Characterization of Lignanamides from Hemp (Cannabis sativa L

Article pubs.acs.org/JAFC

Characterization of Lignanamides from Hemp (Cannabis sativa L.) Seed and Their Antioxidant and Acetylcholinesterase Inhibitory Activities Xiaoli Yan,† Jiajing Tang,† Carolina dos Santos Passos,§ Alessandra Nurisso,§ Claudia Avello Simões-Pires,§ Mei Ji,† Hongxiang Lou,† and Peihong Fan*,† †

Department of Natural Product Chemistry, Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China § School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland S Supporting Information *

ABSTRACT: Hemp seed is known for its content of fatty acids, proteins, and fiber, which contribute to its nutritional value. Here we studied the secondary metabolites of hemp seed aiming at identifying bioactive compounds that could contribute to its health benefits. This investigation led to the isolation of 4 new lignanamides, cannabisin M (2), cannabisin N (5), cannabisin O (8), and 3,3′-demethyl-heliotropamide (10), together with 10 known lignanamides, among which 4 was identified for the first time from hemp seed. Structures were established on the basis of NMR, HR-MS, UV, and IR as well as by comparison with the literature data. Lignanamides 2, 7, and 9−14 showed good antioxidant activity, among which 7, 10, and 13 also inhibited acetylcholinesterase in vitro. The newly identified compounds in this study add to the diversity of hemp seed composition, and the bioassays implied that hemp seed, with lignanamides as nutrients, may be a good source of bioactive and protective compounds. KEYWORDS: hemp seed, lignanamides, cannabisin, antioxidant, acetylcholinesterase inhibitor

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INTRODUCTION Cannabis sativa L. is an annual plant in the Cannabaceae family, which is known for its long, thin flowers and spiky leaves. It has been an important source of food, fiber, medicine, and psychoactive drug since ancient times.1 The drug and nondrug varieties of C. sativa L. must be distinguished. The drug type is known as marijuana or hashish, containing Δ9-tetrahydrocannabinol (THC) in concentrations between 1 and 20%, high enough to exhibit psychoactivity.2 The nondrug type has no psychoactive properties because of low THC concentrations and is called industrial hemp.3 According to the European Industrial Hemp Association, hemp plant for industrial purposes should not exceed 0.2% THC (in dry matter of the upper one-third of the crop).4 Due to the legalization of growing industrial hemp and the renewed demand for hemp fiber and seed products, many countries allowed planting this crop or approved its production.5 Hemp seed has been documented as a folk source of food throughout recorded history, raw, cooked, or roasted, and hemp seed oil has been used as a food/medicine in China for at least 3000 years.6,7 Canada, Australia, Austria, China, Great Britain, France, and Spain are among the most important agricultural producers of hemp seed.8 In some countries, milled hemp seed is now consumed as a source of vegetable protein and dietary fiber as hemp flour and in shake drinks.5,9 With the advantage of an attractive nutty taste, hemp seed is also incorporated into many food preparations, often mimicking familiar foods, such as snack bars, bread, cookies, yogurts, frozen dessert, pizza, cheese, and beverages. Hemp food © XXXX American Chemical Society

products currently have a niche market, based particularly on natural food and specialty food outlets.8 Another important factor for the popularity of hemp seed is the increasing knowledge of its excellent nutritional value and health benefits. Hemp seed has high levels of fatty acids, protein, insoluble fiber, and a rich set of minerals and vitamins.7 It is considered to be perfectly balanced with regard to the ratio (3:1) of two essential polyunsaturated fatty acids (PUFAs) for human nutrition, linoleic (ω-6) and α-linolenic acids (ALA) (ω-3).7,9,10 In addition to its nutritional value, hemp seed demonstrated positive health benefits, including alleviating constipation,11 lowering cholesterol,12 cardiovascular health benefits,13 immunomodulatory effects, and dermatological disease amelioration effects.7 Hemp seed extracts showed also strong antioxidant and antiaging effects14−16 and the potential to improve the impaired learning and memory induced by chemical drugs in mice.17 Despite reports relating the extensive uses and multiple functions of hemp seed, its bioactive constituents are still not fully understood. In addition to the above-mentioned nutritional composition, hemp seed is rich in lignanamides.18,19 The lignanamide family displayed interesting and diverse biological activities, including feeding deterrent activity,20 insecticidal effects,21 and anti-inflammatory activity.22 As part of our Received: July 24, 2015 Revised: November 19, 2015 Accepted: November 19, 2015

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DOI: 10.1021/acs.jafc.5b05282 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. 1H and 13C NMR Data for Compounds 2, 5, 8, and 10 in CD3ODa 2 position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″,6″ 3″,5″ 4″ 7″ 8″ 1‴ 2‴,6‴ 3‴,5‴ 4‴ 7‴ 8‴ 1⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗ 7⁗ 8⁗ 9⁗ 1‴″ 2‴″,6‴″ 3‴″,5‴″ 4‴″ 7‴″ 8‴″ 3-OMe 3′-OMe 3⁗-OMe

δH mult (J in Hz) 7.15 d (1.8)

6.95 7.08 7.41 6.42

d (8.4) dd (8.4, 1.8) d (15.7) d (15.7)

6.82 d (1.8)

6.77 6.70 4.97 4.47

d (8.1) dd (8.1, 1.8) d (7.0) d (7.0)

7.03 d (8.4) 6.70 d (8.4) 2.75 t (7.3) 3.44 t (7.3) 6.82 d (8.4) 6.64 d (8.4) 2.51, 2.40 m 3.32, 3.16 m

