Sesqui- and Diterpenoids from the Radix of ... - ACS Publications

Article Cite This: J. Nat. Prod. 2017, 80, 3093−3102

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Sesqui- and Diterpenoids from the Radix of Curcuma aromatica Shengjuan Dong, Baocai Li, Weifeng Dai, Dong Wang, Yi Qin, and Mi Zhang* Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new sesquiterpenoids (1−8), two new diterpenoids (9 and 10), and 17 known sesqui- and diterpenoids (11−27) were isolated from the radix of Curcuma aromatica. Among these compounds, 1 is an unprecedented guaiane with unique cyclopropane and furan functionalities, and 9 is the first atisane diterpenoid isolated from a Curcuma species. Their 2D and 3D structures were established using HRESIMS and spectroscopic methods, including ECD and IECD data. The antioxidant activities of compounds 1−27 were evaluated based on their ability to protect PC12 cells against H2O2-induced damage, with 1, 2, 5−8, 11, 12, 14, 16, 18, and 19 exhibiting notable antioxidant effects on oxidative injury induced by H2O2. damage in PC12 cells, with 1, 2, 5−8, 11, 12, 14, 16, 18, and 19 demonstrating significant antioxidant effects.

Curcuma, one of the genera belonging to the Zingiberaceae family, includes approximately 50 species distributed in southeastern Asia and northern Australia and 12 species in the southern area of China.1 Most of the 12 species found in southern China have been used in traditional Chinese medicine to treat qi stagnation and blood stasis.1 To date, more than 140 compounds, primarily curcumins2 and sesquiterpenes,1,3,4 have been isolated from Curcuma plants, and most of these molecules have shown a broad range of biological activities such as antioxidant,5 anti-inflammatory,6,7 neuroprotective,8 and antitumor effects.5 Curcuma aromatica Salisb. is a perennial herb in the Curcuma genus, and its radix is used in traditional Chinese medicine to treat depression and qi and blood stasis.5 In traditional Chinese medicine, one of the clinical features of qi and blood stasis is memory impairment, which is also a symptom of depression.9,10 Meanwhile, modern pharmacology research has shown that oxidative stress is a major cause of depression.11,12 This development has led to speculation that the radix of C. aromatica is an excellent source of antioxidant components. In a previous study, a 75% ethanol extract from C. aromatica was shown to generate more significant antioxidant activity against H2O2-induced damage in PC12 cells than the 95% ethanol and methanol extracts. To identify the active constituents, the phytochemistry of the 75% ethanol extract of C. aromatica was investigated in this study. Consequently, 27 terpenoids, including 10 new compounds (1−10) and 17 known analogues (11−27), were isolated from its petroleum ether and EtOAc fractions. Notably, 1 is an unprecedented guaiane with a new carbon skeleton, and 9 is the first atisane diterpenoid isolated from a Curcuma species. Compounds 1−27 were tested for their antioxidant activities in the model of H2O2-induced © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The 75% ethanol extract from the radix of C. aromatica was suspended in H2O and successively partitioned with petroleum ether, EtOAc, and n-BuOH. The first two fractions were repeatedly subjected to column chromatography over silica gel, MCI gel, ODS gel, and semipreparative HPLC to obtain 10 new terpenoids (1−10) and 17 known terpenoids (11−27). The known compounds were identified as zedoarolide B (11),13 zedoalactone C (12),13 phaeocaulisin B (13),14 4βhydroxy-5β-H-guai-1(10),7(11),8-trien-12,8-olide (14),13 gweicurculactone (15),15 curdionolide A (16),16 curdionolide B (17),16 7,8-seco-9(10),11(12)-guaiadien-8,5-olide (18),17 4α,8β-dihydroxy-5α-(H)-eudesm-7(11)-en-8,12-olide (19),18 isogermafurenolide (20),19 phacadinane D (21),20 zedoarondiol (22),13 wenyujinin B (23),21 procurcumenol (24),13 curcumadiol (25),22 (E)-3-hydroxylabda-8(17),12-dien-16,15olide (26),23 and labda-8(17),13(14)-dien-15,16-olide (27)24 by comparing their HRESIMS and NMR data with the corresponding data in the cited references, and their structures are shown in Figure S1 in the Supporting Information. Compound 1 was obtained as a colorless powder whose molecular formula was deduced as C15H20O3 by HRESIMS (m/ z 271.1310 [M + Na]+, calcd C15H20O3Na 271.1310), indicating that it had six indices of hydrogen deficiency. The 1 H NMR data (Table 1) revealed four methyl groups at δH 1.25, 1.34, 1.40 (each 3H, each s) and 1.89 (3H, d, 1.4 Hz) and methylene protons at δH 1.06 (1H, d, 5.2 Hz) and δH 1.28 (1H, Received: November 28, 2016 Published: December 13, 2017 3093

DOI: 10.1021/acs.jnatprod.6b01100 J. Nat. Prod. 2017, 80, 3093−3102

Journal of Natural Products

Article

Table 1. 1H NMR Data of Compounds 1−10 position

1a

2b

1

2

3

4 5 6

1.63, m 2.13, dd (12.2, 5.0) 1.73, dd (13.8, 5.0) 2.09, m

1.06, d (5.2)

3a

4b

2.53, m

2.73, d (7.8)

2.69, m 2.84, m

1.25, m 1.68, m

1.90, m 1.94, m

2.02, m

1.06, m

2.25, m

1.75, m 2.02, m

1.60, dd (12.6, 7.0) 2.33, m 2.04, m

7.27, s

2.36, d (13.8) 2.54, m

1.28, d (5.2)

1.65, dd (13.8, 8.6) 2.54, dd (13.8, 8.6)

5b

6b

7b

8b

3.21, d (8.1)

2.99, dd (11.2, 7.3)

4.88, d (10.0)

2.44, m 2.51, m

1.85, m 1.90, m

1.24, m 2.18, m

1.78, m

1.57, m

1.51, m

2.00, d (16.0) 2.24, dd (16.0, 8.0) 1.68, d (16.0)

1.79, m

2.30, m 2.02, m

1.89, m 2.25, m

2.09, m 2.43, m

2.66, d (15.7) 3.06, d (15.7)

2.85, d (16.2) 2.89, d (16.2)

