Isobutylhydroxyamides from Sichuan Pepper and ... - ACS Publications

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Cite This: J. Agric. Food Chem. 2018, 66, 3408−3416

Isobutylhydroxyamides from Sichuan Pepper and Their Protective Activity on PC12 Cells Damaged by Corticosterone Jiahuan Chen,† Tao Zhang,‡ Qiubo Zhang,‡ Yang Liu,‡ Lingyu Li,‡ Jinguang Si,‡ Zhongmei Zou,*,‡ and Huiming Hua*,† †

Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China ‡ Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, People’s Republic of China S Supporting Information *

ABSTRACT: The pericarp of Zanthoxylum bungeanum Maxim., commonly known as Sichuan pepper, is a widely used spice to remove fishy odor and add palatable taste. A phytochemical investigation of the 95% ethanol extract of Sichuan pepper resulted in the isolation of 21 isobutylhydroxyamides, including 8 new ones named ZP-amides G−N, among which the chiral resolution of racemic ZP-amide A and ZP-amide B was successfully accomplished. The protective activity on corticosterone-treated PC12 cells of the isolated isobutylhydroxyamides was also evaluated. The new compounds 3−5 and the known compounds 1, 1a, 2, 2a, 11, and 15 improved the survival rate of PC12 cells. The bioactivity studies disclosed the potential of Sichuan pepper to be developed as new neuroprotective functional food. KEYWORDS: Zanthoxylum bungeanum Maxim., sichuan pepper, chiral resolution, isobutylhydroxyamide, PC12 cells

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INTRODUCTION Zanthoxylum bungeanum Maxim. (Rutaceae) is an aromatic shrub grown in China, Japan, and Korea.1 The ripe dried pericarp of Z. bungeanum Maxim., known as Sichuan pepper, has been used as spicy food additives in East Asia with a long history. Its delicate flavor and the feeling of tingling in the mouth can stimulate saliva production and increase appetite.2 The numbing taste makes it indispensable in hot Sichuan cuisine as well as a key ingredient of the 13-spice powder. It also serves as a natural preservative in the cooking of meat paste, sausage, and pickle. Sichuan pepper has been a commonly used traditional Chinese medicine in China, which is effective in the treatment of abdominal pain, diarrhea, and eczema due to its spleen and stomach warming medical functions.3,4 Modern pharmacological studies have demonstrated that it possesses a series of beneficial biological activities such as anti-type-1 diabetes,5 antitumor,6 and anti-inflammatory1 properties. The phytochemical investigation revealed a variety of secondary metabolites comprising alkaloids, lignans, flavonoids,7−9 and terpenoids10,11 together with essential oils.12,13 As characteristic constituents in the genus Zanthoxylum (Rutaceae), isobutylhydroxyamides have been reported to be the tingling and anesthetic component based on the cis and trans double bonds in the structure.6,14−17 Interestingly, isobutylhydroxyamides with at least one chiral carbon were obtained as racemic mixtures.6,16,18,19 The racemic mixtures have not been separated successfully until now. However, in the areas of pharmaceuticals and food development, chiral enantiomers are receiving much more attention than before, and chiral drugs seem to be very stereoselective.20,21 Thus the chemical and bioactive functions of optically pure isobutylhydroxyamides still remain underexploited. The CH2Cl2 and © 2018 American Chemical Society

EtOAc fractions from the 95% ethanol extract of Sichuan pepper exhibited a protective effect on corticosterone-treated PC12 cells. Therefore, they were subjected to phytochemical investigation, which led to the isolation of 21 isobutylhydroxyamides including 8 new compounds. Among them, racemic ZP-amides A and B16 were separated by preparative HPLC on chiral columns with two pairs of optically pure enantiomers being obtained for the first time. The protective activity of these isobutylhydroxyamides on PC12 cells damaged by corticosterone was evaluated.

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MATERIALS AND METHODS

General. NMR spectra, optical rotations, UV data, and IR spectra were recorded on a 500 instrument (Bruker, Rheinstetten, Germany), a 241 polarimeter (PerkinElmer, Waltham, MA), a UV-2501 PC (Shimadzu, Kyoto, Japan), and an FTIR-8400S spectrophotometer (Shimadzu, Kyoto, Japan), respectively. HRESIMS spectra were acquired with a Q-TOF MS analyzer in a SYNAPT HDMS system (Waters, Milford, MA). Chromatography. HPLC system used for purification included a 2535 instrument and a 2489 UV detector (Waters). Silica gel (60− 100, 100−200, and 200−300 mesh), ODS (50 μm), and Sephadex LH-20 (40−70 μm) used for column chromatography (CC) were supplied by Qingdao Marine Chemical Plant (Qingdao, China), YMC (Kyoto, Japan), and Pharmacia Biotech AB (Uppsala, Sweden), respectively. Chiral columns employed were 15 cm × 3 cm i.d., 5 μm, Chiralpak AD-H column (chiral column 1); 15 cm × 3 cm i.d., 5 μm, Chiralpak AY-H column (chiral column 2); 25 cm × 0.46 cm i.d., 5 μm, Chiralpak AD-H column (chiral column 3); 15 cm × 0.46 cm i.d., Received: Revised: Accepted: Published: 3408

December March 11, March 14, March 14,

24, 2017 2018 2018 2018 DOI: 10.1021/acs.jafc.7b06057 J. Agric. Food Chem. 2018, 66, 3408−3416

