Study of in vitro anticancer activity of the

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Study of in vitro anticancer activity of the jujube seed extracts against the HCT

Study of in vitro anticancer activity of the jujube seed extracts against the HCT 116 colorectal cancer cell line Tille Vanlangendonck

Index Introduction Methodology Results Conclusion Future prospects

Index Introduction Methodology Results Conclusion Future prospects

Introduction Ziziphus mauritiana = jujube o Seeds from an old/ripe jujube o Seeds from

Introduction Ziziphus mauritiana = jujube o Seeds from an old/ripe jujube o Seeds from a young/unripe jujube Pharmaceutical effects o Seeds: sedative o Fruit: relieve cough and fever Triterpenic acids o Betulinic acid (C 30 H 48 O 3) o Maslinic acid (C 30 H 48 O 4) o Oleanolic acid (C 30 H 48 O 3)

Methodology Jujube seed Maceration Extract Cytotoxicityt est IC 50 DAPI No apoptosis Apoptosis

Methodology Jujube seed Maceration Extract Cytotoxicityt est IC 50 DAPI No apoptosis Apoptosis

Methodology: Maceration Soaking the plant material 6 solvents o Water o Ethanol o Dichloromethane

Methodology: Maceration Soaking the plant material 6 solvents o Water o Ethanol o Dichloromethane o Ethylacetate o Chloroform o Hexane Evaporation

Methodology

Methodology

Methodology: HCT 116 cell line Human colon Colorectal carcinoma Morphology Unattached 10 x Attached

Methodology: HCT 116 cell line Human colon Colorectal carcinoma Morphology Unattached 10 x Attached 10 x

Methodology: Cytotoxicitytest Seeding cells o 96 -well plate o 30. 000 cells/well Adding extract

Methodology: Cytotoxicitytest Seeding cells o 96 -well plate o 30. 000 cells/well Adding extract o Diluted in DMSO 0, 6% DMSO = 10% cytotoxicity o Concentration of 500 µg/ml 0, 5% DMSO

Methodology: Neutral red uptake assay NR can penetrate both living and dead cells o

Methodology: Neutral red uptake assay NR can penetrate both living and dead cells o Non-ionic diffusion Living cells take up and bind NR in lysosomes Lyse with 0, 33% HCl in isopropanol Spectrophotometer: 537/650 nm IC 50 calculated with the linear regression equation

Methodology

Methodology

Methodology: Cell death Necrosis Apoptosis Because of cell damage Self-induced by the body Cell

Methodology: Cell death Necrosis Apoptosis Because of cell damage Self-induced by the body Cell debris is released freely in the tissue Cell debris is packed in vacuoles Inflammatory reaction No inflammatory reaction Tissue damage gangrene No tissue damage

Methodology: DAPI-staining Popular nuclear and chromosome counterstain Emits a cyan blue light Binds to

Methodology: DAPI-staining Popular nuclear and chromosome counterstain Emits a cyan blue light Binds to AT regions of DNA 40 x

Results: Cytotoxicitytest Extract from the seeds of the jujube with a chloroform maceration IC

Results: Cytotoxicitytest Extract from the seeds of the jujube with a chloroform maceration IC 50 = 779 µg/ml IC 50 = 290 µg/ml

Results: Cytotoxicitytest Extract from the seeds of the jujube with a hexane maceration Almost

Results: Cytotoxicitytest Extract from the seeds of the jujube with a hexane maceration Almost no cell death, IC 50 is not calculated

Results: DAPI-staining Extract from chloroform maceration of the seeds from the old jujube 40

Results: DAPI-staining Extract from chloroform maceration of the seeds from the old jujube 40 x

Conclusion Chloroform maceration: o Anticancer activity o Seeds from young jujube: IC 50 is

Conclusion Chloroform maceration: o Anticancer activity o Seeds from young jujube: IC 50 is too high o Seeds from old jujube: IC 50= 290 µg/ml Cell death: apoptosis Hexane maceration: o No anticancer activity

Future prospects Flow cytometry o Early or late apoptosis DNA fragment analysis Search the

Future prospects Flow cytometry o Early or late apoptosis DNA fragment analysis Search the mechanism of apoptosis o Intrinsic or extrinsic

Study of in vitro anticancer activity of the jujube seed extracts against the HCT

Study of in vitro anticancer activity of the jujube seed extracts against the HCT 116 colorectal cancer cell line Tille Vanlangendonck