Anticancer evaluation ofCalotropis gigantea Linn. leavesin Dalton’s ascitic lymphoma induced Swiss albino mice.
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore– 560 041
By
Mr. Tailor Ashishkumar ThakorbhaiB. Pharm.
Under the Guidance of
Dr. Kalyani DivakarM.Pharm., Ph.D.
Professor Head of
Department of Pharmacology
2011-2013
Acharya & B.M.Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post),
Hesaraghatta Main Road, Bangalore – 560 090.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTFOR DISSERTATION
1. / Name of the candidate & Address / Mr.Tailor Ashishkumar Thakorbhai.S/o Mr. Tailor Thakorbhai Durlabhbhai.
#8th, Mit-har Soc,Near shamshanbhumi,
Amalsad post,T- Gandevi, D-Navsari,
St-Gujarat-396310.
Email Id:
2. / Name of the Institution / Acharya & B.M. Reddy College Of Pharmacy
Soldevanahalli, Hesaraghatta main Road,
Chikkabanavara Post,Bangalore-560090.
Phone No: 080 28896011
Fax No: 080 28393541
3. / Course of the study& subject / M.Pharm (Pharmacology)
4. / Date of admission / 14/12/2011
5. / Title of the Topic / Anticancer evaluation of Calotropis gigantea Linn. leaves in Dalton’s ascitic lymphoma induced Swiss albino mice.
6. / Brief resume of intended work
6.1Introduction and need of the work
6.2 Review of Literature
6.3 Aim and Objective of the study / Enclosure I
Enclosure II
Enclosure III
7. / Materials & Methods
7.1Source of data
7.2Methods of collection of data
7.3Does the study require investigation on animals?
a. If yes give details
7.4Has ethical clearance been obtained from your institution in case of 7.3 / Enclosure IV
Enclosure V
Yes,The above study requires investigation on Swiss albino mice (76 in number) for in vivo studies.
Enclosed
8. / List of references / Enclosure VI
9. / Signature of the candidate
10. / Remarksof the guide / RECOMMENDED
11. / 11.1 Name & Designation ofGuide
11.2 Signature of Guide / Dr. Kalyani Divakar M.Pharm., Ph.D.
Professor & Head, Dept. of Pharmacology,
Acharya & B.M.Reddy College of Pharmacy.
11.3 Name &Designation of Co-guide
11.4 Signature of Co-guide / Mr. Uday Raj Sharma M.Pharm.
Assistant professor
Acharya & B.M. Reddy College of Pharmacy.
11.5 Name & Designation of HOD
11.6 Signature of HOD / Dr. Kalyani DivakarM.Pharm., Ph.D.
Professor & Head, Dept. of Pharmacology,
Acharya & B.M. Reddy College of Pharmacy.
12. / 12.1 Remarks of the Principal
12.2 Name & Signature of Principal / Dr.Divakar GoliM.Pharm., Ph.D.
Principal,
Acharya & B.M. Reddy College of Pharmacy.
Enclosure-I
6.BRIEF RESUME OF INTENDED WORK
6.1 Introduction and need of work:
Cancer is known medically as a malignant neoplasm andis abroad group of various diseases, in this all involve unregulated cell growth. In cancer cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. Not all tumors are cancerous. Benign tumors do not grow uncontrollably, do not invade neighboring tissues, and do not spread throughout the body. There are over 200 different known cancers that afflict humans1,2.
Determining what causes cancer is complex. Many things are known to increase the risk of cancer, including tobacco use, certain infections, radiation, lack of physical activity, obesity, and environmental pollutants.These can directly damage genes or combine with existing genetic faults within cells to cause the disease.Approximately five to ten percent of cancers are entirely hereditary2.
