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“ANTICANCER EFFECT OF BARK OF DALBERGIA LATIFOLIA AGAINST
EHRLICH ASCITES CARCINOMA INDUCED TUMOR”
Synopsis for registration of M. Pharm Dissertation
Submitted to
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE
KARNATAKA
In partial fulfilment
Of the requirement for the Degree of
Master of Pharmacy in Pharmacology
Under the Guidance of
Dr. Nagarathna P.K.M.
Assistant Professor.
BY
P. SRIRAM REDDY.
Department of Pharmacology
KARNATAKA COLLEGE OF PHARMACY,
BANGALORE-64,
2012- 14.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE .
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
2.
Name of the candidate and address
Palla sriram reddy,
s/o indrasena reddy
c/o venkanna
H:NO:14-99/4,thorrur
warangal(dist),
Andhrapradesh(A.P).
Name of the Institution
Karnataka College of Pharmacy,
Thirumenahalli,
Bengaluru-560064
Karnataka, India
3.
Course of study and subject
M.Pharm-Pharmacology
4.
Date of the admission
07.06.2012
Title of the topic
5.
“ANTICANCER EFFECT OF BARK OF DALBERGIA LATIFOLIA AGAINST
EHRLICH ASCITES CARCINOMA INDUCED TUMOR”
6
BRIEF RESUME ON THE INTENDED WORK
6.1 -NEED FOR THE STUDY:Cancer is one of the leading causes of mortality worldwide and the failure of conventional
chemotherapy to affect major reduction in the mortality indicates that new approaches are
critically needed [1]. Over the past few years, cancer has remained a major cause of death and the
number of individuals affected with cancer is continuing to expand. Hence a major portion of the
current pharmacological research is devoted to anticancer drug design customized to fit new
molecular targets (Xia et al., 2004). Due to enormous propensity of plants, which synthesize a
variety of structurally diverse bioactive compounds. The plant kingdom is a potential source of
chemical constituents with antitumor and cytotoxic activities (Kim et al., 2005; Indap et al.,2006).
The rich and diverse plant sources of I9ndia are likely to provide effective anticancer agents. One
of the best approaches in the search of anticancer agents from plant sources is the selection of
plants based on ethno medical leads (Kintzios, 2006)[2]. The new and recent approaches of
chemotherapy serve as an attractive alternative to control the cancer. Recently, the major focus of
research in chemotherapy for cancer includes the identification, characterization and development
of new and safe cancer chemo preventive agents. A large number of agents including natural and
synthetic compounds have been identified as having some potential cancer chemotherapeutic
value. A number of natural products have been studied for anticancer activity on various
experimental models. This has resulted in the availability of nearly 30 effective anticancer drugs
[1]
. Cancer is a group of diseases where cell growth is aggressive and abnormal, invasive, and /or
metastatic many times leading to death. Carcinoma arises from epithelial cells invading
surrounding tissues and organs and may metastasize, or spread, to lymph nodes and other sites.
The treatment of cancer has undergone major advances, which include benefits of combination
chemotherapy as well as the incorporation of biologic therapy, and yielding significant
improvements in survival over the past decade [3]. Improving the quality of life of patients living
with cancer and dying from cancer is therefore an urgent humanitarian need. This can be
achieved by in depth research and continuous screening of new molecules or natural agents,
which will provide the antitumor activity in Indian traditional system of medicine
(Ayurvedicsystem) uses plant derived medicines in health care from ancient period of time. These
natural medicines have played a great role to treat various disorders in humans including cancer.
Oxygen free radicals are formed in tissue cells by many endogenous and exogenous influences
like cellular metabolism, exposure to chemicals, carcinogens and ionizing radiation. Oxygen free
radicals may attack all macromolecules in cell (proteins, lipids and DNA) giving rise to a wide
variety of damaged products. Free radicals cause DNA double strand breaks, mismatching of
bases and chromosome deletions and rearrangements. Hence, the antioxidants which can quench
these free radicals could act as cancer chemo-preventive agents [4] .Natural products are playing
an important role as a source of effective anticancer a gents and it is significant that 60% of
currently used anticancer agents are derived from natural sources, including plants, marine
organism and micro-organism. The mechanism of interaction between many secondary
metabolites and cancer cells has been studied extensively. In particular, there is growing interest
in the pharmacological evaluation of various plants used in Indian traditional system of medicine.