5 δC 128.9 115.8 143.6 142.5 117.1 121.6 139.7 119.1 167.4 126.7 114.2 145.9 145.9 114.9 118.9 76.4 78.2 167.7 129.9 129.3 114.9 155.4 34.4 41.3 129.6 129.3 114.9 155.5 34.1 40.9

8


7.02 7.10 7.42 6.44

d (8.4) dd (8.4, 1.8) d (15.7) d (15.7)

6.82 s 6.70 s 6.70 s 5.53 d (3.2) 4.83 d (3.2)

6.82 d (8.4) 6.70 d (8.4) 2.74 t (7.3) 3.44 t (7.3) 7.04 d (8.4) 6.64 d (8.4) 2.40, 2.37 m 3.29, 3.16 m

δC

δH mult (J in Hz)

131.2 116.9 144.8 145.1 118.5 122.9 141.1 120.6 168.7 128.3 112.0 148.1 116.0 148.8 121.4 77.1 76.9 168.7 131.3 130.7 116.3 156.9 35.6 42.6 130.8 130.6 116.2 156.9 35.8 41.9

7.32 s

6.92 s 7.26 s

6.89 d (1.8)

6.80 6.75 5.88 4.13

d (8.1) dd (8.1, 1.8) d (8.3) d (8.3)

7.01 d (8.4) 6.75 d (8.4) 2.66 m 3.51 m 7.09 d (8.4) 6.75 d (8.4) 2.79 m 3.45 m 7.21 d (1.8)

6.75 7.05 7.46 6.50

d (8.4) dd (8.4, 1.8) d (15.7) d (15.7)

6.87 d (8.4) 6.62 d (8.4)

3.70 s

56.2

2.68 3.51 3.75 3.82 3.89

m m s s s

10 δC 126.2 114.1 144.3 149.1 127.8 119.0 123.6 140.9 164.0 131.1 109.0 147.8 146.7 114.8 118.6 88.4 57.2 171.3 129.5 129.3 115.0 155.5 34.1 40.5 129.8 129.4 114.8 155.5 34.3 41.2 130.6 111.2 144.3 145.9 114.2 120.8 139.6 119.7 167.2 129.3 129.2 114.8 155.4 34.1 40.8 54.9 54.9 54.9


6.79 d (8.2) 6.87 dd (2.0, 8.2) 7.42 d (2.1)

6.54 d (2.1)

6.77 6.45 4.27 3.84

d (8.1) dd (8.1, 2.1) d (2.4) d (2.4)

6.99 d (8.4) 6.71 d (8.4) 2.55, 2.77 m 2.92, 3.84 m 6.95 d (8.4) 6.71 d (8.4) 2.62 m 3.19, 3.45 m

δC 126.1 116.6 145.2 147.2 115.1 122.4 134.6 125.1 169.3 131.1 112.5 145.8 145.6 115.1 118.2 65.1 53.0 171.3 129.7 129.4 114.8 155.4 32.4 42.9 129.7 129.3 114.9 155.6 34.0 40.8

δ in ppm, frequency was 600 MHz for 1H NMR and 150 MHz for 13C NMR, assignments of 1H, 13C NMR data were based on HSQC and HMBC experiments. a

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ongoing search for antioxidants and acetylcholinesterase (AChE) inhibitors from secondary plant metabolites in medicinal and food plants, the aim of the present study was to conduct a detailed phytochemical characterization of lignanamides in hemp seed and to screen their bioactivities to correlate with those from literature reports to establish a global database of scientific knowledge on this nutritional crop of economic and pharmaceutical importance.

MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured on an MCP 200 (Anton Paar, Shanghai, China). Ultraviolet (UV) spectra were recorded using a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). Infrared (IR) spectra were taken from KBr disks on a Nicolet Nexus 470 FT-IR spectrophotometer (Thermo Scientific, Waltham, MA, USA). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was carried out on an LTQ-

