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Natural Product Sciences - Vol. 29 , No. 1
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Natural Product Sciences - Vol. 29, No. 1, pp. 17-23
Abbreviation: Nat. Prod. Sci.
ISSN: 1226-3907 (Print) 2288-9027 (Online)
Print publication date 31 Mar 2023
Received 11 Jan 2023 Revised 12 Feb 2023 Accepted 13 Feb 2023
DOI: https://doi.org/10.20307/nps.2023.29.1.17

Meliasendanins E-J, Nor-neolignan Constituents from Melia toosendan and their Anti-inflammatory Activity
Jin Woo Lee*
College of Pharmacy, Duksung Women’s University, Seoul 01369, Republic of Korea

Correspondence to : *Jin Woo Lee, College of Pharmacy, Duksung Women’s University, Seoul 01369, Republic of Korea. Tel: +82-2-901-8841; E-mail: jwlee22@duksung.ac.kr

Abstract

A phytochemical investigation of the fruits extract of Melia toosendan afforded the isolation of two new nor-neolignans, meliasendanins E (1) and F (2), as well as twelve known compounds (3 - 14) using various separation technique such as Diaion HP20, silica, RP-18 gel column chromatography and semi-preparative HPLC. Their chemical structures were elucidated by extensive NMR spectroscopic data including 2D-NMR, and HR-ESI-MS as well as ECD data. Among the twelve known compounds, the absolute structures of 3 - 6 were determined first, and given the trivial names as meliasendanins G-J (3 - 6). Based on the evaluation of anti-inflammatory activity, compounds 7 - 8 exhibited inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages with IC50 values of 34.6 and 39.5 μM, respectively.

Keywords: Melia toosendan, Meliaceae, Nor-neolignans, Anti-inflammatory, Limonoid
Introduction

Melia toosendan Sieb. et Zucc. (Meliaceae) is widely distributed in the tropical and subtropical region such as southwestern China. The fruit of Melia sp. has been known to contain mainly limonoids,1 triterpenes,2 sterols,3 lignans and neolignans.4 These structures isolated from M. toosendan have been reported to show antibacterial,5 antiviral,6 and antifeedant activities.7 The fruit of M. toosendan traditionally used as anthelmintics, antimalarials, and antipyretics in China and Africa. In the course of phytochemical search of Meliaceae family, two new nor-neolignans, meliasendanins E (1) and F (2), along with twelve known compounds (3 - 14) (Fig. 1), were isolated from the methanol extract of the fruit of M. toosendan. Herein, this paper describes the purification of all isolated compounds using various separation technique such as Diaion HP20 gel column chromatography, normal phase (NP) silica gel column chromatography, reversed phase (RP) silica gel column chromatography, and semi-preparative HPLC. The absolute structure elucidation of new compounds (1 and 2) and four known neolignans (3 - 6), which had been only reported the relative configurations of C-7 and C-8 in the lignan skeleton, has been determined based on the spectroscopic data interpretation, especially 1D- and 2D-NMR data such as HSQC, HMBC, COSY and NOESY, HR-ESI-MS, and ECD data.


Fig. 1. 
Structures of compounds 1 - 14.

Experimental

General experimental procedures – TLC: Pre-coated silica gel 60 F254 (SiO2, 0.25 mm; Merck); spots were visualized under UV light and by spraying with 10% vanillin-H2SO4 in water reagent followed by heating. Column chromatography (CC): silica gel (SiO2, 70-230 mesh; Merck), Lichroprep RP-18 (40-63 μm, Merck), Diaion HP-20 ion exchange resin (Mitsubishi Chemical Corporation). Medium pressure liquid chromatography (MPLC): Biotage Isolera chromatography system (Biotage). Semi-preparative high-performance liquid chromatography (HPLC) was performed on Waters instruments (pump 515, UV detector 2996) equipped with YMC J’sphere ODS-H80 column (4 μm, 150 × 20 mm i.d.) at a flow rate of 6 mL/min. Optical rotations were measured on a JASCO DIP-1000 polarimeter. UV spectra were recorded on a JASCO UV-550 spectrophotometer, and IR spectra were measured on a JASCO FT-IR 4100 spectrometer. ECD spectra were obtained on a JASCO J-715 spectrometer. 1D- and 2D-NMR spectra were taken on a Bruker AVANCE 400 and 500 MHz spectrometers using CD3OD as a solvent. ESI-MS and HR-ESI-MS were obtained with LCQ Fleet and maXis 4G mass spectrometers, respectively.

