Chemical Constituents of the Leaves of Campanula Takesimana (Korean Bellﬂower) and Their Inhibitory Eﬀects on LPS-Induced PGE2 Production

Article Chemical Constituents of the Leaves of Campanula takesimana (Korean Bellﬂower) and Their Inhibitory Eﬀects on LPS-induced PGE2 Production

Yutong Qi 1 , Se-In Choi 1, So-Ri Son 1, Hee-Soo Han 1, Hye Shin Ahn 2, Yu-Kyong Shin 2, Sun Hee Lee 2, Kyung-Tae Lee 1 , Hak Cheol Kwon 3 and Dae Sik Jang 1,*

1 Department of Life and Nanopharmaceutical Sciences, Graduate School Kyung Hee University, Seoul 02447, Korea; [email protected] (Y.Q.); [email protected] (S.-I.C.); [email protected] (S.-R.S.); [email protected] (H.-S.H.); [email protected] (K.-T.L.) 2 Department of New Material Development, COSMAX BIO, Seongnam 13486, Korea; [email protected] (H.S.A.); [email protected] (Y.-K.S.); [email protected] (S.H.L.) 3 Natural Product Informatics Research Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Gangneung 25451, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-961-0719

Received: 1 September 2020; Accepted: 17 September 2020; Published: 18 September 2020

Abstract: Campanula takesimana Nakai (Campanulaceae; Korean bellflower) is one of the endemic herbs of Korea. The plant has been used as traditional medicines for treating asthma, tonsillitis, and sore throat in Korea. A hot water extract of the leaves of C. takesimana exhibited a significant inhibitory effect on lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) production. Repetitive chromatographic separation of the hot water extract led to the isolation of three new neolignan glucosides, campanulalignans A–C (1–3), with 15 known compounds (4–18). The structures of new compounds 1–3 were elucidated by analyzing nuclear magnetic resonance (NMR) spectroscopic data, along with high resolution quadrupole time of flight mass (HR-Q-TOF-MS) spectrometric data. Among the isolates, simplidin (7), 5-hydroxyconiferaldehyde (11), icariside F (12), benzyl-α-l-arabinopyranosyl-(1” 6 )-β-d-glucopyranoside (13), and kaempferol 2 → 0 3-O-β-d-apiosyl (1 2)-β-d-glucopyranoside (15) were isolated from the Campanulaceae family for → the first time. The isolates (1, 2, and 4–18) were assessed for their anti-inflammatory effects on LPS-stimulated PGE2 production on RAW 264.7 cells. 7R,8S-Dihydrodehydrodiconiferyl alcohol (5), 30,4-O-dimethylcedrusin 9-O-β-glucopyranoside (6), pinoresinol di-O-β-d-glucoside (8), ferulic acid (10), 5-hydroxyconiferaldehyde (11), and quercetin (18) showed significant inhibitory effects on LPS-stimulated PGE2 production.

Keywords: Campanula takesimana; Campanulaceae; campanulalignans; phenolic compounds; PGE2 production

1. Introduction Campanula takesimana Nakai (Campanulaceae), known as Korean bellflower, is a native herb of Korea, growing on Ulleng island. The leaves and stems of C. takesimana are shiny, and the flowers have spots on a pale purple background. It is clearly distinguished from C. punctata, commonly distributed in Korea, China, and Japan, by its smaller overall size and lighter purple color of flower [1]. Because it has long flowering period and shape of flower, this plant has been commonly cultivated for a decorative plant. Besides the value of horticulture, roots of C. takesimana have been used for herbal remedies for asthma, tonsillitis, and sore throat in traditional Korean medicine [2].

Plants 2020, 9, 1232; doi:10.3390/plants9091232 www.mdpi.com/journal/plants Plants 2020, 9, 1232 2 of 11

It was reported that the n-butanol and ethyl acetate fractions of C. takesimana exhibit free radical-scavenging, tyrosinase inhibition, and superoxide dismutase (SOD)-like activities [2]. Plants 2020, 9, x FOR PEER REVIEW 2 of 11 Although extensive research has been done on the pharmacological activities and/or phytochemical constituentsIt was of the reported plants belongingthat the n-butanol to family and Campanulaceae, ethyl acetate fractions yet only fewof C. studies takesimana have exhibit been conducted free on theradical-scavenging, genus Campanula ,tyrosinase despite their inhibition, frequent and traditional superoxide use dismutase [3]. Moreover, (SOD)-like chemical activities constituents [2]. of C. takesimanaAlthoughhave extensive never research been reported has been fromdone previouson the pharmacological studies. activities and/or phytochemical Asconstituents part of ourof the continuing plants belonging project to searchfamily Campanulaceae, for bioactive compounds yet only few with studies anti-inflammatory have been activitiesconducted and identify on the chemicalgenus Campanula constituents, despite of highertheir frequent plants, traditio we observednal use that [3]. aMoreover, hot water chemical extract from constituents of C. takesimana have never been reported from previous studies. the leaves of C. takesimana showed a significant inhibitory effect on lipopolysaccharide (LPS)-stimulated As part of our continuing project to search for bioactive compounds with anti-inflammatory prostaglandinactivities and E2 identify(PGE2) chemical production constituents in RAW of 264.7 higher macrophages. plants, we observed LPS that is one a hot of water the most extract potent activatorsfrom ofthe macrophages, leaves of C. takesimana and LPS-stimulated showed a significant macrophages inhibitory and monocytes effect on arelipopolysaccharide known to produce inflammatory(LPS)-stimulated mediators. prostaglandin In this regard,E2 (PGE2 inflammatory) production in mediators,RAW 264.7 macr suchophages. as PGE 2LPS, nitric is one oxide of the (NO), and numerousmost potent pro-inflammatoryactivators of macrophages, cytokines and LP releasedS-stimulated by the macrophages activated and macrophages monocytes are are known important targetsto produce for the treatmentinflammatory of inflammationmediators. In this [4]. regard, Thus, inflammatory compounds mediators, that can regulate such as PGE the2 production, nitric oxide (NO), and numerous pro-inflammatory cytokines released by the activated macrophages are of pro-inflammatory mediators like PGE2 may be promising therapeutic agents for the treatment of variousimportant inflammatory targets for diseases. the treatment of inflammation [4]. Thus, compounds that can regulate the Inproduction the present of pro-inflammatory research, we concentrated mediators like on PGE isolating2 may secondarybe promising metabolites therapeutic from agents the for hot the water treatment of various inflammatory diseases. extract and identifying the active constituents. Three new neolignan glucosides (1–3) and 15 known In the present research, we concentrated on isolating secondary metabolites from the hot water compoundsextract and (4– 18identifying) were identified the active by consti repetitivetuents. Three chromatographic new neolignan separation glucosides of(1– the3) and hot 15 water known extract. The structurecompounds of (4 the–18) new were compounds identified by (1 –repetitive3) was elucidatedchromatographic by analysis separation of one-dimensionalof the hot water (1D) and two-dimensionalextract. The structure (2D) of the nuclear new compounds magnetic resonance(1–3) was elucidated (NMR) spectroscopic by analysis of andone-dimensional high resolution quadrupole(1D) and time two-dimensional of flight mass (2D) (HR-Q-TOF-MS) nuclear magnetic spectrometric resonance data.(NMR) The spectroscopic structures and of the high known compoundsresolution were quadrupole identified time in of comparison flight mass with(HR-Q- previouslyTOF-MS) spectrometric published values. data. The Thereafter, structures the of the isolates known compounds were identified in comparison with previously published values. Thereafter, the were assessed for their inhibitory effects on the production of the pro-inflammatory mediator, PGE2, in LPS-stimulatedisolates were assessed RAW264.7 for macrophages.their inhibitory We effe describects on the in thisproduction paper theof isolationthe pro-inflammatory of the secondary mediator, PGE2, in LPS-stimulated RAW264.7 macrophages. We describe in this paper the isolation metabolites from the leaves of P. japonicus, structure elucidation of the three new neolignan glucosides of the secondary metabolites from the leaves of P. japonicus, structure elucidation of the three new (1–3), and inhibitory effects of the isolates on PGE2 production. neolignan glucosides (1–3), and inhibitory effects of the isolates on PGE2 production. 2. Results 2. Results 2.1. Structure Elucidation of New Compounds 1–3 2.1. Structure Elucidation of New Compounds 1–3 By repetitive chromatographic separation of the hot water extract, three new neolignan glucosides (1–3) wereBy isolated, repetitive along chromatographic with 15 known separation compounds, of the hot including water extract, five lignans three (new4–8), neolignan five phenolic glucosides (1–3) were isolated, along with 15 known compounds, including five lignans (4–8), five compounds (9–13), and five flavonoids (14–18), in the present research (Figure1). phenolic compounds (9–13), and five flavonoids (14–18), in the present research (Figure 1).

