“Evaluation of Anticancer activity of leaves of Basella rubra
Linn. and its influence on anticancer effect of
Cyclophosphamide”
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore– 560 041
By
Ms.Tanu shekhawat B.Pharm
Under the Guidance of
Dr. Divakar Goli M.Pharm. Ph.D
Professor
Department of Pharmacology
Acharya & B.M.Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road, Bangalore – 560 090
2008-09
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.
Name of the candidate &
Address.
Ms. TANU SHEKHAWAT
#120/A,10th main road,2nd cross,
Shankar nagar,
Bangalore-560096
Phone No:09742376279
2.
Name of the Institution.
Acharya & B.M. Reddy College Of Pharmacy
Soldevanahalli, Hesaraghatta Road,
Chikkabanavara Post,
Bangalore-560090.
Phone No: 080 65650815
Fax No: 080 28393541
3.
Course of the study
& subject.
M.Pharm (Pharmacology)
4.
Date of admission.
16th June-2008
5.
Title of the Topic
“Evaluation of Anticancer activity of leaves of Basella rubra
Linn. And its influence on anticancer effect of
cyclophosphamide ”
6.
Brief resume of intended work
6.1 Introduction and need of the work
Enclosure I
6.2 Review of Literature
Enclosure II
6.3 Aim and Objective of the study
Enclosure III
7.
Materials & Methods
7.1 Source of data
Enclosure IV
7.2 Methods of collection of data
Enclosure V
7.3 Does the study require
investigation on animals?
If yes give details
Enclosure VI
7.4 Has ethical clearance been
obtained from your institution in case
of 7.3
Yes (Copy Enclosed)
8.
List of references ( About 1 – 6)
Enclosure VII
9.
Signature of the candidate
10.
Remarks of the guide
11.
Name & Designation of
11.1 Guide
Dr. Divakar Goli M.Pharm. Ph.D
Professor
Department of Pharmacology,
Acharya & B.M.Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road , Bangalore – 560 090
11.2 Signature of Guide
11.5 Head of the Department
Dr.Kalayani Divakar M.Pharm. PhD
Department of Pharmacology,
Acharya & B.M.Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road Bangalore– 560 090
11.6 Signature of HOD
11.7 Principal
Dr. Divakar Goli M.Pharm. PhD
Professor
Department of Pharmacology,
Acharya & B.M.Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road , Bangalore – 560 090
11.8 Remarks and Signature of
Principal
ENCLOSURE - I
6. BRIEF RESUME OF INTENDED WORK
6.1 Introduction and need of work:
Cancer is a general term used frequently to indicate any of the various types of
malignant neoplasms (i.e. abnormal tissue that grows by cellular proliferation more
rapidly than normal), most of which invade surrounding tissue and may metastasize to
several sites1. Cancer arises as a result of genetic and epigenetic changes- inactivation of
tumor suppressor genes and the activation of oncogenes2.
Cancer may arise via multiple pathways that may proceed in parallel at different
rates in many cells. The foundation has been set in the discovery of mutations that
produce oncogenes with dominant gain of function and tumor suppressor genes with
recessive loss of function; both classes of cancer genes have been identified through their
alteration in cancer cells and by their elicitation of cancer phenotypes in experimental
models.
Today, modern medicine is able to treat or control a majority of the cancers.
However, a complete cure of cancer may not be possible in a significant percentage of
those affected as the side effects of therapy, and recurrence being major problems3.
Therefore the current investigations should be directed to find out alternative
drugs which are highly effective at nontoxic doses, inexpensive and accessible to the
common man. This can be achieved by screening newer synthetic molecule or from plant
products.
Cyclophosphamide is an alkylating agent and a derivative of nitrogen mustard.
These compounds produce highly reactive carbonium ion intermediates which transfer
alkyl groups to cellular macromolecules by forming covalent bonds. The position 7 of
guanine residues in DNA is especially susceptible, but other molecular sites are also
involved. This results in cross linking/ abnormal base pairing/ scission of DNA strand.
Cross linking of nucleic acids with proteins can also take place.
Cyclophosphamide is inactive as such; produces few acute effects and is not
locally damaging. Cyclophosphamide is converted by P450 mixed function oxidase
enzyme in the liver to active metabolites (aldophosphamide, phosphoramide mustard)
and a wide range of antitumor action is exerted.4
It is used to treat cancer of the ovaries, breast, blood and lymph system, nerves
(found primarily in children), retinoblastoma (a cancer of the eye found primarily in
children), multiple myeloma (cancer in the bone marrow), and mycosis fungoides
(tumors on the skin). Cyclophosphamide is also used for treatment of some kinds of
kidney diseases. Cyclophosphamide interferes with the growth of cancer cells, which are
eventually destroyed, since the growth of normal body cells may also be affected by
Cyclophosphamide5.