Plant materials – The dried fruits of M. toosendan were purchased from Kyung-dong herbal market, Seoul, Korea, in April 2022 and authenticated by Prof. Jin Woo Lee. A voucher specimen (2022-DFMT) has been deposited at the Laboratory of Pharmacognosy, College of Pharmacy, Duksung Women’s University, Seoul, Republic of Korea.

Extraction and isolation – The dried and powdered fruits of M. toosendan (1 kg) were extracted with 100% MeOH. The crude extract (100 g) was fractionated into five fractions, fractions A (100% water), B (30% MeOH), C (60% MeOH), D (80% MeOH) and E (100% MeOH), by Diaion HP20 gel column chromatography and eluted with H2O-MeOH gradient system (100:0 to 0:100). Fraction D (28 g) was chromatographed on a silica gel column and eluted with CH2Cl2-MeOH gradient system (100:0 to 0:100) to give eight fractions, MTD1 – MTD8. Compound 13 (1 mg) was purified from MTD1 fraction (55 mg) by semi-preparative HPLC (MeCN-H2O, 40:60 to 60:40). MTD4 fraction (2.0 g) was chromatographed on MPLC (RP-18) and eluted with MeOH-H2O (20:80 to 100:0) to obtain nine fractions, MTD4-1 - MTD4-9. MTD4-2 fraction (160 mg) was further separated by semi-preparative HPLC (MeCN-H2O, 30:70 to 50:50) to yield compounds 3 (1.8 mg) and 4 (2.3 mg). MTD4-3 fraction (120 mg) was purified by semi-preparative HPLC (MeCN-H2O, 30:70 to 60:40) to obtain compounds 1 (0.9 mg) and 2 (1.4 mg). MTA4-4 fraction (150 mg) was further separated by semi-preparative HPLC (MeCN-H2O, 40:60 to 60:40) to afford compounds 7 (5.6 mg), and 8 (6.3 mg). Compound 9 (2.2 mg) was purified from MTD4-5 fraction (40 mg) by semi-preparative HPLC (MeCN-H2O, 40:60 to 70:30). MTD4-7 fraction (140 mg) was further purified by semi-preparative HPLC (MeCN-H2O, 50:50 to 70:30) to yield compounds 5 (1.5 mg), 6 (1.9 mg) and 10 (5.0 mg). MTD5 fraction (2.8 g) was chromatographed on MPLC (RP-18) and eluted with MeOH-H2O (20:80 to 100:0) to afford eleven fractions, MTD5-1 – MTD5-11. MTD5-3 fraction (150 mg) was further purified by semi-preparative HPLC (MeCN-H2O, 50:50 to 70:30) to obtain compounds 12 (15.2 mg) and 14 (1.5 mg). Compound 11 (6.1 mg) was purified from MTD5-6 fraction (80 mg) by semi-preparative HPLC (MeCN-H2O, 70:30 to 100:0).

Meliasendanin E (1) – Colorless gum; αD25 : -18 (c 0.1, MeOH); UV (MeOH): 280 (1.25); ECD (c 0.02, MeOH): 236 (+0.56), 298 (-0.16); 1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz), see Table 1; HR-ESI-MS: 385.1258 [M + Na]+, C19H22NaO7, calcd. 385.1258).

