Research ArticleIsolation and Bioactivity Analysis of Ethyl AcetateExtract from Acer tegmentosum Using In Vitro Assay andOn-Line Screening HPLC-ABTS+ System

Kwang Jin Lee Na-Young Song You Chang Oh Won-Kyung Cho and Jin Yeul Ma

Korean Institute of Oriental Medicine (KIOM) KM-Based Herbal Drug Development Group 1672 Yuseongdae-roYuseong-gu Daejeon 305-811 Republic of Korea

Correspondence should be addressed to Jin Yeul Ma jymakiomrekr

Received 23 June 2014 Revised 27 August 2014 Accepted 3 September 2014 Published 15 October 2014

Academic Editor Serban C Moldoveanu

Copyright copy 2014 Kwang Jin Lee et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The Acer tegmentosum (3 kg) was extracted using hot water and the freeze-dried extract powder was partitioned successivelyusing dichloromethane (DCM) ethyl acetate (EA) butyl alcohol (n-BuOH) and water From the EA extract fraction (124 g)five phenolic compounds were isolated by the silica gel octadecyl silica gel and Sephadex LH-20 column chromatography Basedon spectroscopic methods such as 1H-NMR 13C-NMR and LCMS the chemical structures of the compounds were confirmedas feniculin (1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) Moreover a rapid on-linescreening HPLC-ABTS+ system for individual bioactivity of the EA-soluble fraction (five phenolic compounds) was developedThe results indicated that compounds 1 and 2 were first isolated from the A tegmentosum The anti-inflammatory activities andon-line screeningHPLC-ABTS+ assaymethod of these compounds in LPS-stimulatedmurinemacrophages were rapid and efficientfor the investigation of bioactivity of A tegmentosum

1 Introduction

Traditional remedies based on natural products could betraced back over five millennia to written documents of theearly civilizations [1] Particularly traditional Korean andChinese oriental medicines herbs (OMHs) have attractedinterest and acceptance in many countries with the meritsof a few side effects affordability and local availabilityMoreover their long historical clinical practice and reliabletherapeutic efficacy make them excellent sources for discov-ering natural bioactive compounds [2] Among them Acer(A) tegmentosum (Aceraceae Sancheong-mok in Korean) isa type of deciduous tree distributed in the Northeast Asiaincluding Korea Russia and China [3] In Korea the leavesand stem of A tegmentosum have been traditionally used forthe treatment of hepatic disorders such as hepatitis hepaticcancer hepatic cirrhosis and liver detoxification [4] Previousstudies have shown that extracts of the stem of A tegmen-tosum possess various pharmacological properties such as

antioxidative anti-inflammatory antigastrophatic antiadi-pogenic anticancer and cytotoxic activities [5ndash7] Howeverthe phytochemical constituents and efficient method forinvestigating bioactivity of A tegmentosum have not beenreported

Also a variety of approaches have been developed for theextraction of useful components from A tegmentosum forinstance soxhlet extraction (SE) heating reflux extraction(HEE) supercritical fluid extraction (SFE) ultrasonicassisted extraction (UAE) andmicrowave-assisted extraction(MAE) [8ndash10] Water methanol ethanol and ethyl acetate(EA) were commonly used solvents for the extraction ofbioactive compounds from plant materials and OMHsThe identification and relative amounts of five types ofcompounds in A tegmentosum were determined by LC-MS NMR and on-line screening HPLC-ABTS+ assay [1]These techniques included on-line screening with HPLCpost-column assay involving the ABTS+ radical technique

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2014 Article ID 150509 15 pageshttpdxdoiorg1011552014150509

2 Journal of Analytical Methods in Chemistry

[11] allowing spectrophotometric monitoring of bioactivecompounds Generally DPPH (ABTS) radical is anothersimple rapid on-linemethod for the detection of antioxidantsfrom crude plant extracts [12] It combines HPLC with anassay involving a stable radical species [11-diphenyl-2-picrylhydrazyl radical (DPPH) and 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS)]in the HPLC-DPPH (ABTS) method [13 14] Moreoverthis method was successfully applied for the screening andidentification of natural bioactive compounds from complexmixtures particularly for the extracts of OMHs [15ndash17]The chemical structures of the five types of compoundswere confirmed by spectroscopic methods such as 1H-NMR 13C-NMR and LCMS [18] In this study fivephenolic compounds were isolated from hot water extractof A tegmentosum bychromatographic separation Usingspectroscopic methods the structures of these compoundswere determined as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)Moreover we investigated their anti-inflammatory effectson LPS-stimulated RAW 2647 cells We also investigatedthe applications of on-line screening HPLC-ABTS+ assaysfor bioactivity screening to find a more practical approachtoward the use of on-line screening HPLC-ABTS+ assaysfor the rapid pinpointing of peaks in chromatogramscorresponding to bioactive compounds

2 Experimental

21 Reagents and Materials The stem of A tegmentosumwas purchased from Yeongcheon traditional herbal mar-ket (Gyeongsangbuk-do Yeongcheon Korea) All voucherspecimens were deposited in the herbal bank of KM-Based Herbal Drug Development Group Korea Instituteof Oriental Medicine The following reagents were usedfor radical scavenging assays ABTS+ (221015840-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) potassium persulfateand trifluoroacetic acid (TFA) were purchased from SigmaCo (USA) The HPLC grade methanol (MeOH) and ace-tonitrile (ACN) were purchased from J T Baker (Philips-burg NJ USA) The hexane dichloromethane (DCM) ethylacetate (EA) and normal butyl alcohol (n-BuOH) werepurchased from Daejung chemical (Gyeonggi-do ShiheungKorea) The triple distilled water was filtered with a 02120583mmembrane filter (Advantec Tokyo Japan) before analysisMaterials for cell culture were obtained from Lonza (BaselSwitzerland) LPS Bovine serum albumin (BSA) and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) were purchased from Sigma (St Louis MO USA)Antibodies for ELISA were obtained from eBioscience (SanDiego CA USA) The chemical structures of five types ofcompounds are shown in Figure 1

22 Standard Sample Preparation The high purity isolatedcompounds (higher than gt 95) were prepared by dissolving2mg of the standard chemicals feniculin (1) avicularin(2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl

Table 1 LC-MS operating condition

Instrument Condition

Column HECpTOR-M-RS-tech C18(100 times 46mm 5 120583m)

Oven temp (∘C) 40Flow rate (mLmin) 05Injection vol (120583L) 10Ionization source(positive mode) API-ES

Fragmentor voltage (V) 70Quadrupole temp (∘C) 99Capillary voltage (V) 3000Nebulizer pressure (psi) 35Drying gas temp (∘C) 350Mass rang (119898119911) scanmode 200sim500

Mobile phase () A 01 TFA in waterB acetonitrile

Gradient elutioncomposition ()

(B) 10ndash25 (0ndash35min) 25ndash10(35ndash37min) 10 (37ndash45min)

salidroside (5) in 10mL of methanol and adjusting the con-centration to 200 ppm

23 ABTS+ Sample Preparation A 2mM ABTS stock solu-tion containing 35mM potassium persulfate was preparedandwas kept in the dark at room temperature for 16 h to allowthe completion of radical generation and was then dilutedwith water (1 29 vv)

24 Solvent Extraction and Purification Dry samples (3 Kg)from the powders of the A tegmentosum were loaded (10-times volume) in hot water extraction systemThe extractionwas performed by heating for 3 h at 100∘C (GyeongseoExtractor Cosmos-600 Inchon Korea) Then the solutionwas filtered using standard testing sieves (150120583m RetschHaan Germany) freeze-dried andmaintained in desiccatorsat 4∘C prior to use For large amount of extractions 20 gfreeze-dry samples were loaded (1 1 extracted thrice) andextracted successively using DCM EA and n-BuOH Thecontents of feniculin (1) avicularin (2) (+)-catechin (3)(minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) in Ategmentosum were remarkably higher in the EA extract(124 g) Then the samples were filtered through a 02120583mmembrane filter prior to on-line screening HPLC-ABTS+analysis The extraction and purification processes from Ategmentosum are shown in Figure 2

25 Analysis System NMR spectra were obtained using aVarian Inova 400MHz and 600MHz NMR (Varian USA)The isolated compounds feniculin (1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside(5) were confirmed viaLC-MS analysis using Agilent 1100 +G1958 (Agilent USA) LC systems (Table 1) Open columnchromatography was performed using silica gel (Kieselgel 60

Journal of Analytical Methods in Chemistry 3

MW 43435 Isolation no E-6-4Feniculin

O

O

OOH

OH

OH

OH

O

HO

HO

HO

MW 43435 Isolation no E-6-2

O

O

OOH

OH

OH

OH

HO

HOHO

O

Avicularin

O

OH

OH

OH

OH

HO

MW 29027 Isolation no E-7-6-2

O

OH

OH

HO

OH

OH

MW 29027 Isolation no E-7-6-4

HO

OH

OH

OH OH

OO

OHO

HO

O

MW 45241 Isolation no E-9-3-2

(+)-Catechin (minus)-Epicatechin

6998400-O-Galloyl salidroside

Figure 1 Chemical structure of five types of compounds from Acer tegmentosum

MerckGermany) and octadecylsilane (ODS) Li-ChropreRP-18 (Merck Germany) Molecular sieve column chromatog-raphy was performed using Sephadex LH-20 (Fluka USA)Thin layer chromatography (TLC) analysis was performedusing silica gel glass plates (Kieselgel 60 F

254and RP-18

F254S Merck Germany) and developed using a mobile phase

composed of chloroform-methanol-water and stained using10 H

2SO4to detect the EA extract and pure compounds

26 On-Line Screening HPLC-ABTS+ Assay Analysis A teg-mentosum extract was injected into a Dionex Ultimate 3000HPLC system (Thermo Scientific) The chromatographiccolumns used in this experiment were commercially availableand were purchased from RS-tech (046 times 25 cm 5 120583mC18 Daejeon Korea) The injection volume was 10 120583L and

the flow rate of the mobile phase was 10mLmin Thewavelength of the UV detector was fixed at 210 254 280

4 Journal of Analytical Methods in Chemistry

1 3 5 6

109871 2 3 4 5 6

71 3 5 6 10 11 12 139871 2 3 4 5 6

10 11 129871 2 3 4 5 6

1 3 4

CH2Cl2 H2O

H2O

H2OEtOAc fr (124 g)

SiO2 cc

Freeze-dried extract powder of Acer tegmentosum (20 g)

n-BuOH

CH2Cl2H2O times 4(1 1)

EtOAcH2O times 4(1 1)

2

2

2

4

4

n-BuOHH2O times 4(1 1)

Figure 2 Process of isolation five kind compounds of ethyl acetate extract from Acer tegmentosum

Table 2 Analysis of gradient elution conditions with RP-HPLC

Instrument ConditionReverse phase column RS-Tech Optimapak C18 (46 times 250mm 5 120583m)Oven temp (∘C) 40Flow rate (mLmin) 10Mobile phase () A 01 TFA in water B acetonitrileUV absorbance (nm) 210 254 280 320

Time (min) Solvent composition ()01 TFA in water (A) Acetonitrile (B)

0 90 1050 60 4060 60 4070 90 10

and 320 nm The composition of the mobile phases wasA 999 watertrifluoroacetic acid (99901 vol) and B100 acetonitrile The run time was 60min and the solventprogram was the linear gradient method (90 10ndash60 40 A Bvol 70min initial condition) (Table 2) Figure 3 showsa schematic diagram of the on-line coupling of a HPLCto a DAD (diode array detector) and the continuous flowABTS+ assay Then on-line HPLC was connected to a ldquoTrdquopiece where ABTS+ was added The ABTS+ at a flow rateof 05mLmin was delivered using a Dionex ultimate 3000pump Aftermixing through a 1mL loop maintained at 40∘Cthe absorbance was measured using a multiple wavelengthdetector (MWD) at 734 nm Data were analyzed using theChromeleon 7 software

Sample injection

ColumnPump PumpRegents Regents

DADdetector

VWDdetector

Passive splitReaction loop

Figure 3 Schematic of on-line screening HPLC-ABTS+ system

27 Cell Culture and Drug Treatment RAW 2647 cells werepurchased from Korea Cell Line Bank (Seoul Korea) andgrown in RPMI 1640 medium containing 10 FBS and

Journal of Analytical Methods in Chemistry 5

100UmL of antibiotics sulfate The cells were incubatedin humidified 5 CO

2atmosphere at 37∘C To stimulate

the cells the medium was changed with fresh RPMI 1640medium and LPS (200 ngmL) was added in the presence orabsence of five compounds (10 30 50 and 100 120583M) for 24 h

28 MTT Assay for Cell Viability Cytotoxicity was analyzedusing the MTT assay Five compounds were added to thecells and incubated for 24 h at 37∘C with 5 CO

2 MTT

solutionswere added to eachwell and the cells were incubatedfor another 4 h The formazan melted in dimethyl sulfoxide(DMSO) and then the optical density was read at 570 nmusing an ELISA reader (Infinite M200 TECAN MannedorfSwitzerland)

29 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated withfive compounds and stimulated with LPS for 24 h Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

210 Determination of TNF-120572 IL-6 and IL-1120573 CytokineProduction Cells were seeded at a density of 5 times 105 cellsmLin 24-well culture plates and pretreated with various con-centrations of five compounds for 30min before the LPSstimulation ELISA plates (Nunc Roskilde Denmark) werecoated overnight at 4∘C with capture antibody diluted in acoating buffer (01M carbonate pH 95) and then washedfive times with phosphate-buffered saline (PBS) containing005 Tween 20 The nonspecific protein-binding sites wereblocked with assay diluent buffer (PBS containing 10 FBSpH 70) for gt1 h The samples and standards were addedto the wells promptly After 2 hours of incubation at RT orovernight at 4∘C the working detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 h Subsequently the substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in the dark before the reaction was quenchedwith stop solution (NH

3PO4) The optical density was read

at 450 nm [19]

211 Statistical Analysis The results are expressed as mean plusmnSE values for the number of experiments Statistical signif-icance of each treated group was compared to the controland determined by Studentrsquos 119905-tests Each experiment wasrepeated at least thrice to yield comparable results Valueswith 119875 lt 001 and lt0001 were considered significant

