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Prenylated xanthones from the stem bark of Garcinia dulcis
Phytochemistry Letters 21 (2017) 32–37
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Short communication
Prenylated xanthones from the stem bark of Garcinia dulcis a,c
b,c
MARK
d,e
Parichat Thepthong , Souwalak Phongpaichit , Anthony R. Carroll , ⁎ Supayang Piyawan Voravuthikunchaib,c, Wilawan Mahabusarakama,c, a
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Natural Product Research Center of Excellence, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d Environmental Futures Research Institute, Griffith University, Gold Coast, Queensland 4222, Australia e Eskitis Institute, Griffith University, Brisbane, Queensland 4111, Australia b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Clusiaceae Garcinia dulcis Prenylated xanthones Antibacterial activity
Three new prenylated xanthones, named dulcisxanthone J (1), dulcisxanthone K (2), and dulcisxanthone L (3), along with 38 known compounds have been isolated from the stem bark of Garcinia dulcis. Their structures were elucidated mainly by analysis of 1D and 2D spectroscopic data. 12b-Hydroxy-des-D-garcigerrin A (12) and dulcisxanthone J (1) displayed moderate antibacterial activity against both penicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) with minimum inhibitory concentration (MIC) values of 4 and 16 μg/mL, respectively.
1. Introduction Garcinia dulcis Kurz. is a medicinal plant in the Clusiaceae family. This plant species is widely distributed in the southern part of Thailand, Malaysia, Indonesia and Philippines. The fruit juice has been used as an expectorant. The leaves and seeds have been applied as a traditional medicine for the treatment of lymphatitis, parotitis and struma in Indonesia (Kasahara and Henmi, 1986). In Thailand, its stem bark has been used as an anti-inflammatory agent. The various parts of this plant are rich in secondary phenolic metabolites, including xanthones, biflavonoids, flavonoids, chalcones and phloroglucinols. Xanthones, the main chemical components of G. dulcis, have many pharmacological properties. Some xanthones from fruits, flowers and seeds have been reported to possess antioxidant and antibacterial activity (Deachathai et al., 2005, 2006, 2008), while xanthones from stem bark were reported to possess anti-androgenic (Shakui et al., 2014) and antimalarial activity (Likhitwittayawuid et al., 1998). The knowledge that G. dulcis is a rich source of xanthones, together with our test result that the extract of stem bark inhibited the growth of both penicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) with minimum inhibitory concentration (MIC) values of 32 μg/mL led us to examine the active constituents from stem bark as antibacterial agents. 2. Results and discussion Purification of the CH2Cl2 and acetone extracts of the stem bark of ⁎
G. dulcis by chromatographic and crystallization techniques gave 41 compounds. Structural elucidation mainly by analysis of 1D and 2D spectroscopic data and comparison of the data with those of previously reported compounds indicated three new and 38 known compounds. The new compounds were prenylated xanthones named dulcisxanthone J (1), dulcisxanthone K (2), and dulcisxanthone L (3) (Fig. 1). Dulcisxanthone J (1) was a yellow gum. Its molecular formula was determined to be C23H22O6 by HRESIMS at m/z 417.1104 [M + Na]+. The UV spectrum showed absorption maxima at λmax 226, 265 and 296 nm. The IR spectrum showed absorption bands for OeH stretching at 3353 cm−1 and C]O stretching at 1624 cm−1. The 13C NMR spectrum displayed resonances of 23 carbons which were classified as one carbonyl, four methyl, one methylene, five methine and twelve quaternary carbons (Table 1). The 1H NMR spectrum showed resonances of a chelated hydroxyl group (1-OH) at δ 12.80, and singlet aromatic protons, H-3 and H-8 at δ 7.41 and δ 7.55, respectively. The HMBC correlations from H-8, to C-6 (δ 145.1), C-9 (δ 181.9), C-10a (δ 145.0) and from H-3 to C-1 (δ 153.2), C-4a (δ 140.9), C-4 (δ 135.0) located H-8 ortho to a ketone carbonyl, and H-3 meta to hydrogen bonded hydroxyl group (Fig. 2). The resonances for an olefinic methine proton at δ 6.30 (dd, J = 17.5, 10.5 Hz, H-2″), terminal methylene protons at δ 5.04 (br d, J = 17.5 Hz, Ha-3″) and δ 5.05 (br d, J = 10.5 Hz, Hb-3″), and gem-dimethyl groups at δ 1.56 (s, CH3-1″) as well as the HMBC correlations of Ha-3″ and Hb-3″ to C-1″ (δ 40.4), C2″ (δ 147.1) and H-2″ to CH3-1″ (δ 26.9), C-1″ (δ 40.4) were suggestive that an isoprenyl unit was present in the molecule. The non equivalent
Corresponding author at: Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. E-mail address: [email protected] (W. Mahabusarakam).
http://dx.doi.org/10.1016/j.phytol.2017.05.014 Received 9 December 2016; Accepted 11 May 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. The chemical structures of the new compounds 1, 2 and 3.
CH3-1″ and H-2″ to C-2 (δ 129.4), and H-3 to C-1″ (δ 40.4), C-1 (δ 153.2), along with the NOESY correlations between CH3-1″ and H-3, suggested that the isoprenyl unit was attached at C-2 and ortho to H-3. The remaining 1H NMR signals including two doublets of cis-olefinic protons at δ 5.74 and δ 6.46 (J = 10.0 Hz, H-3′ and H-4′) and a singlet of dimethyl protons at δ 1.57 (CH3-2′), as well as the HMBC correlations of H-4′ to C-2′ (δ 79.2); H-3′ to C-2′ (δ 79.2), CH3-2′ (δ 28.6); and CH3-2′ to C-2′ (δ 79.2), C-3′ (δ 130.9) were in agreement with typical signals for a 2,2-dimethylchromene ring. The HMBC correlations of cis-olefinic proton H-4′ to oxygenated aromatic carbon C-6 (δ 145.1), methine aromatic carbon C-8 (δ 114.5) allowed the chromene ring to be attached to C-6 and C-7 of the xanthone skeleton with an ether linkage at C-6. Finally, two hydroxy groups were assigned at C-4 (δ 135.0) and C-5 (δ 141.1) to satisfy the molecular formula derived from MS analysis. Thus dulcisxanthone J (1) was identified as 1,4,5-trihydroxy-2-(1,1dimethyl-2-propenyl)-2′,2′-dimethylpyrano[5′,6′:6,7]xanthone. It was a positional isomer of subelliptenone H (13) (Iinuma et al., 1995a) (Fig. 3). Dulcisxanthone K (2) was a yellow gum. Its molecular formula was determined to be C23H24O6 by analysis of a HRESIMS ion at m/z 419.1471 [M + Na]+. The UV spectrum showed absorption maxima at λmax 255, 280 and 328 nm. The IR spectrum showed absorption bands at 3415 cm−1 (OeH stretching) and 1638 cm−1 (C]O stretching). The 13 C NMR spectrum showed 23 carbon signals which were classified as one carbonyl, four methyl, two methylene, four methine, and twelve quaternary carbons. The 1H NMR spectrum exhibited signals for a chelated hydroxy proton (1-OH) at δ 13.82, and aromatic singlet protons H-4 and H-7 at δ 6.48 and δ 6.77, respectively. The characteristic signals for two prenyl groups were also present. The HMBC correlation allowed assignment of the methylene doublet protons at δ 3.35 (J = 7.2 Hz, H-1′), an olefinic triplet proton at δ 5.28 (J = 7.2 Hz, H-2′), gem-dimethyl singlet protons at δ 1.78 (H-4′) and δ 1.65 (H-5′) for the 2-prenyl side chain, while the methylene protons at δ 3.97 (d, J = 7.2 Hz, H-1″), an olefinic proton at δ 5.39 (t, J = 7.2 Hz, H-2″), gem-dimethyl protons at δ 1.74 (s, H-4″) and δ 1.72 (s, H-5″) for the 8prenyl side chain. The HMBC correlations of 1-OH and H-4 to C-2 (δ 110.2), C-9a (δ 102.6); H-1′ to C-1 (δ 161.0), C-2 (δ 110.2), C-3 (δ 162.1) confirmed the location of H-4 and the 2-prenyl unit attached at
Table 1 1 H and 13C NMR spectral data of compounds 1, 2 and 3. Position
1a δH, (mult., J in Hz)
1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″
4″ 5″ 1-OH (CH3)2-2′ (CH3)2-3′ (CH3)2-1″ a b
7.41 (s)
7.55 (s)
5.74 (d, 10.0) 6.46 (d, 10.0)
6.30 (dd, 17.5, 10.5) 5.04 (br d, 17.5, Ha) 5.05 (br d, 10.5, Hb)
2b δC
153.2 129.4 122.2 135.0 140.9 141.1 145.1 117.9 114.5 120.3 181.9 106.3 145.0 79.2 130.9 121.6 40.4 147.1
3b
δH, (mult., J in Hz)
6.48 (s)
6.77 (s)
3.35 (d, 7.2) 5.28 (t, 7.2) 1.78 1.65 3.97 5.39
(s) (s) (d, 7.2) (t, 7.2)
110.6
δC
161.0 110.2 162.1 92.5 154.8 130.5 150.1 113.8 135.7 111.3 182.5 102.6 147.2 21.1 122.6 130.5 17.0 24.9 32.8 123.5
δH, (mult., J in Hz)
6.22 (d, 2.0) 6.41 (d, 2.0)
6.78 (s)
3.28 (m) 1.77 (m)
δC
164.2 97.9 164.6 93.2 157.1 130.5 150.4 114.7 137.9 111.4 182.3 102.9 147.0 29.9 45.9 69.4
131.6
110.6 1.74 (s) 1.72 (s) 13.82 (s)
12.80 (s) 1.57 (s)
28.6
1.56 (s)
26.9
17.1 25.0 13.56 (s) 1.29 (s)
28.8
recorded in CDCl3. recorded in acetone-d6.
