Rida Thesis

EVALUATION OF HEPATOPROTECTIVE ACTIVITY
OF
Taraxacum officinale
A Thesis Submitted
in Partial Fulfillment of the Requirements

for the Degree of
MASTER OF PHARMACY
in
Pharmacology
by
Rida Arif Qidwai

1419798506
Under the Supervision of
Dr. MOHD. IMTIYAZ AHMAD

(Professor)
Azad Institute of Pharmacy and Research
Lucknow
the
Faculty of Pharmacy
Dr.A.P.J.ABDUL KALAM TECHNICAL UNIVERSITY,
LUCKNOW
(Formerly Uttar Pradesh Technical University)
JANUARY, 2016
Dr.A.P.J ABDUL KALAM TECHNICAL UNIVERSITY, LUCKNOW
CERTIFICATE OF THESIS SUBMISSION FOR EVALUATION
1. Name : Rida Arif Qidwai

2. Enrollment No. : 1419798506
3. Thesis title: “Evaluation Of Hepatoprotective Activity Of Taraxacum officinale ”
4. Degree for which the thesis is submitted : Master Of Pharmacy In Pharmacology

5. Faculty (of the University to which the thesis is submitted) : Faculty of Pharmacy
6. Thesis Preparation Guide was referred to for preparing the thesis. YES NO
7. Specifications regarding thesis format have been closely followed. YES NO
8. The contents of the thesis have been organized based on the YES NO
guidelines.
9. The thesis has been prepared without resorting to plagiarism. YES NO
10. All sources used have been cited appropriately. YES NO
11. The thesis has not been submitted elsewhere for a degree. YES NO
12. Submitted 3 spiral bound copies plus one CD. YES NO
(Signature of the Candidate)

RIDA ARIF QIDWAI
Enrollment No.: 1419798506
Dr.A.P.J.ABDUL KALAM TECHNICAL UNIVERSITY, LUCKNOW
CERTIFICATE OF THESIS SUBMISSION FOR EVALUATION
1. Name : RIDA ARIF QIDWAI

2. Enrollment No. : 1419798506
3. Thesis title: “Evaluation Of Hepatoprotective Activity Of Taraxacum officinale ”
4. Degree for which the thesis is submitted : Master Of Pharmacy In Pharmacology

5. Faculty (of the University to which the thesis is submitted) : Faculty of Pharmacy
6. Thesis Preparation Guide was referred to for preparing the thesis. YES NO
7. Specifications regarding thesis format have been closely followed. YES NO
8. The contents of the thesis have been organized based on the YES NO
guidelines.
9. The thesis has been prepared without resorting to plagiarism. YES NO
10. All sources used have been cited appropriately. YES NO
11. The thesis has not been submitted elsewhere for a degree. YES NO
12. Submitted 5 Hard bound copies plus one CD. YES NO
(Signature of the Candidate)

RIDA ARIF QIDWAI
Enrollment No.: 1419798506
Page |1
CERTIFICATE
This is to certified that Rida Arif (Enrollment no.1419798506 ) has carried out the research work
presented in this thesis entitled “Evaluation of Hepatoprotective Activity of Taraxacum
officinale” for the award of Master of Pharmacy from Dr. A.P.J.Abdul Kalam Technical
University, Lucknow under my guide Dr.Mohd Imtiyaz Ahmad(Director)Azad institute of
Pharmacy & Research ,supervision.The thesis embodies results of original work,and studies are
carried out by the student herself and the content of the thesis do not form the basis for the award of
any other degree to the candidate or to anybody else from this or any other University /Institution.
Dr.Mohd.Imtiyaz Ahmad
(Supervisor)
Department of Pharmacology
Azad Institute of Pharmacy &Research
Lucknow
Page |2
CERTIFICATE
This is to certified that thesis entitlted “Evaluation of Hepatoprotective activity of Taraxacum

officinale “ has been submitted to the faculty of Pharmacy Dr.A.P.J.Abdul Kalam Technical
University ,Lucknow .In fulfillment of requirement for the award of degree of Master of Pharmacy
in Pharmacology embodies the original research work carried out by Rida Arif under our
supervision and has not been submitted in part or full for any degree or diploma of this or any other
university.
It is further certified that the scholar fulfills all the requirement as laid down by the university for the
purpose of submission of M.Pharm thesis.
Signature Signature
Supervisor Director
Dr.Imtiyaz Ahmad Azad Institute of Pharmacy & Research
Co-ordinator(M.Pharm) Lucknow
A.I.P.R,Lucknow
Evaluation of Hepatoprotective activity of Taraxacum officinale

Page |3
Rida Arif Qidwai
ABSTRACT
Liver plays a vital role in metabolism and excretion. Any injury to it or impairment of its function
may lead to many implications on one’s health. Liver ailments need to be treated with outmost care.
Management of liver diseases is still a challenge to modern medicine. The allopathic medicine has
little to offer for the alleviation of hepatic ailments whereas the most important representatives are
of phytoconstituents.
Objective:
To assess the hepatoprotective activity of ethanolic extract of Taraxacum officinale root against
CCl4 and thioacetamide induced liver injury in rats followed by in-vivo antioxidant property.
Methodology:
The model used to evaluate hepatoprotective activity of the plant was “Ethanol induced
Hepatotoxicity” & the following steps were carried out.
1.Acute toxicity studies

2.Hepatoprotective activity
3.Estimation of biochemical parameter
4.Histopathological studies
Results:
This study aimed on evaluating hepatoprotective activity of Taraxacum officinale showed marked
decrease in hepatotoxic effect of ethanol which was evident by study of biochemical parameters.
Page |4
All the biochemical markers, liver weight, liver volume, lipid peroxidation (LPO) of hepatic injury
were elevated and reduced Glutathione (GSH) upon Thioacetamide and CCL4 challenge and they
were brought down to near normal levels by pretreatment with 250 mg/kg and 500 mg/kg of test
extract in both the models. The elevated levels of liver weight, liver volume and destructed liver
architecture were normalized by the treatment with test extract.
Conclusion:
The results are indicating that test extract possesses hepatoprotective property against
Thioacetamide and CCL4 induced hepatic damage. The study was concluded as plant possesses in-
vivo antioxidant and hepatoprotective property. The antioxidant and hepatoprotective property may
be attributing to the phenol and flavonoids content of the plant.
Keywords: Taraxacum officinale ,Hepatoprotective activity,

Page |5
ACKNOWLEDGEMENT
First and foremost, I submit my thesis at feet of Almighty. I thank Almighty with that mercy, it has
been made possible for me to reach this so far.
I am delighted to place on record the invaluable co-operation of certain individuals who supported me
in completing this work. I would like to express my appreciation for all the effort, to everyone who
have directly and indirectly contributed their ideas and energies in successful completion of my
project.
I express with immense respect and deep sense of gratitude my sincere thanks to Dr. Mohd Imtityaz
Ahmad,Supervisor and Director Azad Institute of Pharmacy and Research, Lucknow for the
support, disciplined technical advice, continuous encouragement, functional freedom, invaluable
guidance and constant supervision given throughout my project work.
I gratefully wish to express immense gratitude to Mr.Nishant Kumar Yadav Head of Department of
Pharmacy amd to Miss Amrita Shukla Lecturer of Pharmacolgy .A special thanks to all the staff
and co-workers who co-operated along in completion of this project.
I convey a special thanks to my batch mates Neha Shukla and Rupali Srivastava for their kind help
and cooperation throughout the project work.
I am also very pleased to acknowledge my Abba Mr.Salman Samdi , Mr. Arif Aziz , Mom Shafia
Samdi , Yasmeen Iqbal, my Husband Kamran Samdi and my siblings who supported me
throughout with moral support , valuable suggestions and sincere advice offered to me for carrying
out this project.
Page |6
Rida Arif Qidwai
Ridz786922@gmail.com
LIST OF ABBREVIATIONS USED
b.w. - Body weight.
CPCSEA - Committee for the purpose of control and supervision of
Experiments on animals.
ext. - Extract.
fig - Figure.
gm - Gram.
IAEC - Institutional Animal Ethics Committee.
P.O. - Per oral.
LD50 - Lethal dose.
Mtr - Meter.
No - Number.
ANOVA - Analysis of variance.
c - Degree centigrade.
SEM - Standard error mean.
Con - concentration.
MEAL - ethanolic extract of Tecoma stans .

Page |7
TABLE OF CONTENTS
Page no.
Certificate………………………………………………………………………
Certificate………………………………………………………………………
Abstract…………………………………………………………………………
Acknowledgement……………………………………………………………..
Content…………………………………………………………………………
List of Tables……………………………………………………………………
List of Figures…………………………………………………………………..
List of Symbols & Abbreviations………………………………………………
CHAPTER 1. INTRODUCTION………………………………….1-36
General Introduction
Herbal wealth of India
1.Hepatic injury
2.Metabolic Functions
1.1 Anatomy of Liver
Structure of Liver
Blood Supply
Function of Liver
1.Secretion and Excretion of Bile
2. Metabolic Function
Phases of Biotransformation
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1.2 Liver Disease
1.3 Physiology Of Liver
Role in Production of Binding Proteins
Protective & clearance Functions
Phagocytic and Endocytic Functions Of Kupffer cells
Endocytic Functions of Hepatocytes
1.4 Types of Liver Dysfunction
Hepatocyte Dysfunction
Portal Hypertension
Pathophysiology of Functional Zonation
Manifestation Of Liver Dysfunction
Causes of Liver disease
1.5 Mechanism of Drug Induced hepatotoxicity
1.6 Models to Evaluate Effect of Drugs on Liver
CHAPTER-2 PLANT INTRODUCTION……………………………….
Classification
Vernacular name
Chemical Constituent
Pharmacological Study
Biological study
Antispasmodic effect
Anti-inflammatory Activity
Antimicrobial activity
Antioxidant activity
Wound healing Activity

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Cytotoxic Activity
Antifungal activity
Review of Literature…………………………………………………………
CHAPTER -3 METHODOLOGY…………………………………………..
3.1 List of material and Equipent
3.2 Collection of Plant Material
3.3Preparation of Ethanolic Extract
3.4 Qualitative Phytochemical Screening
3.5 Experimental animals
3.6 Pharmacological Activities
1.Toxicity Studies
2. Hepatoprotective activity
Determination of oral acute toxicity
3.7 Experimental Design
1.Thioacetamide induced Hepatotoxicity in rats
2.CCl4 induced Hepatotoxicity in rats
Histopathology
Statistical analysis
CHAPTER-4 RESULT & DISCUSSION…………………………………….

4.1 Details of Qualitative Phytochemical tests
4.2 Toxicity Studies
4.3 Hepatoprotective activity
1.Physical Parameter
2.Biochemical Parameter
Plant Material
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Preparation of Extract
Determination of Oral acute Toxicity

Result
Disscussion
Conclusion
CHAPTER-5 REFERENCES…………………………………………….
LIST OF TABLES
Sl.No. List Of Tables Page No.
1.1 Classification of hepatotoxins and mechanism of action of each group 57

of hepatotoxins.
1.2 List of hepatotoxic therapeutic agents and chemicals. 42
4.1 List of materials and equipments used during experiment.
5.1 Details of qualitative phytochemical tests. 93-94
5.2 Effect of 70% EETSL on liver weight, liver volume and biochemical 96
markers in thioacetamide induced hepatotoxicity.
5.3 Effect of 70% EETSL on tissue GSH and in-vivo lipid peroxidation 99
levels in thioacetamide induced hepatotoxicity.
5.4 Effects of 70% EETSL on wet liver weight, liver volume and 102
biochemical markers in CCl4 induced hepatotoxicity
5.5 Effect of 70% EETSL on tissue GSH and in-vivo lipid peroxidation 105
levels in CCl4 induced hepatotoxicity
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LIST OF FIGURES
Sl.No. List Of Figures Page.No.
1.1 Liver anatomy. 9
1.2 Structure of liver cells. 11
1.3 Blood supply to the liver. 12
1.4 Drug metabolism in liver. 21
1.5 Pathways for alcohol metabolism. 43
1.6 Taraxacum officinale Flower 56
1.7 Taraxacum officinale roots 57
1.8 Preparation of ethanolic extract 57
5.1 Qualitative phytochemical screening: 97
5.2 Effect of ethanolic extract of Taraxacum officinale roots 97

on Wet liver.
5.3 Effect of ethanolic extract of on Taraxacum officinale SGOT 100

levels in ethanol induced hepatotoxic rats.
5.4 Effect of ethanolic extract of Taraxacum officinale roots on ALP 100

Level in ethanol induced hepatotoxic rats
5.5 Effect of ethanolic extract of Taraxacum officinale on Direct 100

Bilirubin & Total bilirubin levels in ethanol induced hepatotoxic
rats.
5.6 Effect of ethanolic extract of on Taraxacum officinale Total 103
Protein levels in ethanol induced hepatotoxic rats.
5.7 Effect of 70% EETSL on tissue GSH and in-vivo lipid 106
peroxidation levels in thioacetamide induced
hepatotoxicity.
5.8 Histopathological Studies in Thioacetamide and CCL4
induced hepatotoxicity.
P a g e | 12
CHAPTER-1
INTRODUCTION
Azad Institute of Pharmacy & Research Page 12

P a g e | 13
Liver plays an astonishing artay o f vital functions in the T maintenance and

performance of the body. Some o f these major functions include carbohydrate, protein
and fat metabolism, detoxification and secretion of bile which among other things has
an important role in digestion. Liver diseases appear to be on increase in our society.
Pan o f this increase may be due to our frequent contact with chemicals and other
environmental pollutants. The amount of medicine consumed has increased greatly with
resulting dangers to the liver. The liver, the detoxifying factory in the body, has become
an increasingly overworked organ. While those who smoke, abuse alcohol and drugs
and live in severely polluted environments are at great risk, we all suffer some threat o f
damage or disease to the liver (Scott 1998). Though a strict delineation o f various
hepatic disorders is not possible yet from didantic point o f view, liver disorders may be
classified as A cute or Chronic Hepatitis, Hepatosis and Liver Cirrhosis (Morgan, 1985).
The predominant type o f liver disease varies according to country and may be
influenced by local factors. For 1989 it was estimated that there were some 200 million
chronic carriers o f hepatitis B virus o f which 40% were expected eventually to die o f
hepatocellular carcinoma and 15% o f cirrhosis (Evans 1996). In spite o f the
tremendous advances made in allopathic medicine, except for the use o f the appropriate
vaccine for the treatment o f hepatitis caused by viral infection, there are few effective
cures for liver diseases. A considerable interest has therefore, developed in the
examination of traditional plant remedies.
Traditional healers and pharmacists in developing countries are in important
source of information about plant sources of new drugs. Only a fraction of the earth's
natural pharmacopoeia has been analyzed with modern techniques. The threat of
imminent extinction of many plant species, especially in tropical areas, makes it
urgent that scientists learn as much as possible before old remedies are forgotten or
their raw materials are destroyed. This process requires the observation and recording
of medical techniques, identification of plant materials and experimental investigation
of the ingredients and their effects. Ethnopharmacology can also be an important
element of a developing nation's medical and economic system. Third World
governments are being encouraged to seek a synthesis between modern and traditional
medicine.
.
Herbal Wealth of India
Now-a-days natural products are an integral part of human health care system,
because there is popular concern over toxicity and resistance of modern drugs. India is
one of the 12 leading biodiversity centers with presence of over 45,000 different plant

P a g e | 14
species, 15000-18000 flowering plants, 23,000 fungi, 16,000 lichens, 18,000
bryophytes and 13 million marine organisms. From this flora, 15,000 to 20,000 have
good medicinal value. Among those only about 7,000 plants are used in Ayurveda,
600 in Siddha, 700 in Unani and 30 in modern medicines.
Liver is the heaviest gland of the body weighing about 1.4 kg in an average
adult and is inferior to the diaphragm occupying most of the right hypochondriac and
a part of the epigastric region of abdominopelvic cavity. The liver is divided into right
and left lobe constituted by hepatocytes which are arranged in irregular, branching
interconnected plates around a central vein. The liver also consists of sinusoids
through which the liver receives the blood. The sinusoids consists of fixed phagocytes
called stellate reticuloendothelial (kupffer) cells which destroy the worn out white
blood cells, red blood cells, bacterial and other foreign matter in the venous blood
draining from the gastrointestinal tract. Bile is partially excretory product and
partially a digestive secretion from hepatocytes. The sodium and potassium salts of
bile acids play an important role in emulsification and breakdown of large lipid
globules into a suspension of droplets and also in the absorption of lipids following
their digestion.
The liver has an enormous task of maintaining the body‘s metabolic
homeostasis. This includes, the processing of dietary amino acids, carbohydrates,
lipids, and vitamins; synthesis of serum proteins; and detoxification and excretion into
bile of endogenous waste products and pollutant xenobiotics.
In hepatic injury five general responses are seen, viz;
1) Inflammation: Injury to hepatocytes associated with an influx of acute or chronic
Inflammatory cells into the liver are termed hepatitis. Attack of viable antigen-
Expressing liver cells by sensitized T-cells is a common cause of liver damage.