FigureFigure 1. Structures 1. Structures of of compounds compounds 11–18 isolatedisolated from from CampanulaCampanula takesimana takesimana. .

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Compound 1 was isolated as a pale yellow powder. Based on its molecular ion peak [M H] at m/z − − 697.2360 (calcd for C32H41O17, 697.2344) in negative mode of HR-Q-TOF-MS (Figure S1), the molecular formula of 1 was established as C32H42O17. The infrared absorption (IR) spectrum displayed absorption 1 bands at 3278, 2903, and 1580 cm− , implying that compound 1 has phenolic group characteristics. The 1H NMR spectroscopic data of 1 (Table1, Figure S2) revealed one aromatic singlet signal (δH 6.71 (1H, H-6)) and 1,3,4,5-tetrasubstituted aromatic ring protons (δH 6.48 (1H, d, J = 2.0 Hz, H-20) and 6.47 (1H, d, J = 2.0 Hz, H-60)). The existence of two trans-olefinic groups was identified on the basis of the proton signals and coupling constants (δH 6.86 (1H, d, J = 15.5 Hz, H-7), 6.24 (1H, dt, J = 15.0, 5.0 Hz, H-8) and 6.20 (1H, dt, J = 15.0, 5.0 Hz, H-80), 6.14 (1H, d, J = 15.5 Hz, H-70)). Additionally, two methylene (δH 4.24 (2H, dd, J = 5.5, 2.0 Hz, H-9) and 3.55 (2H, d, J = 5.5 Hz, H-90)) and two 1 methoxy proton signals (δH 3.86 (3H, s, OCH3-5), 3.81 (3H, s, OCH3-50)) were also observed in the H NMR. Two anomeric protons (δH 4.67 (1H, d, J = 8.0 Hz, Glc H-1), 4.63 (1H, d, J = 8.0 Hz, Glc H-10)) andPlantsPlants overlapped 20202020,, 99,, xx FORFOR signalsPEERPEER REVIEWREVIEW at δH 3.23–3.82 suggested that 1 has two sugars with β configuration44 ofof [5 1111]. PlantsThe 202013C, NMR9, x FOR exhibited PEER REVIEW 32 signals, containing 12 signals for two glucopyranosyl moieties (Table4 of 111 , Figure S3). EnzymeGlc-6Glc-6 hydrolysis 62.0 62.0 3.68/3.82 3.68/3.82 of 1 overlapoverlapand high 62.4 62.4 performance 3.82 3.82 overlapoverlap liquid chromatography 62.3 62.3 3.86 3.86 (HPLC) mm experiment Glc-6 62.0 3.68/3.82 overlap 62.4 3.82 overlap 62.3 3.86 m led to the determinationGlc-1Glc-1′′ 107.0107.0 of the 4.63 4.63 sugars dd (8.0)(8.0) of 1 as 107.0 107.0β- d -glucopyranose. 4.64 4.64 dd (8.0)(8.0) 107.1In 107.1 combination 4.63 4.63 dd with (7.5)(7.5) 13C NMR and Glc-1Glc-2′ ′ 107.0 71.0 4.63 3.47 d overlap(8.0) 107.0 71.0 4.64 3.47d (8.0) m 107.1 71.0 4.63 3.51 d overlap(7.5) HMBC experimentalGlc-2′ 71.0results, compound 3.47 overlap 1 had 71.0 two phenylpropanoid 3.47 m 71.0 units that 3.51 had overlapπ bond between α Glc-2Glc-3Glc-3′ ′′ 71.0 77.8 77.8 3.47 3.43 3.43 overlap overlapoverlap 71.0 77.9 77.9 3.84 3.84 3.47 overlapoverlap m 71.0 77.8 77.8 3.51 3.45 3.45 overlap overlapoverlap and β carbons (Figure S6). Each phenylpropyl moiety had one methoxy and one β-d-glucopyranose, Glc-3Glc-4Glc-4′ ′′ 77.8 75.5 75.5 3.43 overlap 3.51 3.51 mm 77.9 75.5 75.5 3.84 overlap 3.44 3.44 mm 77.8 75.5 75.5 3.45 3.42 3.42 overlap overlapoverlap approximateGlc-4Glc-5Glc-5 to′ ′ demethyl′ 75.5 78.5 78.5 syringin 3.51 3.30 3.30 m m m (9 ), supported 75.5 78.5 78.5 by 3.44HMBC 3.29 3.29 m m m correlations 75.5 78.7 78.7 (Figure 3.42 3.23 3.23 overlap overlapoverlap2). The di fferences were that oneGlc-5Glc-6Glc-6 of′ ′′ the 78.5 two 62.0 62.0 phenylpropanoid 3.68/3.82 3.68/3.82 3.30 m overlapoverlap 78.5 units 62.2 62.2 in 1 3.22 3.22had 3.29 overlapoverlap m 2-substitued 78.7 62.3 62.3 aromatic 3.23 ringoverlap 3.78 3.78 mm and the other one Glc-6OCHOCH′ 33-5-5 62.0 56.8 56.8 3.68/3.82 3.86 3.86 overlap ss 62.2 62.7 62.7 3.22 overlap 3.87s 3.87s 62.3 56.8 56.8 3.78 3.84 3.84 m ss had modified C-90 into C-C bond instead of hydroxyl group. Furthermore, both of the substituted OCHOCHOCH3-533-5 -5′′ 56.8 56.8 56.8 3.86 3.81 3.81 s ss 62.7 56.8 56.8 3.87s 3.81s 3.81s 56.8 56.7 56.7 3.84 3.81 3.81 s ss partsa connectedOCH3-5′ to each 56.8 other 3.81 were s 1 determined1 56.8 by HMBC 3.81s correlation 56.7 signal1 13 between3.81 s H-90 and C-2. a AllAll assignmentsassignments werewere basedbased onon 1H-H-1HH correlationcorrelation spectroscopyspectroscopy (COSY),(COSY), 1H-H-13CC heteronuclearheteronuclear singlesingle Compounda 1 was very similar to1 the1 known neolignan glucoside, tangshenoside1 13 III [6], except for Allquantumquantum assignments coherencecoherence were spectroscopybasedspectroscopy on H- H(HSQC),(HSQC), correlation andand spectroscopy 11H-H-1313CC heteronuclearheteronuclear (COSY), H-multiplemultipleC heteronuclear bondbond correlationcorrelation single missingquantum one methoxycoherenceb groupspectroscopy in each (HSQC), of the twoand aromatic1H-13C heteronuclear rings. Therefore, multiple the bond structure correlation of the new (HMBC)(HMBC) results.results. b δδHH (multi(multi JJ inin Hz).Hz). compound(HMBC) 1results.was elucidated b δH (multi asJ in 3,3 Hz).0-demethyl tangshenoside III, and named as campanulalignan A.