3 Results and Discussion

31 High Purity Isolation and Analysis The stems of A teg-mentosum were extracted using boiling water and then par-titioned successively using dichloromethane (DCM) ethylacetate (EA) normal butyl alcohol (n-BuOH) and water

(H2O) The EA extract (124 g) was applied to silica gel

cc (120593 4 times 10 cm) and eluted using chloroform- (CHCl3minus)

MeOHndashH2O [15 3 1 (12 L) rarr 13 3 1 (13 L) rarr 10 3 1

(12 L) rarr 7 3 1 (1 L) rarr 4 3 1 (1 L) rarr MeOH] Elutedfractions were monitored by TLC to produce 12 fractions(OS1E-1ndash12) Fraction OS1E-6 (555mg) was subjected toSephadex LH-20 cc [120593 15 times 45 cm 50 MeOH (180mL)rarr 70 MeOH (300mL) rarr 100 MeOH] yielding sevenfractions (OS1E-6-1ndash7) and ultimately produced compounds1 (OS1E-6-2 60mg) and 2 (OS1E-6-4 59mg) FractionOS1E-7 (2603mg) was subjected to Sephadex LH-20 cc [12059315 times 44 cm 80MeOH (400mL) rarr 100MeOH] yielding10 fractions (OS1E-7-1ndash10) Fraction OS1E-7-6 (531mg) wassubjected to ODS cc (120593 15 times 8 cm) and eluted with MeOHndashH2O [1 6 (900mL) rarr MeOH] to afford compounds 3

(OS1E-7-6-2 307mg) and 4 (OS1E-7-6-4 142mg) FractionOS1E-9 (2375mg) was subjected to ODS cc (120593 25 times 8 cm)and eluted withMeOHndashH

2O [1 3 (450mL) rarr 1 2 (300mL)

rarr 1 1 (300mL) rarr MeOH] yielding 13 fractions (OS1E-9-1ndash13) Fraction OS1E-9-3 (778mg) was subjected to silicagel cc (120593 15 times 10 cm) and eluted with CHCl

3ndashMeOHndash

H2O [10 3 1 (22 L) rarr 7 3 1 (16 L) rarr 4 3 1 (14 L) rarr

MeOH] to afford compound 5 (OS1E-9-3-2 603mg) Basedon the spectroscopic methods such as 1H-NMR 13C-NMRand LCMS the chemical structures of the compounds wereconfirmed as feniculin (1) avicularin (2) (+)-catechin (3)(minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) by com-parison of spectral data with those of references Moreoverthe on-line screening HPLC-ABTS+ assay method was rapidand efficient for the investigation of bioactivity of the isolatedcompounds from Acer tegmentosum Compounds 1 2 3 4and 5 were developed on ODS TLC with 10 H

2SO4and the

expected yellow or brown color was observed for the phenoliccompounds

Compound 1 (Feniculin) Yellow amorphous powder (MeOH)ESI-MS mz 4351 [M+H]+ 1H-NMR (400MHz CD

3OD

120575H) 773 (1H d J = 16Hz H-21015840) 755 (1H dd J = 84 16HzH-61015840) 688 (1H d J = 84Hz H-51015840) 639 (1H d J = 16HzH-8) 619 (1H d J = 16Hz H-6) 516 (1H d J = 60Hz H-110158401015840) 389 (1H dd J = 80 60Hz H-210158401015840) 382 (1H H-510158401015840a) 380(1H m H-410158401015840) 364 (1H dd J = 80 28Hz H-310158401015840) 344 (1Hdd J = 132 32Hz H-510158401015840b) 13C-NMR (100MHz CD

3OD

120575c) see Table 3The characterization data of compound 1werecompared to the literature value and combinedwith quercetinand arabinopyranoside to confirm the structure as feniculin[20]

Compound 2 (Avicularin) Yellow amorphous powder(MeOH) ESI-MS mz 4351 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 752 (1H d J = 18Hz H-21015840) 749 (1H dd J =

84 18Hz H-61015840) 690 (1H d J = 84Hz H-51015840) 638 (1Hd J = 18Hz H-8) 620 (1H d J = 18Hz H-6) 546 (1Hs H-110158401015840) 432 (1H d J = 24Hz H-210158401015840) 389 (1H dd J =48 24Hz H-310158401015840) 3861 (1H m H-410158401015840) 3494 (2H t J =42 H-510158401015840) 13C-NMR (150MHz CD

3OD 120575c) see Table 3

1H-NMR spectrum of compound 2 showed a similar patternto compound 1 The structure was confirmed by ESI-MSmz

6 Journal of Analytical Methods in Chemistry

Table 3 13C-NMR chemical shifts of compounds 1ndash5 isolated from Acer tegmentosum

Number of carbon1 2 3 4 5

CD3OD100MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

1 130562 15870 15944 8281 7986 130833 13565 13500 6978 6747 116084 17948 18009 2847 2925 156525 16305 16319 15754 15766 116086 9988 9995 9628 9638 130837 16607 16625 15778 15798 36338 9470 9484 9550 9588 72209 15843 15869 15688 1573510 10563 10569 10081 1000611015840 12289 12309 13219 1322821015840 11745 11654 11523 1158831015840 14595 14647 14619 1457641015840 14996 14996 14621 1459251015840 11617 11654 11608 1153161015840 12303 12320 12003 11938Sugar-110158401015840 10463 10959 10439

210158401015840 7287 8341 7499310158401015840 7413 7876 7784410158401015840 6911 8810 7167510158401015840 6694 6264 7534610158401015840 6470

Galloyl-1101584010158401015840 121372101584010158401015840 110163101584010158401015840 146444101584010158401015840 139785101584010158401015840 146446101584010158401015840 11016

ndashCOOndash 16831

4351 [M+H]+ [21] Compounds 1 and 2 were isolated fromthe plant for the first time

Compound 3 [(+)-Catechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

683 (1H d J = 24Hz H-21015840) 676 (1H d J = 84Hz H-51015840)671 (1H dd J =84 24HzH-61015840) 592 (1H d J =24HzH-6)585 (1H d J = 24Hz H-8) 456 (1H d J = 72Hz H-2) 397(1H m H-3) 250 (1H dd J = 162 84Hz H-4a) 284 (1Hdd J = 162 60Hz H-4b) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3

Compound 4 [(minus)-Epicatechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

696 (1H d J = 18Hz H-21015840) 679 (1H dd J = 84 18HzH-61015840) 675 (1H d J = 84Hz H-51015840) 593 (1H d J = 18HzH-6) 591 (1H d J = 18Hz H-8) 480 (1H brs H-2) 416(1H m H-3) 286 (1H dd J = 168 48Hz H-4a) 273

(1H dd J = 168 30HzH-4b) 13C-NMR (150MHzCD3OD

120575c) see Table 3 Compounds 3 and 4 were compared to theliterature data for (+)-catechin and (minus)-epicatechin for thestructure identification [22] The structure was confirmed bythe presence of mz at 2910 [M+H]+ in the ESI-MS positivemode

Compound 5 (61015840-O-Galloyl salidroside) Pale yellow oil(MeOH) ESI-MS mz 4531 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 710 (2H br s H-2101584010158401015840 H-6101584010158401015840) 696 (2H d J =

84Hz H-2 6) 665 (2H d J = 84Hz H-3 H-5) 665 (1Hd J = 84Hz H-5) 452 (1H dd J = 114 24Hz H-610158401015840a) 445(1H dd J = 114 60Hz H-610158401015840b) 432 (1H d J = 78Hz H-110158401015840) 395 (1H m H-8a) 370 (1H m H-8b) 355 (1H m H-510158401015840) 342 (2H t J = 78Hz H-310158401015840 410158401015840) 322 (1H t J = 84HzH-210158401015840) 277 (2H m H-7) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3 1H-NMR data were consistent with the literaturevalues [23]

Journal of Analytical Methods in Chemistry 7

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

UV wavelength (nm)

Abso

rban

ce

Abso

rban

ce

Abso

rban

ceAb

sorb

ance

Abso

rban

ce

E-6-2

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

UV wavelength (nm)

E-6-4

200 220 240 260 280 300 320 340 360

0

200

400

600

800

1000

UV wavelength (nm)

E-7-6-2

200 220 240 260 280 300 320 340 360

0

100

200

300

400

500

600

700

800

UV wavelength (nm)

E-7-6-4

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

400

UV wavelength (nm)

E-9-3-2

Figure 4 Analysis of UV wavelength from high purity isolation compounds

32 On-Line Screening HPLC-ABTS+ Assay Analysis Thisstudy investigated the bioactivity (using ABTS+ assay rad-ical scavenging activity) and anti-inflammatory activity ofthe five isolated phenolic compounds that were measuredAll the compounds 1ndash5 in the EA fraction (each yieldmg) exhibited antioxidant activities (Table 4) Moreoverthis on-line screening HPLC-ABTS+ assay method wasrapid and efficient for the investigation of bioactivity from

A tegmentosum and was obtained from RS-tech (046 times25 cm 5 120583m C

18 Daejeon Korea) The injection volume was

10 120583L and the flow rate of the mobile phase was 10mLminThe wavelength of the UV detector was fixed at 210 254 280and 320 nmThefive phenolic compoundswere characterizedby comparing the HPLC UV-DAD maximum absorptionpeaks of the samples with those of the pure isolation stan-dards (Figure 4) The determination of antioxidant activity

8 Journal of Analytical Methods in Chemistry

Table4Ex

tractamou

ntandyieldin

ethylacetate(EA)fractionsample

Extractio

nsolvent

Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)

EA phase

1390

6

1321

7

198

7-6

127

9

1631

9-3

125

21018

2lowast60lowast

21640

2lowast307lowast

2228

2lowast603lowast

3657

352

337

332

3778

378

4235

4lowast59lowast

462

4lowast142lowast

486

4123

540

95

185

795

24

585

6555

613

6531

6112

683

72603

722

772

747

8421

856

843

92375

949

940

10975

1021

1057

111324

11103

122552

1290

1327

lowastShow

nforfi

vekind

compo

unds

from

major

extract

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

2 Journal of Analytical Methods in Chemistry

[11] allowing spectrophotometric monitoring of bioactivecompounds Generally DPPH (ABTS) radical is anothersimple rapid on-linemethod for the detection of antioxidantsfrom crude plant extracts [12] It combines HPLC with anassay involving a stable radical species [11-diphenyl-2-picrylhydrazyl radical (DPPH) and 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS)]in the HPLC-DPPH (ABTS) method [13 14] Moreoverthis method was successfully applied for the screening andidentification of natural bioactive compounds from complexmixtures particularly for the extracts of OMHs [15ndash17]The chemical structures of the five types of compoundswere confirmed by spectroscopic methods such as 1H-NMR 13C-NMR and LCMS [18] In this study fivephenolic compounds were isolated from hot water extractof A tegmentosum bychromatographic separation Usingspectroscopic methods the structures of these compoundswere determined as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)Moreover we investigated their anti-inflammatory effectson LPS-stimulated RAW 2647 cells We also investigatedthe applications of on-line screening HPLC-ABTS+ assaysfor bioactivity screening to find a more practical approachtoward the use of on-line screening HPLC-ABTS+ assaysfor the rapid pinpointing of peaks in chromatogramscorresponding to bioactive compounds

2 Experimental

21 Reagents and Materials The stem of A tegmentosumwas purchased from Yeongcheon traditional herbal mar-ket (Gyeongsangbuk-do Yeongcheon Korea) All voucherspecimens were deposited in the herbal bank of KM-Based Herbal Drug Development Group Korea Instituteof Oriental Medicine The following reagents were usedfor radical scavenging assays ABTS+ (221015840-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) potassium persulfateand trifluoroacetic acid (TFA) were purchased from SigmaCo (USA) The HPLC grade methanol (MeOH) and ace-tonitrile (ACN) were purchased from J T Baker (Philips-burg NJ USA) The hexane dichloromethane (DCM) ethylacetate (EA) and normal butyl alcohol (n-BuOH) werepurchased from Daejung chemical (Gyeonggi-do ShiheungKorea) The triple distilled water was filtered with a 02120583mmembrane filter (Advantec Tokyo Japan) before analysisMaterials for cell culture were obtained from Lonza (BaselSwitzerland) LPS Bovine serum albumin (BSA) and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) were purchased from Sigma (St Louis MO USA)Antibodies for ELISA were obtained from eBioscience (SanDiego CA USA) The chemical structures of five types ofcompounds are shown in Figure 1

22 Standard Sample Preparation The high purity isolatedcompounds (higher than gt 95) were prepared by dissolving2mg of the standard chemicals feniculin (1) avicularin(2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl

Table 1 LC-MS operating condition

Instrument Condition

Column HECpTOR-M-RS-tech C18(100 times 46mm 5 120583m)

Oven temp (∘C) 40Flow rate (mLmin) 05Injection vol (120583L) 10Ionization source(positive mode) API-ES

Fragmentor voltage (V) 70Quadrupole temp (∘C) 99Capillary voltage (V) 3000Nebulizer pressure (psi) 35Drying gas temp (∘C) 350Mass rang (119898119911) scanmode 200sim500

Mobile phase () A 01 TFA in waterB acetonitrile

Gradient elutioncomposition ()

(B) 10ndash25 (0ndash35min) 25ndash10(35ndash37min) 10 (37ndash45min)

salidroside (5) in 10mL of methanol and adjusting the con-centration to 200 ppm

23 ABTS+ Sample Preparation A 2mM ABTS stock solu-tion containing 35mM potassium persulfate was preparedandwas kept in the dark at room temperature for 16 h to allowthe completion of radical generation and was then dilutedwith water (1 29 vv)

24 Solvent Extraction and Purification Dry samples (3 Kg)from the powders of the A tegmentosum were loaded (10-times volume) in hot water extraction systemThe extractionwas performed by heating for 3 h at 100∘C (GyeongseoExtractor Cosmos-600 Inchon Korea) Then the solutionwas filtered using standard testing sieves (150120583m RetschHaan Germany) freeze-dried andmaintained in desiccatorsat 4∘C prior to use For large amount of extractions 20 gfreeze-dry samples were loaded (1 1 extracted thrice) andextracted successively using DCM EA and n-BuOH Thecontents of feniculin (1) avicularin (2) (+)-catechin (3)(minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) in Ategmentosum were remarkably higher in the EA extract(124 g) Then the samples were filtered through a 02120583mmembrane filter prior to on-line screening HPLC-ABTS+analysis The extraction and purification processes from Ategmentosum are shown in Figure 2

