicles containing
scanty amount of colloid and increased in inter follicular space. Treatment with ETU, brought
about pronounced interfollicular hemorrhage with severe degenerative changes like sloughing of
follicular epithelial cells into the lumen and necrosis of follicles. Co- administration of ETU with
gingerol can be observed to bring about a change in the appearance of follicular architecture with
little or depleted colloid. Certain degenerative follicles are also seen. Co-administration of ETU
with ginger extract also seems to be restorative in nature with figure containing both exfoliated
follicular epithelial cells expressing degeneration and smaller number of active follicles.
5.4 EFFECT ON SERUM PROTEIN (Table.3 Figure.3)
The level of total protein in liver tissue was observed to be significantly decreased in both
the ginger extract administrated as well as ginger extract co-administrated with ETU. A
significant increase in protein level can be seen in gingerol treated and co-administrated groups
with ETU. Induction of ETU seems to cause a significant increase in total protein levels.
TABLE 1: THE EFFECT OF GINGER EXTRACT AND GINGEROL ON BODY WEIGHT OF
ETHYLENE THIOUREA INDUCED ALBINO RATS
GROUPS
BODY WEIGHT (GRAMS)
INITIAL
FINAL
I
209± 5.26
221± 3.56
II
220 ± 3.21
203± 3.03a*
III
221 ± 6.56
245± 4.14b*
IV
213 ± 6.082
198± 4.39*
V
237± 3.271
256± 4.81*
VI
227± 5.50
241± 4.65c,e*
Mean ± S.E.M of five rats.
I – Control, II – Ginger Extract III- Gingerol IV- Ethylene thiourea V-Ethylene thiourea + Gengerol
VI- Ethylene thiourea + Ginger extract
*Significant level at 5%; I Vs other treater groups, a-IV Vs V, b-IV Vs VI
FIGURE 1: : THE EFFECT OF GINGER EXTRACT AND GINGEROL ON BODY WEIGHT OF
ETHYLENE THIOUREA INDUCED ALBINO RATS
body weight
Grams
400
221
209
203
220
245
221
198
213
256
237
241
227
I
II
III
IV
V
VI
200
0
GROUPS
BODY WEIGHT INITIAL
BODY WEIGHT FINAL
TABLE 2: EFFECT OF GINGER EXTRACT AND GINGEROL ON THYROID HORMONES OF ETHYLENE
THIOUREA INDUCED ALBINO RATS
GROUPS
T3
T4
TSH
GROUP I
101.4 ±2.502
4.5±0.472
2.04±0.186
GROUP II
87.2±4.554*
1.8±0.221*
3.46±0.132
GROUP III
74±3.449*
1.84±0.120*
2.32±0.171
GROUP IV
99.4±2.874*
4.54±0.246*
2.32±0.124
GROUP V
80.2±3.967*a
2.82±0.128a
3.02±0.171
GROUP VI
90±3.701*b
2.84±0.233*
1.76±0.172
Mean ± S.E.M of five rats.
I – Control, II – Ginger Extract, III- Gingerol, IV- Ethylene thiourea, V-Ethylene thiourea + Gengerol,
VI- Ethylene thiourea + Ginger extract
*Significant level at 5%; I Vs other treater groups, a-IV Vs V, b-IV Vs VI
FIGURE 2: EFFECT OF GINGER EXTRACT AND GINGEROL ON THYROID HORMONES OF ETHYLENE
THIOUREA INDUCED ALBINO RATS
T3
T4
T3
100
101.4 87.2
74
99.4
80.2
5
90
4.54
4.5
2.82 2.84
1.8 1.84
U/L
U/L
200
T4
0
I
0
II
III
IV
Groups
V
TSH
TSH
3.02
2.32 2.32
2.04
1.76
0
II
III
IV
Groups
2
I
II
VI
3.46
4
U/L
I
III
IV
Groups
V
VI
V
VI
PLATE 1: NORMAL THYROID CELLS
Active follicles filled with colloid visible and normal thyroid architecture maintained.
PLATE 2: GINGER EXTRACT TREATED THYROID CELLS
Slight focal degenerative changes in follicles visible with appearance of inter follicular spaces.