Many Ayurvedic herbal drugs have been listed with possible antineoplastic
activity.6 Sadafuli (Catharanthus roseus) – vinblastin, vincristin; Talispatra (Taxus
bravifolia) – Taxol; are the few examples of plant based anticancer drugs. Basella rubra
L. is one of the edible herbs among that list, on which very less research work i.e.
antiulcer, antifungal, target for HIV and splenocyte proliferation activities have been
reported but no report about its anticancer activity is available.7 Hence, the present work
is proposed to evaluate the anticancer activity of Basella rubra Linn. as well as its
influence on anticancer effect of Cyclophosphamide.
ENCLOSURE – II
6.2 REVIEW OF LITERATURE:
Plant:
Name of the plant selected for the present studies is “Basella rubra Linn”
belonging to family Basellaceae.
Synonyms:8
Basella rubra L. is locally called by different names in different languages:
Bengali- poi
Hindi- poi, poy, poi shak
Kannada- Basale
Marathi- Poi, Basala
Tamil- Shivappu-vasla-kire
Telugu- Batsal
Description of plant:
The perennial succulent glabrous twinning herb with white or red branches;
leaves simple upto 5 inch by 3 inch, alternate, broadly ovate, acute or acuminate, cordate
at base, thick lamina narrowed into petiole; flowers white or red in spikes, bracteoles
longer than perianth; fruits red, white or black, globose, utricle enclosed in the perianth.
Distribution:
The plant is widely distributed in India, Ceylon and Asia and is grown as a potherb. The red leaved and green leaved varieties are equally common in Bengal, Assam
and South India, while the green leaved ones are found more often in Uttar Pradesh and
adjacent states and Punjab. The plants can be raised either from seeds or from roots or
stem cuttings, and a spacing of 3″ is given between them. They are often made to grow
on shades and will be ready for picking within three months (80-90 days).
Chemical Constituents:
The plant contains calcium 2.32, potassium 5.8, magnesium 0.06, sodium 5.11,
iron 0.04mg/100gm.8 The leaves of the plant contain flavonoids (133.1±26.2 mg QC /100
g FM), β- cyanin and 7, 4΄_ di– ortho methyl kempferol. The flower contain phenolic
compounds (269.0±3.1mg GAE/100 g FM) such as Rutin, Quercetin, Scopoletin,
Coumarin, β-xanthin and β-cyanin pigments and Caffeic-, Homo-protocatechuic-,
Chlorogenic-, trans- and cis-p-coumaric-, p-hydroxy-benzoic-, phloretic-, trans- and cissinapic-, cinnamic- acids; and the fruit consists of β-cyanin, gomphrenin І, gomphrenin
II, and gomphrenin III.9
Therapeutic uses:8
The pounded or ground leaves, on account of the presence of mucilage are used as
poultice. The juice of leaves is prescribed in cases of constipation, particularly in children
and pregnant women.
Basella rubra L. is an edible herb. It is useful as one of the vegetable herbs of
domestic utility. The vegetable is considerably medicated and has potential for certain
ailments as remedy as well as wholesome diet or vegetable suitable to some particular
ailing conditions.
Basella rubra L. with curd is given for pacifying necrosis. Leaves act as laxative
in piles and is used during bleeding hemorrhoids. The oil of Basella rubra L. is
prescribed for local application in cracks of feet. A poultice prepared with leaves is
applied on tumor. The leaves are used to cover the boils and tumor which are also
anointed with the juice of Basella rubra L.
Therapeutic uses with scientific support:
1. Aqueous extract of the leaves of Basella rubra L. was prepared and antiulcer
activity was studied on ethanol and pylorus ligated-induced gastric ulcers in rats.
