review article application of traditional chinese medical

Review ArticleApplication of Traditional Chinese Medical Herbs in Preventionand Treatment of Respiratory Syncytial Virus

Li Li Lin12 Jin Jun Shan123 Tong Xie12 Jian Ya Xu12 Cun Si Shen12 Liu Qing Di13

Jia Bin Chen4 and Shou Chuan Wang123

1 Jiangsu Key Laboratory of Pediatric Respiratory Disease Nanjing University of Chinese Medicine Nanjing 210023 China2Institute of Pediatrics The First Clinical Medical College Nanjing University of Chinese Medicine Nanjing 210023 China3Jiangsu Engineering Research Center for Efficient Delivery System of TCM Nanjing University of Chinese MedicineNanjing 210023 China4Dongzhimen Hospital Beijing University of Chinese Medicine Beijing 100700 China

Correspondence should be addressed to Jin Jun Shan jshannjucmeducn and Shou Chuan Wang wscnj126com

Received 6 January 2016 Accepted 26 May 2016

Academic Editor Troy Carlo

Copyright copy 2016 Li Li Lin et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Respiratory syncytial virus (RSV) is a common viral pathogen of the lower respiratory tract which in the absence of effectivemanagement causesmillions of cases of severe illness per yearMany of these infections develop into fatal pneumonia In a review ofEnglish and Chinesemedical literature recent traditional Chinesemedical herb- (TCMH-) based progress in the area of preventionand treatment was identified and the potential anti-RSV compounds herbs and formulas were explored Traditional Chinesemedical herbs have a positive effect on inhibiting viral attachment inhibiting viral internalization syncytial formation alleviationof airway inflammation and stimulation of interferon secretion and immune system however the anti-RSVmechanisms of TCMHsare complicated which should be further investigated

1 Introduction

Respiratory syncytial virus (RSV) is classified as a nonseg-mented negative-sense membrane-bound RNA virus of thefamily Paramyxoviridae spread by droplets and causingrepeated airway infections [1] Respiratory syncytial virusis the most common cause of hospitalization in infantsand children with severe acute lower respiratory infectionsAdditionally it is the third most common cause of death inchildren through the development of fatal pneumonia afterpneumococcal pneumonia and Haemophilus influenzae typeb infection It had been estimated that between 66 000 and199 000 children younger than 5 years died of RSV-associateddiseases in 2005 with 99 of these deaths occurring indeveloping countries [2] In the 1960s a formalin-inactivatedvaccine (FI-RSV) given intramuscularly did not reduce thefrequency of infection and among infected children (particu-larly in the youngest age cohort immunized between 2 and 7

months of age) 80 percent required hospitalization and 2 died[3ndash5]

Due to the nature of the virus and the mode of infectionreinfections are a common event suggesting that naturallyacquired immunity does not provide long-lasting protectionThis has made the development of an effective vaccineimpossible as yet [6] Currently the Food and Drug Admin-istration (FDA) has approved prophylactic drugs for RSVincluding palivizumab and ribavirin administered alongwith symptomatic treatment and supportive care [7] Whilepalivizumab a humanized monoclonal antibody directedagainst the F protein RSV effectively prevents RSV infectionit is expensive and ineffective in the management of anestablished RSV infection [8] Ribavirin has been shownto exhibit potent activity against RSV in vivo and in vitrohowever comparisons of the results of both animal andcell testing in humans have not yet been completed [9ndash11] Furthermore ribavirin is limited due to the side effects

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 6082729 13 pageshttpdxdoiorg10115520166082729

2 Evidence-Based Complementary and Alternative Medicine

[11 12] There are any antiviral agents against RSV that arecurrently under development

This review aims to explore evidence available on pre-ventativemeasures against RSV infection discuss the currentstatus of anti-RSV research on TCMH-derived active com-pounds TCMHs and traditional Chinese medical formulas(TCMFs) and finally highlight the challenges issues andfuture directions of using TCMHs in clinical practice

2 Evidence Supporting the Efficacy of TCMHs

Traditional Chinese medical herbs are the most importantcomponents of the traditional Chinese medicine (TCM)system which have been reported to cure infectious diseasesin the form of hot water extracts for almost 2000 yearsOver 10000 herbal medicines and 100000 recipes have beendocumented in ancient literature [13]

Nowadays TCMHs are widely used for the preven-tion and treatment of viral infectious diseases in Chinaand many other Asian countries They are associated withadvantages such as lower toxicity economical and multi-functional use in regulating immunological function andviral destruction [14] This reduction in viral load allowsfor the screening of effective medications However theinternational community remains uncertain about the effi-cacy of TCMHs due to the lack of supporting clinical evi-dence collected under international standards (randomizedplacebo-controlled double-blind and multicentered clinicalstudies) Governments have put forward support aimed atthe international regulatory approval of TCMHs Leadingthe pack is compound T89 (also known as Dantonic atraditional Chinese medical product by Tasly Pharmaceuti-cals China) which is used in China for the managementof ischemic heart disease Dantonic may become the firsttraditional Chinese medicine to receive Food and DrugAdministration (FDA) approval in the United States [15]Moreover the Yinhuang granules (tablet) and Xiasangjugranules have been approved by the China Food and DrugAdministration (SFDA) for the treatment of RSV-infectiousdiseases [15] The 2015 Nobel Prize in Medicine was awardedto Youyou Tu for the discovery of artemisinin a drug isolatedfrom a plant which has significantly reduced the mortal-ity rates for patients with malaria This acknowledgementhas increased the optimism of the natural product commu-nity worldwide [16]

3 Prevention

Prevention is the most important aspect of healthcare andbecause of the nature of the respiratory syncytial virus andmode of infection reinfections are a common event [6] LiveRSV can survive on surfaces for several hours moreoverdirect or indirect contact with the nasopharyngeal secretionsor droplets (sneezing coughing and kissing) fomites andfood from infected patients can potentially transmit RSV[7] Therefore without effective preventive measures themortality and economic burden of the disease will increase

31 RSV F Protein Neutralizing Antibodies The respiratorysyncytial virus depends on the host cell to complete its

replication cycle which includes attachment and entry intothe host cell transcription of viral mRNA viral genomereplication protein synthesis and the assembly and buddingof progeny virus particles [15] The F protein of RSV accu-mulates in the host membrane and surrounds the buddingprogeny viruses thus spreading the infection to adjacent cellsand exacerbating the infection The F protein is thereforeconsidered a potential candidate for the development ofpossible preventive measures Palivizumab is a humanizedmonoclonal antibody (IgG) directed against an epitope inthe A antigenic site of the F protein of RSV Palivizumab-N a plant-based F neutralizing antibody was experimentedin phages and plants Successful palivizumab-N productionwas observed in the Nicotiana benthamiana plant systemwhich offered glycosylation and high production at lowupstream and equivalent downstream cost when comparedto mammalian derived palivizumab [7]

32 Boost Resistance against RSV by Modulating ImmuneResponse Following coevolution with the host many viruseshave established sophisticated mechanisms to interact withthe host immune system for immune evasion Among theclasses of antiviral agents immunomodulators are the mostabundant in TCMHs [15] Traditional Chinese medical herbshave been shown to enhance antibody production T cellproliferation the expression of antigen specific CD4+ andCD8+ responses and increase of the titers of IgG1 IgG2a andIgA along with the function to balance Th1 Th2Th17 typeimmune response [17] Interferon- (IFN-) 120574 and interleukin-(IL-) 12 are representatives of Th1 type cytokines interleukin4 and interleukin 5 belong toTh2 type cytokines and IL-6 andIL-17a are representatives ofTh17 type cytokines all of whichplay key roles in Th2Th17 responses [18 19] And recentlymacrophages have been identified as stimulation cells byinteraction with Th2 cytokine IL-4 [20] Traditional Chinesemedical herbs were reported to simultaneously promoteTh1-response including increased levels of Th1 type cytokinesecretion and activation of alveolar macrophages suppressTh2Th17-responses and maintain the balance between Th1and Th2Th17 cells for the prevention and treatment ofdiseases

Moreover TCMHs have been previously reported to offerefficient protection against RSV and significantly decreaseviral load and inflammation [15] It is well-known that theproduction of inflammatory mediators is regulated by atranscription factor NF-120581B which plays a pivotal role ininflammation due to its ability to induce the transcriptionof inflammatory genes [21ndash23] Toll-like receptor 4 (LR4)is also a factor expressed on the respiratory epithelium andmacrophages and appears to play a central role in mediatingboth the antiviral and inflammatory responses of the innateimmunity to combat viral infections [22] Along with directantiviral activity TCMHs could be beneficial in preventingRSV infection due to its immune-modulatory effect produc-ing of IFN-120573 and TNF-120572 Respiratory syncytial viral infectioncan induce cellular production of IFN-120573 and TNF-120572 BothIFN and TNF contribute to the innate immunity againstviral infection [24] and TNF triggers multiple antiviral

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Aqueous extracts of TCMHs in prevention of RSV

Herbs Used part Prevention activity References

Fresh ginger Rhizome

Showed its better effect when given before viral infection viralattachment darr viral internalization darr

[29]In HEp-2 cells IC50 was 2127 120583gmL (2 h before)in A549 cellsIC50 was 26 120583gmL (2 h before)IC50 was 828 120583gmL (1 h before)

