BROCHURE DAUN BETIK to hospitals 130814 - crc.gov.my · Langkah-langkah penyediaan jus daun betik...

10
Langkah-langkah penyediaan jus daun betik Sediakan tempat untuk menyediakan jus daun betik yang bersih. Bilas daun dengan air bersih sekali lagi. Potong daun sehingga menjadi kepingan yang kecil. Dua helai daun betik dapat menghasilkan sebanyak 30 mL atau dua sudu besar. Tuang jus ke dalam sudu besar. Minum jus daun betik sebanyak dua sudu makan. Ambil 2 helai daun betik matang dan buang tangkai daun. Basuh daun dengan air bersih yang mengalir. Rendamkan daun dalam bekas berisi air untuk 15 minit. Jus daun betik yang telah siap. Kisar daun dalam mesin pengisar jus TANPA tambah air Tumbuk kepingan daun betik TANPA tambah air sehingga halus. Tapis untuk asingkan jusnya 2 3 7 atau 11 13 12 8 1 9 4 5 6 10 Tos/keringkan daun untuk menyingkirkan air bilasan. PERINGATAN Amalkan pengambilan jus sekali sehari untuk 3 hari berturut-turut sahaja. Digalakkan mendapat nasihat doktor sebelum memberi jus daun betik kepada kanak-kanak berusia 13 tahun dan ke bawah. Pengambilan jus daun betik adalah rawatan sampingan dan BUKAN rawatan khas untuk demam denggi.

Transcript of BROCHURE DAUN BETIK to hospitals 130814 - crc.gov.my · Langkah-langkah penyediaan jus daun betik...

Langkah-langkah penyediaan

jus daun betik

Sediakan tempat untuk

menyediakan jus daun

betik yang bersih

Bilas daun dengan

air bersih sekali lagi

Potong daun sehingga

menjadi kepingan yang

kecil

Dua helai daun betik

dapat menghasilkan

sebanyak 30 mL

atau dua sudu besar

Tuang jus ke dalam

sudu besar

Minum jus daun

betik sebanyak

dua sudu makan

Ambil 2 helai daun betik

matang dan buang

tangkai daun Basuh

daun dengan air bersih

yang mengalir

Rendamkan daun dalam

bekas berisi air untuk 15

minit

Jus daun betik yang

telah siap

Kisar daun dalam

mesin pengisar jus

TANPA tambah air

Tumbuk kepingan daun

betik TANPA tambah air

sehingga halus

Tapis untuk asingkan

jusnya

2

3

7 atau

11

13

12

8

1

9

4

5

6

10

Toskeringkan daun untuk

menyingkirkan air bilasan

PERINGATAN

Amalkan pengambilan jus sekali sehari

untuk 3 hari berturut-turut sahaja

Digalakkan mendapat nasihat doktor

sebelum memberi jus daun betik kepada

kanak-kanak berusia 13 tahun dan ke

bawah

Pengambilan jus daun betik adalah

rawatan sampingan dan BUKAN rawatan

khas untuk demam denggi

DAUN BETIK SEGAR

Daun betik telah digunakan dalam perubatan

tradisional selama berabad-abad Kajian

klinikal yang telah dijalankan oleh Institut

Penyelidikan Perubatan Kementerian

Kesihatan Malaysia telah menunjukkan

bahawa jus daun betik segar didapati

mempunyai kesan menambahkah sel darah

kuning (platelet) pada pesakit demam denggi

Kajian juga menunjukkan bahawa ia selamat

untuk diminum jika disediakan dengan cara

yang betul dan bersih seperti di lampiran ini

Daun betik

Pusat Penyelidikan Perubatan Herba

Institut Penyelidikan Perubatan

Kementerian Kesihatan Malaysia

wwwimrgovmy

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 616737 7 pageshttpdxdoiorg1011552013616737

Research ArticleCarica papaya Leaves Juice Significantly Acceleratesthe Rate of Increase in Platelet Count among Patients withDengue Fever and Dengue Haemorrhagic Fever

