Korean Journal of Plant Resources (한국자원식물학회지)

The Plant Resources Society of Korea (한국자원식물학회)
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Hepatoprotective Effects of the Extracts of Alnus japonica Leaf on Alcohol-Induced Liver Damage in HepG2/2E1 Cells

알코올로 유도된 간손상 모델 HepG2/2E1 세포에서 오리나무 잎 추출물의 간보호효과

  • Bo-Ram Kim (Honam National Institute of Biological Resources) ;
  • Tae-Su Kim (Honam National Institute of Biological Resources) ;
  • Su Hui Seong (Honam National Institute of Biological Resources) ;
  • Seahee Han (Honam National Institute of Biological Resources) ;
  • Jin-Ho Kim (Honam National Institute of Biological Resources) ;
  • Chan Seo (Honam National Institute of Biological Resources) ;
  • Ha-Nul Lee (Honam National Institute of Biological Resources) ;
  • Sua Im (Honam National Institute of Biological Resources) ;
  • Jung Eun Kim (Honam National Institute of Biological Resources) ;
  • Ji Min Jung (Honam National Institute of Biological Resources) ;
  • Do-Yun Jeong (Honam National Institute of Biological Resources) ;
  • Kyung-Min Choi (Honam National Institute of Biological Resources) ;
  • Jin-Woo Jeong (Honam National Institute of Biological Resources)
  • 김보람 (국립호남권생물자원관) ;
  • 김태수 (국립호남권생물자원관) ;
  • 성수희 (국립호남권생물자원관) ;
  • 한세희 (국립호남권생물자원관) ;
  • 김진호 (국립호남권생물자원관) ;
  • 서찬 (국립호남권생물자원관) ;
  • 이하늘 (국립호남권생물자원관) ;
  • 임수아 (국립호남권생물자원관) ;
  • 김정은 (국립호남권생물자원관) ;
  • 정지민 (국립호남권생물자원관) ;
  • 정도윤 (국립호남권생물자원관) ;
  • 최경민 (국립호남권생물자원관) ;
  • 정진우 (국립호남권생물자원관)
  • Received : 2024.02.20
  • Accepted : 2024.03.20
  • Published : 2024.04.01
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Abstract

Alcoholic liver disease (ALD) is a significant risk factor in the global disease burden. The stem bark of the Betulaceae plant Alnus japonica, which is indigenous to Korea, has been used as a popular folk medicine for hepatitis and cancer. However, the preventive effect of Alnus japonica leaf extracts on alcohol-related liver damage has not been investigated. The objective of this study was to investigate the hepatoprotective effects of the extracts of Alnus japonica leaf (AJL) against ethanol-induced liver damage in HepG2/2E1 cells. Treatment with AJL significantly prevented ethanol-induced cytotoxicity in HepG2/2E1 cells by reducing the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). This protective effect was likely associated with antioxidant potential of AJL, as evidenced by the attenuation of reactive oxygen species (ROS) and malondialdehyde (MDA) production and restoration of the depleted glutathione (GSH) levels in ethanol-induced HepG2/2E1 cells. Our findings suggest that FCC might be considered as a useful agent in the prevention of liver damage induced by oxidative stress by increasing the antioxidant defense mechanism.

