Inhibitory Effects of Chemical Compounds Isolated from the Rhizome of Smilax glabra on Nitric Oxide and Tumor Necrosis Factor-α Production in Lipopolysaccharide-Induced RAW264.7 Cell

The rhizome of Smilax glabra has been used for a long time as both food and folk medicine in many countries. The present study focused on the active constituents from the rhizome of S. glabra, which possess potential anti-inflammatory activities. As a result, nine known compounds were isolated from the rhizome of S. glabra with the bioassay-guiding, and were identified as syringaresinol (1), lasiodiplodin (2), de-O-methyllasiodiplodin (3), syringic acid (4), 1,4-bis(4-hydroxy-3,5-dimethoxyphenyl)-2,3-bis(hydroxymethyl)-1,4-butanediol (5), lyoniresinol (6), trans-resveratrol (7), trans-caffeic acid methyl ester (8), and dihydrokaempferol (9). Among these compounds, 2 and 3 were isolated for the first time from S. glabra. In addition, the potential anti-inflammatory activities of the isolated compounds were evaluated in vitro in lipopolysaccharide- (LPS-) induced RAW264.7 cells. Results indicated that 4 and 7 showed significant inhibitory effects on NO production of RAW264.7 cells, and 1, 2, 3, and 5 showed moderate suppression effects on induced NO production. 1, 7, and 5 exhibited high inhibitory effects on TNF-α production, with the IC50 values less than 2.3, 4.4, and 16.6 μM, respectively. These findings strongly suggest that compounds 1, 2, 3, 4, 5, 7, and 9 were the potential anti-inflammatory active compositions of S. glabra.


Introduction
Inflammation is an important physical response to harmful stimuli, such as infection, injury, and irritation [1]. Activated macrophages play key roles in inflammatory diseases related to overproduction of proinflammatory cytokines, including tumor necrosis factor (TNF)-, interleukin-(IL-) 1 , IL-6, and inflammatory mediators, including nitric oxide (NO), prostaglandin E 2 (PGE 2 ), and reactive oxygen species (ROS). Thus, inhibiting the production of these macrophage mediators is an important target in treating inflammatory diseases [2]. Bacterial lipopolysaccharide (LPS), the structural component of the Gram-negative bacteria outer cell wall, has been reported to be a major initiator of the inflammatory response during most commonly seen bacterial infections. Binding of LPS to its cognate CD14 receptor on the monocyte/macrophage cell membrane induces the release of various proinflammatory cytokines and chemokines, which are implicated in the pathogenesis of the major inflammatory complications [3].
Smilax glabra Roxb, belonging to the family Liliaceae, is a perennial evergreen climbing shrub mainly distributed in China, Japan, Thailand, and so forth [4][5][6]. This plant is generally consumed as a substitute for tea and sugar to prevent scurvy, and for treating a range of different conditions such as chest ailments, rheumatism, leprosy, impotence, syphilis, and so forth [7]. Syrup made by prolonged boiling of the leaves of S. glabra was marketed in Sydney in the early 1900s as a tonic and remedy against catarrh and coughs [8]. The rhizome of S. glabra is named Tufuling in China and commonly consumed in soup, beneficial tea, and herbal medicine. It is also used in folk medicine alone or in combination with other herbal medicines for the treatment of a variety of diseases such as psoriasis and cancer in many other countries.
As mentioned above, S. glabra has demonstrated a potential to be utilized in health products. However, to the best of our knowledge, phytochemical and pharmacological studies on the edible plant S. glabra are limited, and there have been no reports on inhibitory effects of the chemical constituents from S. glabra on the proinflammatory mediators. Our previous study indicated that the phenolic-enriched extracts of S. glabra possessed significant anti-inflammatory activity, in which, astilbin, a known anti-inflammatory compound, was found [27]. The main purpose of the present study was to isolate the chemical constituents of the S. glabra rhizomes with bioassay-guiding and evaluate their in vitro anti-inflammatory activities in LPS-induced RAW264.7 cells. Overall, aim of the present study was to obtain a comprehensive understanding of the anti-inflammatory compounds in S. glabra.

