(Morinda Citrifolia L.) Fruit Juice

molecules

Communication Identiﬁcation of Anti-Inﬂammatory Compounds from Hawaiian Noni (Morinda citrifolia L.) Fruit Juice

1, 2, 3 3 3 Dahae Lee †, Jae Sik Yu †, Peng Huang , Mallique Qader , Arulmani Manavalan , Xiaohua Wu 3, Jin-Chul Kim 4, Changhyun Pang 5, Shugeng Cao 3,* , Ki Sung Kang 1,* and Ki Hyun Kim 2,*

1 College of Korean Medicine, Gachon University, Seongnam 13120, Korea; [email protected] 2 School of Pharmacy, Sungkyunkwan University, Suwon 16419, Korea; [email protected] 3 Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA; [email protected] (P.H.); [email protected] (M.Q.); [email protected] (A.M.); [email protected] (X.W.) 4 Natural Product Informatics Research Center, KIST Gangneung Institute of Natural Products, Gangneung 25451, Korea; [email protected] 5 School of Chemical Engineering, Sungkyunkwan University, Suwon 16419, Korea; [email protected] * Correspondence: [email protected] (S.C.); [email protected] (K.S.K.); [email protected] (K.H.K.); Tel.: +1-808-981-8017 (S.C.); +82-31-750-5402 (K.S.K.); +82-31-290-7700 (K.H.K.) These authors contributed equally to this work. † Academic Editor: Lesław Juszczak Received: 23 September 2020; Accepted: 26 October 2020; Published: 27 October 2020

Abstract: Noni (Morinda citrifolia L.) fruit juice has been used in Polynesia as a traditional folk medicine and is very popular worldwide as a functional food supplement. In this study, compounds present in Hawaiian Noni fruit juice, with anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were identified. Five compounds were isolated using a bioassay-driven technique and phytochemical analysis of noni fruit juice: asperulosidic acid (1), rutin (2), nonioside A (3), (2E,4E,7Z)-deca-2,4,7-trienoate-2-O-β-d-glucopyranosyl-β-d-glucopyranoside (4), and tricetin (5). The structures of these five compounds were determined via NMR spectroscopy and LC/MS. In an anti-inflammatory assay, compounds 1–5 inhibited the production of nitric oxide (NO), which is a proinflammatory mediator, in LPS-stimulated macrophages. Moreover, the mechanisms underlying the anti-inflammatory effects of compounds 1–5 were investigated. Parallel to the inhibition of NO production, treatment with compounds 1–5 downregulated the expression of IKKα/β, I-κBα, and NF-κB p65 in LPS-stimulated macrophages. Furthermore, treatment with compounds 1–5 downregulated the expression of nitric oxide synthase and cyclooxygenase-2. Thus, these data demonstrated that compounds 1–5 present in noni fruit juice, exhibited potential anti-inflammatory activity; these active compounds may contribute preventively and therapeutically against inflammatory diseases.

Keywords: noni; Morinda citrifolia; anti-inﬂammation; NO production; RAW 264.7 cells

1. Introduction Noni is the Hawaiian name given to Morinda citrifolia Linn. (Family: Rubiaceae), also known as beach mulberry or cheese fruit [1]. It is a small tropical evergreen tree or shrub that is native to Southeast Asia and is distributed in Australia and the South Paciﬁc, including Hawaii [1,2]. The phytotherapeutic applications of noni fruit juice have been mentioned in folklore, and the juice has been successfully used as per traditional systems of medicine to treat a broad range of leading diseases

Molecules 2020, 25, 4968; doi:10.3390/molecules25214968 www.mdpi.com/journal/molecules Molecules 2020, 25, 4968 2 of 12 Molecules 2020, 25, x FOR PEER REVIEW 2 of 12 likediseases diabetes, like diabete cancer,s, hypertension, cancer, hypertension, cardiovascular cardiovascular diseases, arthritis,diseases, pathogenic arthritis, pathogenic and worm and infections, worm poorinfections, indigestion, poor indigestion, and HIV–AIDS and HIV and– drugAIDS addiction and drug [3 addiction–5]. [3–5]. The products derived from noninoni are very popular globally,globally, particularlyparticularly the fruit juice,juice, which is used as a functionalfunctional food supplement [[3]3].. Noni juice was introduced intointo markets in the USA in thethe 1990s1990s andand intointo EuropeanEuropean communities since 2003 [[3]3].. By now, the products are very famous in New Zealand, Australia,Australia, andand somesome Asian Asian countries countries [ 6[6]].. The The worldwide worldwide market marke fort for noni-derived noni-derived products products is peakingis peaking each each year; year in; in 2006, 2006 it, reachedit reached USD USD two two billion billion [6]. [6]. Nuclear factor factor kappa kappa B (NF- B (NFκB)-κB) is a transcription is a transcription factor thatfactor mainly that regulates mainly regulates the cellular the responses cellular toresponses inflammation, to inflammation, cancer, and cancer, nervous and system nervous functioning system [7 functioning]. The activation [7]. Th ofe NF- activationκB contributes of NF-κB to thecontributes development to the ofdevelopment various disorders, of various including disorders, rheumatoid including arthritis, rheumatoid atherosclerosis, arthritis, atherosclerosis, inflammatory bowelinflammatory diseases, bowel multiple diseases, sclerosis, multiple and tumors sclerosis, [8]. and The tumors inhibition [8] of. The NF- iκnhibitionB could stopof NF the-κB proliferation could stop ofthe tumor proliferation cells, which of tumor could cells become, which more could sensitive become tomore antitumor sensitive agents to antitumor [9]. Thus, agents the [9] inhibition. Thus, the of NF-inhibitionκB signaling of NF- hasκB potentialsignaling therapeutic has potential applications therapeutic in applications cancer and inflammatory in cancer and diseases inflammatory [8]. It wasdiseases reported [8]. I thatt was patients reported with that stage patients III/IV with invasive stage adenocarcinoma III/IV invasive adenocarcinoma who consumed homemadewho consum nonied fruithomemade juice regained noni fruit their juice body regained weight their after body 4 weight weeks [after10]. 4 This weeks encouraged [10]. This en researcherscouraged researchers to conduct in-depthto conduct studies in-depth on nonistudies fruit on juice. noni Asfruit an juice. initiative As an in initiative 2001, clinical in 2001, studies clinical have studies been carried have been out. However,carried out. after However, 5 years, after they 5 wereyears, unable they were to publish unable any to publish data supporting any data thesupporting relevance the [11 relevance]. In our previous[11]. In our studies previous on noni studies juice, on we noni identified juice, we alkyl identified catechols alkyl (4-methyl catechols catechol, (4-methyl 4-ethyl catechol, catechol, 4-eth andyl 4-vinylcatechol, catechol) and 4-vinyl [12] and catechol scopoletin) [12] [ 13and], among scopoletin which [13] 4-methyl, among catechol, which 4-ethyl 4-methyl catechol, catechol, and 4-vinyl4-ethyl catecholcatechol, are and potent 4-vinyl molecules catechol that are providepotent molecules protection that against provide diseases, protection like cancer against associated diseases, with like oxidativecancer associated stress, by with stimulating oxidative nuclear stress, factor by stimulating erythroid 2-related nuclear factor factor 2 erythroid (Nrf2) transcription 2-related factor factors. 2 In(Nrf2) continuation, transcription the factors. present In study continuation, was undertaken the present to isolatestudy was and undertaken identify inflammatory to isolate and inhibitors identify presentinflammatory in noni inhibitors fruit juice usingpresent a bioassay-guided in noni fruit juicetechnique. usingFive a bioassay compounds-guided (1–5 ) technique (Figure1). wereFive isolatedcompounds using (1 column–5) (Figure chromatography 1) were isolated and HPLC,using column and their chromatography structures were and determined HPLC, andvia NMR their spectroscopicstructures were and determined LC/MS analyses. via NMR Herespectroscopic we report and the LC/MS isolation analys andes. structure Here we characterizationreport the isolation of theseand structure isolates found characterization in noni fruit of juice, these as well isolates as their found anti-inflammatory in noni fruit juice effects, as and well basic as underlying their anti- mechanisminflammatory of effects action. and basic underlying mechanism of action.

