Article pubs.acs.org/jnp
Inflammation Modulatory Phorbol Esters from the Seeds of Aquilaria malaccensis Vitthal D. Wagh,†,◇ Michal Korinek,†,‡,◇ I-Wen Lo,† Yu-Ming Hsu,† Shu-Li Chen,† Hsue-Yin Hsu,§ Tsong-Long Hwang,⊥,∥,▽ Yang-Chang Wu,†,# Bing-Hung Chen,*,‡,⬡,δ Yuan-Bin Cheng,*,†,¶,# and Fang-Rong Chang*,†,¶,□,△ †
Graduate Institute of Natural Products, College of Pharmacy, ‡Department of Biotechnology, College of Life Science, ¶Center for Infectious Disease and Cancer Research, and #Research Center for Natural Products & Drug Development, Kaohsiung Medical University, Kaohsiung 807, Taiwan § Department of Life Sciences, Tzu Chi University, Hualien 970, Taiwan ⊥ Graduate Institute of Natural Products, College of Medicine, and Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan ∥ Research Center for Chinese Herbal Medicine, Research Center for Food and Cosmetic Safety, and Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan ▽ Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan ⬡ The Institute of Biomedical Sciences and △Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan □ Cancer Center and δDepartment of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan S Supporting Information *
ABSTRACT: The tree Aquilaria malaccensis is a valuable source of agarwood, which is used in herbal medicinal preparations. Phytochemical research on A. malaccensis seeds has led to the isolation of four new phorbol esters (1−4), two known phorbol esters (5, isolated from Nature for the first time, and 6), and two known glycerides (7 and 8). The structures of these isolates were elucidated by means of spectroscopic data interpretation. The inflammation-modulatory activities of the isolates on elastase release and superoxide anion generation in human neutrophils were evaluated. Interestingly, phorbol esters 1, 5, and 6 showed potent inhibitory activity on elastase release in human neutrophils, with IC50 values of 2.7, 0.8, and 2.1 μM, respectively. All isolated phorbol esters exerted enhancing activity on superoxide anion generation. The results indicated that phorbol esters may play a bilateral modulatory role in the processes of inflammation. In addition, the compounds were evaluated for their cytotoxic properties against HepG2 (hepatoma), MDA-MB-231 (breast), and A549 (lung) cancer cells, but all compounds were inactive for all cell lines used (IC50 > 10 μM). Aquilaria malaccensis Lam. (family Thymelaeaceae) is a tropical tree native to Malaysia, known locally as “Karas”. The tree is distributed also in the rainforests of Cambodia, India, Indonesia, Laos, the Philippines, Taiwan, and Thailand.1 This species serves as the source of agarwood, a valuable fragrant resinous wood, which is used widely in religious ceremonies as well as in traditional medicinal preparations in Asia.2 Agarwood oil has been used as antiasthmatic, aphrodisiac, astringent, cardiotonic, © 2017 American Chemical Society and American Society of Pharmacognosy
and carminative agents and as a remedy for diarrhea, dysentery, gout, rheumatism, and paralysis.3 Extracts of A. malaccensis have been investigated for their cardiotonic,4 cytotoxic,5 antitrypanosomal,6 antibacterial,7 and antiallergic8,9 activities. Received: November 25, 2016 Published: April 26, 2017 1421
DOI: 10.1021/acs.jnatprod.6b01096 J. Nat. Prod. 2017, 80, 1421−1427
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Chart 1
(Figure 1) showed correlations between H-10 (δ 3.27)/H-1 (δ 7.59)/Me-19 (δ 1.78), H2-5 (δ 2.49 and 2.55)/H-7 (δ 5.69)/H-8 (δ 3.27), H-14 (δ 1.12), and H-12 (δ 5.51)/H-11 (δ 2.23)/Me18 (δ 0.92). The CH3 proton at δH 1.78 (H-19) exhibited HMBC correlations to the vinylic carbon signal at δC 160.5 (C-1), quaternary carbon at δC 133.0 (C-2), and ketone at δC 208.7 (C3), supporting an α,β-unsaturated carbonyl moiety. The H-7 signal showed a HMBC correlation to the oxygenated methylene at δC 67.9 (C-20) and the methylene at δC 38.7 (C-5). The observation of two methyl singlets at δH 1.22 and δH 1.27 (H-16 and H-17) and a methine doublet at δH 1.12 (J = 5.4 Hz, H-14) together with HMBC correlations from the methyl signal [δH 1.27 (H-17)] to δC 25.9 (C-15) and δC 23.9 (C-16) were used to establish the presence of a dimethyl cyclopropane moiety. Furthermore, the connectivity of the H-12 (δ 5.51)/H-11 (δ 2.23)/Me-18 (δ 0.92) moiety with the dimethyl cyclopropane ring was established by means of HMBC correlations from the oxymethine proton signal at δH 5.51 (H-12) to δC 43.1 (C-11), 65.5 (C-13), 36.5 (C-14), 25.9 (C-15), and 14.5 (C-18) (Figure 1). Thus, compound 1 was deduced to possess a backbone of a phorbol-type