Molecular herbal inhibitors of Dengue virus: an...

Int. J. Med. Arom. Plants, ISSN 2249 – 4340REVIEW ARTICLE

Vol. 2, No. 1, pp. 1-21, March 2012

*Corresponding author: (E-mail) dengue.herbal.network<A.T.>gmail.com http://www.openaccessscience.com©2012 Open Access Science Research Publisher [email protected]

Molecular herbal inhibitors of Dengue virus: an update

Shivendra KUMAR1#, Sunil KUMAR2#, Ishita REHMAN3#, Pradeep DHYANI4#, Lalita KU-

MARI5*#, Charu6#, Sudeep ACHARYA7#, Girdhar BORA8#, Prashant DURGAPAL9#, Amit

KUMAR#9

1Recombinant Gene Product Laboratory, International Centre for Genetic Engineering and Biotechnology,New Delhi-110067, India2Biologics Development Center, Dr. Reddy's Laboratories Ltd, Hyderabad, Andra Pradesh 500090, India3Institute of Genomics and Integrative Biology, Delhi-110 007, India4Department of Biochemistry, Lok Nayak Hospital, New Dehli-110002, India5Charitable Society Clinic, Naryana Vihar, New Delhi-110028, India6Department of Pediatrics, All India Institute of Medical Sciences, New Delhi-110029, India7Department of Radiodiagnosis, All India Institute of Medical Sciences, New Delhi-110029, India8Department of Urology, All India Institute of Medical Sciences, New Delhi-110029, India9Department of Pathology, All India Institute of Medical Sciences, New Delhi-110029, India

*Corresponding Author, Tel: +91-011-32403212#all authors have contributed equally.

Article History: Received 23rd February 2012, Revised 18th March, 2012, Accepted 23th March, 2012.

Abstract: Dengue fever is the most prevalent mosquito transmitted viral infection affecting 2.5 billion people across theglobe. Etiological agent (dengue virus) is an enveloped positive sense RNA virus belonging to the family Flaviviridae. Ithas four serotypes which does not provide cross protection immunity against each other making vaccine developmentdifficult due to antibody-dependent enhancement effect. Till now there is no approved vaccine or drug against this virus.Therefore, there is an urgent need of development of alternative solutions for dengue. In traditional medicine systemplants extracts have been used to cure several human aliments. Several plant species have been reported with anti-dengueactivity. However, mechanism of action of only some of the anti-dengue herbal compounds isolated from medicinalplants have been elucidate while other needs further investigations. With the advent of synthetic biology and metagenom-ics and high throughput screening, it have become possible to screen and produce plant based effector molecules intomicroorganism like E. coli at larger amount and at relatively low cost. Networking is the key of success. Networkingamong pure scientist, clinicians, applied scientist and industry people can only make possible to available these naturalbioactive compounds against dengue virus from nature to bedside for humankind.

Keywords: Dengue; medicinal plants; patents; inhibitors; anti-dengue herbal drugs; scientific networking.

Introduction

Dengue is the most prevalent mosquitoborne viral infection in the world (WHO 2009;Guzman et al. 2010; Whitethorn and Farrar2010). The name Dengue virus (DENV) refersto a group of four antigenically and geneticallyrelated viruses called serotypes (DENV1-4)(Halstead 1988; WHO 2009; Whitethorn and

Farrar 2010; Chen et al. 2011; Rothman 2011).Each serotype is capable of causing dengue fev-er and manifests in severe forms (dengue he-morrhagic fever (DHF)/dengue shock syndrome(DSS)) (Gubler 1998; WHO 2009; Whitethornand Farrar 2010; Murphy and Whitehead 2011).According to the World Health Organization(WHO), nearly 2.5 billion people are at the risk

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2Int. J. Med. Arom. Plants Medicinal Plants and Dengue: a molecular herbal update

Kumar et al. http://[email protected]

of dengue virus infection (WHO 2009) (Figure1). Dengue is endemic in more than 100 coun-tries in Asia, Pacific, America, Africa, and Ca-ribbean (Figure 1). Dengue manifests in bothepidemics as well as sporadic forms (Gubler1998; WHO 2009; Thomas and Endy 2011).During epidemics of dengue, infection ratesamong those who have not been previously ex-

posed to the virus are often 40% to 50%, butcan reach up to 80% to 90% (Gibbons et al.2007; Kyle and Harris 2008; WHO 2009). Anestimated 500 000 people with DHF requirehospitalization each year, a significant propor-tion of whom are children with mortality rate2.5% among infected people (Singhi et al. 2007;WHO 2009; Thomas and Endy 2011).