활성산소종은 미토콘드리아 대사활동을 통해 생성되는 반응성이 강한 산소 함유 분자로서 특히 간에서 복잡한 세포 분자와 반응하여 산화적 스트레스를 유발하는 물질이다(Choi et al., 2012). 에탄올 대사 과정에서는 ROS가 생성되어 산화스트레스를 유발하며, 과도한 에탄올은 ROS를 제거하는 항산화 시스템을 억제함으로써 지방, 단백질 또는 DNA와 같은 생체 분자에 산화 손상을 유발하여 세포를 손상시킨다(Albano, 2006; Koch et al., 2004). 본 연구에서는 오리나무 잎 추출물(AJL)에서 알코올(EtOH)로 유발된 산화적 스트레스에 대한 간세포 손상의 보호 효과를 평가하기 위해 HepG2/2E1 세포에서 EtOH와 AJL 및 대조군silymarin을 각각 처리한 후 세포생존율을 평가하고 세포독성이 없는 범위를 설정하여 활성산소종 생성량, 항산화 관련 지표(DPPH, ABTS, MDA, GSH) 및 간 손상 지표(AST 및 ALT)를 측정하였다. 알코올에 대한 간 보호 효과에서는 알코올만 처리한 군의 세포생존율이 53.8%로 감소하였으나 AJL을 함께 처리 군에서 세포생존율이 85.1%의 유의적인 증가로 간 보호효과를 확인하였다. 다음으로 AJL에서 세포 내 ROS 생성 억제능은 에탄올 처리군에서 정상군에 비해 유의적으로 ROS 생성이 증가하였으나 AJL 농도별 처리시 정상군 수준으로 ROS 생성을 현저하게 감소시켰다. MDA 함량은 에탄올 처리군에서 무처리군에 비해 유의적으로 증가하였으나 AJL 처리시 농도 의존적으로 에탄올 처리군에 비해 유의적인 차이를 보이며 MDA 함량을 감소시켰다. GSH 함량은 에탄올 처리군에서 정상군보다 유의적으로 함량이 감소하였으나 AJL 농도별 처리군 모두 에탄올 처리군에 비해 GSH 함량을 증가시켰다. 또한 간손상 지표로 활용되는 AST 및 ALT 활성 변화 확인 시, 에탄올 처리에 따른 간세포 손상군에 비하여 AJL 처리군에서는 농도 의존적으로 현저하게 감소시켰으며, 대조군silymarin과 비교하여 유사한 감소된 결과를 관찰하였다. 이상의 결과로부터 종합해 볼때AJL은 간손상에 대한 보호 및 간손상 억제 가능성을 기대할 수 있으며, 간기능 개선 효과에 대한 건강기능식품개발의 기초자료로서 활용될 수 있을 것이라 사료된다.

Keywords

Acknowledgement

본 결과물은 환경부의 재원으로 국립호남권생물자원관(HNIBR202302115) 및 한국환경산업기술원의 다부처 국가생명연구자원 선진화 사업(RS-2023-00230402)을 지원받아 수행된 연구이며, 이에 감사드립니다.