Leaf aqueous extracts (10 and 20 mg/kg p.o.) showed significant and dosedependent antiulcer activity against ethanol and pylorus ligated-induced ulcer in
rats.10
2. Antiviral activity performed on new single-chain ribosome-inactivating proteins
(RIPs) isolated from the seeds of Basella rubra L. The results indicated that
ribosome-inactivating proteins (RIPs) have antiviral activity, and inhibited
infection of Nicotiana benthamiana by Artichoke Mottled Crinkle Virus
(AMCV).11
3. Two novel antifungal peptides, designated alpha- and beta-basrubrins,
respectively, were isolated from seeds of the Ceylon spinach Basella rubra L., at
a concentration of 40 μM. Both alpha- and beta-basrubrins exerted potent
antifungal activity toward Botrytis cinerea, Mycosphaerella arachidicola, and
Fusarium oxysporum.12
4. The seed oil of Basella rubra L. is a valuable source of both essential as well as
commercially important fatty acids. This necessitates that efforts be made to
process the seeds of Basella rubra L. for human consumption as well as for use in
soap making industry and for its medicinal value.13
ENCLOSURE – III
6.3 AIM AND OBJECTIVE OF THE STUDY
AIM:
The present study is intended to evaluate the anticancer activity of Basella rubra
Linn. and also its influence on anticancer effect of Cyclophosphamide.
OBJECTIVES:
1) To Collect and prepare aqueous extracts of leaves of Basella rubra Linn.
2) To perform preliminary phytochemical analysis of aqueous extract of leaves of
Basella rubra Linn.
3) To study the anticancer activity of aqueous extract of leaves of Basella rubra Linn.
and also its influence on anticancer effect of Cyclophosphamide by using following
methods:
a) MTT assay
b) Clonogenic Assay
c) Cell viability test.
d) DNA fragmentation test.
e) Ames Test.
f) Glutathione estimation
ENCLOSURE-IV
7. MATERIALS AND METHODS
7.1 SOURCE OF DATA:
Data will be obtained from laboratory based studies by finding outA) Phytochemical analysis of aqueous extract of leaves of Basella rubra Linn.
B) Evaluations of the anticancer effect of aqueous extract of leaves of Basella rubra
Linn. and its influence on anticancer effect of Cyclophosphamide by:
a. The estimation of Glutathione using Experimental animals (Swiss albino
mice)
b. MTT assay, Clonogenic assay, and cell Viability test on HL-60 Cell lines.
c. Mutagenicity of Salmonella typhimurium by Ames assay.
d. DNA fragmentation test.
C) National & International Journals
D) Text books.
E) Internet.
ENCLOSURE-V
METHOD OF COLLECTION OF DATA
Drug: Cyclophosphamide is obtained from pharmacy store.
Plant: The leaves of Basella rubra Linn. collected, dried and then coarsely powdered &
stored in an air tight container.
Animals: Swiss albino mice weighing 22-25 g will be selected for glutathione
estimation. The animals will be housed in standard environmental condition and provided
with food and water ad libitum.
Solvent: Distilled water will be used for the extraction of leaves.
Methodology: Preparation of Extraction: - Powdered seeds of Basella rubra L. will be
extracted by using distilled water for 18 h with Soxhlet extractor. The dried extract will
be used for determination of the anticancer activity.
Dose of Cyclophosphamide: 200mg/kg body weight of Swiss albino mice.17
Pharmacological studies: Evaluation of the anticancer activity of aqueous extract of
leaves of Basella rubra Linn. and its influence on anticancer effect of Cyclophosphamide
will be done through the following methods and each experiment will be performed in
triplicate.:
MTT Assay:-14
The 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyltrtrazlium bromide (MTT) assay
is a common method used to assess cell proliferation and cytotoxicity. Briefly, 1x104
exponentially growing cells will be exposed to various concentrations of drug and
incubated and MTT will be added and incubated at 37oC for 4 h. The precipitated
formazan salt will be dissolved in DMSO and the samples will be read at 570 nm. The
50% inhibitory concentration (IC50) of drug will be calculated.
DNA fragmentation analysis:- 14
HL-60 cells will be incubated with 0, 5, 10, 15, 20 and 25 μg/ml of drug for 24 h
at 37oC. The DNA will be isolated and precipitated with ethanol, air-dried and dissolved
in TE buffer (5 mM Tris-Hcl (pH 8.0) and 20mM edetic acid). DNA fragmentation will be
analyzed by agarose gel electrophoresis.
Cell viability:-14
The effect of drug on the HL-60 Cancer cell will be determined by means of
tryphan blue exclusion technique. Briefly, 3x104 cells will be seeded per well in 24 wells
and then treated with various concentrations of drug. The wells will be incubated at 37°C
and the number of cultured cells in the different wells will be counted using a
hemocytometer after staining with 0.4% tryphan blue every 24 h to calculate the doubling
time.