Figure 1: Geographic distribution of dengue (From Centers for Disease Control and Prevention)(http://www.healthmap.org/dengue/index.php)

Several approaches have been employed tofight with DENV including monoclonal antibo-dies (Sukupolvi-Petty et al. 2010; Deng et al.2011; Cockburn et al. 2012), RNAi technology(Sanchez-Vargas et al. 2004; Stein and Shi2008; Wu et al. 2010; Lee et al. 2010; Subra-manya et al. 2010; Alhoot et al. 2011; Burnett etal. 2011; Stein et al. 2011; Aliabadi et al. 2012;Foged 2012;) vaccines (Durbin and Whitehead2011) which include live attenuated vaccine(Guirakhoo et al. 2006; Kitchener et al. 2006;Guy and Almond 2008; Simmons et al. 2010);subunit tetravalent vaccine (Batra et al. 2010;Morrison et al. 2010; Watanaveeradej et al.2011), inactivated vaccines (Maves et al. 2010,2011; Simmons et al. 2010); DNA vaccines(Azevedo et al. 2011; Costa et al. 2011; Dankoet al. 2011). However, till now there is no li-censed vaccine or drug available in the market

for dengue. Prevention measures are insuffi-cient and only supportive therapy is available(Clarke 2002; WHO 2009; Thomas and Endy2011; Whitehorn and Simmons 2011).

Hence, there is an urgent need to find alter-nate solutions to combat dengue. Plants andplant derived compounds remain an importantsource for the discovery and the development ofnew antiviral drugs because of their expectedlow side effects and their high accessibility inthe nature (Balandrin et al. 1985; Manuel et al.1993; Rates 2001; Dewick 2002; Schmidt et al.2007; Saklani and Kutty 2008; Adams et al.2011; Bruno 2011; Cordell 2011). This reviewwill provide an update on our understanding ofpathogenesis of this virus and summarize suc-cess stories of medicinal plants against DENVand how far are they from the target?



http://www.healthmap.org/dengue/index.php



Dengue transmission

The mosquitoes Aedes aegypti and Aedesalbopictus, are main culprits responsible fortransmitting dengue between people and arecosmopolitan in nature (Kyle and Harris 2008;WHO 2009; Guzman and Istúriz 2010; Lam-brechts et al. 2010; Ross 2010; Urdaneta-Marquez and Failloux 2011). The life cycle ofdengue virus involves two hosts: mosquito asvector, and targets humans.

When an infected mosquito bites a person, itinjects the DENV into the bloodstream of thehost. Subsequently virus infects nearby skincells called keratinocytes, the most commoncell type in the skin. In addition dengue virusalso infects and replicates inside a specializedimmune cell located in the skin, a type of den-dritic cell called a Langerhans cell (NATUREEDUCATION). Once infected, humans becomea reservoir and source of more viruses generat-ed during viral replication in host and thus serveas a source of the virus for uninfected mosqui-toes (Adams and Kapan 2009; WHO 2009;Omenn 2010). Patients who are already infectedwith the dengue virus can transmit the infectionvia Aedes mosquitoes. Subsequently, mosquitomainly acquires the virus while feeding on theblood of an infected person (WHO 2009; Guz-man et al. 2010; Whitethorn and Farrar 2010).

Within the mosquito, DENV travels to themid-gut of mosquito and afterward spreads tothe salivary glands in a time period of 8-12 days(WHO 2009). After this incubation period (inmosquito), the virus is ready for being transmit-ted to humans during probing or feeding.

It is worth mentioning here that Aedes mos-quito is a holometabolous insect which meansthat the mosquito goes through a complete me-tamorphosis with an egg, larvae, pupae, and fi-nally adult stage (Urdaneta-Marquez and Fail-loux 2011) (Figure 2). Average life span rangesfrom 14 to 30 days based on environmentalconditions. After taking a human blood meal(Hansen et al. 2004; Bryant et al. 2010) femaleAedes aegypti mosquito can produce on average100 to 200 eggs per batch (Wong et al. 2011).The females can produce up to five batches ofeggs during a lifetime which means 500-1000(Wong et al. 2011) eggs in a lifetime (Figure 2).

This gives a fair idea of chances of getting ofdengue infection in an endemic region.

Figure 2: Life cycle of vector Aedes aegyptiresponsible for transmission of dengue. (Fromhttp://www.denguevirusnet.com/life-cycle-of-aedes-aegypti.html)

Symptoms

Primary infection (for the first time) withdengue virus is usually asymptomatic or withmild symptoms which include high fever, retro-orbital pain (pain behind eyes), low plateletcount, joint and muscles pain which are nonspecific (WHO 2009; Guzman et al. 2010; Whi-tehorn and Farrar 2010; Whitehorn and Sim-mons 2011). Secondary infection with anotherDENV serotype can prove lethal with highmorbidity and mortality and manifests in theform of dengue hemorrhagic fever (DHF) anddengue shock syndrome (DSS) (Gubler 1998;WHO 2009; Faheem et al. 2011). Symptomsusually appear in 3-14 days (average 4–7 days)after the infective female Aedes bite.

