REGISTRATION OF SUBJECT FOR DISSERTATION
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BENGALURU
A PROTOCOL FOR THE PROJECT WORK ENTITLED
“PRE CLINICAL EVALUATION OF ANTICANCER ACTIVITY OF CITRULLUS COLOCYNTHIS FRUIT EXTRACTS”
By,
JEEVAN J HEGDE
M Pharm, Part I
Department of Pharmacology
Shree Devi College of Pharmacy,
Mangalore – 574142
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / NAME OF THE CANDIDATE AND ADDRESS / JEEVAN J HEGDESITHARA HOUSE, 41,SHIROOR HARIKANDIGE POST (VIA) PERDOOR-576124
UDUPI DISTRICT,
KARNATAKA
2. / NAME OF THE INSTITUTION / SHREE DEVI COLLEGE OF PHARMACY
AIRPORT ROAD, KENJAR,
MANGALORE – 574142
3. / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOLOGY
4. / DATE OF ADMISSION OF COURSE / DECEMBER 2012
5. / TITLE OF THE TOPIC
“PRE CLINICAL EVALUATION OF ANTICANCER ACTIVITY OF CITRULLUS COLOCYNTHIS FRUIT EXTRACTS ”
6.
6.1 / BRIEF RESUME OF THE INTENDED WORK
NEED FOR THE STUDY:
The term neoplasm or tumour means new growth 1 .
Cancer is an abnormal type of tissue growth in which the cells exhibit an uncontrolled division leading to a progressive increase in the number of dividing cells.
Lymphoma is a disease of the lymphocytes (a type of white blood cell involved in immune responses) and the lymphatic system, which includes the spleen, thymus, and liver, as well as other lymphatic tissues. Dalton’s Ascites Lymphoma is transplantable, poorly differentiated malignant tumour which appeared originally as lymphocytes in a mouse. It grows in both solid and ascetic form2.
Cancer is responsible for one in eight deaths worldwide, with more than twelve million new cases diagnosed yearly. A large percentage of patients die after developing cancer despite aggressive treatment, indicating a need for new approaches to cancer therapy 3. It becomes the second major cause of death in the human after cardiovascular disease. About 7.6 million people died due to cancer in the world during 2007. Hence there is an urgent and present need for developing new approaches and drugs to prevent as well as cure this devastating disease 4.
Projections are that by the year 2030, the incidence of cancer will increase by threefold and that there will be disproportionate rise in cancer cases and deaths 5.
Chemotherapy, surgery and drugs, which are currently used, are standard methods for treatment of cancer, although not been fully effective and safe. Some progress has been made in cancer diagnosis and treatment, but still the cancer rate is increasing and low survival rate of patient have still been reported. The development of new therapeutic approach remains one of the most challenging thing in cancer research. Many synthetic and chemotherapeutic agents such as paclitaxel, methotrexate , carmustin, fluorouracil, mercaptopurine, mephalan etc are having low bioavailability & adverse effects like alopecia, skin eruptions, reduced immunity, hepatotoxicity etc. There are over 50 chemotherapy drugs that are commonly used with various side effects. Side effects may occur just after treatment (days or weeks) or they may occur later (months or years) after the chemotherapy has been given. Possible side effects includes decrease in blood cell counts , nausea and vomiting, mouth ulcers, skin rashes, photosensitivity, dizziness, headache, drowsiness, kidney damage (with a high-dose therapy) liver damage, hair loss (reversible), seizures 6 .
Increasing recurrence of mammalian tumors and severe side-effects of chemotherapeutic agents reduce the clinical efficacy of a large variety of anticancer agents that are currently being used. Thus, there is always a constant need to develop alternative anticancer drugs with minimal side-effects. One
important strategy to develop effective anticancer agents is to study anticancer agents derived from natural sources. Anticancer agents derived from plants and their derivatives have been proven to be effective for cancer prevention and therapeutics7.
The in vivo anticancer activity of Citrullus colocynthis against Dalton Ascites Lymphoma has not been reported. Hence the present study is aimed to investigate the anticancer activity of the fruit extract of Citrullus colocynthis against Dalton Ascites Lymphoma induced tumour model.
6.2 / REVIEW OF LITERATURE
Cancer cell lines have been extensively used in various cancer researches, mainly Dalton’s Ascites Lymphoma 8, Ehrlich ascites carcinoma 9.
A thorough and complete literature search on Citrullus colocynthis was performed from the chemical abstracts, national and international journals, E-library, internet and other research materials.
Citrullus colocynthis [Family: Cucurbitaceae] commonly found wild in the sandy lands of North West, the Punjab, Sind, and Central and southern India. Commonly Citrullus colocynthis known as - Indravaruni (Sanskrit), Chitrapala or Bitter apple. Fruit is bitter, pungent and used as purgative, anthelmintic, antipyretic, carminative, cures tumour, leucoderma, ulcers, asthma, etc 10.
