Download Title of the Topic - Rajiv Gandhi University of Health Sciences

Evaluation of Anticancer activity of Moringa oleifera Lam.
in Balb/c mice
Protocol of Dissertation Submitted (2008-2009)
By
Mr. MASOOD.MK
To
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA.
Under the guidance of
Mr. CHANDRASHEKHAR V.M.
Assist. Professor.
Department of Pharmacology,
HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,
BAGALKOT- 587101, KARNATAKA
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA-BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
Name of the Candidate and Address
MASOOD.MK
DEPARTMENT OF PHARMACOLOGY,
H.S.K COLLEGE OF PHARMACY,
B.V.V.S CAMPUS,
BAGALKOT-587101, KARNATAKA
2.
Name of the Institution
H.S.K.COLLEGE OF PHARMACY,
B.V.V.S.CAMPUS,
BAGALKOT-587101, KARNATAKA
3.
Course of Study and Subject
MASTER OF PHARMACY IN
PHARMACOLOGY
4.
Date of Admission to Course
5.
Title of the Topic:
16 – 06 - 2008
“Evaluation of Anticancer activity of Moringa oleifera Lam. in Balb/c mice”
1
6.
Brief Resume of the intended work:6.1 Need for the study:
Cancer is one of the major causes of death in developed nations at least one of the five of the
population of Europe and America can expect to die of cancer. In the year 2000, there were 10
million new cases of cancer and 6 million deaths worldwide1. Lung cancer is becoming
increasingly common throughout the world, but at 14/100 000 persons in India the incidence is
much lower than in UK, where it is four times higher2. Cancer is basically a disease of cells
characterized by a shift in the control mechanisms that govern cell proliferation and
differentiation.3. Rheumatoid arthritis, sickle cell anemia, psoriasis are some related diseases
during cancer treatment4. The extent of metastasis and deterioration in metabolic processes,
resulting from cancer, leads to eventual death of patient5. Most carcinomas occur in the later years
of life (≥55 years). Cancer is the main cause of death among women aged 40 to 79 and among
men age 60 to 796. National Cancer Institute Survival Epidemiology and End {NCISEE} results
population data from the senses that 1.36 million new cases of invasive cancers were diagnosed,
5,63700 persons died from cancer i.e. percentage distribution of new cancer and cancer death
increases becoming major disease in the world7. Treatment of cancer with chemotherapeutic
agents, along with radiation and surgery. The chemotherapeutic agents provides a temporary
improve of symptoms and signs of cancers and most of the therapeutic agents are given in
combination with the radiation therapy and surgery8 and patient has to consume for the long
duration and these drugs have more toxic to the patient and economically burden to the patients.
The most of the plant based medicines have shown promising agents for the treatment of the
cancer with safe, efficacy and economical9. Moringa oleifera Lam belongs to the family Moringa
2
has wide spread throughout India. It is used as a traditional medicine for the treatment of Antiinflammatory, Purgative, Antipyretic and Ophthalmic. The phytochemical studies are reported
that it contains Octacosanoic acid, β-Sitosterol, β-Sitostenone, 4-hydroxymelline Sponins,
Alkaloides, Flavonoides10etc. In view of this above information, the present study was designed
for the In vitro and In vivo evaluation of anticancer activity of Moringa oleifera Lam in Balb/c
mice.
6.2 Review of literature:
Plant profile:
Title of plant
: Moringa oleifera Lam.
Family
: Moringaceae
Synonyms
: Drum stick, Sahinjan, Nugge.
Parts used
: Dried Seeds.
Habitat
: Throughout India.
Chemical constituents: Alkaloides, Flavonoides, β-Sitosterol, β-Sitostenone, 4-hydroxymelline,
Octacosanoic and fixed oils10. Proteins-40.19%, Lipids-40.19%, Carbohydrates-41.58%, saponins
-2.052%11.
Medicinal actions and uses: Traditional practitioners used the seeds of Moringa oleifera Lam as
Anti-inflammatory, purgative, Antipyretic, Ophthalmic, Obortifacient, Antifungal, Skin
papillomagenesis and Anti-cancerous11.
Pharmacological activities: - The Hydro-Ethanolic extracts of Seeds of Moringa oleifera Lam
have anti-anaphylactic, anti-arthritic, skin-papillomagenesis, antifungal, hypotensive, and antiinflammatory activities12.
