Garcimultiflorone G, a Novel ... - Wiley Online Library

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)

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Garcimultiflorone G, a Novel Benzoylphloroglucinol Derivative from Garcinia multiflora with Inhibitory Activity on Neutrophil Pro-Inflammatory Responses by Chia-Wei Ting a ), Tsong-Long Hwang b ), Ih-Sheng Chen a ), Ming-Jen Cheng c ), Ping-Jyun Sung d ), Ming-Hong Yen* a ), and Jih-Jung Chen* e ) b

a ) Faculty of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan ) Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan c ) Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI), Hsinchu 300, Taiwan d ) National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan e ) Graduate Institute of Pharmaceutical Technology & Department of Pharmacy, Tajen University, Pingtung 907, Taiwan (phone: þ 886-8-7624002 ext. 2827; fax: þ 886-8-7624002 ext. 5121; e-mail: [email protected])

A novel benzoylphloroglucinol derivative, garcimultiflorone G (1), was isolated from the fruits of Garcinia multiflora. The structure of 1 was determined through extensive 1D- and 2D-NMR, and MS analyses. Garcimultiflorone G (1) showed inhibitory effects against superoxide anion (O .2 ) generation and elastase release by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/ cytochalasin B (fMLP/CB), with IC50 values of 6.97 1.56 and 11.70 1.58 mm, respectively.

Introduction. – Garcinia multiflora Champ. is a small evergreen tree, distributed in South China, Hong Kong, and Taiwan [1]. Benzophenones [2] [3], phloroglucinols [4] [5], terpenoids [4] [5], xanthones [6] [7], biflavonoids [8], and their derivatives are widely distributed in plants of the genus Garcinia (Guttiferae). Many of these isolates possess diverse biological features, including antitubercular [6], trypanocidal [7], antioxidant [3], cytotoxic [2] [3], anti-inflammatory [4] [5], and anti-HIV [8] activities. In a preliminary screening, the MeOH extract of the fruit of this species showed inhibitory effects on O 2. generation and elastase release by human neutrophils in response to fMLP/CB. Previous chemical work on this plant led to the isolation of some benzophenone and benzoylphloroglucinol derivatives [9] [10]. In this study, a new benzoylphloroglucinol derivative, garcimultiflorone G (1), was isolated from this species, and its anti-inflammatory activity, by suppressing fMLP/CB-induced superoxide radical anion (O .2 ) generation and elastase release by human neutrophils, was evaluated. Results and Discussion. – 1. Structure Elucidation. Chromatographic purification of the AcOEt-soluble fraction of the MeOH extract of fruits of G. multiflora by silica-gel column chromatography, MPLC, and preparative TLC afforded a new compound 1, which was isolated as amorphous, optically active powder. Its molecular formula, C38H50O7, was determined on the basis of the positive HR-ESI-MS (m/z 641.3452 2014 Verlag Helvetica Chimica Acta AG, Zrich

