Chemical Constituents from the Fruiting Bodies of Hexagonia apiaria

Article pubs.acs.org/jnp

Chemical Constituents from the Fruiting Bodies of Hexagonia apiaria and Their Anti-inflammatory Activity Tran Dinh Thang,*,†,# Ping-Chung Kuo,‡,# Nguyen Thi Bich Ngoc,† Tsong-Long Hwang,§,⊥ Mei-Lin Yang,‡ Shih-Huang Ta,∥ E-Jian Lee,∥ Dai-Huang Kuo,∇ Nguyen Huy Hung,† Nguyen Ngoc Tuan,† and Tian-Shung Wu*,∇,△ †

Department of Chemistry, Vinh University, Vinh City 42000, Vietnam Department of Biotechnology, National Formosa University, Yunlin 63201, Taiwan, ROC § Graduate Institute of Natural Products, Chang Gung University, Taoyuan 33302, Taiwan, ROC ⊥ Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan 33302, Taiwan, ROC ∥ Neurosurgical Service, Department of Surgery, National Cheng Kung Medical Center and Medical School, Tainan, Taiwan, ROC ∇ Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung 90741, Taiwan, ROC △ School of Pharmacy, National Cheng Kung University, Tainan 70101, Taiwan, ROC ‡

ABSTRACT: A chemical investigation of the fruiting bodies of Hexagonia apiaria resulted in the identification of nine compounds including five new triterpenoids, hexagonins A−E (1−5), along with four known compounds. The purified constituents were examined for their anti-inflammatory activity. Among the tested compounds, hexatenuin A displayed the most significant inhibition of superoxide anion generation and elastase release. These triterpenoids may have potentials as antiinflammatory agents.

5), and four known compounds were reported (Figure 1). The chemical structures of new compounds were determined on the basis of 1D and 2D NMR and mass spectrometric analytical results. Moreover, the purified triterpenoids were examined for inhibition of superoxide anion generation and elastase release, thereby evaluating their anti-inflammatory potential.

Hexagonia is a fungus genus belonged to the Polyporaceae family. Hexagonia species are widely distributed in tropical and subtropical zones, such as South China, Indochina, and Taiwan.1,2 Recent studies on Hexagonia speciosa afforded various oxygenated cyclohexanoids.3,4 These compounds were structurally close to the biologically active siccayne, which was previously reported from the fungus Helminthospoum siccans as well as the marine basidiomycete Halocyphina villosa and exhibited cytotoxicity and antimicrobial activity.5,6 In addition, three lanostane triterpenoids, hexatenuins A−C, with antitrypanosomal activity against Trypanosoma brucei were reported from the fruiting bodies of Hexagonia tenuis.7 These new triterpenoids possessed an unusual malonate half-ester functional group and a spirostructure in the side chain. In our program to discover anti-inflammatory lead drugs from natural sources, the chemical composition of the fruiting bodies of Hexagonia apiaria collected in Vietnam and which in preliminary studies had shown anti-inflammatory activity was investigated in order to identify the bioactive constituents. In the present study, five new triterpenoids, hexagonins A−E (1− © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Air-dried and powdered fruiting bodies of H. apiaria were extracted with methanol, and the combined extracts were concentrated under reduced pressure to give a deep brown syrup. The crude extract was suspended into water and partitioned with ethyl acetate to afford ethyl acetate- and watersoluble fractions, respectively. Purification of the ethyl acetate fraction by a conventional combination of column chromatographies yielded five new triterpenoids (1−5) and four known Received: June 1, 2015

A

DOI: 10.1021/acs.jnatprod.5b00449 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Chemical structures of all the purified compounds.

