ResearchArticledownloads.hindawi.com/journals/jchem/2020/3837207.pdfCorrespondence should be addressed to Rajiv Ravi; [email protected] and Mohamad Faiz Mohd Amin; [email protected]

Research ArticleChemical Composition and Larvicidal Activity of FlowerExtracts fromClitoria ternatea againstAedes (Diptera Culicidae)

Darvin R Ravindran1 Madhuri Bharathithasan1 Patmani Ramaiah2

Mohd Sukhairi Mat Rasat3 Dinesh Rajendran45 Shakila Srikumar1 Intan H Ishak45

Abd Rahman Said1 Rajiv Ravi 6 and Mohamad Faiz Mohd Amin 7

1Faculty of Medicine Quest International University Perak Jalan Hj Eusoff Housing Trust Ipoh 30250 Perak Malaysia2Faculty of Medicine Universiti Kuala Lumpur Royal College of Medicine Perak (UniKL RCMP) 30450 Ipoh Perak Malaysia3Faculty of Bioengineering and Technology Universiti Malaysia Kelantan Jeli Campus Jeli Kelantan Malaysia4School of Biological Sciences Universiti Sains Malaysia Minden Penang Malaysia5Vector Control Research Unit School of Biological Sciences Universiti Sains Malaysia Minden Penang Malaysia6School of Biological Sciences Faculty of Science and Technology Quest International University PerakJalan Raja Permaisuri Bainun Ipoh 30250 Perak Malaysia7Faculty of Earth Science Universiti Malaysia Kelantan Jeli Campus Jeli Kelantan Malaysia

Correspondence should be addressed to Rajiv Ravi rajiv_ravi86yahoocom and Mohamad Faiz Mohd Aminmohamadfaizumkedumy

Received 17 June 2020 Accepted 15 September 2020 Published 1 October 2020

Academic Editor Ioannis G Roussis

Copyright copy 2020 Darvin R Ravindran et al 0is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Mosquitoes have always been a human health threat the major global health problems caused by them are malaria dengue feveryellow fever and Zika as well as several other vector-borne outbreaks 0e major problems in controlling these vectors bornediseases are related to resistance to eradication measures Different classes of insecticides used for controlling public health haveraised the concern of resistant problems with mosquitoes and environmental pollution caused by the control measures 0us asearch for alternative natural compounds is necessary for solving the insecticidal resistance problem using pesticides in the larvalstage of vector development as well as creating a chemical-free environment for a healthy society Hence the major focus of thisstudy is to identify the larvicidal mechanisms metabolite antioxidants and chemical compounds and elucidate their structuresfrom C ternatea flower and to test their efficacies against early 4th instar larvae of Aedes aegypti and Aedes albopictus Clitoriaternatea flowers were collected from the garden of the Faculty of Medicine in International Quest University Ipoh Perak andthence used for crude extraction Further on the metabolite test antioxidant test and chromatography techniques wereconducted to identify the chemical composition of extracts and their chemical structures were identified using GCMS-QP2010Ultra (Shimadzu) Next the extracts were evaluated against the early 4th instar larvae of Aedes mosquito vectors following theWHO procedures for larval bioassays 0e larvicidal activity of Clitoria ternatea flower extracts evidently affected the early 4thinstar larvae of Aedes mosquito vectors 0e highest larvicidal activity was observed against the early 4th instar larvae of Aedesaegypti with the LC50 and LC95 values of 1056 and 2491mgL respectively Meanwhile the larvae bioassay test for Aedesalbopictus recorded the LC50 and LC95 values of 1425 and 2753mgL Moreover the results for nontarget organism test on guppyfish Poecilia reticulata showed no mortalities with flower extracts at 2500mgL hence posing no toxic effects on fish In thisstudy we have found a total of 16 chemical compounds and 6 chemical compounds have been reported to possess directinsecticidal larvicidal and pupicidal effects Six chemicals with insecticidal properties were found to be glycerin 2-hydroxy-gamma-butyrolactone neophytadiene n-hexadecanoic acid cis-vaccenic acid and octadecanoic acid with a total of 287efficacy Clitoria ternatea flower extracts also showed different types of phenols such as anthocyanins flavonoids and tannins Ourfindings showed that the crude extract of Clitoria ternatea flower bioactive molecules is effective and may be developed asbiolarvicide forAedesmosquito vector control Furthermore this study also provided a baseline understanding for future researchwork in the field of applications of Clitoria ternatea flower extracts for their long-term effects on human health such as a foodadditive antioxidant and cosmetic

HindawiJournal of ChemistryVolume 2020 Article ID 3837207 9 pageshttpsdoiorg10115520203837207

1 Introduction

Mosquitoes have always been a human health threat fromeons the major health problems caused by them aremalaria dengue fever yellow fever and Zika as well asseveral other vector-borne diseases Based onWorld HealthOrganization [1] western Pacific region northern hemi-sphere reports on January 2020 dengue updates recordedwere as follows Cambodia with a higher average of 1239cases weekly compared to the period of 2019 Malaysia withlower 42 decrease weekly as 2604 cases with 9 deathscompared to the period of 2019 Philippines with 2778 caseswith 10 deaths in 2020 Singapore with 2508 cases highercompared to the period of 2019 Vietnam with 1282 weeklycases at 2020 In the southern hemisphere Australia hasrecorded 78 dengue cases since the beginning of 2020 till 26February 2020 which is lower compared to the period of2019 [1] 0us dengue cases are inevitable in humancommunity and there are many factors contributing to-wards this global issue