methylene protons Ha-3″ and Hb-3″ in the isoprenyl side chain were assigned trans and cis to methine H-2″ base on the coupling constant values of 17.5 and 10.5 Hz, respectively. The HMBC correlations of
Fig. 2. The key HMBC correlations of 1, 2 and 3.
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Fig. 3. Structure of compounds from the stem bark of G. dulcis (4-41).
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that an aromatic proton (H-7) and the 3-hydroxy-3-methylbutyl group were placed at C-7 and C-8, respectively. Finally, the carbon chemical shifts of the remaining unassigned carbon indicated that three hydroxy groups were attached at C-3 (δ 164.6), C-5 (δ 130.5) and C-6 (δ 150.4) to complete the structure. Hence, dulcisxanthone L was established as 1,3,5,6-tetrahydroxy-8-(3-hydroxy-3-methylbuty)xanthone. It was a 3′hydroxy derivative of garcinexanthone C (Chen et al., 2008). The 38 known compounds were identified as one isocoumarin: 8hydroxy-6-methoxy-3-pentylisocoumarin (4) (Kijjoa et al., 1991), six biflavonoids: O-methyl fukugetin (36), (Waterman and Crichton, 1980), volkensiflavone (37), GB-2a (39), morelloflavone (40) fukugeside (41) (Luzzi et al., 1997), amentoflavone (38) (Pelter et al., 1970), and 31 xanthones: brasilixanthone B (5) (Marques et al., 2000), garciniaxanthone C (6), 2,6-dihydroxy-1,5-dimethoxyxanthone (32) (Minami et al., 1994), garciniaxanthone A (7), garciniaxanthone B (10) (Fukuyama et al., 1991), dulcisxanthone H (8), dulcisxanthone I (11) (Mahabusarakam et al., 2016), 6-deoxyjacareubin (9) (Jackson et al., 1967), 12b-hydroxy-des-D-garcigerrin A (12) (Sordat-Diserens et al., 1989), subelliptenone H (13) (Iinuma et al., 1995a), subelliptenone B (14) (Iinuma et al., 1994), 1-O-methylglobuxanthone (15) (Nguyen and Harison, 2000), globuxanthone (16), symphoxanthone (24) (Locksley et al., 1966), 1,2,5,4′-tetrahydroxy-4-(1,1-dimethyallyl)-5′-(2-hydroxypropan-2-yl)-4′,5′-dihydrofurano(2′,3′:6,7)xanthone (17) (Chen et al., 2010), subelliptenone D (19), subelliptenone C (31) (Iinuma et al., 1995c), subelliptenone F (27) subelliptenone G (30) (Iinuma et al., 1995b), 6,11-dihydroxy-2,2-dimethylpyrano[3,2-c]xanthen-7(2H)-one (20) (Sordat-Diserens et al., 1992), euxanthone (21) (Perkin and Hummel, 1896), toxyloxanthone B (22) (Cotterill and Scheinmann, 1975), 1,3,5-trihydroxyxanthone (23) (Locksley and Murray, 1971), 1,7-dihydroxy-2-(3-methylbut-2-enyl)-3-methoxyxanthone (25) (Asai et al., 1995), 1-O-methylsymphoxanthone (18), garciniaxanthone E (26), 2,5-dihydroxy-1-methoxyxanthone (29) (Minami et al., 1996), garciniaxanthone D (28) (Minami et al., 1995), 1,3,7-trihydroxyxanthone (33) (Chawla et al., 1975), garciduol A (34) (Iinuma et al., 1996), griffipavixanthone (35) (Xu et al., 1998). Compounds 4, 5, 9, 13, 14, 15, 17, 20, 35 and 36 were reported from this plant for the first time. The extracts and some of the pure compounds were evaluated for antibacterial activity. The result showed that the CH2Cl2 and acetone extracts inhibited the growth of penicillin-susceptible S. aureus ATCC 25923 with MIC values of 32 and 128 μg/mL, and MRSA SK1 with MIC values of 64 and 128 μg/mL. For pure compounds, 12b-hydroxy-des-Dgarcigerrin A (12) and dulcisxanthone J (1) exhibited antibacterial activity against both test strains with MIC values of 4 and 16 μg/mL, respectively. Compounds 6, 14, 16, 24 and 35 showed activity against S. aureus with MIC values ranging from 32 to 64 μg/mL and against MRSA with MIC values ranging from 32 to 128 μg/mL. Compounds 2, 7, 15, 17, 18, 28 and 30 showed activity against both bacteria test strains with MIC values ranging from 128 to 200 μg/mL, whereas compounds 8-11, 13, 19, 21, 22, 26 and 34 showed no activity at 200 μg/mL (Table 2). The structure activity relationships of the tested xanthones, especially with isoprenyl side chain, on antibacterial activity have been studied. The test result that xanthone 12 was much more effective in inhibiting the growth of bacteria than 30 indicated that the isoprenyl unit at C-2 enhanced the activity. However, more steric hindrance by substituents at position C-6 and C-7 in xanthones 1, 7, 10 and 28 could reduce the activity. Xanthone 16 which contained an isoprenyl unit at C-4 exhibited antibacterial activity, however less effectively than the positional isomer 12. The weak activity of xanthones 13, 14, 17, 19 and 24 as well as xanthones 15 and 18 suggested that the substituents at position C-6 and C-7, and replacement of 1-OH by a methoxy group, affected the antibacterial activity of xanthones containing an isoprenyl group. Compounds 3-5, 20, 23, 25, 27, 29, 31-33 and 41 were not tested due to the lack of material. For biflavonoids 36-40, the antibacterial activities have been previously reported (Deachathai et al., 2005; Saelee et al., 2015).
Table 2 Antibacterial activity of compounds and extracts from the stem bark of G. dulcis. Compounds
1 2 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 24 26 28 30 34 35 CH2Cl2 extract Acetone extract Vancomycin
S. aureus ATCC 25923
MRSA SK1
MIC (μg/mL)
MBC (μg/mL)
MIC (μg/mL)
MBC (μg/mL)
16 128 32 200 > 200 > 200 > 200 > 200 4 > 200 32 200 32 128 128 > 200 > 200 > 200 32 > 200 200 128 > 200 32 32 128 1
> 200 > 200 > 200 200 > 200 > 200 > 200 > 200 128 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 128 > 200 > 200 200 > 200 128 200 > 200 2
16 128 32 200 > 200 > 200 > 200 > 200 4 > 200 128 200 32 128 128 > 200 128 > 200 128 > 200 128 128 > 200 64 64 128 1
64 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 128 > 200 128 200 > 200 2
MIC = Minimum Inhibitory Concentrations. MBC = Minimum Bactericidal Concentrations.