diterpene.12,13 Moreover, the NMR data of 1 were similar to those of the known phorbol esters 12-O-(2Z,4E,6E)deca-2,4,6-trienoylphorbol-13-acetate5 and aquimavitalin,9 except for the fatty acid moiety. There were two substituents detected in 1, namely, an acetyl group [δC 173.7 (C-21); δH 2.11 (s, H-22), δC 21.1 (C-22)] and a fatty acid moiety possessing four olefinic protons [δH 6.31 (d, J = 15.6 Hz, H-2′), δC 129.5 (C-2′); δH 7.40 (dd, J = 15.6 and 11.4 Hz, H-3′), δC 140.8 (C-3′); δH 7.17 (dd, J = 15.6 and 11.4 Hz, H-4′), δC 146.8 (C-4′); δH 6.43 (dd, J = 15.6 and 7.8 Hz, H-5′), δC 137.2 (C-5′)], and also a typical formyl group proton [δH 9.68 (d, J = 7.8 Hz, H-6′), δC 192.7 (C6′)] was evident. The structure of the long-chain fatty acid moiety was further assigned by COSY correlations between H-2′ (δ 6.31)/H-3′ (δ 7.40)/H-4′ (δ 7.17)/H-5′ (δ 6.43) and HMBC
In a previously reported study, feruryl glyceride and phorbol esters were isolated from the bark of A. malaccensis.6 Gas chromatography−mass spectrometry analysis (GC-MS) of agarwood (A. malaccensis) revealed the presence of aromatic terpenes, with sesquiterpenes as the main components, and chromones, as well as benzenoids, sterols, and fatty acids.10 GCMS analysis of A. malaccensis seeds showed high amounts of unsaturated (oleic acid 68.5%) and saturated fatty acids.11 Currently, there is scarce information on the chemical composition as well as bioactivities of seeds of A. malaccensis. Previously, potent antiallergic and anti-inflammatory activities of a crude extract and chromatographic fractions were observed, and a promising antiallergic phorbol ester (aquimavitalin) with nanomolar IC50 values was isolated.9 In the current study, eight compounds were isolated, namely, four new phorbol esters (1− 4), two known phorbol esters (5 and 6), and two known diglycerides (7 and 8), from the seeds of A. malaccensis. Their inflammation-modulatory and cytotoxic activities for cancer cell lines have been investigated.
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RESULTS AND DISCUSSION Compound 1 was obtained as a colorless oil. The molecular formula was calculated as C28H34O9 (12 degrees of unsaturation) from the analysis of its HRESIMS (m/z 537.20959 [M + Na]+, calcd 537.20950). Its IR absorbance spectrum showed the presence of hydroxy (3413 cm−1), carbonyl (1713 cm−1), and olefinic (1597 cm−1) groups. The 1H, 13C, and HSQC NMR data of compound 1 (Tables 1 and 2) indicated the occurrence of two vinylic groups [δH 7.59 (s, H-1) and δC 160.5 (C-1), δC 133.0 (C2); δC 140.6 (C-6), δH 5.69, (d, J = 4.2 Hz, H-7), 128.9 (C-7)], an oxygenated methine [δH 5.51 (d, J = 10.2 Hz, H-12), δC 78.2 (C12)], an oxygenated methylene [δH 4.05 (d, J = 12.6 Hz, H-20a), 4.01 (d, J = 12.6 Hz, H-20b), δC 67.9 (C-20)], four methyls, a methylene, and four methines.13 The 1H−1H COSY spectrum 1422
DOI: 10.1021/acs.jnatprod.6b01096 J. Nat. Prod. 2017, 80, 1421−1427
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Table 1. 1H NMR Data of Compounds 1−4 in CDCl3a position 1 5α 5β 7 8 10 11 12α 12β 14 16 17 18 19 20a 20b 22 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′ 18′ a1
1 7.59, s 2.49, d (19.2) 2.55, d (19.2) 5.69, d (4.2) 3.27, t (5.2) 3.27, brs 2.23, m 5.51, d (10.2) 1.12, d (5.4) 1.22, s 1.27, s 0.92, d (6.6) 1.78, brs 4.05, d (12.6) 4.01, d (12.6) 2.11, s 6.31, d (15.6) 7.40, dd (15.6, 11.4) 7.17, dd (15.6, 11.4) 6.43, dd (15.6, 7.8) 9.68, d (7.8)
2
3
7.60, s 2.38, d (18.8) 2.50, d (18.8) 5.70, d (4.4) 2.99, t (5.2) 3.28, brs 2.01, m 1.57, m 2.08, m 0.83, d (5.2) 1.06, s 1.19, s 0.88, d (6.0) 1.78, brs 4.48, d (12.4) 4.43, d (12.4) 2.06, s 2.29, t (7.6) 1.60, m 1.25−1.26, m 1.25−1.26, m 1.25−1.26, m 1.25−1.26, m 2.01, m 5.33, m 5.33, m 2.01, m 1.25−1.26, m 1.25−1.26, m 1.25−1.26, m 1.25−1.26, m 1.25−1.26, m 1.25−1.26, m 0.88, t (6.8)
4
7.60, s 2.49, d (19.2) 2.55, d (19.2) 5.69, d (5.4) 3.26, m 3.28, brs 2.19, m 5.48, d (10.2)
7.60, s 2.49, d (19.2) 2.55, d (19.2) 5.69, d (4.8) 3.26, m 3.28, brs 2.19, m 5.47, d (10.2)
1.10, d (5.4) 1.21, s 1.26, s 0.91, d (6.6) 1.78, brs 4.05, d (12.6) 3.95, d (12.6) 2.10, s 5.92, d (15.6) 7.27, dd (14.4, 12.0) 6.45, m 6.17, dd (15.6, 7.2) 4.24, m 3.74, m 1.42, m 1.27−1.29, m 1.27−1.29, m 1.27−1.29, m 1.27−1.29, m 1.27−1.29, m 0.88, t (7.2)
1.10, d (5.4) 1.21, s 1.26, s 0.90, d (6.6) 1.78, brs 4.05, d (13.2) 4.00, d (13.2) 2.10, s 5.92, d (15.6) 7.27, dd (14.4, 12.0) 6.48, m 6.12, dd (15.6, 6.0) 4.07, m 3.52, m 1.42, m 1.26−1.30, m 1.26−1.30, m 1.26−1.30, m 1.26−1.30, m 1.26−1.30, m 0.88, t (7.2)
H NMR spectra were measured at 600 MHz (1, 3, 4) and at 400 MHz (2) in CDCl3.
