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New Benzophenones and Xanthones from Cratoxylum sumatranum ssp. neriifolium and Their Antibacterial and Antioxidant Activities † † ‡ § Cholpisut Tantapakul, Wisanu Maneerat, Tawanun Sripisut, Thunwadee Ritthiwigrom, ∥ ∥ ⊥ † † Raymond J. Andersen, Ping Cheng, Sarot Cheenpracha, Achara Raksat, and Surat Laphookhieo*, † Natural Products Research Laboratory, School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand ‡ School of Cosmetic Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand § Department of Chemistry, Faculty of Science, Chiang Mai University, Sutep, Muang, Chiang Mai 50200, Thailand ∥ Departments of Chemistry and Earth, Ocean & Atmospheric Sciences, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1 ⊥ School of Science, University of Phayao, Maeka, Muang, Phayao 56000, Thailand

*S Supporting Information

ABSTRACT: Two new benzophenones (1 and 2) and four new xanthones (4−6 and 17) together with 24 known compounds (3, 7−16, and 18−30) were isolated from the roots and twigs of Cratoxylum sumatranum ssp. neriifolium. Their structures were elucidated by spectroscopic methods. Compounds 5 and 26 showed antibacterial activity against Micrococcus luteus, Bacillus cereus, and Staphylococcus epidermis with minimum inhibitory concentrations ranging from 4 to 8 μg/mL, whereas compounds 7, 20, and 26 displayed selective antibacterial activities against Staphylococcus aureus (8 μg/mL), Salmonella typhimurium (4 μg/ mL), and Pseudomonas aeruginosa (4 μg/mL), respectively. The radical scavenging effects of some isolated compounds were investigated. Compounds 11 and 21 exhibited potent activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) with IC50 values of 7.0 ± 1.0 and 6.0 ± 0.2 μM, respectively. KEYWORDS: Cratoxylum sumatranum ssp. neriifolium, benzophenone, xanthone, antibacterial activity, antioxidant activity

■ INTRODUCTION nones,3 and some of them exhibited cytotoxicity.3 In the 1 investigation presented here, we report the isolation and Cratoxylum belongs to the Hypericaceae family that is widely fi distributed in South East Asian countries. In Thailand, only six identi cation of two new benzophenones (1 and 2)(Figure 1), 2 four new xanthones (4−6 and 17), and 24 known compounds species have been found, and some of them have been used as − − traditional medicines and cooking. The roots and stems of (3, 7 16, and 18 30)(Figure 2) from C. sumatranum ssp. Cratoxylum cochinchinense have been used in folk medicine to neriifolium roots and twigs. In addition, the antibacterial and − treat diuretic, stomachic, and tonic effects and diarrhea,3 5 antioxidant activities of some isolated compounds are reported. whereas the roots, bark, and leaves of Cratoxylum sumatranum

Downloaded via UNIV OF PHAYAO on March 29, 2021 at 09:15:26 (UTC). ssp. neriifolium have been used for the treatment of rheumatoid ■ MATERIALS AND METHODS arthritis and musculoskeletal pain and also used as a protective General Experimental Procedures. Melting points were 6 α medicine for women after childbirth. Young leaves of C. determined on a Buchi B-540 visual thermometer. The [ ]D values sumatranum ssp. neriifolium as well as the flowers of Cratoxylum were measured with a Bellingham and Stanley ADP400 or Jasco P- See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. formosum ssp. formosum are chewed for relief of coughs.7,8 The 1010 polarimeter. UV−vis spectra were recorded with a PerkinElmer latex of C. cochinchinense, C. formosum ssp. formosum, and C. UV−vis or BMG LABTECH/SPECTROstar Nano spectrometer. The formosum ssp. pruniflorum has been used to stop bleeding and IR spectra were recorded using a PerkinElmer FTS FT-IR − to treat wound infections.9 11 The fresh shoots, young leaves, spectrometer. Electronic circular dichroism spectra were recorded on fl a JASCO J-815 CD spectrometer. The NMR spectra were recorded and owers of some species of this genus, especially C. using a 400 or 600 MHz Bruker spectrometer. The HREIMS and ESI- formosum ssp. formosum, are traditionally consumed as TOF-MS data were measured on a MAT 95 XL or a Bruker-Hewlett- vegetables, and the taste is sour and slightly astringent because Packard 1100 Esquire-LC system mass spectrometer. Chiral HPLC 12 of the phenolic components. In addition, C. cochinchinense was performed on a CHIRALPAK IA column (10 mm × 250 mm) young leaves and fruits are commonly used as a spice for attached to a Waters 2487 dual λ absorbance detector. Silica gel C60 cooking and a substitute for tea, respectively.13 (Silicycle, 0−20 μm) and silica gel G60 (Silicycle, 60−200 μm) were Cratoxylum species produce various types of secondary used to perform quick column chromatography (QCC) and column metabolites, including anthraquinones,14 benzophenones,15 chromatography (CC), respectively. Analytical thin-layer chromatog- flavonoids,16 xanthones,3,14,17 and triterpenoids,15 and many of them exhibited interesting biological activities as well as Received: August 15, 2016 antioxidant activity.17,18 Previous phytochemical investigations Revised: October 24, 2016 of C. sumatranum leaves and stem bark resulted in the Accepted: October 27, 2016 identification of xanthones17,19 and anthraquinonebenzophe- Published: October 27, 2016

© 2016 American Chemical Society 8755 DOI: 10.1021/acs.jafc.6b03643 J. Agric. Food Chem. 2016, 64, 8755−8762 Journal of Agricultural and Food Chemistry Article

Figure 1. Compounds isolated from C. sumatranum roots.

