Inhibitory Activity of Chemical Constituents from Arenaria serpyllifolia on Nitric Oxide Production

• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Acid hydrolysis of compounds 2 and 3
• Cell culture and viability assay
• Measurement of NO production
• Supporting information
• Results and Discussion
• Acknowledgements
• References

Abstract

Five new compounds, including one new xanthone, 1-hydroxy-5-methoxyxanthone 6-O-β-D-glucopyranoside (1), one new lignan, 3-(β-D-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-acetoxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran (2), and three new γ-pyrones, japonicumone A 4′-O-β-D-glucopyranoside (3), japonicumone B 3′-O-β-D-glucopyranoside (4), and japonicumone B 4′-O-β-D-glucopyranoside (5), together with eight known compounds (6–13) were isolated from the whole plants of Arenaria serpyllifolia. Their structures were elucidated on the basis of extensive spectroscopic analysis (UV, IR, HRESIMS, 1D- and 2D-NMR, and CD) as well as chemical methods. The isolated compounds were evaluated for their inhibitory effects on nitric oxide production in lipopolysaccharide-activated RAW 264.7 macrophages. Compounds 1–5, sacranoside A (9), and pedunculoside (13) showed potential nitric oxide inhibitory activities with IC₅₀ values ranging from 14.92 µM to 52.23 µM.

Introduction

Nitric oxide (NO), one of the important proinflammatory mediators, is speculated to participate in provoking and maintaining some inflammatory disorders, such as rheumatoid arthritis, chronic hepatitis, and pulmonary fibrosis [1], [2], [3]. Suppression of the release of NO and other inflammatory mediators plays a critical role in the treatment of such diseases. Therefore, the in vitro NO production inhibitory screening model has been widely used for searching for bioactive components with potential anti-inflammatory effect from natural medicines [4], [5], [6].

Arenaria serpyllifolia L. (Xiaowuxincai in Chinese) is an annual or biennial herbaceous plant belonging to the Caryophyllaceae family. It has been used in the southeast of China as a traditional Chinese medicine against fever and for detoxification, improving eyesight, and relieving cough [7]. Chemical studies showed that the main components from Arenaria species were flavonoids, triterpenoid saponins, steroids, alkaloids, phenylpropanoids, and cyclic peptides [8], [9], [10], [11], [12]. Up to now, there has been no report on the chemical ingredients of A. serpyllifolia. During our ongoing program of searching for new anti-inflammatory agents from natural sources, the extract of A. serpyllifolia was found to show a potent inhibition against NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, thus, a systematic phytochemical investigation was then carried out in order to clarify the bioactive constituents of A. serpyllifolia. As a result, five new compounds including one new xanthone (1), one new lignan (2), and three new γ-pyrones (3–5), together with eight known compounds (6–13) were obtained ([Fig. 1]). This paper describes the isolation and structural elucidation of compounds 1–5, as well as their inhibitory activities against the NO production induced by LPS in RAW 264.7 cells.

Materials and Methods

General experimental procedures

UV spectra were taken on a Shimadzu UV-2450 spectrophotometer. IR spectra were obtained on a Thermo Nicolet Nexus 470 spectrometer with KBr pellets. HRESIMS spectra were run on a Shimadzu LCMS-IT-TOF System. 1D- and 2D-NMR spectra were recorded on a Varian UNITY INOVA 500 spectrometer. CD spectra were measured on a JASCO J-810 spectrometer. Column chromatographies were performed over silica gel (200–300 mesh; Qingdao Haiyang Chemical Co. Ltd.) and ODS (Merck) on an Agela Technologies CHEETAH Flash System and BÜCHI Flash System [BÜCHI pump module C605, BÜCHI control unit C-620, BÜCHI UV photometer C-635, BÜCHI fraction collector C-660, and BÜCHI Sepacore control software (version 1.0.3000.1)]. Semi-preparative HPLC was carried out on a Waters 600 instrument with GRACE Prevail C18 (5 µm, 10 × 250 mm) and Agela Venusil XBP RP-8 (5 µm, 10 × 250 mm) columns, detected with a Waters 2487 dual λ absorbance detector and ELSD detector (Alltech). Sephadex LH-20 for chromatography was purchased from Pharmacia Fine Chemical Co. Ltd. Fractions were monitored by TLC, and spots were visualized by heating silica gel plates after spraying with 10 % H₂SO₄ in EtOH. HPLC analysis was carried out using an Agilent 1200 series liquid chromatography, equipped with a G1322A degasser, a G1311A quatpump, a G1316B column compartment, a G1315C diode array detector, and a G1329A autosampler. Optical rotation was measured on a Chiralyser-MP optical rotation detector produced by IBZ Messtechnik Company. The optical density of cells was measured using a microplate reader (Tecan Trading AG). Quercetin (purity ≥ 98 %) was purchased from the National Institute for Food and Drug Control, China. The purities of all the isolated compounds used for bioassay were more than 95 % and were determined by ¹H NMR and HPLC techniques. Mouse RAW 264.7 monocytic cells were obtained from the Peking Union Medical College (PUMC) Cell Bank. LPS (Escherichia coli 0111:B4) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma. Griess reagent was purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Plant material