2.99 (t, J = 5.2 Hz, H-8), δC 39.4 (C-8); δH 2.01 (m, H-11), δC 36.3 (C-11); δH 1.57 (m, H-12α), δH 2.08 (m, H-12β), δC 31.9 (C-12)].14,15 Furthermore, signals for acetyl [δC 173.2 (C-21); δH 2.06 (s, H-22), δC 21.2 (C-22)] and fatty acid moieties were observed in the NMR spectra. The COSY correlations of compound 2 (Figure 1) showed connectivities of the H-10 (δ 3.28)/H-1 (δ 7.60)/Me-19 (δ 1.78), H2-5 (δ 2.38 and 2.50)/H-7 (δ 5.70)/H-8 (δ 2.99)/H-14 (δ 0.83), and H2-12 (δ 1.57 and 2.08)/H-11 (δ 2.01)/Me-18 (δ 0.88) substituents for the skeleton, as well as H-2′ (δ 2.29)/H-3′ (δ 1.60)/H-4′ (δ 1.25), and H-7′ (δ 1.25)/H-8′ (δ 2.01)/H-9′ (δ 5.33)/H-10′ (δ 5.33)/ H-11′ (δ 2.01)/H-12′ (δ 1.25), for the long-chain fatty acid moiety. To confirm the long-chain fatty acid moiety, compound 2 was subjected to methanolysis using a method reported by Ichihara et al.16 Compound 2 was reacted with 1.2% HCl in methanol for 1.5 h at 100 °C, and the resulting fatty acid methyl ester was identified by GC-MS analysis as (9Z)-octadecenoic (oleic) acid methyl ester (tR 21.30, m/z 296 [M]+). Comparison of the spectra (Table 1) with the literature revealed that the data of compound 2 closely matched those of 12-deoxyphorbol-13(9′Z)-octadecenoate-20-acetate.15 However, the HMBC correlation from H-20 to C-1′ (δC 173.6) indicated that the fatty acid moiety is linked to the backbone at C-20. Therefore, the acetyl
correlations of H-2′/C-4′ (δC 146.8) and H-3′/C-5′ (δC 137.2). The 1H NMR coupling constants and NOESY correlations were used to identify the geometry of double bonds. Accordingly, the NMR data indicated that 1 possesses a (2E,4E)-6-oxohexa-2,4dienoic acid moiety. The fatty acid moiety was attached to the phorbol backbone at C-12 according to the HMBC correlation of H-12 to C-1′ (δC 165.4); so, therefore, the acetyl moiety was attached to C-13. The relative configuration of compound 1 was deduced by comparison of its NOESY correlation data (Figure 2) with literature values.13 The NOESY correlations between H-11/ H-17 and H-11/H-8 suggested that these protons are β-oriented. Further correlations between Me-18/H-12α indicated that the fatty acid moiety is also β-oriented. On the basis of all of the above information, the structure of 1 was determined as 12-O(2′E,4′E)-6-oxohexa-2′,4′-dienoylphorbol-13-acetate. Compound 2 was also obtained as a colorless oil. It was assigned with a molecular formula of C40H62O7 (10 degrees of unsaturation) on the basis of HRESIMS (m/z 677.43884 [M + Na]+, calcd 677.43878). The IR spectrum indicated the presence of hydroxy (3409 cm−1), carbonyl (1717 cm−1), and olefinic (1597 cm−1) groups. A comparison of the 1H and 13C NMR data (Tables 1 and 2) with 1 suggested that compound 2 possesses a 12-deoxyphorbol type of diterpenoid backbone. Moreover, the signals typical of a 12-deoxyphorbol derivative were detected [δH 1423
DOI: 10.1021/acs.jnatprod.6b01096 J. Nat. Prod. 2017, 80, 1421−1427
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Table 2. 13C NMR Data of Compounds 1−4 in CDCl3a position
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′ 18′
160.5, CH 133.0, C 208.7, C 73.7, C 38.7, CH2 140.6, C 128.9, CH 39.1, CH 78.2, C 56.2, CH 43.1, CH 78.2, CH 65.5, C 36.5, CH 25.9, C 23.9, CH3 16.9, CH3 14.5, CH3 10.1, CH3 67.9, CH2 173.7, C 21.1, CH3 165.4, C 129.5, CH 140.8, CH 146.8, CH 137.2, CH 192.7, CH
161.3, CH 132.8, C 209.0, C 73.6, C 39.0, CH2 135.0, C 133.7, CH 39.4, CH 75.9, C 55.7, CH 36.3, CH 31.9, CH2 63.5, C 32.3, CH 22.7, C 23.2, CH3 15.3, CH3 18.5, CH3 10.1, CH3 69.5, CH2 173.2, C 21.1, CH3 173.6, C 34.2, CH2 24.9, CH2 29.1−29.7, CH2 29.1−29.7, CH2 29.1−29.7, CH2 29.1−29.7, CH2 27.2, CH2 129.8, CH 130.0, CH 27.2, CH 29.1−29.7, CH2 29.1−29.7, CH2 29.1−29.7, CH2 29.1−29.7, CH2 31.8, CH2 22.7, CH2 14.1, CH3