In the current situation only physical and chemicalmethods are being used to control mosquito-borne dis-eases Physical methods such as mosquito bed netsmosquito window nets at homes and electrical mosquitorackets are only temporary solutions [2] Besides that thechemicals used for vector control such as temephos andpyrethroids are more well-known with some insecticidesresistant challenges [2] Chemical insecticides are syn-thesized chemically based on single bioactive compoundswith reference to structurally related plant compoundswhich lead to the current resistance problem for allmosquitocide classes [2ndash5] Only one single chemicalcompound can be synthesized at a time due to its hightoxicity effects and the multiple plant biochemical com-pounds are more stable and lower in toxicity due to theirsynergetic effects under natural conditions AdditionallyRavi et al [4] supported the concepts of botany controlmechanisms as a simple and sustainable method com-pared to the conventional insecticides in use

Unlike current synthetic or chemical insecticides theadvantages of plant-based insecticides are their composi-tions of natural blends of multiple chemical compoundsthat may act synergistically on both physiological andbehavioral processes of mosquitoes [2ndash6] 0us botanical-based insecticides are an alternative and accurate solutionagainst dengue vectors of Aedes aegypti and Aedesalbopictus

In tune with the concept of a safer alternative Clitoriaternatea plant has the potential as a bioinsecticide to solvethe problems of resistance from a single-based chemicalcompound Clitoria ternatea is a well-known flower in Asiancountries such as Malaysia 0ailand Philippine India andChina and widely used in Ayurveda and Chinese medicine[7] Additionally Malaysians use the extract of the flower asa food coloring to prepare a popular dish called Nasi Kerabuand Indians from the state of Kerala region consume thisplant as a vegetable on a daily basis [7] 0e potential

compounds of this plant are prevalent in the form ofpolyphenols triterpenoids flavonoids glycosides antho-cyanins tannins and steroids [8] To date only one studyMathew et al [9] has reported the basic use of C ternatea asan Aedes aegypti and Aedes stephensimosquito larvicidal butnone on Ae albopictus According to Mathew et al [9] themethanolic extract of C ternatea showed larvicidal effec-tiveness however no mechanisms of larvicidal wereexplained and hence suggested improvements on furtherinvestigations pertaining to the active principles of thechemical compounds responsible for larvicidal activities0us within this expansion of framework this seems to bethe first study to elucidate larvicidal mechanism metaboliteantioxidant and chemical compound identifications fromC ternatea flower which may be responsible for Aedeslarvicidal activities 0e objective of this study is to identifythe larvicidal mechanism metabolite antioxidant chemicalcompounds and the structure of C ternatea flower and totest its efficacies against the early 4th instar larvae of Aedesaegypti and Aedes albopictus

2 Materials and Methods

21 Statement for Experimental Protocols All experimentalprotocols mentioned in this paper were in accordance withlarval susceptibility test method guidelines by the WorldHealth Organization (WHO)

22 PlantMaterials Clitoria ternatea used for this study wasauthenticated by the Herbarium of the University of Malayawith the herbarium voucher number KLU60080 Clitoriaternatea flowers (Figure 1) were collected from the garden ofthe Faculty of Medicine in International Quest UniversityIpoh Perak 0e petals of C ternatea were washed inrunning tap water to remove adhered debris and soil par-ticles and washed again with distilled water and dried in hotair oven at 70degC until the petals were completely dry [10]0e dried petals were ground into fine powder by using alaboratory grinder and stored in a vacuum-tight containerfor further usage

23 Aqueous Extraction Aqueous extraction was performedby maceration using 250ml distilled water and 50 g of thepowdered C ternatea flowers petal 0ey were mixed in a500ml beaker and stored in a dark area for an hour 0enthe extract was filtered using cotton wool and filter paper0e final volumes were stored in a universal bottle andmaintained at 4degC to 6degC [10]

24 Methanol Extraction Methanol extraction was per-formed in three different concentrations of 50 70 and95 Amixture of 5 g powderedC ternatea flower petals and100ml of respective concentration of methanol was pre-pared in three separate Scott bottles and covered withaluminum foil to avoid exposure to light Later the bottles

2 Journal of Chemistry

were kept overnight in the orbital shaker for the macerationprocess After 24 hours the extract was filtered using cottonwool and filter paper by vacuum filtration method Subse-quently they were concentrated using rotary evaporator atthe temperature of 45degC Finally the remained concentratedamount of extract was stored in a universal bottle andmaintained at 4degC to 6degC [4 11]

25 Ethanol Extraction Ethanol extraction was prepared inthree different concentrations similar to methanol extrac-tions of 50 70 and 95 Maceration process was per-formed using 5 g of powdered C ternatea flower petals and100ml of respective concentrations of ethanol mixed inthree separate Scott bottles then covered with aluminum foilto avoid exposure to light Later the bottles were kept atroom temperature for 24 hours 0e following day all theextracts were filtered using cotton wool and filter paper byvacuum filtration method Next they were concentratedusing rotary evaporator with the temperature at 60degC Fi-nally the concentrated extracts were stored in a universalbottle and maintained at 4degC to 6degC [11]

26 Petroleum Ether Extraction Petroleum ether extractionwas done with 5 g of fresh flowers they were cut into smallpieces and soaked in a Scott bottle with 50ml of petroleumether for 48 hours Later the extract was filtered out usingfilter paper and transferred to a universal bottle and stored at4degC to 6degC [12]

27 Ethyl Acetate Extraction Ethyl acetate extraction wasdone with 5 g of powdered C ternatea flower petals and100ml of ethyl acetate in a Scott bottle and then covered withaluminum foil to avoid exposure to light 0en the bottlewas kept at room temperature for 48 hours and after 48hours the extract was filtered out using cotton wool andfilter paper Finally they were stored in a universal bottle andmaintained at 4degC to 6degC [13]

28 Test for Flavonoid Flavonoids identification was de-termined with two different chemical tests for all nine ex-tracts 1ml of C ternatea extracts was added to a test tubefollowed by a few drops of FeCl3 the color changes wereobserved 0e second confirmation test for the presence of

flavonoids was done in another test tube by adding 1ml ofcrude extract and few drops of 5 of AlCl3 solution and thecolor changes were observed [14]

