Article pubs.acs.org/jnp
Anti-inflammatory Diterpenoids from Croton tonkinensis Ping-Chung Kuo,† Mei-Lin Yang,‡ Tsong-Long Hwang,§,⊥ Yuan-Yu Lai,† Yue-Chiun Li,† Tran Dinh Thang,∥ and Tian-Shung Wu*,‡ †
Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan, Republic of China Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan, Republic of China § Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan, Republic of China ⊥ Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan, Republic of China ∥ Department of Chemistry, Vinh University, Vinh City, Vietnam ‡
S Supporting Information *
ABSTRACT: Phytochemical investigation of the methanolic extract of Croton tonkinensis afforded two known kauranes (1, 2), eight new ent-kauranes (3−10), and 16 known ent-kauranetype diterpenoids (12−27). In addition, 30 known compounds were identified by comparison of their physical and spectroscopic data with reported data. Among the isolated compounds, ent-18-acetoxykaur-16-en-15-one (20) displayed the most significant inhibition of superoxide anion generation and elastase release.
V
the phytochemical constituents of C. tonkinensis are comprehensively studied. The anti-inflammatory bioactivity-monitored fractionation and purification of the MeOH extracts from the whole plant of C. tonkinensis led to the isolation of 26 diterpenoids, including eight new ent-kaurane diterpenoids, crotonkinins C−J (3−10), together with 30 known compounds. In addition, the purified diterpenoids were examined for inhibition of superoxide anion generation and elastase release to evaluate their anti-inflammatory potential. The structural determination of the new compounds and the bioactivity of the series of diterpenoids are reported herein.
arious human diseases, including rheumatoid arthritis, ischemia, reperfusion injury, chronic obstructive pulmonary disease, and asthma, have already been linked to neutrophil overexpression.1−5 In response to diverse stimuli, activated neutrophils secrete a series of cytotoxins, such as superoxide anion and elastase.6 Thus, in infected tissues and organs it was critical to maintain superoxide anion production and elastase release under physiological conditions. Nowadays only a few available agents could directly modulate neutrophil proinflammatory responses in clinical practice. In the preliminary assay, the crude extracts of Croton tonkinensis at 10 μg/mL displayed significant inhibition of superoxide anion generation and elastase release with percentages of 37.50 ± 1.95% and 77.58 ± 1.01%, respectively. In addition, purified diterpenoids from C. tonkinensis exhibited potent NO-reducing activities in microglial cells.7 As part of our continuous program aimed at new anti-inflammatory drug discovery, C. tonkinensis was selected as a target and reinvestigated for its chemical constituents. C. tonkinensis Gagnep. (Euphorbiaceae) is a tropical shrub native to Northern Vietnam and has been used commonly in Vietnam to treat stomachache, abscesses, impetigo, and gastric and duodenal ulcers, as well as to cure malaria, urticaria, leprosy, psoriasis, and genital organ prolapse.8−11 Prior phytochemical investigations showed that C. tonkinensis is a rich source of diterpenoids.7,12−18 In recent studies, antiinflammatory and cancer chemopreventive activities of C. tonkinensis extracts were also demonstrated. These effects were linked to the ability to inhibit the transcription nuclear factor kappa B (NF-κB), which was further correlated to the presence of ent-kaurane diterpenoids.14 Therefore, in the present paper © 2013 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION The air-dried whole plants of C. tonkinensis were extracted with MeOH, and the concentrated extracts (CT) were suspended in MeOH−H2O and extracted with n-hexane to afford an nhexane fraction (CTH). The residues were partitioned between CH2Cl2 and H2O to afford a CH2Cl2 fraction (CTD) and a H2O-soluble fraction (CTW), respectively. The fractions were examined for their inhibition effects of superoxide anion generation and elastase release (data as shown in Table S1). At the tested concentration (10 μg/mL), the CTH and CTD fractions displayed significant inhibition of superoxide anion generation and elastase release with percentages of 62.58 ± 5.41% and 88.04 ± 2.28%; 79.75 ± 2.87% and 107.29 ± 5.11%, respectively. The active fractions were subjected to further purification by a combination of conventional chromatographic Received: October 5, 2012 Published: January 24, 2013 230
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Figure 1. Chemical structures of crotonkinins A−J (1−10) and 11 characterized from C. tonkinensis.
orientation of the acetoxy group at C-11 in 3 as β. The complete assignments (Tables 1 and 2) established by 1D and 2D NMR experiments confirmed the structure of 3 as ent-11βacetoxy-17-oxokaur-15-en-18-oic acid, and 3 was given the trivial name crotonkinin C following the previous convention.7 In addition, detailed comparisons of the HMBC spectra of 2 and 3 showed similar correlations between H-11 and C-10 and the absence of correlations between H-11 and C-15/C-16, indicating that the acetoxy group in 2 was also positioned at C11, rather than at C-14 as previously reported.7 Thus, the structure of crotonkinin B (2) should be revised as shown in Figure 1. These HMBC correlation peaks (H-11/C-10 or H14/C-7, -15, -16) could provide a useful tool to differentiate between the C-11- and C-14-substituted ent-kaurane diterpenoids. Compound 4 was isolated as colorless needles with mp 114− 116 °C, and the HRESIMS data of 4 displayed a pseudomolecular [M − H]− ion peak at m/z 417.2259 corresponding to a molecular formula of C24H34O6. The UV, IR, and 1H NMR data of 4 resembled those of 2 except that an additional acetoxy group at δ 2.07 (3H, s, CH3-24) was present instead of the formyl group. The acetoxy group was positioned at C-18, as evidenced by the downfield shift of the C-18 methylene group at δ 3.63 (1H, d, J = 11.0 Hz) and 3.87 (1H, d, J = 11.0 Hz) compared with those of 2. The HMBC correlations from H-11 to C-13, -10, -8, -21; from H-18 to C19, -3, -5, -23; and from H-15 to C-13, -8, -16, -17 established the planar structure of 4 (Figure S1). The NOESY spectrum also displayed NOE correlations similar to those in 2 and 3; thus the structure of 4 was determined as ent-11β,18diacetoxykaur-15-en-17-oic acid as shown in Figure 1 and named crotonkinin D.
