molecules

Article Article A New ErythrinanErythrinan AlkaloidAlkaloid GlycosideGlycoside fromfrom thethe Seeds ofSeedsErythrina of Erythrina crista-galli crista-galli Qing-Wei Tan 1,2,*, Jian-Cheng Ni 1, Pei-Hua Fang 1 and Qi-Jian Chen 1,2,* Qing-Wei Tan 1,2,*, Jian-Cheng Ni 1 ID , Pei-Hua Fang 1 and Qi-Jian Chen 1,2,* 1 Key Laboratory of Bio-Pesticide and Chemistry-Biology, Ministry of Education, Fujian Agriculture and 1 ForestryKey Laboratory University, of Bio-PesticideFuzhou 350002 and, Fujian, Chemistry-Biology, China; [email protected] Ministry of Education, (J.-C.N.); Fujian [email protected] Agriculture and (P.Forestry-H.F.) University, Fuzhou 350002, Fujian, China; [email protected] (J.-C.N.); 2 [email protected] Laboratory of Plant (P.-H.F.) Virology of Fujian Province, Institute of Plant Virology, Fujian Agriculture and 2 ForestryKey Laboratory University, of PlantFuzhou Virology 350002 of, Fujian, Fujian China Province, Institute of Plant Virology, Fujian Agriculture and * Correspondence:Forestry University, [email protected] Fuzhou 350002, Fujian, (Q. China-W.T.); [email protected] (Q.-J.C.); * Tel.:Correspondence: +86-187-501-627 [email protected] (Q.-W.T); +86-137-993 (Q.-W.T.);-087-16 (Q. [email protected]) (Q.-J.C.); Tel.: +86-187-501-627-80 (Q.-W.T.); +86-137-993-087-16 (Q.-J.C.)

Received: 03 3 August August 2017; 2017; Accepted: Accepted: 13 13 September September 2017; 2017; Published: Published: 16 16 September September 2017 2017

Abstract: AA new newErythrina Erythrinaalkaloid alkaloid glycoside, glycoside, named named erythraline-11 erythralineβ-O--glucopyranoside,11β-O-glucopyranoside was isolated, was fromisolated the from seeds the of Erythrinaseeds of Erythrina crista-galli cristaL., together-galli L., withtogether five with known fiveErythrina known alkaloidsErythrina andalkaloids an indole and alkaloid.an indole The alkaloid. structure The of structure the new alkaloidof the new glycoside alkaloid was glycoside elucidated was by elucidated spectroscopic by methods,spectroscopic and allmethods, of the compoundsand all of the were compounds evaluated were for theirevaluated antiviral for their activity antiviral against activity tobacco against mosaic tobacco virus. mosaic virus. Keywords: Erythrina crista-galli; fabaceae; Erythrinan alkaloid; tobacco mosaic virus; antiviral activity Keywords: Erythrina crista-galli; fabaceae; Erythrinan alkaloid; tobacco mosaic virus; antiviral activity