B


Article

Journal of Agricultural and Food Chemistry

(MeOH/H2O 60:40) to afford 4 (4.51 mg, tR19 min), 5 (5.34 mg, tR 21 min), and 6 (17.21 mg, tR 23 min). Fraction 3 (2.01 g) was subjected to the silica gel column (2 × 15 cm, 200−300 mesh, 150 g; CH2Cl2/MeOH, 100:0 to 85:15) and the Sephadex LH-20 column (MeOH) and then purified over HPLC (MeOH/H2O 65:35) to afford 8 (1.72 mg, tR 9.5 min). Cannabisin M, 2: colorless amorphous powder; [α]20D −5.0 (0.1, MeOH); UV (MeOH) λmax (log ε) 317.5 (3.00), 291.2 (3.30), 243 (3.49); IR (KBr) νmax 3400, 2972, 1655, 1612, 1517, 1443, 1381, 1270, 1046, 815 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data, see Table 1; HR-ESI-MS m/z 597.2237 [M + H]+ (calcd for C34H33N2O8, 597.2231). Cannabisin N, 5: colorless amorphous powder; [α]20D −9.8 (0.1, MeOH); UV (MeOH) λmax (log ε) 315 (3.50), 285 (3.63), 239.9 (3.78); IR (KBr) νmax 3377, 2972, 1659, 1516, 1268, 1047, 822 cm−1; 1 H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data, see Table 1; HR-ESI-MS m/z 611.2390 [M + H]+ (calcd for C35H35N2O8, 611.2388). Cannabisin O, 8: colorless amorphous powder; [α]20D +0.71 (0.07, MeOH); UV (MeOH) λmax (log ε) 225 (4.78), 287 (4.48), 320 (4.46); IR (KBr) νmax 3415, 2925, 2853, 1654, 1613, 1515, 1384, 1258, 1032, 825, 555 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data, see Table 1; HR-ESI-MS m/z 936.3707 [M + H]+ (calcd for C54H53N3O12, 936.3702). 3,3′-Demethyl-heliotropamide, 10: colorless amorphous powder; [α]20D +2.5 (0.1, MeOH); UV (MeOH) λmax (log ε) 337.8 (4.17) 300.7 (3.69) 251.2 (3.73); IR (KBr) νmax 3373, 2926, 1643, 1612, 1514, 1452, 1384, 1243, 1057, 824, 557 cm−1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data, see Table 1; HR-ESIMS m/z 597.2230 [M + H]+ (calcd for C34H32N2O8, 597.2231). DPPH• Radical-Scavenging Assay. The radical-scavenging activities for the compounds were assessed on the basis of the radical-scavenging effect of the stable 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH•).23 The concentration of DPPH• was kept at 300 μM in MeOH. The compounds were dissolved in MeOH. Each compound solution (10 μL) was mixed with 190 μL of DPPH• at 37 °C for 30 min in a 96-well microliter plate in the absence of light. After incubation, the decrease in absorption for each solution was measured at 490 nm using a microplate reader. The corresponding blank reading was also taken, and the remaining DPPH• was calculated. The percentage of radical-scavenging activity by samples was determined by comparison with a 10 μL MeOH-treated blank group. Quercetin, an antioxidant flavone, was used as reference. All of the samples were analyzed in triplicate. Inhibition of free radical DPPH• in percent (I%) was calculated as

Orbitrap XL (ThermoFinnigan, Bremen, Germany). NMR spectra (1H, 13C, HSQC, HMBC) were obtained with a 600 MHz DD2 spectrometer operating at 600 (1H) and 150 (13C) MHz (Agilent Technologies, Santa Clara, CA, USA). Methanol-d4 (CD3OD) and dimethyl-d6 sulfoxide were used as analytical solvents (Sigma-Aldrich, Shanghai, China). High-performance liquid chromatography (HPLC) was performed on a 1260 G1311C quaternary pump equipped with a 1260 G1329B 1260ALS, 1260 G1365D MWD detector, and the column used was a 250 mm × 4.6 mm i.d., 5 μm, ZORBAX SB-C18, with a 4 mm × 4 mm i.d. guard column of the same material (Agilent Technologies). Silica gel (100−300 and 200−300 mesh) for column chromatography was purchased from the Qingdao Marine Chemical Inc. (Qingdao, China). Sephadex LH-20 (40−70 μm) (Amersham Pharmacia Biotech AB, Uppsala, Sweden), reversed phase C18 (octadecylsilyl, ODS) silica gel (YMC*GEL HG, ODS-A, 120 Å, 50 μm) (YMC, Tokyo, Japan), MCI gel (75−150 μm; Mitsubishi Chemical Corp., Tokyo, Japan), and GF254 plates (Qingdao Marine Chemical Inc.) were obtained from the suppliers noted. Thin-layer chromatography (TLC) analysis was performed on plates precoated with silica gel GF254 (0.20−0.25 mm) (Qingdao Marine Chemical Inc.). Bioactivities were measured using a microplate reader. Reagents. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), fluoresescein, K2S2O8, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), glycine, acetylcholinesterase (AChE) from electric eel, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and acetylthiocholine iodide (ATCI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Materials. The hemp seed material was collected from Bama county, Guangxi province, of China in September 2012. It was identified by Professor Lan Xiang, Department of Pharmacognosy, Shandong University. A voucher specimen has been deposited in Dr. Fan’s laboratory (Voucher 201209-1). Extraction and Isolation. The air-dried hemp seed (5.7 kg) was crushed and then defatted with petroleum ether (PE; 50 L, 2 h) at room temperature. Defatted seed biomass was percolated with 75% ethanol (EtOH) three times (60 L, 3 days for each time). The filtrate was concentrated under vacuum to 500 mL, followed by successive partition with PE (3 × 0.5 L), ethyl acetate (EtOAc) (3 × 0.5 L), and n-butanol (n-BuOH) (3 × 0.5 L), respectively. The EtOAc extract (12.12 g) was subjected to reverse phase column liquid chromatography (4 × 40 cm, 1 kg) and eluted successively with MeOH/H2O (1:9 to 1:0; 5 L for each gradient elution) to afford five fractions (fractions 1−5). Fraction 1 (4.30 g) was separated by medium-pressure reverse phase column liquid chromatograph (RPMPLC) and eluted successively with MeOH/H2O (4:6 to 1:0; 1 L for each gradient elution) to afford six subfractions (fractions R1−R6). Fractions R2−R6 were successively separated by Sephadex LH-20 column (elution with MeOH) followed by HPLC (MeOH/H2O as mobile phase) purification to afford a series of compounds, respectively: 7 (9.50 mg, tR 15 min; MeOH/H2O 50:50, 1.8 mL/ min) from fraction R2; 13 (7.60 mg, tR 18.5 min; MeOH/H2O 52:48, 1.8 mL/min) and 14 (13.22 mg, tR 11 min; MeOH/H2O 52:48, 1.8 mL/min) from fraction R3; 10 (5.80 mg, tR 30 min; MeOH/H2O 47:53, 1.8 mL/min) and 11 (2.10 mg, tR 25 min; MeOH/H2O 47:53, 1.8 mL/min) from fraction R4; 9 (35.00 mg, tR 6.5 min; MeOH/H2O 50:50, 1.8 mL/min) from fraction R5; and 12 (4.80 mg, tR 22.5 min; MeOH/H2O 50:50, 1.8 mL/min) from fraction R6. Fraction 2 (3.31 g) was separated by MCI gel column chromatography (2 × 20 cm, 500 g; MeOH/H2O, 2:5 to 1:0) to give seven fractions (fractions M1− M7). Fraction M3 (1.03 g) was chromatographed on a silica gel column (2 × 10 cm, 200−300 mesh, 100 g; PE/EtOAc, 1:0 to 0:1) to give six subfractions (S1−S6). Fraction M3S4 (0.40 g) was separated again by Sephadex LH-20 column (MeOH) to give two subfractions, and each fraction was purified by HPLC to afford 1 (2.04 mg, tR 10.0 min, MeOH/H2O 51:49, 1.8 mL/min) and 2 (6.87 mg, tR 20.0 min, MeOH/H2O 60:40, 1.8 mL/min), respectively. Fraction M4 (200.00 mg) was subjected to silica gel column chromatography (1 × 10 cm, 200−300 mesh, 20 g; CH2Cl2/MeOH, 100:0 to 94:6) to give four subfractions (C1−C4). 3 (22.94 mg) was crystallized from fraction M4C2; fraction M4C3 (60.00 mg) was further purified over HPLC

