젓갈 미생물 Bacillus idriensis로부터 분리된 diketopiperazine 및 합성유도체의 생리활성 탐색

Abstract

The human life span is getting longer and longer, partly due to newly-developed by drug and medical technology. Nevertheless, new diseases difficult to treat by current medical technology are increasing due to a variety of factors, some of which are environmental. Oxidative stress is also one of the important factors related to the increasing incidence of diseases

such stress may cause cell membrane damage, DNA oxidation, and protein denaturation.

As a part of our continuing search for antioxidants from natural resources, a bacterium CGH18 exhibiting antioxidizing and chitin-degrading activities in colloidal chitin culture medium was isolated from crab marinated in soy sauce and identified as Bacillus idriensis by a 16S rDNA sequence homology search.

The culture broth of the bacterium CGH-18 was extracted with EtOAc for 30 minutes using sonication. The extracted solution was evaporated under reduced pressure and then the residue was partitioned between water and n-butanol (n-BuOH). The organic layer was further partitioned between water and methylene chloride (CH2Cl2). Antioxidant activities of the crude extract and its solvent fractions were evaluated using five different activity tests. Among them, the CH2Cl2 fraction showed the strongest antioxidant activities. Further purification of the CH2Cl2 fraction by silica preparative TLC followed by HPLC resulted in the isolation of five 2,5-diketopiperazines (compounds 1-5). The chemical structure of isolated compounds was determined by 1H, 13C and extensive 2D NMR experiments such as 1H COSY, TOCSY, gHMQC, and gHMBC, and by comparison with published spectral data.

Antioxidant activities of the isolated compounds (1-5) were measured on DPPH, hydroxyl, and superoxide radicals, and peroxynitrite. Compounds 1-3 significantly scavenged peroxynitrite induced from SIN-1 in ratios of 80.5, 62.2, and 59.5%, respectively, at the concentration of 50 μM. In addition, compound 1 showed the good scavenging effect on hydroxyl and superoxide radicals with ratios of 74.8% and 77.2% at 500 μM, respectively, while compound 2 showed a significant scavenging effect only for the hydroxyl radical (76.6% at 500 μM).

Antioxidant activities of the synthetic derivatives (6-10) were also evaluated in the same bioassay systems. Among them, compound 8 revealed the potent scavenging effect on DPPH and hydroxyl radicals, and authentic peroxide with scavenging ratios of 82.0% (500 μM), 79.8% (500 μM), and 75.6% (50 μM), respectively. Compounds 6 and 7 inhibited formation of hydroxyl radical in ratios of 81.2 and 80.8% at a concentration of 500 μM, respectively.

We investigated the antiproliferative effects of all compounds (1-10) against the growth of human cancer cells (AGS, HT-29, and MCF-7) as well as the inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. None of the compounds (1-10) significantly inhibited the growth of human cancer cells. Compounds 1, 3, and 5 reduced the production of nitric oxide by more than 50%.