Secrete IFN-120573 that contributed to the inhibition of viral replication

Ginseng (naturallydried) Root

Anti-inflammatory functions less weight loss diminishing pulmonaryinflammatory response

[30]Inducing host immune responses towardTh1 type immunity IgG2aisotype antibodies uarr IFN-120574 uarr IL-4 darrCD4 T cell infiltration darr cell numbers of CD4+ T (CD3+ CD8minus) cells darrthe ratio of CD8+CD4+ T cells uarr

Panax Korean redginseng (fermentedred)

Root

In the prevention of RSV infections improved survival of human lungepithelial cells against RSV infection inhibited RSV-induced cellularoxidative damage

[31]Play a role in priming the host immune system enhanced IFN-120574production following RSV viral infection blocked induction ofRSV-induced proinflammatory gene expression in the human alveolarepithelial cell line

Radix Glycyrrhizae Rhizome

It is more effective when given before viral inoculation (119875 lt 00001)inhibition of viral attachment (119875 lt 00001) and penetration (119875 lt 00001)

[32]

In HEp-2 cellsIC50 was 780 120583gmL (2 h before)IC50 was 997 120583gmL (1 h before)

in A549 cellsIC50 was 188120583gmL (2 h before)IC50 was 338 120583gmL (1 h before)

Stimulate mucosal cells to secrete IFN-120573 to counteract viral infectionIL-4 interleukin-4 IFN-120574 interferon-120574 IFN-120573 interferon 120573 Th1 type immunity T helper type 1 (Th1) immune A549 human lung carcinoma cell HEp-2human larynx epidermoid carcinoma cell

mechanisms and synergizes with IFN in promoting antiviralactivities [25]

Resveratrol a compound synthesized by at least72 plant species [26] exhibited certain abilities in theinhibition of RSV replication inflammation the level of IL6virus-induced TIR-domain-containing adapter inducinginterferon-120573 (TRIF) and TANK binding kinase 1 (TBK1)protein expression [27] Moreover it has been shown toprevent airway inflammationhyperresponsiveness duringRSV infection inmice suggesting its applicability in reducingRSV-induced airway symptoms Additionally it has also beenshown to reduce IFN-120574 levels associated with RSV-mediatedairway inflammation [28]

In Table 1 we have provided evidences of medicinal herbsused in TCM for the prevention of RSV This novel approachseems to be a promising and safe prevention option againstRSV

4 Treatment

41 TCMH-Derived Active Compounds Drug use from nat-ural products has progressed to the isolation of active com-pounds Natural products and particularly medicinal plants

remain an important source of new drugs new drug leadsand new chemical entities [58 59] Drug discovery fromnature led to the isolation of early drugs such as quinineartesunate and arsenic trioxide which are active ingredientseffectively extracted from natural products and have sinceproven to be clinically successful Quinine is made fromcinchona bark and artesunate is derived from Artemisiaannua and both are used for the treatment of severe malaria[60 61] Arsenic trioxide extracted from white arsenic isused for the treatment of acute promyelocytic leukemia [6263]

Traditional Chinese medical herbs have been utilized asanti-infectious medicines for thousands of years in Chinaand offer various anti-infectious compounds particularly forantiviral An increasing number of TCMH-derived activecompounds with antiviral activity are garnering evidenceof experimental efficacy As shown in Green Baersquos research(minus)-(R)-nyasol (minus)-(R)-41015840-O-methylnyasol and broussoninA three structurally known compounds isolated from therhizomes ofA asphodeloides exhibited potent antiviral activ-ities against the RSV-A2 strain propagated in HEp-2 cellsThe results showed IC50 values of 085 039 and 062 120583Mrespectively slightly more potent than that of the positive


control ribavirin (IC50 = 115 120583M) [64] Table 2 details a listof anti-RSV active compounds derived fromTCMHs verifiedby experiments in vivo or in vitro

411 The Characteristics of Active Compounds in TCMHsA vast amount of basic research has been conducted onTCMHs Therefore the isolation and characterization ofpharmacologically active compounds from TCMHs and thetesting of their pharmacological activities in the pursuit ofnew drug discovery are now necessary [65]When comparedto random screening from a combinatorial chemical libraryTCMHs are drugs with a long history of medical use andcould be used as the markers for selecting active ingredientsfromherbalmedicines It would therefore bemore efficient toscreen active compounds fromTCMHs Furthermore in bio-logical screening assays TCMH-derived active compoundsoften have better pharmacokinetics and bioavailability [15]

Previous studies as shown in Table 2 have provided evi-dence of the direct anti-RSV activity of compounds derivedfrom TCMHs Notwithstanding the failure to discover newchemical structures during drug discovery from medicinalplants known compounds with new biological activitiessuch as the inhibition of RSV replication and activation ofthe immune system which can provide important drug leadsmay be found [66] Moreover for countries with limitedresources TCMHs-derived active compounds will serve as agateway for the merging of modern drug discovery

However even with these unique advantages screeningprograms focused on identifying potential anti-RSV agentsfrom TCMHs are using conventional methods In order tocompetewith other drug discoverymethods efforts are ongo-ing to replace these traditional methodologies with modernsample preparation and extraction procedures ThereforeTCMH research needs to continually improve the speedof screening isolation and structure elucidation processesThe most challenging issues are understanding the operativemechanisms to identify TCMH-derived active componentsas each kind of TCMH contains multiple active componentswith multiple targets Some of these components workdirectly on the therapeutic targets whereas others mayenhance the bioavailability [59] Therefore more efforts areneeded in the research of compounds in TCMH assays

It is known that cell-based assays are often initially carriedout for the evaluation of whole extracts that show clinicalevidence of antiviral activity Without larger randomizeddouble-blind placebo-controlled multicenter clinical trialsapproval of TCMH-derived active compounds will be dif-ficult through international regulatory agencies such as theFood and Drug Administration (FDA) of the United States ofAmerica or other equivalent European counterparts

Above all and in long term TCMHs still offer a greatprospect in the field of pharmaceutical developments Thecharacteristics of TCMHs offer opportunities for findingactive and novel chemical structures against a variety oftherapeutic targets

42 Traditional Chinese Medical Herbs (TCMHs) Tradi-tional Chinese medicine theory is in line with seasonscircumstances and individuality For example in summer

ldquoAgastache rugosardquo or ldquoEupatoriumrdquo is commonly used innorthern China (colder district) to ease the summer-heat andldquoephedrardquo and to relieve exterior syndrome (ldquoexterior syn-dromerdquo refers to lesions with mild conditionsThe symptomsare aversion to cold fever headache body pain stuffy noseno sweat and so on) by diaphoresis while in southern China(warmer district) ldquohoney-fired ephedrardquo is frequently usedbecause of its mild function of stimulating the secretion ofsweat Moreover the TCM treatment should vary betweenindividuals For the children with upper respiratory tractinfection the treatmentmethod aims to clear the exterior evil(ldquoevilrdquo is the general name of all kinds of pathogenic factorsldquoexterior syndromerdquo discussed above is caused by exteriorevil) while in the elderly the treatment method aims tostrengthen body resistance and relieve the exterior syndrome

In both 2001 and 2002 approximately one-quarter of thebestselling drugs worldwide were natural products or werederived fromnatural products [59] Recent publication showsthat TCMHs account for 10 of the prescription drugs inChina They are perceived as harmless and natural and arewidely used in several parts of the world individually or incombination [15]

421 The Characteristics of TCMHs Traditional Chinesemedical herbs are the most important components of theTCM system which have long been used for the multiplecombinations of compounds in the form of processed nat-ural products Experimental findings of TCMHs with theidentification of potent antiviral activities against RSV shouldbe made available to the healthcare providers practicingtraditional medicine From a clinical perspective diseasesare complex and variable combined with complex clinicalsymptoms Traditional Chinese medical herbs contain mul-tiple active components with multiple targets which may bea better choice to achieve multiacting treatment

Traditional Chinese medicine theories originate from theprofound experiences of practitioners with understanding ofancient Chinese medicine and the methods of applicationhave been passed down through oral history It is suggestedthat hot water extracted TCMHs would exhibit direct antivi-ral activity In this review we summarized experiments invivo or in vitro (Table 3) that used aqueous extracts ofTCMHs for treating RSV-infectious diseasesThe objective ofthese experiments was to review the potential uses of naturalproducts for the treatment of infections caused by RSV

Ma et al recently tested 44 medicinal herbs used inthe TCM system for treating virus-infectious diseases Themedicinal herbs were tested for antiviral activities againstRSV using a cytopathologic effect (CPE) assay Twenty-sevenof the 44 medicinal herbs showed moderate or potent antivi-ral activities against RSVwith a 50 inhibition concentration(IC50) ranging from 63 to 521 gmL and a selectivity index(SI) ranging from 20 to 321The control group ribavirin hadan SI of 24 [44]

This evidence illustrates that natural products can be arich source for the discovery of potent anti-RSV agents

422 The Bottleneck Problems of TCMHs Although tradi-tional natural products have played an important role in


Table 2 TCMHs-derived compounds inhibiting RSV(a)

Herbs Compounds Anti-RSV activity References

Folium Isatidis Fraction IIIfraction IV

RSV replication darr (SI 1306 gt2433 MTT 1167 gt2232)The ribavirin group (SI lt2804 MTT 4052) [33]