I % = (1 − A sample /A blank ) × 100 where Ablank is the absorbance of the blank group and Asample is the absorbance of the group of compounds. For each compound with a percentage of inhibition >60%, the IC50 value was determined by a dose−response curve with at least six concentrations. ABTS•+ Radical-Scavenging Assay. The free radical-scavenging ability was evaluated spectrophotometrically by the ABTS•+ loss of absorbance at 715 nm,24 using the same procedure as described for the DPPH• assay, by using the ABTS•+ solution to replace DPPH• solution. To form the ABTS•+ radical, 7 mM ABTS was added to 2.45 mM potassium persulfate in water. The mixture was stocked in the dark at room temperature for 16 h before use. To assess the ABTS•+ radical concentration, the absorbance of the radical stock solution was measured at 734 nm and the exact concentration was calculated according to the Beer−Lambert law, given the extinction coefficient of the radical at this wavelength. Radical final concentration was then adjusted to 67 μM. Compounds were tested at the same concentration as for the DPPH• assay. Oxygen Radical Absorbance Capacity (ORAC) Assay. The antioxidant activity was determined by the ability of each compound to preserve the fluorescence of fluorescein exposed to peroxyl radicals generated by 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH).25 Black polypropylene 96-well plates were filled with 60 nM C


Article


Figure 1. Structures of compounds 1−14.

Figure 2. Key HMBC correlations for the new compounds. Tris-HCl buffer (pH 7.8), 20 μL of 0.5 U/mL AChE solution in buffer, and 20 μL of tested solution (samples were dissolved in 0.1 M TrisHCl buffer containing 85% MeOH) were mixed and incubated at 37 °C for 15 min in a 96-well microplate. Then, 40 μL of 0.75 mM DTNB was added followed by 20 μL of 1.5 mM ATCI to initiate the reaction. The hydrolysis of ATCI was monitored using a microplate reader at a wavelength of 405 nm. The reaction system with the test solution replaced by equivalent MeOH buffer was used as blank control, and the AChE replaced by buffer was used as background of compound group. The reaction system without compounds and AChE was used as reagent background. Galanthamine base, an inhibitor of AChE, was used as reference. Inhibition of AChE in percent (I%) was calculated as

fluorescein in glycine buffer, pH 8.3, and tested samples (in buffer with 1% MeOH) and pre-incubated for 15 min at 40 °C with continuous shaking at 150 rpm. Finally, AAPH 5 mM (in buffer) was added to sample and oxidized control wells. Nonoxidized control wells received buffer instead of AAPH. The oxidation of fluorescein by AAPH was obtained by incubation at 40 °C for 90 min with continuous shaking at 150 rpm. The plate was cooled to room temperature (5 min) prior to fluorescence reading at 485/528 nm. The screening of pure compounds was conducted at 10 μM. For each compound with a percentage of fluorescein protection >50%, the IC50 value was determined by a dose−response curve with at least six concentrations. Assay for Acetylcholinesterase Inhibitory Activity. AChE inhibitory activity was measured by slightly modifying a spectrophotometric method described in the literature.23,26 Briefly, 100 μL of 0.1 M D


Article

Journal of Agricultural and Food Chemistry I % = [1 − (A sample − A background )/(A blank − A′background )] × 100

