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

Lee KJ, Song NY, Oh YC, Cho WK, Ma JY - J Anal Methods Chem (2014)

Bottom Line: Based on spectroscopic methods such as (1)H-NMR, (13)C-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).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.

View Article: PubMed Central - PubMed

Affiliation: Korean Institute of Oriental Medicine (KIOM), KM-Based Herbal Drug Development Group, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 305-811, Republic of Korea.

ABSTRACT
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 (1)H-NMR, (13)C-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.

No MeSH data available.


Analysis of UV wavelength from high purity isolation compounds.
© Copyright Policy - open-access
Related In: Results  -  Collection


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fig4: Analysis of UV wavelength from high purity isolation compounds.

Mentions: 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 μm, C18, Daejeon, Korea). The injection volume was 10 μL, 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 from the on-line HPLC–DPPH (ABTS) assay was based on the decrease in absorbance at 517 and 734 nm after the postcolumn reaction of antioxidants separated from HPLC with DPPH (ABTS), and the antioxidants present in a sample would be easily indicated by negative peaks. The composition of the mobile phases was A: 99.9 vol% water/trifluoroacetic acid (99.9/0.1, vol%) and B: 100% acetonitrile. The run time was 60 min, and the solvent program used the linear gradient method (90 : 10–60 : 40 A : B vol%, 70 min: initial condition). The ABTS+ flow rate was 0.5 mL/min. The HPLC separated analytes showed a postcolumn reaction with the ABTS+ and the reduction was detected as a negative peak using a MWD at 734 nm. The combined UV (positive signals) and ABTS+ quenching (negative signals) chromatograms of the different A. tegmentosum extracts (200 ppm) are presented in Figures 5 and 6. Several eluted fractions of the five phenolic compounds in the EA extract were detected as feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5), giving a positive signal on the UV detector at 210 nm. The other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radical at the applied concentration. These results revealed that the method could be applied for quick bioactivity screening, or more precisely, to detect the radical-scavenging activity of compounds, indicating that (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5) exhibited bioactivities. 6′-O-Galloyl salidroside (5) exhibited higher bioactivity, whereas feniculin (1) and avicularin (2) showed low bioactivity (Figure 5). The retention time (Rt) of feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5) was 10.700, 13.884, 15.928, 24.217, and 25.062 min, respectively. The bioactivity appeared to be approximately proportional to the concentration of the five phenolic compounds in the extracts. The HPLC analysis conditions for the best separation of the five compounds were successfully established by varying the open column treatment and solvent purification step (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.


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

Lee KJ, Song NY, Oh YC, Cho WK, Ma JY - J Anal Methods Chem (2014)

Analysis of UV wavelength from high purity isolation compounds.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4216704&req=5

fig4: Analysis of UV wavelength from high purity isolation compounds.
Mentions: 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 μm, C18, Daejeon, Korea). The injection volume was 10 μL, 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 from the on-line HPLC–DPPH (ABTS) assay was based on the decrease in absorbance at 517 and 734 nm after the postcolumn reaction of antioxidants separated from HPLC with DPPH (ABTS), and the antioxidants present in a sample would be easily indicated by negative peaks. The composition of the mobile phases was A: 99.9 vol% water/trifluoroacetic acid (99.9/0.1, vol%) and B: 100% acetonitrile. The run time was 60 min, and the solvent program used the linear gradient method (90 : 10–60 : 40 A : B vol%, 70 min: initial condition). The ABTS+ flow rate was 0.5 mL/min. The HPLC separated analytes showed a postcolumn reaction with the ABTS+ and the reduction was detected as a negative peak using a MWD at 734 nm. The combined UV (positive signals) and ABTS+ quenching (negative signals) chromatograms of the different A. tegmentosum extracts (200 ppm) are presented in Figures 5 and 6. Several eluted fractions of the five phenolic compounds in the EA extract were detected as feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5), giving a positive signal on the UV detector at 210 nm. The other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radical at the applied concentration. These results revealed that the method could be applied for quick bioactivity screening, or more precisely, to detect the radical-scavenging activity of compounds, indicating that (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5) exhibited bioactivities. 6′-O-Galloyl salidroside (5) exhibited higher bioactivity, whereas feniculin (1) and avicularin (2) showed low bioactivity (Figure 5). The retention time (Rt) of feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5) was 10.700, 13.884, 15.928, 24.217, and 25.062 min, respectively. The bioactivity appeared to be approximately proportional to the concentration of the five phenolic compounds in the extracts. The HPLC analysis conditions for the best separation of the five compounds were successfully established by varying the open column treatment and solvent purification step (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.

Bottom Line: Based on spectroscopic methods such as (1)H-NMR, (13)C-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).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.

View Article: PubMed Central - PubMed

Affiliation: Korean Institute of Oriental Medicine (KIOM), KM-Based Herbal Drug Development Group, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 305-811, Republic of Korea.

ABSTRACT
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 (1)H-NMR, (13)C-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.

No MeSH data available.