Table 1. 
1H- (500 MHz) and 13C-NMR (125 MHz) spectroscopic data for 1 and 2 in CD3OD
Position 1 2
δC (125 MHz) δH (500 MHz) δC (125 MHz) δH (500 MHz)
1 132.4 - 132.3 -
2 110.5 7.06 d (2.0) 110.1 7.05 d (2.0)
3 147.2 - 147.4 -
4 145.6 - 145.7 -
5 114.1 6.71 d (8.0) 114.3 6.76 d (8.0)
6 119.7 6.87 dd (8.0, 2.0) 119.1 6.87 dd (8.0, 2.0)
7 72.6 4.82 d (5.5) 72.3 4.91 m
8 83.6 4.64 m 84.2 4.60 m
9 61.0 3.87 m 60.6 3.81 dd (12.0, 5.0)
3.57 dd (12.0, 7.0)
1' 130.2 - 130.3 -
2' 110.8 7.50 d (2.0) 110.8 7.57 d (2.0)
3' 149.7 - 149.6 -
4' 152.9 - 153.1 -
5' 114.2 7.04 d (8.5) 114.1 7.11 d (8.5)
6' 123.0 7.57 dd (8.5, 2.0) 123.1 7.61 dd (8.5, 2.0)
7' 198.0 - 198.0 -
CH3CO 24.9 2.55 s 24.9 2.57 s
3-OCH3 54.8 3.82 s 54.8 3.83 s
3'-OCH3 55.0 3.85 s 55.0 3.93 s

Meliasendanin F (2) – Colorless gum; αD25 : -16 (c 0.1, MeOH); UV (MeOH): 279 (0.85); ECD (c 0.02, MeOH): 240 (+0.71), 290 (-0.14); 1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz), see Table 1; HR-ESI-MS: 385.1258 [M + Na]+, C19H22NaO7, calcd. 385.1258).

Meliasendanin G (3) – Colorless gum; αD25 : -16 (c 0.1, MeOH); ECD (c 0.02, MeOH): 240 (+0.29), 298 (-0.19); 1H-NMR (CD3OD, 400 MHz): δ 9.78 (1H, d, J = 8.0 Hz, H-7'), 7.44 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 7.42 (1H, d, J = 2.0 Hz, H-2'), 7.14 (1H, d, J = 8.0 Hz, H-5'), 7.07 (1H, d, J = 2.0 Hz, H-2), 6.87 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.70 (1H, d, J = 8.0 Hz, H-5), 4.83 (1H, d, J = 6.0 Hz, H-7), 4.66 (1H, m, H-8), 3.89 (2H, m, H-9), 3.87 (3H, s, 3'-OCH3), 3.82 (3H, s, 3-OCH3); 13C-NMR (CD3OD, 100 MHz): δ 191.5 (C-7'), 154.1 (C-4'), 150.4 (C-3'), 147.3 (C-3), 145.7 (C-4), 132.4 (C-1), 130.3 (C-1'), 125.7 (C-6'), 119.8 (C-6), 114.5, 114.2 (C-5,5'), 110.7, 110.2 (C-2,2'), 83.7 (C-8), 72.6 (C-7), 61.1 (C-9), 55.0, 54.9 (3,3'-OCH3); HR-ESI-MS: 371.1100 [M + Na]+, C18H20NaO7, calcd. 371.1101).

Meliasendanin H (4) – Colorless gum; αD25 : -18 (c 0.1, MeOH); ECD (c 0.02, MeOH): 236 (+0.49), 278 (-0.15); 1H-NMR (CD3OD, 500 MHz): δ 9.81 (1H, d, J = 8.0 Hz, H-7'), 7.49 (1H, d, J = 2.0 Hz, H-2'), 7.48 (1H, dd, J = 8.5, 2.0 Hz, H-6'), 7.20 (1H, d, J = 8.0 Hz, H-5'), 7.06 (1H, d, J = 2.0 Hz, H-2), 6.88 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.77 (1H, d, J = 8.0 Hz, H-5), 4.93 (1H, m, H-7), 4.65 (1H, m, H-8), 3.94 (3H, s, 3'-OCH3), 3.84 (3H, s, 3-OCH3), 3.81 (1H, m, H-9), 3.60 (1H, m, H-9); 13C-NMR (CD3OD, 125 MHz): δ 191.5 (C-7'), 154.3 (C-4'), 150.2 (C-3'), 147.4 (C-3), 145.8 (C-4), 132.3 (C-1), 130.3 (C-1'), 125.9 (C-6'), 119.2 (C-6), 114.4, 114.2 (C-5, 5'), 110.2, 110.0 (C-2, 2'), 84.1 (C-8), 72.4 (C-7), 60.7 (C-9), 55.1, 54.9 (3,3'-OCH3); HR-ESI-MS: 371.1100 [M + Na]+, C18H20NaO7, calcd. 371.1101).