29 Test for Anthocyanins Anthocyanins identification wasdetermined with two different chemical tests for all nineextracts First 1ml of crude extract was added to a test tubefollowed by 2ml of HCl and then heated for 5min at 100degCand the color changes were observed For the second test1ml of crude extract was added to a test tube followed by2ml of NaOH and then the color changes were observed[14]

210 Test for Tannins Tannins were determined with achemical test for all nine extracts 1ml of crude extract wasadded to a test tube followed by a few drops of FeCl3 0enthe color changes were observed [14]

211 Test for Polyphenols A UV-visible spectrophotometerwas used to evaluate the concentrations of polyphenolspresent in each extract the absorbance of which wasmeasured at 725 nm wavelength All the measurements wereperformed in triplicate for each sample [13]

212 Paper Chromatography Paper chromatography wasused to study the fractions of the components Chroma-tography paper was used to study the fractions of the extractsby placing 10 μL of crude extract and allowing them to drycompletely before immersing in the solvent 0e solventused in this process was the same solvent that was used forthe extraction processes such as the aqueous 50 methanol70 methanol 95 methanol 50 ethanol 70 ethanol95 ethanol petroleum ether and ethyl acetate Subse-quently the Rf values were calculated and the resultsinterpreted comparing with the controls [15]

213 gtin-Layer Chromatography 0in-layer chromatog-raphy is also a method to study the components based on theprinciple of fractionation 0e solvent used for thin-layerchromatography is a mixture of methanol and chloroformand this process was done in a specific TLC tank First theTLC gel was applied on the plate and was spotted by all the

Figure 1 Picture of Clitoria ternatea flower from the field

Journal of Chemistry 3

crude extracts and allowed to dry for a few minutes and thenthe gel plate was placed into the tank to immerse with thesolvent Lastly 1 FeCl3 was sprayed on the gel plate toidentify the spots and fix the spots and Rf values werecalculated based on the measurements and the resultsinterpreted comparing with the control spots [9]

214 Antioxidant Activity Based on Fu et al [16] antiox-idant scavenging activity of C ternate petals extractions was

measured using DPPH solvent To conduct the study a 96-well plate was used to mix the extract with the DPPHsolvents and other controls Later the plate was covered withaluminum foil and left in a dark area for 30minutes Duringthis test ascorbic acid was used as the positive control andTris-HClL buffer was used as the negative control lastly thecolor changes were observed and OD was read using theELISA reader at 517 nm 0e scavenging activity of DPPHradicals was calculated using the following equation

scavenging activity() (negative control minus sample)

(negative control minus positive control)1113890 1113891 times 100 (1)

215 GC-MS Analysis 0e GC-MS analysis was done onlywith the methanol (CH3OH) solvent extract and was used todetermine the compounds with the analysis performed on aGCMS-QP2010 Ultra Shimadzu0e reason for only choosingmethanol (CH3OH) solvent is due to its extractrsquos best efficaciesagainst Aedes larvae as published before in several plant-basedlarvicidal papers [2ndash5] 0e GCMS-QP2010 Ultra Shimadzusystem was fitted with Rtx-5MS capillary column the mea-surement of 30mtimes 025mm inner diameter and times025μmfilm thicknesses and the maximum temperature that can beused is 370degC coupled with a QP2010 Ultra Shimadzu MSUltra-high-purity helium was used as carrier gas at a constantflow rate of 10mLmin 0e injection transfer line and ionsource temperatures were all at 280degC 0e oven temperaturewas programmed from 80degC and it was held for 2min andraised to 280degC at a rate of 3degCmin 0e crude samples werediluted with an appropriate solvent and filtered 0e particle-free diluted crude extracts about 1μL were taken in a syringeand injected into an injector with a split ratio of 10 1 All datawere obtained by collecting the full-scan mass spectra withinthe scan range of 40ndash550 amu 0e percentage composition ofthe crude extract constituents was expressed as the percentageby peak area 0e identification and characterization ofchemical compounds in various crude extracts were based onthe GC retention time [2 4]

216 Larvae Rearing 0e eggs of Aedes were obtained fromVector Control Research Unit (VCRU) at Universiti SainsMalaysia (USM) Penang Malaysia We followed the methodused by WHO [2ndash5 17] in larvae rearing 0e eggs werehatched in dechlorinated water for 24 hours and weremaintained at 25degC to 30degC (room temperature) pH of 695 to703 and relative humidity of 80plusmn 10 and dissolved oxygenfrom 55 to 61mgL in the laboratory After five days theearly 4th instar larvae will be used for the bioassay test

217 Larvicidal Bioassay Larvicidal bioassays were per-formed in accordance with the standard World HealthOrganization [17] Bioassays were carried out using 25 each

of Aedes aegypti and Aedes albopictus early 4th instar larvae(homogeneous population consisting 5mm to 6mm in bodylength) 0e bioassays were replicated four times using 25larvae for each concentration with methanol (CH3OH) assolvent control 0e methanol (CH3OH) solvent extrac-tion was chosen for larvicidal in this current study due toprevious studies by Ravi et al [2ndash5] which showed thatmethanol (CH3OH) solvent had the best larvicidal effectson Aedes During the larvae testing period fish meal wasprovided Initially before selecting the accurate testingdose all the larvae were subjected to a wide range of testconcentrations 0is step is necessary to determine therange of extract solution for larvicidal activities [2 4 5] Inthis study seven concentrations ranging from 500mgL to2500mgL yielding between 0 and 100 mortality in 24hours of exposure were selected as test concentrationsControl solutions were prepared with 1ml of distilledwater and 10 of respective methanol solvent during eachexperimental replicate [2 4 5] 0e reason for usingsolvent control is to ensure that all test replicates areidentical to plant extract solutions and to ensure thatmortality results were not due to its solvents [2 4 5]Experiments were conducted at room temperature28 plusmn 2degC and larvae mortalities were recorded at intervalsof 24 hours and 48 hours [14] Immobilization and totalabsence of movement from the larvae even aftertouch arethe endpoint of bioassay [2 4 5] 0e data were analyzedby using probit analysis in IBM SPSS Statistics 24

