Secoiridoids from the Seed of Gonocaryum calleryanum and Their Inhibitory Potential on LPS-Induced Tumor Necrosis Factor and Nitric Oxide Production

Three new secoiridoid constituents, goncarin A−C (1–3), and a new derivative, goncarin A monoacetate (4), along with two known lignins, pinoresinol (5) and paulownin (6), were isolated from the seed of Gonocaryum calleryanum (Baill.) Becc. The structures of the new metabolites were determined on the basis of extensive spectroscopic analysis, particularly mass spectroscopy and 2D NMR (1H–1H COSY, HMQC, HMBC, and NOESY) spectroscopy. The aim of this study was to identify the anti-inflammatory effects of compounds 1–6 on lipopolysaccharide (LPS)-stimulated murine macrophage cell lines (RAW 264.7). Following stimulation with LPS, elevated levels of nitric oxide (NO) production were detected in RAW 264.7 cells; however, pretreatment with compounds 1–6 significantly inhibited the production of NO (around 40–80%, p < 0.01–0.05), by suppressing the expression of inducible NO synthase (iNOS). In addition, LPS-stimulated tumor necrosis factor-α (TNF-α) production was significantly reduced by compounds 1–3 (25–40%, p < 0.01–0.05). These results suggested that compounds 1–3 may exert anti-inflammatory activity, and that compounds 1–3 may be considered a potential therapeutic for the treatment of inflammatory diseases associated with macrophage activation.


