The Effect of the Aerial Part of Lindera akoensis on Lipopolysaccharides (LPS)-Induced Nitric Oxide Production in RAW264.7 Cells

Four new secondary metabolites, 3α-((E)-Dodec-1-enyl)-4β-hydroxy-5β-methyldihydrofuran-2-one (1), linderinol (6), 4′-O-methylkaempferol 3-O-α-l-(4″-E-p-coumaroyl)rhamnoside (11) and kaempferol 3-O-α-l-(4″-Z-p-coumaroyl) rhamnoside (12) with eleven known compounds—3-epilistenolide D1 (2), 3-epilistenolide D2 (3), (3Z,4α,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (4), (3E,4β,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (5), matairesinol (7), syringaresinol (8), (+)-pinoresinol (9), salicifoliol (10), 4″-p-coumaroylafzelin (13), catechin (14) and epicatechin (15)—were first isolated from the aerial part of Lindera akoensis. Their structures were determined by detailed analysis of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Lindera akoensis showed that in vitro anti-inflammatory activity decreases the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cell, with IC50 values of 4.1–413.8 μM.

The folk usage of L. akoensis is in the treatment of trauma and inflammation [14]. Butanolides showed anti-inflammation in previous studies [15,16]. In a random screening for inhibitory activity of various Chinese traditional medicines toward nitric oxide (NO) production in vitro by RAW264.7 cells, the EtOH extract of the aerial parts of L. akoensis showed a significant activity. Thus, the constituents of L. akoensis were investigated. This paper deals with the structure elucidation of the new compounds, and the inhibitory activity of the isolates toward nitric oxide (NO) production towards RAW264.7 cells is also discussed.
Compound 11 was a pale yellow amorphous solid, ([α] 2°D ± 0° (c = 8.3, CH 3 OH)). Its molecular formula was determined to be C 31 H 28 O 12 by HR-ESI-MS spectrometry (m/z 592.1576 ([Na] + ; calcd 592.5446). The IR spectrum exhibited bands at 3426 and 1651 cm −1 due to a hydroxyl and a conjugated carbonyl group. The NMR signals of rhamnose were easily assigned by their characteristic multiplicities, especially on the unique proton signal of the methyl, which was up-field at δ H 0.78 (3H, d, J = 6.3 Hz), shielded by a C-ring, the aromatic ring of flavon [24]. An A 2 X 2 coupling system at δ H 7.49 (2H, d, J = 8.6 Hz, H-5''', -9''') and 6.84 (2H, d, J = 8.6 Hz, H-6''', -8'''), as well as two olefinic proton signals at δ H 6.25 and 7.53 (each 1H, d, J = 16.0 Hz) could be observed in the presence of a E-p-coumaroyl moiety. The H-4'' triplets (δ H 4.91, t, J = 9.7 Hz) in this compound appeared at a relatively low field with respect to the corresponding signal of afzelin [24]. Hence, this compound is esterified at this position. The apigenin group could be observed by NMR spectra, matching the literature [27], but the proton signal at H-3 (δ H 6.76, 1H, s) cannot be detected; moreover, a conspicuous difference of the carbon signal between C-3 of 11 (δ C 135.7) and C-3 of apigenin (δ C 103.2) was observed. By this evidence, we speculated that rhamnose connected on apigenin with a C-3-C-1'' linkage, just like the common afzelin; this speculation was certificated by 1-and 2-D NMR. A methoxy, with a resonance at δ H 3.85 (3H, s), correlated with C-4' (δ C 163.6) on the HMBC spectrum, indicating that C-4' was the position where it linked with a methoxy; furthermore, the significant NOE correlation on position 3' (δ H 7.14, 2H, d, J = 8.8) and a methoxy (δ H 3.85, 3H, s) proved this. The rhamnoside and E-p-coumaroyl configurations were decided by the 1D-, 2D-NMR and comparison of the 1 H-and 13 C-NMR spectrum of compound 13 [24]. Based on the above deduction, 11 was designated to be a new compound 4'-O-methylkaempferol 3-O-α-L-(4''-E-p-coumaroyl)rhamnoside.