techniques to yield eight new ent-kaurane diterpenoids, crotonkinins C−J (3−10). In our previous paper, two kaurane-type diterpenoids, crotonkinins A (1) and B (2), from the title plant were reported, and the absolute configuration of 1 and 2 was determined from their positive optical rotation data.7 The UV, IR, and 1H and 13C NMR data indicated that compounds 3−10 (Figure 1) also belong to the kaurane diterpenoid class when compared to reported metabolites of this species.7,12−18 In contrast, the negative optical rotations of 3−10 indicated that these compounds possess the common ent-kaurane skeleton. The HRESIMS of 3 showed a pseudomolecular [M − H]− ion peak at m/z 373.2006, corresponding to the formula C22H30O5. The UV spectrum of 3 exhibited absorption maxima at 259 and 221 nm, compatible with an α,β-unsaturated carbonyl chromophore.19 The IR absorption bands at 3607 and 1732 cm−1 suggested the presence of hydroxy and ester carbonyl functionalities, respectively. In addition, IR signals characteristic for a Fermi resonance of a formyl group were also found at 2862 and 1373 cm−1. The 1H NMR spectrum of 3 displayed characteristic signals for two methyl singlets at δ 1.08 (3H, CH3-20) and 1.18 (3H, CH3-19), one acetyl methyl at δ 1.90 (3H, s, CH3-22), one oxygenated methine proton at δ 5.17 (1H, d, J = 5.4 Hz, H-11), one olefinic proton at δ 6.60 (1H, s, H-15), and one formyl proton at δ 9.75 (1H, s, H-17). Comparison of the UV, IR, and 1H NMR data of 3 with the reported crotonkinin B (2)7 inferred that the hydroxymethyl group at C-4 in 2 was oxidized to a carboxylic function in 3. In the HMBC spectrum, the 3J-HMBC correlations from the oxygenated methine at δ 5.17 (H-11) to carbons at δ 38.3 (C10) and 169.4 (C-21) provided evidence for the presence of an acetoxy function at C-11 in 3 (Figure S1). In the NOESY spectrum, the NOE correlation of H-11/CH3-20 indicated the 231
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showed 2J and 3J correlations from δ 3.61 (H-18) to δ 17.4 (C19), 35.7 (C-3), 50.2 (C-5), and 171.3 (C-21); from δ 2.21 (H13) to δ 23.2 (C-17), 40.4 (C-12), 44.8 (C-8), and 77.9 (C11); from δ 1.97 (H-14) to δ 40.4 (C-12), 57.2 (C-15), and 85.5 (C-16); from δ 1.54 (H-9) to δ 19.0 (C-20), 36.3 (C-10), 40.4 (C-1), 44.8 (C-8), and 77.9 (C-11); from δ 1.38 (H-15) to δ 37.5 (C-7), 45.6 (C-13), 59.1 (C-9), and 85.5 (C-16); and from δ 1.34 (CH3-17) to δ 45.6 (C-13), 57.2 (C-15), and 85.5 (C-16), respectively (Figure S1). The spectroscopic data also evidenced the presence of hydroxy groups at C-11 and C-16 and acetoxy substitution at C-18. The relative configuration of 8 at C-11 was determined by comparison of its NOE correlations with those in 5. Thus, the structure of 8 was deduced as ent-18-acetoxykaurane-11β,16-diol (Figure 1), but the absolute configuration at C-16 remains undefined. The molecular formula of 9 (C24H36O5) was determined from the ESIMS and HRESIMS data. The characteristic IR absorption bands and 1H NMR resonances of 9 were similar with those of 8. The differences in the 1H NMR spectra between 8 and 9 was that a hydroxy group in 8 was changed to an O-acetyl group in 9, which was identified by the downfield shift of H-11 from δ 4.35 to δ 5.10. In the 13C NMR spectrum of 9, the quaternary oxygenated carbon was replaced by one set of epoxide carbons at δ 62.4 (C-16) and 68.1 (C-15). This indicated that the C-16 tertiary hydroxy group was transformed into the C-15/C-16 epoxide, as evidenced by the HMBC correlations from δ 2.83 (H-15) to δ 31.3 (C-14) and 42.6 (C8); from δ 1.46 (CH3-17) to δ 38.2 (C-13), 62.4 (C-16), and 68.1 (C-15); and from δ 1.30 (H-9) to δ 18.0 (C-20), 31.3 (C14), 37.9 (C-10), 42.6 (C-8), and 68.1 (C-15), respectively (Figure S1). Similar NOE correlations characterized the structure of 9 as ent-11β,18-diacetoxykauran-15,16-epoxide, named crotonkinin I, but the absolute configuration at C-16 remains undefined. Crotonkinin J (10) was assigned a molecular formula of C23H34O5 from its HRESIMS analysis, which displayed a pseudomolecular [M + Na]+ ion peak at m/z 413.2291. This molecular formula implied one carbon less than that of crotonkinin E (5). In addition, the IR absorption bands and 1H NMR proton signals of 10 were similar to those of 5. The significant