PLATE.3: GINGEROL TREATED THYROID CELLS
Majority of follicles are normal with scanty amount of colloid and increase in inter follicular
space.
PLATE.4: ETHYLENE THIOUREA TREATED THYROID CELLS
Severe degenerative changes with depletion of colloid and sloughing of follicular epithelial cells
into the lumen. Necrosis of follicles with mono-nuclear cell infiltration.
PLATE.5: EHTYLENE THIOUREA AND GINGEROL TREATED THYROID CELLS
Follicular degeneration with preservation of follicular architecture with few follicles showing
scanty or depleted colloids.
PLATE.6: ETHYLENE THIOUREA AND GINGER EXTRACT TREATED THYROID
CELLS
Recovery of severe vacuolar degenerative changes in follicular epithelial cells observed with
smaller number of active follicles along with clumps of exfoliated follicular epithelial cells.
5.5 EFFECTS OF LIVER MARKER ENZYMES (Table.3 Figure.3)
The level of AST ALT and ALP are observed to be significantly increased all treatment
groups when compared to normal rats. This increase is highest in the ETU treated group. Coadministration of ginger extract and gingerol along with ETU was seen to bring about a
significant decrease from the elevated level of AST and ALP caused by ETU induction.
5.6 EFFECT ON LIVER HISTOLOGY (Plates 7-12)
The control liver showed normal architecture of hepatocytes with parenchyma strands
and sinusoidal space with normal triad. Administration of ginger extract brought about mild
degenerative changes but with normal orientation of hepatocytes. Likewise, administration with
gingerol can be also seem to cause slight infiltration of inflammatory cells in centri-lobular area
but with well-maintained hepatocyte architecture in mid zonal and peri-portal area. Treatment
with ETU brought about severe diffuse necrosis with inflammatory cell infiltrate in peri-vascular
area. Co-administration with gingerol brought about corrective changes in liver parenchyma,
with few focal necrotic areas visible. Co-administration with ginger extract also seems to be
remedial in nature as evidenced by the appearance of normal liver parenchyma exhibiting slight
peri-portal focal inflammatory cell infiltration and few necrotic hepatocytes.
TABLE 3: EFFECT OF GINGER EXTRACT AND GINGEROL ON PROTEIN AND LIVER ENZYME MARKERS
OF ETHYLENE THIOUREA INDUCED ALBINO RATS
GROUPS
PROTEIN
AST
ALT
ALP
I
18.35 ± 1.61
155.31±4.19
57.07±5.68
300.52±4.40
II
15.42 ± 0.47*
178.28±5.18*
74.34±3.30*
481.82±3.28*
III
19.47 ± 0.57
189.84±3.53*
63.83±3.04*
312.92±6.64
IV
22.52 ± 0.55*
295.59±4.31*
118.14±3.68*
584.51±5.24*
V
20.03 ± 0.84
200.15±3.52*a
93.47±3.79*
503.39±5.02*a
VI
16.25 ± 0.61b*
172.42±3.44*b
71.74±3.33*
415±3.24*b
Mean ± S.E.M of five rats.
I – Control, II – Ginger Extract, III- Gingerol, IV- Ethylene thiourea, V-Ethylene thiourea + Gengerol,
VI- Ethylene thiourea + Ginger extract
*Significant level at 5%; I Vs other treater groups, a-IV Vs V, b-IV Vs VI
FIGURE 3: EFFECT OF GINGER EXTRACT AND GINGEROL ON PROTEIN AND LIVER ENZYME MARKERS
OF ETHYLENE THIOUREA INDUCED ALBINO RATS
PROTEIN
AST
PROTEIN
20
22.52 20.03
18.35 15.42 19.47
16.25
g/dl
g/dl
40
400
200
0
I
II
III
IV
V
155.31 178.28
189.84
I
II
ALT
III
IV
Groups
ALP 584.51
ALT
g/dl
118.14
74.34 63.83
93.47
71.74
600
400
481.82
V
503.39
VI
ALP
415
312.92
300.52
u/l
200
57.07
200.15 172.42
0
VI
Groups
100
AST
295.59
200
0
I
II
III
IV
Groups
V
VI
0
I
II
III
IV
Groups
V
VI
PLATE.7: NORMAL HEPATIC CELLS
Normal histoarchitecture of hepatocytes and sinusoidal space observed.
