Cytotoxic and Anti-Inflammatory Prenylated Benzoylphloroglucinols

J. Nat. Prod. , 2014, 77 (7), pp 1700–1707. DOI: 10.1021/np5003498. Publication Date (Web): June 24, 2014. Copyright © 2014 The American Chemical S...
0 downloads 4 Views 1MB Size
Download PDF
Article pubs.acs.org/jnp

Cytotoxic and Anti-Inflammatory Prenylated Benzoylphloroglucinols and Xanthones from the Twigs of Garcinia esculenta Hong Zhang,† Dan-Dan Zhang,§ Yuan-Zhi Lao,† Wen-Wei Fu,† Shuang Liang,† Qing-Hong Yuan,⊥ Ling Yang,|| and Hong-Xi Xu*,†,‡ †

School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Cai Lun Lu 1200, Shanghai 201203, People’s Republic of China ‡ Engineering Research Center of Shanghai Colleges for TCM New Drug Discovery, Cai Lun Lu 1200, Shanghai 201203, People’s Republic of China § Murad Research Center for Modernized Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Cai Lun Lu 1200, Shanghai 201203, People’s Republic of China ⊥ Department of Physics, East China Normal University, Dongchuan Road 500, Shanghai 200241, People’s Republic of China || Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhong-shan Road, Dalian 116023, People’s Republic of China S Supporting Information *

ABSTRACT: Five new prenylated benzoylphloroglucinol derivatives, garciesculentones A−E (1−5), a new xanthone, garciesculenxanthone A (6), and 15 known compounds were isolated from the petroleum ether extract and the EtOAcsoluble fraction of a 80% (v/v) EtOH extract of Garcinia esculenta. The structures of the new compounds were elucidated by 1D- and 2D-NMR spectroscopic analysis and mass spectrometry. Experimental and calculated ECD and a convenient modified Mosher’s method were used to determine the absolute configurations. The cytotoxicity of these compounds were evaluated by MTT assay against three human cancer cell lines (HepG2, MCF-7, and MDA-MB-231) and against normal hepatic cells (HL-7702). In addition, these isolates were evaluated for their inhibitory effects on interferon-γ plus lipopolysaccharide-induced nitric oxide production in RAW264.7 cells.

T

on interferon-γ (IFN-γ) plus lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells are described.

he genus Garcinia is native to southeastern Asia, southern Africa, and Polynesia, with a total of 21 species distributed in mainland China.1 This genus produces secondary metabolites with diverse chemical structures, including xanthones,2 prenylated benzophenones,3 biflavonoids,4 and triterpenoids,5 which have been reported to possess a wide range of biological activities, including cytotoxic, anti-inflammatory,3b,6 antibacterial,5,7 and antienteroviral effects.8 Garcinia esculenta Y. H. Li (Clusiaceae) is an endemic plant of the People’s Republic of China, and it is distributed mainly in the western and northwestern parts of Yunnan Province.1 To date, there are no phytochemical and biological reports on this species. A preliminary study indicated that a petroleum ether extract and an EtOAc-soluble part of the 80% (v/v) EtOH extract of the twigs of G. esculenta showed potent cytotoxic activities against HepG2, MCF-7, and MDA-MB-231 cells (Table S1, Supporting Information). In follow-up work, bioassay-directed fractionation led to the isolation of 21 compounds, including six new (1−6) compounds and 15 known analogues from the active fractions. In this report, the isolation and structure elucidation of these compounds, as well as their inhibitory effects on the proliferation of cancer cells and © 2014 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION The petroleum ether extract and the EtOAc-soluble part of the 80% (v/v) EtOH extract of G. esculenta were purified by separate column chromatography over MCI gel, silica gel, reversed-phase C18 silica gel, and Sephadex LH-20, and also by preparative HPLC to afford 21 compounds, including six new (1−6) and 15 known compounds. The structures of the known compounds were elucidated as garciniagifolone A (7),9 garcimultiflorone E (8),3c cambogin (9),10 guttiferone F (10), 11 5,8-dihydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one (11),12 γ-mangostin (12),13 garcicowin C (13),14 GDPHH-2 (14),15 1,3,7-trihydroxy-2-(3-methylbut-2enyl)-xanthone (15),16 griffipavixanthone (16),17 1,3,5,7tetrahydroxy-8-isoprenylxanthone (17),18 1,3,6,7-tetrahydroxyxanthone (18),19 hyperxanthone E (19),20 toxyloxanthone B (20),13b and 3,5,8-trihydroxy-2,2-dimethyl-3,4,4-trihydroReceived: April 20, 2014 Published: June 24, 2014 1700

dx.doi.org/10.1021/np5003498 | J. Nat. Prod. 2014, 77, 1700−1707

Journal of Natural Products

Article

Chart 1. Compounds 1−6

Table 1. 1H NMR Data of Compounds 1−5 (400 MHz, J in Hz)a 1b

position 7 8 12 15 16 17 18 20 21 22 23 24 25 27 28 29 30 32 33 34 35 37 38 OCH3

1.57, 2.37, 1.99, 7.50, 6.85, 7.53, 2.59, 2.51, 4.79, 1.61, 1.65, 0.99, 1.32, 2.47,

m brd (14.6) m m d (8.7) m m m m s s s s m

4.94, 1.72, 1.69, 2.97, 1.03, 1.46, 1.32, 1.24, 2.10, 1.86, 5.21, 1.77, 1.65,

m s s dd (3.52, 14.1) m m s s m m t (8.28) s s

2b 1.53, 2.20, 2.03, 7.43, 6.91,

3c m m m m m

2.90, m 4.68, 1.45, 1.77, 1.27, 1.11, 2.01, 1.79, 4.79, 1.60, 1.34, 2.22,

m s s s s m m m s s m

2.59, 4.44, 1.62, 1.58, 1.47, 3.85, 4.78, 1.72,

m brs s s m m brs s

1.76, 2.27, 2.08, 7.15, 6.67, 6.93, 2.71, 2.57, 5.04, 1.72, 1.69, 1.01, 1.14, 1.48, 1.40, 3.27, 1.00, 1.02, 1.97,

