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Research Article | Open Access

5-Demethylnobiletin and its major metabolites: efficient preparation and mechanism of their anti-proliferation activity in HepG2 cells

Yanping Xin^{^a^,^b^,^c^,¹}, Ting Zheng^{^c^,¹}, Man Zhang^{^c}, Ruiqiang Zhang^{^a}, Siyue Zhu^{^c}, Dongli Li^{^a^,^b}(), Denggao Zhao^{^a^,^b}, Yanyan Ma^{^a^,^b}, Chi-Tang Ho^{^c}, Qingrong Huang^{^c}()

a

School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China

b

International Healthcare Innovation Institute (Jiangmen), Jiangmen 529020, China

c

Department of Food Science, Rutgers University, New Jersey 08901, USA

¹ These two authors contribute equally.

Peer review under responsibility of KeAi Communications Co., Ltd.

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Abstract

5-Demethylnobiletin (5-DMN), a hydroxylated polymethoxyflavone (OH-PMF) identified in aged citrus peels, has demonstrated health benefiting effects in previous studies. 5-DMN undergoes biotransformation in vivo, yielding 5,3'-didemethylnobiletin (5,3'-DDMN), 5,4'-didemethylnobiletin (5,4'-DDMN) and 5,3',4'-tridemethylnobiletin (5,3',4'-TDMN). However, the anti-cancer effects of 5-DMN and its in vivo metabolites against HepG2 cells remain unclear. In this study, an efficient chemical synthetic method was developed to obtain 5-DMN and its 3 metabolites, and their molecular structures were confirmed by ¹H NMR and LC-MS. Cytotoxicity, cell cycle arrestment, apoptosis and caspase-3 expression were investigated to evaluate the anti-liver cancer effects of these OH-PMFs on HepG2 cells. The results showed that all 4 compounds inhibited the proliferation of HepG2 cells in a concentration-dependent manner. Their anti-proliferative activity was exerted through inducing G2/M phase arrestment, cell apoptosis and promoting expression of a key apoptotic protein called cleaved caspase-3. Our results indicated that 5,3'-DDMN and 5,3',4'-TDMN showed a stronger inhibitory activity on cell proliferation than 5-DMN, followed by 5,4'-DDMN. The expression of cleaved caspase-3 was the highest in cells treated with 5,4'-DDMN, implying that the apoptosis induced by other OH-PMFs might be mediated by other apoptotic execution proteins. Our research reveals the application potential and scientific evidence for the production and functionality of OH-PMFs.

Keywords

Hydroxylated polymethoxyflavones Chemical synthesis HepG2 Cell cycle Apoptosis

References

[1]

L. Kulik, H.B. El-Serag, Epidemiology and management of hepatocellular carcinomam, Gastroenterology 156(2) (2019) 477-491. https://doi.org/10.1053/j.gastro.2018.08.065.

Crossref Google Scholar

[2]

H. Wege, J. Li, H. Ittrich, Treatment lines in hepatocellular carcinoma, Visc. Med. 35(4) (2019) 266-272. https://doi.org/10.1159/000501749.

Crossref Google Scholar

[3]

R. Sasaki, T. Kanda, M. Fujisawa, et al., Different mechanisms of action of regorafenib and lenvatinib on toll-like receptor-signaling pathways in human hepatoma cell lines, Int. J. Mol. Sci. 21(9) (2020) 33-49. https://doi.org/10.3390/ijms21093349.

Crossref Google Scholar

[4]

M. Ikeda, M. Kobayashi, M. Tahara, et al., Optimal management of patients with hepatocellular carcinoma treated with lenvatinib, Expert. Opin. Drug Saf. 17(11) (2018) 1095-1105. https://doi.org/10.1080/14740338.2018.1530212.

Crossref Google Scholar

[5]

N. Personeni, T. Pressiani, A. Santoro, et al., Regorafenib in hepatocellular carcinoma: latest evidence and clinical implications, Drugs Context 7 (2018) 212533. https://doi.org/10.7573/dic.212533.