Dengue hemorrhagic fever (DHF) is charac-terized by increased vascular permeability, hy-povolaemia (low blood volume), and abnormalblood clotting mechanisms. DHF is a potential-ly deadly complication with symptoms similarto those of dengue fever (Gubler 1998; Pottsand Rothman 2008; WHO 2009; Pawitan 2011;Guzman et al. 2010; Guzman and Vazquez2010; Rodenhuis-Zybert et al. 2010; Srikiatkha-chorn and Green 2010; Ranjit and Kissoon2011).

The dengue Shock Syndrome (DSS) is cha-racterized by bleeding that may appear as tinyspots of blood on the skin (petechiae) and largerpatches of blood under the skin (ecchymoses).



http://www.denguevirusnet.com/life-cycle-of-



Shock may result led to death within 12 to 24hours. Patients can recover following timelysupportive medical treatments (Kabra et al.1999; WHO 2009).

Our immunity and Dengue virus

Our adaptive immune response to DENVinfection contributes to resolution of the infec-tion (Murphy and Whitehead 2011). Dengueantibodies against one serotype can prevent theinfection of those specific serotypes and pro-vide lifelong immunity against that particularserotype. That’s why primary Dengue infectionis usually asymptomatic. However, this tempo-rary immunity usually wanes after 6 months,the point at which an individual is susceptible tothe other three DENV serotypes (Sabin 1952;Balakrishnan et al. 2011; Dowd and Pierson2011). Infection with a different serotype leadsto secondary infection as well as antibody de-pendent enhancement (ADE) (Dowd and Pier-son 2011; Murphy and Whitehead 2011).

Diagnosis of Dengue

Diagnosis of dengue is based on serology(ELISA) detection of IgM, IgG in patients(Gowri Sankar et al. 2012). Detection of denguevirus nucleic acid (RNA) in human blood isperformed by Reverse transcriptase PCR. Leu-copenia (decrease in leukocytes counts), throm-bocytopenia (decrease in platelet count) are alsogood indicators of dengue fever but are nonspecific. However, isolation of dengue virus isthe most reliable way to diagnose the dengue.But facility to isolate is not frequently availableespecially in developing countries (Peeling etal. 2010; Peh et al. 2011).

Dengue virus: Molecular organization

Dengue virus (DENV) belongs to the familyFlaviviridae, genus Flavivirus (ICTV 2005), is asmall (~50 nm), enveloped, single-stranded,positive sense RNA virus (~ 11 kb in size) withicosahedral symmetry (Figure 3A).

The 5’ end of the virus is protected by aclass I G cap followed by a short untranslatedregion (~100 nt) (NCR) which plays a critical

role in viral replication as well as translation(Iglesias and Gamarnik 2011). The 3’ NCR ofthe virus, which is relatively longer than the 5’end (~450 bp) has several conserved RNAstructures but lacks a terminal poly A tail (Igle-sias and Gamarnik 2011) (Figure 3A). Theseconserved structures are necessary for DENVreplication and also regulates translation.

Figure 3A: Schematic diagram of dengue virusgenome and polyprotein encoded by singleopen reading frame (From Tomlinson et al.,2009) (for description see text).

Figure 3B: Outline of the dengue virus replica-tion cycle (for description see text) (From Tom-linson et al. 2009)

Replication cycle Dengue Virus in Human:unique protein- potent drug

Briefly, the first step of replication is the at-tachment of virus to the specific receptorpresent on the host cells (Tomlinson et al. 2009;Smit et al. 2011; Smith 2012). The envelope (E)glycoprotein present on DENV membrane acts





as ligand that binds to receptors present on thehost cell membrane. It initiates the interactionbetween E-glycoprotein and the host receptor(s)which leads to receptor mediated endocytosis ofthe virus particle (Tomlinson et al. 2009; Smithet al. 2011) (Figure 3B). Further acidificationof the endocytic vesicle; release the entrappednucleocapsid to the cytoplasm where the virusgenome is released. DENV is a positive senseRNA virus with single open reading frame(ORF) thus behave like messenger RNA anddirectly translates to produce a large single po-lyprotein which subsequently undergoes posttranslational modification mediated by viral as

well as host proteases to produce seven nonstructural proteins (NS1, NS2A, NS2B, NS3,NS4A, NS4B, NS5) which are necessary forvirus replication and three structural proteins(capsid, pre membrane and envelope) which areinvolved in packaging and secretion of theDENV from infected cells (Tomlinson et al.2009) (Table 1). Replication occurs on intercel-lular membranes, and assembly take place onthe endoplasmic reticulum. Newly assembledvirion are transported through trans-golgi net-work and released from cell by exocytosis(Mukhopadhyay et al. 2005; Tomlinson et al.2009) (Figure 3B).

Table 1: Summarizing putative and characterized function of proteins encoded by dengue virus.

