- freely available
Molecules 2014, 19(5), 5863-5875; doi:10.3390/molecules19055863
Published: 6 May 2014
Abstract: Bioassay-guided fractionation of the ethanolic extract of the leaves of Flickingeria flimbriata led to the isolation of two new degraded diterpenoids 1 and 2, a new ent-pimarane type diterpenoid 3, and four known steroids 4–7. The structures of 1–3 were elucidated by spectroscopic analysis, and their absolute configurations were determined by chemical methods, TDDFT quantum chemical calculations of ECD spectra, and CD exiton chirality method. Compounds 1 and 2, named flickinflimilins A and B, possess a rare 15,16-dinor-ent-pimarane skeleton. Compounds 1–7 were screened for the inhibitory activity against lipopolysaccharide (LPS)-induced NO and TNF-α production in RAW264.7 cells. Compounds 1–3 exhibited potent inhibitory activities, with IC50 values of less than 10 µM.
Flickingeria fimbriata (Bl.) Hawkes (Orchidaceae), is widely used as a substitute of the precious and scarce Traditional Chinese Medicine Dendrobium candidum, for treatment of pneumonia, tuberculosis, asthma, and pleurisy . Phytochemical investigations of Flickingeria species have revealed the occurrence of diterpenes [2,3,4,5,6], phenanthrenes , steroids , and bibenzyls . These natural products are reported possessing P-glycoprotein inhibition , anti-inflammation [7,8,9], and antimutagenic activities .
In our continuing search for anti-inflammatory metabolites from medical plants, a fraction of the EtOAc extract partitioned from the ethanolic extract of leaves of F. fimbriata showed an inhibitory activity of 42.2% against NO production and 40.1% against TNF-α production, respectively, at a concentration of 10 µM. Column chromatographic separations led to the isolation of three new diterpenoids 1–3 together with four known steroids 4–7 (Figure 1). Flickinflimilins A and B (compounds 1,2) represent an unusual group of 15,16-dinor-ent-pimarane. The isolated compounds 1–7 were screened for their inhibitory activity against lipopolysaccharide (LPS)-induced NO and TNF-α production in RAW264.7 cells. The results showed that compounds 1–3 exhibited potent inhibitory activities with the IC50 values less than 10 µM. Herein, the details of the isolation, structural elucidation, and inhibitory activities of these compounds are described.
2. Results and Discussion
The air-dried powder of the leaves of F. fimbriata was extracted with 95% EtOH at room temperature to give a crude extract, which was then suspended in H2O and successively partitioned with petroleum ether, EtOAc, and n-BuOH, respectively. Various column chromatographic separations of the petroleum ether, EtOAc extract afforded compounds 1–7.
Compound 1 was obtained as a colorless oil with the molecular formula C18H28O3 as established by an m/z of 315.1943 [M + Na]+ (calcd for C18H28O3Na, 315.1936) from HRESIMS. The 1H-NMR spectrum of 1 showed four tertiary methyls [at δH 2.27, 1.00, 0.94, and 0.87 (each 3H, s)], two oxygenated methines [at δH 4.05 (ddd, J = 2.8, 4.4, and 11.8 Hz, H-2) and 3.45 (d, J = 2.8 Hz, H-3)], and an olefinic proton [at δH 6.65 (t, J = 1.6 Hz, H-14)]. The 13C-NMR spectrum in combination with DEPT experiments showed 18 carbon resonances, including four quaternary carbons (one conjugated carbonyl and one olefinic), six methines (two oxygenated and one olefinic), four methylenes, and four quaternary methyls. The double bond and the carboxyl group accounted for two out of the five degrees of unsaturation, and the leftover double-bond equivalents required a tricyclic nature of 1. The gross structure was constructed by two-dimensional (2D) NMR analysis. Three fragments (Figure 2), a (C-1 to C-3), b (C-5 to C-7), and c (C-14, C-8, C-9, and C-11) were established by the correlations observed in the 1H-1H COSY spectrum of 1. The connectivity of these fragments, quaternary carbons and other substitutes was accomplished mainly by analysis of the HMBC spectrum (Figure 2). Fragment a, C-10 bearing the angular methyl group (C-20), the C-5 methine, and the gem-dimethyl group could construct ring A by the HMBC correlations from H3-20 to C-1, C-5, and C-10 and from H3-18/19 to C-3, C-4, and C-5. The HMBC correlations of H-9 to C-5, C-7, and C-10, H-7 to C-9, and H-8 to C-7, C-9 and C-10 supported that ring B was constructed by C-8–C-10 and fragment b, and fused with ring A via C-5 and C-10. Fragment c and the α,β-unsaturated ketone group provide ring C by the HMBC correlations from H-14 to C-8, C-9, C-12, and C-13. The planar structure of 1 thus emerged from the above spectra analysis.