Flos PuerariaOmeiensis Genistein tectorigenin

Possess potent antiviral activity against RSV (CC50 450500 120583gmL IC50 125 30 120583gmL SI 36 167)the ribavirin group (CC50 625 120583gmL IC50 30 120583gmL SI 208)

[34]

Radix Gentianae RG2-1 [35]RG3-1 [36]

RSV replication darr (TC50 1066 529mgmL EC50 42026295 120583gmL TI 2538 2012) [35 36]the ribavirin group (TC50 118 115mgmL EC50 30881548 120583gmL TI 3821 7492) [35 36]

[35 36]

Herba Patriniae AP3 [37]AP4 [38]

RSV replication darr RSV entry darr (TC50 1145 1089mgmL EC5000986 00801mgmL TI 11612 13595) [37 38]the ribavirin group (TC50 0076 197mgmL EC50 0001430036mgmL TI 5345 5472) [37 38]

[37 38]

Litchi Chinensis Flavonoids RSV replication darr (TC50 1529mgmL IC50 586mgmL TI 26)the ribavirin group (TC50 1549mgmL IC50 571mgmL TI 27) [39]

Radix Glycyrrhizae 18120573-GA [32]GD4 [40]

RSV replication darr (IC50 43ndash45 120583gmL CC50 715ndash763 120583gmLSI 159ndash177) [32] (EC50 2873 120583gmL TI 80) [40]the ribavirin group (EC50 567120583gmL TI 2150) [40]

[32 40]

Ligustrum lucidum OleuropeinSignificant antiviral activities against RSV (TC50 5625 120583gmLIC50 117 120583gmL TI 48) the ribavirin group (TC50 625120583gmLIC50 26 120583gmL TI 24)

[41]

Lophatherumgracile Flavone 6-C-monoglycosides

Potent antiviral activities against RSV (CC50 ranging from 2545 plusmn64 to 3626 plusmn 154 120583gmL IC50 ranging from 57 to 50 120583gmL SIranging from 56 to 636 120583gmL)the ribavirin group (CC50 625 plusmn 32 120583gmL IC50 30 plusmn04 120583gmL SI 208)

[42]

Pithecellobiumclypearia Quercetin Possess potent antiviral activity against RSV (IC50 25120583gmL SI

180) the ribavirin group (IC50 30 120583gmL SI 208) [43]

Sophora flavescens Anagyrine oxymatrinesophoranol

Potent antiviral activities against RSV (IC50 104 104 104 120583gmLCC50 250 125 250 120583gmL SI 240 12 24)the ribavirin group (IC50 26 120583gmL CC50 625 120583gmL SI 24)

[44]

TC50 is 50 of toxic concentration EC50 is the concentration for 50 of maximal effect IC50 is the concentration of the sample required to inhibitvirus-induced CPE 50 CC50 is the concentration of the 50 cytotoxic effect selective index (SI) or therapeutic index (TI) CC50IC50 or TC50EC50or TC50IC50 MTT assay is a colorimetric assay for assessing cell metabolic activity

(b)


Scutellariabaicalensis Wgonin oroxylin A

Potent antiviral activities against RSV (IC50 74 145 120583gmLCC50 1192 581 120583gmL SI 161 40)the ribavirin group (IC50 26 120583gmL CC50 625 120583gmL SI 24)

[44]

Selaginellauncinata

Uncinoside Auncinoside B

Potently inhibit RSV infection (IC50 69 13120583gmL TC50 825833 120583gmL TI 12 64)the ribavirin group (IC50 26 120583gmL CC50 625 120583gmL TI 24)

[45]

Wikstroemia indica Daphnoretin RSV replication darr (IC50 587 120583gmL SI 2817)the ribavirin group (IC50 305120583gmL SI 214) [46]

Radix wikstroemiaeGenkwanol Bgenkwanol Cstelleranol

Potent antiviral activities against RSV (IC50 96 66 102 120583gmLCC50 1061 1453 1615 120583gmL SI 11 219 158)the ribavirin group (IC50 119120583gmL CC50 2561 120583gmL SI 216)

[47]

Youngia japonica

Fractions 10 [26]fractions 11 [26]34-dicaffeoylquinic acid[48]35-dicaffeoylquinic acid[48]

Syncytium formation of RSV darr potent antiviral activities againstRSV (IC50 30ndash60 120583gmL MNCC 200 120583gmL) [26] (IC5005 120583gmL) [48]the ribavirin group (IC50 30 120583gmL MNCC 31 120583gmL) [26] theribavirin group (IC50 25 120583gmL) [48]

[26 48]

TC50 is 50 of toxic concentration EC50 is the concentration for 50 ofmaximal effect IC50 is the concentration of the sample required to inhibit virus-induced CPE 50 CC50 is the concentration of the 50 cytotoxic effect selective index (SI) or therapeutic index (TI) CC50IC50 or TC50EC50 orTC50IC50MNCC themaximal concentration of the sample that did not exert toxic effect detected bymicroscopic monitoring after 72 h of incubation


Table 3 Aqueous extracts of TCMHs inhibiting RSV

Herbs Used part Anti-RSV activity ReferencesIC50 (120583gmL) CC50 (120583gmL) SI

Bupleurum chinense

Root

368 4413 120

[44]

Gardenia jasminoides 210 1121 53Isatis indigotica 500 1000 200Ipomoea cairica 521 8333 160Polygonum multiflorum 521 gt10000 gt192Platycodon grandiflorum lt441 3525 gt80Polygonum cuspidatum Sieb lt130 gt2000 gt154Scutellaria baicalensis lt104 gt625 60Sophora flavescens 521 8333 160Tinospora capillipes 172 2758 160Paeonia suffruticosa Root cortex 260 gt8333 gt320Phragmites communis Rhizome 465 1858 40

Andrographis paniculata

Aerial parts

276 2950 107Artemisia capillaries 130 2083 160Schizonepeta tenuifolia 492 1969 gt40Scutellaria indica 313 3500 112

Perilla frutescens Leaf 376 2556 60Lonicera japonica Flower bud 500 gt10000 gt200Dendranthema morifolium Flower 505 2692 53Forsythia suspense Fruit 500 10000 200Prunella vulgaris L Fruit spike lt104 gt625 60

Callicarpa nudiflora

Whole plant

375 8000 213Patrinia villosa lt130 4167 gt320Sarcandra glabra 500 10000 200Selaginella sinensis 500 gt625 gt200Blumea laciniata Callicarpa 156 2438 155

[49]Laggera pterodonta 156 5000 318Mussaenda pubescens 5125 320 160

Schefflera octophylla Leaf stalk 125 5000 400

Ribavirin [44] mdash 26 625 240 [44]Ribavirin [49] mdash 30 625 208 [49]IC50 is the concentration of the sample required to inhibit virus-induced CPE 50 CC50 is the concentration of the 50 cytotoxic effect selective index (SI)or therapeutic index (TI) CC50IC50

drug discovery in the past few years many pharmaceuticalcompanies have scaled down or terminated their naturalproduct research [59] Drug discovery programs alreadyfollow a challenging process the design determinationand implementation of appropriate clinically relevant high-throughput bioassays of drugs and TCMH discovery pro-grams are more complicated and lengthier than other drugdiscovery methods

Despite the recognized therapeutic benefits and theexpanding use of medicinal plants worldwide TCMHs stilllack robust evidence from evidence-based medicine (EBM)research especially the supporting clinical evidence col-lected under international standards (randomized placebo-controlled double-blind and multicentered clinical studies)As a result it is a challenge for TCMHs to be accepted by the

Western medicine community and mainstream healthcaresystems

Though much debate remains on the feasibility and thevalidity of applying the EBM criteria to this traditionalpractice for the integration of TCM into theWestern health-care system it is essential to demonstrate its efficacy andsafety through high level evidence in accordance with EBMprocedures

43 Traditional Chinese Medicine Formulas (TCMFs) Basedon TCM theory a remedy often has a principle of hierarchythe so-called ldquomonarchrdquo ldquoministerrdquo ldquoassistantrdquo and ldquoguidecomponentsrdquo [53] and the functions differ among the differ-ent rolesThe basic principle of TCM treatment is to keep thebalance between Yin and Yang (two opposite as well as rele-vant agents the main components of the universe in Chinese


philosophy) and Qi and blood (two basic components of thehuman body with the functions of maintaining the life)

A TCM formula contains multiple active herbs withmultiple compounds and targets Some of these components(ldquomonarchrdquo and ldquoministerrdquo) work directly on the therapeutictargets while others (ldquoassistantrdquo and ldquoguide componentsrdquo)enhance the bioavailability or counteract drug toxicity of themedicine [15] This review summarized some TCMFs andChinese patent medicines in Tables 4 and 5 which had anti-RSV activities supporting their possible use in managingRSV infection

