Cytotoxic and anti-inflammatory constituents from Momordica cochinchinensis seeds

Fitoterapia 139 (2019) 104360

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Cytotoxic and anti-inflammatory constituents from Momordica cochinchinensis seeds Mengyue Wanga, Zhibin Zhana,b, Ying Xiongb, Ying Zhanga, Xiaobo Lia, a b

T

⁎

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Gac Momordica cochinchinensis Lignan Cytotoxicity Anti-inflammation

Five new lignans mubiesins A − E (1–5), together with twenty-seven known compounds (6–32), were isolated from the cytotoxicity and anti-inflammation portions of Momordica cochinchinensis seeds which were widely used for various tumors and inflammations. Their structures were elucidated by extensive spectroscopic analyses (HRMS, UV, CD, IR, 1D-NMR, and 2D-NMR). Their cytotoxic and anti-inflammatory activities were evaluated in vitro. Various lignans and saponins showed the significant activities, they could obviously inhibit the growth of tumor cells and the release of NO and TNF-α in RAW 264.7 cells induced by LPS.

1. Introduction Momordica cochinchinensis Sprenger (Mubie in Chinese), is a commonly medical and food plant widely cultured in China, Vietnam, Thailand and India [1]. Its fruit (Gac) is a common food in Vietnam, and India [2]. However, its seed is a traditional medicine to treat various tumors and inflammations, such as hepatoma, melanoma, gastric carcinoma, acute mastitis, and lymphnoditis [3]. Recently, the extracts of M. cochinchinensis seeds were proved to possess the significant anti-inflammation and cytotoxicity [4,5]. Meanwhile, several saponins with cytotoxic and anti-inflammatory activities were isolated [6–8]. However, a systemic investigation of the antitumor and anti-inflammatory constituents was not reported yet. In the present study, the cytotoxic and anti-inflammatory portions of M. cochinchinensis seeds were screened first. And then, the chemical constituents in the active portions and their cytotoxicity and anti-inflammation were investigated. As results, five new lignans (1–5), together with twenty-seven known compounds (6–32), were obtained; various lignans and saponins exhibited the significant cytotoxic and anti-inflammatory activities. 2. Materials and methods 2.1. General experimental procedure 1 H and 13C NMR spectra were recorded on Bruker Avance DRX-600 spectrometer with a 5 mm 13C/1H/15N TCI CryoProbe. UV spectra were

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measured on a UV1102 spectrophotometer. Optical rotations were measured on a JASCO P-2000 polarimeter. IR spectra were recorded on a Nicolet 6700 FT-IR spectrophotometer. CD spectra were measured on a J-815 spectrometer polarimeter. Semi-preparative HPLC was performed on a Shimadzu LC 2010 AHT liquid chromatography system, equipped with an auto-sampler, a UV–Vis detector, and a YMC ODS-AQ column (20 × 250 mm, 5 μm). Optical absorbance was measured using a Bio-Rad 680 microplate reader. Silica gel G (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd.) and ODS (40–63 μm, Merck & Co., Inc.) were used for column chromatography (CC). 2.2. Chemicals and material Petroleum ether (PE, 60–90 °C), dichloromethane (CH2Cl2), ethyl acetate (EtOAc), alcohol, n-butanol (n-BuOH), methanol (MeOH), and methyl cyanide (MeCN) were purchased from China National Medicines CO. Ltd. (Shanghai, China). Cytoxan (CTX), dexamethasone (DEX), 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), lipopolysaccharide (LPS), and fetal bovine serum (FBS) were provided by Shanghai Sangon Bioteck Co. Ltd. (Shanghai, China). Hepg 2, B16, SGC-790, and RAW 264.7 cells were provided by Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. PRMI 1640 medium and DMEM medium were purchased from GIBCOBRL Co. Ltd. (Gaithersburg, MD, USA). Momordica cochinchinensis seeds were collected in Bozhou, Anhui province (China) in October 2017, and authenticated by one of authors Mengyue Wang. A voucher specimen (MC 20171029) was deposited in

Corresponding author. E-mail address: [email protected] (X. Li).

https://doi.org/10.1016/j.fitote.2019.104360 Received 5 August 2019; Received in revised form 18 September 2019; Accepted 22 September 2019 Available online 17 October 2019 0367-326X/ © 2019 Elsevier B.V. All rights reserved.

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the herbarium of School of Pharmacy, Shanghai Jiao Tong University.

ESI-MS m/z: 493.1887 [M-H]− (calcd for C28H29O8, 493.1868).