Crossref Google Scholar

[6]

J. Balogh, D. Victor Ⅲ, E.H. Asham, et al., Hepatocellular carcinoma: a review, J. Hepatocell Carcinoma 3(3) (2016) 41-53. https://doi.org/10.2147/JHC.S61146.

Crossref Google Scholar

[7]

J.X.H. Goh, L.T. Tan, J.K. Goh, et al., Nobiletin and derivatives: functional compounds from citrus fruit peel for colon cancer chemoprevention, Cancers (Basel) 11(6) (2019) 11-46. https://doi.org/10.3390/cancers11060867.

Crossref Google Scholar

[8]

C.S. Lai, S. Li, C.Y. Chai, et al., Anti-inflammatory and antitumor promotional effects of a novel urinary metabolite, 3',4'-didemethylnobiletin, derived from nobiletin, Carcinogenesis 29(12) (2008) 2415-2424. https://doi.org/10.1093/carcin/bgn222.

Crossref Google Scholar

[9]

K.H. Lu, H.Y. Lee, Y.L. Chu, et al., Bitter orange peel extract induces endoplasmic reticulum-mediated autophagy in human hepatoma cells, J. Funct. Foods 60 (2019) 103404. https://doi.org/10.1016/j.jff.2019.06.006.

Crossref Google Scholar

[10]

Y.H. Lo, M.H. Pan, S. Li, et al., Nobiletin metabolite, 3',4'-dihydroxy-5,6,7,8-tetramethoxyflavone, inhibits LDL oxidation and down-regulates scavenger receptor expression and activity in THP-1 cells, Biochim. Biophys. Acta 1801(2) (2010) 114-126. https://doi.org/10.1016/j.bbalip.2009.10.002.

Crossref Google Scholar

[11]

X. Wen, H. Zhao, L. Wang, et al., Nobiletin attenuates dss-induced intestinal barrier damage through the HNF4alpha-claudin-7 signaling pathway, J. Agric. Food Chem. 68(16) (2020) 4641-4649. https://doi.org/10.1021/acs.jafc.0c01217.

Crossref Google Scholar

[12]

N.H. Nguyen, Q.T.H. Ta, Q.T. Pham, et al., Anticancer activity of novel plant extracts and compounds from Adenosma bracteosum (Bonati) in human lung and liver cancer cells, Molecules 25(12) (2020) 2512-2912. https://doi.org/10.3390/molecules25122912.

Crossref Google Scholar

[13]

C. DiMarco-Crook, K. Rakariyatham, Z. Li, et al., Synergistic anticancer effects of curcumin and 3',4'-didemethylnobiletin in combination on colon cancer cells, J. Food Sci. 85(4) (2020) 1292-1301. https://doi.org/10.1111/1750-3841.15073.

Crossref Google Scholar

[14]

A. Nakajima, Y. Aoyama, T.T. Nguyen, et al., Nobiletin, a citrus flavonoid, ameliorates cognitive impairment, oxidative burden, and hyperphosphorylation of tau in senescence-accelerated mouse, Behav. Brain Res. 250 (2013) 351-360. https://doi.org/10.1016/j.bbr.2013.05.025.

Crossref Google Scholar

[15]

K. Feng, X. Zhu, T. Chen, et al., Prevention of obesity and hyperlipidemia by heptamethoxyflavone in high-fat diet-induced rats, J. Agric. Food Chem. 67(9) (2019) 2476-2489. https://doi.org/10.1021/acs.jafc.8b05632.

Crossref Google Scholar

[16]

C. Zhao, F. Wang, Y. Lian, et al., Biosynthesis of citrus flavonoids and their health effects, Crit. Rev. Food Sci. Nutr. 60(4) (2020) 566-583. https://doi.org/10.1080/10408398.2018.1544885.

Crossref Google Scholar

[17]

S. Guo, Y. Zhang, Z. Wu, et al., Synergistic combination therapy of lung cancer: cetuximab functionalized nanostructured lipid carriers for the codelivery of paclitaxel and 5-demethylnobiletin, Biomed. Pharmacother. 118 (2019) 109225. https://doi.org/10.1016/j.biopha.2019.109225.

