A New Germacrane-Type Sesquiterpene from Fermented Curcuma longa L.

Result and Discussion

Compound 1 was isolated as yellow viscous oil. The molecular formula of 1 was determined to be C15H24O5 from a pseudomolecular ion m/z 249.1508 [M + H − 2H2O]+ (calcd. for C15H21O3, 249.1491) in the HRESITOFMS. The 1H NMR spectrum of 1 exhibited signals for one olefinic proton at δ 5.78 (1H, br s, H-9), two oxymethines [δ 4.29 (1H, m, H-1) and 3.52 (1H, dd, J = 2.0, 10.8 Hz, H-5)], one oxymethylene [δ 4.23 and 4.07 (each d, J = 12.4 Hz, H2- 12)], two vinylic methyls [δ 1.73 (3H, s, H3-13) and 1.71 (3H, s, H3-15)], and one tertiary methyl at δ 1.19 (3H, s, H3-14)] (Table 1). The 13C NMR and DEPT spectra displayed 15 carbon signals, including three methyls, four methylenes, three methines, and five quaternary carbons. Analysis of 13C NMR spectrum revealed the presence of one carbonyl carbon [δ 207.8 (C-8)] and two olefinic group carbon signals [δ 145.0 (C-10), 140.7 (C-7), 133.6 (C-11), 126.1 (C-9)]. The positions of two double bonds at C-7 and C-9 were assigned by HMBC correlations from H3-13 to C-7 and C-11, and from H3-15 to C-9 and C-10, respectively (Fig. 2). The 13C NMR spectrum further showed signals for an oxygenated quaternary carbon at δ 87.7 (C-4), two oxymethines at δ 82.4 (C-1) and 78.9 (C-5), one oxymethylene at δ 63.2 (C-12), and three methylenes [δ 39.0 (C-3), 35.9 (C-6), and 31.9 (C-2)]. The HMBC correlations from H3-14 to C-3, C-4, and C-5, and from H3-15 to C-1, along with the 1H-1H COSY cross-peaks between H-1/H2-2, H2-2/H2-3, and H5/H2-6 allows to assign the positions of the two oxymethine groups (C-1 and C-5), three methylene groups (C-2, C-3, and C-6), and one oxygenated quaternary carbon (C-4). Furthermore, the HMBC cross peaks of H-6 with C-7, C-8, and C-11, along with HMBC correlation from H-9 to C-8 clearly indicated that the carbonyl group was located at C-8. The position of oxymethylene group (C-12) was suggested by HMBC correlation from H3-13 to C-12. Based on these analyses, the planar structure of 1 was determined to be a germacrane-type sesquiterpene. The geometry of double bonds and relative configuration of 1 was established by NOESY spectrum. The NOE cross peaks between H2-12/H- 5 and H2-12/H-6b confirmed the position of the oxymethylene group (C-12) and showed E geometry of the double bond at C-7. The NOESY correlation of H-9 with H3-15 indicated Z configuration of the double between C-9 and C- 10. The NOESY correlations between H-1/H3-14 and between H-5/H3-14 suggested that these groups are positioned at the same plane of the ring (Fig. 2). It is noted that the structure of 1 was similar to that of the reported compound, heyneanone A, exept for the presence of hydroxyl group at C-12 in 1.10 The similar NMR data and specific optical rotation values of these two compounds [1: −34.2° (c= 0.19, CHCl3); heyneanone A: −21° (c = 0.23, CHCl3)] suggested that both share the same configuration.10 Thus, the gross structure of compound 1 was established as 7E,9Z- 1β,4β,5β,12-tetrahydroxy-7(11),9-germacradien-8-one, named longanone A.

Table 1.aSpectra were recorded in CD3OD. b400 MHz, c100MHz

Figure 1.Structures of compounds 1-5.

Figure 2.Selected HMBC, COSY, and NOESY correlations of compound 1.

Structures of the known compounds were identified as: 1α,4β-dihydroxyeudesman-8-one (2),11 zedoarondiol (3),12 4-(4-hydroxy-3-methoxyphenyl)-2S-butanol (4),13 and conorarin I (5)14 by analyses of their NMR and MS data, and comparison with those of reported compounds (Fig. 1).

