Research Article Arctigenin from Fructus Arctii...

Research ArticleArctigenin from Fructus Arctii (Seed of Burdock) ReinforcesIntestinal Barrier Function in Caco-2 Cell Monolayers

Hee Soon Shin12 Sun Young Jung1 Su Yeon Back1

Jeong-Ryong Do1 and Dong-Hwa Shon12

1Korea Food Research Institute 1201-62 Anyangpangyo-ro Bundang-gu Seongnam-si Kyeonggi-do 463-746 Republic of Korea2Division of Food Biotechnology Korea University of Science and Technology Daejeon 305-350 Republic of Korea

Correspondence should be addressed to Dong-Hwa Shon dhs95kfrirekr

Received 28 July 2015 Accepted 28 September 2015

Academic Editor Subrata Chakrabarti

Copyright copy 2015 Hee Soon Shin 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

Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries This study aimed toinvestigate effect of F Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal theactive component of F Arctii We measured transepithelial electrical resistance (TEER) value (as an index of barrier function) andovalbumin (OVA) permeation (as an index of permeability) to observe the changes of intestinal barrier function The treatment ofF Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers Furthermore we found that arctigenin as anactive component of F Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but notarctiin In the present study we revealed that F Arctii could enhance intestinal barrier function and its active component was anarctigenin on the functionalityWe expect that the arctigenin from F Arctii could contribute to prevention of inflammatory allergicand infectious diseases by reinforcing intestinal barrier function

1 Introduction

Arctium lappa (burdock) is a biennial plant of the Arctiumgenus in the Asteraceae family Its root is widely used as adietary ingredient and its seed (called Fructus Arctii) is usedas a traditional herbal medicine in oriental countries such asChina Japan and Korea In particular F Arctii has been usedfor the treatment of sore throat urticaria and furuncle Theanti-inflammatory and antioxidant properties of F Arctii aresupported by its ethnopharmacological use for the treatmentof inflammation wherein it inhibits proinflammatory factorssuch as nuclear factor kappa B (NF-120581B) inducible nitric oxidesynthase and oxidative stress [1 2]

Intestinal epithelial cells regulate the influx of nutrientsand the efflux of xenobiotics against substances enteringthe gut intestinal tract [3] The tight junction (TJ) proteinsexpressed in the cells play a key role in intestinal barrierfunction by protecting against toxic substances allergens andmacromolecules derived from food [4] However dysfunc-tion or destruction of TJ may cause inflammatory diseases

such as inflammatory bowel disease irritable bowel syn-drome leaky gut syndrome and food allergy [5ndash8] Recentlyvarious components derived from food and plants have beenknown to enhance intestinal barrier function For exampletheaflavins in black tea could enhance intestinal barrierfunction by increasing the expression of TJ-related proteins(occludin claudin-1 and zonula occludens-1) through theactivation of AMP-activated protein kinase in Caco-2 cells[9]

The Caco-2 cells which are derived from colon carci-noma spontaneously undergo the process of differentiationthat leads to the formation of monolayers The cell mono-layers have been used as an in vitro model that mimicsthe human intestinal epithelium [10 11] In this study weestablished an in vitro system to evaluate the effect of food-and plant-derived extracts including F Arctii on intestinalbarrier function in Caco-2 cell monolayers In particular theeffect of F Arctii was focused on intestinal barrier functionand the permeability was measured using ovalbumin (OVA)as one of substrates absorbed through paracellular diffusion

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 368105 7 pageshttpdxdoiorg1011552015368105

2 Evidence-Based Complementary and Alternative Medicine

Furthermore we investigated what component was an activecompound in the extract by fractionation HPLC analysisand ability to enhance the intestinal barrier function inhuman intestinal epithelial Caco-2 cells

2 Materials and Methods

21 Materials The Caco-2 cell line was obtained from theAmerican Type Culture Collection (Rockville MD USA)Dulbeccorsquos modified Eaglersquos medium (DMEM) fetal bovineserum (FBS) penicillin-streptomycin nonessential aminoacids (NEAA) phosphate-buffered saline (PBS) and Hankrsquosbalanced salt solution (HBSS) were purchased from Wel-GENE (DaeguKorea)OVAandbile salts (mixture of sodiumcholate and sodium deoxycholate) were purchased fromSigma (St Louis MO USA) Arctiin and arctigenin werepurchased from Santa Cruz Biotechnology (Santa Cruz CAUSA)

22 Sample Preparation The 9 samples that were used in thisstudy were purchased from Plant Extract Bank at the KoreaResearch Institute of Bioscience amp Biotechnology (DaejeonKorea) and Kyung-dong Oriental Pharmacy (Seoul Korea)and identified by Professor Y Bu Department of HerbalPharmacology Kyung Hee University The specimen (KFRI-SL-2170) has been kept in Korea Food Research InstituteFructus Arctii (400 g) was pulverized to powder and then theextract from the powder was obtained by reflux extractionin 95 ethanol (4 L) for 3 h twice The ethanol extract wasconcentrated under vacuum in a rotary evaporator and thenfreeze-dried for 3 days (84 g yield 211)

