Research Article A Laboratory Evaluation of Medicinal Herbs Used...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 504563 10 pageshttpdxdoiorg1011552013504563

Research ArticleA Laboratory Evaluation of Medicinal Herbs Used in China forthe Treatment of Hand Foot and Mouth Disease

Xiaoqing Chen12 Chunyang Wang1 Lanfang Xu12 Xiaoshuang Chen13 Wei Wang2

Guang Yang4 Ren Xiang Tan2 Erguang Li12 and Yu Jin14

1 Medical School and Jiangsu Key Laboratory of Molecular Medicine Nanjing University 22 Hankou Road Nanjing 210093 China2 State Key Laboratory of Pharmaceutical Biotechnology and School of Life Sciences Nanjing University 22 Hankou Road Nanjing210093 China

3 Rosalind Franklin University of Medicine and Science North Chicago IL 60064 USA4Nanjing Childrenrsquos Hospital Nanjing Medical University 72 Guangzhou Road Nanjing 210008 China

Correspondence should be addressed to Erguang Li erguangnjueducn and Yu Jin jinyuldyy163com

Received 18 December 2012 Revised 5 February 2013 Accepted 7 February 2013

Academic Editor Wen Chuan Lin

Copyright copy 2013 Xiaoqing Chen et alThis 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

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the causative agents of hand foot and mouth disease (HFMD) Duringrecent epidemics of HFMD inChina medicinal herbals and preparations containing herbal extracts have demonstrated therapeuticefficacy with relative safety profilesThere have been no microbiological studies to validate their usefulness for HFMDWe selected12 commonly used herbs for HFMD from government recommended guidelines as well as published reports and tested for theirantiviral activity and anti-inflammatory activity A water extract of Houttuynia cordata Thunb (HCT) inhibited EV71 infectionsignificantly and was marginally active against CVA16 infection The IC

50(concentration to have 50 inhibitory effect) values

of HCT against a Fuyang strain and a BrCr strain of EV71 were determined at 89120583gmL and 206 120583gmL respectively Menthahaplocalyx Briq (MHB) water extract was active against CVA16 with an IC

50value of 703 120583gmL The extract did not exhibit

activity against EV71 infection Although the majority of the extracts showed no activity against viral infection several extractsdemonstrated activity in blocking proinflammatory response by viral infection This study therefore validates the effectiveness ofChinese herbs for HFMD since some formulations containing the correct combination of the herbs can block viral replication aswell as proinflammatory response of HFMD

1 Introduction

Hand foot and mouth disease (HFMD) is a common viralillness that usually affects infants and children younger than5 years old Sporadic occurrence of the disease has beenreported globally since first reported in New Zealand in 1957but larger outbreaks with higher morbidity and mortalityhave increased recently in the Asian Pacific region includingan outbreak in Fuyang China that affected close to a halfmil-lion children with 126 deaths in 2008 [1] Coxsackievirus A16(CVA16) and enterovirus 71 (EV71) are the causative agentsfor HFMD accounting for over 70 of cases during theoutbreak inChina in 2008 [2]The viruses aremembers of thePicornaviridae family and are spread through contact withvirus-containing body fluids respiratory droplets and feces

There are no vaccine or antiviral drugs available for HFMDAlthough immunoglobulin and antiviral agent ribavirin arecommonly used the efficacy remains uncertain [3 4] Goodpersonal hygiene including handwashing and disinfection ofsurfaces in child care facilities remains therefore as the mosteffective approach to reduce the transmission rate of HFMD[5 6]

During recent epidemics of HFMD in China herbalsand preparations containing herb extracts have demonstratedtherapeutic efficacy against the disease Some of the herbsor herbal preparations have been shown to ameliorate thesymptoms and others to shorten the course of the disease[7] There was no definitive description of symptoms relatedto HFMD in ancient manuscripts and publications in Chinauntil recent outbreaks of the disease To deal with the clinical

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Evaluation of cytotoxicity and antiviral activity of plant materials used for HFMD in Chinalowast

Latin name Abbr Parts traditional usage CC50 (120583gmL) IC50 (120583gmL)EV71 CVA16

Houttuynia cordataThunb HCT Stem and leaves bacterial and viral disease gt1000 89 plusmn 16 (FY)206 plusmn 25 (BrCr) 1863 plusmn 201

Mentha haplocalyx Briq MHB Leaves viral and bacterial disease gt1000 (minus) 703 plusmn 46Glycyrrhiza uralensis Fisch GUF Stem and root coughing gt1000 (plusmn) (plusmn)Gardenia jasminoides Ellis GJE Fruits icteric hepatitis gt1000 (minus) (minus)Anemarrhena asphodeloides Bge AAB Stem and root coughing gt1000 (minus) (minus)Arctium lappa L ALL Fruits coughing gt1000 (minus) (minus)Eupatorium fortunei Turcz EFT Stem and leaves fever gt1000 (minus) (minus)Lonicera japonicaThunb LJT Flowers inflammation gt1000 (minus) (minus)Scutellaria baicalensis Georgi SBG Root common cold 2402 plusmn 154 (minus) (minus)Isatis indigotica Fort IIFL Leaves coughing gt1000 (minus) (minus)Isatis indigotica Fort IIFS Root fever and viral disease gt1000 (minus) (minus)Forsythia suspensa (Thunb) Vahl FSV Fruits viral disease gt1000 (minus) (minus)lowastThe reference of Latin names abbreviations parts used in pharmaceuticals and traditional usages of the medicinal plants and data on antiviral screeningagainst EV71 (Fuyang strain and BrCr strain) and CVA16 are listed Cytotoxicity was assayed on Vero cells at a maximal concentration of 1000120583gmL Thoseshowed toxicity against Vero cells were further tested to determine a CC50 value while those showed no toxic effect at the maximal concentration tested werereported as gt1000120583gmL IC50 50 inhibitory activity against virus infection-induced cytopathic effectData are presented as mean plusmn SE of triplicates All experiments were performed at least twice with triplicate samples FY EV71 Fuyang strain (minus) no activityat 1000120583gmL (plusmn) marginally active

manifestations characterized with mild fever and rash prac-titioners of Chinese medicine reasoned and have effectivelytreated HFMD as dampness and damp heat We compileda list of commonly used herbs with implication for HFMDtherapy (Table 1) from a list of recommended remedies bygovernment agencies in China and from published reports[7ndash9] Most of the herbs in Table 1 have traditionally orfolklorically been used for inflammatory or infective diseasesIn addition most of the herbs listed in Table 1 have not beeninvestigated for their antiviral activity against pathogens ofHFMD disease although some of them have demonstratedantiviral activity Less is known about the active componentsof those plants

The objectives of this study were to validate the effec-tiveness of the herbs microbiologically by measuring theirantiviral activity In addition we also studied whether theherbs possessed anti-inflammatory activity since one of thesymptoms of HFMD is an acute inflammatory responseHere we report the antiviral and anti-inflammatory activityevaluation of the commonly used herbs for HFMD therapyin China

2 Materials and Methods

21 Literature Survey of Plant Materials Plant MaterialAuthentication and Preparation of Hot Water Extracts In aneffort to identifymost commonly used Chinesemedicines forHFMDZhang et al surveyedChinese literatures from 1988 to2009 and identified Forsythia suspense Lonicera japonica andGlycyrrhiza spp as among the most commonly used herbsfor HFMD [8] Although some minerals like gypsum min-eral (CaSO

4sdot2H2O) and talcum powder (magnesium silicate

hydrate) or insect molting from cicada have been prescribed

forHFMDwe focused our investigation onherbalmedicinesWe generated a list of themost frequently used plantmaterialsfor HFMD by combining those identified by Zhang andcolleagues with those recommended by government agencies(Table 1)