431 The Characteristics of TCMFs and Chinese PatentMedicines Traditional Chinese Medicine Formulas havebeen used as remedies against infectious diseases for thou-sands of years due to their significant anti-inflammatoryantimicrobial and antiviral activities with little or no adverseeffects [44 67] Therapeutic effects of mixed formulas arebased on the synergic action of its mass constituents [67]Currently Chinese patent medicines with convenient dosageforms represent the majority of drugs on the market Anti-RSV Chinese patent medicines (during infection) are usuallyconsidered ldquocoldrdquo herbs and are applied to remove theheat-evil (ldquoheat evilrdquo related to the clinical manifestationscharacterized by ulcers local swelling and heat pain) expelsuperficial evils (superficial evils lead to ldquoexterior syndromerdquowhich is explained in previous 17th paragraph) and dispeltoxins from the body such as Shuang Huang Lian oralliquid Jin Xin oral liquid Qing Fei oral liquid or Xiasangjugranules (Table 4) The Xia Sang Ju granule was approvedby the China Food and Drug Administration (SFDA) andJin Xin oral liquid a traditional Chinese patent medicinehas been widely used for decades to treat virus-infectiouspneumonia in the Jiangsu Province Hospital of NanjingUniversity of Traditional Chinese Medicine Nanjing ChinaFollowing recovery from an infection Bu Shen Yi Qi formulais administered which is a traditional Chinese herbal formulacomposed of herbs used to tonify Qi of the kidney that is thestrengthening of the bodyrsquos resistance to eliminate pathogenicfactors The anti-RSV mechanism of Bu Shen Yi Qi formulawas investigated in theTh1 Th2 andTh17 cells [50]

Based on the improvements in experiments in vivo or invitro a number of Chinese patent medicines are currentlyundergoing FDA clinical trials in the hope of fulfillingEBM research criteria This includes Danshen dripping pill(Salvia miltiorrhiza Radix notoginseng and Borneol) forstable angina in a phase III trial Fu zheng Hua yu capsule(Salvia miltiorrhiza peach seed pine pollen Gynostemmapentaphylla and Schisandra chinensis) for liver fibrosis Kanglai te injection (coix seed oil and excipients) for cancer andthe Xue zhi kang capsule (red yeast) for hyperlipidemia inphase II trials [71]

Traditional Chinese Medicine Formulas rely on writtentraditional Chinese medicine and an educational system ofTCM scholars According to Shanghan Lun (Treatise on ColdPathogenic Diseases) written by Zhang Zhongjing TCMFsshould be administered three times A dosage three times aday is recommended to avoid an overdose and to ensure the

better absorption of drugsThis is to ensure a balanced blooddrug concentration according to modern medicine [72]

In TCM theory the quality of the decoction is the keyto cure diseases successfully hence detailed requirements ofthe decocting methods should be illustrated The methodsof decocting medicinal herbs were passed down throughthe form of hot water extracts [44] the oral form of thedecoction would exhibit major effects such as direct antiviralactivity The decocting time differs from herbs to herbs Foraromatic plants such as Mentha and Agastache rugosa thedrug efficacywill be lost with lengthened decocting thereforethese plants should be decocted for only 5ndash10 minutes whilefor plants with a certain level of toxicity such as Aconitumcarmichaelii and Pinellia ternata decocting should be at least1 hour to reduce its toxicity

Compared to Western medicines advantages of TCMFsinclude individual treatment based on ldquomulticomponentsmultichannels and multitargetsrdquo application and syndromedifferentiation The characteristics of TCMFs are to bal-ance the body and improve clinical symptoms of a patientConsidering these unique characteristics development ofTCMFs in Tables 4 and 5 with anti-RSV activity is of greatimportance However this clinical application needs furtherclinical validation

432 The Bottleneck Problems of TCM Formulas With thedevelopment of science and technology and the increas-ing need of modern clinical practice the disadvantages oforal TCM preparations have gradually been realized Theseinclude single administration old formulation large dosageslow effect and outdated preparation and transport methods

The development of Chinese patent medicines has aidedthe classification however in treating acute and severediseases TCM formulas are criticized for their slow effectand large dosage This prevents TCM from being able tosolve urgent medical diagnoses Moreover dosage needs tobe improved for a convenient use of the drug and theimprovement of the therapeutic effect the dosage needsto be improved while upholding the active effect Moreexperiments and clinical trials are needed in the field ofTCMFs

5 Future Directions in the Prevention andTreatment of RSV

51 AttachmentInternalization Inhibitors The spread of RSVis initiated through viral attachment and internalizationTheseverity of the RSV-infectious disease is positively correlatedwith the viral load [44] therefore inhibition of viral spreadbetween airway mucosal cells to minimize viral replicationand the decrease viral load is essential for disease control

Traditional Chinese medical herbs can prevent the res-piratory viral infection induced by RSV by inhibiting viralattachment and penetration and reducing the increasedsusceptibility of the cell to the invasion of RSV Fromdetails inTable 2 to Table 5 conclusions can be made that TCMHs caneffectively inhibit viral attachment and cell internalization inorder to minimize the viral spread and replication


Table 4 Chinese patent medicine in prevention and inhibition of RSV(a)

Names CompositionMechanisms and results References

Plant or mineral Weight (g)( ww) Used part

Bu ShenYi Qi Tang(BSYQF)(granules)

Radix Astragali 4615 Root Airway inflammation darr RSV replication darr Fprotein expression of RSV darr (119875 lt 005)

[50]Herba EpimediiRehmanniae Radix

30772308

Aerial partroot tuber

Regulating the balance betweenTh1 andTh2Th17 responsesTh1-response uarr CD4+ T cell proportion uarrT-Bet uarr macrophages uarr IFN-120574 uarr IL-12 uarrTh2Th17 cell proportions darr GATA3 darr STAT6darr ROR120574T darr IL-4 darr IL-5 darr IL-6 darr IL-17a darr

Shuang HuangLian oral liquid(SHL)

Scutellariaebaicalensis Georgi 2500 Rootstalk

RSV replication darr the virus titer darr airwayinflammation darr IL- 8 darrthe SHL group TC50 gt 160 120583gmLthe ribavirin group was similar to the SHLgroup (119875 gt 005)no adverse reaction was found

[51 52]Lonicera japonica 2500 Flower budForsythia suspensa 5000 Fruit

Sheng Ma GeGen Tang(SMGGT)

Pueraria lobataOhwi 3333 Radix The crude extract of SMGGT was more

effective when given before viral infection(119875 lt 00001) viral attachment darr RSVinternalization darr

[24]

Paeonia lactifloraPallas 2222 Radix

Cimicifuga foetidaL 2222 Rhizoma The SMGGT group CC50 3000 120583gmL IC50

342ndash828 120583gmLthe ribavirin group was similar to the SMGGTgroup (119875 gt 005)

Glycyrrhizauralensis Fischer etDC

1111 Radix

Zingiber officinaleRoscoe 1111 Root-like stem

Epithelial cells secrete IFN-120573 uarr and TNF-120572 uarrbeforehand and after viral inoculation todefense RSV

Xia Sang JuGranule (XSJG)

Prunella vulgaris L 6623 Fruit spikeRSV replication darr the virus titer darrthe XSJG group TC50 800 120583gmL IC5054459 120583gmL TI 147the ribavirin group was similar to the XSJGgroup (119875 gt 005)no adverse reaction was found

[53]Dendranthemaindicum 2318 FlowerMulberry leaf 1060 Leaf

Th1 cell T helper 1 cellTh2 cell T helper 2 cellTh17-cell T helper 17 cell GATA3 transcription factors GATA binding protein 3 STAT6 signal transducer andactivator of transcription 6 ROR120574T retinoid-related orphan receptor gamma t T-Bet T box expressed in T cells IL interleukin A549 human lung carcinomacell TC50 is 50 of toxic concentration IC50 is the concentration of the sample required to inhibit virus-induced CPE 50 CC50 is the concentration of the50 cytotoxic effect IFN-120573 interferon 120573 IFN-120574 interferon-120574 TNF-120572 tumor necrosis factor-120572

(b)

Names Composition Mechanisms and results ReferencesPlant or mineral Weight (g) Used part

Jin Xin oralliquid(JOL)

Ephedra sinicaStapf 3 Stem Lung inflammation darr RSV replication darr

the viral load in the lung tissues darr

[54]

Prunusarmeniaca L 10 Seed

Morus alba L 10 VelamenDuring early stage infection (on days 2 and 4after infection) the TLR3-IRF3-IFN-120573signaling pathway darr IFN-120573 darr TLR3 darr IRF3(p-IRF3) darr the expression of SOCS1 darr

ScutellariaebaicalensisGeorgi

10 Rootstalk

PeucedanumpraeruptorumDunn

10 Root

LepidiumapetalumWilld 10 Seed During the later stage of infection (7 d after

infection) the TLR3-IRF3-IFN-120573 signalingpathway uarr IFN-120573 uarr TLR3 uarr IRF3 (p-IRF3) uarrthe expression of SOCS1 darr

Polygonumcuspidatum Sieb 10 Root

Gypsum(1198621198861198781198744sdot21198672119874)

15 mdash


(b) Continued


Qing Fei oralliquid(QOL)

Ephedra sinicaStapf 4 Stem

A multicentered single-blind randomcontrolled clinical study of 166 children inNanjing in China showed that the curative andthe effective rate of the QOL group 80001938the ribavirin group 4114 4114no adverse reaction was found

[55]


Morus alba L 10 VelamenBombyx moriLinnaeus 6 Bombycidae


10 Root

LepidiumapetalumWilld 6 Seed Airway inflammation darr

IL-6 darrIL-8 darr

[56]Polygonumcuspidatum Sieb 12 Root

Gypsum(1198621198861198781198744sdot21198672119874)

