ent-Kaurane ... - ACS Publications

Chinese People,s Liberation Army 210 Hospital, Dalian 116021, People,s Republic of China. •S Supporting Information. ABSTRACT: Thirteen new ent-kaur...
2 downloads 0 Views 3MB Size
Download PDF
Article Cite This: J. Nat. Prod. 2018, 81, 1225−1234

pubs.acs.org/jnp

ent-Kaurane Diterpenoids with Neuroprotective Properties from Corn Silk (Zea mays) Xiao-Li Qi,†,‡ Ying-Ying Zhang,†,‡ Peng Zhao,†,‡ Le Zhou,†,‡ Xiao-Bo Wang,§ Xiao-Xiao Huang,*,†,‡,§ Bin Lin,*,⊥ and Shao-Jiang Song*,†,‡ †

Department of Natural Products Chemistry, ‡Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, and ⊥School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China § Chinese People’s Liberation Army 210 Hospital, Dalian 116021, People’s Republic of China S Supporting Information *

ABSTRACT: Thirteen new ent-kaurane diterpenoids, stigmaydenes A−M (1-13), together with two known compounds (14, 15), were isolated from the crude extract of corn silk (Zea mays). The structures of the compounds were confirmed by comprehensive spectroscopic analyses. The absolute configuration of compound 1 was defined by single-crystal X-ray diffraction. The absolute configurations of the compounds were also confirmed by comparison of experimental and calculated specific rotations. The compounds were evaluated for their neuroprotective effects against H2O2-induced SH-SY5Y cell injury, and compound 8 was active at 100 μM, as determined by flow cytometry (annexin V-FITC/PI staining) and Hoechst 33258 staining. The results suggested that compound 8 could protect neuronal cells from H2O2-induced injury by inhibiting apoptosis in SHSY5Y cells.

M

using an H2O2-induced SH-SY5Y cell injury model. Compound 8 had the highest activity, and the underlying protective mechanism is discussed.

aize (Zea mays Linn.), belonging to the Gramineae family, is cultivated worldwide and has the largest output of all crops, the production of which surpasses one billion tons per year.1,2 Recent studies reported the presence of a group of ent-kaurane diterpenoids, termed kauralexins, produced by pathogens and insects, in maize.3 The ent-kaurane diterpenoids are a class of typical tetracyclic diterpenoids, being considered as intermediates of the plant hormones gibberellins.4,5 These diterpenoids are found naturally in various plants and have neuroprotective, anti-inflammatory, and cytotoxic effects.6−8 Corn silk, being stigmas of maize, is regarded as the most valuable part of maize for medicinal use, and it has been reported to possess antioxidant and neuroprotective effects.9−11 However, few investigations on ent-kaurane diterpenoids isolated from corn silk have been carried out. In a search for potential bioactive compounds from corn silk, we examined an EtOH extract of corn silk, leading to the isolation of 15 entkaurane-type diterpenoids including 13 new (1−13) and two known (14, 15) compounds. On the basis of reported beneficial effects of corn silk on neuroprotection,9 these compounds were evaluated for their neuroprotective effects © 2018 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Compound 1 was obtained as colorless crystals with [α]20 D −91 (c 0.1, MeOH). Its molecular formula was found to be C20H28O4 via the NMR data and the protonated HRESIMS ion at m/z 333.2067 [M + H]+ (calcd for C20H29O4, 333.2060). The 1H NMR data showed the presence of two methyls at δH 0.99 (3H, s, H3-18) and 0.83 (3H, s, H3-20), a formyl proton at δH 9.65 (1H, s, H-19), an oxygenated methine at δH 3.64 (1H, m, H-2), an sp2 methine at δH 6.41 (1H, s, H-15), a hydroxy group at δH 4.48 (1H, s, HO-2), a carboxy group at δH 12.14 (1H, s, HOOC-17), and 17 shielded proton signals (δH 0.60− 2.77) (Table 1). Analysis of the 13C NMR data (Table 3) and the HSQC spectrum revealed 20 carbon signals. The carbon signals at δC 151.7 (C-15), 138.4 (C-16), and 165.8 (C-17) Received: December 3, 2017 Published: May 15, 2018 1225

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

Article

a hydroxy and a formyl group in 1 instead of a carboxy group in pseudolaric acid E, and had an ent-kaurane diterpenoid skeleton. In the HMBC spectrum, correlations of the methyl protons at δH 0.96 (H3-18) with C-3/C-4/C-5/C-19 and of H320 (δH 0.83) with C-1/C-5/C-9/C-10 were observed. The correlations of an olefinic proton (H-15) with C-8/C-13/C-14/ C-16/C-17 were also observed. In addition, a formyl group was shown to be located at C-4 (δC 49.2) based on the HMBC correlations from H-19 (δH 9.65) to C-3 and C-4. A hydroxy group was located at C-2, based on the HMBC correlations from H-1 to C-2/C-10/C-20 and from H-3 to C-1/C-2/C-4/ C-5 (Figure 1). Thus, the 2D structure of 1 was established. The relative configuration of 1 was determined by analyzing its NOESY data (Figure 2). The β-orientation of HO-2 was indicated by the correlation of H-2 with H3-20α, while the association between H2-14 and H3-20α indicated the αorientation of H2C-14. Similarly, the association between H19 and H3-20α indicated the α-orientation of the 19-formyl group. A crystal of compound 1 obtained from MeOH was subjected to single-crystal X-ray diffraction with Cu Kα radiation to confirm the absolute configuration, and its ORTEP drawing is depicted in Figure 3. In addition, its absolute configuration was reconfirmed by comparison of its

indicated the presence of an α,β-unsaturated carboxylic acid moiety. These data indicated that 1 was similar to the known12 ent-kaur-15-en-17,18-dioic acid (pseudolaric acid E), except for

Table 1. 1H NMR Spectroscopic Data (400 MHz, J in Hz) of Compounds 1−7e 1a

position 1α 1β 2α 2β 3α 3β 5β 6a 6b 7a 7b 9β 11a 11b 12a 12b 13α 14α 14β 15 16 17 18 19a 19b 20 OCH3 1-OH 2-OH 3-OH 4-OH 12-OH 19-OH