Figure 2. Compounds isolated from C. sumatranum twigs. raphy (TLC) was performed with the precoated plates of silica gel 60 Extraction and Isolation. Air-dried roots of C. sumatranum ssp.

F254. neriifolium (2.75 kg) were macerated with CH2Cl2 and acetone, Plant Material. The roots and twigs of C. sumatranum ssp. successively. The crude extract (47.88 g) was subjected to QCC over neriifolium were collected from Mae Hong Son Province, Thailand, in silica gel, eluting with an EtOAc/hexanes solvent gradient (100% June 2010. The plant was identified by J. Maxwell, and the voucher hexanes to 100% EtOAc) to give compounds 6 (94.3 mg) and 10 specimen (MFU-NPR0008) was deposited at the Natural Products (18.9 mg) and 13 fractions (1A−1M). Fraction 1B (1.84 g) was Research Laboratory of Mae Fah Luang University. further separated by QCC (100% hexanes to 100% acetone) to give

8756 DOI: 10.1021/acs.jafc.6b03643 J. Agric. Food Chem. 2016, 64, 8755−8762 Journal of Agricultural and Food Chemistry Article

1 13 Table 1. H (400 MHz) and C (100 MHz) NMR Spectroscopic Data of 1 and 2 in CDCl3 cratosumatranone A (1) cratosumatranone B (2) δ δ δ δ position C H [J (Hz)] HMBC C H [J (Hz)] HMBC 1 104.5 −−105.2 −− 2 161.0 −−164.7 −− 3 93.0 6.06 s 1, 2, 4, 5, 7, 1‴ 92.9 6.12 s 1, 2, 5, 7, 1‴ 4 163.8 −−163.7 −− 5 108.8 −−99.1 −− 6 158.7 −−154.3 −− 7 197.9 −−199.9 −− 1′ 140.2 −−142.7 −− 2′ 127.9 7.63 dd (7.2, 1.3) 7, 4′,6′ 127.2 7.45 br d (6.8) 7, 4′ 3′ 129.0 7.50 dd (7.6, 7.2) 1′,5′ 127.5 7.39 br d (6.8) 1′ 4′ 132.0 7.57 m 2′,6′ 130.1 7.44 m 4′,6′ 5′ 129.0 7.50 dd (7.6, 7.2) 1′,3′ 127.5 7.39 br d (6.8) 1′ 6′ 127.9 7.63 dd (7.2, 1.3) 7, 4′,2′ 127.2 7.45 br d (6.8) 7, 4′ 1″ 65.4 4.57 d (6.4) 4, 2″,3″ 65.5 4.58 d (6.4) 4, 2″,3″ 2″ 118.9 5.47 t (6.4) 1″,4″ 118.7 5.45 t (6.4) 1″,4″,9″ 3″ 141.5 −−141.6 −− 4″ 39.4 2.10 m 5″,9″ 39.5 2.10 m 2″,3″,4″,5″,9″ 5″ 26.0 2.10 m 4″ 26.1 2.10 m 3″,4″,6″,7″ 6″ 123.7 5.10 br t (6.8) 4″,5″,8″ 123.6 5.10 br t (6.4) 4″,5″,8″ 7″ 131.8 −−131.9 −− 8″ 25.8 1.68 s 10″ 25.8 1.68 s 6″,7″,10″ 9″ 16.6 1.73 s 4″ 16.7 1.73 s 2″,3″,4″ 10″ 17.7 1.61 s 8″ 25.7 1.61 s 6″,8″ 1‴ 21.6 3.28 d (6.8) 4, 5, 6, 2‴,3‴ 17.7 2.76 dd (17.2, 5.2), 2.55 dd (17.2, 5.2) 4, 5, 6, 2‴,3‴ 2‴ 122.2 5.17 br t (6.8) 1‴,5‴ 68.6 3.58 t (5.2) 5, 4‴ 3‴ 132.6 −−76.7 −− 4‴ 17.8 1.73 s 5‴ 20.9 0.85 s 2‴,3‴ 5‴ 25.6 1.67 s 4‴ 23.9 0.92 s 2‴,3‴ OH-2 9.07 br s 1, 2, 3 − 12.37 br s 1, 2, 3 OH-6 8.60 br s 1, 5, 6 −− −

