Safe dosage and potential risks of chlorogenic acid: insights from in vitro and in vivo studies

Yilin Pang¹Guoqiang Tan²Mengyao Jiang²Lin Huang³Xiaoyu Rao⁴Binjie Ge²Jun Fang¹Yingli Zhong¹Xueqing Wang²Huaibin Zhou²Hua Yang^1*Jianxin Lyu^5*Zheng Wang^1*

• ¹Hunan Agricultural University, Changsha, China
• ²Wenzhou Medical University, Wenzhou, Zhejiang Province, China
• ³Fudan University, Shanghai, Shanghai Municipality, China
• ⁴Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences (CAS), Shanghai, Shanghai Municipality, China
• ⁵Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang Province, China

As the economy grows, there is a growing emphasis on food safety. While the health Word Count: 6415 benefits of CGA are recognized, safe dosages and potential liver cell damage from excessive CGA consumption are not well studied. This study aims to determine the safe and effective dose range of CGA and understand how it causes toxicity in hepatocyte at IC50. Methods: This study assessed the impact of various CGA concentrations on liver cells, examining growth, viability, toxicity, energy metabolism, and colony formation using RTCA, CCK-8, LDH assays, and Seahorse XF96. It established CGA's IC50 for cell viability and identified differentially expressed proteins via proteomics. Subsequently, GO and KEGG pathway analyses were conducted to elucidate the signaling pathways associated with the differentially expressed proteins. Further validation of the molecular mechanisms was performed using flow cytometry, Western blotting, and RT-qPCR. Results: In this study, 200 µM of CGA significantly reduced LDH release and increased the mitochondrial OCR in hepatocytes, but it did not affect the ECAR. Additionally, 200 µM of CGA slightly promoted hepatocyte growth; however, at 300 µM, CGA nearly completely inhibited the clonogenic capacity of hepatocytes, and at 600 µM, it significantly impeded hepatocyte growth. The IC50 of CGA for hepatocyte activity was determined to be 613.1 µM. In vitro experiments indicated that incubation with CGA at its IC50 concentration for 96 h resulted in the arrest of L-02 cells in the S phase of the cell cycle and induced apoptosis. Further investigation revealed that the IC50 concentration of CGA, through the depletion of free iron within hepatocytes, significantly reduced the expression of iron-sulfur cluster subunits in mitochondrial complexes I-III and disrupted the oxidative-reductive homeostasis of hepatocytes, ultimately leading to hepatotoxicity. Interestingly, N-Acetyl-L-cysteine (NAC) or ferric citrate reduced hepatocyte toxicity from excessive CGA. All mice survived after receiving CGA injections at doses up to 125 mg/kg. The LD50 for Kunming mice was 382.28 mg/kg.These findings suggest that the antioxidant and iron-chelating properties of CGA determine its role in either liver protection or toxicity at varying concentrations, providing valuable insight for its rational dietary and clinical use.