2a

3a

1.99, o 0.60, t (11.8) 3.64, m

2.04, dd, (12.0, 3.2) 0.61, t (12.0) 3.84, m

2.04, o 0.57, t (11.5) 3.66, m

2.21, 0.86, 1.15, 1.83, 1.64, 1.67, 1.63, 1.06, 1.55, 1.46, 1.48, 1.44, 2.77, 2.04, 1.39, 6.41,

2.25, 0.89, 1.04, 1.61, 1.77, 1.57, 1.41, 1.04, 1.55, 1.46, 1.45, 1.45, 2.77, 2.00, 1.39, 6.39,

2.00, 0.67, 0.84, 1.25, 1.57, 1.61, 1.54, 1.04, 1.48, 1.48, 1.48, 1.48, 2.76, 2.03, 1.34, 6.38,

dd (12.0, 3.6) t (12.0) m m o o o d (6.2) o o o o m d (10.4) dd (10.4, 4.7) s

dd (12.0, 2.6) t (12.0) m oc md o o m o o o o m d (10.5) dd (10.5, 4.7) s

12.14, s 0.99, s 9.65, s

12.10, s 1.16, s

0.83, s

0.79, s 3.56, s

4.48, s

4.44, s

o t (12.0) br d (12.0) br q (12.4)c od o o m o o o o m o dd (10.0, 4.6) s

12.09, s 0.89, s 3.39, d (10.5) 3.14, d (10.5) 1.00, s

4a

5a

1.66, o 0.77, td (13.2, 3.2) 1.52, qt (13.2, 3.2) 1.34, o 1.99, br d (13.1) 0.96, o 1.18, o 1.66, oc 1.80, md 1.65, o 1.65, o 1.16, o 1.40, d (16.2)c 1.68, od 3.80, br dd (5.2, 3.6)d 2.68, br t (3.6) 2.41, d (10.7) 1.15, o 6.33, s

1.98, dd (12.1, 4.3) 0.71, t (12.1) 3.45, ddd (12.1, 9.4, 4.3) 2.72, 0.80, 1.28, 1.50, 1.56, 1.67, 1.02, 1.56, 1.47, 1.49, 1.49, 2.77, 2.05, 1.36, 6.39,

0.94, s 9.70, s

12.13, s 0.92, s 0.69, s

0.97, s

1.03, s

4.25, s

d (9.4) br d (12.0) qd (12.0, 2.7)c od oc td (13.0, 3.6)d o oc od o o m d (10.5) dd (10.5, 4.8) s

4.34 or 4.43, s 4.34 or 4.43, s

6b 3.60, 2.42, 1.89, 2.30, 1.19, 1.02, 1.97, 1.88, 1.64, 1.59, 1.52, 3.62, 2.06, 1.71, 1.92, 3.25, 2.19, 1.56, 6.83,

7a

o qm (13.0) o br d (13.0) o br d (12.0) o o o o o oc md oc od m d (10.2) o s

1.18, s

1.28, s 3.70, s 5.94, s

1.70, 0.83, 1.34, 1.50,

dt (13.2, 3.2) td (13.2, 3.2) qd (13.2, 3.2) o

3.17, 0.96, 1.19, 1.79, 1.53, 1.62, 1.03, 1.51, 1.47, 1.48, 1.48, 2.77, 2.05, 1.37, 6.39,

dd (13.2, 4.7) br d (12.6) qd (12.6, 3.5)c br d (12.6)d oc td (12.6, 3.5)d m o o o o m d (10.5) dd (10.5, 4.9) s

12.03, s 0.87, s

0.93, s

4.35, s 3.94, s

4.74, s 4.25, s

Recorded in DMSO-d6. bRecorded in pyridine-d5. cThe proton was α-oriented. dThe proton was β-oriented. eThe assignments were based on HMBC and HSQC NMR experiments. Overlapping signals are expressed as o. a

1226

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

Article

Table 2. 1H NMR Spectroscopic Data (400 MHz, J in Hz) of Compounds 8−13 8a,b

position 1α 1β 2α 2β 3α 3β 5β 6α 6β 7a 7b 9β 11a 11b 12a 12b 13α 14α 14β 15a 15b 16α 17 18 19a 19b 20 OCH3 1-OH 2-OH 3-OH 4-OH 12-OH 19-OH

1.71, 0.73, 1.45, 1.45, 1.58, 1.25, 1.02, 1.14, 1.81, 1.55, 1.64, 1.06, 1.55, 1.48, 1.50, 1.48, 2.76, 2.07, 1.37, 6.40,

dt (12.4, 2.0) o o o dt (12.6, 2.0) o br d (12.6) qd (12.6, 2.4) dt (12.6, 2.4) oc td (12.6, 3.9)d m o o o o m d (10.5) dd, (10.5, 5.1) s

9a 1.68, 0.82, 1.33, 1.50,

dt (12.4, 2.8) td (12.4, 2.8) qd (12.4, 2.8) o

12.06, s 0.96, s

3.15, dd (12.4, 4.0) 0.91, br d (11.6) 1.21, qd (11.6, 5.2) 1.78, br d (11.6) 1.41, o 1.41, o 0.98, m 1.53, o 1.50, o 1.49, o 1.44, o 2.48, o 1.77, d (11.2) 1.03, dd (11.2, 4.6) 1.58, o 1.58, o 2.36, m 11.87, s 0.86, s

0.96, s

0.87, s

3.98, s

4.31, s 3.87, s

10a

11a

12a

13a

2.02, o 0.55, t (11.7) 3.65, tt (11.7, 3.2)

2.03, dd (11.8, 3.0) 0.59, t (11.8) 3.82, m

2.03, dd (11.8, 3.5) 0.59, t (11.8) 3.86, m

2.33, br d (13.2) 2.01, d (13.2)

2.00, o 0.65, t (11.7) 0.80, br d (11.8) 1.27, o 1.58, o 1.42, o 1.42, o 0.99, m 1.58, o 1.51, o 1.50, o 1.44, o 2.49, o 1.75, d (11.2) 1.00, dd (11.2, 5.0) 1.57, o 1.57, o 2.36, m 11.91, s 0.87, s 3.40, d (10.6) 3.12, d (10.6) 0.94, s

2.24, dd (12.0, 3.0) 0.87, t (12.0) 1.00, o 1.60, o 1.78, br d (13.2) 1.43, o 1.43, o 0.98, o 1.56, o 1.53, o 1.44, o 1.44, o 2.51, o 1.73, d (11.7) 1.04, dd (11.7, 4.7) 1.57, o 1.57, o 2.36, m 11.92, s 1.15, s