Figure 1. Chemical structures of compounds 1–5. Figure 1. Chemical structures of compounds 1–5.

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2. Results and Discussion 2. Results Results and and Discussion

2.1.2.1. Isolation IsolationIsolation and and Identification IdentificationIdentification ofof BioactiveBioactive CompoundsCompounds fromfrom NoniNoni FruitFruit JuiceJuice OurOur preliminarypreliminary study study [13] [[13]13] showed showed that that different didifferentfferent noninoni juices,juicesjuices,, includingincluding purchasedpurchased crudecrude noninoni juice, ripe noni juice, and noni juice fermented with Lactobacillus plantarum (ATCC8014) at 22 °C (room juice, ripe ripe noni noni juice, juice, and and noni noni juice juice fermented fermented with with LactobacillusLactobacillus plant plantarumarum (ATCC8014)(ATCC8014) at 22 °C at (room 22 ◦C (roomtemperature)temperature) temperature) at at 24 24 h h [13] at[13] 24 demonstrated demonstrated h [13] demonstrated NF NF--κBκB inhibitio inhibitio NF-κB inhibition.nn.. We We decided decided We to to decided use use the the topurchased purchased use the purchased n nonioni juice juice innoniin thethe juiceanti anti--inflammatory ininflammatory the anti-inflammatory assay assay because because assay of of itsits becausecommercial commercial of its availability, availability, commercial reproducibility reproducibility availability, reproducibilityandand previously previously reportedandreported previously Nrf2Nrf2 nuclearnuclear reported ttranslocationranslocation Nrf2 nuclear activityactivity translocation [13][13].. BioBio--assayassay activity guidedguided [13]. fractionationfractionation Bio-assay guided methodmethod fractionation waswas usedused inin thismethodthis study study was [13 [13–– used1616]].. The The in s thisseparationeparation study of of [13 the the–16 purchased purchased]. The separation n nonioni juice juice ofviavia theHP20 HP20 purchased open open column column noni chromatography chromatography juice via HP20 yieldedopenyielded column sixsix fractionsfractions chromatography (Fr.(Fr. AA––F),F), yieldedandand Fr.Fr. sixEE waswas fractions active active (Fr. against against A–F), NFNF and--κBκB Fr. (Figure(Figure E was 22 active)).. Preparative Preparative against NF- HPLC HPLCκB separation followed by semi-preparative HPLC yielded five compounds: asperulosidic acid (1, tR 18.0, (Figureseparation2). Preparative followed by HPLC semi-preparative separation followed HPLC yielded by semi-preparative five compounds: HPLC asperulosidic yielded five acid compounds: (1, tR 18.0, 1 mg) [17], rutin (2, tR 23.0, 3 mg) [18], nonioside A (3, tR 21.2, 1 mg) [19], (2E,4E,7Z)-deca-2,4,7- 1asperulosidic mg) [17], rutin acid ( (21,, tR 18.0,23.0, 1 3 mg) mg) [ 17[1],8] rutin, nonioside (2, tR 23.0, A ( 3, mg)tR 21.2, [18], 1 nonioside mg) [19], A (2 (3E,,4tRE,721.2,Z)-deca 1 mg)-2,4,7 [19],- trienoate-2-O-β-D-glucopyranosyl-β-D-glucopyranoside (4, tR 21.0, 1 mg) [20], and tricetin (5, tR 20.7, (2trienoateE,4E,7Z-2)-deca-2,4,7-trienoate-2--O-β-D-glucopyranosylO-β--βD--dglucopyranoside-glucopyranosyl- (β4,- dtR-glucopyranoside 21.0, 1 mg) [20], and (4, ttricetinR 21.0, 1(5 mg), tR 20.7, [20], 3 mg) [21] (Figures 1 and 3, see Supplementary Material). The NMR data of the isolated compounds 3and mg) tricetin [21] (Figure (5, tR 20.7,s 1 and 3 mg) 3, see [21 ]S (Figuresupplementary1 and3, Material see Supplementary). The NMR Materials). data of the The isolate NMRd compounds data of the wereisolatedwere consistentconsistent compounds withwith were previousprevious consistent reportsreports with.. previous reports.