m brd (13.9) m d (2.1) d (8.3) dd (2.1, 8.3) m m m s s s s m m m s s m

4c

5c

1.74, 2.29, 2.11, 7.19, 6.70, 6.97, 2.73, 2.58, 5.08, 1.75, 1.71, 1.04, 1.16, 2.04,

m m m d (2.0) d (8.3) dd (2.0, 8.3) m m m s s s s m

1.61, 2.26, 2.19, 7.20, 6.70, 7.01, 2.69, 2.55, 5.06, 1.74, 1.69, 0.99, 1.13, 2.02,

m m m d (2.0) d (8.3) dd (2.0, 8.3) m m m s s s s m

5.06, 1.68, 1.60, 1.99,

m s s m

5.04, 1.66, 1.59, 1.93,

m s s m

2.64, m 4.46, brs 1.58, s 2.03,m

2.65, 4.48, 1.60, 1.52,

m brs s m

2.63, 4.44, 1.57, 1.37,

m brs s m

5.02, 1.65, 1.58, 3.18,

3.84, m 4.91, m 1.63, s

m s s s

3.79, m 4.83, brs 1.61, s

a

Assignments were based on HSQC, HMBC, and NOESY experiments; chemical shifts are given in ppm. bMeasured in CD3OD. cMeasured in CD3OD/0.1% TFA.

2H,6H-pyrano[3,2-b]-xanthen-6-one (21),12 respectively, by comparison of their spectroscopic data with published data. Garciesculentone A (1) was obtained as a colorless gum. The 13 C NMR spectrum showed characteristic resonances for six aromatic carbons and a bicyclic[3.3.1]nonane-2,4,9-trione moiety with three quaternary carbons (δC 69.7, 47.0, and 52.7), a methine (δC 47.0), a methylene (δC 39.3), a

nonconjugated ketone (δC 206.6), and an enolized 1,3-diketone (δC 192.7, 133.8, and 165.6).21 HRESIMS showed an ion peak at m/z 617.3475 [M − H]−, giving the molecular formula C38H50O7. The molecular weight of this compound differed from that of 9 by 16 Da (Figure S1, Supporting Information),10 suggesting the presence of an additional oxygen. Also, the NMR data (Tables 1 and 2) obtained from 1D- and 2D-NMR 1701

dx.doi.org/10.1021/np5003498 | J. Nat. Prod. 2014, 77, 1700−1707

Journal of Natural Products

Article

Table 2. 13C NMR Data of Compounds 1−5 (100 MHz)a position

1b

2b

3c

4c

5c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 OCH3

52.7 165.6 133.8 192.7 69.7 47.0 47.0 39.3 206.6 165.3 121.5 118.0 146.3 152.3 116.1 124.4 26.8 121.4 133.9 26.3 18.0 27.2 21.8 30.68d 125.8 134.2 26.2 18.2 29.4 44.3 88.4 21.8 29.3 30.74d 123.2 134.6 26.0 18.3

63.4 194.7 117.2 176.8 65.5 50.9 47.5 45.1 209.2 173.4 118.1 109.5 147.8 155.2 103.9 151.7 27.0 120.6 136.2 26.2 18.8 23.8 27.7 30.8 125.5 133.9 26.1 18.1 38.9 40.8 149.0 115.1 18.2 39.0 75.2 147.8 113.2 16.6

58.7 196.6 117.9 194.2 70.5 50.4 42.9 42.6 210.7 195.3 129.4 117.3 146.3 152.5 115.1 125.2 27.1 121.3 135.8 26.5 18.3 27.7 23.1 31.5 75.4 78.5 21.2 20.8 37.5 45.2 149.5 113.1 18.2 33.4 124.1 132.7 25.9 18.2 49.6

58.7 196.6 118.2 194.7 71.3 50.4 43.0 43.8 210.7 195.6 129.5 117.4 146.4 152.7 115.1 125.5 27.1 121.4 136.0 26.5 18.5 27.4 23.1 33.6 124.2 132.9 26.1 18.4 37.5 45.6 149.6 113.3 18.3 36.8 74.9 149.3 110.9 18.2

59.0 195.60e 120.3 195.56e 69.4 50.6 43.8 47.2 210.7 196.1 129.8 117.6 146.3 152.6 115.2 125.5 27.0 121.5 135.9 26.6 18.3 27.2 23.1 33.6 124.2 132.8 26.1 17.8 37.2 45.4 149.6 113.2 18.2 37.8 77.8 148.6 111.9 18.3

Figure 1. Key correlations observed in the HMBC and NOESY NMR spectra of 1.

