Bioactive Prenylated Xanthones from the Young Fruits and Flowers of

Feb 4, 2015 - from the young fruits and fresh flowers of Garcinia cowa. The ... bioactive metabolites from Garcinia plants,9,12−14 five new xanthone...
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Bioactive Prenylated Xanthones from the Young Fruits and Flowers of Garcinia cowa Teerayut Sriyatep,† Ittipon Siridechakorn,† Wisanu Maneerat,† Acharavadee Pansanit,† Thunwadee Ritthiwigrom,‡ Raymond J. Andersen,§ and Surat Laphookhieo*,† †

Natural Products Research Laboratory, School of 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 ‡

S Supporting Information *

ABSTRACT: Five new xanthones, garciniacowones A−E (1− 5), together with 14 known xanthones, 6−19, were isolated from the young fruits and fresh flowers of Garcinia cowa. The structures of 1−5 were elucidated by analysis of their 1D and 2D NMR spectra and mass spectrometric data. The compounds 1−19 were tested in vitro for their antimicrobial activity and for their ability to inhibit α-glucosidase. Compounds 16 and 17 showed the most potent α-glucosidase inhibitory activity, with IC50 values of 7.8 ± 0.5 and 8.7 ± 0.3 μM, respectively. Compounds 8, 9, and 19 showed antibacterial activity against Bacillus subtilis TISTR 088 with identical MIC values of 2 μg/mL, while 8, 10, and 19 exhibited antibacterial activity against Bacillus cereus TISTR 688 with identical MIC values of 4 μg/mL. (9).16 The EtOAc-soluble portion of a concentrated MeOH extract of macerated fresh G. cowa flowers was subjected to column chromatography over Si gel to give two new xanthones, garciniacowones D (4) and E (5), together with 10 known xanthones, mangostanin (10), 6-O-methylmangostanin (11),17 fuscaxanthone A (12), fuscaxanthone C (13),18 7-O-methylgarcinone E (14),2 cowaxanthone D (15),6 α-mangostin (16), β-mangostin (17),19 3,6-di-O-methyl-γ-mangostin (18),12 and rubraxanthone (19).20 Garciniacowone A (1) was obtained as a yellow, viscous oil that gave a [M + Na]+ ion at m/z 555.2724 in the ESITOFMS, consistent with a molecular formula of C33H40O6 (calcd for C33H40NaO6, m/z 555.2723). Its UV spectrum revealed absorption bands at λmax 223, 244, 311, and 425 nm,16 while the IR spectrum showed an absorption at 3400 cm−1, indicating the presence of a hydroxy group and absorptions at 1667 and 1653 cm−1, assignable to a conjugated carbonyl group.16 The 1 H and 13C NMR data of 1 (Table 1) showed resonances characteristic of a xanthone framework similar to that of garcinianone A (8), which was isolated previously from G. multif lora.16 These included a chelated hydroxy group (δH 13.25, 1H, s, OH-1), two meta-coupled aromatic protons [δH 6.29 (1H, d, J = 2.0 Hz, H-2)/δC 99.5 and δH 6.34 (1H, d, J = 2.0 Hz, H-4)/δC 93.6], and one singlet olefinic proton [δH 6.47

Garcinia cowa Roxb. belongs to the plant family Clusiaceae, which is distributed in tropical and subtropical countries of Southeast Asia, West Africa, and East Africa. The edible fruits and young leaves of G. cowa are used as vegetables, whereas the roots, barks, and latex have been used in traditional antipyretic medicines for the treatment of fever.1 Previous phyotchemical investigations of different parts of this plant have resulted in the isolation and identification of many bioactive xanthones and benzophenones.2−11 As a continuation of studies on the bioactive metabolites from Garcinia plants,9,12−14 five new xanthones, garciniacowones A−E (1−5), and 14 known compounds (6−19) were isolated from the young fruits and flowers of G. cowa. The α-glucosidase inhibitory properties and antibacterial activity against Bacillus cereus TISTR 688, Bacillus subtilis TISTR 008, Staphylococcus aureus TISTR 1466, Escherichia coli TISTR 780, Salmonella typhimurium TISTR 292, and Pseudomonas aeruginosa TISTR 884 of compounds 1− 19 were evaluated.