2.18, d (12.7) 1.99, td (12.7, 1.7) 2.76, dd (12.7, 1.7)

9

5.57, d (1.4)

10 11

7.03, brs

5.81, brs

5.32, dd (16.8, 8.6) 3.18, d (16.8) 2.58, dt (16.8, 1.6)

2.70, d (14.7) 2.56, m

3.45, m

12

1.34, s

13

1.25, s

14

15

1.37, m

1.81, m 1.44, m 1.79, m

1.46, m 1.35, m 1.51, m

1.03, m

1.11, m

2.20, m

1.31, m

3.24, d (16.0)

1.08, m 1.70, m

0.93, m 1.19, m

3.38, d (16.0)

1.86, m

1.52, m

1.26, t (4.0) 1.57, td (13.4, 4.0)

1.08, m 2.10, dt (12.6, 3.2)

1.48, brs

1.61, m

2.58, m

4.29, t (3.5)

1.06, d (2.7)

2.22, m

2.03, m 2.46, m 6.64, m

5.68, d (1.2)

2.97. d (16.0) 2.23, d (16.0)

1.19, d (6.9) 1.19, d (6.9)

1.83, s 1.95, s

1.85, s

1.83, s

1.87, s

1.08, d (2.7)

1.95, s

1.15, m

1.40, s

1.46, s

0.94, d (6.8)

0.84, d (7.4)

1.39, s

0.84, d (8.1)

1.94, d (1.2)

1.06, d (8.0)

1.94, m 1.97, m

1.89, d (1.4)

2.23, s

1.93, s

1.21, s

1.82, s

1.25, s

1.05, d (6.9)

1.89, s

1.88, m 2.90, s

2.88, m 4.29, m 4.31, m

3.10, m 3.43, m 1.20, t (8.0)

1.23, s

3.28, dd (11.0, 5.0) 3.42, m 0.84, s 0.76, s 0.76, s

16 17

18 19 20 HO-1 HO-4 HO-5 HO-8

3.62, s

10a

1.13, m

7

8

9b

1.30, s 0.98, s

2.80, m

5.15, s 1.42, brs 4.43, s 3.04, br s

4.39, dd (7.7, 5.0)

a Recorded in DMSO-d6. bRecorded in CDCl3. Compounds 1, 2, 3, 4, 6, 8, and 10 were measured at 800 MHz, whereas 5, 7, and 9 were measured at 500 MHz.

d, 5.2 Hz). An olefinic proton at δH 5.57 (1H, d, 1.4 Hz) indicated that a trisubstituted double bond was present in 1. Its 13 C NMR (Table 2) and DEPT data displayed 15 signals, including a ketocarbonyl (δC 193.8), two olefinic carbons (δC 122.7, 161.3), three oxygenated tertiary carbons (δC 75.6, 79.0, 86.0), and four methyl carbons (δC 18.7, 23.7, 24.0, 25.2). Based on HSQC analysis, all protons were unambiguously assigned to their corresponding carbons, except for a proton singlet at δH 5.15 (1H, s), which indicated the presence of a hydroxy group and was further elucidated as an α-oriented hydroxy group located at C-1 based on the HMBC correlation from HO-1 to C-1/C-2 and the ROESY correlation from Me-

14 to HO-1 (Figures 2 and 3). Other HMBC correlations from H-9 and C-1/C-7/C-15 and from H-15 to C-1/C-9/C-10 confirmed the presence of an α,β-unsaturated carbonyl moiety in 1, occupying two of the six indices of deficiency. The remaining indices of hydrogen deficiency indicated that 1 was a tetracyclic sesquiterpenoid. Analysis of the NMR data revealed an ether bond connecting C-4 and C-11, as indicated by the chemical shifts of C-4 and C-11 and the molecular formula of 1. The presence of a cyclopropane ring at C-5 and C-7 was deduced via the HMBC correlations from H-6 to C-1/C-4/C5/C-7/C-8/C-11, the isolated methylene signals, and the indices of hydrogen deficiency. The absolute configuration of 1 3094



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Table 2. 13C NMR Data of Compounds 1−10 position

1a

2b

3a

4b

5b

6b

7b

8b

9b

10a

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

79.0 39.0 35.0 86.0 41.8 17.2 37.2 193.8 122.7 161.3 75.6 23.7 24.0 25.2 18.7

144.6 31.8 40.0 84.5 148.9 128.1 159.6 185.9 140.2 144.6 30.3 22.7 22.7 27.4 24.7

58.3 31.7 32.2 40.3 82.7 38.1 130.3 194.5 128.2 155.6 146.2 23.5 23.2 13.5 27.5

45.5 25.0 33.0 42.2 91.4 46.5 160.9 80.0 33.2 81.1 125.3 174.1 8.9 14.8 22.0

141.3 29.2 39.3 81.7 52.7 27.7 161.7 104.1 43.9 123.4 124.0 171.9 8.3 24.4 23.5

43.0 24.9 32.9 42.6 92.6 31.8 156.1 106.3 80.3 81.2 128.4 171.9 8.9 14.3 19.2

44.7 30.0 31.1 42.7 90.7 43.2 212.3 164.1 113.4 161.4 42.4 17.9 18.2 22.8 11.9

133.9 27.4 36.2 48.0 208.8 40.1 152.5 109.2 49.6 130.7 132.1 171.1 9.7 18.6 16.7 59.2 15.3

41.5 19.1 37.9 43.7 56.8 21.2 34.9 45.6 51.1 37.0 76.9 42.0 38.0 38.6 81.9 84.9 20.5 29.3 182.8 18.0

40.0 18.2 41.4 38.3 55.7 19.7 37.4 73.6 60.8 39.9 25.3 144.0 123.3 24.7 65.3 171.1 62.3 33.3 21.4 15.5

a

Recorded in DMSO-d6. bRecorded in CDCl3. Compounds 1, 2, 3, 4, 6, 8, and 10 were measured at 200 MHz, whereas 5, 7, and 9 were measured at 125 MHz.

