(Havil.) Merr. with Cytotoxic Activity on Hepatocellular Carcinoma Cells

molecules

Article Three New Iridoid Derivatives Have Been Isolated from the Stems of Neonauclea reticulata (Havil.) Merr. with Cytotoxic Activity on Hepatocellular Carcinoma Cells

Fang-Pin Chang 1, Wei Chao 2, Sheng-Yang Wang 3,4, Hui-Chi Huang 5 , Ping-Jyun Sung 6,7 , Jih-Jung Chen 8,† , Ming-Jen Cheng 9,†, Guan-Jhong Huang 5,* and Yueh-Hsiung Kuo 1,5,10,11,12,*

1 The Ph.D Program for Cancer Biology and Drug Discovery, China Medical University and Academia Sinica, Taichung 404, Taiwan; [email protected] 2 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 250, Taiwan; [email protected] 3 Department of Forestry, National Chung Hsing University, Taichung 402, Taiwan; taiwanﬁ[email protected] 4 Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan 5 Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan; [email protected] 6 National Museum of Marine Biology and Aquarium, Pingtung 912, Taiwan; [email protected] 7 Graduate Institute of Marine Biology, National Dong Hwa University, Hualien 97401, Taiwan 8 Faculty of Pharmacy, School of Pharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan; [email protected] 9 Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsinchu 300, Taiwan; [email protected] 10 Department of Biotechnology, Asia University, Taichung 413, Taiwan 11 Chinese Medicine Research Center, China Medical University, Taichung 404, Taiwan 12 Research Center for Chinese Herbal Medicine, China Medical University, Taichung 404, Taiwan * Correspondence: [email protected] (G.-J.H.); [email protected] (Y.-H.K.); Tel.: +886-4-2205-3366 (ext. 5508) (G.-J.H.); +886-4-2205-3366 (ext. 5701) (Y.-H.K.); Fax: +886-4-2207-1693 (Y.-H.K.) † These authors contributed equally to this work

Received: 8 August 2018; Accepted: 6 September 2018; Published: 8 September 2018

Abstract: Three new iridoids, namely neonanin A (1), neonanin B (2) and neoretinin A (3), as well as twelve known compounds, 6-hydroxy-7-methyl-1-oxo-4-carbomethoxyoctahydrocyclopenta[c]pyran (4), 4-epi-alyxialactone (5), loganetin (6), loganin (7), phenylcoumaran-α0-aldehyde (8), cleomiscosin A (9), ﬁcusal (10), balanophonin (11), vanillic acid (12), p-coumaric acid (13), cis,trans-abscisic acid (14), and trans,trans-abscisic acid (15) were isolated from the stems of Neonauclea reticulata (Havil.) Merr. These new structures were determined by the detailed analysis of spectroscopic data and comparison with the data of known analogues. Compounds 1–13 were evaluated using an in-vitro MTT cytotoxic assay for hepatocellular carcinoma (HCC) cells, and the preliminary results showed that ﬁcusal (10), balanophonin (11), and p-coumaric acid (13) exhibited moderate cytotoxic activity, with EC50 values of 85.36 ± 4.36, 92.63 ± 1.41, and 29.18 ± 3.48 µg/mL against Hep3B cells, respectively.

Keywords: Neonauclea reticulate; iridoid; neonanin; Hep3B

Molecules 2018, 23, 2297; doi:10.3390/molecules23092297 www.mdpi.com/journal/molecules Molecules 2018, 23, 2297 2 of 10

1. Introduction Neonauclea reticulate (Havil.) Merr. is a large evergreen tree, which is distributed over the Philippines and Taiwan. Of the forty species of the genus Neonauclea (Rubiaceae), this is the only species that can be found in Taiwan, located in the forests at low elevations of southern Taiwan, such as the Kaohsiung area, as well as Pingtung Mountain or Orchid Island [1]. On Orchid Island, when the Tao people celebrate the flying fish festival, this tree is an important folk plant for building the Tribe’s chinurikuran. Alkaloids [2,3], anthraquinones [4], iridoids [5], triterpenes [6], and saponins [7] have been isolated from the plants of this genus in previous chemical investigations. Anti-bacterial [8], anti-malarial [9], and anti-topoisomerase II effects [4] were shown in previous pharmacological studies. To the best of our knowledge, there have been no studies published that have investigated the chemical structure of N. reticulate. Furthermore, the only pharmacological study published mentions that the leaves of this plant could protect human skin fibroblast cells against the effects of ultraviolet B (UVB) irradiation [10]. Further investigations of the chemical and pharmacological properties of N. reticulate are urgently needed. A global estimation report has proposed that hepatocellular carcinoma (HCC) will be one of the leading causes of cancer-associated deaths in 2018 [11]. In Taiwan, HCC is the second most common cancer in males and fourth most common cancer in females, according to the cancer registry annual report for the year 2015 [12]. HepG2 and Hep3B cell lines are the well-known in vitro cytotoxicity assay models, and they are the most well-characterized liver cancer cell lines. These two cell lines are very similar, except that HepG2 is hepatitis B virus-negative and non-tumorigenic, while Hep3B is hepatitis B virus-positive and tumorigenic. These two cell lines showed different chemo-sensitivity in cytotoxicity, gene expression induction, and cell cycle response and biochemical effects, which may provide investigators further instruction for identifying the mechanism [13]. However, there are only a few medication choices for HCC, including molecularly targeted therapy, such as sorafenib; immunotherapy, such as nivolumab; and cytotoxic chemotherapy, such as doxorubicin [14]. Finding possible compounds for HCC treatment demands immediate attention. In previous studies, natural resources for the treatment of hepatocellular carcinoma have been found in the members of Rubiaceae family, such as Oldenlandia diffusa [15] and Paederia scandens [16]. The phytochemical studies of N. reticulate have not yet been performed. Thus, the aim of this study is to investigate the compounds from the stems of N. reticulate and its preliminary in-vitro cytotoxicity analysis against hepatocellular carcinoma cells.