2.21, dd (11.8, 3.5) 0.80, d (11.8) 0.94, br d (11.6) 1.65, o 1.75, o 1.42, o 1.42, o 0.98, br d (6.4) 1.56, o 1.54, o 1.45, o 1.45, o 2.51, o 1.74, d (11.2) 1.05, dd (11.2, 4.5) 1.57, o 1.57, o 2.36, m 11.99, s 1.13, s 11.99, s

2.40, 2.02, 1.50, 1.50, 1.68, 1.37, 1.48, 1.25, 1.54, 1.46, 1.44, 1.50, 2.50, 1.70, 1.03, 1.59, 1.57, 2.36,

br d (12.8) d (12.8) o o o oc od d (6.4) o o oc od o o o o o m

0.72, s 3.55, s

0.84, s

0.99, 3.28, 3.17, 0.90,

s d (10.6) d (10.6) s

4.23, s

4.42, s

4.37, s

4.23, s

4.43, s

Recorded in DMSO-d6. The assignments were based on HMBC and HSQC NMR experiments. Recorded at 600 MHz. The proton was αoriented. dThe proton was β-oriented. Overlapping signals are expressed as o.

a

b

c

group at C-2. The NOESY correlations of H3-20 with H-2 and CH3O-19 indicated that HO-2 and Me-18 were β-oriented. The absolute configuration was confirmed by experimental and calculated specific rotations. The calculated specific rotation value was −75, while the experimental value was −97, hence consistent with the ent-kaurane type. Therefore, the structure of stigmaydene B (2) was identified as 2β-hydroxy-ent-kaur15(16)-en-17,19-dioic acid 19-methyl ester. Compound 3 was obtained as a white powder, and its molecular formula was established as C20H30O5 from its HRESIMS data. Analysis of the NMR data suggested that the structure of 3 was similar to compound 1, except that the formyl group (δC 205.9, δH 9.65) at C-19 was replaced by a hydroxymethyl group (δC 63.5, δH 3.39 and 3.14) in 3, which was verified by the correlations from H2-19 to C-3/C-4/C-18. The β-orientation of HO-2 was derived from the NOESY crosspeak of H-2 with H3-20. The correlation of H2-19 with H3-20 suggested a β-orientation of Me-18 and an α-orientation of HOCH2-19. The calculated specific rotation of the ent-kaurane type was −55, consistent with the experimental value of −45. Therefore, the structure of stigmaydene C (3) was defined as 2β,19-dihydroxy-ent-kaur-15(16)-en-17-oic acid.

experimental and calculated specific rotations. The specific rotation was calculated by density functional theory (DFT) at the B3LYP/6-311++G(2d,p) level with the CPCM solvation model, where MeOH was used as the solvent to match the experimental conditions. There were two possibilities for the absolute configuration, in which the calculated specific rotation value of the ent-kaurane type was −62, while the kaurane type was +62. The experimental value of −91 was consistent with the ent-kaurane type. Taken together, the structure of stigmaydene A (1) was defined as 2β-hydroxy-19-oxo-entkaur-15(16)-en-17-oic acid. Compound 2 was isolated as colorless needles, and the molecular formula was established as C21H30O5 from its HRESIMS data. The 1H NMR and 13C NMR spectra of 2 were similar to those of stigmaydene A (1), except for replacement of the formyl group by a methoxycarbonyl group. The connectivity of the ester carbonyl at C-19 was suggested by the HMBC correlation between H3-18 (δH 1.16) and C-19 (δC 176.8). Additionally, the location of the methoxy group was indicated by the HMBC correlation of the methoxy protons (δH 3.56) with the C-19 carbonyl group. The HMBC correlations of C-2 (δC 61.9) with H2-1 (δH 0.61, 2.04) and H2-3 (δH 0.89, 2.25) suggested the location of the hydroxy 1227

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

Article

Table 3. 13C NMR Spectroscopic Data (100 MHz) of Compounds 1−13

a

position

1a

2a

3a

4a

5a

6b

7a

8a

9a

10a

11a

12a

13a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OCH3

48.6 61.4 43.0 49.2 54.7 17.8 37.6 49.6 44.5 40.2 18.2 24.9 39.8 43.0 151.7 138.5 165.8 23.7 205.9 16.9

49.5 61.9 46.7 44.3 55.0 19.9 37.5 49.7 45.0 40.3 18.3 25.0 39.8 42.8 151.7 138.4 165.8 28.2 176.8 16.0 51.2

49.7 61.9 44.9 40.0 55.3 18.5 38.3 49.8 46.3 40.5 18.3 25.1 40.0 42.8 152.2 138.4 165.8 27.7 63.5 19.0

38.8 17.8 33.7 47.9 55.2 17.8 37.5 48.9 46.3 37.5 27.4 63.6 47.5 36.1 153.1 139.0 166.0 23.8 205.9 14.0

47.1 67.1 82.2 39.0 54.2 18.3 37.7 49.6 45.9 39.0 18.2 25.1 39.8 42.8 152.2 138.4 165.8 28.9 16.9 18.6

82.8 31.5 37.2 44.4 55.9 21.5 40.0 52.1 47.7 46.3 21.9 36.9 42.0 44.9 152.1 141.4

38.0 27.4 78.1 73.9 54.4 17.1 37.4 49.7 39.1 40.5 18.3 25.1 39.9 42.8 152.1 138.4 165.9 17.0

39.4 19.2 42.5 70.0 56.4 17.1 37.3 49.9 46.0 39.4 18.2 25.1 40.0 42.8 152.3 138.3 165.9 22.7

38.0 27.5 78.3 73.9 54.9 19.1 40.4 44.4 55.1 38.9 18.2 30.8 44.9 37.7 44.1 40.5

16.9

16.9

16.8

49.8 62.3 44.9 40.0 55.9 20.3 41.2 44.4 55.6 40.2 18.1 30.7 44.8 37.7 44.1 40.6 177.9 27.9 63.5 18.8

49.6 62.0 46.7 44.2 55.5 21.9 40.6 44.4 54.2 40.0 18.2 30.6 44.7 37.6 43.9 40.3 177.8 28.3 177.0 16.1 51.2

49.8 62.2 47.0 44.2 55.5 22.0 40.7 44.5 54.3 40.3 18.2 30.6 44.7 37.7 43.9 40.4 177.9 28.6 178.5 16.4

55.5 211.0 49.5 43.1 54.5 20.8 40.6 44.5 54.2 43.4 18.1 30.5 44.9 37.6 43.8 40.4 178.1 27.4 63.9 19.0

29.3 178.3 13.0 51.9

17.1

Recorded in DMSO-d6. bRecorded in pyridine-d5.

