PROJECT ONPROJECT ON

PREVENTIVE AND CURATIVE EFFECT OF PREVENTIVE AND CURATIVE EFFECT OF ELEPHANTOPUS ELEPHANTOPUS SCABERSCABER METHANOLIC METHANOLIC

EXTRACT AGAINST EXTRACT AGAINST NN-NITROSODIETHYLAMINE-NITROSODIETHYLAMINE

INDUCED HEPATOTOXICITY IN RATS.INDUCED HEPATOTOXICITY IN RATS.

LINZA ALI LINZA ALI

M.PHIL BIOSCIENCESM.PHIL BIOSCIENCES

LIVER

A human liver normally weighs between 1.4 – 1.6 kg.

It is located in the right upper quadrant of the abdominal cavity, resting just below the diaphragm.

It is both the largest internal organ and the largest gland in the human body.

IMPORTANCE OF LIVER

It perform several role in Carbohydrate metabolism and Lipid metabolism.

Helps in aminoacid synthesis Break bown of hemoglobin, toxic substances, ammonia, hormones etc. Plasma protein synthesis Hormone production Produces bile It is responsible for immunological effects It produces albumin. It produces coagulation factors. It is the main site of red blood cell production.

DISEASES THAT INTERFERE WITH LIVER FUNCTION

Most common include:

Hepatitis A, B, C and E.

Alcohol damage

Fatty liver

Cirrhosis

Hepatocellular Carcinoma

Jaundice

OTHER CAUSES OF LIVER DAMAGE

Chemical agents

Certain Drugs in over doses

Cancer drugs

NITROSO COMPOUNDS AND HEPATOTOXICITY

It is a potent carcinogenic agent.

Nitroso compounds like N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosopyrrolidine and N-nitrosopiperidine has been widely reported in various foodstuffs such as milk products, meat products, soft drinks and alcoholic beverages.

Nitrosamines are present in tobacco smoke.

N-NITROSODIETHYLAMINE (NDEA)

It produces hepatotoxicity after repeated administration in experimental animals.

Its exposure increases lipid peroxidation (LPO).

It decreases the activity of antioxygenic enzymes.

It have been suggested to cause oxidative stress and cellular injury due to the involvement of free radicals.

DRUD INDUCED HEPATOTOXICITY

Drugs are foreign substances to human body.

Smooth endoplasmic reticulum in liver is the principal "metabolic clearing house" for both endogenous chemicals and exogenous substances.

The central role played by liver in the clearance and transformation of chemicals.

This makes it susceptible to drug induced liver injury.

Cancer chemotherapy has been dominated by potent drugs that either interrupt the synthesis of DNA or destroy its structure.

Their toxicity is not limited to cancer cells and normal cells are also harmed.

Hence there is a need of less toxic drugs.

PLANTS IN TRADITIONAL MEDICINE

Today 80% of the world's people depend on traditional medicine for their primary healthcare needs.

In India reports shows that traditional healers use 2500 plant species and 100 species of plants as regular sources of medicine.

Human societies throughout the world have accumulated a vast body of indigenous knowledge over centuries on medicinal uses of plants.

The Indian system of medicines such as Ayurveda, Siddha and Unani entirely and homeopathy to some extent, depend on plant materials or their derivatives for treating human ailments

HERBAL FORMULATIONS FOR LIVER DISORDERS

Polyherbal or single herbal preparations are frequently considered to be less toxic and free from side effects than synthetic drugs.

Some single herbal preparations: Some polyherbal preparations: Andrographis paniculata Liv-52 Boerhaavia diffusa Kamilari Eclipta alba Hibiscus rosasinensis Phyllanthis maderaspatensis, Phyllanthus amarus Phyllanthus debilis Vitex negundo Picrorrhiza kurroa Glycyrrhiza glabra Silymarin

ELEPHANTOPUS SCABER(ANACHUVADY)

Elephantopus scaber Linn is a small herb, found in tropical conditions, almost throughout the world.

The plant appears to have only a bunch of leaves radiating from a point on ground.

It will also produce two or three bunches of flowers. The leaves are 4-6 inch long and 1-2 inch wide.

The base and roots are used in different Ayurveda medicines.

FIG: 1 ELEPHANTOPUS SCABER THE WHOLE PLANT

FIG: 2 FLOWER OF ELEPHANTOPUS SCABER

Fig: 3. VOUCHER SPECIMEN OF ELEPHANTOPUS SCABER

PLANT PROFILE Kingdom: Plantae

Division : Magnoliophyta

Order : Asterales

Family : Asteraceae

Genus : Elephantopus

Species : scaber

Source : IPNI

Habit : Herb

IMPORTANCE OF ELEPHANTOPUS SCABER

In ancient oriental medicine, root, leaves, aerial part and even the whole

plant have been used to treat various diseases

Studies shown that E. scaber possess:

Hepatic protection (Prevent or treat liver cancer)

It helps in wound healing.

It has Anti-venom, Anti-microbial, Anti-inflammatory, Anti-diabetic and Anti-tumour activities.