PLATE 8: GINGER EXTRACT TREATED LIVER CELLS
Normal orientation of hepatocytes with slight degenerative changes in periportal area
PLATE.9: GINGEROL TREATED LIVER CELLS
The hepatocyte architecture is well maintained with slight infiltration of inflammatory cells on
centrilobular area.
PLATE.10: ETHYLENE THIOUREA TREATED LIVER CELLS
Severe diffuse necrosis with inflammatory cell infiltrate in perivascular space seen with
disruption of cellular morphology.
PLATE.11: ETHYLENE THIOUREA + GINGEROL TREATED LIVER CELLS
Few focal necrotic areas observed in centrilobular zone. Hepatocytes show mild structural
changes.
PLATE.12: EFFECT OF THIOUREA AND GINGER EXTRACT TREATED LIVER
CELLS
Liver parenchyma exhibits perifocal in inflammatory cell infiltration with few disruptions in
cellular morphology.
discussion
6. DISCUSSION
6.1 EFFECT ON BODY WEIGHT
Zingiber officinale brought about an increase in body weight of broiler chicken (Tekeli et
al., 2011). The use of 2% red ginger in the ration of broiler chicken produced higher body
weights. Addition of ginger to basal diet resulted in higher body weights. Treating rats with ADR
for 6 weeks caused significant decrease in body weights, when compared to control groups. But
administration of gingerol and ADR caused a significant increase in body weight of rats. Sakr et
al., (2011) have reported about the significant change in body weight on administration of ginger
to rats.
Ginger was observed to have hypo-cholesterolaemic effect causing decrease in body
weight and blood glucose in adult male rats (Gujral et al., 1978). Cadmium induction brought
about a significant decrease in body weight (Haouem et al., 2013). Similarly, in the present
study, induction with ethylene thiourea also brought about significant reduction in body weight.
But co-administration with ginger extract and gingerol have aided in increasing the body weight
from its decreased state. Individual administration of ginger and gingerol have also significantly
increased the body weight.
6.2
EFFECT ON THYROID
The control of the concentration of thyroid hormone in the blood is regulated by a
negative feedback mechanism involving the hypothalamus, the pituitary, and the thyroid (Hill et
al., 1989). The hypothalamus releases thyrotropin releasing hormone (TRH), which stimulates
the pituitary to produce thyroid-stimulating
hormone (TSH). TSH prompts the thyroid to
produce thyroid hormone. Cells in both the hypothalamus and pituitary respond to levels of
circulating thyroid hormone. When levels of thyroid hormone are high, the output of both TRH
and TSH are low. When levels of thyroid hormone are low, the outputs of TRH and TSH are
raised, prompting the thyroid to increase the output of thyroxin (T4) and triiodothyronine (T3).
The negative feedback loop helps the body to respond to varying demands for thyroid hormone
and to maintain hormone homeostasis.
Hypothyroidism is the most common disorders of thyroid function and can be categorized
as primary hypothyroidism and central hypothyroidism (Brunton et al.,2007). During the chronic
toxicity and carcinogenicity studies of ethyelene thiourea on rats and mice. The thyroid gland
was the major site of ETU carcinogenicity while another major sites of ETU carcinogenicity
were liver and pituitary glands (Chhabra et al., 1991). Intrathyroidal and extrathyroidal sites of
action or found and ethyelene thiourea functions as thyroid peroxidase inhibition disrupting
thyroid primary homeostasis (Hurley et al., 1998).
The potential antithyroid sites of action: inhibition of iodide uptake into the thyroid,
thyroid peroxidase inhibition, damage to thyroid follicular cells, inhibition of thyroid hormone
release from the thyroid, inhibition of 5'-monodeiodinase activity, and enhancement of thyroid
hormone metabolism and excretion by the liver (Hill et al., 1989). Both mancozeb and its
metabolite ethylene thiourea are thionamides, a group of chemicals with antithyroid activity
(Green, 1978).
