1.1 Liver and xenobiotic metabolism

The liver which occupies a pivotal position in body plays an essential role in

drug and xenobiotic metabolism and in maintaining the biological equilibrium of

vertebrates (Godkar, 1994). The role played by this organ in the removal of substances from the portal circulation makes it susceptible to first and persistent attack

by offending agents like viruses, chemicals, toxins in food, peroxides, drugs,

environmental pollutants etc., culminating in liver pathology. Owing to the unique

and considerable regenerative capacity of the liver, even a moderate cell injury is not reflected by measurable change in its metabolic function. However, some of its

functions are so sensitive that abnormalities start appearing depending on the nature

and degree of initial insult (Ram and Goel, 1999).

On exposure to xenobiotics, the liver of vertebrates manage to eliminate such

foreign compounds as early as possible. This is accomplished by making use of the

normally existing biochemical mechanisms in the tissue. Certain enzymes and other

endogenous biomolecules which are actually meant for the metabolism of

endogenous substrates may be utilized for this purpose. Biotransformation of a

xenobiotic compound following its exposure can alter its distribution and action

leading to its detoxification and excretion or enhance its toxicity due to the activation

of the compound (Athar el al., 1997).

Biotransformation of xenobiotics usually occurs in two phases. Phase I

metabolism (detoxification) involves oxidative, reductive and / or hydrolytic reactions that cleave substrate molecules to produce a more polar moiety. Phase 11 reactions

(synthetic reactions) involve conjugation of certain endogenous molecules to the products of Phase I reactions (Remmer, 1970). Cytochrome P450 enzymes are responsible for the metabolic conversion of many drugs to the polar metabolites via

Phase I and Phase I1 reactions to earlier excretion.

Drugs and chemicals (xenobiotics) may affect liver function which stimulate the activity of microsomal enzymes (eg., cyt. P450), a process known as enzyme induction. This is important in determining the degree of hepatotoxicity (Conney,

1967). Induction of cytochrome P450 leads to the depletion of glutathione and in turn to hepatotoxicity (Gadgoli and Mishra, 1997).

During xenobiotic metabolism, highly reactive metabolites like peroxides,

epoxides and other radicals are formed. Various cellular mechanisms for their

inactivation are epoxide hydrolase, superoxide dismutase and glutathione-s-

transferase. However, when the rate of formation of the reactive metabolites is

enhanced or the rate of their removal is diminished, reverse cellular damage may

ensue (Jollow and Smith, 1977).

1.2 Hepatotoxins and their effect

A number of pharmacological and chemical agents act as hepatotoxins and

produce a variety of liver ailments. They include industrial toxins, the heat-stable

toxic bicyclic octapeptides of certain species (Amanita and Galerina), chemicals and , various pharmacological agents. In general, there are two major types of chemical

hepatotoxins (Ram and Goel, 1999).

1.2.1 Direct hepatotoxins

These are agents that damage the membrane of hepatocytes directly

resulting interference in cell metabolism. The most common direct hepatotoxins

and their effect are given in Table 1.1.

Table 1.1 Direct hepatotoxins and their effect

I Carbon tetrachloride I Decreases glycogen and protein levels and increases the ~ Name

I I content of lipid 1

Morphological alterations

I Carbon I

~evere lyde~le tes glycogen level and elevates protein and lipid I 1 tetrachloride-Ethanol contents I 1 Thioacetamide I Decreases the glycogen and protein levels without producing I

1 Galactosamine 1 Decreases the content of glycogen and protein with marked 1 Paracetamol

-

Fulvine

any significant change in lipid level. Decreases the liver glycogen and protein contents severely and elevates lipid level.

parenchyma Phalloidin (Toxin , Damages the plasma membranes of the hepatocytes as well as from mushroom) their active filaments

I Ethyl alcohol 1 Causes hepatocyte degeneration, collagen deposition and 1

I 1 necrosis I Aflatoxins L 1 Lanthanum chloride

I Impair the synthesis of DNA and protein in rat liver and

3 decrease H-thymidine incorporation into DNA of regenerating liver and synthesis of RNA. Elevates the level of lipid, protein, a and y - globulins in liver

, Pyrrolizidine alkaloids

i I I and decreases albumin in serum.

Mostly they produce centrilobular hemorrhagic necrosis, inhibit protein biosynthesis, cause decrease in liver RNA content and enzyme activity.