Clonogenic Assay:- 15
Tumor colony forming units will be cultured in Dulbecco's modified Eagle's
medium (Gibco) supplemented with 0.3% agar and 20% fetal calf serum. The cultures
will be incubated at 37°C in a fully humidified atmosphere containing 10% CO2 in air.
Colonies (greater than 40 cells) will be scored after 10-20 days using a dissecting
microscope at 45×. A linear relation of the number of cells plated and colonies could be
established.
Ames assay:- 16
The salmonella point mutation assay proposed by Maron and Ames was followed
with little amendments as suggested by Bala and Grover to verify the inhibitory activity
of the extract. Constant concentration of two direct acting mutagens, NPD (20 µg/0.1
ml/plate) and sodium azide (2.5 g/0.1 ml/plate) and S9 dependent mutagen, 2AF (20 µg
/0.1 ml/plate) will be used as positive control. 0.1 ml each of bacterial culture (about 1-2
X108 cells/ml), mutagen and extract were added to 2 ml of top agar. Equal quantity of the
mutagen and the extract will be blended and allowed to stand for 30 min at 37°C under
incessant shaking and 0.2 ml of this was added to 2 ml of soft agar with 0.1 ml of fresh
bacterial culture. Soft agar poured on minimal glucose agar plate at 37°C for 48 h.
concurrently, a positive control (where mutagen but no extract was added) and a negative
control (where no mutagen was added) will also be set. All the test sample and mutagen,
i.e. NPD and 2AF, will be dissolved in dimethyl sulfoxide and sodium azide in distilled
water. The activity of each extract will be expressed as percentage decrease of reverse
mutations.
Glutathione estimation:- 17
Inbred Swiss albino mice will be maintained under standard laboratory
conditions. Mice of both sexes, 10 to 12 weeks old, weighing 25 to 30 g will be used for
the experiments. The tumor will be maintained in vivo by serial intraperitoneal (i.p.)
7
transplantation of 1×10 tumor cells (0.25 ml in phosphate-buffered saline, pH 7.4) per
animal. Tumor-transplanted mice usually survive for 19 to 21 days.
Drugs treatment schedule and antitumor activity: The dose of CP (200 mg/kg) was selected based on the report by Czyzewska and
Mazur. Tumor-transplanted mice will be randomly divided into five groups, (I-V) of
24 mice each. Group-I mice served as control and receive normal saline. Group-II and
Group-III mice will be given 1/3 of LD50 and 1/7 of LD50 of aqueous extract of leaves
th
of Basella rubra Linn. from 5
day to the 10
th
day of tumor transplantation
respectively. Group-IV mice will be administered a single dose of CP, i.p., on the
th
10 day post-tumor transplantation. Group-V mice will be given aqueous extract of
th
Basella rubra L. and then will be administered with CP on the 10 day post-tumor
transplantation. The survival patterns of the hosts will be determined and deaths, if
any, in different groups, will be recorded daily. After 1, 3, 6 and 12 days of treatment,
liver, kidneys, spleen, and tumor tissue will be dissected out for biochemical
investigations. The antitumor efficacy of different treatments will be reported as
percent increase in life span (ILS) and will be calculated as per the following formula:
(T/ C × 100) – 100, where T and C are the mean survival days of treated and control
mice, respectively.
Glutathione estimation: Total GSH (TGSH) and non-protein thiol (NPSH) content will be determined using
the method of Sedlak and Lindsay. Briefly, 5% homogenates of tissues will be
prepared in 0.02 mol/ L EDTA, pH 4.7 in a motor-driven Teflon–pestle homogenizer.
TGSH will be determined by adding 100 ml of the homogenate to 1.0 ml of 0.2 mol/L
Tris-EDTA buffer, pH 8.2 and 0.9 ml of 0.02 mol/L EDTA, pH 4.7 followed by 20
μl of Elliman’s reagent (10 mmol/L DTNB in methanol). For the determination of
NPSH, 500 μL of the homogenate will be precipitated with 500 μl of 10%
trichloroacetic acid and centrifuged at 3000 rpm for 15 min. To 800 μL of the
supernatant, 1.6 ml of 0.4 mol/L, Tris-EDTA buffer, pH 8.9, will be added before the
addition of 25 μL of Elliman’s reagent. After 30 min of incubation at room
temperature, the reaction mixture will be centrifuged and the absorbency of
supernatants will be read against a reagent blank at 412 nm using a
spectrophotometer.
