Download In vitro anticancer activity of Betanin, fresh juice and hydro

In vitro anticancer activity of Betanin, fresh juice and hydro-alcoholic
root extract of Beta vulgaris on HL-60 cell lines
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore– 560 041
By
Ms. K. Sirisha, B. Pharm.
Under the Guidance of
Dr. Kalyani Divakar, M. Pharm, Ph. D.
Professor & Head
2010-2012
Department of Pharmacology,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post),
Hesaraghatta Main Road, Bangalore – 560 090.
1
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.
Name of the candidate &
Address
2.
Name of the Institution
3.
Course of the study & subject
4.
Date of admission
5.
Title of the Topic
6.
Brief resume of intended work
Ms. K. Sirisha,
D/o K. Nageswara Rao, D.No. 8-15-2,
Sujana appartment -A, flat no B2, Gandhi nagar,
Kakinada, Andhra Pradesh.
Acharya & B.M. Reddy College of Pharmacy
Soldevanahalli, Hesaraghatta Road,
Chikkabanavara Post, Bangalore-560090.
Phone No: 080 65650815
Fax No: 080 28393541
M.Pharmacy (Pharmacology)
02/09/2010
In vitro anticancer activity of Betanin, fresh
juice and hydro-alcoholic root extract of Beta
vulgaris on HL-60 cell lines
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
2
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?
No
7.4 Has ethical clearance been obtained
from your institution in case of 7.3
Not applicable
8.
List of references (About 1 – 19)
9.
Signature of the candidate
10.
Remarks & Signature of the guide
11.
Name & Designation of Guide
12.
Name & Signature of HOD
Enclosure VII
Dr. Kalyani Divakar, M. Pharm, Ph. D.
Professor & Head, Dept. of Pharmacology,
Acharya & B.M. Reddy College of Pharmacy
Dr. Kalyani Divakar, M. Pharm, Ph. D.
Professor & Head, Dept. of Pharmacology,
Acharya & B.M. Reddy College of Pharmacy
13.
Remarks of the Principal
14.
Signature of Principal
Dr. Divakar Goli, M. Pharm, Ph. D.
Principal
Acharya & B.M. Reddy College of Pharmacy
3
ENCLOSURE-I
6. BRIEF RESUME OF INTENDED WORK
6.1 Introduction and need of work:
6.2 Cancer is a leading cause of death worldwide. The disease accounted for 7.4 million
deaths (or around 13% of all deaths worldwide) in 2008. The main types of cancer
leading to overall cancer mortality each year are:

Lung (1.4 million deaths/year)

Stomach (740 000 deaths)

Colorectal (610 000 deaths)

Liver (700 000 deaths)

Breast (460 000 deaths)
Cancer is a generic term for a large group of diseases that can affect any part of the
body. Other terms used are malignant tumours and neoplasms. One defining feature of
cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries,
and which can then invade adjoining parts of the body and spread to other organs. This
process is referred to as metastasis which is the major cause of death from cancer. [1]
More than 70% of all cancer deaths occurred in low-and middle-income countries.
Deaths from cancer worldwide are projected to continue rising, with an estimated 13
million deaths in 2030. [1]
Several attempts have been made to improve existing treatments to increase the
survival of patient. Hence, there is need of new therapeutic agents that are less toxic to
normal cells and produce an enhanced anti tumor effect. [2, 3]
4
Most cancer patients in India reel under the pressure of expensive treatment. Most
American pharma companies producing and selling these drugs in India have a patent over
them.
The patients have no choice but to pay the market price since none of the Indian
companies are producing those medicines here except one drug, which is much less
expensive and equally efficient manufactured by Dr Reddy. Although the Indian government
does provide free/subsidized treatment at the cancer centers, a patient has to go for expensive
advanced-level treatment to increase his chances of survival. But in India, it is beyond the
reach of most needy patients, hence need to find the cost effective newer drugs. [4]
A high intake of vegetables and fruits can reduce the risk of developing cancer and other
diseases. Beetroot has unique chemicals (e.g. Betalains) and high levels of important
micronutrients, which make it a valuable vegetable to include in the diet as a means of
deterring the onset of cancer and other diseases. The Betalains also act as antioxidants.