Soobitha Subenthiran1 Tan Chwee Choon2 Kee Chee Cheong3

Ravindran Thayan4 Mok Boon Teck1 Prem Kumar Muniandy1

Adlin Afzan1 Noor Rain Abdullah1 and Zakiah Ismail1

1 Bioassay Unit Herbal Medicine Research Center Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia2 Department of Internal Medicine Tengku Ampuan Rahimah Hospital Jalan Langat 41200 Klang Malaysia3 Epidemiology and Biostatistics Unit Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia4Virology Unit Infectious Disease Research Center Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia

Correspondence should be addressed to Soobitha Subenthiran drsoobihotmailcom

Received 7 January 2013 Revised 21 March 2013 Accepted 21 March 2013

Academic Editor Martin Kohlmeier

Copyright copy 2013 Soobitha Subenthiran et al This 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 is properlycited

The study was conducted to investigate the platelet increasing property ofCarica papaya leaves juice (CPLJ) in patients with denguefever (DF) An open labeled randomized controlled trial was carried out on 228 patients with DF and dengue haemorrhagic fever(DHF) Approximately half the patients received the juice for 3 consecutive days while the others remained as controls and receivedthe standard management Their full blood count was monitored 8 hours for 48 hours Gene expression studies were conductedon the ALOX 12 and PTAFR genes The mean increase in platelet counts were compared in both groups using repeated measureANCOVA There was a significant increase in mean platelet count observed in the intervention group (119875 lt 0001) but not inthe control group 40 hours since the first dose of CPLJ Comparison of mean platelet count between intervention and controlgroup showed that mean platelet count in intervention group was significantly higher than control group after 40 and 48 hours ofadmission (119875 lt 001) The ALOX 12 (FC = 1500) and PTAFR (FC = 1342) genes were highly expressed among those on the juiceIt was concluded that CPLJ does significantly increase the platelet count in patients with DF and DHF

1 Introduction

Malaysia is blessed with 12000 species of flowering plantsof which 1300 have medicinal properties [1] There is arapidly growing response to the use of medicinal plants bythe Malaysian population WHO estimates that in manycountries 80 of the rural patients seek alternative treatmentusing medicinal plants

Carica papaya is a member of the Caricaceae and isa dicotyledonous polygamous and diploid species [2] Itoriginated from Southern Mexico Central America and thenorthern part of South America It is now cultivated inmany tropical countries such as Bangladesh India IndonesiaSri Lanka the Philippines and the West Indies including

Malaysia Malaysia is known to be one of the top 5 papayaexporting countries [3] The papaya fruit is globally con-sumed either in its fresh form or the form of juices jamsand crystallized dry fruit [4] The ripe fruit is said to bea rich source of vitamin A C and calcium [5] There aremany commercial products derived from the different partsof the C papaya plant the most prominent being papain andchymopapain which is produced from the latex of the youngfruit stem and the leaves C papaya leaves have been usedin folk medicine for centuries Recent studies have shownits beneficial effect as an anti-inflammatory agent [6] for itswound healing properties [7] antitumour as well as immune-modulatory effects [8] and as an antioxidant [9] A toxicitystudy (acute subacute and chronic toxicity) conducted on

2 Evidence-Based Complementary and Alternative Medicine

Sprague Dawley rats administered with Carica papaya leavesjuice (CPLJ) of the sekaki variant revealed that it was safe fororal consumption [10]

Dengue is an arthropod-borne viral disease carried byAedes aegypti as the vector caused by 4 possible viralserotypes namely serotype 1 2 3 and 4 of the Flaviviridaefamily In Malaysia dengue cases have been on the rise since2002 Total of 18371 cases of dengue fever (DF) and denguehaemorrhagic fever (DHF) were reported last year and hadclaimed 33 lives in the same year [11] There is no specificantiviral drug available for the treatment of dengue infectionInfected patients receive supportive management with fluidsblood and blood products complying to the Ministry ofHealth Clinical Practice Guidelines (CPGs) on Managementof Dengue 2010 Each episode of infection is known toinduce a life-long protective immunity to the homologousserotype but confers only partial and transient protectionagainst subsequent infection by the other serotypes Sec-ondary infection is a major risk factor for DHF possiblydue to antibody-dependent enhancement A patient withdengue fever presents typically with fever headache and rashknown as the dengue triadThere are many other nonspecificsigns and symptoms associated with DF and patient canprogress to DHF and typically manifests as abdominal painbleeding and even circulatory collapse The clinical courseof dengue has an abrupt onset followed by three phasesnamely the febrile phase the critical phase and the recoveryphase It is during the critical phase that thrombocytopaeniacharacterized by a decrease in platelet count below 100000 permm3 from the baseline and haemoconcentrationcharacterized by an increase of haematocrit by 20 or moreis detectable before the subsidence of fever and the onset ofshock [12]