Figure 1. HMBC correlations (H → C) of compounds 1, 7, and 10.

Compound 4 was isolated as a white, amorphous powder, and its molecular formula (C20H28O4) was the same as 1 with seven indices of hydrogen deficiency, which was determined by the NMR and HRESIMS data (m/z 355.1879 [M + Na]+, calcd for C20H28O 4Na, 355.1880). Compared with those of compound 1, 1H and 13C NMR data of 4 showed the same molecular framework as 1, except that the oxygenated carbon at δC 63.6 in 4 had slightly shifted downfield in contrast with that of 1 at δC 61.4. The hydroxy group at C-12 was determined from the HMBC cross-peaks of H-12 with C-9/C-14. Previous studies suggested that H-12β of 12α-hydroxy-ent-isokauren-19oic acid and H-12α of platensimycin ML10 with HO-12β had coupling constants of 5.2 and 3.6 Hz and of 11.9, 6.3, and 3.5 Hz, respectively.13,14 The ROESY correlations of H-12α with

Figure 3. ORTEP drawing of compound 1.

H3-20 and H-14b for platensimycin ML10 were not observed in the NOESY spectrum of 4. Thus, based on coupling constants of H-12 (δH 3.80, br dd, J = 5.2, 3.6 Hz) with H-11 and H-13 and in combination with the NOESY data, the hydroxy group at C-12 for 4 was α-oriented. The absolute configuration of 4 was in agreement with the ent-kaurane type because the

Figure 2. Key NOESY correlations of compounds 1, 7, and 10. 1228

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

Article

experimental specific rotation of −70 was consistent with the calculated value of −26 for the ent-kaurane type. Thus, the structure of stigmaydene D (4) was defined as 12α-hydroxy-19oxo-ent-kaur-15(16)-en-17-oic acid. Compound 5 was similar to 1 by comparison of their NMR data, with the distinctions being the presence of an extra hydroxy group and replacement of the formyl group in 1 by a methyl group. The hydroxy groups were located at C-2 and C3, respectively, which was confirmed by the HMBC correlations of H2-1 with C-2/C-3/C-20, of H-3 with C-2/C-18/C-19, and of H3-18/H3-19 with C-3/C-4/C-5. These correlations also determined that two of the methyl groups (δH 0.92, H3-18; 0.69, H3-19) were connected to C-4. The HO-3 was assigned to be α-oriented by the NOESY correlations of H-3 with H-5/H318. The β-orientation of HO-2 was determined by the crosspeaks of H-2 with H3-19/H3-20. The absolute configuration was assigned by comparison of the experimental and calculated specific rotations, and the result showed that the experimental value of −93 was consistent with the calculated value of −46 for the ent-kaurane type. Thus, the structure of stigmaydene E (5) was defined as 2β,3α-dihydroxy-ent-kaur-15(16)-en-17-oic acid. Compound 6 was isolated as a white, amorphous powder with the same molecular formula C21H30O5 as stigmaydene B (2). The 1H and 13C NMR data, along with the HSQC data, showed a methoxy group [δH 3.70 (3H, s), δC 51.9], an oxygenated methine [δH 3.60 (1H, m), δC 82.8], and an olefinic proton [δH 6.83 (1H, s), δC 152.1]. These findings showed that the structure of 6 was similar to 2, except for the position of the hydroxy group. This conclusion was supported by the HMBC correlations of H3-20 with C-1/C-5/C-9/C-10 as well as of H-1 with C-9/C-10/C-20. Although the carboxy carbon signal was absent in the 13C NMR spectrum, the HRESIMS data and the similar NMR spectra of 6 and 2 indicated the presence of a carboxy group at C-17. In addition, the α-orientation of HO-1 was derived from the NOESY cross-peaks of H-1 with H-5/H9. Similarly, the NOESY cross-peak of CH3O-19 with Me-20 indicated that the Me-18 was β-oriented but the CH3OOC-19 was α-oriented. The experimental specific rotation value of 6 was −102, while the calculated value was −45, suggesting an ent-kaurane type. Thus, the structure of stigmaydene F (6) was defined as 1α-hydroxy-ent-kaur-15(16)-en-17,19-dioic acid 19methyl ester. Compound 7 had a molecular formula of C19H28O4 from the sodium adduct ion at m/z 343.1882 (calcd for C19H28O4Na, 343.1880) in the HRESIMS data. Its 1D NMR data, along with the HSQC spectrum, showed 19 carbon signals including 14 shielded carbons, two oxygenated carbons, two olefinic carbons, and a hydroxycarbonyl carbon. These data were comparable to those of stigmaydene E (5), except for the absence of a methyl group at C-19, suggesting that 7 possessed a 19-nor-ent-kaurane skeleton. The hydroxy substituents were attached at C-3 and C4, as supported by the HMBC correlations of H-3 with C-1/C4/C-5/C-18. The α-orientation of HO-3 was based on the NOESY cross-peak between H-3 and H-5. The NOESY correlation of H-3 with H3-18 showed that HO-4 was αoriented (Figure 2). Its absolute configuration was defined by comparison of the experimental specific rotation (−101) with the calculated value (−30). Therefore, the structure of stigmaydene G (7) was defined as 3α,4α-dihydroxy-19-norent-kaur-15(16)-en-17-oic acid. The HRESIMS data of compound 8 was 16 mass units less than that of 7, indicating the absence of an oxygen atom in 8. Detailed analysis of its NMR data suggested that 8 was an

