Iridoid Glycosides Fraction Isolated from Veronica ciliata ...2 Evidence-BasedComplementaryandAlternativeMedicine and in the cold conditions of high-altitude Tibet. Whole V. ciliata

Research ArticleIridoid Glycosides Fraction Isolated from Veronica ciliata FischProtects against Acetaminophen-Induced Liver Injury in Mice

Shancai Tan12 Qiuxia Lu12 Yueyue Shu12 Yiran Sun12 Fang Chen12 and Lin Tang12

1Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education College of Life Sciences Sichuan UniversityChengdu Sichuan 610065 China2National and Local Joint Engineering Laboratory for Energy Plant Bio-Oil Production and Application ChengduSichuan 610065 China

Correspondence should be addressed to Fang Chen fangchenscu163com and Lin Tang tanglinscueducn

Received 27 September 2016 Revised 16 December 2016 Accepted 12 January 2017 Published 13 February 2017

Academic Editor Nunziatina De Tommasi

Copyright copy 2017 Shancai Tan 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

Veronica ciliata Fisch has traditionally been used in Tibetan medicine for the treatment of hepatitis cholecystitis rheumatismand urticaria We analyzed the chemical composition of the iridoid glycosides fraction (IGF) isolated from V ciliata and evaluatedthe antioxidant and hepatoprotective properties The IGF was separated by high-speed countercurrent chromatography (HSCCC)and the main compounds were identified by ultra-performance liquid chromatography coupled to a photodiode array Wedetermined the in vitro antioxidant ability of the IGF through radical scavenging assays and assessed the in vivo hepatoprotectivepotential in an acetaminophen- (APAP-) induced acute liver injury murine model The IGF was separated by HSCCC and threemajor iridoid glycosides (verproside catalposide and amphicoside) were identified as potent antioxidants and hepatoprotectivecompounds Treatment with the IGF significantly suppressed the APAP-induced elevation in serum alanine aminotransferaseaspartate aminotransferase and tumor necrosis factor-alpha (TNF-120572) improved serum total antioxidant capacity decreasedmalondialdehyde formation elevated superoxide dismutase and glutathione activity and decreased expression of proinflammatoryfactors (TNF-120572 nuclear factor kappa B) in the liver Finally we examined the histopathology of resected livers for evidence ofhepatoprotection The protection conferred by the IGF may be related to the reinforcement of antioxidant defense systems

1 Introduction

The liver is the organ in which metabolic detoxifying andexcretory processes occur It is the major site of xenobi-otic metabolism and numerous toxic chemicals endotoxinsdrugs and virusesmay cause injury for it [1] Acetaminophen(N-acetyl-p-aminophenol APAP) is frequently administeredto adults and infants for its analgesic and antipyretic efficacyAlthough therapeutic doses of APAP are effective and safeadministration of an overdose can lead to acetaminophen-induced acute liver injury (ALI) liver failure and even death[2] Accordingly many murine models of ALI are induced byAPAP

The underlying mechanism of APAP-induced hepatotox-icity is thought to occur via its conversion to the electrophilicspecies N-acetyl-p-benzoquinoneimine (NAPQI) by mem-bers of the cytochrome P450 family most notably CYP2E1

NAPQI then reacts immediately with glutathione (GSH anantioxidant thiol) to form the nontoxic metabolite cysteine[3] In circumstances in which the intracellular pool of GSHis exhausted NAPQI may accumulate in the liver whereit exerts oxidative stresses that may trigger mitochondrialsignaling pathways and lead to cellular injury Furthermoremitochondrial damage can inhibit respiration and decreasethe membrane potential which results in mitochondrialdysfunction exacerbate oxidant stress and increase inflam-matory responses [4] Previous reports have suggestedAPAP-induced hepatotoxicity is characterized by an increase inoxidative stress and a massive impairment of antioxidantdefense systems [5 6] Accordingly the development ofeffective liver-protective agents against APAP-induced ALIfrom natural products has attracted considerable attention

Veronica ciliata Fisch is an annual herb of the Scro-phulariaceae family that grows mainly in northern Sichuan

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 6106572 11 pageshttpsdoiorg10115520176106572

2 Evidence-Based Complementary and Alternative Medicine

and in the cold conditions of high-altitude Tibet WholeV ciliata has been used in traditional Tibetan medicine totreat hepatitis cholecystitis rheumatism and urticaria it isestimated that over 100 types of Tibetan medicine contain Vciliata [7]

The ethyl acetate fraction of the ethanol extract of V cili-atahas been reported to have strong antioxidant activities andtherapeutic efficacy for acute hepatotoxicity induced by car-bon tetrachloride (CCl

4) [8] Meanwhile other reports have

indicated that the iridoidmonomers separated fromV ciliataare potent antioxidants and can inhibit the proliferationof HepG2 hepatocellular carcinoma cells [9 10] Currentlyfifteen iridoid glycosides have been isolated from V ciliata[11] To date however no study has reported the antioxi-dant activity of the iridoid glycosides fraction isolated fromV ciliata using high-speed countercurrent chromatography(HSCCC) or the amelioration of APAP-induced ALI in mice

According to a previous report the hepatoprotectiveeffect of the compounds is related at least in part to theirantioxidant activity [8] Antioxidants have been shown toprevent oxidative stress-related liver pathologies directly byscavenging of reactive oxygen species (ROS) and indirectlyas part of the antioxidant defense system [12] Furthermoreiridoid glycosides are able to neutralize reactive oxida-tive species and are effective in the treatment of carbontetrachloride- (CCl

4-) induced acute hepatotoxicity [13 14]

Accordingly we hypothesized that the IGF isolated from Vciliata may be effective in the treatment of APAP-inducedALI in mice Accordingly the present study aimed to (1)isolate the IGF from V ciliata by HSCCC (2) characterizethe chemical composition of the IGF by ultra-performanceliquid chromatography (UPLC) analysis and (3) assess theantioxidant activity in vitro and the hepatoprotective effectsin vivo using an APAP-induced murine model of ALI

2 Materials and Methods

21 Assays and Chemicals SBC Middle Chromatogram Iso-lated (MCI)Gel (Type F 75ndash150120583m)was purchased fromSci-Bio Chem Co Ltd (Chengdu China)The diagnostic kits foraspartate aminotransferase (AST) alanine aminotransferase(ALT) total antioxidant capacity (T-AOC) malondialde-hyde (MDA) superoxide dismutase (SOD) GSH and totalprotein (TP) were provided by the Nanjing Jiancheng Bio-engineering Institute (Nanjing China) The tumor necrosisfactor (TNF-120572) kit was purchased from Shanghai YuanyeBioengineering Institute (Shanghai China) The GTVisionIII Detection SystemMoampRb kit was provided by GeneTech (Shanghai) Company Limited (Shanghai China) Bifen-date pills (BDP) were obtained from Wanbangde Pharma-ceutical Group Co Ltd (Zhejiang China) 22-Diphenyl-1-picrylhydrazyl (DPPH) 26-di-tert-butyl-4-methylphenol(BHT) 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) the reference standards (verproside catalposide andamphicoside) vitamin C (VC) and acetaminophen (APAP)were purchased from Sigma-Aldrich Chemical Co (St LouisMOUSA)HPLC-grademethanol and acetonitrile were usedfor UPLC analyses All other chemicals and reagents used inthis study were of analytical grade

22 Veronica ciliata Herbs of V ciliata were bought fromthe Tibetan Traditional Medicine Pharmaceutical Factory ofLhasa city (Tibet province of China) and the herbs werecollected in Linzhou of Lhasa (28th July 2015) A voucherspecimen (number 00721478) was identified by Dr Jie Bai ofthe School of Life Sciences at the Sichuan University Chinaand deposited in the herbarium of the Sichuan University