Cancer can be detected in a number of ways, including the presence of certain signs and symptoms, screening tests, or medical imaging. Once a possible cancer is detected it is diagnosed by microscopic examination of a tissue sample. Cancer is usually treated with chemotherapy, radiation therapy and surgery. The chances of surviving the disease vary greatly by the type and location of the cancer and the extent of disease at the start of treatment. While cancer can affect people of all ages, and a few types of cancer are more common in children, the risk of developing cancer generally increases with age. In 2007, cancer caused about 13% of all human deaths worldwide (7.9 million). Rates are rising as more people live to an old age and as mass lifestyle changes occur in the developing world.
Cancer can be reduced and controlled by implementing evidence-based strategies for cancer prevention, early detection of cancer and management of patients with cancer. Several attempts have been made to improve existing treatments to increase the survival of patient by testing number of combination therapy.And hence, there is need for new therapeutic agents that are less toxic to normal cells and which produce an enhanced anticancer effect3.
The herbal medicines occupy distinct position right from the primitive period to present day. Theethnobotanical pharmacology is as old as man himself. These medicines have less side effects and man can gather herbs easily from nature. India being a tropical country is blessed with vast natural resources and ancientknowledge for its judicious utilization. However, in order to make these remedies acceptable to modern medicine,there is a need to scientifically evaluate them, to identify the active principles and to understand the mechanismof action.
Calotropis proceraLinn. in India holds a pride of place largely because of its otheruses and economic values. The genus Calotropis (Asclepiadaceous) is distributed in tropical andsubtropical regions of Asia and Africa. It is represented in India by two species viz. Calotropis procera and Calotropis gigantea.
Calotropis gigantea Linn.(Asclepiadaceae) is a perennial undershrub found chiefly in most parts of the world in dry, sandy and alkaline soils and warm climate andis more common in south western and central India and western Himalayas. It is found in waste lands and grows as a weed in agricultural lands.
Traditionally various part of the plant Calotropis gigantea Linn.is used as analgesic, antipyretic, anti-inflammatory, anti-diabetic, anti-proliferating, asthma, stomachic4, 5.
On literature survey from all the various sources, it revealed that Calotropis gigantea Linn. is having wide therapeutic application and is a potent cytotoxic agent. However there is no scientific evidence regarding the in vivocytotoxic effect of Calotropis gigantea Linn.leavesin treatment of cancer.
Hence, the present researchis planned to studyanticancer activity of leaves extract of Calotropis gigantea Linn. in Dalton’s ascitic lymphoma induced Swiss albino mice by taking 5-Fluorouracil(20mg/kg i.p) as standard drug.
Enclosure-II
6.2 Review of Literature:
Calotropis gigantea Linn.belongs to the family of Asclepiadeceae.
It isa wild growing plant and it’s common names are akra, akanal, madar also known as Giant milkweed(English).
The plant Calotropis giganteaLinn.(Asclepiadeceae), commonly known as Aak is used in many ayurvedic formulations like Arkelavana. The medicinal potential of Calotropis giganteaLinn. has been known to traditional system of medicine for a while now with its leaves being widely used. The use of plants, plant extracts, and pure compounds isolated from natural sources has always provided a foundation for modern pharmaceutical compounds.Calotropis gigantea Linn. is a large, bushy shrub with decussate, obovate, coriaceous, auriculate, leaves with acute, subsessile apices extra axillary, umbellate, panicale inflorecence with purple corolla and erect lobes and leaves to be subsessile, 6-15 cm by 4.5-8 cm, broadly ovate, ovate-oblong, elliptical, or obovate, acute, pubescent when young and glabrous on both sides on maturity.
The leaves of Calotropis giganteaLinn. are said to be valuable as an antidote for snake bite, sinus fistula, rheumatism, mumps, burn injuries, and body pain. The leaves of Calotropisare also used to treat jaundice6.
Calotropis species received special attention because of distinct relevant activities found to be present in its latex, leaves and other parts and in India it is widely used as a folk medicine as a rich source of biologically active compounds capable of promoting diverse benefits such as control for dermal fungal infection and pain relief among other useful properties.