Protein func-tional class

Protein Name Function Reference

Non Structur-al Proteins

Non Structural(NS) protein 1

Essential for virus growth. May play role in pathogenesis ofDengue by contributing to endothelium dysfunction and alsohas immune evasive functions.However, precise function is not known.

Avirutnan et al. (2006),Gutsche et al. (2011),Somnuke et al. (2011)

NS2A Little information is available. NS2A has been associatedwith down-regulation of IFN-β-stimulated gene expression

Muñoz-Jordan et al. (2003)

NS2B Act as a cofactor for viral protease (NS3) Chambers et al. (1990),Zuo et al. (2009), Phong etal. (2011)

NS3 Multifunctional protein. Possess NTPase, helicase, andRTPase activities which help in unwinding of double strandedRNA formed during replication.

Bartelma and Padmanab-han (2002), Zuo et al.(2009), Su et al. (2009)

NS4A Precise function not known. However, it is critical determi-nant of Dengue replication.

McLean et al. (2011),Muñoz-Jordan et al. (2003)

NS4B Function is poorly understood. However, role in innate im-mune signaling and virulence is postulated.

Grant et al. (2011), Kelleyet al. (2011)

NS5 Multifunctional protein having Polymerase (RdRP),Methyltransferase activity.Beside recently NS5 has been shown to bind STAT2 and

inhibits IFN dependent signaling.

Ackermann and Padma-nabhan (2001), Egloff etal. (2002), Ashour et al.(2009), Dong et al. (2010),Iglesias et al. (2011)

StructuralProteins

Capsid Necessary for packaging and release of viral particles fromthe cell

Nowak et al. (1989)

prM protein(precursor ofthe M protein)

Protects the envelope protein from premature fusion duringtransit through the acidic environment of the trans-golgi net-work.

Nowak et al. (1989),Zheng et al. (2010)

Envelope Fusion and entry of virus into host cell and cell attachment Nowak et al. (1989), Crilland Roehrig (2001), Linet al. (2011)

The detailed function of dengue proteins in-volved in the viral replication is largely un-known. The role of NS1 is largely unknown butis essential for virus survival (Table 1). It is asoluble glycoprotein detected very early duringinfection thus considered to be a good diagnos-tic marker for Dengue. NS2A, NS2B, NS4A,

and NS4B are membrane –associated proteinsbelieved to anchor and regulate the replicationcomplex during the viral life-cycle (Table 1).NS2B-NS3 is a serine protease necessary forthe processing of polyprotein critical for virusreplication (Bollati et al. 2010; Keller et al.2006). NS5 is a multifunctional, the most high-





ly conserved of the dengue proteins protein andpossesses RNA Dependent RNA polymeraseactivity, key and unique to the virus as RdRp isabsent in mammals (Keller et al. 2006; Rawlin-son et al. 2006; Malet et al. 2008; Alcaraz-Estrada et al. 2010; Bollati et al. 2010). NS5RdRp domain is responsible for the generationof negative sense RNA which further act astemplate for the production of positive senseRNA for packaging. Viral proteins involved inreplication cycle of the virus have been used astargets for the development of new antiviralagents.

Alternative approach: Medicinal Plants: canthey combat DENV?

There is a long history of use of Plants tocure human diseases throughout the world (vanAgtmael et al. 1999; Li and Vederas 2009).Several plant derivatives have been successfullyentered clinical trials (Butler 2004; Koehn andCarter 2005; Harvey 2008; Saklani and Kutty2008). It is noteworthy that few studies haveshown that extract from different parts of plantsprovide better antiviral results compared to theirsynthetic analogues (Chiang et al. 2005; Bruno2011; Tang et al. 2012). As such, the develop-ment of a plant based antiviral preparationpromises a more potential alternative in com-bating dengue disease. Especially the WorldHealth Organization (WHO) advocated that de-veloping and developed countries should colla-borate with traditional medicine practitioners toexplore new aspects that provide safe, economi-cal, and effective remedies for viral diseases(WHO 2009; Bruno 2011; Tang et al. 2012).

According to WHO in some Asian andAfrican countries, nearly 80% of the populationdepend on traditional medicine for primaryhealth care (Grayson 2011). Out of which theherbal medicines are on the top of list whichalso strengthen the economy of several coun-tries and are quite popular in the internationalmarket. Herbal medicines refer to herbs, herbalmaterials, herbal preparations, and finished her-bal products that contain parts of plants or otherplant materials as active ingredients. However,it is noteworthy that herbal medicine are notrisk free and sometime can be lead to adverse

consequences if preparation is of poor quality(Gilbert 2011; Grayson 2011; Sakurai 2011).Strict regulation should be pose which can helpin developing safe and cheap herbal medicine.Unlike pharmaceuticals drugs for which de-tailed pharmacokinetics and molecular mechan-ism of inhibitor is known, herbal medicinesmostly lack such data (Tu 2011; Xu et al. 2012).