2.3. Isolation and purification

2.3.2. Mubiesin B (2) White amorphous powder; αD25−10.6 (c 0.35, MeOH); UV (MeOH) λmax (nm) (log ε): 224 (3.51), 278 (2.37); IR (KBr) vmax: 3430, 2928, 1613, 1514, 1451, 1264, 1228, 1029 cm−1; CD (MeOH, Δε): 224 (−4.67), 278 (−1.53); 1H and 13C NMR data presented in Table 1; HRESI-MS m/z: 495.2023 [M-H]− (calcd for C28H31O8, 495.2019), 991.4149 [2 M-H]− (calcd for C56H63O16, 991.4116).

The seeds (10 kg) were powdered and extracted with 80 L 95% alcohol by reflux three times (3 h each). The extract was combined and completely evaporated under vacuum to give a green-black residue (1.7 kg). The residue was suspended in water (8 L), successively partitioned with PE, CH2Cl2, EtOAc, and n-BuOH (each 5 L × 3), and then completely evaporated under vacuum to afford portions PE (1194 g), CH2Cl2 (13 g), EtOAc (128 g), and n-BuOH (150 g). The remaining water layer was dried under vacuum to afford water portion (214 g). These portions were subjected to the cytotoxicity and anti-inflammation evaluation. The portions with obvious activities were subjected to the further chemical investigation. Part of EtOAc portion (110 g) was fractioned by silica gel CC (CH2Cl2-MeOH 100:0–75:25) to afford seven fractions (Fr. A1–Fr. A7). Fr. A1 (2.7 g) was further purified by silica gel CC (PE-EtOAc 100:0–90:10) to afford compounds 6 (35.9 mg) and 7 (8.2 mg). Fr. A2 (556 mg) was further purified by silica gel CC (PE-EtOAc 100:0–80:20) and then by semi-preparative HPLC (MeCN-H2O 60:40, 210 nm, 5 mL/ min) to afford compounds 8 (1.6 mg, tR 23.6 min), 9 (1.5 mg, tR 25.2 min), and 10 (29.5 mg, tR 8.4 min). Fr. A4 (4.3 g) was further purified by silica gel CC (CH2Cl2-MeOH 92:8–85:15) and then by semipreparative HPLC (MeCN-H2O 37:63, 280 nm, 6 mL/min) to afford compounds 1 (13.0 mg, tR 15.7 min) and 2 (6.7 mg, tR 18.3 min). Fr. A6 (15.2 g) was further purified by silica gel CC (CH2Cl2-MeOH 100:0–70:30) to afford eight fractions (Fr. A4a–Fr. A4h). Fr. A4b (130 mg) was further purified by semi-preparative HPLC (MeCN-H2O 30:70, 280 nm, 5 mL/min) to afford compounds 3 (3.6 mg, tR 22.7 min), 4 (1.0 mg, tR 25.2 min), 11 (9.3 mg, tR 28.0 min), and 12 (7.5 mg, tR 31.3 min). Fr. A4d (269 mg) was further purified by semi-preparative HPLC (MeCN-H2O 35:65, 210 nm, 6 mL/min) to afford compounds 5 (2.1 mg, tR 12.9 min), 13 (73.6 mg, tR 15.8 min), and 14 (3.4 mg, tR 20.1 min). Fr. A4e (84 mg) was further purified by semi-preparative HPLC (MeCN-H2O 40:60, 210 nm, 4 mL/min) to afford compounds 15 (2.0 mg, tR 19.5 min), 16 (5.8 mg, tR 22.0 min), and 17 (1.5 mg, tR 23.1 min). Fr. A4f (156 mg) was further purified by semi-preparative HPLC (MeCN-H2O 30:70, 210 nm, 8 mL/min) to afford compounds 18 (12.9 mg, tR 16.2 min) and 19 (11.3 mg, tR 20.5 min). Fr. A4h (328 mg) was further purified by semi-preparative HPLC (MeCN-H2O 30:70, 210 nm, 5 mL/min) to 20 (4.0 mg, tR 23.6 min), 21 (4.6 mg, tR 24.1 min), 22 (5.9 mg, tR 25.3 min). Fr. A7 (3.1 g) was further purified by silica gel CC (CH2Cl2-MeOH 90:10–85:15) and semi-preparative HPLC (MeCN-H2O 40:60, 210 nm, 5 mL/min) to afford compounds 23 (31.1 mg, tR 14.1 min) and 24 (5.1 mg, tR 19.8 min). Part of n-BuOH (136 g) was fractioned by silica gel CC (CH2Cl2MeOH-H2O 95:5:0–40:60:5) to afford eight fractions (Fr. B1eFr. B8). Fr. B1 (2.5 g) was further purified by silica gel CC eluted with CH2Cl2MeOH (100:0–95:5) and semi-preparative HPLC (MeCN-H2O 35:65, 210 nm, 6 mL/min) to afford compounds 25 (6.0 mg, tR 17.8 min), 26 (7.3 mg, tR 21.4 min), and 27 (24.4 mg, tR 24.5 min). Fr. B4 (7.1 g) was further purified by silica gel CC (CH2Cl2-MeOH 100:0–70:30) and semipreparative HPLC (MeCN-H2O 30:70, 210 nm, 5 mL/min) to afford compounds 28 (26.1 mg, tR 12.4 min) and 29 (7.6 mg, tR 13.1 min). Fr. B6 (13.7 g) was further purified by silica gel CC (CH2Cl2-MeOH 90:10–70:30) and semi-preparative HPLC (MeCN-H2O 15:85, 210 nm, 6 mL/min) to afford compounds 30 (19.3 mg, tR 8.2 min) and 31 (6.8 mg, tR 9.6 min). Fr. B8 (5.3 g) was further purified by ODS CC (MeOH-H2O 20:80–70:30) to afford compound 32 (1.3 g).