218 Morphological View Aedes aegypti early 4th instarlarvae were observed under the optical microscope (LeicaUSA) at magnification 40ndash400x [4]

219 Nontargeted Organism Test Guppy fish Poeciliareticulata a total of ten fish in three replicates with 120 gmean weight and 35 cm mean length were used in this test(acclimatization period of 12 days in laboratory conditionsbefore the start of the experiment) Each treatment group


has been tested with larvicidal LC95 plant extract concen-tration dissolved in 2000mL dechlorinated water A controlgroup has also been set with only 2000mL of nonchlorine-treated water 0e tests were performed for 24 hours withobservations made at first 10 minutes and then 1 2 3 6 and24 hours 0e mortality and observable abnormalities of fishwere recorded Test conditions of water such as pH watertemperature and dissolved oxygen were recorded during thestart and end of the experiment [4]

3 Results

31 Metabolite Study Based on the S1 and S2 (refer tosupplementary file (available here)) the absorbance showeda higher presence of anthocyanin flavonoids and tannins inthe C ternatea flower extracts of 95methanol compared toother extractions

32 Paper Chromatography All the values in S3 (refer tosupplementary file) show the three-retention factor Rfvalues present in C ternatea flower extracts in all the sol-vents used for the extraction process 0e use of methanolsolvent showed higher retention factor Rf values comparedto other solvents

33 gtin-Layer Chromatography All the values in S4 (referto supplementary file) show the three-retention factor Rfvalues present in C ternatea flower extracts in all the sol-vents used for the extraction process 0e use of methanoland ethanol solvent showed approximately similar retentionfactor Rf values

34 Antioxidant Activity 22-Diphenyl-1-picrylhydrazyl(DPPH) was used to identify the presence of antioxidantproperty in each extract Based on S5 (refer to supple-mentary file (available here)) the graph shows the extractswith high antioxidant property and low antioxidant prop-erty 0e extract that carried the highest antioxidantproperty was aqueous in nature

35 GC-MS Analysis and Identification of CompoundsGC-MS analysis of methanol solvent extracts of C ternateaflower showed 16 peaks which indicate the presence of 16phytochemical compounds S6 (refer to supplementary file)Comparing the mass spectra of the constituents with theNIST 08 library 16 compounds were characterized andidentified (Table 1)0emajor three chemical compounds inthe extracts showing the highest peaks were acetic acid 1-(2-methyltetrazol-5-yl) ethenyl ester (165) 4H-pyran-4-one23-dihydro-35-dihydroxy-6-methy (139) and di-glyc-eraldehyde dimer (124) 0e attached S6 (refer to sup-plementary file) contains the NIST 08 library search forchemical compound structures and details

36 Larvicidal Bioassay 0e bioassay testing frommethanolsolvent extracts of C ternatea flower was tested at 300mgL500mgL 1000mgL 1500mgL 1700mgL 2000mgL

and 2500mgL 0e entire larvae bioassay test withC ternatea flower extracts showed a significant increase inmortality percentage with the increase of concentration Oncomparing the tested flower extracts maximum larvicidalactivity was observed to be against early 4th instar larvae ofAedes aegypti with the LC50 and LC95 values of 1056 and2491mgL respectively (Table 2) Meanwhile the larvaebioassay test for Aedes albopictus recorded the LC50 andLC95 values of 1425 and 2753mgL (Table 2) Figure 2 showsthe graphical representation of larvae mortality rate betweenAedes aegypti and Aedes albopictus Finally the results fornontarget organism test on guppy fish Poecilia reticulatashowed no mortalities with plant extracts at 2500mgLposing no toxic effects on this fish (Figure 3) 0e 95confidence limits LC50 (95 CI) and LC95 (95 CI) chi-square and degree of freedom (df ) values were also cal-culated (Table 2) In the control assay there was no sig-nificant mortality

37 Morphological View 0e morphological view fromFigures 4(a) and 4(b) is indicating the presence ofC ternateaflower extracts in the midgut content by dark greenish colorof extracts

4 Discussion

0e results of this study have shown that the phyto-chemical compounds extracted from C ternatea flowermay have innovative and integrated property of a bioinsecticidal agent 0us this study intends to highlight therole of the extract as an alternative control measureagainst Aedes when compared to the synthetic insecti-cides Additionally C ternatea flower extracts may bemore effective than single-based active compound due totheir active ingredientsrsquo synergisms which may be effectivein managing resistant population of mosquitoes In thisstudy we have found a total of 16 chemical compoundsand 6 chemical compounds have been reported to possessdirect insecticidal larvicidal and pupicidal effects 0e sixchemicals used for their insecticidal properties in previousstudies were glycerin 2-hydroxy-gamma-butyrolactoneneophytadiene n-hexadecanoic acid cis-vaccenic acidand octadecanoic acid constituting a total of 287 Allthese chemical components have direct larvicidal activityand there are many more compounds which may actsynergistically in the larvicidal activity Similarly Raviet al [2 4] have also discussed the synergetic effects ofmultiple chemical compounds from plant extract againstAedes larvae