Cytoprotective effects of the metabolites 1-5 were assessed using glutamate-induced cytotoxicity in mouse hippocampal HT-22 cells.15 In addition, PTP1B inhibitory effects of compounds 1-5 were also evaluated.16 As the result, all the metabolites showed no significant cytoprotective and PTP1B inhibitory effects (data not shown).

Nitrite production, a stable end product of NO oxidation process, was used as a measure of iNOS activity. The inhibitory effects of the isolated compounds on nitrite production were also evaluated in LPS-stimulated RAW264.7 cells (Fig. 3).17 Among the five compounds, compound 1 showed nitrite inhibitory effect at the concentrations of 50 and 100 μM, with the inhibition of 10.6 and 13.8%, respectively. Compound 4 inhibited the nitrite production at the concentrations of 25 μM, 50 μM, and 100 μM in a dosedependent manner, with the inhibition of 15.2, 17.5, and 25.8%, respectively. Compounds 2, 3, and 5 showed no inhibitory effects on nitrite production in LPS-stimulated RAW264.7 cells (data not shown).

Figure 3.Effects of compounds 1 and 4 on the production of nitrite in LPS-stimulated RAW264.7 cells. The values are mean ±SD (n = 3). aStimulated with LPS. bStimulated with LPS in the presence of 1 and 4 (10, 25, 50, and 100 mM), and positive control, sulfuretin (SFT, 10 μM). Statistical significance is indicated as *(p < 0.05) as determined by Dunnett’s multiple comparison test.

 

Experimental

General Procedures. Optical rotations were recorded using a Jasco P-2000 digital polarimeter. NMR spectra (1D and 2D) were recorded in CD3OD or CDCl3 using a JEOL JNM ECP-400 spectrometer (400 MHz for 1H and 100 MHz for 13C), and chemical shifts were referenced relative to the corresponding residual solvents signals (CD3OD: δ 4.80 and 3.30/49.0; CDCl3: δ 7.25/77.0). HMQC and HMBC experiments were optimized for 1JCH = 140 Hz and nJCH = 8 Hz, respectively. ESIMS data were obtained using a Q-TOF micro LC-MS/MS instrument (Waters) at Korea University, Seoul, Korea. TLC was performed on Kieselgel 60 F254 (1.05715; Merck, Darmstadt, Germany) or RP-18 F254s (Merck) plates. Spots were visualized by spraying with 10% aqueous H2SO4 solution, followed by heating. Column chromatography was performed on silica gel (Kieselgel 60, 70-230 mesh and 230- 400 mesh, Merck) and YMC octadecyl-functionalized silica gel (C18).

Plant Material and Fermentation. Raw C. longa L. was harvested and obtained from Jindo, Jellanamdo, Korea, and was authenticated by Dr. Humyoung Baek at Korea INS Pharm Research Institute (Hwasun, Korea). The voucher specimen was deposited in the same institute. The powder of C. longa L. was fermented with 2% (wt/wt) of Aspergillus oryzae at 25 ℃ for 36 h and dried.