FigureFigure 2.2. KeyKey 11H-11HH COSY COSY ( ( ▬▬) ) )and andand HMBC HMBCHMBC ( (( ) ) correlations correlations ofof of compoundscompounds compounds 1 –1–3–3..3. Figure 2. Key 1H-1H COSY (▬) and HMBC ( ) correlations of compounds 1–3. CompoundCompoundCompound2 22was waswas obtained obtainedobtained as asas a aa pale palepale yellow yellowyellow powder. powder.powder. The TheThe molecular molecularmolecular formula formulaformula of of2ofwas 22 waswas assigned assignedassigned as C HCompoundO by its 2 HR-Q-TOF-MSwas obtained as ion a pale peak yellow at m/z powder.697.2354 The [M molecularH] (calcd− formula for C H of O2 was, 697.2344) assigned in asas32 CC323242HH424217OO1717 byby itsits HR-Q-TOF-MSHR-Q-TOF-MS ionion peakpeak atat mm//zz 697.2354697.2354 [M[M−−H]H]− − (calcd(calcd forfor C32C3232HH414141OO171717,, 697.2344)697.2344) inin 1 13− − asnegativenegativenegative C32H42O mode 17modemode by its (Figure (Figure (FigureHR-Q-TOF-MS S7) S7)S7) and andand was ionwaswas the peak same thethe atsamesame as m1/.z The697.2354asas 11.H. TheThe and [M 11HH−CH] andand NMR (calcd 1313C spectraC NMRNMRfor C of 32 spectraspectraH2 41stronglyO17, of697.2344)of resembled22 stronglystrongly in 1 13 negativethoseresembledresembled of mode1. However,thosethose (Figure ofof 11 the.. However,However,S7) carbon and was chemical thethe thecarboncarbon same shift chemicalchemical ofas one1. The methoxy shiftshift H ofof and oneone group methoxymethoxyC inNMR aromatic groupgroupspectra ring inin of aromatic movedaromatic 2 strongly to ringring the 1 resembleddownfield those (δ 62.7) of 1. in However,2 (Table1 ,the Figure carbon S9). chemical In the Hshift NMR, of one the methoxy di11 fference group between in aromatic compounds ring 1 movedmoved toto thetheC downfielddownfield ((δδCC 62.7)62.7) inin 22 (Table(Table 1,1, FigureFigure S9).S9). InIn thethe HH NMR,NMR, thethe differencedifference betweenbetween movedand 2 wasto the shown downfield on the (δ aromaticC 62.7) in singlet 2 (Table signal 1, Figure of 2 shifted S9). Into the the 1H downfield NMR, the ( δdifference6.87 (1H, between s, H-6)), compoundscompounds 11 andand 22 waswas shownshown onon thethe aromaticaromatic singletsinglet signalsignal ofof 22 shiftedshifted toto thetheH downfielddownfield ((δδHH 6.876.87 compoundswhereas two 1 and olefinic 2 was protons shown of on2 shifted the aromatic to the upfieldsinglet (signalδ 6.81 of(1H, 2 shifted d, J = to15.5 the Hz, downfield H-7) and (δδH 6.876.17 (1H,(1H, s,s, H-6)),H-6)), whereaswhereas twotwo olefinicolefinic protonsprotons ofof 22 shiftedshifted totoH thethe upfieldupfield ((δδHH 6.816.81 (1H,(1H, d,d, JJ == 15.515.5 Hz,Hz,H H-7)H-7) (1H,(1H, s, dt, H-6)),J = 15.5, whereas 6.0 Hz, two H-8)) olefinic (Table protons1, Figure of 2 S8). shifted By analyzing to the upfield the HMBC (δH 6.81 spectrum, (1H, d, J the= 15.5 carbon Hz, H-7) that andand δδHH 6.176.17 (1H,(1H, dt,dt, JJ == 15.5,15.5, 6.06.0 Hz,Hz, H-8))H-8)) (Table(Table 1,1, FigureFigure S8).S8). ByBy analyzinganalyzing thethe HMBCHMBC spectrum,spectrum, andlinked δH 6.17 to the (1H, methoxy dt, J = proton 15.5, 6.0 showed Hz, H-8)) correlation (Table with 1, Figure methylene S8). By proton analyzing peaks the at δ HMBC3.55 (2H, spectrum, m, H-9 ) thethe carboncarbon thatthat linkedlinked toto thethe methoxymethoxy protonproton showedshowed correlationcorrelation withwith methylenemethyleneH protonproton peakspeaks atat δδ0HH the(Figure3.553.55 carbon (2H,(2H,2). m,thatm, For H-9H-9 theselinked′′)) (Figure(Figure reasons, to the 2).2). methoxy the ForFor location thesethese proton reasons,reasons, of one showed methoxythethe locationlocation correlation group ofof oneone waswith methoxymethoxy suggested methylene groupgroup to proton be waswas C-3 suggestedpeakssuggested of aromatic at δH toto 3.55ring,bebe (2H,C-3C-3 rather ofofm, aromaticaromaticH-9 than′) C-5(Figure ofring,ring,1. 2). Thus, ratherrather For thethese thanthan structure reasons, C-5C-5 ofof of the11 the.. Thus,Thus, location new compoundthethe of structurestructure one methoxy2 was ofof elucidatedthethe group newnew wascompoundcompound as suggested 5,30-demethyl 22 waswasto betangshenosideelucidatedelucidated C-3 of aromatic asas 5,35,3 III,′′-demethyl-demethyl andring, named rather tangshenosidetangshenoside as than campanulalignan C-5 of ⅢⅢ 1,, .andand Thus, B.namednamed the asstructureas campanulalignancampanulalignan of the new B.B. compound 2 was elucidated as 5,3′-demethyl tangshenoside Ⅲ, and named as campanulalignan B. CompoundCompound 33 hadhad aa molecularmolecular formulaformula ofof CC3232HH4242OO1717,, asas establishedestablished byby itsits molecularmolecular ionion peakpeak Compound− 3 had a molecular formula of C32H42O17, as established by its molecular ion peak [M[M−−H]H]− atat mm//zz 697.2336697.2336 (calcd(calcd forfor CC3232HH4141OO1717,, 697.2344)697.2344) inin negativenegative modemode ofof HR-Q-TOF-MSHR-Q-TOF-MS (Figure(Figure − [MS13)S13)−H] andand at wasmwas/z 697.2336 alsoalso thethe samesame(calcd asas for 11 andandC32H 2241.. OTheThe17, 1697.2344)1HH andand 1313C Cin NMRNMR negative spectraspectra mode ofof 3 3of werewere HR-Q-TOF-MS almostalmost identicalidentical (Figure withwith 1 13 S13)thosethose and ofof was 11 andand also 22 ,,the exceptexcept same forfor as the1the and existenceexistence 2. The ofHof andaa vinylvinyl C NMRalcoholalcohol spectra groupgroup of inin 3 33were insteadinstead almost ofof ananidentical allylallyl alcoholalcohol with those of 1 and 2, except1 for the existence of a vinyl alcohol group in 3 instead of an allyl alcohol groupgroup inin 11 andand 22.. 1HH NMRNMR spectraspectra ofof 33 exhibitedexhibited aa vinylvinyl alcoholalcohol signalssignals atat δδHH 5.405.40 (d,(d, JJ == 5.55.5 Hz,Hz, H-7),H-7), 1 group6.016.01 (ddd, (ddd,in 1 and JJ == 16.5,216.5,. H 10.0,10.0,NMR 5.05.0 spectra Hz,Hz, H-8),H-8), of 3 exhibited5.265.26 (d,(d, JJ ==a 17.017.0vinyl Hz,Hz, alcohol H-9a),H-9a), signals 5.125.12 (d,(d, at JJδ =H= 10.55.4010.5 Hz,(d,Hz, J H-9b)H-9b)= 5.5 Hz, (Table(Table H-7), 1).1). 6.01TheThe (ddd, 1313CC NMRNMR J = 16.5, andand 10.0, HSQCHSQC 5.0 Hz,ofof 33 H-8), indicatedindicated 5.26 (d,thethe J presence presence= 17.0 Hz, ofof H-9a), aa vinylvinyl 5.12 alcoholalcohol (d, J = group,group, 10.5 Hz, whichwhich H-9b) includesincludes (Table 1).oneone The 13C NMR and HSQC of 3 indicated the presence of a vinyl alcohol group, which includes one oxygenatedoxygenated carboncarbon signalsignal ((δδCC 72.172.1 (C-7)),(C-7)), oneone olefinicolefinic carboncarbon signalsignal ((δδCC 141.7141.7 (C-8)),(C-8)), andand oneone oxygenated carbon signal (δC 72.1 (C-7)), one olefinic carbon signal (δC 141.7 (C-8)), and one exomethyleneexomethylene signalsignal ((δδCC 115.2115.2 (C-9)),(C-9)), (Table(Table 1,1, FigureFigure S16).S16). TheThe presencepresence ofof thethe vinylvinyl alcoholalcohol groupgroup exomethyleneinin 33 waswas alsoalso signal supportedsupported (δC 115.2 byby (C-9)), 11H-H-11HH (Table COSYCOSY 1, data,data,Figure whichwhich S16). Theshowedshowed presence thethe correlationcorrelationof the vinyl at atalcohol H-7/H-8H-7/H-8 group andand 1 1 inH-8/H-9a,H-8/H-9a, 3 was also 9b9b supported(Figure(Figure 2,2, by S17).S17). H- TheHThe COSY locationlocation data, ofof which thethe vinylvinylshowed alcoholalcohol the correlationgroupgroup waswas at unambiguously unambiguouslyH-7/H-8 and H-8/H-9a, 9b (Figure 2, S17). The location of the vinyl alcohol group was unambiguously determineddetermined byby thethe HMBCHMBC correlationcorrelation frfromom H-6H-6 toto C-7C-7 toto bebe atat C-1.C-1. TwoTwo ββ--DD-glucopyranosyl-glucopyranosyl groupsgroups determinedandand twotwo methoxymethoxy by the HMBC groupsgroups correlation werewere locatedlocated from atat C-4,C-4,H-6 C-4toC-4 C-7′′,, C-5,C-5, to be andand at C-5C-5C-1.′′,, respectively,Tworespectively, β-D-glucopyranosyl asas 11,, throughthrough groups HMBCHMBC andspectralspectral two methoxy interpretationinterpretation groups (Figure(Figure were located 2).2). TheThe at chemicalchemical C-4, C-4 structure′structure, C-5, and ofof C-5 33 was′was, respectively, establishedestablished as byby 1, thethroughthe resultsresults HMBC notednoted spectralaboveabove andandinterpretation byby comparisoncomparison (Figure withwith 2). tangshenosidestangshenosidesThe chemical structure ⅡⅡ andand ⅢⅢ of [6,7],[6,7], 3 was asas shownestablishedshown inin FigureFigure by the 2,2, results andand namednamed noted asas Ⅱ Ⅲ abovecampanulalignancampanulalignan and by comparison C.C. However,However, with tangshenosides thethe absoluteabsolute configurationconfiguration and [6,7], atat C-7C-7 as shownofof 33 waswas in not notFigure determineddetermined 2, and named duedue toto as thethe campanulalignanlimitedlimited amountamount ofofC. sample sampleHowever, obtained.obtained. the absolute configuration at C-7 of 3 was not determined due to the limited amount of sample obtained.