Figure 1. Structures of compounds 1−10.

elucidated as shown in Figure 1. Aromaticane A is an unprecedented guaiane-type sesquiterpenoid with a cyclopropane moiety and a furanyl unit. Compound 2 was obtained as a pale yellow oil with the molecular formula C15H20O2 as determined by HRESIMS (m/z 255.1371 [M + Na]+, calcd for C15H20O2Na 255.1361). The 1H NMR data (Table 1) of 2 showed two methyl singlets [δH 1.46, 2.23 (each 3H, each s)], two methyl doublets [δH 1.19 (6H, d, 6.9 Hz)], and two olefinic protons [δH 7.03 (1H, brs), δH 7.27 (1H, s)], while its 13C NMR data (Table 2) displayed 15 carbon resonances, which were categorized as a ketocarbonyl (δC 185.9), six olefinic carbons (δC 128.1, 140.2, 144.6, 144.6, 148.9, 159.6), an oxygenated tertiary carbon (δC 84.5), an allylic methine carbon (δC 30.3), two methylene carbons (δC

was determined by comparing the experimental electronic circular dichroism (ECD) spectrum with the computed spectrum (Figure 4).25−28 The calculated ECD curve of (1R,4S,5R,7R)-1 matched the experimental spectrum. To further confirm the structure of 1, its 13C NMR data were computed and compared with its experimental 13C NMR data. The linear regression analysis results showed that the correlation coefficient (R2) between the calculated and experimental data was 0.9959 (Figure S2, Supporting Information), and that the mean absolute error (MAE) and the corrected mean absolute error (CMAE) were 7.29 and 2.79 ppm (Table S1, Supporting Information), respectively, indicating the validity of the structure established via the NMR data. Thus, the structure of aromaticane A (1) was 3095



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Figure 2. Key correlations of compounds 1−10.

Figure 3. Key ROESY correlations of compounds 1 and 3−10.

used to determine the absolute configuration of chiral tertiary alcohols.29 The IECD spectrum of 2 displayed a positive Cotton effect at 352 nm (E band), confirming the (4S) absolute configuration by application of the bulkiness rule. Thus, aromaticane B (2) was identified as (4S)-hydroxy-1(5),6(7),9(10)-guaiatrien-8-one. Compound 3 was a pale yellow oil with a molecular formula of C15H22O2, which was determined by HRESIMS (m/z 235.1698 [M + H]+, calcd for C15H23O2 235.1698). The 1H NMR data (Table 1) of 3 showed one secondary methyl at δH 0.94 (3H, d, 6.8 Hz), three methyl singlets at δH 1.83, 1.93, and 1.95 (each 3H, each s), a hydroxy proton at δH 4.43 (1H, s), and an olefinic proton at δH 5.81 (1H, brs). The 13C NMR data (Table 2) revealed the presence of 15 carbons, including a ketocarbonyl (δC 194.5), four olefinic carbons (δC 128.2, 130.3, 146.2, 155.6), and an oxygenated tertiary carbon (δC 82.7). The HMBC correlations of Me-12 and -13 with the two quaternary

31.8, 40.0), and four methyl carbons (δC 22.7, 22.7, 24.7, 27.4) according to the DEPT and HSQC data. The HMBC correlations from Me-12 to C-7/C-11/C-13 and Me-13 to C7/C-11/C-12 revealed the presence of an isopropyl group in 2. The HMBC correlations from Me-15 to C-1/C-9/C-10 (Figure 2) also confirmed that 2 contained an α,β-unsaturated carbonyl moiety. The HMBC correlations from H-6 to C-1/5/7, H-9 to C-8/10/15, H-11 to C-7/12/13, Me-14 to C-3/C-4/C-5, and Me-15 to C-1/9/10, together with the six indices of hydrogen deficiency, indicated that 2 was a 1(5),6(7),9(10)-guaiatrien-8one. Comparing the NMR data of 2 with those reported for phaeocaulisin D,14 which has an oxygenated tertiary carbon at δC 75.5 instead of a methine carbon at δC 30.3, revealed the absence of a hydroxy group at C-11 in compound 2. The absolute configuration of 2 was elucidated using the bulkiness rule for the Rh2(OCOCF3)4-induced electronic circular dichroism (IECD) data, where the E band at ca. 350 nm is 3096



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suggesting that the absolute configuration at C-8 was R14,21,30−35 based on the empirical rule for an α,β-unsaturated five-membered lactone moiety. Thus, the structure of aromaticane D (4) was defined as (5R,10R)-expoxy-(1R,8R)(H)-guaia-7(11)-en-12,8-olide. Compound 5 had a molecular formula of C15H20O4 by HRESIMS (m/z 287.1254 [M + Na]+, calcd for C15H20O4Na 287.1259) with six indices of hydrogen deficiency. Three methyl singlets at δH 1.39, 1.82, and 1.83 (each 3H, each s) were evident in the 1H NMR data (Table 1), while 15 carbon resonances were present in the 13C NMR data (Table 2): three methyl (δC 8.3, 23.5, 24.4), four methylene (δC 27.7, 29.2, 39.3, 43.9), a methine (δC 52.7), a lactone carbonyl (δC 171.9), two tetrasubstituted double bonds (δC 123.4, 161.7, 124.0, 141.3), one oxygenated tertiary carbon (δ C 81.7), and one dioxygenated secondary carbon (δC 104.1). These features were supported by the DEPT and HSQC data. The HMBC correlations from Me-13 to C-7/C-11/C-12, Me-14 to C-3/C4/C-5, Me-15 to C-1/C-9/C-10, and H-5 to C-1/C-4/C-6/C10 (Figure 2) indicated that 5 was a 4,8-dihydroxyguaia1(10),7(11)-dien-12,8-olide. The ROESY correlations of H-5/ HO-8 and HO-8/HO-4 suggested that H-5, HO-4, and HO-8 were β-oriented (Figure 3). The (8S) absolute configuration was elucidated via the characteristic Cotton effects at 221 nm (Δε +0.029) and 256 nm (Δε −0.009) in the ECD spectrum.14,21,30−35 Consequently, the structure of aromaticane E (5) was identified as (4S,8S)-dihydroxy-(5S)-(H)-guaia1(10),7(11)-dien-12,8-olide. Compound 6, a colorless oil, had a molecular formula of C15H20O5 according to the HRESIMS data (m/z 303.1201 [M + Na]+, calcd for C15H20O5Na 303.1208). The 1H NMR data of 6 (Table 1) displayed two methyl singlets (δH 1.25, 1.87) and one methyl doublet [δH 0.84, (d, 8.1 Hz)]. Comparing the 13C NMR data of 6 and 4 revealed significant similarities, except that two of the carbon signals in 6 were deshielded in 4 (δC 80.0 in 4 to δC 106.3 in 6 and δC 33.2 in 4 to δC 80.3 in 6), suggesting that those two carbons were oxidized. The HMBC correlations from H-1 to C-2/C-3/C-4/C-5/C-9, H-9 to C-1/ C-8/C-10/C-15, Me-13 to C-7/C-11/C-12, Me-14 to C-3/C4/C-5, and Me-15 to C-1/C-9/C-10 revealed that an oxygen atom linked C-5 and C-8 and that hydroxy groups were located at C-9 and C-10 (Figure 2). The 9- and 10-hydroxy groups were tentatively assigned α-orientations based on the ROESY correlations from Me-14 to H-1, H-1 to Me-15, and Me-15 to H-9 (Figure 3). The (9R, 10R) absolute configuration was assigned via Mo2(OAc)4-induced ECD (IECD) (Snatzke’s method),36 in which a negative Cotton effect was observed at 311 nm. Furthermore, the (8R) absolute configuration was deduced via the characteristic Cotton effects at 216 nm (Δε −4.35) and 242 nm (Δε +2.69) in the ECD spectrum.14,21,30−35 Therefore, aromaticane F (6) was identified as (9R,10R)-dihydroxy-(5S,8R)-expoxy-(1S,4R)-(H)-guaia7(11)-en-12,8-olide. Compound 7 was a colorless oil whose molecular formula was determined as C15H22O3 based on the HRESIMS data (m/ z 273.1470 [M + Na]+, calcd for C15H22O3Na 273.1467). In the 1 H NMR data of 7 (Table 1), four methyl doublets [δH 1.05 (3H, d, 6.9 Hz), 1.06 (3H, d, 2.7 Hz), 1.08 (3H, d, 2.7 Hz), 1.94 (3H, d, 1.2 Hz)], two methylene proton doublets [δH 2.85 (H, d, 16.2 Hz), 2.89 (H, d, 16.2 Hz)], and one olefinic proton [δH 5.68 (1H, d, 1.2 Hz] were evident. The 13C NMR data indicated the presence of 15 signals (Table 2), including a ketocarbonyl, an ester carbonyl, and two olefinic carbons. The