It is reported that plant extract of Calotropis giganteaLinn. Showed very strong cytotoxic effect against KB cell line in vitro and isolated cardenolide glycosides, calotropin, frugoside and 4’-O-beta-D-glucopyranosyl frugoside were also found to be toxic to cell lines of human origin.
It also have some bioactive compounds such as giganticine, isorhamnetin-3-O-rutinoside, isorhamnetine-3-O-glucopyranoside, taraxasteryl acetate, calotroposides(A and B) etc. which may contribute to show potent anticancer activity on different cell line.
Recently studies on the two new cardenolides,19-Nor and 18, 20-epoxy-cardenolides isolated from Calotropis gigantea Linn. leaves found to have inhibitory effect against KB, BC and NCI-H187 cancer cell lines7,8.
Reported pharmacological activities ofCalotropis giganteaLinn.:
Harikesh D in 2010concluded that ethanolic root extract of Calotropis giganteaLinn. exhibits potent cytotoxic activity and this might be utilized for the development of novel anticancer drug4.
Recently in 2011 Subramanian S and his colleagues reported that the alcoholic and n-Hexane extract of leaves and latex of Calotropis gigantea Linn. contain Triterpenoids and Lupeol which are immense bioactive compounds and have useful medicinal properties like anti-angiogenic, anti-oxidative, anti-inflammatoryand It inhibits early response of tumor growth induced by benzoyl peroxide, plays important role in normalization of lipid profile8.
Bulani Vsuggestedthat Calotropis gigantea Linn.proved to be potential therapeutic drug for treatment of asthma9.
Usman MRM and colleagues reported that different phytoconstituents like glycosides, sterols, carbohydrates, flavonoids, and terpenoides may be responsible for anti-inflammatory and antipyretic activity of Calotropis gigantea Linn10.
Sayeed MSB and co-researchers reported that the administration of crude extract of Calotropis gigantea Linn.showed significant cytotoxic activity by Brine Shrimp Lethality Bioassay11.
Habib RM in Nov 2009 concluded that highest dose of flower extract of Calotropis gigantea Linn.was most potent and comparable with standard drug bleomycin in mice which show that the various extract of plant parts of Calotropis gigantea Linn. can be used for the treatment of cancer12.
Chitme HR and coauthorsproved anti-diarrheal activity of Calotropis gigantea R.BR in experimental animals by significant reduction in fecal output13.
Rathod NRreportedthat the level of SOD and CAT were significantly improved after administration of Calotropis gigantea extract in STZ-induced diabetic Rats14.
Jayakumar D and coauthorsrevealed that Calotropis gigantea shows the highest zone of inhibition of Escherichia coli, salmonella typhi and shigella sonnei than Vinca rosea and also showed good antioxidant activity15.
Lim YY et al., Concluded that the leaf extracts of Calotropis gigantea and Vallaris glabra showed great promise as potential candidates for anticancer drug as they inhibited the growth of all various cell line16.
Jaliwala YA in 2011 revealed that the aqueous extract of Calotropis gigantea has shown significant antitussive and anti asthmatic activity at appropriate dose and the ethanolic extract showed significant as expectorant activity17.
SutthivaiyakitS and coauthorsreported cytotoxic study of cardenolides from the leaves of Calotropis gigantea by performing cytotoxic assay on different cell lines18.
Mathen C and co-workersCalotropis giganteacytotoxic study revealed that DNA ploidy analysis on indicated that both extracts induced dose dependence apoptosis19.
Srivastava SR et al., showed that ethanolic extract of the roots of Calotropis gigantea Linn. exhibited 100 % pregnancy interceptive activity in rats when administered as a single oral dose on day 1 postcoitum20.
Usmani S and co-researchers states that significant reduction in SGOT, ALP and bilirubin which indicates the potent hepatoprotective activity of extract of Calotropis gigantea Linn21.
Enclosure-III
6.3 Objectives of the study:
- To collect extract of dry powderedleaves of Calotropis giganteaLinn. bysuccessive Soxhlet extraction method.
