Potential Anti-inflammatory Effects of the Fruits of Paulownia

Article Cite This: J. Nat. Prod. 2017, 80, 2659-2665

pubs.acs.org/jnp

Potential Anti-inflammatory Effects of the Fruits of Paulownia tomentosa Hyung Won Ryu,†,⊥ Yhun Jung Park,§,⊥ Su Ui Lee,† Seoghyun Lee,† Heung Joo Yuk,† Kyeong-Hwa Seo,† Yeah-Un Kim,† Bang Yeon Hwang,*,§ and Sei-Ryang Oh*,† †

Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Chungbuk 28116, Republic of Korea § College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea S Supporting Information *

ABSTRACT: As part of an ongoing search for new natural products from medicinal plants to treat respiratory disease, six new compounds, a dihydroflavonol (1) and five C-geranylated flavanones (3, 6, 8, 13, and 14), and 13 known compounds were isolated from mature fruits of Paulownia tomentosa. The structures of the new compounds were determined via interpretation of their spectroscopic data (1D and 2D NMR, UV, IR, ECD, and MS). In biological activity assays with human alveolar basal epithelial cells, the expression of TNF-α-induced proinflammatory cytokines (IL-8 and IL-6) was reduced significantly by the EtOAc fraction of a P. tomentosa extract as well as by the new compounds isolated from this fraction. Furthermore, the majority of the isolates (1−19 except 5−7) were found to inhibit human neutrophil elastase (HNE) activity, with IC50 values ranging from 2.4 ± 1.0 to 74.7 ± 8.5 μM. In kinetic enzymatic assays with the HNE substrate MeOSuc-AAPV-pNA, compound 17 exhibited the highest inhibitory activity (Ki = 3.2 μM) via noncompetitive inhibition. These findings suggest that the flavanone constituents of P. tomentosa fruits may be valuable for the development of new drug candidates to treat airway inflammation.

A

inflammatory responses. Therefore, it is a valuable therapeutic target for preventing tissue damage that results from excessive inflammation in the respiratory system.9 In a recent study, flavonoids with inhibitory activity against HNE,10 such as the biologically active catechin, (−)-epigallocatechin-3-gallate, demonstrated protective effects on lung tissue, which stimulated the present study to find additional HNE-modulating flavonoids.11 The fruits of Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae) have been used to treat or prevent a variety of diseases (e.g., tonsillitis, bronchitis, asthmatic attacks, enteritis, and dysentery),12 and compounds isolated from these fruits have demonstrated the ability to inhibit airway inflammation13 and sialidase,14 in addition to having cytotoxic15 and antioxidant activities.16 Although previous studies have reported anti-inflammatory and HNE inhibitory activities of flavonoids, no study has defined the relationship between the biochemical activity and structural moiety on the A-ring. Reported herein are the isolation and structure elucidation of six new prenylated flavonoids (1, 3, 6, 8, 13, and 14) from the mature fruits of P. tomentosa as well as the evaluation of their anti-inflammatory activities, including effects on cytokine production and in vitro HNE inhibition.

irway inflammation is a major cause of chronic pulmonary diseases, such as asthma and chronic obstructive pulmonary disease (COPD), which are emerging public health threats. The World Health Organization estimates that, by 2030, COPD will become the third leading cause of death globally, and the market burden of treating COPD/asthma will increase to $47 trillion United States dollars.1 The major prognostic factor for the pathogenesis and progression of COPD is cigarette smoke exposure,2 which evokes inflammation, oxidative stress, and protease−antiprotease imbalance in the respiratory system.3 Inhaled corticosteroids, phosphodiesterase-4 inhibitors (e.g., roflumilast), 4 and human neutrophil elastase (HNE) inhibitors (e.g., ONO-5046)5 have been used previously to suppress inflammation in small airways and the lung parenchyma; however, these drugs must be prescribed with caution to avoid the inevitable side-effects that arise with long-term use.6 Therefore, searching for new antiinflammatory compounds from natural sources could be an effective strategy for the development of new drugs to reduce severe side-effects and/or replace currently available pharmaceuticals.7 Human neutrophil elastase is a serine protease that plays a key role in the innate immune response, tissue degradation and remodeling, and onset and resolution of inflammation in COPD patients.8 HNE, found in the azurophil granules of neutrophils, has been reported to enhance mucus secretion and © 2017 American Chemical Society and American Society of Pharmacognosy

