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Bioactive Proteins from Benincasa hispida (Thunb.) Cogn
CHURIYAH,LATIFAH KOSIM DARUSMAN
HAYATI Journal of Biosciences , 2009,
Abstract: The research was carried out to isolate and characterize of bioactive proteins from plant parts of Benincasa hispida (Thunb.) Cogn and to analyse of the toxicity and cytotoxicity of the proteins. The proteins were extracted with phosphate buffer saline, then they were precipitated using 80% saturated ammonium sulphate, continued with the dialysis using pH 7 phosphate buffer. The dialysate was fractionated through gel filtration chromatography and characterized using SDS-PAGE. The toxicity of the proteins was analyzed through brine shrimp lethality test (BSLT), followed with cytotoxicity test using HeLa and K-562 cancer cell lines. Three bioactive protein fractions were isolated from the fruits, the seeds and roots. The lowest yield of proteins was 0.021% from the fruit, then 0.051% from the seed, while the highest was 0.54% from the root. All proteins were toxic on BSLT with LC50 within the range of 24-39 μg. Characterization of proteins using SDS-PAGE indicated the molecular mass of those proteins were approximately 17-29 kDa. The cytotoxicity test of the root protein showed that the protein could inhibit proliferation of HeLa cell up to 28.50% and K-562 cell up to 36.60% compared to that of non treated cell.
ANTIPROLIFERATIVE PROTEIN FROM TRICHOSANTHES CUCUMERINA L. VAR ANGUINA (L.) HAINES
CHURIYAH,WAHONO SUMARYONO
BIOTROPIA : the Southeast Asian Journal of Tropical Biology , 2010,
Abstract: Three proteins were isolated from plant parts of Trichosanthes cucumerina L. var anguina (L.) Haines, they were TF2 from fruit, TS3 from seed and TR3 from root with molecular masses (Mr) approximately 16 - 64 kDa on SDS-PAGE characterization. The proteins were extracted with Phosphate Buffer Saline (PBS), then they were precipitated using 80% saturated ammonium sulphate continued with the dialysis using cellophane. The dialysate was fractionated through gel filtration chromatography. The highest yield of protein was 1.109% from the seed (TS3), then 0.356% from the root (TR3), while the lowest was 0.014% from the fruit (TF2). The Lethal Concentration 50 (LC ) of proteins on brine shrimp lethality test was 50within range of 19 μg/ml - 25 μg/ml. The cytotoxicity test of the TR3 and TS3 proteins on cancer cell lines indicated that both of the proteins could inhibit proliferation of HeLa and K-562 cells with IC up to 45 μg/ml. It is recommended to conduct further researches on 50Trichosanthes plant as a herbal medicine to treat cancer.
Induction of Trichosanthes cucumerina anguina L. var (L. Haines) Hairy Roots Using Agrobacterium rhizogenes ATCC 15834 for Production of Bioactive Protein
CHURIYAH,GUSTAF ADOLF WATTIMENA,BAMBANG PONTJO PRIYOSOERYANTO
Microbiology Indonesia , 2011, DOI: 10.5454/mi.5.3.5
Abstract: In the recent years, hairy roots have become a useful model system for the study of bioactive secondary molecule production and protein expression. Hairy roots of Trichosanthes cucumerina var. anguina were obtained by direct inoculation of the plantlet with Agrobacterium rhizogenes ATCC 15834 strain agropine. Six root clones of Trichosanthes cucumerina var. anguina hairy roots TH1, TH2, TH3, TH4, TH6, and TH8 were obtained from the induced hairy roots. The hairy root proteins were extracted with phosphate buffer saline, then fractionated by ammonium sulphate precipitation (80%), dialysis, and gel filtration. The toxicity of the proteins was analyzed using brine shrimp lethality test followed by cytotoxicity test (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay) using HeLa and K-562 cancer cell line. The TH2 root clone showed the highest protein yield (0.83%) and most toxic on BSLT with Lethal Concentration 50 was 5.76 μg mL-1. PCR analysis indicated the integration of rolB gene to the genome of TH2 root clone which was showed by a DNAband of 780 bp on the electrophoretic agarose gel. Protein fractionation of the TH2 root clone resulted in four fractions, one of which the-TH2-3 protein fraction revealed the highest yield (0.29%) and toxicity on BSLT with LC50 up to 0.92 μg mL-1 . The Cytotoxicity test of the TH2 protein and TH2-3 protein fraction indicated that both of proteins inhibited the proliferation of Hela and K-562 cell withLC up to 49 μg mL-1.
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