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

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-α′-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.


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.
Compound 3 was obtained as a colorless oil.The molecular weight was determined by HR-ESI-MS, which showed an [M + Na] + ion at m/z of 253.0685 (calculated for C 10 H 14 O 6 Na 253.0683), indicating four degrees of unsaturation.The IR spectrum displays the presence of hydroxyl (3361 cm −1 ) and lactone carbonyl (1741 cm −1 ) functionalities.The 1 H and 13 C-NMR spectra of compound 3 (Table 1) shows that there is a doublet methyl group δ After hydrolysis, the formulate was abandoned and formed compound 18, which condensed to obtain diacetal (compound 19).Subsequently, compound 19 was partially oxidized to obtain compound 3. From the literature survey, no report has mentioned this type of structure, so we suggested that this compound 3 was a new skeleton and named it 3-nor(2→4)abeoiridoid.The compound 3 was assigned as neoretinin A.

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% H 2 SO 4 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.

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.

Cytotoxic Assay
The in-vitro cytotoxic activity of MeOH extracts, partition fractions, and pure compounds were determined by the MTT assay.Hep3B (2 × 10 4 /well) and HepG2 (1 × 10 4 /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.

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).

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

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Molecules 2018, 23, x FOR PEER REVIEW 3 of 1D) and two-dimensional (2D)-NMR, as well as HR-ESI-MS data.The known compounds identified were compared with the published NMR spectral data.All structures are shown in Figure1.In this study, we described the detailed structural elucidations of new compounds and the activities of compounds 1-13.

Table 2 .
Effects of compounds isolated from Neonauclea reticulate and the cytotoxicity viability of Hep3B cell.

Table 2 .
Effects of compounds isolated from Neonauclea reticulate and the cytotoxicity viability of Hep3B cell.