6. BRIEF RESUME OF THE INTENDED WORK6.1 NEED FOR STUDY

DNA is the genetic material in all living systems and is responsible for establishing and

maintaining the cellular and biochemical functions of an organism. DNA is made up of individual

units called nucleotides that are linked to each other to form long chains. The simplest functional unit

in a DNA molecule is termed as gene and they are ultimate units of heredity. Their normal

expression and regulation is essential for all physiological functions and their abnormality /deficiency

forms the basis for many clinical conditions. All living organisms store DNA in a safe, stable, duplex form

in which two strands of DNA wrap around each other.1

Even under the best of circumstances, DNA is constantly subjected to chemical

modifications2 and one such agent that causes chemical modification is ionizing radiations (

including X-rays) where the radiations interact with mammalian cells either directly or indirectly.

Direct effect of ionizing radiations on mammalian cells includes molecular damage due to absorption

of them by DNA, or indirectly released free radicals that damage the DNA cross linking and

chromosomal translocation, etc. The DNA of exposed cells undergoes single and double strand

breaks, inter and intra strand cross linking and damage to bases and sugars, ultimately

leading to chromosomal aberrations and chromosomal translocations.2

X-rays are a form of electromagnetic radiations. Their energies range between 120eV-

120keV. Based on their energies and penetrating ability X-rays are classified as soft X-rays

and hard X-rays. Hard X-rays are used extensively in diagnosis and treatment. Since the

penetrative ability of soft X-rays is very less, their usage in the therapy or diagnosis is limited.3Since

x-ray is an ionizing radiation it damages DNA. Protection of normal tissues against this cellular

damage is important in radiotherapy. Hence radiation protection has a significant role in radiotherapy

of cancer.

Several chemicals have been found to provide good radiation protection in different

experimental systems, but their clinical utility is limited due to their toxicity on repeated

administration. The only drug approved for clinical use in radiation treatment is Amifostine, a

synthetic phosphorothiorate compound, which also produces toxicity at the maximum effective dose.

Therefore, there is a need to find non-toxic and inexpensive drugs for clinical radiation protection.4

Phytochemicals are non-nutritive components present in vegetables that possess substantial

anti-carcinogenic, antioxidant and anti-mutagenic properties. A large number of antioxidant

phytochemicals are reported to have radio protective effect in various animal models. Protection

against radiation induced abnormalities by dietary ingredients form an attractive proposition to

prevent radiation-induced abnormalities4. Extracts Azadirachta indica (neem) and Momordica

charantia (bitter gourd) are reported to possess antioxidant property.5, 8The present study is designed

to evaluate the radioprotective potential of extracts of Azadirachta indica and Momordica charantia

against radiation injury induced by x-rays radiation in rats because both of them were reported as

potent antioxidants. The outcome of this study will be highly useful in identifying an efficient, safer

and cheaper agent that can be co-administered with radiation therapy to minimize the radiation

induced injury and toxicity, hence this study is needful and justifiable in the present situation.

6.2 REVIEW OF LITERATURE

Kumar A et al., reported leaf and bark extracts of neem to have significant anti-oxidant

properties. Further in comparative study, the bark was found to be more potent than leaf in the

entire assay with higher phenolic content and concluded both the bark and leaf can be used in

pharmacological application as a valuable antioxidant.5

Sithisaran P et al., assessed in-vitro antioxidant activity of neem leaves, fruits, flowers and stem

bark extracts using the 1,1-diphenyl-2-picryl hydrazyl (DPPH) scavenging assay, total

antioxidant activity and inhibition of lipid peroxidation in Chago K1 cancer cell culture by the

thiobarbituric acid reactive substances (TBARS) method. The results showed that aqueous

extract of leaves, ethanolic extract of flower and stem bark exhibited higher free radical

scavenging effect on the DPPH assay with 50% scavenging activity at 26.5, 27.9 and 30.6

microg/ml, respectively. The total antioxidant activity of these extracts were found to be

