Fitoterapia 136 (2019) 104165
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Diarylheptanoids from the fresh pericarps of Juglans hopeiensis Yi Lin, Xiaogang Peng, Hanli Ruan
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T
School of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Hangkonglu 13, Wuhan 430030, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Juglans hopeiensis Juglandaceae Diarylheptanoids Neuroprotective effects
Ten new diarylheptanoids (1−10), including five diarylether-type (1–5), three biaryl-type (6–8), and two linear type (9–10), along with eighteen known ones, were isolated from the fresh pericarps of Juglans hopeiensis Hu. Their structures were settled by integrated spectroscopic techniques, and their absolute configurations were determined by a combination of circular dichroism analysis, optical rotations, and NOESY experiments. Screening results showed that some isolated diarylheptanoids exhibited moderate neuroprotective effects on H2O2-induced or CoCl2-induced cellular damage in human neuroblastoma SH-SY5Y cells.
1. Introduction The genus Juglans (Juglandaceae) contains approximately 20 species and distributes over the north temperate and subtropical zones of the world [1]. Among them, 5 species grow in China, which are J. mandshurica, J. regia, J. sigillata, J. cathayensis and J. hopeiensis [2]. The fresh pericarps of J. mandshurica and J. regia are well-known as ‘qinglongyi’, which have been used for traditional medical treatments for centuries in China, on the basis of their anti-tumour, anti-inflammatory and antioxidant activities [1]. Phytochemical studies on Juglans have revealed the presence of diarylheptanoids [3–5], naphthoquinones [6,7], tetralones [6,8], terpenoids [9,10], flavonoids [11], and lignans [12]. Diarylheptanoids are characterized by a 1,7-diphenylheptane skeleton, more than 400 of which have been identified so far [13]. Diarylheptanoids can be divided into linear and macrocyclic types. Linear diarylheptanoids are abundant in plants of the genera Curcuma, Alpinia (Zingiberaceae), and Betula (Betulaceae), whereas cyclic diarylheptanoids are mainly distributed in Myrica (Myricaceae), Acer (Aceraceae), and Juglans (Juglandaceae) species [14]. Over the past few decades, diarylheptanoids constitute a group of natural products gaining emerging interest due to their remarkable bioactivities including antioxidant [15–17], estrogenic [18,19], NO, TNF-α and IL-6 production inhibitory [20], cytotoxicity [21,22], anti-influenza [23], anti-emetic [24], anti-adipogenic [25], and anti-microbial activities [26]. Juglans hopeiensis Hu (Ma walnut), narrowly distributed in China [27], is mainly used for artwork with high ornamental value due to their hard and textured shells [28]. So far, none of the phytochemical studies have been carried out on J. hopeiensis. For the sake of searching
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for structurally diverse and biologically significant diarylheptanoids, we put forward the study on the fresh pericarps of J. hopeiensis. Accordingly, ten undescribed diarylheptanoids, jughopnin A-J (1–10), together with eighteen known ones, were isolated and identified. In this paper, the isolation, structural elucidation, and neuroprotective activities of these compounds, are reported. 2. Experimental 2.1. General experimental procedures Optical rotations were acquired on a Autopol IV-T automatic polarimeter. UV spectra were obtained on a Perkin-Elmer Lambda-25 UV–vis spectrophotometer. IR spectra were measured with a Bruker VERTEX 70 FT-IR microscopic spectroscopy. CD spectra were obtained using a JASCO J-810 CD spectrometer. NMR spectra were recorded on a Bruker-AM-400 spectrometer with trimethylsilyl (TMS) as the internal standard. HRESIMS was carried on a Thermo Scientific LTQ-Orbitrap XL mass spectrometer and a Bruker micro TOF II spectrometer running in positive ion mode. MPLC was conducted on an EZ Purifer III chromatography system (Lisui Chemical Engineering Co., Ltd., Shanghai, China) with Spherical C18-ODS column (40–60 μm). Column chromatography was run with silica gel (100–200 or 200–300 mesh; Yantai Jiangyou Chemical Inc., Shandong, China), octadecyl silica (ODS) (50 μm, YMC Co. Ltd., Tokyo, Japan), Sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden) and SBC MCI GEL (type F, 75–150 μm; Chengdu Sci-Bio-Chem Co.Ltd. Sichuan, China). Semi-preparative HPLC separation were performed on Agilent 1260 system and 1100 system with a YMC reversed-phase column (250 × 10 mm, 5 μm, Tokyo,
Corresponding author. E-mail address:
[email protected] (H. Ruan).
https://doi.org/10.1016/j.fitote.2019.05.004 Received 21 March 2019; Received in revised form 28 April 2019; Accepted 5 May 2019 Available online 07 May 2019 0367-326X/ © 2019 Elsevier B.V. All rights reserved.
Fitoterapia 136 (2019) 104165
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silica gel column (CH2Cl2/MEOH, 1:0 to 10:1), followed by semi-preparative HPLC (MeCN/H2O, 55:45) to afford compound 23 (8.0 mg) and 28 (25.3 mg). The CH2Cl2 portion (782 g) was subjected to separation over a silica gel column (Φ 10 × 100 cm, 100–200 mesh, 1.8 kg), eluting with a gradient system of PE/EtOAc (15:1 to 1:3) to obtain six fractions, Frs. A–F, in view of the identical TLC profiles. Fr. C (13.0 g) yielded 4 (6.0 mg) (MeOH/H2O, 50:50), 8 (8.1 mg) (MeCN/H2O, 75:25), 9 (5.3 mg) (MeOH/H2O, 50:50), 10 (22.1 mg) (MeCN/H2O, 40:40), 20 (10.3 mg) (MeOH/H2O, 50:50), 21 (2.1 mg) (MeCN/H2O, 50:50), 26 (21.3 mg) (MeOH/H2O, 50:50), and 27 (93.0 mg) (MeCN/H2O, 40:60) through a series of separation steps using Sephadex LH-20 column chromatography (CH2Cl2/MeOH, 1:1), a silica gel column (CH2Cl2/ MeOH, 1:0 to 5:1), ODS column chromatography (MeOH/H2O, 40:60 to 100:0) and semi-preparative HPLC. Fr. D (10.0 g) yielded 7 (4.2 mg) (MeCN/MeOH, 50:50) by using Sephadex LH-20 column chromatography (CH2Cl2/MeOH, 1:1), preparative TLC (CH2Cl2/MeOH, 20:1) as well as semi-preparative HPLC.