Meliasendanin I (5) – Yellowish gum; αD25 : -24 (c 0.1, MeOH); ECD (c 0.02, MeOH): 236 (+0.49), 296 (-0.34); 1H-NMR (CD3OD, 400 MHz): δ 7.03 (2H, m, H-5',6'), 6.89 (2H, m, H-2,2'), 6.86 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.75 (1H, d, J = 8.0 Hz, H-5), 6.56 (1H, d, J = 16.0 Hz, H-7'), 6.20 (1H, dt, J = 16.0, 6.5 Hz, H-8'), 4.83 (1H, m, H-7), 4.39 (1H, m, H-8), 4.03 (2H, dd, J = 6.5, 1.5 Hz, H-9'), 3.84 (2H, m, H-9), 3.83, 3.82 (6H, s, 3,3'-OCH3), 3.38 (3H, s, 9'-OCH3); 13C-NMR (CD3OD, 100 MHz): δ 150.5 (C-3'), 147.8 (C-4'), 147.3 (C-3), 145.6 (C-4), 132.7, 132.4 (C-1,1'), 131.3 (C-7'), 123.8 (C-8'), 119.7, 119.4 (C-6, 6'), 117.4, 114.3 (C-5,5'), 110.5, 110.0 (C-2,2'), 84.8 (C-8), 72.8 (C-7), 72.7 (C-9'), 60.8 (C-9), 56.6 (9'-OCH3), 55.1, 54.9 (3,3'-OCH3); HR-ESI-MS: 413.1579 [M + Na]+, C21H26NaO7, calcd. 413.1571).

Meliasendanin J (6) – Yellowish gum; αD25 : -14 (c 0.1, MeOH); ECD (c 0.02, MeOH): 239 (+1.28), 290 (-1.51); 1H-NMR (CD3OD, 500 MHz): δ 7.09 (1H, d, J = 2.0 Hz, H-2), 7.05 (1H, d, J = 2.0 Hz, H-2'), 7.02 (1H, d, J = 8.5 Hz, H-5), 6.95 (1H, dd, J = 8.0, 2.0 Hz, H-6'), 6.88 (1H, dd, J = 8.5, 2.0 Hz, H-6), 6.77 (1H, d, J = 8.0 Hz, H-5'), 6.58 (1H, d, J = 16.0 Hz, H-7'), 6.22 (1H, dt, J = 16.0, 6.5 Hz, H-8'), 4.89 (1H, m, H-7), 4.32 (1H, m, H-8), 4.09 (2H, dd, J = 6.5, 1.5 Hz, H-9'), 3.90, 3.84 (6H, s, 3,3'-OCH3), 3.75 (1H, m, H-9), 3.50 (1H, m, H-9), 3.39 (3H, s, 9'-OCH3); 13C-NMR (CD3OD, 100 MHz): δ 150.3 (C-3'), 148.0 (C-4'), 147.4 (C-3), 145.8 (C-4), 132.4 (C-1,1'), 131.3 (C-7'), 123.8 (C-8'), 119.6, 119.3 (C-6,6'), 117.2, 114.4 (C-5,5'), 110.2, 109.8 (C-2,2'), 85.6 (C-8), 72.8 (C-7), 72.6 (C-9'), 60.4 (C-9), 56.7 (9'-OCH3), 55.1, 54.9 (3,3'-OCH3); HR-ESI-MS: 413.1571 [M + Na]+, C21H26NaO7, calcd. 413.1571).