reference compound (Figure S2, Supporting Information). Thus, the planar structure of 1 was determined. The relative configuration of 1 was determined by a NOESY experiment. Key NOE correlations as shown in Figure 1 indicated that CH2-17, CH3-23, and CH2-24 are all oriented on the same side of the 2,2-dimethylbicyclo[3.3.1]nonane ring, whereas CH3-22 and H-7 are oriented on the opposite side. The relative configuration at C-30 could not be exactly assigned from the NOESY spectrum. Thus, there were four possible isomers: (1S,5R,7S,30S)-1, (1R,5S,7R,30R)-1, (1S,5R,7S,30R)1, and (1R,5S,7R,30S)-1. The absolute configuration of 1 was determined by comparison of its experimentally measured electronic circular dichroism (ECD) curve with those predicted using TDDFT theory. The results showed that the calculated ECD curve of (1S,5R,7S,30S)-1 was similar to the experimental ECD spectrum of 1 (Figure 2). Therefore, the structure of garciesculentone A (1) was established as shown. Garciesculentone B (2) gave the molecular formula, C38H48O7, as determined by HRESIMS. This elemental composition indicated 15 degrees of unsaturation, which showed that 2 had one more unit of unsaturation than 8 (Figure S1, Supporting Information). The 1H NMR data of 2 were comparable to those of 8, except for the loss of one aromatic proton. Two unique singlet aromatic proton signals at δH 6.91 and 7.43 were apparent in the 1H NMR spectrum aromatic region of 2, which indicated the presence of a 1,2,4,5tetrasubstituted benzene ring rather than a trisubstituted ring in 8. In addition, there were three oxygen-substituted aromatic carbons in the 13C NMR spectrum, which suggested that one aromatic proton of 8 is substituted by an oxygen in 2. HMBC correlations from the proton signals at δH 6.91 and 7.43 to the carbon resonances at δC 147.8, 151.7, and 155.2, together with from the proton signal at δH 6.91 to the carbon resonance at δC 118.1, and from the proton signal at δH 7.43 to the carbon resonance at δC 173.4 allowed the deduction to be made that C-16 is oxygenated. Moreover, HMBC correlations between the carbon resonance at δC 194.7 and the proton resonances of H-8 (δH 2.03) and H-29 (δH 2.22), and between the carbon resonance at δC 176.8 and the proton resonances of H-17 (δH 2.90) allowed the assignments of the signals at δC 194.7 and 176.8 to C-2 and C-4, respectively. The other key HMBC correlations are shown in Figure 1. Based on the information mentioned above,22 it was evident that the carbonyl at C-4 is enolized and that the oxygen is attached to C-16. Thus, compound 2 represents an oxidized derivative of 8, and its planar structure could be established. To determine the relative configuration of 2, a NOESY experiment was performed. The NOE correlations of CH2−17/ CH3-23, CH3-23/H-7, and CH3-22/H-24a indicated that CH217, CH3-23, and H-7 are all oriented on the same side of the molecule, whereas CH3-22 and CH2-24 are oriented on the opposite side. The configurations of C-30 and C-35 could not be assigned from the NOESY spectrum because of the

a

Assignments were based on DEPT, HSQC, and HMBC experiments; chemical shifts are given in ppm. bMeasured in CD3OD. cMeasured in CD3OD/0.1% TFA. d,eData may be interchangeable in the same column.

spectra exhibited close similarities to those of 9,10 with the exception of differences in some carbon signals due to the presence of an additional oxygen atom. The chemical shifts of C-3 and C-10 were δC 133.8 and 165.3, compared to δC 124.7 and 192.3 in 9, respectively, indicating that the additional oxygen atom was located between C-3 and C-10 or C-10 and C-11.8 The HMBC spectrum showed long-range correlations from the proton signals at δH 7.50 (1H, m) and 7.53 (1H, m) to C-10 (δC 165.3), placing the additional oxygen atom between C-3 and C-10. Other key HMBC correlations are shown in Figure 1. In addition, an alkaline hydrolysis reaction was conducted for 1, and its hydrolysate was analyzed by UPLC-PAD and UPLC-ESI-QTOF-MS (Experimental Section, Supporting Information). A peak in the hydrolysate was assigned unambiguously as protocatechuic acid by comparing the retention time, UV spectrum, and m/z value with a 1702

dx.doi.org/10.1021/np5003498 | J. Nat. Prod. 2014, 77, 1700−1707

Journal of Natural Products

Article

Figure 2. Calculated ECD spectra of 1 and 3 and their experimental curves.

flexibility of the aliphatic chain. Thus, eight possible candidate stereostructures: (1S,5S,7R,30S,35S)-2, (1R,5R,7S,30R,35R)-2, (1S,5S,7R,30S,35R)-2, (1R,5R,7S,30R,35S)-2, (1S,5S,7R,30R,35S)-2, (1R,5R,7S,30S,35R)-2, (1S,5S,7R,30R,35R)-2, and (1R,5R,7S,30S,35S)-2, were considered, and the ECD spectra were calculated. Visual inspection suggested either the (1S,5S,7R,30S,35S)-2 or (1S,5S,7R,30S,35R)-2 curve was similar to the experimental curve (Figure S3, Supporting Information). Accordingly, the absolute configuration for garciesculentone B (2) is proposed as (1S,5S,7R,30S)-2. The absolute configuration of C-35 was not determined by the modified Mosher’s method due to an insufficient amount being available. Garciesculentone C (3) was obtained as a yellow gum and showed a deprotonated molecular ion peak at m/z 649.3723 [M − H]− in the HRESIMS, corresponding to the formula C39H53O8 (calcd 649.3740). The 1H NMR spectrum of 3 revealed the presence of one terminal double bond [δH 4.46 (2H, brs)], two olefinic protons [δH 5.04 (1H, m); δH 5.02 (1H, m)], a methoxy group [δH 3.18 (3H, s)], and a 1,3,4trisubstituted benzene ring [δH 6.67 (1H, d, J = 8.3 Hz), 6.93 (1H, dd, J = 2.1 and 8.3 Hz), and 7.15 (1H, d, J = 2.1 Hz)]. The 13C NMR and DEPT spectra of 3 (Table 2) disclosed a total of 39 carbon signals, corresponding to 10 methyl groups, 6 methylene groups, 8 methane groups, and 15 quaternary carbons. These NMR data (Tables 1 and 2) were nearly identical to those of isogarcimultiflorone F (Supporting Information, Figure S4)3c but showed one more methoxy signal [δH 3.18 (3H, s), δC 49.6]. This additional methoxy group was assigned at C-26 on the basis of the correlations from the methoxy protons at δ 3.18 to C-26 (δC 78.5), from H25 (δH 3.27) to C-26 and C-24 (δC 31.5), as well as from H-24 (δH 1.48) to C-7 (δC 42.9) in the HMBC spectrum. Other key HMBC correlations are shown in Figure 3.

