Fitoterapia 117 (2017) 16–21

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Fitoterapia

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Naphthofuranone derivatives and other constituents from Pachira aquatica with inhibitory activity on superoxide anion generation by neutrophils

Lin-Yang Cheng a,1,Hsiang-RueiLiaob, Li-Chai Chen c,d,1, Shih-Wei Wang e,Yueh-HsiungKuof,g,1, Mei-Ing Chung a,⁎, Jih-Jung Chen c,h,⁎ a School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan b Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan c Department of Pharmacy, Tajen University, Pingtung 90741, Taiwan d Department of Pharmacy, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung 81342, Taiwan e Department of Medicine, Mackay Medical College, New Taipei City 25245, Taiwan f Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 40402, Taiwan g Department of Biotechnology, Asia University, Taichung 41354, Taiwan h Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 40447, Taiwan article info abstract

Article history: Two new naphthofuranone derivatives, 11-hydroxy-2-O-methylhibiscolactone A (1)andO-methylhibiscone D Received 11 October 2016 (2), have been isolated from the stems of Pachira aquatica, together with 18 known compounds (3−20). The Received in revised form 27 November 2016 structures of two new compounds were determined through spectroscopic and MS analyses. Among the isolated Accepted 1 December 2016 compounds, 11-hydroxy-2-O-methylhibiscolactone A (1), isohemigossylic acid lactone-7-methyl ether (4), Available online 22 December 2016 gmelofuran (6), and 5-hydroxyauranetin (8) exhibited inhibition (IC50 ≤ 28.84 μM) of superoxide anion generation by human neutrophils in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Keywords: Pachira aquatica © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license Bombacaceae (http://creativecommons.org/licenses/by-nc-nd/4.0/). Structure elucidation Naphthofuranone Anti-inﬂammatory activity

1. Introduction anti-HIV [5], hypotensive [7], and antiangiogenic [8] activities. In our studies on the anti-inflammatory constituents of Formosan plants, Pachira aquatica Aublet (Bombacaceae) is an evergreen tree [1],dis- many species have been screened for in vitro inhibitory activity on tributed in tropical America and introduced to Guangdong, southern neutrophil pro-inflammatory responses, and P. aquatica has been Yunnan, and Taiwan as a cultivated plant. It is known by the common found to be an active species. The current phytochemical investigation names Malabar chestnut, French peanut, Guiana chestnut, monguba of the stems of this plant has led to the isolation of two new (Brazil), pumpo (Guatemala) and is commercially sold under the naphthofuranones, 11-hydroxy-2-O-methylhibiscolatctone A (1) and names money tree and money plant. P. aquatica is a common house- O-methylhibiscone D (2), along with 18 known compounds. The struc- plant. It is often referred to as a money tree, as it is supposed to bring tural elucidation of 1 and 2 and the anti-inflammatory activity of the iso- good luck and money into the owner's home. In our studies on the lates are described herein. anti-inflammatory constituents of Formosan plants, many species have been screened for in vitro inhibitory activity on neutrophil pro-in- 2. Experimental flammatory responses, and P. aquatica has been found to be an active species. Sesquiterpenoids [2,3], neolignans [4], lignans [5], flavonoids 2.1. General [6,7], triterpenoids [8], and their derivatives are widely distributed in plants of the family Bombacaceae. Many of these compounds exhibit Optical rotations were measured using a Jasco DIP-370 polarimeter

in CHCl3. Ultraviolet (UV) spectra were obtained on a Jasco UV-240 spectrophotometer. Infrared (IR) spectra (neat or KBr) were recorded ⁎ Corresponding authors. E-mail address: [email protected] (J.-J. Chen). on a Perkin Elmer 2000 FT-IR spectrometer. Nuclear magnetic reso- 1 Authors have contributed equally in this manuscript. nance (NMR) spectra, including correlation spectroscopy (COSY),