double bond at C-8/C-9 was determined by 3J-HMBC correlations between CH3-19 and C-9 and between CH3-30 and C-8. The HMBC cross-peaks from H-16 (δ 4.32, 1H, ddd, J = 11.5, 11.5, 5.0 Hz) to C-20 (δ 30.7); from H-3 (δ 4.71, 1H, br s) to C-29 (δ 21.7), C-1 (δ 30.5), C-5 (δ 45.3), and C-1′; from CH2-2′ (δ 3.40, 2H, s) to C-1′ and C-3′; and from CH3-4′ (δ 3.72, 3H, s) to C-3′ evidenced the C-16 to be oxygenated and C-3 to be acetylated by the carbomethoxyacetyloxy group. The above elucidations constructed the chemical skeleton of 1 with 10 IHDs. The last IHD was afforded by the cyclization between C-16 and C-23 through the ether linkage with a spiro structure. These spectra evidenced that 1 was very similar to the reported compound hexatenuin A,7 and the only difference was that 1 was the methyl derivative of hexatenuin A. The coupling constants of H-3 (br s) and H-16 (11.5, 11.5, 5.0 Hz) indicated the orientations of H-3 and H-16 as equatorial and axial. The stereochemical configurations of H-3 and H-16 were further established as β and β according to the NOESY analysis and comparison of spectral data between 1 and hexatenuin A (Figure 3).7 The successive two-dimensional spectral experiments including COSY, NOESY, HMQC, and HMBC accomplished the assignments of all the proton and carbon signals of 1, and therefore its chemical structure was established as shown in Figure 1 and named trivially as hexagonin A. Compounds 2−4 were all obtained as optically active white powder, which displayed similar UV spectra and IR absorption bands to those of 1. Moreover, the proton resonances for eight methyl groups characteristic for the triterpenoid basic skeleton were all observed in their 1H NMR spectra. These data indicated that 1−4 were structurally similar compounds. The HRESIMS analysis of compound 2 exhibited a pseudomolec-

compounds. The known compounds were characterized by comparison of their physical and spectroscopic data with the reported values. Compound 1 was obtained as an optically active white powder, with [α]25D +57 (c 0.6, MeOH). The positive-mode HRESIMS of 1 showed a pseudomolecular ion peak at m/z 605.3451 ([M + Na]+, calcd for C35H50O7Na, 605.3454) corresponding to a molecular formula of C35H50O7 with 11 indices of hydrogen deficiency (IHD). The UV spectrum of 1 exhibited absorption maxima at 262 nm, compatible with an α,β-unsaturated carbonyl chromophore.8 The IR absorption bands at 2946, 1759, and 1693 cm−1 suggested the presence of aliphatic C−H, lactonic carbonyl, and carbon−carbon doublebond functionalities, respectively. The 1H NMR spectrum of 1 (Table 1) displayed five methyl singlets at δ 0.68 (3H, CH318), 0.88 (3H, CH3-28), 0.93 (3H, CH3-29), 1.00 (3H, CH319), and 1.08 (3H, CH3-30), respectively. In addition, one doublet methyl group at δ 0.95 (3H, J = 6.5 Hz, CH3-21) suggested the presence of the lanostane skeleton. Two vinyl methyl signals at δ 1.81 (3H, d, J = 0.5 Hz, CH3-27) and 1.94 (3H, d, J = 0.5 Hz, CH3-31), along with the 13C NMR signals (Table 2) at δ 8.5 (C-27), 10.8 (C-31), 108.2 (C-23), 125.2 (C25), 157.4 (C-24), and 172.2 (C-26), indicated a γ-lactone ring cyclized between C-23 and C-26. This was verified by HMBC correlations (Figure 2) from CH3-31 to C-23, -24, and -25 and from CH3-27 to C-24, -25, and -26, respectively. In the downfield region of the 13C NMR spectrum, there were two oxygenated methines at δ 79.6 (C-3) and 79.8 (C-16), one set of tetrasubstituted double bonds at δ 133.8 (C-8) and 135.1 (C-9), and two ester carbonyl carbons at δ 165.9 (C-1′) and 167.2 (C-3′), respectively. The location of the tetrasubstituted B



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Table 1. 1H NMR Spectroscopic Data of Compounds 1−5 [δH, mult. (J in Hz)] 1a

no. 1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22

a1

1.49, 1.41, 1.89, 1.71, 4.71, 1.41, 2.05, 1.60, 1.49, 2.05, 1.84, 1.60, 2.27, 1.20, 4.32, 1.41, 0.68, 1.00, 2.18, 0.95, 1.60, 1.49,

m m m m br s m m m m m m m dd (14.0, 11.5) dd (14.0, 5.0) ddd (11.5, 11.5, 5.0) m s s m d (6.5) m m

23 25 26 27 28 29 30 31

1.81, 0.88, 0.93, 1.08, 1.94,

2′ 4′

3.40, s 3.72, s

d (0.5) s s s d (0.5)

2a 1.84, 1.50, 1.97, 1.62, 3.43, 1.49, 2.07, 1.63, 1.51, 2.07, 1.58, 1.48, 2.26, 1.19, 4.32, 1.46, 0.68, 0.99, 2.18, 0.95, 1.58, 1.46,