P a g e | 15
Inflammation may be limited to portal tract or may spill over into the parenchyma.
E.g., viral hepatitis due to hepatitis A virus (HAV), HBV, HCV, HDV and HEV.
2) Degeneration: The hepatocytes get damaged due to toxic or immunological insult
and show an edematous appearance. Degeneration can also be in the form of stetosis,
where in there is accumulation of fat droplets within the hepatocytes. E.g., hepatic
degeneration can be due to genetic diseases or exogenous substance such as alcohol.
3) Cell death: Cell death which is toxic or immunologically mediated occurs via
apoptosis wherein the hepatocytes become shrunken, pyknotic, and intensely
eosinophilic. Alternatively, hepatocytes may also undergo lytic necrosis (osmotically
swell and rupture). The other types are centrilobular necrosis, bridging necrosis,
submassive necrosis and massive necrosis.
4) Fibrosis: Fibrotic tissue is formed in response to inflammation or direct toxic insult
to the liver. Deposition of collagen has lasting consequences on hepatic pattern of
blood flow and perfusion of heapatocytes. Initially fibrosis may develop within or
around portal tracts or the central vein or may be deposited directly with in the
sinusoids. Progressively, these fibrous strands link regions of the liver (portal-to-
portal, portal-to-central, central-to-central), a process called bridging fibrosis. Fibrosis
is generally considered as an irreversible consequence of hepatic damage.
5) Cirrhosis: Cirrhosis with continuing fibrosis and parenchymal injury, the liver is
subdivided into nodules of degenerating hepatocytes surrounded by scar tissue,
termed cirrhosis and is an end stage form of liver.
The clinical consequences of liver diseases are hepatic dysfunction in the form
of jaundice, hypoalbuminemia, hyperammonemia, hyperglycemia, fector hepatitis,
palmar erythema, spider angiomas, hypogonadism, gynecomastia, weight loss, muscle
wasting, and portal hypertension from cirrhosis. If these are not treated promptly, they

P a g e | 16
will lead to life threatening complications like hepatic failure in the form of hepatic
encephalopathy, hepatorenal-syndrome; or portal hypertension from cirrhosis,
malignancy with chronic disease and hepatocellular carcinoma .
Liver diseases are among the important diseases affecting mankind.
According to WHO about 18,000 people die every year due to liver diseases.
The common ailments of liver are cirrhosis, cholestasis, hepatitis, portal hypertension,
hepatic encephalopathy, fulminant hepatic failure and certain tumors like hepatoma. It
is estimated that two billion people around the world are infected with hepatitis B.
About 350 million of these have the chronic form of the disease.
A few reports on the hepatoprotective activity are cited here, e.g. Apium graveolens
Linn.
(Umbelliferae), Boerhaaia diffusa Linn. (Nyctagina ceae), Euphorbia antisyphilitica
(Euphorbiaceae), Rubia cordifolia (Rubiaceae), Solanum lyratum

(Solanaceae),Tylophora
indica (asclepiadaceae).
Liver damage is always associated with cellular necrosis, increase in tissue
lipid peroxidation and depletion in the tissue GSH levels. In addition serum levels of
many biochemical markers like SGOT, SGPT, triglycerides, cholesterol, bilirubin,
alkaline phosphatase are elevated. In spite of phenomenal growth of modern
medicine, there are few synthetic drugs available for the treatment of hepatic
disorders. However there are several herbs claimed to have possessed beneficial
activity in treating hepatic disorders.but they need to be validated in the light of
science to ensure their ability to conserve therapeutic effectiveness in the formulation
form.
Plant drugs are known to play a vital role in the management of liver diseases
.There are numerous plants and polyherbal formulations claimed to have

P a g e | 17
hepatoprotective action.
tion. However, numerous medicinal prepration
ations have been
advocated a traditional ssystem of medicine, speciallyin ayurvedic, for

f treating liver
disorders.Only a small prroportion of hepatoprotective plants a s well as formulations
used in traditional
ditional medicine are pharmacologically evaluated for their efficiency.
1.1 Anatomy of Liver:
Liver is the largest gland of the body enclosed within the right
ht lower rib cage
beneath the diaphragm. IIt is almost completely covered by visceral peritoneum

p and a
dense irregular connectiive tissue layer that lies deep to the periton
itoneum. Liver is
divided in two principle lobes, a large right lobe and a smaller left lobe separated by
falciform ligament. The right lobe is considered by many anatomissts to include an
inferior quadrate lobe andd a po

posterior quadrate lobe.
Fig. No.1.1 Liver anatomy:
Structure:
The lobes of liver are ma

made up of many functional units called lobule
les. A lobule
consists of specialized epi

pithelial cells called hepatic cells or hepatoccytes arranged in
irregular, branching, intterconnected plates around the central vein. Rather than
capillaries liver has largeer space lined by endothelium called sinusoid

oids through which
blood passes. The sinussoids are also partly lined with stellate reti
eticuloendothellial
(Kuffer‘s) cells which pha

hagocytes worm, bacteria and toxic substancees. Bile secreted

P a g e | 18
by hepatic cells enters bbile capillaries that empty into small bile ducts.
duct These ducts
eventually merge to form

m the larger right and left hepatic duct, which
hich unite and exit
the liver as the common hepatic duct. Further this common hepatic
tic duct joins the
cystic duct from the galll bladder to form the common hepatic duct. The common
hepatic duct and pancreatic

atic duct enter the duodenum in a common duct called the
hepatopancreatic ampulla.
Fig.1.2
ig.1.2 STRUCTURE OF LIVER CELL
Blood Supply:
The liver receives blood from two sources, from hepatic artery it obtains
oxygenated blood and from hepatic portal vein it receives deox

xygenated blood
containing newly absorbed

bed nutrients. Branches of both the hepatic
tic artery and the
hepatic portal vein carrry blood into liver sinusoids, where oxygeen, most of the
nutrients and certain poiisons are excreted by hepatic cells. The reticul
ticuloendothellial
(Kuffer‘s) cells lining thee sinusoids phagocytes microbes and foreign matter from the
blood. Branches of hepatic

patic portal vein, hepatic artery and bilee duct typically
accompany each other in their distribution through the liver. Colle

ollectively, these
structures are called as ‗Porrtal traid
Fig.
ig. No.1.3 Blood supply to the liver
P a g e | 19
Functions of liver:
1. Secretion and excretioon of bile
Bile is partially an excretory product and partially a digestive

tive secretion. Each
day the hepatic cell secrete

etes 800-1000 ml of bile, a yellow, brownish
h or olive green
liquid.
The principle bile pigment is bilirubin. When worm out red blood cells
c broken
down, iron, globins, and bili

bilirubin (derived from heam) are released.
2. Metabolic functions
A) Carbohydrate Metab
bolism
After a meal, the liver achieves net glucose consumption (eg

g, for glycogen
synthesis and generation

on of metabolic intermediates via glyco
olysis and the
tricarboxylic acid cycle).. This occurs as a result of a confluence off several effects.
First, the levels of substrate

ates such as glucose increase. Second, the levels
level of hormones
that affect the amount and

nd activity of metabolic enzymes change. Thus,
Thu when blood
glucose increases, the rattio of insulin to glucagon in the bloodstream

m increases. The
net effect is increased gluco

ucose utilization by the liver. In times of fassting (low blood
glucose) or stress (whenn higher blood glucose is needed), hormone

mone and substrate
levels in the bloodstream

eam drive metabolic pathways of the liver respo
ponsible for net

P a g e | 20
glucose production (eg, the pathways of glycogenolysis and gluconeogenesis). As a
result, blood glucose levels are raised to, or maintained in, the normal range in spite of
wide and sudden changes in the rate of glucose input (eg, ingestion and absorption)
and output (eg, utilization by tissues) from the bloodstream .
B) Protein Metabolism
Related to its important role in protein metabolism, the liver is a major site for
processes of oxidative deamination and transamination. These reactions allow amino
groups to be shuffled among molecules in order to generate substrates for both
carbohydrate metabolism and amino acid synthesis. Likewise, the urea cycle allows
nitrogen to be excreted in the form of urea, which is much less toxic than free amino
groups in the form of ammonium ions. Impairment of this function in liver disease is
discussed in greater detail later.
C) Lipid Metabolism
The liver is the center of lipid metabolism. It manufactures nearly 80% of the
cholesterol synthesized in the body from acetyl-CoA via a pathway that connects
metabolism of carbohydrates with that of lipids. Moreover, the liver can synthesize,
store, and export triglycerides. The liver is also the site of keto acid production via the
pathway of fatty acid oxidation that connects lipid catabolism with activity of the
tricarboxylic acid cycle.
In the process of controlling the body's level of cholesterol and triglycerides,
the liver assembles, secretes, and takes up various lipoprotein particles. Some of these
particles (very low-density lipoproteins [VLDL]) serve to distribute lipid to adipose
tissue for storage as fat or to other tissues for immediate use. In the course of these
functions, the structure of VLDL particles is modified by loss of lipid and protein
components. The resulting low-density lipoprotein (LDL) particles are then returned

P a g e | 21
to the liver by virtue of their affinity for a specific receptor, the LDL receptor, found
on the surface of various cells of the body, including hepatocytes. Other lipoprotein
particles (high-density lipoproteins [HDL]) are synthesized and secreted from the
liver.
3. Haematological functions (Haematopoeisis and coagulation)
1. Production of fibrinogen, prothrombin, heparin, and other clotting factors VII, VIII,
IC and C.
2. Destruction of erythrocytes.(at the end of their respective life span)
4. Circulatory function
1. Transfer of blood from portal to systemic circulation
2. Blood storage (regulation of blood volume)
5. Detoxification and protective functions
1. Kupffer cells remove foreign bodies from blood (phagocytosis).
2. Detoxiocation by conjugation, methylation, oxidation and reduction.
3. Removal of ammonia.
6. Drug metabolism
Liver plays a vital role in biotransformation of drugs. It converts drug molecule
From non polar to polar. Non polar drugs can be conjugated with more polar
Compounds, which make it water soluble for the urinary excretion.
Phases of Biotransformation
Biotransformation generally occurs in two phases. Phase I reactions involve
Oxidation-reductions in which an oxygen-containing functional group is added to the
Substance to be excreted. While oxidation itself does not necessarily have a major
Effect on water solubility, it usually introduces into the drug a reactive "handle" that
Makes possible other reactions that do render the modified substance water soluble.

P a g e | 22
These phase II reactions usually involve covalent attachment of the drug to a
Water-soluble carrier molecule such as the sugar glucuronic acid or the peptide
Glutathione. Unfortunately, by making substances more chemically reactive, phase I
Oxidation reactions often convert mildly toxic drugs into more toxic reactive
Intermediates. If conjugation by phase II enzymes is impaired for some other reason,
the reactive intermediate can sometimes react with and damage other cellular
structures. This feature of drug detoxification has important clinical implications.
1.2: Liver Diseases:
1. Jaundice
This is the yellow pigmentation of the skin, mucous membrane and deeper
tissues due to increased bilirubin level in blood. The normal serum bilirubin level is
0.5to 1.5 mg%. When this exceeds 2 mg %, jaundice occurs.
Types and causes of Jaundice
Jaundice is classified into three type‘s namely haemolytic jaundice,
hepatocellular jaundice, and obstructive jaundice.
a) Haemolytic Jaundice
Haemolytic jaundice is also called prehepatic jaundice. During this, the
excretory function of liver is normal. But, there is excessive destruction of red blood
cells and thus the bilirubin level in blood is increased the liver cells cannot excrete
much bilirubin rapidly. So, it accumulates in the blood resulting in jaundice. In this
type of jaundice the free bilirubin level increases in blood. Increased in formation of
urobilinogenin resulting in the excretion of more amount of urobilinogenin urine. Any
condition that causes haemolytic anemia can lead to haemolytic jaundice.
b) Hepatocellular Jaundice
The jaundice due to the damage of liver cells is called hepatocellular or hepatic

P a g e | 23
jaundice. It is also called hepatic cholestatic jaundice. Here, bilirubin is conjugated.
But the conjugated bilirubin cannot be excreted. So, it returns to the blood. The
damage of liver cells occurs because of toxic substances (toxic jaundice) or by
infection (infective jaundice). Commonly liver is affected by virus resulting
inhepatitis.
c) Obstructive Jaundice
This is otherwise called extra hepatic cholestatic jaundice or post hepatic
jaundice. It is due to the obstruction of bile flow at any level of biliary system.
The bile cannot be poured into small intestine and bile salts and bile pigments
enter the circulation. In this, blood contains more conjugated bilirubin.
2. Hepatitis
Hepatitis is a liver disease characterize by swelling and inadequate functioning
of liver. Hepatitis may be acute or chronic. In severe conditions, it may lead to liver
failure and death.
Causes and Types

Hepatitis is caused by viruses, bacteria poisons, autoimmune disease drug abuse,
alcohol, some therapeutic drugs and inheritance from mother during parturition.
Viral hepatitis is of five types namely, hepatitis A, B, C, D and E.
Hepatitis A and E are caused mostly by intake of water and food contaminated
with hepatitis virus. Generally these two types of hepatitis are not life threatening.
Hepatitis B, C and D are caused by sharing needles with infected person, accidental
prick by infected needle, having unprotected sex with infected person, inheritance
from mother during parturition and blood transfusion from infected donors.
These three forms of hepatitis are serious diseases when compared to hepatitis
A and E. Among these, hepatitis B is more common and considered more serious

P a g e | 24
because it may lead to cirr

rrhosis and cancer of liver.
3. Cirrhosis
The inflammation and dam

damage of parenchyma of liver is known as cirrhosis of
liver. This may results in degeneration of hepatic cells and dysfun

unction of liver.
Cirrhosis is a diffuse, chrronic, necrotic (degenerative) liver disorder characterized

c by
progressive hepatocyte injury followed by regeneration and fibrrosis leading to
disorganization of lobularr architecture, pseudo lobule formation and accquired vascular
malformation. .
4. Tumours of Liver
a) Benign tumors
i) Benign haemangioma
ii) Cysts
b) Malignant tumors
i) Secondary metastasis iis the most common tumors. It may be from

om breast,
lungs and colon.
ii) Primary tumours
5. Hepatocellular Carcin
noma
It is the most common prima

imary liver cancers (comprising 90% of all tumors).
tumo
Etiology and Pathogenessis
Infection with
ith he
hepatitis B virus.
Cirrhosis
Environmental
onmental to
toxins e.g. alpha toxins B produced by Asp
pergillus flavus.
Oral contrace
ceptive.
6. Hepatocellular Failure
re
It may occur due to-
Ultra structuraal lesions of hepatocytes e.g. Raye‘s syndromee.