On the basis of the frequency of their uses for HFMDtwelve medicinal materials were purchased from local herbalmarkets in Nanjing Voucher specimens were stored atthe Laboratory of Microbial Science School of MedicineNanjingUniversityThe corresponding Latin names followedby abbreviations of the names and parts of the plants used forpharmaceutical and clinical practices are listed in Table 1

We used a modified protocol to prepare water extracts[10] In brief two grams of ground specimen was soakedwith 30mL of distilled water at room temperature (RT) foran hour with occasional shaking Subsequently the materialwas warmed up to 65∘C in a water bath and extracted for4 hr The material was extracted one more time and thepooled liquid fractions were left at RT for overnight tolet insoluble materials settle down Finally water solublematerials were separated from the insoluble debris by cen-trifugation (5000timesg 15min) The major chemical compo-nents in those extracts were validated following protocolsgiven in Chinese Pharmacopeia (2010 ed) Those showedthat antiviral activities were further studied to validate thepresence of characteristic chemicals in the plants followingsome literatures (eg used HPLC profiling for H cordataand M haplocalyx) The water extracts were then dried bylyophilizationThe lyophilized powders were stored at minus70∘Cand dissolved in distilled water to make stocks of 20mgmLbefore experiments

The study design and assays used to validate the activityof the selected herbs are summarized in Figure 7

Evidence-Based Complementary and Alternative Medicine 3

22 Cells and Viruses The rhabdomyosarcoma (RD) cellline and African green monkey kidney epithelial (Vero) cellline were purchased from Cell Bank of Chinese AcademySciences (Shanghai China) The cells were maintained inDulbeccorsquos modified Eaglersquos medium (DMEM) with highglucose supplemented with 10 heat inactivated fetal bovineserum 2mM L-glutamine nonessential amino acids andsodiumpyruvate (themedium and supplements are fromLifeTechnologies) Cells were cultured at 37∘C in a humidifiedatmosphere with 5 CO

2

Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71)BrCr strain were obtained from China Center for TypeCulture Collection (CCTCC) atWuhan University A clinicalisolated EV71 Fuyang strain belongs to C4a cluster of the C4subgenotype as verified through sequence analysis of the VP1region [11] EV71 was propagated in RD cells and CVA16 inVero cells Virus titers were determined in the correspondingcells bymeasuring plaque forming units (PFU) of CVA16 andTCID

50for EV71 infection [12]

23 Antibody and Reagents Antibody against I120581B120572 (9242S)was purchased from Cell Signaling (Beverly MA USA)and anti-GAPDH monoclonal antibody (MB001) waspurchased from Bioworld Technology (Minneapolis MNUSA) The recombinant human TNF120572 was purchased fromSino Biological Inc (Beijing China) HRP-conjugatedsecondary antibodies and chemical reagents includingquercetin quercitrin isoquercitrin acyclovir (ACV) andmethylthiazolyldiphenyl-tetrazolium bromide (MTT) werepurchased from Sigma-Aldrich (Shanghai China)The SuperSignal ECL reagent kit was purchased from Thermo Fisher(Rockford IL USA)

24 Cytotoxicity Assay Vero cells (2 times 104 cells) wereseeded into 96-well plate and incubated for 12 hr Varyingconcentrations of extracts at up to 1mgmL were addedinto the medium Both treated cells and untreated cellswere incubated for 72 hr followed by incubation with MTT(05mgmL) for another 4 hr The ratio of optical density(OD) at 570 nm of treated cells to OD

570of untreated cells

was expressed as the survival rate of cells Triplicate wellswere analyzed for each concentration CC

50was defined as

the concentration of the extract to reduce the viable cell by50 relative to the untreated control cells

25 Antiviral Assays EV71 (Fuyang strain and BrCr strain)or CVA16 at a multiplicity of infection (MOI) of 02 was usedto infect the treated and mock-treated cells To study theantiviral activity of Houttuynia cordata (HCT) and Menthahaplocalyx (MHB) Vero cells infected with EV71 or CVA16were treatedwith or without a herb extract of various concen-trations The uninfected cells were used as negative controlAfter 72 hr of infection cells were stained with crystal violetafter fixation of the cells with 3 formaldehyde and capturedby a camera and then DMSO was added into the wells todissolve crystal violet and the absorbance of 570 nm wasmeasured in a Versa Max microtiter plate reader (MolecularDevices Sunnyvale CA USA) The concentration of an

extract that is necessary for 50 inhibition of the virus-induced CPE was expressed as the IC

50

To verify the antiviral effect secondary infection wasperformed Serial dilutions of culture supernatants collectedfrom EV71 or CVA16 infected cells were employed to infectmonolayers of Vero cells and to determine the titration ofinfectious viruses as described [13] The TCID

50numbers

were calculated using the method of Reed and Muench [12]The plaque formation was assessed 5 days postinfection (PI)after being fixed with 3 formaldehyde for 20min and thenfollowed by crystal violet staining as described [14] Thenumber of plaque was counted manually The infection wasperformed in triplicate samples

In time-of-drug addition experiment HCT extract at40 120583gmL or MHB extract at 200120583gmL were added at timepoints specified in the figures They were either added beforeviral adsorption during adsorption (minus2 to 0 hr PI) or atvarying times after viral adsorption HCT orMHBwas left inthe medium throughout the infection assay The cytopathiceffect was determined by measuring cell viability using MTTassay [15] at 72 hr PI An inhibition rate was calculated asa percentage of (ODtreated minus ODinfected) over (ODuninfected minusODinfected)

To study whether the extracts directly deactivate thevirion EV71 or CVA16was preincubatedwith 40120583gmLHCTand 200120583gmL MHB in 20120583L respectively or remaineduntreated The viruses were then used to infect Vero cellscultured with 1mL culture medium Alternatively Vero cellsin 24 well plates were preincubated with 40120583gmL HCT orwith 200120583gmLMHB at 37∘C for 2 hr or remained untreatedAt the end of the treatment the extract was removed byreplacing the culture mediumwith fresh DMEMor left in theculture and the cells were infectedwith EV71 or CVA16 (MOIwas at 02 but final concentrations of HCT andMHB were at08 120583gmL and 4 120583gmL resp) The samples were harvestedat 48 hr PI for titration of infectious viruses with a secondaryinfection assay

26 Immunoblotting Assays A buffer containing 150mMNaCl 50mMTris-HCl (pH 74) and 1NP-40was employedto encourage lysis of cells and to solubilize proteins Proteaseand phosphatase are also added to prevent the digestion of thesample by its own enzymes Prior to protein immobilizationon a polyvinylidene difluoridemembrane (Millipore) sampleproteins were separated using SDS polyacrylamide gel elec-trophoresis (SDS-PAGE) After blocking the membrane wasincubatedwith a dilute solution of primary antibody and thenwith a horseradish peroxidase-linked secondary antibodyunder gentle agitation Depending on incubation with asubstrate the images were captured using Alpha InnotechFlourChem-FC2 imaging system and developed manually

27 Semiquantification of Proinflammatory Gene ExpressionUsing PCR or qPCR Vero cells infected with EV71 or CVA16were treated withHCT orMHB respectivelyThen total RNAwas extracted by using TRIzol (Invitrogen Carlsbad CAUSA) and following the standard protocol With a Nanodropreader the quantity and purity of total RNA were measured


DNA was synthesized using reverse transcriptase MMLV(RNase Hminus) from 500 ng total RNA with oligo-dT primercDNA was amplified by using gene-specific primers and TaqDNA Polymerase (TaKaRa Tokyo Japan)