The relative configuration of 1 was determined by analysis of the NOESY correlations (Figure 2) and coupling constants. The NOESY spectrum showed cross peaks between the proton pairs H-2/H3-19, H-2/H3-20, indicating that these protons were cofacial and axial oriented, which were arbitrarily assigned as α orientation. Accordingly, a chair conformation for ring A was assigned. Based on the small coupling constant (J = 2.6 Hz) between H-2 and H-3, the OH-3 was assigned as β orientation. The β assignment of coplanar protons H-5 and H-9 were deduced from the NOE correlations of H-1β/H-5, H-5/H-7β, and H-5/H-9.
The absolute configuration of 1 was established by comparing the experimental ECD spectrum with the calculated data. The TDDFT calculations were performed using the B3LYP functional and TZVP basis set. Then conformer of 1 were subjected to TDDFT calculations for solution CD in MeOH and also calculated for the gas phase CD. The simulated CD spectra (Figure 3) exhibited negative Cotton effects (CEs) around 320 nm and 240 nm and a positive CE around 210 nm, all generally consistent with the experimental spectrum, and the weighted spectra in both the gas phase and MeOH solution (Figure 3) provided the excellent fit with the experimental data, giving a firm support to the determined absolute configuration of 1.
Compound 2 was obtained as a white amorphous powder, and its molecular formula was determined to be C18H24O3 (seven degrees of unsaturation) from the quasi-molecular ion peak at m/z 311.1987 [M + Na]+ (calcd for C18H24O3Na, 311.1989) in the HRESIMS spectrum. The IR absorption bands at 3440, 1708, 1600, 1500, and 1476 cm−1 indicated the presence of hydroxyl, carbonyl, and aromatic groups. The 1H-NMR spectrum of 2 (Table 1) showed the signals of four tertiary methyl groups [at δH 2.34, 1.25, 1.19, and 1.03 (each 3H, s)], two oxygenated methines [at δH 4.26 (ddd, J = 2.6, 4.5, and 11.5 Hz, H-2) and 3.61 (d, J = 2.6 Hz, H-3)], and three aromatic protons [at δH 7.80 (d, J = 1.3 Hz, H-14), 7.34 (dd, J = 1.3, 8.0 Hz, H-12), and 7.27 (d, J = 8.0 Hz, H-11)]. The 13C-NMR spectrum in combination with DEPT experiments showed 18 carbon resonances, including six quaternary carbons (one conjugated carbonyl and three olefinic carbons), six methines (two oxygenated and three olefinic carbons), two methylenes, and four methyls. The double bonds and ketone group accounted for four out of the seven degrees of unsaturation, the remaining three double-bond equivalents required 2 to be tricyclic. The aforementioned data resembled those of compound 1. The major structural differences were due to the location of the conjugated ketone group and C-ring aromatized in 2. The structure of 2 was further demonstrated by HMBC and NOESY spectra (See supplementary materials Figure S27). Compound 2 represents the first C-ring aromatized dinor-ent-pimarane reported hitherto.
The absolute configuration of compound 2 was postulated on the basis of comparison between the experimental ECD spectrum and the calculated data. As can be seen in Figure 4, the Boltzmann weighted CD spectra in both the gas phase and MeCN solution, in particular the solution spectrum, are in good agreement with the experimental spectrum.