Crossref Google Scholar

[18]

X. Huang, J. Zhu, L. Wang, et al., Inhibitory mechanisms and interaction of tangeretin, 5-demethyltangeretin, nobiletin, and 5-demethylnobiletin from citrus peels on pancreatic lipase: kinetics, spectroscopies, and molecular dynamics simulation, Int. J. Biol. Macromol. 164 (2020) 1927-1938. https://doi.org/10.1016/j.ijbiomac.2020.07.305.

Crossref Google Scholar

[19]

M. Song, Y. Lan, X. Wu, et al., The chemopreventive effect of 5-demethylnobiletin, a unique citrus flavonoid, on colitis-driven colorectal carcinogenesis in mice is associated with its colonic metabolites, Food Funct. 11(6) (2020) 4940-4952. https://doi.org/10.1039/d0fo00616e.

Crossref Google Scholar

[20]

M. Wang, D. Meng, P. Zhang, et al., Antioxidant protection of nobiletin, 5-demethylnobiletin, tangeretin, and 5-demethyltangeretin from citrus peel in Saccharomyces cerevisiae, J. Agric. Food Chem. 66(12) (2018) 3155-3160. https://doi.org/10.1021/acs.jafc.8b00509.

Crossref Google Scholar

[21]

S.P. Chiu, M.J. Wu, P.Y. Chen, et al., Neurotrophic action of 5-hydroxylated polymethoxyflavones: 5-demethylnobiletin and gardenin A stimulate neuritogenesis in PC12 cells, J. Agric. Food Chem. 61(39) (2013) 9453-9463. https://doi.org/10.1021/jf4024678.

Crossref Google Scholar

[22]

S. Li, Z. Wang, S. Sang, et al., Identification of nobiletin metabolites in mouse urine, Mol. Nutr. Food Res. 50(3) (2006) 291-299. https://doi.org/10.1002/mnfr.200500214.

Crossref Google Scholar

[23]

G.J. Wei, J.F. Sheen, W.C. Lu, et al., Identification of sinensetin metabolites in rat urine by an isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry, J. Agric. Food Chem. 61(21) (2013) 5016-5021. https://doi.org/10.1021/jf3046768.

Crossref Google Scholar

[24]

X. Wu, Z. Li, Y. Sun, et al., Identification of xanthomicrol as a major metabolite of 5-demethyltangeretin in mouse gastrointestinal tract and its inhibitory effects on colon cancer cells, Front. Nutr. 7(10) (2020) 103-118. https://doi.org/10.3389/fnut.2020.00103.

Crossref Google Scholar

[25]

S. Li, H. Wang, L. Guo, et al., Chemistry and bioactivity of nobiletin and its metabolites, J. Funct. Foods 6 (2014) 2-10. https://doi.org/10.1016/j.jff.2013.12.011.

Crossref Google Scholar

[26]

S. Li, M.H. Pan, C.S. Lai, et al., Isolation and syntheses of polymethoxyflavones and hydroxylated polymethoxyflavones as inhibitors of HL-60 cell lines, Bioorgan. Med. Chem. 15(10) (2007) 3381-3389. https://doi.org/10.1016/j.bmc.2007.03.021.

Crossref Google Scholar

[27]

O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem. 31(2) (2010) 455-461. https://doi.org/10.1002/jcc.21334.

Crossref Google Scholar

[28]

M. Imran, A. Rauf, T. Abu-Izneid, et al., Luteolin, a flavonoid, as an anticancer agent: a review, Biomed. Pharmacother. 112(112) (2019) 108612. https://doi.org/10.1016/j.biopha.2019.108612.

Crossref Google Scholar

[29]

J. Wang, Y. Duan, D. Zhi, et al., Pro-apoptotic effects of the novel tangeretin derivate 5-acetyl-6,7,8,4'-tetramethylnortangeretin on MCF-7 breast cancer cells, Cell Biochem. Biophys. 70(2) (2014) 1255-1263. https://doi.org/10.1007/s12013-014-0049-7.