Real-time PCR was carried out using the SYBR qPCRMix (TOYOBO Japan) The protocol was carried out for 40cycles comprising 95∘C for 1min 95∘C for 15 s and 60∘Cfor 30 s The threshold cycle (Ct) values obtained from theseexperiments indicated the fractional cycle numbers at whichthe amount of amplified target reached a fixed thresholdThe2minusΔΔCt method [16] was used to quantify and normalize therelative quantification data Datawere emerged as fold change(2minusΔΔCt) which was the amount of IL-6 mRNA normalizedto the internal control (GAPDH) All PCR reactions wereperformed with equal amounts of cDNA using the Bio-RadC1000 Real-Time PCR System A dissociation curve wasdrawn for each primer pair to assess that therewas no primer-dimer formation

Primer sequences were as follows IL-6 51015840-AACCTGAACCTTCCAAAGATGG and 51015840-TCTGGCTTGTTCCTCACTACTGAPDH 51015840-ATGACATCAAGAAGGTGGTGand51015840-CATACCAGGAAATGAGCTTG

28 HPLC Profiling and Quantitative Determination of theChemical Components in the Extract of HCT The identityof the water extract was characterized by HPLC profilingfor several flavonoids The separations were carried out ona Thermo Scientific Hypersil GOLD column (46mm times250mm 5 120583m)The column temperature was set at 25∘CThemobile phase consisted of 01 trifluoroacetic acid (A) andacetonitrile (B)The gradient conditionwas 15B (VV) at 0ndash16min 15ndash25 B at 16ndash30min 25 B at 30ndash32min 25ndash30B at 32ndash35min 30 B at 35ndash37min 30ndash33 B at 37ndash40min33 B at 40ndash45min 33ndash60 B at 45ndash60min and 60ndash15 Bat 60ndash65minThe flow rate was 08mLmin and the injectionvolume was 20120583L Detection wavelength was set at 360 nmThewater extract was identified by comparing their retentiontimes and UV spectra with those of the markers

3 Results

31 Literature Survey of Plant Materials Used for HFMD Wegenerated a list of themost commonly used or recommendedplantmaterials forHFMDas listed in Table 1 In generalmostof the herbs in the list are bitter or bitter-cold in nature andcapable of expelling dampheat in the lung or spleen-stomachwhich is the principle for treating diseases of dampness anddamp heat More specifically the overwhelming majority ofthe materials have been used for inflammatory and infectivediseases For example Lonicera japonica and pharmaceuticalscontaining extracts from the flower buds are widely usedin China for common cold and for inflammatory diseasesof the upper respiratory track Isatis indigotica Scutellariabaicalensis andGlycyrrhiza uralensiswere found to have anti-Japanese encephalitis virus SARS-coronavirus and respira-tory syncytial virus activity [17ndash20] Although Houttuyniacordata and Glycyrrhiza uralensis have been reported withanti-EV71 activity [21 22] most of the plants have not been

evaluated for their activity against HFMD pathogens Thechemical components for the antiviral activity also remainelusive

32 Sample Preparation and Antiviral Activity EvaluationTo perform systematic evaluation of the materials for theirantiviral and anti-infective activities herbal materials werepurchased from local pharmacy stores and authenticatedfollowing methods listed in Chinese Pharmacopeia Thematerials were extracted in hot water and the water extractswere lyophilizedThe lyophilized powders were reconstitutedin distilled water and tested for cytotoxicity prior to theevaluation for their antiviral activity At up to 1000120583gmLconcentrations all extracts except that from Scutellariabaicalensis showedno cytotoxic effect inVero cells Scutellariabaicalensis extract started to show toxic effect at approxi-mately 100 120583gmL in Vero cells with an estimated CC

50value

of 2402120583gmL To assay for their antiviral activity againstEV71 and CVA16 infection monolayers of Vero cells weretreated with an extract at 1000 120583gmL or with Scutellariabaicalensis extract at a nontoxic concentration of 30 120583gmLfor 2 hr or remained untreated The cells were then infectedwith EV71 or CVA16 (MOI = 02) for 72 hr At the end of aninfection the cells were fixed with formaldehyde and exam-ined visually for cytopathic effect Although recommended orwidely used for HFMD disease only three materials showedmoderate to strong activity against EV71 or CVA16 infection(Table 1) The extract of Houttuynia cordata significantlyblocked the cytopathic effect associated with EV71 Theextract did not exhibit significant activity against CVA16infection In contrast MHB treatment significantly blockedthe cytopathic effect by CVA16 but not by EV71 infectionAn extract from Glycyrrhiza uralensis was reported withstrong activity against EV71 infection [22] We found thatthe extract was marginally active against EV71 and CVA16infection Further studies demonstrated that glycyrrhizicacid possessed anti-EV71 activity and anti-CVA16 activity atmillimolar concentrations

33 Validation and Determination of IC50

Values of PlantMaterials with Antiviral Activities We then focused oureffort on validation of the anti-EV71 activity by HCT andthe anti-CVA16 activity of MHB by determining the IC

50

values Vero cells were treated with HCT or with MHB atvarying concentrations 2 hr prior to inoculation The cellswere then infected with EV71 or CVA16 in the presence orabsence of the extracts for 72 hr As shown in Figures 1(a)and 1(b) pretreatment with the extracts dose dependentlyblocked the cytopathic effect by those viruses The IC

50

value of HCT to block EV71 infection was estimated at89 and 206 120583gmL against a Fuyang strain and the BrCrstrain of EV71 respectively The IC

50value of MHB to block

CVA16 infection was estimated at 703 120583gmL while theIC50

for HCT to block CVA16 infection was determinedat 1863 120583gmL The production of infectious virion wasquantitatively determined in parallel experiments to correlatethe cytopathic effect with infectious virion production Asshown in Figure 1(c) EV71 production (Fuyang) was reduced


EV71

0

20

40

60

80

100

120Re

lativ

e sta

inin

g (

of c

ontro

l)

EV71 5 10 20 40

HCT (120583gmL)

(a)

CVA16

CVA16 50 100 200 ACV0

20

40

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Relat

ive s

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( o

f con

trol)

MHB (120583gmL)

(b)

0

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EV71 10 20 40

HCT (120583gmL)

TCID50m

L (lo

g 10)

(c)

3CVA16 50 100 200

4

5

6

7

8

PFU

mL

(log 10)

MHB (120583gmL)

(d)

Figure 1 Antiviral activity evaluation of HCT and MHB extracts against EV71 and CVA16 (a b) Pretreatment of Vero cells with HCT orMHB blocks the cytopathic effect by virus infection Vero cells were pretreated with or without a herb extract at concentrations as indicatedfor 2 hr and the cells were then infected with EV71 (a Fuyang strain) or CVA16 (b) at anMOI of 02 TCID

50cell or PFUcell After incubation

for 72 hr the cells were fixed with 3 formaldehyde stained with 05 crystal violet The cells were photographed To quantitatively measurethe staining crystal violet was extracted by DMSO and the absorbance at 570 nm was measured colorimetrically An increase in readingcompared with that of an infected control indicates an inhibitory effect of the extract against virus infection Data are presented as mean plusmn SEof duplicate samples (c d) HCT and MHB treatments reduce infectious virion production of EV71 and CVA16 respectively Vero cells wereinfected with EV71 or CVA16 (MOI = 02) in the presence or absence of a herbal extract at indicated concentrations The cells and culturesupernatants were harvested at 48 hr PI and determined for virus titration by TCID

50assay for EV71 (c) and by plaque forming assay for

CVA16 (d) Data are presented as mean plusmn SE of triplicate samples The results are representative of two independent experiments

by approximately 18 37 and 56 logs in samples treated withHCT at 10 20 and 40 120583gmL respectively In comparisonMHB at 50 100 and 200120583gmL reduced CVA16 productionby 11 15 and 31 logs respectively (Figure 1(d))

34 Time-of-Drug-Addition Study Suggests That HCT BlocksEV71 Infection at an Early Stage of Infection While MHB

Targets Multiple Events in Suppression of CVA16 InfectionVirus infection is initiated by virus attachment to host cellsfollowed by virus cell entry and replication intracellularlyTo preliminarily determine at what stage of infection theextracts blocked virus infection monolayers of Vero cellswere treated with HCT at 40 120583gmL or MHB at 200120583gmL2 hr prior to inoculation or at varying times of virus infection