Table 4. AChE Inhibitory Activity and IC50 Values of Selected Compounds

where Asample is the absorbance of the test group, Ablank the absorbance of the blank control group, Abackground the absorbance of the background group of compound, and A′background, the absorbance of the background group of reagents. For each compound with a percentage of inhibition >60%, the IC50 value was determined by a dose−response curve with at least six concentrations. Molecular Modeling Studies on AChE. The crystallographic structure of Torpedo californica AChE in complex with galanthamine (TcAChE; PBD ID 1DX6) was downloaded from the Protein Data Bank (PDB; http://www.rcsb.org/pdb/).27,28 The biopolymer module implemented in Sybyl X-1.3 (Tripos Inc., St. Louis, MO, USA) was used to prepare the protein for docking. The starting geometries of the ligands 10 and 13 were built in Sybyl X-1.3. Bond and atom types were assigned according to the Tripos force field.29 Gasteiger−Marsili partial charges were calculated before minimization.30 Docking calculations were performed with GOLD (CCDC, Cambridge, UK) version 5.2,31 applying the MLP as hydrophobic descriptor.32 The binding site was defined by considering the residues around the cocrystallized ligand (6 Å cutoff). For each ligand, 100 docking solutions were generated by using 100,000 GOLD Genetic Algorithm (GA) iterations (preset option). Docking solutions were ranked according to the GoldScore scoring function and clustered. The predicted interactions were inspected with Sybyl X-1.3.

compound 1 3 5 6 7 9 10 11 12 13 14 galanthaminec

AChE inhibitiona (%)

AChE IC50 (μM)

± ± ± ± ± ± ± ± ± ± ± ±

−b − − − 216 − 46.2 − − 38.7 − 2.76

11.17 22.42 51.99 47.16 83.28 47.13 62.84 13.25 42.89 87.37 13.97 89.26

6.86 5.96 8.99 17.92 0.62 5.56 5.93 9.74 9.28 2.31 7.83 1.09

Tested at 100 μg/mL; mean values ± SD (n = 3) are shown. b−, not tested. cUsed as positive control at 10 μg/mL. a

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RESULTS AND DISCUSSION The ethyl acetate-soluble part of the 75% EtOH extract of hemp seed was fractionated by silica gel, reverse phase silica gel, Table 2. Antioxidant Activities of Selected Compounds (Percent)a compound 1 2 3 5 6 7 8 9 10 11 12 13 14 quercetinb

DPPH (100 μg/mL) 29.1 81.9 25.4 16.8 41.7 84.7 8.5 83.5 81.5 69.1 81.5 86.9 80.0 85.5

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.02 0.02 0.01 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.02 0.01 0.01

ABTS (100 μg/mL) 68.6 94.0 59.0 45.3 76.6 89.0 − 95.1 94.9 − 95.0 84.5 65.9 89.4

± ± ± ± ± ±

0.47 0.22 0.06 0.01 0.46 0.01

± 0.07 ± 0.12 ± ± ± ±

0.12 0.03 0.01 0.02

ORAC (10 μM) 84.5 83.3 34.7 34.4 68.1 109.8 − 71.4 79.1 104.0 66.9 82.9 67.0 98.7

± ± ± ± ± ±

0.99 2.08 1.61 2.53 3.47 3.26

± ± ± ± ± ± ±

4.78 1.08 0.14 2.16 3.88 3.91 2.14

Figure 3. Docking validation. Redocked galanthamine (best-ranked docking solution) is reported in red sticks into the catalytic site of AChE (gray ribbons). Crystallographic galanthamine is represented as black sticks. Residues belonging to the PAS are reported in magenta sticks.

MCI gel, and Sephadex LH-20 column chromatography, further purified by MPLC and HPLC to afford 4 new lignanamides, 2, 5, 8, and 10, and 10 known compounds, 1, 3, 4, 6, 7, 9, and 11−14 (Figure 1), among which, 4 was identified for the first time from hemp seed. Compound 2 was isolated as a colorless amorphous powder. Its molecular formula was identified as C34H32N2O8 on the basis of HR-ESI-MS analysis, suggesting 20 degrees of unsaturation. In the 1H NMR spectrum, the proton signals of a trans-substituted double bond and a trans-oriented aliphatic oxymethine were present as two pairs of doublet peaks at δ 7.41 (J = 15.7 Hz, H-7) and 6.42 (J = 15.7 Hz, H-8) and at δ 4.97 (J = 7.0 Hz, H-7′) and 4.47 (J = 7.0 Hz, H-8′), respectively. Eight ortho-coupled protons of two disubstituted aromatic rings were present as doublets at δ 7.03 (H-2″, 6″), 6.70 (H-3″, 5″), 6.82 (H-2‴, 6‴), and 6.64 (H-3‴, 5‴). Additionally, two pairs of vicinal methylenes δ 2.75 (2H, t, H-7″) and 3.44 (2H, t, H-8″) and δ 2.51, 2.40 (2H, H-7‴) and 3.32, 3.16 (2H, H-8‴) suggested the presence of two NHCH2CH2 segments. HMBC correlations of H-7″/C-2″(C-6″), H-8″/C-1″ and H-7‴/ C2‴(C-6‴), H-8‴/C-1‴ confirmed two p-tyramine moieties present in 2 (Figure 2). Six aromatic protons at δ 7.15 (1H, d, J = 1.8 Hz, H-2), 6.95 (1H, d, J = 8.4 Hz, H-5), and 7.08 (1H, dd, J = 1.8, 8.4 Hz, H-6) and at δ 6.82 (1H, d, J = 1.8 Hz, H-2′), 6.77 (1H, d, J = 8.1 Hz, H-5′), and 6.70 (1H, dd, J = 1.8, 8.1 Hz, H-6′) suggested the presence of two ABX spin systems.