Compound 3, a colorless oil, had a molecular formula of C28H44O10 as determined by HRESIMS m/z 585.2929 [M + HCOO]− (calcd for C29H45O12, 585.2923). Closely inspection of the 1H and 13C-NMR spectra indicated compound 3 was a diterpenoid glycoside. Comparison of the NMR data (Table 1) and molecular formula of 3 with those of ephemeranthoside  demonstrated that 3 had an additional acetyl group. [δH2.10 s (3H); δC 21.3 and 173.0], which was located at C-3 from analysis of the HMBC spectrum. The relative configuration of 3 was established by analysis of the NOESY spectrum.
|Table 1. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) data for compounds 1–3 (δ in ppm).|
|No.||1 a||2 b||3 a|
|δH (J in Hz)||δC, type||δH (J in Hz)||δC, type||δH (J in Hz)||δC, type|
|1α||1.54, m||40.4, CH2||2.31, m||40.3, CH2||1.56, m||41.3, CH2|
|1β||1.44, t (11.8)||1.99, m||1.53, m|
|2||4.05, ddd (2.8, 4.4, 11.8)||66.3, CH||4.26, ddd (2.6, 4.5, 11.5)||66.2, CH||4.00, ddd (2.6, 4.7, 11.7)||65.9, CH|
|3||3.45, d (2.8)||79.2, CH||3.61, d (2.6)||78.8, CH||4.91, d (2.6)||81.5, CH|
|4||38.2, C||38.8, C||39.9, C|
|5||1.37, m||47.6, CH||2.29, m||42.8, CH||1.44, m||49.9, CH|
|6α||1.58, m||21.7, CH2||2.64, d (1.4)||36.0, CH2||1.42, m||22.6, CH2|
|6β||1.37, m||2.62, m||1.57, m|
|7α||2.13, m||30.2, CH2||199.4, C||2.12, m||36.5, CH2|
|7β||2.07, m||2.42, d (14.2)|
|8||2.49, t (12.0)||45.8, CH||131.1, C||142.9, C|
|9||1.59, m||61.3, CH||153.7, C||1.92, t (9.1)||52.0, CH|
|10||37.1, C||39.2, C||40.5, C|
|11α||2.37, dd (3.8, 11.6)||29.2, CH2||7.27, d (8.0)||124.2, C||1.65, m||21.3, CH2|
|11β||2.15 br, s||1.24, m|
|12α||197.4, C||7.34, dd (1.3, 8.0)||135.0, C||1.10, m||33.5, CH2|
|13||145.3, C||135.8, C||48.7, C|
|14α||6.65, t (1.6)||148.0, CH||7.80, d (1.3)||127.7, C||5.54, s||125.5, CH|
|16a||4.49, d (18.4)||72.4, CH2|
|16b||4.88, d (18.4)|
|17||2.27, s||26.1, CH3||2.34, s||20.6, CH3||1.14, s||27.5, CH3|
|18||0.87, s||21.3, CH3||1.03, s||21.5, CH3||0.89, s||28.5, CH3|
|19||1.00, s||28.7, CH3||1.19, s||28.4, CH3||0.98, s||22.4, CH3|
|20||0.94, s||16.0, CH3||1.25, s||24.3, CH3||0.77, s||16.0, CH3|
|CH3CO||2.10, s||21.3, CH3|
|1’||4.24, d (7.6)||104.2, CH|
|2’||3.16, dd (7.6, 9.2)||75.0, CH|
|3’||3.27, dd (2.7, 5.0)||78.2, CH|
|4’||3.28, d (8.8)||71.5, CH|
|5’||3.24, dd (2.7, 5.0)||77.6, CH|
|6’a||3.87, dd (3.6, 12.0)||72.4, CH2|
|6’b||3.64, dd (3.6, 12.0)|
a Measured in CDCl3; b Measured in Pyridine.
To determine the absolute configuration of compound 3, the chemical transformation from 3 to 3c was performed (as shown in Scheme 1), then the exciton chirality method was applied on 2,3,16-tri-p-methoxybenzoate derivative (3c). The negative chirality resulting from the exciton coupling between the two chromophores of p-methoxybenzoate at 267 nm (∆ε −18.41, π-π* transition) and 243 nm (∆ε +2.85, π-π* transition) indicated that the transition dipole moments of the two chromophores were oriented in a counterclockwise manner (Figure 5) . Thus, the absolute configurations of C-2 and C-3 of 3c were determined to be 2S and 3R, respectively. The absolute configuration of the β-glucose was identified to be d-configuration by HPLC analysis. Therefore, compound 3 was established as 2,16-dihydroxyl-15-keto-2-acetoxy-ent-pimar-8(14)-ene-16-O-β-d-glucopyranoside.
The known compounds sitost-5-en-3β-ol acetate (4) , β-sitosterol-3-O-β-d-glucopyranoside (5) , 3,5,6-trihydroxysitostane (6) , stigmastane-3β,5α,6β-triol-3,6-diacetate (7)  were identified by comparison of their spectroscopic data with the literature values.