Received: April 14, 2017 Published: October 2, 2017 2659

DOI: 10.1021/acs.jnatprod.7b00325 J. Nat. Prod. 2017, 80, 2659−2665

Journal of Natural Products

Article

at 280−310 nm and the positive ECD Cotton effect at 320− 360 nm, 1 was assigned with the absolute configuration (2R, 3R).17,25 Thus, 1 was determined as a new compound, (2R)-3,5-dihydroxy-2-(3,4,-dihydroxyphenyl)-8-(4-hydroxy-4methylpentyl)-8-methyl-2,3,7,8-tetrahydropyrano-[3,2-g]chromen-4-(6H)-one, and was named tomentin F (1). Compound 3 was isolated as a brownish powder. The molecular formula was determined to be C26H33O8 from the molecular ion peak [M + H]+ at m/z 473.2091 (calcd for C26H33O8, 473.2175) in the HRESIMS, which was 30 amu higher than that of 4. The 1H NMR spectrum showed a methoxy group proton signal [δH 3.87 (3H, s, OMe)]. In the HMBC spectrum, this methoxy group proton signal (δH 3.87) showed a correlation with an oxygenated olefin quaternary carbon signal [(δC 149.8, H-3′)], suggesting C-3′ as the position of the methoxy group. Accordingly, 3 (tomentin G) was determined to be a new compound, (2S)-5-hydroxy-2-(4,5dihydroxy-3-methoxyphenyl)-8-(4-hydroxy-4-methylpentyl)-8methyl-2,3,7,8-tetrahydropyrano-[3,2-g]-chromen-4-(6H)-one. Compound 6 was isolated as a yellowish powder. The molecular formula was determined to be C25H31O6 from the molecular ion peak [M + H]+ at m/z 427.2163 (calcd for C25H31O6 427.2121) in the HRESIMS, which was 16 amu less than that of 4, indicating that 6 has one less hydroxy group. The 1H NMR spectrum showed four olefinic methine proton signals [δH 6.89 (2H, d, J = 8.4 Hz, H-3′, 5′) and δH 7.31 (2H, d, J = 8.4 z, H-2′, 6′)] due to a para-substitution of the B-ring benzene unit. The structure of the new compound 6 (tomentin H) was determined as (2S)-5-hydroxy-2-(4-hydroxyphenyl)-8(4-hydroxy-4-methylpentyl)-8-methyl-2,3,7,8-tetrahydropyrano[3,2-g]-chromen-4-(6H)-one. Compound 8 was isolated as a yellow powder. The molecular formula was determined to be C26H29O8 from the molecular ion peak [M − H]− at m/z 469.1869 (calcd for C26H29O8 469.1862) in the HRESIMS. The NMR data of 8 were very similar to those of tanariflavanone D (9)26 except for the meta-coupled aromatic protons on a 1,2,3,5-tetrasubstituted benzene ring (except for δH 6.70, H-6′ and δH 6.72, H-2′) instead of an ABX system as the flavanone B-ring. From the HMBC spectrum, the methoxy proton signal (δH 3.84) showed a correlation with the oxygenated olefinic quaternary carbon signal (δC 149.0), suggesting C-4′ as the position where the methoxy group was substituted. The structure of 8 (tomentin I) was determined to be the new compound (2S)-6-(6-hydroxy3,7-dimethylocta-2,7-dienyl)-5,7,3′,5′-tetrahydroxy-4′-methoxyflavanone. Compound 13 was isolated as a yellowish powder. The molecular formula was determined to be C26H31O7 from the molecular ion peak [M − H]− at m/z 455.2100 (calcd for C26H31O7 455.2070) in the HRESIMS, which was 14 amu higher than that of 11, indicating 13 to have one more methyl group. From the HMBC spectrum, the methoxy proton signal (δH 3.88) showed a correlation with the oxygenated olefin quaternary carbon signal (δC 147.9), suggesting C-4′ as the location of the methoxy group. The structure of 13 was determined to be the new (2S)-6-(7-hydroxy-7-dimethyloctyl2-enyl)-5,7,5′-trihydroxy-4′-methoxyflavanone, and this compound was named tomentin J. Compound 14 was isolated as a yellowish-brown powder. The molecular formula was determined to be C27H33O8 from the molecular ion peak [M − H]− at m/z 485.2213 (calcd for C27H33O8 485.2175) in the HRESIMS, which was 14 amu more than in 10, indicating 14 to have one more methyl group. From