References

  1. Albano, E. 2006. Alcohol, oxidative stress and free radical damage. Proc. Nutr. Soc. 65(3):278-290.
  2. Alharbi, K.S., W.H. Almalki, M. Albratty, A.M. Meraya, A. Najmi, G. Vyas, S.K. Singh, K. Dua and G. Gupta. 2022. The therapeutic role of nutraceuticals targeting the Nrf2/HO-1 signaling pathway in liver cancer. J. Food Biochem. 46(10):e14357.
  3. An, S.W., Y.G. Kim, M.H. Kim, B.I. Lee, S.H. Lee, H.I. Kwon, B. Hwang and H.Y. Lee. 1999. Comparison of hepatic detoxification activity and reducing serum alcohol concentration of Hovenia dulsis Thumb and Alnus japonica Steud. Kor. J. Med. Crop Sci. 7:263-268.
  4. Baker, M.A., G.J. Cerniglia and A. Zaman. 1990. Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal. Biochem. 190(2):360-365.
  5. Bueqe, J.A. and S.D. Aust. 1978. Microsomal lipid peroxidation. Methods Enzymol. 52:302-310. https://doi.org/10.1016/S0076-6879(78)52032-6
  6. Buitelaar, R.M., A.A.M. Langenhoff, R. Heidstra and J. Tramper. 1991. Growth and thiophene production by hairy root culture in various two liquid phase bioreactiors. Enz. Microbiol. Technol. 13(6):487-494.
  7. Cederbaum, A.I. 2012. Alcohol metabolism. Clin. Liver Dis. 16:667-685. https://doi.org/10.1016/j.cld.2012.08.002
  8. Cederbaum, A.I. 2013. Nrf2 and antioxidant defense against CYP2E1 toxicity. Subcell. Biochem. 67:105-130. https://doi.org/10.1007/978-94-007-5881-0_2
  9. Cheel, J., C. Theoduloz, J. Rodriguez and G. Schmeda-Hirschmann. 2005. Free radical scavengers and antioxidants from lemongrass (Cymbopogon citratus (DC.) Stapf.). J. Agric. Food Chem. 53(7):2511-2517. https://doi.org/10.1021/jf0479766
  10. Choi, C.H., D.H. Won, J.P. Hwang and S.N. Park. 2012. Antioxidative effect of extracts from different parts of Juncus effusus L. J. Soc. Cosmet. Scientists Korea 38:275-282. https://doi.org/10.15230/SCSK.2012.38.3.275
  11. Contreras-Zentella, M.L., D. Villalobos-Garcia, and R. Hernandez-Munoz. 2022. Ethanol metabolism in the liver, the induction of oxidant stress, and the antioxidant defense system. Antioxidants (Basel) 11(7):1258.
  12. Haque, E., M.R. Karim, A. Salam Teeli, M. Smiech, P. Leszczynski, D. Winiarczyk, E.D. Parvanov, A.G. Atanasov and H. Taniguchi. 2020. Molecular mechanisms underlying hepatocellular carcinoma induction by aberrant NRF2 activation-mediated transcription networks: Interaction of NRF2-KEAP1 controls the fate of hepatocarcinogenesis. Int. J. Mol. Sci. 21(15):5378.
  13. Hoekstra, L.T., W. de Graaf, G.A. Nibourg, M. Heger, R.J. Bennink, B. Stieger and T.M. van Gulik. 2013. Physiological and biochemical basis of clinical liver function tests: a review. Ann. Surg. 257(1):27-36. https://doi.org/10.1097/SLA.0b013e31825d5d47
  14. Hwang, J.Y., J.W. Ham and S.H. Nam. 2004. Effect of Maesil (Prunus mume) juice on the alcohol metabolizing enzyme activities. Korean J. Food Sci. Technol. 36: 329-332.
  15. Jaganjac, M., L. Milkovic, S.B. Sunjic and N. Zarkovic. 2020. The NRF2, thioredoxin, and glutathione system in tumorigenesis and anticancer therapies. Antioxidants (Basel). 9(11):1151.
  16. Janero, D.R. 1990. Malondialdehyde and thiobarbituric acidreactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic. Biol. Med. 9(6):515-540.
  17. Kim, H.J., S.H. Yeom, M.K. Kim, J.G. Shim, I.N. Paek and M.W. Lee. 2005. Nitric oxide and prostaglandin E2 synthesis inhibitory activities of diarylheptanoids from the barks of Alnus japonica Steudel. Arch. Pharm. Res. 28(2):177-179.
  18. Kim, K.A. 2009. Understanding and application of liver function tests. Korean J. Med. 76:163-168.
  19. Kim, S.T., J.D. Kim, S.H. Ahn, G.S. Ahn, Y.I. Lee and Y.S. Jeong. 2004. Hepatoprotective and antioxidant effects of Alnus japonica extracts on acetaminophen-induced hepatotoxicity in rats. Phytother. Res. 18(12):971-975.