1. Introduction 1. IntroductionThe Erythrina genus, including approximately 200 species, is mainly distributed in tropical and subtropicalThe Erythrina areas. genus, Four including local and approximately six introduced 200Erythrina species, isspecies mainly are distributed distributed in intropical China and [1]. Erythrinasubtropical crista-galli areas. FL.our (Fabaceae), local and known six introduced as cockspur Erythrina coral tree, species is a popular are distributedornamental plant in China in South [1]. AmericaErythrina andcrista tropical-galli L. and(Fabaceae), subtropical known regions as cockspur of South coral Asia. tree, Pharmacological is a popular ornamental investigations plant have in demonstratedSouth America that andE. tropical crista-galli andseed subtropical extracts regions possess of sedative, South Asia. hypertensive, Pharmacological laxative, investigations and diuretic activitieshave demonstrated [2]. Phytochemical that E. crista studies-galli seed on this extracts plant possess showed sedative, the presence hypertensive, of Erythrina laxative[3,4], and benzylisoquinolinediuretic activities [2] alkaloids. Phytochemical [5,6]. studies The alkaloids on this ofplant the showedErythrina thetype, presence which of Erythrina possess a[3 unique,4] and spirocyclicbenzylisoquinoline structure, alkaloids are responsible [5,6]. The for alkaloids these activities. of the Erythrina Great efforts type, have which been possess made a to unique reveal thespirocyclic biosynthetic structure, pathway are responsible and mechanism for these for activities. the Erythrina Greatalkaloid efforts have biosynthesis been made in theto reveal past few the decadesbiosynthetic [2]. pathway Furthermore, and mechanism there are for chemical the Erythrina studies alkaloid on its biosynthesis non-alkaloidal in the constituents, past few decades in which [2]. flavonoids,Furthermore cinnamylphenols, there are chemical and studies pterocarpans on its non were-alkaloidal reported constituents, functioning inas which phytoalexins flavonoids, and possessingcinnamylphenols antimicrobial and pterocarpan activity [7s– 11were]. Inreported this paper, functioning we describe as phytoalexins the isolation and and possessing structural characterizationantimicrobial activity of a[7 new–11]. ErythrinaIn this paper,alkaloids we describe glucoside, the isolation erythraline-11 and structuralβ-O-glucopyranoside characterization of (1 ),a togethernew Erythrina with sixalkaloids known glucoside, alkaloids erythraline (2–7) (Figure-11β1)- fromO-glucopyranoside the seeds of Erythrina (1), together crista-galli with ,six and known these isolatedalkaloids compounds (2–7) (Figure were 1) from evaluated the seeds for theirof Erythrina inhibitory crista activity-galli, and against these tobacco isolated mosaic compounds virus (TMV) were usingevaluated the leaf-discfor their method.inhibitory activity against tobacco mosaic virus (TMV) using the leaf-disc method.

Figure 1. Structure of Compounds 1–7.

Molecules 2017, 22, 1558; doi:10.3390/molecules22091558 www.mdpi.com/journal/molecules Molecules 2017, 22, 1558; doi:10.3390/molecules22091558 www.mdpi.com/journal/molecules Molecules 2017, 22, 1558 2 of 7

Molecules 2017, 22, 1558 2 of 6 2. Results and Discussion 2. Results and Discussion Phytochemical study of MeOH extract of the seeds of Erythrina crista-galli led to the isolation of a new ErythrinanPhytochemical alkaloid study glucoside, of MeOH erythraline-11 extract of theβ seeds-O-glucopyranoside of Erythrina crista (1),-galli together led to with the fiveisolation known of Erythrinana new Erythrinan alkaloids, alkaloid erythraline glucoside, (2)[12 erythraline], erythratine-11 (β3-)[O-13glucopyranoside], erysodine (4)[ (141),], together erysotrine with (5)[ five13],

(+)-16knownβ -ErythrinanD-glucoerysopine alkaloids, (6 )[erythral15], andine ( an2) [12], indole erythratine alkaloid, (3) ( −[13],)-hypaphorine erysodine (4 ()7[14],)[16 erysotrine] (Figure1 ().5)

