Article pubs.acs.org/jnp
Biotransformation of Curcumenol by Mucor polymorphosporus Li-Xia Chen,† Qian Zhao,† Meng Zhang,† Yan-Yan Liang,† Jiang-Hao Ma,† Xue Zhang,† Li-Qin Ding,‡ Feng Zhao,§ and Feng Qiu*,†,‡ †
Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China ‡ School of Chinese Materia Medica, Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Nankai District, Tianjin 300193, People’s Republic of China § School of Pharmacy, Yantai University, Laishan District, Yantai, 264005, People’s Republic of China S Supporting Information *
ABSTRACT: Biocatalysis of curcumenol (1) was performed by Mucor polymorphosporus AS 3.3443. Six metabolites including five new compounds were obtained, and their structures were elucidated as 10β-hydroxy-9,10-dihydrocurcumenol (2), 2β-hydroxycurcumenol (3), 15-hydroxycurcumenol (4), 12-hydroxycurcumenol (5), 1-hydroxy-4αH-guai1,6,9-triene-2,8-dione (6), and 5-hydroxycarbonyl-1-oxo-3,7dimethylindane (7) by spectroscopic analysis. M. polymorphosporus catalyzed unusual degradation and rearrangement reactions to generate a ring-contracted metabolite (7) of curcumenol (1). Curcumenol (1) and metabolites 4−7 exhibited inhibitory activities against lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages, with 7 exhibiting more potent activity than curcumenol.
properties. Mucor polymorphosporus AS 3.3443 was able to convert curcumenol (1) completely into multiple products with higher polarity by HPLC analysis (Figure 1) and was thus selected for scale-up studies. The preparative biotransformation of curcumenol (1) by M. polymorphosporus AS 3.3443 led to the isolation and characterization of six metabolites (2−7) (Figure 2). The inhibitory activities of curcumenol (1) and its transformed products against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages were evaluated.
Rhizoma Curcumae is an important traditional Chinese medicine and is used to treat certain blood circulation disorders.1 The Chinese Pharmacopoeia has recorded that Rhizoma Curcumae may be the dried rhizomes derived from Curcuma wenyujin Y.H. Chen et C. Ling, C. phaeocaulis Valeton, or C. kwangsiensis S.G. Lee et C.F. Liang.2 Sesquiterpenoids and diarylheptanoids3−8 are considered to be the primary bioactive constituents of Rhizoma Curcumae, which have shown antiinflammatory,8−10 cytotoxic,11−13 antioxidant,14,15 vasorelaxant,16 hepatoprotective,17 and neuroprotective18 activities. Curcumenol (1), a guaiane-type sesquiterpenoid, is one of the major constituents of Rhizoma Curcumae. It is abundant in C. phaeocaulis Valeton.8 Previous studies have indicated that curcumenol (1) exhibits inhibitory effects against nitric oxide production8 and could therefore be considered to be a promising anti-inflammatory agent. However, the poor water solubility of curcumenol (1) could limit its bioavailability and possible clinical applications.19,20 Chemical approaches are the routine solution for structure modification of natural products but are often hampered by problems such as regio- or stereoselectivity, complex reaction steps, and harsh reaction conditions. Microbial biotransformation, on the contrary, occurs under mild reaction conditions.21−24 This provides a useful alternative to chemical approaches and often selectively modifies specific positions of natural product structures.25−27 Screening of curcumenol (1) was carried out with 30 fungal cultures to select the strains that could transform the substrates to a series of metabolites with improved physicochemical © 2015 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Metabolite 2 was assigned the molecular formula C15H24O3 according to the HRESIMS (m/z 275.1617 [M + Na]+) and 13 C NMR spectroscopic data (Table 1). Comparison of the 13C NMR data of 2 with those of curcumenol (1)28 indicated the absence of two sp2 carbon signals and the presence of two sp3 signals at δC 72.7 and 49.5 in 2. In the 1H NMR spectrum of 2, the olefinic proton at δH 5.76 for curcumenol (1) was replaced by two resonances at δH 1.90 (2H, m), which correlated to the carbon signal at δC 49.5 based on the HSQC spectrum. The NMR data of 2 suggested hydration of a Δ9,10 double bond and the presence of a hydroxy group at C-10. This was corroborated by the HMBC correlations of CH3-15 with C-1/C-9/C-10 and H2-9 with C-7/C-8/C-10/C-15. In the NOESY spectrum, correlations from CH3-15 (δH 1.15) to H-1 (δH 1.63), H-2α Received: October 27, 2014 Published: March 30, 2015 674
DOI: 10.1021/np500845z J. Nat. Prod. 2015, 78, 674−680
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Figure 1. HPLC chromatograms of the blank of Mucor polymorphosporus AS 3.3443 (A), curcumenol (1) (B), and the administration of curcumenol (1) for 6 days (C).