Introduction
The Gonocaryum Miq. plant is widely distributed in tropical and subtropical forests on coral rocks from Indonesia to the Philippines (Luzon and Batan Islands) and Taiwan (Hengchun Peninsula) [1]. Gonocaryum calleryanum (Baill.) Becc. is the only species of Gonocaryum found in tropical forests in southern Taiwan. Its leaves are used in Philippine traditional folk medicine for treating stomach disease [2]. However, there are only a few reports about the chemical composition of Gonocaryum calleryanum, and no reports on the analysis of biological activity and toxicity. Kaneko et al. [3] and Chan and co-workers [4] reported the isolation of secoiridoid glycosides, flavonoids, and flavonoid  The HR-ESI-MS of 1 (Supplementary Materials) revealed a pseudo-molecular-ion peak at m/z 381.1159 ([M + Na] + ), consistent with the molecular formula C 16 H 22 O 9 , having six degrees of unsaturation. The IR spectrum displayed absorption bands diagnostic of OH (3456 cm −1 ), ester (1738 cm −1 ), and C=C bond (1635 cm −1 ) functionalities. The 13 C-NMR spectrum of 1 showed the signals of a β-glucopyranosyl moiety, a trisubstituted double bond (δ C 151.9 and 109.7), a carbomethoxyl group (δ C 51.5 and 166. 7), and a ketal carbon (δ C 92.5). The presence of these partial structures suggested that 1 was an iridoid or secoiridoid. 1 H-and 13 C-NMR data (Table 1) indicated the presence of one OMe unit (δ H 3.74 s, δ C 51.5 q), two lactones, an ester group (δ C 173.6 s, 175.7 s, 166.7 s), and one C=C bond (δ H 7.52 s, δ C 151.9 d, 109.7 s), accounting for 6 degrees of unsaturation and suggesting 2 additional rings. The 1 H-NMR, 13 C-NMR, and DPET spectrums also revealed one CH 2 (δ H 2.60 td, 3.10 td, δ C 35.4 t), three CHO groups (δ H 5.88 br s, δ C 92.5 d; δ H 4.94 q, δ C 72.9 d; δ H 5.05 q, δ C 73.2 d), two CH (δ H 3.36 m, δ C 30.6 d; δ H 1.98 br d, δ C 43.9 d), three additional Me (δ H 1.46 d, δ C 18.3 q; δ H 1.33 d, δ C 12.8 q; δ H 1.35 s, δ C 16.4 q), and one additional quaternary C (δ C 74.5 s). The COSY correlations showed the connection from H-1 to H-9, and H-5 to H-6 and H-9. The HMBC ( Figure 2) showed correlations of H-3 to C-4, C-11, and C-1, H-3 to C-1 and C-5, and, with the aid of 13 C-NMR spectrum, indicated that the COOMe group is attached to C-4 and the OH group is attached to C-1, assuming the position of the atom O is between C-1/C-3. The HMBC and 13 C-NMR data also revealed the connections of H-6 to C-5, C-7, and C-9, H-3 to C-7 and C-2 , Me-5 to C-2 and C-1 , H-8 to C-1 and C-9, and indicated the position of two lactones and the OH groups. The relative configuration of 1 was determined on the basis of NOESY experiment and the references. The NOESY data exhibited the connectivity between H-8 to H-5 and H-9, and Me-10 to H-1. These findings and the references established the β-orientation of H-5, H-8, and H-9, and the α-configuration of H-1 and H-10. The stereochemical of C-2 and C-3 was determined by comparing the 1 H and 13 C-NMR spectra using alkaline hydrolysis of goncaryoside A [3,17]. It is speculated that if goncarin A undergoes alkaline hydrolysis, then it will obtain Kingiside aglycone and 2S, 3S angliceric acid [18][19][20][21][22]. Hence, the structure of goncarin A, one new natural compound, was established as 1. rings were suggested. The COSY correlations showed the connection of from H-1 to H-9, from H-9 to H-5, and from H-5 to H-6. The HMBC correlations of H-11 to C-4, C-5-, and C-3 exhibited that the C=C was at C-4 when the lactone was at C-3. The connections of the HMBC spectrum also showed the correlations of Me-4 to C-3 , C-2 , and C-7, H-8 to C-9 and C-1 , and H-5 to C-1 , C-2 , and C-3 , indicating the assignment of the lactone and the OH groups at C-7, C-1 , and C-2 , and connectivity of HMBC to complete the plane structure of 2. The relative configuration of 2 was determined on the basis of NOESY experiment. The NOESY data exhibits the connectivity of H-8 to H-5 and H-9. These findings and the references established the β-orientation of H-5, H-8, and H-9, and the α-configuration of Me-10. The stereo chemical of C-2 and C-3 was determined by comparing the 1 H and 13 C-NMR spectra of goncarin A. Hence, the structure of the newly discovered natural compound, goncarin B, was established as 2. The HR-ESI-MS of 2 revealed a pseudo-molecular-ion peak at m/z 335.1105 ([M + Na] + ), consistent with the molecular formula C15H20O7, having six degrees of unsaturation. The IR spectrum displayed absorption bands diagnostic of OH (3456 cm −1 ), ester (1734 cm −1 ), and C=C bond (1635 cm −1 ) functionalities. The 1 H-NMR, 13  and one quaternary C (δC 74.3 s). Accounting for 6 degrees of unsaturation, 2 additional rings were suggested. The COSY correlations