Mean values ± SD (n = 3) are shown. −, not tested. bUsed as positive control. a

Table 3. IC50 Values of Selected Compounds in the Antioxidant Assays

a

compound

DPPH (μM)

ORAC (μM)

ABTS (μM)

2 7 9 10 11 12 13 14 quercetinb

69.5 145 32.9 39.3 175 58.3 77.6 23.9 25.5

6.61 1.54 6.61 0.56 1.13 1.88 0.23 0.46 0.40

74.70 33.38 7.25 16.41 −a 25.81 35.80 73.03 9.19

−, not tested. bUsed as positive control. E


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Figure 4. Molecular docking results: (A) compound 10 (yellow ball and sticks) in complex with AchE (gray ribbons); (B) compound 13 (red ball and sticks) in complex with AchE (gray ribbons). Residues belonging to the PAS are reported in magenta sticks. Residues involved in complex stabilization are labeled.

spectrum (Figure 2) further confirmed the linkages between moieties, as with 4. Thus, the structure of 5 was determined as (2,3-cis)-3-(3-hydroxy-5-methoxyphenyl)-N-(4-hydroxyphenethyl)-7-{(E)-3-[(4-hydroxyphenethyl)amino]-3-oxoprop-1enyl}-2,3-dihydrobenzo[b][1,4]dioxine-2-carboxamide and was named cannabisin N. Compound 8 was isolated as a colorless amorphous powder. Its molecular formula was established to be C54H52N3O12 on the basis of its 13C NMR and positive-ion HR-ESI-MS, suggesting 30 degrees of unsaturation. The 1H, 13C, and HSQC spectra indicated that it was a lignanamide compound with three tyramine moieties [δ 7.01 (2H, d, H-2″, 6″), 6.75 (2H, d, H-3″,5″), 2.66 (2H, m, H-7″), 3.51 (2H, m, H-8″); δ 7.09 (2H, d, H-2‴,6‴), 6.75 (2H, d, H-3‴,5‴), 2.79 (2H, m, H7‴), 3.45 (2H, m, H-8‴); δ 6.87 (2H, d, H-2‴″,6‴″), δ 6.62 (2H, d, H-3‴″,5‴″), 2.68 (2H, m, 7‴″), 3.51 (2H, m, 8‴″)]; two ABX-type coupled aromatic moieties [δ 6.89 (1H, d, J = 1.8 Hz, H-2′), 6.80 (1H, d, J = 8.1 Hz, H-5′), 6.75 (1H, dd, J = 8.1, 1.8 Hz, H-6′); δ 7.21 (1H, d, J = 1.8 Hz, H-2⁗), 6.75 (1H, d, J = 8.4 Hz, H-5⁗), 7.05 (1H, dd, J = 8.4, 1.8 Hz, H-6⁗)]; one 1,3,4,5-tetrasubstituted benzene ring [δ 7.32 (1H, s) and 6.92 (1H, s)]; a trans-substituted double bond [δ 7.46 (1H, d, J = 15.7 Hz, H-7⁗) and 6.50 (1H, d, J = 15.7 Hz, H-8⁗)]; a trans-oriented aliphatic oxymethine [δ 5.88 (1H, d, J = 8.3 Hz, H-7′) and 4.13 (1H, d, J = 8.3 Hz, H-8′)];22,34,35 three methoxy groups [δ 3.75 (3-OMe), 3.82 (3′-OMe), and 3.89 (3⁗OMe)], and an olefin proton [δ 7.26 (1H, s, H-7]. The HMBC correlations of H-8⁗/C-9⁗, H-8‴″/ C-9⁗, H-7⁗/C-2⁗, C6⁗, and 3⁗-OMe/C-3⁗ indicated an N-trans-feruloyltyraminelike unit (Figure 2).36 The correlations of H-7′/C-9′, C-2′, C6′, H-8′/C-1′, C-5, H-7/C-2, C-6, C-9, and H-8″/C-9 suggested a grossamide-like unit (Figure 2). However, a slight difference from the grossamide-like unit of 8 from grossamide is the disappearance of the H-8 signal and the downfield shift of the H-7 signal in the 1H spectrum,36 suggesting that the two units were connected by C-8 (δ 140.9) and C-4⁗ (δ 145.9) via an O-atom. This elucidation was supported by the literature,37 because the 8-O-4 linkage is common in dimers of this type. Thus, the structure of 8 was determined as (2,3-trans)-2-(4hydroxy-3-methoxyphenyl)-N-(4-hydroxyphenethyl)-5-((Z)-2(4-((E)-3-(4-hydroxyphenethylamino)-3-oxoprop-1-enyl)-2methoxyphenoxy)-6-(4-hydroxyphenyl)-3-oxohex-1-enyl)-7methoxy-2,3-dihydrobenzofuran-3-carboxamide and was given the name cannabisin O.