Plant-derived natural products like flavonoids, steroids, alkaloids and terpenoids have received
considerable attention in recent years due to their diverse pharmacological activities, including
antioxidant and anticancer activity. Antioxidants play an important role in inhibiting and
scavenging radicals and thus, protecting humans against infection and degenerative diseases [1].
6.2- REVIEW OF LITERATURE:Cancer is developed due to some molecular changes within the cell, and is becoming
the second major cause of death in the human after cardiovascular disease.. About 7.6 million
people died from cancer in the world during 2007(American cancer society, 2007). Hence there is
an urgent need for developing new approaches and drugs to prevent as well as cure this
devastating disease. Scientists are now developing drugs that target the unique makeup
mechanism of cancer cells. A number of natural products have been studied for anti cancer
activity on various experimental models [5] ..
. MATERIALS AND METHODS:
PLANT INFORMATION:
Family:Leguminosae, sub-family:Papilionoideae
Dalbergialatifolia is a premium-quality timber species internationally known as "Indian
Rosewood". It is used to manufacture furniture, panelling, and other ornamental products.
Medicines and an appetizer are made from tannins in the bark. The tree is commonly called sitsal,
beete, shisham or Bombay Blackwood in India, and sonokeling or sonobrits in Indonesia.
Medicinal uses: Tannins from the bark are used to produce medicines for the treatment of
diarrhoea, worms, indigestion, and leprosy. These tannins also produce an appetizer.
Chemical constituents: The plant Dalbergia latifolia mainly contains flavonoids that too
isoflavonoid O-glycosides. The main active constituents of DalbergiaLatifolia are dalbergione and
dalbinol.
Preparation of extract: Powdered leaves were subjected to successive extraction in a Soxhlet
apparatus with methanol. The extract obtained was concentrated in a rotary shaker evaporator to
dryness to get a constant weight
6.3- AIM:
1. Collection, Authentication and Extraction of bark of dalbergia latifolia.
2. Structural elucidation of isolated compound from the bark of dalbergia latifolia.
3. To evaluate the in vivo and invitro anticancer activities of extracts and isolated compounds
of dalbergia latifolia on Ehrlich ascites carcinoma induced solid and breast tumour.
6.4-OBJECTIVE OF THE STUDY:The objective of the proposed study is to evaluate the Anticancer activity using a Methanolic
extract of plant dalbergia latifolia(leguminosae) on Ehrlich ascites carcinoma (In vivo) as well as
Ehrlich ascites cell lines(Invitro)
6.5-SOURCE OF DATA
Whole work is planned to generate data from laboratory studies i.e. experiments are performed as
described in references. Experimental studies in journals and in text books available with college,
IISc and other libraries. Literature is searched from various web sites in the internet.
6.6-MATERIALS AND METHODS
Preparation of extract :
100gms powdered bark was subjected to successive extraction in a Soxhlet extractor with
Methanol. The extract obtained was concentrated in a rotary shaker evaporator to dryness to get a
constant weight [15].
IN-VIVO STUDIES
Ehrlich ascites carcinoma (EAC) cells induced mammory tumours:
Materials:
EAC cells : Amala Cancer Research Institute, Thrisur, Kerala, India.
P.B.S: Himedia, India.
Swiss albino mice were divided into five groups of 30 animals (n=6)each.
EAC cells were collected from the donor mice and were suspended in sterile isotonic saline. The
viable EAC cells were counted (Trypan blue indicator) under the microscope and were adjusted at
106 cells/ml. 0.1 ml of EAC cells per 10g body weight of the animals was injected at mammary fat
pad subcutaneously to whole animals on day zero. A day of incubation was allowed for
multiplication of the cells.
The animals were divided into five groups:
Group 1: Serves as a normal control
Group 2: Serves as a Ehrlich ascites control
Group 3: Test group A recieves low dose of dalbergia latifolia.(orally/day/12days)
Group 4: Test group B recieves high dose of dalbergia latifolia.(orally/day/10days)
Group 5: Mice recieves 5Flouro-uracil serves as a standard (20mg/kg;ip; /day/12days)
5Flouro-uracil was dissolved in normal saline and administered to mice (20mg/kg;ip;
/day/12days).After the 12 days of treatement animals were sacrificed and total number of cells
percentage viability, tumor volume and cell morphology were observed.
Ehrlich ascites carcinoma (EAC) cells induced solid tumors:
Materials:
EAC cells: Amala Cancer Research Institute, Thrisur,Kerala,India.
P.B.S : Himedia,India.
Swiss albino mice were divided into five groups of 30 animals (n=6)each.
