Bangladesh Journal of Pharmacology Volume: 17; Number 3; Year 2022 Cite this article as: Tran DM, Tran TT, Nguyen NTT, Chu HH, Nakahama T, Nguyen NT. Anti-inflammatory activity of 9-hydroxy-canthin-6-one extracted from hairy-root cultures of Eurycoma longifolia potentially via aryl hydrocarbon receptor induction. Bangladesh J Pharmacol. 2022; 17: 102-104. Anti-inflammatory activity of 9-hydroxy- canthin-6-one extracted from hairy-root cultures of Eurycoma longifolia potentially via aryl hydrocarbon receptor induction Sir, Eurycoma longifolia Jack (Simaroubaceae) is a commer- cially important herb and is widely used in Southeast Asian countries (Bhat and Karim, 2010; Rehman et al., 2016). The present group and others reported that pharmaceutical secondary metabolites extracted from E. longifolia showed anti-inflammatory and anti-cancer properties (Tong et al., 2015; Rehman et al., 2016; Ngoc et al., 2016; Nguyen et al., 2016). The bioactivity of 9- hydroxycanthin-6-one, the main compound in E. longifolia, however, is not fully understood. This study aimed to accelerate 9-hydroxycanthin-6-one production from E. longifolia hairy root cultures and to test its anti-inflammatory activity in RAW264.7 macrophage cell line. We determined the 9-hydroxycanthin-6-one compound extracted from the hairy root of E. longifolia. The 9- hydroxycanthin-6-one was obtained as an amorphous yellow powder. Its HR-ESI-MS spectrum revealed the peak at m/z: 237 [M+H]+, corresponding to the molecular formula C14H8N2O2. The 1H-NMR spectrum of 9- hydroxycanthin-6-one showed weak signals of the protons of the aromatic ring and dienes, where the signals characteristic of an ABX aromatic system at H 7.95 (1H, d, J = 2.0 Hz, H-8), 6.98 (1H, dd, J = 2.0; 8.5 Hz, H-10), 8.11 (1H, d, J = 8.5 Hz, H-11). Additionally, two signals of protons at H 8.08 (1H, d, J = 5.0 Hz, H-1), 8.72 (1H, d, J = 5.0 Hz, H-2) were assigned to two protons in the aromatic ring with one heteroatom of nitrogen. Two doublet signals at H 6.93 (H-3)/8.06 (H-4) correspond to two hydrogen atoms (H-4 and H-5) of the aromatic ring with two hetroatoms of carbon and nitrogen. The singlet signal at H 7.95 is assigned to H-8. The 13C-NMR and DEPT spectra of this compound indicate the molecule contains 14 carbon atoms, including seven methines and seven quaternary carbons. The signals appearing in the range from C­ 102.9 to 145.9 indicated the carbon atoms of aromatic rings and dienes. The two signal peaks of the carboxyl group appeared at C­ 158.9, with this quite low value of c caused by the conjugation effect of the carboxyl group and a diene, and the peak of C­ 160.5 is assigned to the carbon atom of the hydroxyl group. Therefore, based on the spectral analysis, it could conclude that this compound is 9-hydroxycathin-6-one. There was growth of hairy root and accumulation of 9- hydroxycathin-6-one in Murashige and Skoog (MS), Shenck and Hildebrandt (SH), and McCown’s woody plant (WP) liquid culture media supplemented with 3% (w/v) of sucrose. After 30 days, the biomass generated by the SH basal medium was the greatest (0.8 ± 0.0 g, A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2022; 17: 102-104 Journal homepage: www.banglajol.info; www.bdpsjournal.org Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v17i3.60373 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 Days Figure 1: Dry weight and 9-hydroxycanthin-6-one production in hairy root of E. longifolia on different basal media (MS, SH, and WP). The mean ± SD data from three independent experiments are shown 0.4 0.3 0.2 0.1 0 D ry w e ig h t (g ) 9 -H y d ro x y c a n th in -6 -o n e ( % d ry w e ig h t) DW), followed by MS (0.7 ± 0.1 g, DW), and WP (0.6 ± 0.1 g, DW). Meanwhile, 9-hydroxycanthin-6-one pro- duction dynamics in WP medium increased after 10 days and reached a maximum value of 0.3 ± 0.0 (% DW) after 30 days. Conversely, 9-hydroxycanthin-6-one pro- duction dynamics in the MS and SH media revealed a long lag phase and no