Fordiane A (7) – Yellowish gum; αD25 : +28 (c 0.1, MeOH); ECD (c 0.02, MeOH): 242 (+0.60), 300 (-0.19); 1H-NMR (CD3OD, 400 MHz): δ 9.60 (1H, d, J = 8.0 Hz, H-9'), 7.59 (1H, d, J = 16.0, H-7'), 7.24 (1H, d, J = 2.0 Hz, H-2'), 7.18 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 7.06 (1H, d, J = 2.0 Hz, H-2), 7.03 (1H, d, J = 8.4 Hz, H-5'), 6.87 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.72 (1H, d, J = 8.0 Hz, H-5), 6.68 (1H, d, J = 16.0, 8.0 Hz, H-8'), 4.83 (1H, d, J = 6.0 Hz, H-7), 4.57 (1H, m, H-8), 3.86 (2H, m, H-9), 3.85, 3.82 (6H, s, 3,3'-OCH3); 13C-NMR (CD3OD, 100 MHz): δ 194.8 (C-9'), 154.2 (C-7'), 151.3 (C-4'), 150.3 (C-3'), 147.2 (C-3), 145.7 (C-4), 132.5 (C-1), 127.8 (C-1'), 126.2 (C-8'), 123.0 (C-6'), 119.7 (C-6), 115.6, 114.2 (C-5,5'), 111.2, 110.5 (C-2,2'), 83.9 (C-8), 72.6 (C-7), 61.0 (C-9), 55.1, 54.9 (3,3'-OCH3); ESI-MS: 397 [M + Na]+.

Fordiane B (8) – Yellowish gum; αD25 : -24 (c 0.1, MeOH); ECD (c 0.02, MeOH): 240 (+0.49), 308 (-0.12); 1H-NMR (CD3OD, 400 MHz): δ 9.61 (1H, d, J = 8.0 Hz, H-9'), 7.62 (1H, d, J = 16.0, H-7'), 7.31 (1H, d, J = 2.0 Hz, H-2'), 7.22 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 7.11 (1H, d, J = 8.4 Hz, H-5'), 7.05 (1H, d, J = 2.0 Hz, H-2), 6.88 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.76 (1H, d, J = 8.0 Hz, H-5), 6.71 (1H, d, J = 16.0, 8.0 Hz, H-8'), 4.89 (1H, m, H-7), 4.53 (1H, m, H-8), 3.93, 3.83 (6H, s, 3,3'-OCH3), 3.80 (1H, m, H-9), 3.55 (1H, m, H-9); 13C-NMR (CD3OD, 100 MHz): δ 194.7 (C-9'), 154.0 (C-7'), 151.6 (C-4'), 150.3 (C-3'), 147.4 (C-3), 145.8 (C-4), 132.4 (C-1), 128.0 (C-1'), 126.3 (C-8'), 123.2 (C-6'), 119.3 (C-6), 115.8, 114.5 (C-5,5'), 111.3, 110.3 (C-2,2'), 84.7 (C-8), 72.5 (C-7), 60.7 (C-9), 55.3, 55.0 (OCH3); ESI-MS: 397 [M + Na]+.

Measurement of LPS-induced NO production and cell viability – RAW 264.7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL. Louis, MO, USA) with 10% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin (100 U/mL) at 37°C humidified air containing 5% CO2. RAW264.7 cells were plated at 2 × 105 cells/well in 96-well culture dishes and incubated with or without LPS (1 μg/mL) in the absence or presence of indicate concentration of the samples for 24 h. The NO production was measured according to the Griess reaction. Cell viability of the remaining cells was determined by MTT (Sigma Chemical Co., St. Louis, MO, USA) based colorimetric assay.

