Seven new neuroprotective sesquineolignans isolated from the seeds of Crataegus pinnatifida

Seven new neuroprotective sesquineolignans isolated from the seeds of Crataegus pinnatifida

Fitoterapia 133 (2019) 225–230 Contents lists available at ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote Seven new neur...

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Fitoterapia 133 (2019) 225–230

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Seven new neuroprotective sesquineolignans isolated from the seeds of Crataegus pinnatifida

T

Zhuo-Yang Chenga,1, Li-Li Loua,b,1, Pei-Yuan Yanga, Xiao-Yu Songa, Xiao-Bo Wangb, ⁎ ⁎ Xiao-Xiao Huanga,b, , Shao-Jiang Songa, a School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research & Development, Liaoning Province, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China b Chinese People's Liberation Army 210 Hospital, Dalian 116021, People's Republic of China

A R T I C LE I N FO

A B S T R A C T

Keywords: Crataegus pinnatifida 8-O-4′ type sesquineolignans Neuroprotective activity

The investigation of the ethanol extract of the seeds of Crataegus pinnatifida led to the isolation of seven new 8-O4′ type sesquineolignans crasesquineolignan A–G (1–7), along with a reported analogue, leptolepisol B (8). The chemical structures of these compounds were elucidated based on complex analysis of their MS, 1D and 2D NMR data. All the isolated compounds were tested for their neuroprotective effects against the damage of human neuroblastoma SH-SY5Y cells induced by H2O2, and most of them showed significant neuroprotective activity. Among them, compound 4 (77.58%) showed the best protective effect, even better than the positive control (69.26%) at 25 μM.

1. Introduction

described.

Hawthorn, a kind of plant belonging to the Crataegus genus of Rosaceae family, is widely distributed in Northern China, Japan, South Korea, Europe and North America [1]. There are approximately 300 species in Crataegus genus, among which Crataegus pinnatifida is the major species in China [2]. The fruits, leaves and flowers of hawthorn have been widely used as medicinal and functional food materials in China and European countries for a long history [2–4]. To date, over 150 chemical constituents have been isolated from this plant, including flavonoids, triterpenoids, steroids, lignans, organic acids, and nitrogencontaining compounds [5]. Earlier investigations on the biological activities of the fruits, leaves and flowers of C. pinnatifida showed that they displayed anti-inflammatory, anti-oxidant, hypolipidemic and anti-obesity activities [1,6]. Following the interest on discovering novel secondary metabolites, an ongoing study of the seeds of the C. pinnatifida was undertaken and led to the identification of seven new sesquineolignans: crasesquineolignan A–G (1–7) and an analogue leptolepisol B (8). All isolated compounds were 8-O-4′ type sequineolignans, which were rarely reported in the previous studies. In the present study, the isolation, structure elucidation, as well as the protective effects on H2O2 induced damage in human neuroblastoma SH-SY5Y cells were

2. Material and methods 2.1. General Optical rotations were measured on a Perkin-Elmer 341 polarimeter (Perkin-Elmer, Waltham, USA). UV spectra were recorded on a Shimadzu UV-1700 spectrophotometer. IR data were generated on a Bruker EQUINOX55 spectrometer (Germany). 1H NMR, 13C NMR, HSQC and HMBC spectra were recorded at room temperature on a Bruker ARX-400 and AV-600 spectrometers (Bruker Corporation, Bremen, Germany) with CD3OD as the solvent. ESIMS spectra were obtained with a HRESIMS Micro TOF spectrometer (Bruker Daltonics, Billerica, USA). HPLC was performed on an instrument composed of an Agilent 1100 instrument with YMC-Park ODS-A columns (250 mm × 10 mm I.D., S-5 μm, 12 nm). Column chromatography (CC) was performed using silica gel (100–200 mesh, 200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, PR China), ODS gel (12 nm S-75 mm, YMC Co., Ltd., Japan).



Corresponding authors at: School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research & Development, Liaoning Province, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China. E-mail addresses: [email protected] (X.-X. Huang), [email protected] (S.-J. Song). 1 Zhuo-Yang Cheng and Li-Li Lou contributed equally to this work. https://doi.org/10.1016/j.fitote.2019.01.008 Received 19 December 2018; Received in revised form 13 January 2019; Accepted 15 January 2019 Available online 17 January 2019 0367-326X/ © 2019 Elsevier B.V. All rights reserved.

