“EVALUATION OF THE ANTITUMOR ACTIVITY OF THE

ETHANOL EXTRACT OF BRASSICA RAPAL. ON EHRLICH ASCITE CARCINOMA IN MICE.”

By

Mr. VASOYA MANOJKUMAR R. B.Pharm

M. PharmacyDissertation Protocol Submitted to the

RajivGandhiUniversity of Health Sciences,

Bangalore– 560 041, Karnataka.

In partial fulfillment

of the requirement for the Degree of

MASTER OF PHARMACY

IN

PHARMACOLOGY

Under the Guidance of

Mr. JOHN WILKING EINSTEIN M.PHARM

ASSISTANT PROFESSOR

Dept. of Pharmacology and Toxicology

St. John’sPharmacyCollege

St. John’s Educational Institutions

Bangalore – 560040.

2009-10.

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. / Name of the candidate &
Address. / Mr. VASOYA MANOJKUMAR RATILAL
POST GRADUATE STUDENT,
DEPT OF PHARMACOLOGY AND TOXICOLOGY,
ST. JOHN’S PHARMACYCOLLEGE,
BANGALORE- 560040
2. / Name of the Institution. / St. John’sPharmacyCollege
No 6, 9th cross, 2nd main, Vijayanagar,
2nd stage (Hampinagar),
Bangalore- 560040
Tel: 91-80-23300958/23300668
Email:
3. / Course of the study
& subject. / Master Of Pharmacy - Pharmacology
4. / Date of admission. / 15-June-2008
5. / Title of the Topic :-
“Evaluation of the antitumor activity of the ethanol extract of Brassica rapa L. on
Ehrlich Ascite Carcinoma in mice.
6.
6.1
6.2
6.3 / Brief resume of intended work
Need of the work
Cancer can be defined very broadly as a disease in which there is uncontrolled multiplication of cells 1.
The most common cancers diagnosed globally are lung (1.35 million), breast (1.15 million) and colorectal (1 million) cancers.The most prevalent cancer in the world is breast cancer 2.
Treatment of cancer is difficult because cancer is not one disease but many disorder that share a profound growth deregulation 3.The chemotherapeutic agent must be able to selectively kill or inhibit growth of neoplastic cells leaving normal cells unharmed. However, most of the currently available drugs damage DNA or interfere with DNA synthesis thereby killing all rapidly dividing cells, both normal and cancerous 4.
The ancient Indian literature has prescribed various herbs and metals for their use in cancer. Various plant materials capable of decreasing the proliferation of tumor cell have been tested in experimental animal models and their effects were confirmed.
Since many medicinal herbs have been reported to possess anti tumor activity, very few such herbs have been in used in the herbal formulation. This has led an increasing demand of research on anti tumor natural product that produces minimal or no side effects.
The present work is aimed to evaluate antitumor activity of ethanol extract of Brassica rapa L. using mice.
Review of Literature
І. BOTANICAL INFORMATION 5
a. Botanical Name : Brassica rapa L.
b. Family : Brassicaceae
c. Vernacular Names :
English : Wild turnip
d. Botanical Characters :
Leaves : Radical, lyrate- pinnatifid, long and narrow
Root : Globular soft – fleshed tuber
Flowers : Bright yellow
Fruits : Divaricate ascending pedicels with slender beak
II.ETHNOMEDICAL INFORMATION5
The leaf is traditionally used for stomatic and turnip is used as diuretic, in hemorrhage, tumors , carcinoma ,fever and seed oil is used for fever, bronchitis ,rheumatism.
III.BIOLOGICAL ACTIVITY
Yang. et al. (2006) studied protective effect of the ethanol extract of the roots of Brassica rapa (EBR) on Cisplatin-Induced Nephrotoxicity in LLC-PK1 Cells and Rats. After conducting the study it was determine that Pretreatment of cells with EBR prevented cisplatin-induced decreases in cell viability and cellular GSH content 6 .
Un. et al. (2008) has screened the effect of the ethanol extract of the roots of Brassica rapa on glucose and lipid metabolism in C57BL/KsJ-db/db mice and he reported that in despite hyperinsulinemia, the glucokinase activity was lower in the liver of the db/db mice than the db/+ mice, while the glucose-6-phosphatase activity was higher 7.
IV. CHEMICAL REVIEW
  • The plant contains Isothiocyanate glycosides 8.
  • The leaf essential oil was found to contain 48 volatile components, representing 94.0-96.6% of the oil, which were characterized.
  • The main constituents were 3-butenylisothiocyanate (1.4-29.2%),
4-pentenyl isothiocyanate (8.2-23.5%),
2-methyl 5-hexenenitrile (1.3-16.8%),
2-phenylethyl isothiocyanate (7.0-13.7%) and
Phytol (6.1-23.5%).
  • The essential oil from the leaves of B. rapa was characterized by a high content of sulphur- and nitrogen-containing compounds 9.
Objective of the study
  1. Preparation ofEthanolic extract of Brassica rapa L.(EBR)
  1. To investigate preliminary phytochemical constituents of EBR
  1. Determination of LD50 of the EBR. as per OECD guideline.
  1. To establish the pharmacological profile of prepared extract for its anti- tumor activity in Swiss albino mice model.

