Article pubs.acs.org/jnp
Caryophyllene Sesquiterpenes from the Marine-Derived Fungus Ascotricha sp. ZJ-M‑5 by the One Strain−Many Compounds Strategy Wen-Jing Wang,†,‡ Dan-Yi Li,†,‡ Yan-Chun Li,§ Hui-Ming Hua,†,‡ En-Long Ma,*,§ and Zhan-Lin Li*,†,‡ †
Key Laboratory of Structure-Based Drug Design & Discovery, Shenyang Pharmaceutical University, Ministry of Education, Shenyang 110016, People’s Republic of China ‡ School of Traditional Chinese Materia Media, Shenyang Pharmaceutical University, Wenhua Road 103, Box 49, Shenyang 110016, People’s Republic of China § School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China S Supporting Information *
ABSTRACT: The marine-derived fungus Ascotricha sp. ZJ-M-5, which can produce cyclonerodiol analogues, a 3,4-seco lanostane triterpenoid, and diketopiperazines in an eutrophic medium, was subjected to a one strain−many compounds (OSMAC) analysis. It was found to produce three new caryophyllene derivatives (1−3) and the known 1,3,6-trihydroxy-8-methylxanthone (4) in an oligotrophic medium, Czapek Dox broth with or without Mg2+. (+)-6-O-Demethylpestalotiopsin A (1) and (+)-6-Odemethylpestalotiopsin C (2), which have a five-membered hemiacetal structural moiety, showed growth inhibitory effects against HL-60 and K562 leukemia cell lines with the lowest GI50 value of 6.9 ± 0.4 μM. It can be concluded that modification of the culture media is still effective in the discovery of novel bioactive fungal secondary metabolites.
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During the course of this work, it was discovered that 10 times as much MgSO4 was added to the medium than was intended. Instead of replicating of the experiment, we regarded this as an opportunity to apply the OSMAC strategy to this fungus to investigate if this strain squanders the nutrients provided and requires oligotrophic conditions to produce bioactive secondary metabolites. The impact of Mg2+ on the secondary metabolism of ZJ-M-5 could also be examined. To avoid factors arising from complex ingredients, modified Czapek Dox broth, composed of sucrose and NaNO3 as the sole sources of carbon and nitrogen, respectively, and four inorganic salts including MgCl2 instead of MgSO4, was selected as an oligotrophic medium. The secondary metabolites of ZJM-5 in Czapek Dox broth without Mg2+ or with Mg2+ at different concentrations were compared to those produced in the eutrophic medium by HPLC profiling. This led to the isolation of three new caryophyllene derivatives (1−3) and the known compound, 1,3,6-trihydroxy-8-methylxanthone (4). The details of the OSMAC analyses, isolation and identification of these compounds, and the growth inhibitory effects of 1−3 are reported here.
icrobial secondary metabolites are a major source of lead compounds used in drug development. Recently, whole genome sequencing of microbes has revealed the existence of silent pathways, which are not always expressed under standard culture conditions. These pathways allow researchers to explore the chemical diversity of microbes by physically or chemically triggering these silent gene clusters. This strategy used to increase the diversity and the yield of microorganism-derived natural products from a single strain was termed one strain− many compounds (OSMAC) by Zeeck.1 One successful application is the isolation of a series of spirobisnaphthalenes and mutolide from Sphaeropsidales sp. F-24′707 via modification of the culture media and vessels,2 addition of the enzyme inhibitors ancymidole3 and tricyclazole,4 and exposure to UV radiation.5 Ascotricha sp. ZJ-M-5, a fungus isolated from a mud sample collected on a coastal beach in Fenghua County, Zhejiang Province, China, was found to produce cyclonerodiol derivatives,6 a 3,4-seco lanostane triterpenoid, ascotrichic acid B, and several diketopiperazines7 when placed in a medium containing complex nutrients including yeast extract, peptone, and corn syrup. The yield of cyclonerodiol was more than 23 mg/L. © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 5, 2014