Certain genes have been shown to influence plateletproduction and platelet aggregation namely the Arachido-nate 12-lipoxygenase (ALOX 12) also known as the Platelet-type Lipoxygenase as well as the Platelet-Activating FactorReceptor (PTAFR) An increase in activity of these genes isrequired for platelet production and activationThe ALOX 12gene is strongly expressed in megakaryocytes and has beenknown to be responsible for the 12-Hydroxyeicosatetraenoicacid (12-HETE) production of platelets [13]The PTAFR genewas been found to be expressed inmegakaryocytes indicatingthat it could be a precursor for platelet production in additionto its well known role in platelet aggregation [14]

Safety studies based on OECD guidelines for acutesubacute and chronic toxicity were conducted on C papayaextract and showed that it was found to be safe for humanconsumption [10] The present study was conducted todetermine and investigate the traditional claim that CPLJincreases the platelet count in patients with DF and DHF

2 Materials and Method

21 Plant Material and Sample Preparation C papaya leavesof the the sekaki variant were chosen for the study basedon fingerprinting and safety analysis which also containedallowable limit of heavy metals and microbial content For

the purpose of the study a private plantation certified by theMinistry of Agriculture in Semenyih Selangor was identifiedto provide the leaves for the entire duration of the studyto ensure similar source of authenticated raw material usedThe trees in this plantation were kept free of herbicidespesticides and insecticides Juice was prepared fresh from theleaves that were washed thoroughly with an organic vegetablecleaning agent and reverse osmosis water few times Juice wasextracted from 50 grams of fresh leaves using a juice extractorwithout any addition of water under sterile conditions Thefresh juice was aliquoted at a volume of approximately 30mLsin sterile glass vials and transported daily in an icebox andkept at a temperature of below 4∘C to the study site at themale and female dengue wards of Hospital Tengku AmpuanRahimah Klang Selangor

The juice was characterized and standardized using aHigh Performance Liquid Chromatography Diode ArrayDetector according to three markers manghaslin clitorinand rutin The chemical fingerprinting of the leaves wasconsistent throughout the study

22 Subjects An open labeled randomized controlled trialwas conducted to provide clinical data in support of theproposed claimsThe number of patients involved was deter-mined based on the following calculation

With plan to have a continuous response variable fromindependent control and experimental subjects with 1 con-trol(s) per experimental subject expecting the platelet countto increase by 20000 after administration of CPLJ for threeconsecutive days setting the standard deviation for plateletcount at 40000 in a normal population [15] type I error(alpha) = 001 and power of study of 90 the sample sizecalculated by using PS software [16] was 242 with each armof 121 intervention group and control group Anticipatingdropout rate to be 20 the final sample sizewas 290 (145 eachfor intervention and control group)

All the patients meeting the inclusion criteria wererecruited from theDengueWard ofHospital TengkuAmpuanRahimah Klang Selangor Malaysia Subjects were randomlyassigned to either intervention or control group using theblock of 10 method This method was used to ensure that anequal number of participants were allocated to interventionaland control groups Therefore with the block size of tenof every ten consecutively enrolled participants five will beallocated to one interventional group and five to the controlgroup For the randomization 10 identical opaque envelopeswith five envelopes each contained a card labelled as ldquointer-ventional grouprdquo and other 5 envelopes each contained a cardlabelled as ldquocontrol grouprdquo These envelopes were given tothe field investigator during the patient enrolment The fieldinvestigator then blindly selected an envelope for each patientthus assigning the patient to interventional or control groupsThis procedure was repeated until the targeted sample sizewas achieved

The inclusion and exclusion criteria were used to selectpatients who volunteered to be enrolled into the studyInclusion criteria were (a) male and female patients above18 years and below 60 years old (b) all patients who could