Figure 4. Comparison of the experimental ECD spectrum of (−)-1 with the calculated ECD spectrum of (1R,4S,5R,7R)-1 in MeOH. σ = 0.25 eV; shift = −12 nm.

olefinic carbons (C-7 and C-11) indicated that an isopropylidene group was present in 3. The HMBC correlations from Me-15 to C-1/C-9/C-10 (Figure 2) confirmed the presence of an α,β-unsaturated carbonyl moiety, which occupied two of the five indices of hydrogen deficiency in 3. Combined with analysis of the HMBC correlations from HO to C-5, H-1 to C2/C-5/C-10, H-9 to C-8/C-10/C-15, Me-12 to C-7/C-11/C13, Me-13 to C-7/C-11/C-12, and Me-14 to C-3/C-4/C-5 and the remaining indices of hydrogen deficiency, these data revealed that 3 was a 5-hydroxy-7(11),9(10)-guaiadien-8-one. The relative configuration of 3 was determined by a ROESY experiment, where the correlations of H-1/Me-14 and H-1/ HO-5 revealed that H-1/HO-5/Me-14 were α-oriented. Furthermore, the (5R) absolute configuration was again determined by the Rh2(OCOCF3)4-induced ECD method via the negative Cotton effect at 352 nm.29 Hence, the structure of aromaticane C (3) was defined as (5R)-hydroxy-7(11),9(10)guaiadien-8-one. Compound 4 has a molecular formula of C15H20O3 according to the HRESIMS (m/z 271.1310 [M + Na]+, calcd for C15H20O3Na 271.1310). Two methyl singlets at δH 1.85 and 1.21 and a secondary methyl doublet at δH 0.84 (d, 7.4 Hz) appeared in the 1H NMR data of 4 (Table 1). The 13C NMR data (Table 2) displayed 15 carbon resonances, which were categorized as three methyl carbons (δC 8.9, 14.8, 22.0), four methylene carbons (δC 25.0, 33.0, 33.2, 46.5), three methine carbons (δC 42.2, 45.5, 80.0), a lactone carbonyl carbon (δC 174.1), a tetrasubstituted double bond (δC 125.3, 160.9), and two oxygenated tertiary carbons (δC 81.1, 91.4) by HSQC. In the HMBC spectrum, the correlations from H-1 to C-3/C-4/C5/C-10, H-4 to C-1/C-3/C-5/C-14, H-8 to C-6/C-7/C-11, Me-13 to C-7/C-11/C-12, Me-14 to C-3/C-4/C-5, and Me-15 to C-1/C-9/C-10 (Figure 2) revealed that 4 had a guaiane skeleton with a 5,10-expoxy-7(11)-en-12,8-olide structural moiety, which was further supported by the six indices of hydrogen deficiency in 4. ROESY correlations from Me-14 to H-1 and from H-1 to Me-15 indicated the β-orientation of H1/Me-14/Me-15 and the α-orientation of the oxygen bridging between C-5 and C-10 (Figure 3). The absolute configuration of 4 was determined by the characteristic Cotton effects at 215 (Δε −4.16) and 240 nm (Δε +1.78) in the ECD spectrum, 3097



