Physalis Minima

CHAPTER III

RESEARCH METHOLOGY

1.0 Plant extraction of Physalis minima

1.1 Preparation of fresh sample extract

Physalis minima L. at maturity state was harvested from Kepala Batas, identified and washed with a distilled water. The dried specimen was submitted to the USM Herbarium for future reference. About 200 g weight of leaves were blended finely with 300 mL distilled water using a blender until become a mixture with a small size of leaves. The water mixture of leaves were macerated with 1200 mL methanol and shaken continuously at 250 rpm for 24 hours. The mixtures was filtered with Whatman #1 filter paper and the filtrate was used to repeat the extraction for another two times. Then, the methanol solvent in mixtures was removed using

MCF-7 breast cancer cell line is an ER-positive cell line. It was originally obtained from human breast adenocarcinoma with epithelial morphology. Normal breast cell line used was MCF-10A, an ER-alpha negative normal breast cell line. Both of the cell lines were purcahsed from the American Type Culture Collection (ATCC) (Rockville, USA). MCF-7 and MCF-10A cells were maintained in RPMI-1640 and DMEM media, respectively. These cells were grown and maintained in suitable condition and passaged every three days.

2.2. Reagents for cell culture work

2.2.1 Complete Roselle’s Park Memorial Institute (RPMI-1640) Media

RPMI-1640 was prepared by mixing the 500 mL of RPMI purchased from GIBCO Company with 25 mL of 10% (v/v) FBS and 5 mL of Penstrap.

2.2.2 Complete Dulbecco Modified Eagle’s Media (DMEM)

Complete DMEM medium was prepared by mixing the 500 mL of DMEM purchased from GIBCO Company with 25 mL of 5% Horse serum, 1 mL of 20 mg/mL EGF, 0.25 mL of 0.5 mg/mL Hydrocortison, 0.5 mL of 10 ug/mL Insulin and 5 mL of Penstrap.

2.2.3 Heat-inactivated fetal bovine serum (FBS)

A bottle of sterile FBS (100 ml) (GIBCO) was placed in a 37 °C water bath (JEIO TECH) for 30 min to heat and inactivate the serum. The serum was kept at -20 °C until use.

2.2.4 Phosphate-buffered saline

1 tablet of PBS were dissolved in 100 mL deionized water and kept at 4 °C.

2.2.5 Penicilin/streptomycin stock solution (PenStrap)

The PenStrap solution was ready-made use purchased from GIBCO Company.

2.2.6 Trypsin (0.25 %, w/v)/EDTA 1X solution

A 0.25% trypsin from bovine pancreas was used to detach the cultured cells from the flask. The trypsin solution was obtained from GIBCO Company.

2.2.7 Dimethyl sulfoxide (DMSO) ≥99%

The DSMO acquired from Merck acts as cryoprotectant agent to reduce the formation of ice so that the cell death during the freezing process will prohibited.

2.2.8 Horse Serum 5%

Horse serum 5% was purchased from GIBCO Company and it was ready-to-use.

2.2.9 Epidermal Growth Factor (EGF) (20 mg/mL)

The EGF used in this study was obtained from GIBCO. To prepare the 20 mg/mL of the working solution of EGF, the stock was diluted with distilled water and it was kept in -20 °C until used.

2.2.10 Hydrocortison (0.5 mg/mL)

The hydrocortison of 0.5 mg/mL final concentration was prepared by diluting the stock solution purchased from GIBCO with