Evidence-Based Complementary and Alternative Medicine 3

understand or read Bahasa Malaysia or English irrespectiveof race (c) patients who were confirmed to have DF or DHFgrade I and II (d) patients with a platelet count of less thanor equal to 100000120583L (e) patients with a baseline alaninetransaminase (ALT) level of not more than 3 times of theupper limit of the normal range (not more than 165UL) and(f) patients with a creatinine kinase (CK) value of less than500UL

The exclusion criteria were (a) dengue hemorrhagic fevergrade III and IV (b) pregnant or lactatingwomen (c) patientswho have received blood or blood products transfusionduring the current hospital stay (d) patients with underlyingcomorbids (e) patients who developed Hepatitis with aserum ALT level 3 times higher than the upper limit of thenormal range (gt165UL) and (f) patients with a creatininekinase (CK) value of more than 500UL

The study was conducted in accordance with the ethicalprinciples as outlined in the declaration of Helsinki October2008 following approval from the Medical Review andEthical Committee (MREC) Ministry of Health Malaysiaand was consistent with Good Clinical Practice (GCP) andapplicable regulatory requirements The National MedicalResearch Registry number is NMRR-09-883-4768 Writteninformed consent was obtained from every volunteer priorto clinical trial participation

23 Diagnosis of DF or DHF and Dengue Serotyping Aclinical diagnosis of DF and DHF was made by the clinicianbased on patientsrsquo presentation and blood investigations Inaddition a rapid dengue bedside test (SD Dengue Duo NS1Ag + Ab Combo Standard Diagnostics Korea) was usedto determine dengue status before the subjects were giventhe Carica papaya leave juice This test is able to detect thepresence of NS1 antigen or dengue IgM or IgG antibodiesHowever for ease of interpretation of dengue status onlypatients who had either NS1 or IgM or both detected wereincluded in the studyThe detection of theNS1 antigen or IgMantibodies within the first five to 7 days of onset of symptomsusually indicates a current dengue infection Subsequently allthe samples were subjected tomultiplex real time RT-PCR fordetermination of dengue serotypes

24 Treatment of the Subjects Once a current dengue infec-tion was confirmed a thorough screening of the patientwas conducted Baseline investigations included full bloodcount bleeding profile renal as well as liver function testand cardiac enzymes Patients in the intervention groupreceived fresh juice from 50 grams of C papaya leavesonce daily 15 minutes after breakfast for 3 consecutive dayswhile receiving the standardmanagement as per the NationalClinical Practice Guidelines for the Management of DengueThe controls received the standard management

25 Study Parameters Full blood count was monitored 8hours for the first 48 hours during the study to deter-mine the changes in platelet count and haematocrit levelswhile bleeding profile renal profile and liver function testwere monitored daily to ensure the safety of the juice

The haematological and biochemical tests were conductedby the Pathology Department at Hospital Tengku AmpuanRahimah Klang and the validated results were later tracedand recorded Mean and standard deviation of the baselinehaematological and biochemical parameters were then calcu-lated as shown in Table 1

Blood for RNA extractionwas taken on day 3 after the lastdose of the juice to conduct expression studies on the PTAFRand the ALOX 12 genes RNALater by Ambion (United Statesof America) was added to the whole blood in an EDTA tubeto prevent degradation of RNA

The RNA extraction was carried out at the Institute forMedical research using an RNA extraction kit by AmbionThe RNA concentration and purity were determined usingtheNanodrop 2000 Spectrophotometer byThermo ScientificTheRNAwas then converted to cDNAusing theHighCapac-ity RNA to cDNA kit by Applied Biosystems The cDNAconcentrationwas then determinedusing theNanodrop 2000Spectrophotometer byThermo Scientific

Gene expression was determined using gene expressionassays by Applied Biosystems on the ABI 7500 Fast Systemon 12 RNA samples from the experimental group and 12 RNAsamples from the control group Specific predesigned MGBprobes were used for ALOX 12 and the PTAFR gene The18S Ribosome was used as an endogenous control A 10 120583Lreaction volume was used using the TaqMan Fast UniversalPCR Mastermix (2X) The probe product ID and sequenceused were ALOX 12 (Hs 00167524 M1 NM 000697251015840-FAM-ATTGCCATCCAGCTCAACAAATCC-MGB-31015840)and PTAFR (Hs 00265399 S1 NM 0011647231 51015840-FAM-GCCCGTAATTTATCGCGCTTACTAT MGB-21015840)