2.3.3. Mubiesin C (3) White amorphous powder; αD25−18.9 (c 0.10, MeOH); UV (MeOH) λmax (nm) (log ε): 225 (3.36), 284 (3.27); IR (KBr) vmax: 3429, 2925, 1725, 1663, 1604, 1517, 1488, 1249, 1122 cm−1; CD (MeOH, Δε): 225 (−0.78), 284 (−0.57); 1H and 13C NMR data: see Table 1; HR-ESI-MS m/z: 315.1234 [M + H]+ (calcd for C18H19O5, 315.1232). 2.3.4. Mubiesin D (4) White amorphous powder; αD25+9.8 (c 0.24, MeOH); UV (MeOH) λmax (nm) (log ε): 226 (3.60), 278 (2.44); IR (KBr) vmax: 3441, 2926, 1623, 1517, 1451, 1384, 1217, 1140, 1025 cm−1; CD (MeOH, Δε): 226 (+0.82), 278 (−0.40); 1H and 13C NMR data: see Table 1; HR-ESI-MS m/z: 603.2266 [2M-H]− (calcd for C34H35O10, 603.2230), 905.3359 [3 M-H]− (calcd for C51H53O15, 905.3384). 2.3.5. Mubiesin E (5) White amorphous powder; αD25−7.9 (c 0.32, MeOH); UV (MeOH) λmax (nm) (log ε): 225 (3.41), 276 (2.29). IR (KBr) vmax: 3429, 2924, 1614, 1516, 1451, 1237 cm−1; CD (MeOH, Δε): 225 (+1.37), 276 (−2.31); 1H and 13C NMR data: see Table 1; HR-ESI-MS m/z: 271.0979 [M-H]− (calcd for C16H15O4, 271.0970), 543.2031 [2 M-H]− (calcd for C32H31O8, 543.2019). 2.4. Cytotoxicity evaluation Tumor cell lines were cultured in RPMI 1640 medium supplemented with heat-inactivated 10% FBS, 100 units/mL penicillin, and 100 units/ mL streptomycin in a humidified incubator, at 37 °C and a 5% CO2 atmosphere. Cultured cells (1 × 10 [5]) were seeded in 96-well microplates, incubated, and treated with samples dissolved in 2.5% DMSO solution (2.5, 5, 10, 20, 40, 80, and 160 μg/mL for extracts; 1.56, 3.13, 6.25, 12.5, 25.0, 50, and 100 μM for compounds isolated and CTX). Each plate was incubated for 48 h, and then 20 μL of 5 mg/mL MTT was added to the well. The microplate was further incubated for 4 h, after which the optical density of each well was recorded by the ELISA reader at 570 nm. All experiments were carried out in triplicate. The cytotoxicity of each sample was expressed as IC50, the concentration resulted in 50% inhibition [9]. 2.5. Anti-inflammation evaluation 2.5.1. Cell viability assay RAW 264.7 cells were cultured at 1 × 106 cells/mL in 96-well tissue culture plates for 18 h, and then pretreated with samples (40, 80, and 160 μg/mL for extracts; 50, 100, and 200 μM for compounds isolated) 1 h before LPS (1 μg/mL) stimulation 24 h in incubator. Cell viability was evaluated by the MTT methods. 2.5.2. NO and TNF-α production assay RAW 264.7 cells were cultured in 96-well tissue culture plates, and then pretreated with samples prepared in 2.5% DMSO solution (2.5, 5, 10, 20, 40, 80, and 160 μg/mL for extracts; 1.56, 3.13, 6.25, 12.5, 25.0, and 50 μM for compounds isolated and DEM) for 1 h. Subsequently, cells were stimulated with LPS (1 μg/mL) stimulation for 24 h. NO accumulated in the culture medium was measured by NO assay kit using Griess reaction as an indicator of NO production. TNF-α production was

