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Stereo- and regiospecific biotransformation of curcumenol by four fungal strains
Journal of Molecular Catalysis B: Enzymatic 115 (2015) 13–19
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Stereo- and regiospecific biotransformation of curcumenol by four fungal strains Meng Zhang a,1 , Qian Zhao a,1 , Yan-Yan Liang a , Jiang-Hao Ma a , Li-Xia Chen a,∗∗ , Xue Zhang a , Li-Qin Ding b , Feng Zhao c , Feng Qiu a,b,∗ a 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, China b School of Chinese Materia Medica, Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China c School of Pharmacy, Yantai University, No. 32 Road QingQuan, Laishan District, Yantai 264005, China
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 2 January 2015 Accepted 9 January 2015 Available online 20 January 2015 Keywords: Curcumenol Biotransformation Fungal strains Nitric oxide
a b s t r a c t Biotransformations of curcumenol (1) were performed by four fungal strains, Mucor spinosus AS 3.2450, Penicillium urticae IFFI 04015, Cunninghamella echinulata AS 3.3400, Aspergillus carbonarius IFFI 02087. Five metabolites were prepared in the biotransformation process of 1, and their structures were elucidated as 15-hydroxycurcumenol (2), 1˛-hydroxycurcumenol (3), 14-hydroxycurcumenol (4), 3ˇhydroxycurcumenol (5) and 12-hydroxycurcumenol (6) by spectroscopic data analysis. Among them, metabolites 2–5 are novel. All of these four fungal strains showed the ability of highly stereo- and regiospecific hydroxylation for the substrate (1), which could be used as tools for preparing the hydroxylated derivatives and in vivo metabolites of curcumenol. In addition, the inhibitory effects of substrate and obtained products on nitric oxide production in lipopolysaccaride-activated macrophages were evaluated. The substrate (1) and metabolites 2, 5, and 6 showed significant inhibitory effects. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Rhizoma Curcumae is an important crude drug frequently listed in prescriptions of traditional Chinese medicine for the treatment of Oketsu syndromes [1] which are caused by the obstruction of blood circulation, such as arthralgia, psychataxia, and dysmenorrhea. Three species of Rhizoma Curcumae (Curcuma phaeocaulis Valeton, C. kwangsiensis S.G. Lee & C.F. Liang, and C. wenyujin Y.H. Chen & C. Ling) are officially recorded as traditional Chinese medicine in the Chinese Pharmacopoeia [2]. Sesquiterpenoids and diarylheptanoids [3–8] are considered as the main bioactive constituents of Rhizoma Curcumae, possessing anti-inflammatory [8–10], anti-tumor [11–13], antioxidant [14,15], vasorelaxant [16], hepatoprotective [17] and neuroprotective [18] activities.
∗ Corresponding author at: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China. Tel.: +86 22 59596223; fax: +86 22 59596223. ∗∗ Corresponding author at: Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China. Tel.: +86 24 23986515; fax: +86 24 23986515. E-mail addresses: [email protected] (F. Qiu), [email protected] (L.-X. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.molcatb.2015.01.005 1381-1177/© 2015 Elsevier B.V. All rights reserved.
Curcumenol is one of the major guaiane-type sesquiterpenoid constituents of Rhizoma Curcumae, having an especially high content in the species of C. phaeocaulis Valeton [8]. Our previous studies indicated that curcumenol displayed strong inhibitory effects on nitric oxide production [8], which could be considered as a potential anti-inflammatory agent. However, the poor water-solubility would limit its bioavailability, absorption and clinical application [19,20]. Up to now, no any investigation on the structural derivatization of curcumenol has been reported owing to its limited reaction points for chemical modification. Microbial transformation provides an important tool for structure modification of natural products. And many new chemical derivatives with potent bioactivities and improved physicochemical characteristics were found in the process of biotransformation [21,22]. Microbial transformation can also be used as an in vitro model to prepare the in vivo metabolites [23,24]. This method exhibited many advantages, such as its high stereo- and regioselectivity, ease of handling, low cost, and environmental-friendly nature [25–28]. In the present work, the biocatalysis ability of 30 fungal strains to convert curcumenol (1) was screened in an attempt to obtain new and bioactive derivatives of sesquiterpenoid and improve its physicochemical properties. Four fungal strains, Mucor spinosus AS 3.2450, Penicillium urticae IFFI 04015, Cunninghamella
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echinulata AS 3.3400 and Aspergillus carbonarius IFFI 02087 were chosen because they displayed the characteristics of high stereoand regioselectivity for the biotransformations of curcumenol. Five metabolites, four of which have not previously been reported, were isolated and identified in the biotransformation processes of 1. In addition, the inhibitory effects of the substrate and obtained products on nitric oxide production in lipopolysaccaride-activated macrophages were also evaluated. 2. Experimental 2.1. General experimental procedures The NMR spectra were performed on Bruker ARX-600 spectrometer, using TMS as internal standard. Chemical shifts were expressed in ı (ppm) and coupling constants (J) were reported in Hertz (Hz). Optical rotation values were measured on a PerkinElmer 241 MC polarimeter. UV spectra were measured with a Shimadzu UV-1700 spectrophotometer. IR spectra were recorded with a Bruker IFS 55 spectrometer. HRESIMS spectra were obtained on an Agilent 6210 TOF mass spectrometer, in m/z. Melting points were determined with an X-5 hot stage microscope melting point apparatus (uncorrected). Silica gel GF254 prepared for TLC and silica gel (200–300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, People’s Republic of China). Sephadex LH-20 was a product of Pharmacia. All the analytic reagents were analytical grade and purchased from Tianjin DaMao Chemical Company (Tianjin, China). Spots were detected on TLC plates under UV light or by heating after spraying with anisaldehyde-H2 SO4 reagent. 2.2. Substrates Curcumenol was isolated from the rhizomes of C. phaeocaulis by the author (Jiang-Hao Ma), and was characterized by comparison of the NMR data with those reported in Refs. [8,29]. Its purity was determined to be 98% by HPLC analysis. 2.3. Microorganism 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 IFFI 02360, A. flavus 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 in Beijing, China. They were screened for their abilities to transform curcumenol (1) in the preliminary test. 2.4. Medium All culture and biotransformation experiments were performed in potato medium as following procedure: 200 g of minced husked potato was boiled in water for 1 h, then the extract was filtered and the filtrate was added with water to 1 L after addition of 20 g of glucose. The broth was autoclaved in individual Erlenmeyer flask at 121 ◦ C and 15 psi for 20 min and cooled before incubation.