C-2. The HMBC correlations between H-1″ and C-7 (δ 113.8), C-8a (δ 111.3); H-7 and C-5 (δ 130.5), C-6 (δ 150.1), C-8a (δ 111.3), C-1″ (δ 32.8) confirmed the position of H-7 and the 8-prenyl unit attached at C8. Finally, the hydroxy groups were assigned at C-3 (δ 162.1), C-5 (δ 130.5) and C-6 (δ 150.1) to complete the structure. Dulcisxanthone K (2) was therefore identified as 1,3,5,6-tetrahydroxy-2,8-bis(3-methyl-2butenyl)xanthone. It was a positional isomer of cratoxyarborenone B (Seo et al., 2002). Dulcisxanthone L (3) was a yellow gum. Its molecular formula was determined to be C18H18O7 from analysis of a pseudomolecular ion in HRESIMS at m/z 369.0956 [M + Na]+. The UV spectrum showed absorption maxima at λmax 252, 280 and 326 nm. The IR spectrum showed absorption bands at 3301 cm−1 (OeH stretching) and 1649 cm−1 (C]O stretching). The 13C NMR spectrum showed 18 carbon signals which were classified as one carbonyl, two methyl, two methylene, three methine and ten quaternary carbons. The 1H NMR spectrum exhibited signals for a chelated hydroxy proton (1-OH) at δ 13.56, meta-coupled (d, J = 2.0 Hz) aromatic protons at δ 6.22 (H-2) and δ 6.41 (H-4) and an isolated aromatic proton at δ 6.78 (s, H-7). The remaining signals were for benzylic methylene protons at δ 3.28 (m, H1′), methylene protons at δ 1.77 (m, H-2′) and gem-dimethyl protons at δ 1.29 (s, CH3-3′). These signals, along with the HMBC correlations of H-1′ to C-2′ (δ 45.9), C-3′ (δ 69.4); H-2′ to C-1′ (δ 29.9), C-3′ (δ 69.4), CH3-3′ (δ 28.8) and CH3-3′ to C-2′ (δ 45.9), C-3′ (δ 69.4) corresponded to a 3-hydroxy-3-methylbutyl unit. The deshielded resonance of H-1′ (δ 3.28) suggested that the side chain was at C-8. Furthermore, irradiation of the methylene proton H-1′ caused a NOE enhancement of H-7 and these results suggested that the side chain was ortho to H-7. The assignments of aromatic protons and substituents were deduced from the HMBC spectra. The 3J correlations of 1-OH to C-1 (δ 164.2), C-2 (δ 97.9), C-9a (δ 102.9); H-2 to C-9a (δ 102.9), C-4 (δ 93.2) and H-4 to C-2 (δ 97.9), C-9a (δ 102.9) strongly supported the position of H-2 and H-4. The HMBC correlations of H-7 to C-5 (δ 130.5), C-8a (δ 111.4), C-1′ (δ 29.9) and H-1′ to C-7 (δ 114.7), C-8 (δ 137.9), C-8a (δ 111.4) indicated 35
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crystals and 14 (13.9 mg) as a yellow solid, respectively. The filtrates of fractions D6 and D7 were combined (211.4 mg) and further purified by CC using 20% acetone in hexane yielding 13 (5.2 mg), 15 (2.8 mg) and 16 (10.8 mg) as yellow solid. Fraction E (0.26 g) was further purified by CC using 25% acetone in hexane as an eluent to give 17 (3.0 mg) and 18 (3.1 mg) as a light yellow solid. Yellow solid 19 (3.0 mg) and 20 (2.5 mg) were obtained from fractions F (1.00 g) by repeated CC using 1% MeOH in CH2Cl2 as an eluent. Fraction G (1.07 g) was separated by repeated CC using 1% MeOH in CH2Cl2 as an eluent to give yellow solid 21 (3.5 mg), 22 (4.2 mg), 23 (2.2 mg), 24 (4.5 mg) and a yellow gum 1 (2.5 mg). Fraction H (0.51 g) was separated by CC using 35% acetone in hexane as an eluent to give yellow solid 25 (2.3 mg) and brown solid 26 (24.0 mg). Fraction I (0.55 g) was purified by CC using 2.5% MeOH in CH2Cl2 as an eluent to give yellow gum 2 (3.1 mg) and 27 (2.0 mg). A yellow solid 28 (3.0 mg) was obtained from fraction J (0.09 g) by CC with 3% MeOH in CH2Cl2. The acetone extract was subjected on Sephadex LH-20 eluting with 100% MeOH to give two fractions (IV, V). Fraction V (15.00 g) was further subjected to a QCC and eluted with a stepwise gradient from 10% acetone in CH2Cl2 to acetone gave nine fractions (K–S). Fraction N (0.78 g) was fractionated by CC employing 20% acetone in hexane resulting in nine fractions (N1–N9). Fractions N3 (16.1 mg) and N5 (17.2 mg) were further purified by CC using 15% acetone in hexane to yield a light yellow solid 29 (2.0 mg) and yellow gum 30 (3.0 mg), respectively. Yellow gum 31 (2.0 mg) was obtained from fraction N8 (41.6 mg) by repeated CC with 50% CH2Cl2 in hexane. Fraction O (342.2 mg) was further fractionated by CC with 20% acetone in hexane to give ten fractions (O1–O10). Fractions O5 (24.3 mg) was further purified by preparative TLC using 15% acetone in hexane as an eluent to yield yellow gum 32 (2.0 mg). Yellow solid 33 (1.5 mg) was obtained from fraction O8 (53.8 mg) by repeated CC, eluted with CH2Cl2, and was further purified by Sephadex LH-20 column using MeOH as an eluent. Fraction Q (54.6 mg) was subjected to CC using isocratic of 5% acetone in CH2Cl2 to provide six fractions (Q1–Q6). Fraction Q5 (26.2 mg) was further purified by repeated CC using isocratic of 5% acetone in CH2Cl2 gave yellow gum 3 (1.5 mg). Fraction R (1.06 g) was further fractionated by CC with 7% acetone in CH2Cl2 to give nine fractions (R1–R9). Fraction R5 (24.3 mg) was further purified by repeated CC using 7% acetone in CH2Cl2 as an eluent to yield 34 (3.5 mg) as a light yellow solid. Yellow solid 35 (3.5 mg) was obtained from fraction R7 (48.7 mg) by repeated CC eluting with 10% acetone in CH2Cl2. Fraction S (1.00 g) was preabsorbed on C-18 and loaded into preparative guard column (30 mm × 10 mm i.d.), in line with a semiprep C18 HPLC column employing a gradient of H2O–MeOH resulting in five fractions (S1–S5). Fraction S1 (0.10 g) was further isolated by Diol HPLC and eluted with a gradient of 5–15% MeOH in CH2Cl2 gave light yellow solid 41 (2.5 mg). Fraction S3 (0.10 g) was further isolated by Diol HPLC and eluted with 100% CH2Cl2 to 15% MeOH in CH2Cl2 gave light yellow solid 37 (14.5 mg), 38 (6.5 mg) and 40 (26.3 mg). Fraction S4 (0.50 g) was further isolated by Diol HPLC and employing a gradient of 50% Hexane in CH2Cl2 to 15% MeOH in CH2Cl2 gave light yellow solid 36 (7.4 mg). Fraction S5 (0.40 g) was further isolated by Diol HPLC and employing a gradient of 90% Hexane in CH2Cl2 to 15% MeOH in CH2Cl2 gave light yellow solid 39 (4.2 mg). Dulcisxanthone J (1): yellow gum; UV (MeOH) λmax nm (log ε): 226 (3.46), 265 (3.70), 296 (3.58); IR (neat) νmax (cm−1): 3353, 1624; 1H NMR and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; HRESIMS (positive) m/z 417.1104 [M + Na]+ (calcd. for C23H22O6Na, 417.1314). Dulcisxanthone K (2): yellow gum; UV (MeOH) λmax nm (log ε): 255 (4.51), 280 (3.97), 328 (4.23); IR (neat) νmax (cm−1): 3415, 1638; 1H NMR and 13C NMR data (acetone-d6, 300 and 75 MHz), see Table 1; HRESIMS (positive) m/z 419.1471 [M + Na]+ (calcd. for C23H24O6Na, 419.1471). Dulcisxanthone L (3): yellow gum; UV (MeOH) λmax nm (log ε): 252 (4.23), 280 (3.79), 326 (3.97); IR (neat) νmax (cm−1): 3301, 1649; 1H
