High hydrostatic pressure extract of mulberry leaves ameliorates hypercholesterolemia via modulating hepatic microRNA-33 expression and AMPK activity in high cholesterol diet fed rats

  • Eunyoung Lee Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, South Korea
  • Mak-Soon Lee Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, South Korea
  • Eugene Chang Department of Food and Nutrition, Gangneung-Wonju National University, Gangneung-si, Gangwon-do, South Korea
  • Chong-Tai Kim R&D Center, EastHill Corporation, Gwonseon-gu, Suwon-si, Gyeonggi-do, South Korea
  • Ae-Jin Choi Functional Food & Nutrition Division, National Institute of Agricultural Science (NIAS), Rural Development Administration (RDA), Wanju, jeolabuk-do, South Korea
  • In-Hwan Kim Department of Integrated Biomedical and Life Sciences, Korea University, Seoul, South Korea
  • Yangha Kim Department of Nutritional Science and Food Management, Ewha Womans University, Seoul; and Graduate Program in System Health Science and Engineering, Ewha Womans University, Seoul, South Korea
Keywords: adenosine monophosphate-activated protein kinase (AMPK); bile acid; cholesterol; mulberry leaf extract; microRNA-33


Background: Mulberry leaf (Morus alba L.) contains multiple bioactive ingredients and has been used in the treatment of obesity, diabetes, inflammation, and atherosclerosis. High hydrostatic pressure (HHP) processing has been developed for the extraction of bioactive compounds from plants. However, the hypocholesterolemic effect of the HHP extract from mulberry leaves and its underlying mechanism have never been investigated.

Objective: The specific aim of the present study was to investigate the hypocholesterolemic property of a novel extract obtained from mulberry leaves under HHP in rats.

Design: Sprague–Dawley rats were divided into four groups and fed either a normal diet (NOR), a high cholesterol diet containing 1% cholesterol and 0.5% cholic acid (HC), an HC diet containing 0.5% mulberry leaf extract (ML), or a 1% mulberry leaf extract (MH) for 4 weeks.

Results: High hydrostatic pressure extract of mulberry leaves significantly reduced the HC-increased serum levels of triglyceride (TG), cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), and hepatic contents of TG and TC. The HHP extraction from mulberry leaves also increased the HC-decreased fecal TC and bile acid levels without changing body weight, food intake, liver weight, and serum activities of alanine transaminase (ALT) and aspartate transaminase (AST) (P < 0.05). The mulberry leaf extract significantly enhanced the expression of hepatic genes such as cholesterol 7 alpha-hydroxylase (CYP7A1), liver X receptor alpha (LXRα), and ATP-binding cassette transporters, ABCG5/ABCG8, involved in hepatic bile acid synthesis and cholesterol efflux (P < 0.05). In addition, the HHP extraction of mulberry leaves significantly suppressed hepatic microRNA(miR)-33 expression and increased adenosine monophosphate-activated protein kinase (AMPK) activity.

Conclusion: These results suggest that the HHP extract of mulberry leaves lowers serum cholesterol levels by partially increasing hepatic bile acid synthesis and fecal cholesterol excretion through the modulation of miR- 33 expression and AMPK activation in the liver.


Download data is not yet available.


  1. Barquera S, Pedroza-Tobias A, Medina C, Hernandez-Barrera L, Bibbins-Domingo K, Lozano R, et al. Global overview of the epidemiology of atherosclerotic cardiovascular disease. Arch Med Res 2015; 46(5): 328–38. doi: 10.1016/j.arcmed.2015.06.006

  2. Stone NJ, Robinson JG, Lichtenstein AH, Bairey Merz CN, Blum CB, Eckel RH, et al. 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol 2014; 63(25 Pt B): 2889–934. doi: 10.1016/j.jacc.2013.11.002.

  3. Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Mangelsdorf DJ. Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta. J Biol Chem 2002; 277(21): 18793–800. doi: 10.1074/jbc.M109927200

  4. Zelcer N, Hong C, Boyadjian R, Tontonoz P. LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor. Science 2009; 325(5936): 100–4.