http://dx.doi.org/10.1016/j.fitote.2016.12.008 0367-326X/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). L.-Y. Cheng et al. / Fitoterapia 117 (2017) 16–21 17 nuclear Overhauser effect spectrometry (NOESY), heteronuclear (235 mg) by recrystallization with acetone. Fraction A6-6 (352 mg) multiple-bond correlation (HMBC), heteronuclear single-quantum was purified by MPLC (230–400 mesh, 15 g, n-hexane/acetone, 5:1, coherence (HSQC) experiments, were recorded on a VNMRS-400 spec- 300 mL fractions) to obtain 9 subfractions: A6-6-1–A6-6-9. Part trometer operating at 400 MHz (1H) and 100 MHz (13C), respectively, (28 mg) of fraction A6-6-2 was purified by preparative TLC (silica gel, with chemical shifts given in ppm (δ) using tetramethylsilane (TMS) n-hexane/acetone, 3:1) to obtain 14 (3.9 mg) (Rf =0.31).Part as an internal standard. Electrospray ionization (ESI) and high-resolu- (146 mg) of fraction A6-6-5 was purified by preparative TLC (silica tion electrospray ionization (HRESI)-mass spectra were recorded on a gel, n-hexane/acetone, 1:1) to yield 11 (3.7 mg) (Rf = 0.85) and 12 Bruker APEX II mass spectrometer. Silica gel (70–230, 230–400 mesh) (3.2 mg) (Rf = 0.93). Part (182 mg) of fraction A6–6-6 was purified (Merck) was used for column chromatography (CC). Silica gel 60 F- by preparative TLC (silica gel, CHCl3/acetone, 10:1) to afford 15 254 (Merck) was used for thin-layer chromatography (TLC) and prepar- (4.8 mg) (Rf = 0.55). Fraction A9 (6.2 g) was chromatographed further ative thin-layer chromatography (PTLC). on silica gel (230–400 mesh, 280 g) eluting with CHCl3/MeOH (20:1– 0:1) to give 9 fractions (each 500 mL, A9-1–A9-9). Part (120 mg) of frac- 2.2. Plant material tion A9-4 was purified by preparative TLC (silica gel, n-hexane/acetone,

2:1) to obtain 1 (3.5 mg) (Rf =0.29). The stems of P. aquatica was collected from Tajen University, 11-Hydroxy-2-O-methylhibiscolactone A (1): colorless needles

Pingtung County, Taiwan, in August 2009 and identiﬁed by Prof. J.-J. (CH2Cl2-MeOH); m.p. 185–187 °C; UV (MeOH) λmax (log ε) 221 Chen. A voucher specimen (PA-200908) was deposited in the Depart- (4.50), 254 (4.41), 358 (3.98) nm; IR (KBr) υmax 3367 (OH), 1729 −1 1 ment of Pharmacy, Tajen University, Pingtung, Taiwan. (C = O) cm ; H NMR (acetone-d6, 400 MHz): δ 1.71 (6H, s, H-12 and H-13), 2.32 (3H, s, H-10), 4.22 (3H, s, OMe-2), 7.12 (1H, s, H-6), 13 2.3. Extraction and isolation 7.90 (1H, s, H-4); C NMR (acetone-d6, 100 MHz): δ 18.7 (C-10), 32.6 (C-12), 32.6 (C-13), 60.6 (OMe-2), 73.9 (C-11), 98.9 (C-8), 115.8 (C- The dried stems (3.2 kg) of P. aquatica were pulverized and extracted 4a), 121.3 (C-6), 124.5 (C-4), 126.2 (C-3), 133.3 (C-1), 135.3 (C-8a), three times with MeOH (40 L each) for 3 days. The MeOH extracts were 140.2 (C-2), 156.6 (C-5), 165.4 (C-9), 168.8 (C-7); ESI-MS m/z 287 − − concentrated under reduced pressure at 35 °C, and the residue (166 g) [M − H] ; HR-ESI-MS m/z 287.0913 [M − H] (calcd for C16H15O5Na, was partitioned between n-hexane and H2O(1:1).Then-hexane layer 287.0919). 23 was concentrated to give a residue (fraction A, 75 g). The water layer O-Methylhibiscone D (2): colorless oil; [α] D +40.6(c 0.16, was further extracted with n-BuOH, and the n-BuOH-soluble part (frac- CHCl3); UV (MeOH): λmax (log ε) 208 (4.47), 239 (3.91), 291 (3.26) −1 1 tion B, 33 g) and the water-solubles (fraction C, 47 g) were separated. nm; IR (neat) υmax 1694 (C = O) cm ; H NMR (CDCl3, 400 MHz): δ Fraction A (75 g) was chromatographed on silica gel (70–230 mesh, 0.83 (3H, d, J = 6.8 Hz, H-13), 0.89 (3H, d, J = 6.8 Hz, H-12), 2.08 (1H, 3.8 kg), eluting with n-hexane, gradually increasing the polarity with qqd, J = 6.8, 6.8, 4.4 Hz, H-11), 2.37 (3H, s, H-10), 2.77 (1H, dd, J = EtOAc or MeOH to give 10 fractions: A1 (5 L, n-hexane), A2 (3 L, n-hex- 16.4, 4.4 Hz, Hα-6), 2.84 (1H, dd, J = 16.4, 6.4 Hz, Hβ-6), 3.28 (1H, ane/EtOAc, 50:1), A3 (4 L, n-hexane/EtOAc, 30:1), A4 (3 L, n-hexane/ ddd, J = 6.4, 4.4, 4.4 Hz, H-5), 4.24 (3H, s, OMe-2), 6.98 (1H, s, H-4), 13 EtOAc, 10:1), A5 (4 L, n-hexane/EtOAc, 5:1), A6 (5 L, n-hexane/EtOAc, 7.99 (1H, s, H-9); C NMR (CDCl3, 100 MHz): δ 16.5 (C-10), 18.5 (C- 3:1), A7 (4 L, n-hexane/EtOAc, 1:1), A8 (5 L, n-hexane/EtOAc, 1:2), A9 13), 20.1 (C-12), 33.7 (C-11), 43.6 (C-6), 45.3 (C-5), 59.9 (OMe-2), (2 L, EtOAc), A10 (2 L, MeOH). Fraction A3 (4.2 g) was chromatographed 118.3 (C-8), 124.7 (C-4), 124.8 (C-4a), 125.4 (C-3), 128.4 (C-8a), 141.3 further on silica gel (230–400 mesh, 205 g) eluting with n-hexane/ (C-2), 142.2 (C-9), 143.1 (C-1), 194.7 (C-7); ESI-MS m/z 281 EtOAc (30:1–0:1) to give 8 fractions (each 1 L, A3-1–A3-8). Part [M + Na]+; HR-ESI-MS m/z 281.1155 [M + Na]+ (calcd for