Extraction and Isolation. Fermented C. longa L. was sterilized at 121 ℃ for 15 min, and was extracted with distilled water at room temperature. The dried extracts (600 g) was dissolved then applied to open column chromatography filled with amberlite XAD-2 resin, eluting with a stepwise gradient of 100% H2O, 25% MeOH/H2O, 50% MeOH/H2O, 75% MeOH/H2O, and 100% MeOH. The MeOH 25% fraction was concentrated (10 g) and suspended in H2O, and then partitioned successively with CH2Cl2, EtOAc, and BuOH to give CH2Cl2, EtOAc, BuOH, and aqueous extracts, respectively. The CH2Cl2, EtOAc, BuOH extracts were combined and concentrated in vacuo to give a fraction FCLA (900 mg). Fraction FCLA was subjected to reversed phase C18 column chromatography (CC), stepwise eluted with MeOH-H2O (1:3 to 2:1) to provide 12 fractions: FCLA1 (160 mg), FCLA2 (45 mg), FCLA3 (32 mg), FCLA4 (64 mg), FCLA5 (66 mg), FCLA6 (63 mg), FCLA7 (79 mg), FCLA8 (50 mg), FCLA9 (110 mg), FCLA10 (52 mg), FCLA11 (38 mg), and FCLA12 (31 mg). Fraction FCLA6 was chromatographed over silica gel column, eluting with CH2Cl2-MeOH (10:1 to 3:1) to give 3 subfractions, FCLA61-FCLA63. Subfraction FCLA62 was further purified by preparative TLC using CH2Cl2-MeOH (5:1) as eluent to obtain compound 5 (1.2 mg). Fraction FCLA7 was subjected to silica gel CC, eluting with n-Hexane:Acetone (2:1) to give compound 3 (15 mg) and five subfractions FCLA7A-E. Fraction FCLA7E was further chromatographed over silica gel column, using CH2Cl2- MeOH (15:1) to obtain compound 1 (1.5 mg). Fraction FCLA9 was subjected to Sephadex LH-20 CC, eluted with CH2Cl2-MeOH (5:1) to give 5 subfractions, FCLA91-FCLA95. From subfraction FCLA92, by using silica gel CC and eluting with n-Hexane-EtOAc (1:1 to 1:5), compound 4 (1.5 mg) was isolated along with three subfractions, FCLA922- FCLA924. Subfraction FCLA923 was further purified by silica gel CC, using n-Hexane-EtOAc-MeOH (1:5:0.1) as eluent to give compound 2 (1.4 mg).

Longanone A (1): Yellow viscous oil;−34.2 (c 0.19, CHCl3). HR-ESITOFMS: m/z 249.1508 [M+H-2H2O]+ (calcd. for C15H21O3, 249.1491). 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD): see Table 1.

1α,4β-Dihydroxyeudesman-8-one (2): Colorless gum;−9.9 (c 0.18, MeOH). ESIMS m/z 253 [M+H]+. 1H NMR (400 MHz, CD3OD) δ 3.37 (m, H-1), 3.03 (dd, J = 4.4, 15.6 Hz, H-6a), 2.54 (d, J = 15.6 Hz, H-6b), 1.97 (d, J = 1.6 Hz, H3-12), 1.86 (d, J = 1.6 Hz, H3-13), 1.19 (s, H3-14), 0.87 (d, J = 1.2 Hz, H3-15). 13C NMR (100 MHz, CD3OD) δ 79.0 (C-1), 29.2 (C-2), 41.6 (C-3), 72.3 (C-4), 51.6 (C-5), 27.1 (C-6), 132.1 (C-7), 205.4 (C-8), 58.1 (C-9), 42.8 (C- 10), 145.7 (C-11), 22.4 (C-12), 22.6 (C-13), 23.5 (C-14), 13.2 (C-15).

Zedoarondiol (3): Colorless gum; −40.7 (c 0.9, MeOH). ESIMS m/z 253 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 2.93 (d, J = 12.8 Hz, H-9a), 2.56 (d, J = 12.8 Hz, H-9b), 1.79 (s, H3-12), 1.89 (s, H3-13), 1.14 (s, H3-14), 1.16 (s, H3-15). 13C NMR (100 MHz, CDCl3) δ 55.9 (C-1), 22.9 (C-2), 28.5 (C-3), 80.0 (C-4), 51.9 (C-5), 39.7 (C-6), 134.7 (C-7), 203.2 (C-8), 59.9 (C-9), 72.7 (C-10), 142.2

4-(4-Hydroxy-3-methoxyphenyl)-2S-butanol (4): Colorless viscous liquid; +33.9 (c 0.15, MeOH). ESIMS m/z 197 [M+H]+. 1H NMR (400 MHz, CD3OD) δ 1.17 (d, J = 6.0 Hz, H3-1), 3.71 (m, H-2), 6.76 (d, J = 2.0 Hz, H-6), 6.68 (d, J = 8.0 Hz, H-9), 6.61 (dd, J = 2.0, 8.0 Hz, H-10), 3.82 (s, 7-OMe). 13C NMR (100 MHz, CD3OD) δ 23.5 (C- 1), 67.9 (C-2), 32.7 (C-3), 42.3 (C-4), 135.2 (C-5), 113.1 (C- 6), 148.8 (C-7), 145.4 (C-8), 116.0 (C-9), 121.7 (C-10), 56.3 (7-OMe).