  20. Koch, O.R., G. Pani, S. Borrello, R. Colavitti, A. Cravero, A. Cravero, S. Farre and T. Galeotti. 2004. Oxidative stress and antioxidant defenses in ethanol-induced cell injury. Mol. Aspects Med. 25(1-2):191-198. https://doi.org/10.1016/j.mam.2004.02.019
  21. Kuroyanagi, M., M. Shimomae, Y. Nagashima, N. Muto, T. Okuda, N. Kawahara, T. Nakane and T. Sano. 2005. New diarylheptanoids from Alnus japonica and their antioxidative activity. Chem. Pharm. Bull. 53(12):1519-1523.
  22. Lee, G.H., K.H. Park and S.E. Choi. 2018. Modulative effect of human hair dermal papilla cell apoptosis by oregonin from the braches of Alnus japonica. Korean J. Plant Res. 31(4):322-329.
  23. Lee, M.W., J.H. Kim, D.W. Jeong, K.H. Ahn, S.H. Toh and Y.J. Surh. 2000a. Inhibition of cyclooxygenase-2 expression by diarylheptanoids from the bark of Alnus hirsuta var. Sibrica. Biol. Pharm. Bull. 23(4):517-518. https://doi.org/10.1248/bpb.23.517
  24. Lee, Y.A., K.H. Kim, J.S. Kim, S.M. Cho, S.W. Kim and M.W. Lee. 2000b. Antioxidative effects of diarylhepatonoids from Alnus hirsuta. Arch. Pharm. Res. 44(2):193-196.
  25. Lee, M.W., T. Tanaka, G.I. Nonaka and I. Nishioka. 1992. Dimeric ellagitannins from Alnus japonica. Phytochemistry 31(8):2835-2839. https://doi.org/10.1016/0031-9422(92)83642-C
  26. Li, S., H.Y. Tan, N. Wang, Z.J. Zhang, L. Lao, C.W. Wong and Y. Feng. 2015. The role of oxidative stress and antioxidants in liver diseases. Int. J. Mol. Sci. 16(11):26087-26124.
  27. Lin, R.C. and T.K. Li. 1998. Effects of isoflavones on alcohol pharmacokinetics and alcohol-drinking behavior in rats. Am J Clin Nutr 68: 1512S-1515S. https://doi.org/10.1093/ajcn/68.6.1512S
  28. Lu, Y. and A.I. Cederbaum. 2008. CYP2E1 and oxidative liver injury by alcohol. Free Rad. Biol. Med. 44(5):723-738.
  29. Na, C.S., S.B. Lee, J.B. Kim, H.S. Chung and M.S. Dong. 2012. Effect of hot water extract of Alnus japonica Steud on the experimentally-induced acute gastritis and peptic ulcers in rats. Kor. J. Pharmacogn. 43(1):72-78.
  30. Park, Y.M., J.A Kim, C.H. Kim, J.H. Lim and E.W. Seo. 2015. Effects of cultured Acer tegmentosum cell extract against hepatic injury induced by D-galactosamine in SD-Rats. Korean J. Plant Res. 28(5):551-560.
  31. Sheth, S.G., S.L. Flamm, F.D. Gordon and S. Chopra. 1998. AST/ALT ratio predicts cirrhosis in patients with chronic hepatitis C virus infection. Am. J. Gastroenterol. 93(1):44-48.
  32. Stevic, T., K. Savikin, G. Zdunic, T. Stanojkovic, Z. Juranic, T. Jankovic and N. Menkovic. 2010. Antioxidant, cytotoxic, and antimicrobial activity of Alnus incana (L.) ssp. incana Moench and A. viridis (Chaix) DC ssp. viridis extracts. J. Med. Food 13(3):700-704. https://doi.org/10.1089/jmf.2009.0111
  33. Wada, H., H. Tachibana, H. Fuchino and N. Tanaka. 1998. Three new diarylheptanoid glycosides from Alnus japonica. Chem. Pharm. Bull. 46(6):1054-1055.
  34. Williams, J.A. and W.X. Ding. 2020. Role of autophagy in alcohol and drug-induced liver injury. Food Chem. Toxicol. 136:111075.
  35. Wu, G., Y.Z. Fang, S. Yang, J.R. Lupton and N.D. Turner. 2004. Glutathione metabolism and its implications for health. J. Nutr. 134(3):489-492.
  36. Wu, H., P. Cai, D.L. Clemens, T.R. Jerrells, G.A. Shakeel Ansari and B.S. Kaphalia. 2006. Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: Role of nonoxidative metabolism. Toxicol. Appl. Pharmacol. 216(2):238-247.
  37. Yang, H.K., J.Y. Jin, H.J. Hong, C.H. Han and Y.J. Lee. 2012. Activity of the ethanolic extract from fruit of Empetrum nigrum var. japonicum on CCl4 induced liver injury in BALB/c mice. Korean J. Plant Res. 25(5):633-639.
  38. Yen, G.C. and H.Y. Chen. 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem. 43(1):27-32.
  39. Zhang, L.X., Y. Lv, A.M. Xu and H.Z. Wang. 2019. The prognostic significance of serum gamma-glutamyltransferase levels and AST/ALT in primary hepatic carcinoma. BMC Cancer 19:841.