Japan). Thin-layer chromatography (TLC) was worked on silica gel plates (GF-254, Yantai Chemical Industry Research Institute). MTT assays were accomplished on a Bio-Tek Synergy 2 multimode microplate reader (Gene Company Ltd.), and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT) were purchased from WuHanGoodti meBio-Technology Co., Ltd. 2.2. Plant material The green pericarps of Juglans hopeiensis Hu (Juglandaceae) were collected in September 2015 in Laishui city, Hebei province, P.R. China and confirmed taxonomically by Prof. Hanli Ruan (Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology). A voucher specimen (20151020) has been deposited in the Herbarium of Materia Medica, Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, P. R. China. 2.3. Extraction and isolation
2.3.1. Jughopnin A (1) Yellowish oil; [α]20 D ± 0 (c 0.45, MeOH); UV (MeOH) λmax (log ε) 206 (3.03), 250 (2.79), 300 (2.39) nm; IR (KBr) νmax 3385, 2938, 1700, 1499, 1414, 1253, 1098, 1052, 820 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 347.1257 [M + Na]+ (calcd for C20H20O4Na, 347.1259).
The air-dried and powdered pericarps of J. hopeiensis (150 kg) were extracted three times with 95% EtOH (v/v) at room temperature and the combined extracts were evaporated to dryness under vacuum to give the crude extract (58 kg). The crude extract was suspended in water and partitioned successively with a serious of solvents to give fractions of petroleum ether (PE), CH2Cl2 and EtOAc. The petroleum ether portion (1038 g) was chromatographed on a silica gel column (Φ 10 × 100 cm, 100–200 mesh, 2 kg) eluting with a PE/EtOAc (20:1 to 1:1) gradient to yield seven fractions, Frs. 1–7, on the basis of TLC analysis. Frs. 4–6 (15 g, 40 g and 54 g separately) were subjected to MCI gel column with MeOH/H2O (30:70 to 100:0) to give 5 (Frs. 4.1–4.5), 4 (Frs. 5.1–5.4) and 4 (Frs. 6.1–6.4) fractions respectively. Fr. 4.3 (5.2 g) was separated by silica gel column chromatography with a gradient of PE/EtOAc (1:0 to 1:10) to afford 7 subfractions (Frs. 4.3.1–4.3.7). Fr. 4.3.3 (1.6 g) was further loaded over ODS column with MeOH/H2O (40:60 to 100:0) to give subfractions Fr. 4.3.3.1 to Fr. 4.3.3.13. Fr. 4.3.3.2 was finally purified by semi-preparative HPLC (MeOH/H2O, 75:25) to yield compound 3 (17.8 mg), 19 (9.7 mg), and Fr. 4.3.3.3 was separated via semi-preparative HPLC (MeCN/H2O, 60:40) as well, resulting in 25 (3.2 mg). Fr. 5.2 and Fr. 5.4 were applied to Sephadex LH-20 column eluted with CH2Cl2/MeOH (1:1) to respectively obtained Frs. 5.2.1–5.2.3 and Frs. 5.4.1–5.4.2. Fr. 5.2.1 (6.3 g) was chromatographed over a silica gel column eluting with PE/EtOAc (1:0 to 1:10) to afford fourteen subfractions, Fr. 5.2.1.1 to Fr. 5.2.1.14, and 15 (19.5 mg) had been separated out as a colorless acicular crystal in this progress. Fr. 5.2.1.4 (213.2 mg) and Fr. 5.2.1.10 (121.7 mg) were purified by semi-preparative HPLC (MeOH/H2O, 60:40) to yield 6 (2.0 mg), 12 (8.8 mg), 16 (11.6 mg) and 17 (5.9 mg). Fr. 5.2.1.11 (1.3 g) and Fr. 5.2.1.12 (0.7 g) were chromatographed by ODS column (MeOH/H2O, 40:60 to 100:0) and semi-preparative HPLC (MeOH/H2O, 70:30) to give pure 2 (8.2 mg), 11 (17.9 mg), 24 (39.7 mg). Fr. 5.4.2 (24.4 g) was fractionated on silica gel CC eluted with PE/EtOAc (1:0 to 1:3) to afford nine sub-fractions (Frs. 5.4.2.1–5.4.2.9). Sub-fraction Fr.5.4.2.5 (13.8 g) was continuously separated through a silica gel column (PE/CH2Cl2/MeOH, 1:1:0, 40:40:1, 20:20:1, 10:10:1, 5:5:1, 1:1:1, 0:1:1), ODS column (MeOH/H2O, 60:40 to 100:0) and finally purified by semi-preparative HPLC (MeOH/H2O, 80:20) to give 18 (19.7 mg). Sub-fraction Fr. 5.4.2.6 (223.5 mg) was purified by semi-preparative HPLC (MeOH/H2O, 75:25) to yield 1 (5.9 mg), 5 (3.3 mg) and 14 (2.8 mg). Fr. 6.1 (1.8 g) was chromatographed over a silica gel column eluting with PE/EtOAc (20:1 to 1:1) gradient to afford nine subfractions, and Fr. 6.1.3 further purified by a semi-preparative HPLC (MeOH/H2O, 80:20) to give 13 (19.4 mg) and 22 (83.4 mg). Fr. 6.4 (17.7 g) was separated by a series of purification steps using Sephadex LH-20 column chromatography (CH2Cl2/MeOH, 1:1), ODS column chromatography (MeOH/H2O, 40:60 to 100:0), a
