Gracilaria chorda-derived compounds improve lipid metabolism and insulin sensitivity in 3T3-L1 adipocytes and high-fat diet-induced obese zebrafish larvae

Abstract

Objective: To isolate and identify active constituents from Gracilaria chorda extract prepared under subcritical water conditions at 210 °C (GCSW210) and evaluate their anti-obesity and anti-diabetic effects in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese zebrafish larvae.

Methods: GCSW210 was fractionated through solvent partitioning, ion-exchange chromatography, and silica gel medium-pressure liquid chromatography, followed by preparative high performance liquid chromatography. At each step, bioactivities were assessed in 3T3-L1 adipocytes by Oil Red O staining and 2-NBDG uptake assays. The most active fractions were further purified, and isolated compounds were tested in HFD-induced obese zebrafish larvae.

Results: Liquid chromatography-hyphenated analysis with reference standards identified two major compounds in GCSW210: 5-hydroxymethylfurfural and bis(5-formylfurfuryl) ether. Both compounds significantly inhibited lipid accumulation in 3T3-L1 adipocytes and modulated gene expression associated with adipogenesis, glucose metabolism, and inflammation in zebrafish. They also enhanced glucose uptake, reduced circulating glucose levels, and improved insulin sensitivity. Notably, the effects were comparable to those of the crude GCSW210 extract. In silico docking studies confirmed stable interactions of both compounds with key metabolic and inflammatory targets, with bis(5-formylfurfuryl) ether showing stronger binding affinities.

Conclusions: These findings suggest that 5-hydroxymethylfurfural and bis(5-formylfurfuryl) ether are key contributors to the therapeutic activity of Gracilaria chorda, highlighting its potential as a functional food ingredient for the prevention or management of metabolic disorders.

Keywords

5-Hydroxymethylfurfural / Bis(5-formylfurfuryl) ether / Gracilaria chorda / Obesity / Zebrafish larvae / Anti-diabetes

Conflict of interest statement

We declare that we have no conflict of interest.

Funding

This research was supported by the Basic Science Research Program of the National Research Foundation of Korea, funded by the Ministry of Education (2018R1D1A1B07050273). This work was supported by the National Research Foundation of Korea grant funded by the Korea government (Grant no.: RS-2022-NR070862). This research was supported (in part) by Glocal University Project of Mokpo National University in 2025.

Data availability statement

The data supporting the findings of this study are available from the corresponding authors upon request.

Authors’ contributions

LST conducted the main investigation, including experimental design and analysis, and wrote original draft of manuscript. DDL was responsible for investigation and analysis. HJK also contributed to classification, identification, and data interpretation. JJK supervised aspects of the project and edited the manuscript. JBS contributed to editing. JCP and ML provided overall supervision. DYR conceptualized and supervised the study and contributed to manuscript review and editing. All authors reviewed and approved the final manuscript.

Publisher’s note

The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

[1] Hristov I, Mocanu V, Zugun-Eloae F, Labusca L, Cretu-Silivestru I, Oboroceanu T, et al. Association of intracellular lipid accumulation in subcutaneous adipocyte precursors and plasma adipokines in bariatric surgery candidates. Lipids Health Dis 2019; 18:8. doi: 10.1186/s12944-019-1081-1089.

[2] Lin X, Li H. Obesity: Epidemiology, pathophysiology, and therapeutics. Front Endocrinol (Lausanne) 2021; 12. doi: 10.3389/fendo.2021.706978.

[3] Martyn JA, Kaneki M, Yasuhara S, Warner DS, Warner MA. Obesity-induced insulin resistance and hyperglycemia: Etiologic factors and molecular mechanisms. Anesthesiology 2008; 109:137-148.

[4] Hruby A, Hu FB. The epidemiology of obesity: A big picture. Pharmacoeconomics 2015; 33:673-689.

[5] Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 2006; 444:840-846.