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UV (MeOH) λmax (logε): 280 nm (3.92); IR (KBr) νmax: 3384, 2939, 1595, 1462, 1027 cm−1; the 1H and 13C NMR data were shown in Tables 1 and 2. Compound 2: yellowish oil (MeOH); HRESIMS (m/z): 625.2587 [M + Na]+ (C32H42O11Na, calc. 625.2619); [α]D20+ 2.5 (c 0.2, MeOH); UV (MeOH) λmax (logε): 230 nm (4.11), 280 nm (3.69); IR (KBr) νmax: 3396, 2938, 1603, 1425, 1029 cm−1; the 1H and 13C NMR data were shown in Tables 1 and 2. Compound 3: yellowish oil (MeOH); HRESIMS (m/z): 611.2593 [M + Na]+ (C31H40O11Na, calc. 611.2463); [α]D20 - 4.0 (c 0.2, MeOH); UV (MeOH) λmax (logε): 279 nm (3.75); IR (KBr) νmax: 3385, 2938, 1603, 1425, 1028 cm−1; the 1H and 13C NMR data were shown in Tables 1 and 2. Compound 4: yellowish oil (MeOH); HRESIMS (m/z): 627.2479 [M + Na]+ (C31H40O12Na, calc. 627.2412); [α]D20 - 4.0 (c 0.2, MeOH); UV (MeOH) λmax (logε): 279 nm (3.71); IR (KBr) νmax: 3386, 2940, 1603, 1425, 1030 cm−1; the 1H and 13C NMR data were shown in Tables 1 and 2. Compound 5: yellowish oil (MeOH); HRESIMS (m/z): 627.2141 [M + Na]+ (C31H40O12Na, calc. 627.2412); [α]D20 + 2.0 (c 0.2, MeOH); UV (MeOH) λmax (logε): 231 nm (4.21), 279 nm (3.77); IR (KBr) νmax: 3406, 2940, 1594, 1424, 1026 cm−1; the 1H and 13C NMR data were shown in Tables 1 and 2. Compound 6: yellowish oil (MeOH); HRESIMS (m/z): 629.2548 [M + Na]+ (C30H38O13Na, calc. 629.2205); [α]D20- 1.5 (c 0.2, MeOH); UV (MeOH) λmax (logε): 230 nm (4.22), 279 nm (3.81); IR (KBr) νmax: 3408, 2939, 1595, 1460, 1028 cm−1; the 1H and 13C NMR data were shown in Tables 1 and 2. Compound 7: yellowish oil (MeOH); HRESIMS (m/z): 629.2187 [M + Na]+ (C30H38O13Na, calc. 629.2205); [α]D20 - 1.5 (c 0.2, MeOH); UV (MeOH) λmax (logε): 230 nm (4.00), 279 nm (3.58); IR (KBr) νmax: 3391, 2940, 1594, 1462, 1029 cm−1; the 1H and 13C NMR data were shown in Tables 1 and 2. Compound 8: yellowish oil (MeOH); HRESIMS (m/z):629.2180 [M + Na]+ (C30H38O13Na, calc. 629.2205); [α]D20 - 2.0 (c 0.2, MeOH); UV (MeOH) λmax (logε): 231 nm (3.66), 280 nm (3.32); IR (KBr) νmax: 3398, 2939, 1606, 1462, 1029 cm−1; the 1H and 13C NMR data were shown in Tables 1 and 2.

2.2. Plant material The dried seeds of Crataegus pinnatifida were collected from Shijiazhuang, Hebei province, PR China, in 2011 and identified by professor Jincai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). 2.3. Extraction and isolation The dried seeds (30 kg) of Crataegus pinnatifida were extracted with 80% EtOH for three times. The extract after evaporation was partitioned by EtOAc, n-BuOH and water. The n-BuOH extract (1000 g) was chromatographed on macroporous resin D101, eluted with EtOH:H2O (40:60–90:10) to yield Fr-1 (300 g). Fr-1 was separated on a silica gel column and eluted with CH2Cl2:MeOH to yield seven subfractions, Fr.1 to Fr.7. Fr.2 was applied to octadecylsilyl (ODS) column and eluted with a 10:90 → 40:60 (v/v) gradient elution of MeOH/H2O to yield four subfractions, Fr.2–1–Fr.2–4. Compound 1 (7 mg, tR = 24.8 min) was purified from Fr.2–3 using silica gel column eluted with CH2Cl2:MeOH (30:1 → 5:1), followed by RP-HPLC. Fr.3 was fractionated by ODS column using a 10:90 → 40:60 (v/v) gradient elution of MeOH/H2O to yield six subfractions, Fr.3–1–Fr.3–6. Fr.3–2 was further purified by silica gel column to obtain six fractions (Fr.3–2-1 to Fr.3–2-6). Fr.3–2-4 was then purified by preparative RP-HPLC to afford compounds 2 (6 mg, tR = 31.2 min), 3 (10 mg, tR = 42.7 min) and 4 (41 mg, tR = 45.6 min). Fr.4 was subjected to octadecylsilyl ODS column and eluted using a 10:90 → 40:60 (v/v) gradient elution of MeOH/H2O to yield five subfractions, Fr.4–1–Fr.4–5. Fr.4–3 was separated on a silica gel column and eluted with CH2Cl2:MeOH to yield nine subfractions, Fr.4–3-1–Fr.4–3-9. Fr.4–3-1 was purified by semipreparative HPLC to afford compound 8 (47 mg, tR = 55.1 min). Fr.5 was also applied to ODS column and eluted with a 10:90 → 40:60 (v/v) gradient elution of MeOH/H2O to yield six subfractions, Fr.5–1–Fr.5–6. Fr.5–2 was separated on a silica gel column and eluted with CH2Cl2:MeOH (15:1 → 1:1) to yield six subfractions, Fr.5–2-1–Fr.5–2-6. And Fr.5–2-4 was purified by semipreparative RP-HPLC to afford compounds 5 (60 mg, tR = 51.3 min), 6 (10 mg, tR = 55.7 min) and 7 (4 mg, tR = 59.8 min) (Fig. 1). 2.4. Identification of compounds 1–8