(185 mg) of fraction A3-3 was purified by preparative TLC (silica gel, C16H18O3Na, 281.1154). n-hexane/EtOAc, 30:1) to afford 17 (5.9 mg) (Rf = 0.42). Fraction A5 (11.8 g) was chromatographed further on silica gel (230–400 mesh, 2.4. Biological assay 540 g) eluting with n-hexane/acetone (10:1–0:1) to give 12 fractions (each 1.2 L, A5-1–A5-12). Compound 8 (12.2 mg) was obtained from The effect of the isolated compounds on neutrophil pro-inflammato- fraction 5-1 (452 mg) by recrystallization with n-hexane/EtOAc. Com- ry response was evaluated by monitoring the inhibition of superoxide pound 19 (6.2 mg) was yielded from fraction 5-2 (201 mg) by recrystal- anion generation in fMLP-activated human neutrophils in a concentra- lization with CHCl3/MeOH. Part (185 mg) of fraction A5-3 was purified tion-dependent manner. by preparative TLC (silica gel, n-hexane/acetone, 4:1) to afford 4

(6.5 mg) (Rf = 0.26) and 16 (4.7 mg) (Rf = 0.68). Part (150 mg) of frac- 2.4.1. Preparation of human neutrophils tion A5-4 was puriﬁed by preparative TLC (silica gel, CHCl3/MeOH, 20:1) Human neutrophils from the venous blood [9] of healthy, adult vol- to obtain 2 (4.4 mg) (Rf = 0.93). Part (188 mg) of fraction A5-6 was pu- unteers (20–35 years old) were isolated using a standard method of riﬁed by preparative TLC (silica gel, n-hexane/acetone, 3:1) to obtain 5 dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradi-

(4.2 mg) (Rf = 0.55). Part (205 mg) of fraction A5-7 was puriﬁed by pre- ent and hypotonic lysis of erythrocytes as previously described [10].Pu- parative TLC (silica gel, n-hexane/acetone, 3:1) to obtain 7 (3.5 mg) riﬁed neutrophils containing N98% viable cells, as determined by the

(Rf = 0.82) and 3 (5.2 mg) (Rf = 0.28). Fraction A5-10 (435 mg) was trypan blue exclusion method, were resuspended in HBSS buffer at puriﬁed by medium pressure liquid chromatography (MPLC) (230– pH 7.4 and were maintained at 4 °C prior to use.