0.959,

0.988 and 1.064 mM to standard trolox, respectively.6

Ajibade et al., reported Azadirachta indica (200 mg/kg body wt. p.o.) inhibited paracetamol (2

g/kg body wt. p.o.)-induced lipid peroxidation and prevented depletion of sulfhydryl groups in

rat liver cells.7

Semiz A and Sen demonstrated the antioxidant and chemoprotective activities of Momordica

charantia fruit extract by investigating antioxidant enzymes, cytochrome p450 and

gluthathione-s-transferases as well as its protective effect on hepatocellular damage in CCl4

intoxicated rats.8

Thenmozhi and Subramanian. P reported ethanolic extract of Momordica charantia has

protective activity in ammonium chloride induced hyperammonemic rats against oxidative

stress.9

Abbas et al., reported extracts of Momordica charantia, A.sativum, O.sanctum and

Azadirachta indica has not only possess hypoglycemic activity but these extracts were also

effective in reducing the plasma lipid peroxide levels in diabetic rats. Enzyme of anti-oxidant

activity superoxide dismutase enzyme and serum albumin content were also found increased

in this study.10

Alenzi et al., reported that Nigella sativa oil and Thymoquinone has shown to lower

Cyclophosphamide-induced toxicity by an up-regulation of antioxidant mechanism, indicating

a potential clinical application for these agents to minimize the effect of anticancer drugs.11.

Natividad Sebastia et al., reported that the pretreatment of curcumin and trans resveratrol

would significantly protect normal lymphocytes against ɤ-radiation induced cellular damage,

due to the presence of polyphenols.12

Eunjin park et al., investigated the radio protective effect of eckol against the gamma ray

induced damage in vivo and proposed that eckol can be used as a candidate for adjuvant

therapy to allievate radiation injury to cancer patients.13

Lei yao et al., reported the protective effects of polysaccharides from soya bean meal (SBM)

against x-ray induced damage in mouse spleen lymphocytes and suggested that SBM may be

a good candidate as a radioprotective agent.14

PLANT PROFILE.

AZAD IRACHTA IND ICA

Scientific name

Family

=

=

Azadirachta indica A.Juss

Meliaceae

Genus

Species

=

=

Azadirachta

A. indica

Common name = Neem

The neem tree (Azadirachta indica) is a tropical evergreen tree native to india and is also found in

other southeast countries. In india, neem is known as “the village pharmacy” because of its

healing

versatility, and it has been used in ayurvedic medicine for more than 4,000 years due to its medicinal

properties

Description: it is a hardy, fast-growing evergreen tree with a straight trunk, long spreading branches

and moderately thick, rough, longitudinally fissured bark (plate 10). Mature trees attain a height of 7-

20 m with a spread of 5-10 m. The tree starts producing the yellowish ellipsoidal drupes (fruits) in

about 4 years, becomes fully productive in 10 years and may live for more than 200 years. The leaves

are compound, imparipinnate, comprising up to 15 leaflets arranged in alternate pairs with terminal

leaflets. The leaflets are narrow, lanceolate, up to 6 cm long.

Active constituents: good number of chemicals isolated from the plant belong to the classes

diterpenes: sugiol, nimbiol (bark), triterpenes : β-sitosterol, stigmasterol(leaf), liminoids :

meliantriol(seed oil), nimbidine(seed oil),nimbendiol(seed oil) and azadiractin, sulphurous

compounds: number of cyclic tri and tetrasulphides(leaves), flavonol glycosides: nimaton, quercetin,

myrecetin, kaempferol.15

Medicinal uses: Almost all parts of the neem tree have been used as traditional ayurvedic, unani and

sidhha medicine in india. Neem oil, bark and leaf extracts have been therapeutically used as folk

medicine to control leprosy, intestinal helminthiasis, respiratory disorders, constipation, blood

purifier and also as a general health tonic. It also used for the treatment of rheumatism, chronic

syphilitic sores and indolent ulcer. Neem oil is used to control various skin infections. Bark, leaf, root,

flower and fruit together cure blood morbidity, biliary afflictions, itching, skin ulcers, burning

sensations and pthysis. Neem contained various compounds which showed various biological

activities such as anti- inflammatory, antiarthritic, antipyretic, hypoglycemic, antigastric ulcer,

spermicidal, antifungal; antibacterial, diuretic, antimalarial, antitumor, immunomodulatory etc.16