2.5. Neuroprotective activities assay

Compound 1: yellowish oil (MeOH); HRESIMS (m/z): 625.2587 [M + Na]+ (C32H42O11Na, calc.625.2619); [α]D20- 2.0 (c 0.2, MeOH);

The ability of compounds 1–8 to protect human neuroblastoma SHSY5Y cells against oxidative stress induced by H2O2 was evaluated by

Fig. 1. Structures of compounds 1–8. 226

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Table 1 The 1H NMR data (400 MHz, CD3OD) for compounds 1–8. Position

1

2

3

4

1 2 3 4 5 6 7 8 9

― 6.98 (d, 2.3) ― ― 6.78 (d, 8.0) 6.82 (dd, 8.0, 2.3) 4.54 (d, 6.6) 4.34 (m) 3.67 (dd, 11.8, 3.1) 3.45 (m) ― 7.09 (d, 2.3) ― ― 6.99 (d, 8.2) 6.93 (d, 8.2, 2.3) 4.92 (d, 5.5) 4.24 (m) 3.75 (dd, 11.8, 4.2) 3.49 (dd, 11.8, 5.1) ― 6.85 (d 1.6) ― ― 6.94 (d, 8.2) 6.70 (dd, 8.2, 1.6) 2.62 (2H, m) 1.81 (2H, m) 3.56 (2H, t, 6.5)

― 6.99 (br.s) ― ― 6.80 (d, 8.3) 6.83 (d, 8.3) 4.45 (d, 5.7) 4.32 (m) 3.87 (dd, 11.9, 5.7) 3.78 (m) ― 7.07 (br.s) ― ― 6.94 (d, 8.3) 6.90 (d, 8.3) 4.85 (d, 5.9) 4.29 (m) 3.66 (dd, 11.8, 4.9) 3.43 (dd, 11.8, 4.2) ― 6.79 (br.s) ― ― 6.80 (d, 8.2) 6.66 (dd, 8.2, 2.3) 2.61 (2H, m) 1.80 (2H, m) 3.56 (2H, t, 5.9)

― 7.03 (d, 1.5) ― ― 6.89 (d, 8.3) 6.85 (dd, 8.3, 1.5) 4.94 (d, 5.2) 4.18 (m) 3.54 (dd, 12.0, 3.6) 3.89 (m) ― 7.04 (d, 1.2) ― ― 6.74 (d, 8.1) 6.85 (dd, 8.1, 1.2) 4.85 (d, 5.9) 4.36 (m) 3.87 (m) 3.80 (m) ― 6.55 (s) ― ― ― 6.55 (s) 2.65 (2H, m) 1.84 (2H, m) 3.59 (2H, t, 6.4)

― 7.07 ― ― 7.01 6.88 4.95 4.19 3.55 3.90 ― 7.04 ― ― 6.77 6.87 4.93 4.29 3.75 3.49 ― 6.54 ― ― ― 6.54 2.64 1.83 3.58

3.82 3.83 3.85 3.39 1.13 ― ―

3.82 (3H, s) 3.77 (3H, s) 3.84 (3H, s) ―

3.80 (3H, s) 3.81 (3H, s) 3.80 (3H, s) ―

3.26 (3H, s) ―

― 3.80 (3H, s)

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 7′′ 8′′ 9′′ 3-OCH3 3′-OCH3 3′′-OCH3 7-OCH2CH3 7-OCH3 5′′-OCH3

(3H, (3H, (3H, (2H, (3H,

s) s) s) m) t, 6.9)

5

6

3.81 (3H, s) 3.86 (3H, s) 3.79 (3H, s) ―

― 7.03 (br.s) ― ― 6.76 (d, 8.0) 6.85 (d, 8.0) 4.88 (d, 5.6) 4.28 (m) 3.69 (m) 3.49 (m) ― 7.10 (br.s) ― ― 7.00 (d, 8.3) 6.93 (dd, 8.3, 1.3) 4.93 (d, 5.1) 4.32 (m) 3.76 (dd, 12.0, 4.2) 3.48 (dd, 12.0, 3.8) ― 7.05 (br.s) ― ― 6.98 (d, 7.7) 6.85 (d, 7.7) 4.57 (d, 5.7) 3.66 (m) 3.49 (m) 3.36 (m) 3.80 (3H, s) 3.83 (3H, s) 3.85 (3H, s) ―