Article

Journal of Agricultural and Food Chemistry Table 1. NMR Data of Compounds 1a−10 in Methanol-d4 (δ in ppm and J in Hz) 1a

1b

1

position

13

H

C

1 2 3 4

6.03 dt (15.4, 1.6) 6.81 dt (15.4, 6.9) 2.29 m

5

1.70 m

6 7 8 9 10 11 12 13 14 1′ 2′ 3′, 4′ 1′′

4.23 6.28 6.45 7.29 6.16

169.0 125.0 145.4 29.0 36.5

m dd (15.4, 5.7) dd (15.4, 10.8) dd (15.7, 10.8) d (15.7)

2.29 s

71.7 148.0 129.0 145.2 131.2 201.5 27.0

3.25 s

51.1 71.6 27.2

1.17 s

2a

1

13

H

6.03 dt (15.4, 1.6) 6.81 dt (15.4, 6.9) 2.29 m

4.23 6.28 6.45 7.29 6.16

C

169.0 125.0 145.4 29.0

1.70 m

36.5

m dd (15.4, 5.7) dd (15.4, 10.8) dd (15.7, 10.8) d (15.7)

2.29 s

71.7 148.0 129.0 145.2 131.2 201.5 27.0

3.25 s

51.1 71.6 27.2

1.17 s

1

H

6.02 dt (15.4, 1.6) 6.79 dt (15.3, 6.8) 2.49 m 2.81 t (7.2)

6.22 7.28 6.41 6.28 4.37 1.27

2b 13

C

168.9 125.2 144.5 27.4 39.4

d (15.6) dd (15.6, 10.8) dd (15.4, 10.8) dd (15.4, 5.4) m d (6.5)

3.24 s

201.5 130.2 144.4 128.0 149.4 68.4 23.2

51.1 71.6 27.2

1.17 s

1

13

H

6.02 dt (15.4, 1.6) 6.78 dt (15.3, 6.8) 2.49 m 2.81 t (7.2)

6.22 7.28 6.41 6.28 4.36 1.26

C

168.9 125.2 144.5 27.4 39.4

d (15.6) dd (15.6, 10.8) dd (15.4, 10.8) dd (15.4, 5.4) m d (6.5)

3.24 s

201.5 130.2 144.4 128.0 149.4 68.4 23.2

51.1 71.6 27.2

1.17 s

2′′ −OCH3 3 13

H

6.01 d (15.3) 6.79 dt (15.3, 6.9) 2.29 overlappedb

5

1.71 m

6 7 8 9 10 11 12 13 14 1′ 2′ 3′, 4′ 1′′

3.78 6.12 6.45 7.29 6.19

169.0 125.0 145.2 28.8 34.8

m dd (15.3, 7.3) dd (15.3, 10.8) dd (15.7, 10.8) d (15.7)

2.29 s

81.9 145.0 131.7 144.8 131.7 201.5 27.1

3.25 s 1.17 s

3.30 s

position 1 2 3 4 5 6

H

6.02 6.82 2.41 2.27 1.70 1.51 3.49

d (15.3) dt (15.3, 6.9) m m m m m

13

H

6.02 d (15.3) 6.79 dt (15.3, 6.9) 2.49 m

6.24 7.28 6.42 6.14 3.91 1.25

39.4

d (15.7) dd (15.7, 10.8) dd (15.7, 10.8) dd (15.7, 6.9) m d (6.4)

51.1 71.6 27.2

3.24 s

57.0

3.29 s

201.5 130.7 143.9 130.5 146.5 78.4 21.0

51.1 71.6 27.2

1.17 s

1

C

168.9 125.2 144.4 27.4

2.82 t (7.2)

6.03 d (15.3) 6.79 dt (15.3, 6.9) 2.36 m 2.36 m 6.15 6.28 7.20 5.82

dt (15.3, 6.9) dd (15.3, 10.8) dd (15.3, 10.8) d (15.3)

1

C

169.2 124.7 145.8 29.4 32.4 74.9

6.01 6.81 2.40 2.27 1.66 1.49 3.44

168.9 125.3 144.6 32.2

143.3 130.6 145.8 122.2 a

3.25 s

51.1 71.6 27.2

1.17 s

1

C

32.5

8 13

6 13

H

13

H

C

5.99 d (15.3) 7.12 dd (15.3, 10.8) 6.23 dd (15.3, 10.8)

169.5 123.2 142.4 130.3

6.11 dt (15.3, 6.7)

143.1

2.27 2.34 5.42 6.01 6.50 5.64 4.01 1.53 0.91 3.25

34.0 28.0 131.4 130.0 126.4 137.6 74.7 31.2 10.2 51.2 71.7 27.2

m m dt (10.8, 7.5) t (10.8) dd (15.3, 10.8) dd (15.3, 6.6) q (6.6) m t (7.4) s

1.17 s

56.7

7 1

5

1

C

1 2 3 4

2′′ −OCH3

4

1

position

H

d (15.3) dt (15.3, 6.9) m m m m m

9 13

C

169.2 124.8 145.7 29.4

1

H

10 13

C

5.99 d (15.3) 7.13 dd (15.3, 10.8) 6.21 dd (15.3, 10.8)

169.6 122.9 142.6 129.8

32.4

6.10 dt (15.3, 6.9)

144.2

74.9

2.17 q (7.2)