Statistical analysis
The significance of difference between groups will be tested using one-way
ANOVA followed by Dunnet’s test.
Animals Required:
12 Swiss albino mice will be sacrificed for acute toxicity studies.
100 Swiss albino mice will be sacrificed for Glutathione estimation test.
Group
I
Period of
study
1st day
No. of
animals
5
3rd day
5
6th day
5
12th day
5
1st day
5
1/7th dose of Aqueous extract
3rd day
5
of Basella rubra L.
6th day
5
12th day
5
1st day
5
1/3rd dose of Aqueous extract
3rd day
5
of Basella rubra L.
6th day
5
12th day
5
1st day
5
3rd day
5
6th day
5
12th day
5
1st day
5
Aqueous extract of Basella
3rd day
5
rubra L.+ Cyclophosphamide
6th day
5
12th day
5
Treatment
Distilled water
II
III
IV
Cyclophosphamide
V
ENCLOSURE-VI
7.3 Does the study require any investigation or intervention to be conducted on
patients or other humans or animals? If so, please describe briefly.
The above study requires investigation on Swiss albino mice (112no.) for acute
toxicity study and anticancer activity.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
The study is cleared from Ethical Committee of the institution. (Certificate
enclosed).
ENCLOSURE – VII
8. REFERENCES:
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2. Rang HP, Dale MM, Ritter JM, Moore P K. Pharmacology. 5th ed. India: Elsevier
India Private Limited Publication; 2003. p. 693-94.
3. Bishop JM, Weinberg RA. Molecular Oncology. New York: Scientific American;
1996.
4. Tripathi KD, Essentials of Medical Pharmacology.5th ed. New Delhi: Jaypee
brothers medical publishers; 2004. p. 771.
5. http://www.mayoclinic.com; Drugs and supplements, Cyclophosphamide (oral
route, intravenous route. Date of retrival: 10-10-2008.Time:14:00.
6. Premalatha Balachandan, Rajgopal Govindarajan. Cancer- An Ayurvedic
perspective. Pharmacol Res 2005;51:19-30.
7. Smit HF, Woerdenbag HJ, Singh RH, Meulenbeld GJ, Labadie RP, Zwaving JH.
Ayurvedic herbal drugs with possible cytostatic activity. J Ethnopharmacol.1995;
47:75-84.
8. Dr.Gyandendra Pandey. Dravyaguna Vijnana. Varanasi: Krishnadas Academy;
2003 vol (3). p. 728-32.
9.
The Wealth of India, First supplement series (Raw material vol 1 A-C). p. 118.
10. Deshpande S, Shah G B, Deshpande I, Parmar, NS. Antiulcer activity of aqueous
extract of Basella rubra L. albino rats. J Natural Remedies. 2003;3(2): p. 212-14.
11. Bolognesi A, Polito L, Olivieri F, Valbonesi P, Barbieri L, Battelli MG et al. New
ribosome-inactivating proteins with polynucleotide: adenosine glycosidase and
antiviral activities from Basella rubra L. and bougainvillea spectabilis Willd.
Planta. 1997;203(4):422-9.
12. Hexiang Wang, Tzi Bun Ng. Antifungal peptides, a heat shock protein-like peptide
and a serine–threonine kinase-like protein from Ceylon spinach seeds. Peptides.
2004;25(7):1209-14.
13. Shahid Mahmud, Akhtar Javed M, Yamin, ShafiqMalik. Fatty acids composition
of lipid classes of Basella rubra linn. Proc.Pakistan Acad.Sci.2004;41(2):109-12.
14. Sharma M, Sharma PD, Bansal MP and singh J. Lantadene A-induced apoptosis
in human leukemia HL-60 cells. Int J Pharmacol 2007;39(3):140-44.
15. Yuan-Li Li, Guo-Ping Gan. A flavonoid glycoside isolated from Smilax china L.
rhizome in vitro anticancer effects on human cancer cell lines. J Ethnopharmacol
2007;113:115–24.
16. Bhatia A, Arora S, Nagpal A, Singh B, Ahuja PS. Evaluation of in vitro
antimutagenic activity of Seabuckthorn Hippophae rhamnoides Linn. in Ames
assay. J Chinese Clinical Med 2007;2(8):428-34.
17. Nicol
BM, Prasad SB. The effect of cyclophosphamide alone and in combination
with ascorbic acid against murine ascites Dalton’s lymphoma. Indian J
Pharmacol 2006; 38(4):260-5.