(δH 1.76), and H-6α (δH 2.00) indicated an α-orientation for CH3-15 and a β-orientation for OH-10. Therefore, the
structure of 2 was proposed as 10β-hydroxy-9,10-dihydrocurcumenol. 675
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Figure 2. Structures of biotransformed products of curcumenol (1) by Mucor polymorphosporus AS 3.3443 and their proposed biotransformation pathways.
Table 1. 1H and 13C NMR Data for Metabolites 2−4, 6, and 7 in Methanol-d4a 2 no.
δC
1 2
56.4 26.1
3
32.1
4 5 6
41.7 87.1 38.7
7 8 9 10 11 12 13 14 15
135.7 104.0 49.5 72.7 127.5 22.7 19.4 13.0 31.1
a1
3
δH (J in Hz) 1.63, 1.62, 1.76, 1.33, 1.78, 1.89,
m m m m m m
2.48, dt (15.0, 2.0) 2.00, d (15.0)
1.90, m
1.56, 1.73, 0.91, 1.15,
s s d (6.6) s
4
δC
δH (J in Hz)
δC
57.4 72.7
1.99, d (6.6) 4.11, dt (6.6, 3.0)
48.5 28.8
44.4
1.49, m 2.42, m 1.88, m
32.6
39.2 87.2 38.2 137.2 103.4 130.4 135.1 122.7 22.5 19.0 13.1 21.4
2.56, d (16.0) 2.09, d (16.0)
5.77, brs
1.51, 1.73, 0.98, 1.65,
s s d (6.6) s
41.4 87.3 38.2 138.1 102.9 127.2 143.4 123.2 22.6 19.3 12.4 64.6
6
δH (J in Hz) 2.09 (m) 1.88 (m) 1.43 (m) 1.82, m 1.51, m 1.85, m 2.58, brd (15.6) 2.07, brd (15.6)
5.86, brs
1.49, 1.70, 0.92, 3.87,
s s d (6.3) s
δC
7
δH (J in Hz)
138.1 208.5 44.9 37.9 171.7 130.8 163.6 189.5 139.0 144.6 74.7 30.1 29.2 21.2 23.3
δC 209.1 47.2
2.16, dd (18.9, 2.5) 2.78, dd (18.9, 7.2) 3.26, m 7.84, s
6.60, s
1.53, 1.49, 1.29, 2.43,
s s d (7.2) s
33.6 125.3 137.9 131.5 139.7 137.5 162.6 21.8 169.4 18.5
δH (J in Hz) 2.20, dd (19.1, 3.6) 2.86, dd (19.1, 7.6) 3.35, m 7.91, brs 7.68, brs
1.31, d (7.1) 2.53, s
H NMR spectra were measured at 600 MHz. 13C NMR spectra were measured at 150 MHz.