five fractions (1BA−1BE). Compound 4 (8.1 mg) was obtained from Purification of subfraction 2GB (57.1 mg) by CC (10% acetone/ subfraction 1BA (737.1 mg) by Sephadex LH-20 (10% CH2Cl2/ hexanes) gave compound 26 (6.0 mg). Fraction 2H (3.51 g) was MeOH). Fraction 1C (4.39 g) was obtained by repeated QCC (15% further separated by QCC (100% hexanes to 100% CH2Cl2) to give acetone/hexanes) to yield four subfractions (1CA−1CD). Subfraction compounds 22 (9.8 mg) and 25 (7.7 mg) and eight subfractions 1CA (126.6 mg) was purified by CC (15% acetone/hexanes), yielding (2HA−2HH). Compound 29 (8.1 mg) was obtained from subfraction compound 1 (79.6 mg). Compounds 2 (29.2 mg) and 3 (4.0 mg) 2HD (79.9 mg) by Sephadex LH-20 (100% MeOH). Subfraction 2HG were obtained from subfraction 1CD (220.0 mg) by CC (10% EtOAc/ (72.7 mg) was obtained by repeated CC (10% acetone/hexanes), hexanes). Purification of fraction 1D (838.0 mg) by repeated CC (10% yielding compounds 13 (4.0 mg), 23 (1.5 mg), and 28 (11.2 mg). acetone/hexanes) yielded compound 13 (10.5 mg). Fraction 1F (5.47 Purification of subfraction 2HH (99.9 mg) was achieved by CC (10% g) was further subjected to QCC (100% hexanes to 100% acetone) acetone/hexanes) to give a mixture of compounds 27 and 30 that was giving six fractions (1FA−1FF). Compounds 11 (11.6 mg) and 12 further purified by CC (25% EtOAc/hexanes) to afford compounds 27 (168.6 mg) were obtained from subfraction 1FB (1.37 g) by repeated (1.7 mg) and 30 (3.3 mg). Fraction 2I (27.32 g) was subjected to CC (20% acetone/hexanes) followed by Sephadex LH-20 (100% QCC (100% hexanes to 100% acetone) to give six subfractions (2IA− MeOH). The separation of subfractions 1FE (537.4 mg) and 1FF 2IF). Compounds 19 (7.3 mg) and 24 (6.9 mg) were obtained from ff (471.4 mg) by Sephadex LH-20 (10% CH2Cl2/MeOH) a orded subfraction 2ID (2.06 g) by CC (10% acetone/hexanes). Fraction 2K compounds 9 (13.2 mg) and 16 (76.4 mg), respectively. Fraction 1G (5.64 g) was further separated by Sephadex LH-20 (100% MeOH) to (1.60 g) was chromatrographed by CC (25% acetone/hexanes) to give nine subfractions (1KA−1KI). Subfractions 1KA (151.5 mg), 1KB produce five subfractions (1GA−1GE). Subfraction 1GD (657.1 mg) (58.9 mg), and 1KC (35.5 mg) were combined and further purified by was obtained by repeated CC (20% acetone/hexanes), giving CC (10% EtOAc/hexanes), yielding compounds 17 (1.0 mg) and 18 compounds 7 (150.7 mg) and 8 (82.3 mg). Purification of subfraction (6.1 mg). Subfractions 2KG (60.7 mg) and 2KF (66.1 mg) were fi 1K (1.66 g) by CC (20% acetone/hexanes) produced compound 14 combined and puri ed by CC (7% EtOAc/CH2Cl2), yielding an (80.4 mg). Fraction 1L (1.93 g) was subjected to CC (20% acetone/ additional amount of compounds 5 (11.2 mg) and 7 (11.3 mg). hexanes) followed by Sephadex LH-20 (100% MeOH) to afford Compound 20 (2.0 mg) was obtained from subfraction 2KI (27.8 mg) compound 5 (11.9 mg). Fraction 1M (1.44 g) was subjected to by CC (10% acetone/hexanes), whereas compound 21 (11.7 mg) was Sephadex LH-20 (10% CH2Cl2/MeOH) followed by repeated CC isolated from fraction 2M (4.36 mg) by Sephadex LH-20 (10% (40% acetone/hexanes), yielding compound 15 (22.5 mg). CH2Cl2/hexanes). The air-dried twigs of C. sumatranum ssp. neriifolium (6.57 kg) were Cratosumatranone A (1). Yellow solid: mp 69−71 °C; UV λ ε ν macerated with methanol. The MeOH extract (192.99 g) was (MeOH) max (log ) 233 (4.33), 289 (4.54) nm; IR (neat) max 3328, subjected to QCC (100% hexanes to 100% EtOAc), providing 13 2915, 1626, 1591, 1421, 1171, 1081, 1001, 953, 817, 699 cm−1; see − 1 13 fractions (2A 2M). Fraction 2G (4.48 g) was subjected to CC (15% Table 1 for H NMR (CDCl3, 400 MHz) and C NMR (CDCl3, 100 ff + acetone/hexanes) to a ord two subfractions (2GA and 2GB). MHz); HREIMS m/z 434.2452 [M] (calcd for C28H34O4, 434.2452).

8757 DOI: 10.1021/acs.jafc.6b03643 J. Agric. Food Chem. 2016, 64, 8755−8762 Journal of Agricultural and Food Chemistry Article