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RESULTS AND DISCUSSION The concentrated MeOH extract of the air-dried young fruits harvested from G. cowa was suspended in H2O and then extracted sequentially with CH2Cl2 and EtOAc to give CH2Cl2and EtOAc-soluble fractions, respectively. The CH2Cl2-soluble material was subjected to column chromatography over Si gel to give three new xanthones, garciniacowones A−C (1−3), and four known xanthones, cowaxanthone (6),1 3-O-methylmangostenone D (7),15 garcinianone A (8), and garcinianone B © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 27, 2014

A

DOI: 10.1021/np5008476 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

configurations. 13C NMR signals assigned to C-14 and C-20 had significantly different chemical shifts in the geranyl units of 8 (δC 39.6, C-14; 16.2, C-20) and 9 (δC 31.9, C-14; 23.3, C20), which had Δ12,13 E and Z configurations, respectively.16 Thus, the structure of garciniacowone A was established as 1. 1 H and 13C NMR assignments and a listing of the major HMBC correlations observed for 1 are shown in Table 1. Garciniacowone B (2) was obtained as a yellow, viscous oil that gave a [M]+ ion at m/z 532.2820 in the HREIMS, consistent with a molecular formula of C33H40O6 (calcd for C33H40O6, m/z 532.2819). Its UV and IR spectra were similar to those of compound 1. The 1H and 13C NMR data (Table 1) recorded for compound 2 were also closely related to the data obtained for 1. The main difference was that the 1H and 13C NMR signals assigned to the gem-geranyl units of compound 2 were found to be split into two distinct sets. A set of NMR resonances assigned to the first geranyl fragment was found at δH/δC 2.80 (1H, m, H-11a), 3.45 (1H, m, H-11b)/37.6, 4.63 (1H, m, H-12)/118.2, 139.1 (C-13), 1.92 (1H, m, H-14)/31.9, 1.87 (1H, m, H-15)/26.5, 5.04 (1H, m, H-16)/124.1, 131.7 (C17), 1.68 (3H, s, H-18)/25.7, and 1.58 (3H, s, H-19)/17.6, 1.49 (3H, s, H-20)/23.3. The configuration of the Δ12,13 alkene in this fragment was assigned as Z on the basis of NOESY correlations observed between H-11 (δH 2.80 and 3.45) and H14 (δH 1.92) and between H-12 (δH 4.63) and H-20 (δH 1.49) (Figure 2). In addition, the 13C NMR chemical shifts of C-14 (31.9) and C-20 (δC 23.3) in compound 2 were similar to those reported for garcinianone B (9)16 (the chemical shifts of C-14

(1H, s, H-5)/δC 108.5]. HMBC correlations observed between H-2 (δH 6.29) and C-1 (δC 162.9), C-4 (δC 93.6), and C-9a (δC 105.1); between H-4 (δH 6.34) and C-2 (δC 99.5), C-3 (δC 161.8), C-4a (δC 156.8), and C-9a (δC 105.1); and between H5 (δH 6.47) and C-6 (δC 151.9), C-7 (δC 201.4), C-8a (δC 116.9), C-9 (δC 179.3), and C-10a (δC 159.3) were consistent with this core structure (Table 1 and Figure 1). The main difference in the NMR data of compounds 1 and garcinianone A16 was that the spectra of compound 1 showed NMR resonances that could be assigned to a pair of geminal geranyl units [δH/δC 2.81 and 3.46 (4H, dd, J = 13.6, 7.6 Hz, H-11a, H21a, H-11b, and H-21b/37.8, 4.66 (2H, t, J = 7.6 Hz, H-12, 22)/117. 7, 1.83 (4H, m, H-14, 24)/39.6, 1.79 (4H, m, H-15, 25)/26.5, 4.86 (2H, t, J = 6.8 Hz, H-16, 26)/123.8, 1.60 (6H, s, H-18, 28)/25.5, and 1.49 (12H, s, H-19, H-29/17.7 and H-20, 30/16.2), δC 139.0 (C-13 and C-23), and δC 131.4 (C-17 and C-27)], instead of the resonances assigned to the geminal prenyl and geranyl groups in garcinianone A.16 HMBC correlations observed between the methylene protons H-11/ H-21 (δH 2.81 and 3.46) and C-8 (δC 56.0) and C-7 (δC 201.4) confirmed the attachment of the gem-digeranyl groups at C-8 (Figure 1). The configurations of the Δ12,13 and Δ22,23 alkene units were assigned as E on the basis of NOESY correlations observed between H-11/H-20 and H-21/H-30 and between H12/H-14 and H-22/H-24 (Figure 2). Comparison of 13C NMR chemical data for 1 with those of the previously reported analogues garcinianone A (8) and garcinianone B (9) confirmed the assignments of the Δ12,13 and Δ22,23 alkene B

DOI: 10.1021/np5008476 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR (400 MHz) Data of Garciniacowones A (1) and B (2) in CDCl3 1 position 1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11a 11b 12 13 14 15 16 17 18 19 20 21a 21b 22 23 24 25 26 27 28 29 30 OH-1 OH-3 OH-6

δC, type

2

δH, (J in Hz)

162.9 99.5 161.8 93.6 156.8 108.5 151.9 201.4 56.0 116.9 179.3 105.1 159.3 37.8

C CH C CH C CH C C C C C C C CH2

117.7 139.0 39.6 26.5 123.8 131.4 25.5 17.7 16.2 37.8

CH C CH2 CH2 CH C CH3 CH3 CH3 CH2

117.7 139.0 39.6 26.5 123.8 131.4 25.5 17.7 16.2

CH C CH2 CH2 CH C CH3 CH3 CH3

6.29 d (2.0)