Chart 1

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RESULTS AND DISCUSSION From the methanol-soluble extract of dried P. tomentosa mature fruits, six new (1, 3, 6, 8, 13, and 14) and 13 previously reported dihydroflavonol (2, 4, 5, and 7) and flavanone (9−12 and 15−19) derivatives were isolated. The known compounds were identified by comparison of their physical and spectroscopic data with literature values as tomentin E (2), tomentin A (4), tomentin D (5), tomentin B (7),17 tanariflavanone D (9),18 isopaucatalinone B (10),19 prokinawan (11),20 5,7-dihydroxy-6(7-hydroxy-3,7-dimethyloct-2-en-1-yl)-2S-(4-hydroxyphenyl)3,4-dihydro-2H-1-benzopyran-4-one (12),21 4′-O-methyldiplacol (15), diplacone (16), 6-geranyl-5,7,3′,5′-tetrahydroxy-4′-methoxyflavanone (17),22 3′-O-methyl-5′-O-methyldiplacone (18), and 3′-O-methyldiplacone (19).23 Compounds 3, 6, 8, 13, and 14 showed common infrared absorbance bands at about 3715, 2968, and 1634 cm−1, suggesting the presence of hydroxy, carbonyl, and olefin groups. The 1H NMR signal at about δH 12.3 (1H, s) was assigned to the phenolic OH-5, which was strongly hydrogen-bonded to the C-4-carbonyl group in each case. These data suggested the aglycone of all of the isolated compounds to be a flavanone moiety. The electronic circular dichroism (ECD) spectra of all of compounds 3, 6, 8, 13, and 14 revealed a negative Cotton effect for the π → π* electronic transition at approximately 280−310 nm and a positive Cotton effect for the n → π* electronic transition at approximately 320−360 nm, which confirmed an S absolute configuration of C-2.24,25 Compound 1 was isolated as a yellowish powder. The molecular formula was determined to be C25H31O8 from the ion peak [M + H]+ at m/z 459.2077 (calcd for C25H31O8, 459.2019) in the HRESIMS, which was 16 amu higher than 4, indicating that 1 has one more hydroxy group than 4. The NMR spectrum of 1 (Table 1) was similar to that of 4, with the exception of the C-ring moiety. The presence of a 2,3-dihydroflavonol skeleton was deduced from the large coupling constant (J = 11.2 Hz) between the two oxygenated methine proton signals [δH 4.88 (1H, overlapped, H-2) and 4.48 (1H, d, J = 11.2 Hz, H-3)], which indicated a trans-diaxial relationship, and therefore, the absolute configuration had to be (2R, 3R) or (2S, 3S).17,25 Based on the negative ECD Cotton effect 2660


6.80, 6.84, 2.58, 1.64, 1.23, 1.48, 1.48, 1.48, 1.20, 1.13, 1.20,

4.88, 4.48, 5.92, 6.96,

3a

6b

8b

13b

14b

overlapped 5.26, brd (12.8) 5.41, dd (12.8, 2.4) 5.36, dd (12.8, 3.2) 5.40, dd (12.8, 2.8) 5.39, brd (12.0) d (11.2) 3.08, dd (17.2, 12.8) 2.70, brd (17.2) 3.16, dd (17.2, 13.2) 2.71, dd (17.2, 2.4) 3.15, dd (17.2, 12.8) 2.71, dd (17.2, 3.2) 3.19, dd (17.2, 12.8) 2.71, dd (17.2, 2.8) 3.19, dd (16.8, 12.0) 2.71, brd (16.8) s 6.01, s 6.05, s 6.06, s 6.08, s brs 7.31, d (8.4) 6.72, brs 7.17, d (1.6) 6.87, brs 6.64, brs 6.89, d (8.4) 6.87, d (8.4) d (8.0) 6.89, d (8.4) 6.70, brs 6.99, dd (8.4, 1.6) brd (8.0) 6.61, brs 7.31, d (8.4) 3.27, d (7.2) 3.27, d (7.2) 6.87, brs t-like (8.4) 2.58, t-like (8.0) 2.63, dd, (10.2, 5.6) 5.27, t (7.2) 5.25, t (7.2) 3.27, d (7.2) m 1.65, m 1.66, dd (10.2, 5.6) 1.98, m 1.94, t (7.2) 5.25, t (7.2) s 1.24, s 1.22, s 1.78, s 1.76, s 1.76, s m 1.50, m 1.51, m 1.58, m 1.94, t (7.2) 1.94, t (6.8) m 1.50, m 1.51, m 1.98, m 1.47, m 1.47, m m 1.50, m 1.45, m 3.97, t (6.4) 1.39, m 1.38, m s 1.22, s 1.17, s 4.87, d (1.6) s 1.13, s s 1.22, s 1.17, s 1.67, s 1.13, s 1.13, s 3.87, s 3.86, s 3.84, s 3.88, s 3.86, s

1a

a1 H NMR data were measured in methanol-d4 at 400 MHz. b1H NMR data were measured in acetone-d6 at 400 MHz. Chemical shifts (δ) are in ppm, and coupling constants (J in Hz) are given in parentheses. The assignments made were based on DEPT, COSY, and HMBC experiments.

