Chemical constituents of hemp (Cannabis sativa L.) seed with potential anti-neuroinflammatory activity

Phytochemistry Letters 23 (2018) 57–61

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Chemical constituents of hemp (Cannabis sativa L.) seed with potential antineuroinflammatory activity Yuefang Zhou, Shanshan Wang, Hongxiang Lou, Peihong Fan

T

⁎

Department of Natural Product Chemistry, Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Hemp seed Cannabis sativa L. Lignanamides Glycoside Anti-neuroinflammatory activity

A new lignanamide (1) and a new coumaroylamino glycoside derivative (2) were isolated and identified from hemp (Cannabis sativa L.) seed along with eighteen known compounds (3-20), eight (9, 10, 14-18, 20) of which were isolated for the first time from hemp seed. Their anti-neuroinflammatory activity on LPS (lipopolysaccharide)-induced BV2 microglia cells was evaluated. Compounds 1, 2, 5-9, 12, 13, 15-17, 19 and 20 exhibited significant inhibitory effects on TNF-α release from LPS-induced BV2 microglia cells, especially the new glucoside 2. The results laid a solid foundation for additional research on hemp seed related to its value against neurodegenerative diseases.

1. Introduction Cannabis sativa L., an annual plant in the cannabaceae family, has been an important source of food, fiber, oil, and medicine (non-drug varieties) as well as a psychoactive drug since ancient times (Russo, 2007). The non-drug varieties of C. sativa L. for industrial use are also called hemp (Montserrat-de la Paz et al., 2014). Hemp seed has been utilised as a folk source of food and an important source of nutrition in China for thousands of years (Callaway, 2004). It is also used in traditional Chinese medicine for the prevention of constipation, lowering cholesterol, cardiovascular health, immunomodulatory effects, dermatological disease, amelioration effects, and the treatment of gastrointestinal disease (Cheng et al., 2011; Mustafa et al., 1999; RodriguezLeyva and Pierce, 2010). Recent studies have also reported that hemp seed extracts showed strong antioxidant and anti-ageing effects as well as the potential to improve impaired learning and memory induced by chemical drugs in mice (Cai et al., 2010; Lin et al., 2016; Luo et al., 2003). In addition to its nutritional composition, which includes fatty acids and protein, hemp seed is rich in lignanamides (Sakakibara et al., 1992, 1995, 1991). Previously, we reported 14 lignanamides and their antioxidant and acetylcholinesterase inhibitory activities (Yan et al., 2015). To explore the potential of hemp seed against neurodegenerative disease, we further tested its anti-neuroinflammatory activity by establishing a neuroinflammatory model using lipopolysaccharide (LPS)-

stimulated BV2 microglia cells. Most of these compounds demonstrated good activity, and the preliminary mechanism study showed that such compounds could inhibit the NF-κB signaling pathway (Luo et al., 2017). The activation of NF-κB can induce a large number of inflammatory factors, which will stimulate microglia in return and lead to a vicious circle of additional inflammation (Chen et al., 1999; May and Ghosh, 1998). During the activation of microglia cells, NF-κB will coordinate with other inflammatory-related channels to trigger a variety of signal cascades, which together regulate the inflammatory response (Karin and Ben-Neriah, 2000; Ghosh et al., 1998). Therefore, the discovery of effective compounds that can regulate the activation of the NF-κB signaling pathway is a potential way to combat neurodegenerative disease. Our previous results compelled us to further investigate the chemical constituents of hemp seed in order to identify additional bioactive compounds. Thus, in the current study, we continue to carry out phytochemical research on hemp seed with slightly different technology. This has resulted in the isolation of one new lignanamide (1) and one new glycoside (2) along with eighteen known compounds (3–20), eight of which were isolated for the first time from hemp seed. Anti-neuroinflammatory activity against LPS-induced inflammatory response in BV2 microglia cells was tested by ELISA. We herein report the isolation, structural elucidation and anti-neuroinflammatory activity of the isolated compounds.