25 Analysis System NMR spectra were obtained using aVarian Inova 400MHz and 600MHz NMR (Varian USA)The isolated compounds feniculin (1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside(5) were confirmed viaLC-MS analysis using Agilent 1100 +G1958 (Agilent USA) LC systems (Table 1) Open columnchromatography was performed using silica gel (Kieselgel 60

Journal of Analytical Methods in Chemistry 3

MW 43435 Isolation no E-6-4Feniculin

O

O

OOH

OH

OH

OH

O

HO

HO

HO

MW 43435 Isolation no E-6-2

O

O

OOH

OH

OH

OH

HO

HOHO

O

Avicularin

O

OH

OH

OH

OH

HO

MW 29027 Isolation no E-7-6-2

O

OH

OH

HO

OH

OH

MW 29027 Isolation no E-7-6-4

HO

OH

OH

OH OH

OO

OHO

HO

O

MW 45241 Isolation no E-9-3-2

(+)-Catechin (minus)-Epicatechin

6998400-O-Galloyl salidroside

Figure 1 Chemical structure of five types of compounds from Acer tegmentosum

MerckGermany) and octadecylsilane (ODS) Li-ChropreRP-18 (Merck Germany) Molecular sieve column chromatog-raphy was performed using Sephadex LH-20 (Fluka USA)Thin layer chromatography (TLC) analysis was performedusing silica gel glass plates (Kieselgel 60 F

254and RP-18

F254S Merck Germany) and developed using a mobile phase

composed of chloroform-methanol-water and stained using10 H

2SO4to detect the EA extract and pure compounds

26 On-Line Screening HPLC-ABTS+ Assay Analysis A teg-mentosum extract was injected into a Dionex Ultimate 3000HPLC system (Thermo Scientific) The chromatographiccolumns used in this experiment were commercially availableand were purchased from RS-tech (046 times 25 cm 5 120583mC18 Daejeon Korea) The injection volume was 10 120583L and

the flow rate of the mobile phase was 10mLmin Thewavelength of the UV detector was fixed at 210 254 280

4 Journal of Analytical Methods in Chemistry

1 3 5 6

109871 2 3 4 5 6

71 3 5 6 10 11 12 139871 2 3 4 5 6

10 11 129871 2 3 4 5 6

1 3 4

CH2Cl2 H2O

H2O

H2OEtOAc fr (124 g)

SiO2 cc

Freeze-dried extract powder of Acer tegmentosum (20 g)

n-BuOH

CH2Cl2H2O times 4(1 1)

EtOAcH2O times 4(1 1)

2

2

2

4

4

n-BuOHH2O times 4(1 1)

Figure 2 Process of isolation five kind compounds of ethyl acetate extract from Acer tegmentosum

Table 2 Analysis of gradient elution conditions with RP-HPLC

Instrument ConditionReverse phase column RS-Tech Optimapak C18 (46 times 250mm 5 120583m)Oven temp (∘C) 40Flow rate (mLmin) 10Mobile phase () A 01 TFA in water B acetonitrileUV absorbance (nm) 210 254 280 320

Time (min) Solvent composition ()01 TFA in water (A) Acetonitrile (B)

0 90 1050 60 4060 60 4070 90 10

and 320 nm The composition of the mobile phases wasA 999 watertrifluoroacetic acid (99901 vol) and B100 acetonitrile The run time was 60min and the solventprogram was the linear gradient method (90 10ndash60 40 A Bvol 70min initial condition) (Table 2) Figure 3 showsa schematic diagram of the on-line coupling of a HPLCto a DAD (diode array detector) and the continuous flowABTS+ assay Then on-line HPLC was connected to a ldquoTrdquopiece where ABTS+ was added The ABTS+ at a flow rateof 05mLmin was delivered using a Dionex ultimate 3000pump Aftermixing through a 1mL loop maintained at 40∘Cthe absorbance was measured using a multiple wavelengthdetector (MWD) at 734 nm Data were analyzed using theChromeleon 7 software

Sample injection

ColumnPump PumpRegents Regents

DADdetector

VWDdetector

Passive splitReaction loop

Figure 3 Schematic of on-line screening HPLC-ABTS+ system

27 Cell Culture and Drug Treatment RAW 2647 cells werepurchased from Korea Cell Line Bank (Seoul Korea) andgrown in RPMI 1640 medium containing 10 FBS and

Journal of Analytical Methods in Chemistry 5

100UmL of antibiotics sulfate The cells were incubatedin humidified 5 CO

2atmosphere at 37∘C To stimulate

the cells the medium was changed with fresh RPMI 1640medium and LPS (200 ngmL) was added in the presence orabsence of five compounds (10 30 50 and 100 120583M) for 24 h

28 MTT Assay for Cell Viability Cytotoxicity was analyzedusing the MTT assay Five compounds were added to thecells and incubated for 24 h at 37∘C with 5 CO

2 MTT

solutionswere added to eachwell and the cells were incubatedfor another 4 h The formazan melted in dimethyl sulfoxide(DMSO) and then the optical density was read at 570 nmusing an ELISA reader (Infinite M200 TECAN MannedorfSwitzerland)

29 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated withfive compounds and stimulated with LPS for 24 h Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

210 Determination of TNF-120572 IL-6 and IL-1120573 CytokineProduction Cells were seeded at a density of 5 times 105 cellsmLin 24-well culture plates and pretreated with various con-centrations of five compounds for 30min before the LPSstimulation ELISA plates (Nunc Roskilde Denmark) werecoated overnight at 4∘C with capture antibody diluted in acoating buffer (01M carbonate pH 95) and then washedfive times with phosphate-buffered saline (PBS) containing005 Tween 20 The nonspecific protein-binding sites wereblocked with assay diluent buffer (PBS containing 10 FBSpH 70) for gt1 h The samples and standards were addedto the wells promptly After 2 hours of incubation at RT orovernight at 4∘C the working detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 h Subsequently the substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in the dark before the reaction was quenchedwith stop solution (NH

3PO4) The optical density was read

at 450 nm [19]

211 Statistical Analysis The results are expressed as mean plusmnSE values for the number of experiments Statistical signif-icance of each treated group was compared to the controland determined by Studentrsquos 119905-tests Each experiment wasrepeated at least thrice to yield comparable results Valueswith 119875 lt 001 and lt0001 were considered significant

3 Results and Discussion

31 High Purity Isolation and Analysis The stems of A teg-mentosum were extracted using boiling water and then par-titioned successively using dichloromethane (DCM) ethylacetate (EA) normal butyl alcohol (n-BuOH) and water

(H2O) The EA extract (124 g) was applied to silica gel

cc (120593 4 times 10 cm) and eluted using chloroform- (CHCl3minus)

MeOHndashH2O [15 3 1 (12 L) rarr 13 3 1 (13 L) rarr 10 3 1

(12 L) rarr 7 3 1 (1 L) rarr 4 3 1 (1 L) rarr MeOH] Elutedfractions were monitored by TLC to produce 12 fractions(OS1E-1ndash12) Fraction OS1E-6 (555mg) was subjected toSephadex LH-20 cc [120593 15 times 45 cm 50 MeOH (180mL)rarr 70 MeOH (300mL) rarr 100 MeOH] yielding sevenfractions (OS1E-6-1ndash7) and ultimately produced compounds1 (OS1E-6-2 60mg) and 2 (OS1E-6-4 59mg) FractionOS1E-7 (2603mg) was subjected to Sephadex LH-20 cc [12059315 times 44 cm 80MeOH (400mL) rarr 100MeOH] yielding10 fractions (OS1E-7-1ndash10) Fraction OS1E-7-6 (531mg) wassubjected to ODS cc (120593 15 times 8 cm) and eluted with MeOHndashH2O [1 6 (900mL) rarr MeOH] to afford compounds 3

(OS1E-7-6-2 307mg) and 4 (OS1E-7-6-4 142mg) FractionOS1E-9 (2375mg) was subjected to ODS cc (120593 25 times 8 cm)and eluted withMeOHndashH

2O [1 3 (450mL) rarr 1 2 (300mL)

rarr 1 1 (300mL) rarr MeOH] yielding 13 fractions (OS1E-9-1ndash13) Fraction OS1E-9-3 (778mg) was subjected to silicagel cc (120593 15 times 10 cm) and eluted with CHCl

3ndashMeOHndash

H2O [10 3 1 (22 L) rarr 7 3 1 (16 L) rarr 4 3 1 (14 L) rarr

MeOH] to afford compound 5 (OS1E-9-3-2 603mg) Basedon the spectroscopic methods such as 1H-NMR 13C-NMRand LCMS the chemical structures of the compounds wereconfirmed as feniculin (1) avicularin (2) (+)-catechin (3)(minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) by com-parison of spectral data with those of references Moreoverthe on-line screening HPLC-ABTS+ assay method was rapidand efficient for the investigation of bioactivity of the isolatedcompounds from Acer tegmentosum Compounds 1 2 3 4and 5 were developed on ODS TLC with 10 H

2SO4and the

expected yellow or brown color was observed for the phenoliccompounds

Compound 1 (Feniculin) Yellow amorphous powder (MeOH)ESI-MS mz 4351 [M+H]+ 1H-NMR (400MHz CD

3OD

120575H) 773 (1H d J = 16Hz H-21015840) 755 (1H dd J = 84 16HzH-61015840) 688 (1H d J = 84Hz H-51015840) 639 (1H d J = 16HzH-8) 619 (1H d J = 16Hz H-6) 516 (1H d J = 60Hz H-110158401015840) 389 (1H dd J = 80 60Hz H-210158401015840) 382 (1H H-510158401015840a) 380(1H m H-410158401015840) 364 (1H dd J = 80 28Hz H-310158401015840) 344 (1Hdd J = 132 32Hz H-510158401015840b) 13C-NMR (100MHz CD

3OD

120575c) see Table 3The characterization data of compound 1werecompared to the literature value and combinedwith quercetinand arabinopyranoside to confirm the structure as feniculin[20]

Compound 2 (Avicularin) Yellow amorphous powder(MeOH) ESI-MS mz 4351 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 752 (1H d J = 18Hz H-21015840) 749 (1H dd J =

84 18Hz H-61015840) 690 (1H d J = 84Hz H-51015840) 638 (1Hd J = 18Hz H-8) 620 (1H d J = 18Hz H-6) 546 (1Hs H-110158401015840) 432 (1H d J = 24Hz H-210158401015840) 389 (1H dd J =48 24Hz H-310158401015840) 3861 (1H m H-410158401015840) 3494 (2H t J =42 H-510158401015840) 13C-NMR (150MHz CD

3OD 120575c) see Table 3

1H-NMR spectrum of compound 2 showed a similar patternto compound 1 The structure was confirmed by ESI-MSmz

6 Journal of Analytical Methods in Chemistry

Table 3 13C-NMR chemical shifts of compounds 1ndash5 isolated from Acer tegmentosum

Number of carbon1 2 3 4 5

CD3OD100MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

1 130562 15870 15944 8281 7986 130833 13565 13500 6978 6747 116084 17948 18009 2847 2925 156525 16305 16319 15754 15766 116086 9988 9995 9628 9638 130837 16607 16625 15778 15798 36338 9470 9484 9550 9588 72209 15843 15869 15688 1573510 10563 10569 10081 1000611015840 12289 12309 13219 1322821015840 11745 11654 11523 1158831015840 14595 14647 14619 1457641015840 14996 14996 14621 1459251015840 11617 11654 11608 1153161015840 12303 12320 12003 11938Sugar-110158401015840 10463 10959 10439

210158401015840 7287 8341 7499310158401015840 7413 7876 7784410158401015840 6911 8810 7167510158401015840 6694 6264 7534610158401015840 6470

Galloyl-1101584010158401015840 121372101584010158401015840 110163101584010158401015840 146444101584010158401015840 139785101584010158401015840 146446101584010158401015840 11016

ndashCOOndash 16831

4351 [M+H]+ [21] Compounds 1 and 2 were isolated fromthe plant for the first time

Compound 3 [(+)-Catechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

683 (1H d J = 24Hz H-21015840) 676 (1H d J = 84Hz H-51015840)671 (1H dd J =84 24HzH-61015840) 592 (1H d J =24HzH-6)585 (1H d J = 24Hz H-8) 456 (1H d J = 72Hz H-2) 397(1H m H-3) 250 (1H dd J = 162 84Hz H-4a) 284 (1Hdd J = 162 60Hz H-4b) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3

Compound 4 [(minus)-Epicatechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

696 (1H d J = 18Hz H-21015840) 679 (1H dd J = 84 18HzH-61015840) 675 (1H d J = 84Hz H-51015840) 593 (1H d J = 18HzH-6) 591 (1H d J = 18Hz H-8) 480 (1H brs H-2) 416(1H m H-3) 286 (1H dd J = 168 48Hz H-4a) 273

(1H dd J = 168 30HzH-4b) 13C-NMR (150MHzCD3OD

120575c) see Table 3 Compounds 3 and 4 were compared to theliterature data for (+)-catechin and (minus)-epicatechin for thestructure identification [22] The structure was confirmed bythe presence of mz at 2910 [M+H]+ in the ESI-MS positivemode

Compound 5 (61015840-O-Galloyl salidroside) Pale yellow oil(MeOH) ESI-MS mz 4531 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 710 (2H br s H-2101584010158401015840 H-6101584010158401015840) 696 (2H d J =

84Hz H-2 6) 665 (2H d J = 84Hz H-3 H-5) 665 (1Hd J = 84Hz H-5) 452 (1H dd J = 114 24Hz H-610158401015840a) 445(1H dd J = 114 60Hz H-610158401015840b) 432 (1H d J = 78Hz H-110158401015840) 395 (1H m H-8a) 370 (1H m H-8b) 355 (1H m H-510158401015840) 342 (2H t J = 78Hz H-310158401015840 410158401015840) 322 (1H t J = 84HzH-210158401015840) 277 (2H m H-7) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3 1H-NMR data were consistent with the literaturevalues [23]

Journal of Analytical Methods in Chemistry 7

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

UV wavelength (nm)

Abso

rban

ce

Abso

rban

ce

Abso

rban

ceAb

sorb

ance

Abso

rban

ce

E-6-2

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

UV wavelength (nm)