The anti-inflammatory activity of all isolates were evaluated. The results (Table 2) showed that compounds 1–3 exhibited potent inhibitory activity against NO and TNF-α production in RAW264.7 cells and the steroids were inactive with the IC50 values more than 20 µM.
|Table 2. IC50 values of the active compounds 1–3 against NO and TNF-α production in RAW264.7 cells.|
a Positive control for NO and TNF-α production.
Optical rotations were measured on a Rudolph Autopol I automatic polarimeter. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. ESIMS was measured on a Finnigan LC QDECA instrument, and HRESIMS was performed on a Waters-Micromass Q-TOF. A Shimadzu LC-20 AT equipped with a SPD-M20A PDA detector was used for HPLC. A YMC-pack ODS-A column (250 × 10 mm, S-5 µM, 12 nm) was used for semipreparative HPLC separation. Silica gel (300–400 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao city, China), C18 reversed-phase silica gel (12 nm, S-50 µM, YMC Co., Ltd., Taiwan Chu-Pei city, China), Sephadex LH-20 gel (Amersham Biosciences, Piscataway, NJ, USA) and MCI gel (CHP20P, 75–150 µM, Mitsubishi Chemical Industries Ltd., Tokyo, Japan.) were used for column chromatography. All solvents used were of analytical grade (Guangzhou Chemical Reagents Company, Ltd., Guangzhou, China). All cell lines were obtained from the China Center for Type Culture Collection of the Chinese Academy of Sciences.
3.2. Plant Material
Plant of F. fimbriata were collected in July 2010 from Yunnan Province, China, and were identified by one of the authors (Prof. D. P. Yang). A voucher specimen (accession number: LSJSH201010) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University.
3.3. Extraction and Isolation
The air-dried powder of the leaves of F. fimbriata (1 kg) was extracted with 95% EtOH (3 × 10 L) at room temperature (rt) to give 80 g of crude extract. The extract was suspended in H2O (1 L) and successively partitioned with petroleum ether (PE, 3 × 1 L), EtOAc (3 × 1 L) and n-BuOH (3 × 1 L), respectively. The PE extract (15 g) was subjected to silica gel column chromatography (CC) and eluted with PE/dichloromethane (0:10→10:0) successively to afford three fractions (I–III). Fraction I (2.5 g) was chromatographed over an silica gel column eluting with PE/CH2Cl2 (0:10→10:0) to afford five fractions (Fr. Ia–Ie), Fr. Ia was then subjected to silica gel column eluting with PE/CH2Cl2 (0:10→10:0) and obtained compound 4 (113 mg). Fraction III (3.5 g) was chromatographed over an silica gel column eluting with PE/Acetone (0:10→10:0) to afford five fractions (Fr. IIIa–IIIe). Fr. IIIa (1.0 g) was separated by silica gel CC (PE/CH2Cl2, 50:1→10:1) to afford five fractions (Fr. IIIaa–IIIae). Fr. IIIac was subjected to Rp-C18 CC using a gradient of MeOH/H2O (v/v from 7:3 to 10:0) to yield 6 (22 mg) and another fraction, which after chromatography on a Sephadex LH-20 column using CHCl3/MeOH (1:1) as eluent to obtain 7 (23 mg). The EtOAc extract (42 g) was subjected to MCI gel CC eluted with a MeOH/H2O gradient (3:7→10:0) to afford three fractions (XI–XIII). Fraction XI (6.5 g) was subjected to silica gel CC (PE/EtOAc, 2:1→0:1) to give three fractions (XIa–XIc). Fr. XIa (2.1 g) was separated by silica gel CC (PE/EtOAc, 2:1), followed by semi-preparative HPLC (CH3OH/H2O, 8:2, 3 mL/min) to give 1 (6 mg). Fr. XIc (1.3 g) was separated by Rp-C18 silica gel CC (MeOH/H2O, 5:5→0:0) to yield 2 (8 mg). Fraction XII (5.5 g) was subjected to silica gel CC (CHCl3/MeOH, 30:1→0:1) to give three fractions (XIIa–XIIc). Fr. XIIb (2.5 g) was subjected to silica gel CC (PE/CHCl3, 1:1→0:1) to give three fractions (XIIb1–XIIb4). Fr. XIIb1 (0.9 g) was subjected to Rp-C18CC (MeOH/H2O, 2:8→10:0) and followed by silica gel CC (EtOAc/acetone, 3:1→0:1) to afford 5 (38 mg). Fr. XIIb4 (1.2 g) was subjected to Rp-C18 CC (MeOH/H2O, 0:10→10:0) and Sephadex LH-20 (EtOH) to yield 3 (25 mg).
