NETWORK TOXICOLOGY–EXPLORATION OF SWEETENER EXPOSURE AND DEPRESSION WITH IN VITRO EXPERIMENTAL VALIDATION

Authors

  • SUJATHA DODOALA Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati, Andhra Pradesh-517502, India https://orcid.org/0000-0003-1374-7077
  • LATHA PUJARI Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati, Andhra Pradesh-517502, India
  • MADHAVI DUVURU Department of Home Science, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati, Andhra Pradesh-517502, India https://orcid.org/0009-0008-1023-1765
  • SAI SUNEEL ADEM School of Engineering and Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati, Andhra Pradesh-517502, India https://orcid.org/0000-0002-0989-2522
  • SWETHA PETLU Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati, Andhra Pradesh-517502, India https://orcid.org/0009-0007-2540-0894
  • MOHITHA PILLA Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati, Andhra Pradesh-517502, India
  • CHANDNA SHRINIVAASINY THATHAPPAGARI Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati, Andhra Pradesh-517502, India

DOI:

https://doi.org/10.22159/ijap.2026v18i3.57747

Keywords:

Depression, EFSA, Food additives, Network pharmacology, Sweetener, Toxicology

Abstract

Objective: Sweeteners, a category of food additives are increasingly being incorporated into foods, yet their potential neurobiological safety issues remain underexplored. This study investigated the possible link between the European Food Safety Authority (EFSA)-approved sweeteners and depression by integrating network toxicology, molecular docking, and experimental validation, with a particular focus on steviol glycoside.

Methods: Sweeteners collected from EFSA were initially screened using SwissADME and ADMETlab 3.0 for toxicity profiles. The potential targets, protein–protein interactions (PPI), molecular pathways, and binding affinities of the screened compounds were further analyzed using various suitable in silico tools (including SwissTargetPrediction, PharmMapper, STRING, shinyGO, cytoscape, cytoHubba, molecular docking) and in vitro studies.

Results: A total of 31 sweeteners were retrieved from the EFSA database and evaluated for their ADMET characteristics. Among them, 14 compounds were predicted to exhibit toxicological properties. Integration of target prediction data for these compounds with depression-related genes obtained from DisGeNET, GeneCards, Swiss Target Prediction and PharmMapper identified 225 overlapping targets, with network analysis highlighting AKT1, SRC, TP53, ALB, and ESR1 as major hub genes. Functional enrichment analysis using ShinyGO revealed significant associations with neuroactive ligand–receptor interactions, oxidative stress pathways, and mood-related signaling processes. Molecular docking analysis of the top five sweeteners indicated that steviol glycoside displayed the highest affinity and selectivity toward ESR1. Supporting these, differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy confirmed steviol-induced conformational alterations in bovine serum albumin. Additionally, biochemical assays demonstrated increased protein carbonylation (p<0.05), elevated amadori product formation, reduced thiol content, and dose-dependent modulation of monoamine oxidase enzyme.

Conclusion: These findings suggest that certain EFSA-approved sweeteners, especially steviol glycoside, may influence depression-related pathways through modulation of key molecular targets. It also induced oxidative stress and altered activity of monoamine oxidases. These results underscore the need for further mechanistic and in vivo studies to evaluate potential neurobiological risks associated with increased sweetener exposure.

References

1. Pozdnyakova Y, Murzatayeva A. Neuroprotective potential of Stevia rebaudiana and Stachys sieboldii: effects on oxidative stress and locomotor activity in male rats fed a high-fat, high-sucrose diet. Biology (Basel). 2025;14(4):359. doi: 10.3390/biology14040359, PMID 40282224.

2. Harismah K, Fazeli F, Amini I, Dai M, Mirzaei M. In silico effects of steviol on depression, inflammation and cancer biomarkers. Biointerface Res Appl Chem. 2021;12(6):8385-93. doi: 10.33263/BRIAC126.83858393.

3. Straits Research. Sweeteners market report. Available from: https://straitsresearch.com/report/sweeteners-market. [Last accessed on 13 Mar 2026].

4. Lindseth GN, Coolahan SE, Petros TV, Lindseth PD. Neurobehavioral effects of aspartame consumption. Res Nurs Health. 2014;37(3):185-93. doi: 10.1002/nur.21595, PMID 24700203.

5. EBSCO Research Starters. Sweetener. Available from: https://www.EBSCO.com/research-starters/health-and-medicine/sweetener.

6. Sardesai V. Natural and synthetic intense sweeteners. J Nutr Biochem. 1991;2(5):236-44. doi: 10.1016/0955-2863(91)90081-F.

7. Palomar Cros A, Straif K, Romaguera D, Aragones N, Castano Vinyals G, Martin V. Consumption of aspartame and other artificial sweeteners and risk of cancer in the Spanish multicase-control study (MCC-Spain). Int J Cancer. 2023;153(5):979-93. doi: 10.1002/ijc.34577, PMID 37323037.

