plants Communication Anti-Inflamatory Activity of Neolignan Compound Isolated from the Roots of Saururus chinensis Sae-Rom Yoo , Hyekyung Ha , Hyeun-Kyoo Shin and Chang-Seob Seo * Korea Institute of Oriental Medicine, Daejeon 34054, Korea; [email protected] (S.-R.Y.); [email protected] (H.H.); [email protected] (H.-K.S.) * Correspondence: [email protected]; Tel.: +82-42-868-9361 Received: 6 July 2020; Accepted: 22 July 2020; Published: 23 July 2020 Abstract: Saururus chinensis (Lour.) Baill. is a perennial herb and grows in Korea, China, and Japan. 8 Interestingly, (7S,8S)-D 0 -3,4-methylenedioxy-30,5,50-trimethoxy-7-monoacetate-8.O.40-neolignan (MTMN), one of the active neolignans, was first isolated from the roots of Saururus chinensis. The compound was screened for anti-inflammatory activity using a RAW264.7 murine macrophage cell line. The dried roots of S. chinensis (9.7 kg) were extracted with 70% methanol and then solvent fractionation. From the ethyl acetate fraction, MTMN was purified through silica gel column and reverse-phase column chromatography and its structure was identified by spectroscopic analysis with nuclear magnetic resonance, circular dichroism, and mass spectrometry. RAW264.7 cells were induced using lipopolysaccharide (LPS) and treated with or without MTMN. Production of nitric oxide (NO) and prostaglandin E2 (PGE2) levels were measured and protein expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were analyzed by immunoblotting. The isolated 8 neolignan was (7S,8S)-D 0 -3,4-methylenedioxy-30,5,50-trimethoxy-7-monoacetate-8.O.40-neolignan. This compound suppressed the LPS-induced iNOS and COX-2 protein expressions, which led to a decrease in the production of NO and PGE2 levels. Further studies, including in animal models, will be required to establish the precise pharmacological effect of MTMN. Keywords: Saururus chinensis; neolignan; inflammation 1. Introduction Inflammation is a biological response that protects the body against harmful stimuli. Inflammatory abnormalities underlie a number of diseases including atherosclerosis [1], mental illness [2,3], sarcopenia [4], and obesity [5]. For over a decade, nonsteroidal anti-inflammatory drugs (NSAIDs) have been commonly used to alleviate inflammatory abnormalities. Although their side effects depend on the specific drug, the adverse effects of long-term use of NSAIDs include an increased risk of gastrointestinal disorders, myocardial infarction, and renal disease [6]. Therefore, recent studies in search of anti-inflammatory agents have focused on plant-derived medications, which are known for having relatively fewer side effects than conventional therapy. Saururus chinensis (Lour.) Baill. (family: Saururaceae), commonly known as “Asian lizard’s tail” [7] is a perennial herb and distributed in parts of East Asia, including Korea, China, and Japan. In Korean folk remedy, it is widely used for the treatment of edema, bladder infection, and several inflammatory diseases [8]. The root of S. chinensis contains various kinds of lignans including saucerneol, di-O-methyltetrahydrofuriguaiacin B, machilin D, and sauchinone [9–12], some of which have anti-inflammatory or antioxidant effects [10,13,14]. 8 In this study, we isolated a new 8-O-40 type neolignan, (7S,8S)-D 0 -3,4-methylenedioxy-30,5,50- trimethoxy-7-monoacetate-8.O.40-neolignan (MTMN), from the roots of S. chinensis. This article presents the isolation, structural elucidation, and anti-inflammatory effects of this compound. Plants 2020, 9, 932; doi:10.3390/plants9080932 www.mdpi.com/journal/plants Plants 2020, 9, 932 2 of 5 Plants 2020, 9, x 2 of 5 2. Results2. Results and and Discussion Discussion 2.1.2.1. Structure Structure Determination Determination of MTMNof MTMN CompoundCompound1 was 1 was obtained obtained as a as sticky a sticky solid, solid, with with a molecular a molecular formula formula of C24 Hof28 CO248Hdetermined28O8 determined by high-resolutionby high-resolution electrospray electrospray ionization ionization mass spectrometry mass spectrometry (HRESIMS, (HRESIMS,m/z; found m/z; 447.1683 found [M +447.1683Na]+; calcd[M+Na] 447.1682)+; calcd (Figure 447.1682) S1). (Fig Sevenure methineS1). Seven groups, methine three groups, methylene three methylene groups, two groups, methyl two groups, methyl ninegroups, quaternary nine carbons,quaternary and carbons, three methoxyl and three groups methoxyl were identified groupsin were the 1 identifiedH-NMR, 13 inC-NMR, the 1H DEPT135,-NMR, 13C- andNMR, DEPT90 DEPT135, spectrum and of DEPT90 1 (Figure spectrum S2). The of1 1H-NMR (Figure spectrumS2). The 1H exhibited-NMR spectrum two distinct exhibited methyl two groups distinct (H-9methyl and OAc-CH groups 3),(H three-9 and methoxyl OAc-CH groups3), three (5-CH methoxyl3, 30-CH 3,groups and 50 -CH(5-CH3), one3, 3’ methylenedioxy-CH3, and 5´-CH group3), one (OCHmethylenedioxy2O-3,4), one methylene group (OCH group2O- (H-93,4),0 ),one and methylene four aromatic group protons (H-9´), (H-2, and H-6, four H-2 aromatic0, and H-6 protons0). In