NFNF--kkBB inhibitoryinhibitory activityactivity 500500 450450 400400 350350 300300 250250 200200 150150

100100 Relative Luminescene Units LuminesceneRelative Relative Luminescene Units LuminesceneRelative 5050 00 CrudeCrude Fr.Fr. A A Fr.Fr. B B Fr.Fr. C C Fr.Fr. D D Fr.Fr. E E Fr.Fr. F F DMSODMSO SampleSample treatment treatment

FigureFigure 2.2. TheThe effect effecteffect of ofof the thethe crude crudecrude extract extractextract (5 mg (5(5 /mg/mL)mL)mg/mL) and and theand HP20 thethe HP20HP20 open columnopenopen columncolumn fractions fractionsfractions (Fr. A–F, (Fr.(Fr. 5 mg AA–/–mL)FF, , 55 mg/mLofmg/mL noni) fruit) of of n noni juiceoni fruit fruit on NF-juice juiceκ Bon on in NF NF HepG2--κBκB in in cells. HepG2 HepG2 The cells. cells. negative The The negative negative control was control control 0.2% was was DMSO 0.2% 0.2% (mean DMSO DMSO (mean SD).(mean ± ± SD) SD). . ±

FigureFigure 3. 3. NF NF-NF--κBκκBB assay assay-guidedassay--guidedguided separation separation of of compounds compounds 1 1––55. .

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2.2. NF-κB Inhibitory Activity of Compounds 1–5 The five isolated compounds (1–5) together with their parent fraction (Fr. E) were evaluated in a mammalian cell-based assay designed to monitor TNF-α-induced NF-κB activity. Compounds 1–5 showed NF-κB inhibitory activity with IC50 values of 12.8, 59.0, >100, 15.8 and 18.6 µg/mL, respectively, while the parent fraction (Fr. E) had an IC50 value of 205.7 µg/mL. When tested using the same conditions as the NF-κB assay, the five compounds (1–5, at 100 µg/mL each) and the parent fraction (Fr. E, at 1 mg/mL) did not exhibit any toxicity. The results demonstrated that compounds 1–5 possess anti-inflammatory properties. In the absence of cytotoxicity, inhibition of TNF-α-induced NF-κB activity indicates a chemopreventative potential.

2.3. Effects of Compounds 1–5 on Nitric Oxide Production In the immune response, macrophages, as effector cells, detect pathogenic substances and are responsible for the inflammatory response [22]. Macrophages activated by lipopolysaccharide (LPS), one of the most potent activators of macrophages, release free radicals and inflammatory mediators [23]. The overproduction of inflammatory mediators such as nitric oxide (NO) has been correlated with inflammatory disease [24]. Many natural compounds have been identified as inhibitors of NO synthase and they have been reported to have potential anti-inflammatory effects. Polyphenolic Citrus sinensis fraction has been reported to inhibit pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as NO [25]. In addition, some flavonoids are known to act as inhibitors of NF-κB, that plays a vital role in inflammatory responses [26]. According to the previous study, noni fruit juice is well-known to exhibit anti-inflammatory effects [27]. Administration of noni fruit juice alleviates inflammation symptoms of lung inflammation in rats [28] and a carrageenan-induced paw edema in rats [29]. These previous studies suggested that it is worthwhile to evaluate the anti-inflammatory effects of compounds isolated from noni fruit juice. Thus, to evaluate the anti-inflammatory effects of compounds 1–5, their effects on NO production in LPS-activated RAW 264.7 macrophages were investigated. As shown in Figure4A–E, compared with the LPS-alone treatment group (22.75 0.05 µM), after coincubation of 50 µM of the compounds with ± LPS, the nitrite concentration was found to decrease by 14.73 0.66 µM; 11.15 0.09 µM with an IC ± ± 50 value of 38.21 0.44 µM; 7.11 0.06 µM with an IC value of 2.01 0.14 µM; 6.07 0.13 µM with an ± ± 50 ± ± IC value of 2.14 0.61 µM; and 6.03 0.02 µM with an IC value of 2.15 0.03 µM, respectively, 50 ± ± 50 ± in compounds 1–5. As shown in Figure4F, after coincubation of 50 µM of the NO synthase inhibitor l-NG-nitroarginine methyl ester (L-NAME) with LPS, the nitrite concentration was found to decrease by 12.90 0.09 µM compared with the LPS-alone treatment group (19.13 0.14 µM). Compounds ± ± 2–5 were more efficient than L-NMMA (used as a positive control) in inhibiting NO production in LPS-activated RAW 264.7 macrophages. In the previous studies, asperulosidic acid (1) isolated from Hedyotis diffusa has been proven to inhibit NO production [30] and rutin (2) was also found to inhibit NO production [31]. Compound 5 was found to be the most effective compound in inhibiting NO production in LPS-activated RAW 264.7 macrophages. Previously, the anti-inflammatory effect of tricetin (5) has been evaluated in a mouse model of acute pulmonary inflammation induced by the LPS [32]. To investigate the mechanism underlying the inhibitory effects of compounds 1–5 on NO production in LPS-activated RAW 264.7 macrophages, Western blot analysis was performed. MoleculesMolecules 20202020,,25 25,, 4968x FOR PEER REVIEW 55 of 1212