differences in the 1H NMR spectra between 10 and 5 were that the terminal methylene protons were absent and the H-13 proton signal was shifted upfield from δ 3.05 to δ 2.40. These features suggested the structural variations of 10 and 5 to be only in the D-ring. HMBC analysis showed 2J and 3 J correlations from H-11 to C-12, -10, -8, -13, -9, and -23; from H-13 to C-12 and -16; from H-15 to C-7, -9, and -16; and from H-12 to C-11, -13, and -16, respectively (Figure S1). These indicated the presence of 11,18-diacetoxy and 16-oxo functionalities in 10. The full assignments were confirmed by COSY, NOESY, HMQC, and HMBC experiments, and consequently the structure of 10 was established as ent11β,18-diacetoxynorkauran-16-one. Compound 11 was obtained as a colorless optically active powder, and its optical rotation data, UV absorption maxima, IR absorption bands, and 1H NMR signals were characteristic of the known (R)-N-(1′-methoxycarbonyl-2′-phenylethyl)-4hydroxybenzamide.21 However, the HRESIMS analytical data of 11 displayed a pseudomolecular [M + H]+ ion peak at m/z 284.1282, corresponding to the molecular formula C17H17NO3. NMR signals in the aromatic region at δ 7.13 (2H, d, J = 7.6 Hz, H-2′, -6′), 7.29 (3H, m, H-3′, -4′, -5′), 7.42 (2H, dd, J = 7.4, 7.3 Hz, H-3, -5), 7.50 (1H, t, J = 7.3 Hz, H-4), and 7.72
On the basis of the HRESIMS data (m/z 425.2298 [M + Na]+ for 5 and m/z 403.2475 [M + H]+ for 6), crotonkinins E (5) and F (6) were considered to have the same molecular formula, C24H34O5, and this suggested that these two compounds were isomers. Both their UV and IR data indicated the presence of an α,β-unsaturated carbonyl chromophore19 and carbonyl functionalities. In the 1H NMR spectra of 5 and 6, there were similar characteristic signals for two methyl groups, two acetoxy methyl groups, one oxymethylene group, one oxymethine proton, and two terminal olefinic protons. The only difference between 5 and 6 involved the oxymethines at δ 5.13 (1H, d, J = 4.6 Hz) in 5 and at δ 5.13 (1H, dd, J = 11.1, 4.6 Hz) in 6. In the HMBC spectrum, there were 3J correlations from the proton at δ 5.13 to C-10 in 5, indicating C-11 substitution; however, in 6 the proton at δ 5.13 displayed 2J and 3 J correlations to C-8 and -14, respectively (Figure S1) and indicated C-7 substitution in 6. The NOESY correlations of H11/CH3-20 in 5 and H-7/H-9 and H-7/H-5 in 6 determined their stereochemistry unambiguously. The complete assignments of the proton and carbon signals were furnished by comprehensive 2D NMR experiments. The structure of 5 was established as ent-11β,18-diacetoxykaur-16-en-15-one, and that of 6 as ent-7α,18-diacetoxykaur-16-en-15-one. The physical and spectroscopic data of crotonkinin F (6) are in agreement with those reported20 for the semisynthetic compound resulting from acetylation of ent-18-acetoxy-7α-hydroxykaur-16-en-15one. The pseudomolecular [M + H]+ ion at m/z 393.2632 from HRESIMS analysis revealed that the molecular formula of crotonkinin G (7) was C23H36O5. The IR absorption band at 1728 cm−1 suggested the presence of a carbonyl functionality. In the 1H NMR spectrum, there were characteristic signals for two methyl singlets at δ 0.85 (3H, CH3-19) and 1.13 (3H, CH3-20), one acetoxy methyl singlet at δ 2.10 (3H, CH3-22), two methine protons at δ 2.61 (1H, ddd, J = 11.4, 9.6, 5.7 Hz, H-16) and 2.72 (1H, m, H-13), one methoxy group at δ 3.38 (3H, s, OCH3-23), and one oxymethine at δ 3.91 (1H, dd, J = 11.6, 4.3 Hz, H-7). COSY analysis showed there were three sets of methylene groups located at δ 2.02 and 2.11 (1H each, br d, J = 12.3 Hz, and d, J = 12.3 Hz, H-14), 3.45 and 3.81 (1H each, dd, J = 9.9, 9.6 Hz, and dd, J = 9.9, 5.7 Hz, H-17), and 3.64 and 3.88 (1H each, d, J = 11.1 Hz, H-18), respectively. In addition, the 2J- and 3J-HMBC correlations could be found among H-7/ C-6, -14, -8, -15; H-18/C-19, -3, -4, -5, -21; H-17/C-13, -16, -23, -15; OCH3-23/C-17; H-16/C-12, -13, -15; and H-14/C12, -13, -9, -16, -8, -15, and these correlations revealed the substitution pattern of 7 (Figure S1). Similar NOESY correlations of H-7/H-9 and H-7/H-5 in 7 to those in 6 determined the C-7 hydroxy orientation as α. Accordingly, the structure of crotonkinin G (7) was characterized as