In the present study, the levels of T3 and T4 were consistently decreased in all the
treatment groups when compared to control except for the ginger extract co-administrated
groups. TSH secretion of all the groups seems to be significantly increased. ETU inhibits thyroid
peroxidase causing perturbations in thyroid and pituitary hormone levels. The iodine uptake is
reduced and this may be due to a specific block in the active transport of inorganic iodide into
the cell. It is a manifestation of inhibition of thyroid peroxidase, as iodide is not trapped within
the cell in an organic form (Doerge and Takazawa, 1990).
Ginger seems to interfere with thyroid function. It seems to have stimulating action of
thyroid gland (Sanabi & Afshar., 2010) providing an ameliorative effect (Alizadeh-Navael et al.,
2008). The main antioxidant active principle in ginger are the polyphenolic compound gingerol
and related phenolic ketone derivatives. They act by inhibiting of scavenging radicals of rat body
of by increasing the antioxidative defense mechanism of liver cells (Ali et al., 2008). But in the
present study, both ginger extract and gingerol individually as well as in combination with ETU
exhibits an inhibiting effect on T3 and T4 concentration. The pituitary hormone TSH level is
seen to be increased significantly. Thus, both ginger extract and gingerol individually and in
combination seem to have produced antithyroid effect by reducing the circulating thyroid
hormones increasing TSH, thus increasing the thyroid cancer potential in rats. The damage to
follicular cells and inhibition of thyroid and excretion of thyroid hormone by the liver, mainly
through action of uridine diphosphate glucuronosyltransferase.
Referring to thyroid histology, the thyroid in control rats show normal follicular structure
filled with colloid and inter-follicular space. Administration of ginger extract brought about
slight degenerative changes in certain follicles while other follicles are normal. Administration of
gingerol brought about slight changes in follicular architecture with major follicles appearing
normal containing scanty colloid. Inter follicular space is increased with hemorrhage in stroma.
Induction with ethylene thiourea brought about severe degenerative changes like depletion of
colloid, sloughing of follicular epithelial cells into lumen and necrosis of follicles with
mononuclear cell infiltration. Co-administration with gingerol brought about slight amelioration
as evidenced by the presence of follicular architecture with coalescence with adjacent follicles
and decreased intensity of colloid staining also follicular inflammatory cell infiltration also seen.
Likewise, ginger extracts co-administration also brought about slight constructive changes in
follicular cells are seen by the presence of active follicles along with sloughing of follicular
epithelial cells into the lumen in certain areas with clumps of exfoliated cells in the colloid. Thus,
thyroid follicular cell injury and inhibition of thyroid hormone release can be reported.
In mammals, the liver is the major organ responsible for the metabolism and
detoxification of xenobiotic compounds, and is performed by the specific phases I and II
enzymes (Nirmala et al., 2010). The phase I reactions increase polarity of the xenobiotic
compounds by adding new functional groups to xenobiotic molecules, and is principally
catalyzed by cytochrome P-450 monooxygenase system. During phase II reaction, conjugation to
endogenous hydrophilic molecules (like GSH by GST), and the resulting reaction increases the
polarity and water solubility to eliminate the xenobiotic metabolite out of the body (Nirmala et
al., 2010). Studies have shown that the extracts of ginger and some of its phytochemicals
modulate the activity of both phase I and II enzymes, and to mediate their hepatoprotective
affects, at least in part, through this mechanism. With respect to the phase I enzymes, studies
have shown that feeding ginger causes an increase in the levels of microsomal cytochrome P
450-dependent aryl hydroxylase, cytochrome P 450 and cytochrome b5, and this effect would
have increased the polarity of non-polar xenobiotic compounds (Sambaiah and Srinivasan,
1989). Innumerable studies have shown that ginger extracts, oleoresins and the volatile oils
possess free radical scavenging effects, and to be effective in scavenging superoxide, hydroxyl,
nitric oxide in vitro (Al-Tahtawy et al., 2011)
In the present study, the assessment of liver function by measuring serum ALP, AST and
ALT activities have disclosed the following observations. The activities of all the three enzymes
were significantly increased in all the treated groups on comparison with control. In several
organs, cell damage is followed by release of a number of cytoplasmic enzymes to the blood, a
phenomena which provides the basis for clinical diagnosis. Therefore, the increase in AST and
ALT activities may be explained by the leakage of these enzymes from the liver cytosol into the
blood stream through production of free radicals, which affect the cellular permeability of
hepatocytes leading to elevated level of biochemical parameters like ALT, AST and ALP. The
enzymatic disturbances produced by the combined treatment are lower than those produced by
ethylene thiourea, thus reducing the toxicant induced increase in serum enzymes. The
administration of ginger extract and gingerol alone also seems to cause disturbance in activities
of serum AST, ALT and ALP. Oral ingestion of paracetamol significantly increased serum ALT,
AST and LDH enzyme activities with insignificant increase of serum ALP. Administration of
ginger individually and co-administration with paracetamol decreased serum enzymatic activities
of ALT, AST, LDH and ALP (Lebda et al.,2013). Antioxidant enzymes within normal levels and
increased level of reduced glutathione. The discrepancy in result may be due to the dosages used.