160.7, CH 132.9, C 208.8, C 73.7, C 38.7, CH2 140.5, C 129.2, CH 39.1, CH 78.2, C 56.2, CH 43.1, CH 77.1, CH 65.7, C 36.4, CH 25.8, C 23.8, CH3 16.7, CH3 14.4, CH3 10.0, CH3 68.0, CH2 173.8, C 21.1, CH3 165.4, C 121.7, CH 143.9, CH 129.9, CH 140.0, CH 74.9, CH 74.3, CH 32.2, CH2 29.2, CH2 29.4, CH2 29.7, CH2 31.8, CH2 22.6, CH2 14.0, CH3
160.7, CH 132.9, C 208.8, C 73.7, C 38.7, CH2 140.5, C 129.2, CH 39.1, CH 78.2, C 56.2, CH 43.1, CH 77.2, CH 65.7, C 36.4, CH 25.6, C 23.8, CH3 16.8, CH3 14.4, CH3 10.1, CH3 68.0, CH2 173.8, C 21.1, CH3 165.4, C 121.9, CH 143.8, CH 129.7, CH 141.5, CH 75.0, CH 74.3, CH 33.1, CH2 29.2, CH2 29.5, CH2 29.7, CH2 31.8, CH2 22.6, CH2 14.1, CH3
Compound 3 was isolated as another colorless oil. HRESIMS analysis indicated this compound to have a molecular formula of C36H52O10 (m/z 667.34515 [M + Na]+; calcd 667.34527), 10 degrees of unsaturation). The IR absorption bands of 3 showed hydroxy group (3392 cm−1), carbonyl (1710 cm−1), and olefinic (1632 cm−1) functionalities. The 1D (Tables 1 and 2) and 2D (Figure 1) NMR data of the skeleton of compound 3 closely matched those of compound 1, as a phorbol derivative, with there being two acyl substituents detected in 3. In addition to an acetyl group, the 1D NMR data indicated the presence of a long-chain fatty acid substituent possessing two double bonds along with two hydroxy groups. The 1H and 13C NMR data showed four vinylic protons at δH 5.92, 7.27, 6.45, and 6.17 with corresponding carbons at δC 121.7, 143.9, 129.9, and 140.0. The COSY cross-peaks between the vinylic protons suggested that the two double bonds are conjugated. The chemical shifts of H-2′ to H-5′ and C-2′ to C-5′ closely matched literature chemical shifts for methyl (2E,4E,8E)-6,13-dihydroxytetradeca2,4,8-trienoate, indicating a 2′,4′-diene moiety.17 The positions of two hydroxy groups in the side chain were established according to 2D NMR data analysis (Figure 1). The COSY correlations of H-2′ (δ 5.92)/H-3′ (δ 7.27)/H-4′ (δ 6.45)/H-5′ (δ 6.17)/H-6′ (δ 4.24)/H-7′ (δ 3.74)/H-8′ (δ 1.42) and HMBC correlations of H-2′/C-4′ (δC 129.9), H-4′/C-6′ (δC 74.9), and H-5′/C-6′ (δC 74.9) revealed the connectivity of conjugated double bonds with a 6′-hydroxy methine proton [δH 4.24 (H-6′), δC 74.9, (C-6′)] and a 7′-hydroxy proton [δH 3.74 (H-7′), δC 74.3, (C-7′)]. By comparing the chemical shifts of the 6′,7′vicinal diol units of 3 and 4 (see below) with the reference compounds, methyl 9,10-(erythro)-dihydroxy-11(E)-octadecenoate and methyl 9,10-(threo)-dihydroxy-11(E)-octadecenoate,18 two 6′- and 7′-hydroxymethine protons in 3, δH 4.24 (H-6′) and 3.74 (H-7′), were downfield shifted when compared to those of compound 4, δH 4.07 (H-6′) and 3.52 (H-7′). Thus, the structure of the fatty acid moiety was assigned in 3 as 12-O(2E,4E)-6,7-(erythro)-dihydroxytetradeca-2,4-dienoic acid. The long-chain fatty acid was attached to the phorbol backbone at C12 according to the HMBC correlation of H-12/C-1′ (δC 165.4), and the acetyl moiety was attached to C-13. The NOESY correlations of 3 indicated the same relative configuration as in 1 (Figure 2). Therefore, the structure of 3 was proposed as 12-O(2′E,4′E)-6′,7′-(erythro)-dihydroxytetradeca-2′,4′-dienoylphorbol-13-acetate. Compound 4 was isolated as a further colorless oil. Its IR (3385 cm−1, 1632 cm−1), HRESIMS (m/z 667.34550 [M + Na]+, calcd 667.34527), and 13C NMR data were identical to those of compound 3. Therefore, the basic structure was assigned as a C-12 long-chain fatty acid and C-13 acetyl-disubstituted phorbol ester. However, in a comparison of the 1H NMR data with those of compound 3, the upfield chemical shifts of the 6′,7′vicinal diol were evident [δH 4.07 (m, H-6); δH 3.52 (m, H-7)],
a13 C NMR spectra were measured at 150 MHz (1, 3, 4) and at 100 MHz (2) in CDCl3.