2 3 8 2′ 3′ 5′ 6′ 1″ 2″ 4″ 5″ 6″ 7″ 9″ 4.72, d (1.6) 10″ MeO-3′ MeO-4′ MeO-5′

position

Table 1. 1H NMR Spectroscopic Data of Compounds 1, 3, 6, 8, 13, and 14

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significant inhibition than quercetin (Ki 11.9 μM), used as the positive control, followed by 8 > 3 > 14. Taken together, these results suggest those dihydroflavonols and flavanones, including the newly isolated compounds from P. tomentosa fruits, that exhibited potent anti-inflammatory activities and HNE inhibition may be good lead compounds for certain inflammation-related respiratory diseases.

the HMBC spectrum, the methoxy proton signal (δH 3.86) showed a correlation with the oxygenated olefin quaternary carbon signal (δC 148.7), suggesting C-5′ as the position of the methoxy group substitution. Therefore, the structure of the new compound 14 (tomentin K) was determined to be (2S)-6(7-hydroxy-7-dimethyloctyl-2-enyl)-5,7,4′-trihydroxy-3′,5′-dimethoxyflavanone. C-Geranylated flavonoids from P. tomentosa were reported earlier to have anti-inflammatory effects in vitro.27 The compounds isolated herein were tested for inhibitory effects on TNF-α-induced expression of interleukin 8 (IL-8), which is related to respiratory inflammatory diseases such as COPD in human alveolar basal epithelial cells.28 These results showed that the newly identified compounds inhibited TNF-α-induced IL-8 level significantly at a concentration of 2.5 μM, without detectable cell toxicity (Figure 1). Previous studies have shown