The whole plants of Arenaria serpyllifolia were collected in Pingnan, Fujian province, PR China, in October 2009, and identified by one of the authors (PF Tu). A voucher specimen (No. GS20091008) was deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine.

Extraction and isolation

The air-dried whole plants of A. serpyllifolia (4.5 kg) were cut and extracted thrice with 70 % aqueous ethanol, 3 h for each time. After evaporation of the solvent under reduced pressure, the residue (950 g) was suspended in water (5 L) and partitioned with petroleum ether (5 L) and EtOAc (5 L) successively, each for three times, to afford petroleum ether extract (9 g) and EtOAc extract (285 g).

The EtOAc extract (280 g) was subjected to silica gel column (15 × 100 cm, 4 kg) eluted with CHCl₃-MeOH (19 : 1, 9 : 1, 8 : 2, 7 : 3, 1 : 1, v/v, each 40 L) to yield 5 fractions (Frs. EA-EE). Fr. EA (5 g) was further fractionated into 8 subfractions (Frs. EA-1 → EA-8) by chromatography over a silica gel column (3 × 30 cm, 100 g) eluted with petroleum ether-EtOAc (2 : 1, 1 : 1, v/v, each 1.5 L). Fr. EA-5 (100 mg) was separated on Sephadex LH-20 (2 × 50 cm, 30 g) using CHCl₃-MeOH (1 : 1, v/v) as the eluent and then applied to semi-preparative HPLC (MeCN-H₂O, 67 : 33, v/v, flow rate: 2 mL/min) on a GRACE Prevail C18 column to yield compound 8 (5 mg). Fr. EB (50 g) was separated by MPLC (silica gel, 100 × 460 mm Agela glass column, solvent system: CHCl₃-MeOH, 98 : 2 → 50 : 50, V/V, flow rate: 50 mL/min, max pressure: 10 bar) to give 6 subfractions (Frs. EB-A → EB-F). Fr. EB-A was separated on MCI column (2.5 × 36 cm, 100 g), with gradient of MeOH-H₂O (6 : 4, 7 : 3, 8 : 2, 9 : 1, 10 : 0, v/v, each 200 mL) as eluent to give 7 fractions (Frs. EB-AA → EB-AG). Fr. EB-AC was applied to semi-preparative HPLC (MeCN-H₂O, 67 : 33, v/v, flow rate: 3 mL/min) on a GRACE Prevail C18 column to yield compounds 4/5 (2 mg) and compound 3 (4 mg). Fr. EB-AD was purified by Sephadex LH-20 (2 × 50 cm, 30 g) to yield compound 13 (5 mg). Fr. EB-AF was subjected to semi-preparative HPLC (MeCN-H₂O, 52 : 48, v/v, flow rate: 3 mL/min) on a GRACE Prevail C18 column to yield compound 12 (5 mg). Fr. EB-C was separated on ODS C18 column (2.5 × 37 cm, 100 g), using MeOH-H₂O (2 : 8, 3 : 7, 4 : 6, 5 : 5, v/v, each 200 mL) as eluent to give 17 subfractions (Frs. EB-C01 → EB-C17). Compound 11 (2 mg) was obtained by recrystallization from Fr. EB-C13. Fr. EB-E was separated on ODS C18 column (2.5 × 37 cm, 100 g), using MeOH-H₂O (2 : 8, 3 : 7, 4 : 6, 5 : 5, v/v, each 200 mL) as eluent to give 6 subfractions (Frs. EB-EA → EB-EF). Compound 10 (5 mg) was obtained by recrystallization from Fr. EB-EB.