Extraction and Isolation.
The extraction and isolation of the compounds are shown in Figure 2. In brief, the dried and powdered rhizomes of S. glabra (7.0 kg) were extracted with 70% ethanol (90 L × 3) by heating-reflux to give a black crude extract (marked as ESG, 1169.0 g, semidry). ESG (1000 g) was subjected to a HP-20 macroporous resin column by elution with water and 30%, 60%, and 95% ethanol in sequence to give four fractions: ESG-1 (490.0 g), ESG-2 (262.5 g), ESG-3 (116.6 g), and ESG-4 (40.8 g). ESG-2 and ESG-3 showed a significant inhibitory effect on LPS-induced NO production in RAW264.7 cells. ESG-2 and ESG-3 were merged and subjected to column chromatography on silica gel using CH 2 Cl 2 as the primary eluent with gradual increases in eluent polarity with MeOH to produce 7 subfractions (Frc. 1-7). Further separation of these subfractions using RP-C18 MPLC, preparation HPLC, or/and Sephadex LH-20 chromatography yielded 9 compounds.

Identification of Compounds 1-9.
The NMR data of the isolated compounds were recorded on a Bruker AVANCE-500 instrument using TMS as an internal standard. Electrospray ionization mass spectra (ESI-MS) were measured on a Thermo Scientific Finnigan LTQ mass spectrometer, and Preparative HPLC was conducted using a Waters 2545 Binary gradient module instrument with 2998 Photodiode Array Detector and YMC-Pack ODS-A column (250 × 20 mm, 5 m). Column chromatography (CC) was performed with macroporous adsorption resin Diaion HP-20 (Mitsubishi Chemical Holdings, Japan), silica gel (100-200 mesh, Qingdao Marine Chemical Inc., Qingdao, China), ODS-A (50 m YMC, Japan), and Sephadex LH-20 (GE Healthcare Bio-Science AB, Sweden). TLC was carried out on glass precoated silica gel GF 254 plates, and spots were visualized under UV light (254 and/or 366 nm) or by spraying with 10% (v/v) sulfuric acid in ethanol followed by heating to 105 ∘ C.

Cell
Culture. The mouse macrophage-derived RAW264.7 cell line was purchased from Sun Yat-Sen University, Guangzhou, China, and maintained at 37 ∘ C in a humidified atmosphere containing 5% CO 2 in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 g/mL). Cells in exponential growth phase were used for experiments.

Cells Viability Assay.
The cytotoxicity of the isolated compounds toward RAW264.7 was evaluated by a conventional MTT assay as reported previously [28]. RAW264.7 cells (1 × 10 5 cells/well) were inoculated to 96-well plates and incubated for 12 h and then treated with different concentrations of compounds. After additional 24 h incubation, 10 L of MTT solution (5 g/mL) was added to each well, and the plate was incubated for another 4 h. The medium was discarded and 150 L of dimethyl sulfoxide (DMSO) was added to each well, solubilizing formazan. After 15 min incubation, absorbance at 570 nm was read using a microplate reader. The percent viability was calculated using the following formula:  . Dexamethasone (DMS) was employed as a positive control. # and ###, respectively, mean P < 0.05 and P < 0.001 compared with control group. * , * * , and * * * , respectively, mean P < 0.05, P < 0.01, and P < 0.001 compared with group treated with LPS alone.

Measurement of Nitric Oxide Production in LPS-
concentration by reference to a standard curve generated with sodium nitrite.

Determination of TNF-Production in LPS-Induced
RAW264.7 Cells. RAW264.7 cells (3 × 10 6 cells/mL) were seeded onto 24-well culture plate and incubated for 12 h. The cells were then pretreated with various concentrations of the isolated compounds for 2 h before stimulation with LPS (100 ng/mL) with or without samples for 12 h. Supernatants were then collected and the TNF-concentrations in the medium were determined using commercially available ESISA kits according to the manufacturer's instructions as described in previous study [29].