26 Statistical Analysis Repeated measure ANCOVA wasused to determine the effect of CPLJ on the mean plateletcount over 48 hours (time effect) Multiple comparisonsof mean platelet count 8 hours after admission with meanplatelet count at the 16 24 32 40 and 48 hours after admis-sion for interventional and control group were performedMultiple paired t-test was conducted to demonstrate if thereis any significant different in mean platelet count for eachcomparison Hence Bonferroni correction was applied toreduce the possibility of rejecting a true null hypotheses(commit a type 1 error) Therefore Bonferroni correctionwas used to adjust the level of significance to 119875 lt 001(005number of comparison = 0055 = 001) The efficacyof treatment of CPLJ (treatment effect) and treatment overtime (time-treatment interaction) in increasing the meanplatelet count was analysed by comparing mean differencein platelet count between interventional and control groupAll the statistical analyses were done using PASW 180 (SPSSInc Chicago USA)The comparative CT methodwas used todetermine the relative quantification of the genes expressed

3 Results

A total of 145 patients were recruited into the interventionalgroup while 145 patients were recruited into the control

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Demographic characteristics and baseline biochemistry investigation of respondents by treatment

Demographic characteristics Interventional group119899 ()

Control group119899 () 120594

2 (df)lowast 119875 value

GenderMen 91 (469) 103 (531) 1644 (1) 0200Women 20 (588) 14 (412)

EthnicityMalay 77 (481) 83 (519)Chinese 4 (571) 3 (429)Indian 11 (500) 11 (500)Others 19 (487) 20 (513)

Age (meanSD) 304 (103)dagger 264 (73)dagger minus3370daggerdagger 0001Type of dengue fever

Classical dengue fever 42 (532) 37 (468) 0971 (1) 0324Dengue haemorrhagic fever 69 (463) 80 (537)

Baseline Haematology investigationPlatelet count (times103120583L) 664 (228)dagger 690 (226)dagger 0847daggerdagger 0398Total white blood cell (times103120583L) 34 (17)dagger 33 (16) minus0297daggerdagger 0766Haemoglobin (g) 139 (15)dagger 142 (14)dagger 1595daggerdagger 0112Haematocrit () 415 (42)dagger 429 (60)dagger 1878daggerdagger 0062Lymphocytes () 418 (142)dagger 409 (148)dagger minus0459daggerdagger 0646Neutrophils () 421 (177)dagger 421 (194)dagger 0017daggerdagger 0987

lowastPearson Chi-Square Df (degree of freedom) daggermean and standard deviation daggerdagger119905-value for independent samples 119879-test

Table 2 Comparison of platelet count in each group based on time

Type of laboratory investigation Interventional group (119899 = 111) Control group (119899 = 117)Platelet count Mean difference (95 CI) 119905 119875 value Mean difference (95 CI) 119905 119875 value8ndash16 hours 0993 (minus1660 3645) 0740 0460 minus1411 (minus3961 1140) minus1094 02768ndash24 hours minus0432 (minus4422 3558) minus0214 0831 2213 (minus0523 4948) 1600 01128ndash32 hours minus2716 (minus7540 2107) minus1114 0267 2775 (minus0796 6347) 1537 01278ndash40 hours minus7890 (minus14472 minus1310) minus2374 0019 0867 (minus3472 5207) 0395 06938ndash48 hours minus16764 (minus24566 minus8964) minus4256 lt0001 minus7703 (minus14055 1351) minus2399 0018Repeated measure ANCOVA within group analysis was applied followed by multiple paired 119905-testslowastBonferroni correction was applied by correcting the level of significance (0055 = 001) Potential covariate (age) was controlled by using repeated measuresANCOVA (ANCOVA controlled for age)

group At the end of the study 111 patients from the interven-tional group and 117 controls were included in the statisticalanalysis Sixty-two patients were excluded from the analysisas 38 patients were lost to followup and 24 patients hadincomplete data (missing results due to sample rejection)

Table 1 shows demographic characteristics and baselinebiochemistry investigation of respondents by treatment Interms of dengue status all patients recruited had eitherdengue NS1 or IgM or both detected while the percentagedistribution of the dengue serotypes among them was DEN1(304) DEN2 (284) DEN 3 (206) andDEN 4 (206)Hence all serotypes were well represented in the study