Crossref Google Scholar

[30]

X. Ma, S. Jin, Y. Zhang, et al., Inhibitory effects of nobiletin on hepatocellular carcinoma in vitro and in vivo, Phytotherrpy Research 28 (2013) 560–567. https://doi.org/10.1002/ptr.5024.

Crossref Google Scholar

[31]

M.L. Tan, J.P. Ooi, N. Ismail, et al., Programmed cell death pathways and current antitumor targets, Pharm. Res. 26(7) (2009) 1547-1560. https://doi.org/10.1007/s11095-009-9895-1.

Crossref Google Scholar

[32]

J. Zhang, X. Zheng, P. Wang, et al., Role of apoptosis repressor with caspase recruitment domain (ARC) in cell death and cardiovascular disease, Apoptosis 26(1/2) (2021) 24-37. https://doi.org/10.1007/s10495-020-01653-x.

Crossref Google Scholar

[33]

A.G. Porter, R.U. Jänicke, Emerging roles of caspase-3 in apoptosis, Cell Death Differ. 6 (1999) 99-104. https://doi.org/10.1038/sj.cdd.4400476.

Crossref Google Scholar

[34]

J. Zheng, J. Bi, D. Johnson, et al., Analysis of 10 metabolites of polymethoxyflavones with high sensitivity by electrochemical detection in high-performance liquid chromatography, J. Agric. Food Chem. 63(2) (2015) 509-516. https://doi.org/10.1021/jf505545x.

Crossref Google Scholar

[35]

H. Zhang, G. Tian, C. Zhao, et al., Characterization of polymethoxyflavone demethylation during drying processes of citrus peels, Food Funct. 10(9) (2019) 5707-5717. https://doi.org/10.1039/c9fo01053j.

Crossref Google Scholar

[36]

F.P. Guengerich, Comparisons of catalytic selectivity of cytochrome P450 subfamily enzymes from different species, Chem. Biol. Interact. 106 (1997) 161-182. https://doi.org/10.1016/S0009-2797(97)00068-9.

Crossref Google Scholar

[37]

S.E. Nielsen, V. Breinholt, U. Justesen, et al., In vitro biotransformation of flavonoids by rat liver microsomes, Xenobiotica 28 (1998) 389-401. https://doi.org/doi.org/10.1080/004982598239498.

Crossref Google Scholar

[38]

N. Koga, C. Ohta, Y. Kato, et al., In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450, Xenobiotica 41(11) (2011) 927-933. https://doi.org/10.3109/00498254.2011. 593208.

Crossref Google Scholar

[39]

M. Zhang, Y. Xin, K. Feng, et al., Comparative analyses of bioavailability, biotransformation, and excretion of nobiletin in lean and obese rats, J. Agric. Food Chem. 68(39) (2020) 10709-10718. https://doi.org/10.1021/acs.jafc.0c04425.

Crossref Google Scholar

[40]

Z. Gao, W. Gao, S.L. Zeng, et al., Chemical structures, bioactivities and molecular mechanisms of citrus polymethoxyflavones, J. Funct. Foods 40 (2018) 498-509. https://doi.org/10.1016/j.jff.2017.11.036.

Crossref Google Scholar

[41]

L. Wang, J. Wang, L. Fang, et al., Anticancer activities of citrus peel polymethoxyflavones related to angiogenesis and others, Biomed. Res. Int. 2014 (2014) 453972. https://doi.org/10.1155/2014/453972.

Crossref Google Scholar

[42]

B. Pucci, M. Kasten, A. Giordano, Cell cycle and apoptosis, Neoplasia 2 (2000) 291-299. https://doi.org/10.1038/sj.neo.7900101.

Crossref Google Scholar

[43]

D.C.H. Tobias Engel, Apoptosis, Bcl-2 family proteins and caspases: the ABCs of seizure-damage and epileptogenesis? Int. J. Physiol. Pathophysiol. Pharmacol. 1 (2009) 97-115. https://doi.org/hdl.handle.net/10016/18264.

[44]

L.R.M. Matthew Brentnall, R.L. De Guevara, E. Cepero et al., Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis, BMC Cell Biol. 14 (2013) 1471-2121. https://doi.org/10.1186/1471-2121-14-32.