The ORAC test (oxygen radical absorbance capacity), shows that all forms of neem are

extraordinarily high in antioxidants and far higher than foods on the high-antioxidant lists. Along

with its immune- boosting properties, high antioxidant levels may help to explain why neem is

known around the world as “the village pharmacy.”17

MOMORD ICA CH ARA NT IA

It consist of fresh green fruits of plant known as momordica charantia Linn.

Scientific name = Momordica charantia.

Family:

Genus:

=

=

Cucurbitaceae.

Momordica.

Species:

Common names

=

=

charantia.

Bitter Melon, bitter gourd, balsam apple, balsam pear, karela.

The Bitter Melon is believed to be originated in the tropics of the old world. It is widely grown in

India and other parts of the Indian subcontinent, Southeast Asia, China, Africa, and the Caribbean

and South America as a food and medicine.

Description: The plant is a slender climbing annual vine with long-stalked leaves and yellow, solitary

male and female flowers borne in the leaf axils (Bailey et al. 1985). It is related to squash and cucumber

plants. The fruit looks like a warty gourd. The young fruit is emerald green, turning to orange-yellow

when ripe.

Chemical constituents: Chemical constituents from whole plants, fruits, and seeds of bitter melon

have been isolated and described. Bitter melon fruit contains triterpene lycosides, including the

characteristic mormordin and charantin. Other triterpene glycosides (the momordicosides), vitamins,

including beta carotene, ascorbic acid, niacin, and thiamin, elemental compounds (eg, iron, iodine,

magnesium, sodium, calcium), and fatty acids, including stearic, palmitic, and oleic, are also present.

Insulin-like compounds, or compounds exerting hypoglycemic activity, have been described.

Bitter melon seeds and the pericarp contain the phenolics catechin and epicatechin, gallic, gentisic,

and vanillic acids, as well as lutein, lycopene, carotenes, xanthins, momordicosides, and vicine. The

seed essential oil contains sesquiterepene, phenylpropanoids, and monoterpenes, including

nerolidol.17

Medicinal uses: Fruits of Momordica charantia used in asthma, burning sensation, colic,

constipation, cough, diabetes, fever (malaria), gout, helminthiases, inflammation, leprosy, skin

diseases, ulcer and wound. It has also been shown to have hypoglycaemic properties (antidiabetic) in

animal as well as human studies. Juice of the Karela leaves used to treat piles completely. It is used

as a blood purifier due to its bitter tonic properties. It can heal boils and other blood related problems

that show up on the skin. Juice of karela is also beneficial in treating and preventing the liver damage.

Free-radical scavenging activity, attributed to the phenolic content, has been demonstrated in vitro.18

In hyperammonemic rats, the oxidant-antioxidant imbalance was restored by administration of bitter

melon fruit extract.19

7. OBJECTIVE OF THE STUDY:

To evaluate the radioprotective effect of bark extract of azadirachta indica and fruit extract of

momordica charantia in experimental animal models with the following specific objectives:

1. Collection and authentication of Azadirachta indica bark and Momordica charantia fruits.

2. Extraction of plant material

3. Determination of LD50 of bark extract of Azadirachta indica and fruit extract of Momordica

charantia as per OECD guidelines

4. To establish the pharmacological profile of prepared extracts for its radioprotective activity by

a. Evaluation of bark extract of Azadirachta indica and fruit extract of Momordica charantia for

in vivo micronucleus assay.

b. Evaluation of bark extract of Azadirachta indica and fruit extract of Momordica charantia for

in vivo chromosomal aberration test.

c. Evaluation of bark extract of Azadirachta indica and fruit extract of Momordica charantia by

Comet assay.