1, 4, 9a

6.34 d (2.0)

2, 3, 4a, 9a

6.47 s

6, 7, 8a, 9, 10a

2.81 dd (13.6, 7.6) 3.46 dd (13.6, 7.6) 4.66 t (7.6)

7, 8, 12, 13, 21 7, 8, 12, 13, 21 8, 11, 14, 20

1.83 m 1.79 m 4.86 t (6.8)

12, 13, 15, 16, 20 14, 16, 17 14, 18, 19

1.49 1.60 1.49 2.81 3.46 4.66

16, 17, 19 16, 17, 18 12, 13, 14 7, 8, 11, 22, 23 7, 8, 11, 22, 23 8, 21, 24, 30

s s s dd (13.6, 7.6) dd (13.6, 7.6) t (7.6)

δC, type

HMBC

1.83 m 1.79 m 4.86 t (6.8)

22, 23, 25, 26, 30 24, 26, 27 24, 28, 29

1.49 s 1.60 s 1.49 s 13.25 s

26, 27, 29 26, 27, 28 22, 23, 24 1, 2, 9a

6.99 s

5, 6, 7

δH, (J in Hz)

163.0 99.5 161.8 93.6 156.8 108.5 151.8 201.4 55.9 116.9 179.6 105.1 159.3 37.6

C CH C CH C CH C C C C C C C CH2

118.2 139.1 31.9 26.5 124.1 131.7 25.7 17.6 23.3 38.1

CH C CH2 CH2 CH C CH3 CH3 CH3 CH2

117.6 139.1 39.6 26.5 123.8 131.4 25.7 17.6 16.2

CH C CH2 CH2 CH C CH3 CH3 CH3

HMBC

6.29 m

1, 4, 9a

6.34 m

2, 3, 4a, 9a

6.49 s

6, 7, 8a, 10a

2.80 m 3.45 m 4.63 m

7, 8, 12, 13 7, 8, 12, 13 8, 14, 20

1.92 m 1.87 m 5.04 m

12, 13, 15 17 15

1.68 1.58 1.49 2.80 3.45 4.63

16, 17, 19 16, 17, 18 12, 13, 14 7, 8, 22, 23 7, 8, 22, 23 8, 24, 30

s s s m m m

1.82 m 1.79 m 4.86 m

24, 27 28, 29

1.49 s 1.60 s 1.46 s 13.22 s

26, 27, 29 26, 27, 28 22, 23, 24 1, 2, 9a

6.99 brs

5, 6, 7

absorption bands at λmax 219, 289, 334, and 368 nm,17,18 while the IR spectrum showed absorption bands at 3344 and 1650 cm−1 assigned to a hydroxy group and a conjugated carbonyl group,17,18 respectively. The 1H and 13C NMR data (Table 2) of 3 showed the characteristics of a xanthone with one chelated hydroxy group proton (δH 13.30, 1H, s, OH-1), a free hydroxy proton (δH 6.37, 1H, brs, OH-6), three singlet aromatic protons [δH 6.31 (1H, s, H-4)/δC 94.7, 6.94 (1H, s, H-5)/δC 102.7 and 7.57 (1H, s, H-8)/δC 104.5], and one methoxy group (δH 4.01, 3H, s, OMe-7)/δC 56.5). The location of the methoxy group at C-7 was indicated by a cross-peak in the NOESY spectrum between the OMe resonance at δH 4.01 and the H-8 aromatic resonance at δH 7.57. In addition, compound 3 also displayed NMR signals at δH/δC 6.77 (1H, d, J = 10.4 Hz, H-11)/116.0, 5.53 (1H, d, J = 10.4 Hz, H-12)/126.3, 5.09 (1H, m, H-16)/ 123.8, 2.10 (2H, m, H-15)/22.7, 1.80 (2H, m, H-14)/41.7, 1.65 (3H, s, H-18)/25.7, 1.57 (3H, s, H-19)/17.7, and 1.44 (3H, s, H-20)/27.2 that could be assigned to a 2-methyl-2-(4methylpent-3-enyl)pyran moiety found in the known cooccurring compounds 10−12.17,18 This pyran unit was placed