3.4. Spectral Data
Flickinflimilin A (1). Colorless oil; −46.2° (c 0.24, CHCl3); UV (MeOH) λmax (log ε) 242 (2.18) nm, 203 (2.46) nm; CD (c 3.4 × 10−3 M, CH3CN), λmax (∆ε) 242 (−0.26), 278 (−0.01), and 323 (−0.07); IR (KBr) νmax 3437, 1718, 1591, 1461, 1378, 1129, 1038, 949, and 764 cm−1; 1H and 13C-NMR data, see Table 1; positive ESIMS m/z 315.3 [M + Na]+, 607.6 [2M + Na]+; HREIMS m/z 315.1943 [M + Na]+ (calcd for C18H8O3Na, 315.1936).
Flickinflimilin B (2). White amorphous powder; +16.0° (c 0.50, MeOH); UV (MeOH) λmax (log ε) 207 (2.94), 251 (2.45) nm; CD (c 1.7 × 10−3 M, CH3CN), λmax (∆ε) 207 (−6.64), 251 (+2.29), 270 (−0.34), 300 (+2.19), and 337 (−3.08); IR (KBr) νmax 3440, 1708, 1600, 1500, 1476, 1129, 767, and 750 cm−1; 1H and 13C-NMR data, see Table 1; positive ESIMS m/z 289.1 [M + H]+; negative ESIMSm/z 333.2 [M + HCOO]−; HRESIMS m/z 311.1987 [M + Na]+ (calcd for C18H24O3Na, 311.1989).
Flickinflimilin C (3). Colorless oil; −55.0° (c 0.22, CHCl3); IR (KBr) νmax 3445, 1714, 1646, 1461, 1379, 1257, 1126, 1085, and 950 cm−1; 1H and 13C-NMR data, see Table 1; positive ESIMS m/z 563.3 [M + Na]+, 1103.5 [2M + Na]+; negative ESIMS m/z 585.3 [M + HCOO]−, 1125.8 [2M + HCOO]−; HRESIMS m/z 585.2929 [M + HCOO]− (calcd for C29H45O12, 585.2923).
3.5. Computational Methods for Electronic Circular Dichroism
Molecular mechanics calculations were carried out with Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA, 2013) and quantum chemical computations were run with Gaussian 09 program package (Gaussian, Inc., Pittsburgh PA, USA, 2011) using default grids and convergence criteria. MMFF conformational search generated conformers within a 10 kcal/mol energy window were optimized using DFT method at B3LYP/6-31G (d) level. Frequency calculations were run at the same level to verify that each optimized conformer was a true minimum and to estimate their relative thermal free energies (∆G) at 298.15K. The TDDFT calculations were performed using the hybrid B3LYP functional, and Ahlrichs’ basis sets SVP (split valence plus polarization) and TZVP (triple zeta valence plus polarization). The number of excited states per each molecule was 20–30. Solvent effects were taken into account by using polarizable continuum model (PCM). CD spectra were generated by the program SpecDis (University of Würzburg, Würzburg, Germany, 2012) using a Gaussian band shape with 0.28 eV exponential half-width from dipole-length dipolar and rotational strengths; the difference with dipole-velocity values was negligible (<10%) for most transitions.
3.6. Determination of Sugar Configuration 
Compound 3 (6 mg) was refluxed with 2 M HCl (2 mL, dioxane/H2O, 1:1) at 100 °C for 4 h. After removing the dioxane under vacuum, the solution was then diluted with H2O and then extracted with EtOAc (3 × 1 mL). The EtOAc layer was evaporated under vacuum, then subjected to CC over silica gel eluteing with CHCl3:MeOH (30:1) to afford 3a. The aqueous layer was evaporated under vacuum, diluted repeatedly with H2O, evaporated under vacuum to obtain neutral residue, and then analyzed by TLC over silica gel (Me2CO/n-BuOH/H2O, 6:3:1) together with authentic sugar sample (glucose, Rf = 0.49). The remaining residue was dissolved in pyridine (200 µL), to which 2 mg of l-cysteine methyl ester hydrochloride was added. The mixture was stirred at 60 °C for 1 h; then 50 µL of o-tolyl isothiocyanate was added, and the mixture was stirred at 60 °C for another 1 h. The reaction mixture was directly analyzed by standard C18 HPLC [a YMC-pack ODS-A column (250 × 10 mm, S-5 µM, 12 nm), CH3CN/H2O, 25:75, 3 mL/min]. The peak (tR = 19.0 min) coincided with a derivative of d-glucose, as compared with authentic d-glucose with tR at 19.1 min.