― 7.03 ― ― 6.74 6.84 4.82 4.33 3.84 3.76 ― 7.04 ― ― 6.87 6.84 4.85 4.33 3.84 3.76 ― 7.01 ― ― 6.87 6.84 4.53 3.72 3.65 3.58 3.78 3.81 3.81 ―

― 3.79 (3H, s)

― ―

― ―

(br.s)

(d, 8.4) (dd, 8.4, 1.7) (d, 5.2) (m) (dd, 12.1, 3.5) (dd, 12.1, 5.3) (br.s)

(d, 8.2) (dd, 8.2, 1.6) (overlap) (m) (dd, 11.9, 4.2) (dd, 119, 5.8) (s)

(s) (2H, m) (2H, m) (2H, t, 6.4)

(br.s)

(d, 8.3) (m) (d, 5.9) (m) (m) (m) (br.s)

(m) (m) (d, 5.9) (m) (m) (m) (br.s)

(m) (m) (d, 5.7) (m) (m) (m) (3H, s) (3H, s) (3H, s)

7

8

― 7.03 (d, 1.2) ― ― 6.74 (d, 8.2) 6.83 (d, 8.2, 1.2) 4.83 (d, 5.8) 4.33 (m) 3.84 (m) 3.76 (m) ― 7.04 (d, 1.2) ― ― 6.88 (d, 8.2) 6.86 (dd, 8.2, 1.2) 4.82 (d, 6.1) 4.32 (m) 3.84 (m) 3.76 (m) ― 6.98 (d, 1.3) ― ― 6.86 (d, 8.3) 6.80 (dd, 8.3, 1.3) 4.54 (d, 8.5) 4.17 (m) 3.65 (m) 3.48 (m) 3.77 (3H, s) 3.78 (3H, s) 3.82 (3H, s) ―

― 7.04 ― ― 6.76 6.86 4.90 4.24 3.74 3.47 ― 7.11 ― ― 7.02 6.95 4.93 4.29 3.76 3.50 ― 6.84 ― ― 6.93 6.69 2.62 1.81 3.56

3.83 (3H, s) 3.82 (3H, s) 3.83 (3H, s) ―

― ―

― ―

(d, 1.0)

(d, 8.1) (d, 8.1, 1.0) (d, 5.7) (m) (m) (m) (d, 1.8)

(d, 8.3) (m) (d, 5.4) (m) (m) (m) (d, 1.7)

(d, 8.2) (dd, 8.2, 1.7) (2H, m) (2H, m) (2H, t, 6.5)

(2H, m, H-10) and 1.13 (3H, t, J = 6.9 Hz, H-11), three oxygen-bearing methane groups at δH 3.67 (1H, dd, J = 11.8, 3.1 Hz, H-9a), 3.45 (1H, m, H-9b), 3.75 (1H, dd, J = 11.8, 4.2 Hz, H-9'a), 3.49 (1H, dd, J = 11.8, 5.1 Hz, H-9'b), and 3.56 (2H, t, J = 6.5 Hz, H-9″), four oxymethines at δH 4.54 (1H, d, J = 6.6 Hz, H-7), 4.34 (1H, m, H-8), 4.92 (1H, d, J = 5.5 Hz, H-7′), 4.24 (1H, m, H-8′), and two methenes at δH 2.62 (2H, m, H-7″) and 1.81 (2H, m, H-8″), which were all confirmed by the 13C NMR data (Table 2). The 13C NMR spectrum of 1 revealed 32 carbons, which were resolved into 18 aromatic carbons arising from three benzene rings and 14 aliphatic carbons. The HMBC correlations (Fig. 2) of H-7 at δH 4.54 with C-1, C-2, C-6, C-8 and C-9 and of H-7′ at δH 4.92 with C-1′, C-2′, C-6′, C-8′ and C-9′ and of H-7″ at δH 4.54 with C-1″, C2″, C-6″, C-8″ and C-9″ confirmed the presence of two guaiacylglycerol units and one propanolguaiacol unit. These NMR spectroscopic data suggested that 1 might be an 8-O-4′ system sesquineolignan [8,9]. The HMBC correlation from 7-OCH2CH3 to C-7 indicated that the ethoxy group was located at C-7. Besides, the positions of three methoxy groups were also confirmed by HMBC correlations. Previous reports revealed that compounds with a threo-aryl glycerol unit could be distinguished from its erythro isomer with the chemical shift difference between C-8 and C-7. Some researchers in our group found that when the 7-OH was substituted by alkyl, the ΔδC8-C7 values of erythro were larger than threo [7]. The relative stereochemistry of C-7 and C-8 of 1 was established as an erythro configuration based on the larger ΔδC8-C7 value (3.6 ppm) compared with compound 2 (1.6 ppm). When 7-OH wasn't substituted, the ΔδC8-C7 value of threo configuration was larger than the erythro configuration [10–13]. Thus, the C-7′ and C8′ was established as a threo configuration based on the larger ΔδC8-C7 value (13.5 ppm) when compared with compound 3 (12.1 ppm).

