Bioactive Sesquiterpenoid and Polyacetylene Glycosides from

May 26, 2016 - Nine new sesquiterpenoids (1–9), five new polyacetylenes (10–14), and six known compounds were isolated from the rhizomes of Atract...
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Bioactive Sesquiterpenoid and Polyacetylene Glycosides from Atractylodes lancea Kuo Xu, Jian-Shuang Jiang, Zi-Ming Feng, Ya-Nan Yang, Li Li, Cai-Xia Zang, and Pei-Cheng Zhang* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new sesquiterpenoids (1−9), five new polyacetylenes (10−14), and six known compounds were isolated from the rhizomes of Atractylodes lancea. These new chemical structures were established using NMR, MS, and ECD data. Notably, compounds 3−5, the aglycone of which possesses two stereogenic centers (C-5 and C-7), exhibited similar ECD spectra to compounds 1 and 2, the aglycone of which possesses one stereogenic center (C-7). Such a difference was supported by the experimental and calculated ECD data and single-crystallographic analyses of 3a. In addition, compound 3 inhibited lipopolysaccharide-induced NO production in BV2 cells with an IC50 value of 11.39 μM (positive control curcumin, IC50 = 4.77 μM); compound 4 showed better hepatoprotective activity against N-acetyl-p-aminophenol-induced HepG2 cell injury than the positive drug (bicyclol) at a concentration of 10 μM (p < 0.001). C 10-polyacetylenes (10−14) were isolated. These new compounds were elucidated via NMR, MS, and electronic circular dischroism (ECD) data. In addition, all compounds were assessed for inhibitory effects on the lipopolysaccharide (LPS)-induced NO production of BV2 microglial cells and hepatoprotective activities against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell injury.

Atractylodes lancea (Thunb.) DC., a perennial herb that is widely distributed in eastern Asia, belongs to the genus Atractylodes, which comprises seven species, five of which are unique to China.1,2 The rhizomes of A. lancea and A. chinensis, known as “Cangzhu” in Traditional Chinese Medicine, are widely used to treat nyctalopia, stomachache, and influenza.3 Previous pharmacological studies demonstrated that the extract and chemical constituents of A. lancea also have promising antiinflammatory and hepatoprotective effects.4−6 Over the last several decades, phytochemical studies of A. lancea led to identifying nearly 100 compounds, containing sesquiterpenoids, polyacetylenes, monoterpenes, and steroids.7−9 Among these, eudesmane- and guaiane-type sesquiterpenoids and polyacetylenes are considered to be the characteristic phytochemicals. Only four spirovetivane-type sesquiterpenoids have been characterized to date.10−12 In a search for the biological active compounds from the n-BuOH part of an aqueous EtOH extract of A. lancea, eight new spirovetivane-type sesquiterpenoids (1− 8), one new guaiane-type sesquiterpenoid (9), and five new © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The molecular formula C21H32O7 of compound 1 {m/z 397.2228 [M + H]+} was determined by HRESIMS and 13C NMR data. The IR spectrum showed absorptions attributable to hydroxy (3344 cm−1), carbonyl (1666 cm−1), and olefinic (1619 cm−1) groups. Its 1H NMR data (Table 1) were indicative of two olefinic protons at δH 5.88 and 5.89, four methyl resonances at δH 1.20, 1.21, 2.03, and 2.04, and other Received: January 26, 2016

A

DOI: 10.1021/acs.jnatprod.6b00066 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

aliphatic resonances at δ H 1.70−2.40. The remaining resonances were assigned to a glucosyl unit. The 13C NMR data (Table 3) showed four olefinic carbons at δC 124.5, 124.8, 165.7, and 166.3 and one carbonyl at δC 185.1 in the downfield region. With the exception of the glucosyl signals, one oxygenated tertiary carbon (δC 76.7), three methylene carbons (δC 29.2, 35.8, and 37.3), and four methyl carbons (δC 20.2, 20.3, 24.4, and 26.0) were also observed. HMBC correlations (Figure 2) of H-1 with C-3 and C-5, H-3 with C-5, H-6 with C4 and C-10, H-7 with C-5 and C-9, and H-1′ with C-11 revealed the presence of a cyclohexadienone and a cyclopentane unit. Therefore, compound 1 was characterized as a spirovetivane-type sesquiterpenoid with a glucopyranosyl moiety substituted at C-11. Snailase hydrolysis of 1 produced the corresponding aglycone (1a) and glucose. The βconfiguration of the glucopyranosyl moiety was deduced based on the 3J1′,2′ value (7.5 Hz), whereas the D-configuration was determined by GC analyses (retention time of 20.55 min) after chiral derivatization. Considering the numerous conformations stemming from the single glucosyl bonds, a simplified structure (1a) was used for ECD calculations. The systematic conformational analysis was completed using a molecular mechanics force field (MMFF94) calculation. Two optimized conformations for the (7R)- and (7S)-configured 1a were obtained using the timedependent density functional theory (TDDFT) method at the B3LYP/6-31G(d) level. The overall calculated ECD spectra (Figure 1) were generated by Boltzmann weighting of their lowest energy conformers. Throughout the entire range of wavelengths, the calculated spectrum of (7R)-1a matched with the experimental data for 1 and 1a (Figure 1).13 Thus, the structure of compound 1 was defined as (7R)-3,4-dehydrohinesolone-11-O-β-D-glucopyranoside. Compound 2 was found to have the molecular formula C26H40O11, according to an HRESIMS ion at m/z 529.2646 [M + H]+ and 13C NMR data. The IR spectrum exhibited typical absorptions for hydroxy (3399 cm−1), carbonyl (1655 cm−1), and olefinic (1600 cm−1) groups. Comparison of the 1D NMR data (Table 3) of 2 with the data of 1 suggested that 2 possessed an apiosyl moiety via resonances at δC 109.2, 76.0,