2.5. Biotransformation procedures The four fungal strains (Mucor spinosus AS 3.2450, Penicillium urticae IFFI 04015, Cunninghamella echinulata AS 3.3400 and Aspergillus carbonarius IFFI 02087) had been sub-cultured for three times on potato dextrose agar slants before use to obtain maximal enzyme activities. Preliminary screening biotransformations of curcumenol (1) by microorganisms were carried out in 250 mL Erlenmeyer flasks containing 100 mL of potato medium. The flasks were placed on rotary shakers, operating at 180 rpm and 28 ◦ C. After 1 day of culture, the acetone (0.2 mL) containing curcumenol (2 mg) was added into each biotransformation flask, and the biotransformations were continued under the same conditions for an additional 5 days. Both substrate and organism controls were incubated under the same conditions in order to demonstrate that substrate was stable in the control culture. Similarly, preparative scale biotransformations were carried out in 500 mL Erlenmeyer flasks containing 200 mL of potato medium, and the microorganisms were pre-cultured under culture conditions for 2 days, respectively. The acetone (0.5 mL) containing curcumenol (5 mg) was added into each biotransformation flask, and the biotransformations were continued under the same conditions for an additional 7 days. A total of 200 mg of 1 was transformed by the four strains, respectively. When the biotransformation finished, the broths of substrate were filtered and the filtrates were extracted with the equal volume of ethyl acetate for three times, respectively. The organic phase was collected and concentrated to dry under reduced pressure at 40 ◦ C for further isolation. 2.6. Isolation and purification of metabolites The crude transformation residues of curcumenol (1) by M. spinosus AS 3.2450 were subjected to column chromatography (CC) over silica gel and eluted with the mixtures of petroleum ether–acetone (100:1, 50:1, 20:1, 15:1, 6:1, 2:1), which yielded ten fractions (1–10). Fraction 6 was purified by preparative TLC (CH2 Cl2 /EtOAc/petroleum ether, 1.2:1:0.5) to afford 2 (11.2 mg, 5.6%) and 6 (16.6 mg, 8.3%). Fraction 9 was submitted to a silica gel column eluted with petroleum ether–ethyl acetate (20:1, 15:1, 10:1, 5:1) to give 3 (156.6 mg, 78.3%). The crude transformation residues of curcumenol (1) by P. urticae IFFI 04015 were submitted to silica gel CC and eluted with the mixtures of petroleum ether–acetone (50:1, 30:1, 15:1, 10:1, 6:1, 3:1, 1:1), which yielded twelve fractions (1–12). Fraction 5 was purified by preparative TLC (CH2 Cl2 /EtOAc/petroleum ether, 1.2:1:0.1) to obtain 2 (137.6 mg, 68.8%) and 6 (36.4 mg, 18.2%). Fraction 6 was purified by preparative TLC (CH2 Cl2 /EtOAc/petroleum ether, 1.2:1:0.5) to afford 4 (7.8 mg, 3.9%). The crude transformation residues of curcumenol (1) by C. echinulata AS 3.3400 were subjected to silica gel CC and eluted with the mixtures of petroleum ether–ethyl acetate (30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1), which yielded fourteen fractions (1–14). Fraction 6 was chromatographed on a Sephadex LH-20 column eluted with MeOH to yield 5 (47.6 mg, 23.8%). Fraction 7 was purified by preparative TLC (CH2 Cl2 /EtOAc/petroleum ether, 1.2:1:0.5) to afford 6 (108.0 mg, 54.0%) and 2 (20.2 mg, 10.1%). The crude transformation residues of curcumenol (1) by A. carbonarius IFFI 02087 were subjected to silica gel CC and eluted with the mixtures of petroleum ether–acetone (100:1, 50:1, 30:1, 15:1, 10:1, 5:1, 3:1), which yielded nine fractions (1–9). Fraction 7 was recrystallized with acetone to afford 6 (186.6 mg, 93.3%). Metabolite 2: colorless needles (acetone); mp 197–198 ◦ C; [␣]25 D +3.46 (c 0.1, MeOH); UV (MeOH) max (log ε): 217 (4.36) nm;
M. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 115 (2015) 13–19
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Table 1 1 H NMR (ı) spectroscopic data (600 MHz, J in Hz) of metabolites 2–5. No.
1a
2a
3a
3b
4a
5a
H-1 H-2
H-7 H-8 H-9 H-10 H-11 H-12 H-13 H-14
1.96 (m) 1.90 (m) 1.57 (m) 1.86 (m) 1.71 (m) 1.87 (m) – 2.65 (dt, 15.5) 2.11 (brd, 15.5) – – 5.76 (brs) – – 1.60 (s) 1.80 (s) 1.00 (d, 6.2)
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 (s) 1.70 (s) 0.92 (d, 6.3)
– 1.94 (m) 1.74 (m) 1.87 (m) 1.42 (m) 2.14 (m) – 2.57 (brd, 16.5) 2.34 (brd, 16.5) – – 5.60 (q, 1.5) – – 1.54 (s) 1.69 (s) 0.91 (d, 6.9)
– 2.25 (m) 2.12 (m) 2.13 (m) 1.70 (m) 2.55 (m) – 3.00 (brd, 16.0) 2.52 (brd, 16.0) – – 6.14 (brs) – – 1.34 (s) 2.02 (s) 1.12 (d, 6.9)
1.86 (m) 2.31 (m) 1.44 (m) 3.94 (ddd, 12.6, 6.6, 2.4) 1.83 (m) – 2.57 (brd, 15.5) 2.03 (brd, 15.5) – – 5.61 (brs) – – 1.47 (s) 1.65(s) 0.94 (d, 7.2)
H-15
1.66 (s)
3.87 (s)
1.61 (s)
1.84 (s)
1.90 (m) 1.92 (m) 1.45 (m) 1.87 (m) 1.64 (m) 2.05 (m) – 2.80 (brd, 15.6) 2.18 (brd, 15.6) – – 5.59 (brs) – – 1.49 (s) 1.68 (s) 3.71 (dd, 11, 5.4) 3.56 (dd, 11, 6.9) 1.55 (s)
H-3 H-4 H-5 H-6
a b
IR max (KBr, cm−1 ): 3414, 3246, 2974, 2959, 2924, 1691, 1665, 1451, 995; HRMS (ESI) (positive) m/z: 273.1467 [M+Na]+ (calcd. for C15 H22 O3 Na, 273.1461); 1 H NMR (600 MHz, CD3 OD) and 13 C NMR (150 MHz, CD3 OD), see Tables 1 and 2. Metabolite 3: colorless oil; [␣]25 D +2.87 (c 0.1, MeOH); UV (MeOH) max (log ε): 217 (4.31) nm; IR max (KBr, cm−1 ): 3406, 2932, 2873, 1631, 1447, 1383, 1104, 974; HRMS (ESI) (positive) m/z: 273.1466 [M+Na]+ (calcd. for C15 H22 O3 Na, 273.1461); 1 H NMR (600 MHz, CD3 OD) and 13 C NMR (150 MHz, CD3 OD), see Tables 1 and 2. Metabolite 4: colorless oil; [␣]25 D +1.60 (c 0.05, MeOH); UV (MeOH) max (log ε): 211 (4.19) nm; IR max (KBr, cm−1 ): 3403, 2932, 2873, 1649, 1442, 1383, 1138, 966; HRMS (ESI) (positive) m/z: 251.1644 [M+H]+ (calcd. for C15 H23 O3 , 251.1642); 1 H NMR (600 MHz, CD3 OD) and 13 C NMR (150 MHz, CD3 OD), see Tables 1 and 2. Metabolite 5: white powders (MeOH); [␣]25 D +2.03 (c 0.16, MeOH); UV (MeOH) max (log ε): 213 (4.20) nm; IR max (KBr, cm−1 ): 3461, 2929, 2855, 1657, 1453, 1383, 1119, 963; HRMS (ESI) (positive) m/z: 273.1466 [M+Na]+ (calcd. for C15 H22 O3 Na, 273.1461); 1 H NMR (600 MHz, CD3 OD) and 13 C NMR (150 MHz, CD3 OD), see Tables 1 and 2.
Table 2 13 C NMR (ı) spectroscopic data (150 MHz) of metabolites 2–5. No.
1a
2a
3a
3b
4a
5a
C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15
51.4 27.8 31.4 40.6 85.9 37.4 139.2 101.7 125.9 137.5 122.4 22.5 19.1 12.1 21.4
48.5 28.8 32.6 41.4 87.3 38.2 138.1 102.9 127.2 143.4 123.2 22.6 19.3 12.4 64.6
83.5 37.5 30.4 38.3 89.9 32.2 136.5 102.8 128.9 140.4 123.2 22.6 19.1 12.5 17.0
82.4 37.5 30.5 37.9 88.9 32.3 137.7 102.4 129.5 139.9 121.3 22.5 19.4 13.0 17.5
53.4 29.8 28.4 48.2 86.9 39.8 137.9 103.0 127.6 139.1 122.7 22.5 19.2 62.9 23.3
50.6 40.5 76.7 45.7 87.0 38.6 137.6 103.4 128.1 139.6 123.2 22.6 19.3 7.1 21.1
a b
1.54 (3H)
Measured in CD3 OD. Measured in C5 D5 N.
Measured in CD3 OD. Measured in C5 D5 N.
