Metabolism of asperchalasine A, the secondary metabolite of Aspergillus flavipes

Abstract

Cytochalasans는 구조적으로 다양한 곰팡이의 polyketide-amino acid hybrid 대사체들의 그룹이며, 다양한 생리활성 기능을 보인다. Asperchalasine A는 해양 유래 곰팡이인 Aspergillus flavipes 추출물에 서 확인되어 분리 동정되었다. Asperchalasine A는 cytochalasan의 이합체로, epicoccine으로 연결된 두 개의 cytochalasan 분자로 구성되어 있다. 하지만, Asperchalasine A에 대한 생리활성 및 대사에 대한 연구는 보고되어 있지 않았다. 따라서 asperchalasine A와 Aspergillus flavipes 추출물 생리활성과 in vitro 대사 연구 및 평가를 하고자 한다. Aspergillus flavipes 및 asperchalasine A의 항산화 활성은 DPPH scavenging 측정법을 이용하여 평가하였다. Aspergillus flavipes 추출물은 ascorbic acid과 비교하였을 때, 강한 용량 의존적 항산화 활성이 나타났다. 하지만, asperchalasine A는 항산화 활성이 약했다. Aspergillus flavipes 추출물과 ascorbic acid의 IC50 값은 각각 8.79 μg/mL, 11.37 μM이었다. 그러므로, Aspergillus flavipes 추출물은 항산화 특성을 가지고 있으며 산화 예방제로서의 가능성을 가지고 있다. 또한, 항암 활성을 평가하였다. 암세포 억제는 MTT assay를 통해 평가하였다. Aspergillus flavipes 추출물과 asperchalasine A에서는 암세포에서 용량 의존적 억제 활성이 나타났지만, 약한 활성이 나타났다. 또 한, 세포 실험을 통해 혈관 신생 억제 효과를 평가하였다. Aspergillus flavipes 추출물과 asperchalasine A는 인간 탯줄 정맥 내피세포 (HUVECs)에서 세포관 형성을 강하게 억제하였다. Aspergillus flavipes 추출물과 asperchalasine A는 choriollantoic membrane 측정법으로 수정된 닭 난자의 혈관 형성량을 유의적으로 억제하였다. 이 결과는 Aspergillus flavipes 추출물과 asperchalasine A의 새로운 생리활성을 암시하였다. 그러므로, Aspergillus flavipes 추출물과 이차대사산물인 asperchalasine A는 암질환을 위한 신약 후보 물질로서 개발 가능할 것으로 사료되었다. Asperchalasine A의 약물간의 상호작용 평가를 수행하였다. Aspergillus falvipes 추출물과 asperchalasine A의 in vitro cytochrome P450(CYP450) 효소에 대한 억제 효과는 human liver microsomes 및 recombinant CYP2D6 isozyme에서 CYP isozyme-specific 기질과 함께 반응을 시켰으며, CYP 효소 활성에 대한 억제 가능성을 평가하기 위해 marker 대사체의 형성량을 측정하였다. 그 결과 asperchalasine A는 농도 의존적으로 CYP2D6에 강한 억제 효과를 나타내었지만, Aspergillus flavipes 추출물은 낮은 억제 효과를 나타내었다. 따라서 asperchalasine A는 CYP2D6 기질 약물과의 상호작용을 유발할 것으로 사료되었다. Asperchalasine A는 5종 간 마이크로좀에서 대사 안정성 평가를 하였다. Human에서의 asperchalasine A의 대사 안정성은 dog, monkey와 유사하였다. 또한 asperchalasine A의 biotransformation을 평가하였다. Asperchalasine A는 NADPH 의존적 반응을 시켰으며, asperchalasine A의 대사체 구조는 LC-QTOF/MS로 측정하였다. 그 결과 asperchalasine A는 biotransformation에 의해 5개의 대사체가 나타났다. Hydroxylation을 통해 생성된 대사체들의 hydroxyl기는 dehydroxylation이 되어 각각의 product ion을 생성하였으며 주요 대사경로는 1상대사인 hydroxylation으로 사료되었다. 이번 연구를 통해 Aspergillus flavipes 추출물과 asperchalasine A는 항암 및 항산화 활성이 나타났으므로 암 예방제로 개발될 가능성이 있다. 하지만, asperchalasine A는 CYP2D6와 강한 상호 작용을 나타내어 추가적인 상호작용 연구가 필요할 것이다. 키워드: Aspergillus flavipes, Asperchalasine A, 대사, 생리활성; Cytochalasans are a group of structurally diverse fungal polyketide-amino acid hybrid metabolites that exhibit diverse biological functions. Asperchalasine A was identified and isolated from an extract of the marine-derived fungus, Aspergillus. Asperchalasine A is a cytochalasan dimer which consists of two cytochalasan molecules connected by an epicoccine. However no research on bioactivities and metabolism for asperchalasine A has been reported. Therefore, the bioactivities and in vitro metabolism studies of asperchalasine A (a new secondary metabolite) and Aspergillus flavipes were evaluated. Antioxidant potential of Aspergillus flavipes extract and asperchalasine A was evaluated by using DPPH scavenging assay, reducing total antioxidant capacity. The extract showed significant activities in all antioxidant assays compared to the reference antioxidant ascorbic acid in a dose dependent manner. However, asperchalasine A showed weak antioxidant activity. In DPPH scavenging assay the IC50 value of the extract was found to be 8.79 μg/mL while the IC50 value of the reference standard ascorbic acid