RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

M. PHARM SYNOPSIS
YEAR OF ADMISSION-JULY 2010

TITLE OF THE SYNOPSIS

EFFECT OF BIXIN ISOLATED FROM
BIXA ORELLANA LINN. ON ADRIAMYCIN INDUCED
CARDIOTOXICITY AND NEPHROTOXICITY IN WISTAR RATS
BY

Mr. KHAISER MEHDI KHAN

M.PHARM, PART-I
DEPARTMENT OF PHARMACOLOGY

UNDER THE GUIDANCE OF

Dr. THIPPESWAMY B. S. M.Pharm., Ph.D.
Professor and Head
DEPARTMENT OF PHARMACOLOGY

INSTITUTION
SREE SIDDAGANGA COLLEGE OF PHARMACY
B.H. ROAD,TUMKUR-572102.
KARNATAKA

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA
ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DESSERTATION

NAME OF THE CANDIDATE AND

ADDRESS

Mr. KHAISER MEHDI KHAN

Sree Siddaganga College of Pharmacy,
B.H.Road Tumkur-572 102.

NAME OF THE GUIDE

Dr. THIPPESWAMY B S. M.Pharm., Ph.D.

Professor & Head

NAME OF THE INSTITUTION

SREE SIDDAGANGA COLLEGE OF

PHARMACY,
B.H.Road, Tumkur- 572 102

COURSE OF STUDY AND SUBJECT

M.Pharm. (Pharmacology)

DATE OF ADMISSION TO COURSE

July-2010

TITLE OF THE TOPIC:

EFFECT OF BIXIN ISOLATED FROM BIXA ORELLANA LINN. ON ADRIAMYCIN
INDUCED CARDIOTOXICITY AND NEPHROTOXICITY IN WISTAR RATS

BRIEF RESUME OF INTENDED WORK:

6.1 Need for the study:
Despite major therapeutic advances cardiovascular disease (CVD) remains the leading cause
of morbidity and mortality world wide.1 CVD is the major cause of death in India, and its
contribution to mortality is rising; deaths due to CVD are expected to double between 1985
2015.2 Many drugs are available for the treatment of heart diseases and equally they produce lot
of adverse effect. Studies shows that cardiovascular and anticancer drugs are among the most
commonly cause of adverse events in hospitalized patients.3
Cardiac and nephrotoxicity are the two major adverse effect caused by adriamycin.
Adriamycin (ADR) is an anthracycline chemotherapeutic agent that has been commonly used in
the treatment of a wide range of cancers. The use of adriamycin has been shown to cause
development of cardiomyopathy, ultimately leading to congestive heart failure known as
adriamycin induced cardiomyopathy and nephropathy.4
Adriamycin induced cardiac toxicity and nephrotoxicity has been believed to be mediated
through different mechanism like inducing semiquinone free radical formation, ion dependent
oxidative damage to biological macromolecules and membrane lipid peroxidation. Tissue with
less developed activities of anti-oxidant enzymes like super oxide dismutase (SOD), catalase
(CAT) and glutathione peroxide (GHS-px) have been reported in adriamycin induced cardiac
and nephrotoxicity.5
Many plants are useful for the treatment of cardiac and nephrotoxicity related ailments.6
Synthetic flavonoides like fallerenol, lycopene, caffeic acid, vitamin A, Silymarin, and plants
containing flavonoides like Phyllanthus Urinaria and Centella Asiatica have been shown to
protect against adriamycin induced cardiac and nephrotoxicity. Even though modern drugs are
effective in the control of cardiac and nephrotoxicity disorders, their use is often limited because
of their side effects. The search for an effective medicine to treat cardiac and nephrotoxicity
disorders without any side effects has led to the use of traditional plant based medication.7-10
Bixa orellana Linn. (family Bixiceae) is an evergreen shrub or small tree native to Argentina,
Bolivia, Brazil, Chile, Colombia and exotic to India, USA, Thailand .11
Bixa orellana Linn. have been used traditionally for various purposes, like the seeds for heart
diseases, as antidote for prussic acid and prescribed in epilepsy and skin diseases. Twigs are
used in liver diseases. Roots are used as antimicrobial, Seed fatty oil is used in leprosy.12,13