E-6-4

200 220 240 260 280 300 320 340 360

0

200

400

600

800

1000

UV wavelength (nm)

E-7-6-2

200 220 240 260 280 300 320 340 360

0

100

200

300

400

500

600

700

800

UV wavelength (nm)

E-7-6-4

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

400

UV wavelength (nm)

E-9-3-2

Figure 4 Analysis of UV wavelength from high purity isolation compounds

32 On-Line Screening HPLC-ABTS+ Assay Analysis Thisstudy investigated the bioactivity (using ABTS+ assay rad-ical scavenging activity) and anti-inflammatory activity ofthe five isolated phenolic compounds that were measuredAll the compounds 1ndash5 in the EA fraction (each yieldmg) exhibited antioxidant activities (Table 4) Moreoverthis on-line screening HPLC-ABTS+ assay method wasrapid and efficient for the investigation of bioactivity from

A tegmentosum and was obtained from RS-tech (046 times25 cm 5 120583m C

18 Daejeon Korea) The injection volume was

10 120583L and the flow rate of the mobile phase was 10mLminThe wavelength of the UV detector was fixed at 210 254 280and 320 nmThefive phenolic compoundswere characterizedby comparing the HPLC UV-DAD maximum absorptionpeaks of the samples with those of the pure isolation stan-dards (Figure 4) The determination of antioxidant activity

8 Journal of Analytical Methods in Chemistry

Table4Ex

tractamou

ntandyieldin

ethylacetate(EA)fractionsample

Extractio

nsolvent

Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)

EA phase

1390

6

1321

7

198

7-6

127

9

1631

9-3

125

21018

2lowast60lowast

21640

2lowast307lowast

2228

2lowast603lowast

3657

352

337

332

3778

378

4235

4lowast59lowast

462

4lowast142lowast

486

4123

540

95

185

795

24

585

6555

613

6531

6112

683

72603

722

772

747

8421

856

843

92375

949

940

10975

1021

1057

111324

11103

122552

1290

1327

lowastShow

nforfi

vekind

compo

unds

from

major

extract

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 3

MW 43435 Isolation no E-6-4Feniculin

O

O

OOH

OH

OH

OH

O

HO

HO

HO

MW 43435 Isolation no E-6-2

O

O

OOH

OH

OH

OH

HO

HOHO

O

Avicularin

O

OH

OH

OH

OH

HO

MW 29027 Isolation no E-7-6-2

O

OH

OH

HO

OH

OH

MW 29027 Isolation no E-7-6-4

HO

OH

OH

OH OH

OO

OHO

HO

O

MW 45241 Isolation no E-9-3-2

(+)-Catechin (minus)-Epicatechin

6998400-O-Galloyl salidroside

Figure 1 Chemical structure of five types of compounds from Acer tegmentosum

MerckGermany) and octadecylsilane (ODS) Li-ChropreRP-18 (Merck Germany) Molecular sieve column chromatog-raphy was performed using Sephadex LH-20 (Fluka USA)Thin layer chromatography (TLC) analysis was performedusing silica gel glass plates (Kieselgel 60 F

254and RP-18

F254S Merck Germany) and developed using a mobile phase

composed of chloroform-methanol-water and stained using10 H

2SO4to detect the EA extract and pure compounds

26 On-Line Screening HPLC-ABTS+ Assay Analysis A teg-mentosum extract was injected into a Dionex Ultimate 3000HPLC system (Thermo Scientific) The chromatographiccolumns used in this experiment were commercially availableand were purchased from RS-tech (046 times 25 cm 5 120583mC18 Daejeon Korea) The injection volume was 10 120583L and

the flow rate of the mobile phase was 10mLmin Thewavelength of the UV detector was fixed at 210 254 280

4 Journal of Analytical Methods in Chemistry

1 3 5 6

109871 2 3 4 5 6

71 3 5 6 10 11 12 139871 2 3 4 5 6

10 11 129871 2 3 4 5 6

1 3 4

CH2Cl2 H2O

H2O

H2OEtOAc fr (124 g)

SiO2 cc

Freeze-dried extract powder of Acer tegmentosum (20 g)

n-BuOH

CH2Cl2H2O times 4(1 1)

EtOAcH2O times 4(1 1)

2

2

2

4

4

n-BuOHH2O times 4(1 1)

Figure 2 Process of isolation five kind compounds of ethyl acetate extract from Acer tegmentosum

Table 2 Analysis of gradient elution conditions with RP-HPLC

Instrument ConditionReverse phase column RS-Tech Optimapak C18 (46 times 250mm 5 120583m)Oven temp (∘C) 40Flow rate (mLmin) 10Mobile phase () A 01 TFA in water B acetonitrileUV absorbance (nm) 210 254 280 320

Time (min) Solvent composition ()01 TFA in water (A) Acetonitrile (B)

0 90 1050 60 4060 60 4070 90 10

and 320 nm The composition of the mobile phases wasA 999 watertrifluoroacetic acid (99901 vol) and B100 acetonitrile The run time was 60min and the solventprogram was the linear gradient method (90 10ndash60 40 A Bvol 70min initial condition) (Table 2) Figure 3 showsa schematic diagram of the on-line coupling of a HPLCto a DAD (diode array detector) and the continuous flowABTS+ assay Then on-line HPLC was connected to a ldquoTrdquopiece where ABTS+ was added The ABTS+ at a flow rateof 05mLmin was delivered using a Dionex ultimate 3000pump Aftermixing through a 1mL loop maintained at 40∘Cthe absorbance was measured using a multiple wavelengthdetector (MWD) at 734 nm Data were analyzed using theChromeleon 7 software

Sample injection

ColumnPump PumpRegents Regents

DADdetector

VWDdetector

Passive splitReaction loop

Figure 3 Schematic of on-line screening HPLC-ABTS+ system

27 Cell Culture and Drug Treatment RAW 2647 cells werepurchased from Korea Cell Line Bank (Seoul Korea) andgrown in RPMI 1640 medium containing 10 FBS and

Journal of Analytical Methods in Chemistry 5

100UmL of antibiotics sulfate The cells were incubatedin humidified 5 CO

2atmosphere at 37∘C To stimulate

the cells the medium was changed with fresh RPMI 1640medium and LPS (200 ngmL) was added in the presence orabsence of five compounds (10 30 50 and 100 120583M) for 24 h

28 MTT Assay for Cell Viability Cytotoxicity was analyzedusing the MTT assay Five compounds were added to thecells and incubated for 24 h at 37∘C with 5 CO

2 MTT

solutionswere added to eachwell and the cells were incubatedfor another 4 h The formazan melted in dimethyl sulfoxide(DMSO) and then the optical density was read at 570 nmusing an ELISA reader (Infinite M200 TECAN MannedorfSwitzerland)

29 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated withfive compounds and stimulated with LPS for 24 h Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

210 Determination of TNF-120572 IL-6 and IL-1120573 CytokineProduction Cells were seeded at a density of 5 times 105 cellsmLin 24-well culture plates and pretreated with various con-centrations of five compounds for 30min before the LPSstimulation ELISA plates (Nunc Roskilde Denmark) werecoated overnight at 4∘C with capture antibody diluted in acoating buffer (01M carbonate pH 95) and then washedfive times with phosphate-buffered saline (PBS) containing005 Tween 20 The nonspecific protein-binding sites wereblocked with assay diluent buffer (PBS containing 10 FBSpH 70) for gt1 h The samples and standards were addedto the wells promptly After 2 hours of incubation at RT orovernight at 4∘C the working detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 h Subsequently the substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in the dark before the reaction was quenchedwith stop solution (NH

3PO4) The optical density was read

at 450 nm [19]

211 Statistical Analysis The results are expressed as mean plusmnSE values for the number of experiments Statistical signif-icance of each treated group was compared to the controland determined by Studentrsquos 119905-tests Each experiment wasrepeated at least thrice to yield comparable results Valueswith 119875 lt 001 and lt0001 were considered significant

3 Results and Discussion

31 High Purity Isolation and Analysis The stems of A teg-mentosum were extracted using boiling water and then par-titioned successively using dichloromethane (DCM) ethylacetate (EA) normal butyl alcohol (n-BuOH) and water

(H2O) The EA extract (124 g) was applied to silica gel

cc (120593 4 times 10 cm) and eluted using chloroform- (CHCl3minus)

MeOHndashH2O [15 3 1 (12 L) rarr 13 3 1 (13 L) rarr 10 3 1

(12 L) rarr 7 3 1 (1 L) rarr 4 3 1 (1 L) rarr MeOH] Elutedfractions were monitored by TLC to produce 12 fractions(OS1E-1ndash12) Fraction OS1E-6 (555mg) was subjected toSephadex LH-20 cc [120593 15 times 45 cm 50 MeOH (180mL)rarr 70 MeOH (300mL) rarr 100 MeOH] yielding sevenfractions (OS1E-6-1ndash7) and ultimately produced compounds1 (OS1E-6-2 60mg) and 2 (OS1E-6-4 59mg) FractionOS1E-7 (2603mg) was subjected to Sephadex LH-20 cc [12059315 times 44 cm 80MeOH (400mL) rarr 100MeOH] yielding10 fractions (OS1E-7-1ndash10) Fraction OS1E-7-6 (531mg) wassubjected to ODS cc (120593 15 times 8 cm) and eluted with MeOHndashH2O [1 6 (900mL) rarr MeOH] to afford compounds 3

(OS1E-7-6-2 307mg) and 4 (OS1E-7-6-4 142mg) FractionOS1E-9 (2375mg) was subjected to ODS cc (120593 25 times 8 cm)and eluted withMeOHndashH

2O [1 3 (450mL) rarr 1 2 (300mL)

rarr 1 1 (300mL) rarr MeOH] yielding 13 fractions (OS1E-9-1ndash13) Fraction OS1E-9-3 (778mg) was subjected to silicagel cc (120593 15 times 10 cm) and eluted with CHCl

3ndashMeOHndash

H2O [10 3 1 (22 L) rarr 7 3 1 (16 L) rarr 4 3 1 (14 L) rarr

MeOH] to afford compound 5 (OS1E-9-3-2 603mg) Basedon the spectroscopic methods such as 1H-NMR 13C-NMRand LCMS the chemical structures of the compounds wereconfirmed as feniculin (1) avicularin (2) (+)-catechin (3)(minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) by com-parison of spectral data with those of references Moreoverthe on-line screening HPLC-ABTS+ assay method was rapidand efficient for the investigation of bioactivity of the isolatedcompounds from Acer tegmentosum Compounds 1 2 3 4and 5 were developed on ODS TLC with 10 H

2SO4and the

expected yellow or brown color was observed for the phenoliccompounds

Compound 1 (Feniculin) Yellow amorphous powder (MeOH)ESI-MS mz 4351 [M+H]+ 1H-NMR (400MHz CD

3OD

120575H) 773 (1H d J = 16Hz H-21015840) 755 (1H dd J = 84 16HzH-61015840) 688 (1H d J = 84Hz H-51015840) 639 (1H d J = 16HzH-8) 619 (1H d J = 16Hz H-6) 516 (1H d J = 60Hz H-110158401015840) 389 (1H dd J = 80 60Hz H-210158401015840) 382 (1H H-510158401015840a) 380(1H m H-410158401015840) 364 (1H dd J = 80 28Hz H-310158401015840) 344 (1Hdd J = 132 32Hz H-510158401015840b) 13C-NMR (100MHz CD

3OD

120575c) see Table 3The characterization data of compound 1werecompared to the literature value and combinedwith quercetinand arabinopyranoside to confirm the structure as feniculin[20]

Compound 2 (Avicularin) Yellow amorphous powder(MeOH) ESI-MS mz 4351 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 752 (1H d J = 18Hz H-21015840) 749 (1H dd J =

84 18Hz H-61015840) 690 (1H d J = 84Hz H-51015840) 638 (1Hd J = 18Hz H-8) 620 (1H d J = 18Hz H-6) 546 (1Hs H-110158401015840) 432 (1H d J = 24Hz H-210158401015840) 389 (1H dd J =48 24Hz H-310158401015840) 3861 (1H m H-410158401015840) 3494 (2H t J =42 H-510158401015840) 13C-NMR (150MHz CD

3OD 120575c) see Table 3

1H-NMR spectrum of compound 2 showed a similar patternto compound 1 The structure was confirmed by ESI-MSmz

6 Journal of Analytical Methods in Chemistry

Table 3 13C-NMR chemical shifts of compounds 1ndash5 isolated from Acer tegmentosum

Number of carbon1 2 3 4 5

CD3OD100MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

1 130562 15870 15944 8281 7986 130833 13565 13500 6978 6747 116084 17948 18009 2847 2925 156525 16305 16319 15754 15766 116086 9988 9995 9628 9638 130837 16607 16625 15778 15798 36338 9470 9484 9550 9588 72209 15843 15869 15688 1573510 10563 10569 10081 1000611015840 12289 12309 13219 1322821015840 11745 11654 11523 1158831015840 14595 14647 14619 1457641015840 14996 14996 14621 1459251015840 11617 11654 11608 1153161015840 12303 12320 12003 11938Sugar-110158401015840 10463 10959 10439

210158401015840 7287 8341 7499310158401015840 7413 7876 7784410158401015840 6911 8810 7167510158401015840 6694 6264 7534610158401015840 6470

Galloyl-1101584010158401015840 121372101584010158401015840 110163101584010158401015840 146444101584010158401015840 139785101584010158401015840 146446101584010158401015840 11016

ndashCOOndash 16831

4351 [M+H]+ [21] Compounds 1 and 2 were isolated fromthe plant for the first time

Compound 3 [(+)-Catechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

683 (1H d J = 24Hz H-21015840) 676 (1H d J = 84Hz H-51015840)671 (1H dd J =84 24HzH-61015840) 592 (1H d J =24HzH-6)585 (1H d J = 24Hz H-8) 456 (1H d J = 72Hz H-2) 397(1H m H-3) 250 (1H dd J = 162 84Hz H-4a) 284 (1Hdd J = 162 60Hz H-4b) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3

Compound 4 [(minus)-Epicatechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