and C-20 in 9 were δC 31.9 and 23.3, respectively). A set of NMR resonances assigned to the second geranyl fragment appeared at δH/δC 2.80 (1H, m, H-21a), 3.45 (1H, m, H-21b)/ 38.1, 4.63 (1H, m, H-22)/117.6, 139.1 (C-23), 1.82 (1H, m, H24)/39.6, 1.79 (1H, m, H-25)/26.5, 4.86 (1H, m, H-26)/123.8, 131.4 (C-27), 1.49 (3H, s, H-28)/25.7, 1.60 (3H, s, H-29)/ 17.6, and 1.46 (3H, s, H-30)/16.2. The 1H and 13C NMR shifts assigned to this second geranyl fragment in 2 were nearly identical to the assignments for the geranyl fragments in 1 (Table 1), confirming that the Δ22,23 alkene in 2 also has the E configuration.16 Correlations between H-22 and H-24 and between H-21 and H-30 in the NOESY spectrum confirmed the E configuration of the Δ22,23 alkene (Figure 2). Thus, the structure of garciniacowone B was assigned as 2. 1H and 13C NMR assignments and a listing of the major HMBC correlations observed for this compound are shown in Table 1. Garciniacowone C (3) was obtained as a yellow, viscous oil that gave a [M + Na]+ ion at m/z 431.1473 in the ESITOFMS, corresponding to the molecular formula C24H24O6 (calcd for C24H24NaO6, m/z 431.1471). Its UV spectrum contained C

DOI: 10.1021/np5008476 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. NMR (400 MHz) Data of Garciniacowones C−E (3−5) in CDCl3 3a position 1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 OH-1 OH-6 OH-7 OMe-6 OMe-7 a

δC, type 160.1 103.0 160.0 94.7 157.4 102.7 152.4 144.0 104.5 113.3 179.8 103.0 152.6 116.0 126.3 80.5 41.7 22.7 123.8 131.8 25.7 17.7 27.2

4b δH, (J in Hz)

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

6.31 s 6.94 s

7.57 s

6.77 d (10.4) 5.53 d (10.4) 1.80 m 2.10 m 5.09 m 1.65 s 1.57 s 1.44 s

δC, type 157.9 103.5 156.9 93.9 151.2 102.8 150.7 136.5 119.4 108.2 182.6 102.2 152.2 115.7 127.3 77.6 28.5 28.5 121.6 131.6 79.7 40.4 22.6 123.5 132.4 25.6 17.6 25.8

δH, (J in Hz) C C C CH C CH C C C C C C C CH CH C CH3 CH3 CH CH C CH2 CH2 CH C CH3 CH3 CH3

13.30 s 6.37 brs

5b

6.26 s 6.82 s

6.72 d (10.0) 5.57 d (10.0) 1.46 1.46 8.07 5.79

s s d (10.4) d (10.4)

1.80 m 2.14 m 5.08 br t (7.0) 1.66 s 1.58 s 1.44 s 13.65 s 6.21 s

δC, type 157.9 104.3 159.5 93.8 156.2 97.1 151.6 140.3 127.0 112.2 182.3 103.9 152.7 115.7 127.2 77.8 28.2 28.2 25.5 122.4 132.4 18.0 25.9

δH, (J in Hz) C C C CH C CH C C C C C C C CH CH C CH3 CH3 CH2 CH C CH3 CH3

6.23 s 6.75 s

6.73 d (10.0) 5.56 d (10.0) 1.45 1.45 4.15 5.29

s s d (7.0) brt (7.0)

1.84 s 1.68 s

13.84 s 5.66 s 3.99 s

56.5

CH3

4.01 s

Recorded at 400 MHz. bRecorded at 500 MHz.

at C-2/C-3 because of the 2J and 3J HMBC correlations observed between H-11 (δH 6.77) and both C-2 (δC 103.0) and C-3 (δC 160.0) and of H-12 (δH 5.53) with C-2 (δC 103.0) and C-14 (δC 41.7) (Table 2 and Figure 1). Thus, the structure of garciniacowone C was assigned as 3. The detailed assignments of the 1H, 13C, and HMBC NMR data are shown in Table 2. Garciniacowone D (4) was obtained as a yellow, viscous oil that gave a [M]+ peak at m/z 460.1889 in the HREIMS, consistent with a molecular formula of C28H28O6 (calcd for C28H28O6, m/z 460.1880). Its UV and IR spectra were similar to those of compound 3, indicating the presence of a xanthone skeleton. The 1H NMR spectra of 4 (Table 2) displayed a chelated hydroxy group (δH 13.65, 1H, s, OH-1), two singlet aromatic protons [δH 6.82 (1H, s, H-5) and 6.26 (1H, s, H-4)], a group of resonances assigned to a 2-methyl-2-(4-methylpent3-enyl)pyran moiety [δH 8.07 (1H, d, J = 10.4 Hz, H-16), 5.79 (1H, d, J = 10.4 Hz, H-17), 5.08 (1H, brt, J = 7.0 Hz, H-21), 1.80 (2H, m, H-19), 2.14 (2H, m, H-20), 1.66 (3H, s, H-23), 1.58 (3H, s, H-24), and 1.44 (3H, s, H-25)], and a group of resonances assigned to a 2,2-dimethylpyran fragment [δH 6.72 (1H, d, J = 10.0 Hz, H-11), 5.57 (1H, d, J = 10.0 Hz, H-12), and 1.46 (6H, s, H-14 and 15)]. The 2-methyl-2-(4methylpent-3-enyl)pyran moiety was situated at C-7/C-8 due to 2J and 3J HMBC correlations observed between H-16 (δH