  5. Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE, et al. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha. Cell 1998; 93(5): 693–704. doi: 10.1016/s0092-8674(00)81432-4

  6. Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA, et al. Synthetic LXR ligand inhibits the development of atherosclerosis in mice. Proc Natl Acad Sci U S A 2002; 99(11): 7604–9. doi: 10.1073/pnas.112059299

  7. Katsube T, Yamasaki M, Shiwaku K, Ishijima T, Matsumoto I, Abe K, et al. Effect of flavonol glycoside in mulberry (Morus alba L.) leaf on glucose metabolism and oxidative stress in liver in diet-induced obese mice. J Sci Food Agric 2010; 90(14): 2386–92. doi: 10.1002/jsfa.4096

  8. Ann JY, Eo H, Lim Y. Mulberry leaves (Morus alba L.) ameliorate obesity-induced hepatic lipogenesis, fibrosis, and oxidative stress in high-fat diet-fed mice. Genes Nutr 2015; 10(6): 46. doi: 10.1007/s12263-015-0495-x

  9. Yang MY, Huang CN, Chan KC, Yang YS, Peng CH, Wang CJ. Mulberry leaf polyphenols possess antiatherogenesis effect via inhibiting LDL oxidation and foam cell formation. J Agric Food Chem 2011; 59(5): 1985–95. doi: 10.1021/jf103661v

  10. Peng CH, Lin HT, Chung DJ, Huang CN, Wang CJ. Mulberry leaf extracts prevent obesity-induced NAFLD with regulating adipocytokines, inflammation and oxidative stress. J Food Drug Anal 2018; 26(2): 778–87. doi: 10.1016/j.jfda.2017.10.008

  11. Sheng Y, Liu J, Zheng S, Liang F, Luo Y, Huang K, et al. Mulberry leaves ameliorate obesity through enhancing brown adipose tissue activity and modulating gut microbiota. Food Funct 2019; 10(8): 4771–81. doi: 10.1039/c9fo00883g

  12. Kim GN, Jang HD. Flavonol content in the water extract of the mulberry (Morus alba L.) leaf and their antioxidant capacities. J Food Sci 2011; 76(6): C869–73. doi: 10.1111/j.1750-3841.2011.02262.x

  13. Park E, Lee S-M, Lee EJ, Kim J-H. Anti-inflammatory activity of mulberry leaf extract through inhibition of NF-κB. J Funct Foods 2013; 5(1): 178–86. doi: 10.1016/j.jff.2012.10.002

  14. Ren C, Zhang Y, Cui W, Lu G, Wang Y, Gao H, et al. A polysaccharide extract of mulberry leaf ameliorates hepatic glucose metabolism and insulin signaling in rats with type 2 diabetes induced by high fat-diet and streptozotocin. Int J Biol Macromol 2015; 72: 951–9. doi: 10.1016/j.ijbiomac.2014.09.060

  15. Kobayashi Y, Miyazawa M, Kamei A, Abe K, Kojima T. Ameliorative effects of mulberry (Morus alba L.) leaves on hyperlipidemia in rats fed a high-fat diet: induction of fatty acid oxidation, inhibition of lipogenesis, and suppression of oxidative stress. Biosci Biotechnol Biochem 2010; 74(12): 2385–95. doi: 10.1271/bbb.100392

  16. Huang J, Wang Y, Ying C, Liu L, Lou Z. Effects of mulberry leaf on experimental hyperlipidemia rats induced by high-fat diet. Exp Ther Med 2018; 16(2): 547–56. doi: 10.3892/etm.2018.6254

  17. Lopes ML, Valente Mesquita VL, Chiaradia AC, Fernandes AA, Fernandes PM. High hydrostatic pressure processing of tropical fruits. Ann N Y Acad Sci 2010; 1189: 6–15. doi: 10.1111/j.1749-6632.2009.05177.x

  18. Yamamoto K. Food processing by high hydrostatic pressure. Biosci Biotechnol Biochem 2017; 81(4): 672–9. doi: http://dx.doi.org/10.1111/j.1749-6632.2009.05177.x

  19. Butz P, Garcı́a AF, Lindauer R, Dieterich S, Bognar A, Tauscher B. Influence of ultra high pressure processing on fruit and vegetable products. J Food Eng 2003; 56(2–3): 233–6. doi: 10.1016/S0260-8774(02)00258-3

  20. Wang F, Du BL, Cui ZW, Xu LP, Li CY. Effects of high hydrostatic pressure and thermal processing on bioactive compounds, antioxidant activity, and volatile profile of mulberry juice. Food Sci Technol Int 2017; 23(2): 119–27. doi: 10.1177/1082013216659610

  21. Jung S, Lee MS, Choi AJ, Kim CT, Kim Y. Anti-inflammatory effects of high hydrostatic pressure extract of mulberry (Morus alba) fruit on LPS-stimulated RAW264.7 Cells. Molecules 2019; 24(7): 1425. doi: 10.3390/molecules24071425

  22. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18(6): 499–502. doi: 10.1093/clinchem/18.6.499

  23. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37(8): 911–17. doi: 10.1139/o59-099

  24. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta C(T)) method. Methods 2001; 25(4): 402–8. doi: 10.1006/meth.2001.1262