The relative configuration of 3 was revealed by NOE correlations obtained from the NOESY spectrum. The CH3-23, H-7, and CH2-17 functionalities were determined to be on the same side of the molecule, with CH3-22 and CH2−24 oriented on the opposite side, from the NOE correlations of CH3-23/H17a and CH3-22/CH2−24. However, the configurations at C25 and C-30 could not be assigned from the NOESY spectrum. Therefore, a convenient Mosher ester method23 combined with calculated ECD data were used to determine the absolute configuration of 3. Treatment of 3 with (R)-(−)-α- and (S)(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride gave the (S)- and (R)-MTPA esters 3S and 3R, respectively. The 1H NMR signals of the two MTPA esters were assigned unambiguously based on their DEPT, HSQC, and 1H−1H COSY spectra, and the ΔδH (S−R) values were then calculated (Figure 4). The results indicated the 25S absolute config-

Figure 4. Δδ (δS − δR) values (in ppm) for the MTPA esters of 3.

uration. All four possible isomers, (1R,5R,7S,25S,30S)-3, (1S,5S,7R ,25S,30R)-3 , (1R,5R,7S,25S,30R)-3, and (1S,5S,7R,25S,30S)-3, were then considered, and the ECD spectra were calculated. The experimental ECD spectrum of 3 was in accordance with the calculated ECD spectra for (1R,5R,7S,25S,30S)-3 or (1R,5R,7S,25S,30R)-3, thus establishing the assignment of the absolute configuration of 3 as (1R,5R,7S,25S)-3. The absolute configuration of C-30 could not be determined by ECD calculations. Garciesculentone D (4) was isolated as a yellow gum. The molecular formula, C38H50O7, was deduced by HRESIMS. The 1 H NMR data of 4 were similar to those of guttiferone F (10),11 except for differences at H-35 [δH 3.84 (1H, m)] and H-37 [δH 4.91 (2H, m)] relative to [δH 5.03 (1H, m)] and [δH 1.65 (3H, s)], respectively, in 10. This result indicated the presence of a 2-hydroxy-3-methylbutenyl group at C-30 of 4.3c This finding was further supported by the 13C NMR signals for C-35 (δC 74.9), C-36 (δC 149.3), and C-37 (δC 110.9), and the HMBC correlations of H-35/C-37, H-38/C-35, H-38/C-37, and H-34/C-35. The connectivity of garciesculentone D was

Figure 3. Key correlations observed in the HMBC NMR spectra of 2 and 3. 1703

dx.doi.org/10.1021/np5003498 | J. Nat. Prod. 2014, 77, 1700−1707

Journal of Natural Products

Article

confirmed by DEPT, HSQC, and HMBC experiments. Other key HMBC correlations are shown in Figure S5 (Supporting Information). The relative and absolute configurations of 4 were assigned in a similar manner to 3. Key NOE correlations are shown in Figure S5 (Supporting Information). The absolute configuration at C-35 was determined to be R by a convenient Mosher ester method (Figure 5). Furthermore, the ECD

Figure 6. Calculated ECD spectra of 5 and its experimental curve.

NMR spectrum suggested the presence of a 1,2,3,4,5pentasubstituted ring A rather than the tetrasubstituted ring as in nigrolineaxanthone K. In addition, there were significant differences in the chemical shifts at C-5, C-6, and C-7 (δC 131.2, 151.7, and 114.6 for 6, respectively, versus δC 142.5, 119.4, and 125.2 for nigrolineaxanthone K, respectively).2a The information mentioned above indicated the presence of an additional hydroxy group in the ring A. The HMBC correlations of H-7/C-8a, C-6, and C-5, as well as CH2−16/ C-8a, C-8 and C-7 were used to assign the additional hydroxy group at C-6. Therefore, garciesculenxanthone A (6) was determined as 1,5,6-trihydroxy-13,13-dimethyl-8-(3-methylbut2-enyl)-2H,6H-pyrano[2,3-b]xanthen-6-one. In this study, all isolates were evaluated for their cytotoxic activity against the HepG2 human hepatocellular carcinoma cell line, two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231), and HL-7702 human normal hepatic cells using the MTT method, with paclitaxel used as the positive control. As shown in Table 3, compounds 7−12 exhibited

Figure 5. Δδ (δS − δR) values (in ppm) for the MTPA esters of 4 and 5.

experiment and ECD calculation of 4 were conducted. Visual inspection suggested either the (1R,5R,7R,30S,35R)-4 or (1R,5R,7R,30R,35R)-4 curve was similar to the experimental curve (Figure S6, Supporting Information). The configuration of C-30 could not be determined by ECD calculations. Thus, the absolute configuration for garciesculentone D (4) is determined as depicted. Garciesculentone E (5) had the same molecular formula as 4 (C 38H50O7), which was deduced from HRESIMS. On comparing the 1D- and 2D-NMR spectra of 5 with those of 4, it appeared that they might be a pair of epimers with a prenylated benzoylphloroglucinol skeleton. 13C NMR chemical shift differences between these two compounds were observed for the C-30 side chain and the bicyclo[3.3.1]nonane-2,4,9trione moiety (as shown in Table 2). These differences resulted from different configurations at C-35 and different hydrogen bonds between OH-35 and carbonyl groups at C-4 and C-9.3c Thus, the only difference between 5 and 4 was deduced to be the configuration at C-35. The absolute configuration of C-35 was determined to be S by a convenient Mosher ester method (Figure 5). The structure of 5 was confirmed by NOESY, DEPT, HSQC, and HMBC experiments. The key HMBC correlations are shown in Figure S5 (Supporting Information). Furthermore, the ECD experiment and ECD calculation of 5 were conducted. The experimental ECD spectrum of 5 was in accordance with the calculated ECD spectra for (1R,5R,7R,30R,35S)-5 or (1R,5R,7R,30S,35S)-5 (Figure 6), thus establishing the assignment of the absolute configuration of 5 as depicted. The configuration of C-30 could not be determined by ECD calculations. Garciesculenxanthone A (6) was isolated as a pale yellow amorphous powder, with a molecular formula of C23H22O6, as determined by HRESIMS (m/z 393.1342 [M − H]−, calcd for 393.1338). Comparison of the spectroscopic data of 6 with those of the known xanthone nigrolineaxanthones K (Supporting Information, Figure S4)2a showed that they have the same B ring, in which the dimethylchromene ring unit could be located at C-2 and C-3, and the aromatic proton was attributed to H-4. This was confirmed by comparing the NMR data of ring B with those of nigrolineaxanthone K and the HMBC correlations from H-11 (δH 6.60) to C-1 (δC 157.5), C-2 (δC 103.8), and C-3 (δC 159.3), as well as from H-4 (δH 6.30) to C3 and C-2. The remaining singlet aromatic proton in the 1H