The[13], known (+)-16β structures-D-glucoerysopine were identified (6)[15], by and comparison an indole ofalkaloid, their spectroscopic (−)-hypaphorine data with(7) [16] those (Figure reported 1). The in theknown literature. structures were identified by comparison of their spectroscopic data with those reported in the literature.Compound 1 was isolated as a white amorphous powder. It was assigned with a molecular Compound 1 was isolated as a white amorphous powder−. It was assigned with a molecular formula of C24H29NO9 by HR-ESI-MS at m/z = 510.1537 [M + Cl] (Calcd for C24H29ClNO9, 510.1536). − Theformula IR spectrum of C24H29 (SupplementaryNO9 by HR-ESI-MS Materials) at m/z = exhibited 510.1537 absorption[M + Cl] (Calcd bands for due C24 toH an29ClNO aromatic9, 510.1536). moiety andThe IR a conjugate spectrum olefin(Supplementary (1627, 1503, Materials) 1482 cm− exhibited1). The 1 H-NMRabsorption and bands HSQC due spectrum to an aromatic of 1 indicated moiety and a conjugate olefin (1627, 1503, 1482 cm−1). The 1H-NMR and HSQC spectrum of 1 indicated the the presence of a tetrasubstituted aromatic ring [δH 6.73 (1H, s) and 7.15 (1H, s)], a dioxy-methylene presence of a tetrasubstituted aromatic ring [δH 6.73 (1H, s) and 7.15 (1H, s)], a dioxy-methylene [δH [δH 5.91 (1H, d, J = 1.2 Hz), 5.90 (1H, d, J = 1.2 Hz)], two olefins including a trisubstituted one [δH 6.59 5.91 (1H, d, J = 1.2 Hz), 5.90 (1H, d, J = 1.2 Hz)], two olefins including a trisubstituted one [δH 6.59 (1H, (1H, dd, J = 10.2, 2.1 Hz), 6.04 (1H, d, J = 10.2 Hz), 5.78 (1H, br s)], three methylene groups [δH 3.89 (1H, brdd, d, J J= =10.2, 15.5 2.1 Hz), Hz), 3.70 6.04 (1H, (1H, dd, d,J =J = 15.5, 10.2 3.0Hz) Hz),, 5.78 3.57 (1H, (1H, br dd,s)], threeJ = 14.1, methylene 4.8 Hz), 3.37–3.30groups [δ (1H,H 3.89 overlap), (1H, br d, J = 15.5 Hz), 3.70 (1H, dd, J = 15.5, 3.0 Hz), 3.57 (1H, dd, J = 14.1, 4.8 Hz), 3.37–3.30 (1H, overlap), 2.46 (1H, dd, J = 11.5, 5.5 Hz), 1.73 (1H, dd, J = 11.5, 10.4 Hz)], a methoxy group [δH 3.34 (3H, s)], and a 2.46 (1H, dd, J = 11.5, 5.5 Hz), 1.73 (1H, dd, J = 11.5, 10.4 Hz)], a methoxy group [δH 3.34 (3H, s)], and glucopyranosyl moiety [δH 4.61 (1H, d, J = 7.8 Hz), 3.92 (1H, dd, J = 11.8, 2.0 Hz), 3.73 (1H, dd, J = 11.8, 5.4a glucopyranosyl Hz), 3.42 (1H, t,moietyJ = 8.7 Hz),[δH 4.61 3.39–3.28 (1H, d, (2H, J = overlap),7.8 Hz), 3.92 3.23 ( (1H,1H, dd, J = 11.8, 9.2, 7.8 2.0 Hz)]. Hz), Its3.7313 C-NMR(1H, dd, and J = 13 DEPT11.8, 5.4 spectra Hz), 3.42 showed (1H, 24t, J carbon = 8.7 Hz), resonances, 3.39–3.28 including (2H, overlap), one methoxy, 3.23 (1H, five dd, methylenes, J = 9.2, 7.8 Hz) twelve]. Its methines,C-NMR and sixDEPT quaternary spectra showed carbons (including24 carbon resonances, one olefinic). including The 13C-NMR one methoxy, data of 1 fivewere methylenes, similar to those twelve of methines, and six quaternary carbons (including one olefinic). The 13C-NMR data of 1 were similar to erythraline (2), except for the additional signals for a glucopyranosyl group at δC 105.9 (C-1’), 78.2 (C-3’), those of erythraline (2), except for0 the additional signals for a glucopyranosyl group at δC 105.9 (C- 78.0 (C-5’), 75.4 (C-2’), 71.6 (C-4 ), and 62.8 (C-6’), as well as an oxygenated methine appearing at δC 1’), 78.2 (C-3’), 78.0 (C-5’), 75.4 (C-2’), 71.6 (C-4′), and 62.8 (C-6’), as well as an oxygenated methine 74.5 instead of a methylene carbon appearing at δC 25.3 (C-11) in that of 2. The data above suggested a glucoseappearing moiety at δC attached 74.5 instead through of a anmethylene oxygen to carbon C-11. Theappearing glucoside at δ mustC 25.3 have (C-11) a β inattachment, that of 2. The since data the above suggested a glucose0 moiety attached through an oxygen to C-11. The glucoside must have a β anomeric proton H-1 (δH 4.61) exhibits a diaxial coupling (J = 7.8 Hz) with H-2’ (δH 3.23). The HMBC andattachment,1H-1H COSY since the correlations anomeric (Figureproton2 H) confirmed-1′ (δH 4.61) thatexhibits1 was a diaxial a typical coupling Erythrinan (J = 7.8 alkaloid Hz) with having H-2’ 1 1 a(δ characteristicH 3.23). The HMBC spiro-carbon and H positioned- H COSY in correlations the center of (Figure ringsA, 2)B, confirmed and C with that two 1 olefinswas a of typical∆ 1,2 andErythrinan∆ 6,7 and alkaloid a dioxy-methylene having a characteristic group attached spiro-carbon to the positioned C-15 and C-16.in the Thecenter presence of rings of A, a B, glucose and C 1,2 6,7 moietywith two at C-11olefins was of further∆ and confirmed ∆ and bya d HMBCioxy-methylene correlation group observed attached from to the the anomeric C-15 and proton C-16. H-1’The presence of a glucose moiety at C-11 was further confirmed by HMBC correlation observed from the to C-11. The HMBC correlation from methoxy protons H-18 (δH 3.32) to C-3 (δC 77.4) indicated that C-3anomeric was substituted proton H-1 with’ to C a- methoxy11. The HMBC group. correlation The relative from configuration methoxy protons of 1 was H determined-18 (δH 3.32 by) to NOESY C-3 (δC experiment.77.4) indicated The that observed C-3 was cross-peaks substituted (Figure with 3a) methoxy between H-3/H-14,group. The H-4 relativβ, ande configuration correlations betweenof 1 was H-4determinedβ/H-11, by H-10 NOESYα implied experiment. that the The protons observed at C-3 cross and-peaks C-11 were(Figure placed 3) between at β- and H-α3/H-orientation,-14, H-4β, respectively.and correlations Therefore, between the H-4 structureβ/H-11, H of-101αwas implied established that the asproton erythraline-11s at C-3 andβ- OC--glucopyranoside.11 were placed at Biogeneticβ- and α-orientation, considerations respectively on Erythrina. Therefore,alkaloids the and structure the positive of 1 was optical established rotation as value erythraline suggested-11β that-O- 1glucopyranosidehas an S configuration. Biogenetic at C-5 considerations [6,14,17]. on Erythrina alkaloids and the positive optical rotation value suggested that 1 has an S configuration at C-5 [6,14,17].