3
6.3 Objectives of the study:
Traditionally well known seeds of Moringa oleifera Lam will be used for the present study
and objectives of the present study are to evaluate:
A] In vitro Anticancer activity of extracts.
In vitro Micronucleus Assay of extracts.
B] In vivo anticancer activity of extract:
1. Effect of the extracts on physiological parameters associated with anticancer activity.
2. Effect of the extracts on Hematological parameter.
3. Effect of the extracts on Biological parameters.
7.
Methods and Materials:7.1. Source of data: All data will be collected from the experimental animal model and eight
animals in each group will be subdivided follows:
Group I
- Normal
Group II
- Tumor control / Negative control
Group III
- Cyclophosphamide
Group IV
- Low dose of Hydro-Ethanolic extract
Group V
- Moderate dose of Hydro-Ethanolic extract
Group VI
- High dose of Hydro-Ethanolic extract
Group VII
- Low dose of Aqueous extract
Group VIII
- Moderate dose of Aqueous extract
Group IX
- High dose of Aqueous extract.
7.2. Materials:
Plant material
: Moringa oleifera Lam.
Animals
: Female Balb/ c mice
4
Chemicals
: All the chemicals are AR grade
Instruments
: Hemocytometer, Binocular microscope, refrigerator centrifuge machine etc.
7.3. Methods:
Preparation of Moringa oleifera Lam extract:Moringa oleifera Lam seeds were obtained from Bagalkot, locally after authentification by
taxonomist of Ayurvedic Medical College, Bagalkot. 5kg of seeds were powdered to fine texture
and passed through sieve no. # 40 to obtain uniform texture and extracted successively using
soxhlet extractor by Hydro-Ethanolic and Aqueous with distilled water for 24 hrs cycle, the
extract obtained was concentrated using rotatory evaporator at 500c. Extract was dried in freeze
dryer preserved in aseptic condition before the experiment. The percentage yield of the hydroalcohalic and aqueous extract calculated respectively13. The dose of the extract can be obtained
from acute toxicity (LD50) which will be done according to OECD/OCED Guidelines.
In vitro Studies: Tryphan Blue Exclusion Method (Cell viability test):
This is one of the methods to assess cytotoxicity of anticancer compounds. It comes under
preliminary screening of anticancer compounds. This test is based on the principle that living cell
membrane has the ability to prevent the entry of dye. Hence, they remain unstained and can be
easily distinguished from dead cells, which take the dye. The percentage of viable cells was
determined as follows,
Method:
The Ascitic fluid was withdrawn from animals which were induced with EAC cell lines, 0.1
ml of Ascitic fluid was aspirated, and it was diluted with 2ml of phosphate buffer saline (PBS).
To this equal volume of different extracts were mixed and this mixture incubated for 3 hrs at
370C. Then the mixture was added to 0.5 ml of tryphan blue and mixed thoroughly. The diluted
5
suspension was charged into hemocytometer. The viable cells(unstained) were counted in a WBC
chamber under a microscope and the mean number of cells in four chambers were calculated as
follows:
Total number of cells = Mean number of cells Dilution factor (2) × 104
In vitro Micronucleus test:
Genetic effects were evaluated in the mouse bone marrow micronucleus test according to
Schmid14. The bone marrow cells from both femurs were flushed in the form of a fine suspension
into a centrifuge tube containing human AB serum. This cell suspension was centrifuged at 2000
rpm for 10 minutes and pellet was resuspended in a drop of serum before being used for preparing
slides. Air dried slides were stained with May Grunwald and Giemsa as described by Schmid. For
each experimental point, six mice were used and 2500 polychromatic erythrocytes (PCEs) were
scored per animal per slide to determine the frequency of micro nucleated polychromatic
erythrocytes (Mn-PCEs). Special care was taken to ensure that pretreatment with the three doses
of Moringa oleifera did not lead to suspension of cell proliferation. This was done by monitoring
the ratio of PCEs to NCEs (Normochromatic erythrocytes). All the slides were scored by the same
observer unaware of the experimental protocol.
In vivo Anticancer activity of extracts:
Induction of Ehrlich Ascites Carcinoma:
The Ascitic carcinoma bearing mice (donor) was taken 15 days after tumor transplantation.