696 (1H d J = 18Hz H-21015840) 679 (1H dd J = 84 18HzH-61015840) 675 (1H d J = 84Hz H-51015840) 593 (1H d J = 18HzH-6) 591 (1H d J = 18Hz H-8) 480 (1H brs H-2) 416(1H m H-3) 286 (1H dd J = 168 48Hz H-4a) 273

(1H dd J = 168 30HzH-4b) 13C-NMR (150MHzCD3OD

120575c) see Table 3 Compounds 3 and 4 were compared to theliterature data for (+)-catechin and (minus)-epicatechin for thestructure identification [22] The structure was confirmed bythe presence of mz at 2910 [M+H]+ in the ESI-MS positivemode

Compound 5 (61015840-O-Galloyl salidroside) Pale yellow oil(MeOH) ESI-MS mz 4531 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 710 (2H br s H-2101584010158401015840 H-6101584010158401015840) 696 (2H d J =

84Hz H-2 6) 665 (2H d J = 84Hz H-3 H-5) 665 (1Hd J = 84Hz H-5) 452 (1H dd J = 114 24Hz H-610158401015840a) 445(1H dd J = 114 60Hz H-610158401015840b) 432 (1H d J = 78Hz H-110158401015840) 395 (1H m H-8a) 370 (1H m H-8b) 355 (1H m H-510158401015840) 342 (2H t J = 78Hz H-310158401015840 410158401015840) 322 (1H t J = 84HzH-210158401015840) 277 (2H m H-7) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3 1H-NMR data were consistent with the literaturevalues [23]

Journal of Analytical Methods in Chemistry 7

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

UV wavelength (nm)

Abso

rban

ce

Abso

rban

ce

Abso

rban

ceAb

sorb

ance

Abso

rban

ce

E-6-2

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

UV wavelength (nm)

E-6-4

200 220 240 260 280 300 320 340 360

0

200

400

600

800

1000

UV wavelength (nm)

E-7-6-2

200 220 240 260 280 300 320 340 360

0

100

200

300

400

500

600

700

800

UV wavelength (nm)

E-7-6-4

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

400

UV wavelength (nm)

E-9-3-2

Figure 4 Analysis of UV wavelength from high purity isolation compounds

32 On-Line Screening HPLC-ABTS+ Assay Analysis Thisstudy investigated the bioactivity (using ABTS+ assay rad-ical scavenging activity) and anti-inflammatory activity ofthe five isolated phenolic compounds that were measuredAll the compounds 1ndash5 in the EA fraction (each yieldmg) exhibited antioxidant activities (Table 4) Moreoverthis on-line screening HPLC-ABTS+ assay method wasrapid and efficient for the investigation of bioactivity from

A tegmentosum and was obtained from RS-tech (046 times25 cm 5 120583m C

18 Daejeon Korea) The injection volume was

10 120583L and the flow rate of the mobile phase was 10mLminThe wavelength of the UV detector was fixed at 210 254 280and 320 nmThefive phenolic compoundswere characterizedby comparing the HPLC UV-DAD maximum absorptionpeaks of the samples with those of the pure isolation stan-dards (Figure 4) The determination of antioxidant activity

8 Journal of Analytical Methods in Chemistry

Table4Ex

tractamou

ntandyieldin

ethylacetate(EA)fractionsample

Extractio

nsolvent

Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)

EA phase

1390

6

1321

7

198

7-6

127

9

1631

9-3

125

21018

2lowast60lowast

21640

2lowast307lowast

2228

2lowast603lowast

3657

352

337

332

3778

378

4235

4lowast59lowast

462

4lowast142lowast

486

4123

540

95

185

795

24

585

6555

613

6531

6112

683

72603

722

772

747

8421

856

843

92375

949

940

10975

1021

1057

111324

11103

122552

1290

1327

lowastShow

nforfi

vekind

compo

unds

from

major

extract

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Analytical Methods in Chemistry

1 3 5 6

109871 2 3 4 5 6

71 3 5 6 10 11 12 139871 2 3 4 5 6

10 11 129871 2 3 4 5 6

1 3 4

CH2Cl2 H2O

H2O

H2OEtOAc fr (124 g)

SiO2 cc

Freeze-dried extract powder of Acer tegmentosum (20 g)

n-BuOH

CH2Cl2H2O times 4(1 1)

EtOAcH2O times 4(1 1)

2

2

2

4

4

n-BuOHH2O times 4(1 1)

Figure 2 Process of isolation five kind compounds of ethyl acetate extract from Acer tegmentosum

Table 2 Analysis of gradient elution conditions with RP-HPLC

Instrument ConditionReverse phase column RS-Tech Optimapak C18 (46 times 250mm 5 120583m)Oven temp (∘C) 40Flow rate (mLmin) 10Mobile phase () A 01 TFA in water B acetonitrileUV absorbance (nm) 210 254 280 320

Time (min) Solvent composition ()01 TFA in water (A) Acetonitrile (B)

0 90 1050 60 4060 60 4070 90 10

and 320 nm The composition of the mobile phases wasA 999 watertrifluoroacetic acid (99901 vol) and B100 acetonitrile The run time was 60min and the solventprogram was the linear gradient method (90 10ndash60 40 A Bvol 70min initial condition) (Table 2) Figure 3 showsa schematic diagram of the on-line coupling of a HPLCto a DAD (diode array detector) and the continuous flowABTS+ assay Then on-line HPLC was connected to a ldquoTrdquopiece where ABTS+ was added The ABTS+ at a flow rateof 05mLmin was delivered using a Dionex ultimate 3000pump Aftermixing through a 1mL loop maintained at 40∘Cthe absorbance was measured using a multiple wavelengthdetector (MWD) at 734 nm Data were analyzed using theChromeleon 7 software

Sample injection

ColumnPump PumpRegents Regents

DADdetector

VWDdetector

Passive splitReaction loop

Figure 3 Schematic of on-line screening HPLC-ABTS+ system

27 Cell Culture and Drug Treatment RAW 2647 cells werepurchased from Korea Cell Line Bank (Seoul Korea) andgrown in RPMI 1640 medium containing 10 FBS and

Journal of Analytical Methods in Chemistry 5

100UmL of antibiotics sulfate The cells were incubatedin humidified 5 CO

2atmosphere at 37∘C To stimulate

the cells the medium was changed with fresh RPMI 1640medium and LPS (200 ngmL) was added in the presence orabsence of five compounds (10 30 50 and 100 120583M) for 24 h

28 MTT Assay for Cell Viability Cytotoxicity was analyzedusing the MTT assay Five compounds were added to thecells and incubated for 24 h at 37∘C with 5 CO

2 MTT

solutionswere added to eachwell and the cells were incubatedfor another 4 h The formazan melted in dimethyl sulfoxide(DMSO) and then the optical density was read at 570 nmusing an ELISA reader (Infinite M200 TECAN MannedorfSwitzerland)

29 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated withfive compounds and stimulated with LPS for 24 h Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

210 Determination of TNF-120572 IL-6 and IL-1120573 CytokineProduction Cells were seeded at a density of 5 times 105 cellsmLin 24-well culture plates and pretreated with various con-centrations of five compounds for 30min before the LPSstimulation ELISA plates (Nunc Roskilde Denmark) werecoated overnight at 4∘C with capture antibody diluted in acoating buffer (01M carbonate pH 95) and then washedfive times with phosphate-buffered saline (PBS) containing005 Tween 20 The nonspecific protein-binding sites wereblocked with assay diluent buffer (PBS containing 10 FBSpH 70) for gt1 h The samples and standards were addedto the wells promptly After 2 hours of incubation at RT orovernight at 4∘C the working detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 h Subsequently the substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in the dark before the reaction was quenchedwith stop solution (NH

3PO4) The optical density was read

at 450 nm [19]

211 Statistical Analysis The results are expressed as mean plusmnSE values for the number of experiments Statistical signif-icance of each treated group was compared to the controland determined by Studentrsquos 119905-tests Each experiment wasrepeated at least thrice to yield comparable results Valueswith 119875 lt 001 and lt0001 were considered significant

3 Results and Discussion

31 High Purity Isolation and Analysis The stems of A teg-mentosum were extracted using boiling water and then par-titioned successively using dichloromethane (DCM) ethylacetate (EA) normal butyl alcohol (n-BuOH) and water

(H2O) The EA extract (124 g) was applied to silica gel

cc (120593 4 times 10 cm) and eluted using chloroform- (CHCl3minus)

MeOHndashH2O [15 3 1 (12 L) rarr 13 3 1 (13 L) rarr 10 3 1

(12 L) rarr 7 3 1 (1 L) rarr 4 3 1 (1 L) rarr MeOH] Elutedfractions were monitored by TLC to produce 12 fractions(OS1E-1ndash12) Fraction OS1E-6 (555mg) was subjected toSephadex LH-20 cc [120593 15 times 45 cm 50 MeOH (180mL)rarr 70 MeOH (300mL) rarr 100 MeOH] yielding sevenfractions (OS1E-6-1ndash7) and ultimately produced compounds1 (OS1E-6-2 60mg) and 2 (OS1E-6-4 59mg) FractionOS1E-7 (2603mg) was subjected to Sephadex LH-20 cc [12059315 times 44 cm 80MeOH (400mL) rarr 100MeOH] yielding10 fractions (OS1E-7-1ndash10) Fraction OS1E-7-6 (531mg) wassubjected to ODS cc (120593 15 times 8 cm) and eluted with MeOHndashH2O [1 6 (900mL) rarr MeOH] to afford compounds 3

(OS1E-7-6-2 307mg) and 4 (OS1E-7-6-4 142mg) FractionOS1E-9 (2375mg) was subjected to ODS cc (120593 25 times 8 cm)and eluted withMeOHndashH

2O [1 3 (450mL) rarr 1 2 (300mL)

rarr 1 1 (300mL) rarr MeOH] yielding 13 fractions (OS1E-9-1ndash13) Fraction OS1E-9-3 (778mg) was subjected to silicagel cc (120593 15 times 10 cm) and eluted with CHCl

3ndashMeOHndash

H2O [10 3 1 (22 L) rarr 7 3 1 (16 L) rarr 4 3 1 (14 L) rarr

MeOH] to afford compound 5 (OS1E-9-3-2 603mg) Basedon the spectroscopic methods such as 1H-NMR 13C-NMRand LCMS the chemical structures of the compounds wereconfirmed as feniculin (1) avicularin (2) (+)-catechin (3)(minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) by com-parison of spectral data with those of references Moreoverthe on-line screening HPLC-ABTS+ assay method was rapidand efficient for the investigation of bioactivity of the isolatedcompounds from Acer tegmentosum Compounds 1 2 3 4and 5 were developed on ODS TLC with 10 H

2SO4and the

expected yellow or brown color was observed for the phenoliccompounds

Compound 1 (Feniculin) Yellow amorphous powder (MeOH)ESI-MS mz 4351 [M+H]+ 1H-NMR (400MHz CD

3OD

120575H) 773 (1H d J = 16Hz H-21015840) 755 (1H dd J = 84 16HzH-61015840) 688 (1H d J = 84Hz H-51015840) 639 (1H d J = 16HzH-8) 619 (1H d J = 16Hz H-6) 516 (1H d J = 60Hz H-110158401015840) 389 (1H dd J = 80 60Hz H-210158401015840) 382 (1H H-510158401015840a) 380(1H m H-410158401015840) 364 (1H dd J = 80 28Hz H-310158401015840) 344 (1Hdd J = 132 32Hz H-510158401015840b) 13C-NMR (100MHz CD

3OD

120575c) see Table 3The characterization data of compound 1werecompared to the literature value and combinedwith quercetinand arabinopyranoside to confirm the structure as feniculin[20]

Compound 2 (Avicularin) Yellow amorphous powder(MeOH) ESI-MS mz 4351 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 752 (1H d J = 18Hz H-21015840) 749 (1H dd J =

84 18Hz H-61015840) 690 (1H d J = 84Hz H-51015840) 638 (1Hd J = 18Hz H-8) 620 (1H d J = 18Hz H-6) 546 (1Hs H-110158401015840) 432 (1H d J = 24Hz H-210158401015840) 389 (1H dd J =48 24Hz H-310158401015840) 3861 (1H m H-410158401015840) 3494 (2H t J =42 H-510158401015840) 13C-NMR (150MHz CD

3OD 120575c) see Table 3

1H-NMR spectrum of compound 2 showed a similar patternto compound 1 The structure was confirmed by ESI-MSmz

6 Journal of Analytical Methods in Chemistry

Table 3 13C-NMR chemical shifts of compounds 1ndash5 isolated from Acer tegmentosum

Number of carbon1 2 3 4 5

CD3OD100MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

1 130562 15870 15944 8281 7986 130833 13565 13500 6978 6747 116084 17948 18009 2847 2925 156525 16305 16319 15754 15766 116086 9988 9995 9628 9638 130837 16607 16625 15778 15798 36338 9470 9484 9550 9588 72209 15843 15869 15688 1573510 10563 10569 10081 1000611015840 12289 12309 13219 1322821015840 11745 11654 11523 1158831015840 14595 14647 14619 1457641015840 14996 14996 14621 1459251015840 11617 11654 11608 1153161015840 12303 12320 12003 11938Sugar-110158401015840 10463 10959 10439

210158401015840 7287 8341 7499310158401015840 7413 7876 7784410158401015840 6911 8810 7167510158401015840 6694 6264 7534610158401015840 6470

Galloyl-1101584010158401015840 121372101584010158401015840 110163101584010158401015840 146444101584010158401015840 139785101584010158401015840 146446101584010158401015840 11016

ndashCOOndash 16831

4351 [M+H]+ [21] Compounds 1 and 2 were isolated fromthe plant for the first time

Compound 3 [(+)-Catechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

683 (1H d J = 24Hz H-21015840) 676 (1H d J = 84Hz H-51015840)671 (1H dd J =84 24HzH-61015840) 592 (1H d J =24HzH-6)585 (1H d J = 24Hz H-8) 456 (1H d J = 72Hz H-2) 397(1H m H-3) 250 (1H dd J = 162 84Hz H-4a) 284 (1Hdd J = 162 60Hz H-4b) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3

Compound 4 [(minus)-Epicatechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

696 (1H d J = 18Hz H-21015840) 679 (1H dd J = 84 18HzH-61015840) 675 (1H d J = 84Hz H-51015840) 593 (1H d J = 18HzH-6) 591 (1H d J = 18Hz H-8) 480 (1H brs H-2) 416(1H m H-3) 286 (1H dd J = 168 48Hz H-4a) 273