23 Preparation of IGF Using HSCCC

231 HSCCC Apparatus A preparative TBE-300C HSCCCsystem (Shanghai Tauto Biotech Co Ltd Shanghai China)was equipped with three sequential multilayer coil separationcolumns a 20mL sample loop and a TBP-5002 pump(Shanghai Tauto BiotechCo Ltd) ADC-0506 constant tem-perature circulating implement (Shanghai Sunny HengpingScientific Instrument Co Ltd Shanghai China) was used tomaintain the separation temperature An AFB750D0-10A30air compressor pump was used to generate the stationaryphase flow The UV absorbance of the eluent was monitoredwith a UV-2000 detector (Shanghai Sanotac Scientific Instru-ment Co Ltd Shanghai China) All data were collectedby an Easy Chrom v22123 workstation (Beijing QingbohuaLtd Beijing China)

232 Preparation of Crude Extract The whole plant of Vciliata (10 kg) was powdered and macerated three times in15 L reagent barrels containing 95 ethanol (1 6 wv) at 20ndash25∘C for 24 h The extracted solutions were evaporated todryness using a rotary evaporator under reduced pressureat 40ndash50∘C for 2 days The extract was then dissolved inultrapure water (03 L) and sequentially extracted six timeswith an equal volume of petroleum ether to remove the pig-ments and lipids Finally chlorophyll was removedusingMCIGEL column chromatography and eluted with MeOH-H

2O

(9 1 vv 65 L) which yielded the crude extract (1071 g) Analiquot of 400 g was subjected to HSCCC separation

233 HSCCC Separation The crude extract was separatedby using HSCCC according to a protocol previously reportedwith slight modifications [10 15] A two-phase solvent systemcomposed of ethyl acetatendashn-butanolndashwater (2 1 3 vvv)was chosen The solvents were shaken and equilibrated ina separation funnel at 25∘C The upper and lower phaseswere separated and degassed by ultrasonication for 30minbefore use The temperature of the separation column wasmaintained at 28∘C The column was first entirely filledwith the lower aqueous phase at a flow rate of 30mLminSubsequently the apparatus was rotated at 800 rpm in reverseand the upper mobile phase was pumped into the columnat a flow rate of 10mLmin The system reached equilibriumwhen the mobile phase eluted from the tail at which pointthe retention of stationary phase was calculated (667)The sample solution (20mL of the lower phase containing200mg of the extracts) was injected into the column throughthe sample loop The effluent with a flow rate of 5mLminwas monitored by using a UV detector at 260 nm and thefractions were collected manually according to the HSCCCchromatogram Each fraction was collected as a unit Four

Evidence-Based Complementary and Alternative Medicine 3

fractions were collected and fractions 2 3 and 4 werecombined to provide the iridoid glycosides fraction (IGF) forUPLC analysis

24 UPLC-Photodiode Array Analysis of IGF The chem-ical composition of the IGF was analyzed using UPLCCrude extracts were dissolved in methanol to produce afinal concentration of 05mgmLThe standards (verprosidecatalposide and amphicoside) were dissolved in methanolto produce a final concentration of 01mgmL respectivelyOur UPLC-photodiode array (PDA) used a Waters AcquitySystem (Waters Co Milford MA USA) that consisted ofa PDA binary autosampler online degasser and columnoven The UPLC system was fitted with an Acquity UPLCHSS T3 column (100 times 21mm 17 120583m Waters Co) Thecolumn and autosampler temperatures were maintained at40∘C and 25∘C respectivelyThemobile phases A and B wereultrapure water and acetonitrile respectively The followinggradient profile was used 10ndash25 B (0ndash08min) 25ndash35 B(08ndash24min) 35ndash95 B (24ndash28min) 95 B (28ndash4min)and 95ndash100 B (4ndash8min)The flow rate was 05mLmin andthe injection volume was 10 120583L Compounds were identifiedby a comparison of the retention times between samplesand standards The PDA detection was conducted at 260 nmLevels of the IGF were quantitated using Empower softwareversion 30 (Waters) [10 16]

25 Antioxidant Activity of IGF In Vitro

251 DPPH Radical Scavenging Assay The free radical scav-enging activity of the IGF was determined according to aconventional DPPH assay protocol with slight modificationsas previously described [17] Briefly 120120583L of the IGF wasdissolved in methanol at various concentrations (10 20 4080 160 or 200120583gmL) and incubated with 100120583L of DPPHsolution (02mM inmethanol) for 30min at 25∘C in the darkThe absorbance was measured at 517 nm with a MultiskanGO spectrophotometer (Thermo Fisher Scientific WalthamMassachusetts USA) and the radical scavenging activity wascalculated using

DPPH radical scavenging activity ()= [1 minus (119860 i minus 119860 s)119860 c ] times 100

(1)

where 119860 c represents the absorption of the negative control119860 i is the absorption of the experimental group and 119860 srepresents the absorption of the sample background VitaminC (Vc) was used as a reference standard

252 ABTS Radical Scavenging Assay The ability of the IGFto scavengeABTSwas evaluatedwithminormodification of apreviously reported protocol [18] ABTS cation radicals wereprepared by reacting 7mM ABTS with 245mM potassiumpersulfate (described as final concentrations both dissolvedin phosphate buffer [02M pH 74]) at 25∘C in the dark for12ndash16 h Next 100 120583L of 4 8 16 32 64 or 128 120583gmL IGF inmethanol was incubated with an equal volume of the ABTS

cation radical solution diluted in phosphate buffer at 25∘C for30min to obtain an absorbance of 07 plusmn 002 at 734 nm Thelevel of the ABTS radical scavenging was calculated using (1)Vc was used as a reference

253 Nitrite-Scavenging Assay The ability of the IGF toscavenge nitrites was evaluated with minor modificationsto a previously reported protocol [19] Briefly 1mL of IGFsolution of 10 20 40 80 160 or 320 120583gmL was mixed with03mL of NaNO

2(5 120583gmL) after which the pHwas adjusted

to 20 by dropwise addition of 01M HCl The reaction wasincubated at 37∘C for 30min and then immediately blendedwith 03mL of sulfanilic acid (04) at 25∘C for 5min Next03mL of N-ethylenediamine (02) and 20mL of ultrapurewater were added to the reaction and left to stand for 15minat 25∘C Finally 220120583L of the reaction mixture was pipettedinto well on a Multiskan GO (Thermo Fisher Scientific)and the absorbance was measured at 538 nm The nitrite-scavenging activity was calculated using (1) and Vc was usedas a reference

254 Reducing Power Assay The reducing power of the IGFwas determined according to a method previously described[20 21] Briefly 50 120583L aliquots of 10 20 40 80 160 or320 120583gmL IGF were mixed with 50120583L of phosphate buffersaline (02M pH 66) and 25 120583L of 1 (wv) potassiumferricyanide [K

3Fe(CN)

6] solution After incubation at 45∘C

for 30min 50120583L of 10 (wv) trichloroacetic acid (TCA)and 60 120583L of 01 (wv) ferric chloride (FeCl

3) solution were

added The absorbance was measured at 700 nm and Vc wasused as a reference An increase in absorbance was correlatedwith a greater reducing power

26 Effects of IGF on Murine Models of APAP-InducedHepatotoxicity

261 Experimental Mice Male Kunming mice (22 plusmn 116 gbody weight) were purchased from DOSSY BiotechnologyCo Ltd (Chengdu China) The mice were housed in astandard animal laboratory in strictly controlled conditionstemperature 25 plusmn 2∘C humidity 60 plusmn 5 lightdark cycle12 12 hThemicewere given free access towater and standardcommercial pellets and subjected to a 1-week acclimatizationperiod before the experiments were commenced All experi-ments were approved by the Animal Experimentation EthicsCommittee at Sichuan University (approval number SCXK[Chuan] 0000387) and were conducted in accordance withthe National Institute of Health (China) Guide for the Careand Use of Laboratory Animals