Figure 4. Comparison of inhibitory effect of the isolated compounds 1–5 and L-NMMA on LPS-induced Figure 4. Comparison of inhibitory effect of the isolated compounds 1–5 and L-NMMA on LPS- nitric oxide (NO) production in RAW 264.7 macrophages. (A–F) The inhibitory effect of compounds induced nitric oxide (NO) production in RAW 264.7 macrophages. (A–F) The inhibitory effect of on NO production was investigated using the Griess reagent (mean SD, * p < 0.05 compared to compounds on NO production was investigated using the Griess reagent± (mean ± SD, * p < 0.05 the LPS-treated group). In the graphs description, “+/-” means with/without LPS in the RAW 264.7 compared to the LPS-treated group). In the graphs description, “+/-” means with/without LPS in the macrophages treatment for production of NO. RAW 264.7 macrophages treatment for production of NO. 2.4. Effects of Compounds 1–5 on the LPS-Induced Expression of IKKα/β, I-κBα, and NF-κB p65 in RAW 264.7 Mouse2.4. Effects Macrophages of Compounds 1–5 on the LPS-Induced Expression of IKKα/β, I-κBα, and NF-κB p65 in RAW 264.7 Mouse Macrophages IκB kinase (IKK) contains two catalytic subunits including IKKα and IKKβ. The kinase is necessary for theIκB phosphorylation kinase (IKK) of contains IκBα, which two catalytic is associated subunits with the including activation IKKα of NF- andκB[ IKKβ33]. NF-. TheκB regulates kinase is thenecessary expression for the of phosphorylation inflammatory cytokine of IκBα, genes which such is associated as iNOS with and COX-2.the activation IKKα of/β NFis activated-κB [33]. NF by- proinflammatoryκB regulates the stimuliexpression such of as inflammatory LPS [34]. Following cytokine the stimulationgenes such ofas RAWiNOS 264.7 and cellsCOX with-2. IKKα/β LPS, the is eactivatedffects of compounds by proinflammatory1–5 on the stimuli expression such ofas IKKLPSα [3/β4,]. inhibitor Following of the kappa stimulation B alpha (I- ofκ RAWBα), and 264.7 NF- cellsκB p65with were LPS, examined the effects via of Western compounds blot analysis.1–5 on the As expression shown in Figure of IKKα/β,5A, under inhibitor unstimulated of kappa conditions, B alpha (I- theκBα cells), and did NF not-κB express p65 IKKwereα / examinedβ, I-κBα, and via NF- WesternκB p65; blot however, analysis. the expression As shown of in IKK Figureα/β, I-5A,κB α under, and NF-unstimulatedκB p65 was conditions, increased considerably the cells did in not response express to LPS.IKKα/β, Coincubation I-κBα, and of NF the-κB cells p65; with however, compounds the 4expressionand 5 dramatically of IKKα/β, inhibited I-κBα, the and LPS-induced NF-κB p65 expressionwas increased of IKK considerablyα/β, I-κBα, and in NF- responseκB p65. toThus, LPS. compoundsCoincubation4 andof the5 inhibitedcells with thecompounds inflammatory 4 and response 5 dramatically through inhibited NF-κB the by interactingLPS-induced with expression IKKα/β andof IKKα/β, I-κBα in I- LPS-stimulatedκBα, and NF-κB RAW p65. 264.7 Thus, cells compounds (Figure5B). 4 and 5 inhibited the inflammatory response through NF-κB by interacting with IKKα/β and I-κBα in LPS-stimulated RAW 264.7 cells (Figure 5B).

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FigureFigure 5.5. Comparison of eeffectsffects of isolated compounds 11––55 on the LPS-inducedLPS-induced protein expressionexpression ofof I-I-κκBα,Bα,I IκBκB kinasekinase (IKK)(IKK)α/βα/β andand NF-NF-κκBB p65p65 inin RAWRAW 264.7264.7 macrophages.macrophages. ((AA)) RepresentativeRepresentative WesternWestern blotsblots for for I- κ I-BκBα,α, IKK IKKα/β,α/β, NF- NFκB-κB p65, p65, and andGAPDH GAPDH protein protein expression. expression. (B) Quantitative (B) Quantitative graph for graph Western for blots (mean SD, * p < 0.05 compared with the LPS-treated group). In the graphs description, “+/-” Western blots± (mean ± SD, * p < 0.05 compared with the LPS-treated group). In the graphs description, means“+/-” means the protein the proteinexpression expression of RAW 264.7 of RAW macrophages 264.7 macrophages with/without with/without LPS or sample LPS (compounds or sample 1(com–5) respectively.pounds 1–5) respectively. 2.5. Effects of Compounds 1–5 on the LPS-Induced Expression of iNOS and COX-2 in RAW 264.7 Mouse2.5. Effects Macrophages of Compounds 1–5 on the LPS-Induced Expression of iNOS and COX-2 in RAW 264.7 Mouse Macrophages Inflammatory cytokine genes such as iNOS and COX-2 are coding genes regulated by NF-κB p65. TheseInflammatory coding genes cytokine increase genes NO synthesis such as underiNOS theand stimulationCOX-2 are ofcoding inflammatory genes regulated cytokines by [NF35,36-κB]. Quercetinp65. These isolated coding genesfrom noni increase fruit NO juice synthesis collected under in Tahiti the stimulation has been reported of inflammatory as an inhibitor cytokines of COX-2[35,36]. [Quercetin37]. In addition, isolated infrom the noni rat chondrocytes,fruit juice collected tricetin in Tahiti (5) was has found been toreported suppress as an production inhibitor of COX-2 [37]. In addition, in the rat chondrocytes, tricetin (5) was found to suppress production of of NO and prostaglandin E2 (PGE2) and expression of iNOS and COX-2 [38]. Asperulosidic acid (NO1) and and rutin prostaglandin (2) were reported E2 (PGE2) to and inhibit expression LPS-induced of iNOS iNOS and COX and- COX-22 [38]. Asperulosidic expression in acid RAW (1 264.7) and macrophagesrutin (2) were [30 reported,31], which to displaysinhibit LPS an e-inducedffect consistent iNOS withand COXour study.-2 expression However, in the RAW detailed 264.7 molecularmacrophages mechanisms [30,31], which of asperulosidic displays an acid effect and consistent rutin involved with in our the study downregulation. However, of the iNOS detailed and molecular mechanisms of asperulosidic acid and rutin involved in the downregulation of iNOS and COX-2 in LPS-activated RAW 264.7 macrophages, have been previously unknown. In this study, the

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Molecules 2020, 25, x FOR PEER REVIEW 7 of 12 COX-2 in LPS-activated RAW 264.7 macrophages, have been previously unknown. In this study, the effects of compounds 1–5 on iNOS and COX-2 expression in RAW 264.7 macrophages were examined effects of compounds 1–5 on iNOS and COX-2 expression in RAW 264.7 macrophages were examined via Western blot analysis. As shown in Figure 6A, under unstimulated conditions, the cells did not via Western blot analysis. As shown in Figure6A, under unstimulated conditions, the cells did not express iNOS and COX-2; however, the expression of iNOS and COX-2 was greatly increased in express iNOS and COX-2; however, the expression of iNOS and COX-2 was greatly increased in response to LPS. Coincubation of the cells with compounds 1–5 dramatically inhibited the LPS- response to LPS. Coincubation of the cells with compounds 1–5 dramatically inhibited the LPS-induced induced expression of iNOS and COX-2. Thus, compounds 1–5 regulated LPS-induced NO expression of iNOS and COX-2. Thus, compounds 1–5 regulated LPS-induced NO production through production through the inhibition of LPS-induced iNOS and COX-2 expression (Figure 6B). These the inhibition of LPS-induced iNOS and COX-2 expression (Figure6B). These results suggested that results suggested that compounds 1–5 might be considered as a potential anti-inflammatory agent. compounds 1–5 might be considered as a potential anti-inflammatory agent. However, the fact that the However, the fact that the bioavailability and bioaccessibility of compounds 1–5 have not been bioavailability and bioaccessibility of compounds 1–5 have not been verified is a limitation of this study, verified is a limitation of this study, and the issue should be verified by additional studies including and the issue should be verified by additional studies including the results of animal experiments. the results of animal experiments.