(1H dd J = 168 30HzH-4b) 13C-NMR (150MHzCD3OD

120575c) see Table 3 Compounds 3 and 4 were compared to theliterature data for (+)-catechin and (minus)-epicatechin for thestructure identification [22] The structure was confirmed bythe presence of mz at 2910 [M+H]+ in the ESI-MS positivemode

Compound 5 (61015840-O-Galloyl salidroside) Pale yellow oil(MeOH) ESI-MS mz 4531 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 710 (2H br s H-2101584010158401015840 H-6101584010158401015840) 696 (2H d J =

84Hz H-2 6) 665 (2H d J = 84Hz H-3 H-5) 665 (1Hd J = 84Hz H-5) 452 (1H dd J = 114 24Hz H-610158401015840a) 445(1H dd J = 114 60Hz H-610158401015840b) 432 (1H d J = 78Hz H-110158401015840) 395 (1H m H-8a) 370 (1H m H-8b) 355 (1H m H-510158401015840) 342 (2H t J = 78Hz H-310158401015840 410158401015840) 322 (1H t J = 84HzH-210158401015840) 277 (2H m H-7) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3 1H-NMR data were consistent with the literaturevalues [23]

Journal of Analytical Methods in Chemistry 7

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

UV wavelength (nm)

Abso

rban

ce

Abso

rban

ce

Abso

rban

ceAb

sorb

ance

Abso

rban

ce

E-6-2

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

UV wavelength (nm)

E-6-4

200 220 240 260 280 300 320 340 360

0

200

400

600

800

1000

UV wavelength (nm)

E-7-6-2

200 220 240 260 280 300 320 340 360

0

100

200

300

400

500

600

700

800

UV wavelength (nm)

E-7-6-4

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

400

UV wavelength (nm)

E-9-3-2

Figure 4 Analysis of UV wavelength from high purity isolation compounds

32 On-Line Screening HPLC-ABTS+ Assay Analysis Thisstudy investigated the bioactivity (using ABTS+ assay rad-ical scavenging activity) and anti-inflammatory activity ofthe five isolated phenolic compounds that were measuredAll the compounds 1ndash5 in the EA fraction (each yieldmg) exhibited antioxidant activities (Table 4) Moreoverthis on-line screening HPLC-ABTS+ assay method wasrapid and efficient for the investigation of bioactivity from

A tegmentosum and was obtained from RS-tech (046 times25 cm 5 120583m C

18 Daejeon Korea) The injection volume was

10 120583L and the flow rate of the mobile phase was 10mLminThe wavelength of the UV detector was fixed at 210 254 280and 320 nmThefive phenolic compoundswere characterizedby comparing the HPLC UV-DAD maximum absorptionpeaks of the samples with those of the pure isolation stan-dards (Figure 4) The determination of antioxidant activity

8 Journal of Analytical Methods in Chemistry

Table4Ex

tractamou

ntandyieldin

ethylacetate(EA)fractionsample

Extractio

nsolvent

Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)

EA phase

1390

6

1321

7

198

7-6

127

9

1631

9-3

125

21018

2lowast60lowast

21640

2lowast307lowast

2228

2lowast603lowast

3657

352

337

332

3778

378

4235

4lowast59lowast

462

4lowast142lowast

486

4123

540

95

185

795

24

585

6555

613

6531

6112

683

72603

722

772

747

8421

856

843

92375

949

940

10975

1021

1057

111324

11103

122552

1290

1327

lowastShow

nforfi

vekind

compo

unds

from

major

extract

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

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Carbohydrate Chemistry

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Journal of

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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CatalystsJournal of

Journal of Analytical Methods in Chemistry 5

100UmL of antibiotics sulfate The cells were incubatedin humidified 5 CO

2atmosphere at 37∘C To stimulate

the cells the medium was changed with fresh RPMI 1640medium and LPS (200 ngmL) was added in the presence orabsence of five compounds (10 30 50 and 100 120583M) for 24 h

28 MTT Assay for Cell Viability Cytotoxicity was analyzedusing the MTT assay Five compounds were added to thecells and incubated for 24 h at 37∘C with 5 CO

2 MTT

solutionswere added to eachwell and the cells were incubatedfor another 4 h The formazan melted in dimethyl sulfoxide(DMSO) and then the optical density was read at 570 nmusing an ELISA reader (Infinite M200 TECAN MannedorfSwitzerland)

29 Measurement of NO Production NO production wasanalyzed by measuring the nitrite in the supernatants ofcultured macrophage cells The cells were pretreated withfive compounds and stimulated with LPS for 24 h Thesupernatant was mixed with the same volume of Griessreagent (1 sulfanilamide 01 naphthylethylenediaminedihydrochloride and 25 phosphoric acid) and incubatedat room temperature (RT) for 5min [19] The absorbance at570 nm was read

210 Determination of TNF-120572 IL-6 and IL-1120573 CytokineProduction Cells were seeded at a density of 5 times 105 cellsmLin 24-well culture plates and pretreated with various con-centrations of five compounds for 30min before the LPSstimulation ELISA plates (Nunc Roskilde Denmark) werecoated overnight at 4∘C with capture antibody diluted in acoating buffer (01M carbonate pH 95) and then washedfive times with phosphate-buffered saline (PBS) containing005 Tween 20 The nonspecific protein-binding sites wereblocked with assay diluent buffer (PBS containing 10 FBSpH 70) for gt1 h The samples and standards were addedto the wells promptly After 2 hours of incubation at RT orovernight at 4∘C the working detector solution (biotinylateddetection antibody and streptavidin-HRP reagent) was addedand incubated for 1 h Subsequently the substrate solution(tetramethylbenzidine) was added to the wells and incubatedfor 30min in the dark before the reaction was quenchedwith stop solution (NH

3PO4) The optical density was read

at 450 nm [19]

211 Statistical Analysis The results are expressed as mean plusmnSE values for the number of experiments Statistical signif-icance of each treated group was compared to the controland determined by Studentrsquos 119905-tests Each experiment wasrepeated at least thrice to yield comparable results Valueswith 119875 lt 001 and lt0001 were considered significant

3 Results and Discussion

31 High Purity Isolation and Analysis The stems of A teg-mentosum were extracted using boiling water and then par-titioned successively using dichloromethane (DCM) ethylacetate (EA) normal butyl alcohol (n-BuOH) and water

(H2O) The EA extract (124 g) was applied to silica gel

cc (120593 4 times 10 cm) and eluted using chloroform- (CHCl3minus)

MeOHndashH2O [15 3 1 (12 L) rarr 13 3 1 (13 L) rarr 10 3 1

(12 L) rarr 7 3 1 (1 L) rarr 4 3 1 (1 L) rarr MeOH] Elutedfractions were monitored by TLC to produce 12 fractions(OS1E-1ndash12) Fraction OS1E-6 (555mg) was subjected toSephadex LH-20 cc [120593 15 times 45 cm 50 MeOH (180mL)rarr 70 MeOH (300mL) rarr 100 MeOH] yielding sevenfractions (OS1E-6-1ndash7) and ultimately produced compounds1 (OS1E-6-2 60mg) and 2 (OS1E-6-4 59mg) FractionOS1E-7 (2603mg) was subjected to Sephadex LH-20 cc [12059315 times 44 cm 80MeOH (400mL) rarr 100MeOH] yielding10 fractions (OS1E-7-1ndash10) Fraction OS1E-7-6 (531mg) wassubjected to ODS cc (120593 15 times 8 cm) and eluted with MeOHndashH2O [1 6 (900mL) rarr MeOH] to afford compounds 3

(OS1E-7-6-2 307mg) and 4 (OS1E-7-6-4 142mg) FractionOS1E-9 (2375mg) was subjected to ODS cc (120593 25 times 8 cm)and eluted withMeOHndashH

2O [1 3 (450mL) rarr 1 2 (300mL)

rarr 1 1 (300mL) rarr MeOH] yielding 13 fractions (OS1E-9-1ndash13) Fraction OS1E-9-3 (778mg) was subjected to silicagel cc (120593 15 times 10 cm) and eluted with CHCl

3ndashMeOHndash

H2O [10 3 1 (22 L) rarr 7 3 1 (16 L) rarr 4 3 1 (14 L) rarr

MeOH] to afford compound 5 (OS1E-9-3-2 603mg) Basedon the spectroscopic methods such as 1H-NMR 13C-NMRand LCMS the chemical structures of the compounds wereconfirmed as feniculin (1) avicularin (2) (+)-catechin (3)(minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) by com-parison of spectral data with those of references Moreoverthe on-line screening HPLC-ABTS+ assay method was rapidand efficient for the investigation of bioactivity of the isolatedcompounds from Acer tegmentosum Compounds 1 2 3 4and 5 were developed on ODS TLC with 10 H

2SO4and the

expected yellow or brown color was observed for the phenoliccompounds

Compound 1 (Feniculin) Yellow amorphous powder (MeOH)ESI-MS mz 4351 [M+H]+ 1H-NMR (400MHz CD

3OD

120575H) 773 (1H d J = 16Hz H-21015840) 755 (1H dd J = 84 16HzH-61015840) 688 (1H d J = 84Hz H-51015840) 639 (1H d J = 16HzH-8) 619 (1H d J = 16Hz H-6) 516 (1H d J = 60Hz H-110158401015840) 389 (1H dd J = 80 60Hz H-210158401015840) 382 (1H H-510158401015840a) 380(1H m H-410158401015840) 364 (1H dd J = 80 28Hz H-310158401015840) 344 (1Hdd J = 132 32Hz H-510158401015840b) 13C-NMR (100MHz CD

3OD

120575c) see Table 3The characterization data of compound 1werecompared to the literature value and combinedwith quercetinand arabinopyranoside to confirm the structure as feniculin[20]

Compound 2 (Avicularin) Yellow amorphous powder(MeOH) ESI-MS mz 4351 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 752 (1H d J = 18Hz H-21015840) 749 (1H dd J =

84 18Hz H-61015840) 690 (1H d J = 84Hz H-51015840) 638 (1Hd J = 18Hz H-8) 620 (1H d J = 18Hz H-6) 546 (1Hs H-110158401015840) 432 (1H d J = 24Hz H-210158401015840) 389 (1H dd J =48 24Hz H-310158401015840) 3861 (1H m H-410158401015840) 3494 (2H t J =42 H-510158401015840) 13C-NMR (150MHz CD

3OD 120575c) see Table 3

1H-NMR spectrum of compound 2 showed a similar patternto compound 1 The structure was confirmed by ESI-MSmz

6 Journal of Analytical Methods in Chemistry

Table 3 13C-NMR chemical shifts of compounds 1ndash5 isolated from Acer tegmentosum

Number of carbon1 2 3 4 5

CD3OD100MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

1 130562 15870 15944 8281 7986 130833 13565 13500 6978 6747 116084 17948 18009 2847 2925 156525 16305 16319 15754 15766 116086 9988 9995 9628 9638 130837 16607 16625 15778 15798 36338 9470 9484 9550 9588 72209 15843 15869 15688 1573510 10563 10569 10081 1000611015840 12289 12309 13219 1322821015840 11745 11654 11523 1158831015840 14595 14647 14619 1457641015840 14996 14996 14621 1459251015840 11617 11654 11608 1153161015840 12303 12320 12003 11938Sugar-110158401015840 10463 10959 10439

210158401015840 7287 8341 7499310158401015840 7413 7876 7784410158401015840 6911 8810 7167510158401015840 6694 6264 7534610158401015840 6470

Galloyl-1101584010158401015840 121372101584010158401015840 110163101584010158401015840 146444101584010158401015840 139785101584010158401015840 146446101584010158401015840 11016

ndashCOOndash 16831

4351 [M+H]+ [21] Compounds 1 and 2 were isolated fromthe plant for the first time

Compound 3 [(+)-Catechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

683 (1H d J = 24Hz H-21015840) 676 (1H d J = 84Hz H-51015840)671 (1H dd J =84 24HzH-61015840) 592 (1H d J =24HzH-6)585 (1H d J = 24Hz H-8) 456 (1H d J = 72Hz H-2) 397(1H m H-3) 250 (1H dd J = 162 84Hz H-4a) 284 (1Hdd J = 162 60Hz H-4b) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3

Compound 4 [(minus)-Epicatechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

696 (1H d J = 18Hz H-21015840) 679 (1H dd J = 84 18HzH-61015840) 675 (1H d J = 84Hz H-51015840) 593 (1H d J = 18HzH-6) 591 (1H d J = 18Hz H-8) 480 (1H brs H-2) 416(1H m H-3) 286 (1H dd J = 168 48Hz H-4a) 273

(1H dd J = 168 30HzH-4b) 13C-NMR (150MHzCD3OD

120575c) see Table 3 Compounds 3 and 4 were compared to theliterature data for (+)-catechin and (minus)-epicatechin for thestructure identification [22] The structure was confirmed bythe presence of mz at 2910 [M+H]+ in the ESI-MS positivemode

Compound 5 (61015840-O-Galloyl salidroside) Pale yellow oil(MeOH) ESI-MS mz 4531 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 710 (2H br s H-2101584010158401015840 H-6101584010158401015840) 696 (2H d J =

84Hz H-2 6) 665 (2H d J = 84Hz H-3 H-5) 665 (1Hd J = 84Hz H-5) 452 (1H dd J = 114 24Hz H-610158401015840a) 445(1H dd J = 114 60Hz H-610158401015840b) 432 (1H d J = 78Hz H-110158401015840) 395 (1H m H-8a) 370 (1H m H-8b) 355 (1H m H-510158401015840) 342 (2H t J = 78Hz H-310158401015840 410158401015840) 322 (1H t J = 84HzH-210158401015840) 277 (2H m H-7) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3 1H-NMR data were consistent with the literaturevalues [23]

Journal of Analytical Methods in Chemistry 7

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

UV wavelength (nm)

Abso

rban

ce

Abso

rban

ce

Abso

rban

ceAb

sorb

ance

Abso

rban

ce

E-6-2

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

UV wavelength (nm)

E-6-4

200 220 240 260 280 300 320 340 360

0

200

400

600

800

1000

UV wavelength (nm)

E-7-6-2

200 220 240 260 280 300 320 340 360

0

100

200

300

400

500

600

700

800

UV wavelength (nm)