262 Murine Model of APAP-Induced Hepatotoxicity Sixtyacclimatized mice were randomly divided into six groups often animals and received daily oral administration of eithersaline (normal and model groups) 150mgkg BDP (positivegroup) or 150 300 or 450mgkg IGF (IGF groups 150 300and 450 resp) for 2 weeks On day 14 mice were fastedfor 6 h (with unrestricted access to water) and then injectedintraperitoneally with 180mgkg acetaminophen (with theexception of group containing normal mice which received


an equal volume of normal saline) [6 22] After APAP treat-ment for 12 h [6] mice were sacrificed by cervical dislocationand their blood was collected for the determination the levelsof serum AST ALT T-AOC and TNF-120572 The livers wereretrieved and levels of SOD GSH and MDA were measured

263 Serum ALT AST and T-AOC Blood samples werecollected and centrifuged at 587119892 for 15min at 4∘C SerumALT AST and T-AOC levels were then measured usingcommercial assay kits in accordance with the manufacturerrsquosprotocols

264 Serum Cytokines Levels of serum tumor necrosisfactor TNF-120572 were measured with a commercial ELISA kitin accordance with the manufacturerrsquos instructions (YuanyeBioengineering Institute Shanghai China)

265 Liver Enzymatic Activity Harvested livers (02 g) werewashed immediately with ice-cold normal saline and homog-enized with ice-cold normal saline (10 wv) using anautomatic homogenizer Homogenates were then centrifugedat 587119892 for 20min at 4∘C The supernatant was collectedand GSH SOD and MDA levels were determined usingcommercial assay kits in accordance with the manufacturerrsquosinstructions The results were expressed as mgg proteinUmg protein and nmolmL respectivelyThe concentrationof the total protein in the homogenates was determined usinga bicinchoninic acid (BCA) assay kit in accordance withthe manufacturerrsquos instructions (Jiancheng BioengineeringInstitute) [23]

266 Liver Histopathology Livers harvested from the samelocation were immersed in fixative (5 formalin 5 aceticacid and 70 ethanol 1 1 18 vvv) for 48 h and thenembedded in paraffin The paraffin blocks were sliced into5 120583m thick serial sections and stained with HampE for gen-eral histopathological examination under a light microscope(Olympus Tokyo Japan)

267 Immunohistochemistry Murine livers were stained forimmunohistochemical (IHC) analysis of TNF-120572 and NF-120581Bp65 as per the instructions provided with the GTVision IIIDetection SystemMoampRb immunohistochemical stainingkit as previously reported [24 25] Briefly paraffin-embeddedsectionswere deparaffinizedwith xylene and dehydratedwithgraded alcohols and endogenous peroxidase was blockedwith 03 fresh hydrogen peroxide in methanol Antigenswere retrieved during a 4-minute incubation with 01M citricacid at 120∘C and then cooled to 25∘C Nonspecific bindingsites were blocked with normal goat serum The sectionswere then incubated with primary polyclonal antibodiesraised against NF-120581B (1 200 dilution) or TNF-120572 (1 300dilution) at 37∘C for 1 h and overnight at 4∘C The next dayslides were washed twice with PBS (pH 72ndash74) and thenincubated with the secondary antibody (GTVision III Detec-tion SystemMoampRb GK500705) for 40min at 25∘C DAB(3-31015840-diaminobenzidine tetrahydrochloride) was applied todevelop the color and removed by rinsingwith distilled waterThe nuclei were stained with hematoxylin and deparaffinized

with xylene Finally the staining was visualized and capturedunder a light microscope

Immunohistochemical stains were scored separately bytwo experienced pathologists who were not exposed to anyclinical information In cases of disagreement a third senioradvisor was consulted for consensus The presence of yellowor brown staining in the cytoplasm for TNF-120572 and NF-120581Bwas classified as positive which was quantitatively definedaccording to a method reported elsewhere with minormodifications [25] Briefly TNF-120572 and NF-120581B expressionswere assessed semiquantitatively (immunostaining intensityand positivity) in ten randomly selected fields containingapproximately 1000 cells at 200x magnification The scoringof the stain intensity was defined as follows 0 no notablestain 1 barely detectable stain 2 notable brown stains and3 dark-brown stainsThe scoring values for positivity wereas follows 0 no liver cells positively stained 1 lt10 of livercells stained 2 10ndash50 of liver cells stained 3 50ndash75 ofliver cells stained and 4 gt75 of cells stained Finally theintensity and fractions scores were multiplied to obtain acombined score in the range 0ndash12 A score of 0ndash3 was definedas negativemild expression while a score of 4ndash12 indicatedpositive expression 4ndash6 low 7ndash9 moderate 10ndash12 strong

27 Statistical Analyses Data were expressed as mean plusmnstandard deviation (SD) Comparisons between multiplegroups were evaluated using ANOVA followed by a t-test of the least significant differences A P value lt 005was considered statistically significant All analyses wereconducted with SPSS Version 150 (SPSS Inc Chicago ILUSA) with the exception of IC

50(the IGF concentration at

which 50 of radicals were scavenged) which was calculatedusing GraphPad Prism software (GraphPad Software IncCA USA)

3 Results and Discussion

31 HSCCC Separation The crude extract of V ciliatawas separated into four fractions by using ethyl acetatendashn-butanolndashwater (2 1 3 vvv) at a flow rate of 5mLmin Asfractions 2 and 4 have been identified as catalposide andverproside respectively [10] we combined fractions 2 3 and4 to yield 148 g of the IGF Compared with previous methods[10] our method of the separation of the IGF shortenedthe separation time to 26min The one-step elution for theseparation of the IGF from crude extracts of V ciliata byHSCCC was an effective and efficient method

32 Chemical Components of IGF Based on our establishedUPLC chromatographic conditions three iridoid glycosides(verproside catalposide and amphicoside) were identified inthe IGF by comparison of the individual UPLC peak reten-tion times with those of the authentic reference standards(Figure 1)

33 Antioxidant activity of IGF In Vitro DPPH is a stableradical that is widely used to measure the antioxidant activityof compounds The ability of the IGF to neutralize DPPH


1

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3

StandardsSamples

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020000

040060080100120140160180200220240260280

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Figure 1 Ultra-performance liquid chromatography (UPLC) chro-matograms detected at 260 nmThe iridoid glycosides fraction (IGF)was compared with a sample of mixed standards (verproside catal-poside and amphicoside) The concentrations of the samples andmixed standards were 05mgmL and 01mgmL respectively Redline samples black line standards (1) Verproside (2) catalposideand (3) amphicoside

Table 1 Antioxidant activities of the iridoid glycosides fraction(IGF) of V ciliata

Samples Radical scavenging IC50(120583gmL) Reducing power

DPPH ABTS NitriteIGF 3199 plusmn 151 912 plusmn 096 5563 plusmn 175 0003Vc 1173 plusmn 107 304 plusmn 048 1547 plusmn 102BHT 0004

radicals was influenced in a dose-dependent manner simi-lar to Vc (Figure 2(a)) The IC

50values (the concentration

required to scavenge 50 of radicals) for IGF and Vc were3199 plusmn 151 and 1173 plusmn 107 120583gmL respectively (Table 1) Atthemaximumdose tested (200120583gmL) 9488 and 9941ofDPPH radicals were scavenged by IGF and Vc respectively