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting point was obtained using a Stuart SMP40 automatic melting point apparatus (Bibby Scientific, Stone, Staffordshire, UK). Optical rotations were measured on a JASCO P-1020 digital polarimeter (JASCO, Easton, MD, USA) with a 100 mm glass microcell. UV spectra were recorded on a SpectraMax M5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). ECD spectra were recorded on a JASCO J-815 CD spectrometer. IR spectra were obtained on a Tensor 27 FT-IR spectrometer (Bruker, Billerica, MA, USA) with KBr pellets. 1D (1H, 13C, and DEPT) and 2D (COSY, HMQC, and HMBC) NMR spectra were obtained on Bruker AM 400 and Bruker DRX 500 spectrometers (Bruker) with tetramethylsilane as the internal standard. HRESIMS were measured on an ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometer (UPLC-QTOFMS, Waters, Milford, MA, USA) in the negative-ion mode. Mediumpressure liquid chromatography (MPLC) was performed with a Spot Prep II 250 instrument (Armen, Paris, France). Preparative HPLC was performed using a PLC 2020 personal purification system (Gilson, Middleton, WI, USA). Column chromatography (CC) was performed using a silica gel (40−63 μm, Silicyle, Québec, Canada) and a Cosmosil 75 C18 Prep resin (75 μm, Nacalai Tesque, Kyoto, Japan). Preparative chromatography was performed using a YMC Actus Triart C18 column (5 μm, 250 × 20 mm, YMC, Kyoto, Japan), YMC Pack Pro C8 column (5 μm, 250 × 20 mm, YMC), YMC Pack ODS AQ HG column (10 μm, 250 × 20 mm, YMC), Inno C18 column (5 μm, 250 × 20 mm, Young Jin Biochrom, Seongnam, Korea), Kinetex Biphenyl column (5 μm, 250 × 21.2 mm, Phenomenex, Torrance, CA, USA), or a Synergi polar RP column (4 μm, 250 × 21.2 mm, Phenomenex). Plant Material. Mature fruits of Paulownia tomentosa were collected at Sancheong, Republic of Korea, in June 2015 and identified by Dr. Joong Ku Lee (Korea Research Institute of Bioscience & Biotechnology). A voucher specimen (KRIB 0059121-0059123) was deposited at the Plant Extract Bank of KRIBB in Daejeon, Korea. Extraction and Isolation. Dried fruits of P. tomentosa (3.8 kg) were extracted in 100% methanol (18 L × 3) at room temperature for 24 h. The extracts were combined and concentrated in vacuo at 40 °C to produce a dried extract (333 g, 8.8%), which was further extracted with n-hexane (86.1 g), CHCl3 (54.3 g), EtOAc (42.4 g), n-BuOH (31.6 g), and H2O (101.4 g). The EtOAc layer (2 g) was subjected to MPLC reversed-phase silica gel (YMC ODS-AQ, 10 μm, 220 g) using a stepwise MeOH−H2O gradient (0−5 min 50% MeOH, 5−65 min 50−100% MeOH, 65−80 min 100% MeOH, 15 mL/min, 80 min) to give 12 fractions (EA Frs. 1−12). This MPLC procedure was repeated 15 times using the same conditions before further isolation. Subfraction EA Fr. 2+3 (3.0 g) was further separated with a Synergi polar RP column using a gradient of CH3CN−H2O (30% → 40% → 100%) to give compounds 1 (13.0 mg) and 2 (36.5 mg). Subfraction EA Fr. 4+5 (8.0 g) was further separated with a YMC ODS AQ HG column using a gradient of MeOH−H2O (60% → 70% → 100%) to give six subfractions (Frs. 4+5-1 to 4+5-6). Subfraction EA Fr. 6 (382 mg) was further separated with a Synergi Polar RP column using a gradient of MeOH−H2O (55% → 60% → 100%) to give compounds 3 (5.5 mg) and 4 (25.6 mg). Subfraction EA Fr. 7 (2.7 g) was further separated with a YMC Triart C18 column using a gradient of CH3CN−H2O (35% → 100%) to give seven subfractions (EA Frs. 7-1 to 7-7). Subfraction EA Fr. 7-2 (39.7 mg) was further separated with a YMC Triart C18 column using a gradient of CH3CN−H2O (45% → 100%) to give compound 5 (10.4 mg). Subfraction EA Fr. 8 (45.3 mg) was further separated with a YMC Triart C18 column using a gradient

Figure 1. Effects of the new compounds isolated from Paulownia tomentosa mature fruits on cell viability and IL-8 production in TNF-αstimulated A549 cells. Bar graphs represent the means ± SD of three independent experiments (*p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control, TNF-α alone).

a strong correlation between HNE activity and the development of respiratory disease.29 In HNE enzyme assays, all of the compounds tested displayed significant HNE inhibition with IC50 values ranging from 2.4 to 74.7 μM (Figure 2 and Table 3). Compounds 4, 10, 11, 13, 17, and 19 displayed potent inhibition with IC50 values of 8.4, 2.4, 6.7, 6.3, 3.3, and 7.8 μM, respectively, and were compared with the positive controls quercetin (IC50 14.3 μM), luteolin (IC50 12.7 μM), and apigenin (IC50 46.1 μM). The activities of isolated compounds (1−19) demonstrated a positional requirement for the C-geranyl derivatives. However, hydroxylation at C-3 and methoxylation at C-5′ might tend to reduce the resultant HNE inhibitory potencies. Subsequently, a kinetic study was performed to examine the inhibition modes of the compounds showing significant HNE inhibition (IC50 < 25 μM; 3, 4, 8−11, 13, 14, 16, 17, 19, and quercetin) using the double-reciprocal Lineweaver−Burk and Dixon plots (Figure 2 and Figure S9, Supporting Information).30 All of the tested compounds were found to be noncompetitive inhibitors, as the Vmax values decreased without changing the Km with increasing concentrations of inhibitor. This effect can be seen directly from each graph, where −1/Km (the x-intercept) was intersected at a negative value of 1/[S] (Figure 2 and Table 3). According to kinetic enzymatic assays with the HNE substrate MeOSuc-AAPV-pNA, compound 17 exhibited the highest inhibitory activity (Ki 3.2 μM) via noncompetitive inhibition. Among the newly structurally characterized compounds, 13 (Ki 6.3 μM) showed a more 2662