3.7. Chemical Transformation of 3a to 3c 
To a stirred solution of 3a (10 mg) in MeOH (2 mL), NaOH (1 mg) was added. The mixture was stirred at room temperature for 0.5 h to obtain 3b, and then 3b (7.2 mg) was transferred into a clean NMR tube and was dried completely under the vacuum of an oil pump. Deuterated pyridine (0.55 mL) and 4-methoxybenzoyl chloride (12 µL) were added into the NMR tube immediately under dry conditions, and then the NMR tube was shaken carefully to mix the sample and 4-methoxybenzoyl chloride evenly. The reaction NMR tube was permitted to stand at 45 °C and monitored by 1H-NMR. The reaction was found to be completed after 2 h. 1H-NMR data of the 4-methoxybenzoate derivative (3c) of 3b was obtained from the reaction NMR tube directly. The reaction mixtures were transferred from the NMR tube and subjected to Sephadex LH-20 CC eluting with CHCl3:MeOH (1:1). CD (CH3OH, ∆ε) 212 (−17.20), 243 (+2.85), 267 (−18.41), 297 (+1.12) nm; 1H-NMR (CDCl3, 400 MHz) δH 5.43, (1H, s, H-14), 5.40, [1H, (ddd, J = 2.1, 4.3, 12.4 Hz, H-2)], 4.15, [1H, (dd, J = 7.6, 14.2 Hz, H-16a)], 3.67, [1H, (d, J = 2.1 Hz, H-3)], 3.15, [1H, (dd, J = 7.6, 14.2 Hz, H-16b)], 2.29–2.42, (4H, m, H-6, H-7), 1.96–2.14, (4H, m, H-1, H-12), 1.83, (1H, m, H-9), 1.44, (1H, m, H-5), 1.07, (3H, s, H-17), 1.01, (3H, s, H-18), 0.92, (3H, s, H-19), 0.88, (3H, s, H-20), [7.97–8.05, (6H, m), 6.91–6.96, (6H, d, J = 8.5 Hz ), 3.86–3.87, (9H, s, CH3O-p), p-methoxybenzoyl]; 13C-NMR (CDCl3, 100 MHz) δC 207.2 (C-15), 142.1 (C-8), 124.1 (C-14), 76.8 (C-3), 71.3 (C-2), 66.7 (C-16), 50.8 (C-9), 47.7 (C-5), 47.3 (C-13), 39.5 (C-10), 38.7 (C-4), 36.2 (C-7), 35.6 (C-12), 32.6 (C-1), 28.6 (C-18), 27.2 (C-17), 22.6 (C-19), 21.6 (C-11), 20.2 (C-6), 15.4 (C-20), (165.7, 165.5, 163.6, 163.5, 132.0, 131.6, 113.7, 113.6, 55.4) (p-methoxybenzoyl); negative ESIMS m/z 737.3 [M − H]−.
3.8. Biological Assays
3.8.1. Cytotoxic Assay 
The RAW264.7 cell line was obtained from ATCC (Manassas, VA, USA), and was cultured in DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by MTT (Sigma, St. Louis, MO, USA). Briefly, 100 µL of adherent cells with an initial density of 1 × 105 cells/mL were seeded into a 96-well plate and allowed to adhere for 24 h. Cells were exposed to the test compounds at various concentrations for 48 h. After the incubation, MTT (5 mg/mL) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 µL of 20% SDS–50% DMF after removal of the medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680, Hercules, CA, USA). The IC50 value of each compound was calculated by Reed and Muench’s method .
