Antioxidant Effects of Diallyl sulfide, Capsaicin, Gingerol and Sulforaphane in H2O2-Stressed HepG2 cells

Abstract

생체는 외부의 에너지를 섭취하여 산화과정을 통하여 체내 유용한 에너지인 고인산 에너지(ATP)를 생성한다. 이러한 생체 산화과정은 무수한 형태의 활성산소(reactive oxygen substrate: ROS)가 생성을 동반하며 이들은 높은 반응성으로 인해 생체 내 거대분자인 단백질, 지질, 유전자에 산화적 손상을 유발하게 된다. 이러한 이유로 산화스트레스에 의한 여러 가지 질병을 예방하기 위해 우리가 자주 섭취하는 천연식품에서 항산화 물질을 찾으려는 노력이 끊임없이 이어지고 있다. 연구에 의하면 천연식품성분 중의 항산화물질들은 생체 내에서 산화적 활성화과정에 관여하는 free radical을 소거하여 지질의 과산화를 억제하며, 결과적으로 DNA손상 즉 돌연변이 발현을 저해하고 여러 가지 퇴행성 질병 등을 예방하고 건강한 삶을 누릴 수 있게 해 준다. 우리나라 대표 향신료 식물인 마늘, 고추, 생강은 김치의 필수 재료이며 주성분은 Diallyl sulfide, Capsaicin과 Gingerol으로 우리나라 식생활에서 중요하게 여겨지고 있으며 이미 오래전부터 음식의 감칠맛을 살리는 향신료, 조미료, 식재료로 사용되어져왔다. 브로콜리의 경우 주성분은 Silforaphane이며, 항산화능이 알려진 이래로 우리나라에서도 비교적 쉽게 섭취할 수 있는 식품이 되었다. 생체 내에서 간은 외부로부터 유입된 물질 뿐 아니라 대사 생성물에 의한 독성으로부터 보호와 해독 기능에 깊이 관여하는 중요한 기관으로 산화스트레스에 의한 간 손상은 여러 가지 질병의 원인이 된다고 알려져 있다. 본 연구에서 선정한 HepG2 cell은 human hepatic carcinoma 로부터 분리된 간암세포로서 인체 간 기능의 실험적 모델로 널리 이용되고 있으며 정상 간과 생화학적 대사가 유사한 범위 안에 있다고 알려져 있다. 따라서 본 연구에서는 HepG2 cell 에 가장 독성이 강한 산화스트레스 인자인 H2O2를 처치하여 산화스트레스를 유발하고 DAS, CAP, GGR과 SFN을 농도별로 각각 또는 함께 처치하여 세포의 보호효과, 항산화효소 활성, 지질과산화 생성억제와 항산화와 관련된 세포내 신호전달과정을 분자적 수준에서 확인하고자 한다. 본 실험에서 H2O2처리는 HepG2 세포의 손상을 직접적으로 유발시켰고, DAS, CAP, GGR and SFN의 첨가는 손상된 세포의 보호효과를 가지는 것이 확인 되었다 (DAS: 100, 200?M, CAP : 25, 50?M, GGR : 50, 100?M, SFN : 12.5, 25?M). 또한 세포 내 ROS의 생성을 강하게 억제시키는 것으로 확인 되었으며, 세포내 항산화 효소활성 증가와 과산물 생성억제 효과를 나타냈다. 또한, Western blot으로 확인한 결과 30분동안 처리하였을 때 DAS(200?M), CAP(25?M), GGR(50?M) and SFN(12.5?M)은 항산화와 관련된 전사인자(Nrf2)의 신호 전달 체계를 활성화시켰고 산화스트레스에 의해 활성화되는 단백질(NFkB)의 발현을 억제시킴으로써 연차적으로 염증성 사이토카인 단백질(TNF-a)의 활성을 저해하여 산화스트레스에 의한 세포손상을 막는 것으로 나타났다. 최종적으로, 우리전통 고유의 음식으로부터 유래된 DAS, CAP, GGR과 브로콜리의 SFN은 항산화효소 활성을 증진시키고 이와 관련된 세포내 단백질발현을 조절함으로써 ROS로부터 유도된 산화적 스트레스에서 간세포를 보호하는 것으로 사료된다. |Oxygen is necessary to sustain life, yet the very process of oxygen metabolism in the cells creates destructive elements called free radicals. Reactive oxygen species (ROS) are generated during oxidative stress, including hydroxyl radical superoxide, peroxyl radical, hydrogen peroxide and singlet oxygen. Free radicals, or oxidants, are chemically unbalanced, carrying free electrons that can damage molecules in our cells while trying to achieve balance ? potentially damaging the cell itself. Fortunately, the body has its own free radical defense system. Virtually every cell produces antioxidant enzymes called Superoxide Dismutase (SOD), catalase and glutathione peroxidase. These enzymes protect the cells during oxygen metabolism, safely breaking down harmful free radicals to balanced elements like H2O. However, our antioxidant defense system can become overwhelmed. Many studies indicate the levels of SOD, catalase and GPx decrease with age. Also, certain conditions are related to the increased production of unstable oxygen derivatives, including physical stress, health challenges and exposure to environmental toxins such as smoking and pollution [1]. For this reason, many antioxidant products, including supplement and functional foods, are being developed. However, the bioactivity of dietary antioxidants is often quite low, and the amounts needed to be consumed daily to remove free radicals and inhibit oxidative stress are expensive and unsafe. In addition, this approach is designed to correct a possible antioxidant deficiency, and does not specifically promote the body?s own endogenous antioxidant defenses. Natural products are rich source of pharmacologically active compounds in which plant materials deserves an important position. Naturally occurring organosulphur compounds from garlic and onion mediate significant chemopreventive activities against the initiation stage of carcinogenesis induced by various chemical carcinogens [2,3]. Therefore, this study examined the effect of Antioxidant capacity in Korea traditional seasoning ingredients that usually eat a lot. Garlic organosulfur compounds are recognized as a group of potential chemopreventive compounds. among these, diallyl sulfide(DAS) is known that can modulate drug metabolism systems, especially various phase II detoxifying enzymes, though the mechanism underlying their inductive effect on these enzymes remains largely unknown. antioxidant enzymes.