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Figure 2. Concentration-dependent inhibition and graphical kinetic determination for the isolated compounds. Double-reciprocal Lineweaver−Burk plots for the inhibition of isolated compounds 3, 4, 8−11, 13, 14, 16, 17, and 19 on the HNE-catalyzed hydrolysis of MeOSuc-AAPV-pNA. The plot is expressed as 1/velocity versus 1/substrate (μM−1) without or with an inhibitor in the reaction solution. Each point represents the mean value of three replicates at substrate concentration. of CH3CN−H2O (50% → 100%) to give compound 6 (17.0 mg). Subfraction EA Fr. 9 (89.8 mg) was further separated with a YMC Triart C18 column using a gradient of CH3CN−H2O (50% → 100%) to give compound 7 (27.1 mg). Subfraction EA Fr. 10 (1.0 g) was further separated with a YMC-Pack Pro C8 column using a gradient of MeOH−H2O (60% → 100%) to give compounds 8 (5.5 mg), 9 (8.4 mg), 10 (10.2 mg), and 11 (83.8 mg). Subfraction EA Fr. 11 (15.0 g) was further separated with a YMC ODS AQ HG column using a gradient of MeOH−H2O (70% → 85% → 100%) to give four subfractions (EA Frs. 11-1 to 11-4). Subfraction EA Fr. 11-2 (2.0 g) was further separated with a Kinetex Biphenyl column using a gradient of MeOH−H2O (63% → 85% → 100%) to give eight subfractions (EA Fr. 11-2-1 to 11-2-8). Subfraction EA Fr. 11-2-5 (65.3 mg) was further separated with a Kinetex Biphenyl column using a gradient of CH3CN−H2O (45% → 50% → 100%) to give compound 12 (13.5 mg). Subfraction EA Fr. 11-2-8 (78.7 mg) was further separated with a Kinetex Biphenyl column using a gradient of CH3CN−H2O (45% → 50% → 100%) to give compounds 13 (15.0 mg) and 14 (11.2 mg). The CHCl3 layer (30.0 g) was fractionated on a silica gel column (10 × 30 cm, 230−400 mesh, 700 g) and eluted using hexane−EtOAc mixtures [15:1 (1.5 L), 10:1 (1.5 L), 8:1 (2.5 L), 5:1 (2 L), 4:1 (2 L), 3:1 (2 L), 1:1 (1 L), and pure EtOAc (2 L)] to give 20 pooled fractions (CHCl3 Fr. 1−20), which were combined based on comparison of their UPLC-PDA profiles. Subfraction CHCl3 Fr.

7+8 (235.3 mg) was further separated with an Inno C18 column using a gradient of MeOH−H2O (75% → 85% → 100%) to give compound 15 (89.2 mg). Subfraction CHCl3 Fr. 17 (1.0 g) was separated by silica gel CC (6 × 42 cm) using a gradient of CHCl3−acetone (20:1 → 10:1 → 5:1 → 2:1 → 1:1) to give compounds 18 (60.6 mg) and 19 (140.9 mg). Subfraction CHCl3 Fr. 18 (800 mg) was separated with a Kinetex Biphenyl column using a gradient of MeOH−H2O (71% → 100%) to give compounds 16 (89.2 mg) and 17 (55.4 mg). The purity (>98%) of each isolated compound (1−19) was confirmed by UPLC-PDA-QTOF-MS. Tomentin F (1): yellowish powder; mp 124−125 °C; [α]20D +4.0 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 232 (sh), 294 (3.52), 336 (sh); ECD (MeOH, c 2.0 × 10−3 M) λmax (Δε) 340 (+3.68), 310 (−3.52), 255 (+3.16) nm; IR (KBr) νmax 3175, 2968, 1634 cm−1; 1 H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 459.2077 [M + H]+, calcd for C25H31O8, 459.2019. Tomentin G (3): brownish powder; mp 144−145 °C; [α]20D −0.7 (c 0.05, CH3OH); UV (MeOH) λmax (log ε) 236 (sh), 292 (3.72), 344 (sh); ECD (MeOH, c 2.1 × 10−3 M) λmax (Δε) 350 (+0.27), 305 (−1.95) nm; IR (KBr) νmax 3175, 2919, 1636 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 473.2091 [M + H]+, calcd for C26H33O8, 473.2175. Tomentin H (6): yellowish powder; mp 133−134 °C; [α]20D +4.8 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 232 (sh), 292 (4.12), 2663



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Table 2. 13C NMR Spectroscopic Data of Compounds 1, 3, 6, 8, 13, and 14a

