EVALUATION OF ANTI-CANCER ACTIVITY OF THE EXTRACT OF
APHANAMIXISPOLYSTACHYA(WALL.) PARKER LEAVES
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE
BY
GARGI MOITRA B.PHARM
Under The Guidance of
MR. KARUNAKAR HEGDE M.PHARM
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY
MANGALORE – 574143
2010 – 2012
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.0 / NAME AND ADDRESS OF THE CANDIDATE / GARGI MOITRASURYAVIHAR COLONY JUNVANI ROAD, BHILAI, CHATTISGARH, 490020
2.0 / NAME OF THE INSTITUTION / SRINIVAS COLLEGE OF PHARMACY
VALACHIL, MANGALORE- 574143
3.0 / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOLOGY
4.0 / DATE OF ADMISSION TO COURSE / 29/10/2010
5.0 / TITLE OF THE TOPIC / “EVALUATION OF ANTI-CANCER ACTIVITY OF THE EXTRACT OF APHANAMIXISPOLYSTACHYA(WALL.) PARKER LEAVES”
6.0 / BRIEF RESUME OF THE INTENDED WORK :
6.1 NEED FOR THE STUDY:
Cancer is the uncontrolled growth and quick division of the abnormal cells in the body. These cells invade and destroy the surrounding tissues. Inspite of all advances in medical sciences, cancer a disease as old as mankind is globally a major health problem1. Recent reports from the International Agency for Cancer Research indicates that, approximately 12.7 million new cancer cases and 7.6 million cancer death occurred and, also that of these 56% of all new cancer cases and 63% of the cancer deaths were in the less developed regions of the world. Projections are that by the year 2020, the incidence of cancer will increase by threefold, and that there will be a disproportionate rise in cancer cases and deaths from the developing countries that have resources to tackle the problem2.
In the treatment of cancer many synthetic and chemotherapeutic agents have been developed, that show various side effects like alopecia, skin eruptions, reduced immunity, secondary carcinogenesis, etc. Hence, to overcome these flaws and to make the course of treatment more convenient herbal drugs have been developed as they are not known for severe side effects. There are good enough plants considered as medicinally useful by the local tribes for the cancer treatment.
One such plant, Aphanamixispolystachya(WALL.) Parker belongs to the family Meliaceae, locally known as Tiktaraj, a large tree grows wild and planted in forests and roadsides all over the country. The plant is extensively used in traditional system of medicine for various ailments like spleenomegaly, liver complaints, tumors, ulcers, haemorrhoids, burning sensations, ophthalmia, nervousness and rheumatism3.
The plant is reported to possess hepatoprotective4, insecticidal, antibacterial, antifungal and immunosuppressive activities5, 6. Further the stem bark and seeds extract were reported to possess anticancer activity7, 8. The leaves of the plant are also used by tribal healers of Western Ghats region of India to ameliorate cancer. However, no such scientific data are available in published form to validate the folklore claim.
Keeping the above information in view, the present study has been designed to evaluate the anticancer activity of the methanolic extract of the leaves of the plant Aphanamixis polystachya.
REVIEW OF LITERATURE:
6.2.1 Aphanamixispolystachya(WALL.) Parker belongs to the family Meliaceae3. Botanical name:Antidesma acidum Retz
Genus:Aphanamixis,
Species:A. polystachya,
Kingdom:Plantae,Order: Sapindales.
Synonymns : Amoora rohituka W& A, A. rohituka.
Common Names: Rohitaka, Harinharra, Tiktaraj
Vernacular Names :
Gujarati: Ragat Rohido
Marathi: Raktharohida
Bengali:Tiktaraj
Kannada:Mullumuttage
Tamil:Malampuluvan, Sem, Semmaram
Distribution:
It is native to tropical areas generally from Asia and Oceania: China, Bhutan, India, Sri Lanka, Indochina, Burma, Thailand, Indonesia, Malaysia,Papua New Guineaand Philippines. Within India, it has been recorded in the Sub-Himalayan tract, from Gonda (Uttar Pradesh) to West Bengal, Sikkim, along the north eastern states, Peninsular India and Andaman & Nicobar Islands3.
Description:
Large canopy tree (up to 25 m high, rarely to 35 m); bole cylindrical or markedly fluted (slightly up to 100 cm diam.); often crooked or straight;barkbrownish red or pale brown, rough, scaly or flaky. Leaves are large imparipinate, 30-75 cm long; leaflets opposite 4-8 pairs and elliptic oblong or oblong lanceolate, acuminate, glabrous on both surfaces. Male flowers numerous, erect, 4 mm long subglobular, in solitary axillary panicles more than half as long as the leaves3.
Active constituents and medicinal uses:
· Agnihotri 9, (1987) isolated aphanamixol, diterpene alcohol polifesterol and beta-sitosterol from the petroleum ether extract of the leaves A. polystachya.
· Md. Mokarram et al. 10, (2009) investigated that the leaves extract of the A. polystachya possesses strong central nervous system depressant and analgesic activity in mice.
