Processing-induced changes in neuroprotective components and mechanisms of gardeniae fructus: integrating UPLC-Q-TOF-MS/MS, network pharmacology, and in vitro analysis

Abstract

Objective

Gardeniae Fructus (GF), the dried fruit of Gardenia jasminoides J. Ellis, has been used in East Asian medicine for centuries. Its carbonized form, Gardeniae Fructus Carbonisatus (GFC), is produced through processing, yet the effects of this transformation on active constituents and neuroprotective mechanisms remain unclear. This study aims to elucidate the key compositional changes induced by processing and explore their relevance to neuroprotective activity.

Methods

After obtaining GF and GFC extracts via CO₂ supercritical fluid extraction (SFE), UPLC-Q-TOF-MS/MS was employed for qualitative analysis of differential compounds. A pathology-specific network pharmacology screening approach, combined with UPLC-UV-DAD, was applied to quantify major bioactive differential components. Finally, in vitro models and molecular pharmacology techniques were utilized to validate the neuroprotective effects of key compounds.

Results

We identified 23 differential compounds and quantified 10 key bioactive constituents. Integrated network pharmacology and quantitative analysis implicated neuroinflammation and ferroptosis in GF’s neuroprotection, with geniposide and crocetin as pivotal compounds. Mechanistic studies confirmed roles for TLR4/NF-κB and Nrf2 pathways.

Conclusion

Geniposide and Crocetin were identified as key compounds responsible for the neuroprotective effects of GF and GFC, primarily through the inhibition of neuroinflammation and ferroptosis. Crocetin is highlighted as a potential marker for GFC.

Graphical Abstract

Processing transforms Gardeniae Fructus into GFC, enhancing glycoside–aglycone conversion and markedly increasing crocetin. Integrated network pharmacology and quantitative analysis reveal geniposide and crocetin as core neuroprotective agents. In vitro analysis, these compounds inhibit neuroinflammation and ferroptosis via TLR4/NF-κB suppression and Nrf2 activation, supporting crocetin as a characteristic marker of GFC.

Keywords

Gardeniae fructus / Gardeniae fructus carbonisatus / Geniposide / Crocetin / Neuroinflammation / Ferroptosis

References

[1] Aktan, F. (2004) iNOS-mediated nitric oxide production and its regulation. Life Sci. 75(6) 639–53. Available at: https://doi.org/10.1016/j.lfs.2003.10.042

[2] Bao WD, Pang P, Zhou XT, Hu F, Xiong W, Chen K, Wang J, Wang F, Xie D, Hu YZ, Han ZT, Zhang HH, Wang WX, Nelson PT, Chen JG, Lu Y, Man HY, Liu D, Zhu LQ. Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease. Cell Death Differ, 2021, 28(5): 1548-1562

[3] Bonnechère B, Amin N, van Duijn C. What are the key gut microbiota involved in neurological diseases? A systematic review. Int J Mol Sci, 2022

[4] Chen G, Zhang W, Chen Q, Dong M, Liu M, Liu G. Geniposide exerts the antidepressant effect by affecting inflammation and glucose metabolism in a mouse model of depression. Chem Biol Interact, 2024, 400: 111182

[5] Cheng Y, Song Y, Chen H, Li Q, Gao Y, Lu G, Luo C. Ferroptosis mediated by lipid reactive oxygen species: a possible causal link of neuroinflammation to neurological disorders. Oxid Med Cell Longev, 2021

[6] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China,, 2020, Place, China Medical Science and Technology PressI

[7] Choi PG, Park SH, Jeong HY, Kim HS, Hahm JH, Seo HD, Ahn J, Jung CH. Geniposide attenuates muscle atrophy via the inhibition of Foxo1 in senescence-accelerated mouse prone-8. Phytomedicine, 2024, 123: 155281

[8] Currais, A. (2015) Ageing and inflammation - A central role for mitochondria in brain health and disease. Ageing Res Rev 21 30–42. Available at: https://doi.org/10.1016/j.arr.2015.02.001

[9] Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep, 2017, 7: 42717

[10] Daina A, Michielin O, Zoete V. SwissTargetPrediction: updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res, 2019, 47(W1): W357-w364

[11] Deleidi M, Gasser T. The role of inflammation in sporadic and familial Parkinson's disease. Cell Mol Life Sci, 2013, 70(22): 4259-4273

[12] Dodson M, Castro-Portuguez R, Zhang DD. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol, 2019, 23: 101107

[13] Dong J, Li S, Zhang J, Liu A, Ren J. Thermal degradation of cyanidin-3-O-glucoside: mechanism and toxicity of products. Food Chem, 2022, 370: 131018