In conclusion, investigation of the active constituents as antibacterial agents from the stem bark of G. dulcis resulted in the isolation of 41 compounds including one isocoumarin, six biflavonoids and 34 xanthones. Three xanthones are new. The most effective antibacterial compound was a xanthone with isoprenyl side chain named 12bhydroxy-des-D-garcigerrin A (12) (MIC 4 μg/mL). The stem bark produced more prenylated xanthones than other plant parts of G.dulcis. 3. Experimental 3.1. General experimental procedures Crude extracts were obtained by sonication technique with S70H Elmasonic (Elma) at 35 °C. Ultraviolet spectra were recorded using a UV-160A spectrophotometer (SHIMADZU). The infrared spectra were obtained on a FTS165 FT-IR spectrometer (Perkin-Elmer). Nuclear Magnetic Resonance (NMR) spectra were recorded with a FT-NMR Ultra Shield™ 300 and 500 spectrometers (Bruker). Electrospray ionization mass spectrometric (ESIMS) data were determined on a Waters Alliance 2690 and a Micromass LCT (United Kingdom) mass spectrometers. Quick column chromatography (QCC) was performed on silica gel 60H (Merck). Column chromatography (CC) was performed on silica gel 100 (Merck) and Sephadex LH-20 (Amersham Biosciences, Sweden). Pre-coated TLC aluminum sheets (layer thickness 0.2 mm, Merck) and preparative TLC plates (layer thickness 1.25 mm, Merck) of silica gel 60 GF254 were used. Altech Davisil 30–40 μm 60 A° C18 silica gel or Altech Davisil 30–40 μm 60 A° diol silica was used to absorb the extract prior to HPLC separation. A Hitachi L7455 PDA detector (Merck) with a Hitachi L7100 pump (Merck) and a L7250 autosampler (Merck) were used for HPLC separations. HPLC columns used were Thermosystems betasil C18 5 μm, 21 mm × 150 mm, YMC Pack Diol NP 5 μm (21 mm × 150 mm) and phenyl bonded silica 5 μm (21 mm × 250 mm). The flow rate of mobile phase was constant at 9.0 mL/min. 3.2. Plant material The stem bark of G. dulcis was collected in Nakorn Si Thammarat province, the southern part of Thailand, and air-dried. The voucher specimen (Coll. No. 02, Herbarium No. 0012652) has been deposited in the Herbarium of Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation The chopped and air-dried stem bark of G. dulcis (1.5 kg) was extracted with CH2Cl2 (5 L) and acetone (5 L) in a sonicator bath for thirty minutes (2 times) and filtered. After removal of solvent, brown viscous CH2Cl2 extract (60.15 g) and acetone extract (44.30 g) were obtained. The CH2Cl2 extract was loaded onto Sephadex LH-20 and eluted with 100% MeOH to give three fractions (I–III). Fraction III (24.06 g) was further subjected to a QCC and eluted with a stepwise gradient from 10% acetone in hexane to pure acetone. The fractions with the same major components were combined on the basis of TLC analysis to give ten fractions (A–J). Fraction C (0.14 g) was separated by repeated CC using isocratic of 5% acetone in hexane to give a white solid 4 (2.0 mg), yellow solid 5 (2.0 mg) and 6 (5.0 mg). Fraction D (1.54 g) was chromatographed on CC using 15% acetone in hexane as an eluent to give ten fractions (D1–D10). Fraction D3 (25.1 mg) was rinsed with hexane to yield a yellow solid 7 (10.1 mg). Fraction D4 (40.8 mg) was separated by CC using 15% acetone in hexane to give five fractions (D4.1–D4.5). Fractions D4.2 and D4.4 were further purified by CC and preparative TLC plate using 15% acetone in hexane as eluent to give a light yellow gum 8 (2.5 mg), yellow solid 9 (3.0 mg), 10 (3.4 mg) and 11 (4.0 mg). Fractions D6 (252.1 mg) and D7 (35.1 mg) were rinsed with hexane to afford 12 (35.3 mg) as orange 36
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NMR and 13C NMR data (acetone-d6, 500 and 125 MHz), see Table 1; HRESIMS (positive) m/z 369.0956 [M + Na]+ (calcd. for C18H18O7Na, 369.0950).
xanthone dimers from the roots of Garcinia dulcis. Chem. Pham. Bull. 44, 1744–1747. Jackson, B., Locksley, H.D., Scheinmann, F., 1967. Extractives from Guttiferae. VII. Isolation and structure of seven xanthones from Calophyllum scribiltifolium. J. Chem. Soc. [Section] C: Organic 23, 2500–2507. Kasahara, S., Henmi, S., 1986. Medicinal Herb Index in Indonesia, Jakarta : Eisai Indonesia. Kijjoa, A., Gonzalez, M.J.T.G., Pinto, M.M.M., Monanondra, I.O., Herz, W., 1991. Constituents of Knema laurina and Knema tenuinervia ssp. setosa. Planta Med. 57, 575–577. Likhitwittayawuid, K., Chanmahasathien, W., Ruangrugsi, N., Krungkra, J., 1998. Xanthones with antimalarial activity from Garcinia dulcis. Planta Med. 64, 281–282. Locksley, H.D., Murray, I.G., 1971. Extractives from Guttiferae. Part XIX. The isolation and structure of two benzophenones six xanthones and two biflavonoids from the heartwood of Allanblackia floribunda Oliver. J. Chem. Soc. C 1332–1340. Locksley, H.D., Moore, I., Scheinmann, F., 1966. Extractives from Guttiferae. Part III. The isolation and structure of symphoxanthone and globuxanthone from Symphonia globulifera L. J. Chem. Soc. C 2186–2190. Luzzi, R., Guimaraes, C.L., Verdi, L.G., Simionatto, E.L., Delle Monache, F., Yunes, R.A., Floriani, A.E., Cechinel-Filho, V., 1997. Isolation of biflavonoids with analgesic activity from Rheedia gardneriana leaves. Phytomedicine 4, 141–144. Mahabusarakam, W., Mecawun, P., Phongpaichit, S., 2016. Xanthones from the green branch of Garcinia dulcis. Nat. Prod. Res. 30, 2323–2328. Marques, V.L.L., De Oliveira, F.M., Conserva, L.M., Brito, R.G.L., Guilhon, G.M.S.P., 2000. Dichromenoxanthones from Tovomita brasiliensis. Phytochemistry 55, 815–818. Minami, H., Kinoshita, M., Fukuyama, Y., Kodama, M., Yoshizawa, T., Sugiura, M., Nakagawa, K., Tago, H., 1994. Antioxidant xanthones from Garcinia subelliptica. Phytochemistry 36, 501–506. Minami, H., Takahashi, E., Fukuyama, Y., Kodama, M., Yoshizawa, T., Nakagawa, K., 1995. Novel xanthones with superoxide scavenging activity from Garcinia subelliptica. Chem. Pharm. Bull. 43, 347–349. Minami, H., Takahashi, E., Kodama, M., Fukuyama, Y., 1996. Three xanthones from Garcinia subelliptica. Phytochemistry 41, 629–633. Nguyen, L.H., Harison, L., 2000. Xanthones and triterpenoids from the bark of Garcinia vilersiana. Phytochemistry 53, 111–114. Pelter, A., Warren, R., Hameed, N., Khan, N.U., Ilyas, M., Rahman, W., 1970. Biflavonyl pigments from Thuja orientalis (Cupressaceae). Phytochemistry 9, 1897–1898. Perkin, A.G., Hummel, J.J., 1896. The colouring principle contained in the bark of Myrica nagi. Part I. J. Chem. Soc. Trans. 69, 1287–1294. Saelee, A., Phongpaichit, S., Mahabusarakama, W., 2015. A new prenylated biflavonoid from the leaves of Garcinia dulcis. Nat. Prod. Res. 29, 1884–1888. Seo, E.K., Kim, N.C., Wani, M.C., Wall, M.E., Navarro, H.A., Burgess, J.P., Kawanishi, K., Kardono, L.B.S., Riswan, S., Rose William, C., 2002. Cytotoxic prenylated xanthones and the unusual compounds anthraquinobenzophenones from Cratoxylum sumatranum. J. Nat. Prod. 65, 299–305. Shakui, T., Iguchi, K., Ito, T., Baba, M., Usui, S., Oyama, M., Tosa, H., Iinuma, M., Hirano, K., 2014. Anti-androgenic activity of hydroxyxanthones in prostate cancer LNCaP cells. Fitoterapia 92, 9–15. Sordat-Diserens, I., Marston, A., Hamburger, M., Rogers, C., Hostettmann, K., 1989. Novel prenylated xanthones from Garcinia gerrardii Harvey. Helv. Chim. Acta. 72, 1001–1007. Sordat-Diserens, I., Rogers, C., Sordat, B., Hostettmann, K., 1992. Prenylated xanthones from Garcinia livingstonei. Phytochemistry 31, 313–316. Supaphon, P., Phongpaichit, S., Rukachaisirikul, V., Sakayaroj, J., 2013. Antimicrobial potential of endophytic fungi derived from three seagrass species: Cymodocea serrulata, Halophila ovalis and Thalassia hemprichii. PLoS One 8, e72520. Waterman, P.G., Crichton, E.G., 1980. Xanthones and biflavonoids from Garcinia densivenia stem bark. Phytochemistry 19, 2723–2726. Xu, Y.J., Cao, S.G., Wu, X.H., Lai, Y.H., Tan, B.H.K., Pereira, J.T., Goh, S.H., Venkatraman, G., Harrison, L.J., Sim, K.Y., 1998. Griffipavixanthone, a novel cytotoxic bixanthone from Garcinia griffithii and Garcinia parvifolia. Tetrahedron Lett. 39, 9103–9106.