2. Results and Discussions

2.1. Isolation and Structural Elucidation In this study, we used the human hepatocellular carcinoma cell lines of HepG2 and Hep3B to evaluate the cytotoxicity of the MeOH extract. The results show that the MeOH extract exhibits a dose-dependent cytotoxicity against the Hep3B cells (effective concentration dose; EC50 = 912.98 ± 3.95 µg/mL). In contrast, there was no significant cytotoxicity in Hep G2 cells. These biological assays suggest that the extract of N. reticulate might inhibit the growth of the Hep3B cell line. Therefore, the extract was suspended in water and partitioned with ethyl acetate (EtOAc). After being suspended in water, this extract was successively partitioned with butanol, with each step being repeated three times. Three different fractions were used to evaluate the cytotoxicity in Hep3B cells. Among them, only the EtOAc fraction revealed a significant inhibitory effect (EC50 = 591.13 ± 4.99 µg/mL). The other fractions (BuOH and H2O) showed no significant cytotoxicity in Hep3B cells. The investigation of the active fraction was isolated by silica gel column chromatography and normal-phase, semi-preparative, high-performance liquid chromatography (HPLC) to obtain fifteen compounds. Among these compounds, we isolated three new iridoids, which are namely neonanin A (1), neonanin B (2) and neoretinin A (3), and twelve known compounds, including 6-hydroxy-7-methyl-1-oxo-4-carbomethoxyoctahydrocyclopenta[c]pyran (4) Molecules 2018, 23, 2297 3 of 10

4-epi-alyxialactone (5), loganetin, loganin (7), phenylcoumaran-α0-aldehyde (8), cleomiscosin A (9), p cis trans ﬁcusalMolecules (10 2018), balanophonin, 23, x FOR PEER REVIEW (11), vanillic acid (12), -coumaric acid (13), , -abscisic acid 3 of 10 (14 ), and trans,trans-abscisic acid (15). The structures of the new compounds were determined through spectral(1D) and analyses, two-dimensional including (2D)-NMR, IR, UV, one-dimensional as well as HR-ESI-MS (1D) and data. two-dimensional The known compounds (2D)-NMR, identified as well as HR-ESI-MSwere compared data. with The the knownpublished compounds NMR spectral identiﬁed data. All were structures compared are shown with thein Figure published 1. In this NMR spectralstudy, data. we described All structures the detailed are shown structural in Figure elucidations1. In this study, of new we compounds described theand detailed the activities structural of elucidationscompounds of 1 new–13. compounds and the activities of compounds 1–13.

Figure 1. The chemical structures of compounds 1–15 from Neonauclea reticulata (Havil.) Merr. Figure 1. The chemical structures of compounds 1–15 from Neonauclea reticulata (Havil.) Merr. Compound 1 was obtained as a colorless oil. The molecular weight was determined by HR-ESI-MS, Compound 1 was obtained+ as a colorless oil. The molecular weight was determined by HR-ESI- which showed an [M + Na] ion at an m/z of 267.0845 (calculated for C11H16O6Na 267.0839), indicating MS, which showed an [M + Na]+ ion at an m/z of 267.0845 (calculated for C11H16O6Na 267.0839), four degrees of unsaturation. The IR spectrum displayed the presence of hydroxyl (3491 cm−1) and indicating four degrees of unsaturation. The IR spectrum displayed the presence of hydroxyl (3491 −1 1 carbonylcm−1) and (1728 carbonyl cm )(1728 functionalities. cm−1) functionalities. The H spectraThe 1H ofspectra compound of compound1 (Table 1 1(Table) showed 1) showed a doublet a methyl group δ 1.23 (d, J = 7.1, H-10); one pair of geminal coupling methylene groups at δ 1.53 doublet methylH group δH 1.23 (d, J = 7.1, H-10); one pair of geminal coupling methylene groups atH δH α δ β δ δ (m,1.53 H-6 (m,) andH-6α)H and2.01 δH(m, 2.01 H-6 (m, H-6β);); two two methine methine protons protons of ofH δH2.88 2.88(dd, (dd, JJ = 9.9, 9.9, 7.1, 7.1, H-9) H-9) and and δH 3.31H 3.31 (ddd,(ddd,J = J 9.9,= 9.9, 8.0, 8.0, H-5); H-5); a a singlet singletcarbomethoxy carbomethoxy group at at δδHH 3.813.81 (s, (s, COOMe); COOMe); one one pair pair of of mutual mutual geminallygeminally coupled coupled oxymethylene oxymethylene signals signals atatδ δHH 3.87 (d, J = 12.0, 12.0, H-3α) H-3α )and and δHδ H3.973.97 (d, (d, J =J 12.0,= 12.0, H-3β); H-3 β); 13 andand an an oxygenated oxygenated methine methine proton protonδ δHH4.22 4.22 (m,(m, H-7).H-7). Eleven carbons carbons were were assessed assessed using using 13C-NMRC-NMR (Table(Table1), and1), and the the heteronuclear heteronuclear single single quantum quantum coherencecoherence spectroscopyspectroscopy (HSQC) (HSQC) spectra spectra revealed revealed thethe presence presence of of methyls methyls δδCC 14.114.1 (C-10) (C-10) and and δC δ52.8C 52.8 (OMe); (OMe); methylenes methylenes δC 37.8δ C(C-6)37.8 and (C-6) δC 67.5 and (C-3);δC 67.5 (C-3);methines methines δC 43.9δC (C-5),43.9 (C-5),δC 44.4 δ(C-8),C 44.4 and (C-8), δC 50.6 and (C-9);δC 50.6oxygenated (C-9); oxygenatedmethine δC 76.9 methine (C-7); oxygenatedδC 76.9 (C-7); quaternary carbon δC 88.4 (C-4); and carbonyl groups δC 169.1 (C-11) and δC 178.5 (C-1). The oxygenated quaternary carbon δC 88.4 (C-4); and carbonyl groups δC 169.1 (C-11) and δC 178.5 (C-1). correlation spectroscopy (COSY) correlations between H-5 (δH 3.31)/H-6 (δH 1.53 and δH 2.01) and H- The correlation spectroscopy (COSY) correlations between H-5 (δH 3.31)/H-6 (δH 1.53 and δH 2.01) 9 (δH 2.88), H-6 (δH 1.53 and δH 2.01)/H-7 (δH 4.22), H-7 (δH 4.22)/H-8 (δH 2.29), H-8 (δH 2.29)/H-9 (δH and H-9 (δH 2.88), H-6 (δH 1.53 and δH 2.01)/H-7 (δH 4.22), H-7 (δH 4.22)/H-8 (δH 2.29), H-8 (δH 2.88), and H-10 (δH 1.23) suggests that there was a cyclopentanyl moiety with a methyl group and a 2.29)/H-9 (δH 2.88), and H-10 (δH 1.23) suggests that there was a cyclopentanyl moiety with a methyl hydroxyl group in the structure. We found that there were heteronuclear multiple bond coherence group and a hydroxyl group in the structure. We found that there were heteronuclear multiple bond (HMBC) correlations between H-3 (δH 3.87 and δH 3.97)/C-4 (δC 88.4) and C-11 (δC 169.1); H-5 (δC coherence (HMBC) correlations between H-3 (δH 3.87 and δH 3.97)/C-4 (δC 88.4) and C-11 (δC 169.1); 3.31)/C-1 (δC 178.5), C-3 (δC 67.5), and C-6 (δC 37.8); H-9 (δC 2.88)/C-1 (δC 169.1), C-6 (δC 37.8), and C- H-5 (δC 3.31)/C-1 (δC 178.5), C-3 (δC 67.5), and C-6 (δC 37.8); H-9 (δC 2.88)/C-1 (δC 169.1), C-6 (δC 10 (δC 14.1); and OMe (δH 3.81)/C-11 (δC 169.1). These correlations indicated that there was another 37.8),pyranyl and C-10moiety (δC with14.1); a andlactone OMe and (δ Ha carbomethoxy3.81)/C-11 (δ Cgroup,169.1). which These included correlations the COSY indicated and thatHMBC there wasinformation. another pyranyl This indicates moiety that with a cyclopentan a lactone and and a a carbomethoxy pyranyl moiety group, were connected which included to each other the COSY in andcompound HMBC information. 1. This spectra This information indicates for that compound a cyclopentan 1 was andvery asimilar pyranyl to those moiety of werethe compound connected 4 to each[17 other]. Thus, in we compound suggested1. thatThis compound spectra information 1 has an iridoid for compoundtype structure.1 was Thevery difference similar between to those these of the two compounds was in the C-4 position. The C-4 position of compound 4 was a tertiary carbon (δC 46.1), while in compound 1 a quaternary carbon with an oxygen atom (δC 88.4) were connected in the