The ability of the IGF to scavenge ABTS cation radicalswas also dose dependent (Figure 2(b)) The IC

50values were

912plusmn 096 and 304plusmn 048 120583gmL for IGF andVc respectively(Table 1) At a maximum dose of 128 120583gmL 9932 and9972 of ABTS radicals were scavenged by IGF and Vcrespectively

Likewise the IGF scavenged nitrite radicals in a dose-dependent manner at concentrations ranging from 10 to320 120583gmL (Figure 2(c)) The IC

50values were 5563 plusmn 175

and 1547 plusmn 102 120583gmL for IGF and Vc respectively (Table 1)At a concentration of 320120583gmL 8817 and 9896of nitriteradicals were scavenged by IGF andVc respectivelyThis dataclearly indicated the ROS-scavenging ability of the IGF

The reducing capacity of the compounds which wemeasured via the transformation of Fe3+ into Fe2+ may alsoserve as a significant indicator of its potential antioxidantactivity We calculated the reducing power slope gradientsin which the steepness of the gradients is correlated withthe reducing power [26] The slope gradients were 0003 and

0004 for IGF and BHT respectively (Table 1) The reducingpower of the IGF is shown in Figure 2(d)

It has been suggested that the hepatoprotection of phy-tochemicals arises from their antioxidant activities [12 26]Our results demonstrated that the IGF exhibited potentantioxidant activity in the neutralization of four radicalsOn the basis of the potent antioxidant activities of the IGFwe hypothesized that it should also be hepatoprotective Wethen established an APAP-induced murine model of ALI toconfirm our hypothesis

34 Effects of IGF on Serum Liver Markers Serum levels ofALT AST and T-AOC were determined in mice given APAPby injection (Figure 3) Serum levels of AST and ALT weresignificantly greater in the model control mice given APAPcompared with normal control mice given saline (P lt 001)These data suggest that the liver functions of APAP-injectedmice were damaged in association with decreased T-AOC (Plt 001) In a similarmanner to the positive control (BDP) 300and 450mgkg IGF significantly decreased the levels of ASTandALT and improved the T-AOC level comparedwith thosein model control mice (P lt 005) Pretreatment of mice witha lower dose (150mgkg) of IGF inhibited the levels of ASTandALT and improved T-AOC capacity but these differenceswere not statistically significant (P gt 005)35 Effects of IGF on Hepatic MDA GSH and SOD Theeffects of the IGF on the levels of MDA GSH and SODin liver tissues were notable (Figure 4) Hepatic levels ofGSH and SODwere significantly lower in APAP-treatedmicecompared with normal control mice (P lt 001) however thehepatic levels of MDA were markedly increased (P lt 001)Hepatic levels of GSH and SOD were significantly greater inmice treated with 300 or 450mgkg IGF and 150mgkg BDPcompared with model control mice (P lt 005) In contrasthepatic MDAwas markedly lower (P lt 001) Pretreatment ofmice with 150mgkg IGF significantly increased the levels ofSOD (Plt 005) and inhibited the levels ofGSHandMDA(Pgt005)

36 Effective Inhibition of Inflammation TNF-120572 is an impor-tant proinflammatory cytokine involved in the progressionof APAP-induced hepatotoxicity [27ndash29]Therefore wemea-sured the serum levels of TNF-120572 inmice (Figure 5)The levelsof serum TNF-120572 in APAP-treated model control mice weresignificantly greater compared with those of normal controlmice whereas pretreatment of mice with 300 and 450mgkgIGF significantly suppressed serum TNF-120572 compared withmodel control mice (P lt 005)37 Liver Histopathology The histopathological changes inthe liver induced by APAP treatment are shown in Figure 6In the normal control mice the structures of the hepaticlobule were normal hepatocytes were well-preserved centralveins were visible and no abnormalities were detected inthe sinusoids (Figure 6(a)) In contrast acute administra-tion of APAP resulted in severe liver injuries characterizedby necrosis serious inflammatory-cell infiltration extensivebridging necrosis and interface hepatitis (Figure 6(b)) In


0

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0 50 100 150 200 250

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc

Concentration (120583gmL)

(a)

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ABT

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(b)

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IGFVc


(c)

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1

12

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0 100 200 300 400

IGFBHT

Redu

cing

pow

er as

say

abso

rban

ce at

700

nm


(d)

Figure 2 Antioxidant properties of the iridoid glycosides fraction (IGF) were determined using (a) a DPPH radical scavenging assay (b) anABTS radical scavenging assay (c) a nitrite-scavenging assay and (d) a reducing power assay Data are expressed as the mean plusmn SD (n = 3)Vc ascorbic acid BHT 26-di-tert-butyl-4-methylphenol

BDP-treated mice compared with model control the liverinjury was clearly alleviated by BDP and only slight infiltra-tion of inflammatory-cell was observed (Figure 6(c)) Similarto BDP treatment 300 and 450mgkg IGF potently inhib-ited APAP-induced liver damage However treatment with150mgkg IGF appeared inadequate owing to the presence ofstrong inflammatory-cell infiltration in some regions of theliver (Figures 6(d) 6(e) and 6(f)) This showed that the IGFtreatment inhibited APAP-induced ALI

38 Effect of IGF on Hepatic TNF-120572 and NF-120581B The livers ofAPAP-treatedmice were examined by immunohistochemicalanalysis of TNF-120572 andNF-120581Bp65 expression (Figure 7) Liver

tissues in APAP-treated mice were stained positive for TNF-120572 and NF-120581Bp65 in all the observed tissues areas and therelative amount of staining was correlated with the severity ofthe toxicity In contrast mice treated with 300 or 450mgkgIGF and 150mgkg BDP presented lower staining (P lt 005)The livers of mice treated with 150mgkg IGF were morecomparable to those of model control mice (P gt 005) overthose treated with greater doses of IGF (Figures 7(a)ndash7(d))These results suggested that the protective effect of the IGFmay arise from its anti-inflammatory activity which wasthought to occur through the suppression of NF-120581Bp65

Modern pharmacological studies have recognized thatiridoid compounds isolated from plant extracts are active


0

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ALT (UL)AST (UL)T-AOC (UmL)

Seru

m b

ioch

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al p

aram

eter

I II III IV V VI

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowastlowast


Figure 3 Effect of the iridoid glycosides fraction (IGF) on the activity of serum ALT AST and T-AOC in APAP-induced ALI Results arepresented as the mean plusmn SD (n = 10) Notes 119875 lt 001 119875 lt 005 compared with group I lowastlowast119875 lt 001 lowast119875 lt 005 compared with group IIGroup I normal control (saline) group II model control (APAP only) group III APAP + 150mgkg BDP group IV APAP + 450mgkg IGFgroup V APAP + 300mgkg IGF group VI APAP + 150mgkg IGF

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GroupI II III IV V VI

lowastlowast

lowastlowast

lowast

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lowastlowast lowastlowast

SOD (10Umg prot)

Figure 4 Effect of the iridoid glycosides fraction (IGF) on the levelsof GSH SOD and MDA in liver tissue with APAP-induced ALIResults are presented as the mean plusmn SD (n = 10) Notes 119875 lt 001119875 lt 005 compared with group I lowastlowast119875 lt 001 lowast119875 lt 005 comparedwith group II Group I normal control (saline) group II modelcontrol (APAP only) group III APAP + 150mgkg BDP group IVAPAP + 450mgkg IGF groupV APAP + 300mgkg IGF groupVIAPAP + 150mgkg IGF

substances with hepatoprotective antioxidant and anti-inflammatory activities [13 14 30ndash32] Our present researchdemonstrated that the IGF isolated from V ciliata possessedstrong antioxidant activity and was significantly protectiveagainst the acute hepatotoxicity induced by APAP To thebest of our knowledge we have made the first report of thehepatoprotective traits of the IGFWe have also demonstrated