3.4. Antibacterial assay This was carried out according to the previously reported procedure of Supaphon et al., 2013. Acknowledgements This work was supported by the Thailand Research Fund (TRF): Senior Research Scholar (Grant No. RTA5880005). We thank the Graduate School, Prince of Songkla University for a partial research grant and a scholarship for support exchange students and international credit transferred through ASEAN community. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.05.014. References Asai, F., Tosa, H., Tanaka, T., Iinuma, M., 1995. A xanthone from pericarps of Garcinia mangostana. Phytochemistry 39, 943–944. Chawla, H.M., Chibber, S.S., Khera, U., 1975. Separation and identification of some hydroxyxanthones by thin-layer chromatography. J. Chromatogr. A. 111, 246–247. Chen, Y., Zhong, F., He, H., Hu, Y., Zhu, D., Yang, G., 2008. Structure elucidation and NMR spectral assignment of five new xanthones from the bark of Garcinia xanthochymus. Magn. Reson. Chem. 46, 1180–1184. Chen, Y., Fan, H., Yang, G.Z., Jiang, Y., Zhong, F.F., He, H.W., 2010. Prenylated xanthones from the bark of Garcinia xanthochymus and their 1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging activities. Molecules 15, 7438–7449. Cotterill, P.J., Scheinmann, F., 1975. A revised structure for toxyloxanthone. B. J. Chem. Soc. Chem. Commun. 16, 664–665. Deachathai, S., Mahabusarakam, W., Phongpaichit, S., Taylor, W.C., 2005. Phenolic compounds from the fruit of Garcinia dulcis. Phytochemistry 66, 2368–2375. Deachathai, S., Mahabusarakam, W., Phongpaichit, S., Taylor, W.C., Zhang, Y.J., Yang, C.R., 2006. Phenolic compounds from the flowers of Garcinia dulcis. Phytochemistry 67, 464–469. Deachathai, S., Phongpaichit, S., Mahabusarakam, W., 2008. Phenolic compounds from the seeds of Garcinia dulcis. Nat. Prod. Res. 22, 1327–1332. Fukuyama, Y., Kamiyama, A., Mima, Y., Kodama, M., 1991. Prenylated xanthones from Garcinia subelliptica. Phytochemistry 30, 3433–3436. Iinuma, M., Tosa, H., Tanaka, T., Shimano, R., Asai, F., Yonemori, S., 1994. Two xanthones from root bark of Garcinia subelliptica. Phytochemistry 35, 1355–1360. Iinuma, M., Tosa, H., Tanaka, T., Asai, F., Shimano, R., 1995a. Two xanthones with a 1,1dimethylallyl group in root bark of Garcinia subelliptica. Phytochemistry 39, 945–947. Iinuma, M., Tosa, H., Tanaka, T., Asai, F., Shimano, R., 1995b. Three xanthones from root bark of Garcinia subelliptica. Phytochemistry 38, 247–249. Iinuma, M., Tosa, H., Tanaka, T., Asai, F., Shimano, R., 1995c. Two new xanthones from the root bark of Garcinia subelliptica. Heterocycles 40, 279–284. Iinuma, M., Tosa, H., Ito, T., Tanaka, T., Riswan, S., 1996. Three new benzophenone-
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Short communication
Prenylated xanthones from the stem bark of Garcinia dulcis a,c
b,c
MARK
d,e
Parichat Thepthong , Souwalak Phongpaichit , Anthony R. Carroll , ⁎ Supayang Piyawan Voravuthikunchaib,c, Wilawan Mahabusarakama,c, a
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Natural Product Research Center of Excellence, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d Environmental Futures Research Institute, Griffith University, Gold Coast, Queensland 4222, Australia e Eskitis Institute, Griffith University, Brisbane, Queensland 4111, Australia b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Clusiaceae Garcinia dulcis Prenylated xanthones Antibacterial activity
Three new prenylated xanthones, named dulcisxanthone J (1), dulcisxanthone K (2), and dulcisxanthone L (3), along with 38 known compounds have been isolated from the stem bark of Garcinia dulcis. Their structures were elucidated mainly by analysis of 1D and 2D spectroscopic data. 12b-Hydroxy-des-D-garcigerrin A (12) and dulcisxanthone J (1) displayed moderate antibacterial activity against both penicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) with minimum inhibitory concentration (MIC) values of 4 and 16 μg/mL, respectively.
1. Introduction Garcinia dulcis Kurz. is a medicinal plant in the Clusiaceae family. This plant species is widely distributed in the southern part of Thailand, Malaysia, Indonesia and Philippines. The fruit juice has been used as an expectorant. The leaves and seeds have been applied as a traditional medicine for the treatment of lymphatitis, parotitis and struma in Indonesia (Kasahara and Henmi, 1986). In Thailand, its stem bark has been used as an anti-inflammatory agent. The various parts of this plant are rich in secondary phenolic metabolites, including xanthones, biflavonoids, flavonoids, chalcones and phloroglucinols. Xanthones, the main chemical components of G. dulcis, have many pharmacological properties. Some xanthones from fruits, flowers and seeds have been reported to possess antioxidant and antibacterial activity (Deachathai et al., 2005, 2006, 2008), while xanthones from stem bark were reported to possess anti-androgenic (Shakui et al., 2014) and antimalarial activity (Likhitwittayawuid et al., 1998). The knowledge that G. dulcis is a rich source of xanthones, together with our test result that the extract of stem bark inhibited the growth of both penicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) with minimum inhibitory concentration (MIC) values of 32 μg/mL led us to examine the active constituents from stem bark as antibacterial agents. 2. Results and discussion Purification of the CH2Cl2 and acetone extracts of the stem bark of ⁎
G. dulcis by chromatographic and crystallization techniques gave 41 compounds. Structural elucidation mainly by analysis of 1D and 2D spectroscopic data and comparison of the data with those of previously reported compounds indicated three new and 38 known compounds. The new compounds were prenylated xanthones named dulcisxanthone J (1), dulcisxanthone K (2), and dulcisxanthone L (3) (Fig. 1). Dulcisxanthone J (1) was a yellow gum. Its molecular formula was determined to be C23H22O6 by HRESIMS at m/z 417.1104 [M + Na]+. The UV spectrum showed absorption maxima at λmax 226, 265 and 296 nm. The IR spectrum showed absorption bands for OeH stretching at 3353 cm−1 and C]O stretching at 1624 cm−1. The 13C NMR spectrum displayed resonances of 23 carbons which were classified as one carbonyl, four methyl, one methylene, five methine and twelve quaternary carbons (Table 1). The 1H NMR spectrum showed resonances of a chelated hydroxyl group (1-OH) at δ 12.80, and singlet aromatic protons, H-3 and H-8 at δ 7.41 and δ 7.55, respectively. The HMBC correlations from H-8, to C-6 (δ 145.1), C-9 (δ 181.9), C-10a (δ 145.0) and from H-3 to C-1 (δ 153.2), C-4a (δ 140.9), C-4 (δ 135.0) located H-8 ortho to a ketone carbonyl, and H-3 meta to hydrogen bonded hydroxyl group (Fig. 2). The resonances for an olefinic methine proton at δ 6.30 (dd, J = 17.5, 10.5 Hz, H-2″), terminal methylene protons at δ 5.04 (br d, J = 17.5 Hz, Ha-3″) and δ 5.05 (br d, J = 10.5 Hz, Hb-3″), and gem-dimethyl groups at δ 1.56 (s, CH3-1″) as well as the HMBC correlations of Ha-3″ and Hb-3″ to C-1″ (δ 40.4), C2″ (δ 147.1) and H-2″ to CH3-1″ (δ 26.9), C-1″ (δ 40.4) were suggestive that an isoprenyl unit was present in the molecule. The non equivalent
Corresponding author at: Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. E-mail address: [email protected] (W. Mahabusarakam).
http://dx.doi.org/10.1016/j.phytol.2017.05.014 Received 9 December 2016; Accepted 11 May 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. The chemical structures of the new compounds 1, 2 and 3.