the(H-2, heteronuclearH-6, H-2´, and multiple H-6´). bond In the correlation heteronuclear spectrum multiple of 1 (Figure bond1 bcorrelation and Figure spectrum S3), long-range of 1 (Fig correlationsure 1b and of C-7Figure with S3 H-2,), long H-6,-range H-8 andcorrelati H-9 wereons of observed. C-7 with C-7 H-02,showed H-6, H long-range-8 and H-9 correlationswere observed. with H-9C-7´0 ,showed H-20, andlong H-6-range0. In addition, correlations the ketonewith H group-9´, H- of2´, 7-monoacetateand H-6´. In addition, showed athe long-range ketone group correlation of 7-monoacetate with H-7. Theshowed circular a dichroism long-range (CD) correlation spectrum (Figurewith H S4)-7. wasThe measured circular dichroism to determine (CD the) configurationsspectrum (Fig ature C-7 S4 and) was C-8measured of compound to determine1. The results the revealedconfigurations that the at CD C- spectrum7 and C-8 of of1 compoundshowed positive 1. The signs results in the revealed region ofthat 240–400the CD nm, spectrum indicating theof 8S-configuration1 showed positive compared signs within the those region of reported of 240 analogs–400 nm, [15– 17indicating]. Furthermore, the 8S- weconfiguration confirmed the compared threo-configuration with those between of reported H-7 and analogs H-8 by [15 confirming–17]. Furthermore, the large coupling we confirmed constant the (J7,8threo= 7.3-configuration Hz) according between to previous H-7 studiesand H- [818 by,19 confirming]. The basis the of theselarge datacoupling and reportedconstant literature, (J7,8 = 7.3 1Hz), 8 wasaccording determined to toprevious be (7S,8S)- studiesD 0 -3,4-methylenedioxy-3 [18,19]. The basis0,5,5 of0 -trimethoxy-7-monoacetate-8.these data and reported literature,O.40-neolignan, 1, was whichdetermined was first to isolated be (7S,8S) and reported-Δ8′-3,4-m inethylenedioxy this study. -3′,5,5′-trimethoxy-7-monoacetate-8.O.4′-neolignan, which was first isolated and reported in this study. (a) (b) FigureFigure 1. (a 1.) Chemical(a) Chemical structure structure and and (b) key(b) key heteronuclear heteronuclear multiple multiple bond bond correlation correlation of compound of compound1. 1. 2.2. Inhibitory Effect of Inflammation Related Mediator of MTMN 2.2.Macrophages Inhibitory Effect are aof major Inflammation component Related of the M innateediator immuneof MTMN system, which modulates inflammatory responsesMacrophages and tissue are homeostasis a major [20component]. Lipopolysaccharide of the innate (LPS) immune is a well-knownsystem, which activator modulates of macrophageinflammatory and activatedresponses macrophage and tissue leadshomeostasis to the production [20]. Lipopolysaccharide of inflammatory ( mediatorsLPS) is a includingwell-known cytokines,activator NO, of macrophage and prostaglandins and activated [20–22]. macrophage In this study, lead wes determinedto the production the anti-inflammatory of inflammatory mediatorsmediators aff ectedincluding by MTMN cytokines, in the NO, LPS-stimulated and prostaglandins mouse murine[20–22]. macrophageIn this study, RAW264.7 we determined cell line. the Weanti evaluated-inflammatory the cytotoxicity mediators of affected MTMN by following MTMN in the the treatment LPS-stimulated of RAW264.7 mouse cellsmurine with macrophage various concentrationRAW264.7 forcell 24line. h. MTMNWe evaluated has no toxicitythe cytotoxicity up to 25 µofM MTMN treatment following (data not the shown). treatment Thus, of nontoxicRAW264.7 concentrationscells with various of MTMN concentration were used for for 24 all h. subsequent MTMN has experiments. no toxicity up to 25 μM treatment (data not shown).Nitric Thus oxide, nontoxic synthase concentrations (iNOS) is produced of MTMN in response were used to bacterial for all subsequent products andexperiments. inflammatory cytokinesNitric [21 ],oxide and synthase iNOS-derived (iNOS NO) is p isroduced closely in related response to inflammatory to bacterial products processes. and NO inflammatory plays a rolecytokines in various [21], physiological and iNOS-derived and pathological NO is closely processes, related excessiveto inflammatory production processes. of NO NO may plays lead a torole inflammation response. As expected, LPS significantly increased the level of NO (9.53 0.34 µM) in various physiological and pathological processes, excessive production of NO ±may lead to andinflammation the expression response. of iNOS As in expected, RAW264.7 LPS cells significantly (Figure2 a,b).increased By contrast, the level MTMN of NO (9.53 downregulated ± 0.34 μM) and thethe expression expression of iNOSof iN atOS a concentrationin RAW264.7 ofcells 25 µ(FigureM. The LPS-stimulated2a and Figure NO2b). level By wascontrast, decreased MTMN by MTMN at a concentration of 25 µM by 6.11 0.43 µM(p < 0.05). L-NG-monomethyl arginine downregulated the expression of iNOS at a concentration± of 25 μM. The LPS-stimulated NO level (L-NMMA),was decreased as a non-selective by MTMN at inhibitor a concentration of NOS, of also 25 significantly μM by 6.11 ± decreased 0.43 μM ( thep < level0.05).