Plants 2020, 9, 1232 4 of 11

1 13 Table 1. H and C NMR spectroscopic data of compounds 1–3 (δ in ppm, CD3OD, 500 and 125 MHz).

1 2 3 Position a b b b δC δH δC δH δC δH 1 134.9 135.4 140.0 2 119.5 123.9 119.0 3 152.4 153.0 150.1 4 134.9 139.3 134.6 5 150.1 150.8 152.5 6 102.5 6.71 s 110.8 6.87 s 102.9 6.71 s 7 129.3 6.86 d (15.5) 128.9 6.81 d (15.5) 72.1 5.40 d (5.5) 8 132.0 6.24 dt (15.0, 5.0) 130.0 6.17 dt (15.5, 6.0) 141.7 6.01 ddd (16.5, 10.0, 5.0) 5.26 d (17.0) 9 62.2 4.24 dd (5.5, 2.0) 63.9 4.21 dd (5.5, 2.0) 115.2 5.12 d (10.5) 10 136.9 136.6 136.9 20 108.2 6.48 d (2.0) 108.2 6.49 d (2.0) 103.1 6.47 d (1.5) 30 151.9 152.0 152.5 40 134.5 134.6 134.6 50 154.1 154.3 154.3 60 103.0 6.47 d (2.0) 103.1 6.48 d (2.0) 108.4 6.48 d (1.5) 70 130.9 6.14 (15.5) 130.4 6.13 d (15.5) 131.0 6.18 d (16.0) 80 129.9 6.20 dt (15.0, 5.0) 131.4 6.24 dt (15.5, 5.5) 130.1 6.24 dt (15.5, 6.0) 3.62 d (7.0) 9 29.9 3.55 d (5.5) 30.2 3.55 d (5.5) 29.5 0 3.54 d (6.0) Glc-1 107.3 4.67 d (8.0) 107.3 4.81 d (8.0) 107.3 4.67 d (8.0) Glc-2 71.0 3.45 overlap 71.0 3.49 m 71.0 3.51 overlap Glc-3 77.8 3.40 overlap 78.0 3.43 overlap 77.8 3.45 overlap Glc-4 75.5 3.49 overlap 75.5 3.43 overlap 75.5 3.42 overlap Glc-5 78.5 3.22 m 78.6 3.22 overlap 78.7 3.23 overlap Glc-6 62.0 3.68/3.82 overlap 62.4 3.82 overlap 62.3 3.86 m Glc-10 107.0 4.63 d (8.0) 107.0 4.64 d (8.0) 107.1 4.63 d (7.5) Glc-20 71.0 3.47 overlap 71.0 3.47 m 71.0 3.51 overlap Glc-30 77.8 3.43 overlap 77.9 3.84 overlap 77.8 3.45 overlap Glc-40 75.5 3.51 m 75.5 3.44 m 75.5 3.42 overlap Glc-50 78.5 3.30 m 78.5 3.29 m 78.7 3.23 overlap Glc-60 62.0 3.68/3.82 overlap 62.2 3.22 overlap 62.3 3.78 m OCH3-5 56.8 3.86 s 62.7 3.87s 56.8 3.84 s OCH3-50 56.8 3.81 s 56.8 3.81s 56.7 3.81 s a All assignments were based on 1H-1H correlation spectroscopy (COSY), 1H-13C heteronuclear single quantum 1 13 b coherence spectroscopy (HSQC), and H- C heteronuclear multiple bond correlation (HMBC) results. δH (multi J in Hz).

Compound 3 had a molecular formula of C32H42O17, as established by its molecular ion peak [M H] at m/z 697.2336 (calcd for C H O , 697.2344) in negative mode of HR-Q-TOF-MS (Figure S13) − − 32 41 17 and was also the same as 1 and 2. The 1H and 13C NMR spectra of 3 were almost identical with those of 1 and 2, except for the existence of a vinyl alcohol group in 3 instead of an allyl alcohol group in 1 and 2. 1 H NMR spectra of 3 exhibited a vinyl alcohol signals at δH 5.40 (d, J = 5.5 Hz, H-7), 6.01 (ddd, J = 16.5, 10.0, 5.0 Hz, H-8), 5.26 (d, J = 17.0 Hz, H-9a), 5.12 (d, J = 10.5 Hz, H-9b) (Table1). The 13C NMR and HSQC of 3 indicated the presence of a vinyl alcohol group, which includes one oxygenated carbon signal (δC 72.1 (C-7)), one olefinic carbon signal (δC 141.7 (C-8)), and one exomethylene signal (δC 115.2 (C-9)), (Table1, Figure S16). The presence of the vinyl alcohol group in 3 was also supported by 1H-1H COSY data, which showed the correlation at H-7/H-8 and H-8/H-9a, 9b (Figure2, Figure S17). The location of the vinyl alcohol group was unambiguously determined by the HMBC correlation from H-6 to C-7 to be at C-1. Two β-d-glucopyranosyl groups and two methoxy groups were located at C-4, C-40, C-5, and C-50, respectively, as 1, through HMBC spectral interpretation (Figure2). The chemical structure of 3 was established by the results noted above and by comparison with tangshenosides II and III [6,7], as shown in Figure2, and named as campanulalignan C. However, the absolute configuration at C-7 of 3 was not determined due to the limited amount of sample obtained. By comparison of their spectroscopic data with those published, the structures of the known compounds were identified as 30,4-O-dimethylcedrusin (4)[8], 7R,8S-dihydrodehydrodiconiferyl Plants 2020, 9, 1232 5 of 11

alcohol (5)[9], 30,4-O-dimethylcedrusin 9-O-β-glucopyranoside (6)[10], simplidin (7)[11], pinoresinol di-O-β-d-glucoside (8)[12], demethyl syringin (9)[13], ferulic acid (10)[14], 5-hydroxyconiferaldehyde (11)[15], icariside F (12)[16], benzyl-α-L-arabinopyranosyl-(1” 6 )-β-d-glucopyranoside (13)[17], 2 → 0 astragalin (14)[18], kaempferol 3-O-β-d-apiosyl(1 2)-β-d-glucopyranoside (15)[19], nicotiﬂorin → (16)[20], kaempferol 3,7-di-O-β-d-glucoside (17)[21], and quercetin (18)[22].