Molecules 2018, 23, 2297 4 of 10 compound 4 [17]. Thus, we suggested that compound 1 has an iridoid type structure. The difference between these two compounds was in the C-4 position. The C-4 position of compound 4 was a tertiary carbon (δC 46.1), while in compound 1 a quaternary carbon with an oxygen atom (δC 88.4) were connected in the C-4 position. It is difficult to elucidate the relative configuration of C-4 in compound 1 using the present spectra data. According to the nuclear overhauser effect spectroscopy (NOESY) spectra, H-6α/H-7/H-8 were in the α-configuration, while H-5, H-6β, and H3-10 were all in the β form. By consolidating the above-mentioned results and comparing them to the literature, compound 1 was assigned to be neonanin A.

Table 1. NMR data (CDCl3) of compound 1–3 in ppm, with J in Hz.

Compound 1 Compound 2 Compound 3

Position δH δC δH δC Position δH δC 1 178.5 173.9 1 176.7 2 2 3α 3.87 (d, J = 12.0) 67.5 5.42 (d, J = 2.4) 101.9 3 100.3 3β 3.97 (d, J = 12.0) 4 3.47 (q, J = 8.5) 44.8 4 88.4 2.55 (dd, J = 11.4, 2.4) 49.8 5α 2.25 (m) 34.6 5 3.31 (ddd, J = 9.9, 8.0) 43.9 3.28 (m) 31.9 5β 1.88 (m) 6α 1.53 (m) 37.8 1.38 (m) 42.1 6 4.29 (m) 76.0 6β 2.01 (m) 2.34 (m) 7 2.36 (m) 44.3 7 4.22 (m) 76.9 4.12 (m) 74.8 8 2.99 (t, J = 8.5) 51.6 8 2.29 (m) 44.4 2.27 (m) 43.1 9 1.23 (d, J = 7.0) 13.6 9 2.88 (dd, J = 9.9, 7.1) 50.6 2.79 (dd, J = 11.4, 9.1) 46.0 10 170.1 10 1.23 (d, J = 7.1) 14.1 1.28 (d, J = 6.9) 14.3 COOMe 3.88 (s) 54.1 11 169.1 169.8 COOMe 3.81 (s) 52.8 3.51 (s) 57.1 3-OMe 3.76 (s) 52.4