analogue of 7 with a 19-nor-ent-kaurane skeleton. They differed in the absence of a C-3 substituent in 8. This deduction was confirmed by the HMBC correlations from H3-18 to C-3/C-4/ C-5. The HO-4 group had an α-orientation by analysis of the NOESY correlations of H-6 (δH 1.81) with H-5/H3-18, as shown in Figure S79 (Supporting Information). The absolute configuration was assigned based on comparison between experimental and calculated specific rotations, in which the experimental value was −56, consistent with the calculated value of −39. Hence, the structure of stigmaydene H (8) was defined as 4α-hydroxy-19-nor-ent-kaur-15(16)-en-17-oic acid. Compound 9 was isolated as a white powder. By comparison with those of 7, the NMR data of 9 revealed that they were analogues, with the difference being the reduction of the Δ15(16) double bond in 9. The NOESY correlations of 9 suggested that the orientations of the hydroxy groups were identical to those of 7. Similar to 6, the hydroxycarbonyl carbon signal was absent in the 13C NMR spectrum, but the 1H NMR (δH 11.87, s, HOOC-17) and HRESIMS data indicated the presence of a carboxy group at C-17. The β-orientation of HOOC-17 was supported by the NOESY cross-peak of H-16 with H-14 (δH 1.03). The experimental specific rotation showed a negative value (−82), similar to the calculated value of −89 for the entkaurane type. Thus, the structure of stigmaydene I (9) was defined as (16S)-3α,4α-dihydroxy-19-nor-ent-kaur-17-oic acid. Compound 10 was obtained as a white powder with [α]20 D −67 (c 0.1, MeOH). Its molecular formula was assigned as C20H32O4 according to the HRESIMS ion at m/z 359.2199 [M + Na]+ (calcd for C20H32O4Na, 359.2193), indicating five indices of hydrogen deficiency. Comparison of the NMR data of 10 with those of 3 suggested that they possessed similar structures, but the signals of the Δ15(16) bond in 3 were replaced by a tertiary carbon (δC 40.6) and a methylene carbon (δC 44.1) in 10. This conclusion was verified by the HMBC crosspeaks from H-13 to C-12/C-14/C-15/C-16/C-17 as well as the HRESIMS data that were two mass units more than that of 3. The NOESY cross-peaks of H3-20 with H-2/H2-19 and of H-16 with H-13/H-14 (δH 1.00) implied β-orientations of HO-2 and HOOC-17, while HOCH2-19 had an α-orientation (Figure 2). The absolute configuration was similar to those of ent-kauranes by comparison of the experimental specific rotation (−67) with the calculated value (−81). Therefore, the structure of stigmaydene J (10) was defined as (16S)-2β,19-dihydroxy-entkaur-17-oic acid. Compound 11, isolated as a white powder, had a similar structure to stigmaydene B (2) by comparison of their 1H and 13 C NMR data, except that the olefinic carbons (δC 151.7, C15; 138.4, C-16) in 2 were replaced by two sp3 carbons (δC 43.9, C-15; 40.3, C-16) in 11. Analysis of the NOESY data of 11 showed the same orientations of the substituents as 2, except for the carboxy group at C-17. The NOESY cross-peak of H-16/H-14 (δH 1.04) indicated a β-orientation of the carboxy group. The calculated specific rotation value was −107 for the ent-kaurane type, consistent with the experimental value of −81. Thus, the structure of stigmaydene K (11) was defined as (16S)-2β-hydroxy-ent-kaur-17,19-dioic acid 19-methyl ester. Compound 12 was isolated as a white powder, and its molecular formula was C20H30O5 from its NMR and HRESIMS data with an [M + Na]+ ion at m/z 373.1997 (calcd for C20H30O5Na, 373.1985). The 1H and 13C NMR data of 12 were similar to those of 11, except for replacement of the methoxycarbonyl by a hydroxycarbonyl group in 11. This was verified by the HRESIMS data showing that 12 had 14 mass 1229

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

Article

Figure 4. Putative biosynthetic pathways toward the formation of compounds 1−15.

cross-peaks of H-16 with H-14 (δH 1.03) as well as of H2-19 with H3-20 indicated that the HOOC-17 group was β-oriented, while the HOCH2-19 group was α-oriented. The absolute configuration was assigned because its experimental value of −104 was consistent with the calculated value of −112. Hence, the structure of stigmaydene M (13) was defined as (16S)-19hydroxy-2-oxo-ent-kaur-17-oic acid. By comparison of their experimental and reported NMR data, the known compounds were identified as 16β,17dihydroxy-ent-kaur-19-oic acid (14)5 and ent-kaur-15(16)-en17,19-dioic acid (15).15 Compound 14 was isolated from the Zea genus for the first time. Corn silk was found to express ZmAn2 (the maize ent-CDP synthase) induced by infection or

units less than 11. The HMBC and NOESY correlations of 12 indicated that the substituents had the same positions and orientations as those of 11. The absolute configuration of 12 was assigned by comparison of the calculated specific rotation value of −107 with the experimental value of −101. Thus, the structrue of stigmaydene L (12) was defined as (16S)-2βhydroxy-ent-kaur-17,19-dioic acid. Compound 13 was obtained as a white powder. The 1H and 13 C NMR data (Tables 2, 3) were similar to those of 10. The difference was that the oxymethine group (δH 3.65; δC 62.3) in 10 was replaced by a ketocarbonyl group (δC 211.0) in 13, which was confirmed by the HMBC correlations of H2-1 with C-2/C-9/C-20 and of H2-3 with C-2/C-18/C-19. The NOESY 1230

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

Article

insects, resulting in the synthesis of the intermediate entCDP.16,17 Subsequently, the KSs (ent-kaurane synthases) mediated the production of ent-kaur-15-ene, ent-kaur-16-ene, and ent-kaur-16β-ol, which was followed by oxidation to give ent-kaur-15-ene-17-oic acid, ent-kaur-17-oic acid, and 14.18 The first two compounds were oxidized at C-17 step by step via the ent-kaurane oxidases (multiple reactive cytochrome P450) to obtain products with a −CH2OH, −CHO, or −COOH group at C-17.18,19 These intermediates also led to the formation of a series of hydroxylated ent-kaurane diterpenoids, involving oxidation, methylation, and decarboxylation reactions. Accordingly, a putative biosynthetic pathway (Figure 4) was proposed. Compounds 1−15 were evaluated for their potential neuroprotective activities against H2O2-induced SH-SY5Y cell damage, using the MTT assay. H2O2 induces cytotoxicity in SH-SY5Y cells, which provides a suitable model system for studying neuronal cell death caused by oxidative stress.20 According to Figure 5, compounds 4, 5, 8, 9, and 13 exhibited