0

20

40

60

80

100

120

minus2 0 2 4 6 8 10 12 14 16 18 20 22 24

Inhi

bitio

n (

)

Time (hr)

EV71 + HCTCVA16 + MHB

InoculationDrug addition

(a)

1

3

5

7

EV71 Mock Virus Cell PlusHCT

TCID50m

L (lo

g 10)

(b)

35

45

55

65

PFU

mL

(log 10)

CVA16 Mock Virus Cell Plus MHB

(c)

Figure 2 HCT directly inactivates EV71 virion whereas MHB blocks CVA16 infection by targeting both the virion and cellular factors (a)Time course study of HCT and MHB antiviral activity HCT at 40 120583gmL and MHB at 200120583gmL were added at 2 hr prior to (minus2 hr) during(0 hr) or postvirus inoculation at times as indicated (2 hr 4 hr 8 hr 10 hr 12 hr and 24 hr PI) Virus infection was determined at 72 hr PI bymeasuring cell viability colorimetrically Data are presented as an inhibition rate which is calculated as described in Materials and Methods(b c) Coincubation of EV71 with HCT abolishes EV71 infectivity andMHB inhibits CVA16 infection likely through viral and cellular factorsEV71 (b) or CVA16 (c) in 20120583L culture mediumwasmock-treated or treated with HCT at a concentration of 40120583gmL orMHB at 200 120583gmLin a 37∘C water bath respectively for 2 hr (virus) The treated samples were then added to 10mL fresh culture medium and used to infectVero cells (final MOIs at 02 and final concentrations of HCT and MHB were at 08 120583gmL and 4 120583gmL resp) In parallel experiments Verocells were pretreated with HCT at 40120583gmL or with MHB at 200120583gmL at 37∘C for 2 hr The drugs were replaced with fresh culture medium(cell) or left in the culture medium (plus drug) The cells were then infected with EV71 or with CVA16 at equal MOIs Virus infection wasassayed by secondary infection assays at 48 hr PI Pretreatment of EV71 but not Vero cells with HCT abolished the infectivity of EV71 virusTreatment of CVA16 or Vero cells with MHB blocked CVA16 infection Data are presented as mean plusmn SE of triplicate samples The results arerepresentative of two independent experiments

The effect of drug addition on virus infection was determinedat 72 hr PI As shown in Figure 2(a) addition of HCT priorto the inoculation or during the inoculation significantlysuppressed EV71 infection The effect became less significantwhen HCT was added at later time points In comparisonMHB was effective when used prior to or within the first fourhours of inoculation Again the effect became less significantwhen added thereafter Receptor-mediated endocytosis ofvirus cell entry is generally a rapid processThe result suggeststhat HCT likely targets EV71 virus by blocking virus cellattachment or by direct inactivation of infectious virionwhereas MHB potentially targets multiple events of CVA16infection

To determine whether HCT extract directly deactivatesEV71 virion 1 times 104 TCID

50EV71 in 20120583L culture medium

was incubated with HCT at 40120583gmL for 2 hr or remaineduntreated The viruses were then used to infect Vero cells atan MOI of 02 TICD

50cell (the final concentration of HCT

was 08 120583gmL) In parallel experiments Vero cells in 24 wellplates were preincubated with 40 120583gmL of HCT at 37∘C for2 hr or remained untreated At the end of the treatment theextract was removed by replacing the culture medium withfresh DMEM or left in the culture and the cells were infectedwith EV71 at 02 TICD

50cell As shown in Figure 2(b)

pretreatment of EV71 but not the host cells with HCTabolished the infectivity of EV71 virus indicating that HCT

Evidence-Based Complementary and Alternative Medicine 7In

tens

ity (m

V)

1

1 2

23

3

4

4

Time of retention (min)

minus20minus10

010

30

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130

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0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Standard

Extract

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

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nsity

(mV

)

minus20minus10

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0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 722 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

(a)

15

20

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EV71 3 10 3 10 3 10 3 10 3 10

Cel

l via

bilit

y (

of c

ontro

l)

Quercetin Quercitrin Isoq Hepe Chlo(120583M)

(b)

Figure 3 Treatment with flavonoids or chlorogenic acid of HCTdoes not block EV71 infection (a) HPLC profiling of water extractof HCT The identity of the water extract of HCT was characterizedbyHPLC profiling on aHypersil GOLD column (46mmtimes 250mm5120583m) and acetonitrile with increasing concentration of 01 triflu-oroacetic acid as a mobile phase Hyperoside (peak 1) isoquercitrin(2) quercitrin (3) and quercetin (4) were used as standards (b)Flavonoids or chlorogenic acid inHCThas no antiviral effect againstEV71 infection Vero cells were pretreatedwith or without quercetinquercitrin isoquercitrin (Isoq) hyperoside (Hepe) or chlorogenicacid (Chlo) at indicated concentrationsThe cells were then infectedwith EV71 at an MOI of 02 TCID

50cell for 72 hr and cell viability

was measured colorimetrically Data are presented as mean plusmn SE oftriplicate samplesThe results are representative of two independentexperiments

directly deactivates EV71 virion In a similar experiment wefound that preincubation of CVA16 or the host cells withMHB reduced infectious CVA16 production by 21 logs and09 logs The result suggests that MHB potentially targetsseveral steps of CVA16 infection (Figure 2(c))

35 Antiviral Activity Evaluation of the Flavonoids fromHCT The chemical contents of HCT and MHB are wellcharacterized HCT contains 2-undecanone in the essentialoil and water soluble chlorogenic acid and flavonoids likequercetin quercitrin isoquercitrin and hyperoside whilethe involatile components of MHB include triterpenoidsand flavonoids like acacetin tilianin and linarin Several

flavonoids like kaempferol [23] and chrysosplenetin [24] haverecently been shown with strong antiviral activity againstEV71 infection In a recent study we also found the flavonoidsaccounted for the anti-HSV activity of H cordata [25] Todetermine whether the flavonoids were responsible for Hcordata anti-EV71 activity flavonoid content in HCTwas firstprofiled by HPLC analysis (Figure 3(a)) The extract con-tained quercetin quercitrin isoquercitrin and hyperosideWhen tested for their antiviral activity against EV71 infectionusing the flavonoids purchased from a commercial sourcethe compounds did not display significant activity againstEV71 infection (Figure 3(b))

36 Anti-Inflammatory Activity of the Herbal Materials Usedfor HFMD Virus infection tends to promote proinflamma-tory cytokine production and elevated production of IL-1120573IL-6 and IL-8 has been detected in patients withHFMD [26]The production of IL-6 and IL-8 is regulated transcriptionallyby NF-120581B pathway whose activation can be assayed bymonitoring the degradation of I120581B120572 the inhibitory proteinof NF-120581B activity We next investigated whether the extractsfrom commonly used herbs for HFMD had activity insuppressing inflammatory response by investigating whetherthe extracts blocked I120581B120572 degradation As shown in Figure 4EV71 and CVA16 infection promoted I120581B120572 degradation atapproximately 10 hr PI (Figures 4(a) and 4(d)) Consistentwith the results from antiviral studies pretreatment of Verocells with HCT or MHB blocked I120581B120572 degradation causedby EV71 and CVA16 infection (Figures 4(b) and 4(e))respectively In addition the extracts also showed inhibitoryactivity against TNF120572-induced I120581B120572 degradation (Figures4(c) and 4(f)) indicating that the extracts had activity againstproinflammatory factor stimulation