EAC cells were collected from the donor mice and were suspended in sterile isotonic saline.the
viable EAC cells were counted (Trypan blue indicator) unde the microscope and Tumor cells
(1×106 cells /mouse) were injected into the right hind limb of all the animals intramuscularly to
whole animals on day zero. A day of incubation was allowed for multiplication of the cells.
The animals were divided into five groups:
Group 1: Serves as a normal control
Group 2: Serves as a Ehrlich ascites control
Group 3: Test group A recieves low dose of dalbergia latifolia.(orally/day/12days)
Group 4: Test group B recieves high dose of dalbergia latifolia.(orally/day/10days)
Group 5: Mice recieves 5Flouro-uracil serves as a standard (20mg/kg;ip; /day/12days)
5Flouro-uracil was dissolved in normal saline and administered to mice (20mg/kg;ip;
/day/12days). After the 12 days of treatement animals were sacrificed and total number of cells
percentage viability, tumor volume and cell morphology were observed.
The Antitumor activity of dalbergia latifolia.( will be screened with respect to the following
parameters.
(A)Effect on survival time:
Animals will be inoculated with EAC cells (1 X 106cells/mouse) on day ‘0’ and the median
survival time (MST) of each group, consisting of 6 mice will be noted.
MST = (day of first death + day of last death)
2
(B)Percentage increase life span (% ILS)[20]:
The effect of the drugs on tumour growth was monitored by recording the mortality daily for a
period of 6 weeks and percentage increase in life span (%ILS) was calculated.
%ILS = [(T-C)/T] ×100
Where, T = number of days the treated animals survived.
C = number of days the control animals survived.
(C)Tumor volume:
The mice were dissected on the 15th day and the ascitic fluid was collected from the peritoneal
cavity. The volume was measured by taking it in a graduated centrifuge tube and packed cell
volume was determined by centrifuging at 1000 rpm for 5 min.
(D)Tumor cell count:
The ascitic fluid was taken in a WBC pipette and diluted 100 times. Then a drop of the diluted cell
suspension was placed on the Neubauer counting chamber and the number of cells in the 64 small
squares was counted.
(E)Viable/non-viable tumor cell count: The cells were stained with trypan blue (0.4% in normal
saline) dye. The cells that did not take up the dye were viable and those that took the stain were
nonviable. These viable and nonviable cells were counted.
Cell count =
(No.of cells × Dilution)
(Area × Thickness of liquid film)
(F)Effect of extracts on normal peritoneal cells[21]:
To evaluate whether the drugs treatment indirectly inhibited tumour cell growth, the effect was
determined on the peritoneal exudate cells of normal mice. Peritoneal exudate cells were collected
after 24 h treatment by repeated intraperitoneal wash with normal saline and counted in each the
treated groups and compared with those of the untreated group.
(G)Body weight:
Body weights of the experimental mice were recorded both in the treated and control group at the
beginning of the experiment (day 0) and sequentially on every 5th day during the treatment
period.
(H)Effect on hematological parameters:
At the end of the experimental period, 6 mice of each group were killed the next day after an
overnight fast by decapitation. Blood was collected from freely flowing tail vein and used for the
estimation Hemoglobin (Hb) content, red blood cell count (RBC) and white blood cell count
(WBC)[35]. WBC differential count was carried out from Leishman stained blood smears[24].
(I)Histopathological studies[25]:
A portion of Liver and Kidney of animals in all groups were stored in container for 12 hours in
10% formalin solution and subjected to histopathological studies.
(J)Estimation of in vivo Antioxidants:
After collecting the blood samples, the mice were killed by cervical dislocation. The liver was
excised, rinsed in ice-cold normal saline solution followed by cold 0.15 M Tris-HCl (pH 7.4),
blotted dried and weighed. A 10% w/v homogenate was prepared in 0.15 M Tris-HCl buffer and
was used for the estimation of lipid peroxidation (LPO) and reduced glutathione (GSH). The rest
of the homogenate was centrifuged at 1500 rpm for 15 min at 40C. The supernatant thus obtained
was used for the estimation of superoxide dismutase (SOD), catalase (CAT) and total proteins.