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lowastlowastlowastlowastlowast

Figure 5 Effect of the iridoid glycosides fraction (IGF) on serumTNF-120572 levels in APAP-induced ALI Results are presented as themean plusmn SD (n = 8) Notes lowastlowast119875 lt 001 lowast119875 lt 005 compared withgroup II Group I normal control (saline) group II model control(APAP only) group III APAP + 150mgkg BDP group IV APAP +450mgkg IGF groupVAPAP+ 300mgkg IGF groupVI APAP +150mgkg BDP group IV APAP+ 450mgkg IGF groupVAPAP +300mgkg IGF group VI APAP + 150mgkg IGF

that the IGF suppressed the levels of AST and ALT butincreased the level of T-AOC and the activity of antioxidantenzymes Finally we identified the main constituents of theIGF as verproside catalposide and amphicoside

Numerous studies have previously demonstrated that theadministration of APAP is sufficient to cause severe ALI inmice with a notable elevation of serum AST and ALT levels[33] In our study the serum levels of AST and ALT of theAPAP-treated model control mice were notably increasedcompared with normal controlmice that received only salinewhich indicated that the liver functions of the model controlmice were seriously damaged Moreover pretreatment with


CV

(a)

CV

(b)

CV

(c)

CV

(d)

CV

(e)

CV

(f)Figure 6 Histopathological effects of the iridoid glycosides fraction (IGF) on APAP-induced ALI (HampE staining 100x magnification) (a)Normal control group (saline) (b) model control group (APAP only) (c) APAP + 150mgkg BDP (d) APAP + 450mgkg IGF (e) APAP +300mgkg IGF (f) APAP + 150mgkg IGF Black arrows and red ovals indicated interface hepatitis and focal necrosisinflammatory-cellinfiltration respectively CV central vein

high doses of the IGF (300 and 450mgkg) ameliorated theAPAP-induced hepatotoxicity in mice Simultaneously thehepatoprotective activity of the IGF was demonstrated fromhistological observation which showed that mice pretreatedwith the IGF had significantly less liver tissue damageincluding serious inflammatory cells infiltration fragmentednecrosis or interface hepatitis and bridging necrosis

Treatment with APAP induced oxidative stress and mas-sively impaired the antioxidant defense systems as evi-denced by decreased GSH and SOD activities and increasedintrahepatic MDA levels [6 12 34] TNF-120572 is a pleiotropicproinflammatory cytokine that induces cell proliferation celldeath or inflammation [27 35]We demonstrated that APAPadministration raised TNF-120572 level in serum and liver tissuesNF-120581Bp65 has also been associated with the inflammatoryresponse in many different diseases and is upregulated inresponse to inflammatory challenges [36 37] The results ofthe present study showed that the IGF possessed remarkableantioxidant and anti-inflammatory activities which wereconfirmed by the recovery in the levels of GSH and SOD andthe decrease in the levels of hepatic MDA TNF-120572 and NF-120581BP65 These results indicated that the IGF isolated from Vciliata could alleviate the oxidative stress and inflammatoryresponses to APAP-induced ALI

From the biochemical analysis of the expression of proin-flammatory factors (TNF-120572 NF-120581B) and histopathologicalobservation we concluded that the IGF was hepatoprotectiveagainst APAP-induced damage Indeed the IGF exhibitedmore potent protective effects than the drug BDP in tests for

some markers The IGF could also prevent oxidative stressand decrease the inflammatory response and hepatic damagecaused by APAP exposure in mice this was evidenced bythe reduction in serum AST ALT and TNF-120572 activities liverMDA level recovery of hepatic GSH SOD and T-AOC andlowered expression of proinflammatory factors (TNF-120572 NF-120581B)

Our results revealed that the IGF of V ciliata exhibitedstrong antioxidant activities and significant protective effectsonAPAP-inducedALI in amurinemodelThiswork providesscientific evidence for the traditional medicinal uses of Vciliata and a scientific basis for its clinical application

4 Conclusion

Our study is the first report on the separation the IGF fromV ciliata using an efficient one-step HSCCC elution Firstwe demonstrated that the IGF from V ciliata possessedstrong antioxidant activity and second that the IGF washepatoprotective against APAP-induced ALI in mice Theprotection afforded by the IGFmay arise from its inhibition ofoxidative stress and inflammatory response Accordingly theIGF isolated from V ciliatamay prove a promising candidatefor the treatment of many liver diseases

Competing Interests

The authors declare that there is no conflict of interests


Score 0ndash3 negativemild Score 4ndash6 low

200x 400x 200x 400x

200x 400x 200x 400x

Score 7ndash9 moderate Score 10ndash12 strong

(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)

Score 0ndash3 negativemildTNF-120572 NF-120581Bp65

200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e

GroupTNF-120572NF-120581B

I II III IV V VI

lowast lowast

lowast


(d)

Figure 7 The effects of iridoid glycosides fraction (IGF) pretreatment on TNF-120572 and NF-120581Bp65 expression Bp65 expression in APAP-induced ALI Representative immunohistochemical staining of TNF-120572 (a) and NF-120581Bp65 (b) in the liver was divided into negativemild lowmoderate and strong grades Expression in normal control mice (c) was negativemild Each low magnification (200x) image was pairedwith a high magnification (400x) image for clearer observation The immunohistochemical staining scores of TNF-120572 and NF-120581Bp65 weresignificantly lowered by IGF which indicated a reduction in their activation and expression (d) Data are pressed as the mean plusmn SD (n = 8)Notes lowastlowast119875 lt 001 lowast119875 lt 005 compared with group II Group I normal control (saline) group II model control (APAP only) group IIIAPAP + 150mgkg BDP group IV APAP + 450mgkg IGF group V APAP + 300mgkg IGF group VI APAP + 150mgkg IGF


Authorsrsquo Contributions

Shancai Tan performed the experiment and wrote themanuscript Qiuxia Lu and Yueyue Shu participated in theexperimental work Yiran Sun analyzed the data and LinTang designed and guided the experiments and wrote themanuscript Lin Tang and Fang Chen provided the financialsupport for the experiments

Acknowledgments

This work was supported by the Natural Science Foundationof China (no 31570351)

References

[1] V Mihailovic M Mihailovic A Uskokovic et al ldquoHepatopro-tective effects ofGentiana asclepiadea L extracts against carbontetrachloride induced liver injury in ratsrdquo Food and ChemicalToxicology vol 52 pp 83ndash90 2013

[2] H J Zimmerman ldquoEffects of aspirin and acetaminophen on theliverrdquo Archives of Internal Medicine vol 141 no 3 pp 333ndash3421981

[3] M Q Bromer and M Black ldquoAcetaminophen hepatotoxicityrdquoClinics in Liver Disease vol 7 no 2 pp 351ndash367 2003

[4] A M Larson ldquoAcetaminophen hepatotoxicityrdquo Clinics in LiverDisease vol 11 no 3 pp 525ndash548 2007

[5] S T Ahmad W Arjumand S Nafees et al ldquoHesperidin alle-viates acetaminophen induced toxicity in wistar rats by abroga-tion of oxidative stress apoptosis and inflammationrdquoToxicologyLetters vol 208 no 2 pp 149ndash161 2012

[6] M Tai J Zhang S Song et al ldquoProtective effects of luteolinagainst acetaminophen-induced acute liver failure in mouserdquoInternational Immunopharmacology vol 27 no 1 pp 164ndash1702015

[7] State Administration of Traditional Chinese Medicine Chi-nese Materia Medica Shanghai Science and Technology PressShanghai China 1st edition 2002