To extend those findings we investigated whetherextracts that showed no antiviral activities possessed activityagainst proinflammatory response to viral infection Tothis end Vero cells were pretreated with the extracts orremained untreatedThe cells were then infectedwith EV71 orCVA16 I120581B120572 degradation was detected by immunoblottinganalysis As shown in Figures 5(a) and 5(b) several extractsshowed inhibitory activity against infection-induced I120581B120572degradation by EV71 or CVA16 infection indicating thatthose extracts have anti-inflammatory activity

Finally the inhibitory effect of HCT and MHB on proin-flammatory response was verified by examining expression ofdownstream genes (Figure 6) The cells were pretreated withHCT or MHB for 2 hr before infection with EV71 or CVA16at 3 MOI for 16 hr Both extracts significantly reduced theaccumulation of IL-6 mRNA by EV71 and CVA16 infectionrespectively as determined by reverse-transcription PCR(upper panels) as well as quantitative real-time PCR (lowerpanels)

Together we showed here that the herbal materials rec-ommended by government agencies or commonly prescribedby practitioners of Chinesemedicine possess antiviral activityandor anti-inflammatory activity validating the usefulnessof those medicinal materials for HFMD disease


EV71

GAPDH

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(a)

20 40

GAPDH

I120581B120572

(+)

HCT (120583gmL)

Con EV

(b)

20 40

HCT (120583gmL)

Con TNF120572

(c)

C

GAPDH

CVA16

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(d)

CVA

GAPDH

I120581B120572

(+)MHB (120583gmL)

Con 100 200

(e)

MHB (120583gmL)100 200Con TNF120572

(f)

Figure 4 Inhibition of infection-induced I120581B120572 degradation by HCT andMHB (a d) Determination of the time courses of I120581B120572 degradationduring EV71 and CVA16 infections Vero cells were infected with EV71 (a) or CVA16 (d) at an MOI of 3 for indicated times The cytoplasmiclevels of I120581B120572 proteins were determined by immunoblotting assay At 10 hr PI I120581B120572was significantly degraded implying theNF-120581B activation(b c) HCT treatment blocks I120581B120572 degradation induced by EV71 infection and TNF120572 treatment Vero cells were infected with EV71 (b MOI= 3) for 10 hr or treated with 1 ngmL TNF120572 (c) for 20min in presence or absence of HCT at indicated concentrations I120581B120572 degradationwas detected by immunoblotting assay (e f) MHB treatment blocks I120581B120572 degradation induced by CVA16 infection and TNF120572 treatmentVero cells were infected with CVA16 (e MOI = 3) for 10 hr or treated with 1 ngmL TNF120572 (f) for 20min in presence or absence of MHB atindicated concentrations I120581B120572 degradation was detected by immunoblotting assay GAPDH was used as a loading control The results arerepresentative of two independent experiments TNF120572 (+) at 3 ngmL was used as a positive control for I120581B120572 degradation

GAPDH

EV71

EV71

Con mdash

Con mdash

1 2 3 4 5 6

7 8 9 10 11 12

I120581B120572

GAPDH

I120581B120572

(a)

CVA16

CVA16

Con mdash 1 2 3 4 5 6

Con mdash 7 8 9 10 11 12

GAPDH

I120581B120572

GAPDH

I120581B120572

(b)

Figure 5 The effect of herb extracts on virus-induced I120581B120572 degradation Vero cells were pretreated with extracts (1 EFT 2 IIFL 3 GUF 4GJE 5 MHB 6 HCT 7 IIFS 8 LJT 9 FSV 10 ALL 11 SBG 12 AAB) for 2 hr and then infected with EV71 (a) or CVA16 (b) at an MOI of 3for 10 hr I120581B120572 degradation was detected by immunoblotting assay

4 Discussions

There are currently no treatments for HFMD Diuret-ics and intravenous administration of dexamethasone andimmunoglobulin-120574 are among the most commonly recom-mended drugs for patients with more severe syndromesTraditional medicine and the use of herbal medicines forHFMD are also among the recommended approaches forthe management of HFMD in China The effectiveness ofherbs against HFMD has not previously been evaluated ina laboratory setting In this study we selected 12 herbs from

government agency-recommended lists and those of themostcommonly prescribed We focused on their antiviral activityevaluation against EV71 and CVA16 and their activity againstproinflammatory response since viral infections tend to beassociated with proinflammatory response

EV71 and CVA16 are the two major causative agentscausing HFMD We found that a water extract ofHouttuyniacordata has strong activity against EV71 infection whileMen-tha haplocalyx has antiviral activity against CVA16 Althoughmost of the herbs from this list showed no activity againstviral infection several extracts demonstrated activity against


20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)

Figure 6 The herb extracts inhibit EV71 or CVA16 infection induced proinflammatory response Vero cells were infected with EV71 (a) orCVA16 (b) at an MOI of 3 in the presence or absence of HCT or MHB at indicated concentrations The total RNA was isolated by the TRIzolreagent at 16 hr PI Five hundred nanograms of total RNA was reverse transcribed and amplified by PCR (upper panels) or by quantitativereal-time PCR (lower panels) using primers specific for IL-6 and GAPDH PCR products were run on a 20 agarose gel and visualizedwith ethidium bromide staining Data are presented as mean plusmn SE of triplicate samples The results are representative of two independentexperiments

Selection of herbs for HFMD

Specimen authentication

Preparation of water extracts

Assays for activity validation

(1) Cell culture antiviral assay(2) Inhibition of inflammation

(1) Botanical(2) Chromatographical

(2) Literature survey(1) Government recommendation

Figure 7 Summary of study design and methodology applied formicrobiological validation of medicinal herbs commonly used inChina for HFMD

proinflammatory response induced by virus infection Thestudy therefore validates the effectiveness of herbal medicinefor an emerging viral disease

Many herbs have been commonly prescribed or recom-mended for the treatment of HFMD we found Houttuynia

cordata Thunb was the only remedy with potent antiviralactivity against both EV71 and CVA16 Houttuynia cordatahas been widely used as an edible vegetable in Chineseand Vietnamese cuisines and has a long history of use inChinese medicine The medicinal use of this plant includesits antiseptic anti-inflammatory and febrifuge activities Ina previous study Lin et al identified Houttuynia cordata asthe only material with activity against EV71 infection among22 medicinally used herbs from Taiwan Consistent with theobservation by Lin et al [21] we also found that the waterextract from Houttuynia cordata was active against CVA16extending the antiviral spectrum of this plant Unlike otherwidely used herbs Houttuynia cordata was recommendedfor treatment of HFMD with severe syndrome by the Gov-ernment of Guangxi Zhuang Autonomous Region a regionwith relatively low rate of HFMD incidences and deathFurthermore the plant contains flavonoids and undecanoneIn a preliminary screening study we failed to detect anyantiviral activity with those compounds Instead we foundthat activity was stable against heat and can be extracted outto n-butanol fraction Further study is warranted to identifythe active components from this plant

It is worthy of noticing that the herbs were tested assingle identity while clinically they are used in combi-nation or formulation containing several other herbals or


with cicada molting or even inorganic minerals in somecases Combined use of those herbs may adversely or inad-versely affect the effectiveness of those materials for theirantiviral and anti-inflammatory activities Although this isa laboratory study the result from this study neverthelessvalidates the usefulness of herbal medicine for the man-agement of previously undocumented diseases in traditionalmedicines

Authorsrsquo Contribution

X Chen and C Wang contributed equally to this paper

Acknowledgments

This work was supported by Grants from NSFC (9081303681121062) Basic Research Foundations of Central Universi-ties (1114021404) and Jiangsu Province University PhysicianProgram (11KJB320002) X Chen is an awardee of JiangsuInnovation Program of Predoctoral Students

References

[1] F Yang L Ren Z Xiong et al ldquoEnterovirus 71 outbreak inthe Peoplersquos Republic of China in 2008rdquo Journal of ClinicalMicrobiology vol 47 no 7 pp 2351ndash2352 2009