P a g e | 25
Chronic liver di
diseases e.g. chronic hepatitis, cirrhosis, Wilson
on‘s disease
Coma
7. Hepatic Encephalopaathy
Also called as hepatic coma, is a feature of chronic liver failure. It is a metabolic
disorder of the central nervous system and neuromuscular systema

emassociated with
hepatic failure. It is reverrsible condition.
8. Portal Hypertension
n
In this condition, the

here is increased resistance to portal blood flo
low. It may occur
in the conditions of Portal

tal vein th
thrombosis, Splenomegaly, Cirrhosis.
1.3 Physiology of liver:
It is an understatement
tatement to say that the liver is an important organ.
gan. Every second
the liver cells go through

gh thousands of complex biochemical reaction
tions that influence
all the functions of other organs in the body.
The liver has reserrve functional power and can operate effective
tively when most
of the hepatocytes are nott working well. In addition, diseased hepatocy

ytes can actually
regenerate and returnn to no

normal function.
Fig.
ig. no. 1.4: Drug metabolism in liver
The human body identifies almost all drugs as foreign substances (xenobiotics) and
subjects them to various cheemical processes i.e. metabolism to make them suitable for

P a g e | 26
elimination. This involves chemical transformations to (a) reduce fat solubility and (b) to
change biological activity. Although almost all tissue in the body have some ability to
metabolize chemicals, smooth endoplasmic reticulum in liver is the principal
"metabolic clearing house" for both endogenous chemicals (e.g. cholesterol, steroid
hormones, fatty acids, proteins), and exogenous substances (e.g. drugs). The central role
played by liver in the clearance and transformation of chemicals also makes it susceptible
to drug induced injury.
Drug metabolism is usually divided into two phases: phase 1 and phase 2.
Phase 1 reaction is thought to prepare a drug for phase 2. However many compounds can
be metabolized by phase 2 directly. Phase 1 reaction involves oxidation, reduction,
hydrolysis, hydration and many other rare chemical reactions. These processes tend to
increase water solubility of the drug and can generate metabolites, which are more
chemically active and potentially toxic. Most of phase 2 reactions take place in cytosol
and involve conjugation with endogenous compounds via transferase enzymes.
Chemically active phase 1 products are rendered relatively inert and suitable for elimination
by this step. A group of enzymes located in the endoplasmic reticulum, known
asCytochrome P-450, is the most important family of metabolizing enzymes in the liver.
Cytochrome P-450 is the terminal oxidase component of an electron transport chain.
It is not a single enzyme, rather consists of a family of closely related 50 isoforms, six of
them metabolize 90% of drugs.
1. Three important characteristics of the P-450 system have roles in drug induced
toxicity.
1. Genetic diversity:
Each of the p-450 proteins is unique and accounts to some extent for the
variation in drug metabolism between individuals. Genetic variations (polymorphism)
in CYP-450 metabolism should be considered when patients exhibit unusual
sensitivity or resistance to drug effects at normal doses. Such polymorphism is also
responsible for variable drug response among patients of differing ethnic
backgrounds.
2. Change in enzyme activity:
Many substances can influence p-450 enzyme mechanism like drugs interact with the
enzyme family in several ways. Drugs that modify Cytochrome P-450 enzyme are
referred to as either inhibitors or inducers. Enzyme inhibitors block the metabolic activity

P a g e | 27
of one or several P-450 enzymes; this effect usually occurs immediately and on the
other hand inducers increase p-450 activity by increasing its synthesis. Depending on
inducing drug's half-life, there is usually a delay before enzyme activity increases.
3. Competitive inhibition:
Some drugs may share the same P-450 specificity and thus competitively block
their biotransformation. This may lead to accumulation of drugs metabolized by the
enzyme. This type of drug interaction may also reduce the rate of generation of toxic
substrate.
Role in Production of Binding Proteins
Various cells in the liver synthesize proteins that bind certain substances very
tightly (eg, some vitamins, minerals, and hormones). In some cases, this allows their
transport in the bloodstream, where they would otherwise not be soluble (eg, steroids
bound to steroid-binding globulin, which is synthesized and secreted by hepatocytes).
In other cases, binding proteins made by the liver (eg, thyroid hormone–binding
globulin) allow transport of specific substances (eg, thyroxine) in a form not fully
accessible to tissues. In this way, the effective concentration of the substance is
limited to its free concentration at equilibrium, and the tightly bound fraction forms a
reservoir of the substance that is made available slowly as the free fraction is
metabolized, thereby prolonging its half-life.
Control of body iron occurs at the level of the enterocyte in the duodenum . Thus,
the primary defect in the iron overload disorder hemochromatosis probably
involves the enterocyte. Nevertheless, the liver has the responsibility of making a
variety of proteins crucial for the binding and metabolism of iron. Through the actions of
these proteins, the body gets the iron it needs without allowing excess free iron to
cause damage or support pathogens.

P a g e | 28
Transferrin is an iron-binding protein synthesized and secreted into the
bloodstream by the liver. On binding of free iron at normal pH, transferrin undergoes
a conformational change that gives it high affinity for a specific membrane receptor of
the hepatocyte (transferrin receptor). On receptor binding, the transferrin–transferrin
receptor complex is internalized into the endocytic pathway, a progressively more
acidic environment. There, at low pH, iron no longer remains bound to transferrin.
However, conformational changes that occur at low pH allow transferrin to maintain
high-affinity binding to its receptor even in the absence of bound iron. Thus, when the
receptor recycles back to the surface, it brings the "empty" transferrin with it. On
presentation to the pH 7.4 environment of the bloodstream, transferrin lacking bound
iron is released from the receptor, and the cycle can start over again. In this way,
transferrin and its receptor keep the bloodstream free of unbound iron. Meanwhile, the
free iron released from transferrin in the acidic environment of the endosome is
transported into the cytoplasm of the hepatocyte, where it binds to ferritin, a
cytoplasmic iron storage protein. This provides a reservoir that can be mobilized in
response to the body's needs but makes iron inaccessible to pathogens and keeps it
from causing direct toxic effects. Similar dynamics of plasma-binding proteins,
receptors, or cytosolic storage proteins occur for many other substances, including fat-
soluble vitamins and steroid hormones.
Whereas most solubilization functions are performed in hepatocytes, some of the
binding and storage functions involve accessory cells. Thus, vitamin A storage occurs
in fat droplets seen in the lipocytes of the reticuloendothelial system. Lipocytes have
been implicated in the pathogenesis of chronic liver injury and cirrhosis. Injury to
other cells releases cytokines, which activate the lipocytes
Protective & Clearance Functions

P a g e | 29
Many of the functions of the liver already discussed (eg, drug detoxification and
excretion of excess cholesterol by conversion to and solubilization in bile) can also be
considered protective. Nevertheless, it is useful to conceptualize the protective
function as a separate category because of its clinical importance in ameliorating the
consequences of liver disease.
Phagocytic and Endocytic Functions of Kupffer Cells
The liver helps remove bacteria and antigens that breach the defenses of the gut
to enter the portal blood and also participates in clearing the circulation of
endogenously generated cellular debris. It appears that specialized receptors on the
Kupffer cell surface bind to glycoproteins (via carbohydrate receptors), to material
coated with immunoglobulin (via the Fc receptor), or to complement (via the C3
receptor), thus allowing damaged plasma proteins, activated clotting factors, immune
complexes, senescent blood cells, and so on to be recognized and removed.
Endocytic Functions of Hepatocytes
Hepatocytes have a number of specific receptors for damaged plasma proteins
distinct from the receptors present on Kupffer cells (eg, the asialoglycoprotein
receptor that specifically binds glycoproteins whose terminal sialic acid sugar residues
have been removed). The precise physiologic significance of this metabolic action
remains unclear.
1.4 Types of Liver Dysfunction
Most of the clinical consequences of liver disease can be understood either as a
failure of one of the liver's four broad functions or as a consequence of portal
hypertension, the altered hepatic blood flow of cirrhosis.
Hepatocyte Dysfunction
One mechanism of liver disease, particularly in acute liver injury, is dysfunction

P a g e | 30
of the individual hepatocytes that make up the liver parenchyma. The pathway and
extent of hepatocellular dysfunction determine the specific manifestations of liver
disease. The outcomes to be anticipated when normal hepatic functions fail are
described later.
Portal Hypertension
Some consequences of liver disease, particularly of cirrhosis, are best understood
in terms of what we know about hepatic blood flow. Of greatest clinical importance
are the existence under normal circumstances of a low-pressure portal venous
capillary bed throughout the liver parenchyma and the functional zonation of portal
blood flow.
When pathologic processes (eg, fibrosis) result in elevation of the normally low
intrahepatic venous pressure, blood backs up and a substantial fraction of it finds
alternative routes back to the systemic circulation, bypassing the liver. Thus, blood
from the GI tract is, in effect, filtered less efficiently by the liver before entering the
systemic circulation. The consequences of this portal-to-systemic shunting are loss of
the protective and clearance functions of the liver, functional abnormalities in renal
salt and water homeostasis, and a greatly increased risk of GI hemorrhage from the
development of engorged blood vessels carrying venous blood bypassing the liver
(esophageal varices).
Pathophysiology of Functional Zonation
The fact that hepatocytes in the different zones of the acinus "see" blood in a
particular sequence has great pathophysiologic significance. Because zone 1
hepatocytes see blood that has just left the portal venule or hepatic arteriole, they have
access to the highest concentrations of various substances, both good (eg, oxygen and
nutrients) and bad (eg, drugs and toxins absorbed from the GI tract). Zone 2

P a g e | 31
hepatocytes receive blood containing less of these substances, and zone 3 hepatocytes
are bathed in blood largely depleted of them. However, zone 3 hepatocytes see the
highest concentrations of products (eg, drug metabolites) released into the
bloodstream by hepatocytes of zones 1 and 2. Thus, direct poisons have their most
severe impact on zone 1 hepatocytes, whereas poisons that are generated as a result of
hepatic metabolism cause more damage to those of zone 3. Similarly, because
sinusoidal blood around zone 3 has the lowest oxygen concentration, hepatocytes of
this zone are at greatest risk of injury under conditions of hypoxia.
Manifestations of Liver Dysfunction
Whether a result of hepatocyte dysfunction or portal-to-systemic shunting,
prominent features of liver disease are manifestations of failure of normal functions.
An understanding of these mechanisms offers insight into the probable causes of
illness in a patient with acute or chronic liver disease.
Disordered Bile Secretion
The clinical significance of bile synthesis can be seen in the prominence of
Cholestasis—failure to secrete bile—in many forms of liver disease. Cholestasis can
Occur as a result of extrahepatic obstruction (eg, from a gallstone in the common bile
Duct) or selective dysfunction of the bile synthetic and secretory machinery within
The hepatocytes themselves (eg, from a reaction to certain drugs). The mechanisms
Responsible for cholestatic drug reactions are not well understood. Regardless of the
Mechanism, however, the clinical consequences of severe cholestasis may be
Profound: A failure to secrete bile results in a failure to solubilize substances such as
Dietary lipids and fat-soluble vitamins, resulting in malabsorption and deficiency
States, respectively. Retained bile salts are also cytotoxic, but in the setting of
Cholestasis hepatocytes adapt to decrease uptake of bile salts by downregulating
Ntcp while maintaining bile salt excretion. As a result, hepatic necrosis is minimized

P a g e | 32
in predominantly cholestatic syndromes, with the typical laboratory findings of
Minimally elevated levels of AST and ALT in the presence of marked jaundice and
High levels of bilirubin. However, prolonged exposure to bile salts in chronic
Cholestatic diseases such as primary biliary cirrhosis lead to portal tract cytotoxic
Injury and inflammation, leading eventually to fibrosis and cirrhosis.
Similarly, cholesterol is normally excreted either by conversion into bile acids
or by forming complexes, termed micelles, with preexisting (recycled) bile acids. In
Cholestasis, the resultant buildup of bile acids can lead to their deposition in the skin.
This is believed to cause intense itching, or pruritus. Data suggest that in at least
some patients cholestasis results in altered levels of endogenous opioids. Altered
endogenous opioid-mediated neurotransmission may be responsible for pruritus
instead of skin deposition of bile acids
Impaired Drug Detoxification
Two features of the mechanisms of drug detoxification are of particular clinical
Importance. One is the phenomenon of enzyme induction. It is observed that the
Presence in the bloodstream of any of the large class of drugs inactivated by phase I
Enzymes increase the amount and activity of these enzymes in the liver. This
Property of enzyme induction makes physiologic sense (as a response to the body's
need for increased biotransformation) but can have undesired effects as well.
A second clinically important phenomenon in drug metabolism is that phase I
reactions often convert relatively benign compounds into more reactive and hence
more toxic ones. Normally, this heightened reactivity of phase I reaction products
serves to facilitate phase II reactions, making detoxification more efficient. However,
under certain conditions when phase II reactions are impaired (eg, during glutathione
deficiency from inadequate nutrition), continued phase I enzyme activity can cause

P a g e | 33
increased liver injury. This is because the products of many phase I reactions, in the
absence of glutathione, react with and damage cellular components. Such damage
rapidly kills the hepatocyte.
Thus, the combined effects of certain common conditions can make the
individual abnormally sensitive to the toxic effects of drugs. For example, the
combination of induced phase I activity (eg, caused by alcoholism) with low phase II
activity (eg, caused by low glutathione levels from nutritional deprivation) can result
in heightened generation of reactive intermediates with an inadequate capacity to
conjugate and detoxify them. A classic example of this phenomenon is acetaminophen
toxicity. As little as 2.5 g of acetaminophen can produce significant liver damage in
such susceptible individuals, whereas normal individuals have the capacity to detoxify
10 g/d or more.
Lipoprotein Dynamics and Dyslipidemias
The liver's role in lipid metabolism is illustrated by the genetic defect causing
familial hypercholesterolemia. Lack of a functional LDL receptor in such cases
renders the liver unable to clear LDL cholesterol from the bloodstream, resulting in
markedly elevated serum cholesterol and accelerated atherosclerosis and coronary
artery disease. Heterozygotes with one normal LDL receptor allele can be treated with
drugs (eg, HMG-CoA reductase inhibitors) that inhibit endogenous cholesterol
synthesis and thus upregulate LDL receptor levels. However, there is no effective
drug therapy for homozygotes because they have no normal LDL receptors.
In acquired liver diseases, the serum cholesterol is elevated in biliary tract
obstruction as a result of blockage of cholesterol excretion in bile, and it is diminished
in severe alcoholic cirrhosis, in which fat malabsorption prevents cholesterol intake.