A previous study by Mathew et al [9] on Clitoria ternateaextracts did not show the mechanism of larvicidal activityHowever to elucidate the effects of larvicidal mechanisms thiscurrent study has shown the effect of the extract on the midgutafter ingestion as shown in the morphological picture(Figures 4(a) and 4(b) 0e ingested plant extracts shown inFigures 4(a) and 4(b) were quite similar to the previous studydone with Azolla pinnata plant extracts (green color) in thelarvae midgut content after 24 hours of ingestion [4] 0us it


can be congruently observed that the C ternatea extract in-gestion mechanism was used in the killing effect of Aedeslarvae As per the results shown by the C ternatea extract onthe guts of larvae the potential commercialization of thisextract would be done using liquid-based techniques An ex-ample of a similar possible commercial application done in the

past was ldquoDalmatian Powderrdquo dissolved in liquid for its ap-plication [33] Additionally the storage condition of the crudeextract in this study was at minus4degC before its application underroom temperature Similarly Ravi et al [4] have prevented thedegradation of crude extract by storing it at minus4degC before itsapplication

Table 1 Chemical compounds for Clitoria ternatea flower

SN RT Area Area () Compound name Activity1 4256 42912 9631 Glycerin Pupicidal and larvicidal [18]2 465 9713 218 24-Dihydroxy-25-dimethyl-3(2H)-furan-3-one Anthelminthic [19]3 4823 21616 4851 2-Hydroxy-gamma-butyrolactone Insecticidal metabolite [20 21]4 6927 73492 16494 Acetic acid 1-(2-methyltetrazol-5-yl)ethenyl ester Agricultural [22]5 8602 55658 12491 dl-Glyceraldehyde dimer Antioxidant [23]6 884 15893 3567 Alpha-amino-gamma-butyrolactone Antioxidant and cytotoxic [24]

7 9151 61940 13901 4H-pyran-4-one 23-dihydro-35-dihydroxy-6-methy Antioxidant [25]

8 12709 46623 10463 123-Propanetriol monoacetate Antimicrobial [26]

9 14048 16670 3741 12-Dioxolan-3-one 5-ethyl-5-methyl-4-methylene- Antioxidant [27]

10 20099 3546 0796 4-Ethylamino-n-butylamine Herbicidal and insect lighting coating material[28 29]

11 2022 30010 6735 16-Anhydro-24-dideoxy-beta- D-ribo-hexopyranose Pesticidal [30]

12 30899 3872 0869 2-Propenoic acid tridecyl ester Antifungal [31]13 36132 3771 0846 Neophytadiene Larvicidal [4]14 40342 31374 7041 n-Hexadecanoic acid Larvicidal [2 4]15 45991 26925 6043 cis-Vaccenic acid Larvicidal [32]16 46693 1566 0351 Octadecanoic acid Larvicidal [2 4]SN signal noise RT retention time

Table 2 Larvicidal activity of Clitoria ternatea flower extracts against early 4th instar larvae of Aedes aegypti and Aedes albopictus

Larvae LC50 (mgL) (95 LCL-UCL) LC95 (mgL) (95 LCL-UCL) χ2 df RA aegypti 1056 (981ndash1121) Y minus14142 + 4722X 2491 (2094ndash2641) Y minus14142 + 4722X 30lowast 22 0667A albopictus 1425 (1352ndash1491) Y minus19682 + 6282X 2753 (2374ndash2881) Y minus19682 + 6282X 30lowast 22 0882Na total number of mosquitoes larvae used n 25 each with 4 replicates LC50 lethal concentration 50mortality LC95 lethal concentration 95mortalityLCL lower confidence limits UCL upper confidence limits χ2 Pearson chi-square df degrees of freedom R Pearsonrsquos R (note chi-square values with anasterisk are significant (Plt 005))

300 500 1000 1500 1700 2000 2500Concentration (mgL)

100908070605040302010

0

Mea

n m

orta

lity

plusmn SE

()

Larvicidal activity

AealbopictusAeaegpyti

Figure 2 Comparison of larvae mortality rate between Aedes aegypti and Aedes albopictus for various Clitoria ternatea flower extractconcentrations


0e residual activity of C ternatea flower extract showedup to 48 hours in water Moreover Ullah et al [34] recordedthat there are five different plant species with residual ac-tivities against Culex quinquefasciatus up to 72 hours upontheir application Elsewhere a study on Azolla pinnataextract has suggested that 48 hours of residual activity inwater and plant-based applications was much shorter intheir residual effects compared to synthetic pesticides [4]0us it can be concluded that plant-based insecticidespossess lesser residual activity compared to syntheticchemicals Additionally the C ternatea plant extracts didnot possess any toxic effects on fish According to Pereiraand Oliveira [35] Poecilia reticulata has important potentialeffects to predate Aedes aegypti larvae and this will help toeliminate the breeding ground of larvae 0us without thetoxic effects of C ternatea extracts on Poecilia reticulate wecould integrate both applications using C ternatea and fishto control Aedes larvae In addition to that the toxicityresults in this current study were also similar to the previousstudy based on Azolla pinnata plant [2 4] 0us the use ofC ternatea extract is not toxic for the environment and it is

safer to be applied compared to chemical application incontrolling Aedes larvae

In addition to that the metabolite study of C ternateaextracts showed different types of phenols such as antho-cyanin flavonoids and tannins Anthocyanin flavonoidsand tannins were present in both methanol and ethanolextracts of C ternatea compared to other extracts (Table 1)Divya et al [14] reported similar findings on Clitoria ter-natea blue and white flowered leaves which recorded thehighest polyphenol isolation using methanol extractiontechniques Moreover this was further proven with thin-layer chromatography and paper chromatography tech-niques whereby all the extracts were found to have threespots upon fixing with ferric chloride (FeCl3) ConcurrentlySunita et al [36] have also reported the same results withmedicinal plant using ferric chloride (FeCl3) to identify onlyphenolic compounds on chromatography paper Finally theantioxidant test for C ternatea flower extracts showed thataqueous extract has the highest antioxidant propertycompared to other extraction methods 0e aqueous extractwas found to have high antioxidant because water extract

Figure 3 Guppy fish Poecilia reticulata toxicity test with Clitoria ternatea

(a) (b)

