Effect of Thai Medicinal Plant Extracts against Dengue Virus in vitro

Original Article Mahidol University Journal of Pharmaceutical Science 2011; 38 1-2, 13-18Effect of Thai Medicinal Plant Extracts againstDengue Virus in vitroN. Klawikkan 1 , V. Nukoolkarn 2 , N. Jirakanjanakit 3 , S. Yoksan 3 , C. Wiwat 1 and K. Thirapanmethee 1 *1 Department of Microbiology, Faculty of Pharmacy, Mahidol University2 Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University3 Center for Vaccine Development, Institute for Molecular Biosciences, Mahidol UniversityAbstractDengue hemorrhagic fever is one of the major public health problems in Thailand. Thedisease caused by the mosquito-borne dengue virus (DENV). DENV is the member of theFlavivirus genus, Flaviviridae family. There are four genetically related but distinct serotypesDENV 1, 2, 3, and 4. It is transmitted to people through the bite of mosquitoes Aedes aegyptiand Aedes albopictus. It has been reported that dengue virus infects 50 to 100 million peopleannually. However, there is neither vaccine nor effective antiviral drugs to treat dengue virusinfection. Consequently, the development of antiviral drugs licensed for treatment of patientsremains an urgent need to prevent dengue fatalities. Many studies have been conducted forexploring the antiviral activity of chemical compounds against DENV. Among thesecompounds, some are small molecules that can inhibit specific steps of viral intracellularreplication or effect at viral proteins. But none of these compounds have been approved to beused in human. Thailand has many traditional medicinal plants that have been reported onstrong antiviral activity and some of them have already been used to treat people who wereinfected with viruses. Therefore, the aim of this study is to investigate the in vitro anti-dengueactivity from Thai medicinal plants. In this present study, ten medicinal plants were collectedfrom Siri Ruckhachati Natural Park, Salaya campus, Mahidol University. These plants wereextracted with dichloromethane ethanol and subsequently tested for their anti-dengue type 2activities in Vero cell by MTT method. The results showed that the ethanol extracts ofRhizophora apiculata Blume., Flagellaria indica Linn. and Cladogynos orientalis Zipp. at aconcentration of 12.5 µg/ml exhibited inhibitory activity on DENV-2 with 56.14%, 45.52% and34.85%, respectively. Moreover, Houttuynia cordata Thunb. exhibited inhibitory activity againstDENV-2 with 35.99% at a concentration of 1.56 µg/ml. In addition, Cladogynos orientalsZipp., Piper retrofractum Vahl. and Rhizophora apiculata Blume. exhibited an inactivatedviral particle activity with 52.9%, 84.93% and 41.5% at a concentration of 100 µg/ml. Thisstudy indicated that Rhizophora apiculata Blume., Flagellaria indica Linn., Cladogynosorientalis Zipp. and Houttuynia cordata Thunb. have a significant potential effect on thedengue virus in vitro. These medicinal plants could potentially be sources for developing theanti-DENV drug.Key words: Dengue virus type 2, Thai medicinal plants, Anti-dengue activity*Corresponding author: Department of Microbiology, Faculty of Pharmacy, Mahidol University. 447 Sri Ayudthaya Road,Rajthevi, Bankok, 10400, Thailand. Tel.: +66 2644 8677 ext. 1133; fax: +66 2644 8692E-mail address: pyktr@mahidol.ac.th