8. MATERIAL AND METHODS:

8.1 SOURCE OF DATA:

The whole study is planned to generate data from laboratory studies i.e., experiments will be

performed as described in reference, experimental studies in journals and in textbooks available with

college and In addition data is also collected from the literature to interpret the results. Some of the

sources to collect data from the literature are:

Websites: w w w .s c i e n c e d i rect . c o m

w ww . n c b i. n l m . n i h .gov/ pub m e d /

w ww . s h c ol a r .googl e . c o m

www .ijp -onlin e.com

8.2 PLAN OF WORK:

METHODS OF COLLECTION DATA

The whole study is divided into three well designed phases as below

Phase – I

The materials (including animals) required for the study will be collected and stored. In this phase

toxicity studies will be carried out and also dosage regimen for further studies will be established.

Albino mice will be used for toxicological studies.

Phase – II

The core experimentation like irradiation of animals with x-rays at a dose of 0.5Gy/min, for 8 min

at room temperature and protection of animals by using study drugs will be carried out in this

phase. DNA damage is determined by using following methods.

1. Micronucleus assay

2. Chromosomal aberration test

3. Comet assay

Various analytical tools will also be adopted to evaluate the study parameters. The animals used

in this phase will be albino rats.

Phase – III

In this phase data generated will be subjected to statistical analysis to find out significance. In this

phase the data is compiled and promulgated. In addition the data is compiled into dissertation.

A. Experimental animals.

Male albino wistar rats weighing between 150-250g will be used in the study. The animals will be

kept under standard conditions at ambient temp of 25± 20 C with 12hr light/ 12hr dark cycle, with

food and water in the institutional animal house.

B. Plant material

The bark of Azadirachta indica and the fruits of Momordica charantia will be obtained and

authenticated by natural remedies. Aqueous extracts of Azadirachta indica bark and aqueous

extract of momordica charantia fruits will be prepared by maceration process.

Acute Toxicity study

Determination of LD50 of the bark extract of Azadirachta indica and fruit extract of Momordica

charantia as per OECD Guidelines. The guideline described by OECD will be adapted for the

determination of LD50 on adult female mice. Female mice will be used as they are more sensitive

to toxicity compared to male mice. Hence female mice are taken for acute toxicity study

and1/10th of LD50 dose will be taken as effective dose and considered for the further study.

8.3 EXPERIMENTAL DESIGN.

Radioprotective effect of Azadirachta indica (A.I) and Momordica charantia (M.C).

1. MICRONUCLEUS (MN) TEST.20,21

A. P ROCEDUR E :

Albino wistar rats will be randomly divided in to 9 groups of 6 rats in each group. Animals

will be orally administered with extract of drug or distilled water for consecutive 7 days

(pretreatment). One hour after the administration of last dose (on 7th day), the rats will be

exposed to whole body irradiation of x-rays with a total dose of 4 Gy, except rats of control

and extract control groups.

B. GROUP ING O F AN IMA LS :

Group I: Control group (vehicle)

Group II: Positive control group 1. (Irradiation, bone marrow extraction after 24 hrs)

Group III: Positive control group 2. (Irradiation, bone marrow extraction after 48 hrs)

Group IV: Extract alone group 1. (Azadirachta indica extract)

Group V: Extract alone group 2. (Momordica charantia extract)

Group VI: Treatment group 1. (Extract of A.I + Irradiation, bone marrow extraction after 24 hrs.)

Group VII: Treatment group 2. (Extract of A.I + Irradiation, bone marrow extraction after 48 hrs.)

Group VIII: Treatment group 3 (Extract of M.C +Irradiation, bone marrow extraction after 24

hrs.) Group IX: Treatment group 4. (Extract of M.C + Irradiation, bone marrow extraction after

48 hrs.)

After respective treatment, rats will be sacrificed by cervical dislocation and are cut open to excise

femur and tibia. Bone marrow slides will be prepared by using the modified method of Schmid.

Marrow suspensions from femur and tibia bones are prepared in 5% bovine serum albumin (BSA)

and suspensions will be centrifuged at 1000 rpm for 8 min and the pellets are resuspended in BSA.

