New Anti-inflammatory Flavonol Glycosides from Lindera akoensis Hayata

Inflammation is related to many diseases. Lindera akoensis Hayata was often used in folk therapy in Taiwan for inflammation. In this study, three new flavonol acyl glycosides, namely kaempferol-3-O-β-D-4′′,6′′-di-(E)-p-coumaroylglucoside (1), 3′′-(E)-p-coumaroylafzelin (2) and 4′-O- methyl-2′′,4′′-di-(E)-p-coumaroylquercitrin (3), and three components, 3β-dodecyl-4β-hydroxy- 5β-methyldihydrofuran-2-one (4), 2β-acetoxyclovan-9α-ol (5), (1α,4β,6β)-trihydroxyeudesmane (6) that were isolated from the natural product for the first time were obtained along with 25 known compounds from L. akoensis. Their structures were determined by comprehensive spectroscopic analyses (1D and 2D NMR, EI-, ESI- and HRESI-MS). The ability of 1 to decrease the LPS-stimulated production of nitrite in RAW264.7 cell was evaluated, showing an IC50 value of 36.3 ± 3.2 μM. This result supports the value of L. akoensis as a traditional medicine resource.


Introduction
Lindera akoensis Hayata belonging to the Lauraceae family, moreover, it is an endemic species widely distributed in central and southern Taiwan. Traditionally it has been used by local residents to treat various inflammation symptom [1]. The genus Lindera has shown many bioactivities, including antitumor [2][3][4], anti-inflammatory [5,6] and antibacterial [7,8] properties in previous literature reports. Previous phytochemical research of the genus Lindera revealed an abundance of butalactones [8][9][10], sesquiterpenes [11] and flavonoids [9,12,13] in this genus. In our earlier study on the aerial parts of L. akoensis, was explored the isolation of butanolides, flavonols, and lignans [9]. In order to confirm the traditional folk usage of L. akoensis, this study continued our previous work on the isolation and purification of L. akoensis components. Six novel compounds ( Figure 1) and 25 known compounds were isolated and identified. Next their anti-inflammatory activity was evaluated. Based on our experimental results, compound 1 has anti-inflammatory activity by decreasing nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW264.7 cells in IC 50 36.3 ± 3.2 µM, having potential as a lead compound to treat symptoms of inflammation.
Kaempferol-3-O-β-D-4 ,6 -di-(E)-p-coumaroylglucoside (1) was isolated as a pale yellow solid. The IR spectrum showed the presence of hydroxyl (3240 cm −1 ) and carbonyl groups (1651 cm −1 ). Four structural units were observed in the 2D-NMR spectrum: two (E)-p-coumaroyls, glucose and a kaempferol nucleus (Figure 2 [14], the only difference being the fact that the H-3 proton was not detected and the 13 C-NMR signal of C-3 (δ C 135.4) was more down-shifted than the C-3 of apigenin (δ C 103.2) furthermore, a significant HMBC relationship of H-1 to C-3 was detected, so through the above evidence, the glucose-kaempferol linkage was presumed to be in a C-3-O-C-1 configuration.
2β-Acetoxyclovan-9α-ol (5) (Table   S1). A typical acetyl group carbonyl was observed at δ C 171.0 and δ H 2.02 (3H, s). The structure of compound 5 was similar to that of clovandiol (16) [17] that was isolated in this work, the only difference was δ H 4.83 (1H, dd, J= 8.7, 5.9 Hz) of H-4 was down shifted more than the H-4 of clovandiol (δ H 3.79, 1H, dd, J= 10.5, 5.5 Hz), thence the acetyl group is speculated to be linked at this position, and the significant HMBC relationship of H-2/C-1' confirmed this. This work is the first to describe the structure 5 in a natural product. Heymann, et al. previously obtained it by acetylating clovandiol in 1994 [18]. (1α,4β,6β)-Trihydroxyeudesmane (6) was isolated as colorless needle-like crystals, with the molecular formula C 15 H 28 O 3 from HR-EI-MS (m/z 256.2029 [M] + , calcd 256.2010). The absolute stereo configuration of 6 was solved by X-ray single crystal diffraction ( Figure S1). The IR spectrum showed the presence of a hydroxyl (3238 cm −1 ) group. The 13 C-NMR and DEPT spectrum showed compound 6 had a eudesmane skeleton (  (Table S1). The relative stereo configuration was decided by the NOESY spectrum, H-6/H-14/H-15 were in an axial position as defined by their significant NOESY correlations with each other. The significant correlation of H-5/H-1, -12, and -13, and the small coupling constant (4.4 Hz) between H-6 and H-7 decided the relative stereo configuration of H-1, -5, and the isopropyl. According to the literature, compound 6 was never reported as a natural product, but it was prepared by hydrolysis of pumilaside A with hesperidinase by Kitajima et al. in 2000 [19].
Caffeic acid is an effective anti-inflammatory substance. According to the literature [35][36][37][38], it inhibits inflammatory responses in many ways, including nitric oxide (NO) produced by various induction pathways, therefore the anti-inflammatory evaluation in this work used caffeic acid as positive control. linderakoside F (1) showed in vitro anti-inflammatory activity since it decrease the LPS-stimulated production of nitrite in RAW264.7 cell, with the IC 50 value 36.3 ± 3.2 µM a lot better than caffeic acid (162.8 ± 5.6 µM), in addition, they have no obvious cytotoxicity at the concentration of the experiment (Figure 3). Unfortunately, the weights of linderakoside G-H (2-3) was too small to evaluate their anti-inflammatory activity.

Plant Material
The aerial part of L. akoensis was collected in Taichung, Taiwan, in July, 2008. This material was identified by Prof. Yen-Hsueh Tseng, Department of Forestry, National Chung Hsing University, Taichung, Taiwan. A voucher specimen (CMU2008-06-LA) was deposited in the School of Pharmacy, China Medical University.

Bioactivity Assays
The assays of evaluating nitric oxide (NO) production and cell viability on RAW264.7 cells were followed our studies before [9,10] and consulted literature [39]. RAW264.7 cells were seeded at a density of 5 × 10 4 cells/well in 96-well plates for 12 h. Cells were treated with linderakoside F (1) in the presence of LPS (100 ng/mL) for 24 hours. Supernatants were collected and NO levels were determined using the Greiss reagent. Each of 100 µL of supernatant was mixed with 100 µL of Griess reagent (0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride, 1% sulfanilamide, and 5% phosphoric acid) then incubated for 5 min at room temperature. The absorbance of the mixture was measured at 540 nm using a microplate reader (SpectraMax ® M2e, Molecular Devices, Sunnyvale, CA, USA). Culture media were used as blanks and the nitrite levels were determined by using a standard curve obtained from sodium nitrite (0-125 µM).

Conclusions
Despite being an endemic species of Lauraceae in Taiwan, there are not many reports yet on the phytochemistry and bioactivities of L. akoensis. Traditionally, L. akoensis is only used for ornamental purposes and some inflammation treatments. This study obtained three new flavonol acylglycosides, linderakosides F-H (compounds 1-3) and three components 4-6 isolated from a natural product that first time, along with 25 known compounds, including two monoterpenoids 7-8, 17 sesquiterpenoids 9-25, and six steroids 26-31 which were isolated from this plant for the first time. Linderakoside F (1) displayed potential anti-inflammatory activity, with an IC 50 value of 36.3 ± 3.2 µM. In this work, we discovered active components as potential lead compounds and additionally provided a scientific basis for the drug use of L. akoensis.