400 mesh, 20.5 g, CH2Cl2/acetone, 25:1, 250 mL fractions) to obtain 11 •– subfractions: A5-10-1–A5-10-11. Fraction A5-10-2 (23 mg) was puri- 2.4.2. Measurement of O2 generation •– fied further by preparative TLC (silica gel, CH2Cl2/EtOAc, 50:1) to obtain The assay for measurement of O2 generation was based on the SOD- 9 (28.0 mg) (Rf = 0.73). Fraction A5-10-6 (33 mg) was purified by pre- inhibitable reduction of ferricytochrome c [11]. In brief, neutrophils 6 parative TLC (silica gel, CHCl3/acetone, 30:1) to obtain 10 (5.2 mg) (1 × 10 cells/mL) pretreated with the various test agents at 37 °C for (Rf = 0.61) and 13 (3.5 mg) (Rf = 0.80). Compound 18 (8 mg) was ob- 5 min were stimulated with fMLP (1 μmol/L) in the presence of •– tained from fraction 5-11 (253 mg) by recrystallization with MeOH. ferricytochrome c (0.5 mg/mL). Extracellular O2 production was Fraction A6 (9.2 g) was chromatographed further on silica gel (230– assessed with a UV spectrophotometer at 550 nm (Hitachi U-3010, 400 mesh, 415 g) eluting with n-hexane/acetone (6:1–0:1) to give 9 Tokyo, Japan). The percentage of superoxide inhibition of the test com- fractions (each 1.2 L, A6-1–A6-9). Fraction A6–1 (180 mg) was purified pound was calculated as the percentage of inhibition = {(control − by preparative TLC (silica gel, CH2Cl2/MeOH, 50:1) to give 6 (4.5 mg) resting) − (compound − resting)} / (control − resting) × 100. The soft- (Rf = 0.28). Compound 20 (9.11 mg) was obtained from fraction 6-3 ware, SigmaPlot was used for determining the IC50 values. 18 L.-Y. Cheng et al. / Fitoterapia 117 (2017) 16–21

2.4.3. Statistical analysis negative-ion HR-ESI-MS, showing an [M − H]− ion at m/z 287.0913

Results are presented as average ± standard error of the mean (calcd for C16H15O5, 287.0919). The IR absorption bands implied the (SEM) (n = 4), and comparisons were made using Student's t-test. A presence of OH group (3367 cm−1) and carbonyl group (1729 cm−1). probability of 0.05 or less was considered significant. The 1H NMR spectrum of 1 showed the presence of a methyl group [δ 2.32 (3H, s, H-10)], a methoxy group [δ 4.22 (3H, s, OMe-2)], a 2- 3. Results and discussion hydroxypropan-2-yl group [δ 1.71 (6H, s, H-12 and H-13)], and two aromatic protons [δ 7.12, 7.90 (each 1H, each s, H-6 and H-4)]. The 1H Chromatographic purification of the n-haxane-soluble fraction of a NMR spectrum of 1 was similar to those of isohemigossylic acid lac- MeOH extract of stems of P. aquatica on a silica gel column and prepar- tone-2-methyl ether [12], except that the 11-hydroxy group of 1 re- ative thin-layer chromatography (TLC) afforded two new (1 and 2)and placed H-11 of isohemigossylic acid lactone-2-methyl ether [12]. This eighteen known compounds (3–20)(Fig. 1). was supported by (1) H-12/13 (δH 1.71) and C-11 (δC 73.9) of 1 shift to the lower field compared with those of isohemigossylic acid lac- 3.1. Structure elucidation of the new naphthofuranones tone-2-methyl ether [12], (2) NOESY correlations observed between H-12 (δ 1.71) and H-6 (δ 7.12), (3) HMBC correlations observed be- 11-Hydroxy-2-O-methylhibiscolactone A (1) was isolated as color- tween H-12 (δ 1.71) and C-5 (δ 156.6) and C-11 (δ 73.9), and (4) less needles with molecular formula C16H16O5 as determined by HMBC correlations observed between H-6 (δ 7.12) and C-11 (δ 73.9).