P a g e | 34
Altered Hepatic Binding and Storage Functions
Liver disease influences the liver's ability to store various substances. As a
result, patients with liver disease are at high risk for certain deficiency states such as
folic acid and vitamin B12 deficiency. Because these vitamins are needed for DNA
synthesis, their deficiency results in macrocytic anemia (low red blood cell count with
large red cells reflecting abnormal nuclear maturation), a common finding in patients
with liver disease.
Diminished Synthesis & Secretion of Plasma Proteins
The clinical significance of liver protein synthesis and secretion derives from the
Wide range of functions carried out by these proteins. For example, because albumin
Is the major contributor to plasma oncotic pressure, hypoalbuminemia as a
Consequence of liver disease or nutritional deficiency presents with marked edema
Formation. Other important proteins synthesized and secreted by the liver include
Clotting factors and hormone-binding proteins.
Loss of Protective & Clearance Functions
A crucial protective function of the liver is its role as a filter of blood from the
GI tract, by which various substances are removed from portal blood before it reenters
the systemic circulation.
Clearance of Bacteria and Endotoxin
Clearance of bacteria by Kupffer cells of the liver is the final line of defense in
Keeping gut-derived bacteria out of the systemic circulation. Loss of this capacity in
Liver disease as a result of portal-to-systemic shunting may help to explain why, in
Patients with severe liver disease, infections can rapidly become systemic and result
In sepsis.
Altered Hormone Clearance in Liver Disease

P a g e | 35
Normally, the liver removes from the bloodstream the fraction of steroid
hormones not bound to steroid hormone-binding globulin. On uptake by hepatocytes,
these steroids are oxidized, conjugated, and excreted into bile, where a fraction
undergoes enterohepatic circulation. In liver disease accompanied by significant
portal-to-systemic shunting, steroid hormone clearance is diminished, extraction of
the enterohepatic circulated fraction is impaired, and enzymatic conversion of
androgens to estrogens (peripheral aromatization) is increased.
Sodium & Water Balance

Patients with liver disease often display renal abnormalities and complications,
most commonly sodium retention and difficulty excreting water. An intrinsic renal
lesion is apparently not involved, because the kidneys of patients with liver disease
typically function normally when transplanted into patients whose liver is normal.
Instead, renal abnormalities associated with liver disease are functional, occurring
because liver disease induces altered intravascular pressures and perhaps because of
elevated nitric oxide levels or loss of as yet poorly understood factors secreted from
the liver or the endothelium.

Causes of liver disease:
In many cases the dysfunction of liver will be secondary to a problem elsewhere
in the body.
Trauma:
Animals that receive a severe and blunt blow on the abdomen can suffer from
liver disease. The most common cause of this type of blow is being hit by a car there
by a liver lobe fracture and bleeding into the abdomen, even leading to death and a
more common occurrence is a bruise (contusion).Heatstroke, diaphragmatic hernia
and liver lobe torsion can also cause liver problems.

P a g e | 36
Inflammation:
An inflammed condition of liver is called hepatitis. Trauma can cause this, along
with other drugs, viruses, bacteria, bile and toxins.

Pancreatitis:
The severe inflammatory process that occurs with digestive enzymes can spill
over into the liver and cause severe disease.
Anemia:
Hemolytic anemia can decrease the oxygen availability to liver cells and lead to
their death.
Infection:
Bacteria, viruses, and fungi can all cause liver disease. Since bacterial infection
is common in many liver problems it is routine to use antibiotics when treating liver
problems. Specific diseases include infectious canine hepatitis, canine herpes virus,
feline infectious peritonitis (FIP), leptospirosis, abscesses, histoplasmosis,
coccidiomycosis and toxoplasmosis. Acute viral hepatitis is a systemic infection
manifested primarily by an acute attack on the hepatocytes. Five hepatotropic viruses
have been identified (HAV, HBV, HCV, HDV, HEV). Hepatitis A (HAV) causes
acute, self-limited disease that is transmitted orally. Hepatitis B (HBV) and hepatitis
C viruses (HCV) are transmitted by exchange of body fluids such as blood transfusion
or sexual contacts. Hepatitis D (HDV) is a viroid that causes inflammation only in
concrete with HBV. Hepatitis E (HEV) virus is transmitted by enteric route and cause
self limited diseases.
Chronic hepatitis is an uncommon, but important, complication of HBV- HDV
infection. The liver injury also results from an inflammatory immune attack against
hepatocytes.
Toxins:

P a g e | 37
There are literally thousands of chemicals that could be toxic to the liver and a
few examples of these chemicals that are commonly used in the treatment include:
Rimadyl (arthritis treatment) in labradors
Thiacetarsamide (heartworm treatment)
Ketoconazole (fungal treatment)
Tylenol (acetaminophen)
Glucocorticoids (cortisone)
Anthelmintics (deworming medication)
Parasiticides
Cancer:
Cancer can arise directly within the liver (primary) or spread from elsewhere
(Metastatic or secondary) through the circulatory or lymphatic systems. In the
Anatomy section as mentioned the dual blood supply to the liver; the portal vein and
The hepatic artery. This extra blood supply increases the chance that a tumor in a
Different organ that has spread into the bloodstream will end up in the liver. As
mentioned in the physiology section, liver cancer is usually detected only after the
disease is well established, since functional reserve capacity allowed the liver to
function normally for a prolonged period of time.
Some of these liver cancers include:

Primary:
Lymphosarcoma
Hemangiosarcoma
Metastatic:
Adenocarcinoma
Mammary tumors
Oral carcinoma

P a g e | 38
Lymphosarcoma
Hemangiosarcoma
Metabolic diseases that cause secondary liver problems:
Hypothyroidism
Hyperthyroidism
Pancreatitis
Diabetes mellitus
Cushing's syndrome
Inflammatory bowel disease
Hypoadrenocorticis
Protein-losing enteropathy
Table1.1 : Classification of hepatotoxins and mechanism of action of each group
of hepatotoxins.
Histologic
Category Mechanism of action Examples
lesion
Intrinsic toxicity
Direct physiochemical
destruction by per Necrosis and / Carbon tetrachloride,
Direct
oxidation of or Steatosis Phosphorous
hepatocytes
Interference with
Steatosis or Ethionine,ethyl
Indirect cytotoxic hepatocellular
necrosis alcohol, tetracycline
metabolic pathways
Host idiosyncrasy

P a g e | 39
Chlorpromazine,
Necrosis or
Hypersensitivity Drug allergy phenytoin
cholestasis
sulfonamides
Production of Necrosis or Isoniazide, valproic

Metabolic
hepatotoxic metabolites cholestasis acid.

P a g e | 40
Table1.2 : List of hepatotoxic therapeutic agents and chemicals
Therapeutic agents Chemical
Allopurinol Methotrexate
Alcohol
Amiodarone Nicotinic acid
Arsenic
Azathioprine Nitrofurantoin
Carbon tetrachloride
Carbamazepine Paracetamol
Chloroform
Chlorpromazine Phenelzine
Copper
Chloroform Phenytoin
2,4 dichlorophenoacetic
Ciglitazone Pravastatin
acid
Cimetidine Quinidine
Dimethyl formamide
Dantrolene Rifampicin
Flourine
Erythromycine Salicylates
Toluene
Galactosamine Simvastatin
Trichloro ethylene
Halothane Sodium valproate
Valproic acid
Iproniazid Sulphonamides
Vinyl chloride
Isoniazid Tetracyclines
Ketoconazole Ethanol

P a g e | 41
1.5 Mechanism of drug induced hepatotoxicity
1. Ethanol induced Hep

patic necrosis:
Figure N
No.1.5: Pathways for Alcohol metabolism.
Liver damage is explained on basis of following mechanisms:
1. Alcohol toxicity may be associated with increased oxidative

idative stress and free
radical associated
ated injury. Generation of oxygen metabolitess such as super
oxide (O2−) hyddrogen peroxide (H2O2) and hydroxyl rad

adicals (OH−) is
believedd to be impo
important in the pathogenesis of alcoholic liver injury
inju .
2. Acetaldehyde (the
the major intermediate metabolite of alcohol en route to
acetate production
tion) induces lipid peroxidation and aceetaldehyde –
protein adducts fformation, further disrupting cytoskeletal
al and membrane
m
function.
3. Hepatocellular steatosis results from 1) The shunting of normal

substrates away fr
from catabolism and toward lipid biosynthessis, owing to
generation of exccess reduced nicotinamide – adenine dinucleotide by
two major enzym
me of alcohol metabolism, alcohol dehydrrogenase and
acetaldehyde dehhydro-
genase(generating
ating acetate)2)Impaired assembly and secretion
etion of

P a g e | 42
lipoprotein and 3) increased peripheral catabolism of the fat.
4. Neutrophil infiltration of the liver is common in alcoholic hepatitis and it is

thought that activated neutrophils release toxic oxygen metabolites.
Elevated levels of interleukins 8 and a chemoattractants of neutrophils
are seen in plasma of patients with alcoholic hepatitis.
5. Alcohol induced an immunologic attack on hepatocytes that are

antigenically altered by alcohol or by acetaldehyde – induced alteration in
hepatic proteins.
6. Concurrent viral hepatitis, particularly hepatitis C, is a major factor

that accelerates liver disease in alcoholics. The prevalence of hepatitis C in
patients with alcoholic disease is about 30%.
7. Increased redox ratio: Marked increase in NADH: NAD redox ratio

in hepatocytes results in increased redox ratio of lactate – pyruvate,
leading to lactic acidosis. This altered redox potential has been implicated
in a number of metabolic consequences such as in fatty liver, collagen
formation, occurrence of gout, impaired gluconeogenesis and altered
steroid metabolism.
8. Retention of liver cell water and proteins: Alcohol is inhibitory to secretion of

newly synthesized proteins by liver leading to their retention in
hepatocytes. Water is simultaneously retained in cell in proportion to
protein and results in swelling of hepatocytes resulting in hepatomegaly in
alcoholics.
9. Hypoxia: Chronic ingestion of alcohol results in increased oxygen demand

by liver resulting in a hypoxic state which causes hepatocellular
necrosis in centrilobular zone.
10. Increased liver fat: In chronic alcoholism, there is rise in amount of fat
available to liver which could be from exogenous sources, excess mobilization
from adipose tissue or increased lipid synthesis by liver itself. This

may account for lipid accumulation in hepatocytes.

P a g e | 43
11. Immunological mechanism: Cell-mediated immunity is impaired in alcoholic

liver disease. Ethanol causes direct immunologic attack on
hepatocytes. Immunological mechanism may also explain genesis of
Mallory‘s alcoholic hyalin though more favoured hypothesis for its
origin is aggregation of intermediate filaments of prekeratin type
due to alcohol – induced disorganization of cytoskeleton .
12. Fibrogenesis and inflammation: The mechanism of fibrosis and

inflammatory response in alcoholic liver disease are uncertain but
possible mediators are lymphokines and monokines. The major
stimulant for fibrogenesis is cell necrosis. All forms of collagen
are increased and there is increased transformation of fat storing
cells into myofibroblasts and fibrocytes. Leukotrienes which are
important mediators of inflammation are produced by alcohol –damaged
hepatocytes resulting in inflammatory reaction in affected areas.
2. CCl4 induced hepatotoxicity:
Liver injury due to carbontetrachloride in rats was first reported in 1936 and has
been widely and successfully used by many investigators. Carbontetrachloride is
metabolized by cytochrome P-450 in endoplasmic reticulum and mitochondria with
the formation of CCl3O-, a reactive oxidative free radical, which initiates lipid
peroxidation.
Administration of a single dose of CCl4 to a rat produces, within 24 hrs, a
centrilobular necrosis and fatty changes. The poison reaches its maximum
concentration in the liver within 3 hrs of administration. Thereafter, the level falls and
by 24 hrs there is no CCl4 left in the liver. The development of necrosis is associated
with leakage of hepatic enzymes into serum. Dose of CCl4: 0.1 to 3 ml/kg I.P.
3. Thioacetamide induced hepatotoxicity:
Thioacetamide, originally used as a fungicide, is a potent hepatotoxicants,
bioactivated by CYP450 and/or Flavin-Containing Monooxygenase (FMO) systems to
sulfine (sulfoxide) and sulfene (sulfone) metabolites, which cause centrilobular

P a g e | 44
necrosis. Thioacetamide sulfoxide, a relatively stable intermediate of thioacetamide is
the penultimate reactive, is obligatory for the hepatotoxic effects.
The hepatotoxic effects of thioacetamide are expressed only after its metabolism to
thioacetamide s-oxide that further undergoes metabolic conversion to an unidentified
metabolite, probably the reactive unstable thioacetamide sulfone. Since much of the
TA transformation mechanism and toxicity study was conducted largely prior to the
advent of the discovery of CYP450 isozymes and information on specific isozymes
involved in the bioactivation of TA has remained completely blurred and
uninvestigated .
A single large dose 100mg/kg is followed by degenerative changes in 6-8 hrs. By
24 hrs this dose in rats leads to centrizonal necrosis and in 24-30 hr the effect will be
maximal. The prenecrotic changes include loss of glycogen by 6 hrs and acidophilic
degeneration of cells in the central zone by 8 hrs. By 9th hr this is accompanied by the
dilatation of sinusoids to form pathways between the central veins of contiguous
lobules. Hepatocytes of central zone contain a few lipid droplets and repair begins at
37 hr and the liver will return to normal by 7 days.
1.6 Models to Evaluate Effect of Drugs on Liver
As liver is a multifunctional organ, a battery of liver function tests is employed
to evaluate the effect of drug on liver, which are Non-invasive functional methods:
1. Pentobarbitone induced sleeping time
2 .Biochemical analysis of blood for
a. SGPT
b. SGOT

