Determination of Antibacterial Activity of Essential Oil of Myristica 
 fragrans Houtt. using Tetrazolium Microplate Assay and its Cytotoxic Activity 
 Against Vero Cell Line

Objective: The aim of this study was to purify and characterize bacteriocin from the soil isolate Bacillus subtilis GAS101, and to determine its antimicrobial as well as antibiofilm potential. The purified bacteriocin was further analyzed and evaluated for mammalian cell cytotoxicity and the possible mode of action. Material and Methods: Bacteriocin from B. subtilis GAS101 (an animal husbandry soil isolate) was partially purified and checked for antimicrobial and antibiofilm activity against gram-positive and gram-negative bacteria. The molecular weight of bacteriocin was determined using tricine SDS-PAGE gel. The stability of bacteriocin was investigated at various temperatures and pH levels, and its sensitivity towards 8 enzymes and 6 chemicals was determined. Cytotoxicity analysis was performed on a Vero cell line by a tetrazolium dye-based assay. Scanning electron microscopy (SEM) of bacteriocin-treated bacteria was carried out to determine the possible mode of action. Results: Bacteriocin from B. subtilis GAS101 was a potential inhibitor of both the indicator organisms (Staphylococcus epidermidis and Escherichia coli), and had a molecular weight of approximately 6.5 kDa. An in situ gel assay showed a zone of inhibition corresponding to the estimated protein band size. Bacteriocin was stable and showed antibacterial activity in broad ranges of temperature (30–121°C) and pH (2–12). It was sensitive to 4 proteolytic enzymes, which indicated its proteinaceous nature. Bacteriocin showed > 70% cell viability on the mammalian Vero cell line. SEM depicted that the bacteriocin was able to disrupt the bacterial cell membrane as its probable mode of action. Conclusion: Thermostable and pH-tolerant bacteriocin from B. subtilis GAS101, of about 6.5 kDa, showed broad-spectrum antimicrobial and antibiofilm activity.

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CYTOTOXICITY IDENTIFICATION OF THE SUPERNATANT OF Manihot esculenta Crantz RHIZOME AS RIBOSOME-INACTIVATING PROTEIN TO NORMAL CELL

Indonesian Journal of Chemistry ◽

10.22146/ijc.21857 ◽

2010 ◽

Vol 4 (2) ◽

pp. 77-81

Author(s):

Endang Astuti ◽

Sabirin Matsjeh ◽

Winarto Haryadi ◽

Deni Pranowo ◽

Nuning Sri Mulatsih

Keyword(s):

Cytotoxic Activity ◽

Vero Cell ◽

Cell Line ◽

Cancer Cell ◽

Manihot Esculenta ◽

Normal Cell ◽

Cytotoxic Effect ◽

Ribosome Inactivating Protein ◽

Manihot Esculenta Crantz ◽

Vero Cell Line

The supernatant of Manihot esculenta Crantz rhizome has been used as an alternative remedy of infectious and cancer in Jogjakarta. Cytotoxicity assay showed that Manihot esculenta Crantz supernatant had cytotoxic effect to cancer cell line, namely Myeloma (LC50 = 180,24 μg/mL) and HeLa (LC50 = 415,55 μg/mL), but have a little cytotoxic effect to SiHa. This research was aimed to identify cytotoxic activity of Manihot esculenta Crantz's supernatant to normal cell, particularly to human mononuclear cell and Vero cell line, and to compare to the cancer cell lines. The result showed that supernatant of M. esculenta had cytotoxic effect to normal mononuclear cell (LC50 = 564,00 μg/mL) and Vero cell line (LC50 = 686,00 μg/mL). The supernatant of M. esculenta had the highest cytotoxic activity to myeloma and relatively toxic to cervix cancer HeLa and normal cell, but less to SiHa. Keywords: Manihot esculenta Crantz, Ribosome-inactivating Protein (RIP), cytotoxic, normal cell, cancer cell

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