Isolation and Bioactivity Analysis of Ethyl Acetate Extract from Acer tegmentosum Using In Vitro Assay and On-Line Screening HPLC-ABTS+ System

The Acer tegmentosum (3 kg) was extracted using hot water, and the freeze-dried extract powder was partitioned successively using dichloromethane (DCM), ethyl acetate (EA), butyl alcohol (n-BuOH), and water. From the EA extract fraction (1.24 g), five phenolic compounds were isolated by the silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatography. Based on spectroscopic methods such as 1H-NMR, 13C-NMR, and LC/MS the chemical structures of the compounds were confirmed as feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5). Moreover, a rapid on-line screening HPLC-ABTS+ system for individual bioactivity of the EA-soluble fraction (five phenolic compounds) was developed. The results indicated that compounds 1 and 2 were first isolated from the A. tegmentosum. The anti-inflammatory activities and on-line screening HPLC-ABTS+ assay method of these compounds in LPS-stimulated murine macrophages were rapid and efficient for the investigation of bioactivity of A. tegmentosum.


Introduction
Traditional remedies based on natural products could be traced back over five millennia to written documents of the early civilizations [1]. Particularly, traditional Korean and Chinese oriental medicines herbs (OMHs) have attracted interest and acceptance in many countries with the merits of a few side effects, affordability, and local availability. Moreover, their long historical clinical practice and reliable therapeutic efficacy make them excellent sources for discovering natural bioactive compounds [2]. Among them, Acer (A.) tegmentosum (Aceraceae, Sancheong-mok in Korean) is a type of deciduous tree distributed in the Northeast Asia including Korea, Russia, and China [3]. In Korea, the leaves and stem of A. tegmentosum have been traditionally used for the treatment of hepatic disorders such as hepatitis, hepatic cancer, hepatic cirrhosis, and liver detoxification [4]. Previous studies have shown that extracts of the stem of A. tegmentosum possess various pharmacological properties such as antioxidative, anti-inflammatory, antigastrophatic, antiadipogenic, anticancer, and cytotoxic activities [5][6][7]. However, the phytochemical constituents and efficient method for investigating bioactivity of A. tegmentosum have not been reported.
Also, a variety of approaches have been developed for the extraction of useful components from A. tegmentosum, for instance, soxhlet extraction (SE), heating reflux extraction (HEE), supercritical fluid extraction (SFE), ultrasonic assisted extraction (UAE), and microwave-assisted extraction (MAE) [8][9][10]. Water, methanol, ethanol, and ethyl acetate (EA) were commonly used solvents for the extraction of bioactive compounds from plant materials and OMHs. The identification and relative amounts of five types of compounds in A. tegmentosum were determined by LC-MS, NMR, and on-line screening HPLC-ABTS + assay [1]. These techniques included on-line screening with HPLC post-column assay involving the ABTS + radical technique 2 Journal of Analytical Methods in Chemistry [11], allowing spectrophotometric monitoring of bioactive compounds. Generally, DPPH (ABTS) radical is another simple, rapid on-line method for the detection of antioxidants from crude plant extracts [12]. It combines HPLC with an assay involving a stable radical species [1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS)] in the HPLC-DPPH (ABTS) method [13,14]. Moreover, this method was successfully applied for the screening and identification of natural bioactive compounds from complex mixtures, particularly for the extracts of OMHs [15][16][17]. The chemical structures of the five types of compounds were confirmed by spectroscopic methods such as 1 H-NMR, 13 C-NMR, and LC/MS [18]. In this study, five phenolic compounds were isolated from hot water extract of A. tegmentosum bychromatographic separation. Using spectroscopic methods, the structures of these compounds were determined as feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6 -O-galloyl salidroside (5). Moreover, we investigated their anti-inflammatory effects on LPS-stimulated RAW 264.7 cells. We also investigated the applications of on-line screening HPLC-ABTS + assays for bioactivity screening to find a more practical approach toward the use of on-line screening HPLC-ABTS + assays for the rapid pinpointing of peaks in chromatograms corresponding to bioactive compounds.

ABTS + Sample Preparation.
A 2 mM ABTS stock solution containing 3.5 mM potassium persulfate was prepared and was kept in the dark at room temperature for 16 h to allow the completion of radical generation and was then diluted with water (1 : 29, v/v).