Table 3. Inhibitory Effects of Isolated Compounds 1−19 on HNE Enzyme Activity

position

1b

3b

6c

8c

13c

14c

compound

inhibitory effect (IC50 μM)a

2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ MeO-3′ MeO-4′ MeO-5′

85.3 73.8 198.4 162.4 110.9 167.2 95.9 162.3 101.6 130.2 116.0 146.4 147.2 116.2 121.0 18.0 41.1 71.8 27.4 43.0 20.0 45.7 71.8 29.3 29.3

80.9 44.5 198.0 162.6 110.5 166.1 95.6 162.6 103.2 131.3 103.2 149.8 135.7 146.8 108.4 17.9 41.1 71.8 27.4 43.0 20.0 45.8 71.8 29.3 29.3 56.8

79.9 43.7 197.3 162.3 110.3 165.0 95.5 161.9 103.1 130.9 129.0 116.1 158.6 116.1 129.0 17.2 41.1 72.7 27.5 43.3 19.6 45.6 70.3 29.7 29.7

80.3 43.8 197.3 103.1 162.3 109.1 164.9 95.4 162.0 130.9 108.4 135.0 149.0 146.3 102.9 21.6 123.4 135.2 16.3 34.7 36.5 75.3 149.4 110.3 17.9

80.2 43.8 197.3 103.1 162.3 109.1 164.9 95.4 162.0 131.4 120.5 115. 7 147.9 148.4 111.2 21.6 123.3 135.3 16.1 41.1 23.4 44.4 70.1 29.7 29.7

80.4 44.0 197.3 103.0 162.0 109.1 162.0 95.4 162.3 130.4 105.3 148.7 137.1 148.7 105.3 21.6 123.3 135.4 16.1 41.1 23.4 44.4 70.0 29.7 29.7 56.7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 quercetind luteolind apigenind

35.6 ± 5.3 46.6 ± 4.2 17.9 ± 1.6 8.4 ± 0.2 >100 >100 >100 13.6 ± 2.1 15.4 ± 3.3 2.4 ± 1.0 6.7 ± 2.7 74.7 ± 8.5 6.3 ± 0.9 23.3 ± 3.2 59.1 ± 6.6 22.8 ± 0.7 3.3 ± 0.6 71.3 ± 7.5 7.8 ± 0.2 14.3 ± 0.2 12.7 ± 0.5 46.1 ± 0.9

56.6

56.3

inhibition type (Ki μM)b NTc NT noncompetitive noncompetitive NT NT NT noncompetitive noncompetitive noncompetitive noncompetitive NT noncompetitive noncompetitive NT noncompetitive noncompetitive NT noncompetitive noncompetitive noncompetitive noncompetitive

(13.1 ± 5.8) (12.5 ± 3.5)

(12.4 ± 0.7) (11.7 ± 3.4) (5.3 ± 0.9) (8.3 ± 0.7) (6.3 ± 1.4) (19.5 ± 7.5) (15.7 ± 4.1) (3.2 ± 1.2) (4.3 ± 1.6) (11.9 ± 0.7) (10.1 ± 0.3) (49.9 ± 0.4)

a

All compounds were examined in a set of experiments repeated three times; IC50 values of compounds represent the concentration that caused 50% enzyme activity loss. bValues of inhibition constant. cNT, not tested. dPositive control. and H2O) or the test compounds, A549 cells (1 × 104 cells/cm2) were seeded in the growth medium and incubated for 16 h. Subsequently, the medium was changed to growth medium without FBS, and cells were incubated for another 16 h. Cell Viability Assay. A549 cells were seeded in 96-well plates in the growth medium used at a density of about 1 × 104 cells/well and grown for 16 h. The medium was subsequently changed to growth medium without FBS. After a 16 h incubation cells were treated with solvents of different polarity (hexane, CHCl3, EtOAc, BuOH, and H2O) or the test compounds from P. tomentosa in the presence or absence of TNF-α for 24 h. Cell viability was measured in triplicate using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA) according to the manufacturer’s protocol. The absorbance was measured using a VersaMax microplate reader (Molecular Devices), and the measured absorbance was converted to a percentage (%) of the negative control absorbance value. Analysis of Enzyme-Linked Immunosorbent Assay (ELISA). To evaluate human IL-8 or IL-6 production, human IL-8 and IL-6 ELISA kits (BD Pharmingen, San Diego, CA, USA) were used according to the manufacturer’s protocol.28,31 The absorbance was measured at 450 nm using a VersaMax microplate reader (Molecular Devices). The measured absorbance was converted to a percentage (%) of the positive control absorbance value. Human Neutrophil Elastase Inhibitory Assay. Human neutrophil elastase kinetics was assayed, according to standard procedures, by indirectly measuring the hydrolysis of N-methoxysuccinylAla-Ala-Pro-Val-p-nitroanilide (MeOSuc-AAPV-pNA), involving monitoring spectrometrically the formation of p-nitroanilide.29 Briefly, the reaction mixture contained 20 μL of 1.20 mM substrate (N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide) dissolved in 20 mM Tri-HCl buffer (pH 7.4), with the test sample dissolved in 5% DMSO (5 μL), and 5 μL of enzyme solution (ca. 0.01 units/mL elastase from human neutrophils (EC 3. 4. 21. 37, Athens Research & Technology, Athens, GA, USA). The reaction mixture was incubated for 30 min at 37 °C, and then the absorbance at 405 nm was measured immediately for time zero using a SpectraMax M5 multi-mode microplate reader