A drop of this suspension will be taken on a clean glass slide and smear will be prepared on glass

slide and air dried. The slides will be fixed in absolute methanol, stained with May-Grunwald-

Giemsa and observed for micronucleus formation in polychromatic erythrocytes and

normochromatic erythrocyte.

2. CHROMOSOMAL ABBERATION.22

A. P ROCEDUR E :

Albino wistar rats will be randomly divided in to 9 groups of 6 rats in each group. Animals will

be orally administered with extract of drug or distilled water for consecutive 7 days

(pretreatment). One hour after the administration of last dose (on 7th day), the rats will be exposed

to whole body irradiation of x-rays with a total dose of 4 Gy, except rats of control and extract

control groups.

B. GROUP ING O F AN IMA LS :

Group I: Control group (vehicle)

Group II: Positive control group 1. (Irradiation, bone marrow extraction after 24 hrs)

Group III: Positive control group 2. (Irradiation, bone marrow extraction after 48 hrs)

Group IV: Extract alone group 1. (Azadirachta indica extract)

Group V: Extract alone group 2. (Momordica charantia extract)

Group VI: Treatment group 1. (Extract of A.I + Irradiation, bone marrow extraction after 24 hrs.)

Group VII: Treatment group 2. (Extract of M.C+ Irradiation, bone marrow extraction after 48

hrs.) Group VIII: Treatment group 3. (Extract of M.C+ Irradiation, bone marrow extraction after

24 hrs.) Group IX: Treatment group 4. (Extract of M.C + Irradiation, bone marrow extraction

after 48 hrs.)

After respective treatment rats will be sacrificed by cervical dislocation. 90 min. prior to sacrifice,

each animal is injected with 0.04% colchicine in a dose of 4 mg/kg i.p. for mitotic arrest.

Colchicine solution is prepared in distilled water. Bone marrow will be collected from femur and

tibia and suspended in 5 ml hypotonic solution of 0.075M kcl for 20 minutes at 37o c and will be

fixed by carnoys fixative (methanol: acetic acid, 3:1). Smears will be prepared on clean slide,

stained with

giemsa and metaphase plates will be observed and chromosomal aberrations will be scored using oil

immersion (with 100x object lens) under a light microscope.

3. COMET ASSAY.23

A. P ROCEDUR E :

Albino wistar rats will be randomly divided in to 6 groups of 6 rats in each group and animals will

be orally administered with extract of drug or distilled water for consecutive 7 days

(pretreatment). One hour after the administration of last dose (on 7th day), the rats will be exposed

to whole body irradiation of x-rays with a total dose of 4 Gy, except rats of control and extract

control groups.

B. GROUP ING O F AN IMA LS :

Group I: Control group. (Vehicle)

Group II: positive control group 1.

Group III: extract alone group 1 (Azadirachta indica extract)

Group IV: extract alone group 2 (Momordica charantia extract)

Group V: Treatment group 1. (Extract of A.I+ Irradiation).

Group VI: Treatment group 2. (Extract of M.C+ Irradiation).

After irradiation of animals, the lymphocyte/bone marrow will be isolated and the cells will be subjected to comet assay.

Parameters to be evaluated in comet assay.

i. Tail length.

ii. Tail moment.

iii. Olive tail moment.

8.4. STATISTICAL CALCULATION:Data will be analyzed using SPSS 18 for windows. ANOVA will be done to compare groups with

post hoc Bonferroni correction. 95 % CI (confidence interval) for P will be fixed at P = 0.008 for

post hoc analysis.

8.5. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE

CONDUCTED ON PATIENTS OR THE HUMANS OR ANIMALS..? IF SO PLEASE

DESCRIBE BRIEFLY.

Yes, The study requires investigation on animal, effects of the extracts will be studied on

various parameters using albino wistar rats as experimental animals.

8.6. HAS ETHICAL CLEARANCE HAS BEEN OBTAINED FROM YOUR INSTITUTE?

Yes, the study has been referred to the ethical committee of the institution and clearance is

awaited.

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