8.07) and both C-7 (δC 136.5) and C-8 (δC 119.4), whereas the 2,2-dimethylpyran ring [δH 6.72 (1H, d, J = 10.0 Hz, H-11), 5.57 (1H, d, J = 10.0 Hz, H-12), and 1.46 (6H, s, H-14 and 15)] was situated at C-2/C-3 by the observation of an HMBC correlation between H-11 and C-2 (δC 103.5). Therefore, garciniacowone D was assigned the structure 4. The complete NMR assignments of this compound are shown in Table 2. Garciniacowone E (5) was obtained as a yellow solid (mp 157−158.0 °C) that showed a [M]+ ion at m/z 408.1561 in the HREIMS, consistent with a molecular formula of C24H24O6 (calcd for C24H24O6, m/z 408.1567). Its UV and IR spectra were similar to those of compounds 3 and 4, indicating again the presence of a xanthone skeleton. The 1H NMR data were similar to the data for 4 except that the spectra for 5 showed resonances for methoxy (δH 3.99) and prenyl groups [δH 5.29 (1H, br t, J = 7.0 Hz, H-17), 4.15 (2H, d, J = 7.0 Hz, H-16), 1.84 (3H, s, H-19), and 1.68 (3H, s, H-20)] instead of resonances for a 2-methyl-2-(4-methylpent-3-enyl)pyran moiety. The prenyl group was located at C-8 due to the observation of 2J and 3J HMBC correlations between H-16 and C-7 (δC 140.3) and C-8a (δC 112.2), respectively, while the methoxy group (δH 3.99) was placed at C-6, because the OMe signal showed a cross-peak with C-6 (δC 151.6) in the HMBC spectrum. A cross-peak between the OMe (δH 3.99) and H-5 D

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(δH 6.75) resonanaces in the NOESY spectrum provided further support for the location of the methoxy group at C-6. Therefore, garciniacowone E was assigned the structure 5. Complete assignments of the NMR data for compound 5 are shown in Table 2. α-Glucosidase inhibitors are widely used for the treatment of diabetes mellitus type 2, which work by controlling the release of blood glucose by the enzyme α-glucosidase. These inhibitors reduce the fast breakdown of carbohydrates and thereby control blood sugar level. Compounds 1−19 were evaluated for their ability to inhibit α-glucosidase in vitro (Table 3). Ten (1, Table 3. α-Glucosidase Inhibitory Activities of Compounds 1−19 compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 acarbose

% inhibition at 100 μM 62.9 75.8 >100 >100 >100 >100 >100 66.7 69.1 70.1 62.2 71.7 53.8 >100 50.8 72.0 73.1 56.4 71.2

± 4.8 ± 6.8

IC50 (μM) 36.4 ± 0.5 55.3 ± 0.2

Figure 1. Selected HMBC correlations of garciniacowones A−E (1− 5).

± ± ± ± ± ±

8.6 10.0 9.9 8.1 6.1 21.2

± ± ± ± ±

24.4 3.5 4.8 6.6 1.8

34.0 11.7 34.6 26.2 18.6

± ± ± ± ±

0.8 0.1 0.2 0.9 0.3

Figure 2. Key NOESY correlations of 1 and 2.

7.8 ± 0.5 8.7 ± 0.3

(9), and less than five compounds of this type have been isolated from this genus to date. To the best of our knowledge, garciniacowone A (1) is the first example of a xanthone derivative containing gem-(E)-3,7-dimethylocta-2,6-dienyl (geranyl) units at C-8. The presence of (E)-3,7-dimethylocta-2,6dienyl and (Z)-3,7-dimethylocta-2,6-dienyl units at C-8 in garciniacowone B (2) is also the first example of this structural variation from Garcinia. Preliminary bioassay results obtained suggested that the xanthones 16 and 17 may have potential as lead compounds for development of antidiabetic agents, and xanthones 8−10 and 19 may be good candidates for further evaluation as new antibacterial agents.

23.4 ± 0.9 8.0 ± 1.7

2, 8−12, 16, 17, and 19) showed greater than 60% inhibition of α-glucosidase activity at a concentration of 100 μM, and their IC50 data are given in Table 3. Compounds 16 and 17 showed the most potent α-glucosidase inhibition with IC50 values of 7.8 ± 0.5 and 8.7 ± 0.3 μM, respectively, which is equivalent to the reference standard acarbose (8.0 ± 1.7 μM), a drug used clinically to treat type-2 diabetes. The other compounds tested were less effective α-glucosidase inhibitors, with IC50 values ranging from 11.7 ± 0.1 to 55.3 ± 0.2 μM. Compounds 1−19 were also evaluated for antibacterial activity (Table 4) against the Gram-positive bacteria S. aureus TISTR 1466, B. cereus TISTR 688, and B. subtilis TISTR 088 and the Gram-negative bacteria E. coli TISTR 780, P. aeruginosa, and S. typhimurium. Compounds 1, 2, 8−10, 16, 17, and 19 showed antibacterial activity against B. subtilis TISTR 088, with MIC values ranging from 2 to 8 μg/mL, and of these compounds 8, 9, and 19 exhibited the most potent activity (2 μg/mL). Compounds 8, 10, and 19 also demonstrated antibacterial activity against B. cereus TISTR 688, with all having the same MIC value of 4 μg/mL. The remaining compounds were found to have very weak (MIC 32−128 μg/mL) or no antibacterial activity against the test strains. In summary, while Garcinia species are rich sources of xanthones, they rarely produce the geranylated xanthone derivative core structure found in garcinianones A (8) and B