the MTT assay [7]. And Trolox has been used as the positive control. The human neuroblastoma SH-SY5Y cells (1.2 × 104 cells/well) were grown in 96-well plates for 12 h. At the end of incubation period, the SH-SY5Y cells were incubated with or without tested compounds (25, 50 and 100 μM) for 1 h. In order to measure their cytoprotective effects, 300 μM newly-prepared H2O2 was added to the cells for 3 h. After then, the drug solutions in the wells were discarded and 20 μL MTT dissolved by phosphate-buffered saline medium was added in each well. The plates were gently shaken and incubated for another 4 h at 37 °C in 5% CO2 atmosphere. Then, the medium was cleared and 150 μL of DMSO was added to cells to dissolve the formazan. The results were obtained in absorbance at 490 nm using a Thermo microplate reader and the cell viability of each group was expressed as a percentage relative to the value of the control group (100%).

3. Results and discussion Compound 1 was obtained as yellowish oil. Its molecular formula was established as C30H38O11 with 12 indices of hydrogen deficiency by the positive HRESIMS molecular ion at m/z 625.2587 [M + Na]+ (C32H42O11Na, calcd 625.2619). The 1H NMR spectrum (Table 1) showed six aromatic proton signals at δH 6.98 (1H, d, J = 2.3 Hz, H-2), 6.78 (1H, d, J = 8.0 Hz, H-5), 6.82 (1H, d, J = 8.0, 2.3 Hz, H-6), 7.09 (1H, d, J = 2.3 Hz, H-2′), 6.99 (1H, d, J = 8.2 Hz, H-5′), 6.93 (1H, dd, J = 8.2, 2.3 Hz, H-6′), 6.85 (1H, d, J = 1.6 Hz, H-2″), 6.94 (1H, d, J = 8.2 Hz, H-5″), and 6.70 (1H, dd, J = 8.2, 1.6 Hz, H-6″), indicating the presence of three ABX-coupled benzene rings. The 1H NMR spectrum also displayed signals attributed to three methoxys at δH 3.85 (3H, s), 3.83 (3H, s),and 3.82 (3H, s), one oxygen-bearing ethyl at δH 3.39 227