[2] WHO Report on the Hand Foot and Mouth Disease Outbreakin Fuyang City Anhui Province and the Prevention and Con-trol in China 2008 httpwww2wprowhointNRrdonlyres591D6A7B-FB15-4E94-A1E9-1D3381847D600HFMDCCDC20080515ENG pdf

[3] H Tian Q Z Yang J Liang et al ldquoClinical features andmanagement outcomes of severe hand foot andmouth diseaserdquoMedical Principles and Practice vol 21 no 4 pp 355ndash359 2012

[4] M H Ooi S C Wong P Lewthwaite M J Cardosa andT Solomon ldquoClinical features diagnosis and management ofenterovirus 71rdquo The Lancet Neurology vol 9 no 11 pp 1097ndash1105 2010

[5] E Ma C Fung S H L Yip C Wong S K Chuang andT Tsang ldquoEstimation of the basic reproduction number ofenterovirus 71 and coxsackievirus A16 in hand foot andmouthdisease outbreaksrdquoThe Pediatric Infectious Disease Journal vol30 no 8 pp 675ndash679 2011

[6] T Solomon P Lewthwaite D PereraM J Cardosa PMcMinnand M H Ooi ldquoVirology epidemiology pathogenesis andcontrol of enterovirus 71rdquoThe Lancet Infectious Diseases vol 10no 11 pp 778ndash790 2010

[7] H J Cao Z L Liu P Steinmann et al ldquoChinese herbalmedicines for treatment of hand foot and mouth diseasea systematic review of randomized clinical trialsrdquo EuropeanJournal of Integrative Medicine vol 4 no 1 pp e85ndashe111 2012

[8] G L Zhang Z G Li J B Tong et al ldquoA review of Chinesetraditional prescriptions used in treatment and prevention ofhand foot and mouth diseaserdquo Clinical Journal of TraditionalChinese Medicine vol 22 no 7 pp 574ndash582 2010

[9] B Xue Z Yao and R Yu ldquoStudies on anti-EV71 virus activityof traditional Chinese medicine and its clinical application intreatment of HFMDrdquo Zhongguo Zhong Yao Za Zhi vol 36 no23 pp 3366ndash3370 2011

[10] T Hozumi T Matsumoto H Ooyama et al ldquoAntiviral agentcontaining crude drugrdquo US Patent 5411733 1995

[11] Y Zhang Z Zhu W Yang et al ldquoAn emerging recombinanthuman enterovirus 71 responsible for the 2008 outbreak ofhand foot and mouth disease in Fuyang city of Chinardquo VirologyJournal vol 7 no 1 article 94 2010

[12] L J Reed and H Muench ldquoA simple method of estimating fiftyper cent endpointsrdquo American Journal of Epidemiology vol 27no 3 pp 493ndash497 1938

[13] C C Liu A H Chou S P Lien et al ldquoIdentification andcharacterization of a cross-neutralization epitope of Enterovirus71rdquo Vaccine vol 29 no 26 pp 4362ndash4372 2011

[14] R Dulbecco and M Vogt ldquoPlaque formation and isolation ofpure lines with poliomyelitis virusesrdquo Journal of ExperimentalMedicine vol 99 no 2 pp 167ndash182 1954

[15] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[16] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-DeltaDelta C(T))MethodrdquoMethods vol 25 no 4 pp 402ndash4082001

[17] CW Lin F J Tsai C H Tsai et al ldquoAnti-SARS coronavirus 3C-like protease effects of Isatis indigotica root and plant-derivedphenolic compoundsrdquo Antiviral Research vol 68 no 1 pp 36ndash42 2005

[18] S C Ma J Du P P H But et al ldquoAntiviral Chinese medicinalherbs against respiratory syncytial virusrdquo Journal of Ethnophar-macology vol 79 no 2 pp 205ndash211 2002

[19] J Cinatl BMorgensternG Bauer P ChandraH Rabenau andH W Doerr ldquoGlycyrrhizin an active component of liquoriceroots and replication of SARS-associated coronavirusrdquo TheLancet vol 361 no 9374 pp 2045ndash2046 2003

[20] S J Chang Y C Chang K Z Lu et al ldquoAntiviral activityof Isatis indigotica extract and its derived indirubin againstJapanese encephalitis virusrdquo Evidence-Based Complementaryand Alternative Medicine vol 2012 Article ID 925830 7 pages2012

[21] T Y Lin Y C Liu J R Jheng et al ldquoAnti-enterovirus 71 activityscreening of Chinese herbs with anti-infection and inflamma-tion activitiesrdquo The American Journal of Chinese Medicine vol37 no 1 pp 143ndash158 2009

[22] K K Kuo J S Chang K C Wang and L C Chiang ldquoWaterextract of Glycyrrhiza uralensis inhibited enterovirus 71 in ahuman foreskin fibroblast cell linerdquo The American Journal ofChinese Medicine vol 37 no 2 pp 383ndash394 2009

[23] F J Tsai C W Lin C C Lai et al ldquoKaempferol inhibitsenterovirus 71 replication and internal ribosome entry site(IRES) activity through FUBP and HNRP proteinsrdquo FoodChemistry vol 128 no 2 pp 312ndash322 2011

[24] Q C Zhu Y Wang Y P Liu et al ldquoInhibition of enterovirus71 replication by chrysosplenetin and penduletinrdquo EuropeanJournal of Pharmaceutical Sciences vol 44 no 3 pp 392ndash3982011

[25] X Chen Z Wang Z Yang et al ldquoHouttuynia cordata blocksHSV infection through inhibition of NF-kappaB activationrdquoAntiviral Research vol 92 no 2 pp 341ndash345 2011

[26] T Y Lin S H Hsia Y C Huang C T Wu and L YChang ldquoProinflammatory cytokine reactions in enterovirus 71infections of the central nervous systemrdquo Clinical InfectiousDiseases vol 36 no 3 pp 269ndash274 2003

Submit your manuscripts athttpwwwhindawicom

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


Table 1 Evaluation of cytotoxicity and antiviral activity of plant materials used for HFMD in Chinalowast

Latin name Abbr Parts traditional usage CC50 (120583gmL) IC50 (120583gmL)EV71 CVA16

Houttuynia cordataThunb HCT Stem and leaves bacterial and viral disease gt1000 89 plusmn 16 (FY)206 plusmn 25 (BrCr) 1863 plusmn 201

Mentha haplocalyx Briq MHB Leaves viral and bacterial disease gt1000 (minus) 703 plusmn 46Glycyrrhiza uralensis Fisch GUF Stem and root coughing gt1000 (plusmn) (plusmn)Gardenia jasminoides Ellis GJE Fruits icteric hepatitis gt1000 (minus) (minus)Anemarrhena asphodeloides Bge AAB Stem and root coughing gt1000 (minus) (minus)Arctium lappa L ALL Fruits coughing gt1000 (minus) (minus)Eupatorium fortunei Turcz EFT Stem and leaves fever gt1000 (minus) (minus)Lonicera japonicaThunb LJT Flowers inflammation gt1000 (minus) (minus)Scutellaria baicalensis Georgi SBG Root common cold 2402 plusmn 154 (minus) (minus)Isatis indigotica Fort IIFL Leaves coughing gt1000 (minus) (minus)Isatis indigotica Fort IIFS Root fever and viral disease gt1000 (minus) (minus)Forsythia suspensa (Thunb) Vahl FSV Fruits viral disease gt1000 (minus) (minus)lowastThe reference of Latin names abbreviations parts used in pharmaceuticals and traditional usages of the medicinal plants and data on antiviral screeningagainst EV71 (Fuyang strain and BrCr strain) and CVA16 are listed Cytotoxicity was assayed on Vero cells at a maximal concentration of 1000120583gmL Thoseshowed toxicity against Vero cells were further tested to determine a CC50 value while those showed no toxic effect at the maximal concentration tested werereported as gt1000120583gmL IC50 50 inhibitory activity against virus infection-induced cytopathic effectData are presented as mean plusmn SE of triplicates All experiments were performed at least twice with triplicate samples FY EV71 Fuyang strain (minus) no activityat 1000120583gmL (plusmn) marginally active

manifestations characterized with mild fever and rash prac-titioners of Chinese medicine reasoned and have effectivelytreated HFMD as dampness and damp heat We compileda list of commonly used herbs with implication for HFMDtherapy (Table 1) from a list of recommended remedies bygovernment agencies in China and from published reports[7ndash9] Most of the herbs in Table 1 have traditionally orfolklorically been used for inflammatory or infective diseasesIn addition most of the herbs listed in Table 1 have not beeninvestigated for their antiviral activity against pathogens ofHFMD disease although some of them have demonstratedantiviral activity Less is known about the active componentsof those plants