Bixin is the major constituent in the seeds.14 Bixin is reported for its anticlastogenic,15
An antioxidant,16 anti inflammatory and tumor cell suppressor activities.17
Bixa orellana Linn. seeds are traditionally used to treat cardiac disorders.12 However there is
lack of documented scientific evidence regarding the protective effect of Bixin isolated from
Bixa orellana Linn. on experimentally induced cardiac and nephrotoxicity.
Hence we thought it will be worthwhile to screen the cardio and nephroprotective effect of
Bixin on experimentally induced cardiac and nephrotoxicity.
Therefore the present study is undertaken to investigate the effect of Bixin on adriamycin
induced cardiac and nephrotoxicity in Wistar rats.
6.2 Review of literature:
Bixa orellana Linn. appears like a small bush like tree. It grows to about 5 or 6 meters high
and has a peculiar reddish sap. The leaves are alternate, oval to heart shaped and 10-30 cm long.
The flowers are large, pinkish in color and produced in terminal clusters. The fruits are initially
green but turn reddish brown. They are fully covered by soft spines and, when they are dry,
open into two compartments exposing the enclosed red seeds.18
Kingdom

: Plantae

Division

: Mangnoliophyta

Class

: Magnoliopsida

Family

: Bixiceae

Genus

: Bixa

Species

: orellana L.

Botanical name : Bixa orellana L.

Synonyms
English

: Lipstick tree

Hindi

: Latkan

Kannada

: Kappumankala

Tamil

: Amudadaram

Telugu

: Jabura
3

Medicinal uses
Seeds are used for heart diseases, as antidote for prussic acid and prescribed in epilepsy and
skin diseases. Twigs are used in liver diseases. Roots are used as antimicrobial. Seed fatty oil is
used in leprosy.12,13
Reported activities
Bixin has been reported to be anticlastogenic,15 antioxidant,16 anti inflammatory and tumor
cell supressor.17
Adriamycin (ADR):
Adriamycin is an anthracycline chemotherapeutic agent that has been commonly used in
treatment of a wide range of cancers such as, leukaemias, breast, ovarian cancer, Hodgkin and
non-Hodgkin lymphoma. Unfortunately, the clinical use of adriamycin is associated with severe
cytotoxic side effects including cardiac and nephrotoxicity.19
6.3 Objective of the study:
The objective of the study is to investigate the effect of Bixin isolated from Bixa orellana
Linn. on adriamycin induced cardiac and nephrotoxicity in Wistar rats by measuring the
following parameters;
I. Cardiotoxicity studies:
1. Serum biomarkers :i) Serum glutamic oxaloacetic transaminase (SGOT).
ii) Lactate dehydrogenase (LDH).
iii) Creatinine kinase (CKMB).
2. Electro Cardio Graphic Changes using Bio-pac ( Data Aquisation System).
3. Tissue Antioxidant Level.
i.

Non-Enzymatic oxidants
a. Glutathione
b. Total thiols

ii.

Lipid peroxidation

iii.

Enzymatic oxidants
a. Catalase
b. Superoxide dismutase

4. Histopathological Changes.
5. Measurement of Infarct volume by 2,3,5-triphenyl tetrazolium chloride(TTC) stains

II. Nephrotoxicity studies:

1. Serum urea, Serum creatinine and Total protein.
2. Tissue Antioxidant Levels.
i.

Non-Enzymatic oxidants
a. Glutathione
b. Total thiols

ii.

Lipid peroxidation

iii.

Enzymatic oxidants
a. Catalase
b. Superoxide dismutase

3. Histopathological Changes.

METHODS AND MATERIALS:

7.1 Source and collection of data:
The source of data will be obtained from the laboratory experiments. This involves isolation of
Bixin from Bixa orellana Linn. and their pharmacological activities on experimental animals
such as adult rats.
7.2 Extraction and isolation of Bixin
Fresh seeds of Bixa orellana Linn. will be collected and stored in dark, at room temperature.
Pigments present in the pericarp of Bixa orellana Linn. seeds will be extracted 2-3 times with 3
volumes of ethanol: water (93:7, v/v), at 37C for several hours, with vigorous shaking. Most of
the extracted Bixin molecules will be rendered insoluble by extraction solution. The insoluble
Bixin will be separated from gross particulate material by filtration through gauze, followed by
filtration through filter paper. The Bixin retained on the filter paper will be removed and washed
exhaustively by being stirred in hexane, to eliminate contaminant molecules. The residual
hexane will be evaporated at 50C, to get purified Bixin in a crystallized form.20
7.3 Animals :
Male Wistar rats weighing 150-200g will be used. The animals will be maintained under
controlled condition of temperature (23 20C), humidity (50 5%) and 12-h light-dark cycles.
The animals are randomized into experimental and control groups and housed each in two
sanitized polypropylene cages containing sterile paddy husk as bedding, they will have free
access to standard pellets as diet and water.