Compound 2 was obtained as a white needle with an MP of approximately 82–84 ◦C. The molecular weight was determined by HR-ESI-MS, which showed an [M + Na]+ ion at an m/z of 281.0999 (calculated for C12H18O6Na 281.0996), indicating four degrees of unsaturation. The IR spectrum displayed the presence of hydroxyl (3392 cm−1) and ester carbonyl (1730 cm−1) functionalities. The 1H and 13C-NMR spectra of compound 2 (Table1) showed that there is a doublet methyl group δH 1.28 (d, J = 6.9, H-10); one pair of geminal coupled methylene groups—δH 1.38 (m, H-6α) and δH 2.34 (m, H-6β); two methine protons, δH 2.79 (dd, J = 11.4, 9.1, H-9) and δH 3.28 (m, H-5); two methoxy groups, δH 3.51 (s, OMe) and δH 3.76 (s, COOMe); an oxygenated methine proton δH 4.12 13 (m, H-7); and an acetal proton δH 5.42 (d, J = 2.4, H-3). There were twelve carbons found in C-NMR after being combined with distortionless enhancement by polarization transfer (DEPT). There were two ester carbonyl groups: δC 173.9 (C-1) and δC 169.8 (C-11). The COSY spectra found correlations between H-5 (δH 3.28)/H-6 (δH 1.38 and δH 2.34) and H-9 (δH 2.79); H-6 (δH 1.38 and δH 2.34)/H-7 (δH 4.12); H-7 (δH 4.12)/H-8 (δH 2.27); and H-8 (δH 2.27)/H-9 (δH 2.79) and H-10 (δH 1.28). The HMBC spectrum found correlations between H-3 (δH 5.42)/C-1 (δC 173.9), C-4 (δC 49.8), and C-11 (δC 169.8); H-5 (δH 2.79)/C-1 (δC 173.9), C-3 (δC 101.9), and C-6 (δC 42.1); and H-9 (δH 2.79)/C-1 (δC 173.9), C-6 (δC 42.1), and C-10 (δC 1.28). The correlations found in HMBC spectra indicate the presence of a methoxy group (δH 3.51) in the C-3 position. These spectra and corrections were very similar to compound 4, which indicates that compound 2 is also an iridoid type compound, except for the addition of a methoxy group located in the C-3 position. H-4 exhibited the two coupling constants, with J = 11.4 at 2.4 Hz. The coupling constant of H-4 showed a coupling constant of J = 2.4 Hz. Thus, the coupling constant with J = 11.4 Hz represents the coupling between H-4 and H-5. The evidence concluded that H-4 is located in the α-axial conﬁguration. The results provided support for H-3 being in the α-equatorial orientation. The NOESY spectrum (Figure2) provides further evidence for two protons, which was presented by two α-conﬁguration. The compound 2 was assigned to be neonanin B. MoleculesMolecules 20182018,, 2323,, xx FORFOR PEERPEER REVIEWREVIEW 4 4 ofof 1010 Molecules 2018, 23, x FOR PEER REVIEW 4 of 10 C-4C-4 position.position. ItIt isis difficultdifficult toto elucidateelucidate thethe relativerelative configurationconfiguration ofof C-4C-4 inin compoundcompound 11 usingusing thethe C-4 position. It is difficult to elucidate the relative configuration of C-4 in compound 1 using the presentpresent spectraspectra data.data. AccordingAccording toto thethe nuclearnuclear overhauseroverhauser effecteffect spectroscopyspectroscopy (NOESY)(NOESY) spectra,spectra, H-H- present spectra data. According to the nuclear overhauser effect spectroscopy (NOESY) spectra, H- 6α/H-7/H-86α/H-7/H-8 were were in in the the α-configuration, α-configuration, whilewhile H-5, H-5, H-6β, H-6β, and and H H33-10-10 werewere allall inin thethe ββ form.form. ByBy 6α/H-7/H-8 were in the α-configuration, while H-5, H-6β, and H3-10 were all in the β form. By consolidatingconsolidating thethe above-mentionedabove-mentioned resultsresults andand comparingcomparing themthem toto thethe literature,literature, compoundcompound 11 waswas consolidating the above-mentioned results and comparing them to the literature, compound 1 was Moleculesassignedassigned2018, 23 , toto 2297 bebe neonaninneonanin A.A. 5 of 10 assigned to be neonanin A.