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([M þ Na] þ , C38H50NaO þ7 ; calc. 641.3454)) and this was supported by the 1H-, 13C-, and DEPT-NMR data. The IR spectrum evidenced the presence of OH (3426 cm 1) and C¼O (1735, 1705 cm 1) groups. The 1H-NMR spectrum (Table 1) indicated the presence of a lavandulyl ( ¼ 5methyl-2-(prop-1-en-2-yl)hex-4-en-1-yl) group (d(H) 1.56 (s, Me(35)), 1.61 (s, Me(40)), 1.68 (s, Me(39)), 1.84 (dd, J ¼ 14.5, 4.0, 1 H, CH2(31)), 2.05 – 2.11 (m, CH2(36)), 2.27 (dd, J ¼ 14.5, 9.5, 1 H, CH2(31)), 2.56 – 2.64 (m, HC(32)), 4.61 (br. s, 1 H, CH2(34)), 4.69 (br. s, 1 H, CH2(34)), 5.02 (br. t, J ¼ 6.8, HC(37))), a phenyl group (d(H) 7.29 (br. dd, J ¼ 8.0, HC(27) and HC(29)), 7.31 (br. d, J ¼ 8.0, HC(26) and HC(30)), 7.42 (br. t, J ¼ 8.0, HC(28))), six further Me groups (d(H) 1.10 (s, Me(19)), 1.12 (s, Me(18)), 1.16 (s, Me(21)), 1.30 (s, Me(20)), 1.35 (s, Me(22)), 1.47 (s, Me(23))), a CHO H-atom (d(H) 4.58 (dd, J ¼ 11.5, 3.5, HC(3))), and several CH2 and CH H-atoms. The 13C-NMR (Table 1) and DEPT spectra of 1 revealed the presence of a benzoyl group (d(C) 127.8 (2 ), 128.9 (2 ), 132.2, 135.1, 192.4), three C¼O groups (d(C) 204.0, 205.7, 208.5), nine Me groups (d(C) 18.0 (3 ), 22.6, 24.9, 25.2, 25.7, 26.3, 28.7), five CH2 C-atoms (d(C) 31.4, 31.6, 33.4, 34.6, 45.1), two O-bearing quaternary C-atoms (d(C) 73.1, 88.4), four quaternary aliphatic C-atoms (d(C) 50.7, 65.9, 67.3, 82.1), an Obearing C-atom (d(C) 88.6), and four olefinic C-atoms (d(C) 112.5, 122.5, 132.2, 148.9). Comparison of the 1H- and 13C-NMR data (Table 1) of 1 with those of otogirinin B [11] indicated that their structures were closely related, except that the 14-lavandulyl group of 1 replaced a 14-geranyl group of otogirinin B [11]. This was supported by both HMBCs (Fig. 1) between CH2(31) (d(H) 1.84, 2.27) and C(13) (d(C) 205.7), C(14) (d(C) 67.3), C(15) (d(C) 45.1), C(16) (d(C) 204.0), C(32) (d(C) 43.5), C(33) (d(C) 148.9), and C(36) (d(C) 33.4), and NOESY correlations (Table 1) between CH2(31) (d(H) 1.84, 2.27) and CH2(15) (d(H) 1.80, 2.68), and between HC(32) (d(H) 2.56 – 2.64) and Me(35) (d(H) 1.56) and CH2(36) (d(H) 2.05 – 2.11). The NOESY cross-peaks (Fig. 2) HaC(3)/Me(20), HaC(3)/HaC(7), HaC(7)/ HaC(8), HaC(7)/Me(22), HaC(8)/Me(20), HbC(8)/CH2(15), CH2(15)/Me(21), CH2(15)/Me(23), CH2(15)/CH2(31), and Me(22)/HC(26) evidenced that HC(3), HC(7), Me(20), Me(22), and the benzoyl group were a-oriented, and Me(21), Me(23), the 2-hydroxypropan-2-yl group at C(3), and the C(14)-lavandulyl group were b-oriented. Compound 1 showed similar CD Cotton effects (221 ([q] ¼ þ 1410) and 252 ([q] ¼ þ 1049) nm) compared with those of the analogous benzoylphloroglucinol


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Table 1. 1H- and 13C-NMR (500 and 125 MHz resp., in CDCl3 ) Data of 1. d in ppm, J in Hz. Position 1 2 3 6 7 8a 8b 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 a

d(H ) 1.61 (dd , J ¼ 14.8, 3.5), 3.45 (dd , J ¼ 14.8, 11.5) 4.58 (dd, J ¼ 11.5, 3.5) 2.71 (t, J ¼ 7.0) 1.47 – 1.53 (m) 2.31 – 2.37 (m) 2.04 – 2.10 (m)

1.80 (d, J ¼ 14.0), 2.68 (dd, J ¼ 14.0, 6.0)

1.12 1.10 1.30 1.16 1.35 1.47

(s) (s) (s) (s) (s) (s)

7.31 (br. d, J ¼ 8.0) 7.29 (br. dd, J ¼ 8.0) 7.42 (br. t, J ¼ 8.0) 7.29 (br. dd, J ¼ 8.0) 7.31 (br. d, J ¼ 8.0) 1.84 (dd, J ¼ 14.5, 4.0), 2.27 (dd, J ¼ 14.5, 9.5) 2.56 – 2.64 (m) 4.61 (br. s), 4.69 (br. s) 1.56 (s) 2.05 – 2.11 (m) 5.02 (br. t, J ¼ 6.8) 1.68 (s) 1.61 (s)

d(C ) 65.9 (s) b ) 31.4 (t) 88.6 (d) 88.4 (s) 42.1 (d) 31.6 (t) 44.6 (d) 50.7 (s) 82.1 (s) 208.5 (s) 205.7 (s) 67.3 (s) 45.1 (t) 204.0 (s) 73.1 (s) 24.9 (q) 26.3 (q) 28.7 (q) 18.0 (q) 22.6 (q) 25.2 (q) 192.4 (s) 135.1 (s) 128.9 (d) 127.8 (d) 132.2 (d) 127.8 (d) 128.9 (d) 34.6 (t) 43.5 (d) 148.9 (s) 112.5 (t) 18.0 (q) 33.4 (t) 122.5 (d) 132.2 (s) 25.7 (q) 18.0 (q)