P a g e | 45
c. Alkaline phosphatase
d. Serum bilirubin
e. Total proteins
Morphological test-Wet weight of liver/100 gm body weight
Free radical scavengers
a. Glutathione
b. Lipid peroxidation
c. Superoxide dismutase
d. Catalase
f. Glutathione peroxidase
g. Histopathology of liver .
Hexobarbitone or zoxazolamine induced sleeping time
Toxic liver prolongs duration of sleeping time for pentobarbitone,
hexobarbitone, zoxazolamine etc in mice, rats.
Serum and hepatocyte enzyme AST i.e. Aspartate Transaminase (SGOT), and
ALT i.e. Alanine Transaminase (SGPT), are both sensitive markers of hepatocellular
injury. When the liver cell is injured or dies, these proteins can leak through the liver
cell membrane into the circulation and serum levels will rise. ALT or SGPT is a
cytosolic enzyme primarily present in the liver. Its normal serum level is 10-35
Karmel units/ml. ALT reversibly catalyses amino group from alanine to α-
ketoglutarate.
ALT levels are very high in patients of viral hepatitis and hepatic necrosis, 10 to

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200 fold higher in patients of post hepatic jaundice, intrahepatic cholestasis and below
10 fold in patients of metastatic carcinoma, cirrhosis and alcoholic hepatitis.
AST or SGOT is a mitochondrial enzyme released from heart, liver, skeletal
muscles and kidney. Its normal serum level is 10-40 Karmel units/ml AST reversibly
catalyses transfer of amino group from aspartate to α-ketoglutarate.
AST levels are 10 to 200-fold elevated in patients with acute hepatic necrosis,
viral hepatitis, CCl 4 and drug induced poisoning.
Morphological parameters
Morphological parameters like weight of the animals, weight and volume of the
liver have also been used to evaluate the protective effect of the drug. Hepatotoxicity
causes loss in liver weight/100 gm body weight of rats.
Hepatocyte Viability and Oxygen Uptake tests
Hepatotoxicants reduce the viability of hepatic cells as assessed by trypan blue
exclusion and oxygen uptake tests. In liver, CCl 4 is metabolised to CCl 3 O - by

-
cytochrome P-450 and the reactive oxidative free radical intermediate generated, O
causes further damage. Utilisation of oxygen by hepatocytes gets reduced; therefore
the viability of hepatocytes is reduced.
Free Radical Scavenging
Free radicals are reactive molecules involved in many physiological processes
and human diseases such as cancer, aging, arthritis, Parkinson syndrome, ischaemia,
toxin induced reaction, alcoholism, liver injury etc. The damage to hepatic
parenchymal cells, leading to hepatic injury, is due to oxidative stress within the cells
caused by partially reduced free oxygen (PRFO) species such as O2 (Superoxide
anion),H 2 ,O2 , and OH (hydroxy free radical). The elevation of free radical levels

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seen during the liver damage is due to enhanced production of free radicals and
decreased scavenging potential of the cells. A variety of intrinsic antioxidants
(reduced glutathione, superoxide dismutase, glutathione-S-transferase etc.) are present
in the organism, which protect them from oxidative stress.
Technically, the estimation of free radicals directly is not possible due to the
transient nature of the free radicals. Thus estimations are usually done indirectly by
measuring the ―Antioxidant defense status‖ of the liver microsomes. Hepatoprotection
by enzymatic free quenching is brought about by elevating the levels of antioxidant
enzymes in tissues such as the Superoxide dismutase (SOD), Peroxidase and Catalase.

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CHAPTER-2
PLANT PROFILE
Azad institute of pharmacy and research Page 48

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PLANT INTRODUCTION
Tarxacum officinale
Species Information
Scientific Taraxacum officinale Weber ex F H Wigg

name
Synonyms Priest's Crown, Swine's Snout
Common Dandelion
Name
Conversati Extremely common and widespread

on status
Known Not known

Hazards
Habitat Found in a very wide variety of habitats, but tends to thrive in distributed
sites such as lawns,paths,waste ground, pastures and road verges.
Taxanomy
Class Equisetopsida
Sub-class Magnoliidae
Super order Asternae
Order Asteralaes
Family Asteraceae/compositae
Genus Taraxacum
Geography and distribution

Taraxacum officinale is native to Eurasia. The Dandelion (Taraxacum officinale, Weber, T.
Densleonis, Desf; Leontodon taraxacum, Linn.), though not occurring in the Southern
Hemisphere, is at home in all parts of the north temperate zone, in pastures, meadows and
on waste ground, and is so plentiful that farmers everywhere find it a troublesome weed, for
though its flowers are more conspicuous in the earlier months of the summer, it may be found
in bloom, and consequently also prolifically dispersing its seeds, almost throughout the year

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Description
The common name derives from the French 'dent de lion', meaning 'lion's tooth', which refers
to the deeply toothed, deep green leaves, which are in rosettes.
The bright yellow flower heads are borne on hollow stalks and the fruiting heads have a
distinctive downy appearance.
As most British dandelions produce fruit without being fertilised (they are apomictic),
substantial problems arise with the taxonomy of these plants. This group is a complex
consisting of around 200 microspecies, and is typically treated as a species aggregate,
denoted as Taraxacum officinale agg. In reality, the specimen that was used to describe the
species first of all has turned out to be a microspecies restricted to Lapland, and it is not the
same as the plants seen in grassland, lawns and along path edges in Britain. Trying to identify
the microspecies has turned into a science of its own, with the experts termed
‘taraxacologists’.
The dandelion is a perennial plant, and flowers throughout the year. Dandelions have deep
taproots, and the whole plant contains a milky fluid known as latex. The flowerheads close at
night, and can produce around 2,000 wind-dispersed fruits. Plants can also regenerate from
pieces of the taproot.
Uses
Although generally regarded as a weed, dandelions have many uses, both culinary and
medicinal.
Taraxacum officinale has diuretic and laxative properties. It has been used as a tonic, to treat
rheumatic problems, and as a blood purifier. Young leaves and inflorescences are used as
ingredients in salads and stir-fries.
Diuretic, tonic and slightly aperient. It is a general stimulant to the system, but especially to
the urinary organs, and is chiefly used in kidney and liver disorders.
Dandelion is not only official but is used in many patent medicines. Not being poisonous,
quite big doses of its preparations may be taken. Its beneficial action is best obtained when
combined with other agents.
The tincture made from the tops may be taken in doses of 10 to 15 drops in a spoonful of
water, three times daily.
It is said that its use for liver complaints was assigned to the plant largely on the doctrine of
signatures, because of its bright yellow flowers of a bilious hue.
In the hepatic complaints of persons long resident in warm climates, Dandelion is said to
afford very marked relief. A broth of Dandelion roots, sliced and stewed in boiling water with
some leaves of Sorrel and the yolk of an egg, taken daily for some months, has been known
to cure seemingly intractable cases of chronic liver congestion.

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A strong decoction is found serviceable in stone and gravel: the decoction may be made by
boiling 1 pint of the sliced root in 20 parts of water for 15 minutes, straining this when cold
and sweetening with brown sugar or honey. A small teacupful may be taken once or twice a
day.
Dandelion is used as a bitter tonic in atonic dyspepsia, and as a mild laxative in habitual
constipation. When the stomach is irritated and where active treatment would be injurious,
the decoction or extract of Dandelion administered three or four times a day, will often prove
a valuable remedy. It has a good effect in increasing the appetite and promoting digestion.
Dandelion combined with other active remedies has been used in cases of dropsy and for
induration of the liver, and also on the Continent for phthisis and some cutaneous diseases. A
decoction of 2 OZ. of the herb or root in 1 quart of water, boiled down to a pint, is taken in
doses of one wineglassful every three hours for scurvy, scrofula, eczema and all eruptions on
the surface of the body
Cultivation
Taking the fruits from dandelion clocks can provide hundreds of plants if they are sown onto
an all-purpose compost in deep pots, to accommodate the roots, or straight onto open soil.
Plants are likely to start flowering in their second year. There are seed suppliers who stock a
thick-leaved variety, more suitable for growing as a salad vegetable.
The dandelion is so successful at colonizing abandoned areas of the garden that no help is
really needed to grow it. The plentiful one-seeded fruits are carried on the wind and can
travel relatively long distances so neglecting a garden will usually ensure that this plant
appears. Removing it is more difficult, as the long, thick taproots will delve down deeply into
a border or lawn and even in cracks in paving, making them hard to remove completely.
Chopping off the top of the plant is not enough to kill it as it will sprout again from the
remaining roots. Rabbits love the leaves so if you have one as a pet it may appreciate a patch
of dandelions being allowed to grow in a corner of the garden. The flowers and fruiting heads
are attractive but this plant can soon take over and, because it is so strongly associated with a
poorly kept garden, deliberate cultivation is rare.
Parts Used Medicinally---The root, fresh and dried, the young tops. All parts of the plant
contain a somewhat bitter, milky juice (latex), but the juice of the root being still more
powerful is the part of the plant most used for medicinal purposes.

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GiovanniVidar et al., (1997) reported Desymmetrization of bicyclo [3.3.0] octane-3,

7-dione by the Schmidt reaction: an easy synthesis of tecomanine. The C2v symmetry
of cis-bicyclo[3.3.0]octane-3,7-dione 1 was altered by a selective Schmidt reaction to
give the 3-azabicyclo[4.3.0]nonane building block 3b which was employed in a short
synthesis of(±)-tecomanine.AsymmetricSchmidtreactionon1,employing(2S,4R)-2-
azido-4-hydroxypentane 14 as a chiral inducer, showed encouraging levels of
enantiotopic methylene group stereo differentiation.
Gurnos Jones et al., (1971) have reported the stereochemistry and absolute
configuration of two alkaloids from Tecoma stans, tecomanine and the
oxygenated skytanthine Alkaloid C, have been determined by single-crystal X-ray
diffraction studies.
Harmesh Kumar Sharma et al., (1975) reported a new mode of ring cleavage of 2,3-
Dihydroxybenzoic acid in Tecoma stans. 2, 3-Dihydroxybenzoic acid has been
shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. 2-
carboxy-cis, cis-muconic acid, muconolactone, 3-oxo-adipic acid and carbon dioxide
are formed during its metabolism. The first reaction of the pathway, is the
conversion of 2, 3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid catalysed by
an enzyme 2,3-dihydroxybenzoate 2,3-oxygenase.
The enzyme has been partially purified and is very labile with a half-life of 3 - 4 h. It
is maximally active with 2, 3-dihydroxybenzoate as the substrate and does not exhibit
any activity with catechol,4-methylcatechol,3,4-dihydroxybenzoic acid, etc.
Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by
pre incubation of the enzyme with the substrate. It also affords protection to the
enzyme against thermal inactivation. Data on the effect of metal ions as well as metal
chelating agents suggest that copper is the metal cofactor of the enzyme.
Satya p. Kunapuli et al., (1983) isolated a new indole oxygenase from the leaves of
Tecoma stans was isolated and purified to homogenity. The purified enzyme system
catalyzes the conversion of indole to anthranilic acid. It is optimally active at pH 5.2
and 30°C. Two moles of oxygen are consumed and one mole of anthranilic acid is
formed for every mole of indole oxidized. Dialysis resulted in complete loss of the

P a g e | 53
activity. The inactive enzyme could be reactivated by the addition of concentrated

dialysate. The enzyme is not inhibited by copper specific chelators, non-heme iron
chelators or atebrin. It is not a cuproflavoprotein, unlike the other indole oxygenases
and oxidases.
Nicolas Robert et al., (2007) have reported neat synthesis of six monoterpenic
alkaloids of the actinidine series. A concise enantiopure synthesis of six monoterpenic
alkaloids of the actinidine series possessing a cyclopenta[c] pyridineskeleton,(+)-
deoxyrhexifoline (1), (+)-boschniakinic acid (2), (+)-boschniakine (3), (-)-
plantagonine (4), (-)-indicaine (5) and (-)-tecostidine(6) is reported starting with the
chiral precursor 3-bromo-5-((4R)-phenyloxazolin-2-yl)pyridine. Mixture of (3R)- and
(3S)-7-((4R)-phenyloxazolin-2yl)cyclopenta[c]pyridines was separated by HPLC
before being transformedinto enantiopure natural products (4–9) by modification of
the oxazoline
group.
Luca Costantino et al., (2003) reported isolation and pharmacological activities of the
Tecoma stans alkaloids. The alkaloids isolated from the plant, Tecomine is
responsible for the hypoglycemic action. Given the interest in substances able to treat
type II diabetes, the alkaloids are isolated from the plant are tested them in vivo on
db/db mice. Contrary to previous literature reports on different animal models,
Tecomine was unable to modify glycemia; the only effect seen being a decrease in
plasma cholesterol levels, and when tested in vitro on glucose uptake in white
adipocytes, the compound showed a marked effect. The two other alkaloids isolated,
namely 5b-Hydroxyskitanthine, early called Base C, and Boschniakine were inactive
both in vivo and in vitro assays.
P. Madhusudanan Nair et al., (1964) reported an enzyme system which converts
anthranilic acid to catechol was detected in the leaves of Tecoma stans and its
properties studied. The system is present exclusively in the chloroplast fraction of the
leaves. The optimum pH of the reaction is 5.2 and maximum activity was obtained
with citrate-phosphate buffer. There was good stoichiometry between the amounts of
anthranilic acid disappeared and the amounts of catechol and ammonia formed. It
showed an absolute requirement for oxygen and evidence was obtained for the
probable participation of NADPH and FAD in the hydroxylation step. The optimum