3409

1

34.0

H

6.01 6.80 2.39 2.24 1.68 1.51 3.54

d (15.3) dt (15.3, 6.9) m m m m m

13

C

169.1 124.7 145.7 29.4 32.5 73.9

DOI: 10.1021/acs.jafc.7b06057 J. Agric. Food Chem. 2018, 66, 3408−3416

Article

Journal of Agricultural and Food Chemistry Table 1. continued 7 1

position 7 8 9 10 11 12 13 14 1′ 2′ 3′, 4′ 1′′

H

3.93 5.62 6.22 6.08 5.71 1.75

t-like (6.0) dd (15.3, 7.1) dd (15.3, 10.8) dd (15.3, 10.8) m d (6.7)

3.25 s

8 13

1

C

76.9 130.9 133.6 132.5 130.3 18.2

51.1 71.6 27.2

3.91 5.56 6.23 6.08 5.71 1.74

H

t-like (6.5) dd (15.3, 7.1) dd (15.3, 10.8) dd (15.3, 10.8) m d (6.4)

3.25 s 1.17 s

9 13

C

76.9 130.9 133.7 132.4 130.5 18.2

51.1 71.6 27.2

2′′ −OCH3 a

1

H

1.43 1.31 1.31 1.31 1.31 0.90

m m m m m t (6.8)

3.26 s 1.17 s

10 13

1

C

H

30.0 30.3 30.3 33.0 23.7 14.4

3.59 5.46 6.20 6.09 5.73 1.75

51.2 71.7 27.2

3.24 s 1.16 3.54 3.36 1.14

dd (8.1, 4.8) dd (15.3, 8.1) dd (15.3, 10.8) dd (15.3, 10.8) m d (6.4)

s m m t (6.8)

13

C

85.3 128.9 135.8 132.2 130.8 18.2

51.1 71.6 27.2 65.0 15.6

Signal was not observed. bSignal was overlapped.

3 μm, Chiralcel OX-3 column (chiral column 4); 25 cm × 0.46 cm i.d., 5 μm, Chiralcel OJ-H column (chiral column 5); and 15 cm × 0.46 cm i.d., 3 μm, Chiralcel OJ-H column (chiral column 6) (Daicel Chiral Technologies, Shanghai, China). Chemicals and Reagents. (R)-MTPA-Cl and (S)-MTPA-Cl used for modified Mosher’s method, corticosterone, and MTT were provided by Sigma-Aldrich (St. Louis, MO). 4-Dimethylaminopyridine (DMAP) was purchased from Sinopharm Chemical Reagent Company (Beijing, China). Solvents of HPLC grade were provided by Tianjin SaiFuRui Technology Company (Tianjin, China). Plant Material. The pericarp of Z. bungeanum Maxim. was collected from Sichuan province, China. A voucher sample (no. 1000203) was deposited in the Institute of Medicinal Plant Development and identified by Prof. Ben-gang Zhang. Extraction and Isolation. Sichuan pepper (23 kg) was powdered and soaked in 95% EtOH (100 L) three times at 25 °C. The combined extract was filtered and concentrated with a rotary evaporator under −0.1 MPa at 45 °C to yield crude extract (5.1 kg). The extract was loaded on a silica gel CC (45 cm × 24 cm i.d.) and eluted with petroleum ether (3 × 16 L), CH2Cl2 (3 × 16 L), EtOAc (3 × 16 L), nBuOH (3 × 16 L), and MeOH (3 × 16 L). The CH2Cl2 soluble fraction (470 g) was chromatographed on a silica gel column (20 cm × 24 cm i.d.) using petroleum ether/acetone (50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1 v/v, each 25 L) to give 10 fractions (Fr.C1−Fr.C10). Fr.C1 (15 g) was fractionated on a silica gel CC (15 cm × 4 cm i.d.) eluted with petroleum ether/EtOAc (20:1, 10:1, 5:1, 2:1, and 0:1 v/v, each 600 mL) to get four subfractions (Fr.C1.1−Fr.C1.4). Fr.C1.4 was applied to an ODS CC followed by semipreparative HPLC (40−95% CH3CN/H2O for 30 min, v/v, 2 mL/min) to provide 18 (17.2 mg), 19 (16.8 mg), and 9 (12.1 mg) with retention time at 28.0, 28.8, and 29.2 min. Separation of Fr.C3 (22 g) on a silica gel CC (20 cm × 4 cm i.d.) using CH2Cl2/MeOH (50:1 v/v, 1.5 L) as eluent gave five fractions (Fr.C3.1−Fr.C3.5), of which Fr.C3.2 was fractionated by the ODS CC (20 cm × 3 cm i.d., MeOH/H2O, 40, 45, 50, 55, and 100% v/v, each 300 mL), and semipreparative HPLC (50−95% CH3CN/ H2O for 30 min, v/v, 2 mL/min) was further performed to furnish 10 (16.7 mg) and 6 (15.8 mg) with retention time at 19.8 and 23.1 min. Fr.C7 (13 g) was repeatedly recrystallized in petroleum ether/EtOAc (10:1 v/v) to afford 17 (1158 mg). Fr.C8 (25 g) was subjected to a 145 cm × 4.5 cm i.d. Sephadex LH-20 CC flushed with MeOH to give 10 subfractions (Fr.C8.1−Fr.C8.10). Fr.C8.3 (520 mg) was then purified using a semipreparative HPLC (30−65% MeOH/H2O for 30 min, v/v, 2 mL/min) to afford 14 (220 mg), 3 (7.8 mg), and 4 (6.5 mg) with retention time at 18.3, 22.4, and 24.8 min. Fr.C9 (40 g) was loaded on a 25 cm × 5 cm i.d. silica gel column and eluted with a stepwise gradient of CH2Cl2/MeOH (200:1, 50:1, 30:1, 10:1 v/v, each 1 L) to obtain subfractions Fr.C9.1−Fr.C9.7. Fr.C9.2 was isolated by