Table 2 presents the multiple comparisons of meanplatelet count 8 hours after admission with mean platelet

count at 16 24 32 40 and 48 hours after admission forinterventional and control group Multiple paired t-test wasconducted to demonstrate if there was any significant dif-ference in mean platelet count for each comparison HenceBonferroni correction was applied to reduce the possibility ofrejecting a true null hypothesis (committing a type 1 error)Based on the number of patients recruitedwith complete data(111 patients from the intervention group and 117 control)the power of study was 870 (standard deviation of plateletcount of 40000 type I error probability of 001 and thetrue difference in mean platelet count of 20000 betweenthe intervention and control group) Overall there was asignificant increase in mean platelet count over 40 hours inboth groups (Wilkrsquos Lambda = 0939 119875 = 0015 effect size

Evidence-Based Complementary and Alternative Medicine 5

646644

625

644 631

608623

586

708

607

796

694

50

55

60

65

70

75

80

85

90

Experimental control (hours)

Mea

n pl

atel

et co

unt

8 16 24 32 40 48

Figure 1 Comparison of mean platelet count between intervention and control group based on time (time-treatment effect)

Table 3 Comparison of platelets count between experiment andcontrol group (treatment effect regardless of time)

Type of lab Investigation Mean difference (95 CI) 119875 valuePlatelet count 2349 (minus5151 9850) 0538Repeated measure ANCOVA between group analyses was applied Level ofsignificance was set at 005 (two-tailed)

= 006 and power = 840) after adjusting for age Furtheranalysis by using multiple paired t-test on each of the groupsshowed that there was a significant increase in mean plateletcount at 40 hours compared to 8 hours after intervention inthe intervention group (119905 = minus4256 119875 le 0001) but not inthe control group (119905 = minus2399 119875 = 0018) after adjustment ofBonferroni correction (119875 = 0055 = 001)

Table 3 presents the overall efficacy of CPLJ supplementa-tion by comparing the mean platelet count of interventionaland control group regardless the time period Study on thetreatment effect of CPLJ on platelet count regardless of timedid not show any significant difference inmean platelet countbetween intervention and control group (119865 = 1128 119875 =0289) However analysis of the effect of CPLJ over the studyperiod (time and treatment effect) showed that there was asignificant interaction between treatment groups and time(Wilkrsquos Lambda = 0934 effect size = 006 and power =870)

Figure 1 shows the time-treatment effect of CPLJ Theintervention group had a significantly higher mean plateletcount than control group at 40 hours and 48 hours ofintervention

Data from the gene expression study were analyzed usingthe ldquoPCR Array Data Analysis versus 33rdquo software recom-mended by Applied Biosystems and showed that ALOX12(ΔCT mean = 1602 FC = 1500) and PTAFR genes (ΔCTmean= 1487 FC= 1342)were found to be highly expressed inthe intervention group when compared to the control group

4 Discussion

The study conducted shows that there is a rationale behindthe use of CPLJ in the treatment of some of DF and DHFIt is definitely worth investigating this plant for its potentialmedicinal benefits With rapid urbanization and global travelleading to drastic demographic changes dengue is a threatto almost 40 of the worldrsquos population There is still nospecific treatment for dengue Previous attempts to identify apotential antiviral for the treatment of dengue has been facedwith several challenges such as the presence of four distinctviral serotypes which frequently undergo mutations findingan appropriate model for infection and protective action ofa given drug as well as yield interesting therapeutic avenuesfor tailored response modifier drugs The current availablemouse model (AG129) available has its limitations such aslow viral load and a short period of viraemia The journeyto drug discovery through the study of immune-modulatoryeffects against dengue infection lies on the research of genericcompounds and natural products [17]

Research groups around Asia have attempted to studythe efficacy of CPLJ in rapidly increasing platelet counts inDF as well as DHF induced thrombocytopenia but therehas been no conclusive evidence drawn from those studiesDengue is generally a self-limiting disease and the diseaseinduced thrombocytopenia usually reverses itself after takinga slight dip during the phase of defervescence However asignificant number of patients succumb to the disease duringthe thrombocytopenic period Many mechanisms come intoplay during the critical phase of the disease to help reversethe disease state at this point Animal studies in elucidatingsafety data have been conducted on normal Sprague Dawleyrats using freeze dried CPLJ however no significant increasein platelet count was observed among the rats given thejuice and the rats kept as control [10] This was probablydue to the fact that the juice was freeze dried and certainessential compounds could have been lost during the processof freeze drying or perhaps the right disease model was not