Result and Discussion

Meliasendanin E (1) was obtained as a colorless gum. Its HR-ESI-TOF-MS spectrum revealed a molecular formula of C19H22O7 based on the ion peak at m/z 385.1258 [M + Na]+ (calcd. for C19H22NaO7, 385.1258). In the 1H NMR spectrum of 1, six aromatic proton signals at δH 7.57 (1H, dd, J = 8.5, 2.0 Hz, H-6'), 7.50 (1H, d, J = 2.0 Hz, H-2'), 7.06 (1H, d, J = 2.0 Hz, H-2), 7.04 (1H, d, J = 8.5 Hz, H-5'), 6.87 (1H, dd, J = 8.0, 2.0 Hz, H-6) and 6.71 (1H, d, J = 8.0 Hz, H-5) were observed, corresponding to a couple of ABX trisubstituted aromatic ring protons, and two methoxy group signals at δH 3.85 (3H, s, 3'-OCH3) and 3.82 (3H, s, 3-OCH3), and one acetyl group signal at δH 2.55 (3H, s, 1'-Ac) were detected as well. The 13C NMR data of 1 displayed 19 carbon resonances including one carbonyl carbon signal (δC 198.0, C-7'), twelve olefinic carbon singnals (δC 152.9, 149.7, 147.2, 145.6, 132.4, 130.2, 123.0, 119.7, 114.2, 114.1, 110.8, 110.5), two oxymethine carbon signals (δC 83.6, 72.6), one oxymethylene carbon signal (δC 61.0), two methoxy group signals (δC 55.0, 54.8) and one methyl group signal (δC 24.9) (Table 1). Based on the interpretation of 1D and 2D NMR spectra, 1 was deduced to be biosynthesized with C6-C3 and C6-C2. The key HMBC correlation between H-8 and C-4' suggested that the two units of 1 were connected with an oxygen bond, indicating that 1 was thought to be an atypical lignan scaffold, called by nor-neolignan (Fig. 2). The two methoxy and the acetyl moieties were attached to C-3, C-3', and C-1', respectively, based on the HMBC correlations of 3-OCH3/C-3, 3'-OCH3/C-3', and CH3(CO)/C-1'. The relative structure and the absolute configuration of 1 were determined by the difference of the chemical shift in the NMR spectrum, and a comparison of ECD analysis with the literatures. According to the literature reporting about the possible staggered conformers of 8-O-4' neolignan diastereomers, the large and small J values of H-7 and 8 are used to determine the relative configuration of C-7 and 8 like threo form and erythro form, respectively.8 In the 1H NMR spectrum of 1, the relative configuration of C-7 and 8 was confirmed as erythro form by a small coupling constant between H-7 and 8 (JH7-H8 = 5.5 Hz). The absolute configuration of C-8 was established as S by a positive Cotton effect at 230-240 nm in its ECD spectrum (Fig. 3).9,10 Thus, the absolute configuration of meliasendanin E (1) was confirmed to be 7R, 8S, and the structure of 1 was turned out as an enantiomer of lycocernuaside D, which has been previously reported in 2017.11


Fig. 2. 
Key HMBC correlations of compounds 1 and 2.


Fig. 3. 
ECD spectrum of compounds 1 and 2.

Meliasendanin F (2) was isolated as a colorless gum with the same molecular formula as that of 2. The interpretation of 1D and 2D NMR experiment suggested that the planar structure of 2 was identical to that of 1. However, since the JH7-H8 value in the 1H NMR spectrum of 2 was difficult and ambiguous to obtain, we applied the ΔδC8-C7 values eliminating the effect of systematic errors [ΔδC8-C7 (threo) > ΔδC8-C7 (erythro)]. These values are only able to be applied to differentiate threo and erythro aryl glycerols without substituents at C-7 or/and C-8 in the same solvent.9,12 The ΔδC8-C7 of 2 (11.9) was larger than that of 1 (11.0) in CD3OD, indicating 2 was determined as threo form, and 1 was erythro, supported by the pattern of the proton signal chemical shifts of H-9. While the two protons of C-9 in the threo forms of these molecules such as compounds 2, 4, 6 and 8 were shown separately in their 1H-NMR spectra (δH 3.81, 3.57 in 2, δH 3.81, 3.60 in 4, δH 3.75, 3.50 in 6, δH 3.80, 3.55 in 8), those of the erythro forms (1, 3, 5, 7) were overwrapped (δH 3.87 in 1, δH 3.89 in 3, δH 3.84 in 5, δH 3.86 in 7) (Table 2). In addition, the absolute configuration of C-8 in 2 was determined as S by a positive Cotton effect at 230-240 nm in its ECD spectrum. Therefore, the absolute configuration of 2 was confirmed as 7S, 8S (Fig. 3).