2 λ 1 13 Cratosumatranone B ( ). Yellow viscous oil: UV (MeOH) max Table 2. H and C NMR Spectroscopic Data of 4 and 17 ε ν (log ) 201 (4.43), 233 (3.98), 285 (4.23) nm; IR (neat) max 3440, 2925, 1705, 1621, 1588, 1447, 1333, 1127, 1126, 1101, 815 cm−1; see cratosumatranone C (4) cratosumatranone F (17) Table 1 for 1H NMR (CDCl , 400 MHz) and 13C NMR (CDCl , 100 δ δ 3 3 H [J H [J + a b c d MHz); HREIMS m/z 450.2406 [M] (calcd for C28H34O5, 450.2401). position δ (Hz)] HMBC δ (Hz)] HMBC − C C Chiral HPLC Separation of ( )-2 and (+)-2. Separation of two 1 156.6 156.5 enantiomers of 2 (3.3 mg) was performed by semipreparative HPLC 2 105.2 137.8 on an enantioselective column [CHIRALPACK IA, 15 μL, 10 mm × 25 mm, 98:2 (v/v) n-hexane/i-PrOH eluent, 2 mL/min]. Compounds 3 159.7 152.7 − α 23 − 4 112.8 99.0 6.74 s 2, 3, 4a, ( )-2 (tR = 53 min) [0.8 mg; [ ] D 5.70 (c 0.4, CHCl3)] and (+)-2 α 23 9, 9a (tR = 72 min) [1.2 mg; [ ] D + 3.7 (c 0.4, CHCl3)] were obtained. 4 − ° α 26 5 145.3 149.6 Cratosumatranone C ( ). Yellow solid: mp 142 144 C; [ ] D λ ε 6 119.6 7.25 dd 5 120.6 7.24 d 4b, 5, 8 0.0 (c 0.06, MeOH); UV (MeOH) max (log ) 243 (4.48), 314 (4.49), ν (7.6, 2.4) (9.0) 373 (3.75) nm; IR (neat) max 3441, 2968, 1648, 1627, 1587, 1497, −1 1 7 124.2 7.22 t (7.6) 5, 8, 8a 105.0 6.80 d 5, 8, 1416, 1255, 889, 757 cm ; see Table 2 for H NMR (CDCl3, 400 13 (9.0) 8a, 9 MHz) and C NMR (CDCl3, 100 MHz); ESITOFMS m/z 447.2174 + 8 116.0 7.75 dd 4b, 6, 9 143.1 [M + H] (calcd for C28H31O5, 477.2171). (7.6, 2.4) 5 − ° α 26 Cratosumatranone D ( ). Yellow solid: mp 211 213 C; [ ] D λ ε 9 181.2 182.4 0.0 (c 0.08, acetone); UV (MeOH) max (log ) 224 (4.48), 296 (3.12) ν −1 4a 154.0 155.5 nm; IR (neat) max 3387, 1649, 1578, 1169, 1092 cm ; see Table 3 for 1 13 4b 144.1 151.4 H NMR (CDCl3, 400 MHz) and C NMR (CDCl3, 100 MHz); + 8a 120.5 109.6 HREIMS m/z 358.1048 [M] (calcd for C19H18O7, 358.1047). 6 − ° α 26 9a 103.5 109.0 Cratosumatranone E ( ). Yellow solid: mp 242 244 C; [ ] D 0.0 λ ε ′ (c 0.05, acetone); UV (MeOH) max (log ) 231 (4.58), 264 (4.00), 1 116.6 6.83 d 1, 2, 3, ν (10.0) 3′ 306 (4.17) nm; IR (neat) max 3294, 2913, 1701, 1649, 1578, 1417, 1306, 1201, 1169, 1092, 1050, 812, 797 cm−1; see Table 3 for 1H 2′ 125.7 5.58 d 2, 3′, 13 (10.0) 4′,9′ NMR (CDCl3, 400 MHz) and C NMR (CDCl3, 100 MHz); + 3′ 81.2 HREIMS m/z 344.0891 [M] (calcd for C18H16O7, 344.0891). Cratosumatranone F (17). Yellow amorphous solid: UV (MeOH) 4′a 41.8 1.91 m 5′,9′ λ ε ′ max (log ) 243 (4.42), 274 (4.37), 321 (4.10), 368 (4.00) nm; IR 4 b 41.8 1.74 m ν ′ ′ (neat) max 3343, 1728, 1670, 1597, 1505, 1446, 1264, 1214, 1114, 5 23.2 2.14 m 4 −1 1 13 817, 756 cm ; see Table 3 for H NMR (CD2Cl2, 600 MHz) and C 6′ 123.6 5.12 br t 5′,8′ + NMR (CD2Cl2, 150 MHz); ESITOFMS m/z 319.0817 [M + H] (7.2) (calcd for C16H15O7, 319.0817). 7′ 132.1 Antibacterial Assay. Micrococcus luteus TISTR 884, Bacillus cereus 8′ 25.7 1.67 s 10′ TISTR 688, Bacillus subtilis TISTR 008, Staphylococcus aureus TISTR 9′ 26.9 1.46 s 4′ 1466, Staphylococcus epidermidis ATCC 12228, Escherichia coli TISTR 10′ 17.6 1.59 s 8′ 780, Salmonella typhimurium TISTR 292, and Pseudomonas aeruginosa 1″ 41.3 TISTR 781 were obtained from the Microbiological Resources Center fi 2″ 155.8 6.70 dd 4, 1″, of the Thailand Institute of Scienti c and Technological Research. The (17.6, 5″ antibacterial assay and the minimum inhibitory concentrations (MICs) 10.4) were determined by a 2-fold serial dilution method using Nutrient ″ ″ ″ 20 3 a 104.0 5.24 dd 1 ,2 broth (NB). In brief, serial 2-fold dilutions of samples in DMSO (10.4, were mixed with MHB in a 96-well microplate, and then 50 μLof 0.8) bacteria was added to each well (final concentration of 1 × 104 colony- 3″b 104.0 5.07 dd 1″ forming units/well). The plates were incubated at 35−37 °C for 16− (17.6, 18 h followed by the addition of 10 μL of resazurin. The MIC was 0.8) ″ ″ ″ determined after added resazurin for 2−3 h. All of the antimicrobial 4 28.1 1.65 s 1 ,5 assays were tested in duplicate, and the standard compounds were 5″ 28.5 1.65 s 4″ vancomycin and gentamycin. OH-1 13.44 s 1, 2, 9a 13.27 s Antioxidant Assay. Briefly, serial dilutions of samples (100 μL) in OH-5 6.67 br s EtOH were mixed with 100 μL of DPPH for 30 min. The mixture was OMe-2 62.2 3.97 s 2 recorded at 517 nm using a microplate reader (SPECTROstar OMe-3 62.2 4.01 s 3 Nano).21,22 The following equation was used for the calculation of the − OMe-8 57.5 3.89 s 8 DPPH radical scavenging capacity: percent inhibition = [(AB AS)/ a b × Measured at 100 MHz in CDCl3. Measured at 400 MHz in CDCl3. AB] 100, where AB and AS are the absorbance of the blank sample c d and sample, respectively. All experiments were performed in triplicate; Measured at 150 MHz in CD2Cl2. Measured at 600 MHz in CD2Cl2. ascorbic acid was used as the positive compound, and the calibration 2 23 24 curve of ascorbic acid was r > 0.9836. The IC50 value of DPPH non-4-O-geranyl ether (3), pruniflorone N (7), neriifolone scavenging activity was calculated by plotting inhibition percentages B(8),19 isocudraniaxanthone B (9),14 10-O-methylmaclurax- against concentrations of the sample. anthone (10),14 macluraxanthone (11),14 5-O-methyl-2-depre- nylrheediaxanthone B (12),25 pancixanthone B (13),26 ■ RESULTS AND DISCUSSION pruniflorone M (14),24 5′-demethoxycadensin G (15),27 Isolation and Identification of Compounds. The crude cochinchinoxanthone (16),28 1,5-dihydroxy-8-methoxyxan- extracts of C. sumatranum ssp. neriifolium (roots and twigs) thone (18),29 1,5-dihydroxy-6,7-dimethoxyxanthone (19),30 were separated by repeated silica gel column chromatography 1,3,6-trihydroxy-7-methoxyxanthone (20),31 1,3,5,6-tetrahy- and Sephadex LH-20 column chromatography to obtain six droxyxanthone (21),27 1,2,8-trihydroxyxanthone (22),32 2,8- new compounds (1, 2, 4−6, and 17) along with 24 known dihydroxy-1-methoxyxanthone (23),33 cratoxyarborenone F compounds that were identified as 2,4,6-trihydroxybenzophe- (24),3 1,7-dihydroxyxanthone (25),34 trapezifolixanthone