growth, with a maximum level of 0.1 ± 0.0 (%DW) after 25 days and 0.1 ± 0.0 (%DW) after 30 days, respectively (Figure 1). These results suggest that the 9-hydroxycanthin-6-one content was greatest in hairy roots cultivated in WP medium, while the maxi- mum biomass of hairy roots was obtained in SH medium. Besides, the present study reported that aryl hydro- carbon receptor (Ahr), a ligand-dependent transcription factor, played a role as an anti-inflammatory agent via modulating various cytokines in such immune cells as macrophages, dendritic cells and t cells which involve in the development of inflammatory and autoimmune disorders (Masuda et al., 2011; Zhu et al., 2018; Neavin et al., 2018). Therefore, looking for new Ahr inducers is crucial for controlling these disorders. This is the first time we showed that Ahr expression was significantly increased in lipopoly- saccharide-stimulated RAW264.7 cells under the induction of 9-hydroxycanthin-6-one. The cells were pre-induced with 9-hydroxycanthin-6-one (1, 3, 10, 30 μM) for 30 min before being stimulated with lipopolysaccharide (1 μg/mL) for 1, 3, 6, and 24 hours. Interestingly, we found that Ahr gene expression was significantly increased, reaching a maximum of 11.5 fold at a concentration of 30 µM, compared to untreated cells at 6 hours (Figure 2). Previously, it has been demonstrated that Ahr suppresses pro-inflammatory cytokine production in macrophages by inhibiting nuclear factor-κB (NF-κB) activation after lipopolysaccharide stimulation Bangladesh J Pharmacol 2022; 17: 102-104 103 12 10 8 6 4 2 0 R e la ti v e e x p re s s io n l e v e l (A h r/ β -a c ti n ) 14 12 10 8 6 4 2 0 R e la ti v e e x p re s s io n l e v e l (I L -6 /β -a c ti n ) 5 4 3 2 1 0 R e la ti v e e x p re s s io n l e v e l (T N F -α /β -a c ti n ) Compound 0 0 1 3 10 30 (µM) Lipopolysaccharide (1 µg/mL) 24 Hours 6 Hours 3 Hours Figure 2: Effect of 9-hydroxycanthin-6-one isolated from the hairy roots of E. longifolia on the expression level of Ahr, IL-6, TNF-α in Raw 264.7 cells treated with lipopolysaccharide. RT-qPCR was utilized to measure the expression level. The mean ± SD data from three independent experiments is shown. ap<0.05; bp<0.01; cp<0.005 compared with lipopolysaccharide only a a a a a b b b b b b c c c c c c c c c c c c (Kimura et al., 2009). We, therefore, measure the expre- ssion of cytokine genes, including IL-6 and TNF-α during this stimulation. Consistently, inflammatory cyto- kine genes such as IL-6 and TNF-α were significantly reduced after treatment with various concentrations of 9-hydroxycanthin-6-one (Figure 2). These findings imply that 9-hydroxycanthin-6-one extracted from the hairy roots of E. longifolia is a potential activator of Ahr in the context of LPS stimulation that thus might inhibit inflammatory cytokines such as IL-6 and TNF-α in the macrophages (Figure 3). This work was supported by Graduate University of Science and Technology, Vietnam Academy of Science and Technology (Grant code: GUST.STS.DT2019-SH01). Duc Minh Tran1, Trang Thu Tran2, Ngan Thi Thuy Nguyen2, Hoang Ha Chu2, Taisuke Nakahama 3 and Nam Trung Nguyen 2 1 Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam; 2 Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam; 3 Department of RNA Biology and Neuroscience, Osaka University, Suita, Osaka, Japan. First two authors contributed equally Corresponding author: Email: nam@ibt.ac.vn References Bhat R, Karim AA. Tongkat Ali (Eurycoma longifolia Jack): A review on its ethnobotany and pharmacological importance. Fitoterapia 2010; 81: 669-79. Kimura A, Naka T, Nakahama T, Chinen I, Masuda K, Nohara K, Fujii-Kuriyama Y, Kishimoto T. Aryl hydrocarbon recep- tor in combination with Stat1 regulates LPS-induced inflammatory responses. J Exp Med. 2009; 206: 2027-35. 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Figure 3: 9-Hydroxycanthin-6-one suppress inflammatory cytokines potentially via Ahr 104 Bangladesh J Pharmacol 2022; 17: 102-104 mailto:nam@ibt.ac.vn