E-7-6-4

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

400

UV wavelength (nm)

E-9-3-2

Figure 4 Analysis of UV wavelength from high purity isolation compounds

32 On-Line Screening HPLC-ABTS+ Assay Analysis Thisstudy investigated the bioactivity (using ABTS+ assay rad-ical scavenging activity) and anti-inflammatory activity ofthe five isolated phenolic compounds that were measuredAll the compounds 1ndash5 in the EA fraction (each yieldmg) exhibited antioxidant activities (Table 4) Moreoverthis on-line screening HPLC-ABTS+ assay method wasrapid and efficient for the investigation of bioactivity from

A tegmentosum and was obtained from RS-tech (046 times25 cm 5 120583m C

18 Daejeon Korea) The injection volume was

10 120583L and the flow rate of the mobile phase was 10mLminThe wavelength of the UV detector was fixed at 210 254 280and 320 nmThefive phenolic compoundswere characterizedby comparing the HPLC UV-DAD maximum absorptionpeaks of the samples with those of the pure isolation stan-dards (Figure 4) The determination of antioxidant activity

8 Journal of Analytical Methods in Chemistry

Table4Ex

tractamou

ntandyieldin

ethylacetate(EA)fractionsample

Extractio

nsolvent

Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)

EA phase

1390

6

1321

7

198

7-6

127

9

1631

9-3

125

21018

2lowast60lowast

21640

2lowast307lowast

2228

2lowast603lowast

3657

352

337

332

3778

378

4235

4lowast59lowast

462

4lowast142lowast

486

4123

540

95

185

795

24

585

6555

613

6531

6112

683

72603

722

772

747

8421

856

843

92375

949

940

10975

1021

1057

111324

11103

122552

1290

1327

lowastShow

nforfi

vekind

compo

unds

from

major

extract

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Journal of Analytical Methods in Chemistry

Table 3 13C-NMR chemical shifts of compounds 1ndash5 isolated from Acer tegmentosum

Number of carbon1 2 3 4 5

CD3OD100MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

CD3OD150MHz

1 130562 15870 15944 8281 7986 130833 13565 13500 6978 6747 116084 17948 18009 2847 2925 156525 16305 16319 15754 15766 116086 9988 9995 9628 9638 130837 16607 16625 15778 15798 36338 9470 9484 9550 9588 72209 15843 15869 15688 1573510 10563 10569 10081 1000611015840 12289 12309 13219 1322821015840 11745 11654 11523 1158831015840 14595 14647 14619 1457641015840 14996 14996 14621 1459251015840 11617 11654 11608 1153161015840 12303 12320 12003 11938Sugar-110158401015840 10463 10959 10439

210158401015840 7287 8341 7499310158401015840 7413 7876 7784410158401015840 6911 8810 7167510158401015840 6694 6264 7534610158401015840 6470

Galloyl-1101584010158401015840 121372101584010158401015840 110163101584010158401015840 146444101584010158401015840 139785101584010158401015840 146446101584010158401015840 11016

ndashCOOndash 16831

4351 [M+H]+ [21] Compounds 1 and 2 were isolated fromthe plant for the first time

Compound 3 [(+)-Catechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

683 (1H d J = 24Hz H-21015840) 676 (1H d J = 84Hz H-51015840)671 (1H dd J =84 24HzH-61015840) 592 (1H d J =24HzH-6)585 (1H d J = 24Hz H-8) 456 (1H d J = 72Hz H-2) 397(1H m H-3) 250 (1H dd J = 162 84Hz H-4a) 284 (1Hdd J = 162 60Hz H-4b) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3

Compound 4 [(minus)-Epicatechin] Brown powder (MeOH) ESI-MS mz 2910 [M+H]+ 1H-NMR (600MHz CD

3OD 120575H)

696 (1H d J = 18Hz H-21015840) 679 (1H dd J = 84 18HzH-61015840) 675 (1H d J = 84Hz H-51015840) 593 (1H d J = 18HzH-6) 591 (1H d J = 18Hz H-8) 480 (1H brs H-2) 416(1H m H-3) 286 (1H dd J = 168 48Hz H-4a) 273

(1H dd J = 168 30HzH-4b) 13C-NMR (150MHzCD3OD

120575c) see Table 3 Compounds 3 and 4 were compared to theliterature data for (+)-catechin and (minus)-epicatechin for thestructure identification [22] The structure was confirmed bythe presence of mz at 2910 [M+H]+ in the ESI-MS positivemode

Compound 5 (61015840-O-Galloyl salidroside) Pale yellow oil(MeOH) ESI-MS mz 4531 [M+H]+ 1H-NMR (600MHzCD3OD 120575H) 710 (2H br s H-2101584010158401015840 H-6101584010158401015840) 696 (2H d J =

84Hz H-2 6) 665 (2H d J = 84Hz H-3 H-5) 665 (1Hd J = 84Hz H-5) 452 (1H dd J = 114 24Hz H-610158401015840a) 445(1H dd J = 114 60Hz H-610158401015840b) 432 (1H d J = 78Hz H-110158401015840) 395 (1H m H-8a) 370 (1H m H-8b) 355 (1H m H-510158401015840) 342 (2H t J = 78Hz H-310158401015840 410158401015840) 322 (1H t J = 84HzH-210158401015840) 277 (2H m H-7) 13C-NMR (150MHz CD

3OD 120575c)

see Table 3 1H-NMR data were consistent with the literaturevalues [23]

Journal of Analytical Methods in Chemistry 7

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

UV wavelength (nm)

Abso

rban

ce

Abso

rban

ce

Abso

rban

ceAb

sorb

ance

Abso

rban

ce

E-6-2

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

UV wavelength (nm)

E-6-4

200 220 240 260 280 300 320 340 360

0

200

400

600

800

1000

UV wavelength (nm)

E-7-6-2

200 220 240 260 280 300 320 340 360

0

100

200

300

400

500

600

700

800

UV wavelength (nm)

E-7-6-4

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

400

UV wavelength (nm)

E-9-3-2

Figure 4 Analysis of UV wavelength from high purity isolation compounds

32 On-Line Screening HPLC-ABTS+ Assay Analysis Thisstudy investigated the bioactivity (using ABTS+ assay rad-ical scavenging activity) and anti-inflammatory activity ofthe five isolated phenolic compounds that were measuredAll the compounds 1ndash5 in the EA fraction (each yieldmg) exhibited antioxidant activities (Table 4) Moreoverthis on-line screening HPLC-ABTS+ assay method wasrapid and efficient for the investigation of bioactivity from

A tegmentosum and was obtained from RS-tech (046 times25 cm 5 120583m C

18 Daejeon Korea) The injection volume was

10 120583L and the flow rate of the mobile phase was 10mLminThe wavelength of the UV detector was fixed at 210 254 280and 320 nmThefive phenolic compoundswere characterizedby comparing the HPLC UV-DAD maximum absorptionpeaks of the samples with those of the pure isolation stan-dards (Figure 4) The determination of antioxidant activity

8 Journal of Analytical Methods in Chemistry

Table4Ex

tractamou

ntandyieldin

ethylacetate(EA)fractionsample

Extractio

nsolvent

Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)

EA phase

1390

6

1321

7

198

7-6

127

9

1631

9-3

125

21018

2lowast60lowast

21640

2lowast307lowast

2228

2lowast603lowast

3657

352

337

332

3778

378

4235

4lowast59lowast

462

4lowast142lowast

486

4123

540

95

185

795

24

585

6555

613

6531

6112

683

72603

722

772

747

8421

856

843

92375

949

940

10975

1021

1057

111324

11103

122552

1290

1327

lowastShow

nforfi

vekind

compo

unds

from

major

extract

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 7

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

UV wavelength (nm)

Abso

rban

ce

Abso

rban

ce

Abso

rban

ceAb

sorb

ance

Abso

rban

ce

E-6-2

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

UV wavelength (nm)

E-6-4

200 220 240 260 280 300 320 340 360

0

200

400

600

800

1000

UV wavelength (nm)

E-7-6-2

200 220 240 260 280 300 320 340 360

0

100

200

300

400

500

600

700

800

UV wavelength (nm)

E-7-6-4

200 220 240 260 280 300 320 340 3600

50

100

150

200

250

300

350

400

UV wavelength (nm)

E-9-3-2

Figure 4 Analysis of UV wavelength from high purity isolation compounds

32 On-Line Screening HPLC-ABTS+ Assay Analysis Thisstudy investigated the bioactivity (using ABTS+ assay rad-ical scavenging activity) and anti-inflammatory activity ofthe five isolated phenolic compounds that were measuredAll the compounds 1ndash5 in the EA fraction (each yieldmg) exhibited antioxidant activities (Table 4) Moreoverthis on-line screening HPLC-ABTS+ assay method wasrapid and efficient for the investigation of bioactivity from

A tegmentosum and was obtained from RS-tech (046 times25 cm 5 120583m C

18 Daejeon Korea) The injection volume was

10 120583L and the flow rate of the mobile phase was 10mLminThe wavelength of the UV detector was fixed at 210 254 280and 320 nmThefive phenolic compoundswere characterizedby comparing the HPLC UV-DAD maximum absorptionpeaks of the samples with those of the pure isolation stan-dards (Figure 4) The determination of antioxidant activity

8 Journal of Analytical Methods in Chemistry

Table4Ex

tractamou

ntandyieldin

ethylacetate(EA)fractionsample

Extractio

nsolvent

Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)

EA phase

1390

6

1321

7

198

7-6

127

9

1631

9-3

125

21018

2lowast60lowast

21640

2lowast307lowast

2228

2lowast603lowast

3657

352

337

332

3778

378

4235

4lowast59lowast

462

4lowast142lowast

486

4123

540

95

185

795

24

585

6555

613

6531

6112

683

72603

722

772

747

8421

856

843

92375

949

940

10975

1021

1057

111324

11103

122552

1290

1327

lowastShow

nforfi

vekind

compo

unds

from

major

extract

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 Journal of Analytical Methods in Chemistry

Table4Ex

tractamou

ntandyieldin

ethylacetate(EA)fractionsample

Extractio

nsolvent

Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)Fractio

n(num

ber)

Yield

amou

nt(m

g)

EA phase

1390

6

1321

7

198

7-6

127

9

1631

9-3

125

21018

2lowast60lowast

21640

2lowast307lowast

2228

2lowast603lowast

3657

352

337

332

3778

378

4235

4lowast59lowast

462

4lowast142lowast

486

4123

540

95

185

795

24

585

6555

613

6531

6112

683

72603

722

772

747

8421

856

843

92375

949

940

10975

1021

1057

111324

11103

122552

1290

1327

lowastShow

nforfi

vekind

compo

unds

from

major

extract

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 9

from the on-line HPLCndashDPPH (ABTS) assay was basedon the decrease in absorbance at 517 and 734 nm after thepostcolumn reaction of antioxidants separated from HPLCwith DPPH (ABTS) and the antioxidants present in a samplewould be easily indicated by negative peaksThe compositionof the mobile phases was A 999 vol watertrifluoroaceticacid (99901 vol) and B 100 acetonitrile The run timewas 60min and the solvent program used the linear gradientmethod (90 10ndash60 40 A B vol 70min initial condition)The ABTS+ flow rate was 05mLmin The HPLC separatedanalytes showed a postcolumn reaction with the ABTS+and the reduction was detected as a negative peak using aMWD at 734 nm The combined UV (positive signals) andABTS+ quenching (negative signals) chromatograms of thedifferent A tegmentosum extracts (200 ppm) are presented inFigures 5 and 6 Several eluted fractions of the five phenoliccompounds in the EA extract were detected as feniculin (1)avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) giving a positive signal on the UVdetector at 210 nmThe other compounds showed hydrogen-donating capacity (negative peak) toward the ABTS+ radicalat the applied concentration These results revealed that themethod could be applied for quick bioactivity screeningor more precisely to detect the radical-scavenging activ-ity of compounds indicating that (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) exhibitedbioactivities 61015840-O-Galloyl salidroside (5) exhibited higherbioactivity whereas feniculin (1) and avicularin (2) showedlow bioactivity (Figure 5)The retention time (119877

119905) of feniculin

(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) was 10700 13884 15928 24217and 25062min respectively The bioactivity appeared to beapproximately proportional to the concentration of the fivephenolic compounds in the extracts The HPLC analysisconditions for the best separation of the five compoundswere successfully established by varying the open columntreatment and solvent purification step (Figure 6) Thisstudy confirms the feasibility of assessing the bioactivity ofspecific phytochemicals using an on-line screening HPLC-ABTS+ assay method (Table 5) This proposed method wassuccessfully applied for the screening and identification ofnatural bioactive compounds from A tegmentosum

33 LC-MS Analysis Condition Figure 7 shows the HPLCprofile and LC-MS spectra of five isolated phenolic com-pounds from A tegmentosum LC-MS analysis indicatedthat the compounds from A tegmentosum were isolated inhighly pure form Each compound was dissolved in MeOHat a concentration of 200 ppm and the LC-MS analysisconditions are listed in Table 1 LC-MS analysis is a powerfultool in metabolic profiling and metabolomics research and itcan accurately determine the content of specific metaboliteseven at low levels in plant samples LC-MS analysis was usedpreviously to identify certain constituents ofA tegmentosumThe comparison of UV spectra mass spectra and retentiontimes of the five types of compounds with the data ofstandard compounds led to their unambiguous assignments

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

minus100

minus50

Avicularin (E-6-4)

Feniculin (E-6-2)

(minus)-Epicatechin (E-7-6-4)

(+)-Catechin (E-7-6-2)

Inte

nsity

(mAU

)

UV 210nm

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 5 Bioactivity screening of ethyl acetate extract from Acertegmentosum using on-line screeningHPLC-ABTS+ assay (injectionvolume 10 120583L UV wavelength positive 210 nm negative 734 nm)

0 5 10 15 20 25 30

0

50

100

150

200

Bioactivity

Time (min)

Inte

nsity

(mAU

)

minus100

minus50

UV 280nmUV 254nm

(+)-Catechin (E-7-6-2)

(minus)-Epicatechin (E-7-6-4)

Avicularin (E-6-4)Feniculin (E-6-2)

6998400-O-Galloyl salidroside (E-9-3-2)