2.7. HPLC analysis The 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 m). Mobile phase: MeOH–H2 O (0.05% HCOOH) (30:70 (v/v)) for 10 min followed by a linear gradient to (60:40 (v/v)) within 40 min and held for an additional 15 min. The flow rate was 1.0 mL/min, and column temperature was set at 30 ◦ C. 2.8. Bioassay for NO Production Mouse monocyte-macrophage RAW 264.7 cells (ATCC TIB-71) were purchased from the Chinese Academy of Science. RPMI 1640 medium, penicillin, streptomycin, and fetal bovine serum were purchased from Invitrogen (New York). Lipopolysaccharide (LPS), dimethylsufoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyltetrazolium bromide (MTT), and indomethacin were obtained from Sigma Co. RAW 264.7 cells were suspended in RPMI 1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 g/mL), and 10% heat-inactivated fetal bovine serum. The cells were harvested with trypsin and diluted to a suspension in fresh medium. The cells were seeded in 96-well plates with 1 × 105 cells/well and allowed to adhere for 2 h at 37 ◦ C in 5% CO2 in air. Then, the cells were treated with 1 g/mL of LPS for 24 h with or without various concentrations of test compounds. DMSO was used as a solvent for the test compounds, which were applied at a final concentration of 0.2% (v/v) in cell culture supernatants. NO production was determined by measuring the accumulation of nitrite in the culture supernatant using Griess reagent [30]. Briefly, 100 L of the supernatant from incubates was mixed with an equal volume of Griess reagent (0.1% N-[1-naphthyl]ethylenediamine and 1% sulfanilamide in 5% H3 PO4 ). Cytotoxicity was determined by the MTT colorimetric assay, after 24 h incubation with test compounds. The concentration of NO2 − was calculated by a working line from 0, 1, 2, 5, 10, 20, 50, and 100 M sodium nitrite solutions, and the inhibitory rate on NO production induced by LPS was calculated by the NO2 − levels as follows: Inhibitory rate (%) = 100 ×
− [NO− 2 ]LPS − [NO2 ]LPS+sample − [NO− 2 ]LPS − [NO2 ]untreated
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Fig. 1. Structures of biotransformed products of curcumenol (1).
Experiments were performed in triplicate, and data are expressed as the mean ± SD of three independent experiments. 3. Results and discussion 3.1. Identification of biotransformation products Metabolite 2 was obtained as colorless needles (acetone). The HRMS (ESI) exhibited the quasi-molecular ion peak [M+Na]+ at m/z 273.1467, accordingly to its molecular formula of C15 H22 O3 in combination with the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2). Comparison of the 13 C NMR data of 2 with those of curcumenol (1) [31] showed that the absence of one methyl signal at ıC 21.4 in 1, and the appearance of one hydroxymethyl signal at ıC 64.6 in 2. The HSQC spectrum exhibited that the proton resonances at ıH 3.87 (2H, s) correlated to the hydroxymethyl carbon signal at ıC 64.6. The HMBC correlations of the proton resonance at ıH 3.87 with C-1 (ıC 48.5), C-9 (ıC 127.2) and C-10 (ıC 143.4), and H-9 (ıH 5.86) with the hydroxymethyl carbon signal at ıC 64.6, confirmed that the hydroxyl group was linked to C-15 in 2. The obvious NOESY correlations from H-9 (ıH 5.86) to H2 -15 (ıH 3.87) and H-1 (ıH 1.99) further corroborated the above conclusion. Therefore, 2 was established as 15-hydroxycurcumenol (Fig. 1). Metabolite 3 was obtained as colorless oil. The molecular formula C15 H22 O3 was drawn from its HRMS (ESI) which gave the ion peak [M+Na]+ at m/z 273.1466, and its 1 H and 13 C NMR spectroscopic data (Tables 1 and 2). By comparing of the 13 C NMR spectrum of 3 with that of curcumenol (1) [31], one bearing-oxygen carbon
signal at ıC 83.5 was observed in 3, while the tertiary carbon signal at ıC 51.4 in 1 was disappeared. And also, the chemical shift value of C-5 shifted downfield to ıC 89.9, and C-4, C-6 and C-15 shifted upfield to ıC 38.3, 32.2 and 17.0, respectively. It is indicated the hydroxylation of C-1. The HMBC correlations of the proton resonances of H-15 (ıH 1.61), H-2 (ıH 1.74), H-9 (ıH 5.60), H-3ˇ (ıH 1.42) and H2 -6 (ıH 2.34, 2.57) with C-1 (ıC 83.5) further confirmed that the hydroxyl group was linked to C-1 in 3. The pyridineinduced solvent shifts [32,33] (Table 1) for H-3˛ (ı methanol-ı pyridine = −0.26 ppm), H-4˛ (−0.41 ppm) and H-6˛ (−0.43 ppm), supported ˛-orientation of the OH group at C-1. On the basis of the above evidence, 3 was established as 1˛-hydroxycurcumenol (Fig. 1). Metabolite 4 was obtained as colorless oil. The HRMS (ESI) exhibited the quasi-molecular ion peak [M+H]+ at m/z 251.1644, accordingly to its molecular formula of C15 H22 O3 by combining the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2). Comparison of the 13 C NMR data of 4 with those of curcumenol (1) [31] showed that the absence of one methyl signal at ıC 12.1 in 1, and the appearance of one hydroxymethyl signal at ıC 62.9 in 4. The 1 H NMR spectra of 4 and 1 were very similar except for the disappearance of a methyl resonance at ıH 1.00 (3H, d, J = 6.2 Hz) in 1, and the presence of two proton signals at ıH 3.71 (1H, dd, J = 11.0, 5.4 Hz) and 3.56 (1H, dd, J = 11.0, 6.9 Hz) in 4. It suggested that C-14 might be hydroxylated. The HMBC correlations of H-4 (ıH 2.05) with C-14 (ıC 62.9), and H2 -14 (ıH 3.71, 3.56) with C-3 (ıC 28.4), C-4 (ıC 48.2) and C-5 (ıC 86.9), as well as the NOESY correlations from H2 -14 (ıH 3.71, 3.56) to H-6 (ıH 2.80) and H-3 (ıH 1.64) confirmed that the
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Fig. 2. HPLC chromatograms of biotransformation of curcumenol (1) by Mucor spinosus AS 3.2450 (A); by Penicillium urticae IFFI 04015 (B); by Cunninghamella echinulata AS 3.3400 (C); by Aspergillus carbonarius IFFI 02087 (D). Peaks 2–6 were: 15-hydroxycurcumenol (2), 1˛-hydroxycurcumenol (3), 14-hydroxycurcumenol (4), 3ˇ-hydroxycurcumenol (5), 12-hydroxycurcumenol (6).
hydroxyl group was linked to C-14 in 4. On the basis of the above evidence, 4 was established as 14-hydroxycurcumenol (Fig. 1). Metabolite 5, white powders (MeOH). The molecular formula of C15 H22 O3 was inferred from positive HRMS (ESI) analysis ([M+Na]+ at m/z 273.1466) in combination with the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2). Comparison of the 13 C NMR data
of 5 with those of curcumenol (1) [31] showed that the absence of one methylene signal at ıC 31.4 in 1, and the appearance of one bearing-oxygen carbon signal at ıC 76.7 in 5. The HSQC spectrum exhibited that the proton resonance at ıH 3.94 (1H, ddd, J = 12.6, 6.6, 2.4 Hz) correlated to the bearing-oxygen carbon signal at ıC 76.7. The HMBC correlations of the proton signal at ıH 3.94 with
Fig. 3. Time courses of biotransformation of curcumenol (1) by Mucor spinosus AS 3.2450 (A); by Penicillium urticae IFFI 04015 (B); by Cunninghamella echinulata AS 3.3400 (C); by Aspergillus carbonarius IFFI 02087 (D).
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Table 3 Inhibitory effects of compounds 1–6 on NO production induced by LPS in macrophages. Compounds
IC50 ± SDa (M)
1 2 3 4 5 6 Indomethacinb
5.42 6.44 33.85 25.37 10.42 9.64 14.1
± ± ± ± ± ± ±
0.64 0.51 2.44 1.49 0.93 0.47 0.69
a
Inhibitory effects of compounds 1–6 against LPS-induced NO production in RAW264.7 macrophages. b Indomethacin was used as positive control.
