Journal of Nutrition and Health

The Korean Nutrition Society (한국영양학회)
권호 표지
DOI QR코드

DOI QR Code

Protective effect of chlorophyll-removed ethanol extract of Lycium barbarum leaves against non-alcoholic fatty liver disease

클로로필 제거 구기엽 추출물의 비알코올성 지방간 보호 효과

  • Hansol Lee (Department of Food and Nutrition, Chungnam National University) ;
  • Eun Young Bae (R&D Center, Elohim) ;
  • Kyung Ah Kim (Department of Food and Nutrition, Chungnam National University) ;
  • Sun Yung Ly (Department of Food and Nutrition, Chungnam National University)
  • Received : 2023.01.18
  • Accepted : 2023.03.17
  • Published : 2023.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose: This study was conducted to establish whether an ethanol extract of Lycium barbarum leaves (LLE) and an ethanol extract of Lycium barbarum leaves from which chlorophyll has been removed, denoted as LLE(Ch-), have a protective effect against hepatic fat accumulation. Methods: The inhibitory effects of LLE and LLE(Ch-) on liver fat accumulation were examined in C57BL/6 mice with non-alcoholic fatty liver disease (NAFLD) induced by an methionine and choline deficient diet and in HepG2 cells with palmitic acid-induced fat accumulation. Results: The plasma triglyceride, aspartate aminotransferase, and alanine aminotransferase levels were lower in the LLE(Ch-) group, whereas the plasma ALT activity decreased significantly in the LLE group. In both the LLE and the LLE(Ch-) groups, the triglyceride and cholesterol contents in the hepatic tissue were significantly reduced. A greater inhibitory effect on tissue fat accumulation was observed in the LLE(Ch-) group than in the LLE group. In HepG2 cells, LLE and LLE(Ch-) were non-toxic up to a concentration of 1,000 ㎍/mL. Compared to the control group, intracellular fat accumulation in the LLE and LLE(Ch-) groups were significantly reduced at concentrations of 200 ㎍/mL and 500 ㎍/mL, respectively. The expression of phosphorylated adenosine monophosphate-activated protein kinase and phosphorylated acetyl-CoA carboxylase in both LLE groups increased at the concentrations of 100 ㎍/mL and 500 ㎍/mL. The fatty acid synthase expression was suppressed in a concentration-dependent manner at 10 ㎍/mL. Conclusion: The examined two ethanol extracts of LLE inhibit hepatic fat accumulation in NAFLD. This effect was more pronounced in the LLE(Ch-) group. Therefore, these 2 extracts have an anti-steatosis effect and can be used for NAFLD treatment.

구기엽 추출물 (LLE)과 클로로필을 제거한 구기엽 추출물 (LLE(Ch-))이 MCD diet로 비알코올성 지방간을 유도한 C57BL/6 mouse와 팔미트산으로 지방 축적을 유도한 HepG2 세포에서 지방축적의 억제에 미치는 영향을 검토하였다. 대조군 대비 LLE(Ch-)는 혈장 TG 농도와 혈장 AST와 ALT의 활성을 유의하게 감소시켰으며 LLE군에서는 혈장 ALT 활성이 유의하게 감소하였다. 두 군 모두 간조직의 TG와 cholesterol 함량을 유의하게 낮추었으며 간조직의 병리학적 변화 결과에서 LLE군에 비해 LLE(Ch-)군에서 지방축적의 억제효과가 더 크게 나타났다. 팔미트산 0.5 mM을 처리한 HepG2 세포에서 LLE와 LLE(Ch-)가 1,000 ㎍/mL 농도까지 독성이 없었으며 대조군에 비하여 각각 200 ㎍/mL과 500 ㎍/mL 농도부터 세포 내 지방 축적량을 유의하게 감소시켰고 pAMPK와 pACC 발현이 농도 의존적으로 증가하였으며 FAS발현은 농도 의존적으로 억제되었다. 결과적으로 구기엽 100% 에탄올 추출물들은 간조직의 지방 축적을 억제할 수 있으며 그 효과는 클로로필 제거 구기엽의 활성이 좀 더 큰 것으로 나타났다. 따라서 이 두 소재 모두 항 지방간 효능이 있는 것으로 판단되며 기능성 소재로서의 개발 가능성을 확인하였다.

Keywords

Acknowledgement

This study was supported by grants from the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea (NRF-2017R1D1A3B03028628).