78.8, 73.3, and 63.3. An HMBC correlation of H-1″ with C-6′ demonstrated that the apiosyl moiety was substituted at C-6′. This was supported by the deshielded C-6′ resonance at δC 67.8. The β-configuration of the apiosyl moiety was deduced by the 3J1″,2″ value (3.0 Hz), whereas the D-configuration was confirmed via GC analyses (retention time of 14.52 min) as above. The similarity of the ECD spectra of 1 and 2 suggested that 2 had a (7R) configuration. Thus, the structure of compound 2 was defined as (7R)-3,4-dehydrohinesolone-11-Oβ-D-apiofuranosyl-(1→6)-β-D-glucopyranoside. Compound 3 was shown to have the molecular formula C21H32O8 by an HRESIMS ion at m/z 435.1986 [M + Na]+ and 13C NMR data. Its IR and NMR data were closely related to those of 1 except for the presence of a hydroxymethyl group (δH 4.20, 4.30) in 3, instead of the methyl group (δH 2.03) in 1. This was supported by the HMBC correlations (Figure 2) of H-1 with C-15 and of H-3 with C-14. From the aforementioned information, 3 was characterized as 14-hydroxy-3,4-dehydrohinesolone-11-O-β-D-glucopyranoside. The ROESY correlations (Figure 3) of H2-14 with H-7 and of H3-15 with H3-12 suggested that H2-14 and H-7 were on the same side of the cyclopentane unit, whereas H3-15 and H3-12 were on the opposite side. Snailase hydrolysis of 3 yielded the aglycone (3a) and glucose. The β-D-configuration of the glucopyranosyl moiety was deduced via the GC method (retention time of 20.54 min) and the 3J1′,2′ value (7.5 Hz). Structurally, compound 3 possesses one more stereogenic carbon (C-5) than 1 due to the presence of the 14-OH group. However, these two compounds exhibited similar ECD spectra. A literature survey indicated that compounds possessing an asymmetric cyclohexadienone unit exhibit a Cotton effect (CE) at approximately 270 nm.14−16 Further analysis of the literature revealed that this CE had a low amplitude (Δε ≈ +2.00). A simplified structure (3a) was used for ECD calculations, and four optimized conformers (5R,7R; 5S,7S; 5S,7R; 5R,7S) were obtained using an MMFF94 calculation and the TDDFT method at the B3LYP/6-31G(d) level. The calculated spectrum (Figure 4) of the (5R,7R)-diastereomer matched the experimental ECD spectra of 3 and 3a. This was supported by the ROESY data, which showed a correlation of H2-14 with B

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Table 1. 1H NMR (500 MHz) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 1−5 in DMSO-d6

Table 2. 1H NMR (500 MHz) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 6−9 in DMSO-d6

position

position

1

1

5.89, d (1.5)

3

5.88, d (1.5)

6a 6b 7 8 9a 9b 12 13 14a

2.00, 1.69, 2.37, 1.89, 1.95, 1.69, 1.21, 1.20, 2.03,

m m m m m m s s s

2 5.89, d (1.5) 5.88, d (1.5) 1.98, m 1.68, m 2.39, m 1.91, m 1.94, m 1.68, m 1.20, s 1.20, s 2.04, s

14b 15 1′ 2′

2.04, s Glc 4.32, d (7.5)

3′

2.89, dd (7.5, 8.5) 3.14, t (8.5)

4′ 5′a

3.01, m 3.07, m

5′b 6′a 6′b

1″ 2″ 4″a 4″b 5″

3.62, dd (5.5, 12.0) 3.38, dd (5.5, 12.0)

2.04, s Glc 4.32, d (7.5) 2.90, dd (7.5, 8.5) 3.14, d (8.5) 2.96, m 3.23, m

3

4

6.20, d (1.5)

2.89, dd (7.5, 8.5) 3.15, t (8.5)

6.20, d (1.5) 5.89, d (1.5) 1.96, m 1.70, m 2.34, m 1.86, m 1.90, m 1.70, m 1.19, s 1.19, s 4.30, overlap 4.20, brd (16.5) 2.03, s Glc 4.32, d (7.5) 2.90, m

3.04, overlap

3.14, m

3.10, t (8.5)

3.01, t (8.5) 3.07, m

2.96, m 3.23, m

3.29, m 3.71, dd (5.0, 11.0) 3.05, overlap

5.89, d (1.5) 1.99, 1.70, 2.31, 1.88, 1.91, 1.70, 1.19, 1.19, 4.30,

m m m m m m s s overlap

4.20, brd (16.5) 2.03, s Glc 4.32, d (7.5)

5 5.92, brs

1 3a

6.38, d (1.5)

3b

1.94, m 1.74, m 2.21, m 1.85, m 1.74, m 1.63, m 1.12, s 1.08, s 4.60, dd (1.5, 16.0) 4.28, dd (1.5, 16.0) 2.03, s Xyl 4.19, d (7.5)

4 6a 6b 7 8a 8b 9a 9b 10 12 13 14a 14b 15a 15b

3.79, m

3.62, m

3.79, m

3.36, overlap Api 4.81, d (3.0) 3.69, brs 3.81, d (9.5) 3.55, d (9.5) 3.31, overlap

3.33, dd (5.5, 12.0)

3.36, overlap Api 4.82, d (3.0) 3.69, brs 3.81, d (9.5) 3.55, d (9.5) 3.31, overlap

1′ 2′ 3′ 4′ 5′a 5′b 6′a 6′b

6

7

6.11, s 2.46, dd (4.0, 16.5) 2.13, dd (8.0, 16.5) 2.05, m

5.66, s 2.57, dd (4.0, 17.0) 2.33, dd (9.0, 17.0) 2.08, m

1.74, 1.43, 1.91, 1.69, 1.59, 1.82, 1.68,

1.83, 1.48, 1.92, 1.70, 1.59, 1.80, 1.73,

overlap t (12.5) m m m m m

1.07, s 1.07, s 0.91, d (6.5)

4.40, dd (1.5, 17.0) 4.26, dd (1.0, 17.0) Xyl 4.17, d (7.5) 3.02, overlap 3.09, t (8.5) 3.28, m 3.70, dd (5.0, 11.0) 3.05, d (11.0)

m t (12.5) m m m m m

8

9

5.63, d (1.0) 3.75, d (12.5)

4.04, d (6.5)

1.74, dd (6.5, 12.5) 2.03, m 1.40, m 2.04, m 1.72, m 1.65, m 1.83, m 1.65, m

2.81, q (6.5)

1.07, s 1.08, s 3.93, dd (4.0, 10.0) 3.27, t (10.0) 1.91, s

1.17, s 1.17, s 1.08, d (6.5)

2.73, 2.17, 1.48, 1.89, 1.45, 1.70, 1.39, 2.71, 1.14, 1.20, 0.90,

1.96, d (1.0)

0.91, s

Xyl 4.06, d (7.5) 2.93, t (8.5) 3.07, t (8.5) 3.24, m 3.67, dd (5.0, 11.0) 3.02, t (11.0)