56.7

a

The assignments were based on DEPT, COSY, and HMBC experiments. b13C NMR data were measured in methanol-d4 at 100 MHz. c13C NMR data were measured in acetone-d6 at 100 MHz. 342 (sh); ECD (MeOH, c 4.6 × 10−5 M) λmax (Δε) 336 (+3.22), 288 (−6.60) nm; IR (KBr) νmax 3175, 2916, 1634 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive) m/z 427.2163 [M + H]+, calcd for C25H31O6, 427.2121. Tomentin I (8): yellow powder; mp 160−161 °C; [α]20D −3.4 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 236 (sh), 292 (3.82), 337 (sh); ECD (MeOH, c 6.3 × 10−5 M) λmax (Δε) 331 (+1.07), 294 (−5.37) nm; IR (KBr) νmax 3358, 2919, 1636 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (negative) m/z 469.1869 [M − H]−, calcd for C26H29O8, 469.1862. Tomentin J (13): yellowish powder; mp 143−144 °C; [α]20D −5.3 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 234 (sh), 290 (4.21), 336 (sh); ECD (MeOH, c 7.0 × 10−5 M) λmax (Δε) 330 (+1.45), 293 (−4.80) nm; IR (KBr) νmax 3220, 2936, 1636 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (negative) m/z 455.2100 [M − H]−, calcd for C26H31O7, 455.2070. Tomentin K (14): yellowish-brown powder; mp 124−125 °C; [α]20D −3.5 (c 0.1, CH3OH); UV (MeOH) λmax (log ε) 234 (sh), 292 (4.08), 340 (sh); ECD (MeOH, c 4.1 × 10−5 M) λmax (Δε) 333 (+0.61), 292 (−3.04) nm; IR (KBr) νmax 3220, 2917, 1634 cm−1; 1 H and 13C NMR data, see Tables 1 and 2; HRESIMS (negative) m/z 485.2213 [M − H]−, calcd for C27H33O8, 485.2175. Cell Preparation and Culture. A549 cells (human alveolar basal epithelial adenocarcinoma) were obtained from the American Type Culture Collection (CCL-185, Manassas, VA, USA). Cells were cultured in growth medium [RPMI 1640 medium (Hyclone, Logan, UT, USA)] supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 units/mL penicillin, and 100 μg/mL streptomycin (Hyclone) at 37 °C in a humidified 5% CO2 atmosphere. For treatment with the polar solvent negative controls (hexane, CHCl3, EtOAc, BuOH, 2664



Article

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(Molecular Devices). The increase in absorbance from time zero was recorded for kinetic analysis. Statistical Analysis. All HNE kinetic measurements were made in triplicate. The results were subject to variance analysis using SigmaPlot. The significance was determined using a two-tailed Student’s t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00325. Spectroscopic data for compounds 1, 3, 6, 8, 13, and 14; UPLC-CAD spectra, anti-inflammatory effects and HNE kinetics (DOCX)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (B. Y. Hwang): [email protected]. Tel: +82-43-261-2814. Fax: +82-43-268-2732. *E-mail (S.-R. Oh): [email protected]. Tel: +82-43-2406111. Fax: +82-43-240-6119. ORCID

Hyung Won Ryu: 0000-0001-5291-3942 Sei-Ryang Oh: 0000-0002-9769-4779 Author Contributions ⊥

H. W. Ryu and Y. J. Park contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program (KGM 1221713) and Ministry for Health and Welfare (HI14C1277) of the Republic of Korea.

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REFERENCES

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