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centers at C-7′ and C-8′ positions. Thus, the structure of 2 was elucidated as (7, 8-threo; 7′, 8′-threo)-3, 3′, 3″-trimethoxy-4-hydroxy-4′, 8oxy-4″, 8′-oxy-sesquineolignan-7-methoxy-9, 7′, 9′, 9″-tetrol and named crasesquineolignan B. Compound 3 was isolated as a yellowish oil and its molecular formula was confirmed as C31H40O12 by positive HRESIMS at m/z 627.2479 [M + Na]+ (C31H40O12Na, calcd for 627.2412). The 1H and 13 C NMR of 3 in CD3OD showed signals attributed to two 1, 3, 4-trisubstituted aromatic rings at δH 7.03 (1H, d, J = 1.5 Hz, H-2), 6.89 (1H, d, J = 8.3 Hz, H-5), 6.85 (1H, dd, J = 8.3, 1.5 Hz, H-6), 7.04 (1H, d, J = 1.2 Hz, H-2′), 6.74 (1H, d, J = 8.1 Hz, H-5′) and 6.85 (1H, dd, J = 8.1, 1.2 Hz, H-6′), a symmetrical 1, 3, 4, 5-tetrasubstituted aromatic ring at δH 6.55 (2H, s, H-2″/6″), and four methoxy groups at 3.81 (3H, s, 3′-OCH3) and 3.80 (12H, s, 3/3″/5″-OCH3). Additionally, a sequence of oxygenated methines and methylenes were observed as same as 1. Overall, the 1H and 13C NMR data of 3 were similar to those of 1 except for the addition of a methoxy group at C-2″ and reduction of an ethoxy group at C-7, indicating another 8-O-4′ system sesquineolignan. The 7, 8-thero configuration of 3 was assigned owing to the larger ΔδC8-C7 value (13.5 ppm) when compared with 6, and the relative configuration of C-7′ and C-8′ was determined to be erythro because of the smaller ΔδC8′-C7′ value (12.1 ppm) when compared with 4. Therefore, the structure of 3 was determined as (7, 8-threo; 7′, 8′-erythro)-3, 3′, 3″, 5″tetramethoxy-4-hydroxy-4′, 8-oxy-4″, 8′-oxy-sesquineolignan-7, 9, 7′, 9′, 9″-pentol and named crasesquineolignan C. Compound 4 was obtained as a yellowish oil and was determined to have a molecular formula C31H40O12 based on HRESIMS analysis, as same as that of 3. The 1H and 13C NMR data was almost identical to be 3, indicating that they are diastereoisomers and shared the same planar structure. By comparing the relevant carbon signals of 3 and 4, the 7, 8thero configuration of 4 was assigned because of the equal value of 13.5 ppm when compared with 3 (ΔδC8-C7 = 13.5 ppm), and the relative configuration of C-7′ and C-8′ of 4 was also determined to be thero configuration according to its larger value of ΔδC8′-C7′ (13.1 ppm) when compared with 3 (12.1 ppm). Therefore, the structure of 4 was determined as (7, 8-threo; 7′, 8′-threo)-3, 3′, 3″, 5″-tetramethoxy-4-hydroxy-4′, 8-oxy-4″, 8′-oxy-sesquineolignan-7, 9, 7′, 9′, 9″-pentol and named crasesquineolignan D. Compound 5 was obtained as a yellowish oil with the molecular formula C30H38O13 established by HRESIMS data, indicating 12 degrees of unsaturation. The 1H NMR spectrum of 5 showed three groups of 1, 3, 4-trisubstituted aromatic rings at δH 7.03 (1H, br s, H-2), 6.76 (1H, d, J = 8.0 Hz, H-5), 6.85 (1H, d, J = 8.0 Hz, H-6), 7.10 (1H, br s, H-2′), 7.00 (1H, d, J = 8.3 Hz, H-5′), 6.93 (1H, dd, J = 8.3, 1.3 Hz, H-6′), 7.05 (1H, br s, H-2″), 6.98 (1H, d, J = 7.7 Hz, H-5″), and 6.85 (1H, d, J = 7.7 Hz, H-6″) and three groups of methoxys at δH 3.85 (3H, s, 3′′OCH3), 3.83 (3H, s, 3′-OCH3),and 3.80 (3H, s, 3-OCH3). The signals at

Table 2 The 13C NMR data (100 MHz, CD3OD) for compounds 1–8. Position

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 7′′ 8′′ 9′′ 3-OCH3 3′-OCH3 3′′-OCH3 7-OCH2CH3

131.6 112.1 149.0 147.4 115.9 121.4 82.4 86.0 62.3 136.3 112.4 151.5 149.5 118.2 120.5 73.8 87.3 61.8 138.2 113.9 151.6 147.5 119.4 122.0 32.7 35.5 62.2 56.5 56.5 56.4 65.7 15.6 ― ―

131.0 112.2 149.1 147.2 116.0 121.6 84.1 85.7 62.2 136.8 112.7 151.4 149.2 117.9 120.9 73.9 86.7 62.0 138.1 114.0 151.8 147.6 119.6 121.8 32.7 35.5 62.2 56.5 56.4 56.5 ―

136.7 112.3 151.5 148.4 118.5 120.5 73.8 87.3 61.4 134.7 111.8 148.7 147.0 115.7 121.0 74.1 86.2 62.2 139.9 106.8 154.3 134.2 154.3 106.8 33.4 35.4 62.2 56.5 56.3 56.6 ―

136.8 112.1 151.3 148.7 118.3 120.7 73.7 87.2 61.4 134.6 111.7 148.8 147.1 115.8 120.8 74.0 87.1 61.8 139.9 106.8 154.3 133.8 154.3 106.8 33.4 35.4 62.1 56.5 56.3 56.6 ―

133.8 111.7 148.7 147.1 115.8 120.6 73.9 87.0 61.8 136.7 112.4 151.3 148.9 118.3 120.7 73.6 86.7 61.8 137.7 112.2 151.4 148.7 118.4 120.5 75.0 77.4 64.2 56.5 56.5 56.4 ―

134.2 111.9 148.4 147.0 115.7 120.9 74.0 86.3 62.2 137.0 112.7 151.5 148.7 118.5 120.9 73.9 86.2 62.2 137.7 112.7 151.6 148.5 118.6 120.9 75.8 76.6 64.5 56.5 56.5 56.4 ―

134.2 111.9 148.5 147.0 115.7 120.9 74.0 86.3 62.2 137.0 112.7 151.5 148.7 118.5 120.9 73.9 86.1 62.2 137.7 112.3 151.6 148.5 118.6 120.4 75.1 77.4 64.2 56.5 56.5 56.4 ―

133.7 111.6 148.7 147.1 115.8 120.7 73.9 87.1 61.7 136.7 112.3 151.3 148.8 118.3 120.7 73.7 87.0 61.7 138.1 113.8 151.5 147.4 119.2 121.9 32.7 35.5 62.2 56.4 56.4 56.3 ―