14N. Klawikkan et al.INTRODUCTIONDengue hemorrhagic fever is one ofthe most important emerging tropicaldiseases in the 21 st century 1 . In the latterpart of the 20 th century, globalization andrapid urbanization of many developingtropical countries produced increasedtransmission and hyperendemicity of thedisease 2 . World Health Organization (WHO)estimates that there are as many as 50million cases of dengue infection worldwideand global warming provide a significantselective advantage for dengue infectionspreading into new areas 3 . Dengue infectionis caused by dengue virus (DENV). DENVbelongs to the genus Flavivirus in thefamily Flaviviridae. There are four distinctserotypes of DENV: 1, 2, 3, and 4. Thevirion contains a positive-sense, singlestrandedRNA molecule of approximately11 kb in length, inserted in an icosahedralnucleocapsid and surrounded by a lipidenvelope-covered with peplomers. The viralgenome is translated into three structuralproteins, capsid (C), pre-membrane (prM),envelope (E) and seven non-structuralproteins (NS1, NS2a, NS2b, NS3, NS4a,NS4b and NS5) 3,4 . DENV is transmittedprincipally by Aedes aegypti mosquito.Other mosquito such as Ae. albopictusand Ae. polynesiensis can also transmitepidemic dengue, but less efficiently 5 .Clinical manifestation of DENV infectionrange from subclinical to a self-limitedfever and rash (dengue fever (DF)) to asevere and sometimes deadly illnesscharacterized by capillary leakage,thrombocytopenia and hypovolemic shock(dengue hemorrhagic fever (DHF) anddengue shock syndrome (DSS)) 6 .At present, no specific treatmentsfor DENV infection are clinically available.Control of DENV by safe, low-cost and longlastingvaccination has not been established.Several types of antiviral agent have beensought intensively, including inhibitorsagainst viral replication, posttranslationalprocessing of viral proteins and E proteinfunctions such as membrane fusion andviral attachment 7 . A number of antiviralcompounds, i.e. ribavirin, mycophenolicacid, 7-deaza-2’-c-methyl-adenosine and 6-O-butanoyl castanospermine have showntheir efficacy in inhibiting DENV replicationin vitro 8 . The only available treatmentis supportive therapy. Therefore, it isnecessary to find new alternative antiviralcompounds against DENV 9 . Plants havelong been used as a source of medicinefrom ancient time to today all over theworld 10 . Many traditional medicinal plantshave been reported to have strong antiviralactivity and some of them have alreadybeen used to treat animals and people whosuffer from viral infection by inhibiting thereplication cycle of various types of DNAand RNA virus 11 . However, very little isknown about potential of plants againstdengue virus. The objective of the presentstudy is to investigate the in vitro inhibitoryactivity of Thai medicinal plants towardDENV2 infection in Vero cells.MATERIALS AND METHODSPreparation of plant extractsAll medicinal plants were collectedfrom Siri-Ruckhachati Natural Park, Salayacampus, Mahidol University. The wholeplants were dried under shade and grindedto powder. Plants powder were soaked indichloromethane at 4°C for 18 hours bythe ratio of plant: solvent at 1:3. The crudeextracts were filtered through Whatmanfilter paper No.1 and evaporated in waterbath at 80°C until dry. The dried extractswere weighed and stored at -20°C untiluse. The residual from dichloromethaneextraction was subsequently extracted againwith 70% ethanol at 4°C for 18 hours bythe ratio of plant: solvent 1:3. The crudeextracts were filtered through Whatman filterpaper No.1 and evaporated in water bathat 80°C until dry. The dried extracts wereweighed and stored at -20°C until use.Cell culture and virusC6/36 mosquito cell line, Africangreen monkey kidney epithelial cells (Verocell) and Dengue virus type 2 strain 16681were kindly provided by Dr. Sutee Yoksan(Center for Vaccine Development, Institutefor Molecular Bioscience, Mahidol University).The C6/36 mosquito cell line was grown

16N. Klawikkan et al.value at wavelength 595 nm. This assay hasseveral advantages: it is easy to perform,the evaluations are objective, it can beautomated using a personal computer andthe toxicity evaluation can be made inparallel with antiviral activity evaluation 11 .The anti-DENV2 activity of each extractswas shown in Table 1. At a concentrationof 12.5 µg/ml, ethanol extracts of Rhizophoraapiculata Blume., Piper retrofractum Vahl.,Flagellaria indica Linn. and Cladogynosorientalis Zipp. exhibited the inhibitoryactivity against DENV2 with 56.14%,53.53%, 45.52% and 34.85% inhibition,respectively. At the same concentration,dichloromethane extract of Piperretrofractum Vahl. showed the inhibitoryactivity against on DENV2 with 32.06%.While, ethanol extract of Houttuynia cordataThunb. exhibited inhibitory activity onDENV2 with 35.99% at a concentrationof 1.56 µg/ml. On the other hand,dichloromethane and ethanol extracts ofAcacia catechu Willd., Piper sarmentosumRoxb., Cassia tora Linn., Phyllanthusurinaria Linn. and Ricinus communisLinn. did not exhibit inhibitory activityagainst DENV2 in Vero cell.From previous experiment, it wasfound that ethanol extracts of Rhizophoraapiculata Blume., Piper retrofractum Vahl.,Cladogynos orientalis Zipp., Houttuyniacordata Thunb., Flagellaria indica Linn.and dichloromethane of Piper retrofractumVahl. showed good antiviral activity againstDENV2. Then, the cytotoxicity of theseextracts were further evaluated by MTTmethod to determine the non-toxicconcentration in Vero cell. Evaluation ofcytotoxicity is an important part of theassessment of the potential antiviral agentsince the beneficial extracts should beselective for virus-specific processes withlittle or no effects on metabolism of hostcells 14 . The results of the cytotoxicityevaluation of the tested extracts were shownin Table 2. The 50% cytotoxic concentration(CC 50 ) of ethanol extracts of Rhizophoraapiculata Blume. and Piper retrofractumVahl. were 625 µg/ml. And, the CC 50 ofethanol extracts of Cladogynos orientalisZipp., Flagellaria indica Linn. and Houttuyniacordata Thunb. were 312 µg/ml. Whereas,the CC 50 of dichloromethane extract of Piperretrofractum Vahl. was 156.25 µg/ml.Table 1. Antiviral activity against DENV2 determined by MTT methodPlantConcentration(µg/ml)% InhibitionDichloromethaneEthanolAcacia catechu 12.5 None NoneCassia tora 12.5 None NoneCladogynos orientalis 12.5 - 34.85Flagellaria indica 12.5 - 45.52Houttuynia cordata 1.56 - 35.99Phyllanthus urinaria 12.5 None NonePiper retrofractum 12.5 32.06 53.53Piper sarmentosum 12.5 None NoneRicinus communis 12.5 None NoneRhizophora apiculata 12.5 - 56.14

-: Not tested; None : No activity