References

  1. Korean Association for the Study of the Liver (KASL). KASL clinical practice guidelines: management of nonalcoholic fatty liver disease. Clin Mol Hepatol 2013; 19(4): 325-348. https://doi.org/10.3350/cmh.2013.19.4.325
  2. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 2016; 64(1): 73-84. https://doi.org/10.1002/hep.28431
  3. Park SH, Plank LD, Suk KT, Park YE, Lee J, Choi JH, et al. Trends in the prevalence of chronic liver disease in the Korean adult population, 1998-2017. Clin Mol Hepatol 2020; 26(2): 209-215. https://doi.org/10.3350/cmh.2019.0065
  4. Michelotti GA, Machado MV, Diehl AM. NAFLD, NASH and liver cancer. Nat Rev Gastroenterol Hepatol 2013; 10(11): 656-665. https://doi.org/10.1038/nrgastro.2013.183
  5. Argo CK, Caldwell SH. Epidemiology and natural history of non-alcoholic steatohepatitis. Clin Liver Dis 2009; 13(4): 511-531. https://doi.org/10.1016/j.cld.2009.07.005
  6. Mundi MS, Velapati S, Patel J, Kellogg TA, Abu Dayyeh BK, Hurt RT. Evolution of NAFLD and its management. Nutr Clin Pract 2020; 35(1): 72-84. https://doi.org/10.1002/ncp.10449
  7. Younossi ZM, Stepanova M, Negro F, Hallaji S, Younossi Y, Lam B, et al. Nonalcoholic fatty liver disease in lean individuals in the United States. Medicine (Baltimore) 2012; 91(6): 319-327. https://doi.org/10.1097/MD.0b013e3182779d49
  8. Jun DW. The role of diet in non-alcoholic fatty liver disease. Korean J Gastroenterol 2013; 61(5): 243-251. https://doi.org/10.4166/kjg.2013.61.5.243
  9. Ipsen DH, Tveden-Nyborg P, Lykkesfeldt J. Does vitamin C deficiency promote fatty liver disease development? Nutrients 2014; 6(12): 5473-5499. https://doi.org/10.3390/nu6125473
  10. Podszun MC, Alawad AS, Lingala S, Morris N, Huang WA, Yang S, et al. Vitamin E treatment in NAFLD patients demonstrates that oxidative stress drives steatosis through upregulation of de-novo lipogenesis. Redox Biol 2020; 37: 101710.
  11. Ghaffari A, Rafraf M, Navekar R, Asghari-Jafarabadi M. Effects of turmeric and chicory seed supplementation on antioxidant and inflammatory biomarkers in patients with non-alcoholic fatty liver disease (NAFLD). Adv Intern Med 2018; 5(3): 89-95. https://doi.org/10.1016/j.aimed.2018.01.002
  12. Woods A, Williams JR, Muckett PJ, Mayer FV, Liljevald M, Bohlooly-Y M, et al. Liver-specific activation of AMPK prevents steatosis on a high-fructose diet. Cell Reports 2017; 18(13): 3043-3051. https://doi.org/10.1016/j.celrep.2017.03.011
  13. Coughlan KA, Valentine RJ, Ruderman NB, Saha AK. AMPK activation: a therapeutic target for type 2 diabetes? Diabetes Metab Syndr Obes 2014; 7: 241-253. https://doi.org/10.2147/DMSO.S43731
  14. Liu JF, Ma Y, Wang Y, Du ZY, Shen JK, Peng HL. Reduction of lipid accumulation in HepG2 cells by luteolin is associated with activation of AMPK and mitigation of oxidative stress. Phytother Res 2011; 25(4): 588-596. https://doi.org/10.1002/ptr.3305
  15. Garcia D, Hellberg K, Chaix A, Wallace M, Herzig S, Badur MG, et al. Genetic liver-specific AMPK activation protects against diet-induced obesity and NAFLD. Cell Rep 2019; 26(1): 192-208.e6. https://doi.org/10.1016/j.celrep.2018.12.036
  16. Herzig S, Shaw RJ. AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol 2018; 19(2): 121-135. https://doi.org/10.1038/nrm.2017.95
  17. Ali MC, Chen J, Zhang H, Li Z, Zhao L, Qiu H. Effective extraction of flavonoids from Lycium barbarum L. fruits by deep eutectic solvents-based ultrasound-assisted extraction. Talanta 2019; 203: 16-22. https://doi.org/10.1016/j.talanta.2019.05.012