57.2 ―

― 56.6

― 56.6

― ―

― ―

― ―

― ―

7-OCH3 5′′-OCH3

Herein, the structure of 1 was defined as (7,8-erythro; 7′,8′-threo)-3, 3′, 3″-trimethoxy-4-hydroxy-4′, 8-oxy-4″, 8′-oxy-sesquineolignan-7ethoxy-9, 7′, 9′, 9″-tetrol, and was named crasesquineolignan A. Compound 2 was obtained as yellowish oil and was determined to have a molecular formula of C31H40O11 based on HRESIMS analysis, indicating 12 degrees of unsaturation. The 1H and 13C NMR data of 2 were similar to those of 1, suggesting an analogue of 1. The only difference between 2 and 1 was that the signals for ethoxy group in the NMR spectra of 1 disappeared, and an additional methoxy group was observed in 2, which was confirmed by the HMBC correlation from 7OCH3 to C-7. The threo configuration between two chiral centers at C-7 and C-8 was determined by its smaller ΔδC8-C7 value (1.6 ppm). Compared with compound 3, the larger ΔδC8′-C7′ value (12.8 ppm) suggested a relative-thero configuration between the other two chiral

Fig. 2. Key HMBC correlations of compounds 1–3 and 5. 228

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Fig. 3. The effect of isolated compounds on the damaged SH-SY5Y cells induced by H2O2 using MTT assay. The cell viability was represented as the percentage of viable cells compared to the control group (cell viability 100%). Values expressed in mean ± standard error of the mean, n = 3. ###P < .001 compared with the Control group. *** P < .001, ** P < .01, and * P < .1 compared with the H2O2 group.

δH 4.57 (1H, d, J = 5.7 Hz, H-7″), 3.66 (1H, m, H-8″), 3.49 (1H, m, H9″a) and 3.36 (1H, m, H-9″b) were attributed to a 1, 2, 3-propane-triol group. Over all, the 1H and 13C NMR data were similar to compound 1, except for the absence of the ethoxy group at C-7 and the presence of the 1, 2, 3-propane-triol group, which could be confirmed by the HMBC correlations from H-7″ to C-1″, C-2″ and C-6″. The 7, 8-threo configuration was assigned owing to the larger ΔδC8-C7 value (13.1 ppm), and the relative configuration of C-7′ and C-8′ was also determined to be threo because of the larger ΔδC8′-C7′ value (13.1 ppm) when compared with 6. It is reported that the relative configuration of arylglycerols without substituents at C-7 or/and C-8 of the glycerol moiety could be determined by the ΔδC8-C7 values (erythro: ΔδC8-C7 < 1.0 ppm; threo: ΔδC8-C7 ≥ 2.0 ppm) [10,12,13]. The relative configuration of the threo configuration between two chiral centers at C-7″ and C-8″ positions were determined by its larger ΔδC8″-C7″ value (2.4 ppm). Therefore, the structure of 5 was determined as (7, 8-threo; 7′, 8′-threo; 7″, 8″-threo)-3, 3′, 3″-trimethoxy-4-hydroxy-4′, 8-oxy-4″, 8′-oxy-sesquineolignan-7, 9, 7′, 9′, 7″, 8″, 9″-heptanol and named crasesquineolignan E. Compound 6 was obtained as yellowish oil and had a molecular formula of C30H38O13 with 12 degrees of unsaturation based on its [M + Na]+ ion peak at m/z 629.2548 in HRESIMS. The NMR spectroscopic data were similar to those of 5, suggesting that 6 was a stereoisomer of 5. The differences were that the smaller ΔδC8-C7 value (12.3 ppm), smaller ΔδC8′-C7′ value (12.3 ppm) and smaller ΔδC8″-C7″ value (0.8 ppm) suggested the erythro relative configurations at C-7/8, C-7′/8′ and C-7″/8″ when compared with 5. Thus, the structure of 6 was deduced as (7, 8-erythro; 7′, 8′-erythro; 7″, 8″-erythro)-3, 3′, 3″-trimethoxy-4-hydroxy-4′, 8-oxy-4″, 8′-oxy-sesquineolignan-7, 9, 7′, 9′, 7″, 8″, 9″-heptanol and named crasesquineolignan F. Compound 7 was isolated as a yellowish oil and had the formula C30H38O13 deriving from its positive HRESIMS. Comparison of NMR data with those of 5 and 6 showed they shared the same planar sturcture. The ΔδC8″-C7″ (2.3 ppm) implied that the relative configuration at C-7″/8″ was threo, as same as compound 5. Thus, the differences between 7 and 5 were the smaller ΔδC8-C7 value (12.3 ppm) and the smaller ΔδC8′-C7′ (12.2 ppm), which suggested the erythro relative configuration at C-7/8 and C-7′/8′ in 7. Therefore, the structure of 7 was deduced as (7, 8-erythro; 7′, 8′-erythro; 7″,8″-threo)-3, 3′, 3″-trimethoxy4-hydroxy-4′, 8-oxy-4″, 8′-oxy-sesquineolignan-7, 9, 7′, 9′, 7″, 8″, 9″heptanol and named crasesquineolignan G. Compound 8 had a molecular formula C30H38O11 determined on the