Table 2. 
Comparison of the 1H- and 13C-NMR chemical shift of erythro and threo-form in C-7 and C-8
erythro (C-7,8) threo (C-7,8)
Compound 1 3 5 7 2 4 6 8
H-9 3.87
(2H)		 3.89
(2H)		 3.84
(2H)		 3.86
(2H)		 3.81 3.81 3.75 3.80
3.57 3.60 3.50 3.55
pattern overwrapped separated
C-7 72.6 72.6 72.8 72.6 72.3 72.4 72.8 72.5
C-8 83.6 83.7 84.8 83.8 84.2 84.1 85.6 84.7
ΔδC8-C7 11.0 11.1 12.0 11.2 11.9 11.7 12.8 11.8

Meliasendanins G (3), H (4), I (5) and J (6) were previously reported as erytrho-guaiacylglycerol-8-vanillic acid ether,13 threo-guaiacylglycerol-8-vanillic acid ether,13 erythro-guaiacylglycerol-β-9'-methylconiferyl ether ether,14 and threo-guaiacylglycerol-β-9'-methylconiferyl ether ether,14 respectively, only with relative configurations of C-8 and C-9. To determine their relative and absolute configurations, the afore-mentioned methods were applied. Based on the ΔδC8-C7 values (11.1 for 3 < 11.7 for 4, and 12.0 for 5 < 12.8 for 6) (Table 2), the pattern of H2-9 in their 1H-NMR spectra, and Cotton effects at 230-240 nm (Fig. S13), the absolute configurations of meliasendanins G-J (3-6) were established as 7R,8S (3), 7S,8S (4), 7R,8S (5), and 7S,8S (6), respectively.

The eight known compounds were identified as fordiane A (7),14,15 fordiane B (8),14,15 vladinol D (9),16 iso-toosendanin (10),17 12-hydroxyamoorastatone (11),18 mesendanin H (12),19 vanillin (13),20 and dihydroconiferyl alcohol (14),21 respectively, by comparing their physicochemical and spectroscopic data with those of published values.

Nitric oxide (NO) generated by inducible NO synthase (iNOS) in macrophage is involved in the action of mechanisms of inflammation. Excessive NO production may cause various inflammatory diseases such as rheumatism and asthma.22 Therefore, inhibition of NO production has been thought to be one of potential therapeutic strategies to control inflammatory diseases. Several neolignans isolated from medicinal plants have been reported to show inhibitory effect of NO production. For example, ficusal, isolated from the seeds of Prunus tomentosa, has been reported to show inhibitory activity of NO with IC50 value of 4.7 μM.23 Other examples include myrislignan (IC50: 21.1 μM) and machilin D (IC50: 4.7 μM), isolated from the seeds of Myristica fragrans, piperkadsin C (IC50: 14.6 μM) and futoquinol (IC50: 16.8 μM), isolated from Piper kadsura.24,25

To find anti-inflammatory compounds, all isolated compounds (1 - 14) were tested for their anti-inflammatory activity on LPS-induced NO production in RAW 264.7 macrophage cells and aminoguanidine was used as positive control (IC50: 15.8 μM). Among them, only fordianes A (7) and B (8), possessing an α,β-unsaturated aldehyde group at C-1', showed mild inhibitory effects with IC50 values of 34.6 and 39.5 μM (Table 3). Although the bioassay results are not enough to mention their structure-activity relationship, because a few aldehyde-bearing neolignans have been previously reported, it is necessary to develop the structures or find more potential nor-neolignan molecules.26

Table 3. 
Inhibitory effects of compounds 1 - 14 on LPS-induced NO production in macrophage RAW 264.7 Cellsa
Compound IC50 (μM) Compound IC50 (μM)
1 >100 8 39.5
2 >100 9 >100
3 >100 10 >100
4 >100 11 >100
5 >100 12 >100
6 >100 13 >100
7 34.6 14 >100
Aminoguanidine was used as the positive control (IC50 = 15.8 μM)
a Results are expressed as the mean IC50 values in μM from triplicate experiments.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

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