23 Cell Culture The Caco-2 cells were cultured at 37∘C inhumidified air containing 5CO

2The cells weremaintained

in a 100mm dish with DMEM containing 1000mgL of glu-cose and supplemented with 10 FBS 1 NEAA 100UmLof penicillin and 100 120583gmL of streptomycin The cells wereseeded at a density of 2 times 105 cellsmL on a 12-transwell plate(Costar Corning NY USA) and allowed to grow for 3 weeksfor the experiments

24 Measurement of Transepithelial Electrical Resistance Theintegrity of the Caco-2 cell monolayers was checked bymeasuring the transepithelial electrical resistance (TEER)by using a Millicell-ERS device (Millipore Bedford MAUSA) The monolayers of Caco-2 cells (300ndash500Ω cmminus2)were preincubated with HBSS for 30min at 37∘C in a CO

2

incubator to stabilize the cell monolayers The cell monolay-ers were treated with each sample and bile salts for 60minand OVA was added for 3 h at 37∘C The presence of bilesalts weakens intestinal barrier function [12] However thebile salts actually exist in gastrointestinal tract in vivo andthe addition of bile salts in experimental in vitro system canmimic in vivo system The TEER value was measured andOVA influx was also detected by ELISA

25 ELISA Coating anti-OVA primary antibodies (ab17290)and horseradish peroxidase- (HRP-) conjugated anti-OVA

Table 1 Yield ( ww) and total phenol content (mgg) of fractionsisolated from Fructus Arctii extract

FAE Yield Total phenols content(Fructus Arctii) ( ww) (mgg)Hexane fraction 3998 1625 plusmn 09Chloroform fraction 41 23744 plusmn 246Ethyl acetate fraction 789 41278 plusmn 1047Butanol fraction 566 27479 plusmn 132Water fraction 547 8462 plusmn 016

secondary antibodies (ab20415) for ELISA were purchasedfrom Abcam (Cambridge MA USA) 96-well immunoplateswere coated by overnight incubation at 4∘C with coatinganti-OVA primary antibodies After blocking for 2 h with1 bovine serum albumin the supernatant was incubatedat RT for 2 h After washing by PBS with 005 Tween20 HRP-conjugated anti-OVA secondary antibodies wereadded for 1 h on plates and then each well was reacted by331015840551015840-tetramethylbenzidine substrate solution in the darkFinally stop solution (2N sulfuric acid) was added andthen absorbance was measured at 450 nm using an Epochmicroplate reader (BioTek Winooski VT USA)

26 Solvent Fractionation of Fructus Arctii Extract The resul-tant ethanol extract of Fructus Arctiiwas further fractionatedwith different solvents Dried Fructus Arctii extract (FAE)was suspended in distilled H

2O (FAE H

2O 1 9) And then

the solution was suspended with hexane solvent (FAE inH2O Hexane 1 1) Each solvent (chloroform ethyl acetate

butanol and water) was sequentially added in FAE solutionand then each fraction was sequentially partitioned with hex-ane chloroform ethyl acetate butanol and water (Figure 1)All solvent fractions were evaporated until dry The detailedmethod and yields of solvent fractions are shown in Table 1

27 Determination of Total Phenolic Content Measurementof total phenolic content (TPC) was based on the methoddescribed by Shin et al [13] TPC was calculated using tannicacid (Sigma-Aldrich) as a standard

28 HPLC Analysis High-performance liquid chromatogra-phy (HPLC) analysis was carried out with a Jasco PU-2080plus liquid chromatography system (JASCO Tokyo Japan)equipped with multiwavelength detector Jasco UV-2075 plus(JASCO Tokyo Japan) Samples were injected to 1120583gmL byautoinjector AS-2057 plus (JASCO Tokyo Japan) and ODSC18 column (250 times 46mm) was used to separate compoundsat 40∘C in column adapter CO-2060 plus (JASCO TokyoJapan) Solvents were used mixture of acetonitrile (A) andwater (B) in gradient mode (eluent A 20 to 40 in 35min)and the flow rate was 1mLmin UVwavelength for detectionwas 280 nm

29 Statistical Analysis Results are expressed as the mean plusmnstandard deviation (SD) A statistical analysis was performedusing the SAS statistical software package (SAS Institute

Evidence-Based Complementary and Alternative Medicine 3

95 ethanol extracts (FAE)

Extracted with 95 ethanol evaporate in vacuum

Ethanol extracts

Hexane layer Water layer

Evaporation

Hexane fractionChloroform layer

Evaporation

Chloroform fraction

Aqueous layer

Ethyl acetate layer Water layer

Extraction with ethyl acetate

Evaporation

Ethyl acetate fraction

Extraction with butanol

Water layer

Evaporation

Water fraction

Butanol layer

Evaporation

Butanol fraction

Extraction with chloroform

Hexane 95 ethanol extracts H2O (10 1 9)

