Evaluation of Aculeatin and Toddaculin Isolated from <i>Toddalia asiatica</i> as Anti-inflammatory Agents in LPS-Stimulated RAW264 Macrophages

Abstract

Anti-inflammatory activity of aculeatin and toddaculin, which are coumarins with a similar structure isolated from Toddalia asiatica (L.) LAM., was evaluated using lipopolysaccharide (LPS)-stimulated RAW264 mouse macrophage cells. Both aculeatin and toddaculin significantly inhibited mRNA expression of inflammatory mediators and nitric oxide production. Furthermore, Toddaculin suppressed LPS-induced phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2 and inhibited LPS-induced activation of nuclear factor-kappaB (NF-κB). However, aculeatin did not exhibit such effects, suggesting that aculeatin and toddaculin suppress LPS-induced inflammation of RAW264 cells via different mechanisms. The cellular uptake of these compounds was also evaluated. Toddaculin was detected in RAW264 cells after 4 and 24 h. However, aculeatin levels were not observed in RAW264 cells at all incubation intervals. These results indicate that de-epoxidation of a prenyl group can increase hydrophobicity of molecule and is thought to accelerate cellular uptake and/or interactions with the phospholipid bilayers of cell membranes. Key words Toddalia asiatica; coumarin; aculeatin; toddaculin; anti-inflammation; macrophageInflammation is an adaptive response by the body to ensure the removal of detrimental stimuli as well as a healing process for repairing damaged tissue.1) However, if left uncontrolled, inflammatory mediators become involved in the pathogenesis of many inflammatory disorders.2) Macrophages play a pivotal role in the host defense against pathogenic microbes by recognizing bacterial constituents resulting in the activation of a variety of antimicrobial effectors and initiation of the inflammatory cascade.3) When activated by stimuli such as lipopolysaccharides (LPS), macrophages produce numerous inflammatory mediators such as nitric oxide (NO) and proinflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1) and IL-6 via toll-like receptor pathways. 4,5) Expression of these inflammatory mediators is regulated by the activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB). 6,7)Toddalia asiatica (L.) LAM. is widely recognized as a traditional medicinal plant in Africa, India, China, and Japan and has been used for treating many diseases. This plant contains a variety of coumarin compounds, and many biological properties have been attributed to it.8-12) Our recent studies have also shown that aculeatin (ACU) derived from T. asiatica enhances the differentiation and lipolysis of 3T3-L1 adipocytes.13) Furthermore, toddaculin (TOD) inhibits osteoclastogenesis in RAW264 cells and enhances osteoblastogenesis in MC3T3-E1 cells.14) On the other hand, another research group showed that ACU and TOD exhibit inducible NO synthase (iNOS) protein expression-inhibitory activity in LPS-stimulated mouse macrophage cells.15) However, detailed mechanisms underlying these results were unclear.Here, we focused on the anti-inflammatory activity of these coumarins. To reve...

Separation of New Coumarin Glycosides from Toddalia asiatica Using Offline Two-Dimensional High-Performance Liquid Chromatography

“…The roots and barks of T. asiatica have been used in Miao minority medicine, mainly in Guizhou, Yunnan, and Guangxi provinces in China, for the treatment of fall injuries, rheumatic arthralgia, stomachache, chronic lumbago, and diarrhea [3,4]. Previous phytochemical and pharmacological investigations of different parts of this plant revealed that coumarins are the main secondary metabolites [5], some of which exhibited cytotoxic, antimalarial [6], antiviral [7], anti-inflammatory [8], antibacterial [9], anti-platelet aggregation [10], anti-plasmodial, larvicidal [11], phosphodiesterase-4 inhibitory [12], and osteoclastogenesis activities [13]. Even though coumarins are rather studied, less attention has been paid to their glycosides.…”

A systematic review on traditional medicine Toddalia asiatica (L.) Lam.: Chemistry and medicinal potential

“…The consequences suggested that omphalocarpin could reduce the release of NO induced by lipopolysaccharide and the secretion of TNF- α and IL-6 inflammatory factors, strongly inhibit the expression and activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and inhibit the transfer of NF- κ B to the nucleus. Kumagai et al, ( Kumagai et al, 2018 ) also found that aculeatin and toddaculin isolated from T. asiatica could also play important roles in anti-inflammation in LPS-stimulated RAW264 macrophages via different mechanisms, which further demonstrated that extracts from T. asiatica own significant anti-inflammatory effect.…”

Rediscovery of Traditional Plant Medicine: An Underestimated Anticancer Drug of Chelerythrine

“…According to the “Chinese Materia Medica” issued by the State Administration of Traditional Chinese Medicine, T. asiatica is used to treat malignant sores and lumps with the efficacy of detoxification, which is closely related to treating cancer. Based on the traditional use of this plant to treat cancer, multiple modern experimental studies have proven its anticancer activity(He and N, 1998; Iwasaki et al, 2010 ; Li et al, 2018 ) and anti-inflammatory activity ( Kumagai et al, 2018 ; Ni et al, 2020 ). In recent years, with in-depth research, the chemical components in T. asiatica have gradually been discovered to be mainly coumarins and alkaloids, and alkaloids in T. asiatica play an important role in antitumor effects, especially benzophenanthridine alkaloids.…”