analysis of its HRESIMS. It was found that the planar sturcture of 8 has been reported in 1979 [14] without attribution of the 1H and 13C NMR data. And the configuration of this compound was not reported either. The larger ΔδC8-C7 value (13.2 ppm) and the larger ΔδC8′-C7′ value (13.3 ppm) established a 7,8-threo; 7′,8′-threo relative configuration of 8. Besides, it was the first time that the relative configuration of this compound had been reported. Thus, compound 8 was identified as (7, 8-erythro; 7′, 8′-erythro)-leptolepisol B. All of the isolated compounds (1–8) were tested for their neuroprotective activities against human neuroblastoma SH-SY5Y cell damage induced by H2O2 using MTT assay. As shown in Fig. 3, most of the compounds showed significant cytoprotective activity, which were comparable to or more potent than the positive control, Trolox. At 25 μM, compounds 1, 2, 4, 5, 6 and 8 showed more remarkable neuroprotective activity, the cell viability of which were 71.10%, 74.22%, 77.58%, 75.90%, 74.78% and 73.34%, respectively, much more potent than Trolox (69.26%). These results demonstrated that these sesquineolignans might be new and effective resource for the treatment of the neurodegenerative diseases. 4. Conclusion In conclusion, seven new 8-O-4′ style sesquineolignans were isolated from the seeds of Crataegus pinnatifida, along with a reported congener. The planar structures and relative configurations of these compounds were determined by HRESIMS, 1D and 2D NMR spectra. All the isolated compounds were tested for their neuroprotective activities. Compounds 1, 2, 4, 5, 6 and 8 showed more potent activity than the positive control at 25 μM. Among them, compound 4 possessed the most significant neuroprotective activity, which might be worth for further evaluation. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements We greatly acknowledge the financial supports from the Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University (ZQN2018006) and the Project of Innovation Team (LT2015027) of Liaoning of P.R. China. 229

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Appendix A. Supplementary data

pharmacology, and potential applications, Molecules 19 (2) (2014) 1685–1712. [6] E.S. Kao, C.J. Wang, W.L. Lin, C.Y. Chu, T.H. Tseng, Effects of polyphenols derived from fruit of Crataegus pinnatifida on cell transformation, dermal edema and skin tumor formation by phorbol ester application, Food Chem. Toxicol. 45 (2007) 1795–1804. [7] X.X. Huang, Q. Ren, X.Y. Song, L. Zhou, G.D. Yao, X.B. Wang, S.J. Song, Seven new sesquineolignans isolated from the seeds of hawthorn and their neuroprotective activities, Fitoterapia 125 (2018) 6–12. [8] S.H. Sung, M.S. Huh, Y.C. Kim, New tetrahydrofuran-type sesquilignans of saururus chinensis root, Chem. Pharm. Bull. 49 (2001) 1192–1194. [9] A. Sakakibara, T. Sasaya, K. Miki, H. Takahashi, Lignans and Brauns' lignins from softwoods, Holzforschung 41 (1987) 1–11. [10] S. Lin, S. Wang, M. Liu, M. Gan, S. Li, Y. Yang, Y. Wang, W. He, J. Shi, Glycosides from the stem bark of Fraxinus sieboldiana, J. Nat. Prod. 70 (2007) 817–823. [11] L. Xiong, C. Zhu, Y. Li, Y. Tian, S. Lin, S. Yuan, J. Hu, Q. Hou, N. Chen, Y. Yang, J. Shi, Lignans and neolignans from Sinocalamus affinis and their absolute configurations, J. Nat. Prod. 74 (2011) 1188–1200. [12] M. Gan, Y. Zhang, S. Lin, M. Liu, W. Song, J. Zi, Y. Yang, X. Fan, J. Shi, L. Hu, L. Sun, N. Chen, Glycosides from the root of Iodes cirrhosa, J. Nat. Prod. 71 (2008) 647–654. [13] R. Guo, L. Zhou, P. Zhao, X.B. Wang, X.X. Huang, S.J. Song, Two new sesquineolignans from the seeds of Crataegus pinnatifida and their β-amyloid aggregation inhibitory activity, Nat. Prod. Res. (2018) 1–7. [14] K. Miki, T. Sasaya, A. Sakakibara, Structures of new lignans from Larix leptolepis Gord, Tetrahedron Lett. 20 (1979) 799–802.

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