NOESY

HMBC a )

3 3 2, 7, 18, 19, 20

1, 12, 13, 17 1, 3, 7, 12, 13, 17 1, 2, 19

3, 8a, 22 7, 20, 22 15 8, 23

1, 2, 6, 8, 9, 12, 13, 20 1, 9, 15 1, 9 11

8b, 21, 23, 31 8b, 21, 23, 31

10, 13 8, 9, 13, 16

3 3 3, 8a 15 7, 8a, 26 15

3, 17, 19 3, 18 6, 7 6, 7, 20 9, 10, 11, 23 9, 10, 11

22 26, 28 27, 29 28, 30 29 15, 32 15, 32 31, 34, 35, 36

24, 25, 28, 30 25, 26, 29 26, 30 25, 27, 30 24, 25, 26, 28 13, 14, 15, 32, 36 13, 14, 15, 16, 33 14, 34, 37

32 32, 35 32, 34 37, 40 36, 39

32, 33, 35 32, 35 32, 33, 34 31, 33, 38 32, 38, 39, 40

37 36

37, 38, 40 37, 39

) From the H- to the C-atom. b ) Multiplicities deduced from DEPT.

derivative otogirinin B [11]. Thus, both possessed the same configuration. On the basis of the evidence above, the structure of garcimultiflorone G (1) was elucidated as depicted. This structure was supported by 1H,1H-COSY (Fig. 1) and NOESY (Fig. 2)

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Fig. 1. The 1H,1H-COSY (— —) and selected key HMB (H ! C) correlations of 1

Fig. 2. Selected key NOESY (H $ H) correlations of 1

experiments, and 13C-NMR assignments were confirmed by DEPT, HSQC, and HMBC (Fig. 1) techniques. 2. Biological Studies. Reactive oxygen species (ROS; e.g., superoxide anion (O .2 ), H2O2 ) and granule proteases (e.g., elastase, cathepsin G) produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. The effect on neutrophil pro-inflammatory response of compound 1 was evaluated by suppressing fMet-Leu-Phe/cytochalasin B (fMLP/CB)-induced superoxide anion (O .2 ) generation and elastase release by human neutrophils. The inhibitory-activity data on neutrophil pro-inflammatory responses are compiled in Table 2. Diphenyleneiodonium and phenylmethanesulfonyl fluoride were used as positive controls for O .2 generation and elastase release, respectively. Compound 1 exhibited inhibitory activities with IC50 Table 2. Inhibitory Effects of 1 on O .2 Generation and Elastase Release by Human Neutrophils in Response to N-Formyl-l-methionyl-l-leucyl-l-phenylalanine/Cytochalasin B Compound

IC50 [mm] a ) O .2 Generation

Garcimultiflorone G (1) Diphenyleneiodonium b ) Phenylmethanesulfonyl fluoride b ) a b

6.97 1.56 1.69 0.72

Elastase release 11.70 1.58 202.3 31.8

) Concentration necessary for 50% inhibition ( IC50 ); results are presented as means S.E.M. (n ¼ 4). ) Diphenyleneiodonium and phenylmethanesulfonyl fluoride were used as positive controls.