[14] Fang S, Dong L, Liu L, Guo J, Zhao L, Zhang J, Bu D, Liu X, Huo P, Cao W, Dong Q, Wu J, Zeng X, Wu Y, Zhao Y. HERB: a high-throughput experiment- and reference-guided database of traditional Chinese medicine. Nucleic Acids Res, 2021, 49(D1): D1197-d1206

[15] Goodsell DS, Burley SK. RCSB protein data bank tools for 3D structure-guided cancer research: human papillomavirus (HPV) case study. Oncogene, 2020, 39(43): 6623-6632

[16] Gouthami K, Veeraraghavan V, Rahdar A, Bilal M, Shah A, Rai V, Gurumurthy DM, Ferreira LFR, Américo-Pinheiro JHP, Murari SK, Kalia S, Mulla SI. Molecular docking used as an advanced tool to determine novel compounds on emerging infectious diseases: A systematic review. Prog Biophys Mol Biol Available at:, 2022

[17] Grill JD, Cummings JL. Current therapeutic targets for the treatment of Alzheimer's disease. Expert Rev Neurother, 2010, 10(5): 711-728

[18] Guo DA, Lu A, Liu L. Modernization of traditional Chinese medicine. J Ethnopharmacol, 2012, 141(2): 547-548

[19] Hammond TR, Marsh SE, Stevens B. Immune signaling in neurodegeneration. Immunity, 2019, 50(4): 955-974

[20] Herrmann N, Chau SA, Kircanski I, Lanctôt KL. Current and emerging drug treatment options for Alzheimer's disease: a systematic review. Drugs, 2011, 71(15): 2031-2065

[21] Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol, 2021, 22(4): 266-282

[22] Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, Li Q, Shoemaker BA, Thiessen PA, Yu B, Zaslavsky L, Zhang J, Bolton EE. PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Res, 2021, 49(D1): D1388-d1395

[23] Leng F, Edison P. Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here?. Nat Rev Neurol, 2021, 17(3): 157-172

[24] Li S, Wu Z, Le W. Traditional Chinese medicine for dementia. Alzheimers Dement, 2021, 17(6): 1066-1071

[25] Li W, Ren C, Fei C, Wang Y, Xue Q, Li L, Yin F, Li W. Analysis of the chemical composition changes of gardeniae fructus before and after processing based on ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. J Sep Sci, 2021, 44(5): 981-991

[26] Mir WR, Bhat BA, Kumar A, Dhiman R, Alkhanani M, Almilaibary A, Dar MY, Ganie SA, Mir MA. Network pharmacology combined with molecular docking and in vitro verification reveals the therapeutic potential of Delphinium roylei munz constituents on breast carcinoma. Front Pharmacol, 2023, 14: 1135898

[27] Narra F, Piragine E, Benedetti G, Ceccanti C, Florio M, Spezzini J, Troisi F, Giovannoni R, Martelli A, Guidi L. Impact of thermal processing on polyphenols, carotenoids, glucosinolates, and ascorbic acid in fruit and vegetables and their cardiovascular benefits. Compr Rev Food Sci Food Saf, 2024, 23(6): e13426

[28] Nogales C, Mamdouh ZM, List M, Kiel C, Casas AI, Schmidt H. Network pharmacology: curing causal mechanisms instead of treating symptoms. Trends Pharmacol Sci, 2022, 432136-150

[29] Oner E, Al-Khafaji K, Mezher MH, Demirhan I, Suhail Wadi J, Belge Kurutas E, Yalin S, Choowongkomon K. Investigation of berberine and its derivatives in Sars Cov-2 main protease structure by molecular docking, PROTOX-II and ADMET methods: in machine learning and in silico study. J Biomol Struct Dyn, 2023, 41(19): 9366-9381

[30] Sies H. Oxidative stress: a concept in redox biology and medicine. Redox Biol, 2015, 4: 180-183

[31] Stockwell BR. Ferroptosis turns 10: emerging mechanisms, physiological functions, and therapeutic applications. Cell, 2022, 185(14): 2401-2421

[32] Szklarczyk D, Nastou K, Koutrouli M, Kirsch R, Mehryary F, Hachilif R, Hu D, Peluso ME, Huang Q, Fang T, Doncheva NT, Pyysalo S, Bork P, Jensen LJ, von Mering C. The STRING database in 2025: protein networks with directionality of regulation. Nucleic Acids Res, 2025, 53(D1): D730-d737