8758 DOI: 10.1021/acs.jafc.6b03643 J. Agric. Food Chem. 2016, 64, 8755−8762 Journal of Agricultural and Food Chemistry Article

1 13 δ Table 3. H (400 MHz) and C (100 MHz) NMR carbon at C 104.5 (C-1), OH-2 was ortho to the methine δ Spectroscopic Data of 5 and 6 in CDCl3 carbon at C 93.0 (C-3), and OH-6 was ortho to the quaternary δ carbon at C 108.8 (C-5). The remaining signals in the NMR cratosumatranone D (5) cratosumatranone E (6) δ data of 1 were assigned to an oxygeranyl unit [ H 5.47 (1H, t, J δ [J ″ ″ δ H δ δ = 6.4 Hz, H-2 ), 5.10 (1H, br t, J = 6.8 Hz, H-6 ), 4.57 (2H, d, position C (Hz)] HMBC C H [J (Hz)] HMBC J = 6.4 Hz, H-1″), 2.10 (4H, m, H-4″/H-5″), 1.73 (3H, s, H- 1 161.2 162.0 9″), 1.68 (3H, s, H-8″), 1.61 (3H, s, H-10″)] and a 2 99.1 6.14 s 1, 4, 9a 98.8 6.12 s 3, 4, 9a δ ‴ dimethylallyl group [ H 5.17 (1H, br t, J = 6.8 Hz, H-2 ), 3 160.2 160.9 3.28 (2H, d, J = 6.8 Hz, H-1‴), 1.73 (3H, s, H-4‴), 1.67 (3H, s, 4 109.4 110.3 H-5‴)]. The oxygeranyl and dimethylallyl units were placed at 5 135.0 133.0 δ ″ δ C-4 and C-5, respectively, because H-3 ( H 6.06), H-1 ( H 6 156.4 150.9 ‴ δ 4.57), and H-1 ( H 3.28) showed HMBC correlations with C- 7 113.9 7.04 d 5, 6, 8a 112.6 7.02 d (8.8) 5, 7, 8a δ (8.8) 4( C 163.8). Once the oxygeranyl and dimethylallyl units had 8 121.1 7.81 d 4b, 6, 9 116.1 7.60 d (8.8) 4b, 6, 9 been situated, the remaining benzoyl fragment had to be (8.8) attached to C-1. This location for the benzolyl fragment was 4 9 180.2 181.5 consistent with the observation of a J HMBC correlation δ 4a 155.7 156.7 (Supporting Information and Table 1) between H-3 ( H 6.06) δ 4b 150.5 147.0 and the carbonyl carbon ( C 197.9), and the chemical shifts and δ 8a 113.9 114.8 sharpness of the two hydrogen-bonded hydroxy signals at H 39 9a 103.5 104.2 9.07 (1H, br s, OH-2) and 8.60 (1H, br s, OH-6), which 2′ 92.9 5.55 d 3, 3′, 92.9 5.52 br d (8.0) 3′,4′ implied they were hydrogen bonded to the benzoyl carbonyl (8.0) 4′ group. Completed assignment of 1H and 13C NMR spectral 3′ 45.7 2.03 m, 4, 2′, 45.9 2.00 dd (13.6, 4, 2′, data and additional HMBC correlations of cratosumatranone A 1.92 m 4′,5′ 2.0), 1.91 br 4′, d (8.0) 5′,6′ (1) are summarized in Table 1. 4′ 31.7 32.7 Cratosumatranone B (2) gave a molecular ion peak at m/z + 5′ 28.4 1.73 s 4, 3′ 28.2 1.73 s 4, 3′, 450.2406 [M] (calcd, 450.2401) in its HREIMS spectrum, ′ 6 suggesting a molecular formula of C28H34O5. The UV and IR 6′ 28.3 1.62 s 4, 3′ 28.1 1.62 s 4, 3′, spectra displayed the same pattern as those of benzophenones ′ 5 1 and 3.23 The 1H and 13C NMR data of 2 were also similar to OH-1 13.09 s 1, 2, 9a 13.16 s 1, 2, 9a those of 1 (Table 1), except that compound 2 displayed a 2- OMe-5 61.1 4.02 s 5 δ hydroxy-3,3-dimethylchromane unit [ H 3.58 (1H, t, J = 5.2 Hz, H-2‴), 2.76 (1H, dd, J = 17.2, 5.2 Hz, H-1‴), 2.55 (1H, dd, J = (26),35 5-O-methylisojacareubin (27),23 2,4,6-trimethoxyben- 17.2, 5.2 Hz, H-1‴), 0.92 (3H, s, H-5‴), 0.85 (3H, s, H-4‴)] zophenone (28),36 4-hydroxy-2,6-dimethoxybenzophenone instead of the dimethylallyl and hydroxy groups at C-5 and C-6 (29),37 and annulatomarin (30).38 in 1. Completed assignment of 1H and 13C NMR spectral data Cratosumatranone A (1) showed a molecular ion peak at m/ and additional HMBC