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Control Figure A-i	100 µg/ml Figure A-ii
300 µg/ml Figure A-iii	500 µg/ml Figure A-iv

Figure A (i,ii,iii,iv) & B shows the Cell Cycle Arrested by different concentration of ayurvedic medicine (LAS 02) in Ovary or cervical cancerous cell line (HeLa)

APOPTOSIS MEASUREMENT BY FLOW CYTOMETRY

Lavanaya R&D division has been done the experiment apoptosis induction of Ayurvedic medicine (LAS 02) against cervix cancer cell line (HeLA).For Apoptosis induction measurement of ayurvedic medicine,using three staining procedures H& E, acriydine orange and propidium iodide respectively

Haematoxylin and Eosin (H&E) Staining

Control Figure A	100 µg/ml Figure B
300 µg/ml Figure C	500 µg/ml Figure D

Fluorescence micrographs of HeLacells treated with different concentrations of LAS02 and stained with a hematoxylin &Eosin stain(A) Control cells not treated with LAS02 (B) 100 μg, (C) 300 μg, and (D) 500 μg for 24 hours.

Finding shows that normal cells appear well nucleus and large numbers without treated with drug (slide A). After treatment with different concentration of drug LAS02 , the number of cells , morphological changes and completely necrotic cells were found at concentration 500 μg of drug after 24 hour treatment. It means that drug at 500 μg concentration causes apoptosis induction againest cervix cancer cell line HeLa (slide D)

Acrydine Orange (A&O) Staining

Control Figure A	100 µg/ml Figure B
300 µg/ml Figure C	500 µg/ml Figure D

Fluorescence micrographs of HeLa cells treated with different concentrations of LAS02 and stained with a Acradine orange stain.(A) Control cells not treated with LAS02, (B) 100 μg, (C) 300 μg, (D) 500 μg and treated with LAS02 for 24 hours.

Results clear showed that viable cells displayed in green florescence with normal shape and size, intact circular cells, intact DNA with nucleus give a round and green nuclei (Slide A) After treatment with drug at different concentration of LAS02, the apoptotic and necrotic cells DNA fragmented and cell bledding are occurred maximum at concentration of drug 500 μg after treatment for 24 hours (slide D)

Propidium Iodide (PI) staining

Control Figure A	100 µg/ml Figure B
300 µg/ml Figure C	500 µg/ml Figure D

Fluorescence micrographs of HeLa cells treated with different concentrations of LAS02and stained with a PI stain (A) Control cells not treated with LAS02, and treated with LAS02 at concentration (B) 100 μg, (C) 300 μg, and (D) 500 μg for 24 hours.

Apoptosis is an important homeostatic mechanism that balances cell division and cell death and maintains the appropriate number of cells in the body. Apoptosis induction by using PI staining in HeLa cell line appeared chromatin condensation, cell and nuclear shrinkage, membrane blebbing and oligonucleosomal DNA fragmentation (Slide A). After treatment with ayurvedic medicine at concentration 500 μg for 24 hours showed that apoptotic and necrotic cells DNA fragmented and cell bledding are found maximum

So on the basis these staining procedure by flow cytometry concluded that our ayurvedic medicine most effective for induction of apoptosis against human cervix cancer cell line HeLA

RESULT

Our findings clear suggested that ayurvedic medicine (LAS 02) is most effective for inhibition of cervical cancer cell line from the cancerous cell growth .