24 mdashRSV replication darrregulating immunabitityINF-120574 uarr

[57]Salviamiltiorrhiza Bge 6 Rootstalk

Polygonumbistorta L 12 Rootstalk

TLR3 toll-like receptors 3 IRF3 interferon regulatory factor 3 IFN-120573 interferon 120573 SOCS1 suppressor of cytokine signaling 1 IL-6 interleukin-6 IL-8interleukin-8 IFN-120574 interferon-120574 A549 human lung carcinoma cell TC50 is 50 of toxic concentration IC50 is the concentration of the sample requiredto inhibit virus-induced CPE 50 therapeutic index (TI) TC50IC50

52 Enhancement of Immunity Traditional Chinese medicalherbs can counteract RSV infection not only through thedirect inhibition of viral attachment internalization or repli-cation but also by the enhancement of immunity (Table 6)They are considered to be less toxic and may warrant furtherevaluation as a possible agent for RSV prevention

Antiviral cytokines play an important role in the immunesystem process of preventing and inhibiting RSV infectionTraditional Chinese medical herbs can stimulate antiviralcytokines such as IFN-120573 and TNF-120572 before and after RSVinoculation TCMHs have also been shown to keep the bal-ance between Th1 and Th2Th17 immune response enhanceantibody production and T cell proliferation enhance theexpression of antigen specific CD4+ and CD8+ responsesand increase the titers of IgG1 IgG2a and IgA [17]

53 Future Directions Despite the evident successes ofdrug discovery from medicinal plants future endeav-ors face many challenges Efforts are needed to con-tinuously improve the quality and quantity of TCMH-derived compounds herbs and formulas that enter thedrug development phase in an effort to compete withother drug discovery efforts [59] The future of currentTCMH research is to meet international standards includ-ing evidence-based efficacy (particularly through multicen-ter randomized double-blind placebo-controlled clinicaltrials) safety assessment and quality and quantity con-trol The aims for the future are that large TCM-focuseddatabases become available and innovative strategies areapplied to improve the process of plant collection Moreoverit is necessary to establish a standardized and centralized

research system aimed at achieving a better understandingof medicinal chemistry and the mechanism of action ofTCHMs The challenges associated with anti-RSV herbs aremany However at the current pace of scientific research anddevelopment traditional Chinese medical herbs can play arole in effective prevention and treatment of RSV-infectiousdiseases

6 Conclusions

Prevention is the most important aspect of healthcare how-ever due to the nature of the RSV and its mode of infectiona safe and effective RSV vaccine has remained elusiveTraditional Chinese medical herbs can enhance immunity toprevent RSV infection and furthermore TCMHs can formpart of anti-RSV medications by inhibiting viral attachmentviral penetration and reducing the increased susceptibility ofthe cell to the invasion of RSV This review details evidencethat TCMH can be effective as preventative or treatmentmedications for RSV the advantages of lower cost betterpatient outcomes and fewer adverse reactions From theresearch that has been conducted in this review we cansee great potential value of conducting trials using TCMHHowever further research especially focused on the toxicityissues is required to develop the level of evidence to supportTCMH to be applied to practice

Competing Interests

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


Table 5 TCM formulas in prevention and inhibition of RSV(a)


Modified DingChuan Tang(MDD)

Salviae miltiorrhizaeRadix 12 Root

MDD exhibited significant antiviral andanti-inflammatory effects lung viral loads darrthe MDD group IC50 272 120583gmL CC50gt1000120583gmL SI gt368the ribavirin group IC50 28 120583gmL CC50416 120583gmL SI 149eotaxin darr IL-4 darr IFN-120574 darr in serum and lungtissue TLR4 darr NF-120581B darr in the lung tissue

[67]Scutellariae Radix 9 RootFarfarae Flos 10 FlowerEphedra sinica Stapf 4 Stem

Ge Gen Tang(GGT)

Cinnamomum cassiaBlume 30 Twig GGT was more effective given before than after

viral inoculation (119875 lt 00001)viral attachment darr viral internalization darrin A549 cellsIC50 was 88 120583gmL (2 h before)IC50 was 271 120583gmL (1 h before)IC50 was 1847120583gmL (1 h after)the immune-modulatory effect IFN-120573 uarrGGT will not increase TNF-120572 secretion butrather inhibit TNF-120572 secretion in HEp-2 cells

[1]

Ephedra sinica Stapf 45 StemGlycyrrhiza uralensisFischer et DC 30 Radix

Paeonia lactiflora Pallas 30 RadixPueraria lobata Ohwi 60 RadixZingiber officinale Roscoe 45 Root-like stemZiziphus jujuba Mill varinermis (Bge) Rehd 40 Fruit

Jia Wei Yu PingFeng Tang (JYT)

Radix Astragali 277 RootViral attachment darr RSV internalization darr lunglesions and complication darrinhibiting the expression of ICAM1in A549 cellsthe JYT group CC50 137337 plusmn 28 120583gmLIC50 970 120583gmLthe ribavirin group CC50 57947 plusmn 74 120583gmLthe IC50 value of ribavirin was similarJYD could inhibit RSV infection in all threemodes of treatment but was more effective inthe simultaneous and posttreatment modes(119875 lt 005)

[68]

Rhizoma AtractylodisMacrocephalae 167 Rhizome

Radix Saposhnikoviae 167 RootFlos Lonicerae japonicae 167 Flower budRhizoma DryopterisCrassirhizoma 111 Rhizome

Pericarpium CitriReticulatae 111 Fruit

IC50 is the concentration of the sample required to inhibit virus-induced CPE 50 CC50 is the concentration of the 50 cytotoxic effect selective index(SI) or therapeutic index (TI) CC50IC50 NF-120581B nuclear factor-kappa B IL interleukin IFN-120574 interferon-120574 IFN-120573 interferon 120573 TLR4 inhibitor toll-likereceptor 4 A549 human lung carcinoma cell HEp-2 human larynx epidermoid carcinoma cell ICAM-1 intercellular cell adhesion molecule-1

(b)


Liu He Tang(LHT)

Agastache rugosa 20 Whole plant

LHT was more effective when given beforeviral inoculation (119875 lt 00001)viral attachment (119875 lt 00001) darrRSV internalization (119875 lt 00001) darrCC50 3000 120583gmL IC50 342ndash828 120583gmLIC50 value of ribavirin was similarLHT stimulated epithelial cells to secreteIFN-120573 and TNF-120572 to counteract HRSVinfection before infection becomes established

[69]

Amomum villosum Lour 15 seedAtractylodes macrocephalaKoidz 20 Root-like

stemChaenomeles sinensis 20 FruitDolichos lablab L 20 SeedGlycyrrhiza uralensisFischeret DC

15 Radix

Magnolia officinalis Rehdet Wils 20 Bark

Panax ginseng CA Meyer 15 RootPinellia ternata (Thunb)Breit 15 Root and

stemPoria cocos (Schw) Wolf 25 SclerotiumPrunus armeniaca L 15 Seed

Zingiber officinale Roscoe 15 Root-likestem

Ziziphus jujuba MillVar inermis (Bge) Rehd 15 Fruit


(b) Continued


Xiao Qing LongTang (XQLT)

Ephedra sinica Stapf 90 Stem

Hot water extract of XQLT dose-dependentlyinhibited HRSV-induced plaque formationwhen given before viral inoculation(119875 lt 00001)viral attachment darr (119875 lt 00001)RSV internalization darr (119875 lt 00001)CC50 300 120583gmL IC50 342ndash828 120583gmLIC50 value of ribavirin was similarin A549 cellsIC50 was 226 120583gmL (2 h before)IC50 was 504 120583gmL (1 h before)IC50 was 1281 120583gmL (1 h after)secreting IFN-120573 uarr before and after viralinoculation to counteract viral infection(119875 lt 00001)

[70]

Cinnamomum cassiaBlume 60 Twig

Paeonia lactiflora Pall 90 RootGlycyrrhiza uralensis Fisch 60 RootZingiber officinale Rosc 30 RhizomePinellia ternata (Thunb)Breit 90 Tuber

Asarum heterotropides FMaekawa 30 Whole plant

Schisandra chinensis(Turcz) Baill 30 Fruit

IC50 is the concentration of the sample required to inhibit virus-induced CPE 50 CC50 is the concentration of the 50 cytotoxic effect selective index (SI)or therapeutic index (TI) CC50IC50 IFN-120573 interferon 120573 TNF-120572 tumor necrosis factor-120572 A549 human lung carcinoma cell

Table 6 The mechanisms of TCMHs-based prevention and treat-ment of RSV

Prevention TreatmentViral attachment darr[1 24 29 32 68ndash70]viral internalization darr[1 24 29 32 68ndash70]

RSV entry darr [24 32ndash49]

IFN-120573 TNF-120572 productionrarrviral replication darr[1 24 29 32 54 69 70]

RSV replication darr[32ndash49 51ndash55 57]

IFN-120574 productionrarr RSVviral infection darr[30 31 50 57 67]

Syncytium formation of RSVdarr [26]

The survival of human lungepithelial cells uarr [31]

Lung inflammation darr[30 50ndash52 54ndash56 67 68]

Activate the immune system[30 50 57]

F protein expression of RSV darr[50]

Blocked proinflammatorygene expression [31 54 68](in the human alveolarepithelial cell line)