P a g e | 54
concentration of anthranilic acid was 10−4 M at higher concentrations the reaction

was inhibited to a considerable extent. Cyanide, pyrophosphate, and EDTA also
caused inhibition indicating a requirement for metal ions.
Armandodoriano Bianco et al., (1981) have reported 5-deoxystansioside, an iridoid
glucoside from Tecoma stans. In addition to plantarenaloside and stansioside, a new
iridoid glucoside with a formyl group at C-4 has been isolated from Tecoma stans.
The new glucoside was shown to be 5-deoxystansioside by 13C NMR and 1H NMR
spectroscopy.
P. Madhusudanan Nair et al., (1964) reported a purified anthranilic acid oxidase
system of Tecoma stans and its separation into different enzymic components. The
conversion of anthranilic acid to catechol has been purified 20-fold from a cell-free
leaf extract of Tecoma stans. The optimum substrate concentration was 10−3 M and
optimum temperature for the reaction was 45°c. The presence of a multi-enzyme
system was inferred from inhibition studies. The formation of catechol was inhibited
by Mg2+, Zn2+, and Co 2+ ions, PCMB, metal chelating enzymes (EDTA,Cyanide)
whereas anthranilic acid disappearance was not affected to the same extent. The
overall reaction is markedly activated by NADPH and THFA.
Harmesh K. Sharma et al., (1975) have reported 2, 3-Dihydroxybenzoate-2, 3-
oxygenase is mainly localized in the soluble and the chloroplast fractions of Tecoma
leaves. The chloroplast enzyme has properties similar to those of the soluble enzyme,
but it has a longer half-life and is more stable to dialysis than the soluble enzyme. It is
inhibited by sulfhydryl reagents and is reversed by the addition of reduced
glutathione. It is insensitive to iron-chelating agents. The enzyme loses activity on
dialysis against copper-chelating agents and the activity is completely recovered on
the addition of copper; addition of iron does not restore the activity. Polyphenol
oxidase is probably present only in the actiive form in the Tecoma chloroplast but it is
not involved in the intradiol cleavage of 2,3-dihydroxybenzoic acid.
Costantino L et al., (2003) isolated Tecostanine was isolated from Tecoma stans
leaves. Its sterochemistry was elucidated as well as its anti-hyperglycemic activity and
its affinity to opioid and nicotinic receptors. The oxalate salt of Tecostanine did not
significantly affect blood glucose levels in normoglycaemic and hyperglycaemic rats.
It did not appear to interact with opioid receptors (mu type) and showed only
moderate affinity to the nicotinic receptor.
Pranay Dogra et al., (2009) reported the Antibacterial and Anticancer activity of

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selected trifoliate plants. The four trifoliate plants are Glycine max, Cajanuscajans,
Phaseolus vulgaris and Tecoma stans. The antibacterial activity of the extracts was
evaluated against seven bacterial strains Bacillus subtilis,Escherichia coli,
Staphylococcus aureus, Proteus mirabilis, Proteus vulgaris, Pseudomonas
aeruginosa and Salmonella typhi by agar well diffusion method. The anticancer
activity was evaluated against MCF7 and Hela cancer cell lines first by visual
examination followed MTT based Cytotoxicity assay and Cell Viability Count.
Chloroform extracts showed the best antimicrobial activity. Methanol extracts of all
the selected plants demonstrated the best anticancer activity. Tecoma stans exhibited
anticancer activity over a range of solvents fractions.
Marzouk M et al., (2006) have reported the Anti-proliferative and antioxidant
constituents from Tecoma stans. Biological investigations of a Tecoma stans fruits
extract and compounds 1, 2, 4, and 8 indicated that the extract, 1, 2, and 4 possessed a
strong scavenging activity to DPPH, peroxyl and hydroxyl radicals. Unlike 4, which
potentially induced NO generation in bacterial lipopolysaccharide-stimulated raw
murine macrophage, the extract, 1, 2, and 8 significantly inhibited the NO generation.
The extract 2 and 4 exhibited a cytotoxic effect on human hepatocarcinoma cells
(Hep-G2), while the extract, 2 and 8 were potent growth inhibitors of human breast
carcinoma cells (MCF-7). 1 and 2 were remarkable growth inducers of human
lymphoblastic leukemia cells, whereas the extract, 2, and 8 stimulated the macrophage
proliferation rate. Taken together, the novel compound 8 is effective as anti-
proliferative agent against MCF-7 cells and as NO inhibitor, whereas 2 exhibited
multi-functional properties as antioxidant and anti-proliferative agent against both
solid tumor cell lines Hep-G2 and MCF-7 cells.
Qureshi S et al., (1997) in vitro evaluation of inhibitory nature of extracts of 18-plant
species against 3-keratinophilic fungi. Effect of extract of 18 plant species, viz.,
Acorus calamus, Adhatoda vasica, Amomum subulatum, Andrographis paniculata,
Boerhaavia diffusa, Cassia occidentalis, Centella asiatica, Cymbopogon citratus,
Hemidesmus indicus, Hyptis suaveolens, Malvestrum sp., Passiflora edulis,
Pergularia daemia, Peristrophe bicalyculata, Shuteria hirsuta, Solanum nigrum,
Tecoma stans, and Verbascum chinense on the growth of Microsporum gypseum,
Chrysosporium tropicum and Trichophyton terrestre was evaluated and discussed.
The sensitivity of the keratinophilic fungi was evaluated by dry-weight method.
Chauhan SV et al., (2004) have reported Quantitative and qualitative analysis of

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phenolics and boron in stigma of transient sterile Tecoma stans during seedless,
partially seed bearing and seedbearing periods was made. UV absorption profile of
stigmatic exudates indicated the presence of simple phenolics. Total phenolics were
higher in stigma during seedless period. Thin layer chromatographic analysis of
stigmatic extracts exhibited only three principle spots. Mass spectrophotometry
showed the presence of derivatives of cinnamic acid, namely, caffeic acid in these
spots. Quantity of boron in stigma during seedless period was lowest but the
difference with other periods was not significant. It was suggested that the
accumulation of higher quantity of caffeic acid in the stigma during seedless period
due to high temperature (400c-450c) could lead to inhibition of pollen germination in
vivo, thereby rendering the plants seedless. This was confirmed by inhibition of in
vitro pollen germination in the basal medium containing higher quantity of caffeic
acid.
M. Lozoya-mecke et al., (1985) reported the intravenous administration of Tecoma

stans infusion in normal dogs produces an early hyperglycemic response and
arterial hypotension followed by a slow decrease of the glucose blood values with a
concomitant hypertriglyceridemia. Heart frequency was gradually increased after the
first 60 min of drug administration and persisted for several hours. The effects
observed on blood parameters seem to be related to hepatic glycogen metabolism,
involving an activation of glycogenolysis. The late hypoglycemic effect of Tecoma
stans infusion could be considered secondary to the observed hepatic glucose output.
Binutu Oa et al., (1994) reported antimicrobial potentials of some plant species of the
Bignoniaceae family The methanol extracts of the leaves and stem bark of four
Bignoniaceae plants Jacaranda mimosifolia, Tecoma stans, Tabebuia rosea , and
Crescentia cujete were studied for their antimicrobial activity using a wide range of
Gram- positive and Gram-negative bacteria and fungi. Extracts of both the leaves and
stem bark of majority of plant species studied showed variable but remarkable broad
spectrum antimicrobial activity. Methanol extracts of Tecoma stans leaves was found
to be effective against only Candida albicans. It was observed that the extracts of stem
bark generally showed better antimicrobial activity than those of the leaves.
Angel Josabad Alonso-Castro et al., (2010) have reported antidiabetic plants Tecoma
stans (Bignoniaceae) and Teucrium cubense (Lamiaceae) induce the incorporation of

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glucose in insulin-sensitive and insulin-resistant murine and human adipocytes. Both

extracts stimulated 2-NBDG uptake by insulin- sensitive and insulin-resistant
adipocytes in a concentration-dependent manner.
Alma R.et al., (2009) repoted comparison of metabolite levels in callus of Tecoma
stans cultured in photoperiod and darkness Tecoma stans cultured in either a set
photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem,
and leaf) were established on B5 culture medium. While leaf-derived callus grew
slower under a 16-h photoperiod than in darkness it accumulated the highest amount
of both phenolics and flavonoids The antioxidant activity was significantly higher
when callus was cultured in period light than when grown in extended darkness.

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3. METHODOLOGY
3.1 List of materials and equipments used during experiment:
Table 3.1: List of materials and equipments used during experiment
S.No Name of the materials and equipments
1 Taraxacum officinale roots : From Ambedkar University by Field Botanist Dr. R.B.Ram
2 Anaesthetic ether (Sigma solvents and Pharmaceuticals- Mumbai)
3 Absolute alcohol (Nice – Cochin)
4 Formalin (Nice–Cochin, Fischer Scientific. Lot No:- 91026906-5., Pdt No.24005))
5 Methanol (Leo chemicals, Bangalore, India)
6 Chloroform (S.D. Fine – Chem Ltd. Mumbai. Mole. Wt-119.5.
7 Heparin (HEP-5, Gland Pharma Ltd, Hyderabad. Batch – No. UJ918)
8 Kcl (S.D. Fine – Chem Ltd. Mumbai.Batch-No:- 200Z-0200-1612-09.Mole.Wt:-74.5)
9 Nacl (LeoChem, Bangalore. Mole.Wt:- 58.44, Lot No. 126012)
10 EDTA(S.D.Fine Chem Ltd. Pdt No:- LO4/10204/2611/13)
11 KH2PO4 (Qualigens fine Chemicals, Mumbai. Lot No:- 18986711)
12 K2HPO4 (Leochem, Bangalore. Lot No- 125176, P-2V829)
13 H2O2 (SDFCL-38694L05,Batch No- G09A/2209/0807/13)
14 Chem. Kit for SGOT estimation (Coral clinical systems, Verna Goa, India)
15 Chem. Kit for SGPT estimation (Coral clinical systems, Verna Goa, India)
16 Chem. Kit for SALP estimation (Coral clinical systems, Verna Goa, India)

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17 Chem. Kit for Bilirubin estimation (Coral clinical systems, Verna Goa, India)
18 Chem.Kit for Albumin estimation(Coral clinical systems, Verna Goa, India)
19 Chem. Kit for Cholesterol estimation (Coral clinical systems, Verna Goa, India)
20 Chem. Kit for Triglycerides estimation (Coral clinical systems, Verna Goa, India)
21 Chem. Kit for Total protiens estimation (Coral clinical systems, Verna Goa, India)
22 Autoanalyser (ARTOS, The versatile Autoanalyser, Sl No- SBPL/188/06-07)
23 Incubator (REMI Cooling Centrifuge. C-24 BL)
24 Digital balance (ACCULAB – Sartorious group)
25 Rotary flash evaporator (SUPERFIT, Rotary “Vaccum Digital Bath”, PMTc – 3040)
26 Deep freezer (BLUE STAR, Model No.- CHE 400, Sr No.- 67771)
27 Albino rats (Wistar strain) ( Naka,Lucknow)
3.2 Collection of plant material
Taraxacum officinale were collected from the garden of Ambedkar University. The plant was
identified and authenticated by Prof. R.B Ram, Head, Dept of applied plant
science(Horticulture),Baba sahib Bhimrao Ambedkar University,Lucknow. A herbarium
specimen No. SCSCOP.Ph.Col Herb.No. 012/2006-2007 was preserved in Ambedkar
University.
3.3 Preparation of Ethanolic Extract:
The dried powder of roots were defatted with petroleum ether, chloroform and then extracted
with 70% ethanol using soxhlet apparatus. The extracts was concentrated under reduced
pressure using rota flash evaporator and stored in airtight container in refrigerator below
10ºC.

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Weighing Dried Powder of roots
Soxhlet Apparatus
3.4 Qualitative phytochemical screening:
The following tests were carried out on the standardized herbal extract to detect
various phytoconstituents present in them.
1.Detection of carbohydrates
Small quantity of the extract was dissolved in distilled water and filtered.
The filterate was subjected to
i.Molisch’s test

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ii.Fehling’s test
iii.Barfoed’s test
i. Molisch’s test
To the filterate few drops of alcoholic α-napthol was added and 2ml of conc
sulphuric acid was added slowly through the slides of the test tube. No purple colored
ring was formed at junction of the two layers, which indicates absesnce of
carbohydrates.
ii. Fehling’s test
Small portion of the extract was treated with fehling’s solution I and II and then heated
on water bath. No brick red colored precipitate was formed, which indicates absence of
carbohydrates
iii. Barfoed,s test
Small portion of the extract was treated with barfoed’s reagent. No red
precipitate formed, which indicates absence of carbohydrates.
2. Test of starch
A small amount of powdered drug was treated with diluted iodine solution. No
blue color was observed, which indicates absence of starch.
3. Detection of proteins and amino acids
Small quantity of extract was dissolved in few ml of water and was subjected to
million’s, biuret and ninhydrin test.
a) Million’s test:
The extract was treated with million’s reagent.white precipitate was

produced, shows
the Presence of proteins and free aminoacids.

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b) Biuret test:
To the extract equal volume of 5%w/v NaOH and four drops of 1%w/v CuSO4
solution were added. purple color was formed indicating the Presence of
proteins.
c) Ninhydrin test:
The extract was treated with ninhydrin reagent.purple color was

produced,indicating
the presence of proteins.
4. Detection of phenolic compounds and tannins
The decoction were diluted with distilled water and filtered. The filtrates were
treated with following reagent.
a) Ferric chloride test:
The filtrate was treated with 5% of ferric chloride solution.black precipitate was
found
in the decoction of the plant, indicating the Presence of tannins and phenolic
compounds.
b) Test with Lead acetate Solution:
Few ml of filtrate were treated with lead acetate solution.white precipitate
was produced in the decoction of plant.
c) Gelatin test:
To the filtrate of decoction, add 1ml of 1% solution of gelatin.white
precipitate was seen, which indicates Presence of tannin in plant.
5. Test for phytosterols
Small quantity of decoction were dissolved in 5ml of chloroform separately.

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Then these chloroform layer subjected to,
a) Salkowski test
b) Libermann – Burchards test
a)Salkowski test:
To 1ml of the above prepared chloroform solutions, few drops of conc H2SO4
was added. Red color produced in the lower layer, shows the presence of phytosterols.
b)Libermann – Burchards test:
The above chloroform solution was treated with few drops of conc H2SO4
followed by 1ml of acetic anhydride solution. Green color was produced, shows the
presence of phytosterols.
6. Test for fixed oils and fats
a) Spot test:
A small quantity of extract was pressed between two filter papers. Oil stain was
observed, show presence of fixed oils.
b) Saponification:
Few drops of 0.5N alcoholic potassium hydroxide was added to extract along
with a few drops of phenolphthalein. The mixture was heated on a water bath for
about 1 – 2 hours. Formation of soap or a partial neutralization of alkali indicated the
presence of fixed oils and fats.
7. Test for alkaloids
Small amount of extract was stirred with a few ml of dil HCl and filtered. The
filtrate was tested with various alkaloidal reagents such as Mayer’s, Dragendroff’s,
Wagner’s and Hager’s reagent.

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a) Mayer’s test:
To the small amount of filtrate add few drops of Mayer’s reagent. A white color
precipitate was formed, indicating the presence of alkaloids.
b) Dragendroff’s test: (potassium bismuth iodide)
To the small amount of filtrate add few drops of Dragendroff’s reagent. An
orange red color precipitate was formed, indicating the presence of alkaloids.
c) Wagner’s test:
To the small amount of filtrate add few drops of Wagner’s reagent. A brown
color precipitate was formed, indicating the presence of alkaloids.
d) Hager’s test: (picric acid)
To the small amount of filtrate add few drops of Hager’s reagent. An yellow
crystalline precipitate was formed, indicating the presence of alkaloids.
8. Test for glycosides
A small amount of the extract was hydrolyzed with hydrochloric acid for one
hour on a water bath and hydrolysate was subjected to
a) Legal’s test
b) Balget’s test
c) Borntrager’s test
d) Modified borntrager’s test.
a) Legal’s test:
To the hydrolysate 1ml pyridine few drops of sodium nitroprusside solution was
added and then made alkaline with NaOH solution. Pink color was obtained showing
the presence of glycosides.