FigureFigureFigure 2. (a )2.2. Signiﬁcant ((aa)) SignificantSignificant correlation correlationcorrelation spectroscopy spectroscopyspectroscopy (COSY) (COSY) (bold(bold (bold line)line) line) andand and heteronuclearheteronuclear heteronuclear multiplemultiple multiple bondbond Figure 2. (a) Significant correlation spectroscopy (COSY) (bold line) and heteronuclear multiple bond bond coherencecoherence (HMBC) (HMBC) ( ( ))) correlations correlations correlations for for for compounds compounds compounds 11––33 ( (bb).). SigniﬁcantSignificant Significant NOESY NOESY ( ( ( )) coherence (HMBC) ( ) correlations for compounds 1–3 (b). Significant NOESY ( ) correlationscorrelationscorrelations of compounds ofof compoundscompounds1–3. 11––33.. correlations of compounds 1–3. CompoundCompoundCompound3 was 22 obtained was was obtained obtained as a colorless as as a a white white oil. The needle needle molecular with with an an weight MP MP of of was approximately approximately determined by 82–84 82–84 HR-ESI-MS, °C. °C. The The Compound 2 was obtained as a white needle with an MP of approximately 82–84 °C. The whichmolecularmolecular showed an weight weight [M + wasNa] was + determined determinedion at m/z of by by 253.0685 HR-ESI-MS, HR-ESI-MS, (calculated which which showed showed for C H an an O [M [MNa + + Na] Na] 253.0683),++ ion ion at at indicating an an m/zm/z of of 10 14 6 + molecular weight was determined by HR-ESI-MS, which showed an [M + Na] ion at an m/z− of four degrees281.0999281.0999 of (calculated (calculated unsaturation. for for C C The1212HH1818 IROO66 spectrumNaNa 281.0996), 281.0996), displays indicating indicating the presence four four degrees degrees of hydroxyl of of unsaturation. unsaturation. (3361 cm The The1) and IR IR 281.0999 (calculated for C12H18O6Na 281.0996), indicating four degrees of unsaturation. The IR lactonespectrumspectrum carbonyl displayed displayed (1741 cm − the the1) functionalities. presence presence of of hydroxyl hydroxyl The 1H and (3392 (339213C-NMR cm cm−1−1)) and and spectra ester ester of compound carbonyl carbonyl (1730 (17303 (Table cm cm−11−1)) spectrum displayed the presence of hydroxyl (3392 cm−1) and ester carbonyl (1730 cm−1) functionalities.functionalities. TheThe 11HH andand 1313C-NMRC-NMR spectraspectraδ ofof compoundcompound 22 (Table(Table 1)1) showedshowed thatthat therethere isis aa doubletdoubletδ showsfunctionalities. that there isThe a doublet1H and 13 methylC-NMR groupspectra ofH 1.23compound (d, J = 2 7.0, (Table H-9); 1) showed two methylene that there protons is a doubletH 2.25 methylmethylα groupgroupδ δδHH 1.281.28 (d,(d, JJ =β= 6.9,6.9, H-10);H-10); oneone pairpair ofof geminalgeminalδ coupledcoupled methylenemethylene groups—δgroups—δHH 1.381.38 (m,(m, (m,methyl H-5 ) group and HδH1.88 1.28 (m,(d, J H-5 = 6.9,); H-10); one carbomethoxy one pair of geminal group coupledH 3.88 methylene (s, COOMe); groups—δ a methineH 1.38 proton(m, H-6α)H-6α) andand δδHH 2.342.34 (m,(m, H-6β);H-6β); twotwo methinemethine protons,protons, δδHH 2.792.79 (dd,(dd, JJ == 11.4,11.4, 9.1,9.1, H-9)H-9) andand δδHH 3.283.2813 (m,(m, H-H- δH H-6α)2.99 (t, andJ = δ 8.5,H 2.34 H-8); (m, H-6β); and a hydroxyltwo methine group protons,δH 4.92 δH 2.79 (brs). (dd, Ten J = carbons11.4, 9.1, wereH-9) and shown δH 3.28 in (m,C-NMR H- 5);5); twotwo methoxymethoxy groups,groups, δδHH 3.513.51 (s,(s, OMe)OMe) andand δδHH 3.763.76 (s,(s, COOMe);COOMe); anan oxygenatedoxygenated methinemethine protonproton δδHH after5); beingtwo methoxy combined groups, with δH DEPT. 3.51 (s, There OMe)was and aδH quaternary 3.76 (s, COOMe); carbon anδ oxygenatedC 100.3 (C-3) methine and two proton carbonyl δH 4.124.12 (m,(m, H-7);H-7); andand anan acetalacetal protonproton δδHH 5.425.42 (d,(d, JJ == 2.4,2.4, H-3).H-3). ThereThere werewere twelvetwelve carbonscarbons foundfound inin 1313C-C- groups4.12 (m,δ H-7);176.7 and (C-1) an andacetalδ proton170.1 δ (C-10).H 5.42 (d, The J = 2.4, COSY H-3). spectra There were showed twelve correlations carbons found between in 13C- H-4 NMRNMRC afterafter beingbeing combinedcombinedC withwith distortionlessdistortionless enhancementenhancement byby polarizationpolarization transfertransfer (DEPT).(DEPT). ThereThere (δHNMR3.47)/H-5 after being (δH 1.88combined and 2.25) with anddistortionless H-8 (δH 2.99);enhancement H-5 (δH by1.88 polarization and 2.25)/H-6 transfer (δ (DEPT).H 4.29); There H-6 ( δH werewere two two ester ester carbonyl carbonyl groups: groups: δ δCC 173.9 173.9 (C-1) (C-1) and and δ δCC 169.8 169.8 (C-11). (C-11). The The COSY COSY spectra spectra found found 4.29)/H-7were two (δ ester2.36); carbonyl and H-7 groups: (δ 2.36)/H-8 δC 173.9 (C-1)(δ 2.99) and and δC 169.8 H-9 ( (C-11).δ 1.23). The This COSY indicates spectra that found there correlationscorrelationsH betweenbetween H-5H-5 (δ(δHH 3.28)/H-63.28)/H-6 (δ(δHH 1.381.38H andand δδHH 2.34)2.34) andandH H-9H-9 (δ(δHH 2.79);2.79); H-6H-6 (δ(δHH 1.381.38 andand δδHH wascorrelations also a cyclopentane between H-5 ring (δH in 3.28)/H-6 the structure (δH 1.38 of and compound δH 2.34) and3. The H-9 key (δH HMBC2.79); H-6 spectra (δH 1.38 correlations and δH 2.34)/H-72.34)/H-7 (δ(δHH 4.12);4.12); H-7H-7 (δ(δHH 4.12)/H-84.12)/H-8 (δ(δHH 2.27);2.27); andand H-8H-8 (δ(δHH 2.27)/H-92.27)/H-9 (δ(δHH 2.79)2.79) andand H-10H-10 (δ(δHH 1.28).1.28). TheThe 2.34)/H-7 (δH 4.12);δ H-7 (δH 4.12)/H-8δ (δH 2.27); andδ H-8 (δH 2.27)/H-9 (δH 2.79)δ and H-10 (δαHδ 1.28). The were betweenHMBCHMBC spectrumspectrum H-4 ( H foundfound3.47)/C-1 correlationscorrelations ( C 176.7), betweenbetween C-3 H-3 (H-3C 100.3),(δ(δHH 5.42)/C-15.42)/C-1 and C-10 (δ(δCC 173.9),173.9), ( C 170.1); C-4C-4 (δ(δC H-5C 49.8),49.8),( andHand2.25)/C-3 C-11C-11 (δ(δCC HMBC spectrum found correlations between H-3 (δH 5.42)/C-1 (δC 173.9), C-4 (δC 49.8), and C-11 (δC (δC 100.3);169.8);169.8); and H-5H-5 H-8 (δ(δHH 2.79)/C-1 (2.79)/C-1δH 2.99)/C-1 (δ(δCC 173.9),173.9), (δC 176.7). C-3C-3 (δ(δCC A101.9),101.9), possible andand C-6C-6 biosynthetic (δ(δCC 42.1);42.1); andand pathway H-9H-9 (δ(δH ofH 2.79)/C-12.79)/C-1 compound (δ(δCC 173.9),173.9),3 was 169.8); H-5 (δH 2.79)/C-1 (δC 173.9), C-3 (δC 101.9), and C-6 (δC 42.1); and H-9 (δH 2.79)/C-1 (δC 173.9), proposedC-6C-6 (δ(δ asCC 42.1), illustrated42.1), andand C-10C-10 in Figure (δ(δCC 1.28).1.28).3. Loganetin TheThe correlationscorrelations ( 6) was foundfound oxidized inin HMBCHMBC by spectra aspectra dioxygenase indicateindicate enzymethethe presencepresence to yield ofof aa C-6 (δC 42.1), and C-10 (δC 1.28). The correlations found in HMBC spectra indicate the presence of a dioxetanemethoxymethoxy (compound group group (δ (δ16HH ), 3.51)3.51) before inin beingthethe C-3C-3 spontaneously position.position. TheseThese cleaved spectra spectra to andyield and correctionscorrections the intermediate werewere veryvery compound similarsimilar 17 to to . methoxy group (δH 3.51) in the C-3 position. These spectra and corrections were very similar to After hydrolysis,compoundcompound 4 the4,, whichwhich formulate indicatesindicates was thatthat abandoned compoundcompound and 22 formed isis alsoalso compoundanan iridoidiridoid typetype18, which compound,compound, condensed exceptexcept to forfor obtain thethe compound 4, which indicates that compound 2 is also an iridoid type compound, except for the diacetaladditionaddition (compound ofof aa methoxymethoxy19). Subsequently, groupgroup locatedlocated compound inin thethe C-3C-3 position.position.19 was partially H-4H-4 exhibitedexhibited oxidized thethe twotwo to obtain couplingcoupling compound constants,constants,3 . addition of a methoxy group located in the C-3 position. H-4 exhibited the two coupling constants, Fromwith thewith literature JJ == 11.411.4 atat survey, 2.42.4 Hz.Hz. The noThe report couplingcoupling has constantconstant mentioned ofof H-4H-4 this showedshowed type of aa structure,couplingcoupling constantconstant so we suggested ofof JJ == 2.42.4 Hz.Hz. that Thus,Thus, this with J = 11.4 at 2.4 Hz. The coupling constant of H-4 showed a coupling constant of J = 2.4 Hz. Thus, thethe couplingcoupling constantconstant withwith JJ == 11.411.4 HzHz representsrepresents the→the couplingcoupling betweenbetween H-4H-4 andand H-5.H-5. TheThe evidenceevidence compoundthe coupling3 was constant a new skeletonwith J = 11.4 and Hz named represents it 3-nor(2 the coupling4)abeoiridoid. between The H-4 compound and H-5. The3 was evidence assigned concludedconcluded thatthat H-4H-4 isis locatedlocated inin thethe α-axialα-axial configuration.configuration. TheThe resultsresults providedprovided supportsupport forfor H-3H-3 beingbeing as neoretininconcluded that A. H-4 is located in the α-axial configuration. The results provided support for H-3 being inin thethe α-equatorialα-equatorial orientation.orientation. TheThe NOESYNOESY spectrumspectrum (Figure(Figure 2)2) providesprovides furtherfurther evidenceevidence forfor twotwo in the α-equatorial orientation. The NOESY spectrum (Figure 2) provides further evidence for two protons,protons, whichwhich waswas presentedpresented byby twotwo α-configuration.α-configuration. TheThe compoundcompound 22 waswas assignedassigned toto bebe neonaninneonanin protons, which was presented by two α-configuration. The compound 2 was assigned to be neonanin B.B. B.

