ANNA UNIVERSITY
This presentation is the property of its rightful owner.
Sponsored Links
1 / 13

Senthil.V. *, Jis.C.Joy, Keerthivasan.A, Giridharan.P and Balakrishnan.A PowerPoint PPT Presentation


  • 113 Views
  • Uploaded on
  • Presentation posted in: General

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. Senthil.V. *, Jis.C.Joy, Keerthivasan.A, Giridharan.P and Balakrishnan.A

Download Presentation

Senthil.V. *, Jis.C.Joy, Keerthivasan.A, Giridharan.P and Balakrishnan.A

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

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.

Senthil.V.*, Jis.C.Joy, Keerthivasan.A, Giridharan.P and Balakrishnan.A

Centre for Biotechnology, Anna University, Chennai-600 025, India.


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

INTRODUCTION

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.


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

OVERVIEW

Plant powder

B

I

O

A

S

S

A

Y

Non polar

Hexane

Active Extract

S E

E X

Q T

U R

E A

N C

T T

I I

A O

L N

DCM

Chromatography

Ethyl acetate

Active Fraction

Methanol

Water

Chromatography

Polar

Pure fraction

Structure Elucidation


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

Medicinal Plants

Lead molecule

Preliminary - Thymidine Incorporation assay

Bioassays

Confirmation of mechanism using analysis of apoptotic markers

Confirmation of apoptosis

DNA Fragmentation

  • p53 Tumor suppressor

  • Bcl-2 Anti-apoptotic

  • Bax Pro-apoptotic

  • Cytochrome c Pro-apoptotic

  • Caspase 8 Apoptosis initiator

  • Caspase 3 Apoptosis executioner

OBJECTIVE

Traditional medicine


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

Procaspase 3

Apaf -1

dATP

dATP

Caspase 3

Caspase 9

MAJOR APOPTOTIC PATHWAYS

Death Receptor Pathway

FasL

TNF

Mitochondrial Pathway

oxidants

ceramide

others

DNA damage

D

D

Bcl-2

D

D

D

Procaspase 8

Apaf -1

BID

Procaspase 9

Cytochrome c

Caspase 8

Cellular targets

apoptosome

Apoptosis


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

Andrographis paniculata

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


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

THYMIDINE INCORPORATION ASSAY

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


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

1 2 3 4 5 6

514 bp

p53

388bp

bcl 2

BAX

364 bp

TRANSCRIPTIONAL EXPRESSION PROFILE

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.

  • 100bp ladder

  • Control

  • Positive control

  • A.paniculata Crude

  • A.paniculata Pure

  • Negative control

597 bp

GAPDH


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

Actin Actin

1 2 3 4

Bcl-2

32 kDa

24 kDa

28 kDa

17 kDa

CYT C

14 kDa

CAS 3

42 kDa

ACTIN

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.

  • Control

  • Positive control

  • A. paniculata Crude

  • A. paniculata Pure

  • Actin control


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

Lane 1:

Lane 2:

Lane 3:

Lane 4:

Lane 5:

100bp ladder

Control

Positive control

A. paniculata Crude extract

A. paniculata Pure extract

DNA FRAGMENTATION ASSAY

1 2 3 4 5

Apoptosis is confirmed by DNA fragmentation, observed in cells treated with crude extract and pure compound of Andrographis paniculata


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

RESULTS AND CONCLUSION

  • 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 activemolecular 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.


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

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


Senthil v jis c joy keerthivasan a giridharan p and balakrishnan a

Caspase 9

Caspase 3

Apaf 1

Bcl2

Bax

Cyt C

Cyt C

APOPTOSIS

(DNA Fragmentation)

p 53

p 53


  • Login