3.8.2. Nitric Oxide Inhibitory Assay 
Inhibition of NO production was determined in a LPS-stimulated RAW264.7 macrophage cell line. Cells were seeded in 96-well plates (1 × 105 cells/well) and allowed to adhere for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. The medium was then replaced with fresh medium containing LPS (2 µg/mL) and test compounds at 10 µM, and the cells were incubated for 24 h. NO production was determined by measuring the accumulation of nitrite in the culture supernatant with Griess reagent (0.5% sulfanilamide and 0.05% naphthylene-diamide dihydrochloride in 2.5% H3PO4) and then allowed to stand for 5 min at rt. The absorbance at 540 nm was measured using a HTS 7000 microplate reader. The nitrite concentration in the medium was determined from the calibration curve (r = 0.9998) obtained by using different concentrations of sodium nitrite (NaNO2) in the culture medium as the standard. Blank correction was performed by subtracting the absorbance due to medium only from the absorbance reading of each well.
3.8.3. Assay for the Production of Pro-inflammatory Cytokines (TNF-α) 
Compounds were dissolved in DMSO, and suspensions of RAW264.7 cells were cultured in complete RPMI 1640 medium (Hyclone) containing 10% FBS. The cultured cells were incubated with the tested compounds (10 µM) for 24 h, followed by LPS stimulation (2 µg/mL). Supernatants were collected to analyze cytokine levels. The mouse TNF-α ELISA Kit was used to determine the cytokine concentration in the culture supernatants.
Three new diterpenoids including two 15,16-dinor-ent-pimaranes and one diterpenoid glycoside, and four steroids were isolated from the leaves of F. flimbriata. The absolute configurations of the new compounds were determined by calculated ECD method, chemical transformation and exiton chirality method. All the isolates were screened for the anti-inflammation, the new compounds exhibited potent inhibitory against NO and TNF-α production in RAW264.7 cells.
Supplementary material can be accessed at http://www.mdpi.com/1420-3049/19/5/5863/s1.
We gratefully acknowledge financial support for this project from the National Natural Science Foundation of China (No. 81102782), the China Postdoctoral Science Foundation (No. 2011M501368), and the Priming Scientific Research Foundation for Junior Teachers of Medicine at Sun Yat-sen University.
Jin-Long Chen, De-Po Yang and Lin Jiang designed research; Jin-Long Chen, Wen-Jun Zhong, Jing Li and Zhi-Min Zhao performed research and analyzed the data; Jin-Long Chen and Gui-Hua Tang wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
- Song, L.R. Chinese Materia Medica (Zhonghua BenCao); Shanghai Science and Technology Press: Shanghai, China, 1999; Volume 8, pp. 711–712.
- Ma, G.X.; Wang, T.S.; Yin, L.; Pan, Y.; Guo, Y.L.; LeBlanc, G.A.; Reinecke, M.G.; Waston, W.H.; Krawiec, M. Two pimarane diterpenoids from ephemerantha lonchophylla and their evaluation as modulator of the multidrug resistance phenotype. J. Nat. Prod. 1998, 61, 112–115, doi:10.1021/np970065o.
- Ma, G.X.; Yin, L.; Wang, T.S.; Pan, Y.; Guo, L.W. A 19-carbon pimarane-type diterpenoid from Ephemerantha fimbriata. Pharm. Biol. 1998, 36, 66–68, doi:10.1076/phbi.18.104.22.16823.
- Tezuka, Y.; Hirano, H.; Kikuchi, T.; Xu, G.J. Constituents of Ephemerantha lonchophylla; Isolation and structure elucidation of new phenolic compounds, ephemeranthol-A, ephemeranthol-B and ephemeranthoquinone and of a new diterpene glucoside, ephemeranthoside. Chem. Pharm. Bull. 1991, 39, 593–598, doi:10.1248/cpb.39.593.
- Chen, H.Y.; Shiao, M.S.; Huang, Y.L.; Shen, C.C.; Lin, Y.L.; Kuo, Y.H.; Chen, C.C. Antioxidant principles from Ephemerantha lonchophylla. J. Nat. Prod. 1999, 62, 1225–1227, doi:10.1021/np990025f.
- Chen, C.C.; Huang, Y.L.; Tang, C.M. Antipatelet aggregation principles from Ephemerantha lonchophylla. Planta Med. 2000, 66, 372–373, doi:10.1055/s-2000-8553. 10865460
- Yang, L.; Qin, L.H.; Bligh, S.W.A.; Bashall, A.; Zhang, C.F.; Zhang, M.; Wang, Z.T.; Xu, L.S. A new phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-inflammatory activies. Bioorg. Med. Chem. 2006, 14, 3496–3501, doi:10.1016/j.bmc.2006.01.004.