Figure 1 Fraction separation from ethanolic extract of Fructus Arctii

Cary NC USA) Differences between the experimental datawere assessed by 1-way analysis of variance (ANOVA) fol-lowed by Duncanrsquos multiple-range test a value of 119875 lt 005was considered significant

3 Results

31 Effects of Natural Material Extracts on Intestinal BarrierFunction in Monolayers of Caco-2 Cells We investigated theeffects of 95 ethanolic extracts from nine natural materialsderived from plants (Cynomorii HerbaMaydis Stigma Poly-goniMultiflori RadixAcori Graminei Rhizoma F ArctiiMoriRadicis Cortex Tritici Immatri SemenGleditsiae Fructus andIsatidis Radix) on intestinal barrier function by measuringTEER in Caco-2 cell monolayers Among the nine naturalmaterials the FAE significantly increased the TEER value by289 compared to the control in Caco-2 cell monolayers(Figure 2(a)) In contrast the other eight natural materialextracts had no effect on the TEER value Furthermorethe FAE dramatically enhanced the TEER value in a dose-dependentmanner (Figure 2(b)) Next we examinedwhetherthe FAE-induced enhancement of intestinal barrier functioncould suppress allergen permeation via paracellular diffusionpathway The results showed that FAE treatment reducedOVA permeation across Caco-2 cell monolayers by enhanc-ing the intestinal barrier function (Figures 2(c) and 2(d))

These results indicated that F Arctii could enhance intestinalbarrier function preventing allergens and toxic and xenobi-otic substrates from entering the intestine

32 Effects of FAE Fractions on TEER and OVA Flux in Caco-2 Cell Monolayers To investigate the active components ofFAE FAE was fractionated using different polar solventssuch as hexane chloroform ethyl acetate butanol and waterAmong the five fractions extracted fromFAE chloroformandethyl acetate fractions significantly increased TEER value inCaco-2 monolayers and also inhibited the OVA permeationacross Caco-2 cell monolayers (Figure 3) Although TEERvalues were significantly increased by hexane and butanolfractions the increases were similar to the level of TEER valuein initial control (normal condition) Therefore we thoughtthat the hexane and butanol fractions recovered TEER valuesreduced by bile salts in Caco-2 cell monolayers These resultsdemonstrated that the FAE-induced enhancement of intesti-nal barrier function was owing to compounds in chloroformand ethyl acetate fractions Thus we investigated TPC of thefractions to determine the properties of these componentsTable 1 shows the TPC of fractions the ethyl acetate fractioncontained the highest amount of phenolic compounds (41278plusmn 1047) Chloroform and butanol fractions also containedlarge quantities of phenolic compounds however the hexaneand water fractions had relatively low content of phenolic


0 50 100 150 200 250 300 350TEER ()

Control

Isatidis RadixGleditsiae

FructusTritici Immatri

SemenMori Radicis

CortexFructus Arctii

RhizomeAcori Graminei

PolygoniMultiflori RadixMaydis Stigma

CynomoriiHerba

lowastlowast

lowast

(a)

0 500 1000

Control

FAE 100

FAE 200

FAE 400

TEER ()

lowastlowast

lowastlowast

lowastlowast

(b)

0

100

200

300

400

500

OVA FAE

TEER

()

lowastlowast

(c)

0

01

02

03

04

05

OVA FAE

OVA

(120583g

mL)

lowastlowast

(d)

Figure 2 Fructus Arctii extract increased TEER value and decreased OVA influx in Caco-2 cells The monolayers of Caco-2 cells werepreincubated with HBSS for 30min at 37∘C in a CO

2

incubator to stabilize the cell monolayers The cell monolayers were treated with eachsample and bile salts for 60min and OVA was added for 3 h at 37∘C (a) Changes of TEER were measured by treating the cell monolayerswith nine extracts (400120583gmL) and (b) Fructus Arctii extract (FAE) at various doses (100 200 and 400 120583gmL) for 3 h (c and d) Changes inTEER value and OVA influx induced by FAE were detected by ELISA Each value is presented as mean plusmn SD (119899 = 3) Bars are significantlydifferent from the control at lowast119875 lt 005 and lowastlowast119875 lt 001 Data were analyzed using ANOVA followed by Duncanrsquos multiple-range test

compounds Therefore we suggest that the active compo-nents of chloroform and ethyl acetate fractions might benonpolar polyphenols

33 Effects of Single Component (Arctiin and Arctigenin) onTEER and OVA Flux in Caco-2 Cell Monolayers Next weanalyzed the components in chloroform and ethyl acetatefractions using HPLCThe results showed four peaks (P1 P2P3 and P4) which included chloroform and ethyl acetatefractions (Figures 4(a)-4(b)) The two main peaks P2 andP4 were associated with arctiin and arctigenin respectivelyWe then investigated the effects of arctiin and arctigeninon intestinal barrier function and OVA permeation acrossCaco-2 cell monolayers Arctigenin not arctiin enhancedthe TEER value and inhibited OVA permeation (Figures 4(c)and 4(d)) Moreover the TEER increased and the OVA flux

decreased in a dose-dependent manner in cells incubatedwith increasing concentrations (1ndash200120583molL) of arctigenin(Figures 4(e) and 4(f)) Thus we suggest that arctigenin isan active component of F Arctii and enhances the intestinalbarrier function and suppresses OVA permeation acrossCaco-2 cell monolayers

