Senthil.V.*, Jis.C.Joy, Keerthivasan.A, Giridharan.P and Balakrishnan.A
Centre for Biotechnology, Anna University, Chennai-600 025, India.
ANNA UNIVERSITY CENTRE FOR BIOTECHNOLOGY
Screening of biological properties of Medicinal plants for anticancer Activity using In vitro techniques - An approach towards Anticancer Drug Development.
Medicinal plants are the most exclusive source of life saving
drugs for the majority of the world’s population. Medicinal
herbs have been widely used for treatment of diseases in
traditional way for several generations. An interaction
between traditional medicine and modern biotechnological
tools is to be established towards New Drug development. The
interface between cell biology, in vitro assays and structural
chemistry will be the best way forward to obtain valuable
leads.
INTRODUCTION
OVERVIEW
Plant powder
Hexane
DCM
Ethyl acetate
Methanol
Water
Non polar
S E E X
Q T U R E A N C T TI I
A O L N
Polar
BIOASSAY
Active Extract
Chromatography
Active Fraction
Structure Elucidation
Chromatography
Pure fraction
OBJECTIVETraditional medicine
Medicinal Plants
Lead molecule
Bioassays
Confirmation of apoptosis Confirmation of mechanism using analysis of apoptotic markers • p53 Tumor suppressor• Bcl-2 Anti-apoptotic• Bax Pro-apoptotic• Cytochrome c Pro-apoptotic • Caspase 8 Apoptosis initiator• Caspase 3 Apoptosis executioner
Preliminary - Thymidine Incorporation assay
DNA Fragmentation
Mitochondrial Pathway
Death Receptor Pathway
Caspase 3
DDD D
Procaspase 8
Caspase 8
BID
oxidants ceramide others
Bcl-2D
Cytochrome c
dATP
Procaspase 9Apaf -1
dATPApaf -1
Caspase 9
Procaspase 3
apoptosome
DNA damage
Cellular targets
MAJOR APOPTOTIC PATHWAYS
Apoptosis
FasL TNF
Andrographis paniculata
O
O
O
OH
CH2OHH
Structure of Andrographis paniculata pure compound
3,19- Dihydroxy-11-oxo-8(17),13-labdadien-15,16-olide
Ref: Balmain.A , et al, J.Chem.Soc, Perkin Trans 1,1973,1247
Thymidine incorporation studies showed a significant anti-proliferative activity of the Crude and Pure compounds isolated from Andrographis paniculata at a dose concentration of Crude 25µg/ml and Pure 12.5 µg/ml at a time point of 24 and 48 hours
THYMIDINE INCORPORATION ASSAY
Dose response curve of Andrographis paniculata Methanol Crude and Pure on HeLa cell line.
0
200
400
600
800
1000
Concentration ug/ml
CP
M/m
g p
rote
in x
1
00
0
Day 1 Day 2
597 bpGAPDH
388bpbcl 2
p53 514 bp
364 bpBAX
1 2 3 4 5 6
1. 100bp ladder 2. Control 3. Positive control4. A.paniculata Crude 5. A.paniculata Pure6. Negative control
RT-PCR studies reveal the activation of p53 followed by the activation of bax and downregulation of bcl-2 in HeLa cells treated with Crude extract and Pure compound of the Andrographis paniculata at a time point of 12 hours.
TRANSCRIPTIONAL EXPRESSION PROFILE
TRANSLATIONAL EXPRESSION PROFILE
The western blot analysis confirmed the down regulation of the anti-apoptotic protein Bcl-2 leading to Cytochrome c release and activation of Caspase 3.
1. Control 2. Positive control3. A. paniculata Crude 4. A. paniculata Pure5. Actin control
Actin Actin
24 kDa
Bcl-2
1 2 3 4
14 kDa
CYT C
CAS 3 32 kDa28 kDa17 kDa
ACTIN 42 kDa
DNA FRAGMENTATION ASSAY
Lane 1:
Lane 2:
Lane 3:
Lane 4:
Lane 5:
100bp ladder
Control
Positive control
A. paniculata Crude extract
A. paniculata Pure extract
1 2 3 4 5
Apoptosis is confirmed by DNA fragmentation, observed in cells treated with crude extract and pure compound of Andrographis paniculata
Apoptosis or programmed cell death is a genetically regulated process occurring naturally in a response to a variety of signals. We found that the lead molecule obtained from
Andrographis paniculata leaf, was able to induce apoptosis in human HeLa cells.
In this work the extraction, purification and elucidation of active molecular lead based on the bioactivity directed screening is described.
Induction of apoptosis was confirmed by assessing cellular markers involved in apoptotic signals.
This work is an example of a natural product with interesting anti- proliferative activity, in which the basic skeleton can be further, used as a template for the production of New Chemical Entity.
RESULTS AND CONCLUSION
Activated p53
Suppressed Bcl-2 expression
Up-regulation of pro-apoptotic protein Bax
Cytochrome c release from mitochondria
Activation of Caspase-3
DNA fragmentation
APOPTOSIS
MECHANISM OF ACTION
O
O
O
OH
CH2OHH
p 53
Bax
Cyt C Cyt C
Bcl2
p 53
APOPTOSIS
(DNA Fragmentation)
Caspase 9
Apaf 1
Caspase 3