Figure 6. Comparison in effects of isolated compounds 1–5 (untreated (-) or treated (+)) on the FigureLPS-induced 6. Comparison protein expressionin effects of of isolated iNOS and compounds COX-2 in RAW1–5 (untreated 264.7 macrophages. (-) or treated (A (+))) Representative on the LPS- inducedWestern blots protein for expressionprotein expressions of iNOS of and iNOS, COX COX-2,-2 in and RAW GAPDH. 264.7 macrophages. (B) Quantitative (A graph) Representative for Western Westernblots (mean blots SD, for *proteinp < 0.05 expressions compared with of iNOS, the LPS-treated COX-2, and group). GAPDH. (B) Quantitative graph for Western blots± (mean ± SD, * p < 0.05 compared with the LPS-treated group). 3. Materials and Methods 3. Materials and Methods 3.1. General Experimental Procedures 3.1. GeneralThermo Experimental Scientific Ultimate Procedures 3000 preparative and semi-preparative HPLC systems equipped with a diodeThermo array Scientific detector (Germany) Ultimate 3000 were preparativeused for the isolationand semi of-preparative compounds HPLC using HPLC-grade systems equipped MeOH with(Fisher a diode Chemicals, array detector Hampton, (Germany) NH, USA) were and used Milli-Q for the water isolation as the of mobile compounds phases. using 1D andHPLC 2D-grade NMR MeOHdata were (Fisher recorded Chemicals in deuterated, Hampton, methanol NH, USA (Cambridge) and Milli Isotope-Q water Laboratories as the mobile Inc, Tewksbury,phases. 1D and MA) 2D on NMRa Bruker data 400 were MHz recorded NMR system, in deuterated and LC-ESIMS methanol data (Cambridge were obtained Isotope on Laboratories an Agilent 1260 Inc, HPLC Tewksbury, system MAcoupled) on a toBruker a 6120 400 quadrupole MHz NMR LC-MS system, spectrometer and LC-ESIMS in dual data ionizationwere obtained modes on (column:an Agilent Phenomenex 1260 HPLC systemC18, 100 coupled Å, 100 to4.6 a mm, 6120 5 quadrupoleµm; flow rate: LC 0.2-MS mL spectrometer/min; solvent: in 10–100% dual ionization acetonitrile–H modes2O with(column 0.1%: × Phenomenexformic acid, forC18, 20 100 min). Å, 100 × 4.6 mm, 5 μm; flow rate: 0.2 mL/min; solvent: 10–100% acetonitrile– H2O with 0.1% formic acid, for 20 min).

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3.2. Noni Juices Commercially available noni juice was purchased from Virgin Noni Juice (http://www. virginnonijuice.com), order number 809979. Virgin Noni juice was prepared using a standardized traditional Hawaiian method known as “dripping.” First, the ﬁnest ripe noni fruits were carefully hand-picked, washed thoroughly with water, and placed in large containers. The juice was further fermented for several weeks to allow the juice to seep out of the fruits. Stacked noni fruits develop a natural positive pressure that further allows the juice to ﬂow out under the weight of the ripe fruits [6]. The pure noni juice was further strained several times and pasteurized to preserve most nutrients, vitamins, and important active enzymes.

3.3. HP20 Open Column, Preparative, and Semi-Preparative HPLC Purification of Noni Fruit Juice Extract Filtered noni juice (500 mL) was loaded into an open column packed with Diaion HP20 polymaric matrix (HP20 6.6 g, 1.5 6.0 cm) and eluted with MeOH–H O (0%, 10%, 30%, 50%, × 2 70%, and 100% MeOH) solvent systems. The resulting six fractions (A–F) were dried using SpeedVac. The fraction E (70%) was purified on a Thermo Scientific Ultimate 3000 preparative HPLC system (C18 column, 5 µm; 100.0 L 21.2 mm i.d.; flow rate 10 mL/min; with 0.1% × formic acid in mobile phases) and eluted with 10–100% MeOH–H2O to obtain 30 subfractions (E1–E30). Finally, in total, seven subfractions were obtained after combining them according to their chemical profiles (Figure3). Further, a Thermo Scientific Ultimate 3000 semi-preparative HPLC system quipped with a HPLC/diode array detector [column: Phenomenex Luna C18 (100 Å, 250 10 mm L i.d., 5 µm) at a flow rate of 3 mL/min] was employed to obtain pure compounds: × × asperulosidic acid (1, tR 18.0, 1 mg/L), rutin (2, tR 23.0, 3 mg/L), nonioside A (3, tR 21.2, 1 mg/L), (2E,4E,7Z)-deca-2,4,7-trienoate-2-O-β-d-glucopyranosyl-β-d-glucopyranoside (4, tR 21.0, 1 mg/L), and tricetin (5, tR 20.7, 3 mg/L) from subfractions E-11, 14, 19, 21, and 26, respectively (Figure1). In both HPLC systems, 210 and 254 nm wavelengths were used for the isolation. The structural characterization of the isolated compounds was performed using extensive analysis of 1D and 2D NMR and LC-MS data, along with published data.

3.4. NF-κB Assay Human embryonic kidney cells HEK 293 (20 103 cells/well) were maintained in Dulbecco’s × modified Eagle’s medium (DMEM) (Invitrogen Co., New York, NY, USA) supplemented with 10% of fetal bovine serum (FBS), 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 2 mM of l-glutamine and incubated for 48 h. After the incubation the culture media was replaced and treated with the test samples with different concentrations. Then the activator TNF-α (human, recombinant, E. coli, Calbiochem, San Diego, CA, USA) at 2 ng/mL was added and incubated for another 6 h, the spent media was removed and the cells were washed with phosphate-buffered saline. The cells were lysed on a shaker for 5 min using 50 mL of reporter lysis buffer (Promega, Madison, WI, USA) and were then stored at 80 C. The luciferase assay was performed using the Luc assay system from Promega, USA − ◦ and followed the manufacturer instructions. NF-κB inhibition was expressed in terms of IC50 values (i.e., the concentration required to inhibit TNF-α-induced NF-κB activity by 50%). The NF-κB inhibitor TPCA-1 was used as the positive control (IC 48.2 0.8 nM) [39]. All experiments were performed 50 ± in triplicate.