4 Discussion

Arctigenin and arctiin are lignans found in many plantsof the Asteraceae family and their physiological functionshave been identified by researchers Typical functions ofarctiin include protective [14] anti-inflammatory [15] andantidiabetic [16] effects whereas those of arctigenin wereanti-inflammatory [17] anti-cancer [18] and neuroprotective[19] effects In the present study we showed that arctigenin


0

50

100

150

200

250

Hexane fraction Chloroformfraction

Ethyl acetatefraction

Butanol fraction Water fraction

TEER

()

Control FAE

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(a)

0

05

1

15

2

25

Ethyl acetate fractionControl

OVA

(120583g

mL)

lowastlowast

(b)

0

05

1

15

2

Chloroform fractionControl

ND

OVA

(120583g

mL)

(c)

Figure 3 Ethyl acetate and chloroform fractions isolated from Fructus Arctii extract reinforced TEER and reduced OVA influx in Caco-2cells FAE was fractionated to five fractions hexane chloroform ethyl acetate butanol and water fractions Effects of fractions (200120583gmL)on TEER values were measured in the cell monolayers and amount of permeated OVA was also detected in the basolateral side Each valueis presented as mean plusmn SD (119899 = 3) Bars are significantly different from the control at lowastlowast119875 lt 001 Data were analyzed using ANOVA followedby Duncanrsquos multiple-range test

from F Arctii enhanced the intestinal barrier function inhuman intestinal epithelial Caco-2 cells but arctiin has nosuch effect Both arctigenin and arctiin were well-knownactive components of F Arctii Furthermore arctiin is theglycoside of arctigenin Nevertheless they have differentphysiological and pharmacological functions and mecha-nisms For example arctigenin effectively inhibited intestinalinflammation (body weight loss proinflammatory cytokinesand crypt destruction) by suppressing the activation ofmitogen-activated protein kinases and NF-120581B in dextransulfate sodium-induced colitis model [20] However arctiindid not significantly ameliorate dextran sulfate sodium-induced symptoms such as body weight loss inflammatoryindex colon length andmyeloperoxidase activityOur resultsalso showed different functions of arctigenin and arctiin onintestinal barrier function Therefore although arctigenin isan aglycon of arctiin the physiological effects of arctigenindiffer with arctiin

Recently the major components of F Arctii have beenidentified such as chlorogenic acid arctiin and arctigenin[21] Besides these components it containsmany constituentssuch as dicaffeoylquinic acid lappaol H lappaol C arctignanEmatairesinol lappaolAmatairesinoside lappaol B diarcti-genin and methyl arctate-b

Our result showed that arctigenin from F Arctii enhancedintestinal barrier function However we suggest that othercomponents also may have the potential on enhancementof intestinal barrier function For example chlorogenic aciddecreased intestinal permeability through increasing intesti-nal expression of tight junction proteins such as occludinand zonula occludens-1 in weaned rats challenged withlipopolysaccharide [22] Therefore we will investigate theeffects of other components fromFArctii on intestinal barrierfunction in further study

5 Conclusions

In summary F Arctii enhanced intestinal barrier function inhuman intestinal epithelial Caco-2 cells Active componentof the extract was arctigenin We expect that the reinforcingeffect of arctigenin from F Arctii could contribute to preven-tion of inflammatory allergic and infectious diseases

Abbreviations

DMEM Dulbeccorsquos modified Eaglersquos mediumFAE Fructus Arctii extractFBS Fetal bovine serum


00 50 100 150 200 250 300 350

0

5000

10000


P1 P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(a)00 50 100 150 200 250 300 350

0

5000

10000

Chloroform fraction

P1P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(b)

TEER

()

400

300

200

100

0

Control 50 100

Arctiin

50 100

Arctigenin(120583molL)

lowast

lowastlowast lowastlowast

(c)

ND

Control 50 100

Arctiin

50 100


lowastlowastOVA

(120583g

mL)

25

2

15

1

05

0

(d)

Arctigenin

Control(120583molL)

lowastlowastlowastlowastlowastlowast

lowastlowast


TEER

()

300

250

200

150

100

50

0

1 5 10 50 100 200

(e)

Arctigenin

lowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowastlowastlowast

OVA

(120583g

mL)

500

400

300

200

100

000

Control(120583molL)

1 5 10 50 100 200

(f)