2.3.2. Jughopnin B (2) White solid; [α]25 D ± 0 (c 0.46, CHCl3); UV (MeOH) λmax (log ε) 217 (2.49), 298 (1.83) nm; IR (KBr) νmax 3433, 2928, 1703, 1506, 1408, 1260, 1220, 1045, 805 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS 347.1222 [M + Na]+ (calcd for C20H20O4Na, 347.1259), m/z 671.2614 [2 M + Na]+ (calcd for C40H40O8Na, 671.2621). 2.3.3. Jughopnin C (3) Yellowish oil; [α]20 D ± 0 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 214 (3.29), 252 (2.78), 297 (2.60) nm; IR (KBr) νmax 3391, 2939, 1701, 1502, 1233, 987, 822 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 349.1420 [M + Na]+ (calcd for C20H22O4Na, 349.1416). 2.3.4. ( ± )-Jughopnin D (4) White solid; [α]20 D ± 0 (c 0.30, CHCl3); UV (MeOH) λmax (log ε) 211 (2.80), 296 (2.08) nm; IR (KBr) νmax 3426, 2928, 1704, 1503, 1418, 1247, 1097, 821 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 379.1544 [M + Na]+ (calcd for C21H24O5Na, 379.1521). 2.3.5. Jughopnin E (5) White solid; [α]20 D − 12.2 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 216 (3.38), 252 (2.87), 295 (2.68) nm; IR (KBr) νmax 3339, 2931, 1586, 1500, 1264, 1240, 1135, 1058, 996, 820 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 365.1733 [M + Na]+ (calcd for C21H26O4Na, 365.1729). 2.3.6. Jughopnin F (6) Yellowish oil; [α]20 D ± 0 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 204 (2.91), 280 (2.10) nm; IR (KBr) νmax 3428, 2934, 1710, 1515, 1263, 1124, 1024, 825 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 377.1362 [M + Na]+ (cacld for C21H22O5Na, 377.1365). 2.3.7. Jughopnin G (7) White solid; [α]20 D + 10 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 204 (2.82), 281 (1.78) nm; IR (KBr) νmax 3425, 2930, 1704, 1513, 1382, 1275, 1228, 1107, 1025, 820 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 351.1295 [M + Na]+ (cacld for C19H20O5Na, 351.1208). 2
Fitoterapia 136 (2019) 104165
Y. Lin, et al.
Table 1 1 H NMR data of compounds 1-5a (400 MHz, δH in ppm, J in Hz). Position
1
1
3.04, 3.04, 2.74, 2.74,
d (7.0) d (7.0) s s
3.08, 2.75, 3.08, 2.75,
m m m m
3.04, 3.04, 2.79, 2.79,
m m t (5.9) t (5.9)
3.04, 3.04, 2.77, 2.77,
m m m m
6
3.04, 3.04, 2.54, 2.54, 5.83,
d (6.8) d (6.8) q (8.5) q (8.5) dt (11.1, 8.7)
7
6.59, d (11.1)
2.79, 2.79, 1.89, 1.89, 1.89, 1.89, 2.75, 2.75, 6.84, 6.89, 7.09, 6.77, 6.47,
t (5.9) t (5.9) m m m m d (6.8) d (6.8) d (2.4) d (8.3) dd (8.3, 2.4) d (2.1) m
m m m m m m dd (8.0, 4.0)
6.79, 6.76, 7.01, 6.78, 6.77, 3.89,
m m m m m m m m d (2.4) d (8.2) dd (8.2, 2.4) d (1.7) d (1.7)
2.83, 2.83, 1.73, 1.73, 2.04, 2.04, 4.43,
2′ 5′ 6′ 2″ 6″ 3″-OCH3 4″-OCH3 7-OCH3 3′,4′-OCH2O-
2.70, 2.70, 1.83, 1.83, 1.81, 1.81, 2.64, 2.64, 7.00, 7.20, 7.14, 6.70, 6.63,
6.60, 6.89, 7.05, 6.88, 6.74,
d (2.4) d (8.2) dd (8.2, 2.4) d (2.0) d (2.0)
2 3 4 5
2
d (2.4) d (8.1) dd (8.1, 2.4) d (2.1) d (2.1) s
3
4
3.72, s
5 2.98, 1.86, 2.28, 1.66, 3.98, 1.86, 1.66, 1.66, 1.54, 1.99, 1.86, 2.54, 2.54, 7.17, 6.80, 7.07, 6.77, 6.83, 3.80, 3.88,
3.95, s 3.38, s
m m m m t (9.9) m m m m m m m m d (2.4) d (8.3) dd (8.3, 2.4) d (2.1) d (2.1) s s
6.03, s
a 1
H NMR data were gauged in CD3OD for 1, and 5, in Pyridine‑d5 for 2, in CDCl3 for 3, and 4.
HRESIMS m/z 381.1679 [M + Na]+ (calcd for C21H26O5Na, 381.1678).
2.3.8. Jughopnin H (8) White solid; [α]20 D + 16 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 204 (3.04), 280 (2.03) nm; IR (KBr) νmax 3435, 2930, 2941, 1511, 1442, 1260, 1123, 1024, 830, 797 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 351.1575 [M + Na]+ (calcd for C20H24O4Na, 351.1572).