ent-18acetoxy-7α-hydroxy-17-methoxykauran-15-one, but the absolute configuration at C-16 remains undefined. On the basis of the ESIMS and HRESIMS analyses, crotonkinin H (8) was considered to have the molecular formula C22H36O4. The 1H and 13C NMR spectra of 8 revealed different signals from those described above, including three methyl singlets at δ 0.85 (3H, CH3-19), 1.12 (3H, CH3-20), and 1.34 (3H, CH3-17), rather than two in the 1H NMR spectrum of 6, and one quaternary oxygenated carbon signal at δ 85.5 (C-16) in the 13C NMR spectrum. These features indicated that the C-16/C-17 terminal methylene group in 6 was hydrated to form a tertiary hydroxy functionality at C-16. This assumption was verified by the HMBC spectrum, which 232
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Table 1. 1H NMR Data of Diterpenoids 3−10 [(400 MHz, CDCl3, δ ppm (J = Hz)] H
3
1
0.99, m; 1.99, m 1.49, m; 2.01, m 1.76, m 1.76, m
2 3 5 6
13 14
0.99, m; 1.59, m 1.59, m; 2.18, br d (11.0) 1.37, br s 5.17, d (5.4) 1.66, m; 1.88, m 2.99, m 1.88, m
15
6.60, s
7 9 11 12
4
5
6
7
8
9
10
0.86, m; 1.98, m
0.85, m; 1.92, m
0.77, m; 1.81, m
1.05, m; 1.90, m
0.90, m; 1.97, m
0.96, m; 2.00, m
1.36, m; 1.48, m
1.61, m
1.65, m
0.69, td (11.9, 5.7); 1.78, m 1.47, m
1.41, m
1.14, m; 1.20, m
1.55, m; 1.65, m
1.36, m 1.15, d (10.7)
1.34, m 1.34, m
1.31, m; 1.40, m 1.40, m
1.35, m 1.25, m
1.38, m 1.09, m
1.34, m 1.12, m
1.48, m; 1.65, m
1.09, m; 1.61, m
1.40, m; 1.81, m
1.40, m; 1.66, m
1.47, m; 1.53, m
1.71, m
1.34, m; 1.92, m
3.91, dd (11.6, 4.3)
1.53, m; 1.79, m
1.65, m
1.25, br s 5.17, d (3.7) 1.94, m
1.45, m 5.13, d (4.6) 1.45, m; 2.41, d (12.2) 3.05, m 1.92, m
5.13, dd (11.1, 4.6) 1.29, m 1.51, m; 1.62, m 1.71, m; 1.97, m
1.48, br d (3.6); 1.54, m 1.41, m
1.38, m 1.18, dd (11.3, 2.4) 1.49, m
1.30, br s 5.10, br s 1.86, m
3.10, m 2.00, m; 2.16, m
2.72, m 2.02, br d (12.3); 2.11, d (12.3)
1.54, m 4.35, br s 1.76, br d (12.3); 2.03, d (12.3) 2.21, m 1.25, m; 1.97, dd (11.7, 3.5) 1.38, m
1.43, m 5.20, d (4.1) 1.49, m; 2.27, dd (12.1, 4.1) 2.40, m 2.00, m; 2.06, m
1.34, s
1.46, s
3.61, d (10.9); 3.85, d (10.9) 0.85, s 1.12, s 2.06, s
3.64, d (11.0); 3.85, d (11.0) 0.81, s 0.99, s 2.01, s 2.07, s
2.89, m 1.65, m; 2.17, d (10.9) 6.64, s
16 17
9.75, s
18 CH3-19 CH3-20 CH3-22 CH3-24 OCH3
3.63, d (11.0); 3.87, d (11.0) 0.82, s 1.08, s 1.93, s 2.07, s
1.18, s 1.08, s 1.90, s
5.22, s; 5.88, s
5.28, s; 5.95, s
3.63, d (11.0); 3.86, d (11.0) 0.79, s 1.09, s 1.85, s 2.09, s
3.58, d (11.2); 3.85, d (11.2) 0.80, s 1.14, s 1.90, s 2.13, s
1.13, m 1.25, m; 1.66, m 1.78, m
2.61, ddd (11.4, 9.6, 5.7) 3.45, dd (9.9, 9.6); 3.81, dd (9.9, 5.7) 3.64, d (11.1); 3.88, d (11.1) 0.85, s 1.13, s 2.10, s
2.15, m 1.17, m; 1.44, m 2.83, s
1.25, d (18.4); 2.44, d (18.4)
3.63, d (11.0); 3.89, d (11.0) 0.85, s 1.08, s 1.97, s 2.07, s
3.38, s
Table 2. 13C NMR Data of Diterpenoids 1−10 (100 MHz, CDCl3) C
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 OCH3
40.4 18.4 41.7 33.6 53.8 37.9 212.9 62.5 59.9 39.2 16.8 32.3 50.4 77.6 38.2 151.7 108.3 32.8 21.0 16.7
39.1 17.7 35.1 37.3 48.4 18.3 42.3 49.4 55.3 38.8 68.5 37.6 36.1 34.5 162.5 150.7 189.2 71.8 17.5 18.1 169.3 21.4
38.6 21.7 36.8 47.3 49.4 17.5 42.2 49.5 55.4 38.3 68.1 37.1 36.1 34.4 162.1 150.7 189.2 182.7 16.2 18.0 169.4 21.4
39.0 18.5 35.6 36.5 49.3 17.5 37.3 49.1 55.0 38.8 68.6 34.4 38.5 42.3 156.9 139.6 169.0 72.6 17.4 18.1 169.6 21.4 171.3 21.0
38.9 18.2 35.3 36.4 48.6 17.8 33.1 50.5 59.5 38.7 68.2 36.3 36.6 38.5 209.0 149.9 113.2 72.3 17.8 18.4 169.5 21.4 171.3 21.1
38.9 17.9 35.7 36.3 45.8 24.3 73.1 56.2 51.9 39.6 17.6 32.6 37.5 29.2 207.5 148.7 115.3 72.0 17.7 18.2 169.6 21.0 171.5 21.1
38.5 17.4 35.4 36.3 46.3 28.2 71.0 58.7 51.8 39.1 18.0 25.3 32.0 28.2 221.1 54.2 69.2 72.2 17.4 18.2 171.3 21.1
40.4 19.9 35.7 36.7 50.2 17.6 37.5 44.8 59.1 36.3 77.9 40.4 45.6 43.8 57.2 85.5 23.2 73.0 17.9 19.0 171.3 21.0
39.4 18.6 35.6 36.5 49.6 17.5 34.7 42.6 58.0 37.9 68.4 34.3 38.2 31.3 68.1 62.4 15.4 72.8 17.4 18.0 169.4 21.4 171.2 21.0
39.4 17.6 35.5 36.6 49.6 18.9 40.4 41.3 60.7 38.2 68.4 37.3 46.1 36.9 54.3 220.4 72.6 17.6 18.3 169.6 21.5 171.3 21.0
58.9
(2H, d, J = 7.4 Hz, H-2, -6) indicated the presence of two sets of monosubstituted benzene rings. Thus, the corresponding signal at δ 133.9 indicated the absence of a C-4 group.