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secondary metabolite of ZJ-M-5 in the eutrophic medium, in the Czapek Dox broth without MgCl2 (Figure 1i). Mg2+ was found to slightly stimulate the production of cyclonerodiol (Figure 1d−f), but the yield was significantly lower than that in the eutrophic medium (Figure 1b). The fungus synthesized (+)-6-O-demethylpestalotiopsin A (1) and (+)-6-O-demethylpestalotiopsin C (2) in the Czapek Dox broth without Mg2+ (Figure 1i), as well as 4, which was also detected in the modified Czapek Dox broth with MgCl2 (Figure 1f−i). The presence of Mg2+ markedly inhibited the production of 1 and 2 and stimulated the biosynthesis of (−)-6-O-demethylpestalotiopsin B (3) (Figure 1h). As the concentration of MgCl2 in the modified Czapek Dox broth increased, ZJ-M-5 stopped the production of compounds 1−4 stepwise (Figure 1d). The peak at 69 min (Figure 1d−h) represents a mixture of fatty acid esters and was not investigated further and the peak at 79 min (Figure 1d−i) was not consistently observed in repeated fermentations. Additionally, highly polar secondary metabolites, mainly the diketopiperazines produced in the eutrophic medium (Figure 1b), were not found in the modified Czapek Dox broth, which simplified the isolation of compounds 1−4. Compound 1, C17H26O6 by HRESIMS, showed the existence of four methyl singlets at δH 2.02, 1.90, 1.55, and 1.14, one olefinic proton at δH 5.88 (1H, d, J = 11.5 Hz), and three oxymethine protons at δH 5.57 (1H, dd, J = 10.4, 5.7 Hz), 4.91 (1H, dd, J = 11.5, 5.8 Hz), and 4.75 (1H, dd, J = 5.8, 2.2 Hz) in its 1H NMR spectrum (Table 1). The 13C NMR spectrum (Table 1) showed 17 carbon resonances, comprising two signals for an acetyl at δC 170.3 and 21.3, and 15 carbons constituting a sesquiterpene scaffold including two olefinic carbons at δC 132.1 and 129.4, four oxygenated carbons at δC 97.3, 80.7, 74.7, and 74.2, and one distinctive carbon for a hemiacetal moiety at δC 109.6, guiding us to a related analogue of compound 1, pestalotiopsin A.8 The skeleton of 1 was confirmed to be the same as that of pestalotiopsin A by careful comparison of their NMR data, excepting only that 1 lacked a methoxy group at C-6. HSQC and HMBC experiments were used to confirm the planar structure of 1. The relative configuration and conformation of compound 1 were
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RESULTS AND DISCUSSION The fermentation products of ZJ-M-5 in the eutrophic medium, and in the modified Czapek Dox broth without MgCl2 and with MgCl2 at 0.04, 0.4, 0.8, 2, and 4 g per 100 mL, were subjected to HPLC analyses. The chromatograms are displayed in Figure 1. The fungus did not biosynthesize cyclonerodiol, the major
Figure 1. HPLC chromatograms for cyclonerodiol (a), ZJ-M-5 in eutrophic medium (b), compound 3 (c), ZJ-M-5 in Czapek Dox with MgCl2 at 4.0 (d), 2.0 (e), 0.8 (f), 0.4 (g), 0.04 ((h), the peak for compound 3 is marked), and 0 ((i), the peaks for compounds 1, 2, and 4 are marked) g/100 mL. Although HPLC chromatogram (b) has peaks with retention times similar to compounds 1 and 4, further analyses revealed that 1 and 4 were not present.
Table 1. NMR Data for Compounds 1 and 2 in Pyridine-d5 1 position
δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
97.3, C 74.2, CH 41.3, CH2 132.1, C 129.4, CH 74.7, CH 80.7, CH 68.1, CH 39.1, CH 43.1, CH2 39.9, C 27.4, CH3 24.7, CH3 109.6, CH 17.2, CH3 170.3, C 21.3, CH3
2 δH (J in Hz)
δC
5.57, dd (10.4, 5.7) β 2.73, t (10.4) α 2.69, dd (10.4, 5.7) 5.88, d (11.5) 4.91, dd (11.5, 5.8) 4.75, dd (5.8, 2.2) 3.25, brd (1.6) 3.06, brt (7.4) ∼2.0, overlapped 1.14, 1.55, 6.56, 1.90,
s s d (2.5) s
2.02, s
B
97.3, C 74.1, CH 41.4, CH2 131.8, C 129.4, CH 72.6, CH 90.8, CH 63.4, CH 39.2, CH 43.0, CH2 40.0, C 27.4, CH3 24.8, CH3 108.7, CH 17.1, CH3 170.3, C 21.3, CH3 57.5, CH3