showed the connection of from H-1 to H-9, from H-9 to H-5, and from H-5 to H-6. The HMBC correlations of H-11 to C-4, C-5-, and C-3 exhibited that the C=C was at C-4 when the lactone was at C-3. The connections of the HMBC spectrum also showed the correlations of Me-4′ to C-3′, C-2′, and C-7, H-8 to C-9 and C-1′, and H-5′ to C-1′, C-2′, and C-3′, indicating the assignment of the lactone and the OH groups at C-7, C-1′, and C-2′, and connectivity of HMBC to complete the plane structure of 2. The relative configuration of 2 was determined on the basis of NOESY experiment. The NOESY data exhibits the connectivity of H-8 to H-5 and H-9. These findings and the references established the β-orientation of H-5, H-8, and H-9, and the αconfiguration of Me-10. The stereo chemical of C-2′ and C-3′ was determined by comparing the 1 H and 13 C-NMR spectra of goncarin A. Hence, the structure of the newly discovered natural compound, goncarin B, was established as 2. The HR-ESI-MS of 2 revealed a pseudo-molecular-ion peak at m/z 335.1105 ([M + Na] + ), consistent with the molecular formula C15H20O7, having six degrees of unsaturation. The IR spectrum displayed absorption bands diagnostic of OH (3456 cm −1 ), ester (1734 cm −1 ), and C=C bond (1635 cm −1 ) functionalities. The 1 H-NMR, 13 C-NMR (Table 1), and DPET spectrum indicated the presence of three Me (δH 1.23 s, δH 1.31 d, 1.38 d), one C=CH2 (δH 5.68 s, δH 6.40 s; δC 128.8 t, 138.4 s), one CH2 (δH 2.33 m, 2.70 t, δC 36.0 t), one OCH2 group (δH 4.64 d, δC 64.5 t), two CH (δH 3.48 d, δC 40.4 d; δH 2.30 m, δC 41.6 d), two OCH groups (δH 4.82 q, δC 73.8 d; δH 5.06 q, δC 73.5 d), three lactones (δC 171.6 s, 174.9 s, 163.7 s), and one quaternary C (δC 74.3 s). Accounting for 6 degrees of unsaturation, 2 additional rings were suggested. The COSY correlations showed the connection of from H-1 to H-9, from H-9 to H-5, and from H-5 to H-6. The HMBC correlations of H-11 to C-4, C-5-, and C-3 exhibited that the C=C was at C-4 when the lactone was at C-3. The connections of the HMBC spectrum also showed the correlations of Me-4′ to C-3′, C-2′, and C-7, H-8 to C-9 and C-1′, and H-5′ to C-1′, C-2′, and C-3′, indicating the assignment of the lactone and the OH groups at C-7, C-1′, and C-2′, and connectivity of HMBC to complete the plane structure of 2. The relative configuration of 2 was determined on the basis of NOESY experiment. The NOESY data exhibits the connectivity of H-8 to H-5 and H-9. These findings and the references established the β-orientation of H-5, H-8, and H-9, and the αconfiguration of Me-10. The stereo chemical of C-2′ and C-3′ was determined by comparing the 1 H and 13 C-NMR spectra of goncarin A. Hence, the structure of the newly discovered natural compound, goncarin B, was established as 2.  The molecular formula C 15 H 22 O 8 (five degrees of unsaturation) of 3 was deduced from the HR-ESI-MS data (m/z 353.1209 ([M + Na] + )). Its IR spectrum showed absorption bands suggesting the functionalities of OH (3433 cm −1 ) and ester (1738 cm −1 ). The 1 H-NMR, 13 C-NMR, and DEPT spectroscopic data (Table 1) indicated the presence of three CH 3 , two OCH 2 , two OCH, and three CH, and four quaternary C including one lactone, one ester group, and one acid group. Accounting for 5 degrees of unsaturation, 2 additional rings were suggested. In comparison with goncarin B, two sets of signals (δH 2.70 d, δH 3.75 m; δC 48.9 d, 61.1 t) were found from 1 H-NMR and 13 C-NMR. According to the COSY correlations, it showed the connection of H-1 to H-9, H-5 to H-6, and H-4 to H-11. The HMBC correlations of H-1 to C-3, C-5, and C-9, H-4 to C-3, C-5, and C-11, and H-6 to C-7, established lactones at C-3, the ester group at C-7, and the assignments of the right part of structure. The connections of HMBC spectrum also showed the correlations of H-3 to C-7, C-2 , Me-4 , and Me-5 , Me-5 to C-1 , C-2 , and C-3 , and Me-4 to C-2 and C-3 , which exhibited the location and the assignments of the left part of structure. From the above precise spectral data, it can be inferred that the oxygen-containing open ring at position C-8 of goncarin B is linked to the double bond on C-11. The NOESY spectrum showed the correlations of H-8 to H-5, H-4 to H-5, H-5 to H-9, and H-8 to H9. The relative configuration of 3, elucidated mainly from the nuclear Overhauser effect spectroscopy (NOESY) spectrum, was compatible with that of 3 ascertained using molecular mechanics calculations (MM2). It is suggested to be the most stable conformations, as shown in Figure 3. These findings and the references established the β-orientation of H-4, H-5, H-8, and H-9, and the α-configuration of Me-10. Hence, the structure of newly discovered natural compound, goncarin C, was established as 3.