Therefore, the complete assignments established by 1D and 2D NMR experiments indicated that the structure of 11 should be 233
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Table 3. Inhibitory Effects of Purified Samples from C. tonkinensis on Superoxide Anion Generation and Elastase Release by Human Neutrophils in Response to N-Formyl-Lmethionylphenylalanine/Cytochalasin B (FMLP/CB)
revised to (R)-N-(1-methoxycarbonyl-2-phenylethyl)benzamide as shown in Figure 1. In addition to crotonkinins A−J (1−10) and 11, 45 additional compounds were characterized from the MeOH extracts of C. tonkinensis, including 16 diterpenoids, (16S)-ent18-acetoxy-7β-hydroxykaur-15-one (12), ent-18-acetoxy-7α-hydroxykaur-16-en-15-one (13), ent-1β-acetoxy-7α,14β-dihydroxykaur-16-en-15-one (14), ent-14β-acetoxy-7α-hydroxykaur-16en-15-one (15), ent-7α,14β-dihydroxykaur-16-en-15-one (16), crotonkinensin A (17), ent-7β,18-dihydroxykaur-16-en-15-one (18), ent-15-oxokaur-16-en-18-oic acid (19), ent-18-acetoxykaur-16-en-15-one (20), ent-7β-hydroxykaur-16-en-15-one (21), ent-14β-hydroxykaur-16-en-15-one (22), ent-11α-acetoxykaur-16-en-18-oic acid (23), ent-18-hydroxykaur-16-en-15-one (24), ent-18-acetoxy-7α-hydroxykaur-16-ene (25), ent-11α,18diacetoxy-7β-hydroxykaur-16-en-15-one (26), and ent-18-acetoxy-7α,14β-dihydroxykaur-16-en-15-one (27); nine steroids, i.e., a mixture of β-sitosterol and stigmasterol, a mixture of stigmasta-4-en-3-one and stigmasta-4,22-dien-3-one, a mixture of stigmastan-3-one and stigmast-22-en-3-one, ergosterol peroxide, and a mixture of 3β-hydroxysitost-5-en-7-one and 3β-hydroxystigmasta-5,22-dien-7-one; one triterpenoid, 3-acetylaleuritolic acid; four ionones, dehydrovomifoliol, (S)-2-cisabscisic acid, phaseic acid, and blumenol-A; two alkaloids, indole-3-carboxylic acid methyl ester and corydaldine; three lignans, sesamin, pinoresinol, and syringaresinol; and 10 benzenoids, methylparaben, p-hydroxybenzoic acid, (E)-methyl ferulate, vanillin, p-hydroxybenzaldehyde, syringaldehyde, 4isopropylbenzoic acid, 3,4-dimethoxybenzoic acid, vanillic acid, and benzoic acid, respectively. These known compounds were identified by comparison of their physical and spectral data with those reported. The purified diterpenoids isolated in sufficient quantities were examined for their inhibition of superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB (Table 3), and most of them at 10 μM concentration exhibited inhibition percentages higher than 50%. Among these, ent-18-acetoxy-7α-hydroxykaur-16-en-15one (13), ent-1β-acetoxy-7α,14β-dihydroxykaur-16-en-15-one (14), ent-7α-hydroxy-14β-acetoxykaur-16-en-15-one (15), crotonkinensin A (17), ent-7β-hydroxy-16-kauren-15-one (21), and ent-11α,18-diacetoxy-7β-hydroxykaur-16-en-15-one (26) displayed significant inhibition of superoxide anion generation and elastase release with IC50 values ranging from 1.12 ± 0.06 to 2.64 ± 0.12 μM, compared with the reference compound LY294002,22 which displayed an IC50 value of 1.12 ± 0.20 and 1.92 ± 0.22 μM toward superoxide anion generation and elastase release, respectively. In addition, diphenyleniodonium (DPI), an NADPH oxidase inhibitor, was also used as a positive control for superoxide anion generation, with an IC50 value of 0.93 ± 0.52 μM. Moreover, ent-18-acetoxykaur-16-en-15-one (20) displayed 1-fold higher inhibition of superoxide anion generation and elastase release than the reference compounds, with an IC50 of 0.13 ± 0.02 and 0.24 ± 0.04 μM, respectively. In our previous report, compound 20 exhibited only moderate cytotoxicity.7 Therefore, the extracts and purified compounds of C. tonkinensis have potential to be developed as new antiinflammatory drugs or health foods.