8. Zhang Y, Tang Z, Shi Y, Li L. Associations between artificial sweetener intake from cereals coffee and tea and the risk of type 2 diabetes mellitus: a genetic correlation, mediation and mendelian randomization analysis. PLOS One. 2024;19(2):e0287496. doi: 10.1371/journal.pone.0287496, PMID 38324548.

9. Lopez Meza MS, Otero Ojeda G, Estrada JA, Esquivel Hernandez FJ, Contreras I. The impact of nutritive and non-nutritive sweeteners on the central nervous system: preliminary study [The impact of nutritive and non-nutritive sweeteners on the central nervous system: preliminary study]. Nutr Neurosci. 2022;25(8):1623-32. doi: 10.1080/1028415X.2021.1885239, PMID 33641634.

10. Xiong J, Wang L, Huang H, Xiong S, Zhang S, Fu Q. Association of sugar consumption with risk of depression and anxiety: a systematic review and meta-analysis. Front Nutr. 2024;11:1472612. doi: 10.3389/fnut.2024.1472612, PMID 39479195.

11. Said Elshama SS. Synthetic and natural food additives: toxicological hazards and health benefits. OAJT. 2020 Oct 1;4(4):555643. doi: 10.19080/OAJT.2020.04.555643.

12. Kim S, Thiessen PA, Bolton EE, Chen J, Fu G, Gindulyte A. PubChem substance and compound databases. Nucleic Acids Res. 2016;44(D1):D1202-13. doi: 10.1093/nar/gkv951, PMID 26400175.

13. Ahmed SR, Al-Sanea MM, Mostafa EM, Qasim S, Abelyan N, Mokhtar FA. A network pharmacology analysis of cytotoxic triterpenes isolated from Euphorbia abyssinica latex supported by drug-likeness and ADMET studies. ACS Omega. 2022;7(21):17713-22. doi: 10.1021/acsomega.2c00750, PMID 35664578.

14. Daina A, Michielin O, Zoete V. Swiss target prediction: updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res. 2019 Jul 2;47(W1):W357-64. doi: 10.1093/nar/gkz382, PMID 31106366.

15. Tipugade O, Sawale JA, Jadhav N. Network pharmacology and molecular docking-based exploration of Rubiaceous plants for breast cancer: phytochemicals preclinical studies and regulatory perspectives. Asian J Pharm Clin Res. 2025;18(7):52-71. doi: 10.22159/ajpcr.2025v18i7.54934.

16. Bhosale VB, Akshada Amit Koparde, Vandana M Thorat, Mayuri V Bhosale. Network pharmacology and molecular docking-based investigation of Calotropis gigantea Linn. leaf extract against vulvovaginal candidiasis. Asian J Pharm Clin Res. 2025;18(12):54-66. doi: 10.22159/ajpcr.2025v18i12.56803.

17. Adhish M, Madhan B, Manjubala I. Exploring capsaicin as a multi-target agent for osteoporosis through computational insights. In Silico Pharmacol. 2025;13(2):112. doi: 10.1007/s40203-025-00400-x, PMID 40761847.

18. Bibi M, Baboo I, Majeed H, Kumar S, Lackner M. Molecular docking of key compounds from Acacia honey and Nigella sativa oil and experimental validation for colitis treatment in albino mice. Biology (Basel). 2024;13(12):1035. doi: 10.3390/biology13121035, PMID 39765702.

19. Li Y, Yang JM, Cui WH, Wang JK, Chen X, Zhang C. Prediction of active ingredients and mechanism of Siwei Jianbu decoction in the treatment of atherosclerosis by network pharmacology. Eur Rev Med Pharmacol Sci. 2022;26(15):5436-46. doi: 10.26355/eurrev_202208_29412, PMID 35993639.

20. Giordano D, Biancaniello C, Argenio MA, Facchiano A. Drug design by pharmacophore and virtual screening approach. Pharmaceuticals (Basel). 2022 May 23;15(5):646. doi: 10.3390/ph15050646, PMID 35631472.

21. Chaturvedi SK, Ahmad E, Khan JM, Alam P, Ishtikhar M, Khan RH. Elucidating the interaction of limonene with bovine serum albumin: a multi-technique approach. Mol Biosyst. 2015;11(1):307-16. doi: 10.1039/c4mb00548a, PMID 25382435.

22. Alhazmi HA, Al Bratty M, Meraya AM, Najmi A, Alam MS, Javed SA. Spectroscopic characterization of the interactions of bovine serum albumin with medicinally important metal ions: platinum (IV), iridium (III) and iron (II). Acta Biochim Pol. 2021;68(1):99-107. doi: 10.18388/abp.2020_5462, PMID 33596034.

23. Colombo G, Clerici M, Garavaglia ME, Giustarini D, Rossi R, Milzani A. A step-by-step protocol for assaying protein carbonylation in biological samples. J Chromatogr B Analyt Technol Biomed Life Sci. 2016;1019:178-90. doi: 10.1016/j.jchromb.2015.11.052, PMID 26706659.