2.2. Inhibitory Eﬀects of Compounds on LPS-Induced PGE2 Production in RAW 264.7 Macrophages

The isolates 1, 2, and 4–18 were evaluated for their inhibitory effects of LPS-stimulated PGE2 production in RAW 264.7 macrophages. Compound 3 was not subjected to this experiment due to the limited amount obtained. Except for 2, 3, and 4, all other compounds did not exhibit cytotoxicity in cell viability tests at concentrations up to 100 µM. The effects were assessed using the inhibitory rate on PGE2, which are listed in Table2. Quercetin ( 18) showed the most potent inhibitory effect on LPS-induced PGE2 production in RAW 264.7 macrophages (97% inhibitory rate at 50 µM concentration), among isolated compounds from C. takesimana. 5-Hydroxyconiferaldehyde (11) also decreased remarkably the PGE2 production with 85.17% inhibitory rate. Additionally, 7R,8S-dihydrodehydrodiconiferyl alcohol (5), simplidin (7), pinoresinol di-O-β-d-glucoside (8), and ferulic acid (10) showed inhibitory effects over 50% on the PGE2 production at 50 µM concentration while the new compounds 1 and 2 did not exhibit a significant effect (<50% inhibitory rate). Among the active compounds, quercetin (18), 7R,8S-dihydrodehydrodiconiferyl alcohol (5), pinoresinol di-O-β-d-glucoside (8), and ferulic acid (10) have already been reported to have anti-inflammatory effects, whereas there is no report on biological activities related to anti-inflammatory effects for simplidin (7) and 5-hydroxyconiferaldehyde (11). Therefore, inhibitory effects of 7 and 11 on LPS-induced PGE2 production at various concentrations were investigated.

Table 2. The cytotoxicities and inhibitory eﬀects of the isolates from C. takesimana on lipopolysaccharide

(LPS)-induced prostaglandin E2 (PGE2) production in RAW 264.7 macrophages.

IC (µM) Inhibition IC (µM) Inhibition Compound 80 Compound 80 Cytotoxicitiy Rate (%) a Cytotoxicitiy Rate (%) a 1 >50 23.92 10 >50 62.53 2 33.66 33.07 11 >50 85.17 3 -- 12 >50 31.07 4 30.69 7.89 13 >50 33.12 5 >50 69.35 14 >50 23.55 6 >50 20.01 15 >50 43.49 7 >50 60.01 16 >50 36.73 8 >50 58.08 17 >50 34.51 9 >50 37.44 18 >50 97.01 a Cells were pretreated with 50 µM concentration of compounds for 1 h, then with LPS (100 ng/mL), and incubated for 24 h.

As shown in Figure3A,B, simplidin ( 7) and 5-hydroxyconiferaldehyde (11) significantly and concentration-dependently suppressed LPS-stimulated PGE2 production. Compounds 7 and 11 did not influence the RAW 264.7 cell viability in either the presence or absence of LPS at concentrations up to 50 µM for 24 h, suggesting that 7 and 11 may be major constituents of the leaves of C. takesimana showing anti-inflammatory activities. Accordingly, further pharmacological studies are demanded to investigate the mechanisms responsible for the inhibitory effects of 7 and 11 on inflammatory mediator in RAW 264.7 macrophages. Plants 2020, 9, 1232 6 of 11 Plants 2020, 9, x FOR PEER REVIEW 6 of 11

(A) (B)

Figure 3. Inhibitory effects of compounds 7 (A) and 11 (B) on LPS-stimulated PGE2 production in Figure 3. Inhibitory eﬀects of compounds 7 (A) and 11 (B) on LPS-stimulated PGE2 production RAWin RAW 264.7 264.7 macrophages. macrophages. Cells were Cells pretreated were pretreated with different with concentrations diﬀerent concentrations (20, 40, or 80 (20,μM) 40,of compoundsor 80 µM) of7compounds and 11 for7 and1 h,11 forthen 1 h,with then LPS with (100 LPS (100ng/mL), ng/ mL),and andincubated incubated for for 24 24 h. h. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamideN-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398, (NS-398, 10 10 μµM)M) was was used used as as a a positive

PGEPGE22 productionproduction inhibitor. inhibitor. Level Level of of PGE PGE22 inin culture culture media media was was quantified quantified using using enzyme-linked immunosorbentimmunosorbent assay assay (ELISA) (ELISA) kits. kits. (#: p < 0.05 as compared with the untreated group, ** p < 0.05, and *** p < 0.0002). 3. Discussion 3. Discussion The investigation on secondary metabolites in the leaves of C. takesimana was carried out for the firstThe time. investigation Consequently, on secondary three new metabolites neolignan in glucosides, the leaves ofcampanulalignansC. takesimana was A–C carried (1–3), out were for isolatedthe first by time. repeated Consequently, chromatography. three new Additionally, neolignan glucosides, five lignans campanulalignans (4–8), five phenolic A–C compounds (1–3), were (isolated9–13), and by repeated five flavonoids chromatography. (14–18) were Additionally, obtained five from lignans the (4present–8), five study. phenolic To compounds the best of (9 –our13), knowledge,and five flavonoids 3′,4-O (14-dimethylcedrusin–18) were obtained from9-O- theβ-glucopyranoside present study. To the(6), best ofsimplidin our knowledge, (7), 30,4-O-dimethylcedrusin 9-O-β-glucopyranoside (6), simplidin (7), 5-hydroxyconiferaldehyde (11), 5-hydroxyconiferaldehyde (11), icariside F2 (12), benzyl-α-L-arabinopyranosyl-(1 ″ icariside F2 (12), benzyl-α-L-arabinopyranosyl-(1” 60)-β-d-glucopyranoside (13), and kaempferol →6′)-β-D-glucopyranoside (13), and kaempferol 3-→O-β-D-apiosyl-(1→2)-β-D-glucopyranoside (15) 3-O-β-d-apiosyl-(1 2)-β-d-glucopyranoside (15) were isolated from the Campanulaceae family for the were isolated from→ the Campanulaceae family for the first time in this work. first time in this work. All the isolates from the leaves of C. takesimana except for compound 3 were estimated for their All the isolates from the leaves of C. takesimana except for compound 3 were estimated for their inhibitory effects on the production of PGE2 in the LPS-induced RAW 264.7 macrophages. inhibitory effects on the production of PGE2 in the LPS-induced RAW 264.7 macrophages. Inflammatory Inflammatory mediators, such as NO and PGE2, and numerous pro-inflammatory cytokines released mediators, such as NO and PGE , and numerous pro-inflammatory cytokines released by the activated by the activated macrophages 2are important targets for the treatment of various inflammatory macrophages are important targets for the treatment of various inflammatory diseases, including diseases, including multiple sclerosis, rheumatoid arthritis, and obesity [23,24]. iNOS and COX-2 multiple sclerosis, rheumatoid arthritis, and obesity [23,24]. iNOS and COX-2 protein expression are protein expression are remarkably elevated in the inflammatory processes, resulting in increased remarkably elevated in the inflammatory processes, resulting in increased production of NO and PGE2, production of NO and PGE2, respectively. These overexpressed pro-inflammatory mediators can respectively. These overexpressed pro-inflammatory mediators can lead to damage in living cells and lead to damage in living cells and tissues, resulting in various physiological disorders associated tissues, resulting in various physiological disorders associated with inflammation [4,23]. Therefore, with inflammation [4,23]. Therefore, compounds that inhibit the production of pro-inflammatory compounds that inhibit the production of pro-inflammatory mediators like NO and PGE2 can be mediators like NO and PGE2 can be candidates for anti-inflammatory drugs [4]. candidates for anti-inflammatory drugs [4]. Among the isolates, well known quercetin (18) showed the strongest inhibitory effect [25]. In Among the isolates, well known quercetin (18) showed the strongest inhibitory effect [25]. addition, 7R,8S-dihydrodehydrodiconiferyl alcohol (5), pinoresinol di-O-β-D-glucoside (8), and In addition, 7R,8S-dihydrodehydrodiconiferyl alcohol (5), pinoresinol di-O-β-d-glucoside (8), ferulic acid (10) exhibited remarkable inhibitory activity on LPS-stimulated PGE2 production in and ferulic acid (10) exhibited remarkable inhibitory activity on LPS-stimulated PGE production RAW264.7 cells and their inhibitory effects were well correlated with previous reports2 [26–28]. in RAW264.7 cells and their inhibitory effects were well correlated with previous reports [26–28]. 7R,8S-Dihydrodehydrodiconiferyl alcohol (5), a neolignan, showed ABTS free radical scavenging 7R,8S-Dihydrodehydrodiconiferyl alcohol (5), a neolignan, showed ABTS free radical scavenging and and LPS-stimulated nitric oxide (NO) production inhibitory activities in a previous report [26]. LPS-stimulated nitric oxide (NO) production inhibitory activities in a previous report [26]. Pinoresinol Pinoresinol di-O-β-D-glucoside (8) possesses protective activity on human endothelial cells by di-O-β-d-glucoside (8) possesses protective activity on human endothelial cells by inhibiting oxidized inhibiting oxidized low density lipoprotein (oxLDL)-induced SOD activity, NO production, and low density lipoprotein (oxLDL)-induced SOD activity, NO production, and endothelial nitric oxide endothelial nitric oxide synthase (eNOS) expression [27]. A prior study revealed that ferulic acid synthase (eNOS) expression [27]. A prior study revealed that ferulic acid (10) significantly reduced (10) significantly reduced LPS-induced PGE2 levels [28]. LPS-induced PGE levels [28]. Simplidin (7) 2and 5-hydroxyconiferaldehyde (11) also showed significant inhibitory effects on Simplidin (7) and 5-hydroxyconiferaldehyde (11) also showed significant inhibitory effects PGE2 production in this study, but there was no investigation on biological activities related to on PGE production in this study, but there was no investigation on biological activities anti-inflammatory2 effects in previous studies. Simplidin (7), first isolated from Firmiana simplex (Sterculiaceae) [11], exhibited cytotoxicities against human cancer cell lines [29]. 5-Hydroxyconiferaldehyde (11) was discovered in the biosynthetic pathway for syringyl

