Protein-Protein Interaction Analysis to Identify Nuclear Factor-Erythroid-2 Factor 2 (Nrf2) Inhibition by Extracellular Enzymes From Water Kefir Organisms

Abstract

Objective: This research was conducted to screen the anticancer activity of bitter herbs that contains Andrographis paniculata (Brum. f) leaves (AP) and Tinospora crispa L. stems (TC) in form of fresh materials and extracts using a mechanism-based yeast bioassay.
 Methods: Samples tested by mechanism-based yeast bioassay (MBYB) were single extract, mixed extract, and jamu gendong pahitan from a traditional market and made in the laboratory. Fresh sample of jamu gendong pahitan from the market and a single extract (AP and TC) was tested at one dose. While fresh jamu gendong pahitan made in the laboratory and the mixed extract (AP: TC) was tested at three different doses, doses 1 (3:10), dose 2 (1:1), and dose 3 (10:3). The leaves and stems were extracted by 70% ethanol for 3x24 h, each day the solvent was changed then every macerate was evaporated using a rotavapor and water bath. By this MBYB method, noted that the active sample must have an IC12 value of<8000µg/ml, so all the samples or doses were tested using final concentration varying at around 8,000; 4,000; 2,000; 1,000; 500, 250, and 125µg/ml.
 Results: The percentage yield of Andrographis paniculata (Brum. f) leaves was 11.2% and Tinospora crispa L. stems was 19.%. The activity assay for jamu gendong pahitan from the traditional market was inactive as a topoisomerase inhibitor (IC12>8000µg/ml). Samples showed topoisomerase I inhibitor activity were jamu gendong pahitan made in laboratory doses 1 and 2. While samples showed topoisomerase I and II inhibitor activities were jamu gendong pahitan made in laboratory dose 3, single and mixed extracts.
 Conclusion: The fresh material of jamu gendong pahitan (bitter herbs) bought from the market is inactive, while the fresh material of samples of jamu gendong pahitan made in laboratory doses 1 and 2 have topoisomerase I inhibitor activity. Based on the IC12, value, it is known that the sample that gave the best activity was the mixed extract of bitter herbs dose 3 that contain extract of A. paniculata and T. crispa (10:3), with IC12 values in strains 1138, 1140, and 1353 were 926.28±173, 576.75±42, and 865.5±135µg/ml respectively.