In Vitro Mechanistic Study of the Anti-inﬂammatory Activity of a Quinoline Isolated from Spondias pinnata Bark Nikhil B. Ghate, †,∥ Dipankar Chaudhuri, †,∥ Sourav Panja, † Sudhir S. Singh, ‡ Gajendra Gupta,* ,§ Chang Yeon Lee, § and Nripendranath Mandal* ,† † Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VIIM, Kolkata 700054, India ‡ TCG Lifesciences Limited, Block BN, Plot 7, Salt Lake Electronics Complex, Sector V, Kolkata 700091, India § Department of Energy and Chemical Engineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea *S Supporting Information ABSTRACT: The search for new plant-based anti-inﬂamma- tory drugs continues in order to overcome the detrimental side eﬀects of conventional anti-inﬂammatory agents, both steroidal and nonsteroidal. This study involves the quinoline SPE2, 7-hydroxy-6-methoxyquinolin-2(1H)-one, isolated from the EtOAc fraction of Spondias pinnata bark. Structure elucidation was done using analytical spectroscopic methods including Fourier transform infrared spectroscopy, high-resolution electro- spray ionization mass spectrometry, nuclear magnetic resonance spectroscopy, and single-crystal X-ray crystallography. The anti-inﬂammatory activity of SPE2 was evaluated in a lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 model. SPE2 eﬀectively suppressed LPS-induced overproduction of pro-inﬂammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β, and reactive oxygen species. Expression levels of NO synthesizing enzyme, cyclooxygenase-2, TNF-α, IL-6 and IL-1β were also determined to return to normal after SPE2 treatment. Localization of NF-κB was evaluated by confocal microscopy and Western blotting, which showed a dose-dependent reduction of NF-κB inside the nucleus and an increase in cytoplasmic NF-κB with SPE2 treatment. Collectively, the results suggest that SPE2 has anti-inﬂammatory activity via inhibition of NF-κB activation. I nﬂammation is a central biological protective response of tissues to damaging impulses including tissue injury, irritants, and pathogenic infection. 1 Inﬂammation is the immune system’s immediate response and primarily activates macrophages, which in turn release molecular mediators such as pro-inﬂammatory molecules, including prostaglandins, cytokines, and free radicals such as nitric oxide (NO). 2 Although the function of inﬂam- mation is vital to eliminate microbial infections and damaged tissues and to initiate tissue repair, uncontrolled chronic inﬂam- matory responses may inﬂuence the onset of several diseases like atherosclerosis, Alzheimer’s disease, type 2 diabetes, and can- cer. 3 Diﬀerent synthetic nonsteroidal or steroidal anti-inﬂammatory drugs are regularly used to suppress chronic inﬂammatory responses but exhibit several adverse eﬀects such as immunode- ﬁciency, gastrointestinal disorders, and humoral disturban- ces. 4−6 Therefore, anti-inﬂammatory agents of natural origin that cause fewer side eﬀects have received much attention as promising new drug candidates. Several phenolics and ﬂavonoids acquired from natural sources exhibit excellent free radical scavenging and anticancer potentials. Earlier reports have suggested that these types of compounds may be useful in treating inﬂammation-related diseases. 7 Spondias pinnata (Linn. f.) Kurz (Anacardiaceae family), known as “Wild mango” in English, “Amra” in Hindi, and “Aamada” in Bengali, is a caducous tree distributed throughout India, Sri Lanka, China, and other South-East Asian countries. Phytochemical studies have shown that it contains a signiﬁcant amount of ﬂavonoids, tannins, saponins, and terpenoids. The fruit pulp yielded an essential oil composed of carboxylic acids and esters, alcohols, and aromatic hydrocarbons. The major compounds extracted were 9,12,15-octadecatrien-1-ol, hexade- canoic acid, and furfural. The fruits and gum contain galact- uronic acid, D-galactose, and L-arabinose. 8,9 The bark of this plant is used to treat burns, sores, and wounds as well as articular and muscular rheumatism, diabetes, gonorrhea, dysentery, diarrhea, and to prevent vomiting. 10−13 The crude extract of S. pinnata was previously investigated for its antioxidant and iron-chelating potential and also reported to contain signiﬁcant amounts of ﬂavonoids and other phenolic compounds. 14,15 Phenolics and glycosides obtained from S. pinnata bark pos- sess antioxidant and iron-chelating activity. 16,17 The bark of S. pinnata was also evaluated for its potential anticancer activity against lung and breast carcinomas. 18 From previous studies, it was observed that targeting the tumor microenvironment including inﬂammation might be a successful approach to enhance the eﬃcacy of cancer treatment. 19 Therefore, the Received: January 11, 2018 Article pubs.acs.org/jnp Cite This: J. Nat. Prod. 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