Molecules 2018, 23, 2297 6 of 10 Molecules 2018, 23, x FOR PEER REVIEW 6 of 10

FigureFigure 3. 3.Proposed Proposed biosyntheticbiosynthetic sequence of of neoretinin neoretinin A A (3 ().3 ). 2.2. Structural Identification of Known Isolates 2.2. Structural Identification of Known Isolates The known isolates were readily identified by the comparison of their spectroscopic data The known isolates were readily identified by the comparison of their spectroscopic data with with those of the corresponding authentic samples or literatures. They include the following those of the corresponding authentic samples or literatures. They include the following twelve 4 twelvecompounds: compounds: 6-hydroxy-7-methyl-1-oxo-4-carbomethoxyoctahydrocyclopenta[c]pyran 6-hydroxy-7-methyl-1-oxo-4-carbomethoxyoctahydrocyclopenta[c]pyran (4) [17], 4- ( epi)[-17], 0 4-epialyxialactone-alyxialactone (5) (5 [18],)[18 loganetin], loganetin (6) (6 [19],)[19 loganin], loganin (7) ( 7 [20],)[20 ], phenylcoumaran-α′-aldehyde phenylcoumaran-α -aldehyde (8) ( [21],8)[21 ], cleomiscosincleomiscosin A (A9 )[(922) [22],], ficusal ficusal (10 (10)[) 23[23],], balanophonin balanophonin ( 11))[ [24],24], vanillic vanillic acid acid (12 (12) [25],)[25 p],-coumaricp-coumaric acid acid (13)[(1326) [26],], cis,trans cis,trans-abscisic-abscisic acid acid (14 (14),), and andtrans,trans trans,trans-abscisic-abscisic acid acid ( (1515))[ [27].27].

2.3.2.3. In-Vitro In-Vitro Cytotoxic Cytotoxic Activity Activity Against Against Hep3B Hep3B CellsCells InIn this this study, study, the the cytotoxic cytotoxic abilities abilities ofof sevenseven iridoidsiridoids ( (11––77),), four four neolignanes neolignanes (8– (811–),11 and), and two two aromaticaromatic rings rings (12 (12–13–13) against) against Hep Hep 3B 3B cells cells werewere shown in in Table Table 2.2. The The three three compounds, compounds, which which are are namelynamely ﬁcusal ficusal (10 ), (10 balanophonin), balanophonin (11 ), (11 and), and p-coumaric p-coumaric acid acid (13), ( exhibited13), exhibited moderate moderate inhibitory inhibitory effects, witheffects, EC50 values with EC of50 85.36 values± 4.36, of 85.36 92.63 ± ± 4.36,1.41, 92.63 and ±29.18 1.41,± and3.48 29.18µg/mL, ± 3.48 respectively. µg/mL, respectively. Doxorubicine wasDoxorubicine used as a positive was used control, as which a positive is an control,anthracycline which antibiotic is an anthracycline used for hepatocellular antibiotic used carcinoma for therapy.hepatocellular The main carcinoma mechanism therapy. is intercalant The main transcription, mechanism is which intercalant inhibits transcription, the effectivity which and inhibits inhibition the effectivity and inhibition of topoisomerase II activity [28]. The EC50 value of doxorubicine was of topoisomerase II activity [28]. The EC50 value of doxorubicine was 0.31 ± 0.08 µg/mL. Only one study0.31 published ± 0.08 µg/mL. recently Only one mentioned study published that balanophonin recently mentioned (10) has that cytotoxic balanophonin ability (10 with) has cytotoxic regard to a ability with regard to a Hep3B cell with IC50 = 29.3 ± 0.2 µM (equal to 10.44 ± 0.2 µg/mL). These Hep3B cell with IC50 = 29.3 ± 0.2 µM (equal to 10.44 ± 0.2 µg/mL). These inconsistent ﬁndings might inconsistent findings might be related to different study designs, laboratory performing skills, and be related to different study designs, laboratory performing skills, and experimental environment [29]. experimental environment [29]. Table 2. Effects of compounds isolated from Neonauclea reticulate and the cytotoxicity viability of Table 2. Effects of compounds isolated from Neonauclea reticulate and the cytotoxicity viability of Hep3B cell. Hep3B cell.