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137.5, 131.5, and 125.3, and four sp3 carbons at δC 33.6, 47.2, 18.5, and 21.8. The NMR data of 7 were analogous to those of 6. The main differences included the absence of two methyl groups (δC 30.1, 29.2; δH 1.53, 1.49) and an oxygenated quaternary carbon (δC 74.7) in 6, suggesting the absence of the 2-hydroxyisopropyl group in 7. In the HMBC spectrum, the correlations of CH3-10 with C-2/C-3/C-9 and H2-2 with C-1/ C-3/C-8/C-9/C-10 (Figure 3) indicated the presence of a 2,3-
Metabolite 3 was deduced to have the molecular formula C15H22O3 from its HRESIMS and 13C NMR data (Table 1). The 1H and 13C NMR spectra closely matched those of curcumenol (1).28 The main differences between these two compounds were the absence of the C-2 methylene signal in 1 and the appearance of an oxymethine carbon signal at δC 72.7 and a one-proton resonance at δH 4.11 (1H, dt, J = 6.6, 3.0 Hz) in 3, indicating hydroxylation at C-2. This finding was confirmed by the HMBC correlations of the H-2 resonance with C-4/C-5 and H-3β with C-14/C-4/C-2. The NOESY correlations from H-2 (δH 4.11) to H-3α (δH 2.42), H-15 (δH 1.65), and H-1 (δH 1.99) and the absence of a correlation from H-2 to CH3-14 (δH 0.98) corroborated the β-orientation of OH-2. On the basis of the above evidence, the structure of 3 was established as 2β-hydroxycurcumenol. Metabolite 4 gave the molecular formula C15H22O3, as indicated by the HRESIMS and 13C NMR data (Table 1). Comparison of the 1H and 13C NMR data of 4 with those of curcumenol (1)28 showed the disappearance of the C-15 methyl signal in 1 and the presence of a hydroxymethyl singlet (δC 64.6; δH 3.87, s) in 4. The HMBC correlations of H2-15 with C-1/C-9/C-10 and H-9 with C-15 established the position of the hydroxy group at C-15. The NOESY correlations from H-9 (δH 5.86) to H2-15 (δH 3.87) and H-1 (δH 1.99) confirmed the above conclusions. Therefore, the structure of 4 was determined as 15-hydroxycurcumenol. Metabolite 6 was determined to have the molecular formula C15H18O3 from its HRESIMS and 13C NMR data (Table 1). Its molecular formula showed two additional indices of hydrogen deficiency when compared to curcumenol (1), suggesting the formation of new double bonds in 6. The 13C NMR spectrum indicated the presence of two carbonyl carbons at δC 208.5 and 189.5, six olefinic carbons at δC 171.7, 163.6, 144.6, 139.0, 138.1, and 130.8, and one oxygenated carbon at δC 74.7. Comparison of the 13C NMR data of 6 with those of phaeocaulisin D8 showed that an oxygen-bearing carbon at δC 84.9 (C-4) and a methylene signal at δC 32.7 (C-2) in the latter compound were replaced by a methine signal at δC 37.9 (C-4) and a carbonyl carbon at δC 208.5 (C-2) in 6, respectively. The HMBC correlations of H-4 with C-14/C-3/C-5/C-1/C-2, H2-3 with C-5/C-14/C-4/C-2, and H-6 with C-4 confirmed the carbonyl group to be located at C-2, and the methine carbon resonance at δC 37.9 was assigned to C-4. In the NOESY spectrum, correlations from H-4 (δH 3.26) to H-3α (δH 2.78) and from CH3-14 (δH 1.29) to H-3β (δH 2.16) indicated a βorientation for CH3-14, identical to that in curcumenol (1). Thus, the structure of 6 was defined as 11-hydroxy-4αH-guai1,6,9-triene-2,8-dione. The molecular formula of metabolite 7 was defined as C12H12O3 from the positive-ion HRESIMS ([M + H]+ at m/z 205.0859) in combination with the 13C NMR spectroscopic data (Table 1). Reaction with Bromocresol Green produced a positive result, confirming the presence of a carboxylic group. The molecular formula and indices of hydrogen deficiency suggested a nor-sesquiterpenoid derivative and the presence of new double bonds in 7 as compared to 1. The 1H NMR spectrum showed two methyl groups at δH 1.31 (3H, d, J = 7.1 Hz) and 2.53 (3H, s), two aromatic proton signals at δH 7.68 (1H, brs) and 7.91 (1H, brs), two methylene resonances at δH 2.20 (1H, dd, J = 19.1, 3.6 Hz) and 2.86 (1H, dd, J = 19.1, 7.6 Hz), and a methine group at δH 3.35 (1H, m). The 13C NMR spectrum exhibited the presence of two carbonyl carbons at δC 209.1 and 169.4, six olefinic carbons at δC 162.6, 139.7, 137.9,
Figure 3. Key NOESY and HMBC correlations of 7.