was 11.37 μM. Total antioxidant activity was also found to increase in a dose dependent manner. Aspergillus flavipes extract showed strong reducing power. These results suggest that Aspergillus flavipes may act as a chemopreventative agent, providing antioxidant properties and offering effective protection from free radicals. In addition, anti-cancer activity was evaluated through the cancer cell inhibition and angiogenesis inhibition. The cancer cell inhibition was evaluated using the MTT assay. In Aspergillus flavipes and asperchalasine A, dose-dependent inhibitory activity was observed in cancer cells, but weak activity was observed. On the other hand, the potential antiangiogenic effects of Aspergillus flavipes and asperchalasine A was investigated. Aspergillus flavipes and asperchalasine A significantly inhibited cell tube formation in human umbilical vein endothelial cells (HUVECs). Aspergillus flavipes and asperchalasine A significantly inhibited the amount of blood vessel formation in fertilized chicken eggs using a chorioallantoic membrane assay. Our results provide experimental evidence of this novel biological activity of the potential antiangiogenic substances, Aspergillus flavipes, and asperchalasine A. This study also suggests that Aspergillus flavipes and its active component asperchalasine A are excellent candidates as adjuvant therapeutic substances for cancer prevention and treatment. Asperchalasine A exhibits various pharmacological effects such as anticancer activities and has been predicted as an anti-cancer drug candidate. Drug-drug interaction needs to be evaluated. The inhibitory effects of Aspergillus flavipes extract and asperchalasine A on the cytochrome P450(CYP) enzymes in vitro. Asperchalasine A was incubated with CYP isozyme-specific substrates in human liver microsomes and recombinant CYP2D6 isozyme, and the formation of the marker metabolites was measured to investigate the inhibitory potential on CYP enzyme activities. The results showed that asperchalasine A has significant inhibitory effects on CYP2D6 activity in a concentration-dependent manner. Meanwhile, Aspergillus flavipes extract, did not have strong inhibition effects on CYP enzyme activities. Asperchalasine A could modulate the pharmacokinetics of CYP2D6 substrate drugs and lead to interactions with those drugs. The metabolic stability of asperchalasine A in rat, mouse, dog, monkey and human liver microsomes was evaluated. The metabolic stability of asperchalasine A in human liver microsomes was similar to that in dog and monkey liver microsomes. In addition, The biotransformation of asperchalasine A was investigated. Asperchalasine A was reacted by NADPH, and the resultant metabolites were structurally characterized by LC-QTOF/MS. As a result, asperchalasine A was biotransformed to yield metabolites: M1, M2 , M3, M4 and M5. A major metabolic routes were hydroxylation; the hydroxyl group of asperchalasine A were dehydroxylated to generate M1, M2, M3, M4, and M5 respectively. Among these metabolites, M5 was reported for the major metabolite in this study. Therefore, the main metabolism pathway of asperchalasine A was predicted as phase 1 metabolism. In this study, the anti-cancer effect and antioxidant effect were confirmed by Aspergillus flavipes and asperchalasine A. Therefore, asperchalasine A would be able to be developed as cancer prevention agent. However, asperchalasine A has a strong interactions with CYP2D6, so further interaction studies will be required.