Anti-Inflammatory Activity
NO, produced from L-arginine by NO synthase, has various biological actions, e.g., as a defense and regulatory molecule for homeostatic equilibrium [28]. However, in pathophysiologic conditions, such as inflammation, there is an increased production of NO by inducible NO synthase (iNOS) [29]. Macrophages have been expected to be an origin of inflammation, because they contain various chemical mediators that may be responsible for several inflammatory stages [30]. The inhibitory activity toward NO production, induced by lipopolysaccharides (LPS), by murine macrophage-derived RAW264.7 cells, was assayed. These compounds from L. akoensis were screened by anti-inflammatory activity in vitro with a decrease in nitrite of the LPS-stimulated production in RAW 264.7 cells with IC 50 values of 4.1-413.8 µM ( Table 2). Values are expressed as mean ± SD of three replicates.

General
UV spectra were obtained with a Shimadzu Pharmaspec-1700 UV-Visible spectrophotometer. Optical rotations were obtained with a Jasco P-1020 polarimeter. Infrared spectra were obtained with a Shimadzu IRprestige-21 Fourier transform infrared spectrophotometer. 1D-and 2D-NMR spectra were recorded with a Bruker DRX-400 FT-NMR spectrometer. Mass spectrometric (HR-EI-MS and HR-ESI-MS) data were generated at the Mass Spectrometry Laboratory of the Chung Hsing University. Column chromatography was performed using Merck Si gel (30-65 µM), and TLC analysis was carried out using aluminum pre-coated Si plates; the spots were visualized using a UV lamp at λ = 254 nm. HPLC chromatograms were obtained with a Shimadzu LC-6A and a IOTA-2 RI-detector with a Phenomenex luna silica(2) 250 × 10 column.

Plant Material
Lindera akoensis was collected and identified by Dr. Yen-Hsueh Tseng (Department of Forestry, National Chung Hsing University) at Taichung, Taiwan, in July, 2008.

Extraction and Isolation
The materials were totally dried under dark in air. The dried aerial part of L. akoensis (5.9 kg) was cut into small pieces and soaked in 95% ethanol (60 liter, 7 days × 3). After filtration, the crude extract was concentrated and stored under vacuum to yield an brown thick paste (337.8 g) that was suspended in H 2 O (1000 mL) and extracted with ethyl acetate (1000 mL, 3 times). The resulting ethyl acetate extract was concentrated to yield 127.8 g of a brown thick oil that was purified by 1900 g silica gel with a particle size 0.063-0.200 mm and an internal diameter of the column, 15 cm packed height, 25 cm chromatography, using a gradient of increasing polarity with n-hexane/ethyl acetate (99:1-0:100) as the mobile phase and separated into 21 fractions on the basis of TLC analysis for random isolation of compounds. Fraction 11 (5.08 g) was re-separated by chromatography and semi-preparative HPLC with 40% EtOAc in n-hexane to afford pure, butanolide 1 (6.1 mg, 0.00104‰), 2 (7.7 mg, 0.00131‰), 3 (7.8 mg, 0.00132‰) and 4 (2.1 mg, 0.00036‰) and 5 (1.6 mg, 0.00027‰). Fraction 15 (6.82 g) was re-separated by chromatography and semi-preparative HPLC with 50% EtOAc in n-hexane to afford pure lignans 7 (2.3 mg, 0.00039‰), 9 (1.5 mg, 0.00025‰) and 10 (1.2 mg, 0.00020‰). Fraction 16 (7.15 g) was re-separated by chromatography and semi-preparative HPLC with 60% EtOAc in n-hexane to afford pure lignans 6 (8.  Table 1.

Measurement of Nitric Oxide/Nitrite
NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction. The cells were incubated with butanolides (0, 3.125, 6.25, 12.5, 25 and 50 µg/mL) in the presence of LPS (100 ng/mL) at 37 °C for 24 h. Then, cells were dispensed into 96-well plates, and 100 µL of each supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min; the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices Orleans Drive, Sunnyvale, CA, USA). Serum samples were diluted four times with distilled water and deproteinized by adding 1/20 volume of zinc sulfate (300 g/L) to a final concentration of 15 g/L. After centrifugation at 10,000× g for 5 min at room temperature, 100 μL supernatant was applied to a microtiter plate well, followed by 100 μL of Griess reagent. After 10 min of color development at room temperature, the absorbance was measured at 540 nm with a Micro-Reader. By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured form absorbance at 540 nm.

Cell Viability
Cells (2 × 105) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with compounds 1-5 in the presence of 100 ng/mL LPS (lipopolysaccharide) for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 µL of 0.5 mg/mL MTT for 2 h at 37 °C testing for cell viability. The medium was