Plants 2020, 9, 1232 7 of 11 related to anti-inflammatory effects in previous studies. Simplidin (7), first isolated from Firmiana simplex (Sterculiaceae) [11], exhibited cytotoxicities against human cancer cell lines [29]. 5-Hydroxyconiferaldehyde (11) was discovered in the biosynthetic pathway for syringyl monolignol in angiosperms [30] and identified from the methanol extract of Carthamus tinctorius seed [15]. However, to the best of our knowledge, there have been no reports of any biological or pharmacological activities for 5-hydroxyconiferaldehyde (11). In our investigation and for the first time, simplidin (7) and 5-hydroxyconiferaldehyde (11) are suggested as active natural products with inhibitory effects on a pro-inflammatory mediator, PGE2 production.

4. Materials and Methods

4.1. Plant Material The leaves of Campanula takesimana Nakai (Campanulaceae; Korean bellﬂower) used in present study were provided by COSMAX BIO, Gyeonggi-do, Republic of Korea, in July 2019. The origin of the material was identiﬁed by D.S.J., one of authors, and a voucher specimen (CATA-2019) has been deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea.

4.2. General Experimental Procedures General experimental procedures are in the Supplementary Materials.

4.3. Extraction and Isolation

Dried leaves of C. takesimana (2.5 kg) were extracted with distilled water (60 L) at 100 ◦C for 5 h, and the solvent was evaporated by rotary evaporator at 45 ◦C. The hot water extract (1.0 kg) was separated over column chromatography (CC) using Diaion HP-20 as the stationary phase with a gradient system of acetone-H2O (0:100 to 100:0, v/v) to give 17 fractions (F1~F17). Fraction F10 was fractionated further using Sephadex LH-20 CC (5.5 45.9 cm) with 40% acetone × to produce eight fractions (F10-1~F10-8). Fraction F10-4 was subjected to silica gel CC (230–400 mesh; 5.1 33.0 cm, CH Cl -MeOH-H O = 80:18:2 to 70:27:3, v/v/v), yielding 9 (78.5 mg, yield 0.00314% by × 2 2 2 dry weight of the plant material). Fraction F10-5 was separated through silica gel CC (230–400 mesh; 5.1 33.0 cm, CH Cl -MeOH-H O = 80:18:2 to 70:27:3, v/v/v) to get 12 (3.7 mg, 0.000148%) and 13 × 2 2 2 (3.0 mg, 0.00012%). Fraction F12 was subjected to Sephadex LH-20 CC (4.5 45.9 cm) with MeOH-H2O × mixture (70:30 v/v) to give 10 subfractions (F12-1~F12-10). Subfraction F12-2 was fractionated further by using silica gel CC (230–400 mesh; 3.1 25.0 cm, CH Cl -MeOH-H O = 85:13.5:1.5 to 70:27:3, v/v/v) × 2 2 2 to produce 14 subfractions (F12-2-1~F12-2-14). Compound 6 (175.3 mg, 0.00701%) was isolated from subfraction F12-2-6 by a flash chromatographic system with a Redi Sep-C18 cartridge (43 g, MeOH-H2O, from 15:85 to 45:55, v/v). Subfraction F12-2-9 was also subjected to a flash chromatographic system by a Redi Sep-C18 cartridge (43 g, MeOH-H2O, from 13:87 to 40:60, v/v) to afford 8 (14.3 mg, 0.000572%). Subfraction F12-2-10 was fractioned by a flash chromatographic system with a Redi Sep-C18 cartridge (43 g, MeOH-H2O, 27:72 to 50:50, v/v) to isolate 1 (20.2 mg, 0.000808%), 2 (8.0 mg, 0.00032%), and 17 (8.7 mg, 0.000348%). Fraction F13 was separated into six subfractions (F13-1~F13–6) using Sephadex LH-20 CC (5.5 47.9 cm) with 50% acetone. Compounds 1 (3.0 mg, 0.00012%) and 3 (2.5 mg, 0.0001%) × were purified from subfraction F13-2 using a preparative HPLC with Gemini 5 µm NX-C18 110A column (acetonitirle-H2O = 15:85 to 30:70, v/v). Fraction F15 was subjected to Sephadex LH-20 CC (5.6 58.6 cm) with 60% acetone, and produced six fractions (F15-1~F15-6). Fraction F15-2 was × fractionated using silica gel CC (230–400 mesh; 5.1 33.0 cm, CH Cl -MeOH-H O = 90:9:1 to 60:36:4, × 2 2 2 v/v/v), and produced 16 subfractions (F15-2-1~F15-2-16). Compounds 6 (28.0 mg, 0.00112%) and 7 (7.0 mg, 0.00028%) were obtained from subfraction F15-2 by a flash chromatographic system with a Redi Sep-C18 cartridge (30 g, MeOH-H2O = 20:80 to 40:60, v/v). Compound 11 (3.7 mg, 0.000148%) was purified from subfraction F15-5-2 by silica gel CC (230–400 mesh; 2.5 20.0 cm, CH Cl -MeOH-H O = × 2 2 2 Plants 2020, 9, 1232 8 of 11

95:4.5:0.5 to 60:36:4 v/v/v). Fraction F15-5 was subjected to silica gel CC (230–400 mesh; 5.1 33.0 cm, × CH2Cl2-MeOH-H2O = 90:9:1 to 60:36:4, v/v/v) to isolate 10 (11.2 mg, 0.000448%), 14 (91.2 mg, 0.00365%), 15 (17.0 mg, 0.000704%), and 16 (32.4 mg, 0.0013%). Fraction F16 was separated into nine subfractions (F16-1~F16-9) using Sephadex LH-20 CC (4.5 45.9 cm) with 70% MeOH. Subfraction F16-1 was × subjected to silica gel CC (230–400 mesh; 5.1 33.0 cm, CH Cl -MeOH-H O = 90:9:1 to 60:36:4, v/v/v) × 2 2 2 to aﬀord 4 (293.0 mg, 0.0117%) and 5 (79.5 mg, 0.00318%). Compound 18 (10.1 mg, 0.000404%) was puriﬁed by recrystallizing subfraction F17 with MeOH.