· Jagetia and Venkatesha11 (2005), reported that the ethanolic extract of A. polystachya protected albino mice transplanted with ehrlich ascites carcinoma and exposed to various doses of gamma-radiation.
· Srivastava et al.6, (2003), reported that the extract Amoora rohituka (stem bark) exhibited significant in vitro antibacterial and mild antifungal effect.
· Prasad8 (1999), reported that Prieurianin, a limonoid isolated from the seeds of Amoora rohituka, exhibited anticancer activity in human beings and in mice.
· Gole et. al.12, (1997), reported that the alcoholic extract of Amoora rohituka exhibited hepatoprotective activity against carbon tetrachloride induced hepatic injury in rats.
· Rabi and Gupta13 (1995), reported the in vivo antitumor activity of amooranin (a triterpene acid from Amoora rohituka) against N-nitrosomethyl urea- induced mammary adenocarcinoma in Sprague-Dawley rats.
· Rabi7 (1996), also reported that an ethyl acetate extract of the stem bark of A. rohituka exhibited antitumor activity on mice inoculated with Dalton’s lymphoma ascites cells.
6.2 OBJECTIVES OF THE STUDY:
The main objective of the present study are as follows:
1) Authentication and collection of the plant Aphanamixis polystachya (Wall.) Parker leaves.
2) To prepare the methanolic extract of the leaves of A. polystachya.
3) Preliminary phytochemical investigation of the methanolic extract.
4) To study the effect of methanolic extract for following activities:
i) In vitro antioxidant activity by Diphenyl Picryl Hydrazyl (DPPH) and Super oxide radical scavenging assay.
ii) In vitro anticancer activity by Microculture Tetrazolium (MTT) assay.
iii) In vivo anticancer activity against Ehrlich Ascites Carcinoma (EAC) cell lines by liquid tumor model.
7.0 / MATERIALS AND METHODS:
7.1 SOURCE OF DATA:
Experiment will be performed as described in the standard bibliography, literatures and text books. The reputed journals and publications are obtained from college library and through web search.
7.2 COLLECTION OF MATERIAL:
7.2.1 Collection of plant material and extraction:
The leaves of A. polystachya will be collect from the local region of Mangalore district and authenticated by Taxonomist. The herbarium of the plant will be kept in our institution. The dried leaves will be coarsely powdered and subjected to soxhlet extraction with a mixture of methanol: water (7:3 v/v) at 60°C. The solvent will be completely evaporated using rotary flash evaporator and obtained dried extract will be used for experimental investigation.
7.2.2 Animals
Wistar albino rats (150-200 g) and Swiss albino mice (20-30 g) of either sex will be used for the experiment. They will be maintained under standard conditions (temperature 22 ± 2oC, relative humidity 50±5% and 12 h light/dark cycle) and have free access to standard pellet diet and water ad libitum. All experimental protocols will be reviewed and approved by the Institutional Animal Ethical Committee (IAEC) prior to the initiation of the experiment and the care of the laboratory animals will be taken as per the CPCSEA regulations.
7.3 EXPERIMENTAL METHODS :
7.3.1 In vitro Antioxidant activity
1. DPPH radical scavenging assay14
To 1 ml of various concentrations of extract, 1 ml solution of DPPH (0.1 mM) will be added. An equal amount of methanol and DPPH served as control. After 20 min of incubation in the dark, absorbance will be recorded at 517 nm and the experiment will be performed in triplicate14.
2. Superoxide radical scavenging assay15
The potassium superoxide and dry DMSO will be allowed to stand in contact for 24 h and the filtrate (200 µl) will be added to 2.8 ml of an aqueous solution containing NBT (56 µM), EDTA (10 µM) and potassium phosphate buffer (10 mM). the methanolic extract (1 ml) at various concentrations will be added and the absorbance will be record at 560 nm against a control in which pure DMSO will be added instead of alkaline DMSO15.
7.3.2 In vitro Anticancer activity
Determination of mitochondrial synthesis by micro culture tetrazolium (MTT) assay16
Cell lines used:, HeLa (Human Cervical cancer cell line).
Standard drugs: Cisplatin (1.25-15 μg/ml).
Method:
The monolayer cell culture will be trypsinized and the cell count will be adjusted to 1.0x105 cells/ml using medium containing 10% new born calf serum. To each well 0.1 ml of the diluted cell suspension (approximately 10,000 cells) will be added. After 24 hours, the supernatant will be flicked off and 100 µl of test extract will be added to the cells. The plates will be then incubated at 370C for 3 days, and microscopic examination will be carried out and observations recorded every 24 hours. After 72 hours, the drug solutions in the wells will be discarded and 50µl of MTT will be added to each well. The plates shall be gently shaken and incubated for 3 hours at 37oC. The supernatant has to be removed and 50 µl of 1- propanol will be added and the plates will be gently shaken to solubilize the formed formazan. The absorbance will be measured at a wavelength of 540nm16. The percentage growth inhibition will be calculated.