Viral loads in serum and lungtissue darr [51ndash54 67]no adverse reaction was found[53 55]

Authorsrsquo Contributions

All the authors wrote the first draft of the paper togetherAll the authors contributed to the review of all papers andconstructed the final report

Acknowledgments

This study was supported by National Natural Science Foun-dation of China (no 81373688) the Postgraduate Researchand InnovationProject of Colleges andUniversities of JiangsuProvince (Grant no SJLX 0432) and The project of YouthFoundation of Jiangsu Province (Grant no BK 20151004)

References

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[2] H Nair D J Nokes B D Gessner et al ldquoGlobal burden of acutelower respiratory infections due to respiratory syncytial virusin young children a systematic review and meta-analysisrdquo TheLancet vol 375 no 9725 pp 1545ndash1555 2010

[3] P F Wright R B Belshe H W Kim L P Van Voris andR M Chanock ldquoAdministration of a highly attenuated liverespiratory syncytial virus vaccine to adults and childrenrdquoInfection and Immunity vol 37 no 1 pp 397ndash400 1982

[4] H W Kim J G Canchola C D Brandt et al ldquoRespiratorysyncytial virus disease in infants despite prior administrationof antigenic inactivated vaccinerdquo American Journal of Epidemi-ology vol 89 no 4 pp 422ndash434 1969

[5] R B Belshe L P Van Voris and M A Mufson ldquoParenteraladministration of live respiratory syncytial virus vaccine resultsof a field trialrdquo Journal of Infectious Diseases vol 145 no 3 pp311ndash319 1982

[6] M J Polak ldquoRespiratory syncytial virus (RSV) overview treat-ment and prevention strategiesrdquo Newborn and Infant NursingReviews vol 4 no 1 pp 15ndash23 2004

[7] S S Bawage P M Tiwari S Pillai V Dennis and S R SinghldquoRecent advances in diagnosis prevention and treatment ofhuman respiratory syncytial virusrdquo Advances in Virology vol2013 Article ID 595768 26 pages 2013

[8] K A Shadman and E R Wald ldquoA review of palivizumaband emerging therapies for respiratory syncytial virusrdquo ExpertOpinion on BiologicalTherapy vol 11 no 11 pp 1455ndash1467 2011

[9] R Rudraraju B G Jones R Sealy S L Surman and JL Hurwitz ldquoRespiratory syncytial virus current progress invaccine developmentrdquo Viruses vol 5 no 2 pp 577ndash594 2013

[10] K M Empey R S Peebles Jr and J K Kolls ldquoPharmacologicadvances in the treatment and prevention of respiratory syncy-tial virusrdquo Clinical Infectious Diseases vol 50 no 9 pp 1258ndash1267 2010


[11] R C Welliver ldquoPharmacotherapy of respiratory syncytial virusinfectionrdquo Current Opinion in Pharmacology vol 10 no 3 pp289ndash293 2010

[12] R W Sidwell and D L Barnard ldquoRespiratory syncytial virusinfections recent prospects for controlrdquo Antiviral Research vol71 no 2-3 pp 379ndash390 2006

[13] K Chen and B Yu ldquoCertain progress of clinical research onChinese integrativemedicinerdquoChineseMedical Journal vol 112no 10 pp 934ndash937 1999

[14] N Wiseman ldquoTraditional Chinese medicine a brief outlinerdquoJournal of Chemical Information and Computer Sciences vol 42no 3 pp 445ndash455 2002

[15] T Li and T Peng ldquoTraditional Chinese herbal medicine as asource of molecules with antiviral activityrdquo Antiviral Researchvol 97 no 1 pp 1ndash9 2013

[16] B Shen ldquoA new golden age of natural products drug discoveryrdquoCell vol 163 no 6 pp 1297ndash1300 2015

[17] D M Lindell S B Morris M P White et al ldquoA novel inac-tivated intranasal respiratory syncytial virus vaccine promotesviral clearance without TH2 associated Vaccine-Enhanced dis-easerdquo PLoS ONE vol 6 no 7 Article ID e21823 pp 1ndash12 2011

[18] A S Barbas T E Downing K R Balsara et al ldquoChronicaspiration shifts the immune response from Th1 to Th2 ina murine model of asthmardquo European Journal of ClinicalInvestigation vol 38 no 8 pp 596ndash602 2008

[19] D C Newcomb and R S Peebles Jr ldquoTh17-mediated inflamma-tion in asthmardquo Current Opinion in Immunology vol 25 no 6pp 755ndash760 2013

[20] F O Martinez L Helming R Milde et al ldquoGenetic programsexpressed in resting and IL-4 alternatively activated mouse andhuman macrophages similarities and differencesrdquo Blood vol121 no 9 pp e57ndashe69 2013

[21] S L Cyr I Angers L Guillot et al ldquoTLR4 and MyD88control protection and pulmonary granulocytic recruitment ina murine intranasal RSV immunization and challenge modelrdquoVaccine vol 27 no 3 pp 421ndash430 2009

[22] S L Cyr T Jones I Stoica-Popescu D Burt and B J WardldquoC57Bl6 mice are protected from respiratory syncytial virus(RSV) challenge and IL-5 associated pulmonary eosinophilicinfiltrates following intranasal immunization with Protollin-eRSV vaccinerdquo Vaccine vol 25 no 16 pp 3228ndash3232 2007

[23] N Dey T Liu R P Garofalo and A Casola ldquoTAK1 regulatesNF-120581B and AP-1 activation in airway epithelial cells followingRSV infectionrdquo Virology vol 418 no 2 pp 93ndash101 2011

[24] K-C Wang J-S Chang L-C Chiang and C-C LinldquoSheng-Ma-Ge-Gen-Tang (Shoma-kakkon-to) inhibited cyto-pathic effect of human respiratory syncytial virus in cell linesof human respiratory tractrdquo Journal of Ethnopharmacology vol135 no 2 pp 538ndash544 2011

[25] E Bartee M R Mohamed and GMcFadden ldquoTumor necrosisfactor and interferon cytokines in harmonyrdquo Current Opinionin Microbiology vol 11 no 4 pp 378ndash383 2008

[26] L M Ooi H Wang C-W Luk and V E Ooi ldquoAnticancerand antiviral activities of Youngia japonica (L) DC (AsteraceaeCompositae)rdquo Journal of Ethnopharmacology vol 94 no 1 pp117ndash122 2004

[27] X-H Xie N Zang S-M Li et al ldquoResveratrol inhibitsrespiratory syncytial virus-induced IL-6 production decreasesviral replication and downregulates TRIF expression in airwayepithelial cellsrdquo Inflammation vol 35 no 4 pp 1392ndash1401 2012

[28] N Zang X Xie Y Deng et al ldquoResveratrol-mediated gammainterferon reduction prevents airway inflammation and air-way hyperresponsiveness in respiratory syncytial virus-infectedimmunocompromisedmicerdquo Journal of Virology vol 85 no 24pp 13061ndash13068 2011

[29] J S Chang K CWang C F Yeh D E Shieh and L C ChiangldquoFresh ginger (Zingiber officinale) has anti-viral activity againsthuman respiratory syncytial virus in human respiratory tractcell linesrdquo Journal of Ethnopharmacology vol 145 no 1 pp 146ndash151 2013

[30] J S E Lee M K Y Cho H S U Hwang et al ldquoGinsengdiminishes lung disease in mice immunized with formalin-inactivated respiratory syncytial virus after challenge by modu-lating host immune responsesrdquo Journal of Interferon amp CytokineResearch vol 34 no 11 pp 902ndash914 2014

[31] J S Lee E-J Ko H S Hwang et al ldquoAntiviral activity ofginseng extract against respiratory syncytial virus infectionrdquoInternational Journal of Molecular Medicine vol 34 no 1 pp183ndash190 2014

[32] C F Yeh K C Wang L C Chiang D E Shieh M H Yenand J S Chang ldquoWater extract of licorice had anti-viral activityagainst human respiratory syncytial virus in human respiratorytract cell linesrdquo Journal of Ethnopharmacology vol 148 no 2pp 466ndash473 2013

[33] Y-P Deng Y-Y Liu Z Liu et al ldquoAntiviral activity of FoliumIsatidis derived extracts in vitro and in vivordquo American Journalof Chinese Medicine vol 41 no 4 pp 957ndash969 2013

[34] M X Su Y L Li Y H Zhou and R Z Chen ldquoStudy onthe the antiviral and antibacterial activities of the flowers ofFlos Pueraria omeiensis (China)rdquo Journal of Jinan University(Medicine Edition) vol 27 no 2 pp 204ndash208 2006

[35] X Li H Y Li Z P Xie Z H Chen and J J Zhang ldquoThe anti-respiratory syncytial virus effect of an active compound RG2-1 from Radix Gentianae in vitrordquo Chinese Journal of DiseaseControl amp Prevention vol 11 no 2 pp 154ndash156 2007

[36] Z H Chen H Y Li Z P Xie J J Zhang X Li and J XWang ldquoThe anti-respiratory syncytial virus effect of an activecompound RG3-1 from Radix Gentianae in vitrordquo Journal ofHarbinMedical University vol 41 no 4 p 344 2007 (Chinese)