[8] L Yin L Wei R Fu et al ldquoAntioxidant and hepatoprotec-tive activity of veronica ciliata fisch Extracts against carbontetrachloride-induced liver injury in micerdquo Molecules vol 19no 6 pp 7223ndash7236 2014

[9] L Yin Q Lu S Tan et al ldquoBioactivity-guided isolation ofantioxidant and anti-hepatocarcinoma constituents fromVeronica ciliatardquo Chemistry Central Journal vol 10 no 1 article27 2016

[10] Q Lu Y Sun Y Shu et al ldquoHSCCC separation of the two iri-doid glycosides and three phenolic compounds from Veronicaciliata and their in vitro antioxidant and anti-hepatocarcinomaactivitiesrdquoMolecules vol 21 no 9 article no 1234 2016

[11] L Yin The studies on chemical constituents and bioactivity ofV ciliata Fisch as a traditional Tibetan medicine [PhD thesis]College of Life Sciences Sichuan University Chengdu China2014

[12] E Madrigal-Santillan E Madrigal-Bujaidar I Alvarez-Gonza-lez et al ldquoReview of natural products with hepatoprotectiveeffectsrdquo World Journal of Gastroenterology vol 20 no 40 pp14787ndash14804 2014

[13] J Quan L Piao H Xu T Li and X Yin ldquoProtective effectof iridoid glucosides from Boschniakia rossica on acute liver

injury induced by carbon tetrachloride in ratsrdquo BioscienceBiotechnology amp Biochemistry vol 73 no 4 pp 849ndash854 2009

[14] W Peng X-Q Qiu Z-H Shu et al ldquoHepatoprotective activityof total iridoid glycosides isolated from Paederia scandens(lour) Merr var tomentosardquo Journal of Ethnopharmacologyvol 174 pp 317ndash321 2015

[15] Y Wang Y Chen L Deng et al ldquoSystematic separationand purification of iridoid glycosides and crocetin derivativesfrom gardenia jasminoides ellis by high-speed counter-currentchromatographyrdquo Phytochemical Analysis vol 26 no 3 pp202ndash208 2015

[16] Y Lin W Xu M Huang et al ldquoQualitative and quantitativeanalysis of phenolic acids flavonoids and iridoid glycosides inYinhua Kanggan tablet by UPLC-QqQ-MSMSrdquoMolecules vol20 no 7 pp 12209ndash12228 2015

[17] M S Aslam M S Ahmad A S Mamat M Z Ahmad and FSalam ldquoAntioxidant and wound healing activity of polyherbalfractions of Clinacanthus nutans and Elephantopus scaberrdquoEvidence-based Complementary and Alternative Medicine vol2016 Article ID 4685246 14 pages 2016

[18] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[19] R Fu Y Zhang Y Guo and F Chen ldquoAntioxidant andtyrosinase inhibition activities of the ethanol-insoluble fractionof water extract of Sapium sebiferum (L) Roxb leavesrdquo SouthAfrican Journal of Botany vol 93 pp 98ndash104 2014

[20] K I Berker K Guclu I Tor and R Apak ldquoCompara-tive evaluation of Fe(III) reducing power-based antioxidantcapacity assays in the presence of phenanthroline batho-phenanthroline tripyridyltriazine (FRAP) and ferricyanidereagentsrdquo Talanta vol 72 no 3 pp 1157ndash1165 2007

[21] N Dekdouk N Malafronte D Russo et al ldquoPhenolic com-pounds from olea europaea l possess antioxidant activity andinhibit carbohydrate metabolizing enzymes in vitrordquo Evidence-Based Complementary and Alternative Medicine vol 2015Article ID 684925 9 pages 2015

[22] S L Michael N R Pumford P R Mayeux M R Niesmanand J A Hinson ldquoPretreatment of mice with macrophageinactivators decreases acetaminophen hepatotoxicity and theformation of reactive oxygen and nitrogen speciesrdquoHepatologyvol 30 no 1 pp 186ndash195 1999

[23] J M Walker ldquoThe bicinchoninic acid (BCA) assay for proteinquantitationrdquo in The Protein Protocols Handbook pp 11ndash14Humana Press Totowa NJ USA 2002

[24] B Chen Z Tan J Gao et al ldquoHyperphosphorylation ofribosomal protein S6 predicts unfavorable clinical survival innon-small cell lung cancerrdquo Journal of Experimental andClinicalCancer Research vol 34 no 1 article 126 2015

[25] R Ikarashi K Shibasaki and A Yamaguchi ldquoImmunohisto-chemical studies of organic anion transporters and urate trans-porter 1 expression in human salivary glandrdquoActa OdontologicaScandinavica vol 71 no 2 pp 312ndash316 2013

[26] R Fu Y Zhang Y Guo T Peng and F Chen ldquoHepato-protection using Brassica rapa var Rapa L Seeds and itsbioactive compound Sinapine thiocyanate For CCl4-inducedliver injuryrdquo Journal of Functional Foods vol 22 pp 73ndash81 2016

[27] C Nastevska E GerberMHorbach E Rohrdanz and R KahlldquoImpairment of TNF-120572 expression and secretion in primary ratliver cell cultures by acetaminophen treatmentrdquo Toxicology vol133 no 2-3 pp 85ndash92 1999


[28] A Gandhi T Guo and R Ghose ldquoRole of c-Jun N-terminalkinase (JNK) in regulating tumor necrosis factor-alpha (TNF-120572) mediated increase of acetaminophen (APAP) and chlor-promazine (CPZ) toxicity in murine hepatocytesrdquo Journal ofToxicological Sciences vol 35 no 2 pp 163ndash173 2010

[29] F Hussan A S Mansor S N Hassan T N T Tengku NorEffendy Kamaruddin S B Budin and F Othman ldquoAnti-inflammatory property of plantago major leaf extract reducesthe inflammatory reaction in experimental acetaminophen-induced liver injuryrdquoEvidence-Based Complementary andAlter-native Medicine vol 2015 Article ID 347861 7 pages 2015

[30] E J Jeong K Y Lee S H Kim S H Sung and Y C KimldquoCognitive-enhancing and antioxidant activities of iridoid gly-cosides from Scrophularia buergeriana in scopolamine-treatedmicerdquo European Journal of Pharmacology vol 588 no 1 pp 78ndash84 2008

[31] J H Kwak H J Kim K H Lee S C Kang and O PZee ldquoAntioxidative iridoid glycosides and phenolic compoundsfrom Veronica peregrinardquo Archives of Pharmacal Research vol32 no 2 pp 207ndash213 2009

[32] S J An H O Pae G S Oh et al ldquoInhibition of TNF-120572IL-1120573 and IL-6 productions and NF-120581B activation in lipopo-lysaccharide-activated RAW2647macrophages by catalposidean iridoid glycoside isolated from Catalpa ovata G Don(Bignoniaceae)rdquo International Immunopharmacology vol 2 no8 pp 1173ndash1181 2002

[33] A B Imaeda AWatanabeMA Sohail et al ldquoAcetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and theNalp3 inflammasomerdquo Journal of Clinical Investigation vol 119no 2 pp 305ndash314 2009

[34] J-H Zhu J P McClung X Zhang et al ldquoComparative impactsof knockouts of two antioxidant enzymes on acetaminophen-induced hepatotoxicity in micerdquo Experimental Biology andMedicine vol 234 no 12 pp 1477ndash1483 2009

[35] H Kamata S S Honda L Chang et al ldquoReactive oxygenspecies promote TNF alpha-induced death and sustained JNKactivation by inhibiting MAP kinase phosphatasesrdquo Cell vol120 no 5 pp 649ndash661 2005