The water layer (700 g) was separated on a D101 macroporous adsorption resin eluted with aqueous EtOH in gradient (0 %, 30 %, 50 %, 95 %, v/v, each 40 L). The fraction of 50 % aqueous EtOH (70 g) was separated on MPLC (silica gel, 15 × 460 mm, Agela glass column; solvent system: CHCl₃-MeOH-HOAc, 9 : 1 : 0.1 → 1 : 1 : 0.1, V/V, flow rate: 50 mL/min, max pressure: 10 bar) to give 7 subfractions (Frs. W5-1 → W5–7). Fr. W5-2 was separated further on MPLC (flash column C18, 40 g; solvent system: MeOH-H₂O, 3 : 7 → 10 : 0, v/v, flow rate: 5 mL/min, max pressure: 50 bar) to give 5 subfractions (Frs. W5-2A → W5-2E). Fr. W5-2D was separated by semi-preparative HPLC (MeCN-H₂O, 31 : 69, v/v, flow rate: 2 mL/min) on an Agela Venusil XBP RP-8 column and then purified by Sephadex LH-20 to yield compound 6 (2 mg). Fr. W5-3 was separated on MPLC (C18 flash column, 40 g; solvent system: MeOH-H₂O 25 : 75 → 100 : 0, v/v, flow rate: 5 mL/min, max pressure: 50 bar) to give 6 subfractions (Frs. W5-3A → W5-3F). Fr. W5-3D was separated by semi-preparative HPLC on a GRACE Prevail C18 column (MeCN-H₂O, 27 : 73, v/v, flow rate: 2 mL/min) and then subjected to Sephadex LH-20 to yield compound 1 (1 mg). Fr. W5-5 was separated by MPLC (C18 flash column, 40 g; solvent system: MeOH-H₂O, 2 : 8 → 10 : 0, v/v, flow rate: 5 mL/min, max pressure: 50 bar) to give 7 subfractions (Frs. W5-5A → W5-5G). Fr. W5-5D was purified by Sephadex LH-20 and eluted with MeOH to give 11 fractions (Frs. W5-5DA → W5-5DK). Fr. W5-5DB was fractioned by semi-preparative HPLC on a GRACE Prevail C18 column (MeCN-H₂O, 28 : 72, v/v, flow rate: 2 mL/min) into 7 fractions (Frs. W5-5DBA → W5-5DBG). Fr. W5-5DBD was separated by semi-preparative HPLC (MeCN-H₂O, 18 : 82, v/v, flow rate: 2 mL/min) on GRACE Prevail C18 column to yield compound 9 (5 mg). Fr. W5-5DBG was separated by semi-preparative HPLC (MeCN-H₂O, 34 : 66, v/v, flow rate: 2 mL/min) on a GRACE Prevail C18 column to yield compound 2 (4 mg). Fr. W5-5F was separated on MPLC (C18 flash column, 20 g; solvent system: MeOH-H₂O, 5 : 5 → 10 : 0; V/V, flow rate: 5 mL/min, max pressure: 50 bar) to give 10 subfractions (Frs. W5-5FA → W5-5FJ). Fr. W5-5FE was separated further by semi-preparative HPLC (MeCN-H₂O, 42 : 58, v/v, flow rate: 3 mL/min) on GRACE Prevail C18 column to yield compound 7 (2 mg).