In further in this review, we have groupedmedicinal plants on the basis of their inhibitoryeffect on replication cycle of dengue viruswhich will help the reader to understand theirmolecular basis. However, several relevant stu-dies are present where anti- Dengue activity hasbeen demonstrated by plant extract by severalgroups. But their systemic pharmacological ex-amination and molecular basis have not yetbeen well characterized and needs further inves-tigations.

Medicinal Plants: Virus Entry Inhibitor

As described earlier, first step of viral infec-tious replication cycle is the attachment to thesusceptible host via receptor mediated interac-tions followed by entry into host cell (Figure3B) (Hase et al. 1989). Inhibiting virus host in-teraction at this step would be the most efficientstrategy against any viral infection. In dengueEnvelope protein Domain III is the receptor-binding motif thus critical for virus infection asit mediate host virus interaction (Crill and Roe-hrig, 2001).

Tong et al. (2010) in their study have dem-onstrated the inhibitory potential of the com-pound WSS45 (sulfate derivative of an alpha -D-glucan) derived from Gastrodia elata Bl. (asaprophytic perennial herb which is widely usedin traditional Chinese medicine preparation) hasdocumented antiviral potential against denguevirus serotype 2 in vitro using BHK cell line(Baby hamster kidney fibroblast cells). Usingvirus adsorption and virus cell penetration assaythey clearly demonstrate that WSS45 interferewith adsorption of the DENV to the host cellwithout significant cytotoxicity (Tong et al.2010). However, study was purely in vitro innature and restricted to DENV-2 only. Further-more, the ineffectiveness of WSS45 after virus





enters the susceptible cell strongly suggests thatit is a viral entry inhibitor (Tong et al. 2010).

Carrageenans: as virus entry inhibitor

The term Carrageenans refers to the familyof natural polysaccharides extracted from redsea weeds (Irish moss, Chondrus crispus).Chemically they are linear sulfate polysaccha-rides. There are several varieties of carragee-nans (kappa, lambda, Iota) which have been re-ported with potent antiviral effect e.g. papillo-mavirus virus (Buck et al. 2006; Roberts et al.2007). Recently clinical trial of iota carragee-nans based nasal spray has been evaluatedagainst common cold (Eccles et al. 2010).

In another significant study, Talarico andDamonte (2007) investigated the effect oflambda and iota carrageenans sulfate polysac-charides containing linear chains of galactopy-ranosyl residues on multiplication of DENV-2and DENV-3 in Vero (African green monkeyKidney epithelium cells) and HepG2 cells (hu-man liver hepatocellular carcinoma cell line).Their revealed that tested carrageenans werepotent inhibitors of DENV-2 in. Virus growthinhibition was demonstrated by plaque reduc-tion, virus yield inhibition and antigen expres-sion assays. Further using virus internalizationassay and real time PCR they demonstrated thatlambda-carrageenans hinder with the virus ad-sorption to the host cell as well in internaliza-tion step (Talarico and Damonte 2007). It isnoteworthy that lambda- carrageenans are hepa-ran sulfate imitative compound. Heparan sul-fate has been known as receptor or co receptorfor several viruses (Spillmann 2001; Liu andThorp 2002). In addition using infectious centerand virion uncoating assays, they demonstratethat carrageenan-treated virions remain trappedwithin endosomes (Talarico and Damonte2007). Furthermore, no inhibition in virusgrowth was observed when entry step was elim-inate using DENV 2 RNA transfection stronglyindicates that lambda-carrageenan is an effec-tive plant derived dengue virus entry inhibitor.

NS2B-NS3 inhibitors

NS2B acts as a cofactor for the NS2B-NS3active serine protease. The protease domain ofNS3 needs the presence of 40 aa of NS2B toform an active protease site. Polyproteinprocessing is an important and the foremost stepin the replication cycle of DENV (Figure 3B).Even chances of defective virus are not much asDENV has single ORF. That is the reason whyNS2b/NS3 protease has been the first dengueprotein target actively used in drug design pro-grams (Chambers et al, 1990; Bartelma andPadmanabhan 2002; Su et al. 2009; WHO 2009;Tomlinson et al. 2009; Zuo et al. 2009; Phonget al. 2011; Yang et al. 2011).

Kiat et al (2006) studied the effect of groupsof falvanones and their chalcones against pro-tease of DENV-2. Source of these bioactivecompounds was Boesenbergia rotunda (L.),commonly known as Chinese ginger or Finge-root. Their results indicate that cyclohexenylchalcone derivatives (4-hydroxypanduratin Aand panduratin A) were potent competitive in-hibitors of DENV-2 NS3 protease in vitro.Whereas pinostrobin and cardamonin were ob-served to be non-competitive inhibitor (Kiat etal. 2006). Same group further support their databy automated docking studies which is in ar-rangement with their experimental data (Oth-man et al. 2008).