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suggested that the ethoxy group was positioned at C-8 (Figure 2). ROESY correlations of H-4 with H-2 (δH 2.24), H-3 (δH 1.68), and H-6 (δH 3.38) indicated these protons to be βoriented. Furthermore, the ROESY correlation of H-6 (δH 3.38) with H-4/H-16 (δH 3.10)/Me-17 suggested the βorientation of the ethoxy group (Figure 3). Similar to compounds 4−6, the (8S) absolute configuration was confirmed by the Cotton effects at 225 nm (Δε +2.791) and 253 nm (Δε −3.453) in the ECD spectrum.14,21,30−35 Hence, the structure of aromaticane H (8) was defined as depicted in Figure 1. Compound 9 was a white, amorphous powder and had a molecular formula of C20H30O4 with six indices of hydrogen deficiency, as deduced from the HRESIMS data (m/z 333.2054 [M − H]+, calcd for C20H29O4 333.2066). The 1H NMR data of 9 (Table 1) indicated the presence of three methyl groups [δH 0.98, 1.23, 1.30 (each 3H, each s)] and two oxymethine protons [δH 2.90 (1H, s), δH 4.29 (1H, t, 3.5 Hz)]. The 13C NMR data (Table 2) revealed 20 carbon resonances, including three methyl (δC 18.0, 20.5, 29.3), seven methylene (δC 19.1, 21.2, 34.9, 37.9, 38.0, 38.6, 41.5), three methine (δC 42.0, 51.1, 56.8), two oxymethine (δC 76.9, 81.9), an oxygenated tertiary carbon (δC 84.9), three quaternary carbons (δC 37.0, 43.7, 45.6), and a hydroxy carbonyl carbon (δC 182.8), as confirmed by DEPT and HSQC data. The 1H−1H COSY correlations of H-11 with H-9/H-12 and H-6 with H-5/H-7 and HMBC correlations of Me-17 with C-12/C-15/C-16, Me-18 with C-3/ C-4/C-5, Me-20 with C-1/C-5/C-9/C-10, and H-5 with C-19 revealed that 9 had an atisan-19-oic acid diterpenoid skeleton (Figure 2). Comparing the 13C NMR chemical shift of C-15 in 9 with ent-15α-hydroxytrachyloban-19-oic acid38 revealed that the chemical shift of C-15 (δC 81.9) in 9 was slightly shielded compared with the corresponding shift in ent-15α-hydroxytrachyloban-19-oic acid (δC 82.7). This shift, together with the atisan-19-oic acid diterpenoid skeleton accounting for five of the six indices of hydrogen deficiency of 9, indicated that an oxirane moiety was located at C-15/C-16, which was further elucidated by the HMBC correlations of Me-17 with C-15/C16 and H-15 with C-16/C-17. Furthermore, the HMBC correlations of H-11 with C-8/C-12/C-16 and ROESY correlation of H-5 with H-9/H-18 and H-9 with H-11 corroborated that a β-hydroxy group was linked at C-11, thus establishing the α-orientations of H-5/H-9/H-11/Me-18. Other key ROESY correlations of H-12 with Me-17, H-15 with Me-17, and Me-17 with Me-20 suggested the βorientations of H-12, H-15, and Me-17/Me-20 and the αorientation of the oxirane group (Figure 3). The (11R) absolute configuration was determined by Rh2(O2CCF3)4induced ECD data,25 in which a negative Cotton effect was observed at 363 nm, in contrast to 2. Hence, the structure of aromaticane I (9) was established as shown. The molecular formula of 10 was C20H32O4, as deduced from the HRESIMS data (m/z 359.2201 [M + Na]+, calcd for C20H32O4Na 359.2198). Its 1H NMR data (Table 1) indicated three methyl singlets [δH 0.76 (6H, s), δH 0.84, (3H, s)], an olefinic proton [δH 6.64 (1H, m)], and two oxymethylene protons [δH 3.28 (1H, dd, 11.0, 5.0 Hz), δH 3.42 (1H, m)], whereas the 13C NMR and HSQC data (Table 2) revealed 20 carbon resonances, including three methyl (δC 15.5, 21.4, 33.3), seven methylene (δC 18.2, 19.7, 24.7, 25.3, 37.4, 40.0, 41.4), two oxymethylene (δC 62.3, 65.3), and three methine carbons (δC 55.7, 60.8, 144.0), a carbonyl (δC 171.1), a quaternary olefinic carbon (δC 123.3), an oxygenated tertiary carbon (δC

HMBC correlations of H-9 with C-1/C-8/C-10 and Me-14 with C-1/C-9/C-10 indicated that an α,β-unsaturated carbonyl moiety was present in 7. Further, an isobutyryl group linked at C-5 via a methylene function was elucidated by the HMBC correlations of H-6 with C-1/C-4/C-5/C-7/C-11, H-11 with C-7/C-12, and Me-12/C-13 with C-7/C-11 (Figure 2). Comparing the above spectroscopic characteristics with those of curcuzedoalide17 revealed that the main difference between the two was the absence of the NMR signals assigned to the Δ11(12) double bond in curcuzedoalide17 compared with 7. Instead, methyl and methylene signals were observed in 7, suggesting that reduction of the Δ11(12) double bond had occurred. The relative configuration of 7 was confirmed by a ROESY experiment in which correlation of H-4 with H-1 was present, revealing that those protons were cofacial (Figure 3). The (1S, 4S, 5S) absolute configuration of 7 was determined by comparing the calculated and experimental ECD data (Figure 5), as described for 1.25−28 In this way, the skeleton of 7 was

Figure 5. Comparison of the experimental ECD spectrum of (+)-7 with the calculated ECD spectra for (1S,4S,5S)-7 in MeOH. σ = 0.3 eV; shift = 8 nm.

identified as 7,8-seco-guaia-9(10)-en-8,5-olide, and this compound was named aromaticane G, a rare 7,8-seco-guaiane-type sesquiterpenoid isolated from a natural source. Compound 8 had a molecular formula of C17H24O4, as deduced by the HRESIMS data (m/z 315.1564 [M + Na]+, calcd for C17H24O4Na 315.1572). Four methyl groups were shown in the 1H NMR data (Table 1), and 17 carbons were displayed in the 13C NMR spectrum (Table 2), which were categorized as four methyl, five methylene, and two methine carbons, a dioxygenated secondary carbon, three quaternary olefinic carbons, a lactone carbonyl, and a ketocarbonyl, as confirmed by the DEPT and HSQC data. HMBC correlations of Me-13 with C-7/C-11/C-12, Me-14 with C-3/C-4/C-5, Me15 with C-1/C-9/C-10, Me-17 with C-16, H-4 with C-3/C-5/ C-14, and H-16 with C-8/C-15 indicated that 8 was a germacrane sesquiterpenoid with an α,β-unsaturated carbonyl residue (Figure 2). Its NMR data resembled those of souliene A37 except for the presence of an ethoxy group in 8, which was supported by an HMBC correlation from a methyl triplet [δH 1.20 (3H, t, 8.0 Hz)] to an oxymethylene carbon (δC 59.2). Furthermore, an HMBC correlation from one of the oxymethylene protons [δH 3.43 (1H, m)] to C-8 (δC 109.2) 3098



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Figure 6. Putative biosynthesis pathway for 1−8.