C-1 (ıC 50.6) and C-5 (ıC 87.0), H-2ˇ (ıH 1.44) with C-1 (ıC 50.6), C-4 (ıC 45.7) and the carbon signal at ıC 76.7, and H-14 (ıH 0.94) with C-4 (ıC 45.7), C-5 (ıC 87.0) and the carbon signal at ıC 76.7, confirmed that the hydroxyl group was linked to C-3 in 5. The obvious NOESY correlations from H-3 (ıH 3.94) to H-2˛ (ıH 2.31) and H-4 (ıH 1.83) corroborated the ˇ-orientation of 3-OH. Therefore, 5 was established as 3ˇ-hydroxycurcumenol (Fig. 1). Metabolite 6 was established as 12-hydroxycurcumenol (Fig. 1) by comparison of its spectroscopic data with those reported in the literature [34]. 3.2. Metabolic profile by HPLC analysis After 7 days of incubation, HPLC analysis showed a total of five metabolites in the broths containing four fungal strains. The HPLC profiles are given in Fig. 2. The analysis results revealed that metabolites 2 and 3 were the dominant biotransformation products of curcumenol (1) by P. urticae IFFI 04015 and M. spinosus AS 3.2450, respectively, while metabolite 6 is the common dominant products of C. echinulata AS 3.3400 and A. carbonarius IFFI 02087. Although the yield of metabolite 6 was low in the transformation process of curcumenol (1) by M. spinosus AS 3.2450 and P. urticae IFFI 04015, it is a common product of these four fungal strains. The stereo- and regiospecific hydroxylation at C-15, C-1 and C-14 by these four fungal strains yielded metabolites 2, 3 and 6, respectively. It is well known that numerous cytochrome P450 monooxygenases (P450s) play a key role in the process of bioconversion and metabolism of natural products by fungi [35,36]. The biotransformation time-course curves of 1 by four fungal strains (Fig. 3) demonstrated that all of the fungi began to exhibit strong bioconversion abilities from the third day. In our previous investigation [37], another similar guaiane-type sesquiterpenoid from Rhizoma Curcumae, curcumol, undergoes the extensive hydroxylation at C-2 or C-3 or C-15 catalyzed by hydroxylase in rats. We had successfully obtained its in vivo metabolite intermediate, 3-hydroxycurcumol, by the method of microbial transformation [38,39]. The hydroxylated derivative of curcumenol at C-15 was prepared in this research, which implied that they could be used as the standard references for investigating in vivo metabolism of curcumenol in the further study. 3.3. Inhibitory effects on NO production induced by LPS in macrophages Nitric oxide (NO) plays an important role in the inflammatory process, and an inhibitor of NO production may be considered as a potential anti-inflammatory agent [40–42]. The substrate 1 and its metabolites 2–6 were examined for their inhibitory effects on NO production induced by LPS in macrophages (Table 3). The substrate 1, and metabolites 2, 5 and 6 showed strong inhibition of NO production induced by LPS. Metabolites 2 and 6 had a hydroxyl group
substitution at the side chain of the seven-member ring, and 5 had a hydroxyl group substitution at C-3 position of the five-member ring. However, the hydroxylation of C-1 or C-14 in metabolite 3 or 4 reduced their bioactivities of NO inhibition. The above results indicated that the location of hydroxyl group substitution played an important role in rendering the NO inhibitory effects. 4. Conclusion Five metabolites, four of which have not previously been reported, were obtained and identified after incubation of curcumenol by M. spinosus AS 3.2450, P. urticae IFFI 04015, C. echinulata AS 3.3400 and A. carbonarius IFFI 02087. All of these metabolites are the hydroxylated products of the substrate. The highly stereoand regiospecific hydroxylation of curcumenol by these four fungal strains was discovered, which could be used as tools for obtaining hydroxylated derivatives of curcumenol to improve its watersolubility, and preparing its in vivo metabolites. The difference of hydroxylated sites of curcumenol catalyzed by four fungal strains remarkably affects their anti-inflammatory activity, which would provide vital information in developments of anti-inflammatory agent. 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. References [1] Y. Sasaki, H. Goto, C. Tohda, F. Hatanaka, N. Shibahara, Y. Shimada, K. Terasawa, K. Komatsu, Biol. Pharm. Bull. 26 (2003) 1135–1143. [2] Pharmacopoeia Commission of People’s Republic of China, Pharmacopoeia of the People’s Republic of China (Part 1), Chinese Medical Science and Technology Press, Beijing, China, 2010, pp. 257–258. [3] Y. Lou, F. Zhao, H. He, K.F. Peng, X.H. Zhou, L.X. Chen, F. Qiu, J. Asian Nat. Prod. Res. 11 (2009) 737–747. [4] Y. Lou, F. Zhao, Z.H. Hu, K.F. Peng, X.C. Wei, L.X. Chen, F. Qiu, Helv. Chim. Acta 92 (2009) 1665–1672. [5] Y. Lou, F. Zhao, H. He, K.F. Peng, L.X. Chen, F. Qiu, Chem. Biodivers. 7 (2010) 1245–1253. [6] J. Li, F. Zhao, M.Z. Li, L.X. Chen, F. Qiu, J. Nat. Prod. 73 (2010) 1667–1671. [7] J. Li, C.R. Liao, J.Q. Wei, L.X. Chen, F. Zhao, F. Qiu, Bioorg. Med. Chem. Lett. 21 (2011) 5363–5369. [8] Y. Liu, J.H. Ma, Q. Zhao, C.R. Liao, L.Q. Ding, L.X. Chen, F. Zhao, F. Qiu, J. Nat. Prod. 76 (2013) 1150–1156. [9] C. Tohda1, N. Nakayama, F. Hatanaka1, K. Komatsu, Evid.-Based Complement. Alternat. 3 (2006) 255–260. [10] J.S. Jurenka, Altern. Med. Rev. 14 (2009) 141–153. [11] M.X. Xu, L.L. Zhao, C.Y. Deng, L. Yang, Y. Wang, T. Guo, L.F. Li, J.P. Lin, L.R. Zhang, Int. J. Oncol. 43 (2013) 1951–1959. [12] L. Yan, J.A. Yee, J. Cao, Anticancer Res. 33 (2013) 3153–3162. [13] Q. Du, B. Hu, H.M. An, K.P. Shen, L. Xu, S. Deng, M.M. Wei, Oncol. Rep. 29 (2013) 1851–1858. [14] Q.F. Tao, Y. Xu, R.Y.Y. Lam, B. Schneider, H. Dou, P.S. Leung, S.Y. Shi, C.X. Zhou, L.X. Yang, R.P. Zhang, Y.C. Xiao, X. Wu, J. Stockigt, S. Zeng, C.H.K. Cheng, Y. Zhao, J. Nat. Prod. 71 (2008) 12–17. [15] C.L. Lu, H.Y. Zhao, J.G. Jiang, Int. J. Food Sci. Nutr. 64 (2013) 28–35. [16] H. Matsuda, T. Morikawa, K. Ninomiya, M. Yoshikawa, Tetrahedron 57 (2001) 8443–8453. [17] H. Matsuda, K. Ninomiya, T. Morikawa, M. Yoshikawa, Bioorg. Med. Chem. Lett. 8 (1998) 339–344. [18] R. Wang, Y.H. Li, Y. Xu, Y.B. Li, H.L. Wu, H. Guo, J.Z. Zhang, J.J. Zhang, X.Y. Pan, X.J. Li, Prog. Neuropsychopharmacol. 34 (2010) 147–153. [19] C. Lipinski, Am. Pharm. Rev. 5 (2002) 82–85. [20] T. Heimbach, D. Fleisher, A. Kaddoumi, Prodrugs, Springer, New York, 2007, pp. 157–215. [21] X. Lv, D. Liu, J. Hou, P.P. Dong, L.B. Zhan, L. Wang, S. Deng, C.Y. Wang, J.H. Yao, X.H. Shu, K.X. Liu, X.C. Ma, Food Chem. 138 (2013) 2260–2266. [22] W.Z. Yang, M. Ye, F.X. Huang, W.N. He, D.A. Guo, Adv. Synth. Catal. 354 (2012) 527–539.