- To carry out qualitative analysis of the extract for phytoconstituents.
- To carry out acute toxicity studies of extractfollowing OECD guide line 425.
- To investigatethe influence of leaves extract of Calotropis gigantea Linn.in Dalton’s ascitic lymphoma cancer induced Swiss albino mice on following parameters:
a)Observation of mean survival time12.
b)Observation of weight variation12.
c)Haematological studies include Total leukocyte, RBC and Platelet count12.
d)Glutathione estimation22.
e)Nitric oxide estimation23.
f)Super Oxide Dismutase24.
g)Lactate Dehyrogenase estimation25.
h)Measuring the volume of peritoneal fluid and volume of packed cell26.
Enclosure-IV
7. Materials and Methods
7.1 Source of Data:
The data will be obtained from experiments which involve:-
1)Laboratory based studies.
2)Literature survey, abstracts.
i)International Journals of Drug Development and Research.
ii)Ethnobotanical Leaflets.
iii)International Journal of Ayurveda Research.
iv)International Journal of Pharmaceutical Scienceand Research.
v)Journal of Natural Product Plant Resource.
vi)Asian journal of Pharmaceutical and Clinical Research.
vii)Pakistan Journal of Biological Science.
3)Text books.
4)Internet sources
i)
ii)RGUHS internet site ‘Helinet’
iii)
iv)
Enclosure-V
7.2 Method of Collection of Data
7.2.1Drugs and chemicals:
- 5-Fluorouracil will be brought from reliable source.
- All the chemicals usedin this research are of analytical grade.
- Collection, authentication and extract preparation:
Fresh and mature leaves of Calotropis gigantea Linn.will becollected and authenticated and collected leaves will be shade dried.The dried leaves will be powdered and stored in airtight container and powder will be subjected for successive extraction using Soxhlet extraction method.
7.2.3 Animals:
Swiss albino mice weighing 25-30g will be selected for in vivo studies. The animals will be housed in standard environmental condition and provided with food and waterad libitum.
The experiment will be carried out according to guidelines of CPCSEA, New Delhi, India and approved by the Institutional Animal Ethical Committee of Acharya and B.M Reddy College of Pharmacy.
Methodology:
- Powder leaves of Calotropis gigantea Linn. will be extracted by using Soxhlet extractor.
- Preliminary Phytochemical analysis will be carriedout on the leaves extract of Calotropis gigantea Linn.
- Dose will be established by oral acute toxicity studies by following OECD guideline 425.
- The dried extract will be used for evaluation of the anticancer activity.
Cancer induction22:
The Dalton’s ascitic lymphoma cells will be used for induction of cancer in Swiss albino mice. Dalton’s ascitic lymphoma cells will be obtained courtesyof Amala cancer Research centre, Thrissur, Kerala, India. The cells will be maintained in vivo in Swiss albino mice by intra-peritoneal transplantation which will be aspirated from peritoneal cavity of the mice using saline. The cell count will be done and further dilutions will be made, so that total cells will be 1 X 106 cells/ml/mouse. This volume will be given intraperitoneally and tumour will be allowed to grow in the mice for a minimum of seven days before starting the study.
Drugs treatment schedule to cancer induced mice:
Dalton’s ascitic lymphomacells induced mice will be randomly divided into 12groups of 6 mice each, after confirmation of cancer induction. After 8th and 16thday of treatment, spleen, and peritoneal cells will be taken out for biochemical investigations. The survival patterns of the hosts willbe determined and deaths, if any, in different groups, will be recorded daily. The anticancer efficacy of leaves extract of Calotropis giganteaLinn. will be reported based on change in various parameters and percent increase in life span (ILS) and will be compared with standard drug 5- Fluorouracil (20mg/kg i.p).