Figure 4 Morphological midgut content induced by Clitoria ternatea (a) Midgut content view in larvae of Aedes aegypti (b) Midgutcontent view in larvae of Aedes albopictus Note Arrows indicate the plant extracts (dark greenish color) GC gut content (after 24 hours)


can absorb antioxidant compound better than chemicals[37]

0us it can be concluded that C ternatea flower extract-based insecticides may possess lesser residual activitycompared to synthetic chemicals and contain multiplenatural chemical compounds which may solve the single-based insecticide-resistant problems Finally althoughC ternatea flower extracts may be used as bio-larvicidesfuture testing would have to be conducted to validate theirlong-term effects on human health and other organisms inthe environment

5 Conclusion

We can conclude that this current study has shown thecolossal effectiveness of Clitoria ternatea flower extractsagainst major dengue fever vectors during the early 4thinstar larval stages Additionally current findings showedthe bioactive molecules even from crude extracts to be ef-fective and may be developed as biolarvicides for Aedesmosquito vector control programs Furthermore this studyalso provided a baseline understanding for future researchwork on the field of applications of C ternatea flower ex-tracts which could be tested for their long-term effectsapplicable to other industries encompassing all aspects ofhuman health

Data Availability

0e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

0e authors declare no conflicts of interest

Acknowledgments

0e authors would like to acknowledge the Quest Inter-national University internal funds and also thank universitystaffs and research students who have contributed to thiswork

Supplementary Materials

0e NIST 08 library search for chemical compound struc-tures and details (Supplementary Materials)

References

[1] WHO Update on the Dengue Situation in the Western PacificRegion Northern Hemisphere Cambodia World Health Or-ganization Geneva Switzerland 2020

[2] R Ravi H Zulkrnin N Shaida et al ldquoEvaluation of twodifferent solvents for Azolla pinnata extracts on chemicalcompositions and larvicidal activity against Aedes albopictus(Diptera Culicidae)rdquo Journal of Chemistry vol 2018 ArticleID 7453816 8 pages 2018

[3] H Zulkrnin N Shaida N N Rozhan et al ldquoLarvicidal ef-fectiveness of Azolla pinnata against Aedes aegypti (DipteraCulicidae) with its effects on larval morphology and

visualization of behavioural responserdquo Journal of ParasitologyResearch vol 2018 Article ID 1383186 5 pages 2018

[4] R Ravi N S H Zulkrnin N N Rozhan et al ldquoChemicalcomposition and larvicidal activities ofAzolla pinnata extractsagainst Aedes (Diptera Culicidae)rdquo PLoS One vol 13 no 11Article ID e0206982 2018

[5] R Ravi I H Ishak M F Mohd Amin M S M RasatM A Salam and M A Mohd Amin ldquoNano-synthesizedsilver particles and Azolla Pinnata extract Larvicidal effectsagainst aedes aegypti (diptera culicidae)rdquo InternationalJournal of Advanced Science and Technology vol 29 no 4pp 3420ndash3427 2020

[6] A Ghosh N Chowdhury and G Chandra ldquoPlant extracts aspotential mosquito larvicidesrdquo gte Indian Journal of MedicalResearch vol 135 no 5 p 581 2012

[7] G Arumugam P Manjula and N Paari ldquoA review antidiabetic medicinal plants used for diabetes mellitusrdquo Journalof Acute Disease vol 2 no 3 pp 196ndash200 2013

[8] P K Mukherjee V Kumar N S Kumar and M Heinrichldquo0e ayurvedic medicine Clitoria ternateamdashfrom traditionaluse to scientific assessmentrdquo Journal of Ethnopharmacologyvol 120 no 3 pp 291ndash301 2008

[9] N Mathew M G Anitha T S L Bala S M SivakumarR Narmadha and M Kalyanasundaram ldquoLarvicidal activityof Saraca indica Nyctanthes arbor-tristis and Clitoria ter-natea extracts against three mosquito vector speciesrdquo Para-sitology Research vol 104 no 5 pp 1017ndash1025 2009

[10] S Wong L Leong and J Williamkoh ldquoAntioxidant activitiesof aqueous extracts of selected plantsrdquo Food Chemistryvol 99 no 4 pp 775ndash783 2006

[11] S Anand A Doss and V Nandagopalan ldquoAntibacterialstudies on leaves of Clitoria ternatea linnmdasha high potentialmedicinal plantrdquo International Journal of Applied Biology andPharmaceutical Technology vol 2 no 3 pp 453ndash456 2011

[12] T K Mathew and P Benny ldquoFlowers with antibacterialpropertymdasha survey in Meenachil Talukrdquo Journal of Phar-maceutical Chemical and Biological Sciences vol 4 no 2pp 252ndash261 2016

[13] A H Ismail A N Idris M M Amina A S Ibrahim andS A Audu ldquoPhytochemical studies and thin layer chroma-tography of leaves and flower extracts of Senna siamea lam forpossible biomedical applicationsrdquo Journal of Pharmacognosyand Phytotherapy vol 7 no 3 pp 18ndash26 2015

[14] A Divya J Anbumalarmathi and S Sharmili ldquoPhyto-chemical analysis antimicrobial and antioxidant activity ofClitoria ternatea blue and white flowered leavesrdquo Advances inResearch vol 14 no 5 pp 1ndash13 2018

[15] J Ferry and R A Larson ldquoA mixed solvent for rapid TLCanalysis of phenolic compoundsrdquo Journal of ChromatographicScience vol 29 no 11 pp 476-477 1991

[16] M Fu Z He Y Zhao J Yang and L Mao ldquoAntioxidantproperties and involved compounds of daylily flowers inrelation to maturityrdquo Food Chemistry vol 114 no 4pp 1192ndash1197 2009