δH (J in Hz) 5.55, dd (10.2, 5.8) β 2.72, t (10.2), α 2.67, dd (10.2, 5.8) 5.85, d (11.5) 4.85, dd (11.5, 4.9) 4.08, dd (4.9, 2.4) 3.05, brs 2.89, brt (7.6) ∼2.0, overlapped 1.14, 1.56, 6.52, 1.85,
s s d (1.9) s
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The molecular formula of compound 3 was determined to be C17H26O5 by HRESIMS. However, 34 carbons were observed in the 13C NMR spectrum. The 1H NMR spectrum also indicated the existence of two equilibrating atropisomers in the ratio 1:1 at room temperature, which had been reported for fuscoatrol A11 and pestalotiopsin B.8 The two sets of NMR data were distinguished by HSQC, HMBC, and NOESY experiments and analyses of the coupling constants of protons in the same spin systems and were assigned to conformers A and B (Table 2) of compound 3, respectively. The 1H NMR signals of conformer B included two olefinic protons at δH 6.54 (1H, s) and 6.20 (1H, d, J = 7.6 Hz), two oxymethine protons at δH 5.64 (1H, dd, J = 10.4, 7.6 Hz) and 5.16 (1H, d, J = 7.6 Hz), two oxymethylene protons at δH 4.50 (1H, d, J = 10.9 Hz) and 4.26 (1H, d, J = 10.9 Hz), one hydroxy at δH 6.82 (1H, s), one acetyl at δH 2.00 (3H, s), and one methyl attached to an sp2 carbon at δH 2.27 (3H, s). The 13C NMR data of conformer B were similar to those of one conformer of fuscoatrol A except for the downfield shift of C-2 by about 4 ppm and the significant upfield shift of C-6 by 10 ppm, indicating that C-2 of 3 was acetylated and C-6 was substituted with a hydroxy instead of the methoxy in fuscoatrol. These assignments were unambiguously confirmed by the HMBC experiment, leading to the establishment of the planar structure of compound 3. The relative configuration of conformers A and B was then determined using a NOESY experiment (Figure 4). Although the absolute configuration of fuscoatrol or pestalotiopsin B has not yet been determined, compound 3 ([α]D20 −150), named (−)-6-O-demethylpestalotiopsin B, was assigned the 1S, 2S, 6R, and 9R configuration because of its biogenetic similarity to compounds 1 and 2. Compound 4 was identified as 1,3,6-trihydroxy-8-methylxanthone by 1H and 13C NMR, NOESY, and MS. The resonances of the protons adjacent to 3-OH and 6-OH were slightly more upfield by 0.1−0.3 ppm than those in the literature.12 The signals of 3-OH and 6-OH were not observed in the 1H NMR spectrum. This was attributed to the slightly alkaline environment (pH = 9) of the DMSO-d6 used for NMR which favored the dissociation of the hydroxy groups at C-3 and C-6. The growth inhibitory effects of compounds 1−3 against HL-60 and K562 cells were assayed, and 3 was found to show no activity (GI50 > 100 μM). Compounds 1 and 2 exhibited higher activities than the positive control cisplatin (13.4 ± 1.9 μM against HL-60 and 19.1 ± 2.3 μM against K562) with GI50 values of 6.9 ± 0.4 μM (HL-60) and 10.1 ± 0.9 μM (K562) for 1 and 8.5 ± 0.7 μM (HL-60) and 12.3 ± 1.1 μM (K562) for 2. This is the first time that caryophyllene derivatives have been isolated from the Ascotricha genus. Analogues of compounds 1−3 have been isolated from various fungi including Humicola fuscoatra,11 Pestalotiopsis sp.,8,13,14 and Cytospora sp.9 Only pestalotiopsin A has attracted attention from medicinal chemists for its in vitro cytotoxicity against P388 murine leukemia cells.10 As the yields of pestalotiopsins were low and variable in batch culture,13 continuous synthetic efforts have been made, but these were costly and involved stereocontrolled approaches.10,15 The isolation of compounds 1−3 from Ascotricha sp. ZJ-M-5 creates a new opportunity for the production of pestalotiopsin derivative via a bioreactor. Analysis of the chromatograms in Figure 1 showed us that ZJ-M-5 produces different metabolites in the eutrophic and oligotrophic media, respectively. The difference in the sources of nitrogen and carbon might be the key. Sucrose acts as the
established (Figure 2) in a NOESY experiment in which correlations of H-2/H-9/H3-12/H3-15, H3-15/H-6, and H-6/
Figure 2. Key NOESY correlations of compound 1.