Abbreviations: LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; CC, column chromatography; HSCCC, high-speed countercurrent chromatography; RP-MPLC, medium-pressure reverse phase column liquid chromatograph; PA, polyamide; TNF-α, tumor necrosis factor-α ⁎ Corresponding author. E-mail address: [email protected] (P. Fan). https://doi.org/10.1016/j.phytol.2017.11.013 Received 8 October 2017; Received in revised form 13 November 2017; Accepted 14 November 2017 Available online 20 November 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 1. Structures of compounds 1-2.

H-5′), and 6.61 (dd, J = 1.4, 8.1 Hz, H-6′). The signals at δ 7.32 (1H, d, J = 15.7 Hz, H-7) and 6.33 (1H, d, J = 15.7 Hz, H-8) indicated that 1 possessed a trans-substituted double bond. The signals at δ 4.89 (1H, d, J = 7.0 Hz, H-7′) and 4.39 (1H, d, J = 7.0 Hz, H-8′) indicated the presence of a pair of trans-oriented aliphatic methane groups. Additionally, the 1H and 13C NMR data indicated the presence of two NHCH2CH2 segments from the signals at δC 34.1 (C-7‴), 34.4 (C-7″), 40.9 (C-8‴), 41.2 (C-8″), δH 2.64 (2H, t, J = 7.4 Hz), 3.35 (2H, t, J = 7.4 Hz) and 2.32(1H, m), 3.42 (1H, m), 3.08(1H, m), 3.23 (1H, m). In the HMBC spectrum, the correlations between H-7″/C-2″(C-6″), H8″/C-1″ and H-7‴/C-2‴ (C-6‴), H-8‴/C-1‴ suggested the presence of two p-tyramine moieties, the correlations of H-8/C-1 and H-7/C-2, C-6, C-9 (δC 167.4) suggested the presence of a caffeoyl-like unit, and the correlations of the vicinal aliphatic methine proton H-8′/C-9′ (δC 167.5) and H-7′/C-1′, C-2′, C-6′ suggested a phenyl propionyl moiety. Furthermore, the linkages of C-7′-O-C-3 and C-8′-O-C-4 were determined by the correlations between H-7′/C-3 and H-8′/C-4. The correlations between H-8″/C-9 and H-8‴/C-9′ revealed that two p-tyramine moieties were linked to C-9 and C-9′, respectively (Fig. 2). Therefore, the structure of compound 1 was determined to be (2,3-trans)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-6-{(E)-3-[(4-hydroxyphenethyl)amino]-3-oxoprop-1-en-1-yl}-2,3-dihydrobenzo[b][1,4] dioxine-2-carboxamide and was given the trivial name Cannabisin Q. Compound 2 (Fig. 1) was isolated as a white amorphous powder with a molecular formula identified as C19H27NO8 by HR-ESI–MS at m/ z 398.1807 [M+H]+ (calcd for C19H28NO8, 398.1770). Its 1H NMR and 13 C NMR data (Table 1) combined with HMBC correlations indicated that compound 2 has a coumaroyl moiety from the signals of a paradisubstituted aromatic ring [δH 7.46 (2H, d, J = 8.5 Hz), 7.02 (2H, d, J = 8.5 Hz)], a trans-substituted double bond [δH 7.33 (1H, d, J = 15.7 Hz), 6.47 (1H, d, J = 15.7 Hz)] and a carbonyl group [δC 165.4, C-9], as well as a β-D-glucopyranosyl moiety [δC 100.5 (C-1″), 73.6 (C-2″), 77.0 (C-3″), 70.0 (C-4″), 77.5 (C-5″), 61.0 (C-6″)] (Wang et al., 2010). The signals of four pairs of vicinal methylenes at δ 3.14 (2H, m, H-1′), 1.39-1.47 (2H, m, H-2′), 1.39-1.47 (2H, m, H-3′), 3.37 (2H, t, J = 5.9 Hz, H-4′) proposed a NHCH2CH2CH2CH2OH segment. The linkage of C-9 to the NHCH2CH2CH2CH2OH segment was determined by the HMBC correlations of H-1′/C-9 (Fig. 2). The correlations of H-1″/C-4 in HMBC suggested the glucopyranosyl moiety was at position C-4. Thus, the structure of compound 2 was determined to be (E)-N-(4-hydroxybutyl)-3-(4-hydroxyphenyl)acrylamide-4-O-β-D- glucopyranoside and was given the trivial name coumaroylaminobutanol glucopyranoside. The eighteen known compounds 3-20 were identified by comparing NMR and MS data with the reported literatures as Cannabisin A (3) (Sakakibara et al., 1991), Cannabisin B (4) (Sakakibara et al., 1992), Cannabisin M (5) (Yan et al., 2015), 3,3′-demethyl-grossamide (6) (Lesma et al., 2014), Cannabisin F (7) (Sakakibara et al., 1995),