Cyclotides and Dengue

Cyclotides are low molecular weight (28-47aa) plant defense proteins (Craik et al. 2004,2010; Mulvenna et al. 2004). Beside insecticid-al properties (Barbeta et al. 2008) these mini-proteins have wide range of other pharmaceuti-cal activities (Craik et al. 2004, 2010). They areexpressed in leaves, stem, and roots of severalplants species belonging to families like Rubia-ceae, Violaceae, Cucurbitaceae, and Fabaceae(Craik et al. 2004). Cyclotides have two charac-teristic features. First is their N and C terminalmoiety fused to form cyclic backbone. Secondlythey have cysteine knot motif made up of sixconserved cysteine residues (Craik et al. 2004).Presence of these features makes these proteinsthermal, chemical, and enzymatic stable andresistant to protease (Craik et al. 2004). Kalata





B1 was the first cyclotides isolated from Afri-can plant Oldenlandia affinis (Gran 1973).

Recently Gao et al. (2010) tested a panel ofchemically synthesized kalata B1 analogueswith varying the amino acid sequence againstdengue NS2B-NS3 protease. Their search re-vealed a cyclopeptide whose two full oxidizedforms were able to inhibit of dengue viralNS2B-NS3 protease. Inhibition was substratespecific and competitive in nature (Gao et al.2010) make it reliable candidate for the designof stable pharmaceuticals against dengue.

Medicinal Plant with AntiRdRp activity

NS5 is a multifunctional protein with RNAdepend RNA polymerase (RdRp) and methyl-transferase activity. RdRp is unique to the virus(as it is absent in humans) and thus a potentialtarget for drug discovery against the virus. Re-cently, Allard and colleagues during in vitroscreening of New Caledonian (world’s smallestyet diverse hot spot) plants they found severalspecies with significant AntiRdRp activityagainst dengue virus. Out of several species,they choose to perform chemical analysis ofCryptocarya chartacea. Results led to isolationof several new mono- and falvanones namedchartaceones A-F (1-6), along with pinocem-brin. Using plaque reduction assay they discov-ered that dialkylated flavanone; chartaceones C-F (3-6) has the most significant NS5 RdRp in-hibitory effect (Allard et al. 2011).

Virus assembly inhibitors

Dengue virus assembly occurs at the endop-lasmic reticulum (ER) (Chambers et al. 1990).In the ER pRM and Envelope proteins under-goes modification in which an oligosaccharide(14 residue (Glc)3(Man)9(GlcNAc)2) is added tothe specific asparagines residues. Further, thisresidue is sequentially modified by ER loca-lized enzyme called α- glucosidase. This step iscrucial for proper folding, assembly and secre-tion of the virus (Figure 3B). Glucosidase inhi-bitors have been shown to interfere with thedengue virus growth.

Castanospermine, a natural alkaloid (a plantsecondary metabolite) had reported to have α -

glucosidase inhibitory action. It is extractedfrom the black bean or Moreton Bay chestnuttree (Castanospermum australae). Courageotand colleges have demonstrated the molecularbasis of effect of inhibitory effect of Castanos-permine/ deoxynojirimycin (isolated fromleaves of Morus multicaulis) on dengue virusserotype 1 (in vitro). Their finding suggests thatthis alkaloid may disrupt the proper interactionof prM and Envelope protein which is crucialfor virus assembly (Courageot et al. 2000). Infollow up study (Whitby et al. 2005) showedthat Castanospermine is effective against allserotype of Dengue (DENV 1-4) both in vitroas well as in vivo (mice). Further they tested theeffect of this alkaloid against two clinically im-portant members of Flaviviruses viz. West NeilVirus (WNV) and Yellow Fever Virus (YNV).Their results indicate that Catanospermine hadalmost no effect on WNV and mild inhibitoryeffect on YFV. Virus growth inhibition wasshown by plaque assay (Huh7 cells and BHK-21 cells) and flow cytometric assays (Whitby etal. 2005). Further they validated their findingsin mice infection model. Interestingly, the com-pound is safe in mice and protects them specifi-cally and efficiently against all DENV sero-types (Whitby et al. 2005).

Synthetic analogues of this glucosidase in-hibitor have been shown to have protective ef-fect against dengue infection in animal model(mice) (Chang et al. 2011). List of natural alka-loids with anti glucosidase activity is quite long.For example Calystegins an alkaloid extractfrom roots of naturally growing Convolvulusarvensis, had potent anti-glucosidase activity(Molyneux et al. 1993). However there is nostudy so far (to our knowledge) where its inhi-bitory potential has been demonstrated againstdengue virus.

Unclassified Replication inhibitors: Anti-dengue Medicinal Plants

There are several other plant species whichinhibit dengue virus growth. But their precisemechanism of action has not been elucidatedyet. A study by Sánchez et al. (2000) reportedthe effect of flavonoids extracted from theMexican plants Tephrosia madrensis, Tephrosiaviridiflora, and Tephrosia crassifolia. Out of





four only glabranine and 7-O-Methyle glabra-nine exhibited significant antiviral activitiesagainst dengue virus (in vitro) in dose depen-dent manner. Whereas while methyl-hildgardtolA, hildgardtol A and elongatine had no effecton DENV (Sanchez et al. 2000).