1-Hydroxy-5-methoxyxanthone 6-O-β-D-glucopyranoside (1): yellow amorphous powder; [α]_D ²⁰ − 19.18 (c 0.04, MeOH); UV (MeOH): λ _max (log ε): 302 (3.22), 354 (2.89) nm; IR (KBr) υ _max 3445, 2970, 1648, 1055, 1033, 1012 cm⁻¹; ¹H-NMR (DMSO-d ₆, 500 MHz): δ 12.66 (1-OH), 6.83 (1H, d, J = 8.2 Hz, H-2), 7.72 (1H, t, J = 8.2 Hz, H-3), 7.13 (1H, d, J = 8.2 Hz, H-4), 7.36 (1H, d, J = 9.1 Hz, H-7), 7.88 (1H, d, J = 9.1 Hz, H-8), 3.96 (3H, s, 5-OCH₃), 5.16 (1H, d, J = 8.5 Hz, H-1′), 3.35 (1H, overlapped, H-2′), 3.32 (1H, overlapped, H-3′), 3.21 (1H, t, J = 7.0 Hz, H-4′), 3.42 (1H, m, H-5′), 3.70 (1H, dd, J = 12.0, 4.0 Hz, H-6′a), 3.48 (1H, dd, J = 12.0, 7.0 Hz, H-6′b). ¹³C-NMR (DMSO-d ₆, 125 MHz): δ 161.0 (C-1), 110.4 (C-2), 137.2 (C-3), 107.4 (C-4), 155.8 (C-4a), 150.1 (C-4b), 137.2 (C-5), 156.1 (C-6), 112.8 (C-7), 120.6 (C-8), 115.3 (C-8a), 181.1 (C-9), 107.9 (C-9a), 61.2 (5-OCH₃), 100.2 (C-1′), 73.2 (C-2′), 76.2 (C-3′), 69.5 (C-4′), 77.3 (C-5′), 62.7 (C-6′); HRESIMS: m/z 419.1003 [M – H]⁻ (calcd. for C₂₀H₁₉O₁₀, 419.0997).

3-(β-D-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-acetoxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran (2): light yellow amorphous powder; [α]_D ²⁰ − 24.03 (c 0.26, MeOH); UV (MeOH): λ _max (log ε): 295 (2.88) nm; IR (KBr) υ _max 3744, 3419, 2928, 1727, 1602, 1514, 1456, 1248, 1031 cm⁻¹; CD (MeOH): λ _max (Δε) 292 (− 2.40), 241 (− 3.67), 224 (+ 0.44); ¹H-NMR (DMSO-d ₆, 500 MHz): δ 5.50 (1H, d, J = 6.2 Hz, H-2), 3.55 (1H, m, H-3), 6.76 (1H, d, J = 2.0 Hz, H-4), 6.72 (1H, d, J = 2.0 Hz, H-6), 3.62 (1H, t, J = 9.5 Hz, 3-CH₂O-), 4.05 (1H, dd, J = 9.5, 5.5 Hz, 3-CH₂O-), 2.56 (1H, t, J = 8.0 Hz, H-1′), 1.85 (1H, dt, J = 8.0, 6.5 Hz, H-2′), 4.00 (1H, t, J = 6.5 Hz, H-3′), 6.94 (1H, d, J = 1.9 Hz, H-2″), 6.74 (1H, d, J = 8.2 Hz, H-5″), 6.78 (1H, dd, J = 8.2, 1.9 Hz, H-6″), 4.24 (1H, d, J = 7.8 Hz, H-1′″), 3.10 (1H, m, H-2′″), 3.00 (1H, m, H-3′″), 3.02 (1H, m, H-4′″), 3.15 (1H, m, H-5′″), 3.43 (1H, m, H-6′″a), 3.67 (1H, d, J = 11.7 Hz, 6′″b), 3.74 (3H, s, 3-OCH₃), 3.77 (3H, s, 7-OCH₃), 2.01 (3H, s, CH₃ CO-); ¹³C-NMR (DMSO-d ₆, 125 MHz): δ 86.7 (C-2), 51.0 (C-3), 116.8 (C-4), 134.1(C-5), 112.6 (C-6), 143.4 (C-7), 145.7 (C-1a), 128.3 (3a), 70.1 (3-CH₂O-), 31.3 (C-1′), 30.1 (C-2′), 63.3 (C-3′), 132.4 (C-1″), 110.3 (C-2″), 147.4 (C-3″), 146.2 (C-4″), 115.2 (C-5″), 118.2 (C-6″), 103.0 (C-1′″), 73.6 (C-2′″), 76.8 (C-3′″), 70.6 (C-4′″), 77.0 (C-5′″), 61.1 (C-6′″). 55.7 (3-OCH₃), 55.6 (7-OCH₃), 170.4 (CH₃ CO-), 20.7 (CH₃ CO-); HRESIMS: m/z 587.2095 [M + Na]⁺ (calcd. for C₂₈H₃₆O₁₂Na, 587.2099).