disubstituted 4-methyl-2-cyclopenten-1-one moiety (ring A), identical to that in 6. The HMBC correlations of H-12 with C6/C-7/C-8, H-6 with C-4/C-8/C-11/C-12, and H-4 with C-3/ C-6/C-8/C-11 (Figure 3) revealed the presence of a 4,5disubstituted 3-methylbenzoic acid moiety (ring B). Thus, metabolite 7 possesses an indane skeleton.29 The NOESY spectrum showed correlations from H-3 (δH 3.35) to H-2α (δH 2.86) and from CH3-10 (δH 1.31) to H-2β (δH 2.20) (Figure 3), suggesting CH3-10 should be β-oriented. In conclusion, the structure of 7 was characterized as 5-hydroxycarbonyl-1-oxo3,7-dimethylindane. Metabolite 5 was established as 12-hydroxycurcumenol, a known compound, by comparison of its spectroscopic data with reported data.30 Possible metabolic pathways are outlined in Figure 2. The regio- and stereoselective hydration of the Δ9,10 double bond and oxygenation at C-2, C-15, and C-12 yielded metabolites 2, 3, 4, and 5 in the biotransformation process, respectively. It is well known that numerous cytochrome P450 monooxygenases (P450s) play a key role in the bioconversion of natural products by fungi, since they have the capability of catalyzing regio- and stereoselective oxygenation reactions.31,32 The formation of metabolite 6 is proposed to involve oxidation, rearrangement, epoxidation, and epoxide hydrolysis reactions. Although the intermediate products of epoxidation and epoxide hydrolysis were not isolated, similar reactions of the exocyclic double bond have been reported for the biotransformation of guaiane-type sesquiterpenoid by this fungus.33 M. polymorphosporus AS 3.3443 has previously been reported to transform other terpenoids, such as dehydrocostuslactone,33 costunolide,33 curdione,34 glycyrrhetinic acid,35,36 dehydrocostuslactone,37 alantolactone,38 and artemisinin.39 However, the rare degradation and rearrangement reactions of curcumenol (1) are now described for the first time. The isopropyl group is removed via the diol intermediate. Subsequent rearrangement affords the aromatic metabolite 7. This reaction is interesting because it permits the formation of ring-contracted derivatives of substrates possessing a seven-membered ring. This type of catalytic capability of M. polymorphosporus to cause degradation and rearrangement of sesquiterpenoid deserves further investigation to enhance the chemical diversity of sesquiterpenoids. Nitric oxide, a signaling molecule, plays an important role in the pathogenesis of inflammation. Inhibitors of NO production 677
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IFFI 02360, A. f lavus AS 3.3554, Cunninghamella elegans AS 3.1207, C. echinulata AS 3.3400, C. blakesleana lender AS 3.970, Fusarium avenaceum AS 3.4594, Mucor polymorphosporus AS 3.3443, M. subtilissimus AS 3.2454, M. spinosus AS 3.3450, M. spinosus AS 3.2450, Paecilomyces varioti IFFI 04024, Penicillium adametzii AS 3.4470, P. janthinellum AS 3.510, P. notatum IFFI 04013, P. urticae IFFI 04015, P. melinii AS 3.4474, Rhizopus chinensis IFFI 03043, R. stolonifer AS 3.2050, Sporotrichum sp. AS 3.2882, Syncephalastrum racemosum AS 3.264, and Trichoderma viride AS 3.2942, were purchased from China General Microbiological Culture Collection Center, Beijing, People’s Republic of China. They were screened for their abilities to convert curcumenol (1) in preliminary work. Culture Medium. All biotransformation experiments were carried out in potato dextrose medium by the following procedure: 200 g of minced husked potato was prepared in 1 L of boiling water for 1 h, the extract was filtered, and to the filtrate (1 L) was added 20 g of glucose. The medium was sterilized in individual Erlenmeyer flasks at 121 °C and 15 psi for 20 min and cooled before incubation. Extraction and Isolation. The fungal strain M. polymorphosporus AS 3.3443 was subcultured three times on potato dextrose agar slants prior to use in order to get maximal enzyme activities. Preliminary screening biotransformation of curcumenol (1) by M. polymorphosporus AS 3.3443 was performed in 250 mL Erlenmeyer flasks containing 100 mL of potato dextrose medium. The flasks were shaken on rotary