1 1 FigureFigure 2. 2. 1H-H-1H COSY (−)) and and key key HMBC HMBC ( (→→)) correlations correlations of of Compound Compound 11..

Molecules 2017, 22, 1558 3 of 6

Molecules 2017, 22, 1558 3 of 7 Molecules 2017, 22, 1558 3 of 6

Figure 3. Key NOESY correlations of Compound 1. Figure 3. Key NOESY correlationscorrelations of Compound 1.. Alkaloids 1–7 showed potent inhibition against the replication of TMV at a concentration of 500 μg/mL, withAlkaloids inhibitory 1–7 showed rates potent varying inhibition from against 52.1% the to replicationreplicati79.7% on(Figure of TMV 4). at A a concentrationconcentrationcommercial of antiviral 500 μg/mL, with inhibitory rates varying from 52.1% to 79.7% (Figure 4). A commercial antiviral agent, ningnanmycin,500 µg/mL, with exhibited inhibitory 91.7% rates varying inhibition from 52.1%as tested to 79.7% under (Figure the4 same). A commercial condition. antiviral Furthermore, agent, ningnanmycin, exhibited 91.7% inhibition as tested under the same condition. Furthermore, the IC50 valuesthe IC50 of values alkaloids of alkaloids 1–7 were 1–7 were determined determined as as 0.59, 0.59, 1.52,1.52, 1.04, 1.04, 1.48, 1.48, 1.28, 1.28, 0.74, 0.74, and 1.69 and mM 1.69 using mM using the IC50 values of alkaloids 1–7 were determined as 0.59, 1.52, 1.04, 1.48, 1.28, 0.74, and 1.69 mM using the leaf-discthe leaf meth-discod, meth whileod, while the positive the positive control, control, ningnanmycin, ningnanmycin, possess possessed aned IC an50 ofIC 0.1850 of mM 0.18 during mM during the leaf-disc method, while the positive control, ningnanmycin, possessed an IC50 of 0.18 mM during a a test undertesta test underthe under same the the same condition. same condition. condition. The The The new new new alkaloid alkaloid alkaloid glycoside, glycoside, erythraline-11erythraline erythraline-11ββ---11βOO-glucopyranoside-glucopyranoside-O-glucopyranoside ( 1), (1), showed showeda noticeable a noticeable increase increase in thein the inhibition inhibition against against TMV TMV as as comparcompared compared wit withedh wit its h aglycone, its aglycone, erythraline (2). erythralineerythraline (2). (2). 100