The HMBC correlations of H-8/C-1 and H-7/C-2, C-6, C-9 suggested the presence of a caffeoyl-like unit (Figure 2). Two vicinal aliphatic oxymethine signals were linked to C-1′ and C9′, respectively, by the HMBC correlations from H-7′ to C-1′, C-2′, C-6′, and C-9′ and from H-8′ to C-1′ and C-9′. The linkages of C-9 and C-9′ to the p-tyramine moieties were determined by the HMBC correlations of H-8″/C-9 and H-8‴/ C-9′. The NMR data of 2 (Table 1) were similar to those of (2,3-trans)-3-(3-hydroxy-5-methoxyphenyl)-N-(4-hydroxyphenethyl)-7-{(E)-3-[(4-hydroxyphenethyl)amino]-3-oxoprop-1enyl}-2,3-dihydrobenzo[b][1,4]dioxine-2-carboxamide, 4,33 except for an ABX spin system at δH 6.82 (H-2′), 6.77 (H-5′), and 6.70 (H-6′) in the 1H spectrum of 2 replacing a 1,3,5trisubstituted aromatic moiety of 4, together with the disappearance of the methoxy group signal of 4. Thus, the structure of 2 was determined as (2,3-trans)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-7-{(E)-3-[(4hydroxyphenethyl)amino]-3-oxoprop-1-enyl}-2,3dihydrobenzo[b][1,4]dioxine-2-carboxamide and was given the trivial name cannabisin M. Compound 5 was isolated as an optically inactive, colorless amorphous powder. Its molecular formula was identified as C35H34N2O8 on the basis of HR-ESI-MS analysis, suggesting 20 degrees of unsaturation. The 1H, 13C, and HSQC spectra indicated that it was a lignanamide compound with two tyramine moieties [δ 6.82 (2H, d, H-2″,6″), 6.70 (2H, d, H3″,5″), 2.74 (2H, t, H-7″), 3.44 (2H, t, H-8″); δ 7.04 (2H, d, H-2‴,6‴), 6.64 (2H, d, H-3‴,5‴), 2.40, 2.37 (2H, m, H-7‴), 3.29, 3.16 (2H, m, H-8‴)]; one ABX-type coupled aromatic moiety [δ 7.18 (1H, d, J = 1.8 Hz, H-2), 7.02 (1H, d, J = 8.4 Hz, H-5), 7.10 (1H, dd, J = 8.4, 1.8 Hz, H-6)]; one 1,3,5trisubstituted benzene ring [δ 6.82 (1H, s, H-2′), 6.70 (1H, s, H-4′), 6.70 (1H, s, H-6′)]; a trans-substituted double bond [δ 7.42 (1H, d, J = 15.7 Hz, H-7), 6.44 (1H, d, J = 15.7 Hz, H-8)]; and a cis-oriented aliphatic oxymethine [δ 5.53 (1H, d, J = 3.2 Hz, H-7′), 4.83 (1H, d, J = 3.2 Hz, H-8′)].22 The 1H and 13C NMR data of 5 were very similar to those of (2,3-trans)-3-(3hydroxy-5-methoxyphenyl)-N-(4-hydroxyphenethyl)-7-{(E)-3[(4-hydroxyphenethyl)amino]-3-oxoprop-1-enyl}-2,3dihydrobenzo[b][1,4]dioxine-2-carboxamide (compound 4) except that a small coupling constant (J7′,8′ = 3.2 Hz) at 5.53 (H-7′) and 4.83 (H-8′) in 5 replaced the large coupling constant (J7′,8′ = 7.0 Hz) in 4,33 indicating also that the two protons were cis-oriented. The correlation signals in the HMBC F


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compounds aiming at preventing and treating this disease.47 The AChE inhibitory activities of compounds 1, 3, 5−7, and 9−14 at the concentration of 100 μg/mL were assessed (Table 4), and the known AChE inhibitor galanthamine (10 μg/mL) was used as positive control. Among the tested molecules, compounds 7, 10, and 13 had moderate AChE inhibitory activity, whereas the others showed weak activities. The IC50 values of compounds 7, 10, and 13 were 216, 46.2, and 38.7 μM, respectively (Table 4). To understand the interactions between the most active compounds, 10 and 13, and the target of interest (AChE), we performed molecular docking calculations, following our previously validated protocol.30 As expected, by using the GOLD software, the crystallographic binding mode of galanthamine was reproduced into the TcAChE binding site: the best ranked solution presented a RMSD value of 0.5 Å (GoldScore 63.70, 100% convergence of docking solutions at 2 Å) with respect to the experimental pose (Figure 3). We then selected the best-ranked poses of 10 and 13 for visual inspection. Compound 10 was found to favorably interact with AChE (GoldScore 88.46, 85% convergence of docking solutions at 3 Å), occupying the galanthamine binding site and also part of the peripheral anionic site (PAS, Tyr 121, Trp 279, and Tyr 334),48 as shown in Figure 4A. One of the phenolic hydroxyl groups made a hydrogen bond with the carbonyl group of Trp 84, and the lateral chain of Trp 84 stabilized the aromatic ring through hydrophobic contacts. The five-membered heterocyclic ring was found to interact with the Phe 330 lateral chain through van der Waals contacts. From this ring, a double bond guided one dihydroxyphenyl moiety toward the PAS: the two hydroxyl groups interacted with Asn 85 lateral and main chains, whereas the cycle made van der Waals interactions with the Tyr 121 lateral chain. The PAS was also occupied by the second phenol moiety, and the interaction was stabilized by van der Waals contacts with Ile 297 and Tyr 334 residues. The second dihydroxyphenyl group was involved in a hydrogen bond with the Ser 200 lateral chain and in hydrophobic interactions with Phe 331 and His 440 rings (Figure 4A). Compounds 10 and 13 share a dihydroxyphenyl moiety linked to a phenol via an amide-containing alkyl chain. Despite this common scaffold, compound 13 interacted with the AChE pocket in a different way (Figure 4B). The dihydroxyphenyl moiety of the bestranked pose (GoldScore 94.36, 8% convergence of docking solutions at 3 Å) was stabilized by hydrophobic interactions with Trp 84 and His 440 side chains. The tetrahydrofuran moiety was involved in a hydrophobic sandwich between Phe 290, Phe 330, and Phe 331 side chains, whereas the adjacent hydroxyphenyl ring made a hydrogen bond with the Gly119 main chain. The apolar niche composed by Tyr 70, Trp 279, and Tyr 334 accommodated the phenol-containing linker through van der Waals contacts and a hydrogen bond with the Gln 74 main chain. The second phenol ring was stabilized by van der Waals interactions with Tyr 121, whereas its hydroxyl group created polar contacts with the Ser 122 lateral chain. The bioassay results of the current study suggest that hemp seed, with lignanamides as nutrients, may be exploited for their bioactive potential, because compounds with both antioxidant and acetylcholinesterase inhibitory activities are good choices for multitarget anti-Alzheimer’s disease candidates.49