Compounds EC50 (µg/mL) in 48 h Compoundsneonanin A (1) >100 EC50 (µg/mL) in 48 h neonanin B (2) >100 neonaninneoretinin A A (3) (1) >100 >100 6-hydroxy-7-methyl-1-oxo-4-carbomethoxyoctahydrocyclopenta[c]pyran (4) >100 4-neonaninepi-alyxialactone B ( 52)) >100 >100 neoretininloganetin (6 A) (3) >100 >100 loganin (7) >100 6-hydroxy-7-methyl-1-oxo-4-carbomethoxyoctahydrocyclopenta[c]pyranphenylcoumaran-α0-aldehyde (8) ( >1004) >100 cleomiscosin A (9) >100 4-epi-alyxialactoneﬁcusal (10) (5) 85.36 ± 4.36 >100 balanophonin (11) 92.63 ± 1.41 loganetinvanillic acid (12 ()6) >100 >100 p-coumaric acid (13) 29.18 ± 3.48 loganinDoxorubicin (7) 0.31 ± 0.08 >100 Values are expressed as mean ± SD of three replicates. Molecules 2018, 23, 2297 7 of 10

3. Materials and Methods

3.1. General The mass spectrometric (HR-ESI-MS) data were generated at the Mass Spectrometry Laboratory of the Chung Hsing University with a Thermo LTQ Orbitrap XL™ Hybrid Ion Trap-Orbitrap Mass Spectrometer (Thermo Scientific Inc., Waltham, MA, USA). The melting point data were obtained with the melting point apparatus MP-S3 (YANACO Inc, Kyoto, Japan). The specific rotation data were obtained with a Jasco P-2000 Polarimeter (JASCO Inc., Tokyo, Japan). The infrared spectra were obtained with a Shimadzu IRAffinity-1S Fourier Transform Infrared Spectrophotometer (Shimadzu Inc., Kyoto, Japan). The UV spectra were obtained with a Shimadzu 160A UV–Visible recording spectrophotometer. The 1D and 2D-NMR spectra were recorded with a Bruker Avance 500 FT-NMR spectrometer (Bruker Inc., Bremen, Germany). Column chromatography was performed using LiChroCART Si 5 µM gel (Merck, Darmstadt, Germany) and Sephadex LH-20 (GE Healthcare Life Sciences Inc., Marlborough, MA, USA). The TLC (thin-layer chromatography) analysis was carried out using aluminum pre-coated Si plates (Silica Gel 60 F-254; Merck). The spots were visualized using a UV lamp at λ = 254 nm and detected by spraying with 10% H2SO4 alcohol solution, before heating at 125 ◦C. Semi-preparative HPLC was performed using a normal phase column (Luna 5µm Silica 100 Å, 250 × 10 mm; Phenomenex Inc., Torrance, CA, USA) on a Precision Instruments IOTA 2 Refractive Index Detector system.

3.2. Chemicals The solvents used to open the column isolation (Silica gel and Sephadex LH 20 gel column) in the study, such as n-hexane, chloroform, ethyl acetate, acetone, and methanol, were of ACS grade. The n-hexane, chloroform, and acetone used for HPLC isolation, which was of HPLC grade, and the deuterated solvents for NMR measurement (CDCl3 and CD3COCD3) were purchased from the branch of Merck in Taipei, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Doxorubicin and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Minimum essential media (MEM), trypsin–EDTA (ethylenediaminetetraacetic acid), fetal bovine serum (FBS), penicillin/streptomycin, non–essential amino acids (NEAA), and sodium pyruvate were obtained from Gibco (BRL life Technologies, Grand Island, NY, USA).

3.3. Plant Material The stems of Neonauclea reticulate were collected from Nan Ren Mountain, Pingtung, Taiwan, in August 2012, and identiﬁed by Yau Lun Kuo (Professor, Department of Forestry, National Pingtung University of Science and Technology, Pingtung, Taiwan). A voucher specimen (CMU-NR-201208) was deposited at the School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources.

3.4. Extraction and Isolation The dried stems of Neonauclea reticulate (9.0 kg) were extracted with MeOH (50 L each for seven days) three times. The MeOH extract was concentrated under reduced pressure at 35 ◦C, before the residue (365 g) was partitioned between EtOAc and H2O (1:1) to provide the EtOAc-soluble fraction (100 g). The water suspension was partitioned again with butanol (1:1) to get the BuOH-soluble fraction (152 g) and the H2O-soluble fraction (98 g). The EtOAc-soluble fraction was purified by column chromatography (CC) (2.0 kg of SiO2, 70–230 mesh; n-hexane/EtOAc/methanol gradient) to allow 32 fractions, Fr.1–Fr.32. Fr.22 (4.6 g) was re-separated by sephadex LH-20 (250 g; CHCl3/MeOH = 3/7) to produce 11 fractions of Fr.22-1–Fr.22-11. Fr.22-6 (1.47 g) was re-separatecd by silica gel column chromatography (30 g of SiO2, 70–230 mesh; CHCl3/EtOAc (25%)) and purified by normal-phase HPLC (n-hexane/acetone (35%)) to afford pure compounds 2 (9.5 mg, tR = 11 min), 3 (1.7 mg, tR = 16 min), and 4 (6.1 mg, tR = 14 min). Fr.22-7 (275.0 mg) was re-separatecd by silica gel column chromatography (5.5 g of SiO2, 70–230 mesh; CHCl3/acetone (20%)) to afford fourteen fractions of Fr.22-7-1–Fr.22-7-14. Molecules 2018, 23, 2297 8 of 10