4.3.1. Campanulalignan A (1)

Pale yellow powder; HR-Q-TOF-MS (negative mode) m/z = 697.2360 [M–H]− (calcd for C32H41O17, 20 693.2344); [α]D : 12.7◦ (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 224 (3.31), 265 (3.08); IR (ATR) − 1 1 13 νmax 3278, 2903, 1580, 1448, 1348 cm− ; H and C NMR data, see Table1.

4.3.2. Campanulalignan B (2)

Pale yellow powder; HR-Q-TOF-MS (negative mode) m/z = 697.2354 [M–H]− (calcd for C32H41O17, 20 693.2344); [α]D : 14.5◦ (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 222 (3.24), 263 (3.00); IR (ATR) − 1 1 13 νmax 3302, 2920, 1580, 1421, 1347 cm− ; H and C NMR data, see Table1.

4.3.3. Campanulalignan C (3)

Brown amorphous powder; HR-Q-TOF-MS (negative mode) m/z = 697.2336 [M–H]− (calcd for 20 C32H41O17, 693.2344); [α]D : 24.5◦ (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 230 (3.47), 267 (3.22); − 1 1 13 IR (ATR) νmax 3266, 2922, 1585, 1342, 1017 cm− ; H and C NMR data, see Table1.

4.4. Enzymatic Hydrolysis of Compound 1 Compound 1 (1.0 mg) was incubated together with 2 drops of toluene, β-glucosidase (2.5 mg), and H2O (1.5 mL) in a CO2 incubator at 35 ◦C for 3 days. The reaction was stopped by addition EtOH to the reaction mixture and β-glucosidase was removed by ﬁltration. The presence of glucose in 1 was conﬁrmed by co-TLC (Rf 0.3, n-BuOH:acetic acid:H2O = 2:1:1, v/v/v) with a standard compound.

4.5. Absolute Configurations of β-glucose in Compound 1 The absolute configuration of β-glucose in 1 was confirmed by the method from Tanaka et al. [31]. Hydrolysate was dissolved in pyridine (500 µL) and l-cysteine methyl ester hydrochloride (1.2 mg) was added and heated at 60 ◦C for 1 h. The mixture was heated again at 60 ◦C for 1 h after adding σ-tolyl isothiocyanate (100 µL) and analyzed directly by HPLC under a gradient system (A: 0.1% (v/v) formic acid in water, B: 0.1% (v/v) formic acid in acetonitrile, 10 to 50% B, 45 min). The reaction mixture of 1 was detected at 27.0 min. The retention times of authentic l- and d-glucoses were 26.1 and 27.0 min, respectively, under the same HPLC conditions. Thus, the absolute configuration of β-glucose in 1 was confirmed as the D configuration.

4.6. Cell Culture RAW 264.7 macrophages were purchased from the Korea Cell Line Bank (Seoul, South Korea). Cells were incubated in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with penicillin–streptomycin sulfate (100 units/mL and 100 µg/mL) and 10% FBS at 37 ◦C humidiﬁed incubator containing of 5% CO2.

4.7. Cell Viability Test Cell viability test was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dipheyl tetrazoliumbromide; Sigma-Aldrich) assay. RAW 264.7 macrophages were grown with each isolated compound and LPS (100 ng/mL) for 24 h. After 24 h, cells were treated with MTT solution for 4 h. Plants 2020, 9, 1232 9 of 11

MTT formazan was resolved by adding dimethyl sulfoxide (DMSO), and the absorbance of each well at 540 nm was read by a microplate reader.

4.8. Measurement of PGE2 Production RAW 264.7 cells were treated with compounds (50 µM) 1 h prior to LPS (100 ng/mL) stimulation for 24 h. Cells were pretreated with positive controls (NS398) for 1 h, and then stimulated with LPS (100 ng/mL) for 24 h. The supernatant was collected and PGE2 production detected by using PGE2 ELISA Kits (R&D Systems, MN, USA).

5. Conclusions Three new neolignan glucosides (1–3) and 15 known compounds were isolated from the leaves of C. takesimana in the present study. We found that 7R,8S-dihydrodehydrodiconiferyl alcohol (5), simplidin (7), pinoresinol di-O-β-d-glucoside (8), ferulic acid (10), 5-hydroxyconiferaldehyde (11), and quercetin (18) inhibit production of an inflammatory mediator PGE2. Among these, the inhibitory effect of simplidin (7) and 5-hydroxyconiferaldehyde (11) on PGE2 production was reported for the first time in this work. Thus, simplidin (7) and 5-hydroxyconiferaldehyde (11) are worthy of further pharmacological evaluation for their potential as anti-inflammatory drugs.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/9/1232/s1, 1 Figure S1: HR-Q-TOF-MS spectrum of compound 1, Figure S2: The H-NMR (500 MHz, CD3OD) spectrum of 13 compound 1, Figure S3: The C-NMR (125 MHz, CD3OD) spectrum of compound 1, Figure S4: The HSQC spectrum of compound 1 in CD3OD, Figure S5: The COSY spectrum of compound 1 in CD3OD, Figure S6: The HMBC spectrum of compound 1 in CD3OD, Figure S7: HR-Q-TOF-MS spectrum of compound 2, Figure 1 13 S8: The H-NMR (500 MHz, CD3OD) spectrum of compound 2, Figure S9: The C-NMR (125 MHz, CD3OD) spectrum of compound 2, Figure S10: The HSQC spectrum of compound 2 in CD3OD, Figure S11: The COSY spectrum of compound 2 in CD3OD, Figure S12: The HMBC spectrum of compound 2 in CD3OD, Figure S13: 1 HR-Q-TOF-MS spectrum of compound 3, Figure S14: The H-NMR (500 MHz, CD3OD) spectrum of compound 13 3, Figure S15: The C-NMR (125 MHz, CD3OD) spectrum of compound 3, Figure S16: The HSQC spectrum of compound 3 in CD3OD, Figure S17: The COSY spectrum of compound 3 in CD3OD, Figure S18: The HMBC spectrum of compound 3 in CD3OD Author Contributions: Conceptualization, D.S.J.; investigation, Y.Q., S.-I.C., S.-R.S., H.-S.H., H.S.A., and Y.-K.S.; providing plant material, H.S.A, Y.K.S., and S.H.L.; performing experiment, Y.Q.; interpreting data, Y.Q. and S.R.S.; data analysis, Y.Q.; writing—original draft preparation, Y.Q. and S.R.S.; writing—review and editing, K.-T.L. and D.S.J.; supervision, S.H.L, K.-T.L., and D.S.J.; project administration, H.C.K. and D.S.J.; funding acquisition, D.S.J. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Korea Institute of Science and Technology (KIST) Institutional Program (Project No. 2E30650-20-154), and by a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT), Republic of Korea (grant number: NRF-2019R1A2C1083945). Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Park, K.R.; Jung, H.J. Isozyme and morphological variation in Campanula punctata and C. takesimana (Campanulaceae). Korean J. Plant Taxon. 2000, 30, 1–16. [CrossRef] 2. Kim, M.; Kim, K.; Yook, H. Antioxidative eﬀects of Campanula takesimana Nakai extract. J. Korean Soc. Food Sci. Nutr. 2012, 41, 1331–1337. [CrossRef] 3. Kim, H.J.; Kang, S.H. Ethnobotany, Phytochemistry, pharmacology of the Korean Campanulaceae: A comprehensive review. Korean J. Plant Res. 2017, 30, 240–264. [CrossRef] 4. Yang, G.; Lee, K.; Lee, M.; Ham, I.; Choi, H.Y. Inhibition of lipopolysaccharide-induced nitric oxide and prostaglandin E 2 production by chloroform fraction of Cudrania tricuspidata in RAW 264.7 macrophages. BMC Complement. Altern. Med. 2012, 12, 250. [CrossRef] 5. Agrawal, P.K. NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 1992, 31, 3307–3330. [CrossRef] 6. Yuda, M.; Ohtani, K.; Mizutani, K.; Kasai, R.; Tanaka, O.; Jia, M.R.; Ling, Y.R.; Pu, X.F.; Saruwatari, Y.I. Neolignan glycosides from roots of Codonopsis tangshen. Phytochemistry 1990, 29, 1989–1993. [CrossRef] Plants 2020, 9, 1232 10 of 11