6 Evidence-Based Complementary and Alternative Medicine

used for the study Haematocrit level which is an importantparameter which is usually monitored to determine the rateof improvement in haemoconcentration was found to besignificantly reduced in both groups of people White Bloodcell count which is found to be reduced in viral infections wasalso found to increase in both groups

The RNA was extracted from the blood of the patientsrecruited and gene expression of two genes namely theALOX 12 and the PTAFR which were conducted so farThere was a 15-fold increase in the ALOX 12 gene activityamong the patients in the experimental group as comparedto those in the control group at the end of the 3 days ALOX12 is known to be associated with increased megakaryocyteproduction as well as its conversion to platelets through 12-HETE mediated pathway which in turn leads to increasedplatelet production A study was conducted at the RoyalCollege of Surgeons Ireland to determine the platelet specificgenes The Alox 12 gene was highly expressed in platelets andfound to be a platelet specific gene byMcRedmond et al [18]A study conducted in Temple University School of MedicinePhiladelphia provided evidence that ALOX12 is a directtarget of transcription factor RUNX1 in megakaryocytes andplatelets RUNX1 is a transcription factor that regulates theexpression of haemopoietic-specific genes When there isRUNX1 haplodeficiency it affects overall haemopoiesis andhence ALOX 12 expression in platelets is decreased Therewas also an agonist-induced decreased 12-HETE productionin platelets with the decrease in ALOX 12 expression Thisprovides further evidence that platelet production is associ-ated with ALOX 12 expression [13]

This finding supports the claim that the juice consump-tion during the course of dengue infection has the potentialto induce the rapid production of platelets This was clearlydemonstrated by the significant increase in the mean plateletcount after 40 hours and 48 hours of juice consumptionThe PTAFR gene which is known to be responsible forincreased platelet production and aggregation was expressed1342-folds among the patients who consumed the juice ascompared to the control group indicating that the juicehad played an important role in addressing the arresting ofbleeding tendencies among these patients A study conductedin Brazil showed that injection of Platelet Activating Factor(PAFPTAFR) in mice induced an increase in platelet countHowever after a certain level further administration of PAFfailed to induce platelet production indicating autosensitiza-tion These findings show that PAFPTAFR can induce therelease of platelets which may be relevant to thrombocytosis[19] We are currently investigating many other genes todetermine other roles of theCPLJ other than its role in plateletproduction and activation

As all plants C papaya leaves are rich in compounds ofdifferent properties Further studies need to be conductedbefore determining the inflammatory pathways affected bythe juice unopposed However it can be concluded thatthe administration of CPLJ in DF and DHF is safe anddoes induce the rapid increase in platelet count It mayplay a valuable role in the management of DF in the nearfuture

Conflict of Interests

Theresearchwas conducted in the absence of any commercialor financial relationships that could be construed as a poten-tial conflict of interests

Acknowledgments

The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and the Director of theInstitute forMedical Research for giving them the permissionto publish this paper This research was funded by theMinistry ofHealthMalaysia (project code JPP-IMR09-003)They would like to thank Dr Nor Asiah biostatistician ofIMR for reviewing their statistical analysis the Director ofHospital Tengku Ampuan Rahimah for allowing them touse the hospital facilities and the staff of The Departmentof Medicine and the Department of Pathology for theirguidance support and cooperation during the study

References

[1] I H BurkillADictionary of the Economic Products of theMalayPeninsula vol 1 The Crown Agents for the Colonies LondonUK 1935

[2] K Arumuganathan and E D Earle ldquoNuclear DNA contentof some important plant speciesrdquo Plant Molecular BiologyReporter vol 9 no 3 pp 208ndash218 1991

[3] FAOSTATPapaya marketing-general information GFruit Website 2012 httpwwwitfnetorggfruitTemplates20Englishpapayamarketinfohtm

[4] V N Villegas ldquoCarica papaya Lrdquo in Plant Resources of South-East Asia 2 Edible Fruits and Nuts E M W Verheij and R ECoronel Eds vol 2 pp 223ndash225 PROSEA Wageningen TheNetherlands 1997