H-9 showed H-2, H-6, H-9, CH3-12, and CH3-15 to be cofacial and randomly assigned as the α-orientation, and correlations of H-5/H-7 and H-8/H3-13 suggested these protons were βoriented. Additionally, NOEs from H-14 to H-5 and H-7 oriented the hemiacetal proton toward the nine- instead of the four-membered ring. An in situ dimolybdenum CD method9 was used to establish the absolute configuration for the 6,7-diol moiety in 1. The negative Cotton effect at 310 nm observed in the CD spectrum (Figure 3) of the complex of 1 and Mo2(OAc)4 in anhydrous
Figure 3. Conformation of the Mo24+ complex of compound 1 and its ICD spectrum in anhydrous DMSO with the inherent CD subtracted.
DMSO permitted the assignment of the 6R and 7R configurations for 1. Taking the relative configuration into consideration, the 1S, 2S, 6R, 7R, 8S, 9R, and 14R configuration was assigned for 1, named (+)-6-O-demethylpestalotiopsin A. This configuration is the same as determined for (+)-pestalotiopsin A via total synthesis.10 Compound 2 was given the molecular formula of C18H28O6 by HRESIMS, one CH2 unit more than in 1. Comparison of the 13C NMR data (Table 1) of 2 to those of 1 showed a new methoxy carbon at δC 57.5 and the resonance of C-7 of 2 shifted downfield by 10.1 ppm, while C-6 and C-8 shifted upfield by 2.1 and 4.7 ppm, respectively, indicating that etherification occurred at C-7 of compound 2. The methoxy group at δH 3.56 was assigned to C-7 by its long-range correlation to C-7 at δC 90.8 observed in HMBC spectrum. Because compound 2 was dextrorotary ([α]20D +92), has a shared biogenesis with 1, and exhibited the same relative configuration as 1 as established by a NOESY experiment, it was given the trivial name of (+)-6-O-demethylpestalotiopsin C and was assigned the same absolute configuration as for 1. C
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Table 2. NMR Data for Compound 3 in Pyridine-d5 conformer A position
δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1-OH
81.9, C 77.0, CH 40.8, CH2 128.5, C 134.3, CH 67.0, CH 143.1, CH 137.4, C 41.6, CH 34.0, CH2 42.1, C 27.5, CH3 25.2, CH3 66.4, CH2 17.4, CH3 170.3, C 21.4, CH3
conformer B
δH (J in Hz)
δC
5.60, dd (11.2, 4.1) β 3.00, t (10.9), α 2.56, dd (10.4, 4.1) 5.93, d (10.1) 5.33, brd (10.1) 6.64, d (1.5) 3.44, dd (10.6, 7.8) β 2.32, dd (12.4, 7.8), α 1.73, dd (12.4, 10.6) 1.18, 1.42, 4.66, 1.96,
s s d (11.2) s
1.96, s 6.28, s
5.64, dd (10.4, 7.6) β 3.23, dd (13.6, 10.4), α 2.14, dd (13.6, 7.6) 6.20, d (7.6) 5.16, d (7.6) 6.54, s 3.56, m β 2.50, t (10.9), α 1.63, dd (10.6, 8.7) 1.25, 1.30, 4.50, 2.27,
s s d (10.9) s
2.00 6.82, s
with tetramethyl silane as the internal standard. HRESIMS was performed on a Bruker micrOTOF-Q mass spectrometer. ESIMS was conducted on an Agilent 1100 mass spectrometer. Packing materials for column chromatography were silica gel (200−300 mesh, Qingdao Haiyang Chemical Co., Ltd.) and Sephadex LH-20 (GE Healthcare). Isolation and Identification of the Fungus. The fungal strain, ZJ-M-5, was isolated from a mud sample collected on the coastal beach in Fenghua County, Zhejiang Province, China. The incubation was carried out at 28 °C on potato dextrose agar (PDA) prepared by seawater, and ZJ-M-5 was isolated after 9 days of incubation. This strain was submitted for authorized identification (report no. 2011227) at the Institute of Microbiology, the Chinese Academy of Sciences, on Aug 15, 2011. The identification, established by Associate Professor You-Zhi Wang, was performed by the morphological evaluation and the ITS1-5.8S-ITS2 sequence of the rRNA. The sequence was submitted to GenBank (accession no. JX088707). The fungal strain ZJ-M-5 was deposited in the China General Microbiological Culture Collection Center (CGMCC) as CGMCC no. 8278. OSMAC Analyses. The eutrophic medium (100 mL) was prepared by dissolving 2.0 g of