CH3-1″ and H-2″ to C-2 (δ 129.4), and H-3 to C-1″ (δ 40.4), C-1 (δ 153.2), along with the NOESY correlations between CH3-1″ and H-3, suggested that the isoprenyl unit was attached at C-2 and ortho to H-3. The remaining 1H NMR signals including two doublets of cis-olefinic protons at δ 5.74 and δ 6.46 (J = 10.0 Hz, H-3′ and H-4′) and a singlet of dimethyl protons at δ 1.57 (CH3-2′), as well as the HMBC correlations of H-4′ to C-2′ (δ 79.2); H-3′ to C-2′ (δ 79.2), CH3-2′ (δ 28.6); and CH3-2′ to C-2′ (δ 79.2), C-3′ (δ 130.9) were in agreement with typical signals for a 2,2-dimethylchromene ring. The HMBC correlations of cis-olefinic proton H-4′ to oxygenated aromatic carbon C-6 (δ 145.1), methine aromatic carbon C-8 (δ 114.5) allowed the chromene ring to be attached to C-6 and C-7 of the xanthone skeleton with an ether linkage at C-6. Finally, two hydroxy groups were assigned at C-4 (δ 135.0) and C-5 (δ 141.1) to satisfy the molecular formula derived from MS analysis. Thus dulcisxanthone J (1) was identified as 1,4,5-trihydroxy-2-(1,1dimethyl-2-propenyl)-2′,2′-dimethylpyrano[5′,6′:6,7]xanthone. It was a positional isomer of subelliptenone H (13) (Iinuma et al., 1995a) (Fig. 3). Dulcisxanthone K (2) was a yellow gum. Its molecular formula was determined to be C23H24O6 by analysis of a HRESIMS ion at m/z 419.1471 [M + Na]+. The UV spectrum showed absorption maxima at λmax 255, 280 and 328 nm. The IR spectrum showed absorption bands at 3415 cm−1 (OeH stretching) and 1638 cm−1 (C]O stretching). The 13 C NMR spectrum showed 23 carbon signals which were classified as one carbonyl, four methyl, two methylene, four methine, and twelve quaternary carbons. The 1H NMR spectrum exhibited signals for a chelated hydroxy proton (1-OH) at δ 13.82, and aromatic singlet protons H-4 and H-7 at δ 6.48 and δ 6.77, respectively. The characteristic signals for two prenyl groups were also present. The HMBC correlation allowed assignment of the methylene doublet protons at δ 3.35 (J = 7.2 Hz, H-1′), an olefinic triplet proton at δ 5.28 (J = 7.2 Hz, H-2′), gem-dimethyl singlet protons at δ 1.78 (H-4′) and δ 1.65 (H-5′) for the 2-prenyl side chain, while the methylene protons at δ 3.97 (d, J = 7.2 Hz, H-1″), an olefinic proton at δ 5.39 (t, J = 7.2 Hz, H-2″), gem-dimethyl protons at δ 1.74 (s, H-4″) and δ 1.72 (s, H-5″) for the 8prenyl side chain. The HMBC correlations of 1-OH and H-4 to C-2 (δ 110.2), C-9a (δ 102.6); H-1′ to C-1 (δ 161.0), C-2 (δ 110.2), C-3 (δ 162.1) confirmed the location of H-4 and the 2-prenyl unit attached at
Table 1 1 H and 13C NMR spectral data of compounds 1, 2 and 3. Position
1a δH, (mult., J in Hz)
1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″
4″ 5″ 1-OH (CH3)2-2′ (CH3)2-3′ (CH3)2-1″ a b
7.41 (s)
7.55 (s)
5.74 (d, 10.0) 6.46 (d, 10.0)
6.30 (dd, 17.5, 10.5) 5.04 (br d, 17.5, Ha) 5.05 (br d, 10.5, Hb)
2b δC
153.2 129.4 122.2 135.0 140.9 141.1 145.1 117.9 114.5 120.3 181.9 106.3 145.0 79.2 130.9 121.6 40.4 147.1
3b
δH, (mult., J in Hz)
6.48 (s)
6.77 (s)
3.35 (d, 7.2) 5.28 (t, 7.2) 1.78 1.65 3.97 5.39
(s) (s) (d, 7.2) (t, 7.2)
110.6
δC
161.0 110.2 162.1 92.5 154.8 130.5 150.1 113.8 135.7 111.3 182.5 102.6 147.2 21.1 122.6 130.5 17.0 24.9 32.8 123.5
δH, (mult., J in Hz)
6.22 (d, 2.0) 6.41 (d, 2.0)
6.78 (s)
3.28 (m) 1.77 (m)
δC
164.2 97.9 164.6 93.2 157.1 130.5 150.4 114.7 137.9 111.4 182.3 102.9 147.0 29.9 45.9 69.4
131.6
110.6 1.74 (s) 1.72 (s) 13.82 (s)
12.80 (s) 1.57 (s)
28.6
1.56 (s)
26.9
17.1 25.0 13.56 (s) 1.29 (s)
28.8
recorded in CDCl3. recorded in acetone-d6.
methylene protons Ha-3″ and Hb-3″ in the isoprenyl side chain were assigned trans and cis to methine H-2″ base on the coupling constant values of 17.5 and 10.5 Hz, respectively. The HMBC correlations of
Fig. 2. The key HMBC correlations of 1, 2 and 3.
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Fig. 3. Structure of compounds from the stem bark of G. dulcis (4-41).
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that an aromatic proton (H-7) and the 3-hydroxy-3-methylbutyl group were placed at C-7 and C-8, respectively. Finally, the carbon chemical shifts of the remaining unassigned carbon indicated that three hydroxy groups were attached at C-3 (δ 164.6), C-5 (δ 130.5) and C-6 (δ 150.4) to complete the structure. Hence, dulcisxanthone L was established as 1,3,5,6-tetrahydroxy-8-(3-hydroxy-3-methylbuty)xanthone. It was a 3′hydroxy derivative of garcinexanthone C (Chen et al., 2008). The 38 known compounds were identified as one isocoumarin: 8hydroxy-6-methoxy-3-pentylisocoumarin (4) (Kijjoa et al., 1991), six biflavonoids: O-methyl fukugetin (36), (Waterman and Crichton, 1980), volkensiflavone (37), GB-2a (39), morelloflavone (40) fukugeside (41) (Luzzi et al., 1997), amentoflavone (38) (Pelter et al., 1970), and 31 xanthones: brasilixanthone B (5) (Marques et al., 2000), garciniaxanthone C (6), 2,6-dihydroxy-1,5-dimethoxyxanthone (32) (Minami et al., 1994), garciniaxanthone A (7), garciniaxanthone B (10) (Fukuyama et al., 1991), dulcisxanthone H (8), dulcisxanthone I (11) (Mahabusarakam et al., 2016), 6-deoxyjacareubin (9) (Jackson et al., 1967), 12b-hydroxy-des-D-garcigerrin A (12) (Sordat-Diserens et al., 1989), subelliptenone H (13) (Iinuma et al., 1995a), subelliptenone B (14) (Iinuma et al., 1994), 1-O-methylglobuxanthone (15) (Nguyen and Harison, 2000), globuxanthone (16), symphoxanthone (24) (Locksley et al., 1966), 1,2,5,4′-tetrahydroxy-4-(1,1-dimethyallyl)-5′-(2-hydroxypropan-2-yl)-4′,5′-dihydrofurano(2′,3′:6,7)xanthone (17) (Chen et al., 2010), subelliptenone D (19), subelliptenone C (31) (Iinuma et al., 1995c), subelliptenone F (27) subelliptenone G (30) (Iinuma et al., 1995b), 6,11-dihydroxy-2,2-dimethylpyrano[3,2-c]xanthen-7(2H)-one (20) (Sordat-Diserens et al., 1992), euxanthone (21) (Perkin and Hummel, 1896), toxyloxanthone B (22) (Cotterill and Scheinmann, 1975), 1,3,5-trihydroxyxanthone (23) (Locksley and Murray, 1971), 1,7-dihydroxy-2-(3-methylbut-2-enyl)-3-methoxyxanthone (25) (Asai et al., 1995), 1-O-methylsymphoxanthone (18), garciniaxanthone E (26), 2,5-dihydroxy-1-methoxyxanthone (29) (Minami et al., 1996), garciniaxanthone D (28) (Minami et al., 1995), 1,3,7-trihydroxyxanthone (33) (Chawla et al., 1975), garciduol A (34) (Iinuma et al., 1996), griffipavixanthone (35) (Xu et al., 1998). Compounds 4, 5, 9, 13, 14, 15, 17, 20, 35 and 36 were reported from this plant for the first time. The extracts and some of the pure compounds were evaluated for antibacterial activity. The result showed that the CH2Cl2 and acetone extracts inhibited the growth of penicillin-susceptible S. aureus ATCC 25923 with MIC values of 32 and 128 μg/mL, and MRSA SK1 with MIC values of 64 and 128 μg/mL. For pure compounds, 12b-hydroxy-des-Dgarcigerrin A (12) and dulcisxanthone J (1) exhibited antibacterial activity against both test strains with MIC values of 4 and 16 μg/mL, respectively. Compounds 6, 14, 16, 24 and 35 showed activity against S. aureus with MIC values ranging from 32 to 64 μg/mL and against MRSA with MIC values ranging from 32 to 128 μg/mL. Compounds 2, 7, 15, 17, 18, 28 and 30 showed activity against both bacteria test strains with MIC values ranging from 128 to 200 μg/mL, whereas compounds 8-11, 13, 19, 21, 22, 26 and 34 showed no activity at 200 μg/mL (Table 2). The structure activity relationships of the tested xanthones, especially with isoprenyl side chain, on antibacterial activity have been studied. The test result that xanthone 12 was much more effective in inhibiting the growth of bacteria than 30 indicated that the isoprenyl unit at C-2 enhanced the activity. However, more steric hindrance by substituents at position C-6 and C-7 in xanthones 1, 7, 10 and 28 could reduce the activity. Xanthone 16 which contained an isoprenyl unit at C-4 exhibited antibacterial activity, however less effectively than the positional isomer 12. The weak activity of xanthones 13, 14, 17, 19 and 24 as well as xanthones 15 and 18 suggested that the substituents at position C-6 and C-7, and replacement of 1-OH by a methoxy group, affected the antibacterial activity of xanthones containing an isoprenyl group. Compounds 3-5, 20, 23, 25, 27, 29, 31-33 and 41 were not tested due to the lack of material. For biflavonoids 36-40, the antibacterial activities have been previously reported (Deachathai et al., 2005; Saelee et al., 2015).