1.2.2 Indirect hepatotoxins

These are agents that produce hepatic injury as a result of reactive interference with metabolic pathways or selective binding to or alteration of a

specific component. A list of the common indirect hepatotoxins and their effect is

given in Table 1.2. Table 1.2

Indirect hepatotoxins and their effect I

Drugs and Chemicals Class of agent Morphological change Methyl testosterone Methimazole

I I

Anabolic steroid Antithyroid

Erythromycin estolate

Norethynodrel with mestranol

1 Tetracvcline 1 Chemotheraoeutic

Chlorpropamide Chlorpromazine

Valproic acid

Chemotherapeutic

Oral contraceptive

Oral hypoglycemic Tranquilizer

I Anticonvulsant

Cholestasis

Isoniazid 1 Chemotherapeutic I I

Halothane I Phenvtoin

Chlorothiazide 1 Diuretic I I

Anaesthetic Anticonwlsant

I

Oxyphenisatin 1 Laxative I I

Yellow phosphorus 1 Metal I I

Amanita phalloides 1 Mushroom 1 Necrosis I I

Acetaminophen 1 Analgesic I I I

Phenylbutazone 1 Anti-inflammatory I I

! I Allopurinol 1 Xanthine oxidase inhibitor 1 I !

i Rifampicin 1 Antitubercular i Cholestasis and necrosis /

1.3 Carbon tetrachloride as a hepatotoxin

Carbon tetrachloride is a colourless, non-inflammable liquid with a

sweetish odour. Its physical and chemical properties include:

Molecular weight 153.8

Melting point 23 "C

Boiling point 76.5"C

Specific gravity 1.54

Vapour pressure 91.3 mm Hg at 20C

Vapour density 5.32

Solubility in water 800 mgll at 20C

Carbon tetracholoride is lipophilic and because of this property CC14 is

absorbed from the skin and gastrointestinal tract as well as the lungs, although the

rate of absorption by the separate routes is different. CC14 is slowly eliminated

from the body with over 50% being exhaled unchanged (Stewart et al., 1963).

Inhalation studies in monkeys (Mc Collister et al., 195 1) and rats (Paustenbach el al., 1986a and Paustenbach et al., 1986 b) show that the highest CC14 concentration occur in fat and in tissues with high fat content such as bone

marrow, liver, brain and kidney.

CC14 is metabolised in the liver by a specific type of cytochrome P45U to

highly reactive metabolites (the trichloromethyl free radical and phosgene).

Metabolically activated CC14 may bind to both lipid and protein cellular structures

in vivo and in vitro (Rocchi et al., 1973). CC14 and its metabolites are excreted in

exhaled air, urine and faeces in animals but the data are largely unavailable on the

excretion of CC14 in humans. Studies in animals confirm that liquid CC14 is

absorbed through the skin (Jakobsen et al., 1982), though the absorption of

vapours through the skin is very low.

Carbon tetrachloride (CC14) is a toxic chemical agent (Junnila et al., 2000).

CC14 had been widely used as a solvent for some organic compounds, such as oil,

resin or tar and as a raw material of halo-fluorocarbon for refrigeration or as a fire

extinguishing agent. Since some cases of acute toxic injury caused by CC14 used as a solvent have been reported, the above mentioned use of CC14 is now not

common (Ishii et al., 1997). It had also been used in human medicine as an

anaesthetic agent (Jones, 1983). The high incidence of hepatic and renal problems due to the administration led to the discontinuation of its use. Now, it is used as a

model for liver injury.

Excessive absorption of CC14 results in a clinical complex manifesting in

depression of the central nervous system followed by hepatic and renal damage.

Low level exposure may produce nausea, dizziness and vomiting. These

symptoms have been reported at a concentration as low as 5 ppm. Hepatotoxicity

has been shown in workers exposed to CC14 at a level as low as 5 - 10 ppm.

Chronic exposure to CC14 may also cause optic nerve damage and impaired vision.

Alcohol intake potentiates the hepatotoxicity of CC14. CC14 is absorbed through

the skin and may be absorbed in toxic concentrations by these routes. It may

, produce slight eye irritation which is usually transient. There is evidence of

carcinogenicity of CC14 in experimental animals.