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IC50 (μM)a or (Inh %)b compound 1 2 3 4 5 6 7 8 10 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 LY294002d DPIe
superoxide anion generation ± 3.72) ± 0.69 ± 1.15 ± 0.99 ± 1.01 ± 0.52 ± 1.31 ± 6.48) ± 1.22 ± 4.81)** ± 0.39 ± 0.64 ± 0.06 ± 0.72 ± 0.23 ± 0.54 ± 1.07 ± 0.02 ± 0.35 ± 7.29)** ± 0.45 ± 1.69)*** NTc 1.33 ± 0.15 (19.00 ± 6.12)* 1.12 ± 0.20 0.93 ± 0.52
(1.65 3.20 4.96 2.93 3.11 2.88 4.29 (−10.87 4.76 (40.43 1.55 1.94 1.12 1.96 2.06 3.95 3.27 0.13 1.74 (33.63 4.12 (29.89
elastase release ± 3.85) ± 1.43 ± 0.81)*** ± 5.58)* ± 1.60 ± 1.45 ± 6.98) ± 5.31) ± 5.02)*** ± 2.86) ± 0.12 ± 0.37 ± 0.27 ± 0.28 ± 0.18 ± 3.30)*** ± 1.48 ± 0.04 ± 0.10 ± 4.82) ± 0.30 ± 3.82)** NT 1.65 ± 0.29 (20.09 ± 3.57)** 1.92 ± 0.22
(−9.32 4.38 (41.75 (20.27 4.66 4.44 (11.14 (23.26 (48.75 (−0.19 2.64 2.48 2.36 3.22 1.63 (45.77 5.56 0.24 2.23 (3.64 2.58 (17.96
a
Concentration necessary for 50% inhibition. bPercentage of inhibition (Inh %) at 10 μM concentration. Results are presented as mean ± SD (n = 3−4). *p < 0.05. **p < 0.01. ***p < 0.001 compared with the control value. cNT: not tested due to the low solubility. dA phosphatidylinositol-3-kinase inhibitor was used as a positive control for superoxide anion generation and elastase release. eA NADPH oxidase inhibitor was used as a positive control for superoxide anion generation. measured using a Jasco DIP-370 digital polarimeter. The UV spectra were obtained on a GBC Cintra 101 UV−vis spectrophotometer. The IR spectra were recorded on a Shimadzu FT-IR DR-8011 spectrophotometer. 1H and 13C NMR, COSY, HMQC, HMBC, and NOESY spectra were recorded on Bruker Avance III-400 and Avance 300 NMR spectrometers, using TMS as the internal standard. Standard pulse sequences and parameters were used for the NMR experiments, and all chemical shifts were reported in parts per million (ppm, δ). The low- and high-resolution ESI mass spectra were obtained on a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer (San Jose, CA, USA) equipped with an atmosphericpressure chemical ionization (APCI) source operated in both the positive-ion and negative-ion modes. Chemicals were purchased from Merck KGaA (Darmstadt, Germany) unless specifically indicated. Column chromatography was performed on silica gel (Kieselgel 60, 70−230 mesh, Merck KGaA). TLC was conducted on precoated Kieselgel 60 F 254 plates (Merck), and the compounds were visualized by UV light or spraying with 10% (v/v) H2SO4 followed by heating at 110 °C for 10 min. Plant Materials. The whole plant of C. tonkinensis Gagnep. (Euphorbiaceae) was collected from Vietnam in August 2009, and the plant material was identified and authenticated by Assoc. Prof. Dr. Vu Xuan Phuong, Institute of Ecology and Biological Resources,
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points of purified compounds were determined by a Fisher Scientific melting point measuring apparatus without corrections. Optical rotations were 234
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C NMR (100 MHz, CDCl3), see Table 2; ESIMS m/z 373 [M − H]− (100), 339 (16), 335 (12); HRESIMS m/z 373.2006 [M − H]− (calcd for C22H29O5, 373.2010). Crotonkinin D (4): colorless needles (CHCl3); mp 114−116 °C; [α]25D −195 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 233 (3.66) nm; IR (neat) νmax 3300, 2932, 2859, 1732, 1632, 1454, 1377, 1253, 1030 cm−1; 1H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2; ESIMS m/z 417 [M − H]− (100), 375 (20); HRESIMS m/z 417.2259 [M − H]− (calcd for C24H33O6, 417.2272). Crotonkinin E (5): colorless crystals (CHCl3); mp 143−145 °C; [α]25D −81 (c 1.0, CHCl3); UV (MeOH) λ max (log ε) 231 (3.38) nm; IR (neat) νmax 3499, 2940, 2882, 1728, 1450, 1381, 1250, 1026 cm−1; 1 H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2; ESIMS m/z 425 [M + Na]+ (100), 360 (72); HRESIMS m/z 425.2298 [M + Na]+ (calcd for C24H34O5Na, 425.2298). Crotonkinin F (6): colorless needles (CHCl3); mp 161−163 °C; [α]25D −70 (c 2.4, CHCl3); UV (MeOH) λmax (log ε) 229 (3.36) nm; IR (neat) νmax 3499, 2940, 2889, 1732, 1454, 1377, 1250, 1022 cm−1; 1 H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2; ESIMS m/z 425 [M + Na]+ (100), 360 (7); HRESIMS m/z 403.2475 [M + H]+ (calcd for C24H35O5, 403.2479). Crotonkinin G (7): white powder (CHCl3); mp 155 °C (dec); [α]25D −244 (c 1.2, CHCl3); IR (neat) νmax 3507, 2928, 2882, 1728, 1454, 1377, 1246, 1030 cm−1; 1H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2; ESIMS m/z 415 [M + Na]+ (100), 393 [M + H]+ (33), 375 (21); HRESIMS m/z 393.2632 [M + H]+ (calcd for C23H37O5, 393.2636). Crotonkinin H (8): colorless