correlations of cratosumatranone B (2) z 434.2452 (calcd, 434.2452) [M]+ in its HREIMS spectrum, are summarized in Table 1. Attempts to identify the absolute fi ‴ ’ corresponding to a molecular formula of C28H34O4. The UV con guration at C-2 by applied Mosher s method were not and IR spectra displayed the same pattern as those of successful. After the treatment of compound 2 with either (R)- benzophenone 3.23 The 1H and 13C NMR spectra of 1 MTPA-Cl or (S)-MTPA-Cl, a mixture of two diastereomers δ ± (Table 1) had signals for a monosubstituted aromatic ring at H was formed. Therefore, the mixture of ( )-2 was further 7.63 (2H, dd, J = 7.2, 1.3 Hz, H-2′ and H-6′), 7.57 (1H, m, H- analyzed with using a chiral HPLC column. The HPLC ′ ′ ′ δ ± 4 ), and 7.50 (2H, dd, J = 7.6, 7.2 Hz, H-3 and H-5 ) and C chromatogram of ( )-2 showed well-resolved peaks for the two 127.9 (C-2′ and C-6′), 132.0 (C-4′) 129.0 (C-3′ and C-5′), and enantiomers, (−)-2 and (+)-2. Pure compounds (−)-2 and 3 fi α 23 − 140.2 (C-1′). H-2′ (and H-6′) showed J HMBC correlations (+)-2 displayed speci c rotations with [ ] D 5.7 (c 0.4, δ α 23 with C-7 ( C 197.9) connecting this unit to the carbonyl group CHCl3) and [ ] D + 3.7 (c 0.4, CHCl3), respectively. The to give a benzoyl fragment. An EIMS fragment observed at m/z absolute configuration at C-2‴ of (+)-2 was assumed to be R + fi α 23 105 [C7H5O ] supported the presence of a benzoyl group. A because it displayed the same sign of speci c rotation [ ] D + α 22 second major structural unit of the molecule was identified as a 3.7 (c 0.4, CHCl3) with those of lomatin [[ ] D + 12.4 (c 0.10, 23 40,41 − phloroglucinol unit similar to those of benzophenone 3. CHCl3)], while compound ( )-2 was proposed to be S fi δ fi α 23 − Three down eld carbon resonances at C 163.8 (C-4), 161.0 because its speci c rotation ([ ] D 5.7) was the opposite of (C-2), and 158.7 (C-6) indicated trioxygenation.17,39 Quater- that of (+)-2. δ nary carbon resonances at C 108.8 (C-5) and 104.5 (C-1) and Cratosumatranone C (4) displayed a pseudomolecular ion δ δ + methine carbon resonances at C 93.0 (C-3; H 6.06, s) were peak at m/z 447.2174 [M + H] (calcd, 447.2171) in its also assigned to the phloroglucinol ring. 1H NMR resonances at ESITOFMS spectrum, suggesting a molecular formula of δ 39 H 9.07 (1H, br s, OH-2) and 8.60 (1H, br s, OH-6) were C28H31O5. The UV and IR spectra showed the same pattern δ 14,24,26 1 assigned to hydroxy groups. The hydroxy resonances at H 9.07 as that of xanthone core structures. The H NMR (OH-2) showed HMBC correlations to the carbon resonances spectroscopic data (Table 2)of4 exhibited a hydrogen-bonded δ δ at C 161.0 (C-2), 104.5 (C-1), and 93.0 (C-3), while the hydroxy proton at H 13.44 (1H, s, OH-1) and a set of AMX δ δ hydroxy resonances at H 8.60 (OH-6) showed HMBC splitting pattern of aromatic protons at H 7.75 (1H, dd, J = 7.6, δ correlations to the carbon resonances at C 158.7 (C-6), 2.4 Hz, H-8), 7.25 (1H, dd, J = 7.6, 2.4 Hz, H-6), and 7.22 (1H, 108.8 (C-5), and 104.5 (C-1), indicating that OH-2 and OH-6 t, J = 7.6 Hz, H-7). The presence of a 2-methyl-2-(4- were meta to each other and both ortho to the quaternary methylpent-3-en-1-yl)-2H-chromene unit was suggested by 1H