1.4 Types of hepatic diseases (Ram and Goel, 1999) -

Mainly there are three types of hepatic diseases:

Hepatitis (an inflammatory liver disease)

Hepatosis (non inflammatory disease)

Cirrhosis (a degenerative disease)

1.4.1 Morphological classzjication of liver diseases (Ram and Goel, 1999)

I . Hepatitis (viral, drug-induced, toxic)

a. acute

b. chronic

2. Cirrhosis

a. alcoholic (portal, nutritional, Laennec's cirrhosis)

b. post necrotic

c. biliary

d. hemochromatosis

e. rare types (Wilson's disease, galactosemia, cystic fibrosis of pancreas, a- antitrypsin deficiency).

3. Infiltrations

a. glycogen

b. fat (neutral fat, cholesterol, gangliosides, cerebrosides)

t. amyloid

d. lymphoma, leukemia

e. granuloma (sarcoidosis, tuberculosis, idiopathic)

4. Space occupying lesions

a. hepatoma, metastatic tumor

b. abscess (pyogenic, amoebic)

c. cysts

d. gummas

5 - , Hepatobiliary

a. Extrahepatic biliary dysfunction (by stone, tumor, stricture)

b. cholangitis

c. chronic passive congestion and cardiac cirrhosis

d. hepatic vein thrombosis

e. pylephlebitis

1.5 Management of Hepatic diseases

Despite the tremendous strides in modern medicine, there is hardly any

drug that stimulates liver function, offers protection to the liver from damage or

helps regeneration of hepatic cells (Venkateswaran el al., 1997). As it is the

function of hepatoprotective agents to interfere with these pathological processes

by blocking their evolution and helping recovery, the development of new

antihepatotoxic drugs is the need of the hour.

Since increase in the use of synthetic drugs in therapy leads to many side

effects and undesirable hazards, there is a world wide trend to go back to natural

resources (mainly traditional plants) which is both culturally acceptable and economically viable. Thousands of these plants are used in the world to prevent or

cure diseases but the biochemical basis of protective action which is necessary for

the rational development of safe and potent drugs, is lacking in most of the cases.

Ayurveda, an indigenous system of medicine in India, has a long tradition

of treating liver disorders with plant drugs (De et al., 1993). This ancient system

of medicine make use of active principles present in plants for treating diseases.

Medicinal herbs provide protection against hepatotoxins in various ways:

by enhancing the functioning of the hepatic glutathione antioxidant system (Ip and

KO, 1996; Ip et al., 1996); inhibiting cytochrome P450, promoting glucuronidation,

stimulating hepatic regenerat'ion, activating functions of reticulo-endothelial

systems, inhibiting biosynthesis of ~ y t . P ~ ~ ~ (Rao and Mishra, 1998) preventing

lipid peroxidation, stabilizing hepatocellular membrane, enhancing protein

biosynthesis (Lin el al., 1997); accelerating the regeneration of parenchymal cells

and thus protecting against membrane fragility decreasing the leakage of marker

enzymes into the circulation, interfering with the microsomal activation of CC14

and i or accelerating detoxification (Bishayee et al., 1995); counteracting the

hepatic lysosomal enzymes (Slater and Greenbaum, 1965; Saxena et al., 1993).

In India, numerous medicinal plants and their formulations are used to

combat liver diseases in traditional systems of medicine and folklore medicines

(Asha and Pushpangadan, 1998). So in this study, the antihepatotoxic effect of

Kamilari, a polyherbal formulation and four selected medicinal plants were

evaluated to validate their traditional use. The plants selected were : Elephantopus

scaber Linn., Glycyrrhizu glabra Linn, Leucas aspera (Wild.) Spr, Woodfordia fmrticosa (L.) Kurz.

1.5.1 Kamilari

In the Kerala market, Kamilari, a polyherbal formulation is available

claiming to be a Liver tonic. It is recommended as an effective drug in the

treatment of jaundice, acute and chronic inflammatory liver disorders, dyspepsia, loss of appetite, alcoholism etc. The ingredients of this herbal preparation are

given in Table 1.3. Table 1.3

Ingredients of the herbal preparation (Kamilari)

Plant species

I Cordia myxa 1 Boraginaceae 1 Fruit 1 Berberis aristata

Family Medicinal part

Berberidaceae

Curculigo orchioides

1 Glycyrrhira glabra 1 Fabaceae I Root 1

Leaf

Elettaria cardamomum

1 I > I ~ C I * I O I I ~ I I I I I I Piperaceae 1 Fruit ~

Hypoxidaceae

1 Thesyesia y opulnea

Stem

Zingiberaceae

Malvaceae

Seed

Leaf

1.5.2 Eleplzantopus scaber Linn.

Zingiber oflcinale Zingiberaceae

Eleplzaniopus scaber of the family Asteraceae, is a perennial herb. It is

Rhizome

, distributed throughout India, especially in dry localities (Sivarajan and Indira, 1994). Roor of this plant is used in liver troubles (Hammer and Johns, 1993).