1.81, 0.88, 0.97, 1.06, 1.94,

m m m m br s m m m m m m m dd (14.0, 11.5) dd (14.0, 5.0) ddd (11.5, 11.5, 5.0) m s s m d (6.0) m m

d (0.5) s s s d (0.5)

3b 1.40, m 1.92, 1.62, 4.62, 1.59, 1.92, 1.68, 1.55, 2.19, 1.88, 1.64, 2.35, 1.18, 4.23, 1.58, 0.76, 1.05, 1.53, 0.98, 1.64, 1.58,

1.75, 0.88, 0.95, 1.10, 1.96,

m m t (3.0) m m m m m m m dd (14.0, 11.0) dd (14.0, 5.0) ddd (11.5, 11.0, 5.0) m s s m d (6.5) m m

d (1.0) s s s d (1.0)

4c

5d

1.63, 1.50, 1.88, 1.62, 3.83, 2.25, 1.85, 2.13,

m m m m br s m m m

2.13, 1.63, 1.50, 2.27, 1.21, 4.32, 1.50, 0.69, 1.03, 1.85, 0.95, 1.63, 1.50,

m m m m dd (14.0, 5.0) ddd (11.5, 11.5, 5.0) m s s m d (6.5) m m

1.80, 9.59, 1.07, 1.10, 1.94,

d (1.1) s s s d (1.1)

1.02, m 3.42, m 2.73, 1.02, 1.91, 1.87, 1.26, 1.99, 1.52, 1.43, 1.50, 1.13, 1.50, 1.90, 0.67, 0.94, 2.04,

d (9.5) m m m m m m m m m m m s s m

1.50, m 1.90, 2.16, 0.95, 0.94, 0.71, 0.91, 0.81, 4.69, 4.61,

m heptet (7.0) d (6.5) d (6.5) s s s s s

2.00, s

H NMR data (δH) were measured in CDCl3 at 500 MHz. bAcetone-d6 at 500 MHz. cCDCl3 at 400 MHz. dDMSO-d6 at 500 MHz.

ular ion peak at m/z 505.3290 ([M + Na]+, calcd for C31H46O4Na, 505.3294), which represented a molecular formula of C31H46O4. Comparison of the 1H NMR spectra between 2 and 1 (Table 1) displayed that the carbomethoxyacetyloxy group was reduced, and this was also supported by the molecular formula. The HMBC correlations evidenced the basic chemical structure of 2 to be the same as that of 1. As described above, the configurations of H-3 and H-16 were established as β and β, respectively, by the NOESY analysis and the coupling constants of H-3 (br s) and H-16 (ddd, J = 11.5, 11.5, 5.0 Hz). All the proton and carbon signals were completely determined by the 2D spectral experiments, and consequently the chemical structure of 2 was constructed as shown in Figure 1 and named trivially as hexagonin B. The molecular formula of compound 3 was determined as C33H48O5 with the aid of the HRESIMS analytic data, which exhibited a pseudomolecular ion peak at m/z 547.3394 ([M + Na]+, calcd for C33H48O5Na, 547.3399). Comparison of the 1H NMR spectra (Table 1), molecular formulas, and HMBC correlations between 3 and 2 indicated that 3 may be the acetyl ester of 2. The acetoxy substitution was attached to the C-3, evidenced by the 3J-HMBC correlation between H-3 (δ 4.62, 1H, t, J = 3.0 Hz) and C-1′ (δ 170.5). Successive twodimensional spectral experiments furnished the assignments of all the proton and carbon signals of 3, and conclusively its