General pharmacological studies revealed antimicrobial , anti diabetic , anti inflammatory local anesthetic11 ,antioxidant12.
The phytoconstituents present in this plant are Cucurbitane type triterpene glycoside viz Colocynthoside A & B Flavonoid glycoside quercetin Fatty acid like Stearic, Myristic, Palmitic, Oleic, Linoleic, Linolenic acid Vitamin B1 , B2 and Niacin Mineral like Ca, Mg, K, Mn, Fe, P and Zn10,13.
Cucurbitacins and their derivatives are triterpenoids, found in medicinal plants, known for their diverse pharmacological and biological activities, including anticancer effects, throughout human history. Although initial attention to Cucurbitacin as a potential anticancer drug withered for decades, recent discoveries showing that Cucurbitacin is a strong STAT3 (Signal Transducers and Activators of Transcription-3) inhibitor have reclaimed the attention of the drug industry one more time. There is increasing evidence showing that some Cucurbitacins not only inhibit the JAK-STAT pathway, but also affect other signalling pathways, such as the MAPK pathway, which are also known to be important for cancer cell proliferation and survival 14. Also the plants having terpinoids and flavonoids are screened for anticancer activity. The in vitro anticancer activity of Citrullus colocynthis been reported15,16, 17,18. Thus this study has been undertaken to evaluate the anticancer activity of Citrullus colocynthis, fruit extract using animal model.
6.3 / OBJECTIVE OF THE STUDY:
1. Extraction- ethanolic extraction of fruits of Citrullus colocynthis 19.
2. To arrive at the therapeutic dose range after Acute toxicity study.
3. To carry out in-vivo anticancer effect of fruit extract of Citrullus colocynthis against Dalton Ascites lymphoma cells, Brine shrimp lethality (BSL) bioassay and in vitro cytotoxic study using (Trypan blue dye exclusion method).
7.
7.1
7.2 / MATERIALS AND METHODS:
SOURCE OF DATA:
The source of data for this study is based on the following. Swiss albino mice of either sex will be used during the course of the evaluation studies. Experiment will be performed as described in the standard bibliography, literatures, text books. The reputed journals and publications are obtained from college library and through web search. The model selected for the study include Dalton Ascites Lymphoma induced tumor model.
METHOD OF COLLECTION OF DATA:
MATERIALS:
Dalton ascites lymphoma will be procured from Amala cancer research center, Thrissur, Kerela, India. All other drugs & chemicals are of pure analytical grade will be obtained from local suppliers.
GROUPING:
Number of animals in each group will be 6
SL NO / GROUP NUMBER / GROUP CODE / DESCRIPTION
1
2
3
4
5 / GROUP 1
GROUP 2
GROUP 3
GROUP 4
GROUP 5 / N
D
STD
LDCC
HDCC / Normal
Diseased (positive control)
Treatment with standard drug 5 fluro uracil.
Treatment with low dose of “Citrullus colocynthis”.
Treatment with high dose of
“Citrullus colocynthis”.
.
DOSE SELECTION STUDY:
Therapeutic dose of “Citrullus colocynthis” will be fixed on the basis of data obtaining from oral acute toxicity studies .This study will be performed according to the guidelines of the Organization for Economic Co-operation and Development(OECD). Guideline No – 423 20.
DESCRIPTION OF MODEL:
DALTON ASCITES LYMPHOMA INDUCED TUMOR MODEL 8, 21, 22, 23, 24
PROCEDURE:
Swiss albino mice will be divided into 5 groups of six animals each. The animals belonging to groups 2–5 will be injected with DLA cells (1×106 cells/ml/mouse) intraperitoneally, while the remaining group 1 serves as the normal control group. This will be taken as day 0. On the 1st day normal saline (0.9%w/v NaCl, 5mL/kg/mouse/day) administered into normal group (Group 1) and diseased control group (Group 2). The different doses of ethanolic extract of citrullus colocynthis will be administered in groups 3 and 4 respectively for 14 days intra-peritoneally and the standard drug 5 flurouracil will be administered in group 5 for 14 days. On 15th day, the animals will be sacrificed accordingly from each group for the study of antitumor activity. The following parameters will be evaluated accordingly. The evaluating parameters will be
Haematological parameters (estimated using blood)- Hemoglobin (Hb) content, Red Blood cell count (RBC), White Blood Cell count (WBC) differential leucocyte count (DLC) and Platelets.
Tumor progression will be assessed on the basis of Mean Survival Time and Percent increase in life span20, 21.
Biochemical parameters (estimated using serum)- The effects of EECC on serum enzyme and lipid profiles including Total cholesterol, Triglycerides (TG), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline Phosphatase ( ALP) are evaluated.