compounds exhibited no neuroprotective effects at the tested concentrations. Compound 8, exhibiting the highest neuronal protection among these compounds, was selected to study the effect on the morphology of H2O2-induced SH-SY5Y cells at concentrations of 25, 50, and 100 μM. As shown in Figure 6A, in contrast with the control group, SH-SY5Y cells treated with H2O2 alone exhibited significant cell shrinkage, and the morphologic changes were improved by treatment with 8 in a concentration-dependent manner. After staining with Hoechst 33258, a nuclear dye that binds to DNA, H2O2induced SH-SY5Y cells showed significant chromatin condensation, compared with the control group, suggesting the induction of apoptosis. When treated with 8, the number of fragmented nuclei in H2O2-induced SH-SY5Y cells decreased, indicating that 8 protected SH-SY5Y cells from apoptosis (Figure 6B). In addition, flow cytometry (annexin V-FITC/ propidium iodide (PI) staining) was used to further explore the protection of 8 from H2O2-induced apoptosis in SH-SY5Y cells (Figure 7). The apoptosis ratios were significantly reduced from 21.4% to 12.51, 9.27, and 8.52%, when treated with 8 at concentrations of 25, 50, and 100 μM, respectively, showing that compound 8 could attenuate H2O2-induced apoptosis in SH-SY5Y cells.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using an Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) at 20 °C. Melting points were obtained on an X-4 micro melting-point apparatus (Gongyi City Kerui Instrument Company, China) and are uncorrected. 1D and 2D NMR experiments were recorded on Bruker ARX-400 and AV-600 spectrometers (Bruker Company, Billerica, MA, USA) in DMSO-d6 or pyridine-d5. Chemical shifts are expressed as δ values (ppm) relative to tetramethylsilane. HRESIMS data were acquired on a Bruker micro QTOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA). HPLC separations were achieved using the following instruments as well as reversed-phase chromotography columns: Shimadzu LC-10AD pump system equipped with a Shimadzu RID-20A refractive index detector (Shimadzu Corporation, Tokyo, Japan), YMC Pack ODS-A column (250 × 10 mm, 5 μm, YMC Company, Kyoto, Japan), or Waters preparative C18 OBD column (150 × 19 mm, 10 μm, Waters Corporation, MA, USA). Analytical HPLC was performed on a Waters 1525 binary pump equipped with a Waters 2707 autosampler and a Waters 2489 ultraviolet−visible detector (Waters Corporation). Separations (column chromatography) were carried out on silica gel (100−200 or 200−300 mesh) and polyamide (100−200 mesh)

Figure 5. Neuroprotective effects of compounds 1−15 against H2O2induced injury in SH-SY5Y cells. Data (cell viability) was expressed as means ± SEM. Trolox was used as a positive control. ***p < 0.001 versus model, **p < 0.01 versus model, *p < 0.05 versus model.

moderate activities against H2O2-induced SH-SY5Y cell injury, improving cell viabilities by more than 10% at 100 μM compared with the model group. However, the other

Figure 6. Morphological changes of H2O2-induced SH-SY5Y cells generated by stigmaydene H (8). SH-SY5Y cells were (A) detected and (B) stained by Hoechst 33258 by treatment with H2O2 (200 μM) with or without 8 (25, 50, and 100 μM), respectively. 1231

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

Article

Figure 7. Analysis of compound 8 against apoptosis in H2O2-induced SH-SY5Y cells through flow cytometry. The cells were treated with or without 8 for 24 h at concentrations of 25, 50, and 100 μM, respectively. The apoptosis ratio is shown as means ± SD; ##p < 0.01 versus model, #p < 0.05 versus model, *p < 0.05 versus control. tR 48 min). Fraction B4-3-6 contained crystals, which were repeatedly washed with MeOH, resulting in the isolation of 1 (30.0 mg, tR 33 min). Fractions B4-3-3 and B4-3-7 were purified using semipreparative reversed-phase HPLC with MeCN−H 2O (11:89, 26:74, v/v respectively) to yield 13 (11.7 mg, tR 154 min) and 4 (11.7 mg, tR 40 min). Fraction B4-5 (1.8 g) was subjected to preparative reversedphase HPLC (MeOH−H2O, 46:52, v/v) to afford 12 fractions. Fraction B4-5-12 contained crystals that were repeatedly washed with MeOH, leading to the isolation of 5 (8.0 mg, tR 75 min). Fraction B45-6 was subjected to semipreparative reversed-phase HPLC with MeCN−H2O (14:86, v/v) to afford 7 (21.1 mg, tR 70 min). Fraction B4-5-1 was separated using semipreparative reversed-phase HPLC (MeCN−H2O, 13:87, v/v), followed by washing the crystals with MeOH, to afford 10 (6.1 mg, tR 36 min) and 12 (3.3 mg, tR 46 min). Fraction B4-5-7 was subjected to semipreparative reversed-phase HPLC (MeCN−H2O, 16:84, v/v), followed by washing the crystals with MeOH, to afford 9 (2.1 mg, tR 63 min). Fraction B5 (2.1 g) was chromatographed on a silica gel column eluted with a gradient of CH2Cl2−MeOH (from 10:0 to 3:1, v/v) to afford 12 fractions. Fractions B5-3 and B5-12 were separated by semipreparative reversed-phase HPLC with MeCN−H2O (25:75, v/v) to yield fractions B5-3-1 (tR 45 min), B5-3-2 (tR 66 min), and B5-12-1 (tR 26 min). Fractions B5-3-1 and B5-3-2 were purified by washing the crystals with MeOH to afford 8 (1.4 mg) and 15 (2.8 mg), respectively. Fraction B5-12-1 was purified by washing the crystals with MeOH to afford 14 (1.5 mg). Stigmaydene A (1): colorless, block crystals (MeOH); mp 189− 1 13 190 °C; [α]20 D −91 (c 0.1, MeOH); H and C NMR data, see Table 1 and Table 3; HRESIMS m/z 333.2067 [M + H]+ (calcd for C20H29O4, 333.2060). Stigmaydene B (2): colorless, needles (MeOH); mp 146−147 °C; 1 13 [α]20 D −97 (c 0.1, MeOH); H and C NMR data, see Table 1 and Table 3; HRESIMS m/z 385.1982 [M + Na]+ (calcd for C21H30O5Na, 385.1985). Stigmaydene C (3): white, amorphous powder; [α]20 D −45 (c 0.1, MeOH); 1H and 13C NMR data, see Table 1 and Table 3; HRESIMS m/z 357.2040 [M + Na]+ (calcd for C20H30O4Na, 357.2036).