The therapeutic use of beetroot in cancer treatment came to prominence with the work of
the Hungarian physician Alexander Ferenczi in the 1950s. He introduced a revolutionary new
treatment for cancer using nothing but raw beetroot juice. In his papers from the late 1950s
and early 1960s, he reported remarkable success in treating cancer patients. His patients
suffered from a range of different cancers. His reputation grew and beetroot juice became a
sought-after treatment for cancer. Ferenczi’s treatment was based on consuming a liter of
beetroot juice daily, for at least two to three months.
Rosenberg concluded that beetroot’s effect on cancer cells is probably due to the
combined effects of betanine, allantoine, vitamin C and other compounds present, such as
farnesol and rutine.[5]
5
There are two distinct classes of betalains, the red/purple betacyanins and the yellow
betaxanthins.[6]
The present protocol is proposed to find out the in-vitro anticancer activity of Betanine,
fresh juice and hydro-alcoholic extract of root of Beta vulgaris against HL-60 cell lines.
Further this study is also aimed to find out the difference in the activity between Betanine and
fresh juice and hydro-alcoholic extract of root of Beta vulgaris. Since Betanine is main
pigment of beet root whether itself is sufficient to show the anticancer activity (or) any other
phyto-constituents of root of Beta vulgaris is also responsible for its anticancer activity, is
also a part of the evaluation.
6
ENCLOSURE-II
6.2 Review of Literature:
Plant selected for study:[7]
Name of the plant selected for the present study is “Beta vulgaris”
Description:
Botanical name: Beta vulgaris
Family: Amaranthaceae
Synonyms: Garden beet, Table beet, Red beet.
Beetroots are an excellent source of red and yellow pigments, and this trait has fostered
research and industrial interest in the context of natural colorants. Beet pigments, collectively
known as betalains.[6] The betalains found in beetroot peel extract were vulgaxanthin I,
vulgaxanthin II, indicaxanthin, betanin, prebetanin, isobetanin and neobetanin. Also
cyclodopa glucoside, N-formylcyclodopa glucoside, glucoside of dihydroxyindolcarboxylic
acid, betalamic acid, l-tryptophan, p-coumaric acid, ferulic acid and traces of unidentified
flavonoids were detected. [8]
Betanine content in beetroot varies from 100 mg/100 g fresh product to 16-38 mg/100 g
dried vegetable product. Therefore, the plant species and harvesting conditions are important
factors to be considered when selecting the suitable raw material. [9]
About 0.5-1% of extracted beet juice solids are betalains. In addition to the pigments, the root
contains about 700-800 mg oxalic acid and 5-6 mg ascorbic acid per 100 g beetroot .[10]
The beet juice may inhibit or enhance carcinogenic MA (N-nitrosodimethylamine)
formation in human gastric juice depending on the pH of gastric juice. In acid medium, there
was a trend to inhibit MA synthesis, while in neutral and alkaline medium, MA synthesis is
activated.[11]
7
Beetroot is also rich in ferulic acid derivatives. The specific betalains routinely
identified in these fractions were vulgaxanthin I, vulgaxanthin II (glutamic acid replaces
glutamine, and (iso) betanin, all of which have been reported as major betalains in
beetroots.[6]
In the 1990s, cell culture and animal studies, such as those conducted by Edenharder et
al., and Kapadia et al., respectively, confirmed that beetroot juice had significant
antimutagenic effects against Salmonella typhimurium TA98 and TA100 and tumorinhibiting againt prostate and breast cancer cell lines,[12] and in-vivo mice skin and lung
cancer.[13] Beetroot crude extracts contained multiple antioxidant and phase II enzymeinducing activities.
In some studies, antioxidant function has been attributed directly to betalains in terms of
reducing power and inhibition of iron-induced lipid peroxidation.[6]
Beetroot extract able to show anti inflammatory and immunosuppressive activity in
peripheral blood mononuclear cells (PBMC), because inflammation is strongly involved in
the development and progression of several clinical conditions including coronary heart
disease and cancer, beneficial effect of beetroot extract may relate to this anti-inflammatory
capacity.[14]
8
ENCLOSURE-III
6.3 Objectives of the study
a) To find out the invitro anticancer activity of Betanine, fresh juice and hydroalcoholic extract of root of Beta vulgaris.
b) To compare the activity between Betanine and juice and hydro-alcoholic extract of
root of Beta vulgaris.
c) To explore the mechanism involved in the anticancer activity of Betanine, fresh juice
and hydro-alcoholic extract of root of Beta vulgaris through the following
parameters.
d) In-vitro estimations using HL-60 cell lines by conducting the following tests:
a. Clonogenic assay [15]
b. MTT assay [16]
c. Trypan Blue exclusion technique [16]
d.