Plant Material
The seeds of the plant Gomocaryum calleryanum were collected from tropical forests in southern Taiwan in July 2011. Plant identification and collection was conducted by Sheng-feng Hong of the Hengchun Research Center, Forestry Research Institute. Samples were collected and stored in the Specimen Room at Meiho University, Taiwan (Sample No.: 2011-07-2).

Reaction
Acetylation of 1 (100 mg) was treated with acetic anhydride/pyridine (1:1) and left at room temperature for 24 h. Meanwhile, the work continued on the product with HPLC and resulted in the goncarin A monoacetate (4) 61 mg.  and one quaternary C (δC 74.3 s). Accounting for 6 degrees of unsaturation, 2 additional rings were suggested. The COSY correlations showed the connection of from H-1 to H-9, from H-9 to H-5, and from H-5 to H-6. The HMBC correlations of H-11 to C-4, C-5-, and C-3 exhibited that the C=C was at C-4 when the lactone was at C-3. The connections of the HMBC spectrum also showed the correlations of Me-4′ to C-3′, C-2′, and C-7, H-8 to C-9 and C-1′, and H-5′ to C-1′, C-2′, and C-3′, indicating the assignment of the lactone and the OH groups at C-7, C-1′, and C-2′, and connectivity of HMBC to complete the plane structure of 2. The relative configuration of 2 was determined on the basis of NOESY experiment. The NOESY data exhibits the connectivity of H-8 to H-5 and H-9. These findings and the references established the β-orientation of H-5, H-8, and H-9, and the αconfiguration of Me-10. The stereo chemical of C-2′ and C-3′ was determined by comparing the 1 H and 13 C-NMR spectra of goncarin A. Hence, the structure of the newly discovered natural compound, goncarin B, was established as 2.

Plant Material
The seeds of the plant Gomocaryum calleryanum were collected from tropical forests in southern Taiwan in July 2011. Plant identification and collection was conducted by Sheng-feng Hong of the Hengchun Research Center, Forestry Research Institute. Samples were collected and stored in the Specimen Room at Meiho University, Taiwan (Sample No.: 2011-07-2).

Extraction and Isolation
The fresh seeds were dried by cold drying and smashed, and then a 5.5 kg dry sample was collected. The dry sample was extracted with acetone (3 × 4 L) at room temperature, and then the acetone extract was concentrated. The dark-brown crude extract (520 g) was partitioned between EtOAc and H 2 O (1:1). The EtOAc layer (220 g) was subjected to CC (SiO 2 , n-hexane/EtOAc 20:1~1:1) and got 11 fractions.

Reaction
Acetylation of 1 (100 mg) was treated with acetic anhydride/pyridine (1:1) and left at room temperature for 24 h. Meanwhile, the work continued on the product with HPLC and resulted in the goncarin A monoacetate (4) 61 mg.

Cell Culture and Treatment
RAW 264.7 mouse macrophage cells were obtained from the Bioresources Collection and Research Center (Hsinchu, Taiwan; BCRC No. 60001). The macrophages were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactive fetal bovine serum (FBS) in a humidified atmosphere CO 2 incubator (5% CO 2 in air, ESCO, Singapore) at 37 • C. For the experiments, cells (1 × 10 5 ) were seeded in a culture plate with 24 wells and maintained within the incubator. RAW 264.7 macrophages were pre-treated with LPS (Escherichia coli O111:B4) and culture medium mixture (100 ng/mL) for 6 h in a 37 • C incubator [26]. Subsequently, the isolated compounds (compounds 1-6) were dissolved in dimethyl sulfoxide (DMSO) and added into LPS/medium mixture with final concentrations of 0.5, 1.0, and 2.0 µg/mL overnight. The positive and negative control were LPS/medium mixture and culture medium only.