2.3.1. Mubiesin A (1) White amorphous powder; αD25−11.8 (c 0.17, MeOH); UV (MeOH) λmax (nm) (log ε): 224 (3.76), 278 (2.53); IR (KBr) vmax: 3434, 2927, 1614, 1514, 1451, 1265, 1228, 1034 cm−1; CD (MeOH, Δε): 224 (−5.20), 278 (−1.86); 1H and 13C NMR data presented in Table 1; HR2

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Table 1 1 H (600 MHz) and No.

C NMR (150 MHz) data of compounds 1–5 in Pyridine-d5.

1 δC

1 2 3 4 5

132.3 128.1 116.1 158.1 116.1

6 7 8 9

128.1 85.9 54.6 71.8

1′ 2′ 3′ 4′ 5′ 6′ 7′

135.0 111.0 150.9 149.0 117.6 119.0 86.0

8′ 9′

54.4 71.5

1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″

133.3 128.9 115.7 158.4 115.7 128.9 73.1 87.4 61.5

OCH3

55.7

a

13

2 δH (J in Hz)

7.47 d (8.5) 7.25 d (8.5) 7.25 (8.5) 7.47 d (8.5) 4.96 d (4.2) 3.18 m 4.30 m 3.98 m 7.22 d (1.9)

7.57 d (8.3) 7.07 dd (1.9, 8.3) 4.93 d (4.2) 3.18 m 4.31 dd (2.3, 6.7) 3.98 m 7.82 d (8.4) 7.25 d (8.4) 7.25 d (8.4) 7.82 d (8.4) 5.61 d (6.0) 4.99 m 4.40 dd (3.6, 11.8) 4.11 dd (5.7, 11.8) 3.79 s

δC 133.3 129.0 115.7 158.1 115.7 129.0 73.1 87.5 61.4 131.7 130.0 116.1 157.0 116.1 130.0 32.6 43.0 73.0 138.6 110.7 150.9 148.5 117.8 118.7 83.1 53.4 59.8 55.6

3 δH (J in Hz)

7.87 d (8.1) 7.30 d (7.4) 7.30 d (7.4) 7.87 d (8.1) 5.67 d (6.1) 4.88 dd (5.6, 9.4) 4.44 dd (3.1, 11.7) 4.14 m 7.32 d (7.3) 7.25a 7.25a 7.32 d (7.3) 3.24 dd (5.2, 13.6) 2.85 dd (10.6, 13.6) 3.06 m 4.34 dd (6.9, 8.1) 4.10 t (7.8)

4

δC

δH (J in Hz)

131.4 126.0 129.7 164.5 109.0 131.0 197.7 41.9 58.2 132.4 128.0 116.3 158.9 116.3 128.0 89.0 53.4 63.8

8.31 s

7.02 d (8.4) 8.11 d (8.4)

δC 136.6 112.0 129.9 147.9 144.5 116.1 64.4

5 δH (J in Hz)

δC

δH (J in Hz)

7.22 s

83.6 74.9 59.6 76.8

5.70 4.74 3.43 4.63 4.44

7.30 s 4.97a

3.37 m 4.36 t (6.0)

7.19 d (8.4) 7.49 d (8.4) 6.08 d (6.1)

133.3 127.8 116.1 158.6 116.1 127.8 88.1

7.15 d (8.5) 7.57a 6.07 d (6.2)

3.85 m 4.17 m

54.9 64.3

3.90 m 4.22 dd (5.2, 10.5)

7.49 d (8.4) 7.19 d (8.4)

7.57a 7.15 d (8.5)

132.8 128.3 115.7 157.6 115.7 128.3

127.4 131.7 115.8 158.2 115.8 131.7

7.37 d (1.6)

7.62 dd (1.6, 8.3) 7.26a 5.45 dd (1.3, 5.5) 2.79 m 4.29 dd (6.9, 10.2) 4.17 m 3.77 s

d (10.6) brs dd (4.1, 10.6) dd (4.1, 9.2) d (9.2)