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Stereo- and regiospecific biotransformation of curcumenol by four fungal strains Meng Zhang a,1 , Qian Zhao a,1 , Yan-Yan Liang a , Jiang-Hao Ma a , Li-Xia Chen a,∗∗ , Xue Zhang a , Li-Qin Ding b , Feng Zhao c , Feng Qiu a,b,∗ a 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, China b School of Chinese Materia Medica, Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China c School of Pharmacy, Yantai University, No. 32 Road QingQuan, Laishan District, Yantai 264005, China
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 2 January 2015 Accepted 9 January 2015 Available online 20 January 2015 Keywords: Curcumenol Biotransformation Fungal strains Nitric oxide
a b s t r a c t Biotransformations of curcumenol (1) were performed by four fungal strains, Mucor spinosus AS 3.2450, Penicillium urticae IFFI 04015, Cunninghamella echinulata AS 3.3400, Aspergillus carbonarius IFFI 02087. Five metabolites were prepared in the biotransformation process of 1, and their structures were elucidated as 15-hydroxycurcumenol (2), 1˛-hydroxycurcumenol (3), 14-hydroxycurcumenol (4), 3ˇhydroxycurcumenol (5) and 12-hydroxycurcumenol (6) by spectroscopic data analysis. Among them, metabolites 2–5 are novel. All of these four fungal strains showed the ability of highly stereo- and regiospecific hydroxylation for the substrate (1), which could be used as tools for preparing the hydroxylated derivatives and in vivo metabolites of curcumenol. In addition, the inhibitory effects of substrate and obtained products on nitric oxide production in lipopolysaccaride-activated macrophages were evaluated. The substrate (1) and metabolites 2, 5, and 6 showed significant inhibitory effects. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Rhizoma Curcumae is an important crude drug frequently listed in prescriptions of traditional Chinese medicine for the treatment of Oketsu syndromes [1] which are caused by the obstruction of blood circulation, such as arthralgia, psychataxia, and dysmenorrhea. Three species of Rhizoma Curcumae (Curcuma phaeocaulis Valeton, C. kwangsiensis S.G. Lee & C.F. Liang, and C. wenyujin Y.H. Chen & C. Ling) are officially recorded as traditional Chinese medicine in the Chinese Pharmacopoeia [2]. Sesquiterpenoids and diarylheptanoids [3–8] are considered as the main bioactive constituents of Rhizoma Curcumae, possessing anti-inflammatory [8–10], anti-tumor [11–13], antioxidant [14,15], vasorelaxant [16], hepatoprotective [17] and neuroprotective [18] activities.
∗ Corresponding author at: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China. Tel.: +86 22 59596223; fax: +86 22 59596223. ∗∗ Corresponding author at: Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China. Tel.: +86 24 23986515; fax: +86 24 23986515. E-mail addresses: [email protected] (F. Qiu), [email protected] (L.-X. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.molcatb.2015.01.005 1381-1177/© 2015 Elsevier B.V. All rights reserved.
Curcumenol is one of the major guaiane-type sesquiterpenoid constituents of Rhizoma Curcumae, having an especially high content in the species of C. phaeocaulis Valeton [8]. Our previous studies indicated that curcumenol displayed strong inhibitory effects on nitric oxide production [8], which could be considered as a potential anti-inflammatory agent. However, the poor water-solubility would limit its bioavailability, absorption and clinical application [19,20]. Up to now, no any investigation on the structural derivatization of curcumenol has been reported owing to its limited reaction points for chemical modification. Microbial transformation provides an important tool for structure modification of natural products. And many new chemical derivatives with potent bioactivities and improved physicochemical characteristics were found in the process of biotransformation [21,22]. Microbial transformation can also be used as an in vitro model to prepare the in vivo metabolites [23,24]. This method exhibited many advantages, such as its high stereo- and regioselectivity, ease of handling, low cost, and environmental-friendly nature [25–28]. In the present work, the biocatalysis ability of 30 fungal strains to convert curcumenol (1) was screened in an attempt to obtain new and bioactive derivatives of sesquiterpenoid and improve its physicochemical properties. Four fungal strains, Mucor spinosus AS 3.2450, Penicillium urticae IFFI 04015, Cunninghamella
14
M. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 115 (2015) 13–19
echinulata AS 3.3400 and Aspergillus carbonarius IFFI 02087 were chosen because they displayed the characteristics of high stereoand regioselectivity for the biotransformations of curcumenol. Five metabolites, four of which have not previously been reported, were isolated and identified in the biotransformation processes of 1. In addition, the inhibitory effects of the substrate and obtained products on nitric oxide production in lipopolysaccaride-activated macrophages were also evaluated. 2. Experimental 2.1. General experimental procedures The NMR spectra were performed on Bruker ARX-600 spectrometer, using TMS as internal standard. Chemical shifts were expressed in ı (ppm) and coupling constants (J) were reported in Hertz (Hz). Optical rotation values were measured on a PerkinElmer 241 MC polarimeter. UV spectra were measured with a Shimadzu UV-1700 spectrophotometer. IR spectra were recorded with a Bruker IFS 55 spectrometer. HRESIMS spectra were obtained on an Agilent 6210 TOF mass spectrometer, in m/z. Melting points were determined with an X-5 hot stage microscope melting point apparatus (uncorrected). Silica gel GF254 prepared for TLC and silica gel (200–300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, People’s Republic of China). Sephadex LH-20 was a product of Pharmacia. All the analytic reagents were analytical grade and purchased from Tianjin DaMao Chemical Company (Tianjin, China). Spots were detected on TLC plates under UV light or by heating after spraying with anisaldehyde-H2 SO4 reagent. 2.2. Substrates Curcumenol was isolated from the rhizomes of C. phaeocaulis by the author (Jiang-Hao Ma), and was characterized by comparison of the NMR data with those reported in Refs. [8,29]. Its purity was determined to be 98% by HPLC analysis. 2.3. Microorganism 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 IFFI 02360, A. flavus 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 in Beijing, China. They were screened for their abilities to transform curcumenol (1) in the preliminary test. 2.4. Medium All culture and biotransformation experiments were performed in potato medium as following procedure: 200 g of minced husked potato was boiled in water for 1 h, then the extract was filtered and the filtrate was added with water to 1 L after addition of 20 g of glucose. The broth was autoclaved in individual Erlenmeyer flask at 121 ◦ C and 15 psi for 20 min and cooled before incubation.