O O O O O 9 9 C HO 8a OCH3 O 8a OR R OH 8 1 8 1 7 B 2 7 2 6 A 3 6 3 5 4 4a 4 5 4a 10 10

11 11 12 13 12 13 OH 2 R=CH3 3 R=OH 1 5 R=H 4 R=OCH3

O CHO O

O O HO OCH3

O 67

R5 8 R =OH,R =R =R =R =OCH,R =R =H R6 1 2 3 4 6 3 5 7 R 3' 4 2' 4' 9 R1 =OH,R2 =R4 =R5 =R7 =H,R3 =R6 =OCH3 1' 5' 10 R3 O 6' R1 =OH,R2 =R3 =R6 =OCH3,R4 =R5 =R7 =H 8 R 7 2 7 11 R1 =CH3,R2 =R3 =R4 =R6 =R7 =OCH3,R5=H 6 3 5 12 R1 =CH3,R2 =R3 =R4 =OCH3,R5 =R7 =H,R6 =OH R 4 OCH 2 3 13 R1 =OH,R2 =R4 =R7 =H,R3 =R5 =R6 =OCH3 R1 O 14 R1 =R6 =OH,R2 =R4 =R5 =R7 =H,R3 =OCH3

H3CO

HO OO O 15 16

O HO 17 18 OH OH

HO HO 19 20

Fig. 1. The chemical structures of compounds 1–20 from the stems of P. aquatica. L.-Y. Cheng et al. / Fitoterapia 117 (2017) 16–21 19

O O

9 O 9 O

HO 8a OCH3 HO 8a OCH3 8 1 1 7 2 7 6 3 3 5 5 4a 4 4a 10 10 11

12 13 12 13 OH OH

(a) (b)

Fig. 2. Key NOESY (2a) and HMBC (2b) correlations of 1.

The full assignment of 1Hand13C NMR resonances was confirmed by 3H-naphtho[1,8-bc]furan-3-one, named O-methylhibiscone D. This 1H–1HCOSY,NOESY(Fig. 2),DEPT,HSQC,andHMBC(Fig. 2) techniques. was further confirmed by the 1H-1H COSY, NOESY (Fig. 3), DEPT, According to the evidence above, the structure of 1 waselucidatedas3-hy- HSQC, and HMBC (Fig. 3) techniques. droxy-5-(2-hydroxypropan-2-yl)-8-methoxy-7-methyl-2H-naphtho[1,8- bc]furan-2-one, named 11-hydroxy-2-O-methylhibiscolactone A. 3.2. Structure identification of the known isolates O-Methylhibiscone D (2) was obtained as colorless oil. Its molecular formula, C16H18O3, was determined on the basis of the positive HR-ESI- The known isolates were readily identified by a comparison of phys- + 1 MS at m/z 281.1155 [M + Na] (calcd 281.1154) and supported by the ical and spectroscopic data (UV, IR, HNMR,[α]D, and MS) with 1H, 13C, and DEPT NMR data. The presence of carbonyl group was re- corresponding authentic samples or literature values, and this included vealed by the band at 1694 cm−1 in the IR spectrum, and was confirmed two 2H-naphtho[1,8-bc]furan-2-ones, hibiscolactone A (3) [13] and by the resonance at δ 194.7 in the 13C NMR spectrum. The 1H NMR spec- isohemigossylic acid lactone-7-methyl ether (4) [14], a 4,5-dihydro- trum of 2 displayed the presence of a methyl group [δ 2.37 (3H, s, H- 3H-naphtho[1,8-bc]furan-3-one, hibiscone D (5) [13],a 10)], a methoxy group [δ 4.24 (3H, s, OMe-2)], two aromatic protons sesquiterpenoid, gmelofuran (6) [15], a naphthalene-1,4-dione, 2-O- [δ 6.98 (1H, s, H-4), 7.99 (1H, s, H-9)], an isopropanyl group [δ 0.83, methylisohemigossypolone (7) [16], seven flavonoids, 5- 0.89 (each 3H, each d, J = 6.8 Hz, H-13 and H-12), 2.08 (1H, qqd, J = hydroxyauranetin (8) [17], kaempferol 3,7,4′-trimethyl ether (9) [18], 6.8, 6.8, 4.4 Hz, H-11)], and two methylene protons [δ 2.77 (1H, dd, santin-7-methyl ether (10) [17], 3,5,6,7,8,3′,4′-heptamethoxyflavone J = 16.4, 4.4 Hz, Hα-6) and 2.84 (1H, dd, J = 16.4, 6.4 Hz, Hβ-6)] cou- (11) [19],5,4′-dihydroxy-3,6,7,8-tetramethoxyflavone (12) [20], pling with a methine proton [δ 3.28 (1H, ddd, J = 6.4, 4.4, 4.4 Hz, H- retusin (13) [21], and 5,4′-dihydroxy-3,7-dimethoxyflavone (14) [22], 5)]. The 1H NMR data of 2 were similar to hibiscone D (5) [13],except a coumarin, scopoletin (15) [23], a benzophenone, benzophenone that the 2-methoxy group [δ 4.24 (3H, s)] of 2 replaced 2-hydroxy (16) [24], and four triterpenoids, lupenone (17) [25],2α,3β- group [δ 5.70 (1H, br s, D2O exchangeable)] of hibiscone D (5) [13]. dihydroxylup-20(29)-ene (18) [26], (23Z)-cycloart-23-en-3β,25-diol This was supported by the HMBC correlations between OMe-2 (δH (19) [27], and (24R)-cycloart-25-en-3β,24-diol (20) [27]. 4.24)/C-2 (δC 141.3), Me-3 (δH 2.37)/C-2 (δC 141.3), and H-4 (δH 6.98)/C-2 (δC 141.3) of 2. In addition, the ring B of 2 was unaromatized 3.3. Biological studies and established by the following evidences: (a) A tertiary C-5 carbon (δC 45.3), a secondary C-6 carbon (δC 43.6), and a quaternary C-7 carbon (δC Human neutrophils are known to play an significant role in the host 194.7) of ring B of 2 was similar to those of hibiscone D (5) [13] and dif- defense against microorganisms and in the pathogenesis of various dis- ferent from those of 1. (b) Compound 2 showed a dextrorotatory optical eases such as rheumatoid arthritis, ischemia-reperfusion injury, asthma, 23 26 activity with [α]D = + 40.6 as in the cases of hibiscone D (5)([α]D = and chronic obstructive pulmonary disease [28,29].Inresponsetodif- +37)[13] and the absolute configuration of C-5 in 2 was proposed to be ferent stimuli, activated neutrophils secrete a series of cytotoxins, such •– R. Conversely, compound 1 is optically inactive. Thus, the structure of 2 as superoxide anion (O2 ), a precursor of other reactive oxygen species was elucidated as (R)-5-isopropyl-8-methoxy-7-methyl-4,5-dihydro- (ROS), granule proteases, bioactive lipids [9,28,30]. Suppression of the