The objectives of this study were to validate the effec-tiveness of the herbs microbiologically by measuring theirantiviral activity In addition we also studied whether theherbs possessed anti-inflammatory activity since one of thesymptoms of HFMD is an acute inflammatory responseHere we report the antiviral and anti-inflammatory activityevaluation of the commonly used herbs for HFMD therapyin China

2 Materials and Methods

21 Literature Survey of Plant Materials Plant MaterialAuthentication and Preparation of Hot Water Extracts In aneffort to identifymost commonly used Chinesemedicines forHFMDZhang et al surveyedChinese literatures from 1988 to2009 and identified Forsythia suspense Lonicera japonica andGlycyrrhiza spp as among the most commonly used herbsfor HFMD [8] Although some minerals like gypsum min-eral (CaSO

4sdot2H2O) and talcum powder (magnesium silicate

hydrate) or insect molting from cicada have been prescribed

forHFMDwe focused our investigation onherbalmedicinesWe generated a list of themost frequently used plantmaterialsfor HFMD by combining those identified by Zhang andcolleagues with those recommended by government agencies(Table 1)

On the basis of the frequency of their uses for HFMDtwelve medicinal materials were purchased from local herbalmarkets in Nanjing Voucher specimens were stored atthe Laboratory of Microbial Science School of MedicineNanjingUniversityThe corresponding Latin names followedby abbreviations of the names and parts of the plants used forpharmaceutical and clinical practices are listed in Table 1

We used a modified protocol to prepare water extracts[10] In brief two grams of ground specimen was soakedwith 30mL of distilled water at room temperature (RT) foran hour with occasional shaking Subsequently the materialwas warmed up to 65∘C in a water bath and extracted for4 hr The material was extracted one more time and thepooled liquid fractions were left at RT for overnight tolet insoluble materials settle down Finally water solublematerials were separated from the insoluble debris by cen-trifugation (5000timesg 15min) The major chemical compo-nents in those extracts were validated following protocolsgiven in Chinese Pharmacopeia (2010 ed) Those showedthat antiviral activities were further studied to validate thepresence of characteristic chemicals in the plants followingsome literatures (eg used HPLC profiling for H cordataand M haplocalyx) The water extracts were then dried bylyophilizationThe lyophilized powders were stored at minus70∘Cand dissolved in distilled water to make stocks of 20mgmLbefore experiments

The study design and assays used to validate the activityof the selected herbs are summarized in Figure 7



2







50was defined as




50


50numbers











3 Results




50value




50


50






EV71

0

20

40

60

80

100

120Re

lativ

e sta

inin

g (

of c

ontro

l)

EV71 5 10 20 40

HCT (120583gmL)

(a)

CVA16

CVA16 50 100 200 ACV0

20

40

60

80

100

120

Relat

ive s

tain

ing

( o

f con

trol)

MHB (120583gmL)

(b)

0

2

4

6

8

EV71 10 20 40

HCT (120583gmL)

TCID50m

L (lo

g 10)

(c)

3CVA16 50 100 200

4

5

6

7

8

PFU

mL

(log 10)

MHB (120583gmL)

(d)










0

20

40

60

80

100

120

minus2 0 2 4 6 8 10 12 14 16 18 20 22 24

Inhi

bitio

n (

)

Time (hr)



(a)

1

3

5

7


TCID50m

L (lo

g 10)

(b)

35

45

55

65

PFU

mL

(log 10)


(c)











tens

ity (m

V)

1

1 2

23

3

4

4


minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Standard

Extract

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

Inte

nsity

(mV

)

minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 722 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

(a)

15

20

25

30

EV71 3 10 3 10 3 10 3 10 3 10

Cel

l via

bilit

y (

of c

ontro

l)


(b)












EV71

GAPDH

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(a)

20 40

GAPDH

I120581B120572

(+)

HCT (120583gmL)

Con EV

(b)

20 40

HCT (120583gmL)

Con TNF120572

(c)

C

GAPDH

CVA16

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(d)

CVA

GAPDH

I120581B120572

(+)MHB (120583gmL)

Con 100 200

(e)

MHB (120583gmL)100 200Con TNF120572

(f)


GAPDH

EV71

EV71

Con mdash

Con mdash

1 2 3 4 5 6

7 8 9 10 11 12

I120581B120572

GAPDH

I120581B120572

(a)

CVA16

CVA16

Con mdash 1 2 3 4 5 6

Con mdash 7 8 9 10 11 12

GAPDH

I120581B120572

GAPDH

I120581B120572

(b)


4 Discussions





20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)


















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2







50was defined as




50


50numbers











3 Results




50value




50


50






EV71

0

20

40

60

80

100

120Re

lativ

e sta

inin

g (

of c

ontro

l)

EV71 5 10 20 40

HCT (120583gmL)

(a)

CVA16

CVA16 50 100 200 ACV0

20

40

60

80

100

120

Relat

ive s

tain

ing

( o

f con

trol)

MHB (120583gmL)

(b)

0

2

4

6

8

EV71 10 20 40

HCT (120583gmL)

TCID50m

L (lo

g 10)

(c)

3CVA16 50 100 200

4

5

6

7

8

PFU

mL

(log 10)

MHB (120583gmL)

(d)










0

20

40

60

80

100

120

minus2 0 2 4 6 8 10 12 14 16 18 20 22 24

Inhi

bitio

n (

)

Time (hr)



(a)

1

3

5

7


TCID50m

L (lo

g 10)

(b)

35

45

55

65

PFU

mL

(log 10)


(c)











tens

ity (m

V)

1

1 2

23

3

4

4


minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Standard

Extract

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

Inte

nsity

(mV

)

minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 722 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

(a)

15

20

25

30

EV71 3 10 3 10 3 10 3 10 3 10

Cel

l via

bilit

y (

of c

ontro

l)


(b)












EV71

GAPDH

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(a)

20 40

GAPDH

I120581B120572

(+)

HCT (120583gmL)

Con EV

(b)

20 40

HCT (120583gmL)

Con TNF120572

(c)

C

GAPDH

CVA16

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(d)

CVA

GAPDH

I120581B120572

(+)MHB (120583gmL)

Con 100 200

(e)

MHB (120583gmL)100 200Con TNF120572

(f)


GAPDH

EV71

EV71

Con mdash

Con mdash

1 2 3 4 5 6

7 8 9 10 11 12

I120581B120572

GAPDH

I120581B120572

(a)

CVA16

CVA16

Con mdash 1 2 3 4 5 6

Con mdash 7 8 9 10 11 12

GAPDH

I120581B120572

GAPDH

I120581B120572

(b)


4 Discussions





20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)


















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results




50value




50


50






EV71

0

20

40

60

80

100

120Re

lativ

e sta

inin

g (

of c

ontro

l)

EV71 5 10 20 40

HCT (120583gmL)

(a)

CVA16

CVA16 50 100 200 ACV0

20

40

60

80

100

120

Relat

ive s

tain

ing

( o

f con

trol)