2.5. Biotransformation procedures The four fungal strains (Mucor spinosus AS 3.2450, Penicillium urticae IFFI 04015, Cunninghamella echinulata AS 3.3400 and Aspergillus carbonarius IFFI 02087) had been sub-cultured for three times on potato dextrose agar slants before use to obtain maximal enzyme activities. Preliminary screening biotransformations of curcumenol (1) by microorganisms were carried out in 250 mL Erlenmeyer flasks containing 100 mL of potato medium. The flasks were placed on rotary shakers, operating at 180 rpm and 28 ◦ C. After 1 day of culture, the acetone (0.2 mL) containing curcumenol (2 mg) was added into each biotransformation flask, and the biotransformations were continued under the same conditions for an additional 5 days. Both substrate and organism controls were incubated under the same conditions in order to demonstrate that substrate was stable in the control culture. Similarly, preparative scale biotransformations were carried out in 500 mL Erlenmeyer flasks containing 200 mL of potato medium, and the microorganisms were pre-cultured under culture conditions for 2 days, respectively. The acetone (0.5 mL) containing curcumenol (5 mg) was added into each biotransformation flask, and the biotransformations were continued under the same conditions for an additional 7 days. A total of 200 mg of 1 was transformed by the four strains, respectively. When the biotransformation finished, the broths of substrate were filtered and the filtrates were extracted with the equal volume of ethyl acetate for three times, respectively. The organic phase was collected and concentrated to dry under reduced pressure at 40 ◦ C for further isolation. 2.6. Isolation and purification of metabolites The crude transformation residues of curcumenol (1) by M. spinosus AS 3.2450 were subjected to column chromatography (CC) over silica gel and eluted with the mixtures of petroleum ether–acetone (100:1, 50:1, 20:1, 15:1, 6:1, 2:1), which yielded ten fractions (1–10). Fraction 6 was purified by preparative TLC (CH2 Cl2 /EtOAc/petroleum ether, 1.2:1:0.5) to afford 2 (11.2 mg, 5.6%) and 6 (16.6 mg, 8.3%). Fraction 9 was submitted to a silica gel column eluted with petroleum ether–ethyl acetate (20:1, 15:1, 10:1, 5:1) to give 3 (156.6 mg, 78.3%). The crude transformation residues of curcumenol (1) by P. urticae IFFI 04015 were submitted to silica gel CC and eluted with the mixtures of petroleum ether–acetone (50:1, 30:1, 15:1, 10:1, 6:1, 3:1, 1:1), which yielded twelve fractions (1–12). Fraction 5 was purified by preparative TLC (CH2 Cl2 /EtOAc/petroleum ether, 1.2:1:0.1) to obtain 2 (137.6 mg, 68.8%) and 6 (36.4 mg, 18.2%). Fraction 6 was purified by preparative TLC (CH2 Cl2 /EtOAc/petroleum ether, 1.2:1:0.5) to afford 4 (7.8 mg, 3.9%). The crude transformation residues of curcumenol (1) by C. echinulata AS 3.3400 were subjected to silica gel CC and eluted with the mixtures of petroleum ether–ethyl acetate (30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1), which yielded fourteen fractions (1–14). Fraction 6 was chromatographed on a Sephadex LH-20 column eluted with MeOH to yield 5 (47.6 mg, 23.8%). Fraction 7 was purified by preparative TLC (CH2 Cl2 /EtOAc/petroleum ether, 1.2:1:0.5) to afford 6 (108.0 mg, 54.0%) and 2 (20.2 mg, 10.1%). The crude transformation residues of curcumenol (1) by A. carbonarius IFFI 02087 were subjected to silica gel CC and eluted with the mixtures of petroleum ether–acetone (100:1, 50:1, 30:1, 15:1, 10:1, 5:1, 3:1), which yielded nine fractions (1–9). Fraction 7 was recrystallized with acetone to afford 6 (186.6 mg, 93.3%). Metabolite 2: colorless needles (acetone); mp 197–198 ◦ C; [␣]25 D +3.46 (c 0.1, MeOH); UV (MeOH) max (log ε): 217 (4.36) nm;
M. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 115 (2015) 13–19
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Table 1 1 H NMR (ı) spectroscopic data (600 MHz, J in Hz) of metabolites 2–5. No.
1a
2a
3a
3b
4a
5a
H-1 H-2
H-7 H-8 H-9 H-10 H-11 H-12 H-13 H-14
1.96 (m) 1.90 (m) 1.57 (m) 1.86 (m) 1.71 (m) 1.87 (m) – 2.65 (dt, 15.5) 2.11 (brd, 15.5) – – 5.76 (brs) – – 1.60 (s) 1.80 (s) 1.00 (d, 6.2)
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 (s) 1.70 (s) 0.92 (d, 6.3)
– 1.94 (m) 1.74 (m) 1.87 (m) 1.42 (m) 2.14 (m) – 2.57 (brd, 16.5) 2.34 (brd, 16.5) – – 5.60 (q, 1.5) – – 1.54 (s) 1.69 (s) 0.91 (d, 6.9)
– 2.25 (m) 2.12 (m) 2.13 (m) 1.70 (m) 2.55 (m) – 3.00 (brd, 16.0) 2.52 (brd, 16.0) – – 6.14 (brs) – – 1.34 (s) 2.02 (s) 1.12 (d, 6.9)
1.86 (m) 2.31 (m) 1.44 (m) 3.94 (ddd, 12.6, 6.6, 2.4) 1.83 (m) – 2.57 (brd, 15.5) 2.03 (brd, 15.5) – – 5.61 (brs) – – 1.47 (s) 1.65(s) 0.94 (d, 7.2)
H-15
1.66 (s)
3.87 (s)
1.61 (s)
1.84 (s)
1.90 (m) 1.92 (m) 1.45 (m) 1.87 (m) 1.64 (m) 2.05 (m) – 2.80 (brd, 15.6) 2.18 (brd, 15.6) – – 5.59 (brs) – – 1.49 (s) 1.68 (s) 3.71 (dd, 11, 5.4) 3.56 (dd, 11, 6.9) 1.55 (s)
H-3 H-4 H-5 H-6
a b
IR max (KBr, cm−1 ): 3414, 3246, 2974, 2959, 2924, 1691, 1665, 1451, 995; HRMS (ESI) (positive) m/z: 273.1467 [M+Na]+ (calcd. for C15 H22 O3 Na, 273.1461); 1 H NMR (600 MHz, CD3 OD) and 13 C NMR (150 MHz, CD3 OD), see Tables 1 and 2. Metabolite 3: colorless oil; [␣]25 D +2.87 (c 0.1, MeOH); UV (MeOH) max (log ε): 217 (4.31) nm; IR max (KBr, cm−1 ): 3406, 2932, 2873, 1631, 1447, 1383, 1104, 974; HRMS (ESI) (positive) m/z: 273.1466 [M+Na]+ (calcd. for C15 H22 O3 Na, 273.1461); 1 H NMR (600 MHz, CD3 OD) and 13 C NMR (150 MHz, CD3 OD), see Tables 1 and 2. Metabolite 4: colorless oil; [␣]25 D +1.60 (c 0.05, MeOH); UV (MeOH) max (log ε): 211 (4.19) nm; IR max (KBr, cm−1 ): 3403, 2932, 2873, 1649, 1442, 1383, 1138, 966; HRMS (ESI) (positive) m/z: 251.1644 [M+H]+ (calcd. for C15 H23 O3 , 251.1642); 1 H NMR (600 MHz, CD3 OD) and 13 C NMR (150 MHz, CD3 OD), see Tables 1 and 2. Metabolite 5: white powders (MeOH); [␣]25 D +2.03 (c 0.16, MeOH); UV (MeOH) max (log ε): 213 (4.20) nm; IR max (KBr, cm−1 ): 3461, 2929, 2855, 1657, 1453, 1383, 1119, 963; HRMS (ESI) (positive) m/z: 273.1466 [M+Na]+ (calcd. for C15 H22 O3 Na, 273.1461); 1 H NMR (600 MHz, CD3 OD) and 13 C NMR (150 MHz, CD3 OD), see Tables 1 and 2.
Table 2 13 C NMR (ı) spectroscopic data (150 MHz) of metabolites 2–5. No.
1a
2a
3a
3b
4a
5a
C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15
51.4 27.8 31.4 40.6 85.9 37.4 139.2 101.7 125.9 137.5 122.4 22.5 19.1 12.1 21.4
48.5 28.8 32.6 41.4 87.3 38.2 138.1 102.9 127.2 143.4 123.2 22.6 19.3 12.4 64.6
83.5 37.5 30.4 38.3 89.9 32.2 136.5 102.8 128.9 140.4 123.2 22.6 19.1 12.5 17.0
82.4 37.5 30.5 37.9 88.9 32.3 137.7 102.4 129.5 139.9 121.3 22.5 19.4 13.0 17.5
53.4 29.8 28.4 48.2 86.9 39.8 137.9 103.0 127.6 139.1 122.7 22.5 19.2 62.9 23.3
50.6 40.5 76.7 45.7 87.0 38.6 137.6 103.4 128.1 139.6 123.2 22.6 19.3 7.1 21.1
a b
1.54 (3H)
Measured in CD3 OD. Measured in C5 D5 N.
Measured in CD3 OD. Measured in C5 D5 N.