73.6), and two quaternary carbons (δC 38.3, 39.9). Combined analysis of the above NMR data and the HMBC correlations of Me-18 with C-3/C-5/C-19/C-20, Me-19 with C-1/C-4/C-5, Me-20 with C-4/C-5/C-9/C-10, and H-15 with C-13/C-14/C16 indicated that 10 was a labdane-type diterpenoid with an α,β-unsaturated carbonyl moiety and structurally similar to (E)labda-8(17),12-dien-15,16-olide (Figure 2).39 A comparison of the NMR data of 10 with those of (E)-labda-8(17),12-dien15,16-olide39 revealed that the exocyclic double bond at C-8 in (E)-labda-8(17),12-dien-15,16-olide39 was oxidized to a vicinal diol in 10, which was verified by the HMBC correlations from H-7 to C-8/C-17 and H-11 to C-8, and the HRESIMS data indicated the presence of hydroxy groups at C-8 and C-17. The relative configuration of 10 was established by the ROESY correlations of H-5/H-9/Me-18 and Me-19/Me-20, indicating that H-5/H-9/Me-18 were β-oriented and Me-19/Me-20/HO8 were α-oriented (Figure 3). The (8R) absolute configuration was determined via the negative Cotton effect at 315 nm in the Mo2(OAc)4-induced ECD spectrum.36 Therefore, aromaticane J (10) was identified as (E)-labda-(8R,17)-dihydroxy-12-en15(16)-olide. To date, hundreds of guaianes have been isolated from natural materials by chemists, most of which originated from the Compositae family.40−42 In terms of the structural types of those guaianes, many of them are 12,8-olide or 12,6-olideguaiane types, and a few of them are 12,5-olide-, 1,2-seco, 4,5seco, 7,8-seco, or 1,10-seco-guaiane types.13,43−47 In the current study, the majority of the isolates were guaiane-type sesquiterpenoids, including an unprecedented guaiane sesquiterpenoid with cyclopropyl and furanyl groups (1) and eight guaianes with 12,8-olide moieties (4, 5, 6, and 11−15). From the viewpoint of biosynthesis, classic guaianes (e.g., 2 and 3), guaian-12,8-olides (e.g., 4−6), and a 7,8-seco-guaiane (e.g., 7) are presumably formed by oxidation from farnesyl pyrophosphate (FPP), which is the common precursor to a variety of sesquiterpenoids via intramolecular cyclization at different positions, such as guaiane and germacrane (Figure 6). However, during the biosynthesis process of 1, the carbocation, as a key active intermediate, participated in the formation of the cyclopropyl moiety in 1.

Considering the traditional medicinal function and antioxidant effects of the constituents found in the genus Curcuma, the antioxidant activities of some extracts from the radix of C. aromatica were evaluated using a model of H2O2-induced oxidative stress, which is an effective cell damage model for screening antioxidants and oxidative stress inhibitors.48,49 This study showed that in this model the 75% EtOH extract of C. aromatica exhibited a stronger antioxidant activity than did other extracts (Table 3). To further study the antioxidant Table 3. Antioxidant Effects of the Extracts of C. aromatica on H2O2-Induced Damage in PC12 Cellsa viability % 1.0 μg/mL control model VE (100 μM) 95% EtOH extract 75% EtOH extract MeOH extract

100 66.68 83.41 80.33 91.87 74.07

± ± ± ± ±

1.52d 0.59b 0.73b 1.28c 0.89

10.0 μg/mL

50.0 μg/mL

91.94 ± 0.97c 102.13 ± 2.65c 80.10 ± 1.87b

60.58 ± 2.01 75.10 ± 2.22 76.86 ± 0.86

a Data are expressed as the mean ± SD (n = 6). bp < 0.05. cp < 0.01 vs model. dp < 0.01 vs control.

agents in the active extract, isolates 1−27 were also tested for their antioxidant activities using the same model, and vitamin E (VE) served as the reference. The results showed that 1, 2, 5− 8, 11, 12, 14, 16, 18, and 19 at different concentrations (1.0, 5.0, or 10.0 μM) could increase H2O2-damaged PC12 cell viability significantly (p < 0.001), indicating their antioxidant effects (Table 4).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 spectropolarimeter. UV spectra were measured on a Shimadzu UV-2401PC spectrophotometer. ECD spectra were determined on an APP Chirascan spectropolarimeter. 1H and 13C NMR spectra were measured on Bruker Ascend TM-500 MHz and Bruker Ascend TM-800 MHz instruments, respectively. HRESIMS spectra were recorded with an Agilent UPLC-Q-TOF 3099