The antitumor activity will be evaluated using following parameters- Tumor volume, Body weight, Cancer cell count, Percentage increase in life span, Estimation of viable tumor cell count.
7.3 / Brine shrimp lethality (BSL) bioassay25
The assay is carried out as a preliminary test for the investigation of cytotoxicity of the selected extracts/fractions. The chamber will be divided into two equal parts. Aeration is given in both the compartments. One part will be illuminated with a bulb (60 W), while the other is darkened. Brine shrimp eggs will be sprinkled in the dark side and incubated at room temperature for 48 hrs. As the hatching occurs, the nauplii will swim towards the illuminated side, where they are collected by suitable pipette. Samples of the extracts are prepared by dissolving 5 mg of extract in 5 ml of DMSO to get 500 ppm stock solution and further diluted with sea water to get the required concentrations (10, 50, 100 and 150 ppm). 5 fluro uracil is used as standard. Dried vials are taken and ten shrimps are transferred in each vial and then volume will be made upto 5 ml with sea water. A drop of dry yeast suspension (3 mg in 5 ml sea water) is added to each vial as food for shrimps. For each concentration test was done in triplicate. Control vials will be prepared by adding equal volumes of distilled water. The vials are maintained under illumination. After 24 hours, survivors, percentage deaths and accordingly other parameters will be counted 25.
In vitro cytotoxic study (Trypan blue dye exclusion method)25
Trypan blue dye exclusion method is used to assess the in vitro cytotoxicity of extracts and fractions in DAL cells. Ascitic fluid withdrawn from the peritoneum of DAL inoculated mouse was washed with phosphate buffer saline. Stock cell suspension was adjusted to(1×106 cells) by phosphate buffer saline using hemocytometer.The cells will be incubated with desired test drug concentration in a final volume of 1 ml for 3 h at 37 ◦C. 5 fluro uracil is used as positive control. After incubation, 0.1 ml of trypan blue will be added and mixed well in the incubation mixture. The total number of dead and living cells are counted using haemocytometer and the percentage viability/cytotoxicity is calculated.
DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.
Yes. The investigation will be done as mentioned in 7.2.
7.4 / HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION?
Ethical committee approval letter has been enclosed.
8. / REFERENCE:
1) Harsh M. Neoplasia. Jaypee. Text book of pathology. 6th ed.India:2010.p.192-235.
2) Hemamalini K, Soujanya GL, Anurag B, Uma V. In vivo Anticancer activity of Tabebuia Rosea (Betrol) DC .leaves on Daltons ascetic lymphoma in mice. Int J Pharm Sci Res 2012;3(11): 4496-502.
3) Erin MG, Wen HL. From bench to bedside: The growing use of translational research in cancer medicine. Am J Transl Res 2010;2(1): 1–18.
4) Sobhan BK, Sivakumar P, Karthiyayini T, Rajkumar M, Senthil Kumar KL. In-Vitro Anti Cancer Activity of Ethanolic extract of the leaves of Achyranthes bidentata (Blume). Res J Pharm Biol Chem 2012; 3(2): 519-23.
5) Chandrakanth A, Madhuri A, Hemanth R, Vijayakumar M, Lois C, Hugh S ,et al. A survey of the educational environment for oncologists as perceived by surgical oncology professionals in India. World J Surg Oncol 2012: 1-4.
6) Richard AH, Pamela CC, Richard F, Luigi XC, Michelle AC, editors. Pharmacology 4th edition. Anticancer drugs. Lippincott Williams & Wilkins; 2009. P. 457-88.
7) Ali R, Mirza Z, Ashraf GM, Kamal MA, Ansari SA, Damanhouri GA, et al. New anticancer agents: recent developments in tumor therapy. Anticancer Res 2012 Jul; 32(7): 2999-3005.
8) Manokaran K , Rajasekaran N, Paramasivam R, Ganesan R, Duraisamy G, Dominic S, ,et al. In vivo and in vitro antitumor activity of jasminum sambac (Linn) Oleaceae flower against Dalton ascites lymphoma induced swiss albino mice .Int J Pharm Sci 2012;4(1): 144-47.
9) Prerona S, Mazumder UK, Haldar PK, Sagar N, Sriparna K, Asis B,et al. Anticancer activity of methanol extract of Cucurbita maxima against Ehrlich as-cites carcinoma. Int J Res Pharm Sci 2011; 2(1): 52-59.
10) Borhade P, Deshmukh T, Patil V and Khandelwal K. Review on Citrullus colocynthis. Int J Phar chem 2013; 3(1): 46-53.
11) Gurudeeban S, Satyavani K, Ramanathan T .Bitter Apple(cittrus colocynthis): An over view of Chemical Composition and Biomedical Potential. Asian J of Plant Sci 2010; 9(7): 394-401.