7. Mizutani, K.; Yuda, M.; Tanaka, O.; Saruwatari, Y.I.; Jia, M.R.; Ling, Y.K.; Pu, X.F. Tanghenosides i and ii from chuan-dangshen, the root of Codonopsis tangshen oliv. Chem. Pharm. Bull. 1998, 36, 2726–2729. [CrossRef] 8. Pieters, L.; de Bruyne, T.; Claeys, M.; Vlietinck, A.; Calomme, M.; vanden Berghe, D. Isolation of a dihydrobenzofuran lignan from South American dragon’s blood (Croton spp.) as an inhibitor of cell proliferation. J. Nat. Prod. 1993, 56, 899–906. [CrossRef] 9. Li, L.; Seeram, N.P. Maple syrup phytochemicals include lignans, coumarins, a stilbene, and other previously unreported antioxidant phenolic compounds. J. Agric. Food Chem. 2010, 58, 11673–11679. [CrossRef] 10. Calis, I.; Kirmizibekmez, H.; Beutler, J.A.; Donmez, A.A.; Yalçin, F.N.; Kilic, E.; Ozalp, M.; Ruedi, P.; Tasdemir, D. Secondary metabolites of Phlomis viscosa and their biological activities. Turk. J. Chem. 2005, 29, 71–81. 11. Son, Y.K.; Lee, M.H.; Han, Y.N. A New antipsychotic effective neolignan from Firmiana simplex. Arch. Pharm. Res. 2005, 28, 34–38. [CrossRef][PubMed] 12. Schumacher, B.; Scholle, S.; Hölzl, J.; Khudeir, N.; Hess, S.; Müller, C.E. Lignans isolated from valerian: Identification and characterization of a new olivil derivative with partial agonistic mactivity at A1 adenosine receptors. J. Nat. Prod. 2002, 65, 1479–1485. [CrossRef][PubMed] 13. Tan, R.X.; Ma, W.G.; Wei, H.X.; Zhang, L.X. Glycosides from Wahlenbergia marginata. Phytochemistry 1998, 48, 1245–1250. [CrossRef] 14. Yawadio, R.; Tanimori, S.; Morita, N. Identification of phenolic compounds isolated from pigmented rices and their aldose reductase inhibitory activities. Food Chem. 2007, 101, 1616–1625. [CrossRef] 15. Sakakibara, N.; Nakatsubo, T.; Suzuki, S.; Shibata, D.; Shimada, M.; Umezawa, T. Metabolic analysis of the cinnamate/monolignol pathway in Carthamus tinctorius seeds by a stable-isotope-dilution method. Org. Biomol. Chem. 2007, 5, 802–815. [CrossRef][PubMed] 16. Peng, W.; Fu, X.; Li, Y.; Xiong, Z.; Shi, X.; Zhang, F.; Huo, G.; Li, B. Phytochemical study of stem and leaf of Clausena lansium. Molecules 2019, 24, 3124. [CrossRef] 17. Lee, S.Y.; Kim, K.H.; Lee, I.K.; Lee, K.H.; Choi, S.U.; Lee, K.R. A new flavonol glycoside from Hylomecon vernalis. Arch. Pharm. Res. 2012, 35, 415–442. [CrossRef] 18. Chiang, H.; Lo, Y.; Lu, F. Xanthine oxidase inhibitors from the leaves of Alsophila spinulosa (Hook) Tryon. J. Enzym. Inhib. 1994, 8, 61–71. [CrossRef] 19. Cheng, X.; Tang, X.; Guo, C.; Zhang, C.; Yang, Q. New flavonol glycosides from the seeds of Desmodium styracifolium. Chem. Nat. Compd. 2018, 54, 846–850. [CrossRef] 20. Kazuma, K.; Noda, N.; Suzuki, M. Malonylated flavonol glycosides from the petals of Clitoria ternatea. Phytochemistry 2003, 62, 229–237. [CrossRef] 21. Markham, K.R.; Ternai, B.; Stanley, R.; Geiger, H.; Mabry, T.J. Carbon-13 NMR Studies of flavonoids—III: Naturally occurring flavonoid glycosides and their acylated derivatives. Tetrahedron 1978, 34, 1389–1397. [CrossRef] 22. Aisyah, L.S.; Yun, Y.F.; Herlina, T.; Julaeha, E.; Zainuddin, A.; Nurfarida, I.; Hidayat, A.T.; Supratman, U.; Shiono, Y. Flavonoid compounds from the leaves of Kalanchoe prolifera and their cytotoxic activity against P-388 murine leukimia cells. Nat. Prod. Sci. 2017, 23, 139–145. [CrossRef] 23. Nathan, C. Points of control in inflammation. Nature 2002, 420, 846–852. [CrossRef][PubMed] 24. Hotamisligil, G.S. Inflammation and metabolic disorders. Nature 2006, 444, 860–867. [CrossRef] 25. Hammer, K.D.; Hillwig, M.L.; Solco, A.K.; Dixon, P.M.; Delate, K.; Murphy, P.A.; Wurtele, E.S.; Birt, D.F.

Inhibition of prostaglandin E2 production by anti-inflammatory Hypericum perforatum extracts and constituents in RAW264. 7 mouse macrophage cells. J. Agric. Food Chem. 2007, 55, 7323–7331. [CrossRef][PubMed] 26. Liu, Q.; Huang, X.; Bai, M.; Chang, X.; Yan, X.; Zhu, T.; Zhao, W.; Peng, Y.; Song, S. Antioxidant and anti-inflammatory active dihydrobenzofuran neolignans from the Seeds of Prunus tomentosa. J. Agric. Food Chem. 2014, 62, 7796–7803. [CrossRef] 27. Yao, J.; Zou, Z.; Wang, X.; Ji, X.; Yang, J. Pinoresinol diglucoside alleviates oxLDL-induced dysfunction in human umbilical vein endothelial cells. Evid. Based Complement. Alternat. Med. 2016, 2016, 10. [CrossRef] 28. Yoo, S.; Jeong, S.; Lee, N.; Shin, H.; Seo, C. Simultaneous determination and anti-inflammatory effects of four phenolic compounds in Dendrobii herba. Nat. Prod. Res. 2017, 31, 2923–2926. [CrossRef] 29. Wu, X.; Zhang, L.; He, J.; Li, G.; Ding, L.; Gao, X.; Dong, L.; Song, L.; Li, Y.; Zhao, Q. Two new diterpenoids from Excoecaria acerifolia. J. Asian Nat. Prod. Res. 2013, 15, 151–157. [CrossRef] Plants 2020, 9, 1232 11 of 11

30. Li, L.; Popko, J.L.; Umezawa, T.; Chiang, V.L. 5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms. J. Biol. Chem. 2000, 275, 6537–6545. [CrossRef] 31. Tanaka, T.; Tatsuya, N.; Toshihisa, U.; Kenji, T.; Isao, K. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 2007, 55, 899–901. [CrossRef][PubMed]

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