[17] World Health Organization Guidelines for Laboratory andField Testing of Mosquito Larvicides World Health Organi-zation Geneva Switzerland 2005

[18] M A Principato ldquoPupicidal and larvicidal compositionrdquoUS20100310685A1 2010

[19] K G Koorse S Samraj P John et al ldquoAnthelmintic activityof fruit extract and fractions of Piper longum L In vitrordquoPharmacognosy Journal vol 10 no 2 pp 333ndash340 2018

[20] P Bharali Y Gamo A K Das H Tag A M Baruah andD Kakati ldquoPhytochemical and biochemical study of four


legume plants with detergent and anti-lice properties from theEastern Himalayan region of Indiardquo Current Science vol 113no 7 p 1434 Article ID 00113891 2017

[21] S Vats and T Gupta ldquoEvaluation of bioactive compoundsand antioxidant potential of hydroethanolic extract of Mor-inga oleifera Lam from Rajasthan Indiardquo Physiology andMolecular Biology of Plants vol 23 no 1 pp 239ndash248 2017

[22] S Fujita T Takayanagi and S Kato ldquoAgricultural chemicalsformulation for rice paddy field preparation thereof and themethod for scattering the samerdquo US6486095B1 2002

[23] S H Ahmed ldquoEvaluation of antioxidant and anti-mutagenicactivity of naturally fortified honey with curcuma longa ex-tractrdquo International Journal of Biosciences (IJB) vol 13 no 1pp 248ndash256 2018

[24] M Padmashree R Ashwathanarayana and R B Raja NaikaldquoAntioxidant cytotoxic and nutritive properties of Ipomoeastaphylina Roem amp Schult plant extracts with preliminaryphytochemical and GCMS analysisrdquo Asian Journal of Phar-macy and Pharmacology vol 4 no 4 pp 473ndash492 2018

[25] L Cechovska K Cejpek M Konecny and J Velisek ldquoOn therole of 23-dihydro-35-dihydroxy-6-methyl-(4H)-pyran-4-one in antioxidant capacity of prunesrdquo European Food Re-search and Technology vol 233 no 3 pp 367ndash376 2011

[26] J Heeres L J Backx J B 0ijssen and A G Knaeps ldquo[[4-[4-(4-phenyl-1-piperazinyl)phenoxymethyl]-1 3-dioxolan-2-yl]methyl]-1h-imidazoles and 1h-124-triazoles having anti-microbial propertiesrdquo PT78156A 1988

[27] S Prabakaran L Ramu S Veerappan B Pemiah andN Kannappan ldquoEffect of different solvents on volatile andnon-volatile constituents of red bell pepper (Capsicum ann-uum L) and their in vitro antioxidant activityrdquo Journal ofFood Measurement and Characterization vol 11 no 4pp 1531ndash1541 2017

[28] P Van Kleef ldquoInsect control lighting devicerdquo US Patent20180116196A1 2019

[29] J D Cleveland ldquoHerbicidal N-substituted-dithioamino andN-substituted-oxythioamino triazinesrdquo US Patent 3909237A1975

[30] R F Henzell R H Furneaux and P C Tyler ldquoAn evaluationof the herbicidal and plant growth regulatory activity of anovel class of carbohydrate-derived 68-dioxabicyclo[321]octanesrdquo Pesticide Science vol 30 no 1 pp 59ndash66 1990

[31] C Sangeetha A Krishnamoorthy and D Amirtham ldquoAn-tifungal bioactive compounds from Chinese caterpillar fungus(Ophiocordyceps sinensis (Berk) GH Sung et al) against plantpathogensrdquo Madras Agricultural Journal vol 102 2015

[32] M Nyamoita Z Mbwambo B Ochola E InnocentW Lwande and A Hassanali ldquoChemical composition andevaluation of mosquito larvicidal activity of Vitex payoffsextracts against Anopheles gambiae Giles SS larvaerdquo SpatulaDD vol 3 no 3 pp 113ndash120 2013

[33] R Pavela ldquoHistory presence and perspective of using plantextracts as commercial botanical insecticides and farmproducts for protection against insectsmdasha reviewrdquo PlantProtection Science vol 52 no 4 pp 229ndash241 2016

[34] Z Ullah A Ijaz T K Mughal and K Zia ldquoLarvicidal activityof medicinal plant extracts againstCulex quinquefasciatus Say(Culicidae Diptera)rdquo International Journal of Mosquito Re-search vol 5 no 2 pp 47ndash51 2018

[35] B B Pereira and E A d Oliveira ldquoDeterminacao do potenciallarvofago de Poecilia reticulata em condicoes domesticas decontrole biologicordquo Cadernos Saude Coletiva vol 22 no 3pp 241ndash245 2014

[36] A Sunita M Sonam and K Ganesh ldquoGas chromatography-mass spectrometry analysis of an endangered medicinal plantSarcostemma viminale (L) R BR From thar desert Rajasthan(India)rdquo Asian Journal of Pharmaceutical and Clinical Re-search vol 10 no 9 p 210 2017

[37] K Bouabid F Lamchouri H Toufik and M E A FaouzildquoPhytochemical investigation in vitro and in vivo antioxidantproperties of aqueous and organic extracts of toxic plantAtractylis gummifera Lrdquo Journal of Ethnopharmacologyvol 253 p 112640 2020


1 Introduction

Mosquitoes have always been a human health threat fromeons the major health problems caused by them aremalaria dengue fever yellow fever and Zika as well asseveral other vector-borne diseases Based onWorld HealthOrganization [1] western Pacific region northern hemi-sphere reports on January 2020 dengue updates recordedwere as follows Cambodia with a higher average of 1239cases weekly compared to the period of 2019 Malaysia withlower 42 decrease weekly as 2604 cases with 9 deathscompared to the period of 2019 Philippines with 2778 caseswith 10 deaths in 2020 Singapore with 2508 cases highercompared to the period of 2019 Vietnam with 1282 weeklycases at 2020 In the southern hemisphere Australia hasrecorded 78 dengue cases since the beginning of 2020 till 26February 2020 which is lower compared to the period of2019 [1] 0us dengue cases are inevitable in humancommunity and there are many factors contributing to-wards this global issue