2.7. HPLC analysis The 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 m). Mobile phase: MeOH–H2 O (0.05% HCOOH) (30:70 (v/v)) for 10 min followed by a linear gradient to (60:40 (v/v)) within 40 min and held for an additional 15 min. The flow rate was 1.0 mL/min, and column temperature was set at 30 ◦ C. 2.8. Bioassay for NO Production Mouse monocyte-macrophage RAW 264.7 cells (ATCC TIB-71) were purchased from the Chinese Academy of Science. RPMI 1640 medium, penicillin, streptomycin, and fetal bovine serum were purchased from Invitrogen (New York). Lipopolysaccharide (LPS), dimethylsufoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyltetrazolium bromide (MTT), and indomethacin were obtained from Sigma Co. RAW 264.7 cells were suspended in RPMI 1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 g/mL), and 10% heat-inactivated fetal bovine serum. The cells were harvested with trypsin and diluted to a suspension in fresh medium. The cells were seeded in 96-well plates with 1 × 105 cells/well and allowed to adhere for 2 h at 37 ◦ C in 5% CO2 in air. Then, the cells were treated with 1 g/mL of LPS for 24 h with or without various concentrations of test compounds. DMSO was used as a solvent for the test compounds, which were applied at a final concentration of 0.2% (v/v) in cell culture supernatants. NO production was determined by measuring the accumulation of nitrite in the culture supernatant using Griess reagent [30]. Briefly, 100 L of the supernatant from incubates was mixed with an equal volume of Griess reagent (0.1% N-[1-naphthyl]ethylenediamine and 1% sulfanilamide in 5% H3 PO4 ). Cytotoxicity was determined by the MTT colorimetric assay, after 24 h incubation with test compounds. The concentration of NO2 − was calculated by a working line from 0, 1, 2, 5, 10, 20, 50, and 100 M sodium nitrite solutions, and the inhibitory rate on NO production induced by LPS was calculated by the NO2 − levels as follows: Inhibitory rate (%) = 100 ×
− [NO− 2 ]LPS − [NO2 ]LPS+sample − [NO− 2 ]LPS − [NO2 ]untreated
16
M. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 115 (2015) 13–19
Fig. 1. Structures of biotransformed products of curcumenol (1).
Experiments were performed in triplicate, and data are expressed as the mean ± SD of three independent experiments. 3. Results and discussion 3.1. Identification of biotransformation products Metabolite 2 was obtained as colorless needles (acetone). The HRMS (ESI) exhibited the quasi-molecular ion peak [M+Na]+ at m/z 273.1467, accordingly to its molecular formula of C15 H22 O3 in combination with the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2). Comparison of the 13 C NMR data of 2 with those of curcumenol (1) [31] showed that the absence of one methyl signal at ıC 21.4 in 1, and the appearance of one hydroxymethyl signal at ıC 64.6 in 2. The HSQC spectrum exhibited that the proton resonances at ıH 3.87 (2H, s) correlated to the hydroxymethyl carbon signal at ıC 64.6. The HMBC correlations of the proton resonance at ıH 3.87 with C-1 (ıC 48.5), C-9 (ıC 127.2) and C-10 (ıC 143.4), and H-9 (ıH 5.86) with the hydroxymethyl carbon signal at ıC 64.6, confirmed that the hydroxyl group was linked to C-15 in 2. The obvious NOESY correlations from H-9 (ıH 5.86) to H2 -15 (ıH 3.87) and H-1 (ıH 1.99) further corroborated the above conclusion. Therefore, 2 was established as 15-hydroxycurcumenol (Fig. 1). Metabolite 3 was obtained as colorless oil. The molecular formula C15 H22 O3 was drawn from its HRMS (ESI) which gave the ion peak [M+Na]+ at m/z 273.1466, and its 1 H and 13 C NMR spectroscopic data (Tables 1 and 2). By comparing of the 13 C NMR spectrum of 3 with that of curcumenol (1) [31], one bearing-oxygen carbon
signal at ıC 83.5 was observed in 3, while the tertiary carbon signal at ıC 51.4 in 1 was disappeared. And also, the chemical shift value of C-5 shifted downfield to ıC 89.9, and C-4, C-6 and C-15 shifted upfield to ıC 38.3, 32.2 and 17.0, respectively. It is indicated the hydroxylation of C-1. The HMBC correlations of the proton resonances of H-15 (ıH 1.61), H-2 (ıH 1.74), H-9 (ıH 5.60), H-3ˇ (ıH 1.42) and H2 -6 (ıH 2.34, 2.57) with C-1 (ıC 83.5) further confirmed that the hydroxyl group was linked to C-1 in 3. The pyridineinduced solvent shifts [32,33] (Table 1) for H-3˛ (ı methanol-ı pyridine = −0.26 ppm), H-4˛ (−0.41 ppm) and H-6˛ (−0.43 ppm), supported ˛-orientation of the OH group at C-1. On the basis of the above evidence, 3 was established as 1˛-hydroxycurcumenol (Fig. 1). Metabolite 4 was obtained as colorless oil. The HRMS (ESI) exhibited the quasi-molecular ion peak [M+H]+ at m/z 251.1644, accordingly to its molecular formula of C15 H22 O3 by combining the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2). Comparison of the 13 C NMR data of 4 with those of curcumenol (1) [31] showed that the absence of one methyl signal at ıC 12.1 in 1, and the appearance of one hydroxymethyl signal at ıC 62.9 in 4. The 1 H NMR spectra of 4 and 1 were very similar except for the disappearance of a methyl resonance at ıH 1.00 (3H, d, J = 6.2 Hz) in 1, and the presence of two proton signals at ıH 3.71 (1H, dd, J = 11.0, 5.4 Hz) and 3.56 (1H, dd, J = 11.0, 6.9 Hz) in 4. It suggested that C-14 might be hydroxylated. The HMBC correlations of H-4 (ıH 2.05) with C-14 (ıC 62.9), and H2 -14 (ıH 3.71, 3.56) with C-3 (ıC 28.4), C-4 (ıC 48.2) and C-5 (ıC 86.9), as well as the NOESY correlations from H2 -14 (ıH 3.71, 3.56) to H-6 (ıH 2.80) and H-3 (ıH 1.64) confirmed that the
M. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 115 (2015) 13–19
17
Fig. 2. HPLC chromatograms of biotransformation of curcumenol (1) by Mucor spinosus AS 3.2450 (A); by Penicillium urticae IFFI 04015 (B); by Cunninghamella echinulata AS 3.3400 (C); by Aspergillus carbonarius IFFI 02087 (D). Peaks 2–6 were: 15-hydroxycurcumenol (2), 1˛-hydroxycurcumenol (3), 14-hydroxycurcumenol (4), 3ˇ-hydroxycurcumenol (5), 12-hydroxycurcumenol (6).
hydroxyl group was linked to C-14 in 4. On the basis of the above evidence, 4 was established as 14-hydroxycurcumenol (Fig. 1). Metabolite 5, white powders (MeOH). The molecular formula of C15 H22 O3 was inferred from positive HRMS (ESI) analysis ([M+Na]+ at m/z 273.1466) in combination with the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2). Comparison of the 13 C NMR data
of 5 with those of curcumenol (1) [31] showed that the absence of one methylene signal at ıC 31.4 in 1, and the appearance of one bearing-oxygen carbon signal at ıC 76.7 in 5. The HSQC spectrum exhibited that the proton resonance at ıH 3.94 (1H, ddd, J = 12.6, 6.6, 2.4 Hz) correlated to the bearing-oxygen carbon signal at ıC 76.7. The HMBC correlations of the proton signal at ıH 3.94 with
Fig. 3. Time courses of biotransformation of curcumenol (1) by Mucor spinosus AS 3.2450 (A); by Penicillium urticae IFFI 04015 (B); by Cunninghamella echinulata AS 3.3400 (C); by Aspergillus carbonarius IFFI 02087 (D).
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M. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 115 (2015) 13–19
Table 3 Inhibitory effects of compounds 1–6 on NO production induced by LPS in macrophages. Compounds
IC50 ± SDa (M)
1 2 3 4 5 6 Indomethacinb
5.42 6.44 33.85 25.37 10.42 9.64 14.1
± ± ± ± ± ± ±
0.64 0.51 2.44 1.49 0.93 0.47 0.69
a
Inhibitory effects of compounds 1–6 against LPS-induced NO production in RAW264.7 macrophages. b Indomethacin was used as positive control.