The Ascitic fluid is drawn using an 18-gauge needle into sterile syringe. A small amount tested
for microbial contamination. The Ascetic fluid was suitably diluted in normal saline to get a
concentration of 106 cells / ml of the tumor cell suspension. This was injected intraperitonially to
6
obtain Ascitic tumor. The mice were weighed on the of tumor inoculation and then for each three
days. Treatment was started 24 hours after tumor inoculation. Cyclophosphamide was injected on
two alternate day’s first and third day, by intraperitonial route. Extracts were administered till the
ninth day by intraperitonial route15.
Evaluation:
A] Physical parameters:
1. Percentage increase in body weight as compared to day-016.
2. Median survival time (MST).
3. Percentage increase in life span (%ILS) 17.
4. Mean survival time (MEST).
5. Cell viability test (% survivors of malignant cells in Ascitic fluid)18
B] Hematological parameters:
Total WBC, RBC, Hemoglobin content, PCV, MCH and MCHC19.
In order to detect the influence of plant extracts on the status of cancer bearing mice,
comparison was made amongst for groups of mice for each extract on the fourteenth day after
transplantation. These comprised as :
1. Normal mice
2. Tumor bearing mice
3. Tumor bearing mice is treated with one dose of Cyclophosphamide.
4. Tumor bearing mice treated with plant extracts for 14 days.
C] Biochemical parameters:
After the collection of the blood samples, the mice were sacrificed and their liver were
excised, rinsed in ice-cold normal saline followed by cold 0.15 mol / L. Tris-Hcl buffer (pH 7.4)
7
blotted dry and weighed 10% w/v homogenate was prepared in 0.15 mol / L of Tris-Hcl buffer
and a portion utilized for the estimation of lipid peroxidation20 and other portion of the same after
precipitating with TCA was used for the estimation of Glutathione21. The remaining homogenate
was centrifuged at 1500 rpm from the estimation of Superoxide dismutase, Catalase and Protein
Content and analyze LDH and ALP in blood serum22, 23.
Statistical analysis:
All the data were expressed in mean ± SEM. The significance of differences in mean
between control and treated animals for different parameters determined by using one way
ANOVA followed by Dunnet’s Comparison and Post-hoc test using Graph prism computer
package and percentage tumor inhibition can be calculated by Chi-Square Test.
7.4 Does the study require any investigations or interventions to be conducted on patients
or other humans/animals? If so describe briefly:
Yes, the above study requires to be carried out on mice. The effects of preparation on cancer will
be studied by considering physiological, pathological, biochemical parameters using animal
models.
7.5 Has ethical clearance been obtained from your institution for performing various tests
on animals?
Yes, the study is cleared from Institutional Animal’s Ethics Committee and the copy is enclosed
with the protocol.
8
8.
References:
1. Rang H.P., Dale M.M., Ritter J.M., Moore P.K. Pharmacology 5th ed , Elsevier
Publication, New Delhi, India 2003; 693.
2. Cameron D.A., Smyth J.F., Christopher Haslett., Edwin R. Chilvers., Nicholas A.Boon.,
Nicki Colledge., John A.A.Hunter. Davidson’s Principles and practice of Medicine, 19th
ed, Churchill Livingstone, Toronto 2008;214
3. Bertran G. Katzung., Edward Chu.,Alan C. Sartorelli. Basic and clinical Pharmacology
9th ed, Mc Graw Hill, Singapore 2004; 848.
4. Bruce A. Chabner., Philip C. Amrein., Brain J. Druker., M.drov Michael., Constantine S.
Mitsiades., Paul E. Gross., David P.Ryan., Sumant Ramachand., Paul G.Richardson.,
Jeffrey G Supko., Wyndham H. Wilson., Brunton Lazo Parker. Goodman and Gilman’s
The Pharmaco logical Basis of Therapeutics, 11th ed, Mc Graw-Hill, USA 2005; 1315.
5. Sharma H.L., Sharma K.K. Principles of Pharmacology, Paras Publishing, Hyderabad,
India; 2007: 862.
6. Robbins., Cotran. Pathologic basis of Disease by Kumar-Abbas-Fausto. 7th ed, Elsevier
Publ ication, New Delhi, India 2007; 284.