  18. Bae S, Kim J, Bae E, Kim K, Ly SY. Anti-inflammatory effects of fruit and leaf extracts of Lycium barbarum in lipopolysaccharide-stimulated RAW264.7 cells and animal model. J Nutr Health 2019; 52(2): 129-138. https://doi.org/10.4163/jnh.2019.52.2.129
  19. Kim JE, Bae SM, Nam YR, Bae EY, Ly SY. Antioxidant activity of ethanol extract of Lycium barbarum's leaf with removal of chlorophyll. J Nutr Health 2019; 52(1): 26-35. https://doi.org/10.4163/jnh.2019.52.1.26
  20. Xia G, Xin N, Liu W, Yao H, Hou Y, Qi J. Inhibitory effect of Lycium barbarum polysaccharides on cell apoptosis and senescence is potentially mediated by the p53 signaling pathway. Mol Med Rep 2014; 9(4): 1237-1241. https://doi.org/10.3892/mmr.2014.1964
  21. Mao F, Xiao B, Jiang Z, Zhao J, Huang X, Guo J. Anticancer effect of Lycium barbarum polysaccharides on colon cancer cells involves G0/G1 phase arrest. Med Oncol 2011; 28(1): 121-126. https://doi.org/10.1007/s12032-009-9415-5
  22. Zhu CP, Zhang SH. Lycium barbarum polysaccharide inhibits the proliferation of HeLa cells by inducing apoptosis. J Sci Food Agric 2013; 93(1): 149-156. https://doi.org/10.1002/jsfa.5743
  23. Wang J, Hu Y, Wang D, Zhang F, Zhao X, Abula S, et al. Lycium barbarum polysaccharide inhibits the infectivity of Newcastle disease virus to chicken embryo fibroblast. Int J Biol Macromol 2010; 46(2): 212-216. https://doi.org/10.1016/j.ijbiomac.2009.11.011
  24. Olatunji OJ, Chen H, Zhou Y. Lycium chinense leaves extract ameliorates diabetic nephropathy by suppressing hyperglycemia mediated renal oxidative stress and inflammation. Biomed Pharmacother 2018; 102: 1145-1151. https://doi.org/10.1016/j.biopha.2018.03.037
  25. Xiao X, Ren W, Zhang N, Bing T, Liu X, Zhao Z, et al. Comparative study of the chemical constituents and bioactivities of the extracts from fruits, leaves and root barks of Lycium barbarum. Molecules 2019; 24(8): 1585.
  26. Zhao XQ, Guo S, Lu YY, Hua Y, Zhang F, Yan H, et al. Lycium barbarum L. leaves ameliorate type 2 diabetes in rats by modulating metabolic profiles and gut microbiota composition. Biomed Pharmacother 2020; 121: 109559.
  27. Senklang P, Anprung P. Optimizing enzymatic extraction of Zn-chlorophyll derivatives from pandan leaf using response surface methodology. J Food Process Preserv 2010; 34(5): 759-776. https://doi.org/10.1111/j.1745-4549.2009.00393.x
  28. Hsiao CJ, Lin JF, Wen HY, Lin YM, Yang CH, Huang KS, et al. Enhancement of the stability of chlorophyll using chlorophyll-encapsulated polycaprolactone microparticles based on droplet microfluidics. Food Chem 2020; 306: 125300.
  29. Olatunde OO, Benjakul S, Huda N, Zhang B, Deng S. Ethanolic Noni (Morinda citrifolia L.) leaf extract dechlorophyllised using sedimentation process: Antioxidant, antibacterial properties and efficacy in extending the shelf-life of striped catfish slices. Int J Food Sci Technol 2021; 56(6): 2804-2819. https://doi.org/10.1111/ijfs.14917
  30. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37(8): 911-917. https://doi.org/10.1139/y59-099
  31. Huynh FK, Green MF, Koves TR, Hirschey MD. Measurement of fatty acid oxidation rates in animal tissues and cell lines. Methods Enzymol 2014; 542: 391-405. https://doi.org/10.1016/B978-0-12-416618-9.00020-0
  32. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72(1-2): 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  33. Anstee QM, Goldin RD. Mouse models in non-alcoholic fatty liver disease and steatohepatitis research. Int J Exp Pathol 2006; 87(1): 1-16. https://doi.org/10.1111/j.0959-9673.2006.00465.x
  34. Pacana T, Cazanave S, Verdianelli A, Patel V, Min HK, Mirshahi F, et al. Dysregulated hepatic methionine metabolism drives homocysteine elevation in diet-induced nonalcoholic fatty liver disease. PLoS One 2015; 10(8): e0136822.