group was assigned to C-13. The relative configuration of 2 was assigned according to the ROESY spectrum (Figure 2). The correlations between H-11/H-8 and H-17/H-8 indicated that these protons are β-oriented. On the basis of the interpretation of the above spectroscopic data, compound 2 was assigned as 12deoxy-13-O-acetylphorbol-20-(9′Z)-octadecenoate.
Figure 1. COSY (bold bonds) and selected HMBC (arrows) correlations of 1−3. 1424
DOI: 10.1021/acs.jnatprod.6b01096 J. Nat. Prod. 2017, 80, 1421−1427
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Figure 2. Key NOE (left right arrow) correlations of 1−3.
indicating a threo configuration. On the basis of the obtained data, compounds 3 and 4 were determined as stereoisomers, and compound 4 was assigned as 12-O-(2′E,4′E)-6′,7′-(threo)dihydroxytetradeca-2′,4′-dienoylphorbol-13-acetate. Compound 5, 12-O-deoxyphorbol 13-decanoate, was isolated as a natural product for the first time.19 Compound 6 was identified as the phorbol ester, mellerin A.14 In addition, known glycerides were purified, 1,3-dioleoyl glyceride, a major component of A. malaccensis seeds (7),20 and 1-oleoyl-2palmitoyl glyceride (8).21 The NMR spectroscopic data of the known compounds were in agreement with the literature. To confirm the fatty acid moiety, compound 7 was subjected to methanolysis using the method reported by Ichihara et al.16 The resulting fatty acid methyl ester was determined by GC-MS analysis as an oleic acid methyl ester. Using the same procedure, two long-chain substituents of compound 8 were identified by GC-MS analysis as oleic acid methyl ester and hexadecanoic (palmitic) acid methyl ester. All isolated compounds were evaluated for their potential antiinflammatory and cytotoxic activities. In the anti-inflammatory assay, 1 (IC50 2.7 μM), 5 (IC50 0.8 μM), and 6 (IC50 2.1 μM) showed potent inhibitory activity on N-formyl-L-methionyl-Lleucyl-L-phenylalanine (fMLF)/cytochalasin B (CB)-induced elastase release by human neutrophils, comparable to a positive control, the PI3K inhibitor LY294002 (IC50 3.3 μM) (Table 3). On the other hand, phorbol 12-myristate 13-acetate (PMA) showed an enhancing effect on elastase release. The conjugated aldehydic fatty acid substituent in 1 and saturated fatty acid substituents in 5 and 6 may play a role in their bioactivity. All phorbol esters (1−6 and PMA) showed enhancing activities on superoxide anion generation. In general, phorbol esters trigger superoxide anion generation and cause pro-inflammatory effects. However, the extracts of the seeds of A. malaccensis exhibited an excellent superoxide anion generation inhibitory effect,9 which could be due to a high content of free anti-inflammatory fatty acids in the seed oil of A. malaccensis.11,22 Also, it may be suggested that the fatty acid substituents, which themselves show significant anti-inflammatory effects,22 play an important role in mediating the effect of phorbol esters. In addition, for the first time, it was discovered in the present study that some of the phorbol esters inhibit elastasereleasing effects. These findings may provide insights for further research related to the inflammation-modulating activities of phorbol esters.