chemical structure was determined as shown and named trivially as hexagonin C. The HRESIMS of 4 showed a pseudomolecular ion peak at m/z 519.3083 ([M + Na]+, calcd for C31H44O5Na, 519.3086), corresponding to the formula C31H44O5. The typical IR signals for Fermi resonances of a formyl group were found at 2928 and 1375 cm−1. In addition, the 1H NMR spectrum of 4 (Table 1) displayed characteristic signals for five aliphatic methyl groups at δ 0.69 (3H, s, CH3-18), 0.95 (3H, d, J = 6.5 Hz, CH3-21), 1.03 (3H, s, CH3-19), 1.07 (3H, s, CH3-29), and 1.10 (3H, s, CH3-30); two vinyl methyl signals at δ 1.80 (3H, d, J = 1.1 Hz, CH3-27) and 1.94 (3H, d, J = 1.1 Hz, CH3-31); one oxygenated methine proton at δ 3.83 (1H, br s, H-3); and one formyl proton at δ 9.59 (1H, s, CHO-28), respectively. Comparison of the UV, IR, and 1H NMR data of 4 with hexagonin B (2) inferred that the methyl group at C-28 in 2 was oxidized to a formyl function in 4. In the HMBC spectrum (Figure 4), the 3JHMBC correlations from the CH3-29 (δ 1.07) to carbons at δ 38.7 (C-5), 52.0 (C-4), 72.6 (C-3), and 209.2 (C-28) provided strong evidence for the presence of one formyl function located at C-4 in 4. In addition, the HMBC cross-peaks from H-16 (δ 4.32, 1H, ddd, J = 11.5, 11.5, 5.0 Hz) to C-20 (δ 30.6); from H3 to C-5 (δ 38.7); from CH3-18 to C-12, -13, -14, and -17; from CH3-30 to C-8, -13, -14, and -15; from CH3-21 to C-17, -20, and -22; from CH3-31 to C-23, -24, and -25; and from CH3-27 C



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Table 2. 13C NMR Spectroscopic Data of Compounds 1−5 [δC, Type] no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1′ 2′ 3′ 4′

1a 30.5, 23.1, 79.6, 36.8, 45.3, 26.5, 17.9, 133.8, 135.1, 37.1, 20.2, 30.1, 43.5, 48.6, 35.4, 79.8, 54.6, 16.5, 18.8, 30.7, 19.4, 41.1, 108.2, 157.4, 125.2, 172.2, 8.5, 27.6, 21.7, 28.0, 10.8, 165.9, 41.8, 167.2, 52.3,

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

2a 30.2, 25.7, 76.0, 37.2, 44.2, 26.6, 18.1, 133.5, 135.3, 37.6, 20.2, 29.9, 43.5, 48.6, 35.4, 79.7, 54.6, 16.5, 18.8, 30.7, 19.4, 41.1, 108.2, 157.5, 125.2, 172.3, 8.5, 28.0, 22.2, 28.0, 10.8,

3b

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

31.4, 23.6, 78.1, 37.4, 46.2, 23.9, 18.7, 134.7, 136.1, 37.9, 27.2, 31.2, 44.3, 49.3, 36.2, 80.2, 55.3, 16.8, 19.1, 31.7, 19.6, 41.7, 108.7, 158.3, 125.3, 172.1, 8.5, 28.0, 22.2, 28.3, 10.7, 170.5, 20.9,

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

4c 29.1, 26.1, 72.6, 52.0, 38.7, 26.1, 20.1, 134.0, 134.8, 36.5, 20.2, 30.0, 43.4, 48.6, 35.3, 79.5, 54.5, 16.5, 19.2, 30.6, 19.4, 41.1, 108.2, 157.5, 125.1, 172.3, 8.5, 209.2, 14.8, 28.0, 10.9,

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

44.0, 67.8, 82.2, 39.1, 50.2, 20.7, 26.8, 133.7, 134.2, 37.8, 26.0, 28.4, 49.2, 44.2, 30.2, 18.1, 46.7, 15.8, 20.2, 48.7, 177.8, 31.1, 31.9, 155.5, 33.5, 21.9, 21.8, 28.8, 17.1, 24.3, 106.7,

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

a13

C NMR data (δC) were measured in CDCl3 at 125 MHz. bAcetone-d6 at 125 MHz. cCDCl3 at 100 MHz. dDMSO-d6 at 125 MHz.

Figure 2. Significant HMBC correlations of compound 1.


as β and β, respectively, which was also indicated by the coupling constants of H-3 (br s) and H-16 (ddd, J = 11.5, 11.5, 5.0 Hz). All other proton and carbon signal assignments were established by 2D NMR techniques and confirmed the structure of 4 as shown in Figure 1. Therefore, it was named trivially as hexagonin D. Compound 5 was afforded as an optically active white powder with [α]25D +27 (c 0.3, MeOH). The positive-mode HRESIMS of 5 showed a pseudomolecular ion peak at m/z 509.3599 ([M + Na]+, calcd for C31H50O4Na, 509.3607) corresponding to a molecular formula of C31H50O4. The 1H NMR spectrum of 5 (Table 1) displayed five methyl singlets at