3.5. Cell Culture RAW 264.7 mouse macrophages were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in DMEM medium (Cellgro Thomas Scientiﬁc, Manassas, VA, USA) containing 1% penicillin/streptomycin, 10% FBS (Invitrogen Co., New York, NY, USA), and 4 mM l-glutamine in a 5% CO2 atmosphere at 37 ◦C. Molecules 2020, 25, 4968 9 of 12

3.6. Measurement of Cell Viability RAW 264.7 cells were seeded in 96-well plates at 3 104 cells/well. To determine the cell viability, × cells were treated with compounds 1–5 for 24 h at 37 ◦C and further treated with Ez-Cytox solution for 40 min (Daeil Lab Service Co., Seoul, Korea) [40]. The absorbance values were measured at 450 nm using a PowerWave XS microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

3.7. Measurement of NO Production NO production was measured using Griess reagent (2% sulfanilamide, 5% phosphoric acid, and 0.2% naphthylethylenediamine dihydrochloride). NG-methyl-l-arginine acetate salt (L-NMMA; nitric oxide synthase inhibitor) was used as the positive control compound. RAW 264.7 cells were seeded on 96-well plates at 3 104 cells/well. Then, the cells were treated with compounds 1–5 or L-NMMA for × 24 h at 37 ◦C and further treated with LPS (1 µg/mL) for 24 h. The absorbance values were measured at 540 nm using a PowerWave XS microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

3.8. Western Blot Analysis Proteins in the samples (20 µg protein/lane) were separated by electrophoresis on a 10% sodium dodecyl sulfate–polyacrylamide gel and further transferred onto a polyvinylidene ﬂuoride (PVDF) membrane. The PVDF membrane was incubated with primary antibodies (Cell Signaling, Beverly, MA, USA) against IKKα, phospho-IKKα/β, IKKβ, I-κBα, phospho-I-κBα, NF-κB p65, phospho-NF-κB p65, iNOS, COX-2, and GAPDH for 1 h at room temperature. After binding with HRP-conjugated anti-rabbit antibodies (Cell Signaling, Beverly, MA, USA) for 1 h, the PVDF membranes were developed using enhanced chemiluminescence (ECL) Advance Western Blotting Detection Reagents (GE Healthcare, Little Chalfont, UK) and visualized using a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Germany).

3.9. Statistical Analysis Statistical signiﬁcance was assessed using a one-way analysis of variance (ANOVA) and multiple comparisons with a Bonferroni correction. A p value < 0.05 indicated statistical signiﬁcance. All analyses were conducted using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA).

4. Conclusions We demonstrated that compounds 1–5, present in noni fruit juice, inhibited the LPS-induced inflammatory response in RAW 264.7 macrophages. The anti-inflammatory effects were determined to be mediated through the IKKα/β, I-κBα, and NF-κB p65 signaling pathways, which activate iNOS and COX-2 and regulate NO production (Figure7). Our findings reveal the mechanisms underlying the anti-inflammatory action of compounds from noni fruit juice and their potential therapeutic use against inflammation-related disorders. Furthermore, this study provides supporting evidence that noni fruit juice can be a potential anti-inflammatory agent against inflammation-related disorders. Molecules 2020, 25, 4968 10 of 12 Molecules 2020, 25, x FOR PEER REVIEW 10 of 12

FigureFigure 7.7. SchematicSchematic pathwaypathway ofof thethe rolerole ofof isolatedisolated compoundscompounds 11––55in in inﬂammatoryinflammatory responses.responses.

SupplementarySupplementary Materials:Materials:The TheSupplementary Supplementary MaterialsMaterials areare availableavailable online,online,Figure Figure S1: S1:1 1HNMRHNMR spectrumspectrum ofof 11 compoundcompound1 1(in (in MeOH- MeOHd-d4);4); Figure Figure S2: S2: H-NMRH-NMR spectrumspectrum ofof compoundcompound2 2(in (in D D22O-O-dd22);); FigureFigure S3:S3: HSQCHSQC spectrumspectrum 11 1 1 ofof compoundcompound2 2(in (in D D2O-2Od-d22);); FigureFigure S4:S4: H-NMRH-NMR spectrumspectrum ofof compoundcompound3 3(in (in MeOH- MeOHd-d4);4); Figure Figure S5: S5: H-NMRH-NMR 1 spectrumspectrum ofof compoundcompound4 4(in (in MeOH- MeOHd-d44);); FigureFigure S6:S6: 1H-NMRH-NMR spectrum of compound 5 (in(in MeOH-MeOH-dd44)).. Author Contributions: Conceptualization, S.C., K.S.K., and K.H.K.; formal analysis: D.L., J.S.Y., S.C., J.-C.K., C.P., K.S.K.,Author and Contributions K.H.K.; investigation,: Conceptualization, D.L., J.S.Y., S.C., P.H., M.Q.K.S.K. A.M.,, and andK.H.K.; X.W.; formal writing: analysis original: D.L. draft, J.S.Y., preparation, S.C., J.- D.L.,C.K., M.Q.,C.P., K.S.K. S.C., K.S.K.,, and K.H.K.; and K.H.K.; investigation, writing: D.L. review, J.S.Y and., P.H. edit,, M.Q. K.S.K. A.M. and, and K.H.K.; X.W.; project writing administration,: original draft preparation, K.S.K. and K.H.K.;D.L., M.Q., funding S.C., acquisition, K.S.K., and K.H.K. K.H.K.; All writing authors: review have readand andedit, agreed K.S.K. toand the K.H.K.; published project version administration, of the manuscript. K.S.K. Funding:and K.H.K.;This funding work was acquisition, supported K.H.K by grants. All from authors the National have read Research and agreed Foundation to the of published Korea (NRF), version funded of theby themanuscript Korean government. (MSIT) (2018R1A2B2006879 and 2019R1A5A2027340), and the Bio and Medical Technology Development Program of the National Research Foundation (NRF), funded by the Korean government (MSIT) (NRF-2012M3A9C4048775).Funding: This work was supported by grants from the National Research Foundation of Korea (NRF), funded by the Korean government (MSIT) (2018R1A2B2006879 and 2019R1A5A2027340), and the Bio and Medical Acknowledgments: We would like to thank Young Hye Kim (KBSI) for the ESI-MS analysis. Technology Development Program of the National Research Foundation (NRF), funded by the Korean Conﬂictsgovernment of Interest: (MSIT) (NRFThe authors-2012M3A9C4048775). declare no conﬂict of interest.