Table 3. Effects of Pure Compounds on Superoxide Anion Generation and Elastase Release in FMLF/CB-Induced Human Neutrophils superoxide anion compound 1 2 3 4 5 6 7 8 PMAe LY294002f
elastase release
a
IC50 (μM)a
b
2.7 ± 0.6 >10 enhancingc enhancingc 0.8 ± 0.3 2.1 ± 0.7 >10 >10 enhancingc 3.3 ± 0.7
IC50 (μM)
enhancing enhancingb enhancingb enhancingb enhancingb enhancingb >10 enhancingd enhancingb 1.8 ± 0.7
a IC50 values express the concentration of the sample required to inhibit superoxide anion generation or elastase release by 50%. Results are presented as means ± SEM (n = 3−5). bThe compounds showed enhancing effects on superoxide generation at 10 μM (1 106.1%, 2 18.1%, 3 106.0%, 4 107.1%, 5 95.3%, 6 109.6%, PMA 102.7%) in the absence of fMLF/CB. Compared with fMLF/CB (as 100%). cThe compounds showed enhancing effects on elastase release at 10 μM (3 10.5%, 4 9.2%, PMA 44.2%) in the absence of fMLF/CB. Compared with fMLF/CB (as 100%). dThe compound showed enhancing effects on superoxide generation at 10 μM (8 74.5%) in the presence of CB. Compared with fMLF/CB (as 100%). ePhorbol 12-myristate 13acetate (PMA), a PKC activator, was used as a positive control exerting enhancing effects. fLY294002, the PI3K inhibitor, was used as a positive control exerting suppressive effects.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured using a JASCO P-2000 digital polarimeter (JASCO Inc., Tokyo, Japan). A JASCO V-530 ultraviolet spectrophotometer (JASCO) was used to run UV spectra. IR spectra were obtained on an FT-IR-4100 JASCO spectrophotometer (JASCO). NMR spectra were obtained on a Varian VNMR-600 FT-NMR spectrometer (Varian Inc., Palo Alto, CA, USA) and on a JEOL JNM-ECS 400 NMR spectrometer (JEOL Ltd., Tokyo, Japan). Electrospray-ionization mass spectrometry (ESIMS) data were collected on a Waters micromass ZQ mass spectrometer (Waters Corporation, Milford, MA, USA). Highresolution ESIMS data were determined using a Bruker Daltonics APEX II mass spectrometer (FT-ICR/MS, FTMS) (Bruker Daltonics, Billerica, MA, USA). GC-MS data were collected using a DSQ II single quadrupole GC/MS (Thermo Fisher Scientific, Waltham, MA, USA)
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2925, 2851, 1710, 1632, 1455, 1375, 1261, 1024, 802, 755 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Tables 1 and 2; ESIMS m/z 645 [M + H]+; HRESIMS m/z 667.34515 [M + Na]+ (calcd for C36H52O10Na, 667.34527). 12-O-(2′E,4′E)-6′,7′-(threo)-Dihydroxytetradeca-2′,4′-dienoylphorbol-13-acetate (4): colorless oil; [α]25 D +13.2 (c 0.13, CHCl3); UV (MeOH) λmax (log ε) 289 (2.79), 249 (2.84) nm; IR (neat) νmax 3385, 2929, 2855, 1632, 1455, 1375, 1261, 1124, 802, 755 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Tables 1 and 2; ESIMS m/z 645 [M + H]+; HRESIMS m/z 667.34550 [M + Na]+ (calcd for C36H52O10Na, 667.34527). 1,3-Dioleoyl glyceride (7): C39H72O5; ESIMS m/z 621 [M + H]+; GC-MS analysis (tR 21.30, m/z 296 [M]+, for oleic acid methyl ester, fragmentation ions observed at m/z 264, 222, 137, 123, 110, 98, 74, 69, 55). 1-Oleoyl-2-palmitoyl glyceride (8): C37H70O5; ESIMS m/z 595 [M + H]+; GC-MS analysis (tR 21.31, m/z 296 [M]+, fragmentation ions observed at m/z 264, 180, 137, 123, 110, 98, 74, 69, oleic acid methyl ester; tR 17.71, m/z 270 [M]+, fragmentation ions observed at m/z 227, 185, 143, 87, 74, palmitic acid methyl ester). Methylation of Samples for GC-MS Analysis. The method of Ichihara et al. was utilized.16 Lipid sample was placed in a screw-capped glass test tube and dissolved in 0.20 mL of toluene. To the lipid solution were added 1.50 mL of methanol (HPLC grade) and 0.30 mL of the 8.0% HCl solution, in order. The final HCl concentration was 1.2%. The reaction mixture was heated at 100 °C for 1 h. On cooling, the reaction was quenched by 1 mL of 6% aqueous K2CO3 solution. Then, 1−2 mL of hexane (HPLC grade) was added, and the mixture was partitioned to yield a hexane layer (upper layer). The hexane layer was filtered through a 0.22 μm filter (ChromTech, Apple Valley, MN, USA) into a GC-MS vial and then subjected to GC-MS analysis. GC-MS Analysis. The methylated samples were analyzed by gas chromatography−mass spectrometry (DSQ II single quadrupole GC/ MS, Thermo Fisher Scientific). Derivatized samples were vaporized at 250 °C in standard split mode (1:50) and separated on a 30 m × 0.25 mm HP-5MS capillary column with a 0.25 μm coating (Agilent). The column oven temperature was set at 70 °C, held for 3 min, then increased to 180 °C at 20 °C/min, held for 5 min, then increased to 280 °C at 5 °C/min and held for 5 min. A helium gas carrier flow of 1 mL/ min was used. The interface and ion source temperature were set to 250 °C. An electron-impact