Japonicumone A 4′-O-β-D-glucopyranoside (3): light yellow amorphous powder; [α]_D ²⁰ − 30.53 (c 0.09, MeOH); UV (MeOH): λ _max (log ε): 338 (3.59) nm; IR (KBr) υ _max 3460, 2959, 2867, 1674, 1511, 1459, 1401, 1247, 1215, 1057, 1033 cm⁻¹; ¹H-NMR and ¹³C-NMR data, see [Tables 1] and [2]; HRESIMS: m/z 435.1677 [M + H]⁺ (calcd. for C₂₂H₂₇O₉, 435.1679).

Japonicumone B 3′-O-β-D-glucopyranoside (4) and japonicumone B 4′-O-β-D-glucopyranoside (5): light yellow amorphous powder; [α]_D ²⁰ − 62.00 (c 0.10, MeOH); UV (MeOH): λ _max (log ε): 342 (5.12) nm; IR (KBr) υ _max 3392, 2947, 2839, 1656, 1453, 1410, 1032, 692 cm⁻¹; ¹H-NMR and ¹³C-NMR data, see [Tables 1] and [2]; HR-ESI-MS: m/z 451.1610 [M + H]⁺ (calcd. for C₂₂H₂₇O₁₀, 451.1599).

Acid hydrolysis of compounds 2 and 3

Compound 2 or 3 (1 mg for each compound) was heated in 2 mol/L aqueous CF₃COOH (5 mL) at 110 °C for 6 h in a sealed tube. Then, the sugar and aglycone were separated by liquid-liquid partitioning between chloroform and water. The aqueous layer was repeatedly concentrated with MeOH until neutral, and the residue was separated by Sep-Pak C18 cartridge column (H₂O → MeOH). The H₂O-eluted fraction was subjected to HPLC analysis under following conditions: HPLC column, Alltech Prevail carbohydrate ES, 5 µm, 4.6 mm i. d. × 250 mm; detection, Chiralyser-MP optical rotation detector (IBZ Messtechnik Company); mobile phase, CH₃CN–H₂O (85 : 15, v/v); flow rate: 1.0 mL/min. Detection of D-glucose from 2 and 3 was carried out by comparison of their retention times and optical rotations with those of the authentic sample (glucose, t_R 16.02 min, positive).

Cell culture and viability assay

Cell maintenance, experimental procedures, and viability assay were the same as previously described [13]. Briefly, cells were seeded at the density of 5 × 10⁴ cells per well in 48-well culture plate, then treated with various concentrations of each compound and LPS (1.0 µg/mL). The cell viability of the cultured cells was detected by MTT method. Briefly, RAW 264.7 cells were incubated with 500 µL MTT solution (0.5 mg/mL in medium) for 4 h at 37 °C, and then the supernatants were removed and the residues were dissolved in 500 µL DMSO. The absorbance was detected at 540 nm using a microplate reader. The results were displayed in percentage of control samples. Data are presented as the mean ± SD (n = 3) for three independent experiments. Quercetin was used as the positive control [13].

Measurement of NO production

The NO concentration was detected by the Griess reagent [14]. Briefly, RAW 264.7 cells were treated with LPS (1.0 µg/mL) and compounds for 24 h. After that, 400 µL of culture supernatant was allowed to react with 100 µL of Griess reagent (1 % sulfanilamide/0.1 % naphthylethylene diamine dihydrochloride/2 % phosphoric acid) for 10 min at room temperature in the dark. Then, the optical density (100 µL per well) was measured at 540 nm using a microplate reader. Sodium nitrite in medium was used to calculate a standard curve in the assay.

Inhibition (%) = (A_{LPS treated} – A_untreated)/(A_{LPS treated} – A_{LPS + sample treated}) × 100.

The experiments were performed in triplicate. Quercetin was used as the positive control.

Supporting information

The 1D- and 2D-NMR, and IR spectra of compounds 1–5, as well as the CD spectrum of compound 2 are available as Supporting Information.