(Qingdao Marine Chemical Inc., Qingdao, China) and octadecyl silica gel (50 μm, YMC Company). All reagents used were analytical or HPLC grade and were purchased from Tianjin Damao Chemical Company (Tianjin, China). TLC was performed on silica gel GF254 on glass plates (Qingdao Marine Chemical Inc., Qingdao, China) and eluted with different solvent systems. Spots were monitored by heating the silica gel plates after spraying with anisaldehyde−H2SO4 reagent. Plant Material. The stigmas of Z. mays were collected from Anhui Province, People’s Republic of China, in June 2016 and identified by Professor Jincai Lu, Department of Pharmaceutical Botany, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (no. 20160607) has been deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University. Extraction and Isolation. The air-dried stigmas of Z. mays (50 kg) were extracted three times with 70% aqueous EtOH, and the EtOH extract obtained was subsequently concentrated to 25 L under reduced pressure. The aqueous solution was partitioned with n-BuOH three times to obtain an n-BuOH fraction (400 g). The n-BuOH fraction was separated via vacuum flash chromatography on a silica gel column (200−300 mesh) using a gradient eluent of CH2Cl2−MeOH from 10:0 to 1:1 to afford four fractions (I, II, III, IV). Fraction II was subjected to vacuum flash chromatography over polyamides with EtOH−H2O (0, 30%, 60%, 90%, v/v) as eluent to provide three fractions (A−C). Fraction B was further chromatographed on an open-type ODS column eluted with MeOH−H2O (0, 30%, 50%, 70%, 90%, v/v) to give fractions B1−B6. Fraction B4 (6.9 g) was fractioned over silica gel eluted with CH2Cl2−MeOH of increasing polarity to obtain 10 fractions, B4-1−B4-10. The crystals appearing in fraction B4-6 were repeatedly washed with MeOH to give 3 (30.0 mg). Fraction B4-2 (482.0 mg) was separated using preparative reversedphase HPLC with MeOH−H2O (47:53, v/v) to yield 11 fractions, B42-1−B4-2-11. Fractions B4-2-5 and B4-2-11 were separated using semipreparative reversed-phase HPLC under isocratic elution with MeCN−H2O (16:84, 22:78, v/v, respectively) to afford 11 (6.3 mg, tR 90 min) and 6 (7.7 mg, tR 138 min). Fraction B4-3 (718.0 mg) was chromatographed on preparative reversed-phase C18 HPLC eluted with MeOH−H2O (47:53, v/v) to afford six fractions and 2 (20.0 mg, 1232

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

■

Stigmaydene D (4): white, amorphous powder; [α]20 D −70 (c 0.1, MeOH); 1H and 13C NMR data, see Table 1 and Table 3; HRESIMS m/z 355.1879 [M + Na]+ (calcd for C20H28O4Na, 355.1880). Stigmaydiene E (5): white, needles (MeOH); mp 236−237 °C; 1 13 [α]20 D −93 (c 0.1, MeOH); H and C NMR data, see Table 1 and Table 3; HRESIMS m/z 357.2040 [M + Na]+ (calcd for C20H30O4Na, 357.2036). Stigmaydene F (6): white, amorphous powder; [α]20 D −102 (c 0.1, MeOH); 1H and 13C NMR data, see Table 1 and Table 3; HRESIMS m/z 385.1985 [M + Na]+ (calcd for C21H30O5Na, 385.1985). Stigmaydene G (7): white, amorphous powder; [α]20 D −101 (c 0.1, MeOH); 1H and 13C NMR data, see Table 1 and Table 3; HRESIMS m/z 343.1882 [M + Na]+ (calcd for C19H28O4Na, 343.1880). Stigmaydene H (8): white, amorphous powder; [α]20 D −56 (c 0.1, MeOH); 1H and 13C NMR data, see Table 2 and Table 3; HRESIMS m/z 327.1934 [M + Na]+ (calcd for C19H28O3Na, 327.1931). Stigmaydene I (9): white, amorphous powder; [α]20 D −82 (c 0.1, MeOH); 1H and 13C NMR data, see Table 2 and Table 3; HRESIMS m/z 345.2036 [M + Na]+ (calcd for C19H30O4Na, 345.2036). Stigmaydene J (10): white, amorphous powder; [α]20 D −67 (c 0.1, MeOH); 1H and 13C NMR data, see Table 2 and Table 3; HRESIMS m/z 359.2199 [M + Na]+ (calcd for C20H32O4Na, 359.2193). Stigmaydene K (11): white, amorphous powder; [α]20 D −81 (c 0.1, MeOH); 1H and 13C NMR data, see Table 2 and Table 3; HRESIMS m/z 387.2140 [M + Na]+ (calcd for C21H32O5Na, 387.2142). Stigmaydene L (12): white, amorphous powder; [α]20 D −101 (c 0.1, MeOH); 1H and 13C NMR data, see Table 2 and Table 3; HRESIMS m/z 373.1997 [M + Na]+ (calcd for C20H30O5Na, 373.1985). Stigmaydene M (13): white, amorphous powder; [α]20 D −104 (c 0.1, MeOH); 1H and 13C NMR data, see Table 2 and Table 3; HRESIMS m/z 357.2035 [M + Na]+ (calcd for C20H30O4Na, 357.2036). X-ray Crystallography Analysis of 1. The crystals of compound 1 was obtained from MeOH, and the absolute configuration was determined using the data collected on a Bruker APEX-2 CCD diffractometer with Cu Kα (radiation λ = 1.541 78) at T = 296(2) K. The structure was solved by the SHELXS method and refined based on full-matrix least-squares on F2 using SHELXL-2014/7 (Sheldrick 2014). Crystallographic data for 2β-hydroxy-19-oxo-ent-kaur-15(16)en-17-oic acid (1): blocks, colorless, crystal size 0.30 × 0.22 × 0.10 mm, C20H28O4, M = 332.42, orthorhombic space group, P212121, a = 8.8766(2) Å, b = 10.9773(3) Å, c = 18.1040(5) Å, V = 1764.07(8) Å3, Z = 4, Dcalcd = 1.252 g/m3, 11 107 collected reflections (4.71° ≤ θ ≤ 58.92°), μ(Cu Kα) = 0.688 mm−1, R1 = 0.0642 and wR2 = 0.1545 for I ≥ 2σ(I), S = 1.115, Flack parameter = 0.1(2), the Hooft parameter is 0.1(2) for 1053 Bijvoet pairs. The crystallographic data of stigmaydene A (1) have been deposited with the Cambridge Crystallographic Data Centre (CCDC number 1566097). The data can be obtained, free of charge, from the Cambridge Crystallographic Data Centre (http:// www.ccdc.cam.ac.uk/data_request/cif). Neuroprotective Activity Assay. The neuroprotective effects of compounds 1−15 against H2O2-induced neurotoxicity in SH-SY5Y cells were examined adopting the same procedures as reported previously.21 Morphological Analyses of SH-SY5Y Cells. In the absence or presence of compound 8 (25, 50, and 100 μM), the SH-SY5Y cells were incubated with H2O2 for 4 h, and the morphologic changes were observed by a phase contrast microscope (Leica, Nussloch, Germany) or a fluorescence microscope (Olympus, Tokyo, Japan) after staining with Hoechst 33258 (Sigma Chemical, MO, USA). Annexin V-FITC/PI Staining Assay. SH-SY5Y cells were seeded in six-well culture plates, treated with or without compound 8 (25, 50, and 100 μM) for 1 h, and incubated with H2O2 for another 4 h. The collected cells were washed twice with PBS and stained with annexin V-FITC and PI at room temperature for 15 min. Finally, the apoptotic ratio of cells was checked by flow cytometry.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01017. NMR and HRESIMS spectra for new compounds 1−13 and figures of the HMBC and NOESY correlations of compounds 2−6, 8, 9, and 11−13 (PDF) Crystallographic data of 1 (CIF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail (X.-X. Huang): [email protected]. *E-mail (B. Lin): [email protected]. *E-mail (S.-J. Song): [email protected]. ORCID