Glc 4.29, 2.87, 3.14, 3.01, 3.06,

Glc 4.28, 2.91, 3.13, 3.02, 3.04,

d (7.5) t (8.5) t (8.5) overlap overlap

3.75, d (12.0) 3.62, brd (12.0)

m m m m m m m m s s s

d (7.5) t (8.5) m overlap overlap

3.57, d (11.5) 3.37, brd (11.5)

enzymatic hydrolysis and chiral derivatization. In combination with HMBC correlations of H-1′ with C-11 in 4 and H-1′ with C-14 in 5, the structure of the former compound was assigned as 14-hydroxy-3,4-dehydrohinesolone-11-O-β-D-apiofuranosyl(1→6)-β-D-glucopyranoside, and the latter as 14-hydroxy-3,4dehydrohinesolone-14-O-β-D-xylopyranoside. The ROESY correlation (Figure 3) revealed that H2-14 and H-7 were on the same side of the cyclopentane unit in both 4 and 5. Thus, the similar ECD curves to those of 1−3 favored (7R) configurations, and the absolute configurations were assigned as (5R,7R). Compound 6 had the molecular formula C 20 H 32 O 7 established by an HRESIMS ion at m/z 407.2034 [M + Na]+ and 13C NMR data. The IR spectrum showed absorption bands of hydroxy (3401 cm−1) and carbonyl (1656 cm−1) groups. Comparison of these 1D NMR and HRESIMS data (Tables 2 and 3) with those of 15-hydroxy-2-oxohinesol11 suggested that 6 possessed an additional xylopyranosyl moiety. The β-Dconfiguration of the xylopyranosyl moiety was established by the same method as for 5. HMBC correlations (Figure 2) from H-1 and H-1′ to C-15 demonstrated that the β-D-xylopyranosyl moiety was substituted at C-15. Therefore, the structure of compound 6 was defined as 15-hydroxyhinesolone-15-O-β-Dxylopyranoside. ROESY correlations (Figure 3) of H-4 with H29, H3-14 with H-7, and H2-15 with H3-12 indicated that H3-14

H-7, but no correlation of H3-15 and H-7. Comparison of the calculated ECD data of 3a with 1a revealed that the lowamplitude positive CE at approximately 275 nm is contributed by the cyclohexadienone moiety, while the high-amplitude positive CE approximately at 235 nm is derived from both the five-membered spirocycle and the cyclohexadienone units. Obviously, the positive CE at 252 nm in the experimental ECD spectrum of 3a may be the additive contribution of the respective chromophores.17,18 An X-ray single-crystal structure of 3a (CCDC 1448943), obtained using Cu Kα radiation, permitted the unambiguous assignment of the absolute configurations as (5R,7R) (Figure 5). Consequently, the structure of compound 3 was defined as (5R,7R)-14-hydroxy3,4-dehydrohinesolone-11-O-β-D-glucopyranoside. Compounds 4 and 5 had the same aglycone moieties as 3, as determined through 1D NMR analyses. However, the sugar units in 4 and 5 were confirmed to be β-D-apiofuranosyl-(1→ 6)-β-D-glucopyranose and xylopyranose. Similarly, the βconfiguration of the xylopyranosyl moiety was confirmed by the 3J1′,2′ value (7.5 Hz), whereas the D-configuration was established by GC analyses (retention time of 14.92 min) after C

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Table 3. 13C NMR (125 MHz) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 1−9 in DMSO-d6 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″

1 124.8, 185.1, 124.5, 165.7, 52.4, 37.3, 53.0, 29.2, 35.8, 166.3, 76.7, 26.0, 24.4, 20.2, 20.3, Glc 97.1, 73.8, 77.1, 70.3, 76.6, 61.2,

2 CH C CH C C CH2 CH CH2 CH2 C C CH3 CH3 CH3 CH3 CH CH CH CH CH CH2

124.8, 185.1, 124.6, 165.7, 52.3, 37.3, 53.1, 29.1, 35.8, 166.3, 77.0, 25.9, 24.3, 20.2, 20.3, Glc 97.1, 73.8, 76.8, 70.5, 75.1, 67.8, Api 109.2, 76.0, 78.8, 73.3, 63.3,

3 CH C CH C C CH2 CH CH2 CH2 C C CH3 CH3 CH3 CH3 CH CH CH CH CH CH2 CH CH C CH2 CH2

120.8, 185.3, 124.7, 166.1, 50.9, 37.5, 52.9, 28.9, 35.7, 169.5, 76.7, 26.0, 24.4, 59.8, 20.0, Glc 97.1, 73.8, 77.1, 70.4, 76.6, 61.2,

4 CH C CH C C CH2 CH CH2 CH2 C C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2

120.8, 185.3, 124.7, 166.1, 50.9, 37.5, 53.0, 28.9, 35.8, 169.5, 77.1, 25.8, 24.4, 59.7, 20.0, Glc 97.0, 73.8, 76.8, 70.5, 75.1, 67.8, Api 109.2, 76.0, 78.8, 73.3, 63.3,

5 CH C CH C C CH2 CH CH2 CH2 C C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2

124.9, 185.2, 121.7, 165.4, 50.9, 37.4, 53.0, 29.0, 35.8, 165.2, 69.1, 29.1, 29.1, 66.1, 20.0, Xyl 103.4, 73.4, 76.5, 69.6, 65.8,

6 CH C CH C C CH2 CH CH2 CH2 C C CH3 CH3 CH2 CH3 CH CH CH CH CH2

121.8, 198.0, 42.5, 36.0, 48.6, 31.3, 49.6, 26.3, 34.2, 165.8, 69.4, 28.7, 28.7, 15.8, 66.1, Xyl 103.3, 73.4, 76.5, 69.5, 65.8,

7 CH C CH2 CH C CH2 CH CH2 CH2 C C CH3 CH3 CH3 CH2 CH CH CH CH CH2

125.4, 197.5, 37.6, 42.4, 48.4, 31.7, 50.1, 26.8, 34.8, 168.2, 69.5, 28.5, 28.7, 69.5, 20.1, Xyl 104.4, 73.4, 76.6, 69.6, 65.7,

8 CH C CH2 CH C CH2 CH CH2 CH2 C C CH3 CH3 CH2 CH3 CH CH CH CH CH2

122.1, 199.0, 73.8, 45.5, 50.5, 30.9, 52.1, 28.2, 35.3, 170.2, 76.6, 24.2, 26.0, 12.1, 20.0, Glc 97.1, 73.8, 77.2, 70.4, 76.6, 61.2,

9 CH C CH CH C CH2 CH CH2 CH2 C C CH3 CH3 CH3 CH3 CH CH CH CH CH CH2

140.9, 207.5, 72.9, 43.3, 176.0, 32.8, 47.6, 25.9, 32.6, 27.3, 79.2, 22.3, 24.8, 14.9, 17.9, Glc 97.3, 73.7, 77.0, 70.2, 76.6, 61.3,

C C CH CH C CH2 CH CH2 CH2 CH C CH3 CH3 CH3 CH3 CH CH CH CH CH CH2

CH CH C CH2 CH2

of 7 was defined as (4R,5S,7R)-14-hydroxyhinesolone-14-O-βD-xylopyranoside.