[6] Kivimäki M, Strandberg T, Pentti J, Nyberg ST, Frank P, Jokela M, et al. Body-mass index and risk of obesity-related complex multimorbidity: An observational multicohort study. Lancet Diabetes Endocrinol 2022; 10:253-263.

[7] Ormazabal V, Nair S, Elfeky O, Aguayo C, Salomon C, Zuñiga FA. Association between insulin resistance and the development of cardiovascular disease. Cardiovasc Diabetol 2018; 17:122. doi: 10.1186/s12933-018-0762-4.

[8] Brown AE, Walker M. Genetics of insulin resistance and the metabolic syndrome. Curr Cardiol Rep 2016; 18:75. doi: 10.1007/s11886-016-0755-4.

[9] Boucher J, Kleinridders A, Kahn CR. Insulin receptor signaling in normal and insulin-resistant states. Cold Spring Harb Perspect Biol 2014; 6. doi: 10.1101/cshperspect.a009191.

[10] Kahn BB. Glucose transport: Pivotal step in insulin action. Diabetes 1996; 45:1644-1654.

[11] Vlavcheski F, MacPherson RE, Tsiani E. Rosemary extract activates AMPK, inhibits mTOR and attenuates the high glucose and high insulin-induced muscle cell insulin resistance. Appl Physiol Nutr Metab 2021; 46(7):819-827.

[12] Mune Mune MA, Miyabe Y, Shimizu T, Matsui W, Kumagai Y, Kishimura H. Characterisation of bioactive peptides from red alga Gracilariopsis chorda. Mar Drugs 2023; 21(1):49. doi: 10.3390/md21010049.

[13] Thakuri LS, Park CM, Park JW, Kim HA, Rhyu DY. Subcritical water extraction of Gracilaria chorda abbreviates lipid accumulation and obesity-induced inflammation. Algae 2023; 38(1):81-92.

[14] Thakuri LS, Park CM, Kim HA, Kim HJ, Park JW, Park JC, et al. Gracilaria chorda subcritical water ameliorates hepatic lipid accumulation and regulates glucose homeostasis in a hepatic steatosis cell model and obese C57BL/6J mice. J Ethnopharmacol 2024; 320. doi: 10.1016/j.jep.2023.117395.

[15] Thakuri LS, Park CM, Park JW, Rhyu DY. Gracilaria chorda subcritical-water extracts as ameliorant of insulin resistance induced by high-glucose in zebrafish and dexamethasone in L6 myotubes. J Tradit Complement Med 2024; 14(1):82-90.

[16] Park CH, Rhyu DY, Sharma BR, Yokozawa T. Inhibition of preadipocyte differentiation and lipid accumulation by 7-O-galloyl-D-sedoheptulose treatment in 3T3-L1 adipocytes. Biomed Prev Nutr 2013; 3(4):319-324.

[17] Thakuri LS, Kim JE, Choi JY, Rhyu DY. Protective effect of Capsosiphon fulvescens on oxidative stress-stimulated neurodegenerative dysfunction of PC12 cells and zebrafish larva models. Fish Aquat Sci 2023; 26(1):24-34.

[18] Den Broeder MJ, Moester MJ, Kamstra JH, Cenijn PH, Davidoiu V, Kamminga LM, et al. Altered adipogenesis in zebrafish larvae following high fat diet and chemical exposure is visualised by stimulated Raman scattering microscopy. Int J Mol Sci 2017; 18(4):894. doi: 10.3390/ijms18040894.

[19] Le DD, Kim E, Dang T, Lee J, Shin CH, Park JW, et al. Chemical investigation and regulation of adipogenic differentiation of cultivated Moringa oleifera. Pharmaceuticals 2024;17. doi: 10.3390/ph17101310.

[20] Deepa VS, Kumar PS, Vadivukkarasi B, Tilton FL. Molecular docking studies on selected phytocompounds from different Andrographis sp against PPAR-γ and C/EBP-α receptors for type 2 diabetes. Asian J Pharm Clin Res 2013; 6(2):196-199.