7.4 Dose of Bixin

Bixin will be administered at doses of 2.5 and 5 mg/kg. The doses of the bixin are selected on
the basis of the previous studies of bixin conducted in experimental animals.16
I.

Experimental designs :

1. Adriamycin induced cardiotoxicity:

The male Wistar rats will be selected and randomized into six groups containing six animals in
each group.
Groups

Treatment

Normal (Vehicle).

II

Normal + Bixin (2.5 mg/kg), (p.o.).

III

Adriamycin (10 mg/kg), (i.p.).

IV

Adriamycin (10 mg/kg), (i.p.) + Bixin (2.5 mg/kg), (p.o.).

Adriamycin (10 mg/kg), (i.p.) + Bixin (5 mg/kg), (p.o.).

VI

Adriamycin (10 mg/kg), (i.p.) + Vitamin C, (p.o.).

Group II, IV and V pre-treated with bixin orally for 11 days and vitamin C used as standard. On
the 11th day adriamycin (10 mg/kg) will be injected intra peritoneal to III, IV, V and VI groups
of animals which is well documented to induce cardiotoxicity.5
Evaluation of cardiotoxicity :
On the 14th day, six animals from each group will be used to record ECG and subjected to
decapitation. Blood samples and heart specimens were collected for the estimation of various
biomarkers in serum as well as heart. Formalin fixed heart specimens were used for
histopathological examinations.
2. Adriamycin induced Nephrotoxicity:
The male Wistar rats will be selected and randomized into six groups containing six animals in
each group.
Groups

Treatment

Normal (Vehicle).

II

Normal + Bixin (2.5 mg/kg), (p.o.).

III

Adriamycin (10 mg/kg), (i.p.).

IV

Adriamycin (10 mg/kg), (i.p.) + Bixin (2.5 mg/kg), (p.o.).

Adriamycin (10 mg/kg), (i.p.) + Bixin (5 mg/kg), (p.o.).

VI

Adriamycin (10 mg/kg), (i.p.) + Vitamin C, (p.o.).

6

Group II, IV and V pretreated with bixin orally for 11 days, and vitamin C used as standard. On
the 11th day Adriamycin (10 mg/kg) will be injected intra peritoneal to III, IV, V and VI groups
of animals which is well documented to induce nephrotoxicity .4
Evaluation of Nephrotoxicity :
Similarly the above group of the animals daily treated with Bixin orally for 30 days, after
administration of adriamycin injection. Animals will be subjected to decapitation, blood samples
and kidney will be collected for the estimation of various biomarkers in serum as well as kidney.
Formalin fixed kidney specimens will be used for histopathological examination.
Assessment of myocardial infarct size by TTC staining method:
The heart is removed and the atria along with the root of the aorta is excised and the ventricles
are kept overnight at a temperature of -4C. Frozen ventricles are sliced into uniform sections of
about 1-2 mm thickness. The slices are incubated in 1% w/v TTC at 37C in 0.2M tris-chloride
buffer for 30 min. The normal myocardium is stained brick red while the infarcted portion
remained unstained. Infarct size is measured by the volume method.21
7.5 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO
BE CONDUCTED ON PATIENTS OR OTHER HUMANS /ANIMALS? IF SO PLEASE
DESCRIBED BRIEFLY.
Yes, the above study requires investigation to be done on the rats for the determination of
cardiotoxicity.
7.6 HAS ANIMAL ETHICAL COMMITTEE CLEARANCE BEEN OBTAINED FROM
YOUR INSTITUTION IN CASE?
The study has been referred to the ethical committee of the institution and clearance has been
obtained (Approval No: SSCPT/IAEC. Clear/ 97/ 2010-11, Date 7/12-2010) Enclosed copy
Anexure-I.