An HRESIMS adduct ion of compound 8 at m/z 459.2235 [M + COOH]− indicated a molecular formula of C21H34O8. With the exception of the β-D-glucopyranosyl unit, 8 possessed similar elementary compositions to those of 6. The most notable differences of their NMR data were that the resonances at δH 4.26, 4.40, and δC 66.1 attributable to C-15 of 6 were not observed in the corresponding upfield region of 8. The presence of a carbonyl group (δC 199.0), two quaternary carbons (δC 50.5 and 170.2), an oxygenated tertiary carbon (δC 76.6), and two spin systems [C(6)H2−C(7)H−C(8)H2− C(9)H2 and C(3)H−C(4)H−C(14)H3] suggested that 8 was also a spirovetivane-type sesquiterpenoid glycoside and that its aglycone was a hydroxylated product of hinesolone.12 HMBC correlations established the structure of compound 8 as 3hydroxyhinesolone-11-O-β- D -glucopyranoside. NMR and ROESY experiments were used to designate the absolute configuration of 8. The 3J3,4 value (12.5 Hz) suggested the 3,4trans configuration. ROESY correlations (Figure 3) revealed that H-3 and H2-6 were on the same side of the cyclohexenone unit, whereas H3-14 and H-7 were on the same side of the cyclopentane unit. The positive CE at 313 nm contributed by the n → π* transition of the α,β-unsaturated cyclohexenone unit facilitated assignment of the absolute configuration as (3S,4S,5S,7R).21 Compound 9 possessed an HRESIMS adduct ion at m/z 459.2244 [M + COOH]−, which corresponded to the molecular formula of C21H34O8. The 1H NMR data (Table 2) exhibited four methyl groups at δH 0.90, 0.91, 1.14, and 1.20. Its 13C NMR data (Table 3) were indicative of an α,β-

Figure 1. Experimental and calculated ECD spectra of 1 and 1a.

and H-7 were on the same side of the cyclopentane unit, whereas H2-15 and H3-12 were on the opposite side. The similar ECD data of 6 to (4S,5S,7R)-15-hydroxy-2-oxohinesol11 permitted the assignment of the absolute configurations as (4S,5S,7R). Compounds 7 and 6 shared a similar skeleton except for the locations of the sugar units, as established by 1D NMR analyses (Tables 2 and 3). The HRESIMS ion at m/z 429.2137 [M + COOH]− corresponded to a molecular formula of C20H32O7. An HMBC correlation from H-1′ to C-14 suggested that the βD-xylopyranosyl moiety was located at C-14 in 7. The similar ECD and ROESY (Figure 3) data to those of 6 permitted assignment of the (4R,5S,7R) configuration. Thus, the structure D

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Figure 2. Key HMBC and 1H−1H COSY correlations of compounds 3, 6, 9, 12, and 13.

Figure 3. Key ROESY correlations of compounds 3−9.

+ COOH]− and 13C NMR data. The IR spectrum showed absorptions attributable to hydroxy (3321 cm−1), acetylenic (2208 cm−1), and olefinic (1599 cm−1) groups. In the 1H NMR data (Table 4), the 3J2,3 value (11.0 Hz) revealed a cisconfigured Δ2,3 double bond, whereas the Δ8,9 double bond was trans-configured (J = 16.0 Hz). Its 13C NMR data (Table 5) were indicative of four olefinic carbons at δC 105.9, 109.3, 144.6, and 149.9, four acetylenic carbons at δC 73.0, 77.3, 79.1, and 82.3, two oxygenated carbons at δC 60.8 and 66.5, and a βD-glucopyranosyl moiety at δC 102.6, 73.4, 76.7, 69.9, 77.0, and 60.9. In the HMBC spectrum, long-range correlations of H-1′ with C-1, H-1 with C-3, H-2 with C-4, H-3 with C-5, H-8 with C-6, H-9 with C-7, and H-10 with C-8 facilitated definition of the structure of compound 10 as (2Z,8E)-deca-2,8-diene-4,6diyne-1,10-diol-1-O-β-D-glucopyranoside. Compound 11 shared the same aglycone moiety and molecular formula of C16H20O7 with 10, as established by an HRESIMS adduct ion at m/z 369.1191 [M + COOH]− and 13 C NMR data (Table 5). In the HMBC experiment, H-1′ correlated with C-1, and H-1 correlated with C-2 and C-3, suggesting the β-D-glucopyranosyl unit was substituted at C-1 in 11. Consequently, the structure of compound 11 was defined as (2E,8Z)-deca-2,8-diene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside.

unsaturated carbonyl moiety at δC 140.9, 176.0, and 207.5. These 13C NMR resonances shifted to lower fields than those of 6, possibly due to the presence of a cyclopentenone rather than a cyclohexenone unit. HMBC correlations (Figure 2) of H-14 with C-5, H-15 with C-1, and H-1′ with C-11 suggested that 4-CH3 and 10-CH3 were located on opposite sides of the olefinic bond and that the β-D-glucopyranosyl moiety was substituted at C-11. Further analyses of 1D NMR and 1H−1H COSY spin systems [C(6)H2−C(7)H−C(8)H2−C(9)H2− C(10)H−C(15)H3 and C(3)H−C(4)H−C(14)H3] confirmed that compound 9 was a guaiane-type sesquiterpenoid glycoside and defined as 2-hydroxypancherione-11-O-β-D-glucopyranoside. The coupling constant (3J3,4 = 6.5 Hz) indicated a 3,4-cis configuration,19,20 which was also supported by a ROESY correlation (Figure 3). In addition, H-6b correlated with H3-12 and H3-14 and H3-12 correlated with H-8a and H-10 in the ROESY spectrum, suggesting that H-6b and H-10 were on the same side of the guaiane ring, whereas H-3, H-4, and H-7 were on the opposite side. A positive CE at 328 nm in the ECD spectrum defined the absolute configuration of 9 as (3R,4S,7R,10R).21 Compound 10 exhibited UV data characteristic of an enediyne chromophore.22 Its molecular formula of C16H20O7 was deduced via an HRESIMS adduct ion at m/z 369.1194 [M E