Shao-Jiang Song: 0000-0002-9074-2467 Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was financially supported by the Project of Innovation Team (LT2015027) of Liaoning of P.R. China. REFERENCES

(1) Sabiu, S.; O’Neill, F. H.; Ashafa, A. O. T. J. Ethnopharmacol. 2016, 183, 1−8. (2) Food and Agriculture Organization of the United Nations. http://faostat.fao.org/site/567/default.aspx (accessed Nov 20, 2017). (3) Fu, J. Y.; Ren, F.; Lu, X.; Mao, H. J.; Xu, M. M.; Degenhardt, J.; Peters, R. J.; Wang, Q. Plant Physiol. 2016, 170, 742−751. (4) Hansen, N. L.; Heskes, A. M.; Hamberger, B.; Olsen, C. E.; Hallstr, B. M.; Andersen-Ranberg, J.; Hamberger, B. Plant J. 2017, 89, 429−441. (5) Yang, Y. L.; Chang, F. R.; Wu, C. C.; Wang, W. Y.; Wu, Y. C. J. Nat. Prod. 2002, 65, 1462−1467. (6) Kim, H. S.; Lim, J. Y.; Sul, D.; Hwang, B. Y.; Won, T. J.; Hwang, K. W.; Park, S. Y. Eur. J. Pharmacol. 2009, 622, 25−31. (7) Wang, R.; Liu, Y. Q.; Ha, W.; Shi, Y. P.; Hwang, T. L.; Huang, G. J.; Wu, T. S.; Lee, K. H. Bioorg. Med. Chem. Lett. 2014, 24, 3944−3947. (8) Qiu, M. S.; Yang, B.; Cao, D.; Zhu, J. P.; Jin, J.; Chen, Y. Y.; Zhou, L.; Luo, X.; Zhao, Z. X. Phytochem. Lett. 2016, 16, 156−162. (9) Choi, D. J.; Kim, S. L.; Choi, J. W.; Park, Y. I. Life Sci. 2014, 109, 57−64. (10) Ren, S. C.; Qiao, Q. Q.; Ding, X. L. Czech J. Food Sci. 2013, 31, 148−155. (11) Wang, G. Q.; Xu, T.; Bu, X. M.; Liu, B. Y. Inflammation 2012, 35, 822−827. (12) Li, Z. L.; Chen, K.; Pan, D. J. Acta Chim. Sinica. 1989, 47, 258− 261. (13) Jakupovic, J.; Schuster, A.; Bohlmann, F.; Ganzer, U.; King, R. M.; Robinson, H. Phytochemistry 1989, 28, 543−551. (14) Rudolf, J. D.; Dong, L. B.; Manoogian, K.; Shen, B. J. Am. Chem. Soc. 2016, 138, 16711−16721. (15) Gao, F.; Wang, H. P.; Mabry, T. J. Phytochemistry 1987, 26, 779−781. (16) Schmelz, E. A.; Kaplan, F.; Huffaker, A.; Dafoe, N. J.; Vaughan, M. M.; Ni, X. Z.; Rocca, J. R.; Alborn, H. T.; Teal, P. E. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5455−5460. (17) Schmelz, E. A.; Huffaker, A.; Sims, J. W.; Christensen, S. A.; Lu, X.; Okada, K.; Peters, R. J. Plant J. 2014, 79, 659−678. (18) Mafu, S.; Jia, M.; Zi, J. C.; Morrone, D.; Wu, Y. S.; Xu, M. M.; Hillwig, M. L.; Peters, R. J. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 2526−2531. 1233

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234

Journal of Natural Products

Article

(19) Mao, H. J.; Liu, J.; Ren, F.; Peters, R. J.; Wang, Q. Phytochemistry 2016, 121, 4−10. (20) Tian, X.; Guo, L. P.; Hu, X. L.; Huang, J.; Fan, Y. H.; Ren, T. S.; Zhao, Q. C. Cell. Mol. Neurobiol. 2015, 35, 335−344. (21) Huang, S. W.; Qiao, J. W.; Sun, X.; Gao, P. Y.; Li, L. Z.; Liu, Q. B.; Sun, B.; Wu, D. L.; Song, S. J. J. Funct. Foods 2016, 24, 183−195.

1234

DOI: 10.1021/acs.jnatprod.7b01017 J. Nat. Prod. 2018, 81, 1225−1234