55.9

7.50 d (7.5) 7.10 d (7.5) 7.10 d (7.5) 7.50 d (7.5)

7.56a 7.12 d (7.3) 7.12 d (7.3) 7.56a

3.79 s

Overlap.

trihydroxy- 7, 9′-epoxy- 8, 8′-lignan (19), chushizisin I (20), chushizisin A (21), chushizisin E (22), 3-[2-(4-hydroxyphenyl)-3-hydroxyphenyl-2, 3-dihydro-1-benzofuran-5-yl] propane-1-ol (23), chushizisin G (24), threo-1-(4-hydroxyphenyl)-1-ethoxy-2, 3-propanediol (25), juglanin D (26), gypsogenin (27), gypsogenin-3-O-β-D-(6′-methyl)-glucuronopyranoside (28), gypsogenin-3-O-β-D-glucuronopyranoside (29), gypsogenin-3-O-β-D-galactopyranosyl (1 → 2)-[α-L-rhamnopyranosyl (1 → 3)]-β-D-glucuronopyranoside (30), lariciresinol-4,4′-di-O-β-D-glucopyranoside (31), momordica saponin I (32). Compound 1, obtained as a white amorphous powder, was assigned the molecular formula C28H30O8 by its HR-ESI-MS, which gave quasimolecular ion peak at m/z 493.1887 [M – H]− (calcd for C28H29O8, 493.1868). The 1H and 13C NMR (Table 1) was similar with those of 3demethoxypinoresinol [10], except for the existence of a guaiacylglyceryl moiety. The observed HMBC correlation between H-8″ (δH 4.99) to C-4′ (δC 149.0) further revealed that the guaiacylglyceryl moiety was connected to C-4′ (Fig. 2). The small coupling constants of J7, 8 = J7′, 8′ = 4.2 Hz and the chemical shifts for bridge carbons C-8/C-8′ (δC 54.6/54.4) indicated that two aryl substitutes are equatorial in compound 1 [11]. This was further confirmed by the cross-peaks from H-8 (δH 3.18) to H-2/H-6 (δH 7.47), and from H-8′ (δH 3.18) to H-2′ (δH 7.22)/H-6′ (δH 7.07) in NOESY spectrum. The weak negative Cotton effect at 278 nm (Δε −1.86) opposite with (+)-aschantin and (+)-yangambin revealed that the absolute configuration of the furofuran unit was 7 R, 8 S, 7′ R, 8′ S [12]. The relative configuration at H7″ and H-8″ was deduced as erythro from the small coupling constant of J7″, 8″ = 6.0 Hz. The CD spectrum of compound 1 also showed strong negative Cotton effect at 224 nm (Δε −5.20), indicating that the absolute configurations at C-7″ and C-8″ were to be 7″ S and 8″ R form [13]. Based on the above evidence, the structure of 1 was determined as

measured by enzyme-linked immunosorbent assay. All experiments were carried out in triplicate and the anti-inflammation activities of samples were expressed as IC50 [9]. 3. Results and discussion 3.1. Chemical constituents in the cytotoxicity and anti-inflammation portions In order to clarify the active constituents in M. cochinchinensis seeds, the cytotoxicity and anti-inflammation portions were screened firstly. The result indicated that EtOAc and n-BuOH portions possessed the significant cytotoxicity, they both obviously inhibited the growth of tumor cells, with IC50 values less than 36 μg/mL (Fig. S1). EtOAc and nBuOH portions also exhibited the anti-inflammation activities. They didn't influence RAW 264.7 cells vitalities at the concentrations of 160 μg/mL (cells vitalities above 91.3%, Fig. S2); meanwhile, they could obviously inhibited the NO and TNF-α release of RAW 264.7 cells induced by LPS, with IC50 values less than 45 μg/mL (Fig. S3). So, EtOAc and n-BuOH portions were taken as the active portions for the subsequent chemical research. By sequentially using silica gel CC, ODS CC and semi-preparative HPLC, 32 compounds were separated from the active portions. These compounds included 5 new compounds (1–5, Fig. 1) and 27 known compounds palmitic acid (6), α-spinasterol (7), viscumamide (8), clavatustide C (9), p-hydroxybenzoic acid (10), laxanol (11), threo-1-(4hydroxyphenyl)-2-{4-[2-formyl-(E)-vinyl]-2-methoxyphenoxyl}-propane-1, 3-diol (12), α-spinasterol-3-O-β-D-glucoside (13), chushizisin F (14), ehletianol C (15), tanegool (16), (7R, 8R, 8′R)-4′-guaiacylglyceryl-evofolin B (17), ligballinone (18), (7R, 8S, 8′R)- 4, 4′, 93