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured with a Buchi melting point B-540. The optical rotation [α]D values were measured with a Bellingham & Stanley APD440 polarimeter. The UV spectra were recorded with a PerkinElmer UV− vis spectrophotometer, whereas the IR spectra were obtained using a PerkinElmer FTS FT-IR spectrophotometer. NMR spectroscopic data were obtained on a 400 MHz Bruker FT-NMR Ultra Shield and a 500 MHz Varian Unity INOVA spectrometer. Chemical shifts are recorded in parts per million (δ) in CDCl3 (δH 7.24 and δC 77.0 ppm) and/or acetone-d6 (δH 2.05 and δC (CO) 206.2 and (CH3) 30.6 ppm), with TMS as an internal reference. TOFESIMS data were measured from a MicroTOF, Bruker Daltonics mass spectrometer, and HREIMS were obtained on a MAT 95 XL mass spectrometer. Quick column chromatography (QCC) and column chromatography (CC) were carried out on Si gel 60 H (Merck, 5−40 μm) and Si gel 100 (Merck, 63−200 μm), respectively. Sephadex LH-20 was also used for CC. Precoated plates of Si gel 60 F254 were used for analytical purposes. E

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Table 4. Antibacterial Activity of Compounds 1−19 MIC (μg/mL)a

a

compound

B. cereus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 gentamycin vancomycin

8 8 32 64 128 64 128 4 2 4 128 64 inactive 32 128 8 16 128 4 not tested 0.25

B. subtilis

S. aureus

E. coli

S. typhimurium

P. aeruginosa

8 8 64 128 128 64

inactive 64 inactive inactive inactive inactive 128 64 64 64 inactive 128 128 inactive 128 64 inactive inactive inactive not tested 0.25

inactive 64 inactive inactive inactive inactive inactive 64 64 inactive inactive 64 64 inactive inactive 64 inactive inactive 64 0.25 not tested

64 64 128 64 128 128 128 64 64 128 64 128 128 128 128 64 128 128 64 0.125 not tested

128 128 128 128 128 128 inactive 128 128 128 128 128 128 128 128 128 128 128 64 2 not tested

2 2 8 128 64 128 32 128 8 4 128 2 not tested 0.25

Data shown are from triplicate experiments and inactive at >128 μg/mL. CH2Cl2−hexanes yielded compounds 12 (21.4 mg) and 14 (2.2 mg). Compounds 5 (23.1 mg) and 10 (171.7 mg) along with three subfractions (D6A−D6C) were isolated from subfraction D6 (728.9 mg) by CC with 60% CH2Cl2−hexanes. Subfraction D6B (13.9 mg) was further purified by CC with 50% CH2Cl2−hexanes to give compound 15 (2.3 mg). Fraction E (1.65 g) was fractionated by Sephadex LH-20 with 100% MeOH to afford two subfractions (E1 and E2). Compounds 18 (2.6 mg) and 17 (4.1 mg) were obtained from subfraction E2 (52.6 mg) by CC with 50% CH2Cl2−hexanes. Fraction F (395.3 mg) was purified further by Sephadex LH-20 with 100% MeOH to yield compound 11 (2.8 mg). Fraction H (2.23 g) was subjected to QCC and eluted with a gradient of hexanes−acetone (100% hexanes to 100% acetone) to give five subfractions (H1−H5). Compound 16 (22.5 mg) was isolated from subfraction H3 (150.2 mg), whereas compound 19 (3.0) was purified from subfraction H4 (146.0 mg) by CC with 20% acetone−hexanes. Finally, fraction I (2.91g) was purified by QCC with a gradient of CH2Cl2−acetone (100% CH2Cl2 to 100% acetone) to give six subfractions (I1−I6), and compound 4 (1.2 mg) was isolated from subfraction I3 (128.1 mg) by CC with 2% MeOH−CH2Cl2. Garciniacowone A (1): yellow, viscous oil; [α]25D 0 (c 0.034, CHCl3); UV (MeOH) λmax (log ε) 223 (4.25), 244 (4.16), 311 (4.11), 425 (3.94) nm; IR (neat) νmax 3400, 1667, 1653 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR data, see Table 1; ESITOFMS m/z 555.2724 [M + Na]+, calcd for C33H40NaO6, 555.2723. Garciniacowone B (2): yellow, viscous oil; [α]25D 0 (c 0.021, CHCl3); UV (MeOH) λmax (log ε) 223 (4.49), 243 (4.35), 312 (4.29), 422 (4.12) nm; IR (neat) νmax 3389, 1667, 1647 cm−1; 1H and 13C NMR data, see Table 1; HREIMS m/z 532.2820 [M]+, calcd for C33H40O6, 532.2819. Garciniacowone C (3): yellow, viscous oil; [α]23D +38.2 (c 0.020, CHCl3); UV (MeOH) λmax (log ε) 219 (6.11), 289 (6.36), 334 (5.99), 368 (5.82) nm; IR (neat) νmax 3344, 1650 cm−1; 1H and 13C NMR data, see Table 2; ESITOFMS m/z 431.1473 [M + Na]+, calcd for C24H24NaO6, 431.1471. Garciniacowone D (4): yellow, viscous oil; [α]23D −27.7 (c 0.010, CHCl3); UV (MeOH) λmax (log ε) 219 (4.00), 289 (3.91), 334 (3.60), 395 (299) nm; IR (neat) νmax 3398, 1647 cm−1; 1H and 13C NMR data, see Table 2; HREIMS m/z 460.1889 [M]+, calcd for C28H28O6, 460.1880.