In the current situation only physical and chemicalmethods are being used to control mosquito-borne dis-eases Physical methods such as mosquito bed netsmosquito window nets at homes and electrical mosquitorackets are only temporary solutions [2] Besides that thechemicals used for vector control such as temephos andpyrethroids are more well-known with some insecticidesresistant challenges [2] Chemical insecticides are syn-thesized chemically based on single bioactive compoundswith reference to structurally related plant compoundswhich lead to the current resistance problem for allmosquitocide classes [2ndash5] Only one single chemicalcompound can be synthesized at a time due to its hightoxicity effects and the multiple plant biochemical com-pounds are more stable and lower in toxicity due to theirsynergetic effects under natural conditions AdditionallyRavi et al [4] supported the concepts of botany controlmechanisms as a simple and sustainable method com-pared to the conventional insecticides in use

Unlike current synthetic or chemical insecticides theadvantages of plant-based insecticides are their composi-tions of natural blends of multiple chemical compoundsthat may act synergistically on both physiological andbehavioral processes of mosquitoes [2ndash6] 0us botanical-based insecticides are an alternative and accurate solutionagainst dengue vectors of Aedes aegypti and Aedesalbopictus

In tune with the concept of a safer alternative Clitoriaternatea plant has the potential as a bioinsecticide to solvethe problems of resistance from a single-based chemicalcompound Clitoria ternatea is a well-known flower in Asiancountries such as Malaysia 0ailand Philippine India andChina and widely used in Ayurveda and Chinese medicine[7] Additionally Malaysians use the extract of the flower asa food coloring to prepare a popular dish called Nasi Kerabuand Indians from the state of Kerala region consume thisplant as a vegetable on a daily basis [7] 0e potential

compounds of this plant are prevalent in the form ofpolyphenols triterpenoids flavonoids glycosides antho-cyanins tannins and steroids [8] To date only one studyMathew et al [9] has reported the basic use of C ternatea asan Aedes aegypti and Aedes stephensimosquito larvicidal butnone on Ae albopictus According to Mathew et al [9] themethanolic extract of C ternatea showed larvicidal effec-tiveness however no mechanisms of larvicidal wereexplained and hence suggested improvements on furtherinvestigations pertaining to the active principles of thechemical compounds responsible for larvicidal activities0us within this expansion of framework this seems to bethe first study to elucidate larvicidal mechanism metaboliteantioxidant and chemical compound identifications fromC ternatea flower which may be responsible for Aedeslarvicidal activities 0e objective of this study is to identifythe larvicidal mechanism metabolite antioxidant chemicalcompounds and the structure of C ternatea flower and totest its efficacies against the early 4th instar larvae of Aedesaegypti and Aedes albopictus

2 Materials and Methods

21 Statement for Experimental Protocols All experimentalprotocols mentioned in this paper were in accordance withlarval susceptibility test method guidelines by the WorldHealth Organization (WHO)

22 PlantMaterials Clitoria ternatea used for this study wasauthenticated by the Herbarium of the University of Malayawith the herbarium voucher number KLU60080 Clitoriaternatea flowers (Figure 1) were collected from the garden ofthe Faculty of Medicine in International Quest UniversityIpoh Perak 0e petals of C ternatea were washed inrunning tap water to remove adhered debris and soil par-ticles and washed again with distilled water and dried in hotair oven at 70degC until the petals were completely dry [10]0e dried petals were ground into fine powder by using alaboratory grinder and stored in a vacuum-tight containerfor further usage

23 Aqueous Extraction Aqueous extraction was performedby maceration using 250ml distilled water and 50 g of thepowdered C ternatea flowers petal 0ey were mixed in a500ml beaker and stored in a dark area for an hour 0enthe extract was filtered using cotton wool and filter paper0e final volumes were stored in a universal bottle andmaintained at 4degC to 6degC [10]

24 Methanol Extraction Methanol extraction was per-formed in three different concentrations of 50 70 and95 Amixture of 5 g powderedC ternatea flower petals and100ml of respective concentration of methanol was pre-pared in three separate Scott bottles and covered withaluminum foil to avoid exposure to light Later the bottles




























3 Results









4 Discussion

















100908070605040302010

0

Mea

n m

orta

lity

plusmn SE

()

Larvicidal activity








(a) (b)





5 Conclusion


Data Availability




Acknowledgments




References




































































3 Results









4 Discussion

















100908070605040302010

0

Mea

n m

orta

lity

plusmn SE

()

Larvicidal activity








(a) (b)





5 Conclusion


Data Availability




Acknowledgments




References























































3 Results









4 Discussion

















100908070605040302010

0

Mea

n m

orta

lity

plusmn SE

()

Larvicidal activity








(a) (b)





5 Conclusion


Data Availability




Acknowledgments




References











































3 Results









4 Discussion

















100908070605040302010

0

Mea

n m

orta

lity

plusmn SE

()

Larvicidal activity








(a) (b)





5 Conclusion


Data Availability




Acknowledgments




References























































100908070605040302010

0

Mea

n m

orta

lity

plusmn SE

()

Larvicidal activity








(a) (b)





5 Conclusion


Data Availability




Acknowledgments




References














































(a) (b)





5 Conclusion


Data Availability




Acknowledgments




References












































5 Conclusion


Data Availability




Acknowledgments




References





























































Documents

ResearchArticledownloads.hindawi.com/journals/jchem/2020/3837207.pdfCorrespondence should be addressed to Rajiv Ravi; [email protected] and Mohamad Faiz Mohd Amin; [email protected]