  25. Kim S, Lee MS, Jung S, Son HY, Park S, Kang B, et al. Ginger extract ameliorates obesity and inflammation via regulating MicroRNA-21/132 expression and AMPK activation in white adipose tissue. Nutrients 2018; 10(11): 1567. doi: 10.3390/nu10111567

  26. Sharrett AR, Ballantyne CM, Coady SA, Heiss G, Sorlie PD, Catellier D, et al. Coronary heart disease prediction from lipoprotein cholesterol levels, triglycerides, lipoprotein(a), apolipoproteins A-I and B, and HDL density subfractions: the Atherosclerosis Risk in Communities (ARIC) study. Circulation 2001; 104(10): 1108–13. doi: 10.1161/hc3501.095214

  27. Tontonoz P, Mangelsdorf DJ. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol 2003; 17(6): 985–93. doi: 10.1210/me.2003-0061

  28. Doi K, Kojima T, Makino M, Kimura Y, Fujimoto Y. Studies on the constituents of the leaves of Morus alba L. Chem Pharm Bull (Tokyo) 2001; 49(2): 151–3. doi: 10.1248/cpb.49.151

  29. Kojima Y, Kimura T, Nakagawa K, Asai A, Hasumi K, Oikawa S, et al. Effects of mulberry leaf extract rich in 1-deoxynojirimycin on blood lipid profiles in humans. J Clin Biochem Nutr 2010; 47(2): 155–61. doi: 10.3164/jcbn.10-53

  30. Aramwit P, Petcharat K, Supasyndh O. Efficacy of mulberry leaf tablets in patients with mild dyslipidemia. Phytother Res 2011; 25(3): 365–9. doi: 10.1002/ptr.3270

  31. Aramwit P, Supasyndh O, Siritienthong T, Bang N. Mulberry leaf reduces oxidation and C-reactive protein level in patients with mild dyslipidemia. Biomed Res Int 2013; 2013: 787981. doi: 10.1155/2013/787981

  32. Groen AK, Bloks VW, Verkade H, Kuipers F. Cross-talk between liver and intestine in control of cholesterol and energy homeostasis. Mol Aspects Med 2014; 37: 77–88. doi: 10.1016/j.mam.2014.02.001

  33. Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, et al. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 2000; 6(3): 507–15. doi: 10.1016/s1097-2765(00)00050-2

  34. Yu L, Hammer RE, Li-Hawkins J, Von Bergmann K, Lutjohann D, Cohen JC, et al. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc Natl Acad Sci U S A 2002; 99(25): 16237–42. doi: 10.1073/pnas.252582399

  35. Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, et al. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest 2002; 110(5): 671–80. doi: 10.1172/jci16001

  36. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009; 136(2): 215–33. doi: 10.1016/j.cell.2009.01.002

  37. Goedeke L, Fernandez-Hernando C. Regulation of cholesterol homeostasis. Cell Mol Life Sci 2012; 69(6): 915–30. doi: 10.1007/s00018-011-0857-5

  38. Hardie DG, Pan DA. Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase. Biochem Soc Trans 2002; 30(Pt 6): 1064–70. doi: 10.1042/bst0301064

  39. Wu C-H, Chen S-C, Ou T-T, Chyau C-C, Chang Y-C, Wang C-J. Mulberry leaf polyphenol extracts reduced hepatic lipid accumulation involving regulation of adenosine monophosphate activated protein kinase and lipogenic enzymes. J Funct Foods 2013; 5(4): 1620–32. doi: 10.1016/j.jff.2013.07.004

  40. Kobayashi Y, Miyazawa M, Araki M, Kamei A, Abe K, Hiroi T, et al. Effects of Morus alba L (Mulberry) leaf extract in hypercholesterolemic mice on suppression of cholesterol synthesis. J Pharmacogn Nat Prod 2015; 2: 1–9. doi: 10.4172/2472-0992.1000113

  41. Yang J, Craddock L, Hong S, Liu ZM. AMP-activated protein kinase suppresses LXR-dependent sterol regulatory element-binding protein-1c transcription in rat hepatoma McA-RH7777 cells. J Cell Biochem 2009; 106(3): 414–26. doi: 10.1002/jcb.22024

How to Cite
Lee, E., Lee, M.-S., Chang, E., Kim, C.-T., Choi, A.-J., Kim, I.-H., & Kim, Y. (2021). High hydrostatic pressure extract of mulberry leaves ameliorates hypercholesterolemia via modulating hepatic microRNA-33 expression and AMPK activity in high cholesterol diet fed rats. Food & Nutrition Research, 65. https://doi.org/10.29219/fnr.v65.7587
Original Articles