D-mannitol, 2.0 g of D-glucose, 0.5 g of yeast extract, 1.0 g of peptone, 0.05 g of KH2PO4, 0.03 g of MgSO4, 0.1 g of corn syrup, and 3.3 g of sea salt in 100 mL of distilled H2O. To prevent the formation of H2O insoluble Mg(OH)2 during autoclaving, 0.04 g of solid MgCl2 was irradiated under UV light for 3 h and then added into 100 mL of the autoclaved broth containing 3.0 g of sucrose, 0.3 g of NaNO3, 0.1 g of K2HPO4, 0.05 g of KCl, and 0.001 g of FeSO4 to afford the modified Czapek Dox broth as the oligotrophic medium. Additionally, Czapek Dox broth without MgCl2 and the modified ones with 0.4, 0.8, 2, and 4 g MgCl2 per 100 mL were prepared. The spores of ZJ-M-5 were inoculated into 100 mL of these seven different media and cultivated at 28 °C on a shaking platform for 7 days. The fermented broth was filtered through cheesecloth, and the supernatant was partitioned three times with an equal volume of EtOAc. The residue was dissolved in 2 mL of MeOH and 10 μL was subjected to HPLC analysis, which was performed on Shimadzu instrument with a diode array detector (SPD-M20A) and LC-20AB pump equipped with an Inertsil ODS-SP column (4.6 mm × 150 mm, 5 μm). The detection wavelength for the chromatogram was 210 nm. The mobile phase comprised CH3CN (A) and H2O (B), and the gradient program was 0−20 min (10%−20% A), 20−50 min (20%−50% A), 50−75 min (50%−90% A), and 75−85 min (90% A). Fermentation and Isolation. The spores of ZJ-M-5 on PDA plates were inoculated into 150 mL of Czapek Dox broth without
Figure 4. Key NOESY correlations of the two atropisomers of compound 3.
sole carbon source and NaNO3 the sole inorganic nitrogen source in the Czapek Dox medium, while mannitol and glucose are the carbon sources in the eutrophic medium and yeast extract and peptone donate both carbon and nitrogen simultaneously. It can be conferred that the sole carbon source and the inorganic nitrogen source in Czapek Dox switched on the silent gene clusters encoding the enzymes necessary to the assembly of compound 4 and the three caryophyllene derivatives. The yield of xanthone 4 remained the same regardless of whether there was any Mg2+ in the Czapek Dox broth or not. Removal of Mg2+ from Czapek Dox stopped production of compound 3 but stimulated synthesis of compounds 1 and 2. The production of compounds 1−4 was inhibited by high concentrations of Mg2+ in the modified Czapek Dox broth. The OSMAC strategy has been applied on microbes by changing the ingredients and pH of the culture media16,17 and by adding copper ions.18 The results in this paper are the first to describe the application of the oligotrophic Czapek Dox broth in the OSMAC approach and the alteration of secondary metabolism in fungi by Mg2+. For ZJ-M-5, the simplest and cheapest medium results in the production of a complex bioactive scaffold.
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83.2, C 80.2, CH 36.1, CH2 139.4, C 131.0, CH 66.7, CH 143.2, CH 134.5, C 46.7, CH 36.4, CH2 41.0, C 24.6, CH3 25.6, CH3 66.8, CH2 25.2, CH3 170.3, C 21.4, CH3
δH (J in Hz)
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were obtained on a PerkinElmer 241MC polarimeter. CD spectra were acquired with Bio-Logic MOS-450 spectrometer. IR spectrum was obtained on a Bruker IFS-55 spectrometer (KBr pellets). NMR spectra were recorded on Bruker ARX-300 or AV-600 NMR spectrometers, D