[21] Choi J, Le DD, Roh N, Lee J, Shrestha D, Dinh T, et al. Chemical composition and biological activities of Lagopsis supina extract: Antioxidant, adipogenic, and anti-inflammatory effects. Pharmaceuticals 2025;18. doi: 10.3390/ph18020150.

[22] Damayanti DS, Utomo DH, Kusuma C. Revealing the potency of Annona muricata leaves extract as FOXO1 inhibitor for diabetes mellitus treatment through computational study. In Silico Pharmacol 2017; 5:3. doi: 10.1007/s40203-017-0023-3.

[23] Zhang H, Jiang Z, Shen C, Zou H, Zhang Z, Wang K, et al. 5-Hydroxymethylfurfural alleviates inflammatory lung injury by inhibiting endoplasmic reticulum stress and NLRP 3 inflammasome activation. Front Cell Dev Biol 2021;9. doi: 10.3389/fcell.2021.782427.

[24] Li W, Qu XN, Han Y, Zheng SW, Wang J, Wang YP. Ameliorative effects of 5-hydroxymethyl-2-furfural (5-HMF) from Schisandra chinensis on alcoholic liver oxidative injury in mice. Int J Mol Sci 2015; 16(2):2446-2457.

[25] Casanova O, Iborra S, Corma A. Chemicals from biomass: Etherification of 5-hydroxymethyl-2-furfural (HMF) into 5,5′(oxy-bis(methylene))bis-2-furfural (OBMF) with solid catalysts. J Catal 2010; 275(2):236-242.

[26] Wang M, Li K, Nie Y, Wei Y, Li X. Antirheumatoid arthritis activities and chemical compositions of phenolic compounds-rich fraction from Urtica atrichocaulis, an endemic plant to China. Evid Based Complement Alternat Med 2012;2012. doi: 10.1155/2012/818230.

[27] Farzaei MH, Farzaei F, Gooshe M, Abbasabadi Z, Rezaei N, Abdolghaffari AH. Potentially effective natural drugs in treatment for the most common rheumatic disorder: Osteoarthritis. Rheumatol Int 2015; 35:799-814.

[28] Long YC, Zierath JR. AMP-activated protein kinase signaling in metabolic regulation. J Clin Invest 2006; 116(7):1776-1783.

[29] Khoramipour K, Chamari K, Hekmatikar AA, Ziyaiyan A, Taherkhani S, Elguindy NM, et al. Adiponectin: Structure, physiological functions, role in diseases, and effects of nutrition. Nutrients 2021; 13(4). doi: 10.3390/nu13041180.

[30] Awazawa M, Ueki K, Inabe K, Yamauchi T, Kaneko K, Okazaki Y, et al. Adiponectin suppresses hepatic SREBP1c expression in an AdipoR1/LKB1/AMPK dependent pathway. Biochem Biophys Res Commun 2009; 382(1):51-56.

[31] Li X, Wan T, Li Y. Role of FoxO1 in regulating autophagy in type 2 diabetes mellitus (Review). Exp Ther Med 2021; 22(1):707. doi: 10.3892/etm.2021.10139.

[32] Luo L, Liu M. Adiponectin: Friend or foe in obesity and inflammation. Med Rev 2022; 2(4):349-362.

[33] Chen S, Villalta SA, Agrawal DK. FOXO 1 mediates vitamin D deficiency-induced insulin resistance in skeletal muscle. J Bone Miner Res 2016; 31(3):585-595.

[34] Su D, Coudriet GM, Kim DH, Lu Y, Perdomo G, Qu S, et al. FoxO1 links insulin resistance to proinflammatory cytokine IL-1β production in macrophages. Diabetes 2009; 58(11):2624-2633.

[35] Agu PC, Afiukwa CA, Orji OU, Ezeh EM, Ofoke IH, Ogbu CO, et al. Molecular docking as a tool for the discovery of molecular targets of nutraceuticals in diseases management. Sci Rep 2023;13. doi: 10.1038/s41598-023-40160-2.