7.
7.1
7.2 / Materials & Methods
Source of data
All experiments are planned to generate data from the loboratory studies i.e.; experiments will be performed as described in reference, experimental studies in journals and in textbooks available with college, IISc library, Bangalore, RGUHS digital library (Helinet) and various institutions.
Methods of collection of data
The data collected will be based on laboratory animal experimentation.
1)Chemical: All chemicals of analytical grade will be procured from Sigma chemical,
USA and S. D. Fine Chem. Ltd. , India.
2) Preparation of the extract:
Material:The fresh turnip of the plant were shade dried for 10 days and then powdered. The powder will be macerated in ethanol for 72 hours. The liquid extract obtained was concentrated in vacuum at 40°C.
Phytochemical screening: Preliminary phytochemical screening will be carried out as described by Khandelwal (2000) 10.
3) Animals: Healthy, adult swiss albino mice of either sex weighing (25-40 g), maintained under standard laboratory conditions, at temperature 25 ± 2°C and a 12 hr light-12 hr dark period will be employed for the experimentation. Food and water will be provided ad libitum.
4) Acute oral toxicity study 11: Acute toxicity study for the ethanolic extract of Brassica rapa L.
will be done according to the OECD guidelines No: 423 and low, medium and high dose will be
selected for treatment.
Method:-The overnight fasted mice will be divided into 04 groups, each group consisting of 3 female animals. The EBR will be given in various doses (5, 50,300,2000) by gastric incubation with a syringe. After administration of the extract, the animal will be observed continuously for the first 2 hours and at 24 hrs to detect changes in behavioral responses and also for tremors, convulsion, salivation, diarrhea, lethargy, sleep, and coma and also will be monitored up to 14 days for the toxic symptoms and mortality.
After 14 days of acute oral toxicity the survival mice will be rehabilitated and reused in anti tumor screening.
5) Antitumor screening (In vivo) :-
5.1Ascitic model -
  • Induction of experimental tumor:Ehrlich ascites carcinoma (EAC) cells will be obtained through the courtesy of AmalaCancerResearchCenter, Thrissur. These preinoculated(1x106 cells/ mice,i.p.) are maintained for 15 days12,13. After two week of inoculation,the peritoneal fluid will be aspirated and diluted to get 1x106 EAC cell / ml in a phosphate buffer. Mice will be reinoculated with 1x106 EAC cell / mice and divided into 5 groups consisting of 5 animals in each group.
Group 1 : Tumor control with solvent
Group 2 : Tumor mice treated with EBR (Low dose)
Group 3 : Tumor mice treated with EBR (Medium dose)
Group 4 : Tumor mice treated with EBR (High dose)
Group 5 : Tumor mice treated with cyclophosphamide (positive control)
Group 1-5 will be treated with solvent & drug samples 24 hrs after the inoculation of EAC for 9 days and the observation will continued until the termination of the study.
5.1.1 Determination of survival time 14.
At terminationsurviving animals of EAC tumour bearing mice will be counted
and the average survival time (MST) and the % increase in life span (% ILS) will be
calculated by following formula.
MST of treated group
Increase of life = 100
MST of control group
  • Body weight analysis: During the same time of study, weekly body weight analysis will be recorded.
5.1.2 Estimation of hematological parameters 15,16.
In order to detect the influence of EBR on the haemotological status of EAC bearing mice, comparison will be made amongst three groups of mice on the 14th day after inoculation. The three groups will be comparised (1) tumour bearing mice (2) tumour bearing mice treated with EBR( p.o. for first 9 days) and (3) control mice. Blood will be drawn from each mouse in the conventional way and the white blood cell count (WBC), red blood cell count (RBC), haemoglobin , protein and packed cellular volume will be determined.
5.2 Determination of solid tumor 17
Induction of experimental tumor:- Fibrosarcoma cells will be obtained through the courtesy of Department of radiobiology, Kasturba Medical college, Manipal and inoculated into Swiss albino mice by injecting 5x105 viable cell subcutaneously into the dorsal region .Fibrosarcoma cellinoculated mice are divided into 5 groups consisting of 5 animals in each group.
.
Group 1 : Tumor control (solvent)
Group 2 : Tumor mice treated with EBR (Low dose)
Group 3 : Tumor mice treated with EBR (Medium dose)
Group 4 : Tumor mice treated with EBR (High dose)
Group 5 : Tumor mice treated with cyclophosphamide
Palpable size will be reached on 6-8 days. Treatment will be
commenced when the tumour volume reached 505mm3 (7-9 days after inoculation)
The diameters D1, D2, D3 of each tumor in three perpendicular planes will be measured
thrice in a week using a vernier caliper. The tumor volume (V) will be calculated
using the formula :

