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