- Lin, Y.; Wang, F.L.; Yang, J.; Chun, Z.; Bao, J.K.; Zhang, G.L. Anti-inflammatory phenanthrene derivatives from stems of Dendrobium denneanum. Phytochemistry 2013, 95, 242–251, doi:10.1016/j.phytochem.2013.08.008.
- Kanekar, Y.; Basha, K.; Duche, S.; Gupte, R.; Kapat, A. Regioselective synthesis of phenanthrenes and evaluation of their anti-oxidant based anti-inflammatory potential. Eur. J. Med. Chem. 2013, 67, 454–463, doi:10.1016/j.ejmech.2013.06.016.
- Miyazawa, M.; Shimamura, H.; Nakamura, S.I.; Sugiura, W.; Kosaka, H.; Kameoka, H. Moscatilin from Dendrobium nobile, a naturally occurring bibenzyl compound with potential antimutagenic activity. J. Agric. Food Chem. 1999, 47, 2163–2167, doi:10.1021/jf970930a.
- Harada, N.; Nakanishi, K. The exiton chirality method and its application to configurational and conformational studies of natural products. Acc. Chem. Res. 1972, 5, 257–263, doi:10.1021/ar50056a001.
- Zhang, X.; Geoffroy, P.; Miesch, M.; Julien-David, D.; Raul, F.; Aoudé-Werner, D.; Marchioni, E. Gram-scale chromatographic purification of β-sitosterol synthesis and characterization of β-sitosterol oxides. Steroids 2005, 70, 886–895, doi:10.1016/j.steroids.2005.06.003.
- Lee, K.H.; Min, Y.D.; Choi, S.Z.; Kwon, H.C.; Cho, O.R.; Lee, K.C.; Lee, K.R. A new sesquiterpene lactone from Artemisia rubripes nakai. Arch. Pharm. Res. 2004, 27, 1016–1019, doi:10.1007/BF02975423.
- Meyer, W.; Jungnickel, H.; Jandke, M.; Dettner, K.; Spiteller, G. On the cytotoxity of oxidized phytosterols from photoautotropic cell cultures of chenopodium rubroum tested on meal-worms tenebrio molitor. Phytochemistry 1998, 47, 789–797, doi:10.1016/S0031-9422(97)00622-5.
- Alvarez, F.S.; Watt, A.N. Steroids. CCCXI the degradation of stigmasterol to 3β-5α-6β-trihydroxyl-23,24-bisnorcholan-22-oic acid. J. Org. Chem. 1968, 33, 2143–2144, doi:10.1021/jo01269a107.
- Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 2007, 55, 899–901, doi:10.1248/cpb.55.899.
- Su, B.N.; Park, E.J.; Mbwambo, Z.H.; Santarsiero, B.D.; Mesecar, A.D.; Fong, H.H.S.; Pezzuto, J.M.; Kinghorn, A.D. New constituents of Euphorbia quinquecostata and absolute configuration assignment by a convenient mosher ester procedure carried out in NMR tubes. J. Nat. Prod. 2002, 65, 1278–1282, doi:10.1021/np0202475.
- Li, F.; Awale, S.; Tezuka, Y.; Kadota, S. Cytotoxic constituents from brazilian red propolis and their structure–activity relationship. Bioorg. Med. Chem. 2008, 16, 5434–5440, doi:10.1016/j.bmc.2008.04.016.
- Reed, L.J.; Muench, H. A simple method of estimating fifty percent endpoint s12. Am. J. Hyg. 1938, 27, 493–497.
- Awale, S.; Tezuka, Y.; Banskota, A.H.; Adnyana, I.K.; Kadota, S. Nitric oxide inhibitory isopimarane-type diterpenes from orthosiphon stamineus of indonesia. J. Nat. Prod. 2003, 66, 255–258, doi:10.1021/np020455x.
- Wen, T.; Li, Y.G.; Wu, M.; Sun, X.L.; Bao, X.C.; Lin, Y.Q.; Hao, J.L.; Han, L.; Cao, G.C.; Wang, Z.W.; et al. Therapeutic effects of a novel tylophorine analog, NK-007, on collagen-induced arthritis through suppressing tumor necrosis factor α production and Th17 cell differentiation. Arthritis Rheum. 2012, 64, 2896–2906, doi:10.1002/art.34528.
- Sample Availability: Samples of the compounds 1–7 are available from the authors.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).