Table 2 Antibacterial activity of compounds and extracts from the stem bark of G. dulcis. Compounds
1 2 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 24 26 28 30 34 35 CH2Cl2 extract Acetone extract Vancomycin
S. aureus ATCC 25923
MRSA SK1
MIC (μg/mL)
MBC (μg/mL)
MIC (μg/mL)
MBC (μg/mL)
16 128 32 200 > 200 > 200 > 200 > 200 4 > 200 32 200 32 128 128 > 200 > 200 > 200 32 > 200 200 128 > 200 32 32 128 1
> 200 > 200 > 200 200 > 200 > 200 > 200 > 200 128 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 128 > 200 > 200 200 > 200 128 200 > 200 2
16 128 32 200 > 200 > 200 > 200 > 200 4 > 200 128 200 32 128 128 > 200 128 > 200 128 > 200 128 128 > 200 64 64 128 1
64 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 128 > 200 128 200 > 200 2
MIC = Minimum Inhibitory Concentrations. MBC = Minimum Bactericidal Concentrations.
C-2. The HMBC correlations between H-1″ and C-7 (δ 113.8), C-8a (δ 111.3); H-7 and C-5 (δ 130.5), C-6 (δ 150.1), C-8a (δ 111.3), C-1″ (δ 32.8) confirmed the position of H-7 and the 8-prenyl unit attached at C8. Finally, the hydroxy groups were assigned at C-3 (δ 162.1), C-5 (δ 130.5) and C-6 (δ 150.1) to complete the structure. Dulcisxanthone K (2) was therefore identified as 1,3,5,6-tetrahydroxy-2,8-bis(3-methyl-2butenyl)xanthone. It was a positional isomer of cratoxyarborenone B (Seo et al., 2002). Dulcisxanthone L (3) was a yellow gum. Its molecular formula was determined to be C18H18O7 from analysis of a pseudomolecular ion in HRESIMS at m/z 369.0956 [M + Na]+. The UV spectrum showed absorption maxima at λmax 252, 280 and 326 nm. The IR spectrum showed absorption bands at 3301 cm−1 (OeH stretching) and 1649 cm−1 (C]O stretching). The 13C NMR spectrum showed 18 carbon signals which were classified as one carbonyl, two methyl, two methylene, three methine and ten quaternary carbons. The 1H NMR spectrum exhibited signals for a chelated hydroxy proton (1-OH) at δ 13.56, meta-coupled (d, J = 2.0 Hz) aromatic protons at δ 6.22 (H-2) and δ 6.41 (H-4) and an isolated aromatic proton at δ 6.78 (s, H-7). The remaining signals were for benzylic methylene protons at δ 3.28 (m, H1′), methylene protons at δ 1.77 (m, H-2′) and gem-dimethyl protons at δ 1.29 (s, CH3-3′). These signals, along with the HMBC correlations of H-1′ to C-2′ (δ 45.9), C-3′ (δ 69.4); H-2′ to C-1′ (δ 29.9), C-3′ (δ 69.4), CH3-3′ (δ 28.8) and CH3-3′ to C-2′ (δ 45.9), C-3′ (δ 69.4) corresponded to a 3-hydroxy-3-methylbutyl unit. The deshielded resonance of H-1′ (δ 3.28) suggested that the side chain was at C-8. Furthermore, irradiation of the methylene proton H-1′ caused a NOE enhancement of H-7 and these results suggested that the side chain was ortho to H-7. The assignments of aromatic protons and substituents were deduced from the HMBC spectra. The 3J correlations of 1-OH to C-1 (δ 164.2), C-2 (δ 97.9), C-9a (δ 102.9); H-2 to C-9a (δ 102.9), C-4 (δ 93.2) and H-4 to C-2 (δ 97.9), C-9a (δ 102.9) strongly supported the position of H-2 and H-4. The HMBC correlations of H-7 to C-5 (δ 130.5), C-8a (δ 111.4), C-1′ (δ 29.9) and H-1′ to C-7 (δ 114.7), C-8 (δ 137.9), C-8a (δ 111.4) indicated 35
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crystals and 14 (13.9 mg) as a yellow solid, respectively. The filtrates of fractions D6 and D7 were combined (211.4 mg) and further purified by CC using 20% acetone in hexane yielding 13 (5.2 mg), 15 (2.8 mg) and 16 (10.8 mg) as yellow solid. Fraction E (0.26 g) was further purified by CC using 25% acetone in hexane as an eluent to give 17 (3.0 mg) and 18 (3.1 mg) as a light yellow solid. Yellow solid 19 (3.0 mg) and 20 (2.5 mg) were obtained from fractions F (1.00 g) by repeated CC using 1% MeOH in CH2Cl2 as an eluent. Fraction G (1.07 g) was separated by repeated CC using 1% MeOH in CH2Cl2 as an eluent to give yellow solid 21 (3.5 mg), 22 (4.2 mg), 23 (2.2 mg), 24 (4.5 mg) and a yellow gum 1 (2.5 mg). Fraction H (0.51 g) was separated by CC using 35% acetone in hexane as an eluent to give yellow solid 25 (2.3 mg) and brown solid 26 (24.0 mg). Fraction I (0.55 g) was purified by CC using 2.5% MeOH in CH2Cl2 as an eluent to give yellow gum 2 (3.1 mg) and 27 (2.0 mg). A yellow solid 28 (3.0 mg) was obtained from fraction J (0.09 g) by CC with 3% MeOH in CH2Cl2. The acetone extract was subjected on Sephadex LH-20 eluting with 100% MeOH to give two fractions (IV, V). Fraction V (15.00 g) was further subjected to a QCC and eluted with a stepwise gradient from 10% acetone in CH2Cl2 to acetone gave nine fractions (K–S). Fraction N (0.78 g) was fractionated by CC employing 20% acetone in hexane resulting in nine fractions (N1–N9). Fractions N3 (16.1 mg) and N5 (17.2 mg) were further purified by CC using 15% acetone in hexane to yield a light yellow solid 29 (2.0 mg) and yellow gum 30 (3.0 mg), respectively. Yellow gum 31 (2.0 mg) was obtained from fraction N8 (41.6 mg) by repeated CC with 50% CH2Cl2 in hexane. Fraction O (342.2 mg) was further fractionated by CC with 20% acetone in hexane to give ten fractions (O1–O10). Fractions O5 (24.3 mg) was further purified by preparative TLC using 15% acetone in hexane as an eluent to yield yellow gum 32 (2.0 mg). Yellow solid 33 (1.5 mg) was obtained from fraction O8 (53.8 mg) by repeated CC, eluted with CH2Cl2, and was further purified by Sephadex LH-20 column using MeOH as an eluent. Fraction Q (54.6 mg) was subjected to CC using isocratic of 5% acetone in CH2Cl2 to provide six fractions (Q1–Q6). Fraction Q5 (26.2 mg) was further purified by repeated CC using isocratic of 5% acetone in CH2Cl2 gave yellow gum 3 (1.5 mg). Fraction R (1.06 g) was further fractionated by CC with 7% acetone in CH2Cl2 to give nine fractions (R1–R9). Fraction R5 (24.3 mg) was further purified by repeated CC using 7% acetone in CH2Cl2 as an eluent to yield 34 (3.5 mg) as a light yellow solid. Yellow solid 35 (3.5 mg) was obtained from fraction R7 (48.7 mg) by repeated CC eluting with 10% acetone in CH2Cl2. Fraction S (1.00 g) was preabsorbed on C-18 and loaded into preparative guard column (30 mm × 10 mm i.d.), in line with a semiprep C18 HPLC column employing a gradient of H2O–MeOH resulting in five fractions (S1–S5). Fraction S1 (0.10 g) was further isolated by Diol HPLC and eluted with a gradient of 5–15% MeOH in CH2Cl2 gave light yellow solid 41 (2.5 mg). Fraction S3 (0.10 g) was further isolated by Diol HPLC and eluted with 100% CH2Cl2 to 15% MeOH in CH2Cl2 gave light yellow solid 37 (14.5 mg), 38 (6.5 mg) and 40 (26.3 mg). Fraction S4 (0.50 g) was further isolated by Diol HPLC and employing a gradient of 50% Hexane in CH2Cl2 to 15% MeOH in CH2Cl2 gave light yellow solid 36 (7.4 mg). Fraction S5 (0.40 g) was further isolated by Diol HPLC and employing a gradient of 90% Hexane in CH2Cl2 to 15% MeOH in CH2Cl2 gave light yellow solid 39 (4.2 mg). Dulcisxanthone J (1): yellow gum; UV (MeOH) λmax nm (log ε): 226 (3.46), 265 (3.70), 296 (3.58); IR (neat) νmax (cm−1): 3353, 1624; 1H NMR and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; HRESIMS (positive) m/z 417.1104 [M + Na]+ (calcd. for C23H22O6Na, 417.1314). Dulcisxanthone K (2): yellow gum; UV (MeOH) λmax nm (log ε): 255 (4.51), 280 (3.97), 328 (4.23); IR (neat) νmax (cm−1): 3415, 1638; 1H NMR and 13C NMR data (acetone-d6, 300 and 75 MHz), see Table 1; HRESIMS (positive) m/z 419.1471 [M + Na]+ (calcd. for C23H24O6Na, 419.1471). Dulcisxanthone L (3): yellow gum; UV (MeOH) λmax nm (log ε): 252 (4.23), 280 (3.79), 326 (3.97); IR (neat) νmax (cm−1): 3301, 1649; 1H