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Fig. 1. Structures of compounds 1–5.

and named mubiesin B. Compound 3 was isolated as an amorphous powder. The HR-ESI-MS gave a quasi-molecular ion peak at m/z 315.1234 [M + H]+ (calcd for C18H19O5, 315.1232), corresponding to the molecular formula C18H18O5. The 1H NMR spectrum showed seven aromatic protons at δH 7.49 (2H, d, J = 8.4 Hz, H-2′, 6′), 7.19 (2H, d, J = 8.4 Hz, H-3′, 5′), 8.31 (1H, s, H-2), 8.11 (1H, d, J = 8.4 Hz, H-6), and 7.02 (1H, d, J = 8.4 Hz, H-5), which indicated the existence of 1, 4-bisubstituted phenyl group and 1, 3, 4-trisubstituted phenyl group (Table 1). NMR data was very similar to those of chushinzisin F [15], the difference was that a methylene at δC 35.8 (C-7) in chushinzisin F was replaced by a ketone at δH 197.7 in compound 3. This was confirmed by HMBC correlations of H-2 (δH 8.31), H-6 (δH 8.11), and H-9 (δH 4.36) with C-7 (δC 197.7) in compound 3. The J7′, 8′ value (6.1 Hz) established the 7′, 8′-trans configuration. The absolute configuration of compound 3 was assigned as 7′R, 8′S by the negative Cotton effects at 284 and 225 nm, in accordance with CD data of compound 9a reported previously [16]. Thus, compound 3 was established as (7′R, 8′S)-7-oxo-9, 4′, 9′-trihydroxy-4, 7′-epoxy −3, 8′-neolignan, and named mubiesin C. Compound 4 was isolated as an amorphous powder and had the formula C17H18O5 derived from its negative HR-ESI-MS. Comparison of

(7 R, 8 S, 7′ R, 8′ S, 7″ S, 8″ R)-4′-guaiacylglyceryl-3-demethoxypinoresinol, and named mubiesin A. Compound 2 obtained as a white amorphous powder and had the molecular formula C28H32O8 derived from its HR-ESI-MS, which gave quasi-molecular ion peak at m/z 495.2023 [M – H]− (calcd for C28H31O8, 495.2019). Absorption maxima at 224 and 278 nm in the UV spectrum and absorption bands at 1613 and 1514 cm−1 in the IR spectrum suggested the presence of a non-conjugated aromatic ring. The 13C NMR spectrum of compound 2 showed 28 carbon signals except for one methoxyl signal, indicating that compound 2 was a sesquilignan. The 1H and 1He1H COSY spectra showed the presence of three sets of an ABX pattern in the aromatic region, a glycerol, and a tetrahydrofuran. The further comparation indicated that compound 2 was very similar with ehletianol C, except for the absence of methoxyl group at C-3 [14]. The stereochemistry of the tetrahydrobenzofuran part was elucidated by a NOESY experiment. The NOE correlations between H-8” (δH 2.79) and H-9’ (δH 4.34)/H-8’ (δH 3.06)/H-2” (δH 7.37) indicated a cis-configuration of 8’/8” and a trans configuration of 7”/8”. CD spectrum of compound 2, which showed the negative Cotton effects at 224 and 278 nm also indicated the same stereochemistry of ethletianol C. Thus, compound 2 was determined to be 3-demethoxyl- ethletianol C,

Fig. 2. Key COSY (e) and HMBC (→) correlations. 4

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Hepg 2

B16

S G C -7 0

110 100 90 80 70 60 50 40

IC 5 0 (μM)

30 20 10 10 8 6 4 2

CT X

32

30

29

28

27

22

21

20

17

15

12

4

2

1

0

Compounds Fig. 3. The cytotoxicities of compounds isolated (mean ± SD, n = 3). The IC50 values above 100 μM not provided.

NO

TNF-α

120 100 80 60

IC 5 0 (mM)

40 20 10 8 6 4 2

X M

D

32

30

29

28

22

21

20

17

15

14

12

9

8

4

3

2

1

0

Compounds Fig. 4. The inhibitory activities on the release of NO and TNF-α in RAW 264.7 cells induced by LPS (mean ± SD, n = 3). The IC50 values above 100 μM not provided.

5

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M. Wang, et al.