semipreparative HPLC (40−70% MeOH/H2O for 30 min, v/v, 2 mL/ min) to produce 15 (35.2 mg, tR 14.5 min). Separation of Fr.C9.5 with preparative HPLC (35−65% MeOH/H2O for 30 min, v/v, 15 mL/ min) resulted in the isolation of 1 (360 mg), 2 (180 mg), 5 (17.7 mg) and 16 (10.5 mg) with retention time at 14.4, 15.8, 20.6, and 23.2 min. A silica gel column (20 cm × 15 cm i.d.) was employed using petroleum ether/acetone (20:1, 20:3, 3:1, 2:1, 1:1, and 1:2 v/v, each 10 L) as eluent to fractionate the EtOAc-soluble fraction (301 g), with eight fractions (Fr.E1−Fr.E8) being obtained. Fr.E4 (50 g) was separated on a 15 cm × 8 cm i.d. silica gel column and eluted with a mixture of CH2Cl2/MeOH (1:0, 30:1, 20:1, 10:1, and 5:1 v/v, each 1.5 L) to get subfractions Fr.E4.1−Fr.E4.5. Fr.E4.2 was loaded on an ODS CC (30 cm × 5 cm i.d.) and eluted with MeOH/H2O (20, 40, 45, 50, 55, 70, and 100% v/v, each 1.8 L) to give six fractions (Fr.E4.2.1− Fr.E4.2.6). Semipreparative HPLC purification (30−70% MeOH/H2O for 30 min, v/v, 2 mL/min) was performed on Fr.E4.2.2 to afford 11 (7.9 mg, tR 16.9 min). Fr.E4.2.5 was purified on semipreparative HPLC (40−70% MeOH/H2O for 30 min, v/v, 2 mL/min) to yield 7 (4.3 mg) and 8 (4.8 mg) with retention time at 16.7 and 17.8 min. Fr.E5 was loaded on a silica gel column (20 cm × 4 cm i.d.) using CH2Cl2/MeOH (15:1, 10:1, 5:1, and 1:1 v/v, each 750 mL) to yield 12 (75.2 mg) and 13 (82.1 mg). Compound 1 (310 mg) was further isolated by preparative HPLC with chiral column 1 (hexane/EtOH 2:3 v/v, 15 mL/min) to obtain 1a (80 mg) and 1b (78 mg) with retention time at 4.2 and 6.8 min. Compound 2 (130 mg) was purified by preparative HPLC with chiral column 2 (hexane/IPA 3:2 v/v, 15 mL/min), which resulted in the isolation of 2a (40 mg) and 2b (45 mg) with retention time at 4.7 and 5.6 min. (−)-(6R)-ZP-amide A, 1a. Colorless oil; [α]D25 −43.3 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 272 nm (4.6); IR (neat) νmax 3350, 2975, 2926, 1633, 1544, 1360, 1265, 1180, 1100, 905, and 767 cm−1; 1 H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 318.1677 [M + Na]+ (calcd for C16H25NO4Na, 318.1681). (+)-(6S)-ZP-amide A, 1b. Colorless oil; [α]D25 +44.5 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 272 nm (4.6); IR (neat) νmax 3350, 2968, 2929, 1633, 1537, 1364, 1258, 1184, 1000, and 912 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 318.1672 [M + Na]+ (calcd for C16H25NO4Na, 318.1681). (−)-(11R)-ZP-amide B, 2a. Colorless oil; [α]D25 −8.4 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 271 nm (4.6); IR (neat) νmax 3346, 2972, 2926, 2360, 2339, 1643, 1541, 1343, 1184, 1000, and 672 cm−1; 1 H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 318.1678 [M + Na]+ (calcd for C16H25NO4Na, 318.1681). 3410


Article

Journal of Agricultural and Food Chemistry

Figure 1. HPLC chromatograms of racemic isobutylhydroxyamides. cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 332.1842 [M + Na]+ (calcd for C17H27NO4Na, 332.1838). ZP-amide I, 5. Colorless oil; UV (MeOH) λmax (log ε) 251 (4.1) nm; IR (neat) νmax 3317, 3138, 2977, 2930, 1688, 1622, 1467, 1259, 934, 821, and 728 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 290.1371 [M + Na]+ (calcd for C14H21NO4Na, 290.1368). (±)-ZP-amide J, 6. Colorless oil; [α]D25 ± 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (4.0) nm; IR (neat) νmax 3377, 2971, 2922, 2851, 1713, 1664, 1635, 1549, 1379, 1266, 1179, 981, 908, and 769 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 330.2063 [M + Na]+ (calcd for C18H29NO3Na, 330.2045).