In conclusion, investigation of the active constituents as antibacterial agents from the stem bark of G. dulcis resulted in the isolation of 41 compounds including one isocoumarin, six biflavonoids and 34 xanthones. Three xanthones are new. The most effective antibacterial compound was a xanthone with isoprenyl side chain named 12bhydroxy-des-D-garcigerrin A (12) (MIC 4 μg/mL). The stem bark produced more prenylated xanthones than other plant parts of G.dulcis. 3. Experimental 3.1. General experimental procedures Crude extracts were obtained by sonication technique with S70H Elmasonic (Elma) at 35 °C. Ultraviolet spectra were recorded using a UV-160A spectrophotometer (SHIMADZU). The infrared spectra were obtained on a FTS165 FT-IR spectrometer (Perkin-Elmer). Nuclear Magnetic Resonance (NMR) spectra were recorded with a FT-NMR Ultra Shield™ 300 and 500 spectrometers (Bruker). Electrospray ionization mass spectrometric (ESIMS) data were determined on a Waters Alliance 2690 and a Micromass LCT (United Kingdom) mass spectrometers. Quick column chromatography (QCC) was performed on silica gel 60H (Merck). Column chromatography (CC) was performed on silica gel 100 (Merck) and Sephadex LH-20 (Amersham Biosciences, Sweden). Pre-coated TLC aluminum sheets (layer thickness 0.2 mm, Merck) and preparative TLC plates (layer thickness 1.25 mm, Merck) of silica gel 60 GF254 were used. Altech Davisil 30–40 μm 60 A° C18 silica gel or Altech Davisil 30–40 μm 60 A° diol silica was used to absorb the extract prior to HPLC separation. A Hitachi L7455 PDA detector (Merck) with a Hitachi L7100 pump (Merck) and a L7250 autosampler (Merck) were used for HPLC separations. HPLC columns used were Thermosystems betasil C18 5 μm, 21 mm × 150 mm, YMC Pack Diol NP 5 μm (21 mm × 150 mm) and phenyl bonded silica 5 μm (21 mm × 250 mm). The flow rate of mobile phase was constant at 9.0 mL/min. 3.2. Plant material The stem bark of G. dulcis was collected in Nakorn Si Thammarat province, the southern part of Thailand, and air-dried. The voucher specimen (Coll. No. 02, Herbarium No. 0012652) has been deposited in the Herbarium of Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation The chopped and air-dried stem bark of G. dulcis (1.5 kg) was extracted with CH2Cl2 (5 L) and acetone (5 L) in a sonicator bath for thirty minutes (2 times) and filtered. After removal of solvent, brown viscous CH2Cl2 extract (60.15 g) and acetone extract (44.30 g) were obtained. The CH2Cl2 extract was loaded onto Sephadex LH-20 and eluted with 100% MeOH to give three fractions (I–III). Fraction III (24.06 g) was further subjected to a QCC and eluted with a stepwise gradient from 10% acetone in hexane to pure acetone. The fractions with the same major components were combined on the basis of TLC analysis to give ten fractions (A–J). Fraction C (0.14 g) was separated by repeated CC using isocratic of 5% acetone in hexane to give a white solid 4 (2.0 mg), yellow solid 5 (2.0 mg) and 6 (5.0 mg). Fraction D (1.54 g) was chromatographed on CC using 15% acetone in hexane as an eluent to give ten fractions (D1–D10). Fraction D3 (25.1 mg) was rinsed with hexane to yield a yellow solid 7 (10.1 mg). Fraction D4 (40.8 mg) was separated by CC using 15% acetone in hexane to give five fractions (D4.1–D4.5). Fractions D4.2 and D4.4 were further purified by CC and preparative TLC plate using 15% acetone in hexane as eluent to give a light yellow gum 8 (2.5 mg), yellow solid 9 (3.0 mg), 10 (3.4 mg) and 11 (4.0 mg). Fractions D6 (252.1 mg) and D7 (35.1 mg) were rinsed with hexane to afford 12 (35.3 mg) as orange 36
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NMR and 13C NMR data (acetone-d6, 500 and 125 MHz), see Table 1; HRESIMS (positive) m/z 369.0956 [M + Na]+ (calcd. for C18H18O7Na, 369.0950).
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3.4. Antibacterial assay This was carried out according to the previously reported procedure of Supaphon et al., 2013. Acknowledgements This work was supported by the Thailand Research Fund (TRF): Senior Research Scholar (Grant No. RTA5880005). We thank the Graduate School, Prince of Songkla University for a partial research grant and a scholarship for support exchange students and international credit transferred through ASEAN community. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.05.014. References Asai, F., Tosa, H., Tanaka, T., Iinuma, M., 1995. A xanthone from pericarps of Garcinia mangostana. Phytochemistry 39, 943–944. Chawla, H.M., Chibber, S.S., Khera, U., 1975. Separation and identification of some hydroxyxanthones by thin-layer chromatography. J. Chromatogr. A. 111, 246–247. Chen, Y., Zhong, F., He, H., Hu, Y., Zhu, D., Yang, G., 2008. Structure elucidation and NMR spectral assignment of five new xanthones from the bark of Garcinia xanthochymus. Magn. Reson. Chem. 46, 1180–1184. Chen, Y., Fan, H., Yang, G.Z., Jiang, Y., Zhong, F.F., He, H.W., 2010. Prenylated xanthones from the bark of Garcinia xanthochymus and their 1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging activities. Molecules 15, 7438–7449. Cotterill, P.J., Scheinmann, F., 1975. A revised structure for toxyloxanthone. B. J. Chem. Soc. Chem. Commun. 16, 664–665. Deachathai, S., Mahabusarakam, W., Phongpaichit, S., Taylor, W.C., 2005. Phenolic compounds from the fruit of Garcinia dulcis. Phytochemistry 66, 2368–2375. Deachathai, S., Mahabusarakam, W., Phongpaichit, S., Taylor, W.C., Zhang, Y.J., Yang, C.R., 2006. Phenolic compounds from the flowers of Garcinia dulcis. Phytochemistry 67, 464–469. Deachathai, S., Phongpaichit, S., Mahabusarakam, W., 2008. Phenolic compounds from the seeds of Garcinia dulcis. Nat. Prod. Res. 22, 1327–1332. Fukuyama, Y., Kamiyama, A., Mima, Y., Kodama, M., 1991. Prenylated xanthones from Garcinia subelliptica. Phytochemistry 30, 3433–3436. Iinuma, M., Tosa, H., Tanaka, T., Shimano, R., Asai, F., Yonemori, S., 1994. Two xanthones from root bark of Garcinia subelliptica. Phytochemistry 35, 1355–1360. Iinuma, M., Tosa, H., Tanaka, T., Asai, F., Shimano, R., 1995a. Two xanthones with a 1,1dimethylallyl group in root bark of Garcinia subelliptica. Phytochemistry 39, 945–947. Iinuma, M., Tosa, H., Tanaka, T., Asai, F., Shimano, R., 1995b. Three xanthones from root bark of Garcinia subelliptica. Phytochemistry 38, 247–249. Iinuma, M., Tosa, H., Tanaka, T., Asai, F., Shimano, R., 1995c. Two new xanthones from the root bark of Garcinia subelliptica. Heterocycles 40, 279–284. Iinuma, M., Tosa, H., Ito, T., Tanaka, T., Riswan, S., 1996. Three new benzophenone-
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