24. Coligan JE, Dunn BM, Speicher DW, Wingfield PT, editors. Current protocols in protein science. Hoboken, NJ, USA: John Wiley & Sons, Inc; 2001. doi: 10.1002/0471140864.

25. Emami L, Khodarahimi E, Mardaneh P, Khoshnoud MJ, Rashedinia M. Binding interaction of sodium benzoate, potassium sorbate and sodium dihydrogen citrate with BSA as food preservatives: in vitro analysis and computational studies. Sci Rep. 2024;14(1):29237. doi: 10.1038/s41598-024-80642-5, PMID 39587338.

26. Huang G, Zhu F, Chen Y, Chen S, Liu Z, Li X. A spectrophotometric assay for monoamine oxidase activity with 2, 4-dinitrophenylhydrazine as a derivatized reagent. Anal Biochem. 2016;512:18-25. doi: 10.1016/j.ab.2016.06.020, PMID 27503749.

27. Mayank JV, Jaitak V. Interaction model of steviol glycosides from Stevia rebaudiana (Bertoni) with sweet taste receptors: a computational approach. Phytochemistry. 2015;116:12-20. doi: 10.1016/j.phytochem.2015.05.006, PMID 26021732.

28. Raj B, Thomas SM, Varghese S, Ruba Priya MG, John S, Ramakrishna P. A mini-review on molecular docking studies and pharmacological activities of Stevia rebaudiana. Asian J Chem. 2021;33(12):2919-23. doi: 10.14233/ajchem.2021.23393.

29. Tabernero A, Granda B, Medina A, Sanchez Abarca LI, Lavado E, Medina JM. Albumin promotes neuronal survival by increasing the synthesis and release of glutamate. J Neurochem. 2002;81(4):881-91. doi: 10.1046/j.1471-4159.2002.00843.x, PMID 12065647.

30. Tanti GK, Goswami SK. SG2NA recruits DJ-1 and Akt into the mitochondria and membrane to protect cells from oxidative damage. Free Radic Biol Med. 2014;75:1-13. doi: 10.1016/j.freeradbiomed.2014.07.009, PMID 25035075.

31. Dankowska K, Nesterowicz M, Lauko KK, Trocka D, Zendzian Piotrowska M, Ladny JR. In vitro and in silico studies and a systematic literature review of antiglycation properties of amlodipine. Sci Rep. 2025;15(1):33277. doi: 10.1038/s41598-025-18925-8, PMID 41006534.

32. Barca Mayo O, Lu QR. Fine-tuning oligodendrocyte development by micro RNAs. Front Neurosci. 2012;6:13. doi: 10.3389/fnins.2012.00013, PMID 22347159.

33. Peng X, Yao D, Pan Y, Yu Q, Ni S, Bian H. Study on the structural changes of bovine serum albumin with effects on polydatin binding by a multitechnique approach. Spectrochim Acta A Mol Biomol Spectrosc. 2011;81(1):209-14. doi: 10.1016/j.saa.2011.06.003, PMID 21723188.

34. Uzbekov MG. Monoamine oxidase as a potential biomarker of the efficacy of treatment of mental disorders. Biochemistry (Mosc). 2021 Jun;86(6):773-83. doi: 10.1134/S0006297921060146, PMID 34225599.

35. Meyer JH, Ginovart N, Boovariwala A, Sagrati S, Hussey D, Garcia A. Elevated monoamine oxidase a levels in the brain: an explanation for the monoamine imbalance of major depression. Arch Gen Psychiatry. 2006;63(11):1209-16. doi: 10.1001/archpsyc.63.11.1209, PMID 17088501.

36. Beucher L, Gabillard Lefort C, Baris OR, Mialet Perez J. Monoamine oxidases: a missing link between mitochondria and inflammation in chronic diseases? Redox Biol. 2024 Nov;77:103393. doi: 10.1016/j.redox.2024.103393, PMID 39405979.

37. Troubat R, Barone P, Leman S, Desmidt T, Cressant A, Atanasova B. Neuroinflammation and depression: a review. Eur J Neurosci. 2021 Jan;53(1):151-71. doi: 10.1111/ejn.14720, PMID 32150310.

Published

2026-05-07

How to Cite

DODOALA, S., PUJARI, L., DUVURU, M., ADEM, S. S., PETLU, S., PILLA, M., & THATHAPPAGARI, C. S. (2026). NETWORK TOXICOLOGY–EXPLORATION OF SWEETENER EXPOSURE AND DEPRESSION WITH IN VITRO EXPERIMENTAL VALIDATION. International Journal of Applied Pharmaceutics, 18(3), 343–351. https://doi.org/10.22159/ijap.2026v18i3.57747

Issue

Vol 18, Issue 3 (May-Jun), 2026

Section

Original Article(s)

Copyright (c) 2026 Sujatha Dodoala, Latha Pujari, D. Madhavi, A. Sai Suneel, P. Swetha, P. Mohita, T.Chandna shrinivaasiny

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