It is also been formulated and patented with other herbs to treat acne.

It is used in the treatment of :

Bronchitis, Leukemia, Rheumatism, Diuresis, Insomnia, Filariasis and Small pox

Root decoction of E. scaber is widely used to treat :

Dysentery, stomach troubles and blood vomiting in tuberculosis

Hot water extract of root has been used to cure fever.

While its fresh roots are chewed to treat cough, cold and headache.

The water extract of the leaves is applied externally to treat eczema and

ulcers.

The roots are also useful for treating cardiac problem and hepatitis.

BIOACTIVITIES OF E. SCABER METHANOLIC EXTRACTS

Methanol extract of E. scaber is an effective hepatoprotective agent.

It significantly increases the reduced serum protein level and decreases the elevated urea, uric cid and creatinine levels.

It had high inhibitory effect on the nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

It possess moderate antioxidant activity.

It also had the hypoglycemic activity.

OBJECTIVES To evaluate the hepatoprotective ability of Elephantopus scaber in animal

models since there is no experimental data available to verify its use in traditional medicine.

a. To study the alterations occuring in the liver of N-

nitrosodiethylamine (NDEA) induced hepatotoxic rats.

b. To study the curative and preventive effect of Elephantopus scaber

on N- nitrosodiethylamine induced hepatotoxic rats.

To study the dose response effect of Elephantopus scaber in experimental animals.

To compare the protective effect of Elephantopus scaber with respect to the protection yielded by standard hepatoprotective drug, Silymarin.

MATERIALS

ANIMALS Female albino rats of Wistar strain weighing 130 – 190g.

These animals were grouped into five groups. Group I - Normal rats Group II - N-nitrosodiethylamine (NDEA) induced hepatotoxic rats

(0.02%) Group III- NDEA rats treated with 70 mg/kg dosage of standard drug,

Silymarin. Group IV- NDEA rats treated with 200 mg/kg dosage of Elephantopus

scaber plant extract. Group V - NDEA rats treated with 100 mg/kg dosage of Elephantopus

scaber plant extract.

PLANT E. scaber plants were collected from its natural habitat in Kerala.

The fresh sample was collected in sufficient quantities (~5 kg) at a time.

Roots of the plant were separated, cleaned, chopped, shade-dried and powdered.

The soxhlet extraction process was carried out using methanol (99.8% assay) as extraction solvent.

CHEMICALS AND BIOLOGICALS

N-Nitrosodiethylamine (NDEA)

Silymarin

5, 5-dithiobis-(2-nitrobenzoic acid)

Bovine serum albumin

1-chloro-2, 4-dinitrobenzene

Thiobarbituric acid

Tween 80

METHODS

1. TISSUE ANALYSIS:

Estimation of Antioxidants levels.

Antioxidants includes: Reduced glutathione(GSH)

Glutathione peroxidase(GPx)

Glutathione-S- transferase (GST)

Thiobarbituric Acid reactive Substances (TBARS) and

Catalase (CAT)

Estimation of Protein levels.

2. SERUM ANALYSIS Estimation of Aspartate aminotransferase (AST) levels.

Estimation of Alanine aminotransferase (ALT) levels.

Blood was collected from the neck blood vessels of the rats and kept for 30

min at room temperature.

Serum was separated by centrifugation at 2000 rpm.

3. ESTIMATION OF THE PREVENTIVE EFFECT OF

ELEPHANTOPUS SCABER

Group I - Normal control

Group II-V - Received a single dose of NDEA (0.02%) 5 days per week for 6 weeks.

Group III - Received Standard drug Silymarin (70 mg/kg) 5 days per week for 6 weeks.

Group IV and V - Received E. scaber methanolic extract (200

mg/kg) and (100 mg/kg doses) 5 days per week for 6 weeks.

Treatment with E. scaber extract and Silymarin were started one week before the onset of NDEA administration and continued up to 6 weeks.

The rats were sacrificed 48 h after the last dose of NDEA administration.

4. ESTIMATION OF THE CURATIVE EFFECT OF

ELEPHANTOPUS SCABER METHANOLIC EXTRACT

Group I - Normal control

Group II-V - Received a single dose of NDEA (0.02%) 5 days per week for 6 weeks.

After 6 weeks exposure to NDEA,

Group III rats were treated with standard drug Silymarin (70 mg/kg) daily for 10 days.

Group IV – V rats received E. scaber methanolic extract (200 and 100 mg/kg) daily for 10 days.

Animals were sacrificed 48 h after the last dose of the extract administration.

5. HISTOPATHOLOGICAL STUDIES

Dissected livers were cut into small pieces.

Fixed in 10% buffered formalin.

Embedded in paraffin.

Stained with hematoxylin and eosin.

Examined for histopathological changes under the microscope.

6. STATISTICAL ANALYSIS

Statistical comparisons were made by means of one way ANOVA test.

p-values less than or equal to 0.05 were considered significant.

RESULTS