7.3.3 In Vivo Anticancer activity against EAC cell lines by liquid tumor model
1. Acute toxicity study17
Acute toxicity study of the methanolic extract will be performed using Wistar albino rats according to OECD guidelines No. 42517. The animals will be fasted overnight and the extract will be administered orally with a starting dose of 2000 mg/kg, to different groups of animals. Animals will be observed continuously for first 3 h and monitored for 14 days for any mortality and general behavior of animals, signs of discomfort and nervous manifestations.
2. Ehrlich ascites carcinoma (EAC) induced tumor model11
Standard drugs: Cisplatin (1.25-15 μg/ml).
Development of EAC:
The ascetic tumor bearing mice (donor) will be taken 12 days after tumor transplantation. The ascetic fluid will be drawn using an 18 gauge needle into a sterile syringe. A small amount of tumor fluid will be tested for microbial contamination. Tumor viability will be determined by tryphan blue exclusion test and cells will be counted using haemocytometer. The ascetic fluid will be suitably diluted with saline to get a concentration of 10 million cells/ml of tumor cell suspension. 250 µl of this fluid will be injected in each mouse by i.p. route to obtain ascetic tumor. The drug treatment will be started 24 hr after the tumor inoculation daily for 7 days. The mice will be weighed every day till 15th day11. Cisplatin will be given intraperitoneally on only 1st day as standard. Tumor response will be assessed on the basis of mean survival time (MST) and % increase in life span (%ILS).
7.4 STATISTICAL ANALYSIS:
The data will be expressed as Mean value + SEM and will be analyzed by the one-way ANOVA.
7.5 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.
Yes. Study requires investigation on albino rats and mice.
7.6 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR
INSTITUTION?
Yes. Ethical clearance has been obtained. (Copy enclosed)
8.0 / REFERENCES
1) Arora R. Herbal Drugs: A Cancer Chemopreventive and Therapeutic Perspective. Jatpee Brothers Medical Publishers (P) Ltd., New Delhi, India, 2010.
2) Are C, Colburn L, Rajaram S, Vijayakumar M. Disparities in cancer care between the United States of America and India and opportunities for surgeons to lead. J Surg Oncol. 2010;102:100-5.
3) Kirtikar KR and Basu BD. Indian medicinal plants. Vol. 1, Bishen Singh, Mahendra Pal Singh, Dehradhun; 2004; pp 551-3.
4) Rao KN, Srinivasan KK, Udupa AL, Krishanand BR. Anti-hepatotoxic activity of Aphanamixis polystachya. Fitoterapia. 1993;64(6):507-9.
5) Chowdhury R, Hasan CM, Rashid MA. Antimicrobial activity of Toona ciliata and Amoora rohituka. Fitoterapia. 2003;74(1-2):155-8.
6) Srivastava SK, Srivastava SD, Srivastava S . New biologically active limonoids and flavonoids from Aphanamixis polystachya. Indian J Chem. 2003;42B(12): 3155-8.
7) Rabi T. Antitumor activity of amooranin from Amoora rohituka stem bark. Current Science. 1996;70(1):80-1.
8) Prasad GC. Critical care of cases with ayurvedic drugs in the management of cancer. South-East Asian seminar on Herbs and Herbal medicines, Patna, 1999; pp 16-19.
9) Agnihotri VK. Poriferasterol-3-rhamnoside, A new saponins from the stem bark of Amoora rohituka. Indian J Pharm Sci.1987;49(4):149-50.
10) Md. Mokarram Hossain, Israt Jahan Biva, Rumana Jahangir, Md. Mynol Islam Vhuiyan. Central nervous system depressant and analgesic activity of Aphanamixis polystachya (Wall) parker leaf extract in mice. African J Pharm and Pharmacol. 2009;3(5):282-6.
11) Jagetia GC, Venkatesha VAK. Enhancement of radiation effect by Aphanamixis polystachya in mice transplanted with Ehrlich ascites carcinoma. Biol Pharma Bull. 2005;28(1):69-77.
12) Gole MK, Dasgupta S, Sur RK, Ghosal J. Hepatoprotective effect of Amoora rohituka. Int J Pharmacog. 1997;35(5):318-22.
13) Rabi T, Gupta RC. Antitumor and cytotoxic investigations of Amoora rohituka. Int J Pharmacog. 1995;33(4):359-61.
14) Sreejayan N, Rao MNA. Free radical scavenging activity of curcuminoids, Drug Res. 1996;46:169-71.
15) Henry LEA, Halliwell B, Hall DO. The superoxide dismutase activity of various photosynthetic organisms measured by a new and a rapid assay technique. FEBS Lett, 1976;66:303-6.
16) Tim Mosmann. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J Immunol Methods. 1983;65(1-2):55-63
17) OECD, Guidelines for testing of chemicals, Acute oral toxicity, Environmental Health and Safety Monograph Series on Testing and Adjustment No. 425, 2001, 1.
9. / SIGNATURE OF THE
CANDIDATE
10. / REMARKS OF THE GUIDE / Recommended and Forwarded
11. / NAME AND DESIGNATION