2.3.10. Jughopnin J (10) Colorless oil; [α]20 D + 1.5 (c 0.41, MeOH); UV (MeOH) λmax (log ε) 204 (3.01), 223 (2.67), 279 (2.09) nm; IR (KBr) νmax 3401, 2938, 1610, 1514, 1235, 1033, 823 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 351.1574 [M + Na]+ (calcd for C20H24O4Na, 351.1572).
2.3.9. Jughopnin I (9) Colorless oil; [α]20 D − 62 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 204 (3.10), 280 (2.08) nm; IR (KBr) νmax 3431, 2934, 1608, 1516, 1270, 1034, 817, 797 cm−1; 1H and 13C NMR data, see Tables 2 and 3; Table 2 1 H NMR data of compounds 6-10a (400 MHz, δH in ppm, J in Hz). Position
6
7
1
2.83, m 3.02, m 2.38, td (7.5, 2.6)
2.95, 2.74, 2.38, 2.28,
2
dd dd dd dd
(16.2, (16.2, (16.9, (16.9,
10.1) 8.4) 10.1) 8.4)
3 4
2.10, m 1.74, m 1.57, m
9
10
2.58, m
4.27, dd (11.0, 2.1)
4.95, t (5.5)
1.51, m
1.83, d (12.9)
1.91, m
3.12, m
1.92, 1.64, 1.64, 1.33, 3.44,
1.67, m
6
2.11, 2.27, 2.27, 2.50, 6.09,
7
6.61, d (10.9)
2′ 4′ 5′ 6′ 2″ 3″ 5″ 6″ 3′-OCH3 4′-OCH3 5′-OCH3 2″-OCH3 3″-OCH3 5″-OCH3
5.32, d (2.1)
3.18, m 2.38, dd (16.0, 9.2) 5.55, s
6.84, d (8.2) 6.72, dd (8.2, 2.1)
6.82, s 6.64, d (8.7)
6.82, d (8.2) 6.72, dd (8.2, 2.0) 6.86, d (2.0)
6.66, d (8.5) 6.89, d (8.5)
6.50, d (8.3) 6.77, d (8.3)
7.05, d (8.1) 6.80, d (8.1, 2.0)
5
m m m m dt (10.9, 7.1)
8
1.74, m
1.22, 0.99, 1.67, 1.56, 1.22, 0.99, 2.69, 2.58, 5.85,
m m m m m m m m d (2.0)
m m m dd (11.1, 3.9) m
1.92, m 1.75, m 2.68, m 6.95, s 6.87, d (2.7) 6.87, d (2.7) 6.69, d (8.9) 6.82, d (8.9) 6.70, s
3.93, s 3.83, s 3.94, s 3.99, s 3.91, s
a 1
H NMR data were gauged in CDCl3 for 6, 7, 8, and 9, in Pyridine‑d5 for 10. 3
1.67, m 1.42, m 3.91, dq (9.1, 4.5) 2.23, 1.75, 2.94, 2.70, 7.32,
m m ddd (14.8, 10.2, 5.1) ddd (13.8, 10.2, 6.4) d (1.9)
7.31, 7.12, 7.28, 7.21, 7.21, 7.28, 3.81,
d (8.1) dd (8.1, 1.9) d (8.4) d (8.4) d (8.4) d (8.4) s
Fitoterapia 136 (2019) 104165
Y. Lin, et al.
Table 3 13 C NMR data of compounds 1–10 (100 MHz, δC in ppm). Position
1b
2c
3a
4a
5b
6a
7a
8a
9a
10c
1 2 3 4 5 6 7 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 3′-OCH3 4′-OCH3 5′-OCH3 2″-OCH3 3″-OCH3 4″-OCH3 5″-OCH3 7-OCH3 3′,4′-OCH2O-
31.8, CH2 44.3, CH2 216.8, C 46.4, CH2 26.6, CH2 131.1, CH 132.7, CH 135.0, C 133.8, CH 127.5, C 153.5, C 117.2, CH 129.9, CH 130.3, C 111.3, CH 149.8, C 142.9, C 126.5, C 128.3, CH
29.9, CH2 43.1, CH2 213.7, C 46.7, CH2 26.4, CH2 22.4, CH2 33.1, CH2 132.2, C 134.8, CH 126.2, C 154.8, C 117.1, CH 129.8, CH 133.6, C 108.9, CH 149.1, C 144.1, C 121.9, C 127.3, CH
29.2, CH2 42.8, CH2 213.8, C 46.6, CH2 25.6, CH2 22.0, CH2 32.2, CH2 132.7, C 132.6, CH 124.9, C 152.0, C 117.0, CH 129.8, CH 137.7, C 115.4, CH 148.6, C 140.8, C 131.0, C 125.9, CH
29.3, CH2 42.9, CH2 213.4, C 45.1, CH2 19.8, CH2 31.5, CH2 81.5, CH 132.6, C 133.6, CH 125.4, C 151.9, C 117.3, CH 129.2, CH 140.8, C 108.9, CH 132.2, C 147.0, C 124.4, C 125.6, CH
26.5, CH2 34.2, CH2 67.8, CH 39.1, CH2 22.6, CH2 26.3, CH2 30.3, CH2 130.5, C 133.7, CH 124.9, C 151.5, C 116.2, CH 129.8, CH 135.6, C 111.9, CH 152.5, C 142.8, C 132.2, C 125.8, CH
26.8, CH2 40.1, CH2 208.8, C 42.3, CH2 19.5, CH2 133.1, CH 127.6, CH 133.7, C 112.8, C 149.2, C 146.5, C 112.3, C 122.1, C 122.4, C 146.5, C 140.6, C 146.6, C 116.6, C 124.4, C
27.3, CH2 41.2, CH2 210.6, C 46.5, CH2 19.1, CH2 24.9, CH2 30.0, CH2 134.2, C 112.6, CH 146.8, C 142.9, C 115.9, CH 123.0, CH 125.7, C 144.3, C 136.5, C 141.0, C 114.5, CH 123.4, CH
28.7, CH2 36.7, CH2 71.9, CH 38.5, CH2 30.6, CH2 22.5, CH2 35.5, CH2 135.4, C 114.7, CH 148.9, C 146.4, C 112.1, CH 123.0, CH 141.7, C 118.6, CH 141.8, C 148.9, C 123.7, CH 122.2, CH
79.6, CH 33.6, CH2 24.2, CH2 31.4, CH2 77.4, CH 38.5, CH2 31.5, CH2 135.9, C 108.8, CH 144.9, C 114.1, CH 146.5, C 118.9, CH 134.5, C 121.1, CH 114.3, CH 143.7, C 146.5, C 111.3, CH
72.5, CH 31.6, CH2 19.9, CH2 30.6, CH2 71.7, CH 36.1, CH2 32.1, CH2 134.9, C 111.6, CH 149.1, C 147.6, C 120.2, CH 120.2, CH 133.7, C 130.3, CH 116.6, CH 157.4, C 116.6, CH 130.3, CH 56.3, CH3
a b c
56.3, CH3 56.1, CH3 56.8, CH3 61.8, CH3
56.5, CH3
60.1, CH3 55.2, CH3
56.4, CH3 61.9, CH3 56.0, CH3
56.2, CH3 101.7, CH2
Data were gauged in CDCl3. Data were gauged in CD3OD. Data were gauged in Pyridine‑d5.