2. Results and discussion 2.1. Structural elucidation Compound 1 (Fig. 1) was isolated as a white amorphous powder with a molecular formula established as C34H32N2O8 based on the peak m/z 597.2233 [M + H]+ (calcd for C34H33N2O8, 597.2192) in HRESI–MS, requiring 20 ° of unsaturation. Its 1H NMR data (Table 1) indicated that compound 1 possessed two para-disubstituted aromatic rings from the signals at δ 6.95 (2H, d, J = 8.4 Hz), 6.61 (2H, d, J = 8.4 Hz), 6.74 (2H, d, J = 8.4 Hz) and 6.56 (2H, d, J = 8.4 Hz), and two tri-substituted aromatic rings from the two ABX spin systems signals at δ 7.06 (d, J = 1.6 Hz, H-2), 6.87 (d, J = 8.4 Hz, H-5), 6.99 (dd, J = 1.6, 8.4 Hz, H-6), 6.73 (d, J = 1.4 Hz, H-2′), 6.69 (d, J = 8.1 Hz,

Table 1 1 H and 13C NMR data of compounds 1 in CD3OD and 2 in DMSO-d6. Position

1

2

δH (J in Hz) 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” 1‴ 2‴,6‴ 3‴,5‴ 4‴ 7‴ 8‴

7.06 d (1.6)

6.87 6.99 7.32 6.33

d (8.4) dd (8.4,1.6) d (15.7) d (15.7)

6.73 d (1.4)

6.69 6.61 4.89 4.39

d (8.1) dd (8.1, 1.4) d (7.0) d (7.0)

6.95 d (8.4) 6.61 d (8.4) 6.61 d (8.4) 6.95 d (8.4) 2.64 t (7.4) 3.35 t (7.4) 6.74 d (8.4) 6.56 d (8.4) 2.32, 2.42 m 3.08, 3.23 m

δC 129.1 115.8 143.6 143.8 117.1 121.6 139.7 119.1 167.4 126.6 114.2 145.2 145.9 114.9 118.9 76.4 78.2 167.5 129.9 129.3 114.8 155.5 114.8 129.3 34.4 41.2 129.5 129.3 114.8 155.5 34.1 40.9

δH (J in Hz)

δC

3.14 m 1.39-1.47 m 1.39-1.47 m 3.37 t (5.9)

129.0 129.2 116.9 158.7 116.9 129.2 138.4 120.8 165.4 39.0 30.4 26.3 60.8

4.89 d (7.5) 3.21 m 3.24 m 3.14 m 3.32 m 3.43 m; 3.66 d (11.5)

100.5 73.6 77.0 70.0 77.5 61.0

7.46 d (8.5) 7.02 d (8.5) 7.02 7.46 7.33 6.47

d d d d

(8.5) (8.5) (15.7) (15.7)

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

inhibitory effects, especially 2 with TNF-α 1644.68 pg/mL compared with resveratrol (the positive control, TNF-α 1479.03 pg/mL).