Crossref Google Scholar

[45]

L.B. Gerard I Evan, M, Whyte, E, Harrington, Apoptosis and the cell cycle, Curr. Opin. Cell Biol. 58 (1995) 825-834. https://doi.org/10.1002/jcb.240580205.

Crossref Google Scholar

[46]

F. Llambi, D.R. Green, Apoptosis and oncogenesis: give and take in the BCL-2 family, Curr. Opin. Genet. Dev. 21(1) (2011) 12-20. https://doi.org/10.1016/j.gde.2010.12.001.

Crossref Google Scholar

Food Science and Human Wellness

Volume 11 Issue 5,
September 2022

Pages 1191-1200

DOI: 10.1016/j.fshw.2022.04.018

Cite this article:

Xin Y, Zheng T, Zhang M, et al. 5-Demethylnobiletin and its major metabolites: efficient preparation and mechanism of their anti-proliferation activity in HepG2 cells. Food Science and Human Wellness, 2022, 11(5): 1191-1200. https://doi.org/10.1016/j.fshw.2022.04.018

Fig. 1 Synthesis of 5-DMN and its metabolites. (a) KOH, ₂H₅OH, 80 ℃, 48 h, yield: 46%‒52%; (b) potassium tert-butoxide, C₂H₅OH, 80 ℃, 6 h, yield: 85%; (c) I₂, DMSO, 100 ℃, 3 h, yield: 85%; (d) 10% Pd/C, NH₄COOH, CH₃OH, 65 ℃, 2 h, yield: 95%; (e) AlCl₃, CH₃CN, 60 ℃, 3 h, yield: 97%.
Fig. 2 Profiles of crude extract of orange peel by HPLC.
Fig. 3 Structure of 5-DMN and its metabolites.
Fig. 4 Growth inhibitory effects of 5-DMN and its metabolites on HepG2 cells. Data were shown as mean ± SD (n = 6), and viable cells of controls were used as 100%.
Fig. 5 Effects of 5-DMN and its metabolites (25 μmol/L) on cell cycle progression of HepG2. (A) DNA histogram of cell cycle distribution of HepG2 cells. (A₁−A₅) Control, 5-DMN, 5,3\'-DDMN, 5,4\'-DDMN and 5,3\',4\'-TDMN, respectively. (B) Percentages of cell population distribution. Different letter symbols in the bar charts indicate statistical significance (P < 0.05, n = 3) by ANOVA.
Fig. 6 Dot plots of apoptotic cells of HepG2 cells (A) and effects of 5 OH-PMFs (12.5‒50 µmol/L) on apoptosis of HepG2 cells (B). (A₁–A₁₃) Control, 12.5 μmol/L 5-DMN, 25 μmol/L 5-DMN, 50 μmol/L 5-DMN, 12.5 μmol/L 5,3\'-DDMN, 25 μmol/L 5,3\'-DDMN, 50 μmol/L 5,3\'-DDMN, 12.5 μmol/L 5,4\'-DDMN, 25 μmol/L 5,4\'-DDMN, 50 μmol/L 5,4\'-DDMN, 12.5 μmol/L 5,3\',4\'-TDMN, 25 μmol/L 5,3\',4\'-TDMN, 50 μmol/L 5,3\',4\'-TDMN, respectively. Data are shown as mean ± SD. Different letter symbols in the bar charts indicate statistical significance (P < 0.05, n = 3) by ANOVA.
Fig. 7 5-DMN and its metabolites (12.5, 25, 50 μmol/L) treatment HepG2 cells induced cleaved caspase-3 protein expression. Data are shown as mean ± SD. Different letter symbols in the bar charts indicate statistical significance P < 0.05, n = 3) by ANOVA.
Fig. 8 5-DMN and its metabolites Molecular docking with caspase-3 was performed by AutoDock Vina program (A) 5-DMN; (B) 5,3\'-DDMN; (C) 5,4\'-DDMN; (D) 5,3\',4\'-TDMN.

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