Solvent Extraction and
Purification. Dry samples (3 Kg) from the powders of the A. tegmentosum were loaded (10times volume) in hot water extraction system. The extraction was performed by heating for 3 h at 100 ∘ C (Gyeongseo Extractor Cosmos-600, Inchon, Korea). Then, the solution was filtered using standard testing sieves (150 m, Retsch, Haan, Germany), freeze-dried, and maintained in desiccators at 4 ∘ C prior to use. For large amount of extractions, 20 g freeze-dry samples were loaded (1 : 1, extracted thrice) and extracted successively using DCM, EA, and n-BuOH. The contents of feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6 -O-galloyl salidroside (5) in A. tegmentosum were remarkably higher in the EA extract (1.24 g). Then, the samples were filtered through a 0.2 m membrane filter prior to on-line screening HPLC-ABTS + analysis. The extraction and purification processes from A. tegmentosum are shown in Figure 2.     and 320 nm. The composition of the mobile phases was A: 99.9% water/trifluoroacetic acid (99.9/0.1 vol%) and B: 100% acetonitrile. The run time was 60 min, and the solvent program was the linear gradient method (90 : 10-60 : 40 A : B vol%, 70 min: initial condition) ( Table 2). Figure 3 shows a schematic diagram of the on-line coupling of a HPLC to a DAD (diode array detector) and the continuous flow ABTS + assay. Then, on-line HPLC was connected to a "T" piece where ABTS + was added. The ABTS + at a flow rate of 0.5 mL/min was delivered using a Dionex ultimate 3000 pump. After mixing through a 1 mL loop, maintained at 40 ∘ C, the absorbance was measured using a multiple wavelength detector (MWD) at 734 nm. Data were analyzed using the Chromeleon 7 software.   100 U/mL of antibiotics sulfate. The cells were incubated in humidified 5% CO 2 atmosphere at 37 ∘ C. To stimulate the cells, the medium was changed with fresh RPMI 1640 medium, and LPS (200 ng/mL) was added in the presence or absence of five compounds (10, 30, 50, and 100 M) for 24 h.

MTT Assay for Cell Viability.
Cytotoxicity was analyzed using the MTT assay. Five compounds were added to the cells and incubated for 24 h at 37 ∘ C with 5% CO 2 . MTT solutions were added to each well and the cells were incubated for another 4 h. The formazan melted in dimethyl sulfoxide (DMSO), and then the optical density was read at 570 nm using an ELISA reader (Infinite M200, TECAN, Männedorf, Switzerland).
2.9. Measurement of NO Production. NO production was analyzed by measuring the nitrite in the supernatants of cultured macrophage cells. The cells were pretreated with five compounds and stimulated with LPS for 24 h. The supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) and incubated at room temperature (RT) for 5 min [19]. The absorbance at 570 nm was read.

Determination of TNF-, IL-6, and IL-1 Cytokine
Production. Cells were seeded at a density of 5 × 10 5 cells/mL in 24-well culture plates and pretreated with various concentrations of five compounds for 30 min before the LPS stimulation. ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at 4 ∘ C with capture antibody diluted in a coating buffer (0.1 M carbonate, pH 9.5) and then washed five times with phosphate-buffered saline (PBS) containing 0.05% Tween 20. The nonspecific protein-binding sites were blocked with assay diluent buffer (PBS containing 10% FBS, pH 7.0) for >1 h. The samples and standards were added to the wells promptly. After 2 hours of incubation at RT or overnight at 4 ∘ C, the working detector solution (biotinylated detection antibody and streptavidin-HRP reagent) was added and incubated for 1 h. Subsequently, the substrate solution (tetramethylbenzidine) was added to the wells and incubated for 30 min in the dark before the reaction was quenched with stop solution (NH 3 PO 4 ). The optical density was read at 450 nm [19].
2.11. Statistical Analysis. The results are expressed as mean ± SE values for the number of experiments. Statistical significance of each treated group was compared to the control and determined by Student's -tests. Each experiment was repeated at least thrice to yield comparable results. Values with < 0.01 and <0.001 were considered significant.  Table 3. The characterization data of compound 1 were compared to the literature value and combined with quercetin and arabinopyranoside to confirm the structure as feniculin [20].  Table 3.   Table 3.  Table 3. Compounds 3 and 4 were compared to the literature data for (+)-catechin and (−)-epicatechin for the structure identification [22].   Table 3. 1 H-NMR data were consistent with the literature values [23].