O O 9 9

O OCH3 O 8a OCH3 8 1 8 1 7 2 2 6 3 6 4a 3 5 4 5 4 10 10

12 13 12 13

(a) (b)

Fig. 3. Key NOESY (3a) and HMBC (3b) correlations of 2. 20 L.-Y. Cheng et al. / Fitoterapia 117 (2017) 16–21 extensive or inappropriate activation of neutrophils by drugs has been Acknowledgements proposed as a way to ameliorate inflammatory diseases. The anti-inflammatory effects of the isolated compounds from the stems of P. This research was supported by a grant from the National Science aquatica were evaluated by suppressing fMet-Leu-Phe (fMLP)-induced Council of the Republic of China (No. NSC 95-2320-B-127-001-MY3), •– O2 generation by human neutrophils. The anti-inflammatory activity awarded to J.-J. Chen. This work was also supported by the grants from data are shown in Table 1. The clinically used anti-inflammatory Zuoying Branch of Kaohsiung Armed Forces General Hospital (ZBH105- agent, ibuprofen, was used as the positive control. From the results of 25 and ZBH105-26). our anti-inflammatory tests, the following conclusions can be drawn: (a) 11-Hydroxy-2-O-methylhibiscolactone A (1), isohemigossylic acid Appendix A. Supplementary data lactone-7-methyl ether (4), gmelofuran (6), and hydroxyauranetin (8) exhibited inhibition (IC50 value ≤28.84 μM) of superoxide anion gener- The 1D, 2D NMR and HRESIMS data of compounds 1 and 2 are avail- ation by human neutrophils in response to formyl-L-methionyl-L- able in the Supplementary data. Supplementary data associated with leucyl-L-phenylalanine (fMLP). 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