MTS Assay
RAW 264.7 macrophages were treated as described. After overnight incubation, the culture medium was removed, and then cells were washed with PBS. Two hundred µL of MTS reagent (Promaga, Madison, WI, USA) was added into each well for 1 h in a 37 • C incubator. The absorbance was measured using a plate reader (BioTek, Winooski, VT, USA) at a wavelength of 490 nm [27].

Nitric Oxide Assay
RAW 246.7 cells (1 × 10 5 ) were seeded in a culture plate with 24 wells and maintained within the incubator. RAW 264.7 macrophages were pre-treated with LPS (Escherichia coli O111:B4) and culture medium mixture (100 ng/mL) for 6 h in a 37 • C incubator. Subsequently, compounds 1-6 were dissolved in dimethyl sulfoxide (DMSO) and added into LPS/medium mixture with final concentrations of 0.5, 1.0, and 2.0 µg/mL overnight. The amount of NO in the supernatants was detected by using the Griess Reagent System (Promaga, Madison, WI, USA) according to the manufacturer's protocol. Data calculations were performed using MS-Excel 2010 software [29].
The pro-inflammatory cytokine TNF-α and the reactive free radical NO synthesized by inducible nitric oxide synthase (iNOS) are important macrophage-derived inflammatory mediators. Thus, the effect on the excessive productions of TNF-α and NO can be employed as criteria to evaluate the anti-inflammatory activity of test compounds. In this study, two inflammatory parameters (NO and TNF-α) were evaluated using macrophages incubated with and without LPS (basal values) and in the absence or presence of different concentrations of compounds 1-6 (0.5, 1.0, and 2.0 µg/mL).
The effect of compounds 1-6 on RAW 264.7 cell viability was determined by an MTS assay. Cells cultured with compounds 1-6 at concentrations of 0.5, 1.0 and 2 µg/mL used in the presence of 100 ng/mL LPS for 24 h did not change cell viability (Figure 4). The results obtained in the NO assay are shown in Figure 5. LPS significantly increased NO with respect to basal cells. Compound 1-6 significantly reduced the effect of LPS stimulation on nitric oxide positively according to concentration (around 40-80%, p < 0.01-0.05). The results obtained for the TNF-α assay are shown in Figure 6. LPS increased the TNF-α basal level significantly. Compounds 1-3 decreased LPS-stimulated TNF-α positively related to concentration (around 25-40%, p < 0.01-0.05), in comparison with the group of cells treated with LPS but without treatment with this compound. Compounds 1-3 exerted an effect not only on NO but also on TNF-α levels in a model of murine macrophages activated with LPS. With increasing concentration of compound added, both NO and TNF-α decreased, suggesting an anti-inflammatory action.
Molecules 2018, 23, x FOR PEER REVIEW 7 of 10 3.5.5. Nitric Oxide Assay RAW 246.7 cells (1 × 10 5 ) were seeded in a culture plate with 24 wells and maintained within the incubator. RAW 264.7 macrophages were pre-treated with LPS (Escherichia coli O111:B4) and culture medium mixture (100 ng/mL) for 6 h in a 37 °C incubator. Subsequently, compounds 1-6 were dissolved in dimethyl sulfoxide (DMSO) and added into LPS/medium mixture with final concentrations of 0.5, 1.0, and 2.0 µ g/mL overnight. The amount of NO in the supernatants was detected by using the Griess Reagent System (Promaga, Madison, WI, USA) according to the manufacturer's protocol. Data calculations were performed using MS-Excel 2010 software [29].
The pro-inflammatory cytokine TNF-α and the reactive free radical NO synthesized by inducible nitric oxide synthase (iNOS) are important macrophage-derived inflammatory mediators. Thus, the effect on the excessive productions of TNF-α and NO can be employed as criteria to evaluate the antiinflammatory activity of test compounds. In this study, two inflammatory parameters (NO and TNFα) were evaluated using macrophages incubated with and without LPS (basal values) and in the absence or presence of different concentrations of compounds 1-6 (0.5, 1.0, and 2.0 μg/mL).
The effect of compounds 1-6 on RAW 264.7 cell viability was determined by an MTS assay. Cells cultured with compounds 1-6 at concentrations of 0.5, 1.0 and 2 μg/mL used in the presence of 100 ng/mL LPS for 24 h did not change cell viability (Figure 4). The results obtained in the NO assay are shown in Figure 5. LPS significantly increased NO with respect to basal cells. Compound 1-6 significantly reduced the effect of LPS stimulation on nitric oxide positively according to concentration (around 40-80%, p < 0.01-0.05). The results obtained for the TNF-α assay are shown in Figure 6. LPS increased the TNF-α basal level significantly. Compounds 1-3 decreased LPSstimulated TNF-α positively related to concentration (around 25-40%, p < 0.01-0.05), in comparison with the group of cells treated with LPS but without treatment with this compound. Compounds 1-3 exerted an effect not only on NO but also on TNF-α levels in a model of murine macrophages activated with LPS. With increasing concentration of compound added, both NO and TNF-α decreased, suggesting an anti-inflammatory action.