tetrasubstituted aromatic ring [δH 6.78 (1H, d, J = 2.1 Hz, H-2″), 6.77 (1H, d, J = 2.1 Hz, H-6″)], a cis-olefinic group [δH 6.59 (1H, d, J = 11.1 Hz, H-7), 5.83 (1H, dt, J = 11.1, 8.7 Hz, H-6)], a methoxy group [δH 3.89 (3H, s)], and a heptanoid chain [δH 3.04 (4H, m, H2–1, H2–4), 2.74 (2H, s, H2–2), 2.54 (2H, q, J = 8.5 Hz, H2–5)]. The 13C NMR spectrum exhibited twenty carbon signals, including twelve aromatic, one ketone carbonyl, one methoxy, and six aliphatic carbons. The above spectroscopic characteristics suggested 1 to be a biaryl-type cyclic diaryheptanoid with two hydroxys, one methoxy, and one double band, which was similar to myricananin C (11) [31]. The double bond was located at C-6 and C-7 by the 1H-1H COSY correlations of H-4/H-5, H-5/H-6, H-6/H-7, and the HMBC correlations of H-4/C-6, H-6/C-1″, H7/C-5, C-1″ (Fig. 2). The locations of the methoxy group at C-3″ and the ketone carbonyl group at C-3 were deduced from the HMBC correlations of H-6″/C-3′, C-2″, C-4″, and H-1, H-5/C-3, respectively. Finally, the HMBC correlations of H-2′, H-6′/C-4′, and H-6″/C-4″ assisted in the linkage of the hydroxyl groups to C-4′ and C-4″, respectively. The preceding analysis permitted the structure of compound 1 to be determined as shown in Fig. 1, named jughopnin A. The 13C NMR and HRESIMS data (m/z 671.2614 [2 M + Na]+, calcd for 671.2621) of compound 2 hint a molecular formula of C20H20O4. Its 1H and 13C NMR data (Tables 1 and 3) displayed that compound 2 was quite similar with (11R)-11,17-dihydroxy-3,4-methylenedioxy-[7,0]-metacyclophane (14) [32], except for the presence of a ketone carbonyl group [δC 213.6] in 2 instead of a hydroxyl group in 14. The long-ranged correlations of H-1, H-2, H-4/C-3 in the HMBC spectrum (Fig. 2), and correlations of H-1/H-2, H-4/H-5, H-5/H-6, and H-6/H-7 in 1H-1H COSY experiment confirmed the above analysis. Thus, the structure of compound 2 was resolved as aforementioned and it was given a trivial name, jughopnin B. Compound 3 was isolated as yellowish oil. Its molecular formula was deduced to be C20H22O4 by HRESIMS (m/z 349.1420 [M + Na]+ , calcd for 349.1416). The NMR data (Tables 1 and 3) of 3 were closely similar to that of 11 (myricananin C) [31]. Detailed analysis of 13C
2.4. Neuroprotective activity assay The neuroprotective activities of compounds 1–28 on the human neuroblastoma SH-SY5Y cells damage induced by H2O2 and CoCl2 were measured according to published methods [29,30]. Cell viability was estimated by MTT assay. Human neuroblastoma SH-SY5Y cells were maintained in DME/F–12 medium supplemented with 15% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin and were incubated at 5% CO2 in a 37 °C incubator. Cells were seeded at 96-well culture plates (1 × 104 cells/well). After 24 h incubation, cells were pretreated with various concentrations of the test compounds for 2 h before incubation in medium containing H2O2 or CoCl2. Then 240 μM freshly prepared H2O2 or 100 μM prepared CoCl2 was added to the cells at 37 °C for 24 h to induce injury. Then, 15 μL of the MTT solution (5 mg/mL) was added into each well followed by 4 h incubation at 37 °C, after that the supernatant was removed and DMSO (100 μL/well) was added. The absorbance was measured at 570 nm with a microplate reader (Bio-Tek Synergy 2 reader). Vitamin-C was used as the positive control. Data (cell viability) were normalized and expressed as a percentage of the control value, which was set to 100%. All values were analyzed by one-way ANOVA (and nonparametric) followed by Newman-Keuls (compare all pairs of columns) with GraphPad Prism 5.0 and expressed as mean ± SD. Three independent experiments were carried out. 3. Results and discussion Compound 1 was obtained as a yellowish oil. Its molecular formula was assigned as C20H20O4 by the HRESIMS (m/z 347.1257 [M + Na]+, calcd for 347.1259). The IR spectrum showed absorption bands at 3385 cm−1 for hydroxy group and at 1700 cm−1 for carbonyl group. The 1H NMR data (Table 1) of 1 showed signals of a 1,3,4- trisubstituted aromatic ring [δH 7.01 (1H, dd, J = 8.1, 2.4 Hz, H-6′), 6.79 (1H, d, J = 2.4 Hz, H-2′), 6.76 (1H, d, J = 8.1 Hz, H-5′)], a 1,3,4,54
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Fig. 1. Structures of the isolated compounds from Juglans hopeiensis.