Results and Discussion

Compound 1 was obtained as yellow amorphous powder, and its molecular formula was elucidated as C₂₀H₂₀O₁₀ from the negative HR-ESI-MS (m/z 419.1003 [M – H]⁻, calcd. 419.0997), indicating eleven degrees of unsaturation. The IR spectrum of 1 suggested the existence of hydroxy groups (3445 cm⁻¹) and a hydrogen-bonded carbonyl group (1648 cm⁻¹). In the ¹H-NMR spectrum of 1, the aromatic signals [δ _H 6.83 (1H, d, J = 8.2 Hz, H-2), 7.13 (1H, d, J = 8.2 Hz, H-4), 7.72 (1H, t, J = 8.2 Hz, H-3), 7.36 (1H, d, J = 9.1 Hz, H-7), and 7.88 (1H, d, J = 9.1 Hz, H-8)] indicated that 1 was a 1,5,6-trisubstituted xanthone [9]. In addition, a hydrogen-bonded hydroxyl [δ _H12.66 (1H, s, 1-OH)], a methoxyl [δ _H 3.96 (3H, s), δ _C 61.2] and an anomeric proton and carbon were also observed from the NMR spectra of 1. The ¹³C-NMR data [δ _C 100.2 (C-1′), 73.2 (C-2′), 76.2 (C-3′), 69.5 (C-4′), 77.3 (C-5′), 62.7 (C-6′)] and the coupling constant (J = 8.5 Hz) of the anomeric proton (δ _H 5.16) indicated the existence of a β-glucopyranosyl moiety. The NMR data of 1 were very similar to those of 1,5-dihydroxyxanthone 6-O-β-D-glucopyranoside isolated from Hypericum japonicum [15], except that a hydroxy group was replaced by a methoxyl. The linkage position of the methoxyl group was elucidated from the HMBC correlation of 5-OCH₃ [δ _H 3.96 (3H, s)] with C-5 [δ _C 137.2]. Consequently, the structure of 1 was elucidated as 1-hydroxy-5-methoxyxanthone 6-O-β-D-glucopyranoside.

Compound 2 was obtained as light yellow amorphous powder, and its molecular formula was determined to be C₂₈H₃₆O₁₂ based on its positive HR-ESI-MS (m/z 587.2095 [M + Na]⁺, calcd. 587.2099), indicating eleven degrees of unsaturation. The IR spectrum of 2 suggested the existence of hydroxy groups (3744, 3419 cm⁻¹), a carbonyl group (1727 cm⁻¹), and phenyl groups (1602, 1514, 1456 cm⁻¹). In the ¹H NMR spectrum of 2, a set of AMX coupled aromatic protons [δ _H 6.74 (1H, d, J = 8.2 Hz), 6.78 (1H, dd, J = 1.9, 8.2 Hz), and 6.94 (1H, d, J = 1.9 Hz)], a pair of meta-coupled aromatic protons [δ _H 6.72 (1H, d, J = 2.0 Hz) and 6.76 (1H, d, J = 2.0 Hz)], two methoxyls [δ _H 3.74 and 3.77 (both s, each 3H)], and an anomeric proton [δ _H 4.24 (1H, d, J = 7.8 Hz)] were observed. The NMR data of 2 were similar to those of 3-(β-D-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran (6) isolated from Lactuca indica [16], except for the presence of a set of additional acetyl group signals [δ _H 2.01 (3H, s), δ _C 170.4, 20.7] in 2, along with 2.3 ppm down-field shift of C-3′ of the aglycone. In the HMBC spectrum, the correlation of H-3′ [δ _H 4.00 (2H, t, J = 6.5 Hz)] with the carbonyl of the acetyl [δ _C 170.4] further supported the linkage position of the acetyl group. The coupling constant (J = 6.2 Hz) of H-2 and H-3 indicated a trans-conformation [17]. Combining with the negative cotton effects at 292 and 241 nm, and the positive cotton effect at 224 nm in the CD spectrum, the absolute configurations of C-2 and C-3 were elucidated to be 2R and 3S [17], [18]. Thus, the structure of compound 2 was deduced as 3-(β-D-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-acetoxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran.