1.5.3 Glycyrrhizaglabra Linn.

Glycyrrhiza glabra of the family Fa

cultivated in Punjab and the sub Himalayan tracts (Warrier et al., 1995). It is used in the treatment of liver diseases (Luper, 1999). In Japan, G.glabra is used in the therapy of hepatitis B and C (Suzuki et al., 1983).

1.5.4 Leucas aspera (Willd) Spr.

(Phlonzis aspera Willd.)

Leucas aspera of the family Larniaceae is an erect herb. It is found as a

weed in cultivated fields, waste lands and road sides. It is used against jaundice (Sivarajan and Indira, 1994).

1.5.5 Woodfordia fruticosa (L.) Kurz.

( Woodfordia floribunda Salisb Lythmnz fmticosum L.)

Woodfordia fmticosa is a bushy shrub found throughout India. Dried

flowers are used in impaired hepatic function ( Chatterjee and Pakrashi, 1994).

1.6 Aims and objectives of the study

This thesis incorporates'the antihepatotoxic effect of Kamilari, a polyherbal

formulation and four medicinal plants (Elephantopus scaber, Glycyrrhiza glabra, Lezrcas , aspera and Woodfordia fmticosa) against CC14-induced hepatic

dysfunction in rats.

Hepatotoxicity can be induced in rats by administering various agents (as

presented In tables 1.1 and 1.2). In the present investigation, CC14 was the

hepatotoxin employed because CC14-induced liver dysfunction in rats simulates

liver cirrhosis in man (Perez-Tamayo, 1983; Lopez- Novoa et al., 1977).

The polyherbal formulation, Kamilari, is available in the Kerala market

claiming to be an effective remedy for liver ailments and its effectiveness in

curing liver diseases is being evaluated in different research laboratories in the

country. A study was conducted in our laboratory to explore the antihepatotoxic

activity of this herbal preparation. Likewise, there is a vast source of literature

which enumerates the medicinal plants which have liver protective activities.

Many of these plants are ingredients of a number of multiherbal preparations. We

selected four medicinal plants: E. scaber, G.glabra, L.aspera and W. fruticosa. The

fact that G.glabra is an ingredient of many polyherbal formulations including

Kamilari justifies the selection of the plant as one of the four different plants. The criteria we adopted for selection of the medicinal plants include: medicinal plants

for which no detailed work has been reported and those plants which are used in

the traditional system of medicine for curing liver diseases.

1.6.1 Biochemical investigations

The aim of this work was to evaluate the antihepatotoxic activity of the

herbal preparation and the above mentioned medicinal plants. For this the

following parameters were investigated:

Changes in the activities of the enzymes like aspartate transaminase (AST),

alanine transaminase (ALT), alkaline phosphatase (ALP), gamma glutamyl

transpeptidase (GGT), lipid profile and changes in the level of bilirubin and protein in the serum of pairfed control, CC14-treated and CCI4+herbal

preparation / medicinal herbs (E. scaber, G.glabra, L.aspera, W. fruticosa) treated male albino rats. Changes in the lipid profile of the tissues of the

above three groups were also investigated.

The free radicals generated during CC14- toxicity can induce peroxidative

changes. So in order to arrive at some conclusions the following aspects were also

studied.

Changes in the level of thiobarbituric acid reactive substances (TBARS),

conjugated dienes (CD), glutathione (GSH), activities of superoxide dismutase (SOD), catalase, glutathione-S-transferase (GST), glutathione

peroxidase (GSH-Px) in the liver and kidney of control, CC14 treated and

CC14+ medicinal herbs (G.glabra, W.@ticosa) treated male albino rats.

In addition to the above biochemical parameters, the changes in the

histopathology of the liver of pairfed control, CC14-treated and CC14+ G.glabra

treated rats were also examined. The results of these investigations are discussed

in this thesis.