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Compound 13 was an enediyne derivative based on the distinctive UV spectrum. Its molecular formula C16H22O7 was established by the HRESIMS adduct ion {m/z 371.1352 [M + COOH]−} and 13C NMR data. In association with the HSQC data, the 1D NMR spectroscopic data (Tables 4, 5) revealed a trans-configured Δ2,3 double bond [δH 5.97, 6.38 (J = 16.0 Hz); δC 108.7, 144.1], two acetylenic bonds (δC 64.8, 73.6, 74.5, and 85.2), two oxygenated methylenes [δH 4.16, 4.34, and 3.42; δC 59.2, 67.4], and a −CH2−CH2− group [δH 1.61, 2.41; δC 15.4, 31.0], as well as a glucosyl unit. In the HMBC experiment (Figure 2), the long-range correlations of H-1′ with C-1, H-1 with C-3, H-2 with C-4, H-3 with C-5, H-8 with C-6 and C-10, and H-9 with C-7 were observed. Thus, the structure of compound 13 was defined as (E)-deca-2-ene-4,6-diyne-1,10diol-1-O-β-D-glucopyranoside. Compound 14 was also an enediyne derivative. A deprotonated molecule at m/z 457.1718 [M − H]− in the HRESIMS spectrum corresponded to the molecular formula C21H30O11. According to the 1D NMR spectroscopic data (Tables 4, 5), 14 possesses an identical aglycone moiety to 13, but contains an additional β-D-apiosyl moiety, as evidenced by the carbon resonances at δC 109.3, 75.9, 78.8, 73.2, and 63.0. The structure of compound 14 was defined as (E)-deca-2-ene4,6-diyne-1,10-diol-1-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside via analyses of the 2D NMR data. In addition to these novel compounds, six known compounds were isolated from A. lancea. The known compounds (15−20) were identified as (2E,8E)-deca-2,8diene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside,23 atractyloside I,24 (−)-lyoniresinol 9α-O-β-D-glucopyranoside,25 codonopilodiynoside A,26 cis-atractyloside I,23 and kankanosides P.27 All compounds were assayed for their anti-inflammatory and hepatoprotective activities. Compound 3 inhibited LPS-induced NO production in BV2 cells with an IC50 value of 11.39 μM (positive control curcumin, IC50 = 4.77 μM). Compared with the model group, compound 4 showed significant hepatoprotective activity (Figure 6) against APAP-induced HepG2 cell injury with a cell survival rate of 58.25% at a concentration of 10 μM (p < 0.001, bicyclol as the positive drug, 52.87%).

Figure 4. Experimental and calculated ECD spectra of 3 and 3a.

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations and UV and ECD spectra were measured on JASCO P-2000, JASCO V650, and JASCO J-815 spectrometers (JASCO, Easton, MD, USA), respectively. The IR data were collected using a Nicolet 5700 spectrometer (Thermo Scientific, Waltham, MA, USA). The NMR spectra were recorded on a Bruker 500 MHz NMR instrument (Bruker-Biospin, Billerica, MA, USA). HRESIMS analyses were performed with an Agilent 1200 series LC/MSD TOF instrument (Agilent Technologies, Waldbronn, Germany). GC analyses were done on an Agilent 7890A system. RP-18 (50 μm, YMC Corp., Kyoto, Japan), Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden), and Diaion HP-20 (Mitsubishi Chemical Corp., Tokyo, Japan) were used as chromatographic substrates. A Shimadzu LC10AT equipped with an ODS-A column (250 × 20 mm, 5 μm, YMC Corp.) was used for preparative HPLC. The HPLC experiments were performed on an Agilent 1260 system equipped with an Apollo C18 column (250 × 4.6 mm, 5 μm). Plant Material. The rhizomes of A. lancea were collected at Huanggang City (Hubei Province, China) in June 2014 and identified by Prof. L. Ma. A voucher specimen (ID-S-2596) was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing. Extraction and Isolation. The rhizomes of A. lancea (100 kg) were crushed and extracted with 80% EtOH (3 × 150 L) at 85 °C for

Figure 5. ORTEP diagram of 3a.

Compound 12 possesses the same skeleton as the known compound (2E,8E)-deca-2,8-diene-4,6-diyne-1,10-diol-1-O-β-Dglucopyranoside (15),23 but 12 had an additional β-D-apiosyl moiety, based on the carbon resonances δC 109.3, 75.9, 78.8, 73.2, and 63.0. This was supported by an anomeric proton at δH 4.85 (J = 3.0 Hz) of the β-D-apiosyl moiety and the HRESIMS ion at m/z 455.1560 [M − H]−. An HMBC correlation of the proton at δH 4.85 with the carbon at δC 67.4 suggested that the β-D-apiosyl moiety was located at C-6′. Consequently, the structure of compound 12 was defined as (2E,8E)-deca-2,8diene-4,6-diyne-1,10-diol-1-O-β-D-apiofuranosyl-(1→6)-β-Dglucopyranoside. F

DOI: 10.1021/acs.jnatprod.6b00066 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 4. 1H NMR (500 MHz) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 10−14 in DMSO-d6 position

10

1a 1b 2 3 8 9 10

4.47, 4.34, 6.33, 5.86, 5.89, 6.53, 4.07, Glc 4.18, 2.95, 3.11, 3.07, 3.08, 3.67, 3.45,

1′ 2′ 3′ 4′ 5′ 6′a 6′b

11

dd (6.0, 14.0) dd (6.5, 14.0) dt (6.5, 11.0) d (11.0) d (16.0) dt (4.0, 16.0) s

4.36, 4.18, 6.46, 6.08, 5.73, 6.27, 4.16, Glc 4.14, 2.98, 3.12, 3.04, 3.08, 3.65, 3.42,

d (7.5) m overlap overlap overlap m m

12

dd (4.5, 16.0) dd (5.0, 16.0) dt (4.5, 16.0) dd (1.0, 16.0) dd (1.0, 11.0) dt (6.0, 11.0) overlap