Compound 3, obtained as light yellow amorphous powder, exhibited a protonated ion at m/z 435.1677 [M + H]⁺ (calcd. 435.1679) in the HR-ESI-MS spectrum, corresponding to a molecular formula of C₂₂H₂₆O₉. The IR spectrum suggested the existence of a hydroxy group (3460 cm⁻¹) and a carbonyl group (1674 cm⁻¹). The ¹H-NMR spectrum of 3 showed a group of AA′BB′ aromatic protons [δ _H 7.13 (2H, d, J = 8.5 Hz) and 7.83 (2H, d, J = 8.5 Hz)], an olefinic proton [δ _H 6.98 (1H, s)], a methine proton [δ _H 4.62 (1H, q, J = 6.6 Hz)], and three methyl proton signals [δ _H 1.12 (s, 3H), 1.32 (s, 3H), and 1.35 (d, 3H, J = 6.6 Hz)]. The ¹³C-NMR data of 3 were similar to those of japonicumone A (hyperbrasilone, 7), a γ-pyrone from Hypericum brasiliense [19], except for the presence of a set of additional glucose signals at δ _C 99.9 (C-1″), 73.2 (C-2″), 76.5 (C-3″), 69.6 (C-4″), 77.1 (C-5″), and 60.6 (C-6″). The C-4′ of the aglycone showed an up-field shift of 0.7 ppm, while C-3′ (5′) and C-1′ presented a down-field shift of 0.8 and 2.8 ppm, respectively. The HMBC correlation between H-1″ of the glucose [δ _H 4.98 (1H, d, J = 7.3 Hz)] and C-4′ of the aglycone (δ _C 159.5) further proved that the linkage position of glucose located at C-4′ of the aglycone. The coupling constant (J = 7.3 Hz) of the anomeric proton of glucose indicated a β configuration. Thus, 3 was elucidated as japonicumone A 4′-O-β-D-glucopyranoside.

Compounds 4 and 5 were obtained as light yellow amorphous powder, and their molecular formulae were deduced both as C₂₂H₂₆O₁₀ from the positive HR-ESI-MS (m/z 451.1610 [M + H]⁺, calcd. 451.1599). The NMR data of 4 and 5 were present in pairs with a ratio of about 1 : 2 and were similar to those of 3, except that the AA′BB′ aromatic protons in 3 were replaced by a set of AMX aromatic protons [δ _H 7.32 (1H, d, J = 2.2 Hz), 7.31 (1H, dd, J = 2.2, 8.5 Hz), 7.18 (1H, d, J = 8.5 Hz)/7.58 (1H, d, J = 2.2 Hz), 7.41 (1H, dd, J = 2.2, 8.5 Hz), 6.89 (1H, d, J = 8.5 Hz], suggesting that 4 and 5 were 3,4-dihydroxyphenyl-substituted γ-pyrone glucosides. The NMR data of 4 and 5 were similar to those of japonicumone B (8) from Hypericum japonicum [20], except for a set of additional glucose signals existing in pairs [δ _C 101.3, 77.2, 75.9, 73.2, 69.7, 60.7/102.1, 77.3, 76.1, 73.4, 69.9, 60.8] in 4 and 5. The glycosidic positions were deduced from the HMBC correlations between H-1 of glucose [δ _H 4.84 (d, 1H, J = 7.0 Hz)/4.80 (d, 1H, J = 7.0 Hz)] and C-3′ (δ _C 147.7) or C-4′ (δ _C 145.5) in 4 and 5, respectively. Thus, 4 and 5 were elucidated as japonicumone B 3′-O-β-D-glucopyranoside and japonicumone B 4′-O-β-D-glucopyranoside, respectively.

In addition, eight known compounds were isolated and identified as 7R,8S-dihydrodehydrodieoniferyl alcohol 9-O-β-D-glucopyranoside (6) [16], japonicumone A (7) [19], japonicumone B (8) [20], sacranoside A (9) [21], epicatechin (10) [22], phelodendrozide (11) [23], 16,23,28-trihydroxyoleana-11,13(18)-diene-3-O-α-L-fucoside (12) [24], and pedunculoside (13) [25], respectively, by comparison of their spectroscopic data with those reported in the literature.