V =  (D1, D2, D3)
6
The tumor response to the different treatments will be assessed
on the basis of tumor regression, growth delay and VDT. The tumour volume will
be measured on 10th day after the completion of drug treatment.
Volume doubling time :- Time required to double the tumor volume from 50 to 500 mm3 will be taken as criterion to assess the anti tumor efficacy of EBR in Fibrosarcoma bearing mice, It will be estimated according to the formula:
VDT = Log 2  (T1-T0) / Log V1 – LogV0
Where, V0 is the volume of the tumor at time T0 and V1 is the volume at the timeT1.
5.3 Assay of peritoneal cells 18.
Three groups of normal mice will be used for the study. One group will be treated with EBR and the second group will be received the same treatment for two consecutive days. The untreated third group will be used as control. Peritoneal exudates cells will be counted 24h after treatment for each of the treated groups and compared with those of the untreated groups.
5.4 In vitro short term cytotocixity 19:-
Short term cytotoxicity will be assessed by incubating 1106 EAC cells in 1 ml phosphate buffer saline with varying concentration of the EBR at 37° C for 3 hours. The viability of the cells will be determined by trypan blue exclusion method. The % viable cells will be calculated by the formula
Total cell counted – Total dead cell
% Viable cells =  100
Total cell counted
Statistical analysis:-
The data obtained will be analyzed using one way ANOVA (Graph pad prism version 5.00 software) followed by suitable post test.
Total No. of animals required:
No of the animal in each group = 05
No of groups = 10
Total no. of animals required = 05 × 10 = 50
Total duration for completion of whole project may be 9 months
1. Literature survey one & half months.
2. Duration of experimentation six months
3. Thesis writing one & half month
7.3
7.4 / Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly.
Yes, The study requires investigation on adult Swiss albino mice.
Has ethical clearance been obtained from your institution in case of 7.3?
Yes, Ethical clearance has been obtained from your institution.(Proposal
No: IJAHSM/IAEC/2008/010)
8. / LIST OF REFERENCES:
  1. Rang HP, Dale MM, Ritter JM and Moore PK. Pharmacology.5th ed. New Delhi: Elsevier-a division of reed elservier India private limited; 2006.
  2. Parkin DM, Bray F, Ferlay J, Pisani P. Global Cancer Statistics, 2002. CA Cancer Journal of Clinicians 2005; 55:74-108.
  3. Robbins SL , Cotran RS, Vijay K. Basic Pathology.8th ed.New Delhi: Elsevier-a division of reed elservier India private limited; 2007.
  4. Bhaswat SC.Cancer drug development key regulatory consideration.Health Administrator 2005;20(1&2) :29-36.
  5. Madhava V, Sivaji K and Tulasi K. Flowering plants of chittor district Andhra Pradesh. Student offset printer, Titupati 2008;2:22.