Conclusions
The inhibitory effects of compounds 1-6 on LPS-induced nitric oxide (NO) production and LPSinduced tumor necrosis factor-α (TNF-α) expression were determined by measuring the levels of nitrite and cytokine enzyme-linked immunosorbent assays (ELISAs). It was identified that compounds 1-3 may suppress NO production in LPS-activated RAW 246.7 macrophages by inhibiting inducible NO synthase (iNOS) expression, and exert curative effects on anti-inflammatory activity by reducing TNF-α expression. The results provide evidence in favor of the use of compounds 1-3 as a potential therapeutic for the treatment of inflammatory diseases.  . Inhibition of tumor necrosis factor-α (TNF-α) production by compounds 1-6 in LPSstimulated RAW 264.7 macrophages. RAW 264.7 cells were stimulated by LPS (100 ng/mL) for 6 h then treated with various concentrations of compounds 1-6 for 24 h. TNF-α production was measured using the corresponding ELISA kits. Values are presented as mean ± standard deviation of three independent experiments. ### p < 0.001 control group as compared to LPS-treated group. * p < 0.05, ** p < 0.01, and *** p < 0.001 were compared with the LPS-alone group. -: cells without treatment, +: cells previously treated with LPS.

Conclusions
The inhibitory effects of compounds 1-6 on LPS-induced nitric oxide (NO) production and LPSinduced tumor necrosis factor-α (TNF-α) expression were determined by measuring the levels of nitrite and cytokine enzyme-linked immunosorbent assays (ELISAs). It was identified that compounds 1-3 may suppress NO production in LPS-activated RAW 246.7 macrophages by inhibiting inducible NO synthase (iNOS) expression, and exert curative effects on anti-inflammatory activity by reducing TNF-α expression. The results provide evidence in favor of the use of compounds 1-3 as a potential therapeutic for the treatment of inflammatory diseases. Figure 6. Inhibition of tumor necrosis factor-α (TNF-α) production by compounds 1-6 in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells were stimulated by LPS (100 ng/mL) for 6 h then treated with various concentrations of compounds 1-6 for 24 h. TNF-α production was measured using the corresponding ELISA kits. Values are presented as mean ± standard deviation of three independent experiments. ### p < 0.001 control group as compared to LPS-treated group. * p < 0.05, ** p < 0.01, and *** p < 0.001 were compared with the LPS-alone group. -: cells without treatment, +: cells previously treated with LPS.

Conclusions
The inhibitory effects of compounds 1-6 on LPS-induced nitric oxide (NO) production and LPS-induced tumor necrosis factor-α (TNF-α) expression were determined by measuring the levels of nitrite and cytokine enzyme-linked immunosorbent assays (ELISAs). It was identified that compounds 1-3 may suppress NO production in LPS-activated RAW 246.7 macrophages by inhibiting inducible NO synthase (iNOS) expression, and exert curative effects on anti-inflammatory activity by reducing TNF-α expression. The results provide evidence in favor of the use of compounds 1-3 as a potential therapeutic for the treatment of inflammatory diseases.
Supplementary Materials: The physical and spectroscopic data of compounds 1-6, as well as NMR and MS spectra of compounds 1-6, are available online.