8759 DOI: 10.1021/acs.jafc.6b03643 J. Agric. Food Chem. 2016, 64, 8755−8762 Journal of Agricultural and Food Chemistry Article

Table 4. Antibacterial Activity of Some Isolated Compounds

antibacterial activity (MIC, μg/mL) Gram-positive bacteria Gram-negative bacteria compound M. luteus B. cereus B. subtilis S. aureus S. epidermidis E. coli Sa. typhimurium P. aeruginosa 1 >128 >128 a >128 >128 >128 >128 >128 2 a 32 32 8 16 >128 >128 128 3 >128 >128 a >128 >128 >128 >128 >128 4 >128 >128 a >128 >128 >128 >128 >128 5 8 8 16 32 8 64 32 16 6 >128 >128 a >128 >128 >128 >128 >128 7 16 32 32 8 16 32 32 16 8 >128 >128 a >128 >128 >128 >128 >128 9 >128 >128 a >128 >128 >128 >128 >128 10 >128 >128 a >128 >128 >128 >128 >128 11 >128 >128 a >128 >128 >128 >128 >128 12 >128 >128 a >128 >128 >128 >128 >128 13 32 16 128 >128 32 32 32 16 14 >128 >128 a >128 >128 >128 >128 >128 15 >128 >128 a >128 >128 >128 >128 >128 16 >128 >128 a >128 >128 >128 >128 >128 17 128 64 128 >128 128 64 32 128 18 32 64 128 128 32 32 32 16 19 32 16 64 128 16 64 32 16 20 128 128 128 >128 128 64 4 128 21 128 64 64 128 128 64 32 128 23 64 64 128 128 64 16 32 64 24 64 64 128 128 64 32 64 128 25 32 64 128 >128 32 16 32 16 26 4 4 64 16 4 32 32 4 28 128 64 128 >128 64 64 32 128 29 128 64 128 >128 64 32 32 128 vancomycin 0.25 0.25 0.25 0.25 0.25 −− − gentamycin −−−− − 0.25 0.125 2 aNot tested. Inactive with a MIC of >128 μg/mL.

δ ′ δ 13 NMR signals at H 6.83 (1H, d, J = 10.0 Hz, H-1 ), 5.58 (1H, d, resonance at C 175.0 in the C NMR spectrum of compound J = 10.0 Hz, H-2′), 5.12 (1H, br t, J = 7.2 Hz, H-6′), 2.14 (2H, 8.19 However, compound 5 showed an additional 1H NMR ′ ′ ′ δ m, H-5 ), 1.91 (1H, m, H-4 a), 1.74 (1H, m, H-4 b), 1.67 (3H, signal at H 5.55 (1H, d, J = 8.0 Hz) that was correlated with a ′ ′ ′ 14 δ s, H-8 ), 1.59 (3H, s, H-10 ), and 1.46 (3H, s, H-9 ). carbon resonance at C 92.9 in the HMQC spectrum. These 1 Moreover, the H NMR spectrum of 4 also showed the results implied that a carbonyl carbon of lactone at C-2′ of 8 δ 1 characteristic signals of the 1,1-dimethylallyl group at H 6.70 was reduced to a hemiacetal moiety in 5. All assignments of H ″ (1H, dd, J = 17.6, 10.4 Hz, H-2 ), 5.24 (1H, dd, J = 10.4, 0.8 and 13C NMR and HMBC correlations of cratosumatranone D ″ ″ Hz, H-3 a), 5.07 (1H, dd, J = 17.6, 0.8 Hz, H-3 b), and 1.65 (5) are given in Table 3. Compound 5 also tried to determine ″ ″ (6H, s, H-4 and H-5 ). The location of the linear chromene the absolute configuration at C-2′ by the applied Mosher’s ring at C-2 and C-3 was confirmed by HMBC correlations in ′ δ δ method. Unfortunately, the NMR data of the Mosher esters of which H-1 ( H 6.83) showed cross peaks with C-1 ( C 156.6), compound 5 showed a mixture of two diastereomers similar to C-2 (δ 105.2), and C-3 (δ 159.7), while a hydrogen-bonded C C that of compound 2. Therefore, compound 5 is also a mixture hydroxy group at δ 13.44 (OH-1) was also correlated with C-1 H of the enantiomers. The specific rotation of compound 5, (δ 156.6), C-2 (δ 105.2), and C-9a (δ 103.5). The C C C [α]26 0.0 (c 0.08, acetone), was also supported by this attachment of a 1,1-dimethylallyl side chain at C-4 was D confirmed by 3J HMBC correlation of the H-2″ methine information. proton (δ 6.70) with C-4 (δ 112.8). Detailed assignment of Cratosumatranone E (6) gave a molecular ion peak at m/z H C 344.0891 [M]+ (calcd, 344.0891) in its HREIMS spectrum, the proton and carbon resonanaces and a listing of HMBC 1 13 correlations observed in the NMR data of 4 are given in Table suggesting a molecular formula of C18H16O7. The H and C 2. NMR spectra (Table 3)of6 were closely related to those of 5, Cratosumatranone D (5) gave a molecular ion peak at m/z except that the OMe group at C-5 of 5 was replaced with an 3 358.1048 [M]+ (calcd, 358.1047) in its HREIMS spectrum, OH group. This change was confirmed by the J HMBC 1 13 δ suggesting a molecular formula of C19H18O7. The H and C correlation observed between H-7 ( H 7.02, d, J = 8.8 Hz) and δ 1 13 NMR data of 5 (Table 3) were similar to those of 8, neriifolone C-5 ( C 133.0). All H and C NMR assignments as well as B.19 The main difference was the absence of a 13C NMR signal HMBC correlations of cratosumatranone E (6) are listed in in the spectrum of 5 corresponding to the lactone carbonyl Table 3. The configuration at C-2′ of 6 was also racemic