Figure 4 Arctigenin arctiin from Fructus Arctii extract enhanced intestinal barrier function in Caco-2 cells (a and b) To identify activecomponents in Fructus Arctii extract HPLC analysis was carried out with a Jasco PU-2080 plus liquid chromatography system equipped withmultiwavelength detector Ethyl acetate and chloroform fractions were injected (1120583gmL each) by autoinjector and an ODS C18 column (250times 46mm) was used to separate compounds at 40∘C in column adapter Solvents used were a mixture of acetonitrile (eluent A) and water(eluent B) in gradient mode (eluent A 20 to 40 in 35min) and the flow rate was 1mLmin UV wavelength for detection was 280 nm (cndashf)The effects of arctiin and arctigenin on TEER and OVA permeability were examined in Caco-2 cells Each value is presented as mean plusmn SD(119899 = 3) Bars are significantly different from the control at lowast119875 lt 005 and lowastlowast119875 lt 001 Data were analyzed using ANOVA followed by Duncanrsquosmultiple-range test

HBSS Hankrsquos balanced salt solutionHPLC High-performance liquid

chromatographyHRP Horseradish peroxidaseNEAA Penicillin-streptomycin nonessential

amino acidsNF-120581B Nuclear factor kappa BOVA OvalbuminPBS Phosphate-buffered salineTEER Transepithelial electrical resistanceTJ Tight junctionTPC Total phenolic content

Conflict of InterestsThe authors have no conflict of interests to declare

AcknowledgmentsThis study was supported by research Grants (E0143033648and E0143033649) from the Korea Food Research Institute

References

[1] M Hwangbo J Y Jung S H Ki et al ldquoU-Bang-Haequi Tanga herbal prescription that prevents acute inflammation through


inhibition of NF-120581B-mediated inducible nitric oxide synthaserdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 542825 12 pages 2014

[2] J Liu Y-Z Cai R N S Wong et al ldquoComparative analysis ofcaffeoylquinic acids and lignans in roots and seeds among var-ious burdock (Arctium lappa) genotypes with high antioxidantactivityrdquo Journal of Agricultural and Food Chemistry vol 60 no16 pp 4067ndash4075 2012

[3] M Shimizu M Tsunogai and S Arai ldquoTransepithelial trans-port of oligopeptides in the human intestinal cell Caco-2rdquoPeptides vol 18 no 5 pp 681ndash687 1997

[4] T Suzuki ldquoRegulation of intestinal epithelial permeability bytight junctionsrdquoCellular andMolecular Life Sciences vol 70 no4 pp 631ndash659 2013

[5] S H Lee ldquoIntestinal permeability regulation by tight junc-tion implication on inflammatory bowel diseasesrdquo IntestinalResearch vol 13 no 1 pp 11ndash18 2015

[6] T Piche ldquoTight junctions and IBSmdashhe link between epithelialpermeability low-grade inflammation and symptom genera-tionrdquo Neurogastroenterology and Motility vol 26 no 3 pp296ndash302 2014

[7] Z Liu N Li and J Neu ldquoTight junctions leaky intestinesand pediatric diseasesrdquo Acta Paediatrica International Journalof Paediatrics vol 94 no 4 pp 386ndash393 2005

[8] T Chen X Liu L Ma et al ldquoFood allergens affect the intestinaltight junction permeability in inducing intestinal food allergyin ratsrdquo Asian Pacific Journal of Allergy and Immunology vol32 no 4 pp 345ndash353 2014

[9] H Park Y Kunitake N Hirasaki M Tanaka and T Mat-sui ldquoTheaflavins enhance intestinal barrier of Caco-2 Cellmonolayers through the expression of AMP-activated proteinkinase-mediated Occludin Claudin-1 and ZO-1rdquo BioscienceBiotechnology and Biochemistry vol 79 no 1 pp 130ndash137 2015

[10] E Grasset M Pinto E Dussaulx A Zweibaum and J FDesjeux ldquoEpithelial properties of human colonic carcinomacell line Caco-2 electrical parametersrdquo American Journal ofPhysiologymdashCell Physiology vol 247 no 3 pp C260ndashC2671984

[11] I J Hidalgo T J Raub andR T Borchardt ldquoCharacterization ofthe human colon carcinoma cell line (Caco-2) as amodel systemfor intestinal epithelial permeabilityrdquo Gastroenterology vol 96no 3 pp 736ndash749 1989

[12] F Raimondi P SantoroMV Barone et al ldquoBile acidsmodulatetight junction structure and barrier function of Caco-2 mono-layers via EGFR activationrdquo American Journal of PhysiologyGastrointestinal and Liver Physiology vol 294 no 4 pp G906ndashG913 2008

[13] H S Shin M-J Bae S Y Jung and D-H Shon ldquoInhibitoryeffect of skullcap (Scutellaria baicalensis) extract on ovalbuminpermeation in vitro and in vivordquo Food Chemistry vol 140 no1-2 pp 22ndash30 2013

[14] H J Cha G T Lee K S Lee et al ldquoPhotoprotective effectof arctiin against ultraviolet B-induced damage in HaCaTkeratinocytes is mediated by microRNA expression changesrdquoMolecular Medicine Reports vol 10 no 3 pp 1363ndash1370 2014

[15] S Lee S Shin K Kim et al ldquoAnti-inflammatory function ofarctiin by inhibiting COX-2 expression via NF-120581B pathwaysrdquoJournal of Inflammation vol 8 article 16 2011