Cannabisin G (8) (Sakakibara et al., 1995), N-trans-caffeoyloctopamine (9) (Sun et al., 2015), N-trans-coumaroyloctopamine (10) (López-Gresa et al., 2011), N-trans-coumaroyltyramine (11) (Al-Taweel et al., 2012), N-trans-feryroyltyramine (12) (Sakakibara et al., 1991), N-trans-caffeoyltyramine (13) (Sakakibara et al., 1991), (S)-N-(2-(4-hydroxyphenyl)-2-methoxyethyl)cinnamamide (14) (Nesterenko et al., 2003), 4-[(E)-p-coumaroylamino]butan-1-ol (15) (Yang et al., 2015), trans-ferulic acid-4-O-β-D-glucopyranoside (16) (Steinmetz and Lin, 2009; Yim et al., 2012), adenosine (17) (Zhang et al., 2004), sucrose (18) (Zhang et al., 2007), p-hydroxybenzaldehyde (19) (Kumar et al., 2017), and 4-hydroxy-3-acid (20) (Dou et al., 2016). Among these isolates (1-20), compounds 9, 10, 14-18, 20 were isolated from hemp (Cannabis sativa L.) seed for the first time.

3. Conclusion In conclusion, we isolated 20 compounds from hemp (Cannabis sativa L.) seed. Most of them are lignanamides. One novel lignanamide, Cannabisin Q (1), and one novel coumaroylamino glucoside derivative (2) were reported, and eight others were isolated from hemp seed for the first time. Based on our previous discovery, we continued to test the anti-neuroinflammatory activity of these isolated compounds and determined that compounds 1, 2, 5-9, 12, 13, 15-17, 19 and 20 exhibited significant inhibitory effects on TNF-α release from LPS-induced BV2 microglia cells, especially the coumaroylamino glucoside derivative 2. These results have laid a solid foundation for additional research on hemp seed related to its value against neurodegenerative diseases.

2.2. Anti-neuroinflammatory activity TNF-α was a representative pro-inflammatory cytokine produced by microglia cells (Park et al., 2011). As shown in Fig. 3, compounds 1, 2, 5-9, 12, 13, 15-17, 19 and 20 exhibited significant inhibitory effects on TNF-α release from LPS-induced BV2 microglia cells, suggesting that they can downregulate the LPS-mediated production of inflammatory molecules to protect cells from inflammation irritation. Among them, most are lignannamides. It is notable that 4-[(E)-p-coumaroylamino] butan-1-ol (15) and its glucoside (2) demonstrated much stronger

4. Experiment 4.1. General experimental procedures Optical rotations were determined on an MCP 200 (Anton Paar, Shanghai, China). UV spectra were measured using a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were measured by Fig. 3. Inhibitory effects on TNF-α release of the isolated compounds in LPS-induced inflammatory response with resveratrol as positive control. The values are presented as the mean ± SD (n = 3). Compared with the LPS group, *** p < 0.001. (con: control group).

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(MeOH) and finally purified by preparative TLC (CH2Cl2/MeOH 8:1) to yield compound 7 (17.84 mg). Compound 6 (10.40 mg, tR 85.4 min) was purified by semi-preparative HPLC (MeOH/H2O 45:55) from Fr.7-5 (150 mg). Fr.8 (1.63 g) was also chromatographed on PA (EtOH/H2O 1:9 to 1:0) to afford three subfractions (Fr.8-1 to Fr.8-3). Fr.8-1 (193 mg) was purified by semi-preparative HPLC (MeOH/H2O 55:45) to yield compounds 8 (5.49 mg) and 14 (4.11 mg). Fr.8-2 (217 mg) was separated by RP-MPLC (MeOH/H2O 1:1 to 1:0) and further purified by HSCCC with n-hexane-EtOAc-EtOH-H2O (3:7:2:8, v/v) to yield compound 11 (1.02 mg). Compounds 1 (9.52 mg, tR 27.2 min) and 5 (3.83 mg, tR 29.5 min) were obtained from Fr.8-3 (105 mg) by semi-preparative HPLC (MeOH/H2O 55:45).

