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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

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

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  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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 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.

  12. 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

  13. Caspase 9 Caspase 3 Apaf 1 Bcl2 Bax Cyt C Cyt C APOPTOSIS (DNA Fragmentation) p 53 p 53

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