  6. Yang-HM, Yong WK, Yong JO, Nam IB, Sun AC, Hae GC et al. Protective Effect of the Ethanol Extract of the Roots of Brassica rapa on Cisplatin-Induced Nephrotoxicity in LLC-PK1 Cells and Rats.Biological & Pharmaceutical Bulletin 2006;29:12.
  7. Un JJ, Nam IB,Hae GC,Myun HB, Tae SJ, Kyung TL et al . Effects of the ethanol extract of the roots of Brassica rapa on glucose and lipid metabolism in C57BL/KsJ-db/db mice.Journals-Elsevier health 2008;27:158-167.
  8. Evans WC.Trease and Evans Pharmacognocy.4th ed.UK: WB Saunders Company Limited;2001.
  9. Miyazawa M, Nishiguchi T, Yamafuji C. Volatile components of the leaves of Brassica rapa L. var. perviridis Bailey. Flavour and Fragrance Journal2004;20(2):158-160.
  10. Khandelwal KR. Practical Pharmacognosy-techniques and experiments.Pune; Nirali Prakashan; 2000.
  11. The Organization of Economic Co-Operation and Development (OECD). The OECD Guideline for Testing of Chemicals: 423 Acute Oral Toxicity,OECD,Paris 2001 ;1-14.
  12. Gothoskar SV , Ranadive KJ . Anticancer screening of SAN-AB: An extract of marking nut semicarpus anacardium. Indian J Exp Biol 1971;9:372-75.
  13. Babu, Kuttan TD, Jose P. Cytotoxic and anti-tumour properties of certain taxa of umbelliferae with special reference to Centella asiatica (L.) Urban J. Ethno. Pharmacol 1995; 48: 53.
  14. Majumdar UK,Gupta M, Maiti S.Antitumour activity of Hygrophila spinosa on Ehrlich ascites carcinoma and sarcoma-180 induced mice. Indian J Exp Biol 1970; 35: 473-76.
  1. D’Amour FF, Blood FR, Belden DA.The Manual for Laboratory Work is Mammalian Physiology, Chicago, The University of Chicago Press1965: 148-150.
  2. Lowry OH, Rosenbrough NT, Farr AL .Protein measurement with Folin - Phenol reagent. J Biol Chem 1951;173:265-75.
  3. Uma D,Sharadh P,Solomon AC. In vivo growth inhibitory and radiosensitizing effects of Withaferin A on mouse Ehrlich ascites carcinoma. J Rad Res 2003;44:1-6.
  4. Sur P, Ganguly DK. Tea plant root extract an antineoplastic agent. Planta Med 1994;60:106-09.
  5. Sheeja KR, Kuttan G, Kuttan R. Cytotoxic and antitumour activity of Berberin. Amala Res Bull 1997;17:73-76.

9. / Signature of the candidate
10. / Remarks of the guide
11.
12. / Name & Designation of
11.1 Guide
11.2 Signature of Guide
11.3 Co – Guide
11.4 Signature of Co Guide
11.5 Head of the Department
11.6 Signature of HOD
12.1 Remarks of the Chairman & Principal
12.2 Signature / MR. JOHN WILKING EINSTEIN,M.PHARM
ASSISTANT PROFESSOR.
DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY.
St. JOHN’S PHARMACYCOLLEGE,
BANGALORE- 560040
MR.KUNTAL DAS, M.PHARM
HEAD.
DEPARTMENT OF PHARMACOGNOSY & PHYTOCHEMISTRY,
St. JOHN’S PHARMACYCOLLEGE,
BANGALORE- 560040
.
DR. E.P. KUMAR,M.PHARM, Ph.d.
PROFFESOR & PRINCIPAL,
HEAD.
DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY.
St. JOHN’S PHARMACYCOLLEGE,
BANGALORE- 560040

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