crystals (CHCl3); mp 90−91 °C; [α]25D −71 (c 0.6, CHCl3); IR (neat) νmax 3507, 2936, 2873, 1736, 1454, 1373, 1242, 1026 cm−1; 1H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2; ESIMS m/z 365 [M + H]+ (89), 333 (100); HRESIMS m/z 365.2652 [M + H]+ (calcd for C22H37O4, 365.2686). Crotonkinin I (9): colorless needles (CHCl3); mp 92−93 °C; [α]25D −186 (c 0.1, CHCl3); IR (neat) νmax 2931, 1738, 1450, 1380, 1244, 1037 cm−1; 1H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2; ESIMS m/z 427 [M + Na]+ (26), 425 (22), 409 (23), 360 (100); HRESIMS m/z 427.2475 [M + Na]+ (calcd for C24H36O5Na, 427.2455). Crotonkinin J (10): white powder (CHCl3); mp 83−85 °C; [α]25D −58 (c 0.2, CHCl3); IR (neat) νmax 2928, 2855, 1740, 1462, 1373, 1242, 1018 cm−1; 1H NMR (400 MHz, CDCl3), see Table 1; 13C NMR (100 MHz, CDCl3), see Table 2; ESIMS m/z 413 [M + Na]+ (100), 371 (36), 361 (40); HRESIMS m/z 413.2291 [M + Na]+ (calcd for C23H34O5Na, 413.2298). (R)-N-(1-Methoxycarbonyl-2-phenylethyl)benzamide (11): colorless needles (CHCl3); mp, [α]D25, UV, IR, see ref 21; 1H NMR (300 MHz, CDCl3) δ 7.72 (2H, d, J = 7.4 Hz, H-2, -6), 7.50 (1H, t, J = 7.3 Hz, H-4), 7.42 (2H, dd, J = 7.4, 7.3 Hz, H-3, -5), 7.29 (3H, m, H-3′, -4′, -5′), 7.13 (2H, d, J = 7.6 Hz, H-2′, -6′), 6.57 (1H, d, J = 7.2 Hz, D2O exchangeable, NH), 5.10 (1H, ddd, J = 7.2, 5.8, 5.5 Hz, H-8′), 3.77 (3H, s, OCH3), 3.30 (1H, dd, J = 12.7, 5.5 Hz, H-7′), 3.23 (1H, dd, J = 12.7, 5.8 Hz, H-7′); 13C NMR, see ref 21; ESIMS m/z 306 [M + Na]+ (100), 284 [M + H]+ (95); HRESIMS m/z 284.1282 [M + H]+ (calcd for C17H18NO3, 284.1281). Preparation of Human Neutrophils. Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. Blood was drawn from healthy human donors (20−30 years old) by venipuncture into heparin-coated vacutainer tubes, using a protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital. Blood samples were mixed gently with an equal volume of 3% dextran solution. The leukocyte-rich plasma was collected after sedimentation of the red cells for 30 min at room temperature. The leukocyte-rich plasma was transferred on top of 20 mL of Ficoll solution (1.077 g/mL) and spun down at 400g for 40 min at 20 °C. The granulocyte/erythrocyte pellets were resuspended in icecold 0.2% NaCl to lyse erythrocytes. After 30 s, the same volume of 13
Vietnamese Academy of Science and Technology. A voucher specimen (Viet-TSWu-2009-0901-001) was deposited in the herbarium of the Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hanoi, Vietnam. Extraction and Isolation. Air-dried and powdered whole plants of C. tonkinensis (10.0 kg) were extracted with MeOH (10 × 25 L) under reflux for 8 h and concentrated to give a brown syrup (700 g). The MeOH extract was suspended in MeOH−H2O (90:10) and partitioned with n-hexane to afford n-hexane solubles (250 g). The solvent was removed under vacuum from the MeOH−H2O fraction, and the residue was fractionated between CH2Cl2 and H2O to afford CH2Cl2 (300 g), and H2O fractions (150 g), respectively. The n-hexane-soluble fraction (CTH) was subjected to silica gel column chromatography (SiO2 CC) eluted with a step gradient of nhexane−acetone (100:1 to 50:50) to afford 19 fractions (H1−H19) based on TLC. Fraction H3 (eluted with n-hexane−acetone, 90:10) was subjected to a second SiO2 CC (eluted with the step gradient of nhexane−acetone, 100:1 to 50:50) and afforded four subfractions (H31−H3-4). Only subfraction H3-3 (eluted with n-hexane−acetone, 80:20) displayed significant spots and was applied to SiO2 CC (eluted with benzene−acetone, 100:1 to 1:1) to afford five minor fractions (H3-3-1−H3-3-5). The minor fraction H3-3-3 (eluted with benzene− acetone, 80:20) was separated by SiO2 CC (eluted with CHCl3− acetone, 100:1) and further purified by preparative TLC (pTLC) to afford crotonkinins E (5) (10.0 mg) and F (6) (24.0 mg), respectively. Fraction H5 (eluted with n-hexane−acetone, 85:15) was subjected to SiO2 CC (eluted with the step gradient of n-hexane−EtOAc, 300:1 to 1:1) to afford four subfractions (H5-1−H5-4). Subfraction H5-2 (eluted with n-hexane−EtOAc, 100:1) was purified by SiO2 CC (eluted with benzene) and further recrystallized with acetone to yield crotonkinins D (4) (3.5 mg) and J (10) (1.7 mg). Subfraction H5-3 (eluted with n-hexane−EtOAc, 85:15) was applied to SiO2 CC (eluted with n-hexane−CHCl3, 1:1) and further recrystallized with CHCl3− MeOH to yield crotonkinin I (9) (1.2 mg). Fractions H6−H10 displayed similar spots under TLC monitoring and were combined. The combined samples were applied to SiO2 CC (eluted with the step gradient of n-hexane−EtOAc, 20:1 to 1:1) to afford five subfractions (H6-1−H6-5). Only subfraction H6-4 (eluted with n-hexane−EtOAc, 3:1) displayed significant spots and was purified with SiO2 CC (eluted with the step gradient of n-hexane−acetone, 100:1 to 1:1) to afford three minor fractions (H6-4-1−H6-4-3). The minor fraction H6-4-2 (eluted with n-hexane−acetone, 85:15) was further purified by pTLC (eluted with benzene−EtOAc, 300:1) to yield crotonkinin H (8) (20.0 mg). Fraction H15 (eluted with n-hexane−acetone, 65:35) was subjected to SiO2 CC (eluted with the step gradient of benzene− acetone, 50:1 to 1:1) to yield four subfractions (H15-1−H15-4). Subfraction H15-2 (eluted with benzene−acetone, 10:1) was isolated by SiO2 CC (eluted with the step gradient of CHCl3−acetone, 50:1 to 1:1) to afford five minor fractions (H15-2-1−H15-2-5). The minor fraction H15-2-4 (eluted with CHCl3−acetone, 10:1) was also purified by pTLC (eluted with n-hexane−acetone, 5:1) to afford crotonkinin C (3) (1.5 mg). Fraction H16 (eluted with n-hexane−acetone, 60:40) was applied to SiO2 CC (eluted with n-hexane−EtOAc, 10:1) to yield five subfractions (H16-1−H16-5). Subfraction H16-4 was purified by SiO2 CC (eluted with the step gradient of CHCl3−acetone, 10:1 to 1:1) and minor fraction H16-4-2 (eluted with CHCl3−acetone, 5:1) was further purified by pTLC (eluted with benzene−acetone, 6:1) to afford crotonkinin G (7) (12.3 mg). The CH2Cl2 solubles (CTD) were subjected to SiO2 CC (eluted with the step gradient of n-hexane−acetone, 50:1 to 1:1) to obtain nine fractions (D1−D9) based on TLC profile. Fractions D4 and D5 (eluted with n-hexane−acetone, 10:1) were combined and purified by SiO2 CC (eluted with CH2Cl2−acetone, 20:1) to result in six subfractions (D4-1−D4-6). Subfraction D4-4 was purified by pTLC (eluted with CHCl3−MeOH, 100:1) to afford crotonkinin F (6) (24.2 mg). Crotonkinin C (3): white powder (CHCl3); mp 117−118 °C; [α]25D −160 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 259 (3.16), 221 (3.64) nm; IR (neat) νmax 3607, 2924, 2862, 2673, 1732, 1458, 1373, 1265, 1192, 1026 cm−1; 1H NMR (400 MHz, CDCl3), see Table 1; 235
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1.6% NaCl solution was added to reconstitute the isotonic condition. Purified neutrophils were pelleted and resuspended in a calcium (Ca2+)-free Hank’s balanced salt solution (HBSS) buffer at pH 7.4 and were maintained at 4 °C before use. Measurement of Superoxide Anion Generation. The assay of the generation of superoxide anion was based on the SOD-inhibitable reduction of ferricytochrome c.6 In brief, after supplementation with 0.5 mg/mL ferricytochrome c and 1 mM Ca2+, neutrophils (6 × 105 cells/mL) were equilibrated at 37 °C for 2 min and incubated with drugs or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min. Cells were activated with 100 nM FMLP during the preincubation of 1 μg/mL cytochalasin B (FMLP/CB) for 3 min. Changes in the absorbance with a reduction in ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer with constant stirring (Hitachi U-3010, Tokyo, Japan). Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm). Measurement of Elastase Release. Degranulation of azurophilic granules was determined by elastase release as described previously.6 Experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), neutrophils (6 × 105/mL) were equilibrated at 37 °C for 2 min and incubated with drugs or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min. Cells were activated by 100 nM FMLP and 0.5 μg/mL cytochalasin B, and changes in absorbance at 405 nm were continuously monitored to assay elastase release. The results were expressed as the percent of elastase release in the FMLP/CB-activated, drug-free control system. Statistical Analysis. Results were expressed as mean ± SE. Computation of 50% inhibitory concentration (IC50) was computerassisted (PHARM/PCS v.4.2). Statistical comparisons were made between groups using Student’s t test. Values of p less than 0.05 were considered to be statistically significant.
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ASSOCIATED CONTENT
* Supporting Information S
Extraction and purification of the methanol extracts of C. tonkinensis, the known compounds, and their references, along with the NMR spectra for 3−11, are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +886-6-2747538. Fax: +886-6-2740552. E-mail: tswu@ mail.ncku.edu.tw. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful for financial support from the National Science Council of Republic of China awarded to T.-S.W.
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REFERENCES
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