Figure 3. Significant NOESY correlations of compound 1.

to C-24, -25, and -26, respectively, constructed the skeleton of 4 as the same as that of 2. In the NOESY spectral analysis, the stereochemical configurations of H-3 and H-16 were assigned D



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δ 0.67 (3H, CH3-18), 0.71 (3H, CH3-28), 0.81 (3H, CH3-30), 0.91 (3H, CH3-29), and 0.94 (3H, CH3-19), respectively. In addition, two methyl doublets at δ 0.94 (3H, J = 6.5 Hz, CH327) and 0.95 (3H, J = 6.5 Hz, CH3-26) suggested the structure of 5 to be a lanostanoid triterpene. In the downfield region of the 13C NMR spectrum (Table 2), there were two oxygenated methines at δ 67.8 (C-2) and 82.2 (C-3), one set of tetrasubstituted double bonds at δ 133.7 (C-8) and 134.2 (C9), one set of gem-disubstituted double bonds at δ 155.5 (C-24) and 106.7 (C-31), and one carboxylic acid group at δ 177.8 (C21), respectively. The position of the tetrasubstituted double bond was located between C-8 and C-9, which was established by 3J-HMBC correlations of CH3-19/C-9 and CH3-30/C-8. Moreover, in the HMBC spectrum of 5 (Figure 5), 2J, 3J-

Table 3. Inhibitory Effects of Purified Samples from H. apiaria on Superoxide Anion Generation and Elastase Release by Human Neutrophils in Response to N-Formyl-Lmethionyl-phenylalanine/Cytochalasin B (FMLP/CB) IC50 (μM)a compound

superoxide anion generation

elastase release

1 2 4 5 hexatenuin A LY294002c

>10 >10 >10 6.0 ± 1.0*** 1.9 ± 0.2*** 0.4 ± 0.02***

−b −b −b >10 4.3 ± 1.4*** 1.5 ± 0.3***

a

Concentration necessary for 50% inhibition. Results are presented as mean ± SD (n = 3−4). ***p < 0.001 compared with the control value. b Increasing effects were observed. cA phosphatidylinositol-3-kinase inhibitor was used as a positive control for superoxide anion generation and elastase release.

significant inhibitory effects in the bioactivity examination. Consequently, the free malonic acid function is important for anti-inflammatory activity. From the above data the purified triterpenoids of H. apiaria are potentially new leads for antiinflammatory drug development and the starting fungus as a health food with a possible known mechanism of action.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined using a Yanagimoto MP-S3 apparatus. Optical rotations were measured using a JASCO DIP-370 polarimeter. The UV spectra were obtained on a Hitachi UV-3210 spectrophotometer, and IR spectra were recorded on a Shimadzu FTIR-8501 spectrophotometer. 1 H and 13C NMR, COSY, NOESY, HMQC, and HMBC spectra were obtained on Bruker AV-500 and Avance III-400 NMR spectrometers, with tetramethylsilane (TMS) as internal standard, and the chemical shifts are reported in δ values (ppm). The electrospray ionization (ESI) mass spectrum was determined using an Agilent 1200 LC-MSD trap spectrometer. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70−230 mesh and 230−400 mesh, E. Merck). Thin-layer chromatography (TLC) was conducted on precoated Kieselgel 60 F 254 plates (Merck), and the compounds were visualized by spraying with 10% (v/v) H2SO4 followed by heating at 110 °C for 10 min. Fungal Material. The basidiomycete of Hexagonia apiaria (Pers.) Fr. was collected at Puhuong National Park of Nghean Province, Vietnam, in October 2012 and identified by Prof. Dr. Ngo Anh, Department of Biology, Hue University. A voucher specimen (VinhTSWu 20120805) was deposited at the herbarium of the Department of Chemistry, Vinh University. Extraction and Isolation. The fruiting bodies of H. apiaria (Pers.) Fr. (8.6 kg) were air-dried, powdered, and extracted with methanol (10 L × 3) at ambient temperature, and the combined extracts were concentrated under reduced pressure to give a deep brown syrup (180 g). The crude extract was suspended in water and partitioned with ethyl acetate to afford ethyl acetate (105 g) and water solubles (75 g), respectively. The ethyl acetate-soluble extracts were applied to silica gel column chromatography with an n-hexane−acetone step gradient system (100:0, 25:1, 15:1, 10:1, 7:1, 5:1) and then eluting with a CHCl3− MeOH step gradient system (10:0, 6:1, 3:1, 2:1, 1:1) to afford minor fractions. These fractions were monitored by TLC to combine into seven fractions. Fraction 7 did not display significant spots based on the TLC profile, so that it was not further purified. Fraction 1 (8.6 g) was purified with the aid of silica gel column chromatography (200 g, 60 × 5 cm) eluting with an n-hexane−acetone step gradient system (100:0, 25:1, 15:1, 10:1, 4:1, each 250 mL) to afford seven subfractions. Subfraction 1.4 (1.0 g) was purified with the