P a g e | 65
b) Balget’s test:
To a solution of extract sodium picrate solution was added. Yellowish orange
color was obtained showing, the presence of glycosides.
c) Borntrager’s test:
Hydrolysate was treated with chloroform and the chloroform layer was
separated. To this equal quantity of dilute ammonia solution was added. Pink color
was observed in ammoniacal layer, confirms the presence of glycosides.
d) Modified borntrager’s test:
The extracts were boiled with few ml of dil HCl and 5ml of ferric chloride
solution. The contents are cooled and shaken with organic solvent. Organic layer was
separated and to this equal volume of ammoniacal solution was added. The
ammoniacal layer showed pink color. In this test, addition of ferric chloride was
addedto break the C – C linking of glycosides which is a stronger than C = O linkage.
9. Test for flavonoids
The extract was dissolved in ethanol and then subjected to the following tests.
a) Ferric chloride test:
To a small quantity of ethanolic solution of extract few drops of neutral ferric
chloride was added. Blackish red color was observed, showing the presence of
flavanoids.
b) Reaction with alkali and acid:
With sodium hydroxide solution the extracts gave yellow color. Extract gave
orange color with conc H2SO4 indicating the presence of flavanoids.

P a g e | 66
c) Zinc, HCl reduction test:
To a small quantity of extract, a pinch of zinc dust was added. Then add few
drops of conc HCl. Magenta color was produced, the presence of flavanoids.
d) Lead acetate solution:
To a small quantity of extract a few drops of 10% lead acetate solution was
added. Yellow precipitate was produced, shows presence of flavanoids.
10. Detection of saponins
The extracts were diluted, with 20ml of distilled water and it was agitated in a
graduated cylinder for 15 minutes. A one centimeter layer of foam was formed,
indicating the presence of saponins.
3.5 Experimental Animals:
Wistar albino rats (weighing 150-250g) and albino mice (weighing 20-25g) of either sex
were used in this study. The animals were acclimatized for one week under laboratory
conditions. They were housed in polypropylene cages and maintained at 270 ± 20 with 12
hour dark/light cycle. They were fed with standard rat feed and water ad libitum. The
husk in the cages was renewed thrice a week to ensure hygeinity and maximum comfort
for animals. The experimental protocols approved by Institutional Animal Ethics
Committee and the guidelines were followed accordingly.
3.6 Pharmacological activities:
1. Toxicity studies (LD50 )
2. Hepatoprotective activity
Determination of oral acute toxicity:
The acute toxicity was determined on albino mice, maintained under standard
conditions. The animals were fasted overnight prior to the experiment. Fixed dose
method of OECD guideline No.420 given by CPCSEA12 was adopted for toxicity

P a g e | 67
studies. Ethical clearance for handling the animals was obtained from the IAEC prior to
the beginning of the project work, the registration no. is as mentioned above.
Experimental Design:
Healthy albino Wistar rats were randomly assigned to 5 different groups having six
animals in each group in all the models.
a.Thioacetamide induced hepatotoxicity in rats
Group 1(normal control) and 2 (positive control) received saline 1ml/kg p.o for 9days. Group
3 received Silymarin 100 mg/kg p.o., for 9 days, group 4 and 5 received 70% EETSL 250 and
500mg/kg b.w p.o for 9 days respectively. On 7th day, 30 min after administration of
scheduled doses to corresponding groups, the animals of groups 2, 3, 4 and 5 Thioacetamide
(100 mg/kg, s.c) was administered which was prepared in distilled water (2% solution). The
animals were sacrificed 48 hr. after the administration of Thioacetamide under mild ether
anaesthesia. The liver sample was dissected out, blotted off blood, washed with saline for wet
liver weight, liver volume, GSH estimation as per the method of Ellman et al, lipid
peroxidation according to the method of Ohkawa et al and stored it in 10% formalin and
proceeded for histopathology to evaluate the details of hepatic architecture in each group
microscopically. The blood so collected was centrifuged immediately to get clear serum and
was subjected to various biochemical studies like SGPT, SGOT, ALP, direct and total
bilirubin.
b.CCL4 induced nephrotoxicity in rats

Group-I received liquid paraffin (1ml/kg) s.c., on 2nd and 3rd day. Group-II, III, IV and V
received CCl4: liquid paraffin (1:1) at a dose of 2ml/kg s.c., on 2nd and 3rd day, after 30 min
of vehicle, 100mg/kg silymarin, 250 mg/kg and 500 mg/kg EETSL administration. Animals
were sacrificed on the 6th day under mild ether anesthesia. Blood samples were collected for
evaluating the serum biochemical parameters as said above. The liver samples were dissected
out, blotted off blood, washed with saline and used to estimate the wet liver weight, liver
volume, GSH estimation and lipid peroxidation estimation and stored it in 10% formalin and
proceeded for histopathology to evaluate the details of hepatic architecture in each group
microscopically.

P a g e | 68
Histopathology:
Pieces of liver from each group in all the 2 experimental models were fixed immediately in
10% neutral formalin for a period of atleast 24 h, dehydrated in graded (50–100%) alcohol,
embedded in paraffin, cut into 4– 5 μm thick sections and stained with hematoxylin- eosin.
The sections were evaluated for the pathological symptoms of hepatotoxicity such as
extensive fatty change, ballooning of hepatocytes etc.
Statistical Analysis
Results were expressed as mean ± SEM, (n=6). Statistical analysis were performed with one
way analysis of variance (ANOVA) followed by Tukey’s Kramer comparison test by using
Graph Pad Instat Software. P value less than 0.05 was considered to be statistically
significant.

P a g e | 69
RESULT AND DISCUSSION

P a g e | 70
RESULTS
4.1 Phytochemical constituents present in ethanolic extract of Leaves

of
Table 4.1: Details of qualitative phytochemical tests.
S. No. Pet ether Chloroform Ethanolic

Test
Extract Extract Extract
1 Carbohydrates
Mohlish's test - - -
Fehling’s test - - -
Proteins and amino

2
acids
Ninhydrin test - - +
Biuret test - - +
3 Alkaloids
Mayer's test - + +
Wagner's test - - +
4 Fixed oils and fats
Spot test + - +
5 Glycosides
Borntrager's test - - +
Legals test - + +
6 Triterpenoids
Tin + thionyl chloride + - -

P a g e | 71
7 Phenolics and tannins
Ferric chloride test - - +
Gelatin test - - +
Lead acetate test - - +
Alkaline reagent test - - +
Dilute HNO3 test - - +
8 Saponins
Foam test - + +
Haemolysis test - + +
Flavones and
9
Flavonoids
Caddy's test + +
Shinoda test - + +
(+) Indicates positive result (–) Indicates negative result.
In, preliminary phytochemical studies of extracts of Taraxxacum officinale confirmed the

strong presence of desired phytochemicals in ethanolic extracts when compared to pet-
ether and chloroform extracts.Hence, for the further studies ethanolic extract of Taraxxacum
officinale have been selected.
4.2: Toxicity study:
In the present study the ethanolic extract of Taraxxacum officinale roots was
subjected for toxicity studies.
For the LD50 dose determination, ethanolic extract was administered upto
dose 4 gm/kg body weight and extract did not produce any mortality, thus
1/10th(250mg), 1/20th(500mg) of maximum dose tested were selected for the present
P a g e | 72
study .
LD50 of extracts of Taraxxacum officinale roots were calculated and found to be

4000mg/kg.
4.3: Hepatoprotective activity:
A) Physical parameters:
Wet liver weight and Wet liver volume:
Ethanol treatment in rats resulted in enlargement of liver which was evident by increase in
the wet liver weight and volume. The groups were treated with Silymarin and ethanolic
extract of Taraxxacum officinale showed significant restoration of wet liver weight and wet
liver volume nearer to normal. The MEAL at 250mg/kg b.wt and 500mg/kg body weight
showed reduction of wet liver weight and wet liver volume significantly at p<0.05.The
results are shown in table no.1.
B) Bio chemical Parameters:
1 Effect of ethanolic extract of Taraxxacum officinale on biochemical parameters

in ethanol induced hepatotoxic rats.
Rats treated with ethanol developed a significant hepatic damage observed as
elevated serum levels of hepatospecific enzymes like SGPT, SGOT and SALP when
compared to normal control. Pretreatment with Silymarin, ethanolic extract had.
showed good protection against ethanol induced toxicity to liver. Test indicates a
significant reduction in elevated serum enzyme levels with extract treated animals
compared to toxic control animals which is evident in table.
Direct bilirubin and total bilirubin:
Elevation of direct and total bilirubin levels after administration of ethanol
indicate its hepatotoxicity. Pretreatment with Silymarin, ethanolic extract
significantly reduced levels of direct and total bilirubin levels when compared to toxic
control group indri citing Hepatoprotective effect of methanolic extract of Anogeissus
latifolia bark which can be seen in the table.

P a g e | 73
Serum Total protein levels:
Ethanol treatment has considerably reduced serum total protein levels.
Pretreatment with Silymarin and methanolic extract of Mallotus philippensis leaves
had showed a significant increase in total protein levels as compared with toxicant
group. Which is evident by table & figure.
Plant material:
Taraxxacum officinale roots were collected from the garden of Ambedkar University. The
plant was identified and authenticated by Prof. R.B Ram, Head, Dept of applied plant
science (Horticulture),Baba sahib Bhimrao Ambedkar University,Lucknow. A Plant
specimen No.
(BSBAU/I-I/ (280)/2014/Tech.II/322) was preserved in Ambedkar University.
Preparation of extract:
The dried powder of root were defatted with petroleum ether, chloroform and then
extracted with 70% ethanol using soxhlet apparatus. The extracts was concentrated
under reduced pressure using rota flash evaporator and stored in airtight container in
refrigerator below 10ºC.
Chemicals:
Thioacetamide and CCL4 is obtained from SD fine chemicals, Mumbai, Silymarin
from Micro labs limited, Bangalore, the biochemical kits from Erba Manheim,
Germany and all other chemicals were of analytical grade.
Animals:
Wistar albino rats (weighing 150-250g) and albino mice (weighing 20-25g) of either
sex were used in this study. They were procured from Naka,Lucknow. The animals
were acclimatized for one week under laboratory conditions. They were housed in
polypropylene cages and maintained at 270 20 with 12 hour dark/light cycle. They
were fed with standard rat feed (Gold Mohur Lipton India Ltd.) and water ad libitum.
The husk in the cages was renewed thrice a week to ensure hygiene and maximum
comfort for animals. The experimental protocols approved by Institutional Animal
Ethics Committee, and the guidelines were followed accordingly.
Determination of oral acute toxicity:

P a g e | 74
The acute toxicity was determined on albino mice, maintained under standard
conditions. The animals were fasted overnight prior to the experiment. Fixed dose
method of OECD guideline No.420 given by CPCSEA12 was adopted for toxicity
studies. Ethical clearance for handling the animals was obtained from the IAEC prior
to the beginning of the project work, the registration no. is as mentioned above.
Experimental Design:
Healthy albino Wistar rats were randomly assigned to 5 different groups having six
animals in each group in all the models.
a.Thioacetamide induced hepatotoxicity in rats:
Group 1(normal control) and 2 (positive control) received saline 1ml/kg p.o for 9days.
Group 3 received Silymarin 100 mg/kg p.o., for 9 days, group 4 and 5 received 70%
EETSL 250 and 500mg/kg b.w p.o for 9 days respectively. On 7th day, 30 min after
administration of scheduled doses to corresponding groups, the animals of groups 2,
3, 4 and 5 Thioacetamide (100 mg/kg, s.c) was administered which was prepared in
distilled water (2% solution). The animals were sacrificed 48 hr. after the
administration of Thioacetamide under mild ether anaesthesia. The liver sample was
dissected out, blotted off blood, washed with saline for wet liver weight, liver volume,
GSH estimation as per the method, lipid peroxidation according to the method and
stored it in 10% formalin and proceeded for histopathology to evaluate the details of
hepatic architecture in each group microscopically. The blood so collected was
centrifuged immediately to get clear serum and was subjected to various biochemical
studies like SGPT, SGOT, ALP, direct and total bilirubin.
b.CCL4 induced nephrotoxicity in rats

Group-I received liquid paraffin (1ml/kg) s.c., on 2nd and 3rd day. Group-II, III, IV
and V received CCl4: liquid paraffin (1:1) at a dose of 2ml/kg s.c., on 2nd and 3rd
day, after 30 min of vehicle, 100mg/kg silymarin, 250 mg/kg and 500 mg/kg EETSL
administration. Animals were sacrificed on the 6th day under mild ether anesthesia.
Blood samples were collected for evaluating the serum biochemical parameters as
said above. The liver samples were dissected out, blotted off blood, washed with
saline and used to estimate the wet liver weight, liver volume, GSH estimation and
lipid peroxidation estimation and stored it in 10% formalin and proceeded for
P a g e | 75
histopathology to evaluate the details of hepatic architecture in each group

microscopically.
Histopathology:
Pieces of liver from each group in all the 2 experimental models were fixed
immediately in 10% neutral formalin for a period of atleast 24 h, dehydrated in graded
(50–100%) alcohol, embedded in paraffin, cut into 4– 5 μm thick sections and stained
with hematoxylin- eosin. The sections were evaluated for the pathological symptoms
of hepatotoxicity such as extensive fatty change, ballooning of hepatocytes etc.
Statistical Analysis
Results were expressed as mean SEM, (n6). Statistical analysis were performed
with one way analysis of variance (ANOVA) followed by Tukey’s Kramer
comparison test by using Graph Pad Instat Software. P value less than 0.05 was
considered to be statistically significant.
Results:
Effects of 70% EETSL on Thioacetamide and CCL4 induced hepatotoxicity
Thioacetamide and CCL4 treated rats showed a significant increase in serum marker
enzymes like SGOT, SGPT, ALP, total bilirubin, direct bilirubin and increased wet
liver weight, liver volume and also there is marked depletion of tissue GSH levels
and increased lipid peroxidation levels when compared with control.
Silymarin and 70% ethanol extract pretreated rats showed significantly (P<0.001)
decreased levels of serum marker enzymes, wet liver weight, liver volume, restoration
of tissue GSH and inhibition of lipid peroxidation levels when compared with the
hepatotoxicants treated rats. The effects are statistically significant in a dose
dependent manner. The results are summarized in table 1 to 4
Table 1. Effect of 70% EETSL on liver weight, liver volume and biochemical markers
in thioacetamide induced hepatotoxicity
Treatment Liver Biochemical parameters Mean  SEM
Volume Weight SGOT SGPT ALP Total Dir

(ml/100g) (g/100g) U/L U/L IU/L Bilirubi ect
n Bili
mg/dl rubi
n
P a g e | 76
mg/
dl
Negative control 6.20.14 6.190. 167.203. 111.062. 149.672. 0.850. 0.24

(1ml vehicle) 27 57 56 73 02 0.
016
Thioacetamide
(positive control) 4.660.2 6.370. 125.660. 129.830. 199.662. 1.780. 0.45
(100mg/kg s.c.) 1 21 98 47 44 03 0.
02
Thioacetamide + 6.020.1 5.970. 26.81.15 32.650.7 94.002.7 0.860. 0.32

Silymarin 1*** 17** *** 4*** 0*** 06*** 0.
(100mg/kg, s.c. + 02*
100mg/kg, p.o.) *
Thioacetamide+70%
EETSL 5.800.1 4.900. 36.443.3 46.214. 101.002. 1.160. 0.38
(100mg/kg s.c.+ 8*** 28** 5*** 10*** 40*** 09*** 0.
250mg/kg p.o.) 03*
**
Thioacetamide+70%
EETSL 4.800.1 4.850. 30.404.5 41.84.03 90.207.1 0.900. 0.36
(100mg/kg s.c.+ 7* 35** 0*** *** 7*** 10*** 0.
500mg/kg p.o.) 04*
*
Values are the mean S.E.M. of six rats/ treatment.