Statistical Analysis.
All values in the figures and text were expressed as means ± SD. The results were analyzed by one-way ANOVA. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software. A P value less than 0.05 was considered significant.

Results and Discussion
The dried rhizomes of S. glabra were extracted with 70% ethanol by heating-reflux to give a black crude extract (marketed as ESG), which was further partitioned into four fractions (ESG-1, ESG-2, ESG-3, and ESG-4) by subjecting to a HP-20 macroporous resin column with gradient elution of ethanol-water. Since fractions ESG-2 and ESG-3 showed a significant inhibitory effect on LPS-induced NO production in RAW264.7 cells (Figure 1), they were merged and further subjected to various column chromatographies to yield 9 known compounds (Figure 2).

trans-Caffeic Acid Methyl Ester
NO is well known as endogenous regulators of cell and tissue function, but excessive production of NO maybe participate in several autoimmune or chronic inflammatory diseases [37]. TNF-is the earliest and primary endogenous mediator of the process of inflammatory reaction and mediates the inflammatory response the local and systemic levels [38]. Therefore, the inhibition of these media can be a very important target for development of anti-inflammation agents.
The anti-inflammatory activities of the isolates were tested in vitro for inhibiting the NO and TNF-production in LPS-induced RAW264.7 cells. Firstly, the cytotoxicity of these compounds cells on the proliferation of RAW264.7 cells was measured by MTT assay. The IC 10 value of each compound (Table 1), at which 10% of cells proliferation was inhibited, was calculated with an improved Karber methodology and defined as the highest noncytotoxic concentration. While the dose is ≤IC 10 , the effect of the isolated compound on  Figure 4(a), all isolated compounds, expect Comp. 8, exhibited dose-dependent inhibitory effects on LPS-induced NO production in RAW264.7 cells. Particularly, Comp. 7 at 4.4 and 0.4 M, respectively, inhibited about 48% and 32% of LPS-induced NO production, while Comp. 4 at 5.1 and 0.5 M inhibited about 25% and 19% of induced NO production, respectively. Compounds 1, 2, 3, and 5 showed moderate suppression effects on induced NO production. But, Compounds 6 and 9 showed the lowest inhibitory effects, at level of IC 10 , respectively, 238.1 and 139.4 M; just 32.7% and 21.3% of NO production were inhibited separately. The inhibitory effects of isolated compounds on TNF-production presented in Figure 4(b) showed that all isolated compounds, expect Compounds 4 and 9, exhibited dose-dependent inhibitory effects on LPS-induced TNFproduction in RAW264.7 cells. Compounds 1, 7, and 5 exhibited high inhibitory effects on TNF-production, with the IC 50 values less than 2.3, 4.4, and 16.6 M, respectively.
The results of the present study were consistent with that reported previously on the anti-inflammatory activities of the isolated compounds. Resveratrol is a natural polyphenolic stilbene derivative found in a variety of edible fruits and is known for its multiple pharmacological activities. Its antiinflammatory activity has been demonstrated in vitro and in vivo, by showing that it could attenuate cytokine production in adipose tissue by repressing TLR2-and TLR4-mediated proinflammatory signaling cascades and decrease COX-2 expression [39]. Syringaresinol isolated from many different plants has been demonstrated to possess significant antiinflammatory activity, which could significantly inhibit NO, PGE2, and TNF-production of LPS-induced RAW264.7 and BV-2 cells, as well as decrease the expression level of iNOS and COX-2 enzyme [40][41][42]. And other compounds, including syringic acid and dihydrokaempferol were also reported previously to exhibit potential anti-inflammatory activities [36,43,44]. Taken together, the present study demonstrated that trans-resveratrol, syringic acid, syringaresinol, lasiodiplodin, de-O-methyllasiodiplodin, and dihydrokaempferol are the potential anti-inflammatory active constituents of the rhizomes of S. glabra. Further studies should be carried out to evaluate their anti-inflammatory effects in vivo and the mechanisms of action by which effects are mediated.