[36] N Silswal A K Singh B Aruna S Mukhopadhyay S Ghoshand N Z Ehtesham ldquoHuman resistin stimulates the pro-inflammatory cytokines TNF-120572 and IL-12 in macrophagesby NF-120581B-dependent pathwayrdquo Biochemical and BiophysicalResearch Communications vol 334 no 4 pp 1092ndash1101 2005

[37] Z-G Liu H Hsu D V Goeddel and M Karin ldquoDissection ofTNF receptor 1 effector functions JNK activation is not linkedto apoptosis while NF-120581B activation prevents cell deathrdquo Cellvol 87 no 3 pp 565ndash576 1996

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and in the cold conditions of high-altitude Tibet WholeV ciliata has been used in traditional Tibetan medicine totreat hepatitis cholecystitis rheumatism and urticaria it isestimated that over 100 types of Tibetan medicine contain Vciliata [7]

The ethyl acetate fraction of the ethanol extract of V cili-atahas been reported to have strong antioxidant activities andtherapeutic efficacy for acute hepatotoxicity induced by car-bon tetrachloride (CCl

4) [8] Meanwhile other reports have

indicated that the iridoidmonomers separated fromV ciliataare potent antioxidants and can inhibit the proliferationof HepG2 hepatocellular carcinoma cells [9 10] Currentlyfifteen iridoid glycosides have been isolated from V ciliata[11] To date however no study has reported the antioxi-dant activity of the iridoid glycosides fraction isolated fromV ciliata using high-speed countercurrent chromatography(HSCCC) or the amelioration of APAP-induced ALI in mice

According to a previous report the hepatoprotectiveeffect of the compounds is related at least in part to theirantioxidant activity [8] Antioxidants have been shown toprevent oxidative stress-related liver pathologies directly byscavenging of reactive oxygen species (ROS) and indirectlyas part of the antioxidant defense system [12] Furthermoreiridoid glycosides are able to neutralize reactive oxida-tive species and are effective in the treatment of carbontetrachloride- (CCl

4-) induced acute hepatotoxicity [13 14]

Accordingly we hypothesized that the IGF isolated from Vciliata may be effective in the treatment of APAP-inducedALI in mice Accordingly the present study aimed to (1)isolate the IGF from V ciliata by HSCCC (2) characterizethe chemical composition of the IGF by ultra-performanceliquid chromatography (UPLC) analysis and (3) assess theantioxidant activity in vitro and the hepatoprotective effectsin vivo using an APAP-induced murine model of ALI

2 Materials and Methods

21 Assays and Chemicals SBC Middle Chromatogram Iso-lated (MCI)Gel (Type F 75ndash150120583m)was purchased fromSci-Bio Chem Co Ltd (Chengdu China)The diagnostic kits foraspartate aminotransferase (AST) alanine aminotransferase(ALT) total antioxidant capacity (T-AOC) malondialde-hyde (MDA) superoxide dismutase (SOD) GSH and totalprotein (TP) were provided by the Nanjing Jiancheng Bio-engineering Institute (Nanjing China) The tumor necrosisfactor (TNF-120572) kit was purchased from Shanghai YuanyeBioengineering Institute (Shanghai China) The GTVisionIII Detection SystemMoampRb kit was provided by GeneTech (Shanghai) Company Limited (Shanghai China) Bifen-date pills (BDP) were obtained from Wanbangde Pharma-ceutical Group Co Ltd (Zhejiang China) 22-Diphenyl-1-picrylhydrazyl (DPPH) 26-di-tert-butyl-4-methylphenol(BHT) 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) the reference standards (verproside catalposide andamphicoside) vitamin C (VC) and acetaminophen (APAP)were purchased from Sigma-Aldrich Chemical Co (St LouisMOUSA)HPLC-grademethanol and acetonitrile were usedfor UPLC analyses All other chemicals and reagents used inthis study were of analytical grade

22 Veronica ciliata Herbs of V ciliata were bought fromthe Tibetan Traditional Medicine Pharmaceutical Factory ofLhasa city (Tibet province of China) and the herbs werecollected in Linzhou of Lhasa (28th July 2015) A voucherspecimen (number 00721478) was identified by Dr Jie Bai ofthe School of Life Sciences at the Sichuan University Chinaand deposited in the herbarium of the Sichuan University

23 Preparation of IGF Using HSCCC

231 HSCCC Apparatus A preparative TBE-300C HSCCCsystem (Shanghai Tauto Biotech Co Ltd Shanghai China)was equipped with three sequential multilayer coil separationcolumns a 20mL sample loop and a TBP-5002 pump(Shanghai Tauto BiotechCo Ltd) ADC-0506 constant tem-perature circulating implement (Shanghai Sunny HengpingScientific Instrument Co Ltd Shanghai China) was used tomaintain the separation temperature An AFB750D0-10A30air compressor pump was used to generate the stationaryphase flow The UV absorbance of the eluent was monitoredwith a UV-2000 detector (Shanghai Sanotac Scientific Instru-ment Co Ltd Shanghai China) All data were collectedby an Easy Chrom v22123 workstation (Beijing QingbohuaLtd Beijing China)

232 Preparation of Crude Extract The whole plant of Vciliata (10 kg) was powdered and macerated three times in15 L reagent barrels containing 95 ethanol (1 6 wv) at 20ndash25∘C for 24 h The extracted solutions were evaporated todryness using a rotary evaporator under reduced pressureat 40ndash50∘C for 2 days The extract was then dissolved inultrapure water (03 L) and sequentially extracted six timeswith an equal volume of petroleum ether to remove the pig-ments and lipids Finally chlorophyll was removedusingMCIGEL column chromatography and eluted with MeOH-H

2O

(9 1 vv 65 L) which yielded the crude extract (1071 g) Analiquot of 400 g was subjected to HSCCC separation

233 HSCCC Separation The crude extract was separatedby using HSCCC according to a protocol previously reportedwith slight modifications [10 15] A two-phase solvent systemcomposed of ethyl acetatendashn-butanolndashwater (2 1 3 vvv)was chosen The solvents were shaken and equilibrated ina separation funnel at 25∘C The upper and lower phaseswere separated and degassed by ultrasonication for 30minbefore use The temperature of the separation column wasmaintained at 28∘C The column was first entirely filledwith the lower aqueous phase at a flow rate of 30mLminSubsequently the apparatus was rotated at 800 rpm in reverseand the upper mobile phase was pumped into the columnat a flow rate of 10mLmin The system reached equilibriumwhen the mobile phase eluted from the tail at which pointthe retention of stationary phase was calculated (667)The sample solution (20mL of the lower phase containing200mg of the extracts) was injected into the column throughthe sample loop The effluent with a flow rate of 5mLminwas monitored by using a UV detector at 260 nm and thefractions were collected manually according to the HSCCCchromatogram Each fraction was collected as a unit Four







(1)








3Fe(CN)



3) solution were






















1

2

3

StandardsSamples

260nm020

040

060

080

100

120

140

160

180

200

220

240

260

280

020000

040060080100120140160180200220240260280

300

320

340

360

380

Time (min)

(AU

)









50values were











0

20

40

60

80

100

120

0 50 100 150 200 250

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(a)

0

20

40

60

80

100

120

0 50 100 150

ABT

S ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(b)

0

20

40

60

80

100

120

0 100 200 300 400

Nitr

ite sc

aven

ging

activ

ity (

)

IGFVc


(c)

0

02

04

06

08

1

12

14

0 100 200 300 400

IGFBHT

Redu

cing

pow

er as

say

abso

rban

ce at

700

nm


(d)







0

20

40

60

80

100

Group


Seru

m b

ioch

emic

al p

aram

eter

I II III IV V VI

lowast

lowast

lowastlowast

lowastlowast




0

5

10

15

20

25

30

35

GSH (mgg prot)