Table 3. Cytotoxicity of Isolated Compounds against Cancer Cell Linesa,b compound

HepG2

MCF-7

MDA-MB-231

7 8 9 10 11 12 paclitaxelc

>10 >10 7.3 ± 0.3 >10 >10 >10 4.6 × 10−3

>10 9.8 ± 1.2 4.8 ± 0.4 >10 >10 >10 1.5 × 10−3

9.1 ± 0.5 >10 5.7 ± 0.8 7.9 ± 0.6 6.1 ± 1.1 6.6 ± 2.5 8.2 × 10−3

HL-7702d >10 >10 9.3 ± 9.6 ± 6.6 ± >10 1.1 ±

0.1 0.5 0.5 0.1

Results are expressed as IC50 (mean values ± SD, n = 3) in μM. Compounds 1−6 and 13−21 were inactive for all tested human cancer cell lines (IC50 > 10 μM). cPositive control. dHuman normal hepatic cells. a b

cytotoxic activity against one or two human cancer cell lines, exhibiting IC50 values below 10 μM. Among these cytotoxic compounds, compound 12 showed no significant cytotoxicity to normal hepatic HL-7702 cells, thereby demonstrating a selective activity toward the cancer cells used. In previous reports, compounds 7 and 9 exhibited cytotoxicity against SGC7901 human gastric carcinoma cell line9 and two human colon cancer cell lines (HT-29 and HCT116),14 respectively. Compounds 8 and 10 showed strong apoptosis induction activity in HeLa cells, which was detected 1704

dx.doi.org/10.1021/np5003498 | J. Nat. Prod. 2014, 77, 1700−1707

Journal of Natural Products

Article

by a fluorescent caspase-3 sensor in our previous study.3c Compound 12 showed cytotoxicity on two human colon cancer cell lines (HCT116 and SW480)24 and could induce apoptosis in HT29 human colon cancer cells25 and human malignant glioma cells (U87 MG and GBM 8401).26 Taken together, compounds 7−12 could inhibit cell proliferation in several cancer cell lines. The detailed mechanims of the active compounds should be further investigated. All isolates were further tested for their inhibitory effect on IFN-γ plus LPS-induced NO in RAW 264.7 cells (Table 4). Only compounds 8, 17, and 19 showed IC50 values below 10 μM in this assay.

Chinese Medicine. A voucher specimen (herbarium no. 20100801) has been deposited at the Innovative Research Laboratory of TCM, Shanghai University of Traditional Chinese Medicine. Extraction and Isolation. Air-dried and powdered twigs of the plant (4 kg) were extracted with petroleum ether (5 × 20 L, each 2 days). The combined extracts were evaporated to dryness under vacuum to produce the petroleum ether-soluble part (fraction I, 40 g). The remaining materials were refluxed with 80% EtOH (v/v, 5 × 20 L). The combined extracts were evaporated to dryness under vacuum, and the residue was suspended in H2O (5 L) and extracted with EtOAc (5 × 5 L) to obtain fractions II (50 g, the EtOAc-soluble part) and III (the remainin H2O portion), respectively. The remaining materials were refluxed with distilled water (5 × 20 L) to afford the H2O-soluble part (fraction IV). Fractions I and II were shown to possess significant cytotoxic activities against the HepG2, MCF-7, and MDA-MB-231 cell lines (Table S1, Supporting Information). Fraction I (37 g) was chromatographed on a silica gel column chromatography (CC) using a gradient of petroleum ether−EtOAc (100:0 to 50:50, v/v) and yielded 15 pooled fractions (IA−IO) based on their TLC profiles, among which three fractions, IL−IN, showed potent cytotoxic activities against three human cancer cell lines. These bioactive fractions were chromatographed separately on MCI gel eluted with 90% and 100% EtOH, successively, to afford two subfractions (IL1 and IL2, IM1 and IM2, IN1 and IN2), repectively. Fraction IL1 (10.5 g) was subjected to reversed-phase C18 silica gel CC and eluted in a step-gradient manner with MeOH−H2O (70:30 to 100:0) to give seven subfractions IL1a−IL1f and compound 10 (350 mg). Fraction IL1e was further separated by preparative HPLC (MeOH−MeCN−H2O, 27.2:40.8:32, with 0.1% formic acid in H2O, 20 mL/min) to give subfractions IL1e1−IL1e7. Compounds 7 (23 mg), 3 (9 mg), 4 (7 mg), and 5 (8.8 mg) were obtained by preparative HPLC from fractions IL1e2− IL1e4 and IL1e6, respectively. Fraction IL1f was separated by preparative HPLC (20 mL/min), eluting with CH3OH−H2O (12:88, with 0.1% formic acid in H2O) to give subfractions IL1f1−IL1f5. Fractions IL1f2 and IL1f3 were purified by preparative HPLC (MeOH−MeCN−H2O, 30:45:25 and MeOH−MeCN−H2O, 32:48:20, repectively, both with 0.1% formic acid in H2O, 20 mL/min) to yield compounds 14 (8 mg) and 1 (8 mg), respectively. Fraction IM1 (1.5 g) was subjected to reversed-phase C18 silica gel CC and eluted in a step gradient manner with MeOH−H2O (70:30 to 100:0) to yield compound 13 (8 mg) and subfraction IM1b, from which compound 9 (88 mg) was obtained by recrystallization in acetone. Fraction IN1 (1.2 g) was first separated using a reversed-phase C18 silica gel column eluted with MeOH−H2O (70:30 to 100:0) as a gradient system to give five subfractions (IN1a−IN1e). Fractions IN1b and IN 1d were further purified by preparative HPLC (MeOH−H2O, 75:25 and MeOH-H2O, 85:15, respectively, both with 0.1% formic acid in H2O, 20 mL/ min) to obtain compounds 8 (15 mg) and 2 (2.8 mg), respectively. Fraction II was subjected to CC on MCI, eluted with 30%, 60%, 90%, 100% EtOH, and EtOAc, successively, to obtain subfractions IIA−IIE, respectively. Fractions IIB−IIC showed significant cytotoxic activities against the human cancer cell lines used. Fraction IIB (18 g) was subjected to CC on reversed-phase C18 silica gel, eluted with MeOH−H2O in a gradient (45:55 to 100:0), to obtain compound 18 (108 mg)