7. Bennet P.N., Brown M.J. Clinical Pharmacology, 9th ed, Churchill Livingstone, New
Delhi, India 2005; 603.
8. Kirsten M., Ducan., Gary Ogawa., Unamarie Clibon., Eric T. Herfindal., Dick R. Gourley.
Text Book of Therapeutics Drug and Disease management, 7th ed, Library of Congress
Cataloging-in-Publication Data, USA 2000; 2025.
9. De Smet P.A. Role of Plant-Derived drugs and Herbal Medicines in Health care, Drugs
1997; 54 (6):801-40.
9
10. The Wealth of India, Raw material, Publication and Information Directorate, CSIR, New
Delhi,India 1962; 6: 426
11. Prajapat., Kumar. Agro’s Dictionary of Medicinal Plants, Jodhpur, India 2003; 218
12. Mahajan S.G., Mehta A.A. Inhibitory action of Ethanolic extract seeds of Moringa
oleifera Lam on Systemic and Local Anaphylaxis, J Immunotoxicology 2007; 4:287-294.
13. Kutung G., Vasudevan D.M., Kuttan R. Effect of a preparation from Viscum album on
tumor development In vitro and In vivo in mice, J Ethnopharmacol 1990; 29: 35-41
14. Schmid W., Protective effect of Spirulina fusiformis on chemical-induced Genotoxicity in
mice. Mutat Res 1975; 31:9
15. Uma Devi P., Rao B.S.S., Soloman F.E., Effect of plumbagin on the radiation induced
Cyto genic and cell cycle changes in mouse Ehrlich Ascites carcinoma In vivo, Ind J Exp
Biol1998;36: 891-895
16. Echardt A.E., Malone B.N., Goldstein I. Cancer Res 1982; 42:2977
17. Umadevi P., Emerson Soloman F., Sharada A.C. Indian J Exp Biol 1994; 32: 523
18. Mazumdar U.K., Gupta M., Suryyendubkas M., Dilip M.
Antitumor
activity of
Hygrophylia Spinosa on Ehrlich Ascites carcinoma and sarcoma- 180 induced mice, Ind J
Biol1997; 35:473-477
19. Naigonkar A.V., Burande M.D. A manual of medical laboratory technology, 2nd ed. Nirali
Prakashan, New Delhi, India 1996; 284
20. Onkava H., Onishi N., Yagi K. Assay for lipid peroxidation in animal tissue by
Thiobarbituric Acid reaction, Anal Biochem 1979; 95:351-358
21. Ellman G.L. Tissue sulphydryl groups, Arch Biochem Biophys 1979; 82:70-77
10
22. Kakkar P., Dos B., Vishwanathan P.N. A modified spectrophotometric assay of
Superoxide Dismutase, Ind J Biochem Biophys 1984; 21:130-132.
23. Lowry Oh., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the
Folinphenol Reagent, Biol Chem 1951; 193:265-275.
11
9.
SIGNATURE OF CANDIDATE
(MASOOD.MK)
10.
REMARKS OF THE GUIDE
The present study has been proposed on the results of
the earlier study, the results obtained from the study
may validate and substantiates the traditional claim.
11.
NAME AND DESIGNATION OF
THE GUIDE
Mr. CHANDRASHEKHAR.V.M
Asst.Professor
12.
SIGNATURE
13.
CO-GUIDE
----------------
14.
SIGNATURE
----------------
15.
HEAD OF THE DEPARTMENT
16.
SIGNATURE
17.
REMARKS OF THE PRINCIPAL
The above mentioned information is correct and I
recommended the same for approval.
18.
NAME OF THE PRINCIPAL
Dr. I.S.MUCHANDI
H.O.D. , Department of Pharmacology
H.S.K.College of Pharmacy,
B.V.V.S. Campus, Bagalkot-587101.
19.
SIGNATURE
Dr. I.S.MUCHANDI
H.O.D., Department of Pharmacology
H.S.K.College of Pharmacy,
B.V.V.S. Campus, Bagalkot-587101.