NMR data (Table 1) with those of compound 3 showed their close structural relationship. The differences were that C-1 was substituted by an hydroxymethyl group, and C-5 was substituted by a methoxyl group in compound 4. This assumption was supported by the observed HMBC correlations from proton signals at δH 4.97 (H-7) to C-1 (δC 136.6), C-2 (δC 112.0), C-6 (δC 116.1), and δH 3.74 (5′-OCH3) to C-4 (δC 147.9). The J7′, 8′ value (6.2 Hz) established a 7′,8′-trans configuration. The absolute configuration of 4 was assigned as 7′ R, 8′ S by the negative Cotton effect at 278 nm and the positive Cotton effect at 226 nm, in accordance with previously reported CD data of chushinzisin E [15]. Therefore, the structure of 4 was defined as (7′S, 8′S)-4-methoxyl-7-hydroxylmethyl9′-hydroxy-4, 7′-epoxy −3, 8′-neolignan, and named mubiesin D. Compound 5 obtained as an amorphous powder. Its molecular formula was determined to be C16H16O4 by its HR-ESI-MS data at m/z 271.0979 [M − H]−(calcd for C16H15O4, 271.0970). The 1H NMR spectrum showed eight aromatic protons at δH 7.50 (2H, d, J = 7.5 Hz, H-2′, 6′), 7.10 (2H, d, J = 7.5 Hz, H-3′, 5′), 7.56 (2H, overlap, H-2″, 6″), 7.12 (2H, d, J = 7.3 Hz, H-3″, 5″), which indicated the existence of two 1, 4-bisubstituted phenyl groups. Two phenolic hydroxyl groups at δH 11.31 and 11.38 (both 1H, brs, 4′, 4″-OH) were also observed, further indicated that two benzene rings were 4-hydroxyl substituted. The remaining oxymethylene [δH 4.63 (1H, dd, H-5a) and 4.44 (1H, d, H-5b)], three methines [δH 5.70 (1H, d, H-2), 4.74 (1H, s, H-3), 3.43 (1H, dd, H4)] were ascribed to a -2CH–3CHOH–4CH–5CH2O– fragment by the 1 He1H COSY spectrum. A 3-hydroxyl furan ring was further deduced from the HMBC analysis. The two aromatic rings were connected to the above moiety at C-2 and C-4 by HMBC cross speaks between H-2 (δH 5.70) and C-1′ (δC 132.8), C-2′/C-6′ (δC 128.3), between H-4 (δH 3.43) and C-1″ (δC 131.7), C-2″/C-6″ (δC 127.4). The absolute configuration was deduced to be 2R, 3S, 4S, according to the positive Cotton effect at 225 nm and the negative Cotton effect 276 nm in the CD spectrum consistent with (−) berchemol [17]. Consequently, compound 5 was determined to be (2R, 3S, 4S)-2, 4-bis-(4-hydroxy-phenyl)-tetrahydrofuran-3-ol, and named mubiesin E.