[16] L-C Lu W Zhou Z-H Li et al ldquoEffects of arctiin onstreptozotocin-induced diabetic retinopathy in sprague-dawleyratsrdquo Planta Medica vol 78 no 12 pp 1317ndash1323 2012

[17] W-Z Zhang Z-K Jiang B-X He and X-B Liu ldquoArctigeninprotects against lipopolysaccharide-induced pulmonary oxida-tive stress and inflammation in a mouse model via suppressionof MAPK HO-1 and iNOS signalingrdquo Inflammation vol 38no 4 pp 1406ndash1414 2015

[18] A Li J Wang MWu X Zhang andH Zhang ldquoThe inhibitionof activated hepatic stellate cells proliferation by arctigeninthrough G0G1 phase cell cycle arrest persistent p27(Kip1)induction by interfering with PI3KAktFOXO3a signalingpathwayrdquo European Journal of Pharmacology vol 747 pp 71ndash872015

[19] D Li Q Liu D Jia D Dou X Wang and T Kang ldquoProtectiveeffect of arctigenin against MPP+ and MPTP-induced neuro-toxicityrdquo Planta Medica vol 80 no 1 pp 48ndash55 2014

[20] X Wu Y Yang Y Dou et al ldquoArctigenin but not arctiin actsas the major effective constituent of Arctium lappa L fruit forattenuating colonic inflammatory response induced by dextransulfate sodium in micerdquo International Immunopharmacologyvol 23 no 2 pp 505ndash515 2014

[21] K Qin Q Liu H Cai et al ldquoChemical analysis of raw and pro-cessed Fructus arctii by high-performance liquid chromatog-raphydiode array detection-electrospray ionization-mass spec-trometryrdquo Pharmacognosy Magazine vol 10 no 40 pp 541ndash546 2014

[22] Z Ruan S Liu Y Zhou et al ldquoChlorogenic acid decreasesintestinal permeability and increases expression of intestinaltight junction proteins in weaned rats challenged with LPSrdquoPLoS ONE vol 9 no 6 Article ID e97815 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Furthermore we investigated what component was an activecompound in the extract by fractionation HPLC analysisand ability to enhance the intestinal barrier function inhuman intestinal epithelial Caco-2 cells

2 Materials and Methods

21 Materials The Caco-2 cell line was obtained from theAmerican Type Culture Collection (Rockville MD USA)Dulbeccorsquos modified Eaglersquos medium (DMEM) fetal bovineserum (FBS) penicillin-streptomycin nonessential aminoacids (NEAA) phosphate-buffered saline (PBS) and Hankrsquosbalanced salt solution (HBSS) were purchased from Wel-GENE (DaeguKorea)OVAandbile salts (mixture of sodiumcholate and sodium deoxycholate) were purchased fromSigma (St Louis MO USA) Arctiin and arctigenin werepurchased from Santa Cruz Biotechnology (Santa Cruz CAUSA)

22 Sample Preparation The 9 samples that were used in thisstudy were purchased from Plant Extract Bank at the KoreaResearch Institute of Bioscience amp Biotechnology (DaejeonKorea) and Kyung-dong Oriental Pharmacy (Seoul Korea)and identified by Professor Y Bu Department of HerbalPharmacology Kyung Hee University The specimen (KFRI-SL-2170) has been kept in Korea Food Research InstituteFructus Arctii (400 g) was pulverized to powder and then theextract from the powder was obtained by reflux extractionin 95 ethanol (4 L) for 3 h twice The ethanol extract wasconcentrated under vacuum in a rotary evaporator and thenfreeze-dried for 3 days (84 g yield 211)

23 Cell Culture The Caco-2 cells were cultured at 37∘C inhumidified air containing 5CO

2The cells weremaintained

in a 100mm dish with DMEM containing 1000mgL of glu-cose and supplemented with 10 FBS 1 NEAA 100UmLof penicillin and 100 120583gmL of streptomycin The cells wereseeded at a density of 2 times 105 cellsmL on a 12-transwell plate(Costar Corning NY USA) and allowed to grow for 3 weeksfor the experiments

24 Measurement of Transepithelial Electrical Resistance Theintegrity of the Caco-2 cell monolayers was checked bymeasuring the transepithelial electrical resistance (TEER)by using a Millicell-ERS device (Millipore Bedford MAUSA) The monolayers of Caco-2 cells (300ndash500Ω cmminus2)were preincubated with HBSS for 30min at 37∘C in a CO

2

incubator to stabilize the cell monolayers The cell monolay-ers were treated with each sample and bile salts for 60minand OVA was added for 3 h at 37∘C The presence of bilesalts weakens intestinal barrier function [12] However thebile salts actually exist in gastrointestinal tract in vivo andthe addition of bile salts in experimental in vitro system canmimic in vivo system The TEER value was measured andOVA influx was also detected by ELISA

25 ELISA Coating anti-OVA primary antibodies (ab17290)and horseradish peroxidase- (HRP-) conjugated anti-OVA

Table 1 Yield ( ww) and total phenol content (mgg) of fractionsisolated from Fructus Arctii extract