Conorarin I (5): Colorless viscous liquid; −9.5 (c 0.11, CHCl3). ESIMS m/z 337 [M+H]+. 1H NMR (400 MHz, CD3OD) δ 3.20 (m, H-3), 1.17 (m, H-5), 3.88 (m, H-7), 1.65 (m, H-9), 5.47 (d, J = 6.0 Hz, H-12), 4.67 (m, H-14), 3.62 (m, H-15a), 3.51 (m, H-15b), 4.11 (d, J = 12.4 Hz, H-16a), 3.97 (d, J = 12.4 Hz, H-16b), 5.21 (br s, H-17a), 4.67 (br s, H-17b), 0.99 (s, H3-18), 0.77 (s, H3-19), 0.72 (s, H3-20). 13C NMR (100 MHz, CD3OD) δ 38.2 (C-1), 28.7 (C-2), 79.4 (C- 3), 40.0 (C-4), 53.6 (C-5), 34.2 (C-6), 74.4 (C-7), 151.1 (C- 8), 56.5 (C-9), 40.1 (C-10), 23.2 (C-11), 131.7 (C-12), 138.9 (C-13), 72.0 (C-14), 66.3 (C-15), 64.3 (C-16), 105.5 (C-17), 28.9 (C-18), 16.2 (C-19), 15.0 (C-20).

Cell Culture. RAW264.7 and HT22 cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air at 5 × 105 cells/mL in DMEM medium supplemented with 10% heat-inactivated FBS, penicillin G (100 units/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM).

Cytoprotective Assay. HT22 cells were pre-treated for 3 h with indicated concentrations of compounds, and then incubated for 12 h with glutamate (5 mM). The effects of various experimental modulations on cell viability were evaluated by determining mitochondrial reductase function with an assay based on the reduction of tetrazolium salt 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into fomazan crystals. The synthesis of formazan is proportional to the number of functional mitochondria in living cells. For the determination of cell viability, 50 mg/ mL of MTT was added to 1 mL of cell suspension (1 × 105 cells/mL in 96-well plates) for 4 h. The formazan synthesized was dissolved in acidic 2-propanol and the optical density was measured at 590 nm. The optical density of the formazan formed in control (untreated) cells was considered as 100% viability.

Nitrite Production Determination. RAW264.7 macrophages were pretreated for 3 h with indicated concentrations of compounds, and treated 24 h with LPS (1 μg/mL). The nitrite concentration in the medium was measured as an indicator of NO production as per the Griess reaction. The nitrite present in the conditioned media was determined spectrophotometrically using the Griess reaction. An aliquot (100 μL) of each supernatant was mixed with an equal volume of Griess reagent (0.1% [w/v] N-(1-naphthyl)-ethylenediamine and 1% [w/v] sulfanilamide in 5% [v/v] phosphoric acid) for 10 min at room temperature. The absorbance of the final reactant at 525 nm was measured using an ELISA plate reader, and the nitrite concentration was determined using a standard curve of sodium nitrite prepared in DMEM without phenol red.

PTP1B Inhibitory Activity Assay. PTP1B (human, recombinant) was purchased from BIOMOL Research Laboratories, Inc. A reaction mixture containing 0.05 μg PTP1B and 2 mM p-nitrophenyl phosphate (p-NPP) in a buffer consisting of 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol with or without a tested compound solution (prepared in the above buffer solution containing 3% DMSO) was incubated at 37℃ for 30 min. The reaction was terminated by addition of 10 N NaOH. The amount of produced p-nitrophenol was evaluated by measuring the increase in absorbance at 405 nm. The blank was measured in the same way except for adding buffer solution instead of the enzyme.

Supporting Information. The HR-ESITOFMS, 1H, 13C, HMQC, and HMBC NMR spectra of compound 1.