ionization of 30 eV was utilized, the injection volume was 1 μL, and the mass range was m/z 38−600. Identification of the compounds was based on a comparison of mass spectra with the data from the Wiley/NBS Registry of Mass Spectral Data (version 5.0)/ National Institute of Standards and Technology (NIST) MS Search (version 2.0). Superoxide Anion and Elastase Release Assays. Blood was obtained from healthy human donors (20−30 years old), using a protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital (protocol number 102-1595A3). Human neutrophils were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll-Hypaque gradient and hypotonic lysis of erythrocytes.23 Purified neutrophils containing >98% viable cells were determined by the trypan-blue exclusion method.24 The neutrophils were resuspended in a Ca2+-free Hank’s buffered salt solution (HBSS) at pH 7.4 and were maintained at 4 °C prior to use. Superoxide Anion Generation Assay. Neutrophil superoxide anion generation was determined using a superoxide dismutase (SOD)inhibitory cytochrome reduction assay according to previously described procedures.25,26 Briefly, after supplementation with 0.5 mg/ mL ferricytochrome c and 1 mM Ca2+, neutrophils (3 × 105 per mL) were equilibrated at 37 °C for 2 min and incubated with different concentrations of test compounds or DMSO (as control) for 5 min. Cells were incubated with cytochalasin B (1 μg/mL) for 3 min prior to the activation with 100 nM N-formyl-L-methionyl-L-leucyl-L-phenylalanine for 10 min. Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continuously monitored in a doublebeam, six-cell positioner spectrophotometer with constant stirring (Hitachi U-3010, Tokyo, Japan). Calculations were based on the differences in the reactions with and without SOD (100 U/mL) divided
equipped with an HP-5MS capillary column (0.25 μm, 30 m × 0.25 mm, Agilent, Santa Clara, CA, USA). Sephadex LH-20 (GE Healthcare, Stockholm, Sweden) and silica gel (Kieselgel 60, 0.063−0.200 mm, Geduran Si 60, 0.040−0.063 mm, Merck KGaA) were used for column chromatography. Silica gel precoated (Kieselgel 60 F254 and RP-18 F254s, Merck KGaA) TLC plates were used. HPLC analyses were performed using a Shimadzu LC-10AT VP (Shimadzu Inc., Kyoto, Japan) pump interface equipped with a Shimadzu SPD-M10A VP diode array detector using a C18 column (5 μm, 250 × 10 mm, Phenomenex Inc.). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), phorbol 12-myristate 13-acetate (PMA), LY294002, and doxorubicin hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Plant Material. The seeds of A. malaccensis were collected and identified by one of the authors (H.-Y.H.) in November 2014. A voucher specimen (no. KMU-AMS 1) has been deposited in the Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. Extraction and Isolation. The dried and powdered seeds of Aquilaria malaccensis (462 g) were extracted with 90% EtOH (3 × 5 L) to give an extract (27.7 g), which was suspended in water and further partitioned between ethyl acetate and H2O (1:1) to obtain an ethyl acetate layer. The ethyl acetate layer was then partitioned between 90% aqueous MeOH and n-hexane to afford a 90% MeOH layer (16.2 g) and an n-hexane partition (7.1 g). The water layer was partitioned with nbutanol to yield a water partition (1.6 g) and an n-butanol layer (0.4 g). The MeOH layer (16.2 g) was submitted to column chromatography (CC) over silica gel 60 (0.063−0.200 mm, Merck), eluted with nhexane/CH2Cl2/MeOH (6:3:1 to 6:10:2), to afford six fractions: Fr.1 (2.9 g), Fr.2 (1.3 g), Fr.3 (6.8 g), Fr.4 (3.2 g), Fr.5 (1.7 g), and Fr.6 (97.9 mg). Fraction 4 was fractionated by Sephadex LH-20 CC with CH2Cl2/ MeOH (1:1) as eluant, to yield eight further fractions (Fr.4-1 to Fr.4-8). Fraction 4-3 (762 mg) was purified using CC on silica gel (30 cm, 1.5 cm, Geduran Si 60, 0.040−0.063 mm, Merck) and eluted with a gradient of EtOAc/n-hexane (from 1:10 to 4:1) to obtain 15 subfractions (4-3-1 to 4-3-15), with compound 2 (39.5 mg) being purified from subfraction 4-3-6. Subfraction 4-3-3 was further fractionated over a Sephadex LH-20 column (CH2Cl2/MeOH, 8:2) to obtain five subfractions (4-3-3-1 to 43-3-5), and compound 7 (130.5 mg) was purified from subfraction 4-33-3. Subfraction 4-3-3-4 was subjected to silica gel CC and eluted with a gradient of n-hexane/CH2Cl2/MeOH (10:1:0 to 3:1:0.5), to yield compound 8 (4.6 mg). Subfraction 4-3-13 (23.5 mg) was separated by RP-HPLC (C18 column, flow rate = 2.0 mL/min), with MeOH/H2O (60:40) used for elution, to afford compounds 1 (0.7 mg), 3 (0.9 mg), and 4 (1.0 mg). Fraction 4-4 (174 mg) was further purified using column chromatography on silica gel (30 cm, 1.5 cm, Geduran Si 60, 0.040− 0.063 mm, Merck), eluted with a gradient of EtOAc/n-hexane (from 1:15 to 4:1), to obtain 11 subfractions (4-4-1 to 4-4-11). Subfraction 44-7 (38 mg) was further purified by RP-HPLC (C18 column flow rate = 2.0 mL/min), with MeOH/H2O (85:15), to yield compounds 5 (7.8 mg) and 6 (0.8 mg). 