REFERENCES:
1. Robinson E, Grieve DJ. Significance of peroxisome proliferator-activated receptors in the
cardiovascular system in health and disease. Pharmacol Ther 2009;122:246-63.
2. Tanuja R, Mario V, Donna S, Reddy KS, Bharathi AV, Stampfer MJ, et al. Physical
activity and risk of coronary heart disease in India. Int J Epidemiol 2004;33:759-67.
3. Gholami K, Ziaie S, Shalviri G. Adverse drug reactions induced by cardiovascular
drugs in outpatients. Pharmacy Practice 2008;6:51-5.
4. El-Shitany NA, El-Haggar S, El-desoky K. Silymarin prevents adriamycin-induced
cardiotoxicity and nephrotoxicity in rats. Food Chem Toxicol 2008;46:2422-8.
5. Minnough EG, Trush MA, Bhatnagar M. Enhancement of reactive oxygen-dependent
mitochondrial membrane lipid peroxidaton by the anticancer drug adriamycin. Bio-Chem
Pharmacol 1985;34:847-56.
6. Mamtani R, Ayurvuda and Yoga in cardiovascular diseases. Cardiol Rev 2005;13:155-62.
7. Rade I, Marija B, Martina P, Borut S. Acute doxorubicin nephrotoxicity in rats with
malignant neoplasma can be successfully treated with fullerenol C60(OH)24 via
suppression of oxidative stress. Pharmacol Reports 2008;60:742-9.
8. Karim S, Bhandari U, Kumar H, Pillai KK. Doxorubicin induced cardiotoxicity and
modulation by drugs. Indian J Pharmacol 2001;33:203-7.
9. Chularojmontri L, Suvara K, Wattanapitayakul, Angkana H, Suphan C. Antioxidant and
cardioprotective effects of Phyllanthus urinaria Linn. on doxorubicin-induced
cardiotoxicity. Biol Pharm Bull 2005;28:1165-71.
10. Stollberger C, Finsterer J. Worsening of heart failure Becker muscular dystrophy after
nonsteroidal anti-inflamatory drugs. South Med J 2005;98:478-80.
11. Available from: URL:http://www.worldagroforestry.org, access date 3/12/2010
12. Kurian JC. Plant that heals. Pune: Oriental watchman publishing house. 2007. Vol 2 p. 26.
13. Asolkar LV, Kakkar KK, Chakre OJ, editors. Glossary of Indian medicinal plants with
active principles. New Delhi: Publication and information directorate. 1992. Part I p. 126.
14. Scotter MJ, Thorpe SA, Reynolds SL, Wilson LA, Strutt PR. Characterization of the
principal coloring components of annatto using high performance liquid chromatography
with photodiode array detection. Food Addit Contam 1994;11:301-15.
8

15. Chengaiah B, Rao KM, Kumar KM, Alagusundaram M, Chetty CM. Medicinal
Importance Of Natural Dyes A Reveiew. Int J PharmTech Res 2010;2:144-54.
16. Silva CR, Antunes LM, Bianchi ML. Antioxidant action of Bixin against cisplatin induced
chromosome aberrations and lipid peroxidation in rats. Pharmacol Res 2001;43:561-6.
17. Reddy MK, Lindo AR, Nair MG. Relative inhibition of lipid peroxidation,
cyclooxygenase enzymes and tumor cell proliferation by natural food colors. J Agric Food
Chem 2005;53:9268-73.
18. Bixa orenella Linn. Available from: URL:http://www.plants.usda.gov/java/profile
access date:1/12/2010.
19. Blum RH, Carter SK. Adriamycin a new anticancer drug with significant clinical activity.
Ann Int Med 1974;80:249-59.
20. Kovary K, Louvain TS, Silva MC, Albano F, Pires BB, Laranja GA, et al. Biochemical
behavior of norBixin during in vitro DNA damage induced by reactive oxygen species. Br
J Nutr 2001;85:431-40.
21. Kavita Garg, Harlokesh N Yadav, Manjeet Singh, PL Sharma. Mechanism of
cardioprotective

effect

of

erythropoietin-induced

Indian J Pharmacol 2010;42:219-23.

preconditioning

in

rat

heart

SIGNATURE OF THE CANDIDATE

10

REMARK OF THE GUIDE:

The above information and literature has been extensively investigated, verified and was found to be
correct. The present study will be carried out under my supervision and guidance.

11

11.1 NAME AND DESIGNATION OF

GUIDE

Dr. THIPPESWAMY B. S. M.Pharm.,. Ph.D

Professor & Head
DEPT.OF PHARMACOLOGY,
SREE SIDDAGANGA COLLEGE OF PHARMACY,
B.H.ROAD, TUMKUR-572 102

11.2 SIGNATURE

12

11.3 CO-GUIDE

NA

11.4 SIGNATURE

NA

12.1 HEAD OF THE DEPARTMENT

Dr. THIPPESWAMY B. S. M.Pharm,. Ph.D

Professor & Head
DEPT.OF PHARMACOLOGY,
SREE SIDDAGANGA COLLEGE OF PHARMACY,
B.H.ROAD, TUMKUR-572 102.

12.2 SIGNATURE

13

13.1 REMARKS OF THE PRINCIPAL

The above mentioned information is correct and I

recommend the same for approval.

13.2 SIGNATURE
Dr. S. BADAMI M.Pharm,. Ph.D
PRINCIPAL AND DIRECTOR
SREE SIDDAGANGA COLLEGE OF PHARMACY,
B.H.ROAD, TUMKUR-572 102.

10