[33] Tikhonova MA, Amstislavskaya TG, Ho YJ, Akopyan AA, Tenditnik MV, Ovsyukova MV, Bashirzade AA, Dubrovina NI, Aftanas LI. Neuroprotective effects of ceftriaxone involve the reduction of Aβ burden and neuroinflammatory response in a mouse model of Alzheimer's disease. Front Neurosci, 2021, 15: 736786

[34] Ward RJ, Dexter DT, Crichton RR. Iron, neuroinflammation and neurodegeneration. Int J Mol Sci, 2022

[35] Wei Z, Gao Y, Meng F, Chen X, Gong Y, Zhu C, Ju B, Zhang C, Liu Z, Liu Q. Idmer: an integrative and mechanism-driven response system for identifying compound interventions for sudden virus outbreak. Brief Bioinform, 2021, 22(2): 976-987

[36] Xia B, Huang X, Sun G, Tao W. Iridoids from Gardeniae fructus ameliorates depression by enhancing synaptic plasticity via AMPA receptor-mTOR signaling. J Ethnopharmacol, 2021, 268: 113665

[37] Xu K, Zhang M, Fang Z, Wang B. Degradation and regulation of edible flower pigments under thermal processing: a review. Crit Rev Food Sci Nutr, 2021, 61(6): 1038-1048

[38] Yang W, Wang Y, Zhang C, Huang Y, Yu J, Shi L, Zhang P, Yin Y, Li R, Tao K. Maresin1 protect against ferroptosis-induced liver injury through ROS inhibition and Nrf2/HO-1/GPX4 activation. Front Pharmacol, 2022, 13: 865689

[39] You LH, Yan CZ, Zheng BJ, Ci YZ, Chang SY, Yu P, Gao GF, Li HY, Dong TY, Chang YZ. Astrocyte hepcidin is a key factor in LPS-induced neuronal apoptosis. Cell Death Dis, 2017, 8(3): e2676

[40] Yu G, Wang LG, Han Y, He QY. ClusterProfiler: an R package for comparing biological themes among gene clusters. OMICS, 2012, 165284-287

[41] Yuan J, Amin P, Ofengeim D. Necroptosis and RIPK1-mediated neuroinflammation in CNS diseases. Nat Rev Neurosci, 2019, 20(1): 19-33

[42] Yuste JE, Tarragon E, Campuzano CM, Ros-Bernal F. Implications of glial nitric oxide in neurodegenerative diseases. Front Cell Neurosci, 2015, 9: 322

[43] Zeng P, Fang M, Zhao H, Guo J. A network pharmacology approach to uncover the key ingredients in Ginkgo Folium and their anti-Alzheimer's disease mechanisms. Aging Albany NY, 2021, 13(14): 18993-19012

[44] Zeng P, Su HF, Ye CY, Qiu SW, Shi A, Wang JZ, Zhou XW, Tian Q. A Tau pathogenesis-based network pharmacology approach for exploring the protections of Chuanxiong Rhizoma in Alzheimer's Disease. Front Pharmacol, 2022, 13: 877806

[45] Zhang H, Wei W, Zhao M, Ma L, Jiang X, Pei H, Cao Y, Li H. Interaction between Aβ and tau in the pathogenesis of Alzheimer's disease. Int J Biol Sci, 2021, 17(9): 2181-2192

[46] Zhang P, Zhang D, Zhou W, Wang L, Wang B, Zhang T, Li S. Network pharmacology: towards the artificial intelligence-based precision traditional Chinese medicine. Brief Bioinform, 2023

[47] Zhang X, Wang Y, Li X, Dai Y, Wang Q, Wang G, Liu D, Gu X, Yu D, Ma Y, Zhang C. Treatment mechanism of gardeniae fructus and its carbonized product against ethanol-induced gastric lesions in rats. Front Pharmacol, 2019, 10 750

[48] Zhao C, Zhang H, Li H, Lv C, Liu X, Li Z, Xin W, Wang Y, Zhang W. Geniposide ameliorates cognitive deficits by attenuating the cholinergic defect and amyloidosis in middle-aged Alzheimer model mice. Neuropharmacology, 2017, 116: 18-29

[49] Zhao Z, Liang Z, Chan K, Lu G, Lee EL, Chen H, Li L. A unique issue in the standardization of Chinese materia medica: processing. Planta Med, 2010, 76(17): 1975-1986

[50] Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, Benner C, Chanda SK. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun, 2019, 1011523

Funding

the Innovation Fund for Medical Sciences of the Chinese Academy of Medical Sciences(CIFMS: 2021-I2M-1-071)

RIGHTS & PERMISSIONS

The Author(s)