correlations from H-31 to C-23, -24, and -25; from H-20 to C21; and from H-22 to C-21, respectively, confirmed the presence of a C-24/C-31 terminal methylene double bond and a C-21 carboxylic acid function. In addition, there were 2J, 3Jcorrelations between H-3 and C-1, -2, -4, -28, and -29, to support the presence of dihydroxylation at C-2 and C-3. The orientations of H-2 and H-3 were characterized as equatorial and axial, respectively, according to NOESY analysis and the coupling constants of H-2 (m) and H-3 (J = 9.5 Hz). Therefore, the configurations of OH-2 and OH-3 were determined as β and β with the aid of the above spectral evidence. All the other proton and carbon assignments were performed with other 2D spectral analyses, and the chemical structure of 5 was elucidated as shown in Figure 1 and named trivially as hexagonin E. In addition to hexagonins A−E (1−5), four known compounds, hexatenuin A,7 ergosterol,9 ergosterol peroxide,10 and ursolic acid,11 were also identified by comparison of their physical and spectral data with those reported. Those purified triterpenoids isolated in sufficient quantity were examined for their inhibition of superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB (Table 3). Among the examined constituents, hexatenuin A displayed the most significant inhibition of superoxide anion generation and elastase release with IC50 values of 1.9 ± 0.2 and 4.3 ± 1.4 μM, compared with the reference compound LY294002,12 with IC50 values of 0.4 ± 0.02 and 1.5 ± 0.3 μM toward superoxide anion generation and elastase release, respectively. In addition, the following structure−activity relationships could be deduced from the bioactivity data. Hexagonins B (2) and D (4), which possess the basic triterpenoid skeleton without the malonyl substitution at C-3, did not show anti-inflammatory bioactivity. Comparatively, hexagonin A (1), with the triterpenoid skeleton and malonyl and methyl ester functions, also did not exhibit significant activity. Hexatenuin A, which had the triterpenoid skeleton and a free malonic acid group, displayed the most E