Table 4. Effects of Compounds on NO Production in IFN-γ Plus LPS-Stimulated RAW 264.7 Cells

a b

compound

IC50a

compound

IC50a

1 2 3 4 5 6 7 8 9 10 11

39.1 ± 4.5 12.9 ± 0.9 24.9 ± 0.7 19.1 ± 0.5 29.1 ± 1.0 >50 >50 1.1 ± 0.1 19.2 ± 0.4 14.1 ± 0.3 >50

12 13 14 15 16 17 18 19 20 21 indomethacinb

>50 21.3 ± 1.0 17.0 ± 0.4 21.3 ± 0.2 41.9 ± 7.9 6.4 ± 0.6 43.0 ± 1.4 4.0 ± 0.1 >50 15.9 ± 0.9 3.9 ± 0.4

Results are expressed as IC50 (mean values ± SD, n = 3) in μM. Positive control.

■

EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 polarimeter. Ultraviolet absorption spectra were recorded on a UV-2401 PC spectrophotometer. ECD spectra were recorded on a JASCO J-810 spectrometer. IR spectra were obtained from a Bio-Rad FtS-135 spectrometer. NMR spectra were measured on a Bruker AV-400 spectrometer and calibrated on the basis of the solvent peak used. Mass spectrometry was performed on a Waters Q-TOF Premier instrument (Micromass MS Technologies, Manchester, U.K.) spectrometer, with an electrospray ion source (Waters, Milford, MA, U.S.A.) connected to a lockmass apparatus performing a real-time calibration correction. Column chromatography was performed with CHP20P MCI gel (75−150 μm, Mitsubishi Chemical Coparation, Tokyo, Japan), silica gel (200−300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, People’s Republic of China), Sephadex LH20 (GE Healthcare Bio-Sciences AB, Sweden), and reversedphase C18 silica gel (50 μm, YMC, Kyoto, Japan). Precoated TLC sheets of silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, People’s Republic of China) were used. A Waters 2535 series machine equipped with a Xbridge C18 column (4.6 × 250 mm, 5 μm) was used for HPLC analysis, and a preparative Xbridge Prep C18 OBD column (19 × 250 mm, 5 μm) was used in sample preparation. Paclitaxel was purchased from Sigma-Aldrich Trading Co. Ltd. (Shanghai, People’s Republic of China). Plant Material. The twigs of Garcinia esculenta Y. H. Li were collected in Nujiang, Yunnan Province, People’s Republic of China, in August 2010. The plant material was identified by Prof. Yuanchuan Zhou, Yunnan University of Traditional 1705

dx.doi.org/10.1021/np5003498 | J. Nat. Prod. 2014, 77, 1700−1707

Journal of Natural Products

Article

Garciesculenxanthone A (6): pale yellow amorphous powder; UV (MeOH) λmax (log ε) 241 (4.27), 279 (4.61), 332 (4.29) nm; IR (KBr) νmax 3423, 2962, 2920, 1649, 1616, 1589, 1516, 1462, 1377, 1315, 1292, 1176, 1153, 1097, 1039, 858, 821 cm−1; 1H NMR (400 MHz, DMSO-d6) δH 14.02 (1H, brs, OH-1), 6.67 (1H, brs, H-7), 6.60 (1H, d, J = 10.0 Hz, H11), 6.30 (1H, s, H-4), 5.73 (1H, d, J = 10.1 Hz, H-12), 5.30 (1H, t, J = 7.0 Hz, H-17), 3.84 (2H, d, J = 7.2 Hz, H-16), 1.68 (6H, s, H-19 and H-20), 1.43 (6H, s, H-14 and H-15); 13C NMR (DMSO-d6, 100 MHz) δC 182.0 (C-9), 159.3 (C-3), 157.5 (C-1), 155.9 (C-4a), 151.7 (C-6), 147.2 (C-10a), 134.7 (C-8), 131.7 (C-18), 131.2 (C-5), 128.1 (C-12), 123.5 (C-17), 115.0 (C-11), 114.6 (C-7), 110.0 (C-8a), 103.8 (C-2), 102.9 (C-9a), 94.1 (C-4), 78.2 (C-13), 32.8 (C-16), 28.1 (C-14 and C-15), 25.8 (C-20), 17.9 (C-19); HRESIMS m/z 393.1342 [M − H]− (calcd for C23H21O6, 393.1338). Cytotoxicity Assays. Cytotoxic activities of all isolates were evaluated by MTT assay using HepG2 (human hepatocellular carcinoma), MCF-7 (human breast adenocarcinoma), MDAMB-231 (human breast adenocarcinoma), and HL-7702 (human normal hepatic cells). Paclitaxel was used as positive control. The detailed methodology for the cytotoxicity assays used was described in a previous report.27 Determination of Nitrite Activity Assay. Anti-inflammatory activities of all isolates were determined by their inhibitory effects on IFN-γ plus LPS-induced NO production in RAW264.7 cells. Indomethacin was used as positive control. The detailed methodology for this assay was described in a previous report.28