4.33, 4.16, 6.41, 6.06, 5.86, 6.50, 4.06, Glc 4.14, 2.98, 3.13, 2.99, 3.25, 3.84, 3.40, Api 4.85, 3.75, 3.84, 3.58, 3.32,

d (7.5) t (8.5) overlap overlap overlap dd (5.0, 11.0) m

1″ 2″ 4″a 4″b 5″

dd (4.5, 16.0) overlap dt (4.5, 16.0) d (16.0) d (16.0) dt (4.0, 16.0) s d (7.5) overlap t (8.5) overlap m d (11.5) dd (6.0, 11.5) d (3.0) d (2.5) d (9.5) d (9.5) overlap

13 4.34, 4.16, 6.38, 5.97, 2.41, 1.61, 3.42, Glc 4.13, 2.97, 3.12, 3.03, 3.07, 3.65, 3.42,

dd (4.5, 16.0) dd (5.0, 16.0) dt (4.5, 16.0) d (16.0) t (7.0) m overlap d (7.5) t (8.5) t (8.5) overlap m dd (6.0, 11.5) overlap

14 4.31, 4.12, 6.36, 5.89, 2.41, 1.60, 3.43, Glc 4.13, 2.97, 3.12, 2.98, 3.25, 3.84, 3.39, Api 4.85, 3.75, 3.85, 3.57, 3.31,

brd (16.0) overlap dt (4.5, 16.0) d (16.0) t (7.0) dq (7.0, 14.0) overlap d (7.5) overlap t (8.5) overlap m d (11.0) overlap d (3.0) brs d (9.5) d (9.5) overlap

Table 5. 13C NMR (125 MHz) Spectroscopic Data (δ in ppm, J in Hz) for Compounds 10−14 in DMSO-d6 position

10

11

12

13

14

1 2 3 4 5 6 7 8 9 10

66.5, CH2 144.6, CH 109.3, CH 77.3, C 79.1, C 73.0, C 82.3, C 105.9, CH 149.9, CH 60.8, CH2 Glc 102.6, CH 73.4, CH 76.7, CH 69.9, CH 77.0, CH 60.9, CH2

67.4, CH2 145.1, CH 108.4, CH 81.6, C 73.6, C 78.3, C 78.0, C 107.1, CH 149.0, CH 59.5, CH2 Glc 102.4, CH 73.5, CH 76.6, CH 70.0, CH 77.0, CH 61.0, CH2

67.4, CH2 144.5, CH 108.7, CH 80.0, C 74.0, C 73.4, C 80.7, C 106.1, CH 149.5, CH 60.8, CH2 Glc 102.2, CH 73.4, CH 76.5, CH 70.2, CH 75.6, CH 67.4, CH2 Api 109.3, CH 75.9, CH 78.8, C 73.2, CH2 63.0, CH2

67.4, CH2 144.1, CH 108.7, CH 73.6, C 74.5, C 64.8, C 85.2, C 15.4, CH2 31.0, CH2 59.2, CH2 Glc 102.3, CH 73.5, CH 76.6, CH 70.0, CH 77.0, CH 61.0, CH2

67.3, CH2 143.9, CH 108.9, CH 73.6, C 74.5, C 64.8, C 85.2, C 15.4, CH2 31.0, CH2 59.2, CH2 Glc 102.1, CH 73.4, CH 76.5, CH 70.2, CH 75.6, CH 67.7, CH2 Api 109.3, CH 75.9, CH 78.8, C 73.2, CH2 63.0, CH2

1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″

Figure 6. Hepatoprotective effects of compounds 2, 4, 13, 15, and 17 (10 μM) against APAP (8 mM)-induced HepG2 cell injury. Results are expressed as the mean ± SD (n = 3). ***p < 0.001 (vs control group), ###p < 0.001, ##p < 0.01, #p < 0.05 (vs model group).

subfractions Fr. C3.5 and Fr. C3.6 by preparative HPLC afforded compounds 3 (50 mg) and 14 (11 mg); the purification of Fr. C3.7− Fr. C3.9 produced compounds 10 (23 mg), 11 (16 mg), and 13 (10 mg); and the purification of Fr. C3.10−Fr. C3.11 yielded (2E,8E)deca-2,8-diene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside (48 mg) and (−)-lyoniresinol 9α-O-β-D-glucopyranoside (44 mg). Fraction C6 (6.3 g) was chromatographed on an LH-20 column using H2O to yield 30 subfractions (Fr. C6.1−Fr. C6.30). These subfractions were separated by preparative HPLC with 40% MeOH−H2O. Subfractions Fr. C6.7−Fr. C6.9 afforded cis-atractyloside I (46 mg); Fr. C6.11−Fr. C6.14 afforded compounds 7 (11 mg), 8 (8 mg), and 9 (33 mg); Fr. C6.15 and Fr. C6.16 afforded compounds 5 (13 mg) and 12 (116 mg); and Fr. C6.17 and Fr. C6.18 afforded kankanoside P (16 mg). Fractions C7−C10 (9.7 g) were separated on an LH-20 column using H2O [subfractions Fr. C (7−10).1−Fr. C (7−10).40] and then chromatographed by preparative HPLC with 40% MeOH−H2O. Finally, Fr. C (7−10).8−Fr. C (7−10).12 afforded compounds 2 (173 mg) and 6 (132 mg), Fr. C (7−10).13−Fr. C (7−10).16 produced compound 1 (180 mg), and Fr. C (7−10).22−Fr. C (7−10).28 afforded codonopilodiynoside A (38 mg).