DNA fragmentation analysis [17]
e. Determination of cell viability in presence of caspase-3 inhibitor [16]
f. Glutathione estimation [18]
g. Lactate dehydrogenase estimation [kit]
h. Nitric oxide estimation [19]
9
ENCLOSURE-IV
7. MATERIALS AND METHODS
7.1 Source of Data:
The data will be obtained from experiments which involve:
A) Laboratory based studies
B) Literature survey, abstracts
C) National & International Journals
D) Text books
E) Internet
10
ENCLOSURE-V
7.2 Methodology:
Collection and Extraction of Root of Beta vulgaris:
a. Clonogenic Assay:[14]
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 X.
A linear relation of the number of cells plated and colonies could be established. The same
procedure will be followed using various concentrations of drug(s).
b. MTT Assay:[15]
The 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay is a
common method used to assess cell proliferation and cytotoxicity. Briefly, 1x104
exponentially growing cells will be seeded per well in 96 well plates and exposed to various
concentrations of drug(s) and incubated to certain period. MTT will be added and incubated
at 37°C 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.
c. Trypan Blue exclusion technique:[15]
The effect of drug on the HL-60 Cancer cell will be determined by means of Trypan Blue
exclusion technique. Briefly, 3x104 cells will be seeded per well in 24 well plates and then
treated with various concentrations of drug(s). The plate 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% Trypan Blue every 24 h to calculate the doubling time.
d. Estimation of percent DNA fragmentation:
Induction of apoptotic mode of cell death in cells was also confirmed by quantitative
determination of DNA fragmentation following a method given by sellins & cohen with
11
slight modification.Treated or untreated tumor cells were lysed in 0.5 ml of Tris-EDTA
buffer, pH 7.4, containing 0.2% (v/v) triton x-100 and the fragmented DNA was separated
from intact chromatin in a microfuge tube (labeled as B) by centrifugation at 13,000× g at
4°C for 10 min. supernatant containing the fragmented DNA was transferred to another
microfuge tube (labeled as T). A volume of 0.5 ml of 25% TCA was added to each T and B
tube and vortexed vigorously. DNA was precipitated overnight at 4°C and collected at
13,000× g at 4°C for 10 min supernatant was discarded and 80 μl of 5% TCA was added to
each pellet. DNA was hydrolyzed by heating at 90°C for 15 min .At this stage, a blank was
included containing 80 µl of 5% TCA. Then 160 µl of freshly prepared diphenylamine (DPA)
reagent (150 mg diphenyleamine in 10 ml glacial acetic acid, 150 µl concentrated H2SO4 and
50 µl of acetaldehyde solution) was added and the tubes were allowed to stand overnight at
room temperature to develop color. 100 µl of this colored solution was transferred to the
wells of a 96-well flat-bottomed ELISA plate and observance was measured at 600 nm in a
microtitre ELISA plate reader percent DNA fragmentation was calculated as:
DNA fragmentation (%) = [T/(T+B)]×100
Where T= absorbance of fragmented DNA and T+B= absorbance of total DNA.
e. Determination of cell viability in presence of caspase-3 inhibitor:[15]
The viability of HL-60 cells treated with caspase-3 inhibitor and drug will be evaluated
using MTT assay. After incubation with caspase-3 inhibitor for 1 h, cells will be treated with
various concentrations of drug(s) for 48 h after which viability will be determined.
f. Glutathione estimation:[16]
Cultured HL 60 cell lines 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 test tubes
with 1-5 ml of 0.2 M Tris buffer, pH 8.2, and 0.1 ml of 0.01 M 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
12
centrifuged at approximately 3000 g at room temperature for 15 minutes. The absorbance of
the supernatants will be read using spectrophotometer at 412 nm in 1 cm quartz cells.
g. Lactate dehydrogenase estimation: [kit]
This is carried out using commercial kit.
h. Nitric oxide estimation:[17]
The cultured cells will be weighed and used for preparation of homogenates in 0.05 M
phosphate buffer. The tubes will centrifuged at 1500 x g 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 (Giuseppina et al., 1999)
which will be expressed in the form of nitrite accumulation. In brief, 100 µl of tissue extract
will be mixed with 100 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 incubated at room temperature for 10 min, and then the absorbance at 550 nm will
be measured in a UV-double beam spectrophotometer. The amount of nitrite in the sample
(µM unit) will be calculated from a sodium nitrite standard curve. The results will be
expressed as µM nitrite/mg of protein.