Plant Material. The young fruits of Garcinia cowa were collected from Khokkhon, Tha Bo District, Nong Khai Province, Thailand, in August 2011, whereas the flowers of G. cowa were collected from Pakook, Muang District, Chiang Rai Province, Thailand, in February 2013. The plant was identified by Mr. James Maxwell, of the Herbarium of Chiang Mai University. Voucher specimens (MFUNPR0014 and MFU-NPR0071) were deposited at Natural Products Research Laboratory, School of Science, Mae Fah Luang University. Extraction and Isolation. Air-dried young fruits of G. cowa (5.27 kg) were extracted with MeOH for 3 days at room temperature and evaporated under reduced pressure to give a crude extract (532.1 g), which was partitioned with CH2Cl2 and EtOAc. The CH2Cl2 portion (115.0 g) was subjected to QCC over silica gel using a gradient of hexanes and EtOAc (100% hexanes to 100% EtOAc) to give 24 fractions (F1−F24). Fraction F11 was separated by CC, eluting with 60% CH2Cl2−hexanes, to afford nine subfractions (F11A−F11I). Subfraction F11H was purified further by CC with 50% CH2Cl2− hexanes to give compound 7 (1.4 mg). Fraction F15 was separated by Sephadex LH-20 with 30% CH2Cl2−MeOH to afford three subfractions (F15A−F15C). Subfraction F15B (200 mg) was purified further by CC with 1% MeOH−CH2Cl2 to give compound 6 (19.7 mg) and three subfractions, F15B1−F15B3. Compound 3 (4.0 mg) was obtained from subfraction F15B1 (30 mg) by CC with 30% EtOAc−hexanes. Fraction F19 was purified by Sephadex LH-20 with 100% MeOH to afford two subfractions (F19A and F19B). Subfraction F19A (200 mg) was separated by CC with 20% EtOAc−hexanes to give compounds 1 (5.9 mg), 2 (3.4 mg), 8 (15.8 mg), and 9 (11.9 mg). The fresh flowers of G. cowa (1.8 kg) were macerated for 3 days at room temperature with MeOH (3 × 5 L); then the solvent was removed under reduced pressure and extracted with hexanes, CH2Cl2, and EtOAc, respectively. All crude extracts were combined (25.0 g) and subjected to QCC over silica gel and eluted with a gradient of hexanes−EtOAc (100% hexanes to 100% EtOAc) to give nine fractions (A−I). Fraction B (1.4 g) was further purified by Sephadex LH-20 with 100% MeOH to afford three subfractions (B1−B3). Compound 13 (5.0 mg) was obtained from subfraction B3 by CC eluted with 10% acetone−hexanes. Fraction D (2.1 g) was isolated by Sephadex LH-20 with 100% MeOH to obtain six subfractions (D1− D6). Purification of subfraction D5 (222.3 mg) by CC with 40% F