2 h. The extract was partitioned with petroleum ether, EtOAc, and nBuOH. The n-BuOH-soluble fraction (1.2 kg) was chromatographed with an HP-20 column using a stepwise gradient to give five fractions: A (H2O, 824 g), B (15% EtOH−H2O, 88.6 g), C (30% EtOH−H2O, 106.4 g), D (50% EtOH−H2O, 53.3 g), and F (95% EtOH−H2O, 19.5 g). Fraction C (106.4 g) was chromatographed on an RP-18 column using 0−100% MeOH (10% stepwise increase of MeOH) to afford subfractions C1−10 via HPLC-DAD analyses. Fraction C3 (10.0 g) was chromatographed on an LH-20 column using H2O to obtain 35 subfractions (Fr. C3.1−Fr. C3.35). Subtraction Fr. C3.4 was purified by preparative HPLC using 30% MeOH−H2O to yield compound 4 (11 mg) and atractyloside I (163 mg). Similarly, the purification of G

DOI: 10.1021/acs.jnatprod.6b00066 J. Nat. Prod. XXXX, XXX, XXX−XXX

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4 and 5; HRESIMS m/z 369.1191 [M + COOH]− (calcd for C17H21O9, 369.1186). (2E,8E)-Deca-2,8-diene-4,6-diyne-1,10-diol-1-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (12): brown, amorphous powder; [α]20 D −142 (c 0.1, MeOH−H2O, 1:3); UV (MeOH) λmax (log ε) 216 (4.37), 261 (3.82), 276 (4.07), 293 (4.22), 312 (4.13) nm; IR (KBr) νmax 3378, 2923, 2884, 2208, 2131, 1631, 1056, 1022 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS m/z 455.1560 [M − H]− (calcd for C21H27O11, 455.1553). (E)-Deca-2-ene-4,6-diyne-1,10-diol-1-O-β- D -glucopyranoside (13): brown, amorphous powder; [α]20 D −21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.67), 240 (3.75), 253 (4.07), 267 (4.19), 283 (4.06) nm; IR (KBr) νmax 3321, 2909, 2235, 1631, 1080, 1050 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS m/z 371.1352 [M + COOH]− (calcd for C17H23O9, 371.1342). (E)-Deca-2-ene-4,6-diyne-1,10-diol-1-O-β-D-apiofuranosyl-(1→ 6)-β-D-glucopyranoside (14): brown, amorphous powder; [α]20 D −62 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.55), 240 (3.60), 252 (3.94), 266 (4.08), 283 (3.96) nm; IR (KBr) νmax 3373, 2931, 2883, 2235, 1050 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS m/z 457.1718 [M − H]− (calcd for C21H29O11, 457.1710). Determination of the Absolute Configuration of Sugars. Compounds 1, 3, 5, and 6 (3−6 mg) were individually mixed with snailase (w/w = 1:2) and then dissolved in H2O (3 mL). The corresponding solutions were maintained at 37 °C for 48 h. Next, 3 mL of MeOH was added. Compound 2 was refluxed in 5 mL of HCl− MeOH (1 mol/L) for 6 h. These reaction mixtures were separated by preparative HPLC and eluted with CH3CN−H2O (v/v = 1:4) to yield the sugar residues and corresponding aglycones (1a−3a, 5a, and 6a). Their 1H NMR data and specific rotations are presented in the Supporting Information. The monosaccharide residues of compounds 1−3, 5, and 6 were processed using a previously reported method.28−30 The samples were analyzed by GC under the following conditions: injection temperature, 300 °C; detector temperature (FID), 300 °C; capillary column, HP-5 (30 m × 0.32 mm, Dikma); start temperature 200 °C, raised to 280 °C at a rate of 10 °C/min, and the final temperature was maintained for 24 min; and N2 was used as the carrier gas. The retention times of Dglucose, D-xylose, and D-apiose were determined to be 20.56, 14.94, and 14.56 min, respectively. Crystallographic Data. Compound 3a was recrystallized from EtOAc to produce colorless needles. The X-ray crystallographic structure of 3a was obtained by anomalous scattering of Cu Kα radiation. C15H24O4, M = 268.34, orthorhombic, a = 6.6770(3) Å, b = 14.8010(8) Å, c = 15.5028(8) Å, U = 1532.08(13) Å3, T = 119.2, space group P212121 (no. 19), Z = 4, μ(Cu Kα) = 0.674, 4971 reflections measured, and 2885 unique reflections (Rint = 0.0269) were used in all calculations. The final wR(F2) was 0.1017. Flack parameter, x = 0.1(2). The complete data were deposited at the Cambridge Crystallographic Data Centre (CCDC 1448943). Anti-inflammatory Activity Assay. The BV2 microglial cells were maintained in DMEM containing 10% FCS (fetal calf serum) and cultured at 37 °C (5% CO2, 100% relative humidity). After incubation for 24 h, the cells in 96-well plates were treated with different samples (three concentrations each), followed by stimulation with LPS (SigmaAldrich) for another 24 h. Then, 100 μL aliquots of the supernatants were added to 100 μL of Griess reagent (0.1% naphthylethylenediamine and 1% sulfanilamide in a 5% H3PO4 solution) at room temperature for 20 min. NO production was measured by the concentration of nitrite in the supernatant. The absorbances were measured at 540 nm, using curcumin as the positive drug. Hepatoprotective Activity Assay. The HepG2 cell line was maintained in DMEM containing 10% FCS and penicillin (100 U/ mL)−streptomycin (100 μg/mL) and cultured at 37 °C (5% CO2, 100% relative humidity). These cells were digested using 0.25% trypsin and then seeded into 96-well plates. After incubation for 12 h, the cells in the 96-well plates were treated with different samples (10 μM) and APAP (8 mM), and the samples were incubated for 48 h. Then, 100 μL of MTT reagent (0.5 mg/mL) was added to each well and incubated for 4 h. After removal of the media, 150 μL of DMSO was