cochinchinensis seeds. Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgement This study was supported by a grant of National Natural Science Foundation of China (No. 81374067) and Shanghai Municipal Commission of Health and Family Planning (2018ZY002). Appendix A. Supplementary data The cytotoxic and anti-inflammatory activities of alcohol extract and its portions partitioned (Figs. S1–S3), influences of compounds isolated on RAW 264.7 cells vitality (Fig. S4), and NMR spectra (1H, 13C NMR, HSQC, 1He1H COSY, HMBC, NOESY) of five new compounds (Figs. S5–S34), are available in supporting information. References [1] A.M. Lu, L.Q. Huang, S.K. Chen, J. Charles, Flora of China, 19 Science Press, Beijing, 2011, p. 30. [2] H.V. Chuyen, M.H. Nguyen, P.D. Roach, J.B. Golding, S.E. Parks, Gac fruit (Momordica cochinchinensis Spreng.): a rich source of bioactive compounds and its potential health benefits, Int. J. Food Sci. Technol. 50 (2015) 567–577. [3] China Pharmacopoeia Committee, Chinese Pharmacopoeia, 1 China Medical Science Press, Beijing, 2015, pp. 65–66. [4] J.S. Yu, H.S. Roh, S. Lee, K. Jung, K.H. Baek, K.H. Kim, Antiproliferative effect of Momordica cochinchinensis seeds on human lung cancer cells and isolation of the major constituents, Rev. Bras. Farmacogn. 27 (2017) 329–333. [5] L. Zheng, Y.M. Zhang, Y.P. Liu, X.O. Yang, Y.Z. Zhan, Momordica cochinchinensis Spreng. seed extract suppresses breast cancer growth by inducing cell cycle arrest and apoptosis, Mol. Med. Rep. B 12 (2015) 6300–6310. [6] J.S. Yu, J.H. Kim, S. Lee, K. Jung, K.H. Kim, J.Y. Cho, Src/Syk-targeted anti-inflammatory actions of triterpenoidal saponins from Gac (Momordica cochinchinensis) seeds, Am. J. Chinese Med. 45 (2017) 459–473. [7] R. Fan, R.R. Cheng, H.T. Zhu, D. Wang, C.R. Yang, M. Xu, Y.J. Zhang, Two new oleanane-type triterpenoids from methanolyzed saponins of Momordica cochinchinensis, Nat. Prod. Commun. 11 (2016) 725–728. [8] K. Jung, Y.W. Chin, K.D. Yoon, H.S. Chae, C.Y. Kim, H. Yoo, J. Kim, Anti-inflammatory properties of a triterpenoidal glycoside from Momordica cochinchinensis in LPS-stimulated macrophages, Imunopharm. Immunot. 35 (2013) 8–14. [9] X.F. Wang, H. Li, K. Jiang, Q.Q. Wang, Y.H. Zheng, W. Tang, C.H. Tan, Anti-inflammatory constituents from Perilla frutescens on lipopolysaccharide-stimulated RAW264.7 cells, Fitoterapia 130 (2018) 61–65. [10] L. Karakaya, Y. Akgul, A. Nalbantsoy, Chemical constituents and in vitro biological activities of Eremurus spectabilis leaves, Nat. Prod. Res. 31 (2017) 1786–1791. [11] D. Qin, L. Li, J.L. Li, J. Li, D. Zhao, Y.T. Li, B. Li, X. Zhang, A new compound isolated from the reduced ribose-tryptophan Maillard reaction products exhibits distinct anti-inflammatory activity, J. Agric. Food Chem. 66 (2018) 6752–6761. [12] H.H. Xiao, Y. Dai, M.S. Wong, X.S. Yao, New lignans from the bioactive fraction of Sambucus williamsii Hance and proliferation activities on osteoblastic-like UMR106 cells, Fitoterapia 94 (2014) 29–35. [13] K.H. Kim, S.Y. Kim, E. Moon, K.R. Lee, Lignans from the tuber-barks of Colocasia antiquorum var. esculenta and their antimelanogenic activity, J. Agric. Food Chem. 58 (2010) 4779–4785. [14] O. Hofer, R. Schölm, Stereochemistry of tetrahydrofurofuran derivative circular dichroism and absolute conformation, Tetrahedron 37 (1981) 1181–1186. [15] R.Q. Mei, Y.H. Wang, G.H. Du, G.M. Liu, L. Zhang, Y.X. Cheng, Antioxidant lignans from the fruits of Broussonetia papyrifera, J. Nat. Prod. 72 (2009) 621–625. [16] H. Kizu, H. Shimana, T. Tomimori, Studies on the constituents of clematis species. VI the constituents of Clematis stans Sieb et Zucc, Chem. Pharm. Bull. 43 (1995) 2187–2194. [17] N. Sakurai, S. Nagashima, K. Kawai, T. Inoue, A new lignan, (−)-berchemol, from Berchemia racemose, Chem. Pharm. Bull. 37 (1989) 3311–3315.

3.2. The activities evaluation of compounds isolated Thirty two compounds isolated from the active portions were subjected to the cytotoxicity evaluation. The results indicated that the steroids (7, 13), fatty acid (6), and phenyl propionic acid (10) didn't show the significant activities (IC50 above than 100 μM). However, lignans 1, 2, 15, 17, and 20 showed the obvious cytotoxicity, they could obviously inhibit the growth of tumor cells Hepg 2, B 16, SGC-790 (IC50 values less than 10 μM, Fig. 3). Subsequently, all compounds isolated were subjected to the antiinflammation analysis. The results indicated that compounds tested didn't obviously influence RAW 264.7 cells vitality at the concentration of 50 μM (cells vitalities above 89.1%, Fig. S4). Seventeen compounds could inhibit the release of NO and TNF-α in RAW 264.7 cells induced by LPS (Fig. 4). In general, lignans, especially 1, 2, 15, 17, and 20, possess the most potent activities. The saponins (27–30, 32) with the same aglycon, all showed the similar activities. Interestingly, the compounds with more sugar chains showed the stronger action, indicating that the glycosylation could enhance the cytotoxicity and antiinflammation activities of saponins with the aglycon of gypsogenin. The results mentioned above revealed that lignans and saponins played the major role in the cytotoxic and anti-inflammatory activities of M.

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