Fig. 2. Key COSY (black bold line) and HMBC (blue arrows) correlations of compounds 1–10. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 5
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NMR data showed that the methoxyl group of 3 was at lower field compared to that of 11, which revealed the difference between 3 and 11 was the linkage site of the methoxyl group. HMBC correlations of 4″OCH3/C-4″ and H-6″/C-3′, C-4″ further verified the methoxyl group to be located at C-4″ (Fig. 2). Therefore, compound 3 was identified as described, and named jughopnin C. The molecular formula of compound 4 was resolved as C21H24O5 by HRESIMS (m/z 379.1544 [M + Na]+, calcd for 379.1521) and the 13C NMR data. The 1H and 13C NMR data (Tables 1 and 3) of 4 resembled that of 1, a typical biaryl-type cyclic diaryheptanoid. A detailed analysis of the NMR data (Tables 1 and 3) disclosed 4 possessed two methoxy groups and two hydroxyl groups. The HMBC correlations of H-7/C-2″, C-3″, C-6″ and the linear connectivity observed in the 1H-1H COSY spectrum of H-4/H-5, H-5/H-6 and H-6/H-7 were used to assign the heptanoid chain, further allowing one methoxy group to be located at C-7. Additionally, the other methoxy group was link to C-4″ on account of HMBC correlation of H-2″/C-4″. Two hydroxyl groups were assigned at C-4′ and C-3″ due to the HMBC correlations of H-6′, H-2′/C-4′ and H2″/C-3″, respectively (Fig. 2). Although there was a chiral carbon in the molecule, the optical rotation value of 4 was very close to zero, suggesting that 4 was most probably a racemic mixture. Subsequently, 4 was analyzed on chiral HPLC using a CHIRALPAK IG column and was taken for two enantiomers, 4a and 4b, at a ratio of approximately 1:1 (Fig. S59, Supplementary data). Accordingly, the structure of compound 4 was established, named ( ± )-jughopnin D. Compound 5 was obtained as a white solid. Its molecular formula was established as C21H26O4 by HRESIMS at m/z 365.1733 (calcd for 365.1729). The NMR data (Tables 1 and 3) of 5 was completely in accord with those of (11S)-11, 17-dihydroxy-3,4-dimethoxy-[7,0]-metacyclophane (12) [33]. Compound 5 exhibited a levorotatory optical activity ([α]20 D − 12.2, c 0.17, MeOH) which was opposite to that of dextrorotatory structure (12) ([α]20 D + 39.4, c 0.17, MeOH), suggesting a 3R-configuration of 5. The absolute configuration was subsequently be corroborated by comparing the experimental ECD spectra of 5 and 12 (Fig. 4). The results showed that 5 exhibited a negative Cotton effect at 227 nm while 12 had an exact opposite one, which unambiguously confirmed the R configuration of C-3. Finally, the structure of compound 5 was validated as the new stereoisomer (11R)11, 17-dihydroxy-3,4-dimethoxy-[7,0]-metacyclophane, namely jughopnin E. Compound 6 was isolated as yellowish oil. It gave a quasi-molecular ion peak at m/z 377.1362 [M + Na]+ (cacld for 377.1365), which was established by HRESIMS to conform to the molecule formula C21H22O5. The 1H NMR data (Table 2) showed signals for five aromatic protons attributed to two aromatic rings: signals at δH 6.84 (1H, d, J = 8.2 Hz, H-5′), 6.72 (1H, dd, J = 8.2, 2.1 Hz, H-6′) and 5.32 (1H, d, J = 2.1 Hz, H-2′), due to a 1,3,4-trisubstituted aromatic ring, and at δH 6.89 (1H, d, J = 8.5 Hz, H-6″) and 6.66 (1H, d, J = 8.5 Hz, H-5″), due to a 1,2,3,4tetrasubstituted aromatic ring. A signal at δH 5.32 (1H, d, J = 2.1 Hz) is the characteristic proton of diarylether-type diarylheptanoid [3,4]. Its 1 H NMR spectrum displayed two olefinic protons at δH 6.61 (1H, d, J = 10.9, H-7), 6.09 (1H, dt, J = 10.9, 7.1, H-6), and two methoxy groups at δH 3.99 (3H, s, 3″-OCH3), 3.94 (3H, s, 2″-OCH3). The 13C NMR spectrum of 6 gave 21 carbon signals that consist of twelve aromatic, one ketone carbonyl, two olefinic, two methoxy and four aliphatic carbons. The HMBC correlations of H-7/C-2″, C-3″, C-5 and the linear connectivity of the heptanoid chain (H-4/H-5, H-5/H-6, H-6/H7) observed in the 1H-1H COSY, allowing the cis-olefinic group to be located at C-6 and C-7 (Fig. 2). The HMBC correlations of H-2, H-4/C-3 allowed the attachment of the ketone carbonyl group to C-3. And the HMBC correlations of H-5″/C-3″, and H-7/C-2″, C-3″ implied that the two methoxy groups were attached to C-2″ and C-3″, respectively. In addition, the hydroxyl group was link to C-4′ due to the correlation of H-6′/C-4′ (Fig. 2). On the basis of the above analysis, the structure of compound 6 was settled and named as jughopnin F. The HRESIMS (m/z 351.1295 [M + Na]+ , cacld for 351.1208) of