FAE Yield Total phenols content(Fructus Arctii) ( ww) (mgg)Hexane fraction 3998 1625 plusmn 09Chloroform fraction 41 23744 plusmn 246Ethyl acetate fraction 789 41278 plusmn 1047Butanol fraction 566 27479 plusmn 132Water fraction 547 8462 plusmn 016

secondary antibodies (ab20415) for ELISA were purchasedfrom Abcam (Cambridge MA USA) 96-well immunoplateswere coated by overnight incubation at 4∘C with coatinganti-OVA primary antibodies After blocking for 2 h with1 bovine serum albumin the supernatant was incubatedat RT for 2 h After washing by PBS with 005 Tween20 HRP-conjugated anti-OVA secondary antibodies wereadded for 1 h on plates and then each well was reacted by331015840551015840-tetramethylbenzidine substrate solution in the darkFinally stop solution (2N sulfuric acid) was added andthen absorbance was measured at 450 nm using an Epochmicroplate reader (BioTek Winooski VT USA)

26 Solvent Fractionation of Fructus Arctii Extract The resul-tant ethanol extract of Fructus Arctiiwas further fractionatedwith different solvents Dried Fructus Arctii extract (FAE)was suspended in distilled H

2O (FAE H

2O 1 9) And then

the solution was suspended with hexane solvent (FAE inH2O Hexane 1 1) Each solvent (chloroform ethyl acetate

butanol and water) was sequentially added in FAE solutionand then each fraction was sequentially partitioned with hex-ane chloroform ethyl acetate butanol and water (Figure 1)All solvent fractions were evaporated until dry The detailedmethod and yields of solvent fractions are shown in Table 1

27 Determination of Total Phenolic Content Measurementof total phenolic content (TPC) was based on the methoddescribed by Shin et al [13] TPC was calculated using tannicacid (Sigma-Aldrich) as a standard

28 HPLC Analysis High-performance liquid chromatogra-phy (HPLC) analysis was carried out with a Jasco PU-2080plus liquid chromatography system (JASCO Tokyo Japan)equipped with multiwavelength detector Jasco UV-2075 plus(JASCO Tokyo Japan) Samples were injected to 1120583gmL byautoinjector AS-2057 plus (JASCO Tokyo Japan) and ODSC18 column (250 times 46mm) was used to separate compoundsat 40∘C in column adapter CO-2060 plus (JASCO TokyoJapan) Solvents were used mixture of acetonitrile (A) andwater (B) in gradient mode (eluent A 20 to 40 in 35min)and the flow rate was 1mLmin UVwavelength for detectionwas 280 nm

29 Statistical Analysis Results are expressed as the mean plusmnstandard deviation (SD) A statistical analysis was performedusing the SAS statistical software package (SAS Institute




Ethanol extracts


Evaporation


Evaporation

Chloroform fraction

Aqueous layer



Evaporation



Water layer

Evaporation

Water fraction

Butanol layer

Evaporation

Butanol fraction





3 Results





0 50 100 150 200 250 300 350TEER ()

Control



SemenMori Radicis




CynomoriiHerba

lowastlowast

lowast

(a)

0 500 1000

Control

FAE 100

FAE 200

FAE 400

TEER ()

lowastlowast

lowastlowast

lowastlowast

(b)

0

100

200

300

400

500

OVA FAE

TEER

()

lowastlowast

(c)

0

01

02

03

04

05

OVA FAE

OVA

(120583g

mL)

lowastlowast

(d)


2





4 Discussion



0

50

100

150

200

250




TEER

()

Control FAE

lowastlowast

lowastlowast


lowastlowast

(a)

0

05

1

15

2

25


OVA

(120583g

mL)

lowastlowast

(b)

0

05

1

15

2


ND

OVA

(120583g

mL)

(c)





5 Conclusions


Abbreviations



00 50 100 150 200 250 300 350

0

5000

10000


P1 P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(a)00 50 100 150 200 250 300 350

0

5000

10000

Chloroform fraction

P1P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(b)

TEER

()

400

300

200

100

0

Control 50 100

Arctiin

50 100


lowast


(c)

ND

Control 50 100

Arctiin

50 100


lowastlowastOVA

(120583g

mL)

25

2

15

1

05

0

(d)

Arctigenin

Control(120583molL)


lowastlowast


TEER

()

300

250

200

150

100

50

0

1 5 10 50 100 200

(e)

Arctigenin

lowastlowast

lowastlowast

lowastlowast


OVA

(120583g

mL)

500

400

300

200

100

000

Control(120583molL)

1 5 10 50 100 200

(f)







References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Ethanol extracts


Evaporation


Evaporation

Chloroform fraction

Aqueous layer



Evaporation



Water layer

Evaporation

Water fraction

Butanol layer

Evaporation

Butanol fraction





3 Results





0 50 100 150 200 250 300 350TEER ()

Control



SemenMori Radicis




CynomoriiHerba

lowastlowast

lowast

(a)