INVITRO STUDIES
Cell Lines :( EAC cell lines)
The cell viability, cell growth as well as cell cytotoxicity of Ehrlich Ascites Carcinoma cell lines
were determined by performing the following assays:
MTT ASSAY:
Cell proliferation activity of the drugs will be carried out by MTT assay which estimate the effect
of the various drugs on the growth of cell in vitro. Measurement of cell in viability and
proliferation forms should be used as basis for this invitro assay. The reduction of terazolium salt
now widely accepted to examine cell proliferation the yellow coloured terazolium, MTT [(3-(4,5dimethylthiazol-2-yl)-2,5,diphenylterazolium bromide],will reduce metabolically active cell in
part by the action of dehydrogenase enzymes to generate reducing equivalents such as NADH
and NADPH.The resulting intracellular purple colour zones will solubilise and quantify by
spectrophotometric method. When metabolic event leads to necrosis or apoptosis in cell, the MTT
method will measures the cell viability. The assay gives low background absorbance values in the
absence of necrosis of the cell.MTT should be dissolved in PBS at a concentration of 5mg/ml.
Then, 50µg of the MTT solution will be added to each well of the 96-well culture plate,
containing the 100-µl medium, and incubated at 37˚c for 4hr. This medium should be removed
carefully without disturbing the purple coloured formazan crystals; 50ml of dimethyl sulphoxide
(DMSO) will be added to each well and mixed thoroughly to dissolve the crystals of the
formazan. Then these plates should be seen on a micro plate reader at a wavelength of 670nm.
The readings should be presented as optical density (OD). The growth inhibition of the cells by
the drugs will be identified.
SULFORHODAMINE B ASSAY:
Sulforhodamine b (SRB) (Sigma–Aldrich Chemie GmbH, Munich, Germany) can be used to test
the effects of active compounds on cell growth and viability, based on the method described by
vichai and kirtikara , compounds 1–8 should be dissolved in dimethylsulfoxide (DMSO) before
diluting with the growth medium to a final DMSO concentration of <0.05%. The cancer cells will
be seeded into 96 well plates in the growth medium at 3000 cells/well. After 24 h of incubation,
the medium to be replaced with a fresh growth medium containing the test compounds 1–8 (0, 25,
50, 100 and 200 lm). The cells will be incubated for another 48 h. The cells should be fixed with
TCA by gently adding 50 µl TCA (50%) to each well to a final TCA concentration of 10% with
subsequent incubation for 1 h at 4˚c. The plates should be washed 5 times with deionised water
and air dried. The dried plates should be stained with 100 µl of 0.4%n (w/v) SRB prepared in 1%
(v/v) acetic acid for 10 min at room temperature. The plates will be rinsed quickly four times with
1% acetic acid to remove unbound dye, followed by air-drying until no moisture should be visible.
The bound dye will be solubilised in 2 mM Tris base (100 µl/well) for 5 min on a shaker. Optical
densities should read on a microplate reader at 562 nm.
STATISTICAL ANALYSIS:
The statistical significance of the results will be analyzed by ANOVA. p<0.05 will indicate the
significance of the result.
7.1- Does The Study Require Any Investigation To Be Conducted On Patients Or
Animals? If So, Please Describe Briefly.
No, the entire experimental models require usage of laboratory animals only.
7.2 - Has Ethical Clearance Been Obtained From Your Institution In Case Of 7.1?
Approved by IAEC
7
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Dhanasekaran, Manonmani Alvin Jose and Chennakesavalu Jothimanivannan. Antitumor
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Mice. Iranian Journal of Pharmaceutical Research (2011);10 (3):505-510.
2. Raju Senthil Kumar, Balasubramanian Rajkapoor, Perumal Perumal. Antitumor and
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9
Signature of the candidate
(SRI RAM REDDY PALLA)
10
Remarks of the Guide: The topic selected for dissertation is satisfactory. Adequate
equipment and chemicals are available to carry out the project work.
11
Name and Designation of Guide:
Dr. NAGARATHNA P.K.M.
ASSISTANT PROFESSOR.
DEPT OF PHARMACOLOGY,
KARNATAKA COLLEGE OF PHARMACY,
BENGALURU-64
Signature of Guide:
[Dr. NAGARATHNA P.K.M.]
Co-Guide:
----------
Signature of Co-Guide:
Head of the Department
Dr. RAJU KONERI
Professor &Dean.
Dept of Pharmacology,
Karnataka college of pharmacy,
Bengaluru-64.
Signature of HOD:
(Dr. RAJU KONERI)
12
Remarks of the Director: All the required facilities will be provided to carry out
dissertation work under the supervision of guide.
Director:
Dr. K.RAMESH.
Director
Karnataka college of pharmacy,
Thirumenahalli,
Bangalore –64.
Signature of the Director:
(Dr. K.RAMESH)