12-O-(2′E,4′E)-6-Oxohexa-2′,4′-dienoylphorbol-13-acetate (1): colorless oil; [α]25 D +4.2 (c 0.17, CHCl3); UV (MeOH) λmax (log ε) 295 (2.80), 249 (2.84) nm; IR (neat) νmax 3413, 2925, 2855, 2360, 2339, 1713, 1625, 1597, 1261, 1184, 755 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Tables 1 and 2; ESIMS found m/ z 515 [M + H]+; HRESIMS m/z 537.20959 [M + Na]+ (calcd for C28H34O9Na, 537.20950). 12-Deoxy-13-O-acetylphorbol-20-(9′Z)-octadecenoate (2): colorless oil; [α]25 D +2.9 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 285 (2.78), 250 (2.83) nm; IR (neat) νmax 3409, 2922, 2855, 1717, 1375, 1332, 1152, 1021 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Tables 1 and 2; ESIMS m/z 655 [M + H]+; HRESIMS m/z 677.43884 [M + Na]+ (calcd for C40H62O7Na, 677.43878); GC-MS analysis (tR 21.30, m/z 296 [M]+, fragmentation ions observed at m/z 264, 222, 137, 123, 110, 98, 83, 74, 69, oleic acid methyl ester). 12-O-(2′E,4′E)-6′,7′-(erythro)-Dihydroxytetradeca-2′,4′-dienoylphorbol-13-acetate (3): colorless oil; [α]25 D +10.4 (c 0.17, CHCl3); UV (MeOH) λmax (log ε) 289 (2.79), 249 (2.83) nm; IR (neat) νmax 3392, 1426
DOI: 10.1021/acs.jnatprod.6b01096 J. Nat. Prod. 2017, 80, 1421−1427
Journal of Natural Products
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by the extinction coefficient for the reduction of ferricytochrome c (21.1 mM−1 cm−1). All experiments were repeated three times. LY294002 and PMA were used as positive controls. Elastase Release Inhibition Assay. Degranulation of azurophilic granules was determined by measuring the elastase release, as described previously.26 Experiments were performed using MeOSuc-Ala-Ala-ProVal-p-nitroanilide as the elastase substrate. After supplementation with MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), neutrophils (3 × 105 per mL) were equilibrated at 37 °C for 2 min and incubated with each test compound for 5 min. Cells were stimulated with fMLF (100 nM)/ CB (0.5 μg/mL), and changes in the absorbance at 405 nm were monitored continuously in order to measure the elastase release. The results were expressed as the percent of elastase release in the fMLF/CBactivated, drug-free control system. All experiments were repeated three times. LY294002 and PMA were used as positive controls. Cytotoxicity Assay. The MTT viability assay was used according to a previously described method.27 HepG2 (1 × 104 cells), A549 (5 × 103 cells), and MDA-MB-231 (1 × 104 cells) were seeded into 96-well plates. The cells were treated with the test compounds at a concentration of 20 μg/mL. After 72 h, the medium was removed and 100 μL of MTT solution (0.5 mg/mL) was added to each well. The plates were incubated at 37 °C for 1 h to form formazan crystals, which were then dissolved by DMSO (100 μL). Plates were gently shaken, and absorbance at 550 nm was measured using a microplate reader. The degree of cell viability of each sample was calculated as the percentage of control value (untreated cells). Doxorubicin hydrochloride was used as a positive control and exhibited IC50 values of 0.5, 0.3, and 1.2 μM against A549, Hep-G2, and MDA-MB-231 cells, respectively.
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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.6b01096. 1 H NMR, 13C NMR, 2D NMR, and HRESIMS spectra of compounds 1−4 and GC-MS data of compounds 2, 7, and 8 (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*Tel: +886-7-3121101, ext. 2676. Fax: +886-7-3125339. E-mail:
[email protected] (B.-H. Chen). *Tel: +886-7-3121101, ext. 2162. Fax: +886-7-3114773. E-mail:
[email protected] (Y.-B. Cheng). *Tel: +886-7-3121101, ext. 2162. Fax: +886-7-3114773. E-mail:
[email protected] (F. R. Chang). ORCID
Yuan-Bin Cheng: 0000-0001-6581-1320 Author Contributions ◇
V. D. Wagh and M. Korinek contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Center for Research Resources and Development at Kaohsiung Medical University for providing instrumentation support. This work was supported by grants from Ministry of Science and Technology, Taiwan (NSC 102-2628-B-037-003MY3, MOST 103-2320-B-037-005-MY2, awarded to F.-R.C., MOST 103-2628-B-037-001-MY3 awarded to Y.-B.C). This study was also supported by Kaohsiung Medical University, Taiwan (Aim for the Top Universities Grant, grant nos. KMUTP105E32, KMU-TP104A26, KMU-M106009), and Ministry of 1427
DOI: 10.1021/acs.jnatprod.6b01096 J. Nat. Prod. 2017, 80, 1421−1427