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0 Figure 4. Inhibitory1 activity2 of the3 isolated4 alkaloids5 1–7 against6 the7 replicationNingnanmycin of TMV. Alkaloids 3. Materials and Methods Figure 4. Inhibitory activity of the isolated alkaloids 1–7 against the replication of TMV. Figure 4. Inhibitory activity of the isolated alkaloids 1–7 against the replication of TMV. 3.1. General Experimental Procedures 3. Materials and Methods 3. Materials andOptical Methods rotations were measured on a JASCO-1020 polarimeter (JASCO, Tokyo, Japan). UV 3.1.spectra General were Experimental measured Procedures using a Perkin Elmer Lambda 800 UV/VIS spectrometer (PerkinElmer, Waltham, MA, USA). IR spectra were obtained with a Thermo Nicolet Avatar 360 FT-IR spectrometer 3.1. General ExperimentalOptical rotations Procedures were measured on a JASCO-1020 polarimeter (JASCO, Tokyo, Japan). UV spectra (Thermo Nicolet Corporation, Madison, WI, USA). 1H- and 13C-NMR spectra were obtained with a were measured using a Perkin Elmer Lambda 800 UV/VIS spectrometer (PerkinElmer, Waltham, MA, Bruker AVANCE III 500 spectrometer (Bruker BioSpin, Rheinstetten, Germany) using OpticalUSA). rotations IR spectra were were measuredobtained with on a a Thermo JASCO Nicolet-1020 Avatar polarimeter 360 FT-IR (JASCO, spectrometer Tokyo, (Thermo Japan). UV tetramethylsilane as an internal standard. HRESIMS were obtained with a Thermo Scientific™ TSQ spectra wereNicolet measured Corporation, using Madison, a Perkin WI, USA). Elmer1H- and Lambda13C-NMR 800 spectra UV/VIS were spectrometer obtained with a( BrukerPerkinElmer, Quantum Access MAX triple stage quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, Waltham,AVANCE MA, USA III 500). IR spectrometer spectra were (Bruker obtained BioSpin, with Rheinstetten, a Thermo Germany) Nicolet using Avatar tetramethylsilane 360 FT-IR spectrometer as an USA). Sephadex LH–20 (25–100 μm, Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden), internal standard. HRESIMS were obtained with a Thermo1 Scientific13 ™ TSQ Quantum Access MAX (Thermo LichroprepNicolet Corp RP-18oration gel (40,– 63Madison, μm, Merck, WI Darmstade,, USA). H Germany),- and C Silica-NMR gel spectra(200–300 weremesh) obtainedand Silica with a triple stage quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA). Sephadex LH–20 Bruker gel AVANCE H (Qingdao III Oceanic 500 Chemical spectrometer Co., Qingdao, (Bruker China) BioSpin, were used Rheinstetten, for column chromatography. Germany) using tetramethylsilaneThin-layer aschromatography an internal standard.(TLC) was performed HRESIMS on wereglass-backed obtaine platesd with coated a Thermo with 0.25 mmScientific layers ™ TSQ Quantum Access MAX triple stage quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA). Sephadex LH–20 (25–100 μm, Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden), Lichroprep RP-18 gel (40–63 μm, Merck, Darmstade, Germany), Silica gel (200–300 mesh) and Silica gel H (Qingdao Oceanic Chemical Co., Qingdao, China) were used for column chromatography. Thin-layer chromatography (TLC) was performed on glass-backed plates coated with 0.25 mm layers

Molecules 2017, 22, 1558 4 of 7

(25–100 µm, Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden), Lichroprep RP-18 gel (40–63 µm, Merck, Darmstade, Germany), Silica gel (200–300 mesh) and Silica gel H (Qingdao Oceanic Chemical Co., Qingdao, China) were used for column chromatography. Thin-layer chromatography (TLC) was performed on glass-backed plates coated with 0.25 mm layers of Silica gel H (Qingdao Oceanic Chemical Co., Qingdao, China). Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 5% H2SO4 in EtOH. All solvents and chemicals used were of analytical reagent grade (Sinopharm Chemical Reagent Co., Ltd., Beijing, China), and water was doubly distilled before use.