Figure 6 Analysis of high purity compounds of ethyl acetate extractin Acer tegmentosum (injection volume 10 120583L UV wavelengthpositive 280 and 254 nm negative 734 nm sample concentration200 ppm)

Simultaneous analysis based on 119877119905of feniculin (1) avicu-

larin (2) (+)-catechin (3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5) of 326 340 121 177 and 213minrespectively was correlated with themolecular mass data andrelative response ESI-MS analysis of compounds feniculin(1) avicularin (2) (+)-catechin (3) (minus)-epicatechin (4) and61015840-O-galloyl salidroside (5) showed intense [M+H]+ signalsat 4351 4351 2910 2910 and 4531mz values respectively

34 Anti-Inflammatory Activities Screening

341 Effect of Five Compounds on RAW 2647 Cell ViabilityWe evaluated the cytotoxicity of five compounds using

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

10 Journal of Analytical Methods in Chemistry

Table5Ex

tractsandbioactivity

efficiency

byon

-line

screeningHPL

C-ABT

Sin

positivea

ndnegativ

epeakarea

Com

poun

dsname

Retentiontim

e(119877119905m

in)

Positivep

eakarea

(mAU

)Negativep

eakarea

(mAU

)Figure

number

Avicularin

E-6-4

10650

123413

57326

Figure

5(EAph

asecomplex

compo

unds)

Feniculin

E-6-2

13870

146317

52711

(+)-Ca

techin

E-7-6-2

15880

205975

140358

(minus)-Ep

icatechinE-7-6-4

2416

736339

04618

61015840-O

-Galloylsalid

rosid

eE-9-3-2

24983

28776

03851

Avicularin

E-6-4

10700

36358

127703

Figure

6(EAph

aseiso

latio

ncompo

unds)

Feniculin

E-6-2

13884

27657

105891

(+)-Ca

techin

E-7-6-2

15928

73008

118252

(minus)-Ep

icatechinE-7-6-4

24217

41437

30740

61015840-O

-Galloylsalid

rosid

eE-9-3-2

25062

5119

641546

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 11

300 350 400 450 5000

20

40

60

80

100

120

4351

0 10 20 30 40

0

20

40

60

80

100

120In

tens

ity (m

AU)

Inte

nsity

(mAU

)

Time (min)

E-6-2

mz350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 40

0

20

40

60

80

100

120

140

160

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

4351

E-6-4

mz

200 250 300 350 400 450 5000

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-2

mz200 250 300 350 400 450 500

0

20

40

60

80

100

120

0 10 20 30 400

20

40

60

80

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

2910E-7-6-4

mz

340 360 380 400 420 440 4600

2

4

6

8

10

12

14

16

4531

0 10 20 30 400

50

100

150

200

250

Inte

nsity

(mAU

)

Inte

nsity

(mAU

)

Time (min)

E-9-3-2

mz

Figure 7 HPLC profile and LC-MS spectra of isolation five kind compounds from Acer tegmentosum

the MTT assay to determine the optimal concentration effec-tive for anti-inflammation with minimum toxicity As shownin Figure 8(a) all the five compounds did not affect cellviability up to 100 120583M indicating that the compounds werenot toxic to cells

342 Effect of Five Compounds on NO Production in LPS-Stimulated RAW2647Macrophages Weevaluated the effectsof five compounds on NO secretion in LPS-stimulated RAW2647 cells The cells were pretreated with five compoundsat various concentrations prior to the LPS stimulation and

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

12 Journal of Analytical Methods in Chemistry

140

120

100

80

60

40

20

010 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

Cel

l via

bilit

y (

)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

LPS (200ngmL)

(a)

Nitr

ic o

xide

(120583M

)

45

40

35

30

25

20

15

10

5

0

lowastlowast

lowast

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(b)

Figure 8 Effect of five compounds on (a) cell viability and LPS-induced (b) NO production in RAW 2647 cells RAW 2647 cells werepretreated with five compounds for 30min before incubation with LPS for 24 h (a) Cytotoxicity was evaluated by an MTT assay (b) Theculture supernatant was analyzed for nitrite production As a control the cells were incubated with vehicle alone Data shows mean plusmn SEvalues of triplicate determination from independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated from comparing with LPS-stimulation value

NO production was measured As a positive control weemployed 10 120583M dexamethasone which is widely employedas an anti-inflammatory agent All the compounds except 61015840-O-galloyl salidroside did not show any inhibitory effect onLPS-induced NO production (Figure 8(b))

343 Effect of Five Compounds on LPS-Induced Inflamma-tory Cytokines Production The inhibitory effect of the fivecompounds on the production of inflammatory cytokinesanother parameter of inflammation was investigated Inthis study we examined the effect of the five compoundson TNF-120572 IL-6 and IL-1120573 expressions Figure 9(a) showsthat feniculin slightly repressed TNF-120572 production at aconcentration of 10 120583M (Figure 9(a)) However avicularin(+)-catechin (minus)-epicatechin and 61015840-O-galloyl salidroside

did not show any inhibitory effect on LPS-induced TNF-120572 production As shown in Figure 9(b) consistent withTNF-120572 results feniculin slightly repressed IL-6 productionat a concentration of 10 120583M Moreover avicularin inhibitedIL-6 production at concentrations of 30 and 50120583M (+)-Catechin inhibited IL-6 production at 30120583M or more (minus)-Epicatechin and 61015840-O-galloyl salidroside did not show anyinhibitory effect on LPS-induced IL-6 secretion Howeverall the compounds except 61015840-O-galloyl salidroside did notinhibit IL-1120573 (Figure 9(c))

4 Conclusions

This study showed that among the soluble fractions from thehot water extract of A tegmentosum the EA-soluble fraction

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 13

TNF-120572

(pg

mL)

60

50

40

30

20

10

0

times103

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast

(minus)-Epicatechin

(a)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

lowastlowast lowast

25

20

15

10

5

0

times103

IL-6

(pg

mL)

(minus)-Epicatechin

(b)

12

10

8

6

4

2

0

times102

lowastlowast

lowastlowast

lowastlowastlowast

IL-1120573

(pg

mL)

LPS

LPS (200ngmL)

10 10 10 101030 30 30 303050 50 50 5050100 100 100 100100

Con Dex Feniculin(guaijaverin)

Avicularin (+)-Catechin

(120583M)

6998400-O-Galloyl salidroside

(minus)-Epicatechin

(c)

Figure 9 Effect of five compounds on the production of (a) TNF-120572 (b) IL-6 and (c) IL-1120573 cytokines in macrophages Cells were pretreatedwith five compounds for 30min before being incubated with LPS for 24 h Production of cytokines was measured by ELISA Data showsmean plusmn SE values of duplicate determinations from three independent experiments lowast119875 lt 001 and lowastlowast119875 lt 0001 were calculated fromcomparing with LPS-stimulation value

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

14 Journal of Analytical Methods in Chemistry

possessed the highest bioactivity and free radical-scavengingactivities Compounds in the dried twigs of A tegmentosumwere extracted with hot water and partitioned successivelyusing DCM EA n-BuOH and water The content of usefulcompounds in A tegmentosum was remarkably higher inEA the extract (124 g) Five phenolic compounds wereisolated by the silica gel octadecyl silica gel and SephadexLH-20 column chromatography The chemical structures ofthe isolated compounds were determined by spectroscopicmethods such as 1H-NMR 13C-NMR and LCMS andwere confirmed as feniculin (1) avicularin (2) (+)-catechin(3) (minus)-epicatechin (4) and 61015840-O-galloyl salidroside (5)by comparison of spectral data with those of referencesThe EA extract of A tegmentosum containing five phenoliccompounds exhibiting the best bioactivity was further mon-itored by an on-line screening HPLC-ABTS+assay methodCompounds 1 and 2 were isolated for the first time and theiranti-inflammatory activities were evaluated Moreover theon-line screening HPLC-ABTS+ assay method was rapid andefficient to search for bioactive compound from A tegmento-sum Furthermore (+)-catechin and 61015840-O-galloyl salidrosideexhibited the inhibitory activities on inflammatory mediatorproduction such as TNF-120572 IL-6 and IL-1120573 cytokines Inconclusion A tegmentosum can be used as a basic materialfor the development of new drugs in OMHs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding to the publication of this paper

Acknowledgments

This study was achieved at KM-Based Herbal Drug Develop-ment Group KIOM The authors also acknowledge the sup-port from the ldquoStudy on Drug Efficacy Enhancement UsingBioconversion for Herbal Medicinesrdquo (K14050) Project

References

[1] B Hazra M Das Sarma and U Sanyal ldquoSeparation methodsof quinonoid constituents of plants used in oriental traditionalmedicinesrdquo Journal of Chromatography B vol 812 no 1-2 pp259ndash275 2004

[2] K J Lee B H Lee P M Jung C Liang and J Y MaldquoAnalysis of bioconversion components of fermentation Hwan-gryunhaedok-tangrdquo Yakhak Hoeji vol 57 pp 293ndash298 2013

[3] T Yu J Lee Y G Lee et al ldquoIn vitro and in vivo anti-inflammatory effects of ethanol extract from Acer tegmento-sumrdquo Journal of Ethnopharmacology vol 128 no 1 pp 139ndash1472010

[4] J M Hur E J Yang S H Choi and K S Song ldquoIsolation ofphenolic glucosides from the Stems of Acer tegmentosumMaxrdquoJournal of the Korean Society for Applied Biological Chemistryvol 49 pp 149ndash152 2006

[5] J-M Hur M Jun E-J Yang S-H Choi C P Jong and S SKyung ldquoIsolation of isoprenoidal compounds from the stemsof Acer tegmentosum maxrdquo Korean Journal of Pharmacognosyvol 38 no 1 pp 67ndash70 2007

[6] H W Seo K J Park G H Guahk et al ldquoAnti-nociceptiveactivity of Acer tegmentosumMeOH extractrdquo Korean Journal ofPharmacognosy vol 44 no 2 pp 188ndash192 2013

[7] Y-H Hwang H Park and J Y Ma ldquoIn vitro and in vivo safetyevaluation of Acer tegmentosumrdquo Journal of Ethnopharmacol-ogy vol 148 no 1 pp 99ndash105 2013

[8] K J Lee C Liang H J Yang and J Y Ma ldquoRapid identificationof homoorientin from Phyllostachys bambusoides leaves byHPLC On-line ABTS+ screening methodrdquo Yakhak Hoeji vol56 pp 217ndash221 2012

[9] K M Park M C Yang K H Lee K R Kim S U Choi and KR Lee ldquoCytotoxic phenolic constituents of Acer tegmentosumMaximrdquo Archives of Pharmacal Research vol 29 no 12 pp1086ndash1090 2006

[10] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQualitycontrol of traditional Chinese medicines A reviewrdquo ChineseJournal of Natural Medicines vol 11 no 6 pp 596ndash607 2013

[11] K J Lee Y K Shin and Y S Kim ldquoEnhanced effect extractionof antioxidant substance homoorientin from Pseudosasa japon-ica and Phyllostachys bambusoides leaves using ultrasonic wavesystemrdquo Korean Society for Biotechnology and BioengineeringJournal vol 24 pp 189ndash194 2009

[12] S-Y Shi Y-P Zhang X-Y Jiang X-Q Chen K-L Huangand H-H Zhou ldquoCoupling HPLC to on-line post-column(bio)chemical assays for high-resolution screening of bioactivecompounds from complex mixturesrdquo Trends in AnalyticalChemistry vol 28 no 7 pp 865ndash877 2009

[13] W He X Liu H Xu Y Gong F Yuan and Y Gao ldquoOn-lineHPLC-ABTS screening andHPLC-DAD-MSMS identificationof free radical scavengers in Gardenia (Gardenia jasminoidesEllis) fruit extractsrdquo Food Chemistry vol 123 no 2 pp 521ndash5282010

[14] Y ZhangQ LiHXing et al ldquoEvaluation of antioxidant activityof ten compounds in different tea samples by means of an on-line HPLC-DPPH assayrdquo Food Research International vol 53no 2 pp 847ndash856 2013

[15] S A Lee and G Moon ldquoEffects of mixture with Hovenia dulcisThunb and Acer tegmentosum Maxim on liver failure inducedby D-galactosamine in ratsrdquo Journal of Korean Oriental InternalMedicine vol 31 pp 11ndash24 2010

[16] N H Tung Y Ding S K Kim K H Bae and Y H KimldquoTotal peroxyl radical-scavenging capacity of the chemicalcomponents from the stems of Acer tegmentosum MaximrdquoJournal of Agricultural and Food Chemistry vol 56 no 22 pp10510ndash10514 2008

[17] I C Shin J H Sa T H Shim and J H Lee ldquoThe physical andchemical properties and cytotoxic effects of Acer tegmentosumMaxim extractsrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 49 pp 322ndash327 2006

[18] Y-M Yoo J-H Nam M-Y Kim J Choi K-T Lee and H-JPark ldquoAnalgesic and anti-gastropathic effects of salidroside iso-lated from Acer tegmentosum heartwoodrdquo The Open BioactiveCompounds Journal vol 2 pp 1ndash7 2009

[19] H-J Choi O-H Kang P-S Park et al ldquoMume Fruc-tus water extract inhibits pro-inflammatory mediators inlipopolysaccharide-stimulated macrophagesrdquo Journal of Medic-inal Food vol 10 no 3 pp 460ndash466 2007

[20] D Fraisse A Heitz A Carnat A-P Carnat and J-L LamaisonldquoQuercetin 3-arabinopyranoside a major flavonoid compoundfrom Alchemilla xanthochlorardquo Fitoterapia vol 71 no 4 pp463ndash464 2000

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 15

[21] H J Kim E-R Woo and H Park ldquoA novel lignan andflavonoids from Polygonum avicularerdquo Journal of Natural Prod-ucts vol 57 no 5 pp 581ndash586 1994

[22] M Watanabe ldquoCatechins as antioxidants from buckwheat(Fagopyrum esculentum Moench) groatsrdquo Journal of Agricul-tural and Food Chemistry vol 46 no 3 pp 839ndash845 1998

[23] K P Latte M Kaloga A Schafer and H Kolodziej ldquoAnellagitannin n-butyl gallate two aryltetralin lignans and anunprecedented diterpene ester from Pelargonium reniformerdquoPhytochemistry vol 69 no 3 pp 820ndash826 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of