a Nicolet Nexus 470 FT-IR spectrophotometer (Thermo Scientific, Waltham, MA, USA). HR-ESI–MS were obtained on a LTQ-Orbitrap XL (ThermoFinnigan, Bremen, Germany). NMR spectra were acquired on a 600 MHz DD2 spectrometer operating at 600 (1H) and 150 (13C) MHz (Agilent Technologies, Santa Clara, CA, USA). CD3OD and DMSO-d6 were used as analytical solvents (Sigma-Aldrich, Shanghai, China). Preparative HPLC was conducted on an Agilent 1200 instrument equipped with a 1100 G1315D DAD detector (Agilent Technologies) and a semi-preparative column (YMC-Pack ODS-A, 250 × 10 mm ID, S–5 μm, 12 nm, Co., Ltd. Japan). High-speed countercurrent chromatography (HSCCC) was used to prepare compounds (TBE-30A, TBE300B, Tauto Biotech, Shanghai, China). Macroporous adsorption resin (AB-8 Crosslinked Polystyrene, Tianjin, China), polyamide (100–200 mesh, Shanghai Macklin Biochemical Co., Ltd., Shanghai, China), silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), octadecylsilyl (ODS-A, 120 Å, 50 μm, YMC, Tokyo, Japan), sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden) and preparative TLC (HSGF 254, 0.4-0.5 mm, Yantai, China) were used in the isolation procedure.

4.3.1. (2,3-trans)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-6{(E)-3-[(4-hy-droxyphenethyl)amino]-3-oxoprop-1-en-1-yl}-2,3dihydrobenzo[b][1,4]dioxine-2-carboxamide (1) White amorphous powder, [α]20 D −2.2 (c 1.0, MeOH). HR-ESI–MS m/z 597.2233 [M+H]+ (calcd for C34H33N2O8, 597.2192). UV (MeOH) λmax 315, 285, 223, 202 nm; IR νmax 3394 (NeH), 2921, 1646 (C]O), 1587, 1510 (C]C), 1467, 1364, 1245, 1118, 1064, 976, 814 cm−1. TLC: Rf = 0.44 (CH2Cl2/MeOH 9:1). 1H NMR (600 MHz) and 13C NMR (150 MHz) data in CD3OD; see Table 1.

4.2. Plant material Hemp (Cannabis sativa L.) seed material was collected from Bama county in the Guangxi province of China in October 2015. It was identified by Professor Lan Xiang, Department of Pharmacognosy, Shandong University. The voucher specimens (201510-1) have been deposited in Dr. Fan’s laboratory.

4.3.2. (E)-N-(4-hydroxybutyl)-3-(4-hydroxyphenyl)acrylamide-4-O-β-Dglucopyrano-side (2) White amorphous powder, [α]20 D −27.4 (c 1.0, MeOH). HR-ESI–MS m/z 398.1807 [M + H]+ (calcd for C19H28NO8, 398.1770). UV (MeOH) λmax 287, 222, 209 nm; IR νmax 3286 (NeH), 2919, 1651 (C] O), 1602, 1544, 1510 (C]C), 1419, 1337, 1228, 1073, 1040, 829 cm−1. TLC: Rf = 0.53 (CH2Cl2/MeOH 2:1). 1H NMR (600 MHz) and 13C NMR (150 MHz) data in DMSO-d6; see Table 1.