(+)-(11S)-ZP-amide B, 2b. Colorless oil; [α]D25 +5.6 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 271 nm (4.6); IR (neat) νmax 3339, 2975, 2926, 2357, 2339, 1643, 1544, 1360, 1180, 1004, and 668 cm−1; 1 H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 318.1670 [M + Na]+ (calcd for C16H25NO4Na, 318.1681). (±)-ZP-amide G, 3. Colorless oil; [α]D25 ± 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 267 (3.1) nm; IR (neat) νmax 3356, 2973, 2933, 1714, 1669, 1633, 1547, 1366, 1257, 1179, 1099, 981, 907, 769, and 623 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 332.1845 [M + Na]+ (calcd for C17H27NO4Na, 332.1838). (±)-ZP-amide H, 4. Colorless oil; [α]D25 ± 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (3.1) nm; IR (neat) νmax 3339, 2974, 2933, 1713, 1669, 1631, 1547, 1369, 1178, 1101, 979, 907, 769, and 648 3411


Article


Figure 2. Structures of isobutylhydroxyamides from Sichuan pepper. (±)-ZP-amide K, 7. Colorless oil; [α]D25 ± 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (4.4) nm; IR (neat) νmax 3336, 2972, 2928, 2851, 1669, 1664, 1627, 1553, 1450, 1367, 1285, 1178, 991, 908, and 768 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 320.1837 [M + Na]+ (calcd for C16H27NO4Na, 320.1838). (±)-ZP-amide L, 8. Colorless oil; [α]D25 ± 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (4.5) nm; IR (neat) νmax 3314, 2973, 2929, 2869, 1713, 1669, 1626, 1553, 1367, 1285, 1178, 991, and 942 cm−1; 1 H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 320.1838 [M + Na]+ (calcd for C16H27NO4Na, 320.1838). ZP-amide M, 9. Colorless oil; UV (MeOH) λmax (log ε) 207 (3.8) nm; IR (neat) νmax 3330, 2954, 2929, 2858, 1713, 1668, 1549, 1463, 1381, 1270, 1179, 980, and 907 cm−1; 1H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1);

HRESIMS (pos.): m/z 290.2110 [M + Na]+ (calcd for C16H29NO2Na, 290.2096). (±)-ZP-amide N, 10. Colorless oil; [α]D25 ± 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (3.9) nm; IR (neat) νmax 3353, 2974, 2929, 2869, 1714, 1669, 1631, 1548, 1370, 1273, 1178, 981, and 908 cm−1; 1 H NMR (500 MHz) and 13C NMR (125 MHz) data were obtained in methanol-d4 (Table 1); HRESIMS (pos.): m/z 348.2144 [M + Na]+ (calcd for C18H31NO4Na, 348.2151). Chiral Analyses of Racemic Isobutylhydroxyamides. The chiral analyses of 3 and 4 were performed on chiral column 3 using SFC system (40% MeOH, 3 mL/min). 6 was analyzed on an HPLC system with chiral column 6 eluted with ethanol/diethylamine (100:0.1 v/v, 0.2 mL/min). 7 was analyzed on an HPLC system with chiral column 3 eluted with n-hexane/isopropanol/diethylamine (85:15:0.1 v/v/v, 1 mL/min). 8 was analyzed on an HPLC system with chiral column 4 eluted with n-hexane/ethanol/diethylamine 3412


Article

Journal of Agricultural and Food Chemistry Table 2. Partial 1H NMR Data of MTPA esters of 1a and 2a in Chloroform-d (δ in ppm and J in Hz) (R)-MTPA ester of 1a 2 3 5 7 8 9 10 12

(S)-MTPA ester of 1a

1

position 5.749 6.779 1.889 1.817 6.048 6.357 7.053 6.159

d (15.6) dt (14.5, 7.1) m m dd (15.6, 7.1) dd (15.6, 10.8) dd (15.6, 10.8) d (15.6)

(R)-MTPA ester of 2a

1

H 5.806 6.817 1.943 1.868 5.976 6.237 7.012 6.087

(S)-MTPA ester of 2a

1

H

1

H

d (15.6) dt (14.5, 7.1) m m dd (15.6, 6.6) dd (15.6, 10.8) dd (15.6, 10.8) d (15.6)

6.182 7.106 6.362 6.131 1.403

d (15.6) dd (15.6, 10.9) dd (15.6, 10.9) dd (15.6, 6.4) d (6.5)

H

6.094 7.058 6.208 6.043 1.464

d (15.6) dd (15.6, 10.9) dd (15.6, 10.9) dd (15.6, 5.9) d (6.5)

Figure 3. Chemical shifts from application of modified Mosher’s method to 1a and 2a. Statistical Analysis. The results were expressed as mean ± standard deviation (SD). The significances (p < 0.05) of the intergroup differences were evaluated by two-tailed Student’s t test.