0 500 1000

Control

FAE 100

FAE 200

FAE 400

TEER ()

lowastlowast

lowastlowast

lowastlowast

(b)

0

100

200

300

400

500

OVA FAE

TEER

()

lowastlowast

(c)

0

01

02

03

04

05

OVA FAE

OVA

(120583g

mL)

lowastlowast

(d)


2





4 Discussion



0

50

100

150

200

250




TEER

()

Control FAE

lowastlowast

lowastlowast


lowastlowast

(a)

0

05

1

15

2

25


OVA

(120583g

mL)

lowastlowast

(b)

0

05

1

15

2


ND

OVA

(120583g

mL)

(c)





5 Conclusions


Abbreviations



00 50 100 150 200 250 300 350

0

5000

10000


P1 P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(a)00 50 100 150 200 250 300 350

0

5000

10000

Chloroform fraction

P1P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(b)

TEER

()

400

300

200

100

0

Control 50 100

Arctiin

50 100


lowast


(c)

ND

Control 50 100

Arctiin

50 100


lowastlowastOVA

(120583g

mL)

25

2

15

1

05

0

(d)

Arctigenin

Control(120583molL)


lowastlowast


TEER

()

300

250

200

150

100

50

0

1 5 10 50 100 200

(e)

Arctigenin

lowastlowast

lowastlowast

lowastlowast


OVA

(120583g

mL)

500

400

300

200

100

000

Control(120583molL)

1 5 10 50 100 200

(f)







References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 50 100 150 200 250 300 350TEER ()

Control



SemenMori Radicis




CynomoriiHerba

lowastlowast

lowast

(a)

0 500 1000

Control

FAE 100

FAE 200

FAE 400

TEER ()

lowastlowast

lowastlowast

lowastlowast

(b)

0

100

200

300

400

500

OVA FAE

TEER

()

lowastlowast

(c)

0

01

02

03

04

05

OVA FAE

OVA

(120583g

mL)

lowastlowast

(d)


2





4 Discussion



0

50

100

150

200

250




TEER

()

Control FAE

lowastlowast

lowastlowast


lowastlowast

(a)

0

05

1

15

2

25


OVA

(120583g

mL)

lowastlowast

(b)

0

05

1

15

2


ND

OVA

(120583g

mL)

(c)





5 Conclusions


Abbreviations



00 50 100 150 200 250 300 350

0

5000

10000


P1 P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(a)00 50 100 150 200 250 300 350

0

5000

10000

Chloroform fraction

P1P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(b)

TEER

()

400

300

200

100

0

Control 50 100

Arctiin

50 100


lowast


(c)

ND

Control 50 100

Arctiin

50 100


lowastlowastOVA

(120583g

mL)

25

2

15

1

05

0

(d)

Arctigenin

Control(120583molL)


lowastlowast


TEER

()

300

250

200

150

100

50

0

1 5 10 50 100 200

(e)

Arctigenin

lowastlowast

lowastlowast

lowastlowast


OVA

(120583g

mL)

500

400

300

200

100

000

Control(120583molL)

1 5 10 50 100 200

(f)







References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250




TEER

()

Control FAE

lowastlowast

lowastlowast


lowastlowast

(a)

0

05

1

15

2

25


OVA

(120583g

mL)

lowastlowast

(b)

0

05

1

15

2


ND

OVA

(120583g

mL)

(c)





5 Conclusions


Abbreviations



00 50 100 150 200 250 300 350

0

5000

10000


P1 P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(a)00 50 100 150 200 250 300 350

0

5000

10000

Chloroform fraction

P1P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(b)

TEER

()

400

300

200

100

0

Control 50 100

Arctiin

50 100


lowast


(c)

ND

Control 50 100

Arctiin

50 100


lowastlowastOVA

(120583g

mL)

25

2

15

1

05

0

(d)

Arctigenin

Control(120583molL)


lowastlowast


TEER

()

300

250

200

150

100

50

0

1 5 10 50 100 200

(e)

Arctigenin

lowastlowast

lowastlowast

lowastlowast


OVA

(120583g

mL)

500

400

300

200

100

000

Control(120583molL)

1 5 10 50 100 200

(f)







References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00 50 100 150 200 250 300 350

0

5000

10000


P1 P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(a)00 50 100 150 200 250 300 350

0

5000

10000

Chloroform fraction

P1P2

Arctiin

ArctigeninInte

nsity

(120583V

)

(b)

TEER

()

400

300

200

100

0

Control 50 100

Arctiin

50 100


lowast


(c)

ND

Control 50 100

Arctiin

50 100


lowastlowastOVA

(120583g

mL)

25

2

15

1

05

0

(d)

Arctigenin

Control(120583molL)


lowastlowast


TEER

()

300

250

200

150

100

50

0

1 5 10 50 100 200

(e)

Arctigenin

lowastlowast

lowastlowast

lowastlowast


OVA

(120583g

mL)

500

400

300

200

100

000

Control(120583molL)

1 5 10 50 100 200

(f)







References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Arctigenin from Fructus Arctii...

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