[37] S S Li H Y Li Y A Piao D L Liu W J Tian and YM Dong ldquoThe anti-respiratory syncytial virus effect of anactive compound (AP3) from aTraditional ChineseHerb-herbapatriniae in vivordquo Chinese Journal of Epidemiology vol 25 no2 pp 150ndash153 2004

[38] F M Zhang L Liu X Li et al ldquoThe extraction purificationand identification of an active compound (AP4) from Herbapatriniae with its effect against the respiratory syncytial virusin vitro (Chin)rdquo Journal of Chinese Medicinal Materials vol 31no 12 pp 1879ndash1881 2008

[39] R G Liang W B Liu Z N Tang and Q Xu ldquoInhibition onrespiratory syncytial virus in vitro by flavonoids extracted fromthe core of Litchi Chinensisrdquo Journal of Fourth Military MedicalUniversity vol 27 no 20 pp 1881ndash1883 2006 (Chinese)

[40] X Q Wang H Y Li X Y Liu et al ldquoThe anti-respiratorysyncytial virus effect of active compound of Glycyrrhiza GD4in vitrordquo Chinese Herbal Medicine Magazine vol 29 no 7 p692 2006

[41] X Q Wang H Y Li X Y Liu et al ldquoIn vitro evaluation ofSecoiridoid Glucosides from the fruits of Ligustrum lucidumas antiviral agentsrdquo Chinese Herbal Medicine Magazine vol 29no 7 p 692 2006


[42] Y Wang M Chen J Zhang et al ldquoFlavone C-glycosides fromthe leaves of Lophatherum gracile and their in vitro antiviralactivityrdquo Planta Medica vol 78 no 1 pp 46ndash51 2012

[43] Y-L Li K-M Li M-X Su K-T Leung Y-W Chen and Y-WZhang ldquoStudies on antiviral constituents in stems and leaves ofPithecellibium clypeariardquo Zhongguo Zhong Yao Za Zhi vol 31no 5 pp 397ndash400 2006

[44] 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

[45] L-Y Ma S-C Ma F Wei et al ldquoUncinoside A and B twonew antiviral chromone glycosides from Selaginella uncinatardquoChemical and Pharmaceutical Bulletin vol 51 no 11 pp 1264ndash1267 2003

[46] W-S Ho J-Y Xue S S M Sun V E C Ooi and Y-L LildquoAntiviral activity of daphnoretin isolated from Wikstroemiaindicardquo Phytotherapy Research vol 24 no 5 pp 657ndash661 2010

[47] WHuang X Zhang YWang et al ldquoAntiviral biflavonoids fromRadix Wikstroemiae (Liaogewanggen)rdquo Chinese Medicine vol5 article 23 pp 2ndash6 2010

[48] L S M Ooi H Wang Z He and V E C Ooi ldquoAntiviralactivities of purified compounds fromYoungia japonica (L) DC(Asteraceae Compositae)rdquo Journal of Ethnopharmacology vol106 no 2 pp 187ndash191 2006

[49] Y Li L S M Ooi H Wang P P H But and V E C OoildquoAntiviral activities of medicinal herbs traditionally used insouthern mainland Chinardquo Phytotherapy Research vol 18 no9 pp 718ndash722 2004

[50] J Wang J F Wu L W Kong et al ldquoBuShenYiQi FormulastrengthensTh1 response and suppressesTh2-Th17 responses inRSV-induced asthma exacerbated micerdquo Journal of Ethnophar-macology vol 154 no 1 pp 131ndash147 2014

[51] C L Wu Z Q Yang W Hou C X Du and F LuoldquoExperimental study on Shuang huang lian oral liquid againstrespiroviruses (Chin)rdquo Journal of Mathernatical Medicine vol18 no 6 pp 592ndash594 2005

[52] H Y Mo K F Lai Y N Jiang J P Xie Y X Tan andN S Zhong ldquoEffect of Shuang huang lian on the releaseof chemokine in RSV-infected A549 airway epithelial cellrdquoAcademic Journal of Guangzhou Medical College vol 33 no 2p 16 2005 (Chinese)

[53] X J Huang W Hou Y L Zhao F Luo and Z Q Yang ldquoAnexperimental study on anti-respiratory syncytial virus with Xiasang ju extractrdquo Chinese Journal of Modern Drug Applicationvol 1 no 8 pp 11ndash14 2007 (Chinese)

[54] Z-G Chen H Luo S-CWang J-Y Xu and J-X Li ldquoAntiviraleffects of Jinxin oral liquid against respiratory syncytial virusinfection in the BALBc mice modelrdquo Journal of Ethnopharma-cology vol 162 no 2 pp 287ndash295 2015

[55] B Yuan X Z Ren YQ Sun et al ldquoMulti-centered single-blindrandom controlled clinical study on Qing fei oral liquid for166 children cases with respiratory syncytial virus pneumonia(Chin)rdquo Journal of Traditional Chinese Medicine vol 50 no 3pp 221ndash223 2009

[56] B Yuan L H Zhou L Li C J Zhao andQ QWu ldquoRegulatoryeffects of Qing fei oral liquid on Interleukin-6 and Interleukin-8 secreted by human epithelial cells infected with respiratorysyncytial virus (Chin)rdquo Journal of Emergency in TraditionalChinese Medicine vol 20 no 4 pp 596ndash598 2011

[57] X Z Ren S C Wang J Q Li K Yao and X L WuldquoExperimental studies on main effects of Qing fei oral liquid

for infant viral pneumoniardquo Chinese Journal of Basic Medicinein Traditional Chinese Medicine vol 8 no 10 pp 22ndash25 2005(Chinese)

[58] D J Newman G M Cragg and K M Snader ldquoNaturalproducts as sources of new drugs over the period 1981ndash2002rdquoJournal of Natural Products vol 66 no 7 pp 1022ndash1037 2003

[59] M S Butler ldquoThe role of natural product chemistry in drugdiscoveryrdquo Journal of Natural Products vol 67 no 12 pp 2141ndash2153 2004

[60] Collaboration Research Group for Qinghaosu ldquoAntimalariastudies on Qinghaosurdquo Chinese Medical Journal vol 92 no 12pp 811ndash816 1979

[61] A Dondorp F Nosten K Stepniewska N Day and N WhiteldquoArtesunate versus quinine for treatment of severe falciparummalaria a randomised trialrdquoThe Lancet vol 366 no 9487 pp717ndash725 2005

[62] G Sun R Ouyang S Chen et al ldquoTreatment of acutepromyelocytic leukemia with all-trans retinoic acid A five-yearexperiencerdquo Chinese Medical Journal vol 106 no 10 pp 743ndash748 1993

[63] S-J Chen G-B Zhou X-W Zhang J-H Mao H DeThe andZ Chen ldquoFrom an old remedy to a magic bullet molecularmechanisms underlying the therapeutic effects of arsenic infighting leukemiardquo Blood vol 117 no 24 pp 6425ndash6437 2011

[64] G Bae J-R Yu J Lee J Chang and E-K Seo ldquoIdentificationof Nyasol and structurally related compounds as the activeprinciples from Anemarrhena asphodeloides against respiratorysyncytial virus (RSV)rdquo Chemistry and Biodiversity vol 4 no 9pp 2231ndash2235 2007

[65] F Y Fung and Y C Linn ldquoDeveloping traditional Chinesemedicine in the era of evidence-based medicine current evi-dences and challengesrdquo Evidence-Based Complementary andAlternative Medicine vol 2015 Article ID 425037 9 pages 2015

[66] R Kramer and D Cohen ldquoFunctional genomics to new drugtargetsrdquo Nature Reviews Drug Discovery vol 3 no 11 pp 965ndash972 2004

[67] L Li C-H Yu H-Z Ying and J-M Yu ldquoAntiviral effectsof modified Dingchuan decoction against respiratory syncytialvirus infection in vitro and in an immunosuppressive mousemodelrdquo Journal of Ethnopharmacology vol 147 no 1 pp 238ndash244 2013

[68] Q Liu L Lu M Hua et al ldquoJiawei-Yupingfeng-Tang a Chineseherbal formula inhibits respiratory viral infections in vitro andin vivordquo Journal of Ethnopharmacology vol 150 no 2 pp 521ndash528 2013

[69] J S Chang K CWang D E Shieh and L C Chiang ldquoLiu-He-Tang inhibited plaque formation by human respiratory syncytialvirus infection in cell lines of the human respiratory tractrdquoJournal of Ethnopharmacology vol 137 no 3 pp 1149ndash1155 2011

[70] J S Chang C F Yeh K C Wang D E Shieh M H Yen andL C Chiang ldquoXiao-Qing-Long-Tang (Sho-seiryu-to) inhibitedcytopathic effect of human respiratory syncytial virus in celllines of human respiratory tractrdquo Journal of Ethnopharmacologyvol 147 no 2 pp 481ndash487 2013

[71] Z Xu ldquoModernization one step at a timerdquoNature vol 480 no7378 pp S90ndashS92 2011

[72] W-H Ma and X-H Sun ldquoDiscussion of time and spacedifferentiation of three-yin and three-yang in Shanghan LunrdquoJournal of Chinese Integrative Medicine vol 3 no 4 pp 257ndash259 2005

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


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Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


[11 12] There are any antiviral agents against RSV that arecurrently under development