In a follow up study, Muhamad et al. (2010)have reported antiviral effect of chemically syn-thesized flavonoid derivatives against dengueserotype 2 in HepG2 and BHK21 cells (Muha-mad et al. 2010).

Recently Zandi et al. (2011) has investi-gated the effect of four types of bioflavonones(quercetin, naringin, daidzein, and hesperetin)on the replication of DENV-2 in Vero cells andC636 cell lines. Virus growth and viral nucleicacid was determined by Foci Forming Unit Re-duction Assay (FFURA) and real time PCR re-spectively. Their result showed that quercetinhad significant antiviral activity against DENV-2. Further, only one serotype was target in thatstudy. Ideal dengue antiviral agent must be ableto handle all serotypes.

Parida et al. (2002) had investigating the an-ti dengue (serotype 2) potential of crudeaqueous extract of neem leaves (Azadirachtaindica Juss) and Azadirachtin in vitro usingC6/36 (cloned cells of larvae of Aedes albopic-tus) cells. Using virus inhibition assay theydemonstrated that crude aqueous extract inhibitsdengue virus-2 growth in a dose dependentmanner. Further they validated their results inanimal model (suckling mice). However, purecompound does not showed any inhibitory ac-tivity against DENV (Parida et al. 2002).

Further, Jiang et al. (2005) had investigatedthe inhibitory effect of four extract (Petroleumether, ethyl acetate, ethyl either and coumane)of Alternanthera philoxeroides (commonlyknown as Alligator weed, is an immersed aqua-tic angiosperm) against dengue virus 2 in vitro(C6/36) without significant cytotoxicity (Jianget al. 2005). Their results indicate that all ex-tracts possess anti-dengue activity. Further, thehighest inhibition of dengue virus was observedwith extracts from petroleum ether and safestwith extracts from coumarin (Jiang et al. 2005;Handa et al. 2008).

In a preliminary study by Klawikkan et al.(2011) investigated the in vitro anti-dengue ac-tivity from ten Thai medicinal plants (Klawik-kan et al. 2011). Plants were extracted withdichloromethane followed by ethanol and testedagainst DENV2 in Vero cells. Cytotoxic effectof plant extract was determined by MTT assay(Mosmann, 1983) and reduction in virus growthwas shown by plaque reduction assay. Theirresults indicate that out of ten, four medicinalplants viz. Rhizophora apiculata Blume., Fla-gellaria indica Linn., Cladogynos orientalisZipp. and Houttuynia cordata Thunb exhibitedpotent antiviral effect against DENV-2 (Kla-wikkan et al. 2011).

Very recently, Tang et al. (2012) in theirprimary study investigated the inhibitory effectof standardized methanolic extracts of six me-dicinal plants viz. Andrographis paniculata,Citrus limon, Cymbopogon citratus, Momordicacharantia, Ocimum sanctum and Pelargoniumcitrosum on dengue virus serotype 1 (DENV-1)in cell culture (Vero E6 cells). Using cytopathicinhibition and MTT assay they demonstrate thatA. paniculata has the most antiviral inhibitoryeffects followed by M. charantia. However,methanolic extracts were tested against onlyone serotype of Dengue (DENV-1) thus studyneeds further extension (Tang et al. 2012).

Vector control

Current mosquito control strategies includethe use of aerial sprays (toxicants) repellents,larvicides, insecticides as well as mosquito netsfor personal protection (WHO 2009). Aerialtoxicants against A. aegypti do not yield signifi-cant results because this mosquito is highly do-mesticated and many adults rest indoors in hid-den places such as closets (WHO 2009).

It is worth mentioning here that effortshave been made to generate genetically mod-ified (GM) mosquito in which gene has beenincorporated into mosquito that kills the insectat the larval stage of its life cycle (Figure 2).The GM mosquito has been developed by scien-tists at Oxford biotechnology company Oxitec(Enserink, 2010). Gene is functionally active atlarvae state and subsequently kills insect at thelarval stage of its life (Figure 2). Speculation





about success of this approach is quite promis-ing. Several reports (Fallatah and Khater 2010)are present where Larvicidal activity of plant

extracts has been demonstrated has been sum-marized in table 2.

Table 2: Few examples of medicinal plants with larvicidal activities against mosquito vector respon-sible for transmission of Dengue.