3.2. Plant Materials The seeds of Erythrina crista-galli L. were collected in July 2016 at the Fujian Agriculture and Forestry University (Fuzhou, China), and identified by Professor Chun-Mei Huang. Herbarium specimens were deposited at the Key Laboratory of Bio-Pesticide and Chemistry-Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, China (Specimen number: EC16701S).

3.3. Extraction and Isolation The seeds of E. crista-galli L. (dried and powdered, 950 g) was extracted with MeOH (2 L, 3 d) at room temperature three times. The MeOH extracts (150 g) were dissolved in H2O and successively partitioned between petroleum ether, EtOAc, and n-BuOH. The EtOAc-soluble materials (7.8 g) were subjected to silica gel (200–300 mesh) column chromatography and eluted with acetone in petroleum ether (from 100:0 to 0:100) to obtained 13 fractions (Fractions 1–13). Fraction 6 (145 mg) and Fraction 7 (2.5 g) were purified with RP–18 gel column chromatography, eluting with MeOH in H2O (80%), and then subjected to silica gel (H, eluted with CHCl3/MeOH, 99:1) column chromatography, respectively, to yield Compound 2 (11.5 mg) and Compound 5 (93.8 mg). Fraction 9 (205.0 mg) was first separated with an ODS column chromatography, eluting with MeOH in H2O (70%), and then subjected to silica gel (H, eluted with CHCl3/MeOH, 98:2) column chromatography, to yield Compound 4 (35.8 mg). Fraction 11 (250 mg) was purified with RP–18 gel column chromatography, eluting with MeOH in H2O (60%), and then subjected to silica gel (H, eluted with CHCl3/MeOH, 95:5) column chromatography, to yield Compound 3 (28.1 mg). Fraction 12 (1.05 g) was purified with ODS column chromatography, eluting with MeOH in H2O (60%), and then subjected to silica gel (H, eluted with CHCl3/MeOH, 90:10) column chromatography, to yield Compound 1 (17.1 mg) and Compound 6 (23.1 mg). Fraction 13 (424 mg) was purified with ODS column chromatography, eluting with MeOH in H2O (40%) to yield Compound 7 (39.2 mg). 25 Erythraline-11β-O-glucopyranoside (1). White amorphous powder. [α]D + 130.6 (c 0.1 MeOH); −1 UV (MeOH) λmax (log ε): 205 (1.97), 226 (0.89), 287 (0.22); IR (KBr) υmax: 1627, 1503, 1482 cm ; 1 HR-ESI-MS m/z: 510.1537 (Calcd for C24H29ClNO9, 510.1536); H-NMR (500 MHz, CD3OD) and 13 C-NMR (125 MHz, CD3OD) data (see Table1).

1 13 Table 1. H- and C-NMR data of Compounds 1 and 2 (δ in ppm, CD3OD).

1 2 Position δC δH δC δH C-1 126.2 6.59 (1H, dd, J = 10.2, 2.1 Hz) 125.4 6.53 (1H, dd, J = 10.1, 2.3 Hz) C-2 132.7 6.04 (1H, d, J = 10.2 Hz) 131.6 5.97 (1H, d, J = 10.1 Hz) C-3 77.4 4.01 (1H, m) 76.2 3.96 (1H, m) 2.46 (1H, dd, J = 11.5, 5.5 Hz), 1.73 2.49 (1H, dd, J = 11.5, 5.5 Hz) C-4 41.2 42.0 (1H, dd, J = 11.5, 10.4 Hz) 1.83 (1H, dd, J = 11.5, 10.4 Hz) C-5 67.8  67.5  C-6 143.7  142.4  C-7 124.5 5.78 (1H, br s) 123.1 5.72 (1H, br s, 1H) 3.89 (1H, br d, J = 15.5 Hz) 3.72 (1H, dd, J = 14.5, 3.0 Hz) C-8 59.3 57.7 3.70 (1H, dd, J = 15.5, 3.0 Hz) 3.53–3.45 (1H, overlap) Molecules 2017, 22, 1558 5 of 7

Table 1. Cont.