Compound 10 was isolated as a colorless amorphous powder. The molecular formula of 10 was determined to be C34H31N2O8 by positive-ion HR-ESI-MS, suggesting 20 degrees of unsaturation. The 1H and 13C NMR spectra of 10 indicated that it was also a lignanamide compound with two tyramine moieties [δ 6.99 (2H, d, H-2″,6″), 6.71 (2H, d, H-3″,5″), 2.55, 2.77 (2H, m, H-7″), 2.92, 3.84 (2H, m, H-8″); δ 6.95 (2H, d, H-2‴,6‴), 6.71 (2H, d, H-3‴,5‴), 2.62, 2.77 (2H, m, H-7‴), 3.19, 3.45 (2H, m, H-8‴)] and two ABX-type coupled aromatic moieties [δ 6.91 (1H, d, J = 2.0 Hz, H-2), 6.79 (1H, d, J = 8.2 Hz, H-5), 6.87 (1H, dd, J = 2.0, 8.2 Hz, H-6); and δ 6.54 (1H, d, J = 2.1 Hz, H-2′), 6.77 (1H, d, J = 8.1 Hz, H-5′), 6.45 (1H, dd, J = 2.1, 8.1 Hz, H-6′)]. In the HMBC spectrum (Figure 2), the correlation signals of H-8″ with C-9 (δ 169.3) and H-8‴ with C-9′ (δ 171.3) indicated that the two tyramine groups were linked to C-9 and C-9′, respectively. The correlation signals between H-8″ (δ 2.92 and 3.84) and C-9 (δ 169.3), C-7′ (δ 65.1), the correlations between H-7′ (δ 4.27) and C-9′ (δ 171.3), C-8 (δ 125.1), C-9 (δ 169.3), and the correlations between H-7 (δ 7.42) and C-9 (δ 169.3), C-8′ (δ 53.0) led to a γ-lactam central moiety as shown in the structure of 10. The small J7′,8′ value (2.4 Hz) of 10 indicated that the two protons were cis-oriented, as shown in Figure 1. The structure of 10 was similar to that of heliotropamide,38 except for the disappearance of the methyl groups. Thus, the structure of 10 was determined as (2,3-cis)-4-(3,4-dihydroxybenzylidene)-2-(3,4-dihydroxyphenyl)-N,1-bis(4-hydroxyphenethyl)-5-oxopyrrolidine-3-carboxamide and was named 3,3′-demethyl-heliotropamide. The known compounds were identified by comparison of their spectroscopic data with those reported in the literature, including cannabisin E33 (1), grossamide22,34−36 (3), (2,3trans)-3-(3-hydroxy-5-methoxyphenyl)-N-(4-hydroxyphenethyl)-7-{(E)-3-[(4-hydroxyphenethyl)amino]-3-oxoprop-1-enyl}2,3-dihydro-benzo[b][1,4]dioxine-2-carboxamide33 (4), cannabisin F39 (6), N-trans-caffeoyltyramine40 (7), cannabisin A41 (9), N-trans-feryroyltyramine40 (11), cannabisin C42 (12), 3,3′demethyl-grossamide43 (13), and cannabisin D42 (14). The new compounds isolated in this study contribute to the structure diversity of lignanamides from hemp seed by providing different modes of coupling of monomeric intermediates, such as the formation of benzodioxine moiety for compounds 2, 4, and 5, the formation of a moiety for 8, and the formation of a lactam moiety for 10. Moreover, compound 8 is the first example of trimeric feruloyltyramine in hemp seed. Free radicals are considered to play key roles in numerous chronic pathologies such as cancer and neurodegenerative diseases and have been implicated in the aging process.44−46 Analysis of the antioxidant ability of selected compounds was conducted on the basis of the free radical-scavenging capacity toward DPPH• and ABTS•+ and also through the ORAC assay. The antioxidant activities of compounds 1−3 and 5−14 were tested using these methods, and the results are shown in Table 2. It was observed that compounds 2, 7, and 9−14 exhibited powerful DPPH• radical-scavenging activity ranging from 69.1 to 86.9% at the concentration of 100 μg/mL, comparable with the positive control quercetin. Their antioxidant activity was further confirmed by two other assays, ABTS•+ and ORAC (Table 2). The IC50 values of compounds 2, 7, and 9−14 for the three antioxidant methods are presented in Table 3. AChE inhibitors are important medications that have received U.S. FDA approval for the treatment of mild to moderate Alzheimer’s disease. The search for AChE inhibitors from traditional medicines is an important way to find G


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05282. 1D and 2D NMR, HR-ESI-MS, and IR spectra of new compounds 2, 5, 8, and 10 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(P.F.) Phone: 0086 531 88382012. E-mail: fanpeihong@sdu. edu.cn. Funding

This work was supported by the National Natural Science Foundation of China (Grant 81473323) and Key R&D program in Shandong Province (No. 2015GSF119025). Notes

The authors declare no competing financial interest.

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REFERENCES

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Characterization of Lignanamides from Hemp (Cannabis sativa L

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