Plant Inhibitorycompound

Inhibitory activity Reference

Anacardium occidentalis, Copaifera langsdorffii,Carapa guianensis, Cymbopogon winterianus andAgeratum conyzoides

Extracts andoils

Larvicidal activity againstAedes aegypti

de Mendonça et al.(2005)

Carum carvi, Apium graveolens, Foeniculum vul-gare, Zanthoxylum limonella and Curcuma ze-doaria

Essential oil larvicidal activity againstA. aegypti

Pitasawat et al. (2007)

Solanum villosum Berry extract Larvicidal activity againstStegomyia aegypti

Chowdhury et al. (2008)

Combretum collinum Shoot barkextract

Larvicidal activity againstA. aegypti

Odda et al. (2008)

Azadirachta indica and Pongamia glabra Herbal formu-lation (PON-NEEM)

Larvicidal, ovicidal andoviposition deterrent activ-ities against A. aegypti andA. albopictus

Maheswaran and Igna-cimuthu (2011)

Nyctanthes arbortistis, Catharanthus roseus Boe-nininghusenia albiflora, Valeriana hardwickii andEupatorium odoratum

Leaf extract Larvicidal propertiesagainst Anopheles stephen-si, A. aegypti and Culexquinquefasciatus

Alam et al. (2011)

Citrus limetta Extracts fromthe peels

Larvicidal Aedes aegypti,and malarial vector, Ano-pheles stephensi

Kumar et al. (2012)

Acalypha alnifolia Leaf extract Larvicidal activity againstA. stephensi, A. aegypti, C.quinquefasciatus

Kovendan et al. (2012)

Delonix elata Leaf and seedextracts

Larvicidal and ovicidalactivities against of A. ste-phensi and A. aegypti

Marimuthu et al. (2012)

Anti dengue herbal patents

Plants are rich source of essential oils(EOs), glyceridic oils with mosquito repellentactivities (Pohlit et al. 2011a&b). Several plantsderived mosquito repellent products have beenpatented which have been extensively reviewedelsewhere (Pohlit et al. 2011a&b).

However, not much patents are availablewhere plant extract has been patented against allserotypes of Dengue virus (DENV1-4). To ourknowledge only one such patent is availablewhere anti Dengue activity of plant Cissampe-los pariere extracts have been patented by in-terdisciplinary team of Indian Scientists fromInternational Centre for Genetic Engineeringand Biotechnology, New Delhi, India , DalichiLife Science Research Centre in India (DSIN)(earlier known as Ranbaxy Research Laborato-

ry) and Department of Biotechnology, Govt. ofIndia. Briefly, based on the literature available,22 medicinal plants were selected and subjectedto extraction and/or fraction (Bhatnagar et al.2009; Raut et al. 2012). Further, 3 different ex-tracted (Methanaolic-D1; Hydroalchoholic-D2and Aqueous- D3) and four fractions of medi-cinal plants were tested against all serotypes ofDENV using a tissue culture based virus neutra-lization assay. In total 200 samples (whichcomprises of extracts, fractions, enriched frac-tions, and pure compounds were subjected toprimary, secondary and tertiary assays (Bhatna-gar et al. 2009; Raut et al. 2012).Their extensiveanalysis revealed that methanolic extract of Cis-sampelos pariera (Cipa/D1) is a potent inhibitorof all serotypes of Dengue virus with insignifi-cant toxicity (WIPO Patent ApplicationWO/2010/084477 A1). Additionally CiPa/D1





extract also showed analgesic/anti-inflammatory activity along with anti-dengueactivity (Raut et al. 2012). This in future couldlead to development of cost effective, safe, andeffective drug against Dengue infection.

Engineering biosynthetic plant pathways inmicroorganism

The secondary metabolites of higher plantsinclude diverse chemicals, such as alkaloids,isoprenoids, and phenolic compounds (phenyl-propanoids and flavonoids) which are generatedfrom primary metabolites such as amino acidsor acetyl-CoA with the help of biosyntheticpathways unique to plants. Although thesecompounds have variety of medicinal propertieshowever, amount of bioactive compound avail-able after extraction from plant is quite low. Inthe era of synthetic biology, several efforts havebeen made in which these secondary metabo-lites are produced in large quantities in engi-neered micro organism by encorporating genesresponsible for synthesis of these metabolitesinto microorganisms (Nakagawa et al. 2011;Chemler et al. 2008). These finding further rais-es hopes for the development of cost effectivedrug against dengue.

Conclusion

Dengue infection has been re-emerging as aserious life threat with increase in the infectioncases each year. It is becoming one of the majorhealth issues across the tropical and the subtrop-ical belt. Various strategies have been adaptedto develop an effective vaccine or drug againstDengue virus. However, till date there is no li-censed vaccine available in the market. There isan urgent need to find alternate solution tocombat dengue infection. Medicinal plants havebeen utilized for the treatment of several humandiseases. Several studies have been made whereanti Dengue potential of medicinal plants hasbeen reported. However, they are still far fromsuccess. There is need of extensive and fruitfulnetworking among academic research groups,clinicians, and industries throughout the globeso that Ethnobotanical knowledge can be circu-lated and finally converted into effective drugagainst Dengue (DENV 1-4).

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