Table 1: TREATMENT PROTOCOL:
GROUP / TREATMENT / NUMBER OF ANIMALSI / Normal control / 6
II / Cancer control / 8th day
Sacrification / 6
III / 16th day
Sacrification / 6
IV / MEAN SURVIVAL / 6
V / Standard drug 5- Fluorouracil
(20mg/kg i.p) treated group / 8th day
Sacrification / 6
VI / 16th day
Sacrification / 6
VII / MEAN SURVIVAL / 6
VIII / Extract of Leaves(dose 1) / 8th day
Sacrification / 6
IX / 16th day
Sacrification / 6
X / MEAN SURVIVAL / 6
XI / Extract of Leaves (dose2) / 8th day
Sacrification / 6
XII / 16th day
Sacrification / 6
XIII / MEAN SURVIVAL / 6
Parameters:
a) Observation of mean survival time12.
b) Observation of weight variation12.
c) Hematological studies include total leukocytes count, platelet count and RBC count12.
d) Glutathione estimation22.
e) Nitric oxide estimation23.
f)Super oxide dismutase estimation24.
g) Lactate dehydrogenase using commercial kit25.
h) Measuring of peritoneal fluid and packed volume cell26.
a) Observation of mean survival time12:
Mean survival time will be observed in cancer control and drugs administered groups of animals.
b) Observation of weigh variation12:
Weight variation will be observed in cancer control and drug administered groups of animals.
c)Haematological studies12:
It includes total leukocytes count, platelet count and RBC count.
d)Glutathione estimation22:
Spleen tissue, peritoneal cells homogenate will be prepared in 0.02 M ethylenediamine tetra acetic acid (EDTA). Aliquots of 0.5 ml of the tissue homogenates will be mixed in testtubes with 1-5 ml of 0.2 M Tris buffer, pH 8.2 and 0.1 ml of 0.01 mM(5,51- Dithio bis- 2-nitro benzoic acid) DTNB. The mixture will be brought to 10.0 ml with 7.9 ml of 0.5% of dodecyl sulphate. The reaction mixture will be centrifuged at approximately 3000 rpm at room temperature for 15 minutes. The absorbance of the supernatants will be read using spectrophotometer at 412 nm in 1 cm quartz cells.
e)Nitric oxide estimation23:
The spleen tissue and peritoneal fluid will be used for nitric oxide estimation. Tissue homogenate will be prepared in 0.05 M phosphate buffer and the tubes will centrifuged at 1500 rpm for 20 min and 40 ml supernatant will be used for nitric oxide (NO) estimation.
Nitric oxide(NO) production will be estimated by Griess reaction which will be expressed in the form of nitriteaccumulation. In brief, 100 µl of tissue extract will be mixed with100 µl of Griess reagent [equal volumes of 1% (w/v)sulphanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v)napthylethylenediamine hydrochloride (NEDH)] and will be incubatedat room temperature for 10 min, and then the absorbance at 550nm will be measured in a UV-double beam spectrophotometer. The amount of nitrite in the sample (µMunit) will be calculated from a sodium nitrite standard curve. Theresults will be expressed as µM of nitrite/mg of protein.
f)Estimation of Superoxide Dismutase24:
The SOD level will be estimated by the method described by Kakkaret al.The assay mixture containing 0.1 ml of heart tissue homogenate (10% w/v), 1.2 ml of sodium pyrophosphate buffer (pH8.3, 0.052 M), 0.1 ml of phenazine methosulphate (186 µM), 0.3 ml of 300 µM nitro blue tetrazolium and 0.2 ml of NADH (750 µM). Reaction starts by the addition of NADH. After incubation at 300C for 90 s, the reaction will be stopped by the addition of 0.1 ml of glacial acetic acid. The reaction mixture is stirred vigorously with 4.0 ml of n-butanol. The mixture is allowed to stand for 10 min, centrifuged and the butanol layer is separated. The colour intensity of the chromogen in the butanol is measured spectrophotometrically at 560 nm and the concentration of SOD will be expressed as units permg of protein.