compound 7 in association with the 13C NMR data suggested a molecular formula of C19H20O5. The 1H and 13C NMR data (Tables 2 and 3) of 7 were similar to those of 6, which suggested 7 was also a diarylether-type diarylheptanoid. Its 1D NMR and HSQC spectra confirmed the same aromatic substitution patterns as in 6. A specific analysis of the NMR data revealed that 7 differed from 6 by the absence of a cisolefinic group and the replacement of two methoxy groups by two hydroxyl groups. The 1H-1H COSY and HMBC spectra further supported the three hydroxy groups at C-4′, C-2″ and C-3″ (Fig. 2). Besides, the 1 H-1H COSY correlations of H-1/H-2 and H-4/H-5/H-6/H-7, illuminated the structural sequences C-1-C-2 and C-4-C-5-C-6-C-7. In consequence, the structure of compound 7 was established as shown in Fig. 1, named jughopnin G. Compound 8 was isolated as an optically active [α]25 D + 16 (c 0.2, MeOH) white solid. The molecular formula C20H24O4 was established by HRESIMS (m/z 351.1575 [M + Na]+ calcd for 351.1572). , Comparing the NMR data (Tables 2 and 3) of 8 with that of 4″-epoxy-1(4′-hydroxyph-enyl)-7-(3″-methoxylphenyl)-heptane-3-hydroxyl (21) [34] indicated that those two structures possessed the same functional groups and aromatic substitution patterns, but the main differences are the substitutional positions of the methoxy and hydroxyl groups. HMBC correlations of 4′-OCH3, H-2′, H-6′/C-4′ and H-5″/C-3″, and cross peaks of H-5′/H-6′ and H-5″/H-6″ in the 1H-1H COSY experiment (Fig. 2) suggested the location of the methoxy group at C-4′ and the hydroxyl group at C-3″, respectively. The planar structure of 8 was then determined. The absolute configuration of 8 was established by comparison of optical rotations of 8 and 21 (8: [α]25 D + 16, c 0.2, MeOH; 21: [α]25 D − 28, c 0.2, MeOH). Therefore, 8 was assigned as shown in Fig. 1, and named jughopnin H. Compound 9 was identified as brownish oil with [α]25 D − 62.0 (c 0.2, MeOH), and its molecular formula was established as C21H26O5 by HRESIMS (m/z 381.1679 [M + Na]+ , calcd for 381.1678). Comparative analysis of the NMR data of 9 (Tables 2 and 3) with those of engelheptanoxide A (24) [35] established that compound 9 could be a linear diarylheptanoid with the skeleton of the tetrahydropyran ring in the carbon chain. In 1H-1H COSY spectrum, the cross peaks of H-1/H-2, H-2/H-3, H-3/H-4, H-4/H-5, H-5/H-6, and H-6/H-7, were in accord with the presence of a spin system corresponding to a CH1–CH2-2–CH23–CH2-4–CH-5–CH2-6–CH2-7 moiety (Fig. 2). Detailed analysis of the 13 C NMR and HSQC spectra of 9 revealed 21 carbon signals corresponding to five methylenes, eight methines, six quaternary carbons, and two methoxyl groups. The two methoxy groups were located at C-5′ and C-5″, and the two hydroxy groups were located at C-3′ and C-4″, respectively, as confirmed by the HMBC correlations (Fig. 2). The NOESY correlation of H-1/H-5, established the relative configurations of the two hydrogens to be the same orientation (Fig. 3). Using the same method as 5, the absolute configurations of 9 was determined. Its experimental ECD spectrum was in agreement with the ECD spectrum of 24, suggesting a 1S,5R-configuration (Fig. 5). Based on the above evidences, structure 9 was established and named jughopnin I.
Fig. 3. Key NOE (blue arrows) correlations of 9. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6
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rotation ([α]25 D + 1.5, c 0.2, MeOH) with that of (+)-epi-Centrolobine ([α]20 D + 3.3, c 0.5, CH2Cl2) [36]. Moreover, the ECD spectrum of 10 was opposite to that of 24, which also indicated the 1R,5R-confgurations in 10 (Fig. 5). Taken together, compound 10 was identified as described, named jughopnin J. Ground on spectroscopic analysis and comparison to literature values, the structures of the known compounds were elucidated as myricananin C (11) [31], (11S)-11,17-dihydroxy-3,4-dimethoxy-[7,0]-metacyclophane (12) [33], juglanin B (13) [3], (11R)-11,17-dihydroxy3,4-methylenedioxy-[7,0]-metacyclophane (14) [32], juglanin A (15) [3], galeon (16) [4], juglanin C (17) [37], pterocarine (18) [38], methylgaleon (19) [39], myrocatomentogenin (20) [40], 4″-epoxy-1-(4′hydroxyph-enyl)-7-(3″-methoxylphenyl)-heptane-3-hydroxy (21) [34], 2-oxatricyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaene-10,16diol (22) [41], jugsigin A (23) [21], engelheptanoxide A (24) [35], yakuchinone A (25) [42], 1,7-bis(4-hydroxy-3-methoxyphenyl)heptan3-one (26) [43], 1-(4-hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl) heptan-3-one (27) [43], 1-(4-hydroxyphenyl)-7-(3-methoxy-4-hydroxyphenyl)-3-heptitol (28) [44]. All of them were firstly obtained from J. hopeiensis. It has been reported that diarylheptanoids isolated from Juglans sinensis showed significant neuroprotective effects [32], therefore, we screened the isolated diarylheptanoids for their neuroprotective effects. All the isolated diarylheptanoids (1–28) were evaluated for their neuroprotective effects against H2O2 induced oxidative injuries and CoCl2 induced hypoxia injuries in SH-SY5Y cells. As a result, compound 7 showed significant neuroprotective effects in vitro and improved cell viability by 30% at 12.5 μM in comparison with the H2O2 treated group. And some other diarylheptanoids showed moderate neuroprotective effects on H2O2 and CoCl2 treated group as shown in Figs. 6 and 7 respectively.