(7R)-3,4-Dehydrohinesolone-11-O-β- D -glucopyranoside (1): white, amorphous powder; [α]20 D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 246 (4.29) nm; ECD (MeOH) λmax (Δε) 210 (−4.25), 241 (+4.02), 325 (−0.44) nm; IR (KBr) νmax 3344, 2970, 2914, 2894, 1666, 1619, 1018 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 397.2228 [M + H]+ (calcd for C21H33O7, 397.2226). (7R)-3,4-Dehydrohinesolone-11-O-β-D-apiofuranosyl-(1→6)-β-Dglucopyranoside (2): white, amorphous powder; [α]20 D −27 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 247 (4.24) nm; ECD (MeOH) λmax (Δε) 209 (−2.99), 244 (+3.41), 327 (−0.38) nm; IR (KBr) νmax 3399, 2971, 2927, 2884, 1655, 1600, 1045 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 529.2646 [M + H]+ (calcd for C26H41O11, 529.2649). (5R,7R)-14-Hydroxy-3,4-dehydrohinesolone-11-O-β-D-glucopyranoside (3): white, amorphous powder; [α]20 D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 246 (4.26) nm; ECD (MeOH) λmax (Δε) 213 (−3.60), 249 (+3.64), 329 (−0.42) nm; IR (KBr) νmax 3374, 2973, 2917, 1657, 1604, 1079, 1037 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 435.1986 [M + Na]+ (calcd for C21H32O8Na, 435.1995). (5R,7R)-14-Hydroxy-3,4-dehydrohinesolone-11-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (4): white, amorphous powder; [α]20 D −14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 246 (4.17) nm; ECD (MeOH) λmax (Δε) 247 (+2.65), 325 (−0.24) nm; IR (KBr) νmax 3371, 2929, 1656, 1601, 1051 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 567.2425 [M + Na]+ (calcd for C26H40O12Na, 567.2417). (5R,7R)-14-Hydroxy-3,4-dehydrohinesolone-14-O-β-D-xylopyranoside (5): white, amorphous powder; [α]20 D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 247 (4.19) nm; ECD (MeOH) λmax (Δε) 213 (−2.80), 253 (+2.48), 331 (−0.25) nm; IR (KBr) νmax 3372, 2971, 2927, 1658, 1609, 1049 cm−1; 1H and 13C NMR see data, Tables 1 and 3; HRESIMS m/z 427.1975 [M + COOH]− (calcd for C21H31O9, 427.1968). (4S,5S,7R)-15-Hydroxyhinesolone-15-O-β-D-xylopyranoside (6): white, amorphous powder; [α]20 D −77 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 240 (4.11) nm; ECD (MeOH) λmax (Δε) 236 (−3.19) nm; IR (KBr) νmax 3401, 2969, 2881, 1656, 1048 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 407.2034 [M + Na]+ (calcd for C20H32O7Na, 407.2046). (4R,5S,7R)-14-Hydroxyhinesolone-14-O-β-D-xylopyranoside (7): white, amorphous powder; [α]20 D −29 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 244 (3.94) nm; ECD (MeOH) λmax (Δε) 236 (−4.51), 307 (+0.14) nm; IR (KBr) νmax 3387, 2969, 2879, 1656, 1613, 1075, 1044 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 429.2137 [M + COOH]− (calcd for C21H33O9, 429.2125). (3S,4S,5S,7R)-3-Hydroxyhinesolone-11-O-β-D-glucopyranoside (8): white, amorphous powder; [α]20 D −117 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 240 (3.98) nm; ECD (MeOH) λmax (Δε) 242 (−14.48), 313 (+1.29) nm; IR (KBr) νmax 3386, 2972, 2881, 1670, 1078, 1030 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 459.2235 [M + COOH]− (calcd for C22H35O10, 459.2230). (3R,4S,7R,10R)-2-Hydroxypancherione-11-O-β-D-glucopyranoside (9): white, amorphous powder; amorphous powder; [α]20 D −16 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 245 (3.99) nm; ECD (MeOH) λmax (Δε) 244 (−11.82), 328 (+0.57) nm; IR (KBr) νmax 3404, 2969, 2926, 1694, 1630, 1078, 1035 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 459.2244 [M + COOH]− (calcd for C22H35O10, 459.2230). (2Z,8E)-Deca-2,8-diene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside (10): brown, amorphous powder; [α]20 D +48 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.38), 261 (3.83), 277 (4.02), 293 (4.17), 313 (4.08) nm; IR (KBr) νmax 3321, 2926, 2208, 1599, 1088, 1042 cm−1; 1H and 13C NMR data, see Tables 4 and 5; HRESIMS m/z 369.1194 [M + COOH]− (calcd for C17H21O9, 369.1186). (2E,8Z)-Deca-2,8-diene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside (11): brown, amorphous powder; [α]20 D −30 (c 0.1, MeOH− H2O, 1:3); UV (MeOH) λmax (log ε) 215 (4.24), 262 (3.70), 277 (3.93), 293 (4.08), 313 (3.99) nm; IR (KBr) νmax 3374, 2923, 2879, 2206, 2128, 1650, 1078, 1041 cm−1; 1H and 13C NMR data, see Tables H

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added to solubilize the residuum. Finally, the absorbances were measured at 570 nm, using bicyclol as the positive contrast.

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(22) He, J.; Shen, Y.; Jiang, J. S.; Yang, Y. N.; Feng, Z. M.; Zhang, P. C.; Yuan, S. P.; Hou, Q. Carbohydr. Res. 2011, 346, 1903−1908. (23) Kitajima, J.; Kamoshita, A.; Ishikawa, T.; Takano, A.; Fukuda, T.; Isoda, S.; Ida, Y. Chem. Pharm. Bull. 2003, 51, 673−678. (24) Yahara, S.; Higashi, T.; Iwaki, K.; Nohara, T.; Marubayashi, N.; Ueda, I.; Kohda, H.; Goto, K.; Izumi, H.; Nuno, M.; Katsuki, S.; Isoda, S.; Satake, M. Chem. Pharm. Bull. 1989, 37, 2995−3000. (25) Achenbach, H.; Lowel, M.; Waibel, R.; Gupta, M.; Solis, P. Planta Med. 1992, 58, 270−272. (26) Jiang, Y. P.; Liu, Y. F.; Guo, Q. L.; Jiang, Z. B.; Xu, C. B.; Zhu, C. G.; Yang, Y. C.; Lin, S.; Shi, J. G. J. Asian Nat. Prod. Res. 2015, 17, 601−614. (27) Morikawa, T.; Pan, Y.; Ninomiya, K.; Imura, K.; Yuan, D.; Yoshikawa, M.; Hayakawa, T.; Muraoka, O. Chem. Pharm. Bull. 2010, 58, 1403−1407. (28) Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35, 501−506. (29) Feng, Z. M.; Song, S.; He, J.; Yang, Y. N.; Jiang, J. S.; Zhang, P. C. Carbohydr. Res. 2013, 380, 59−63. (30) Shen, Y.; Feng, Z. M.; Jiang, J. S.; Yang, Y. N.; Zhang, P. C. J. Nat. Prod. 2013, 76, 2337−2345.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00066. UV, IR, ECD, NMR, and HRESIMS spectra for compounds 1−14 (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail (P.-C. Zhang): [email protected]. Tel: +86-1063165231. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Science and Technology Project of China (2012ZX09301002-001003) and the Fundamental Research Funds for the Central Institutes (No. 2014TD03).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00066 J. Nat. Prod. XXXX, XXX, XXX−XXX