Anticancer activity of Withania somnifera

on HEp-2 cell line

Submitted by Samayaditya Singh B.Tech (Bio.tech) MCSCET

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INTRODUCTION

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Withania somnifera (Ashwagandha)

Ashwagandha is a potent adaptogen

and Immunomodulator

It has anti-agening and anti-

cancerous property.

Chemicals present in leaves are

Steroidal Lactone, withanolides,

withaferin A etc.(P.K.Sharma et

al.2010)

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Cont…Chemical components of Ashwagandha Leaves

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Cont… HEp-2 Larynx carcinoma cell line

• Cells of this line contain HeLa marker chromosomes.

• This line was originally derived from an epidermoid

carcinoma, but recently found line was derived by HeLa

contamination.

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Objective

1. To maintain the Hep-2 cell line for anticancer cell activity.

2. To estimate the cytotoxic effect of Withania somnifera on

Hep-2 cell line.

3. To determine the DNA damaging Potential of Withania

somnifera in Hep-2 cell line.

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Plan of Work

Culture the Hep-2 Larynx carcinoma cell line and maintain there viability

Check viability of cultured cells

Check cell viability after treatment

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Passaging

Trypan blue exclusion test

Preparation of treatments for cell Viability Assay

MTT AssayNRU Assay

Check Apoptosis rate by DNA Fragmentation process

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METHADOLOGY

Passaging

DISCARD PREVIOUS

CULTURE MEDIUM

ADD

INCOMPLETE

MEDIA

ADD TRYPSIN-EDTA

SOLUTION AND WAIT FOR

4-5 MINUTES

BY SCRAPER GENTLY

DETACHED REMAIN

ADHERED CELLS

AFTER ASPIRATING PELLETS

ADD APPROPRIATE ALIQUOTS OF

CELL SUSPENSION TO NEW

FLASKS. INCUBATE AT 37°C

DISCARD SUPERNATANT

AND ADD COMPLETE

MEDIA ON PELLETS

TRANSFER CELLS

SUSPENSION IN FALCON

TUBE AND CENTRIFUGE

TUBE FOR 5 MIN

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Trypan Blue exclusion test

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Mix them homogenously

Blue color cell are deadColorless cell viable

Hemocytometer

Take cell suspension and Trypan blue in 1:1 ratio

Load mixture on Hemocytometer

Microscope (on 10X)

MTT Cell Proliferation Assay(Mosmann, T., 1983) Used to measure rate of reduction in cell viability.

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Extract of LeavesAdd MTT reagent

Incubate for 20hr

Incubate for 2hr

Cell suspension

Add DMSO

Take absorbance

Micro plate reader

NRU( Neutral Red Uptake Assay) (Winckler, 1974) Live cells incorporate neutral red into their lysosomes.

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Incubate for 20hr

Incubate for 2hr

Take absorbance

Micro plate reader

Add Glacial acetic acid

Add NRU reagent

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DNA Fragmentation Assay (Andrew Wyllie,1980)

DNA fragmentation is Hall Mark of Apoptosis in cells.

Add Lysis Buffer

Phenol:Chloroform:Isoamyl alcohal

Centrifuge

DNA

PROTEIN

INCUBATE FOR 24hr

INCUBATE FOR 2hr

Add RNase and Proteinase KLysates

ISOPROPANOL AND SODIUM ACETATE

Centrifuge

ETHANOL(70%)

Centrifuge

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Cont…q

FORM LOADING SAMPLE

FORM GEL BED WITH 6-WELLS

LOAD SAMPLE

GEL DOC System

Electrophoresis system

Dry the sample in air Nanospectro

photometre

Quantitative Analysis of DNA on

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RESULT

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– Under microscope no dead cells are found, test show 50%

viability of cell line.

Viable cell

Trypan blue exclusion test

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MTT Assay

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NRU Assay

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DNA Fragmentation Assay

L C T-1 T-2 T-3 T-4

Where,

L- Ladder Well; T-1, T-2, T-3, T-4- Treatment Well; C- Control Well

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CONCLUSION

Trypan Blue Exclusion test

– Average number of cells in large Square 8.5 cells.

– Total number of cells in 1ml is 1.7X105 cells.MTT Assay– Cells show significant death in T-1 treatment, at conc.

1000µg/ml.NRU Assay– Cells show significant death in T-1 Treatment, at concentration

1000µg/ml.DNA fragmentation Assay• DNA fragmentation assay shows apoptosis at 1000µg/ml concentration of ethanolic extract of Ashwagandha.

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AcknowledgementI would like to express my sincere gratitude towards Prof P.K. Seth,

CEO of Biotech Park, Lucknow, who gives me a opportunity to do my training in the field of Molecular Biology Under Aakaar Biotech.

And a heartiest thanks to my mentor Mr. Ashish Yadav and Dr. Swarnita Dixit, for there guidance in my project work and also for maintaining morale.

Also thanks to Dr. Umashankar Sir, who share it’s pecious knowledge and give more knowledge about Biotechnology.

I would also like to thanks Ms. Namita Tripathi, Mr. Pradeep Singh Chauhan and Mr. Anand Kumar Saroi, for there help in improving lab skills and encouragement.

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THANK YOU