  35. Caballero F, Fernandez A, Matias N, Martinez L, Fucho R, Elena M, et al. Specific contribution of methionine and choline in nutritional nonalcoholic steatohepatitis: impact on mitochondrial S-adenosyl-L-methionine and glutathione. J Biol Chem 2010; 285(24): 18528-18536. https://doi.org/10.1074/jbc.M109.099333
  36. Muriel P, Ramos-Tovar E, Montes-Paez G, Buendia-Montano LD. Experimental models of liver damage mediated by oxidative stress. In: Muriel P, editor. Liver Pathophysiology. London: Academic Press; 2017. p.529-546.
  37. Umemura A, Kataoka S, Okuda K, Seko Y, Yamaguchi K, Moriguchi M, et al. Potential therapeutic targets and promising agents for combating NAFLD. Biomedicines 2022; 10(4): 901.
  38. Bae UJ, Oh MR, Park J, Park JS, Bae EY, Chae SW, et al. Supplementation with Lycium chinense fruit extract attenuates methionine choline-deficient diet-induced steatohepatitis in mice. J Funct Foods 2017; 31: 1-8. https://doi.org/10.1016/j.jff.2017.01.032
  39. Mocan A, Vlase L, Vodnar DC, Bischin C, Hanganu D, Gheldiu AM, et al. Polyphenolic content, antioxidant and antimicrobial activities of Lycium barbarum L. and Lycium chinense Mill. leaves. Molecules 2014; 19(7): 10056-10073. https://doi.org/10.3390/molecules190710056
  40. Kim SB, Kang OH, Lee YS, Han SH, Ahn YS, Cha SW, et al. Hepatoprotective effect and synergism of bisdemethoycurcumin against MCD diet-induced nonalcoholic fatty liver disease in mice. PLoS One 2016; 11(2): e0147745.
  41. Dong JZ, Lu DY, Wang Y. Analysis of flavonoids from leaves of cultivated Lycium barbarum L. Plant Foods Hum Nutr 2009; 64(3): 199-204. https://doi.org/10.1007/s11130-009-0128-x
  42. Lei Z, Chen X, Cao F, Guo Q, Wang J. Phytochemicals and bioactivities of Goji (Lycium barbarum L. and Lycium chinense Mill.) leaves and their potential applications in the food industry: a review. Int J Food Sci Technol 2022; 57(3): 1451-1461. https://doi.org/10.1111/ijfs.15507
  43. Xu XC, Chen WJ, Yu SK, Lei Q, Han LH, Ma WP. Inhibition of preadipocyte differentiation by Lycium barbarum polysaccharide treatment in 3T3-L1 cultures. Electron J Biotechnol 2021; 50: 53-58. https://doi.org/10.1016/j.ejbt.2021.01.003
  44. Jia L, Li W, Li J, Li Y, Song H, Luan Y, et al. Lycium barbarum polysaccharide attenuates high-fat diet-induced hepatic steatosis by up-regulating SIRT1 expression and deacetylase activity. Sci Rep 2016; 6(1): 36209.
  45. Li W, Li Y, Wang Q, Yang Y. Crude extracts from Lycium barbarum suppress SREBP-1c expression and prevent diet-induced fatty liver through AMPK activation. BioMed Res Int 2014; 2014: 196198.
  46. Fang K, Wu F, Chen G, Dong H, Li J, Zhao Y, et al. Diosgenin ameliorates palmitic acid-induced lipid accumulation via AMPK/ACC/CPT-1A and SREBP-1c/FAS signaling pathways in LO2 cells. BMC Complement Altern Med 2019; 19(1): 255.
  47. Yang J, Maika S, Craddock L, King JA, Liu ZM. Chronic activation of AMP-activated protein kinase-alpha1 in liver leads to decreased adiposity in mice. Biochem Biophys Res Commun 2008; 370(2): 248-253. https://doi.org/10.1016/j.bbrc.2008.03.094
  48. Lin J, Zhang Y, Wang X, Wang W. Lycium ruthenicum extract alleviates high-fat diet-induced nonalcoholic fatty liver disease via enhancing the AMPK signaling pathway. Mol Med Rep 2015; 12(3): 3835-3840. https://doi.org/10.3892/mmr.2015.3840
  49. Stephenson K, Kennedy L, Hargrove L, Demieville J, Thomson J, Alpini G, et al. Updates on dietary models of nonalcoholic fatty liver disease: current studies and Insights. Gene Expr 2018; 18(1): 5-17. https://doi.org/10.3727/105221617X15093707969658