13
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.
Not applicable
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Not applicable
14
ENCLOSURE-VII
8. References:
1. World Health Organization [online]. February 2008 [cited 2011 may 8]; URL:
http://www.who.int/mediacentre/factsheets/fs297/en/
2. Manish S, Began G, Suraag P, Dora B, Sunil C, Rajagopal R. Vitamin E succinate in
combination with mda-7 results in enhanced human ovarian tumour cell killing
through modulation of extrinsic and intrinsic apoptotic pathways. Cancer Lett
2007;254:217-26.
3. Faivre S, Djelloul S, Raymond E. New paradigms in anticancer therapy: targeting
multiple signaling pathways with kinase inhibitors, semin. Oncol 2006;33:407-20.
4. Cost of cancer treatment. Citizen journalist [online]. 2007 june 20 [cited 2011 may 9];
[1 screen]. Available from: URL: http://www.ibnlive.in.com/news/money-killscancer-patients/40047-20.html.
5. Stephen Nottingham: Beetroot: Health and Nutrition. Chapter 6. 2004. p. 1-17.
6. Lee CH, Wettasinghe M, Bolling BW, Ji L, Parkin KL. Betalains, Phase II enzymeinducing components from red beetroot (Beta vulgaris L.) Extracts. Nutr cancer
2005; 53(1):91-103.
7. Beta vulgaris. The plant database [online]. 2006 March 6 [Cited 2011 may 11]: URL:
http://www.scientificweb.com/en/Biology/Plants/Magnoliophyta/BetaVulgaris01.html
8. Kujala T, Loponen J, Pihlaja K. Betalains and phenolics in red beetroot (Beta
vulgaris) peel extracts: extraction and characterisation 2001; 56c:343-8.
9. Sturzoiu A, Stroescu M, Stoica A, Dobre T. Betanine extraction from beta vulgaris
experimental research and statistical modelling. U.P.B. Sci Bull 2011;73(1):145-56.
10. Winkler C, wirleitner B, Schroecksnadel K, Schennach H, Fuchs D. In vitro effects of
beet root juice on stimulated and unstimulated peripheral blood mononuclear cells.
Am J Biochem Biotech 2005;1(4):180-85.
15
11. Rajesh K. The wealth of India: A dictionary of Indian raw materials and industrial
products. Newdelhi: NISCAIR; 2007. vol-1(A-Ci). p. 139.
12. Edenharder R, Kurz P, John k, Burgard S, Seeger K. In vitro effect of vegetable and
fruit juices on the mutagenicity of 2-amino-3-methylimidazole[4,5-f]quinoline,
2,amino,3,8-dimethylimide-3-ol[4,5f]quinoxaline. Food Chem Toxicol 1994;32:44359.
13. Kapadia GJ, Tokudab H, Konoshimac T, Nishinod H. Chemoprevention of lung and
skin cancer by Beta vulgaris (beet) root extract. Cancer Lett 1996;100:211-4.
14. Eastwood MA, Nyhlin H. Beeturia and colonic oxalic acid. QJM 1995;88(10):711-7.
15. 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.
16. Sharma M, Sharma PD, Bansal MP, Singh J. Lantadene A-induced apoptosis in
human leukemia HL-60 cells. Ind J Pharmacol 2007;39(3):140-4.
17. Vishvakarma NK, Singh SM. Mechanisms of tumor growth retardation by modulation
of pH regulation in the tumor-microenvironment of a murine T cell lymphoma.
BIOPHA 2010;2957.
18. Ellwart JW, Kremer JP, Dormer P. Drug Testing in Established Cell Lines by Flow
Cytometric
Vitality Measurements
versus
Clonogenic
Assay.
Cancer
Res
1988;48:5722-5.
19. Dolan D, Sandip M, Asankur SD, Maitrayee M, Chandan M. Aqueous extract of
black tea (Camellia sinensis) prevents ethanol + cholecystokinin-induced pancreatitis
in a rat model. Life Sci 2006;78:2194-203.
16