and another 10 subfractions (IIB1−IIB10). Fraction IIB3 was further purified by Sephadex LH-20, eluted with MeOH to obtain compound 19 (9 mg). Fraction IIC (14 g) was separated using a reversed-phase C18 silica gel column eluted with MeOH−H2O (60:40 to 100:0) as a gradient system to give nine subfractions (IIC1−IIC9) and compound 17 (25 mg) as yellow crystals. Compounds 17 (18 mg), 20 (8 mg), and 21 (1.9 mg) were purified from fraction IIC2 by Sephadex LH-20, eluted with MeOH. Fraction IIC6 was chromatographed over reversed-phase C18 silica gel eluted with MeOH−H2O in a gradient (45:55 to 100:0) to afford subfractions IIC6a−IIC6d. Fractions IIC6c, IIC6d, and IIC7 were purified by Sephadex LH-20, eluted with MeOH to obtain compounds 16 (108 mg), 11 (3.5 mg), and 12 (7 mg), respectively. Fraction IIC8 was chromatographed on a Sephadex LH-20 column (MeOH) and further purified by preparative HPLC (MeOH−MeCN−H2O, 30:45:25, with 0.1% formic acid in H2O, 20 mL/min) to obtain compound 7 (1.9 mg). Garciesculentone A (1): yellow gum; [α]20D − 116.5 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 268 (4.26) nm; ECD (c 8.73 × 10−4 M, MeOH) λmax nm (Δε) 190 (− 5.13), 212 (+ 2.91), 243 (− 2.78); IR (KBr) νmax 3423, 2976, 2927, 2858, 1738, 1699, 1660, 1608, 1523, 1444, 1373, 1346, 1292, 1197, 1122, 1089, 960, 825, 756 cm−1; 1H NMR (CD3OD, 400 MHz) data, see Table 1; 13C NMR (CD3OD, 100 MHz) data, see Table 2; HRESIMS m/z 617.3475 [M − H]− (calcd for C38H49O7, 617.3478). Garciesculentone B (2): yellow gum; [α]20D + 18.4 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 264 (4.33), 334 (3.98) nm; ECD (c 1.03 × 10−3 M, MeOH) λmax nm (Δε) 203 (+ 3.42), 230 (− 1.08), 254 (+ 5.88), 323 (− 0.78); IR (KBr) νmax 3421, 2964, 2923, 2879, 1731, 1687, 1618, 1513, 1465, 1379, 1292, 1188, 1143, 895 cm−1; 1H NMR (CD3OD, 400 MHz) data, see Table 1; 13C NMR (CD3OD, 100 MHz) data, see Table 2; HRESIMS m/z 615.3312 [M − H]− (calcd for C38H47O7, 615.3322). Garciesculentone C (3): yellow gum; [α]20D − 24.9 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 279 (4.11) nm; ECD (c 7.07 × 10−4 M, MeOH) λmax nm (Δε) 207 (+ 4.93), 262 (− 3.95), 340 (+ 2.05); IR (KBr) νmax 3424, 2970, 2928, 1686, 1438, 1376, 1291, 1207, 1143, 893, 804, 724 cm−1; 1H NMR (CD3OD/0.1% TFA, 400 MHz) data, see Table 1; 13C NMR (CD3OD/0.1% TFA, 100 MHz) data, see Table 2; HRESIMS m/z 649.3723 [M − H]− (calcd for C39H53O8, 649.3740). Garciesculentone D (4): yellow gum; [α]20D − 15.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 277 (4.17) nm; ECD (c 9.2 × 10−4 M, MeOH) λmax nm (Δε) 215 (+ 3.07), 261 (− 1.43), 341 (+ 1.48); IR (KBr) νmax 3430, 2972, 2923, 1682, 1643, 1554, 1441, 1385, 1290, 1207, 1142, 895, 841, 802, 725 cm−1; 1 H NMR (CD3OD/0.1% TFA, 400 MHz) data, see Table 1; 13 C NMR (CD3OD/0.1% TFA, 100 MHz) data, see Table 2; HRESIMS m/z 617.3480 [M − H]− (calcd for C38H49O7, 617.3478). Garciesculentone E (5): yellow gum; [α]20D − 14.3 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 267 (4.00) nm; ECD (c 1.00 × 10−3 M, MeOH) λmax nm (Δε) 200 (+ 1.21), 264 (− 1.85), 284 (+ 0.62); IR (KBr) νmax 3431, 2970, 2925, 1685, 1554, 1441, 1385, 1292, 1209, 1142, 845, 802, 725 cm−1; 1H NMR (CD3OD/0.1% TFA, 400 MHz) data, see Table 1; 13C NMR (CD3OD/0.1% TFA, 100 MHz) data, see Table 2; HRESIMS m/z 617.3479 [M − H]− (calcd for C38H49O7, 617.3478).

■

ASSOCIATED CONTENT

S Supporting Information *

Computational details for compounds 1−5; alkaline hydrolysis of compound 1 and UPLC-PDA and UPLC-ESI-QTOF-MS analyses; preparation of the (S)- and (R)-MTPA ester derivatives of compounds 3−5; HRESIMS, IR, ECD, and NMR spectra of compounds 1−6. These materials are available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-21-51323089. Tel.: +86-21-51323089. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (nos. 81303188 and 81273403), the Key National Natural Science Foundation of China (no. 81130069), and the Program of Shanghai University of Traditional Chinese Medicine (no. 2012JW05). The authors thank Bo Chen, DongDong Hu, and Wan Hu for technical assistance.

■

REFERENCES

(1) Li, Y. H. Flora Reipublicae Popularis Sinicae; Science Press: Beijing, 1990; Vol. 50, pp 89−110. (2) (a) Rukachaisirikul, V.; Kamkaew, M.; Sukavisit, D.; Phongpaichit, S.; Sawangchote, P.; Taylor, W. C. J. Nat. Prod. 2003, 66, 1531−1535. (b) Tao, S. J.; Guan, S. H.; Wang, W.; Lu, Z. Q.; Chen, G. T.; Sha, N.; Yue, Q. X.; Liu, X.; Guo, D. A. J. Nat. Prod. 2009, 72, 117−124. 1706