8760 DOI: 10.1021/acs.jafc.6b03643 J. Agric. Food Chem. 2016, 64, 8755−8762 Journal of Agricultural and Food Chemistry Article

fi α 26 because of its speci c rotation, [ ] D 0.0 (c 0.05, acetone), as ■ AUTHOR INFORMATION well as the same pattern of NMR data for the Mosher esters. Corresponding Author Cratosumatranone F (17) gave a pseudomolecular ion peak * at m/z 319.0817 [M + H]+ (calcd, 319.0817) in its ESITOFMS Phone: +66-5391-6238. Fax: +66-5391-6776. E-mail: surat. 1 [email protected]. spectrum, suggesting a molecular formula of C16H15O7. The H NMR spectrum (Table 2)of17 showed the character of a Funding xanthone skeleton that displayed a hydrogen-bonded hydroxy This work was supported by the Higher Education Research δ proton ( H 13.27, 1H, s, OH-1), an AB splitting pattern of Promotion (HERP), the Thailand Research Fund through the δ aromatic protons [ H 7.24 (1H, d, J = 9.0 Hz, H-6), 6.80 (1H, TRF-NSFC collaborative project grant (Grant DBG5980001), δ d, J = 9.0 Hz, H-7)], and a singlet aromatic proton [ H 6.74 the NSFC-TRF coproject (Grant 8156114801), and Mae Fah δ (1H, s, H-4)]. The remaining signals at H 4.01 (3H, s, OMe- Luang University through the graduate student research grant. 3), 3.97 (3H, s, OMe-2), and 3.89 (3H, s, OMe-8) indicated A full Ph.D. scholarship (to C.T.) from the Thailand Research three methoxyl groups, which were placed at C-3, C-2, and C-8, Fund through the Royal Golden Jubilee Ph.D. Program (Grant 3 δ respectively, due to the J HMBC correlations of Ome-3 ( H PHD/0109/2554) is also acknowledged. Mae Fah Luang δ δ δ 4.01) with C-3 ( C 152.7), of Ome-2 ( H 3.97) with C-2 ( C University and the University of British Columbia are also δ δ 137.8), and of Ome-8 ( H 3.89) with C-8 ( C 143.1). The full acknowledged for laboratory facilities. 1 13 assignments of H and C NMR spectral data and HMBC Notes correlations of cratosumatranone F (17) are given in Table 2. The authors declare no competing financial interest. Antibacterial Activity. All isolated compounds except compounds 22, 27, and 30 were evaluated for their antibacterial activity against Gram-positive (M. luteus TISTR 884, B. cereus ■ REFERENCES TISTR 688, B. subtilis TISTR 008, S. aureus TISTR 1466, and (1) The Plant List, version 1. http://www.theplantlist.org/, 2010 S. epidermidis ATCC 12228) and Gram-negative (E. coli TISTR (accessed January 1, 2016). 780, Sa. typhimurium TISTR 292, and P. aeruginosa TISTR (2) Smitinand, T. Thai Plant Names (Revised edition); Prachachon 781) (Table 4) bacteria. Compounds 5 and 26 showed good Co.: Bangkok, 2001. activity against M. luteus, B. cereus, and S. epidermis with MIC (3) Seo, E. K.; Kim, N. C.; Wani, M. C.; Wall, M. E.; Navarro, H. A.; μ Burgess, J. P.; Kawanishi, K.; Kardono, L. B. S.; Riswan, S.; Rose, W. values of 8 and 4 g/mL, respectively. Compound 26 also C.; Fairchild, C. R.; Farnsworth, N. R.; Kinghorn, A. D. 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