Fig. 4. The experimental ECD spectra of 5 and 12.
4. Concluding remarks Fig. 5. The experimental ECD spectra of 9, 10 and 24.
In current investigation on the fresh pericarps of J. hopeiensis, ten previously undescribed and eighteen known diarylheptanoids were succeeded in isolating and structurally identifying by various spectroscopic techniques. Some isolated diarylheptanoids showed moderate neuroprotective effects against H2O2-induced oxidative injuries and CoCl2-induced hypoxia injuries in SH-SY5Y cells. These findings enrich the chemical composition of J. hopeiensis, and revealed it might be helpful for the progress in prevention and treatment of the neurodegenerative diseases.
Compound 10 was isolated as brownish oil with [α]25 D + 1.5 (c 0.2, MeOH). Its molecular formula was discriminably deduced to be C20H24O4 by HRESIMS (m/z 351.1574 [M + Na]+ , calcd for 351.1572). The planar structure of 10 was identical to that of 24 due to their similar 1H and 13C NMR data (Tables 2 and 3) and HMBC correlations (Fig. 2). The slight differences in the chemical shifts of H-1 and H-5 hint the different configuration of C-1 or C-5 [36]. The absolute configurations of 10 was assigned to be 1R, 5R by comparison of the specific
Fig. 6. The neuroprotective effects of compounds 1–28 against CoCl2-induced injury in SH-SY5Y cells. 7
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Fig. 7. The neuroprotective effects of compounds 1–28 against H2O2-induced injury in SH-SY5Y cells.
Conflicts of interest
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There are no conflicts to declare. Acknowledgements This project was supported financially by the National Natural Science Foundation of China (No. 21572073, 31770380 and 31270394). We are grateful to the staff at the Analytical and Testing Center of Huazhong University of Science and Technology for collecting the spectroscopic data. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2019.05.004. References [1] Q. Liu, P. Zhao, X.C. Li, M.R. Jacob, C.R. Yang, Y.J. Zhang, New α-tetralone galloylglucosides from the fresh pericarps of Juglans sigillata, Helv. Chim. Acta 93 (2) (2010) 265–271. [2] R.T. Xi, Textural criticism of walnut (Juglans regia L.) origin in China, J. Hebei Agric. Univ. 13 (1) (1990) 89–94. [3] J.X. Liu, D.L. Di, X.Y. Huang, C. Li, Two new diarylheptanoids from the pericarps of Juglans regia L, Chin. Chem. Lett. 18 (8) (2007) 943–946. [4] M. Morihara, N. Sakurai, T. Inoue, K. Kawai, M. Nagai, Two novel diarylheptanoid glucosides from Myrica gale var. tomentosa and absolute structure of plane-chiral galeon, Chem. Pharm. Bull. 45 (5) (1997) 820–823. [5] M. Jin, S. Diao, C. Zhang, S. Cao, J. Sun, R. Li, Z. Jiang, M. Zheng, J.K. Son, G. Li, Two new diarylheptanoids isolated from the roots of Juglans mandshurica, Nat. Prod. Res. 29 (19) (2015) 1839. [6] Y. Zhou, B. Yang, Y. Jiang, Z. Liu, Y. Liu, X. Wang, H. Kuang, Studies on cytotoxic activity against HepG-2 cells of naphthoquinones from green walnut husks of Juglans mandshurica Maxim, Molecules 20 (9) (2015) 15572–15588. [7] H.Y. Yu, X. Li, F.Y. Meng, H.F. Pi, P. Zhang, H.L. Ruan, Naphthoquinones from the root barks of Juglans cathayensis Dode, J. Asian Nat. Prod. Res. 13 (7) (2011) 581–587. [8] K. Machida, E. Matsuoka, T. Kasahara, M. Kikuchi, Studies on the constituents of Juglans species. I. Structural determination of (4S)- and (4R)-4-hydroxy-α-tetralone derivatives from the fruit of Juglans mandshurica MAXIM. var. sieboldiana MAKINO, Chem. Pharm. Bull. 53 (8) (2005) 934–937. [9] D.L. Yao, M. Jin, C.H. Zhang, J. Luo, Z. Jiang, M.S. Zheng, J.M. Cui, G. Li, Chemical constituents of the leaves of Juglans mandshurica, Chem. Nat. Compd. 52 (1) (2016) 93–95. [10] Y.W. Zhang, H. Lin, Y.L. Bao, Y. Wu, C.L. Yu, Y.X. Huang, Y.X. Li, A new triterpenoid and other constituents from the stem bark of Juglans mandshurica, Biochem. Syst. Ecol. 44 (10) (2012) 136–140. [11] C.L. Si, Y. Zhang, Z.Y. Zhu, S.C. Liu, Chemical constituents with antioxidant activity from the pericarps of Juglans sigillata, Chem. Nat. Compd. 47 (3) (2011) 442–445. [12] S. Park, N. Kim, G. Yoo, S.N. Kim, H.J. Kwon, K. Jung, D.C. Oh, Y.H. Lee, S.H. Kim, Phenolics and neolignans isolated from the fruits of Juglans mandshurica Maxim. and their effects on lipolysis in adipocytes, Phytochemistry 137 (2017) 87–93.
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