shakers at 180 rpm and 28 °C for 1 day. Then, 0.2 mL of curcumenol solution in acetone (10 mg/mL) was added to each flask and incubated for 5 days. Both the substrate control (substrate in organism-free culture medium) and culture control (microorganisms in substrate-free culture medium) were incubated under the same conditions to demonstrate the stability of the substrate in the process of incubation. Similarly, a preparative-scale bioconversion was performed in 500 mL Erlenmeyer flasks containing 200 mL of potato dextrose medium, and the microorganisms were precultured for 2 days. Then, 0.5 mL of curcumenol solution in acetone (10 mg/mL) was added to each flask, and the biotransformation was continued for an additional 6 days. A total of 200 mg of 1 was transformed by this strain. The cultures were pooled and filtered, and the filtrates were extracted with EtOAc (3 × 8.0 L). The organic layer was collected and concentrated under vacuum at 40 °C. The crude transformation residues of curcumenol (1) were subjected to silica gel CC (3.0 × 55 cm, 75 g) and eluted with petroleum ether−acetone (30:1, 20:1, 15:1, 10:1, 5:1, 4:1, 1:1 v/v), which yielded 14 fractions (1−14). Fraction 8 (46 mg) was subjected to Sephadex LH-20 CC (1.5 × 30 cm, 10 g) by elution with MeOH to give four subfractions (81−84). Metabolite 2 (6.4 mg, 3.2%) was obtained from fraction 83 through recrystallization from MeOH. Fraction 9 (613 mg) was purified by preparative TLC (CH2Cl2− EtOAc−petroleum ether, 1.2:1:0.5 v/v) to afford 4 (39.6 mg, 19.8%) and 5 (41.2 mg, 20.6%). Fraction 10 (472 mg) was separated by Sephadex LH-20 CC (3.0 × 60 cm, 70 g), eluting with MeOH, to yield five subfractions (101−105). Metabolites 3 (26.6 mg, yield 13.3%) and 7 (29.2 mg, 14.6%) were recrystallized in MeOH from fractions 103 and 105, respectively. Fraction 12 (74 mg) was purified on a Sephadex LH-20 column (1.5 × 30 cm, 10 g) and eluted with MeOH to give 6 (9.0 mg, 4.5%). Metabolite 2: colorless needles (MeOH); mp 142−143 °C; [α]25D +0.3 (c 0.1, MeOH); IR (KBr) νmax 3479, 3344, 2971, 2924, 1690, 1451, 1387, 1113, 974 cm−1; 1H NMR (600 MHz, methanol-d4) and 13 C NMR (150 MHz, methanol-d4), see Table 1; HRESIMS (positive) m/z 275.1617 [M + Na]+ (calcd for C15H24O3Na, 275.1618). Metabolite 3: white powder (MeOH); [α]25D +3 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 217 (4.29) nm; IR (KBr) νmax 3407, 2958, 2930, 1658, 1444, 1375, 1295, 1105, 966 cm−1; 1H NMR (600 MHz, methanol-d4) and 13C NMR (150 MHz, methanol-d4), see Table 1; HRESIMS (positive) m/z 251.1641 [M + H]+ (calcd for C15H23O3, 251.1642). Metabolite 4: colorless needles (acetone); mp 197−198 °C; [α]25D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.36) nm; IR (KBr) νmax 3414, 3246, 2974, 2959, 2924, 1691, 1665, 1451, 995 cm−1; 1H NMR (600 MHz, methanol-d4) and 13C NMR (150 MHz,
represent potential therapeutic agents for inflammatory diseases.40−42 The substrate 1 and its metabolites 2−7 were evaluated for their inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages (Table 2). Curcumenol Table 2. Inhibitory Effects of 1−7 on NO Production Induced by LPS in Macrophages compound 1 2 3 4 5 6 7 indomethacinb
IC50 ± SDa (μM) 5.4 >50 18.5 6.4 9.6 6.4 2.8 14.1
± 0. 64 ± ± ± ± ± ±
1.08 0.51 0.47 0.55 0.16 0.69
a
Inhibitory effects of compounds 1−7 against LPS-induced NO production in RAW264.7 macrophages. bIndomethacin was used as positive control.
(1) and metabolites 4−7 showed inhibitory effects, especially 7, which demonstrated the highest activity, with an IC50 value of 2.8 ± 0.16 μM. By comparison with 2 and 3, metabolites 4 and 5 have a hydroxy group substituent at the side chain of the seven-membered ring, indicating that the hydroxy group location is critical to the inhibition of the NO production. The common structural feature of metabolites 6 and 7 is the presence of an aromatic moiety in their structures, which might also be an important functionality to increase the resultant activity. The above results showed the potential of these compounds as promising anti-inflammatory agents for possible further investigation and development.