Acknowledgments: We would like to thank Young Hye Kim (KBSI) for the ESI-MS analysis. References Conflicts of Interest: The authors declare no conflict of interest. 1. Pandy, V.; Narasingam, M.; Mohamed, Z. Antipsychotic-like activity of Noni (Morinda citrifolia Linn.) in mice. BMC Complement. Altern Med. 2012, 12, 186. [CrossRef][PubMed] References 2. McClatchey, W. From Polynesian healers to health food stores: Changing ethnopharmacology of Morinda 1. citrifolia.Pandy, V.;Integr. Narasingam, Cancer Ther. M.; 2002Mohamed,, 1, 110–120. Z. Antipsychotic [CrossRef][PubMed-like activity] of Noni (Morinda citrifolia Linn.) in 3. Potterat,mice. BMC O.; Complement Hamburger,. Altern M. Morinda Med. citrifolia2012, 12(Noni), 186. fruit-phytochemistry, pharmacology, safety. Planta Med. 2. 2007McClatchey, 73, 191–199., W. From [CrossRef Polynesian][PubMed healers] to health food stores: Changing ethnopharmacology of Morinda 4. Samoylenko,citrifolia. Integr. V.; Zhao,Cancer J.;Ther. Dunbar, 2002, D.C.;1, 110 Khan,–120. I.A.; Rushing, J.W.; Muhammad, I. New constituents from 3. noniPotterat, (Morinda O.; Hamburger, citrifolia) fruit M. juice. MorindaJ. Agric. citrifolia Food (Noni) Chem. 2006fruit,-54phytochemistry,, 6398–6402. [CrossRef pharmacology,][PubMed safety.] Planta 5. Wang,Med. 2007 M.-Y.;, 73 West,, 191– B.J.;199.Jensen, C.J.; Nowicki, D.; Su, C.; Palu, A.K.; Anderson, G. Morinda citrifolia (Noni): A 4. literatureSamoylenko, review V.; andZhao, recent J.; Dunbar, advances D.C.; in Noni Khan, research. I.A.; Rushing,Acta Pharmacol. J.W.; Muhammad, Sin. 2002, 23I. New, 1127–1141. constituents [PubMed from] 6. Nelson,noni (Morinda S.C.; Elevitch, citrifolia)C.R. fruitNoni: juice. TheJ. Agric. Complete Food Chem. Guide 2006 for Consumers, 54, 6398– and6402. Growers ; Permanent Agriculture 5. Resources:Wang, M.-Y.; Holualoa, West, B.J.; HI, Jensen, USA, 2006. C.J.; Nowicki, D.; Su, C.; Palu, A.K.; Anderson, G. Morinda citrifolia (Noni): 7. Albensi,A literature B.C. review What isand nuclear recent factor advances kappa in BNoni (NF- research.κB) doing Acta in andPharmacol. to the mitochondrion?Sin. 2002, 23, 1127Front.–1141. Cell Dev. 6. Biol.Nelson,2019 S.C.;, 7, 154. Elevitch, [CrossRef C.R.] Noni: The Complete Guide for Consumers and Growers; Permanent Agriculture Resources: Holualoa, HI, USA, 2006.

Molecules 2020, 25, 4968 11 of 12

8. Park, M.H.; Hong, J.T. Roles of NF-κB in cancer and inflammatory diseases and their therapeutic approaches. Cells 2016, 5, 15. [CrossRef][PubMed] 9. Escarcega, R.; Fuentes-Alexandro, S.; Garcia-Carrasco, M.; Gatica, A.; Zamora, A. The transcription factor nuclear factor-kappa B and cancer. Clin. Oncol. 2007, 19, 154–161. [CrossRef][PubMed] 10. Wong, D.K. Are immune responses pivotal to cancer patient‘s long term survival? Two clinical case-study reports on the effects of Morinda citrifolia (Noni). Hawaii Med. J. 2004, 63, 182–184. 11. Olaku, O.; White, J.D. Herbal therapy use by cancer patients: A literature review on case reports. Eur. J. Cancer 2011, 47, 508–514. [CrossRef][PubMed] 12. Senger, D.R.; Li, D.; Jaminet, S.-C.; Cao, S. Activation of the Nrf2 cell defense pathway by ancient foods: Disease prevention by important molecules and microbes lost from the modern western diet. PLoS ONE 2016, 11, e0148042. [CrossRef][PubMed] 13. Manavalan, A.; Qader, M.; Wu, X.; Cao, S. Nrf2 activation by Morinda citrifolia L. (Noni) fruit juices. World J. Tradit. Chin. Med. 2020, in press. 14. Cai, Y.-S.; Xu, J.; Chen, M.; Wang, D.; Yang, Y.; Manavalan, A.; Wu, X.; Liu, Y.; Cao, S. Compound Analysis of Jing Liqueur and nrf2 Activation by Jing Liqueur—One of the Most Popular Beverages in China. Beverages 2019, 6, 1. [CrossRef] 15. Yu, J.S.; Roh, H.S.; Baek, K.H.; Lee, S.; Kim, S.; So, H.M.; Moon, E.; Pang, C.; Jang, T.S.; Kim, K.H. Bioactivity-guided isolation of ginsenosides from Korean Red Ginseng with cytotoxic activity against human lung adenocarcinoma cells. J. Ginseng Res. 2018, 42, 562–570. [CrossRef][PubMed] 16. Lee, D.; Lee, D.S.; Jung, K.; Hwang, G.S.; Lee, H.L.; Yamabe, N.; Lee, H.J.; Eom, D.W.; Kim, K.H.; Kang, K.S. Protective effect of ginsenoside Rb1 against tacrolimus-induced apoptosis in renal proximal tubular LLC-PK1 cells. J. Ginseng Res. 2018, 42, 75–80. [CrossRef][PubMed] 17. Tzakou, O.; Mylonas, P.; Vagias, C.; Petrakis, P.V. Iridoid glucosides with insecticidal activity from Galium melanantherum. Z. Naturforsch. Sect. C J. Biosci. 2007, 62, 597–602. [CrossRef] 18. Napolitano, J.G.; Lankin, D.C.; Chen, S.N.; Pauli, G.F. Complete 1H NMR spectral analysis of ten chemical markers of Ginkgo biloba. Magn. Reson. Chem. 2012, 50, 569–575. [CrossRef] 19. Wang, M.; Kikuzaki, H.; Jin, Y.; Nakatani, N.; Zhu, N.; Csiszar, K.; Boyd, C.; Rosen, R.T.; Ghai, G.; Ho, C.-T. Novel glycosides from noni (Morinda citrifolia). J. Nat. Prod. 2000, 63, 1182–1183. [CrossRef] 20. Hu, M.-X.; Zhang, H.-C.; Wang, Y.; Liu, S.-M.; Liu, L. Two new glycosides from the fruits of Morinda citrifolia L. Molecules 2012, 17, 12651–12656. [CrossRef] 21. Lal, C.; Sharma, M.C.; Shakyawar, D.B.; Raja, A.; Sharma, K.K.; Pareek, P.K. Natural Dye constituents from rind of Punica granatum and its application on Pashmina fabrics. Arch. Appl. Sci. Res 2011, 3, 350–357. 22. Carrillo, J.L.M.; García, F.P.C.; Coronado, O.G.; García, M.A.M.; Cordero, J.F.C. Physiology and pathology of innate immune response against pathogens. In Physiology and Pathology of Immunology; InTech: London, UK, 2017. 23. Ruttimann, J. Macrophages and nitric oxide: A deadly combination. J. Exp. Med. 2007, 204, 3057. [CrossRef] 24. Sharma, J.; Al-Omran, A.; Parvathy, S. Role of nitric oxide in inflammatory diseases. Inflammopharmacology 2007, 15, 252–259. [CrossRef] 25. Pepe, G.; Pagano, F.; Adesso, S.; Sommella, E.; Ostacolo, C.; Manfra, M.; Chieppa, M.; Sala, M.; Russo, M.; Marzocco, S. Bioavailable Citrus sinensis extract: Polyphenolic composition and biological activity. Molecules 2017, 22, 623. [CrossRef][PubMed] 26. Shin, S.Y.; Woo, Y.; Hyun, J.; Yong, Y.; Koh, D.; Lee, Y.H.; Lim, Y. Relationship between the structures of flavonoids and their NF-κB-dependent transcriptional activities. Bioorg. Med. Chem. Lett. 2011, 21, 6036–6041. [CrossRef][PubMed] 27. Almeida, É.S.; de Oliveira, D.; Hotza, D. Properties and applications of Morinda citrifolia (noni): A review. Compr. Rev. Food Sci. Food Saf. 2019, 18, 883–909. [CrossRef] 28. Dussossoy, E.; Bichon, F.; Bony, E.; Portet, K.; Brat, P.; Vaillant, F.; Michel, A.; Poucheret, P. Pulmonary anti-inflammatory effects and spasmolytic properties of Costa Rican noni juice (Morinda citrifolia L.). J. Ethnopharmacol. 2016, 192, 264–272. [CrossRef] 29. Dussossoy, E.; Bony, E.; Michel, A.; Boudard, F.; Giaimis, J.; Brat, P.; Vaillant, F. Anti-oxidative and anti-inflammatory effects of the Morinda citrifolia fruit (Noni). Acta Hortic. 2014, 1040, 69–74. [CrossRef] Molecules 2020, 25, 4968 12 of 12

