  Citation: Ma, X.; Okyere, S.K.; Hu, L.; Wen, J.; Ren, Z.; Deng, J.; Hu, Y. Anti-Inflammatory Activity and Mechanism of Cryptochlorogenic Acid from Ageratina adenophora. Nutrients 2022, 14, 439. https:// doi.org/10.3390/nu14030439 Academic Editors: Daniela Rigano and Paola Bontempo Received: 19 December 2021 Accepted: 17 January 2022 Published: 19 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). nutrients Article Anti-Inflammatory Activity and Mechanism of Cryptochlorogenic Acid from Ageratina adenophora Xiaoping Ma 1,2,† , Samuel Kumi Okyere 1,2,† , Liwen Hu 1,2 , Juan Wen 1,2 , Zhihua Ren 1,2 , Junliang Deng 1,2 and Yanchun Hu 1,2, * 1 Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China; mxp886@sicau.edu.cn (X.M.); samuel20okyere@gmail.com (S.K.O.); huliwen197@163.com (L.H.); juanwen881010@163.com (J.W.); zhihua_ren@126.com (Z.R.); dengjl213@126.com (J.D.) 2 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China * Correspondence: yanchunhu@sicau.edu.cn; Tel.: +86-2886291162 † These authors contributed equally to this work. Abstract: Ageratina adenophora is an invasive plant known for its toxicity to livestock. Current research on this plant has shifted from toxicity prevention to the beneficial utilization of plant resources. This study was performed to investigate the effects and mechanisms of cryptochlorogenic acid (CCGA) isolated from Ageratina adenophora on the inflammatory responses induced by lipopolysaccharide (LPS) in RAW264.7 cells. RAW264.7 cells were pretreated with CCGA (200, 100, and 50 μg/mL) and subsequently stimulated with LPS (1 μg/mL) for 16 h. The cytotoxicity of CCGA was tested using the Cell Counting Kit (CCK8). The mechanism of action of CCGA in attenuating inflamma- tion was also identified using enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction, and Western blot. The results showed that CCGA had a maximal safe concentration of 200 mg/mL. Moreover, CCGA reduced the level of nitric oxide (NO) and iNOS in LPS-induced RAW264.7 cells (p < 0.01). In addition, CCGA reduced the levels of pro- inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) and cyclooxygenase-2 (COX-2) in LPS-induced RAW264.7 cells at both the mRNA and protein levels (p < 0.01). CCGA prevented the activation of nuclear factor-kappa B (NF-kB) in LPS-induced RAW264.7 cells via the inhibition of IKK and IκB phos- phorylation and the degradation of IκB proteins (p < 0.01). This finding indicated that CCGA isolated from A. adenophora may be a potential candidate for the treatment of inflammation-related diseases. Keywords: Ageratina adenophora; cryptochlorogenic acid; anti-inflammatory; nuclear factor-kappa B pathway 1. Introduction The chief tool used by most organisms against infection is inducing inflammatory responses [1]. However, elevated levels of inflammatory cytokines in the body circulation or in local inflammatory sites result in severe diseases [2–4]. Chronic inflammation is linked with various diseases, such as diabetes, depression, and cancer [5–13]. Among the inflam- matory inducing agents, lipopolysaccharide (LPS) is reported to enhance the production of pro-inflammatory cytokines in macrophages, fibroblasts, and monocytes [14]. Furthermore, it has been reported that LPS-induced inflammatory reactions can occur via the NF-κB signaling pathway [15]. TNF-α cytokine actively takes part in systemic inflammation as well as immune cell regulation [16]. IL-1β is known for activating the plasmalemmal IL-1R1 receptor in various types of cells [17,18]. IL-6 is involved in numerous cellular activities, including cell proliferation, differentiation, and apoptosis [19]. IL-8 is a pro-inflammatory cytokine that is linked to tumor growth and progression [20]. Cyclooxygenase (COX) is an oxidoreductase enzyme noted for the conversion of arachidonic acid to prostaglandins, Nutrients 2022, 14, 439. https://doi.org/10.3390/nu14030439 https://www.mdpi.com/journal/nutrients