Capsaicin (CAP), N-vanillyl-8- methyl-1-nonenamide, is a pungent ingredient in varieties of red pepper of the genus Capsicum. Gingerol (GGR), a major component of ginger, has antioxidant, anti-apoptotic, and anti-inflammatory activities. also Outstanding antioxidant gingerol inhibited the tumor promotion stage in skin carcinogenesis mice has been reported [4]. Sulforaphane (SFN), is already revealed as biologically active compound extracted from cruciferous vegetables, and possessing potent anti-cancer and anti-inflammatory activities. In the present study, investigated the antioxidnt enzyme levels of superoxide dismutase(SOD), catalase(CAP), glutathione(GSH) contents and glutathione peroxidase and production of Lipid peroxidation (LPO) and the protein level of transcription factor, Nuclear factor-kB(NFkB), nuclear factor E2-related factor 2 (Nrf2), after administration of four natural compound DAS, CAP, GGR and SFN in human hepatoma HepG2 cells. First, appropriate concentration and incubation time were decided 200?M H2O2 to induce 30% cell death and each antioxidant as following ; DAS(100, 200?M), CAP(25, 50?M), GGR(50, 100?M) and SFN(12.5, 25?M) by safe range of the cytotoxicity. And then each antioxidants were treated after H2O2. As a result each groups elevated significantly (p<0.05) the restoration compared to only H2O2-treated cells. But there was no drastic induction of viability. ROS is important mediators of liver damage. The result shown that exposure to H2O2 elevated directly amount of ROS by uo to 220% and all of the treatment significantly reduced intracellular ROS level compared to the negative control by up to almost similar with control group [Fig 12A]. Interestingly, ROS could be more efficiently removed by concentration of each chemicals such as DAS : 200?M, CAP : 25?M, GGR : 50?M and SFN : 12.5?M than another concentration In our experimental conditions, treatment of HepG2 cells with H2O2-induced cells significantly decrease in the activity of antioxidant enzymes such as SOD, CAT and GPx. whereas GSH in cells treated with 200 ?M DAS, 25 ?M CAP, 50?M GGR and 12.5?M SFN for 12, 24hr showed a significant recovery. this result suggest that H2O2-induced oxidative stress in HepG2 cells depleted endogenous antioxidant defense system. LPO concentration was increased significantly in cells treated with H2O2 compared to control cells after 12h and 24hr. However, the LPO level with DAS, CAP, GGR, SFN treatment in the presence of H2O2 caused significantly decreased the level of LPO in 12 than 24hr. In this study confirm that DAS, CAP, GGR and SFN increased transcription factor Nrf2 related with antioxidant system and Nrf2 was regulated by activation of the MAPK-JNK. Futhermore, each antioxidants inhibited activation of TNF? and NF-kB by downregulation of phospholylation of IkB?. The inhibition of TNF? and NF-kB mean that DAS, CAP, GGR and SFN decreased inflammation factor induced by ROS. In conclussion, these results indicate protective effects of DAS CAP, GGR and SFN against H2O2-induced oxidative stress. By increasing the SOD, CAT and GPx activity which removes peroxides and superoxides, also might prevent the accumulation of ROS by removing them. These modulations in the antioxidant enzyme system, which upregulate the host detoxification process, might be associated with reduced risk of pathologies which are related to the ROS-mediated cellular damage. therefore, Garlic, Red pepper, Ginger and Brocoli intake might contribute to the dietary prevention of those.