12
OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)
HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,
BAGALKOT-587101, KARNATAKA
REG NO.821/01/a/CPCSEA, Dated: 6th AUG 2004 UNDER THE RULES 5(a) OF THE
“BREEDING OF AND EXPERIMENTS ON ANIMALS (Control and Supervision)
RULES 1998”
Ref: HSKCP/IAEC, Clear / 2008-09/1-8
CERTIFICATE
This is to certify that Mr.MASOOD.MK a student of FIRST M.Pharma is permitted
to carry out experiments on animals for the dissertation / thesis work entitled as
“Evaluation of Anticancer activity of Moringa oleifera Lam. in Balb/c
mice”, as per details mentioned and after observing the usual formalities laid down by
IAEC as per provision made by CPCSEA.
Animal House in charge
CHAIRMAN
13
Form B
See rule [6 (a) and 8(a)]
PART A
(1)
Name and address of the Establishment:
(2)
Date and Registration Number of the
Establishment:
H.S.K. College Of Pharmacy, B.V.V.S
Campus, Bagalkot.587101,Karnataka
821/01/a CPCSEA
(3)
Name, address and Registration NO. of the
Office OF CPCSEA,
breeder from whom acquired and the date of Ministry of Environment and Forest,
acquisition:
IIIrd Sea Ward road, Valmiki Nagar,
Thiruvanmiyur, Chennai-600041.
Tamil Nadu
(4)
Place where the animals are presently kept:
(5)
(6)
Animal House
H.S.K. College Of Pharmacy, B.V.V.S
Campus, Bagalkot.587101,Karnataka
PG Lab-II, Department of Pharmacology,
Place where the experiment is to be H.S.K. College Of Pharmacy, B.V.V.S
performed:
Campus, Bagalkot.587101,Karnataka
The date on which the experiment is to
commence and the duration of the
experiment:
15-May-2009
The protocol form for the research proposal - PART B in the case of experiments using other
than non-human primate animals for ongoing/new projects, PART C for use of non-human primates
for new projects and PART D for use of non-human primate for extension of ongoing projects –
should be duly filled, singed and annexed with this form.
Signature
Dated:
(Name and Designation)
Place:
14
PART – B
Protocol form for Research Proposal to be submitted to the
Committee on use of small animals / Animals other than non human
Primate in Biomedical Research for ONGOING / NEW PROJECTS
1.
Project Title :
2.
Investigation (s) :
Designation
Mr.CHANDRASHEKHAR.V.M
Asst.Professor
3.
Department (s) :
Department of Pharmacology, H.S.K. College Of
Pharmacy, BVVS Campus,
Bagalkot.587101,Karnataka
4.
Evaluation of Anticancer activity of
Moringa oleifera Lam. in Balb/c mice
(a) Funding Source (s): if any
----------
(b) Are sufficient funds available for
purchase and maintenance of the animals
----------
© Duration of present project :
(1) Number of months :
4 ½ -Months
(2) Date of start of the Project :
(Experiment)
15-May-2009
(3) Date of termination of the
project
:
5.
Date by which approval is needed in case
the project is to be funded by outside
agency (If less than six weeks from the date
of admission, please justify below):
30-September-2009
----------
15
6.
Summary of project briefly summarize in laymen’s term the background, the objective and the
experiment approach.
(a) Background:
Enclosed
(b) Objectives
Enclosed
(c) Experimental procedure:
Enclosed
7.
(a) Name of species
Balb/c Mice
Age
Sex
2-3 Weeks
Female
Weight
25-30 g
(b)
Rationale for selection
Approximate number of animals required during the
first 12 months.
220
Justification of number (define treatment group and
number per group)
220
Number of animals housed per weeks
8.
30 Animals
List all invasive Non Surgical Animal Procedures and Invasive non-surgical
Potentially Stressful Noninvasive procedures to be used procedure
(Example IM injection, foot pad injection , venapunctures).
Procedure and Approximate Frequency:
4 ½ -months
Enclosed
16
Anesthetic and/or Analgesic and Dosage:
No,
Test substance injected and/or applied:
Post orally Administered
9.
Does the protocol prohibit the use of Asnesthetic and analgesic for the conduct of painful
procedures?
NO,
With surgical procedure/Experimental procedure be performed?
10.
NO,
(a) Will the animal be sacrificed after surgery?
NO
11.
Will hazardous agent such as radioisotopes, carcinogens, radiation exposure, microbial and
parasitic agent be administered to animals?
Yes.
INVESTIGATOR SIGNATURE
DATE: ____________________
17