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column with CH2Cl2−MeOH (49:1, 29:1, 19:1, 9:1, and 6:1) to afford Frs. PG1−PG4. Fr. PG2 (40.8 mg) was further separated by semipreparative HPLC with MeOH−H2O (55:45) to obtain 17 (1.0 mg), 7 (1.3 mg), and 23 (2.0 mg). Fr. PG3 (103.5 mg) was chromatographed using a Sephadex LH-20 column and further separated by semipreparative HPLC with MeOH−H2O (55:45) to obtain 3 (0.62 mg), 6 (0.78 mg), and 20 (1.1 mg). Fr. PH (2.3 g) was purified by a silica gel column with CH2Cl2−MeOH (39:1, 29:1, 19:1, and 9:1) to afford Frs. PH1−PH3. Fr. PH2 (41.8 mg) was further purified by semipreparative HPLC with MeCN−H2O (40:60) to yield 2 (5.1 mg), 24 (2.0 mg), and 25 (1.6 mg). Fr. PI (128.9 mg) was purified by a column of Sephadex LH-20 to afford Frs. PI1−PI3. Fr. PI1 (31.7 mg) was further purified by semipreparative HPLC with MeOH−H2O (65:35) to obtain 9 (1.4 mg), 22 (1.8 mg), and 11 (1.2 mg). Fr. PI2 (37.2 mg) was purified by semipreparative HPLC with MeOH−H2O (55:45) to obtain 26 (1.6 mg) and 27 (1.1 mg). Fr. PI3 (21.2 mg) was separated by semipreparative HPLC with MeOH−H2O (50:50) to yield 10 (4.2 mg). The EtOAc extract (20.0 g) was subjected to a column of silica gel eluted with petroleum ether−EtOAc (1:0, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 0:1 v/v) to obtain six fractions (EA−EF), which were combined using TLC analysis. Fr. EC was separated by an open column of ODS (MeOH−H2O, 20−100%). Fr. EC2 (22.3 mg) was further purified by semipreparative HPLC with MeCN−H2O (35:65) to yield 21 (1.1 mg) and 16 (1.8 mg). Fr. ED (5.5 g) was separated by a silica gel column (CHCl3−MeOH, 1:1) followed by Sep-Pak C18 cartridges (MeOH−H2O, 20−80%) and purified by semipreparative HPLC with MeOH−H2O (40:60) to obtain 4 (2.1 mg), 13 (1.4 mg), 15 (4.8 mg), and 19 (1.0 mg). Fr. EE (3.8 g) was separated by an open column of MCI (MeOH−H2O, 20−100%). Fr. EE2 (36.8 mg) was further purified by semipreparative HPLC eluted with MeOH−H2O (40:60) to afford 12 (3.8 mg). Aromaticane A (1): colorless powder; [α]25D −5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (3.59) nm; ECD (c 0.6 × 10−3, MeOH) λmax (Δε) 206 (−0.90), 218 (+1.24) nm; 1H NMR (800 MHz, DMSO-d6), in Table 1; 13C NMR (200 MHz, DMSO-d6), in Table 2; HRESIMS m/z 271.1310 [M + Na]+ (calcd for C15H20O3Na, 271.1310). Aromaticane B (2): pale yellow oil; [α]25D −8 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 238 (4.40), 325 (3.88) nm; Rh2(OCOCF3)4induced ECD (c 2.2 × 10−3, CHCl3) λmax (Δε) 352 (+0.04) nm; 1H NMR (800 MHz, CDCl3), Table 1; 13C NMR (200 MHz, CDCl3), Table 2; HRESIMS m/z 255.1371 [M + Na]+ (calcd for C15H20O2Na 255.1361). Aromaticane C (3): pale yellow oil; [α]25D +9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (3.64), 252 (3.27) nm; Rh2(OCOCF3)4induced ECD (c 2.1 × 10−3, CHCl3) λmax (Δε) 352 (−0.04) nm; 1H NMR (800 MHz, DMSO-d6), Table 1; 13C NMR (200 MHz, DMSOd6), Table 2; HRESIMS m/z 235.1698 [M + H]+ (calcd for C15H22O2H 235.1698). Aromaticane D (4): pale yellow oil; [α]25D +33 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.41) nm; ECD (c 0.8 × 10−3, MeOH) λmax (Δε) 215 (−4.16), 240 (+1.78) nm; 1H NMR (800 MHz, CDCl3), Table 1; 13C NMR (200 MHz, CDCl3), Table 2; HRESIMS m/z 271.1310 [M + Na]+ (calcd for C15H20O3Na 271.1310). Aromaticane E (5): colorless oil; [α]25D +30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.01) nm; ECD (c 0.7 × 10−3, MeOH) λmax (Δε) 221 (+0.029), 256 (−0.009) nm; 1H NMR (500 MHz, CDCl3), Table 1; 13C NMR (125 MHz, CDCl3), Table 2; HRESIMS m/z 287.1254 [M + Na]+ (calcd for C15H20O4Na 287.1259). Aromaticane F (6): colorless oil; [α]25D +11 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 214 (3.37) nm; Mo2(OAc)4-induced ECD (c 1.8 × 10−3, DMSO) λmax (Δε) 311 (−0.68) nm; ECD (c 0.8 × 10−3, MeOH) λmax (Δε) 216 (−4.35), 242 (+2.69) nm; 1H NMR (800 MHz, CDCl3), Table 1; 13C NMR (200 MHz, CDCl3), Table 2; HRESIMS m/z 303.1201 [M + Na]+ (calcd for C15H20O5Na 303.1208). Aromaticane G (7): colorless oil; [α]25D +22 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (3.75) nm; ECD (c 0.6 × 10−3, MeOH) λmax (Δε) 196 (−7.11), 220 (+3.61), 239 (+4.15) nm; 1H NMR (500

Table 4. Antioxidant Effects of the Compounds on H2O2Induced Damage in PC12 Cellsa viability % 1.0 μM control model VE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

100 64.34 80.86 72.67 77.74 69.53 67.44 82.69 73.72 73.43 77.73 68.18 67.58 76.13 73.13 67.54 82.90 68.10 74.55 65.41 69.29 81.20 62.46 65.44 81.91 66.28 66.44 69.49 66.91 67.13

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.02e 1.50d 1.78d 0.88d 0.35d 0.67 2.32d 1.00d 0.98d 1.06d 0.27b 0.46 1.05d 1.75d 1.36 1.15d 0.66b 1.95d 0.19 0.51d 0.81d 1.20 1.02 0.95d 1.96 0.98 0.84d 0.79 0.51

5.0 μM

74.81 76.69 68.85 71.63 76.79 77.67 83.48 80.87 70.77 72.24 77.02 77.28 72.04 83.98 70.57 76.14 77.39 72.42 80.90 63.70 68.66 70.28 63.98 70.04 77.87 67.13 70.24

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.19d 0.83d 1.35c 1.33d 1.04d 0.25d 1.18d 0.35d 0.46d 0.26b 0.46d 0.82d 1.10d 0.64d 1.72d 0.74d 1.52d 1.15d 0.53d 0.61 0.48c 1.33d 0.73 0.55d 1.24d 0.66 0.67d

10.0 μM

77.17 75.21 76.16 79.33 74.30 79.85 89.03 85.24 72.33 74.11 80.73 81.02 76.41 88.66 72.50 78.41 80.16 75.86 90.11 67.18 70.20 72.57 65.23 69.56 77.70 67.43 72.14

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.02d 0.49d 0.96d 1.88d 0.44d 0.76d 0.97d 1.14d 0.36d 0.23b 1.35d 1.02d 0.40d 1.42d 0.52d 0.58d 1.03d 0.88d 1.38d 0.73 1.11d 1.23d 1.33 0.57d 1.74d 1.34 0.68d