This review aims to explore evidence available on pre-ventativemeasures against RSV infection discuss the currentstatus of anti-RSV research on TCMH-derived active com-pounds TCMHs and traditional Chinese medical formulas(TCMFs) and finally highlight the challenges issues andfuture directions of using TCMHs in clinical practice

2 Evidence Supporting the Efficacy of TCMHs

Traditional Chinese medical herbs are the most importantcomponents of the traditional Chinese medicine (TCM)system which have been reported to cure infectious diseasesin the form of hot water extracts for almost 2000 yearsOver 10000 herbal medicines and 100000 recipes have beendocumented in ancient literature [13]

Nowadays TCMHs are widely used for the preven-tion and treatment of viral infectious diseases in Chinaand many other Asian countries They are associated withadvantages such as lower toxicity economical and multi-functional use in regulating immunological function andviral destruction [14] This reduction in viral load allowsfor the screening of effective medications However theinternational community remains uncertain about the effi-cacy of TCMHs due to the lack of supporting clinical evi-dence collected under international standards (randomizedplacebo-controlled double-blind and multicentered clinicalstudies) Governments have put forward support aimed atthe international regulatory approval of TCMHs Leadingthe pack is compound T89 (also known as Dantonic atraditional Chinese medical product by Tasly Pharmaceuti-cals China) which is used in China for the managementof ischemic heart disease Dantonic may become the firsttraditional Chinese medicine to receive Food and DrugAdministration (FDA) approval in the United States [15]Moreover the Yinhuang granules (tablet) and Xiasangjugranules have been approved by the China Food and DrugAdministration (SFDA) for the treatment of RSV-infectiousdiseases [15] The 2015 Nobel Prize in Medicine was awardedto Youyou Tu for the discovery of artemisinin a drug isolatedfrom a plant which has significantly reduced the mortal-ity rates for patients with malaria This acknowledgementhas increased the optimism of the natural product commu-nity worldwide [16]

3 Prevention

Prevention is the most important aspect of healthcare andbecause of the nature of the respiratory syncytial virus andmode of infection reinfections are a common event [6] LiveRSV can survive on surfaces for several hours moreoverdirect or indirect contact with the nasopharyngeal secretionsor droplets (sneezing coughing and kissing) fomites andfood from infected patients can potentially transmit RSV[7] Therefore without effective preventive measures themortality and economic burden of the disease will increase

31 RSV F Protein Neutralizing Antibodies The respiratorysyncytial virus depends on the host cell to complete its

replication cycle which includes attachment and entry intothe host cell transcription of viral mRNA viral genomereplication protein synthesis and the assembly and buddingof progeny virus particles [15] The F protein of RSV accu-mulates in the host membrane and surrounds the buddingprogeny viruses thus spreading the infection to adjacent cellsand exacerbating the infection The F protein is thereforeconsidered a potential candidate for the development ofpossible preventive measures Palivizumab is a humanizedmonoclonal antibody (IgG) directed against an epitope inthe A antigenic site of the F protein of RSV Palivizumab-N a plant-based F neutralizing antibody was experimentedin phages and plants Successful palivizumab-N productionwas observed in the Nicotiana benthamiana plant systemwhich offered glycosylation and high production at lowupstream and equivalent downstream cost when comparedto mammalian derived palivizumab [7]

32 Boost Resistance against RSV by Modulating ImmuneResponse Following coevolution with the host many viruseshave established sophisticated mechanisms to interact withthe host immune system for immune evasion Among theclasses of antiviral agents immunomodulators are the mostabundant in TCMHs [15] Traditional Chinese medical herbshave been shown to enhance antibody production T cellproliferation the expression of antigen specific CD4+ andCD8+ responses and increase of the titers of IgG1 IgG2a andIgA along with the function to balance Th1 Th2Th17 typeimmune response [17] Interferon- (IFN-) 120574 and interleukin-(IL-) 12 are representatives of Th1 type cytokines interleukin4 and interleukin 5 belong toTh2 type cytokines and IL-6 andIL-17a are representatives ofTh17 type cytokines all of whichplay key roles in Th2Th17 responses [18 19] And recentlymacrophages have been identified as stimulation cells byinteraction with Th2 cytokine IL-4 [20] Traditional Chinesemedical herbs were reported to simultaneously promoteTh1-response including increased levels of Th1 type cytokinesecretion and activation of alveolar macrophages suppressTh2Th17-responses and maintain the balance between Th1and Th2Th17 cells for the prevention and treatment ofdiseases

Moreover TCMHs have been previously reported to offerefficient protection against RSV and significantly decreaseviral load and inflammation [15] It is well-known that theproduction of inflammatory mediators is regulated by atranscription factor NF-120581B which plays a pivotal role ininflammation due to its ability to induce the transcriptionof inflammatory genes [21ndash23] Toll-like receptor 4 (LR4)is also a factor expressed on the respiratory epithelium andmacrophages and appears to play a central role in mediatingboth the antiviral and inflammatory responses of the innateimmunity to combat viral infections [22] Along with directantiviral activity TCMHs could be beneficial in preventingRSV infection due to its immune-modulatory effect produc-ing of IFN-120573 and TNF-120572 Respiratory syncytial viral infectioncan induce cellular production of IFN-120573 and TNF-120572 BothIFN and TNF contribute to the innate immunity againstviral infection [24] and TNF triggers multiple antiviral












Root





[32]







4 Treatment


























[34]



[35 36]



[37 38]




[32 40]


[41]



[42]





[44]


(b)




[44]

Selaginellauncinata



[45]




[47]

Youngia japonica



[26 48]





Bupleurum chinense

Root

368 4413 120

[44]



Aerial parts




Whole plant































30772308










[24]





1111 Radix







(b)





[54]




10 Rootstalk


10 Root




Gypsum(1198621198861198781198744sdot21198672119874)

15 mdash


(b) Continued





[55]




10 Root


IL-6 darrIL-8 darr


Gypsum(1198621198861198781198744sdot21198672119874)









6 Conclusions


Competing Interests









Ge Gen Tang(GGT)



[1]





[68]





(b)


Liu He Tang(LHT)



[69]



15 Radix







(b) Continued





[70]





















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Root





[32]







4 Treatment


























[34]



[35 36]



[37 38]




[32 40]


[41]



[42]





[44]


(b)




[44]

Selaginellauncinata



[45]




[47]

Youngia japonica



[26 48]





Bupleurum chinense

Root

368 4413 120

[44]



Aerial parts




Whole plant































30772308










[24]





1111 Radix







(b)





[54]




10 Rootstalk


10 Root




Gypsum(1198621198861198781198744sdot21198672119874)

15 mdash


(b) Continued





[55]




10 Root


IL-6 darrIL-8 darr


Gypsum(1198621198861198781198744sdot21198672119874)









6 Conclusions


Competing Interests









Ge Gen Tang(GGT)



[1]





[68]





(b)


Liu He Tang(LHT)



[69]



15 Radix







(b) Continued





[70]





















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































[34]



[35 36]



[37 38]




[32 40]


[41]



[42]





[44]


(b)




[44]

Selaginellauncinata



[45]




[47]

Youngia japonica



[26 48]





Bupleurum chinense

Root

368 4413 120

[44]



Aerial parts




Whole plant































30772308










[24]





1111 Radix







(b)





[54]




10 Rootstalk


10 Root




Gypsum(1198621198861198781198744sdot21198672119874)

15 mdash


(b) Continued





[55]




10 Root


IL-6 darrIL-8 darr


Gypsum(1198621198861198781198744sdot21198672119874)









6 Conclusions


Competing Interests









Ge Gen Tang(GGT)



[1]





[68]





(b)


Liu He Tang(LHT)



[69]



15 Radix







(b) Continued





[70]





















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Bupleurum chinense

Root

368 4413 120

[44]



Aerial parts




Whole plant































30772308










[24]





1111 Radix







(b)





[54]




10 Rootstalk


10 Root




Gypsum(1198621198861198781198744sdot21198672119874)

15 mdash


(b) Continued





[55]




10 Root


IL-6 darrIL-8 darr


Gypsum(1198621198861198781198744sdot21198672119874)









6 Conclusions


Competing Interests









Ge Gen Tang(GGT)



[1]





[68]





(b)


Liu He Tang(LHT)



[69]



15 Radix







(b) Continued





[70]





















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































30772308










[24]





1111 Radix







(b)





[54]




10 Rootstalk


10 Root




Gypsum(1198621198861198781198744sdot21198672119874)

15 mdash


(b) Continued





[55]




10 Root


IL-6 darrIL-8 darr


Gypsum(1198621198861198781198744sdot21198672119874)









6 Conclusions


Competing Interests









Ge Gen Tang(GGT)



[1]





[68]





(b)


Liu He Tang(LHT)



[69]



15 Radix







(b) Continued





[70]





















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(b) Continued





[55]




10 Root


IL-6 darrIL-8 darr


Gypsum(1198621198861198781198744sdot21198672119874)









6 Conclusions


Competing Interests









Ge Gen Tang(GGT)



[1]





[68]





(b)


Liu He Tang(LHT)



[69]



15 Radix







(b) Continued





[70]





















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Ge Gen Tang(GGT)



[1]





[68]





(b)


Liu He Tang(LHT)



[69]



15 Radix







(b) Continued





[70]





















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(b) Continued





[70]





















Acknowledgments


References

















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article application of traditional chinese medical

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