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values of 6.97 1.56 and 11.70 1.58 mm, respectively, against fMLP/CB-induced O .2 generation and elastase release. Based on these findings, compound 1 is a candidate for treatment of inflammatory diseases. Thus, the detailed mechanism of action of compound 1 seems to be worth of further studies. This research was supported by grants from the National Science Council of the Republic of China (NSC 98-2320-B-127-001-MY3 and NSC 101-2320-B-127-001-MY3) to Prof. J.-J. C. We also thank the National Center for High-Performance Computing (NCHC, Taiwan) for providing computer resources and chemical database services. Experimental Part General. Column chromatography (CC): Silica gel 60 (SiO2 ; 70 – 230 or 230 – 400 mesh; Merck). TLC: Silica gel 60 F254 precoated plates (Merck). Optical rotation: Jasco DIP-370 polarimeter; in CHCl3 . CD Spectra: Jasco J-810 spectropolarimeter; lmax ([q]) in nm. UV Spectra: Jasco UV-240 spectrophotometer; lmax (log e) in nm. IR Spectra: Perkin-Elmer 2000 FT-IR spectrophotometer; ˜n in cm 1. 1H-, 13 C-, and 2D-NMR spectra: Varian Inova 500 spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI- and HR-ESI-MS: Bruker APEX-II mass spectrometer; in m/z. Plant Material. The fruits of G. multiflora were collected from Mudan, Pingtung County, Taiwan, in September 2007, and identified by Dr. I.-S. C. A voucher specimen (Chen 6061) was deposited with the Faculty of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. Extraction and Isolation. The dried fruits (3 kg) of G. multiflora were pulverized and extracted with MeOH (3 10 l) at r.t. for 3 d. The MeOH extract was concentrated under reduced pressure at 358, and the residue (330 g) was partitioned between AcOEt and H2O (1 : 1) to provide the AcOEt-soluble fraction (Fr. A; 167 g). Fr. A (120 g) was purified by CC (SiO2 (4.8 kg), 70 – 230 mesh; CH2Cl2/MeOH gradient) to afford 14 fractions, Frs. A1 – A14. Fr. A3 (7.5 g) was subjected to CC (SiO2 (290 g), 70 – 230 mesh; hexane/acetone 8 : 1, 550-ml fractions) to give twelve subfractions, Frs. A3.1 – A3.12. Fr. A3.9 (318 mg) was purified by MPLC (SiO2 (11 g), 230 – 400 mesh; hexane/AcOEt 20 : 1; 200-ml fractions) to give nine subfractions, Frs. A3.9.1 – A3.9.9. Fr. A3.9.8 (124 mg) was further subjected to prep. TLC (SiO2 ; hexane/AcOEt 8 : 1) to afford 1 (4.8 mg). Garcimultiflorone G ( ¼ (1S,3R,7R,11S,13R)-11-Benzoyl-3-(2-hydroxypropan-2-yl)-6,6,10,10-tetramethyl-13-[5-methyl-2-(prop-1-en-2-yl)hex-4-en-1-yl]-4,5-dioxatetracyclo[9.3.1.19,13.01,7 ]hexadecane12,14,15-trione; 1). Amorphous powder. UV (MeOH): 214 (3.89), 244 (3.95), 380 (sh, 3.28). CD (MeOH): 221 ( þ 1410), 252 ( þ 1049). [a] 25 D ¼ þ 13.2 (c ¼ 0.15, MeOH). IR (KBr): 3426 (OH), 1735 (C¼O), 1705 (C¼O). 1H- and 13C-NMR: see Table 1. ESI-MS: 641 ([M þ Na] þ ). HR-ESI-MS: 641.3452 ([M þ Na] þ , C38H50NaO þ7 ; calc. 641.3454). Biological Assay. The effect of the isolated compounds on neutrophil pro-inflammatory response was evaluated by monitoring the inhibition of O .2 generation and the release of elastase in fMLP/CBactivated human neutrophils in a concentration-dependent manner. Preparation of Human Neutrophils. Human neutrophils from venous blood of healthy, adult volunteers (20 – 28 years old) were isolated using a standard method of dextran sedimentation prior to centrifugation in a FicollHypaque gradient and hypotonic lysis of erythrocytes [12]. Purified neutrophils containing > 98% viable cells, as determined by the trypan-blue exclusion method [13], were re-suspended in a Ca2 þ -free Hanks buffered salt soln. (HBSS) at pH 7.4 and were maintained at 48 prior to use. Measurement of O .2 Generation. The measurement was based on the superoxide dismutase (SOD)inhibitable reduction of ferricytochrome c [14] [15]. Briefly, after supplementation with 0.5 mg/ml ferricytochrome c and 1 mm Ca2 þ , neutrophils (6 105/ml) were equilibrated at 378 for 2 min and incubated with different concentrations (10 – 0.01 mg/ml) of compounds or DMSO (as control) for 5 min. Cells were incubated with cytochalasin B (CB; 1 mg/ml) for 3 min prior to the activation with 100 nm Nformyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) for 10 min. Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell

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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 (e ¼ 21.1 mm 1 cm 1). Measurement of Elastase Release. Degranulation of azurophilic granules was determined by measuring the elastase release as described in [15]. Experiments were performed using MeO-Suc-AlaAla-Pro-Val-p-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-AlaAla-Pro-Val-p-nitroanilide (100 mm), neutrophils (6 105/ml) were equilibrated at 378 for 2 min and incubated with compounds for 5 min. Cells were stimulated with fMLP (100 nm)/CB (0.5 mg/ml), and changes in 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 fMLP/CB-activated, drug-free control system. Statistical Analysis. Results are expressed as the mean S.E.M. (n ¼ 4), and comparisons were made using Students t-test. A probability of 0.05 or less was considered significant. The software SigmaPlot was used for the statistical analysis.

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