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were determined with an X-5 hot stage microscope melting point apparatus (uncorrected). Optical rotations were measured on a PerkinElmer 241 MC polarimeter. UV spectra were recorded on a Shimadzu UV 2201 spectrophotometer, and IR spectra on a Bruker IFS 55 spectrometer. The NMR spectra were recorded on a Bruker ARX-600 spectrometer. The chemical shifts are relative to TMS and expressed in δ (ppm), and coupling constants (J) are reported in hertz (Hz). HRESIMS data were obtained on an Agilent 6210 TOF mass spectrometer. Samples were analyzed on an Agilent 1260 equipped with a diode array detector at 230 nm and an Agilent Zorbax SB-C18 column (4.6 mm × 150 mm, 5 μm). Mobile phase: 30:70 (v/v) MeOH−H2O (0.05% HCOOH) for 10 min followed by a linear gradient to 60:40 (v/v) within 40 min and held for an additional 15 min. The column temperature was set at 30 °C, and the flow rate was 1.0 mL/min. Silica gel GF254 prepared for TLC and silica gel (200−300 mesh) for column chromatography (CC) were purchased from Qingdao Marine Chemical Factory (Qingdao, People’s Republic of China). Sephadex LH-20 was purchased from Pharmacia (Uppsala, Sweden). All reagents were HPLC or analytical grade and were obtained from Tianjin DaMao Chemical Company (Tianjin, People’s Republic of China). Spots were visualized on TLC plates under UV light or by heating after spraying with anisaldehyde−H2SO4 reagent. Substrates. Curcumenol (1) was isolated from the rhizomes of C. phaeocaulis and was authenticated by comparing its physical and spectroscopic data with reported values.8,43 Its purity was determined to be 98% by HPLC analysis. Microorganisms. The 30 fungi, Absidia spinosa AS 3.3391, A. coerulea AS 3.3382, Alternaria alternata AS 3.4578, A. alternata AS 3.577, A. longipes AS 3.2875, Aspergillus niger AS 3.739, A. niger AS 3.795, A. avenaceus AS 3.4454, A. carbonarius IFFI 02087, A. candidus 678
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methanol-d4), see Table 1; HRESIMS (positive) m/z 273.1467 [M + Na]+ (calcd for C15H22O3Na, 273.1461). Metabolite 6: pale yellow oil; [α]25D −0.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 232 (4.47), 245 (4.46) nm; IR (KBr) νmax 3415, 2967, 2929, 1710, 1601, 1578, 1402, 1380, 1276, 991 cm−1; 1H NMR (600 MHz, methanol-d4) and 13C NMR (150 MHz, methanol-d4), see Table 1; HRESIMS (positive) m/z 247.1327 [M + H]+, (calcd for C15H19O3, 247.1329). Metabolite 7: white powder (MeOH); [α]25D −1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 256 (4.11) nm; IR (KBr) νmax 3431, 2959, 2927, 1709, 1632, 1593, 1457, 1241 cm−1; 1H NMR (600 MHz, methanol-d4) and 13C NMR (150 MHz, methanol-d4), see Table 1; HRESIMS (positive) m/z 205.0859 [M + H]+ (calcd for C12H13O3, 205.0856). Bioassay for NO Production. Determination of NO production was performed by measuring the accumulation of nitrite in the culture supernatant using the Griess reagent, as previously described.6,8,44 Briefly, RAW 264.7 cells were seeded into 96-well culture plates with 1 × 105 cells per well and stimulated with 1 μg/mL of LPS in the presence or absence of test compounds. After incubation at 37 °C for 1 day, 100 μL of cell-free supernatant was mixed with 100 μL of Griess reagent. Absorbance values were measured with a microplate reader at 540 nm. Nitrite concentrations and the inhibitory rates were calculated through a calibration curve prepared with sodium nitrite standards. Experiments were operated in triplicate, and data are described as mean ± SD of three independent experiments.
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ASSOCIATED CONTENT
S Supporting Information *
HRESIMS, 1D NMR, and 2D NMR spectra for new metabolites 2−4, 6, and 7 are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86-22-59596223. E-mail: fengqiu20070118@163. com. 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 (NSFC) (Grant No. 30973630), the Medicinal Chemistry Subject Construction Project of Shenyang Pharmaceutical University, and the Program for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University. We thank P. Owusu Donkor (University of Ghana, School of Pharmacy) for the language check and for the editorial assistance.
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