The efficacy in alleviation of bromobenzene induced hepatotoxicity of different doses of
ginger extract has been reported by El-Sharky et al., (2009), have shown that ginger was
beneficial in protecting against mancozeb induced liver toxicity when compared to mancozeb
treated cohorts, Co-administration of ginger reduced the mancozeb induced increase in levels of
ALT, AST, LPx and a contaminant increase in the level of antioxidant enzymes. The
histopathological observations corroborated with the biochemical resu;ts, thus indicating the
usefulness of ginger in preventing moncozeb induced hepato carcinogenesis (Sakr, 2007).
Similarly, recent scientific studies have proved the effectiveness of gingerol its
components in preventing ethionine induced (Yusof et al., 2008), and DEN initiated
hepatocarcinogenesis in rats (Mansour et al., 2010). Molecular studies have also shown that
ginger reduced the elevated expression of NFκB and TNF-α in rats with liver cancer. Suggesting
that the observed chemopreventive effects may be made through inhibitory effects on NFκB,
through suppression of proinflammatory TNF-α (Habib et al., 2008).
Regarding histopathological changes in liver, in control groups, liver shows normal
architecture of hepatocytes and sinusoidal spaces. The portal triad showed normal structure.
Administration of ginger extract did not cause major histological disturbances as evidenced by
normal orientation of hepatocytes. But slight accumulation of infiltration in portal region can be
observed. Similarly, gingerol administration also caused infiltration of inflammatory cells in
centri-lobular area while hepatic architecture is well maintained in mid-zonal and peri-portal
area. ETU induction caused general diffuse with peri-vascular mononuclear cell infiltration as
well as disruption of cellular morphology, karyolysis and distorted sinusoidal space. Coadministration with gingerol as well as ginger extract brought about a slight reversal of hepatic
damage. Few focal necrotic areas can be seen with no significant pathological lesions and
structural changes on ginger extract co-administration while gingerol co-administration resulted
in mid-focal necrotic changes in centrilobular zone with reverting residual hepatocytes showing
mild change in structure.
Thus, it can be observed that ethylene thiourea at the present dosage and duration have
initiated antithyroid as well as hepatotoxic damages to the respective tissues and a melioration of
these changes by both ginger extract and gingerol is comparatively very limited.
Conclusion
CONCLUSION
Ginger has been shown to be a hepatoprotective agent and with a general antitoxidant
effect, and studies with various hepatotoxins validate the property. But considerable work has to
be done to exploit the toxicity alleviating effect of ginger and its constituents. In the present
study, the toxic damages induced by ethylene thiourea at the present dosage and duration is seen
to be ameliorated to a certain extent only. Though biochemical investigations provide near
unresponsive results, histological observations seems to validate the antitoxic effects of ginger
extract and gingerol to a certain extent at the present dose and duration.
Studies should be conducted to assess for possible adverse effects of ginger, at higher
concentrations over longer periods. Further in depth mechanistic studies have to be undertaken,
to elucidate the effective modulation of detoxifying enzymes, specific effective dosages and apt
constituent forms and also to explain the interactions taking place, to promote the alleviating
effect of ginger. Both ginger extract and gingerol have exhibited an equal comparative antitoxic
effect on ETU induction.
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