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aid of silica gel column chromatography (200 g, 60 × 5 cm) eluting with an n-hexane−acetone step gradient system (100:0, 25:1, 15:1, 10:1, 4:1, each 250 mL) to yield ergosterol (153 mg). Subfraction 1.6 (0.3 g) was purified with the aid of silica gel column chromatography (60 g, 60 × 2 cm) eluting with an n-hexane−acetone step gradient system (100:0, 25:1, 15:1, 10:1, 4:1, each 250 mL) to yield hexagonin C (3) (5 mg). Subfraction 1.7 (0.5 g) was subjected to RP-18 column chromatography (100 g, 60 × 3 cm) eluting with MeOH−water to afford hexagonin A (1) (134 mg). Fraction 2 (2.3 g) was subjected to silica gel column chromatography (200 g, 60 × 3 cm) eluting with an n-hexane−acetone step gradient system (9:1, 6:1, each 200 mL) to afford six subfractions. Subfraction 2.6 (0.5 g) was subjected to Sephadex LH-20 column chromatography (50 g, 60 × 3 cm) eluting with MeOH−water to produce hexatenuin A (41 mg). Fraction 3 (2.7 g) was chromatographed with the assistance of a silica gel column (200 g, 60 × 3.2 cm) eluting with an n-hexane−acetone step gradient system (9:1, 6:1, 4:1, 1:1, each 250 mL) to afford four subfractions. Subfraction 3.2 (0.5 g) was subjected to RP-18 column chromatography (100 g, 60 × 3 cm) eluting with MeOH−water to result in hexagonin B (2) (31 mg). Fraction 4 (4.7 g) was isolated by silica gel column chromatography (200 g, 60 × 5 cm) eluting with a CHCl3− MeOH step gradient system (20:1, 10:1, 6:1, 4:1:2:1, each 200 mL) to afford five subfractions. Subfraction 4.1 was further subjected to silica gel column chromatography (200 g, 60 × 3 cm) eluting with a CHCl3−MeOH step gradient system (19:1, 16:1, each 200 mL) to yield hexagonin D (4) (10 mg) and ursolic acid (21 mg). Fraction 5 (1.5 g) was isolated by silica gel column chromatography (200 g, 60 × 3 cm) eluting with a CHCl3−MeOH step gradient system (9:1, 6:1, each 200 mL) to result in ergosterol peroxide (43 mg). Fraction 6 (1.2 g) was subjected to silica gel column chromatography (200 g, 60 × 3 cm) eluting with a CHCl3−MeOH step gradient system (9:1, 6:1, each 200 mL) to afford hexagonin E (5) (13 mg). Hexagonin A (1): white powder (CHCl3); mp 184−185 °C; [α]25D +57 (c 0.6, MeOH); UV (MeOH) λ max (log ε) 262 (2.65) nm; IR (neat) νmax 2946, 1759, 1693, 1455, 1376, 1256, 1219, 1156 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 1 and 2; ESIMS m/z 621 ([M + K]+, 60), 605 ([M + Na]+, 26), 521 (33), 505 (100), 483 (48); HRESIMS m/z 605.3451 ([M + Na]+, calcd for C35H50O7Na, 605.3454). Hexagonin B (2): white powder (CHCl3); mp 214−215 °C; [α]25D +66 (c 0.2, MeOH); UV (MeOH) λ max (log ε) 247 (2.35) nm; IR (neat) νmax 3501, 2941, 1756, 1456, 1375, 1256, 1218, 1156 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 1 and 2; ESIMS m/z 505 ([M + Na]+, 100), 483 ([M + H]+, 86); HRESIMS m/z 505.3290 ([M + Na]+, calcd for C31H46O4Na, 505.3294). Hexagonin C (3): white powder (CHCl3); mp 180−181 °C; [α]25D +57 (c 0.6, MeOH); UV (MeOH) λ max (log ε) 262 (2.65) nm; IR (neat) νmax 3319, 2959, 1697, 1460, 1230, 1202 cm−1; 1H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz, acetone-d6), see Tables 1 and 2; ESIMS m/z 547 ([M + Na]+, 100); HRESIMS m/z 547.3394 ([M + Na]+, calcd for C33H48O5Na, 547.3399). Hexagonin D (4): white powder (CHCl3); mp 187−188 °C; [α]25D +22 (c 0.07, MeOH); UV (MeOH) λ max (log ε) 247 (3.18) nm; IR (neat) νmax 3446, 2928, 1750, 1449, 1375, 1256, 1217, 1156, 1147, 1127 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3), see Tables 1 and 2; ESIMS m/z 519 ([M + Na]+, 100), 473 (60), 457 (25), 429 (49); HRESIMS m/z 519.3083 ([M + Na]+, calcd for C31H44O5Na, 519.3086). Hexagonin E (5): white powder (CHCl3); mp 230−231 °C; [α]25D +27 (c 0.3, MeOH); UV (MeOH) λ max (log ε) 261 (2.40), 248 (2.37) nm; IR (neat) νmax 3259, 2918, 1693, 1454, 1375, 1289, 1228 cm−1; 1 H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSOd6), see Tables 1 and 2; ESIMS m/z 509 ([M + Na]+, 100); HRESIMS m/z 509.3599 ([M + Na]+, calcd for C31H50O4Na, 509.3607). 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 a 20 mL 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 1.6% NaCl solution was added to reconstitute the isotonic condition. Purified neutrophils were pelleted and then 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.12 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.12 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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AUTHOR INFORMATION

Corresponding Authors

*Tel: 84-913049689. E-mail: [email protected] (T.-D. Thang). *Tel: 886-6-2757575, ext 65333. Fax: 886-6-2740552. E-mail: [email protected] (T.-S. Wu). Author Contributions #

T. D. Thang and P.-C. Kuo contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful to the Ministry of Science and Technology (MOST), Taiwan, ROC, and MOST (15/2012/ HĐ−NĐT) (Vietnam) for the financial support of the present research.

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REFERENCES

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Chemical Constituents from the Fruiting Bodies of Hexagonia apiaria

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