1 2 Position δC δH δC δH 3.57 (1H, dd, J = 14.1, 4.8 Hz) 3.53–3.45 (1H, overlap) C-10 50.4 44.6 3.37–3.30 (1H, overlap) 2.92–2.83 (1H, overlap) 2.92–2.83 (1H, overlap) C-11 74.5 4.70 (1H, t, J = 4.3 Hz) 25.3 2.71–2.64 (1H, m) C-12 129.1  128.1  C-13 132.0  132.7  C-14 106.3 6.73 (1H, s) 106.3 6.62 (1H, s) C-15 148.9  146.2  C-16 148.1  145.9  C-17 109.7 7.15 (1H, s) 108.8 6.76 (1H, s) C-18 56.6 3.34 (3H, s) 56.1 3.32 (3H, s) 5.91 (1H, d, J = 1.2 Hz) 5.90 (1H, d, J = 1.5 Hz) C-19 102.3 100.8 5.90 (1H, d, J = 1.2 Hz) 5.87 (1H, d, J = 1.5 Hz) 0 C-1 105.9 4.61 (1H, d, J = 7.8 Hz)   0 C-2 75.4 3.23 (1H, dd, J = 9.2, 7.8 Hz)   0 C-3 78.2 3.42 (1H, t, J = 8.8 Hz)   0 C-4 71.6 3.39–3.28 (1H, overlap)   0 C-5 78.0 3.39–3.28 (1H, overlap)   3.92 (1H, dd, J = 11.8, 2.0 Hz) C-60 62.8 3.73 (1H, dd, J = 11.8, 5.4 Hz)  

3.4. Anti-TMV Assay

3.4.1. Virus and Host Plant Purified TMV (strain U1) was obtained from Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China, whose concentration was determined as 15 mg/mL using an 0.1%, 260 nm ultraviolet spectrophotometer method (virus concentration = (A260 × dilution ration) /E1 cm ). The purified virus was kept at −20 ◦C and was diluted to 30 µg/mL with 0.01 M PBS before use. Nicotiana tabacum cv. K326, which were cultivated and grown to a 5–6-leaf stage in an insect-free greenhouse, were used as an anti-TMV assay, as a systemic TMV infection host. Purified compounds were dissolved in DMSO and diluted with 0.01 M PBS to a certain concentration for test. The final concentration of DMSO in the test solution (≤2%) showed no adverse effect on the plants.

3.4.2. Leaf-Disc Method Growing leaves of N. tabacum cv. K326 were mechanically inoculated with TMV (30 µg/mL in 0.01 M PBS). After 6 h, leaf discs (1 cm diameter) were punched and floated on solutions for test. Discs of healthy and inoculated leaves floated on a solution of 0.01 M PBS with 2% DMSO were used as a mock and negative control, respectively. Three replicates were carried out for each sample. After incubating for 48 h at 25 ◦C in a culture chamber, the leaf discs were grounded in 0.01 M carbonate coating buffer (pH 9.6), and OD405 values were measured using TAS-ELISA method. TAS-ELISA was performed as described in the literature [18,19]. Virus concentration was calculated from a standard curve constructed using OD405 values of purified TMV at concentrations of 1.0, 0.5, 0.25, 0.125, and 0.0625 µg/mL. The inhibition of test solutions on TMV was calculated as follows: inhibition rate = [1 − (virus concentration of treatment) / (virus concentration of negative control)] × 100%.

4. Conclusions A new Erythrina alkaloid glycoside together with five known Erythrina alkaloids and an indole alkaloid have been isolated from the MeOH extract of the seeds of Erythrina Crista-galli L. All the isolated alkaloids showed noticeable inhibition against the replication of TMV but in a relatively higher concentration as compared with that of the positive control agent. Further investigations are needed to reveal the biological action mechanism of Erythrina alkaloids. Molecules 2017, 22, 1558 6 of 7

Supplementary Materials: The following are available online. The 1H- and 13C-NMR of all the isolated compounds, and the IR, UV/Vis, HRESIMS, DEPT, HSQC, HMBC, and NOESY of Compound 1. Acknowledgments: This work was supported by the National Natural Science Foundation of China (grant numbers 31371987 and 31501687). Author Contributions: Qing-Wei Tan and Qi-Jian Chen conceived and designed the experiments. Jian-Cheng Ni and Pei-Hua Fang performed the experiments and test. Qing-Wei Tan analyzed the data and composed the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References

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Sample Availability: Samples of the compounds are available from the authors.

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