(85:15:0.1 v/v/v, 1 mL/min). 10 was analyzed on an HPLC system with chiral column 5 eluted with ethanol/diethylamine (100:0.1 v/v, 0.2 mL/min). HPLC chromatograms are shown in Figure 1. Application of Modified Mosher’s Method to 1a and 2a. Absolutely dried 1a (1.2 mg, 3.8 μmol) was reacted with (R)-MTPACl (3 μL, 15.9 μmol) in 0.8 mL of pyridine and catalyzed by DMAP. The reaction mixture was checked by TLC 5 min later. The result showed that 1a was converted into its (S)-MTPA ester. The crude product was isolated on a silica gel column with a mixture of petroleum ether/acetone (5:4 v/v) to yield (S)-MTPA ester (1.1 mg). Treatment of 1a with (S)-MTPA-Cl and DMAP in a similar way enabled us to get the corresponding (R)-MTPA ester (1.4 mg). Treatment of optically pure 2a was the same as described above. Protective Activity against Corticosterone-Induced PC12 Cells Damage Assay. PC12 cells were routinely maintained in DMEM that contained 100 μg/mL streptomycin, 100 U/mL penicillin, 5% (v/v) fetal bovine serum (FBS), and 10% horse serum and were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The appropriate damage concentration and incubation time of corticosterone were determined based on Gao et al.22 In brief, PC12 cells were incubated with corticosterone in concentration of 10, 20, 40, 100, 200, and 400 μmol/L for 24 and 48 h, respectively. The result showed that after incubation with 200 μmol/L corticosterone for 48 h, the cell viability of PC12 cells decreased significantly, which were used in subsequent experiments. PC12 cells were divided into the corticosterone group, the control group, together with a blank group that only contained cultures and reagents. For corticosterone group, PC12 cells were pretreated with varying concentrations of isobutylhydroxyamides (0, 3.125, 6.25, 12.5, 25, 50, 100 μmol/L) for 24 h before incubation with 200 μmol/L corticosterone for an additional 48 h. The control group was administered with the same amount of DMEM, and the blank group only contained cultures and reagents. Cell viability was evaluated by a MTT method. 80 μL of DMEM and 20 μL of MTT solution (5 g/L) were added and incubated for 4 h at 37 °C. Subsequently, DMSO was added to dissolve the formazan crystals produced. The absorbance at 570 nm was measured with a microplate reader. Cell viability was calculated as the percentage of the control group.

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RESULTS AND DISCUSSION Sichuan pepper was powdered and extracted with 95% ethanol. Phytochemical investigation of the CH2Cl2 and EtOAc fractions results in the isolation of eight new isobutylhydroxyamides and 13 known analogues (Figure 2). Enantiomeric ZPamide A and enantiomeric ZP-amide B were separated by chiral columns for the first time with two pairs of enantiomers being obtained for further study on the influence of stereochemistry on the bioactivity. In addition, with the methods we provided, the other racemic isobutylhydroxyamides can be separated. Compounds 1 and 2 were racemic mixtures identified as ZPamides A and B.13 Fortunately, 1 and 2 were separated successfully by chiral columns to obtain enantiomers 1a, 1b and enantiomers 2a, 2b, respectively. Modified Mosher’s method was further applied to construct the absolute configurations of the optically pure 1a and 2a. The 1H NMR spectra of the MTPA ester derivatives were recorded (Table 2). The difference between chemical shifts was calculated and represented as Δ δSR (ΔδSR = δS − δR). The substituents were located on the model23 according to the sign of the ΔδSR. The absolute configurations of 1a and 2a were established to be 6R and 11R, respectively (Figure 3). Therefore, 1b and 2b were determined to be 6S and 11S, as they are the enantiomers of 1a and 2a. Herein their absolute configurations were reported for the first time and named (−)-(6R)-ZP-amide A, 1a; (+)-(6S)ZP-amide A, 1b; (−)-(11R)-ZP-amide B, 2a; and (+)-(11S)ZP-amide B, 2b, respectively. Compound 3 possessed a molecular formula of C17H27NO4 based on the HRESIMS data. IR absorptions at 3356, 1669, and 1633 cm−1 revealed the existence of the hydroxyl, amide, and olefinic groups. The 1H NMR spectrum suggested compound 3 as an isobutylhydroxyamide with an unsaturated aliphatic chain. Characteristic signals of three pairs of olefinic protons on trans double bonds resonating at δH 7.29 (1H, dd, J = 15.7, 10.8 Hz, H-9), 6.79 (1H, dt, J = 15.3, 6.9 Hz, H-3), 6.45 (1H, dd, J = 15.3, 10.8 Hz, H-8), 6.19 (1H, d, J = 15.7 Hz, H-10), 6.12 (1H, dd, J = 15.3, 7.3 Hz, H-7), and 6.01 (1H, d, J = 15.3 Hz, H-2),

cell viability (%) = [Abs(sample) − Abs(blank)]/[Abs(control) − Abs(blank)] × 100 3413


Article


Figure 4. Selected HMBC and 1H−1H COSY correlations of compounds 3−10.

along with protons of one oxygenated methine at δH 3.78 (1H, m, H-6), one methoxyl signal at δH 3.30 (3H, s), three sets of methylene signals at δH 3.25 (2H, s, H-1′), 2.29 (2H, m, H-4), 1.71 (2H, m, H-5), and three methyl signals at δH 2.29 (3H, s, H-12) and 1.17 (6H, s, H-3′, H-4′) were observed. The 13C NMR spectrum exhibited a ketonic carbon and an amide carbon at δC 201.5 (C-11) and 169.0 (C-1), respectively, six olefinic carbons at δC 145.2 (C-3), 145.0 (C-7), 144.8 (C-9), 131.7 (C-8), 131.7 (C-10), and 125.0 (C-2), together with one oxygenated methine carbon at δC 81.9 (C-6), and three methylene carbons at δC 51.1 (C-1′), 34.8 (C-5), and 28.8 (C4), three methyl carbons at δC 27.2 (C-3′, C-4′) and 27.1 (C12), one quaternary carbon at δC 71.6 (C-2′), and one methoxy group at δC 57.0. These signals of 3 showed similarities with those of 1, except that a methoxyl was observed to replace the hydroxy at C-6 in 3.16 The conclusion was also confirmed by 2D NMR data (Figure 4). Cross peaks from H-1′ to C-1, C-2′, C-3′, and C-4′ demonstrated the isobutylhydroxylamide nature of compound 3. The linkage of the methoxyl group to C-6 and the location of the ketonic carbon at C-11 were built by the cross peaks of the methoxyl proton to C-6 and of H-9, H-10, and H-12 to the ketonic carbon. The 1H−1H COSY spectrum showed corrections from H-2 to H-10. Compound 3 was a racemic mixture due to the optical inactivity. Therefore, the planar structure of 3 was determined as (2E,7E,9E)-6methoxyl-N-(2-hydroxy-2-methylpropyl)-11-oxo-2,7,9-dodecatrienamide, named (±)-ZP-amide G. The molecular formula of compound 4 was the same as that of 3 according to its HRESIMS data. Analysis of the 1D NMR data of compounds 4 and 2 suggested that they were almost similar to each other; the only difference is the appearance of a methoxyl group in 4. The methoxyl group was then attached to C-11 via HMBC connection of the methoxyl proton to C-11