C-1 (ıC 50.6) and C-5 (ıC 87.0), H-2ˇ (ıH 1.44) with C-1 (ıC 50.6), C-4 (ıC 45.7) and the carbon signal at ıC 76.7, and H-14 (ıH 0.94) with C-4 (ıC 45.7), C-5 (ıC 87.0) and the carbon signal at ıC 76.7, confirmed that the hydroxyl group was linked to C-3 in 5. The obvious NOESY correlations from H-3 (ıH 3.94) to H-2˛ (ıH 2.31) and H-4 (ıH 1.83) corroborated the ˇ-orientation of 3-OH. Therefore, 5 was established as 3ˇ-hydroxycurcumenol (Fig. 1). Metabolite 6 was established as 12-hydroxycurcumenol (Fig. 1) by comparison of its spectroscopic data with those reported in the literature [34]. 3.2. Metabolic profile by HPLC analysis After 7 days of incubation, HPLC analysis showed a total of five metabolites in the broths containing four fungal strains. The HPLC profiles are given in Fig. 2. The analysis results revealed that metabolites 2 and 3 were the dominant biotransformation products of curcumenol (1) by P. urticae IFFI 04015 and M. spinosus AS 3.2450, respectively, while metabolite 6 is the common dominant products of C. echinulata AS 3.3400 and A. carbonarius IFFI 02087. Although the yield of metabolite 6 was low in the transformation process of curcumenol (1) by M. spinosus AS 3.2450 and P. urticae IFFI 04015, it is a common product of these four fungal strains. The stereo- and regiospecific hydroxylation at C-15, C-1 and C-14 by these four fungal strains yielded metabolites 2, 3 and 6, respectively. It is well known that numerous cytochrome P450 monooxygenases (P450s) play a key role in the process of bioconversion and metabolism of natural products by fungi [35,36]. The biotransformation time-course curves of 1 by four fungal strains (Fig. 3) demonstrated that all of the fungi began to exhibit strong bioconversion abilities from the third day. In our previous investigation [37], another similar guaiane-type sesquiterpenoid from Rhizoma Curcumae, curcumol, undergoes the extensive hydroxylation at C-2 or C-3 or C-15 catalyzed by hydroxylase in rats. We had successfully obtained its in vivo metabolite intermediate, 3-hydroxycurcumol, by the method of microbial transformation [38,39]. The hydroxylated derivative of curcumenol at C-15 was prepared in this research, which implied that they could be used as the standard references for investigating in vivo metabolism of curcumenol in the further study. 3.3. Inhibitory effects on NO production induced by LPS in macrophages Nitric oxide (NO) plays an important role in the inflammatory process, and an inhibitor of NO production may be considered as a potential anti-inflammatory agent [40–42]. The substrate 1 and its metabolites 2–6 were examined for their inhibitory effects on NO production induced by LPS in macrophages (Table 3). The substrate 1, and metabolites 2, 5 and 6 showed strong inhibition of NO production induced by LPS. Metabolites 2 and 6 had a hydroxyl group
substitution at the side chain of the seven-member ring, and 5 had a hydroxyl group substitution at C-3 position of the five-member ring. However, the hydroxylation of C-1 or C-14 in metabolite 3 or 4 reduced their bioactivities of NO inhibition. The above results indicated that the location of hydroxyl group substitution played an important role in rendering the NO inhibitory effects. 4. Conclusion Five metabolites, four of which have not previously been reported, were obtained and identified after incubation of curcumenol by M. spinosus AS 3.2450, P. urticae IFFI 04015, C. echinulata AS 3.3400 and A. carbonarius IFFI 02087. All of these metabolites are the hydroxylated products of the substrate. The highly stereoand regiospecific hydroxylation of curcumenol by these four fungal strains was discovered, which could be used as tools for obtaining hydroxylated derivatives of curcumenol to improve its watersolubility, and preparing its in vivo metabolites. The difference of hydroxylated sites of curcumenol catalyzed by four fungal strains remarkably affects their anti-inflammatory activity, which would provide vital information in developments of anti-inflammatory agent. 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. References [1] Y. Sasaki, H. Goto, C. Tohda, F. Hatanaka, N. Shibahara, Y. Shimada, K. Terasawa, K. Komatsu, Biol. Pharm. Bull. 26 (2003) 1135–1143. [2] Pharmacopoeia Commission of People’s Republic of China, Pharmacopoeia of the People’s Republic of China (Part 1), Chinese Medical Science and Technology Press, Beijing, China, 2010, pp. 257–258. [3] Y. Lou, F. Zhao, H. He, K.F. Peng, X.H. Zhou, L.X. Chen, F. Qiu, J. Asian Nat. Prod. Res. 11 (2009) 737–747. [4] Y. Lou, F. Zhao, Z.H. Hu, K.F. Peng, X.C. Wei, L.X. Chen, F. Qiu, Helv. Chim. Acta 92 (2009) 1665–1672. [5] Y. Lou, F. Zhao, H. He, K.F. Peng, L.X. Chen, F. Qiu, Chem. Biodivers. 7 (2010) 1245–1253. [6] J. Li, F. Zhao, M.Z. Li, L.X. Chen, F. Qiu, J. Nat. Prod. 73 (2010) 1667–1671. [7] J. Li, C.R. Liao, J.Q. Wei, L.X. Chen, F. Zhao, F. Qiu, Bioorg. Med. Chem. Lett. 21 (2011) 5363–5369. [8] Y. Liu, J.H. Ma, Q. Zhao, C.R. Liao, L.Q. Ding, L.X. Chen, F. Zhao, F. Qiu, J. Nat. Prod. 76 (2013) 1150–1156. [9] C. Tohda1, N. Nakayama, F. Hatanaka1, K. Komatsu, Evid.-Based Complement. Alternat. 3 (2006) 255–260. [10] J.S. Jurenka, Altern. Med. Rev. 14 (2009) 141–153. [11] M.X. Xu, L.L. Zhao, C.Y. Deng, L. Yang, Y. Wang, T. Guo, L.F. Li, J.P. Lin, L.R. Zhang, Int. J. Oncol. 43 (2013) 1951–1959. [12] L. Yan, J.A. Yee, J. Cao, Anticancer Res. 33 (2013) 3153–3162. [13] Q. Du, B. Hu, H.M. An, K.P. Shen, L. Xu, S. Deng, M.M. Wei, Oncol. Rep. 29 (2013) 1851–1858. [14] Q.F. Tao, Y. Xu, R.Y.Y. Lam, B. Schneider, H. Dou, P.S. Leung, S.Y. Shi, C.X. Zhou, L.X. Yang, R.P. Zhang, Y.C. Xiao, X. Wu, J. Stockigt, S. Zeng, C.H.K. Cheng, Y. Zhao, J. Nat. Prod. 71 (2008) 12–17. [15] C.L. Lu, H.Y. Zhao, J.G. Jiang, Int. J. Food Sci. Nutr. 64 (2013) 28–35. [16] H. Matsuda, T. Morikawa, K. Ninomiya, M. Yoshikawa, Tetrahedron 57 (2001) 8443–8453. [17] H. Matsuda, K. Ninomiya, T. Morikawa, M. Yoshikawa, Bioorg. Med. Chem. Lett. 8 (1998) 339–344. [18] R. Wang, Y.H. Li, Y. Xu, Y.B. Li, H.L. Wu, H. Guo, J.Z. Zhang, J.J. Zhang, X.Y. Pan, X.J. Li, Prog. Neuropsychopharmacol. 34 (2010) 147–153. [19] C. Lipinski, Am. Pharm. Rev. 5 (2002) 82–85. [20] T. Heimbach, D. Fleisher, A. Kaddoumi, Prodrugs, Springer, New York, 2007, pp. 157–215. [21] X. Lv, D. Liu, J. Hou, P.P. Dong, L.B. Zhan, L. Wang, S. Deng, C.Y. Wang, J.H. Yao, X.H. Shu, K.X. Liu, X.C. Ma, Food Chem. 138 (2013) 2260–2266. [22] W.Z. Yang, M. Ye, F.X. Huang, W.N. He, D.A. Guo, Adv. Synth. Catal. 354 (2012) 527–539.
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