MDA (nmolL)

Live

r tiss

ues b

ioch

emic

al p

aram

eter


lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast


SOD (10Umg prot)



0

100

200

300

400

500

Group

TNF-

120572se

rum

leve

ls (p

gm

L)

I II III IV V VI






CV

(a)

CV

(b)

CV

(c)

CV

(d)

CV

(e)

CV







4 Conclusion


Competing Interests




200x 400x 200x 400x

200x 400x 200x 400x


(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)


200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e


I II III IV V VI

lowast lowast

lowast


(d)





Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















(1)








3Fe(CN)



3) solution were






















1

2

3

StandardsSamples

260nm020

040

060

080

100

120

140

160

180

200

220

240

260

280

020000

040060080100120140160180200220240260280

300

320

340

360

380

Time (min)

(AU

)









50values were











0

20

40

60

80

100

120

0 50 100 150 200 250

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(a)

0

20

40

60

80

100

120

0 50 100 150

ABT

S ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(b)

0

20

40

60

80

100

120

0 100 200 300 400

Nitr

ite sc

aven

ging

activ

ity (

)

IGFVc


(c)

0

02

04

06

08

1

12

14

0 100 200 300 400

IGFBHT

Redu

cing

pow

er as

say

abso

rban

ce at

700

nm


(d)







0

20

40

60

80

100

Group


Seru

m b

ioch

emic

al p

aram

eter

I II III IV V VI

lowast

lowast

lowastlowast

lowastlowast




0

5

10

15

20

25

30

35

GSH (mgg prot)

MDA (nmolL)

Live

r tiss

ues b

ioch

emic

al p

aram

eter


lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast


SOD (10Umg prot)



0

100

200

300

400

500

Group

TNF-

120572se

rum

leve

ls (p

gm

L)

I II III IV V VI






CV

(a)

CV

(b)

CV

(c)

CV

(d)

CV

(e)

CV







4 Conclusion


Competing Interests




200x 400x 200x 400x

200x 400x 200x 400x


(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)


200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e


I II III IV V VI

lowast lowast

lowast


(d)





Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































1

2

3

StandardsSamples

260nm020

040

060

080

100

120

140

160

180

200

220

240

260

280

020000

040060080100120140160180200220240260280

300

320

340

360

380

Time (min)

(AU

)









50values were











0

20

40

60

80

100

120

0 50 100 150 200 250

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(a)

0

20

40

60

80

100

120

0 50 100 150

ABT

S ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(b)

0

20

40

60

80

100

120

0 100 200 300 400

Nitr

ite sc

aven

ging

activ

ity (

)

IGFVc


(c)

0

02

04

06

08

1

12

14

0 100 200 300 400

IGFBHT

Redu

cing

pow

er as

say

abso

rban

ce at

700

nm


(d)







0

20

40

60

80

100

Group


Seru

m b

ioch

emic

al p

aram

eter

I II III IV V VI

lowast

lowast

lowastlowast

lowastlowast




0

5

10

15

20

25

30

35

GSH (mgg prot)

MDA (nmolL)

Live

r tiss

ues b

ioch

emic

al p

aram

eter


lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast


SOD (10Umg prot)



0

100

200

300

400

500

Group

TNF-

120572se

rum

leve

ls (p

gm

L)

I II III IV V VI






CV

(a)

CV

(b)

CV

(c)

CV

(d)

CV

(e)

CV







4 Conclusion


Competing Interests




200x 400x 200x 400x

200x 400x 200x 400x


(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)


200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e


I II III IV V VI

lowast lowast

lowast


(d)





Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1

2

3

StandardsSamples

260nm020

040

060

080

100

120

140

160

180

200

220

240

260

280

020000

040060080100120140160180200220240260280

300

320

340

360

380

Time (min)

(AU

)









50values were











0

20

40

60

80

100

120

0 50 100 150 200 250

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(a)

0

20

40

60

80

100

120

0 50 100 150

ABT

S ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(b)

0

20

40

60

80

100

120

0 100 200 300 400

Nitr

ite sc

aven

ging

activ

ity (

)

IGFVc


(c)

0

02

04

06

08

1

12

14

0 100 200 300 400

IGFBHT

Redu

cing

pow

er as

say

abso

rban

ce at

700

nm


(d)







0

20

40

60

80

100

Group


Seru

m b

ioch

emic

al p

aram

eter

I II III IV V VI

lowast

lowast

lowastlowast

lowastlowast




0

5

10

15

20

25

30

35

GSH (mgg prot)

MDA (nmolL)

Live

r tiss

ues b

ioch

emic

al p

aram

eter


lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast


SOD (10Umg prot)



0

100

200

300

400

500

Group

TNF-

120572se

rum

leve

ls (p

gm

L)

I II III IV V VI






CV

(a)

CV

(b)

CV

(c)

CV

(d)

CV

(e)

CV







4 Conclusion


Competing Interests




200x 400x 200x 400x

200x 400x 200x 400x


(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)


200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e


I II III IV V VI

lowast lowast

lowast


(d)





Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

0 50 100 150 200 250

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(a)

0

20

40

60

80

100

120

0 50 100 150

ABT

S ra

dica

l sca

veng

ing

activ

ity (

)

IGFVc


(b)

0

20

40

60

80

100

120

0 100 200 300 400

Nitr

ite sc

aven

ging

activ

ity (

)

IGFVc


(c)

0

02

04

06

08

1

12

14

0 100 200 300 400

IGFBHT

Redu

cing

pow

er as

say

abso

rban

ce at

700

nm


(d)







0

20

40

60

80

100

Group


Seru

m b

ioch

emic

al p

aram

eter

I II III IV V VI

lowast

lowast

lowastlowast

lowastlowast




0

5

10

15

20

25

30

35

GSH (mgg prot)

MDA (nmolL)

Live

r tiss

ues b

ioch

emic

al p

aram

eter


lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast


SOD (10Umg prot)



0

100

200

300

400

500

Group

TNF-

120572se

rum

leve

ls (p

gm

L)

I II III IV V VI






CV

(a)

CV

(b)

CV

(c)

CV

(d)

CV

(e)

CV







4 Conclusion


Competing Interests




200x 400x 200x 400x

200x 400x 200x 400x


(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)


200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e


I II III IV V VI

lowast lowast

lowast


(d)





Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

Group


Seru

m b

ioch

emic

al p

aram

eter

I II III IV V VI

lowast

lowast

lowastlowast

lowastlowast




0

5

10

15

20

25

30

35

GSH (mgg prot)

MDA (nmolL)

Live

r tiss

ues b

ioch

emic

al p

aram

eter


lowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast


SOD (10Umg prot)



0

100

200

300

400

500

Group

TNF-

120572se

rum

leve

ls (p

gm

L)

I II III IV V VI






CV

(a)

CV

(b)

CV

(c)

CV

(d)

CV

(e)

CV







4 Conclusion


Competing Interests




200x 400x 200x 400x

200x 400x 200x 400x


(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)


200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e


I II III IV V VI

lowast lowast

lowast


(d)





Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CV

(a)

CV

(b)

CV

(c)

CV

(d)

CV

(e)

CV







4 Conclusion


Competing Interests




200x 400x 200x 400x

200x 400x 200x 400x


(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)


200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e


I II III IV V VI

lowast lowast

lowast


(d)





Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















200x 400x 200x 400x

200x 400x 200x 400x


(a)


200x 400x 200x 400x

200x 400x 200x 400x


(b)


200x 400x 200x 400x(c)

0

2

4

6

8

10

IHC

stai

ning

scor

e


I II III IV V VI

lowast lowast

lowast


(d)





Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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