(Figure 4). 1H−1H COSY spectra further supported the structure proposed. Compound 4 was also a racemic mixture due to its optical inactivity. Thus (2E,7E,9E)-11-methoxyl-N(2-hydroxy-2-methylpropyl)-6-oxo-2,7,9-dodecatrienamide was assigned to compound 4, named (±)-ZP-amide H. Compound 5 had a molecular formula of C14H21NO4 according to the HRESIMS data. It was an analogue of a known compound timuramide C6 due to the same structural fragments observed in the 1D NMR spectra. It is proposed that the cis-olefinic carbons at C-6/C-7 in timuramide C were replaced by the trans-olefinic carbons in 5 and further supported by the large coupling constant (JH‑6/H‑7 = 15.3 Hz) observed in 5.11 In the HMBC spectrum, correlations of H-7 with C-5, C-6, C-8, C-9, and H-3 with C-1, C-4, and C-5 were observed (Figure 4). According to the above data, compound 5 was deduced as (2E,6E,8E)-N-(2-hydroxy-2-methylpropyl)2,6,8-decatrienamid-10-oic acid, named ZP-amide I. The HRESIMS data indicated that compound 6 possessed a molecular formula of C18H29NO3. 6 showed distinctly similar 1 H and 13C NMR data to lanyuamide VI,24 except that 2′hydroxy-isobutyl group in 6 was observed in place of the isobutyl moiety in lanyuamide VI. The HMBC correlations (Figure 4) from H-1′, H-3′, and H-4′ to C-2′ were observed to support this structure as shown. Compound 6 was a racemic mixture due to its optical inactivity. Thus compound 6 was proposed as (2E,4E,8Z,10E)-12-hydroxy-N-(2-hydroxy-2-methylpropyl)-2,4,8,10-teradecatetraenamide, named (±)-ZP-amide J. The HRESIMS data indicated that compounds 7 and 8 shared the same molecular formula as C16H27NO4. The 1H and 13 C NMR spectra indicated 7 as an isobutylhydroxyamide with two chiral centers. The HMBC correlations (Figure 4) of H-3 with C-1, C-4, and C-5 indicated the location of an olefinic 3414


Article

Journal of Agricultural and Food Chemistry Table 3. Cell Viability (% of Control) of PC12 Cells Pretreat with Different Concentrations of the Isolated Isobutylhydroxyamidesa corticosterone plus isobutylhydroxyamide (μmol/L) compounds 1 1a 1b 2 2a 2b 3 4 5 11 15 a

corticosterone group 44.43 43.43 44.73 41.86 46.13 44.04 46.86 43.80 38.92 42.81 46.66

± ± ± ± ± ± ± ± ± ± ±

2.34b 1.92b 1.35b 1.64b 1.55b 1.22b 4.02b 2.33b 1.48b 3.34b 1.90b

3.125 58.35 62.04 40.80 47.30 52.52 43.91 50.80 45.00 37.67 41.56 43.11

± ± ± ± ± ± ± ± ± ± ±

3.98c 3.02c 2.91 4.89d 3.19c 2.98 6.35d 3.42 0.97 0.42 4.09

6.25 60.54 64.97 42.74 49.91 55.19 42.56 55.23 42.95 39.57 42.47 53.43

± ± ± ± ± ± ± ± ± ± ±

12.5

3.78c 3.29c 2.98 1.65c 1.43c 3.42 3.20d 3.19 1.53 0.29 4.50d

61.20 65.75 39.35 47.62 63.47 44.27 51.98 44.22 39.85 48.04 53.59

± ± ± ± ± ± ± ± ± ± ±

4.69c 4.15c 5.29 1.95c 1.13c 4.81 3.99d 4.24 1.87 2.56c 5.93d

25.0 66.61 67.61 34.73 51.43 63.52 42.93 54.44 45.93 46.83 48.21 53.70

± ± ± ± ± ± ± ± ± ± ±

2.53c 3.66c 3.39 2.23c 2.13c 3.49 5.35d 3.18 2.30c 1.14c 3.46c

50.0 68.75 68.44 37.02 50.82 64.04 43.03 53.60 52.21 54.92 50.45 54.72

± ± ± ± ± ± ± ± ± ± ±

2.13c 3.90c 1.64 1.89c 1.98c 3.07 5.97d 3.52c 3.71c 1.03c 3.66c

100.0 67.90 72.47 39.54 56.06 68.06 43.84 56.05 56.28 32.78 54.17 57.38

± ± ± ± ± ± ± ± ± ± ±

2.12c 2.94c 2.24 1.39c 3.07c 5.42 3.73c 2.05c 1.12 0.47c 2.97c

Cell viability expressed as mean ± SD (n = 6). bp < 0.01 compared with control group. cp < 0.01 as compared with corticosterone group. dp < 0.05.