Significance **P <0.01 and *** P<0.001, compared to thioacetamide treatment.
Table 2. Effect of 70% EETSL on tissue GSH and in-vivo lipid peroxidation levels in
thioacetamide induced hepatotoxicity
P a g e | 77
Treatment Absorbance Absorbance

Absorbance Mean  SEM %Increase Mean  SEM %Inhibition
Negative Control 0.85 0.05 -- 0.250.01 --

(1ml vehicle)
Positive Control 0.190.01 -- 0.39 0.016 --

Thioacetamide
(100 mg/kg s.c.)
Thioacetamide +
Standard (Silymarin) 0.78 0.04*** 310.52% % 0.18 53.84%
(100 mg/kg s.c. + 100 0.004***
mg/kg p.o.)
Thioacetamide + 70%
EETSL 0.59 0.02*** 210.52% % 0.27 44.44%
(100 mg/kg s.c. + 250 0.002***
mg/kg p.o.)
Thioacetamide + 70%
EETSL 0.65 242.10% % 0.23 41.02%
(100 mg/kg s.c. + 500 0.019*** 0.006***
mg/kg p.o.)
Values are the mean S.E.M. of six rats/ treatment.,

Significance *** P<0.001, compared to thioacetamide treatment
Table 3. Effects of 70% EETSL on wet liver weight, liver volume and biochemical
markers in CCl4 induced hepatotoxicity
Liver Biochemical parameters

Mean  SEM
Treatment Volume weight SGOT SGPT ALP Total Direct

(ml/100 (gm/100 U/L U/L IU/L Bilirubi Bilirubin
g) g) n mg/dl
P a g e | 78
mg/dl
Negative 6.52 6.250. 138.819. 109.75 134.830 0.830. 0.170.012

Control 0.09 09 65 2.49 6.14 028
(1ml vehicle)
Positive 8.30.1 8.690. 328.684. 166.22 324.805 2.360. 1.50.00

Control 2 18 94 5.80 .15 098
CCl4 + Liq.
Paraffin (1:1)
(2 ml/kg s.c.)
CCl4+
Standard
(Silymarin)
(2 ml/kg 6.650. 6.330. 141.171. 90.081. 215.323 0.920. 0.310.01*
s.c.+100 047* 01** 24*** 03** .45*** 02*** **
mg/kg p.o.)
CCl4 + 70%
EETSL
(2 ml/kg 6.250. 7.350. 288.2830 117.43 203.731 1.410. 1.150.03*
s.c.+250 57** 74* .18*** 10.27** 4.44** 07*** **
mg/kg p.o.)

Significance *P<0.05, **P <0.01 and *** P<0.001, compared to CCl4 treatment
Table 4. Effect of 70% EETSL on tissue GSH and in-vivo lipid peroxidation levels in
CCl4 induced hepatotoxicity
Absorbance %Increase Absorbance %Inhibition

Treatment Mean  SEM Absorbance Mean  SEM
P a g e | 79
Negative 0.940 ± 0.012 -- 0.065 ± 0.016 --

Control
(1ml vehicle)
Positive Control 0.760 ± 0.05 -- 0.40 ± 0.04 --

CCl4 + Liq.
Paraffin (1:1)
(2 ml/kg s.c.)
CCl4 + Standard 0.890 ± 17.10% 0.092 ± 77.0%

(Silymarin) 0.017*** 0.003***
(2 ml/kg s.c.+
100 mg/kg p.o.)
CCl4 + 70% 0.820 ± 0.007* 7.89% 0.33 ± 0.002 ns 17.5%

EETSL
(2 ml/kg s.c. +
250 mg/kg p.o.)
CCl4 + 70% 0.84 ± 0.005** 10.52% 0.29 ± 0.009** 27.5%

EETSL
(2 ml/kg s.c. +
500 mg/kg p.o.)

Significance ***P<0.001, compared to CCl4 treatment.
Histopathological Studies in Thioacetamide and CCl4 induced hepatotoxicity

P a g e | 80
Fig.1 Negative Control Fig.2 Positive Control

(1ml vehicle) Thioacetamide (100 mg/kg s.c.)
Fig.3 Thioacetamide (100 mg/kg s.c.) Fig.4Thioacetamide(100 mg/kg s.c.)

Standard (Silymarin 100 mg/kg p.o.) + 70% EETSL 250 mg/kg p.o
P a g e | 81
Fig. 5 Thioacetamide(100 mg/kg s.c.) Fig.6 Negative Control

+ 70% EETSL 500 mg/kg p.o.) (1ml vehicle)
P a g e | 82
Fig.7 Positive Control Fig.8 CCl4 + Liq. Paraffin (1:1)(2 ml/kg s.c.) F
CCl4 + Liq. Paraffin (1:1)(2 ml/kg s.c.) Standard (Silymarin 100 mg/kg p.o.)
P a g e | 83
Fig.9 CCl4 + Liq. Paraffin ) Fig.9 CCl4 + Liq. Paraffin )

(2 ml/kg s.c.) + 70% EETSL 250 mg/kg p.o) (2 ml/kg s.c.)+ 70% EETSL 500 mg/kg
p.o.)
Histopathological Studies in Thioacetamide and CCl4 induced hepatotoxicity
Fig. 1 and 6 shows the light micrograph of control liver showing hepatic globular structure,
central vein, portal tract and kupffer cells look normal. The hepato-toxicants induced rat liver
showed the extensive fatty change and ballooning of hepatocytes, more around central vein
(Fig.2 and 7). Pretreatment with Silymarin,
70% EETSL 250 and 500mg/kg demonstrated marked improvement with mild fatty change,
with no congestion (Fig. 3, 4, 5, 8, 9 and 10) as compared to hepatotoxicants administered
groups.
Discussion
The aim of current study was to evaluate protective effects of Taraxacum officinale root
extracts on Thioacetamide and CCl4 mediated liver damage in rats. Liver toxicity caused
by Thioacetamide and CCl4 is perhaps widely used animal model for the assessment of
hepatoprotective agent16. Thioacetamide is a potent hepatotoxin, the hepatotoxicity induced
by liver is due to the production of its metabolite thioacetamide-5-oxide, which directly acts
on various cellular component and destroy it. In addition thioacetamide is also metabolised
by CYP 450 2E1 isoenzymes rendering sulfone and sulfoxy derivative, which are apparently
responsible for inactivation of structural proteins and enyzmes17.CCl4 is the inactive
metabolite which is biotransformed by cytochrome P-450 systems to produce the
trichloromethyl free radical (CCl3•) that causes lipid peroxidation and thereby produce liver
damage18-20.
P a g e | 84
The dose dependent reduction of Thioacetamide and CCl4 induced increased plasma
activities of SGPT, SGOT, ALP, total and direct bilirubin levels in animals pre treated with
70 % EETSL demonstrated their ability to restore the normal functional status of the toxic
liver, and also to protection against subsequent Thioacetamide and CCl4 liver damage.
The development of hepatotoxicity induced by Thioacetamide and CCl4 challenge was

exacerbated following the depletion of glutathione. Therefore, in the current study
glutathione content was measured to observe the preventive effects of EETSL in rats. The
results are clearly demonstrated that Thioacetamide and CCl4 intoxication has significantly
reduce the glutathione level compared to normal control animals. Rats on pre treatment with
EETSL have clearly restored the glutathione levels significantly in dose dependant fashion.
The Thioacetamide and CCl4 mediated liver injury was associated with marked increase in
lipid peroxidation levels.This is one of the reliable measure of hepatotoxicity due to oxidative
stress. From the results it is clearly indicated that Thioacetamide and CCl4 intoxicated rats
showed significant increase in the lipidperoxidation levels compared to normal control group.
Rats on pre treatment with EETSL (250 and 500 mg/kg, doses) have clearly decrease the
levels of lipid peroxidation significantly in dose related manner.
The results obtained from the present investigation suggest, leaves extract of the Taraxacum
officinale roots possess significant preventive effects against Thioacetamide and CCl4
damaged rat liver. Further, preliminaryphytochemical investigation revealed that the test
extract showed presence of flavonoids, tannins, alkaloids, saponins and glycosides. Thus, it
revealed that the hepatoprotection offered by titled plant extract may be due to its flavonoid
content.
Conclusion:
In conclusion, 70 % ethanolic extract of Taraxacum officinale roots exhibited dose dependent
significant hepatoprotective effect against Thioacetamide and CCl4 induced hepatotoxicity in
rats. This effect may be attributed to the polyphenolic constituents that are present in
Taraxacum officinale.
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CIRRICULUM VITAE
Rida Arif Qidwai
H.N-336/64 Mansoor Nagar –Samdi House
Cell-7084585858
Email: Ridz786922@gmail.com
Qidwairida@yahoo.com
Objective
Seeking an opportunity as a Pharmacist in a Hospital/Clinic or Organization(s) or

as a faculty in any renowned Institute of Pharmacy for promoting Pharmaceutical
responsibilities.
Summary
A highly talented pharmacy student skilled in clinical pharmacology and

pharmaceutical management. Knowledgeable in preparing and delivering papers
and presentations. Familiar with patient counseling and monitoring. Excellent
verbal and written communication skills.
Commited to continuing education and regularly updating knowledge through

pharmaceutical-related courses.
Professional Qualifications
• Bachelor of Pharmacy (B.PHARM) from Azad Institute of Pharmacy and

Research ,Azadpuram (Lucknow)
• Accolades and Extra- curricular activities
• Secured a place amongst the top 3 Rankers throughout the professional
degree course maintaining an average of 75%marks.
• Organized Seminars, presentations and various college events such as
Pharmacy Day,Farewell to seniors , As a coordinator of Annual function
,Hosted as a anchor with excellent team leader spirit , efficiently and very
impressively.
• Prepared and submitted syllabus oriented Charts and Assignments during
academic sessions.
Educational Qualifications
High School ICSE 67%
Intermediate ISC 70%
B.PHARM UPTU/Dr.APJ ABDUL 75%

KALAM UNIVERSITY
M.PHARM Dr.APJ ABDUL Pursuing
KALAM UNIVERSITY
Professional Experience
• Worked as a lecturer at a recognized Pharmacy institute for a period of

18 months.
• Assisted a hospital pharmacist for a period of 2 months.
• Part time - Home tuitions
Other skills
Working as a Manager and core member of an NGO organization “Cavity Buster

club”.
Major role is to impart awareness in Dental related problems and Presenting

brushing technique.
Strong Points
Fast learner
Adaptable
Strong work ethic
Reliable
Self discipline and ability to deal with day to day
challenges.
“Commitment” and “Challenge” are my assets.
Personal Details
Father’s Name – Arif Aziz Qidwai
Date of Birth – 22nd July ,1992
Sex –Female
Category- General
Marital Status- Married
Language- English ,Hindi and Urdu
Interest and Hobbies
Listening songs, sketching and participating in co-curricular activities.
Declaration
I do hereby declare that what is embedded in my profile is correct and true and best
of my knowledge in belief.

Rida Thesis

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EVALUATION OF HEPATOPROTECTIVE ACTIVITY

in Partial Fulfillment of the Requirements

Rida Arif Qidwai

Under the Supervision of

Dr. MOHD. IMTIYAZ AHMAD

1. Name : Rida Arif Qidwai

4. Degree for which the thesis is submitted : Master Of Pharmacy In Pharmacology

(Signature of the Candidate)

1. Name : RIDA ARIF QIDWAI

4. Degree for which the thesis is submitted : Master Of Pharmacy In Pharmacology

(Signature of the Candidate)

This is to certified that thesis entitlted “Evaluation of Hepatoprotective activity of Taraxacum

Evaluation of Hepatoprotective activity of Taraxacum officinale

Rida Arif Qidwai

1.Acute toxicity studies

Keywords: Taraxacum officinale ,Hepatoprotective activity,

Rida Arif Qidwai

LIST OF ABBREVIATIONS USED

b.w. - Body weight.

CPCSEA - Committee for the purpose of control and supervision of

IAEC - Institutional Animal Ethics Committee.

P.O. - Per oral.

LD50 - Lethal dose.

ANOVA - Analysis of variance.

SEM - Standard error mean.

MEAL - ethanolic extract of Tecoma stans .

List of Symbols & Abbreviations………………………………………………

Herbal wealth of India

1.1 Anatomy of Liver

1.Secretion and Excretion of Bile

1.2 Liver Disease

1.3 Physiology Of Liver

Role in Production of Binding Proteins

Protective & clearance Functions

Phagocytic and Endocytic Functions Of Kupffer cells

Endocytic Functions of Hepatocytes

1.4 Types of Liver Dysfunction

Pathophysiology of Functional Zonation

Manifestation Of Liver Dysfunction

Causes of Liver disease

1.5 Mechanism of Drug Induced hepatotoxicity

1.6 Models to Evaluate Effect of Drugs on Liver

CHAPTER-2 PLANT INTRODUCTION……………………………….

Wound healing Activity

3.2 Collection of Plant Material

3.3Preparation of Ethanolic Extract

3.4 Qualitative Phytochemical Screening

3.5 Experimental animals

3.6 Pharmacological Activities

Determination of oral acute toxicity

3.7 Experimental Design

1.Thioacetamide induced Hepatotoxicity in rats

2.CCl4 induced Hepatotoxicity in rats

CHAPTER-4 RESULT & DISCUSSION…………………………………….

4.2 Toxicity Studies

4.3 Hepatoprotective activity

Determination of Oral acute Toxicity

Sl.No. List Of Tables Page No.

1.1 Classification of hepatotoxins and mechanism of action of each group 57

1.2 List of hepatotoxic therapeutic agents and chemicals. 42

4.1 List of materials and equipments used during experiment.

5.1 Details of qualitative phytochemical tests. 93-94

Sl.No. List Of Figures Page.No.