Fr.22-7-9 affords pure compound 9 (8.5 mg, Rf = 0.2). Fr.22-7-8 (7.4 mg) was purified by normal phase HPLC (n-hexane/acetone (30%)) to form pure compounds 10 (3.2 mg, tR = 15 min) and 11 (2.0 mg, tR = 25 min). Fr.22-9 (141.2 mg) was purified by normal phase HPLC (CHCl3/acetone (20%)), resulting in pure compounds 12 (14.2 mg, tR = 10 min) and 13 (2.8 mg, tR = 9 min). Fr.23 (1.2 g) was re-separated by sephadex LH-20 (140 g; CHCl3/MeOH = 3/7) to afford the six fractions Fr.23-1–Fr.23-6. Fr.23-3 (832.68 mg) was re-separatecd by silica gel column chromatography (30 g of SiO2, 70–230 mesh; CHCl3/EtOAc (25%)) and purified by normal-phase HPLC (n-hexane/acetone (35%)), in order to afford pure compounds 1 (1.1 mg, tR = 28 min), 5 (30.3 mg, tR = 20 min), 6 (27.6 mg, tR = 13 min), 8 (3.0 mg, tR = 32 min), 14 (1.2 mg, tR = 16 min), and 15 (0.8 mg, tR = 11 min). Fr.29 (4.9 g) was washed with acetone and filtered to produce a white residue. The white residue was found to be pure compound 7 (0.5 g).

28 neonanin A (1): colorless oil; [α]D + 17.6 (c 0.10, MeOH); IR (KBr) νmax: 3491, 2962, 1728, 1438, and −1 + 1 1060 cm ; HR-ESI-MS m/z 267.0845 [M + Na] (calculated for C11H16O6Na at 267.0839); H-NMR 13 and C-NMR (500/125 MHz, in CDCl3) are shown on Table1.

◦ 27 neonanin B (2): white needle; MP: 82–84 C; [α]D + 93.6 (c 0.10, MeOH); IR (KBr) νmax: 3392, 1730, 1444, −1 + 1371, and 1172 cm ; HR-ESI-MS m/z 281.0999 [M + Na] (calculated for C12H18O6Na at 281.0996); 1 13 H-NMR and C-NMR (500/125 MHz, in CDCl3) are shown on Table1.

27 neoretinin A (3): colorless oil; [α]D − 73.1 (c 0.10, MeOH); IR (KBr) νmax: 3361, 2939, 1741, 1450, 1205, −1 + 1 and 1024 cm ; HR-ESI-MS m/z 253.0685 [M + Na] (calculated for C10H14O6Na at 253.0683); H-NMR 13 and C-NMR (500/125 MHz, in CDCl3) are shown on Table1.

3.5. Cell Culture Hep3B (Bioresource Collection and Research Center (BCRC) Number: 60434) and HepG2 (BCRC Number: 60364) cells were obtained from Food Industry Research and Development Institute (Hsin Chu, Taiwan). All of the cell lines were cultured in MEM containing 10% (v:v) FBS, penicillin/streptomycin (100 U/mL), 0.1 mM NEAA, and 1.0 mM sodium pyruvate. The cells were ◦ cultured in a humidiﬁed incubator under 5% CO2 at 37 C.

3.6. Cytotoxic Assay The in-vitro cytotoxic activity of MeOH extracts, partition fractions, and pure compounds were determined by the MTT assay. Hep3B (2 × 104/well) and HepG2 (1 × 104/well) cells were seeded in 96-well plates and incubated for 24 h. Both cells were treated with MeOH extracts that contain partition fractions in various concentrations (0, 62.5, 125, 250, 500, and 1000 µg/mL). Furthermore, each pure compound was evaluated only in Hep3B cells, and the dosages used were 0, 6.25, 12.5, 25, 50, and 100 µg/mL. After 48 h, the medium was replaced with a medium containing 0.5 mg/mL MTT solution and incubated at 37 ◦C for 4 h. After the end of the MTT reaction, we used isopropanol/HCl solution to dissolve the formazan crystals. The absorbance was measured spectrophotometrically at 570 nm. The cytotoxicity was calculated and compared with the control group.

3.7. Statistical Analysis The results were presented as mean values ± SD (standard deviations) of at least three independent experiments. Statistical analyses were performed using Microsoft Excel 2010 software (Information Center, China Medical University, Taichung, Taiwan).

4. Conclusions The isolation and structural elucidation of fifteen compounds, including three new compounds—namely neonanin A (1), neonanin B (2) and neoretinin A (3)—as well as twelve Molecules 2018, 23, 2297 9 of 10 known compounds—6-hydroxy-7-methyl-1-oxo-4-carbomethoxyoctahydrocyclopenta[c]pyran (4), 4-epi-alyxialactone (5), loganetin (6), loganin (7), phenylcoumaran-α0-aldehyde (8), cleomiscosin A (9), ficusal (10), balanophonin (11), vanillic acid (12), p-coumaric acid (13), cis,trans-abscisic acid (14), and trans,trans-abscisic acid (15)—were isolated from the stems of Neonauclea reticulata (Havil.) Merr. The structures of these compounds were established based on the spectroscopic data. Three of the compounds, namely ficusal (10), balanophonin (11), and p-coumaric acid (13) exhibited moderate cytotoxicity, with EC50 values of 85.36 ± 4.36, 92.63 ± 1.41, and 29.18 ± 3.48 µg/mL, respectively, against Hep3B cells within 48 h. To the best of our knowledge, this is the first study that has conducted phytochemical investigation of Neonauclea reticulata (Havil.) Merr. and provided preliminary results of cytotoxicity on hepatocellular carcinoma cells.

Supplementary Materials: The following are available online: 1D- and 2D-NMR, as well as HR-ESI-MS spectra of Compounds 1–3. Author Contributions: F.P.C carried out the experimental work, including extraction, partition, chromatographic fractionation, and purification of compounds; performed the structure elucidation of the chemicals; analyzed the spectroscopic data; and prepared the manuscript. W.C carried out experimental work for all samples of the cytotoxicity assay. H.C.H, P.J.S, and J.J.C identified compound structures. S.Y.W and M.J.C analyzed bioassay data. G.J.H. and Y.H.K participated in the design of this study and organized all the research for it. All authors approved the final version of the manuscript. Funding: This work was financially supported by “Chinese Medicine Research Center, China Medical University” from The Featured Areas Research Center Program, within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (CMRC-CHM-4) and Taiwan Ministry of Health and Welfare Clinical Trial Center (MOHW107-TDU-B-212-123004). Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Samples of the compounds 1–15 are available from the authors.

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