12) Sudhanshu, Nidhi R, Sandhya M, Ekta M. Screening of antioxidant potential of Citrullus Colocynthis methanolic extract. J Chem Pharm Res 2012; 4(5):2507-11.
13) Sultan A, Farman UK, Iqbal H, Murad AK, Ihsan UK. Evaluation of Chemical analysis profile of citrullus colocynthis Growing in Southern Areas of Khyber Pukhtunkhwa, Pakistan. World Applied Sci J 2010; 10(4) :402-5.
14) Dhong HL, Gabriela BI, Nils HT. Cucurbitacin: Ancient Compound Shedding New Light on Cancer Treatment. The Scientific World J 2010; 10: 413–18.
15) Tingyan L, Meixia Z, Hongliang Z, Chunyan S, Xiaolin Y, Yihui D, et al. Combined antitumor activity of cucurbitacin B and docetaxel in laryngeal cancer. Eur J Pharmacol 2008;587:78-84.
16) Nizam B , Sajid N, Abdul MK, Zargoona W, Yaseer MSA. In vitro Antileishmanial, antitumor,Cytotoxic activity and phytocemical analysis of citrullus colocynthis fruit exraction and its fraction. Int j Med Arom Plants 2013; 3(1): 78-84.
17) Weikai C, Amanda L, Dong Y, Muriel M, Vernon JL, Phillip KH. Cucurbitacin B inhibits growth ,arrests the cell cycle, and potentiates antiproliferative efficacy of cisplatin in cutaneous squamous cell carcinoma cell lines. Int J Oncol 2010; 37: 737-43.
18) Suwit D, Sumana D, Weena J, Apichart S, Duncan RS, Pimpicha P et al. Antiproliferative Effects of Cucurbitacin B in Breast Cancer Cells: Down-Regulation of the c-Myc/hTERT/Telomerase Pathway and Obstruction of the Cell Cycle. Int J Mol Sci 2010; 11 : 5323-38.
19) Ahmad SA, Ahamed A, Ibrahim M, Mustafa A, lrahim AH, Ahamed El-O ,et al. Cytotoxicity, Mutagenicity and Antimicrobial Activity of Forty Jordanian Medicinal Plants. Informa Healthcare. 1990;28(2):139-44.
20) Available from: http://Iccvam.nieh.nih.gov/suppDocs/OECD/OECD_GL423. [Last accessed on 2013 May 21].
21) Jaslin EJ, Padmaja V. Antitumor activity of Coleus spicatus Benth. Against Dalton’s Ascitic Lymphoma. Int J Pharm Tech Res 2013;5(1):189-92.
22) Rajesh R, Chitra K, Padmaa MP, Chidambaranathan N. Anticancer activity of aerial parts of Aerva lanata Linn Juss ex Schult against Dalton’s Ascitic Lymphoma. Eur.J.Integr.Med 2011;3:e245-e250.
23) Lakshmi KS, Shrinivas SS, Rajesh T, Chitra V. Antitumour activity of ethanolic extract of leaves of Holoptelea integrifolia on Dalton's ascitic lymphoma in Swiss albino mice. Int J Green Pharm 2010;4(1):44-47.
24) Chitra V, Shrinivas S, Nandu K. Evaluation of Anticancer Activity of Vitex negundo in
Experimental Animals: An In Vitro & In Vivo Study. Int J Pharm Tech Res 2009;1(4): 1485-89.
25) Isha D, Nitesh K, Manjula SN, Vipan P ,Manjunath MS, KSR Pai,et al. Preliminary evaluation of in vitro cytotoxicity and in vivo antitumor activity of Premna herbacea Roxb. in Ehrlic ascites carcinoma model and Dalton’s lymphoma ascites model. EXP TOXICOL PATHOL 2013; 65: 235– 42.
9 / SIGNATURE OF THE CANDIDATE
10 / REMARKS 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. The project is viable and is recommended for clearance and approval.
11 / NAME AND DESIGNATION OF 11.1 GUIDE / Mr. Abhilash D
Assistant professor
Dept. of pharmacology
Shree Devi college of Pharmacy,
Mangalore-574142
11.2 SIGNATURE
11.3 HEAD OF THE DEPARTMENT / Dr. J.V KAMATH
Professor
Department of pharmacology
Shree Devi College of Pharmacy,
Mangalore, Karnataka.
11.4 SIGNATURE
12 / 12.1 REMARKS OF PRINCIPAL
The project work has potential implication in the field of pharmacology. The above mentioned information is correct and I recommend the same for approval.
DR.J.V KAMATH
Principal
Shree Devi College of Pharmacy.
Mangalore, Karnataka.
12.2 SIGNATURE
7