30. He, J.; Lu, X.; Wei, T.; Dong, Y.; Cai, Z.; Tang, L.; Liu, M. Asperuloside and asperulosidic acid exert an anti-inflammatory effect via suppression of the NF-κB and MAPK signaling pathways in LPS-induced RAW 264.7 macrophages. Int. J. Mol. Sci. 2018, 19, 2027. [CrossRef] 31. Lee, S.-J.; Lee, S.Y.; Ha, H.J.; Cha, S.H.; Lee, S.K.; Hur, S.J. Rutin attenuates lipopolysaccharide-induced nitric oxide production in macrophage cells. J. Food Nutr. Res. 2015, 3, 202–205. [CrossRef] 32. Geraets, L.; Haegens, A.; Brauers, K.; Haydock, J.A.; Vernooy, J.H.; Wouters, E.F.; Bast, A.; Hageman, G.J. Inhibition of LPS-induced pulmonary inflammation by specific flavonoids. Biochem. Biophys Res. Commun. 2009, 382, 598–603. [CrossRef] 33. Zandi, E.; Rothwarf, D.M.; Delhase, M.; Hayakawa, M.; Karin, M. The IκB kinase complex (IKK) contains two kinase subunits, IKKα and IKKβ, necessary for IκB phosphorylation and NF-κB activation. Cell 1997, 91, 243–252. [CrossRef] 34. Lousse, J.-C.; Van Langendonckt, A.; González-Ramos, R.; Defrère, S.; Renkin, E.; Donnez, J. Increased activation of nuclear factor-kappa B (NF-κB) in isolated peritoneal macrophages of patients with endometriosis. Fertil Steril. 2008, 90, 217–220. [CrossRef][PubMed] 35. Dinarello, C.A. Proinflammatory cytokines. Chest 2000, 118, 503–508. [CrossRef][PubMed] 36. Han, J.M.; Lee, E.K.; Gong, S.Y.; Sohng, J.K.; Kang, Y.J.; Jung, H.J. Sparassis crispa exerts anti-inflammatory activity via suppression of TLR-mediated NF-κB and MAPK signaling pathways in LPS-induced RAW264. 7 macrophage cells. J. Ethnopharmacol. 2019, 231, 10–18. [CrossRef] 37. Deng, S.; Palu, A.K.; West, B.J.; Su, C.X.; Zhou, B.-N.; Jensen, J.C. Lipoxygenase inhibitory constituents of the fruits of noni (Morinda citrifolia) collected in Tahiti. J. Nat. Prod. 2007, 70, 859–862. [CrossRef][PubMed] 38. Sun, F.-F.; Hu, P.-F.; Xiong, Y.; Bao, J.-P.; Qian, J.; Wu, L.-D. Tricetin protects rat chondrocytes against IL-1β-induced inflammation and apoptosis. Oxid. Med. Cell. Longev. 2019, 2019.[CrossRef][PubMed] 39. Kondratyuk, T.P.; Park, E.-J.; Yu, R.; Van Breemen, R.B.; Asolkar, R.N.; Murphy, B.T.; Fenical, W.; Pezzuto, J.M. Novel marine phenazines as potential cancer chemopreventive and anti-inflammatory agents. Mar. Drugs 2012, 10, 451–464. [CrossRef][PubMed] 40. Kee, J.-Y.; Hong, S.-H. Ginsenoside Rg3 suppresses mast cell–mediated allergic inflammation via mitogen-activated protein kinase signaling pathway. J. Ginseng Res. 2019, 43, 282–290. [CrossRef]

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