dx.doi.org/10.1021/np5003498 | J. Nat. Prod. 2014, 77, 1700−1707

Journal of Natural Products

Article

(3) (a) Hamed, W.; Brajeul, S.; Mahuteau-Betzer, F.; Thoison, O.; Mons, S.; Delpech, B.; Nguyen, V. H.; Sevenet, T.; Marazano, C. J. Nat. Prod. 2006, 69, 774−777. (b) Chen, J. J.; Ting, C. W.; Hwang, T. L.; Chen, I. S. J. Nat. Prod. 2009, 72, 253−258. (c) Liu, X.; Yu, T.; Gao, X. M.; Zhou, Y.; Qiao, C. F.; Peng, Y.; Chen, S. L.; Luo, K. Q.; Xu, H. X. J. Nat. Prod. 2010, 73, 1355−1359. (4) Acuña, U. M. O.; Figueroa, M.; Kavalier, A.; Jancovski, N.; Basile, M. J.; Kennelly, E. J. J. Nat. Prod. 2010, 73, 1775−1779. (5) Klaiklay, S.; Sukpondma, Y.; Rukachaisirikul, V.; Phongpaichit, S. Phytochemistry 2013, 85, 161−166. (6) (a) Santa-Cecilia, F. V.; Vilela, F. C.; da Rocha, C. Q.; Dias, D. F.; Cavalcante, G. P.; Freitas, L. A.; dos Santos, M. H.; Giusti-Paiva, A. J. Ethnopharmacol. 2011, 133, 467−473. (b) Santa-Cecília, F. V.; Freitas, L. A. S.; Vilela, F. C.; Veloso, C. d. C.; da Rocha, C. Q.; Moreira, M. E. C.; Dias, D. F.; Giusti-Paiva, A.; dos Santos, M. H. Eur. J. Pharmacol. 2011, 670, 280−285. (7) Tantapakul, C.; Phakhodee, W.; Ritthiwigrom, T.; Cheenpracha, S.; Prawat, U.; Deachathai, S.; Laphookhieo, S. J. Nat. Prod. 2012, 75, 1660−1664. (8) Zhang, H.; Tao, L.; Fu, W. W.; Liang, S.; Yang, Y. F.; Yuan, Q. H.; Yang, D. J.; Lu, A. P.; Xu, H. X. J. Nat. Prod. 2014, 77, 1037−1046. (9) Shan, W. G.; Lin, T. S.; Yu, H. N.; Chen, Y.; Zhan, Z. J. Helv. Chim. Acta 2012, 95, 1442−1448. (10) Shen, J.; Yang, J. S. Acta Chim. Sin. 2007, 65, 1675−1678. (11) Fuller, R. W.; Blunt, J. W.; Boswell, J. L.; Cardellina, J. H., II; Boyd, M. R. J. Nat. Prod. 1999, 62, 130−132. (12) Huang, Z.; Yang, R.; Guo, Z.; She, Z.; Lin, Y. Chem. Nat. Compd. 2010, 46, 348−351. (13) (a) Jefferson, A.; Quillinan, A. J.; Scheinmann, F.; Sim, K. Y. Aust. J. Chem. 1970, 23, 2539−2543. (b) Ishiguro, K.; Fukumoto, H.; Nakajima, M.; Isoi, K. Phytochemistry 1993, 33, 839−840. (14) Xu, G.; Kan, W. L.; Zhou, Y.; Song, J. Z.; Han, Q. B.; Qiao, C. F.; Cho, C. H.; Rudd, J. A.; Lin, G.; Xu, H. X. J. Nat. Prod. 2010, 73, 104−108. (15) Sang, S.; Pan, M. H.; Cheng, X.; Bai, N.; Stark, R. E.; Rosen, R. T.; Lin-Shiau, S. Y.; Lin, J. K.; Ho, C. T. Tetrahedron 2001, 57, 9931− 9938. (16) Harrison, L. J.; Leong, L. S.; Sia, G. L.; Sim, K. Y.; Tan, H. T. W. Phytochemistry 1993, 33, 727−728. (17) 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. Tetrahedron Lett. 1998, 39, 9103−9106. (18) Zhang, Z.; ElSohly, H. N.; Jacob, M. R.; Pasco, D. S.; Walker, L. A.; Clark, A. M. Planta Med. 2002, 68, 49−54. (19) Noro, T.; Ueno, A.; Mizutani, M.; Hashimoto, T.; Miyase, T.; Kuroyanagi, M.; Fukushima, S. Chem. Pharm. Bull. 1984, 32, 4455− 4459. (20) Tanaka, N.; Takaishi, Y.; Shikishima, Y.; Nakanishi, Y.; Bastow, K.; Lee, K. H.; Honda, G.; Ito, M.; Takeda, Y.; Kodzhimatov, O. K.; Ashurmetov, O. J. Nat. Prod. 2004, 67, 1870−1875. (21) Zhang, L. J.; Chiou, C. T.; Cheng, J. J.; Huang, H. C.; Kuo, L. M.; Liao, C. C.; Bastow, K. F.; Lee, K. H.; Kuo, Y. H. J. Nat. Prod. 2010, 73, 557−562. (22) (a) Masullo, M.; Bassarello, C.; Suzuki, H.; Pizza, C.; Piacente, S. J. Agric. Food Chem. 2008, 56, 5205−5210. (b) Huang, S. X.; Feng, C.; Zhou, Y.; Xu, G.; Han, Q. B.; Qiao, C. F.; Chang, D. C.; Luo, K. Q.; Xu, H. X. J. Nat. Prod. 2009, 72, 130−135. (c) Masullo, M.; Bassarello, C.; Bifulco, G.; Piacente, S. Tetrahedron 2010, 66, 139− 145. (23) Su, B. N.; Park, E. J.; Mbwambo, Z. H.; Santarsiero, B. D.; Mesecar, A. D.; Fong, H. H.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod. 2002, 65, 1278−1282. (24) Yoo, J. H.; Kang, K.; Jho, E. H.; Chin, Y. W.; Kim, J.; Nho, C. W. Food Chem. 2011, 129, 1559−1566. (25) Chang, H. F.; Yang, L. L. Molecules 2012, 17, 8010−8021. (26) Chang, H. F.; Huang, W. T.; Chen, H. J.; Yang, L. L. Molecules 2010, 15, 8953−8966.

(27) Xia, Z. X.; Zhang, D. D.; Liang, S.; Lao, Y. Z.; Zhang, H.; Tan, H. S.; Chen, S. L.; Wang, X. H.; Xu, H. X. J. Nat. Prod. 2012, 75, 1459−1464. (28) Tan, X.; Wang, Y. L.; Yang, X. L.; Zhang, D. D. J. Evidence-Based Complementary Altern. Med. 2014, 2014, 681352.

1707

dx.doi.org/10.1021/np5003498 | J. Nat. Prod. 2014, 77, 1700−1707