Data are expressed as the mean ± SD (n = 3). bp < 0.05. cp < 0.01. dp < 0.001 vs model. ep < 0.001 vs control.

a

(6530) spectrometer. Silica gel (200−300 mesh, Qing Dao Ocean Chemical Factory), Sephadex LH-20 (20−100 μm, Pharmacia), SepPak C18 cartridges (Waters), ODS-C18 (40−63 μm, Pharmacia), Agilent 1200 series HPLC, and semipreparative HPLC (Beijing Chuangxintongheng Science & Technology Co., Ltd.) were used for chromatographic separations. All reagents used in bioassays were purchased from Sigma Chemicals. Plant Material. Radix of C. aromatica Salisb., collected from a farm in Luwu town, Lingshan county, Qinzhou city, Guangxi Province, People’s Republic of China, was purchased from the Juhua Traditional Chinese Medicine Market, Kunming, People’s Republic of China, and was identified by Prof. Shiming Guo, Yunnan Institute of Traditional Chinese Medicine and Material Medica, People’s Republic of China. Extraction and Isolation. Dry radix of C. aromatica (10.0 kg) was extracted with 75% ethanol (5 × 10 L × 4 h) to obtain a residue (580.0 g) after evaporation of the solvent. The residue was suspended in H2O (3 L) and partitioned successively with petroleum ether (4 × 3 L), EtOAc (4 × 3 L), and n-BuOH (4 × 3 L). The petroleum ether extract (66.3 g) was subjected to a column of silica gel eluted with petroleum ether−EtOAc (1:0, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 0:1 v/v) to obtain 10 fractions (PA−PJ), which were combined based on TLC analysis. Fr. PE (5.2 g) was subjected to a column of silica gel eluted with a gradient of increasing acetone (0−100%) in petroleum ether to afford Frs. PE1−PE5. Fr. PE3 (60.2 mg) was purified by semipreparative HPLC with MeOH−H2O (55:45) to obtain 1 (1.5 mg), 18 (1.9 mg), and 5 (0.78 mg). Fr. PE5 (71.3 mg) was also purified by semipreparative HPLC with MeOH−H2O (70:30) to yield 8 (0.43 mg) and 14 (12.5 mg). Fr. PG (4.5 g) was separated by a silica gel 3100



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MHz, CDCl3), Table 1; 13C NMR (125 MHz, CDCl3), Table 2; HRESIMS m/z 273.1470 [M + Na]+ (calcd for C15H22O3Na 273.1467). Aromaticane H (8): pale yellow oil; [α]25D +14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (3.99) nm; ECD (c 0.5 × 10−3, MeOH) λmax (Δε) 225 (+2.79), 253 (−3.45) nm; 1H NMR (800 MHz, CDCl3), Table 1; 13C NMR (200 MHz, CDCl3), Table 2; HRESIMS m/z 315.1564 [M + Na]+ (calcd for C17H24O4Na 315.1572). Aromaticane I (9): white, amorphous powder; [α]25D −95 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (3.19) nm; Mo2(OAc)4induced ECD (c 1.5 × 10−3, CHCl3) nm (Δε) 363 (−0.04); 1H NMR (500 MHz, CDCl3), Table 1. 13C NMR (125 MHz, CDCl3), Table 2; HRESIMS m/z 333.2054 [M − H]− (calcd for C20H29O4 333.2066). Aromaticane J (10): colorless oil; [α]25D +47 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (3.84) nm; Mo2(OAc)4-induced ECD (c 2.0 × 10−3, DMSO) λmax (Δε) 315 (−0.58) nm; 1H NMR (800 MHz, DMSO-d6), Table 1; 13C NMR (200 MHz, DMSO-d6), Table 2; HRESIMS m/z 359.2201 [M + Na]+ (calcd for C20H32O4Na 359.2198). Computation of ECD and 13C NMR Data. An MMFF94 conformational search generating low-energy conformers within a 1.0 kcal/mol energy window was performed using CONFLEX 7D. The conformers of 1 and 7 with the highest distribution were selected and further optimized by the density functional theory method at the APFD/6-311+G(2d,p) level. The ECD calculations were conducted using TD-DFT-APFD/6-311+G(2d,p) on optimized geometries through the IEFPCM model (in MeOH). The NMR calculations were conducted using the gauge-including atomic orbital (GIAO) method at the APFD/6-311+G(2d,p) level on optimized geometries through the Polarizable Continuum Model using the integral equation formalism variant (IEFPCM) (in DMSO), and the shieldings obtained were converted into chemical shifts by referencing to tetramethylsilane at 0 ppm (see the Supporting Information for detailed computational methods). Antioxidant Activity Bioassay. Six hundred grams of dry radix of C. aromatica was equally divided into three samples. Two of the samples were extracted separately with 95% EtOH and 75% EtOH (3 × 0.5 L × 6 h). The other was soaked with MeOH at room temperature (3 × 0.5 L × 24 h). The organic solvent was removed from each solution under vacuum to obtain the 95% EtOH, 75% EtOH, and MeOH extracts. Each extract and compound was dissolved in DMSO to prepare different concentrations of the tested samples. Briefly, PC12 cells were treated simultaneously with 400 μM H2O2 and different concentrations of extracts, compounds, and vitamin E (used as positive control) for 24 h. Furthermore, MTT was added into each well, and incubation continued for an additional 4 h. Finally, the culture medium was removed, and DMSO was added to each well, followed by vigorously shaking the plate to dissolve formazan completely. The absorbance of each well was measured at 490 nm on a microplate reader. The results were expressed as a percent of the nontreated control. Statistical Analysis. The data in Tables 3 and 4 were expressed as the mean ± standard deviation (SD) and statistically analyzed via oneway ANOVA followed by Tukey’s HSD test. Differences were accepted as statistically significant at p < 0.05.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (M. Zhang). Tel: +86 871 6592 0738. Fax: +86 871 6592 0738. ORCID

Mi Zhang: 0000-0001-6410-6128 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (31500287) and the Personnel Training Project of Yunnan, China (KKSY201626035).

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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.jnatprod.6b01100. Detailed computational methods, 1D NMR, 2D NMR, and HRESIMS of 1−10, IECD spectra of 2, 3, 6, 9, and 10, ECD spectra of 4, 5, 6, and 8, and structures of known compounds (11−27) (PDF) 3101



Article

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