A. serpyllifolia has been traditionally used as an anti-fever and detoxification agent, and fever is generally related to the inflammation, so an in vitro NO inhibition model was used for screening a possible anti-inflammation activity of A. serpyllifolia. The 70 % aqueous ethanol extract of this plant showed a potent inhibitory effect on NO production (IC₅₀: 48.73 µg/mL). From this active extract, five new compounds were isolated along with eight known compounds, most of which were evaluated for their inhibitory effects against LPS-induced NO production in RAW 264.7 macrophages. Compounds 1–5, 9, and 13 showed moderate inhibitory activities with IC₅₀ values ranging from 14.92 µM to 52.23 µM ([Table 2]). After analysis of the skeletons of these screened compounds, the active compound types were found to include xanthone, lignan, γ-pyrone, monoterpene, and triterpene glycosides. Due to the limited numbers of each type of active compounds, it is difficult to summarize a detailed structure-activity relationship (SAR) for them, but some valuable clues can still be offered. Compound 2 presented a better activity than 6, suggesting that the acetylation at 3′-OH position leads to a higher inhibitory activity. Compounds 3, 4, and 5 have similar inhibitory effects, suggesting that the substitution pattern of the phenyl does not significantly affect the activity, while the glycosidation on the phenyl can obviously increase it, for example, 3 showed a stronger activity than 7. Besides the new compounds, the two known compounds 9 and 13 were for the first time found to also have a potential NO inhibitory effect, which will supply a reference for their wider application, also as anti-histaminic agents or for treating cardiovascular and cerebrovascular diseases [21], [26].

The positive control (quercetin) did not show any significant cytotoxic effects under tested concentrations (cell viability > 90 % by MTT assay). Moreover, the investigated compounds did not induce obvious cell injuries (cell viability > 90 % by MTT assay and microscopic examination).

Acknowledgements

This work was financially supported by the National Key Technology R&D Program “New Drug Innovation” of China (Nos. 2012ZX09301002–002–002 and 2012ZX09304–005) and National Science Fund for Excellent Young Scholars (No. 81222051).

Conflict of Interest

All authors stated no conflict of interest and agreed to publish this paper.

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Correspondence

• References

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  CrossrefPubMedSearch in Google Scholar
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• 4 Qin JJ, Zhu JX, Zeng Q, Cheng XR, Zhang SD, Jin HZ, Zhang WD. Sesquiterpene lactones from Inula hupehensis inhibite nitric oxide production in RAW264.7 macrophages. Planta Med 2012; 78: 1002-1009
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  Thieme ConnectPubMedSearch in Google Scholar
• 6 Shrestha SP, Amano Y, Narukawa Y, Takeda T. Nitric oxide production inhibitory activity of flavonoids contained in trunk exudates of Dalbergia sissoo . J Nat Prod 2008; 98: 98-101
  PubMedSearch in Google Scholar
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  Search in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 12 Liu J, Qin MJ. Chemical constituents from the roots of Arenaria juncea . Chin J Nat Med 2007; 5: 235-236
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  CrossrefPubMedSearch in Google Scholar
• 17 Fang JM, Lee CK, Cheng YS. Lignans from leaves of Juniperus chinensis . Phytochemistry 1992; 31: 3659-3661
  CrossrefPubMedSearch in Google Scholar
• 18 Antus S, Kurtán T, Juhász L, Kiss L, Hollósi M, Májer ZS. Chiroptical properties of 2, 3-dihydrobenzo[b]furan and chromane chromophores in naturally occurring O-heterocyles. Chirality 2001; 13: 493-506
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 20 Wu LQ, Liao YH, Wang SP, Wang LW, Feng YX, Yang JS, Xiao PG. New pyrones from Hypericum japonicum . Chin Chem Lett 1996; 7: 1011-1012
  PubMedSearch in Google Scholar
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• 22 Guo ZH, Gong RG, Wang XB, Wu LJ, Gao HY. A new triucallane derivative from Salacia hainanensis Chun et How. Acta Pharm Sin 2009; 44: 1123-1126
  PubMedSearch in Google Scholar
• 23 Li WK, Xiao PG, Pan JQ. Complete assignment of ¹H and ¹³C NMR spectra of ikarisoside A and epimedoside C. Magn Reson Chem 1998; 36: 303-304
  CrossrefPubMedSearch in Google Scholar
• 24 Zhao YY, Luo HS, Wang B, Ma LB, Tu GZ, Zhang RY. Triterpenoid saponins from Bupleurum smithii var. parvifolium . Phytochemistry 1996; 42: 1673-1675
  CrossrefPubMedSearch in Google Scholar
• 25 Zhao ZX, Jin J, Lin CZ, Xiong TQ, Luo HF, Qin CY, Zhu CC. Study on triterpenoid glycosides from roots of Ilex pubescens . China Pharmacist 2011; 14: 599-601
  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar

• Supplementary Material