On-Line Screening HPLC-ABTS + Assay Analysis.
This study investigated the bioactivity (using ABTS + assay; radical scavenging activity) and anti-inflammatory activity of the five isolated phenolic compounds that were measured. All the compounds 1-5 in the EA fraction (each yield; mg) exhibited antioxidant activities (Table 4). Moreover, this on-line screening HPLC-ABTS + assay method was rapid and efficient for the investigation of bioactivity from A. tegmentosum and was obtained from RS-tech (0.46 × 25 cm, 5 m, C 18 , Daejeon, Korea). The injection volume was 10 L, and the flow rate of the mobile phase was 1.0 mL/min. The wavelength of the UV detector was fixed at 210, 254, 280, and 320 nm. The five phenolic compounds were characterized by comparing the HPLC UV-DAD maximum absorption peaks of the samples with those of the pure isolation standards ( Figure 4). The determination of antioxidant activity   Figure 6). This study confirms the feasibility of assessing the bioactivity of specific phytochemicals using an on-line screening HPLC-ABTS + assay method (Table 5). This proposed method was successfully applied for the screening and identification of natural bioactive compounds from A. tegmentosum. Figure 7 shows the HPLC profile and LC-MS spectra of five isolated phenolic compounds from A. tegmentosum. LC-MS analysis indicated that the compounds from A. tegmentosum were isolated in highly pure form. Each compound was dissolved in MeOH at a concentration of 200 ppm, and the LC-MS analysis conditions are listed in Table 1. LC-MS analysis is a powerful tool in metabolic profiling and metabolomics research and it can accurately determine the content of specific metabolites even at low levels in plant samples. LC-MS analysis was used previously to identify certain constituents of A. tegmentosum. The comparison of UV spectra, mass spectra, and retention times of the five types of compounds with the data of standard compounds led to their unambiguous assignments.

Effect of Five Compounds on RAW 264.7 Cell Viability.
We evaluated the cytotoxicity of five compounds using  the MTT assay to determine the optimal concentration effective for anti-inflammation with minimum toxicity. As shown in Figure 8(a), all the five compounds did not affect cell viability up to 100 M, indicating that the compounds were not toxic to cells.  The culture supernatant was analyzed for nitrite production. As a control, the cells were incubated with vehicle alone. Data shows mean ± SE values of triplicate determination from independent experiments. * < 0.01 and * * < 0.001 were calculated from comparing with LPSstimulation value. NO production was measured. As a positive control, we employed 10 M dexamethasone, which is widely employed as an anti-inflammatory agent. All the compounds, except 6 -O-galloyl salidroside, did not show any inhibitory effect on LPS-induced NO production (Figure 8(b)).

Effect of Five Compounds on LPS-Induced Inflammatory Cytokines
Production. The inhibitory effect of the five compounds on the production of inflammatory cytokines, another parameter of inflammation, was investigated. In this study, we examined the effect of the five compounds on TNF-, IL-6, and IL-1 expressions. Figure 9(a) shows that feniculin slightly repressed TNF-production at a concentration of 10 M (Figure 9(a)). However, avicularin, (+)-catechin, (−)-epicatechin, and 6 -O-galloyl salidroside did not show any inhibitory effect on LPS-induced TNFproduction. As shown in Figure 9(b), consistent with TNF-results, feniculin slightly repressed IL-6 production at a concentration of 10 M. Moreover, avicularin inhibited IL-6 production at concentrations of 30 and 50 M. (+)-Catechin inhibited IL-6 production at 30 M or more. (−)-Epicatechin and 6 -O-galloyl salidroside did not show any inhibitory effect on LPS-induced IL-6 secretion. However, all the compounds except 6 -O-galloyl salidroside did not inhibit IL-1 (Figure 9(c)).

Conclusions
This study showed that among the soluble fractions from the hot water extract of A. tegmentosum, the EA-soluble fraction  Figure 9: Effect of five compounds on the production of (a) TNF-, (b) IL-6, and (c) IL-1 cytokines in macrophages. Cells were pretreated with five compounds for 30 min before being incubated with LPS for 24 h. Production of cytokines was measured by ELISA. Data shows mean ± SE values of duplicate determinations from three independent experiments. * < 0.01 and * * < 0.001 were calculated from comparing with LPS-stimulation value.