4.3. Extraction and isolation The dried hemp seed (about 10.7 kg) was crushed and defatted with n-hexane (2 times, 30 L × 60 h) at room temperature. The defatted seeds were extracted with 95% aqueous ethanol (EtOH) under reflux (3 times, 50 L × 2 h), and the ethanol solution was subsequently evaporated in vacuum to yield 500 mL crude extract solution. This solution was suspended in distilled water and subjected to an AB-8 macroporous adsorption resin column using H2O, 10%, 30%, 50%, 75% and 95% EtOH successively to afford 12 fractions (Fr.1 to Fr.12). Compound 18 (24.00 mg) was crystallized from Fr.1 directly in MeOH. Fr.4 (1.05 g) was separated by a sephadex LH-20 column (MeOH), a medium-pressure reverse phase column liquid chromatograph (RPMPLC) eluted successively with MeOH/H2O (1:9 to 1:0), and HSCCC with the upper phase of solvent systems n-BuOH-ethyl acetate(EtOAc)MeOH-H2O (1:4:0.5:6, v/v) as the stationary phase, successively, to obtain compounds 2 (28.80 mg), 16 (15.58 mg) and 17 (7.46 mg). Fr.5 (1.45 g) was separated with RP-MPLC (MeOH/H2O 1:9 to 1:0) and purified by the semi-preparative HPLC eluted with MeCN/H2O (20:80) to yield compound 15 (2.23 mg, tR 19.6 min). Fr.6 (1.08 g) was chromatographed over polyamide (PA) column chromatography (CC) eluted with a gradient EtOH/H2O from 0:1 to 1:0 and purified using HSCCC with n-hexane-EtOAc-EtOH-H2O (3:7:1:9, v/ v) to yield compounds 9 (20.09 mg) and 10 (1.45 mg). Fr.7 (12 g) was separated by PA CC (EtOH/H2O 3:7 to 1:0) to afford five subfractions (Fr.7-1 to Fr.7-5). Fr.7-1 (32 mg) was separated by a sephadex LH-20 column (MeOH) and purified by preparative TLC (CH2Cl2/MeOH 14:1) to yield compounds 19 (10.20 mg) and 20 (8.42 mg). Fr.7-2 (1.0 g) was separated with RP-MPLC (MeOH/H2O 1:9 to 1:0) and purified by the semi-preparative HPLC eluted with MeOH/ H2O (60:40) to obtain compound 12 (43.70 mg, tR 14.5 min). Fr.7-3 (1.0 g) was separated by RP-MPLC (MeOH/H2O 1:9 to 1:0) to yield three subfractions (Fr.7-3-1 to Fr.7-3-3). Some solid precipitated from Fr.7-3-1 and Fr.7-3-2 in MeOH and were identified as compounds 3 (12.91 mg) and 13 (23.02 mg), respectively. Fr.7-3-3 was purified by the semi-preparative HPLC (MeOH/H2O 37.5:62.5) to yield compound 4 (52.00 mg, tR 60.6 min). Fr.7-4 (920 mg) was successively separated by silica gel CC (CH2Cl2/MeOH 25:1 to 0:1), sephadex LH-20 column

4.4. Anti-inflammatory activity assay The production of TNF-α in the culture supernatants was assessed using ELISA kits (Boster, Wuhan, China) (Wun et al., 2013). Murine BV2 microglia cells were obtained from the China Infrastructure of Cell Line Resources (Beijing, China). Briefly, BV2 cells (8 × 104 cells/mL) were inoculated into 96-well culture plates and incubated overnight. Afterward, the cells were treated or untreated with compounds (15 μM) for 1 h and co-cultured with LPS (100 ng/mL) for another 24 h. Resveratrol was used as a positive control. Cell-free supernatants were collected and stored at −20 °C prior to analysis. TNF-α levels were evaluated with ELISA kit according to the manufacturer’s instructions (Boster), and the absorbance was read at 450 nm on a microplate spectrophotometer (Bio-rad). 4.5. Statistical analysis Experiments were repeated 3 times. All data are expressed as the mean ± standard deviation (SD). Statistical analysis was done by oneway analysis of variance (ANOVA) followed by a Student-NewmanKeuls test using Graphpad Prism v5.0 software (GraphPad, La Jolla, CA, USA). P < 0.05 was perceived to be statistically significant. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81473323) and the Key R&D program in Shandong Province (No. 2015GSF119025). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.phytol.2017.11.013. 60

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