DOI: 10.1021/np5008476 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Garciniacowone E (5): yellow, solid; mp 157−158.0 °C; UV (MeOH) λmax (log ε) 240 (3.78), 291 (4.37), 335 (3.66), 376 (3.30) nm; IR (neat) νmax 3406, 1641 cm−1; 1H and 13C NMR data, see Table 2; HREIMS m/z 408.1561 [M]+ calcd for C24H24O6, 408.1567. α-Glucosidase Assay. A colorimetric α-glucosidase (Sigma, St. Louis, USA, cat. no. G5003) assay was performed according to the method described in the literature.22−26 The tested compounds and a substrate, p-nitrophenyl α-D-glucoside (Sigma, St. Louis, USA; cat. no. N1377), were dissolved in DMSO and were further diluted with 50 mM K2HPO4−KH2PO4 buffer to make the final concentration of DMSO 10%. The mixture of 20 mL of enzyme solution (0.4 U/mL) and 20 mL of the test compound was preincubated at 37 °C for 0.5 h. The enzymatic reaction was started by the addition of 10 mL of substrate (3 mM), and the mixture was further incubated at 37 °C for 1 h. The enzyme activity was terminated by adding 1 mL of 0.1 M Na2CO3, and the absorption at 405 nm was measured immediately and used as the relative rate for the substrate hydrolysis. Acarbose was used as a positive control with an IC50 value of 8.0 ± 1.7 μM. Antibacterial Assays. Bacillus cereus TISTR 688, Bacillus subtilis TISTR 008, Staphylococcus aureus TISTR 1466, Escherichia coli TISTR 780, Salmonella typhimurium TISTR 292, and Pseudomonas aeruginosa TISTR 884 were obtained from the Microbiological Resources Center of the Thailand Institute of Scientific and Technological Research. The minimum inhibitory concentrations (MICs) were determined by a 2fold serial dilution method using Mueller-Hinton broth, according to the Clinical and Laboratory Standards Institute recommendations.21 The test substances were dissolved in DMSO, and vancomycin and gentamycin were used as standard positive-control drugs.

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(10) Trisuwan, K.; Ritthiwigrom, T. Arch. Pharm. Res. 2012, 35, 1733−1738. (11) Na, Z.; Song, Q.; Hu, H. Rec. Nat. Prod. 2013, 7, 220−224. (12) Cheenpracha, S.; Phakhodee, W.; Ritthiwigrom, T.; Prawat, U.; Laphookhieo, S. Heterocycles 2011, 83, 1139−1144. (13) Sriyatep, T.; Maneerat, W.; Sripisut, T.; Cheenpracha, S.; Machan, T.; Phakhodee, W.; Laphookhieo, S. Fitoterapia 2014, 92, 285−289. (14) Tantapakul, C.; Phakhodee, W.; Ritthiwigrom, T.; Cheenpracha, S.; Prawat, U.; Deachathai, S.; Laphookhieo, S. J. Nat. Prod. 2012, 75, 1660−1664. (15) Nguyen, H. D.; Trinh, B. T. D.; Nguyen, L. H. D. Phytochem. Lett. 2011, 4, 129−133. (16) Chiang, Y. M.; Kuo, Y. H.; Oota, S.; Fukuyama, Y. J. Nat. Prod. 2003, 66, 1070−1073. (17) Sen, A. K.; Sarkar, K. K.; Mazumder, P. C.; Benerii, N.; Uusvuori, R.; Hase, T. A. Phytochemistry 1980, 19, 2223−2225. (18) Ito, C.; Itoigawa, M.; Takakura, T.; Ruangrungsi, N.; Enjo, F.; Tokuda, H.; Nishino, H.; Furukawa, H. J. Nat. Prod. 2003, 66, 200− 205. (19) Mahabusarakam, W.; Wiriyachitra, P. J. Nat. Prod. 1987, 50, 474−478. (20) Ampofo, S. A.; Waterman, P. G. Phytochemistry 1986, 25, 2351− 2355. (21) CLSI. Reference Method for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically, Approved Standard M7-A7; Clinical and Laboratory Standards Institute: Wayne, PA, 2006. (22) Liu, Y.; Zou, L.; Ma, L.; Chen, W. H.; Wang, B.; Xu, Z. L. Bioorg. Med. Chem. 2006, 14, 5683−5690. (23) Ryu, H. W.; Cho, J. K.; Curtis-Long, M. J.; Yuk, H. J.; Kim, Y. S.; Jung, S.; Kim, Y. S.; Lee, B. W.; Park, K. H. Phytochemistry 2011, 72, 2148−2154. (24) Lee, D. S.; Lee, S. H. FEBS Lett. 2001, 501, 84−86. (25) Li, G. L.; He, J. Y.; Zhang, A.; Wan, Y.; Wang, B.; Chen, W. H. Eur. J. Med. Chem. 2011, 46, 4050−4055. (26) Liu, Y.; Ke, Z.; Cui, J.; Chen, W. H.; Ma, L.; Wang, B. Bioorg. Med. Chem. 2008, 16, 7185−7192.

ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of compounds 1−5 are provided free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +66-5391-6238. Fax: +66-5391-6776. E-mail: surat.lap@ mfu.ac.th. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Thailand Research Fund through the Advanced Research Scholar (BRG5580008) and the Royal Golden Jubilee Ph.D. scholarship (PHD/0153/ 2556). The authors are grateful to Mae Fah Luang University for partial financial support and laboratory facilities.

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REFERENCES

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DOI: 10.1021/np5008476 J. Nat. Prod. XXXX, XXX, XXX−XXX