MHB (120583gmL)

(b)

0

2

4

6

8

EV71 10 20 40

HCT (120583gmL)

TCID50m

L (lo

g 10)

(c)

3CVA16 50 100 200

4

5

6

7

8

PFU

mL

(log 10)

MHB (120583gmL)

(d)










0

20

40

60

80

100

120

minus2 0 2 4 6 8 10 12 14 16 18 20 22 24

Inhi

bitio

n (

)

Time (hr)



(a)

1

3

5

7


TCID50m

L (lo

g 10)

(b)

35

45

55

65

PFU

mL

(log 10)


(c)











tens

ity (m

V)

1

1 2

23

3

4

4


minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Standard

Extract

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

Inte

nsity

(mV

)

minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 722 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

(a)

15

20

25

30

EV71 3 10 3 10 3 10 3 10 3 10

Cel

l via

bilit

y (

of c

ontro

l)


(b)












EV71

GAPDH

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(a)

20 40

GAPDH

I120581B120572

(+)

HCT (120583gmL)

Con EV

(b)

20 40

HCT (120583gmL)

Con TNF120572

(c)

C

GAPDH

CVA16

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(d)

CVA

GAPDH

I120581B120572

(+)MHB (120583gmL)

Con 100 200

(e)

MHB (120583gmL)100 200Con TNF120572

(f)


GAPDH

EV71

EV71

Con mdash

Con mdash

1 2 3 4 5 6

7 8 9 10 11 12

I120581B120572

GAPDH

I120581B120572

(a)

CVA16

CVA16

Con mdash 1 2 3 4 5 6

Con mdash 7 8 9 10 11 12

GAPDH

I120581B120572

GAPDH

I120581B120572

(b)


4 Discussions





20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)


















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















EV71

0

20

40

60

80

100

120Re

lativ

e sta

inin

g (

of c

ontro

l)

EV71 5 10 20 40

HCT (120583gmL)

(a)

CVA16

CVA16 50 100 200 ACV0

20

40

60

80

100

120

Relat

ive s

tain

ing

( o

f con

trol)

MHB (120583gmL)

(b)

0

2

4

6

8

EV71 10 20 40

HCT (120583gmL)

TCID50m

L (lo

g 10)

(c)

3CVA16 50 100 200

4

5

6

7

8

PFU

mL

(log 10)

MHB (120583gmL)

(d)










0

20

40

60

80

100

120

minus2 0 2 4 6 8 10 12 14 16 18 20 22 24

Inhi

bitio

n (

)

Time (hr)



(a)

1

3

5

7


TCID50m

L (lo

g 10)

(b)

35

45

55

65

PFU

mL

(log 10)


(c)











tens

ity (m

V)

1

1 2

23

3

4

4


minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Standard

Extract

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

Inte

nsity

(mV

)

minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 722 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

(a)

15

20

25

30

EV71 3 10 3 10 3 10 3 10 3 10

Cel

l via

bilit

y (

of c

ontro

l)


(b)












EV71

GAPDH

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(a)

20 40

GAPDH

I120581B120572

(+)

HCT (120583gmL)

Con EV

(b)

20 40

HCT (120583gmL)

Con TNF120572

(c)

C

GAPDH

CVA16

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(d)

CVA

GAPDH

I120581B120572

(+)MHB (120583gmL)

Con 100 200

(e)

MHB (120583gmL)100 200Con TNF120572

(f)


GAPDH

EV71

EV71

Con mdash

Con mdash

1 2 3 4 5 6

7 8 9 10 11 12

I120581B120572

GAPDH

I120581B120572

(a)

CVA16

CVA16

Con mdash 1 2 3 4 5 6

Con mdash 7 8 9 10 11 12

GAPDH

I120581B120572

GAPDH

I120581B120572

(b)


4 Discussions





20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)


















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

minus2 0 2 4 6 8 10 12 14 16 18 20 22 24

Inhi

bitio

n (

)

Time (hr)



(a)

1

3

5

7


TCID50m

L (lo

g 10)

(b)

35

45

55

65

PFU

mL

(log 10)


(c)











tens

ity (m

V)

1

1 2

23

3

4

4


minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Standard

Extract

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

Inte

nsity

(mV

)

minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 722 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

(a)

15

20

25

30

EV71 3 10 3 10 3 10 3 10 3 10

Cel

l via

bilit

y (

of c

ontro

l)


(b)












EV71

GAPDH

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(a)

20 40

GAPDH

I120581B120572

(+)

HCT (120583gmL)

Con EV

(b)

20 40

HCT (120583gmL)

Con TNF120572

(c)

C

GAPDH

CVA16

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(d)

CVA

GAPDH

I120581B120572

(+)MHB (120583gmL)

Con 100 200

(e)

MHB (120583gmL)100 200Con TNF120572

(f)


GAPDH

EV71

EV71

Con mdash

Con mdash

1 2 3 4 5 6

7 8 9 10 11 12

I120581B120572

GAPDH

I120581B120572

(a)

CVA16

CVA16

Con mdash 1 2 3 4 5 6

Con mdash 7 8 9 10 11 12

GAPDH

I120581B120572

GAPDH

I120581B120572

(b)


4 Discussions





20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)


















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















tens

ity (m

V)

1

1 2

23

3

4

4


minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Standard

Extract

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

Inte

nsity

(mV

)

minus20minus10

010

30

50

70

90

110

130

20

40

60

80

100

120

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 722 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70

(a)

15

20

25

30

EV71 3 10 3 10 3 10 3 10 3 10

Cel

l via

bilit

y (

of c

ontro

l)


(b)












EV71

GAPDH

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(a)

20 40

GAPDH

I120581B120572

(+)

HCT (120583gmL)

Con EV

(b)

20 40

HCT (120583gmL)

Con TNF120572

(c)

C

GAPDH

CVA16

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(d)

CVA

GAPDH

I120581B120572

(+)MHB (120583gmL)

Con 100 200

(e)

MHB (120583gmL)100 200Con TNF120572

(f)


GAPDH

EV71

EV71

Con mdash

Con mdash

1 2 3 4 5 6

7 8 9 10 11 12

I120581B120572

GAPDH

I120581B120572

(a)

CVA16

CVA16

Con mdash 1 2 3 4 5 6

Con mdash 7 8 9 10 11 12

GAPDH

I120581B120572

GAPDH

I120581B120572

(b)


4 Discussions





20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)


















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















EV71

GAPDH

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(a)

20 40

GAPDH

I120581B120572

(+)

HCT (120583gmL)

Con EV

(b)

20 40

HCT (120583gmL)

Con TNF120572

(c)

C

GAPDH

CVA16

I120581B120572

Con (+) 05 1 2 4 6 8 9 10 16 24 (hr)

(d)

CVA

GAPDH

I120581B120572

(+)MHB (120583gmL)

Con 100 200

(e)

MHB (120583gmL)100 200Con TNF120572

(f)


GAPDH

EV71

EV71

Con mdash

Con mdash

1 2 3 4 5 6

7 8 9 10 11 12

I120581B120572

GAPDH

I120581B120572

(a)

CVA16

CVA16

Con mdash 1 2 3 4 5 6

Con mdash 7 8 9 10 11 12

GAPDH

I120581B120572

GAPDH

I120581B120572

(b)


4 Discussions





20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)


















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















20EV71Con 40

IL-6

GAPDH

0

15

30

45

60

75

EV71 20 40

Fold

chan

ge (I

L-6)

HCT (120583gmL)

HCT (120583gmL)

(a)

200100CVA16 Con

GAPDH

IL-6

0

20

40

60

80

100

CVA16 100 200

Fold

chan

ge (I

L-6)

MHB (120583gmL)

MHB (120583gmL)

(b)


















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
















Research Article A Laboratory Evaluation of Medicinal Herbs Used...

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