13;16 (2E,6E,8E)-N-(2-hydroxy-2-methylpropyl)-10-oxo-2,6,8decatrienamide, 14;26 (2E,7E,9E)-N-(2-hydroxy-2-methylpropyl)-6,11-dioxo-2,7,9-dodecatrienamide, 15;26 timuramide C, 16;6 hydroxy-β-sanshool, 17;14 bungeanool, 18;14 and isobungeanool, 19.14 Protective Effect of Isobutylhydroxyamides on Corticosterone Induced Damage on PC12 Cells. Cell viability markedly decreased in the present of corticosterone. Pretreated with different concentrations of isobutylhydroxyamide, the protective effect on PC12 cells was shown. Isobutylhydroxyamides (1, 1a, 2, 2a, 3, 4, 5, 11, 15) at a series of concentrations exhibited a protective effect (Table 3). However, the protective effect of enantiomers of the racemic mixture was different. Isobutylhydroxyamides possessing Rconfiguration showed protective activity, while those with Sconfiguration lost activity. The protective effect was found to have some correlations with intrinsic structures. Comparison of the structures of 1, 2, 3, 5, and 15 and 17, 18, and 19 indicated that shorter unsaturated aliphatic chains bearing oxygen functions, especially a carbonyl group, were more likely to have pronounced protect effect. The present results provide a basis for further exploration of this food as a new source of natural neuroprotective agents.

bond at C-2/C-3 followed by a pair of methylenes. Protons of oxygenated methines were observed to show that the HMBC correlations with C-4, C-5, C-7, and C-8 and C-5, C-6, C-8, and C-9, respectively, allowed a reasonable connection of hydroxyl groups attached to C-6 and C-7. Compared with 7, the delicate difference at the shifted signals of H-5 (δH 1.66, 1.49 in 8 and δH 1.70, 1.51 in 7), H-6 (δH 3.44 in 8 and δH 3.49 in 7), H-7 (δH 3.91 in 8 and δH 3.93 in 7), and H-8 (δH 5.56 in 8 and δH 5.62 in 7) was observed in 8 (Table 1). The relative configurations of 7 and 8 were determined as erythro and threo, respectively, due to the coupling constant between H-6 and H-7 (4.8 Hz in 7 and 5.6 Hz in 8) observed in the proton homodecoupling spectra.16,25 Therefore, 8 is the stereoisomer of 7 with different relative configurations at C-6 and C-7. Compounds 7 and 8 were racemic mixtures due to the optical inactivity. According to the above data, the planar structure of compounds 7 and 8 was determined as shown, named (±)-ZPamide K and (±)-ZP-amide L, respectively. The molecular formula C16H29NO2 for compound 9 was established from the HRESIMS data. Compound 9 had an Nisobutylhydroxyl moiety and a 2,4-diene group of a dodecadienamide due to the similar chemical shift values compared with the amide moiety in compounds 18 and 19. The only two double bonds located at C-2/C-3 and C-4/C-5 were readily assigned by the HMBC correlations of H-2 and H3 with C-1 and C-4. In the 1H−1H COSY spectrum, an extended spin system from H-2 to H-12 was observed (Figure 4). Therefore, compound 9 was (2E,4E)-N-(2-hydroxy-2methylpropyl)-2,4-dodecadienamide, named ZP-amide M. The HRESIMS data indicated the molecular formula of compound 10 as C18H31NO4. In the 1H NMR spectrum, protons of an additional methylene at δH 3.54 (H-1′′a) and 3.36 (H-1′′b) and a methyl signal at δH 1.14 (H-2′′) were observed compared with 7, which indicated that a hydroxyl was replaced by an ethoxy group. The location of the ethoxy group at C-7 was determined by the HMBC correlations from H-1′′a and H-1′′b to C-7 (Figure 4). Compared with 7, the same coupling constant between H-6 and H-7 (4.8 Hz) indicated the relative configuration of 10 to be erythro. Compound 10 was a racemic mixture due to its optical inactivity. Thus compound 10 was assigned as (2E,8E,10E)-6-hydroxy-7-ethoxy-N-(2hydroxy-2-methylpropyl)-2,8,10-dodecatrienamide, named (±)-ZP-amide N. The compounds 11−19 were the known analogues of 1−10, identified as zanthoamide, 11;19 ZP-amide E, 12;16 ZP-amide F,

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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.7b06057.

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Spectroscopic and physical data for compounds 1a−10. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*H.H.: Tel: +86 024 23986465. Fax: +86 024 23986465. Email: [email protected]. *Z.Z.: Tel: +86 010 57833290. Fax: +86 010 57833290. E-mail: [email protected]. ORCID

Zhongmei Zou: 0000-0002-8178-4788 Huiming Hua: 0000-0002-0258-3647 3415


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Journal of Agricultural and Food Chemistry Funding

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This work was financially supported by the CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-3-015) and the National Mega-Project for Innovative Drugs (2018ZX09735008). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Prof. Jinwei Ren for the measurement of the NMR data.

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