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MgCl2 in a 500 mL flask, and 77 flasks were incubated on a shaking platform at 28 °C for 7 days. The combined fermentation broth of 11.55 L was filtered through cheesecloth, and the supernatant was partitioned with butanol (11.55 L) three times to afford a light-yellow gum (39 g). The separation of the butanol extract was carried out by silica gel column chromatography (45 mm × 240 mm) eluting with petroleum ether (PE)−acetone (7:1, 5:1, 3:1, 1:1, each 2.1 L, v/v) followed by acetone (2.1 L) and MeOH (2.1 L) to afford six fractions (Fr1−Fr6). Fr2 (PE−acetone 5:1, 75 mg) was subjected to preparative thin-layer chromatography (PTLC) developed with PE−acetone (1:1, v/v, Rf = 0.4), followed by Sephadex LH-20 column chromatography to afford compound 4 (10 mg). Compound 2 (15 mg) was recrystallized from Fr3 (PE−acetone 3:1, 128 mg) in the mixture of MeOH and EtOAc. Recrystallization of Fr4 (PE−acetone 1:1, 194 mg) in MeOH and EtOAc afforded compound 1 (80 mg). The fermentation broth (14.85 L) of ZJ-M-5 in the modified Czapek Dox broth with MgCl2 (0.4 g/100 mL) after 7 days of cultivation at 28 °C was partitioned three times with an equal volume of EtOAc. The extract (1.1 g) was chromatographed on silica gel (18 mm × 45 mm) eluting with a step gradient of PE−acetone (10:1, 7:1, 5:1, 3:1, 1:1, each 60 mL, v/v), acetone (60 mL), and MeOH (60 mL), successively, yielding seven fractions (Fr1−Fr7). Compound 3 (35 mg) was recrystallized from Fr4 (PE−acetone 3:1, 164 mg) in MeOH and EtOAc. (+)-6-O-Demethylpestalotiopsin A (1). Colorless needles (MeOH−EtOAc); mp 193−195 °C; [α]D20 +103 (c 1.0, MeOH); IR (KBr) νmax 3355, 2981, 2936, 2870, 1738, 1239, 1036, 998 cm−1; 1 H (400 MHz) and 13C NMR (150 MHz), see Table 1; HRESIMS m/ z 349.1607 [M + Na]+ (calcd for C17H26NaO6, 349.1627). (+)-6-O-Demethylpestalotiopsin C (2). Colorless needles (MeOH−EtOAc); mp 179−181 °C; [α]D20 +92 (c 0.2, MeOH); IR (KBr) νmax 3412, 2956, 2928, 2869, 1738, 1261, 1240, 1017, 984 cm−1; 1 H (600 MHz) and 13C NMR (100 MHz), see Table 1; HRESIMS m/ z 363.1796 [M + Na]+ (calcd for C18H28NaO6, 349.1784). (−)-6-O-Demethylpestalotiopsin B (3). Colorless needles (MeOHEtOAc); mp 230−232 °C; [α]D20 −150 (c 0.5, MeOH); IR (KBr) νmax 3423, 3321, 2979, 2936, 2881, 1734, 1247 cm−1; 1H (400 MHz) and 13 C NMR (150 MHz), see Table 2; ESIMS m/z 333.0 [M + Na]+, 345.5 [M + Cl]−; HRESIMS m/z 333.1673 [M + Na]+ (calcd for C17H26NaO5, 333.1678). Induced Circular Dichroism of Compound 1. Using a method described in the literature,19 1.7 mg of compound 1 was dissolved in 4 mL of anhydrous DMSO, of which 2 mL were used for the measurement of the CD of 1. Mo2(OAc)4 (1.4 mg) was added into the remaining 2 mL solution. The CD spectrum of the complex was recorded 10 min later, and the inherent CD spectrum was subtracted. CD (c 1.3 mM, DMSO) λmax (Δε) 274 (+0.37), 310 (−3.65), 357 (−0.98), 377 (−1.08) nm. Biological Assay. The growth inhibitory assay was performed as described previously.20 Human leukemia HL-60 and K562 cells (American Type Culture Collection) were cultured in RPMI-1640 medium (Gibco) with 10% fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 1 mmol/L L -glutamine. Cell suspensions (3 × 104 cells/mL) of 1 mL were seeded into 24-well plates, followed by the addition of test samples in DMSO at five concentrations (1, 3, 10, 30, and 100 μM). After coincubation for 72 h, 0.4% trypan blue was added and the cell viability was determined by hemocytometer within 3 min. The growth inhibitory effects of the tested compounds were expressed as GI50 values calculated using LOGIT method, and the results are representative of three individual experiments. Cisplatin (99.0%, Jiutai Pharmaceutical Co., Ltd.) was used as a positive control.
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AUTHOR INFORMATION
Corresponding Authors
*For Z.-L.L.: phone, +86-024-23986465; E-mail, lzl1030@ hotmail.com. *For E.-L.M.: E-mail,
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant no. 21002064) and Program for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
Identification report for ZJ-M-5, and ESIMS and NMR spectra of compounds 1−4. This material is available free of charge via the Internet at http://pubs.acs.org. E
dx.doi.org/10.1021/np500110z | J. Nat. Prod. XXXX, XXX, XXX−XXX