[5] H Y Nakasone and R E Paull Tropical Fruits CAB Interna-tional Walllingford NY USA 1998

[6] B V Owoyele O M Adebukola A A Funmilayo and A OSoladoye ldquoAnti-inflammatory activities of ethanolic extract ofCarica papaya leavesrdquo Inflammopharmacology vol 16 no 4 pp168ndash173 2008

[7] S Gurung and N Skalko-Basnet ldquoWound healing propertiesof Carica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[8] N Otsuki N H Dang E Kumagai A Kondo S Iwata andCMorimoto ldquoAqueous extract of Carica papaya leaves exhibitsanti-tumor activity and immunomodulatory effectsrdquo Journal ofEthnopharmacology vol 127 no 3 pp 760ndash767 2010

[9] N A Imaga G O Gbenle V I Okochi et al ldquoPhytochemicaland antioxidant nutrient constituents of Carica papaya andparquetina nigrescens extractsrdquo Scientific Research and Essaysvol 5 no 16 pp 2201ndash2205 2010

[10] S Z Halim N R Abdullah Z Afzan B A Abdul RashidI Jantan and Z Ismail ldquoAcute toxicity of Carica papaya leafextract in Sprague Dawley ratsrdquo Journal of Medicinal PlantsResearch vol 5 no 10 pp 1867ndash1872 2011

[11] Ministry of Health of Malaysia Vector borne disease controlunit report 2010

[12] World Health Organization Clinical diagnosis 16 2011

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

DAUN BETIK SEGAR

Daun betik telah digunakan dalam perubatan

tradisional selama berabad-abad Kajian

klinikal yang telah dijalankan oleh Institut

Penyelidikan Perubatan Kementerian

Kesihatan Malaysia telah menunjukkan

bahawa jus daun betik segar didapati

mempunyai kesan menambahkah sel darah

kuning (platelet) pada pesakit demam denggi

Kajian juga menunjukkan bahawa ia selamat

untuk diminum jika disediakan dengan cara

yang betul dan bersih seperti di lampiran ini

Daun betik

Pusat Penyelidikan Perubatan Herba

Institut Penyelidikan Perubatan

Kementerian Kesihatan Malaysia

wwwimrgovmy

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 616737 7 pageshttpdxdoiorg1011552013616737

Research ArticleCarica papaya Leaves Juice Significantly Acceleratesthe Rate of Increase in Platelet Count among Patients withDengue Fever and Dengue Haemorrhagic Fever

Soobitha Subenthiran1 Tan Chwee Choon2 Kee Chee Cheong3

Ravindran Thayan4 Mok Boon Teck1 Prem Kumar Muniandy1

Adlin Afzan1 Noor Rain Abdullah1 and Zakiah Ismail1

1 Bioassay Unit Herbal Medicine Research Center Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia2 Department of Internal Medicine Tengku Ampuan Rahimah Hospital Jalan Langat 41200 Klang Malaysia3 Epidemiology and Biostatistics Unit Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia4Virology Unit Infectious Disease Research Center Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia

Correspondence should be addressed to Soobitha Subenthiran drsoobihotmailcom

Received 7 January 2013 Revised 21 March 2013 Accepted 21 March 2013

Academic Editor Martin Kohlmeier

Copyright copy 2013 Soobitha Subenthiran et al This 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 is properlycited

The study was conducted to investigate the platelet increasing property ofCarica papaya leaves juice (CPLJ) in patients with denguefever (DF) An open labeled randomized controlled trial was carried out on 228 patients with DF and dengue haemorrhagic fever(DHF) Approximately half the patients received the juice for 3 consecutive days while the others remained as controls and receivedthe standard management Their full blood count was monitored 8 hours for 48 hours Gene expression studies were conductedon the ALOX 12 and PTAFR genes The mean increase in platelet counts were compared in both groups using repeated measureANCOVA There was a significant increase in mean platelet count observed in the intervention group (119875 lt 0001) but not inthe control group 40 hours since the first dose of CPLJ Comparison of mean platelet count between intervention and controlgroup showed that mean platelet count in intervention group was significantly higher than control group after 40 and 48 hours ofadmission (119875 lt 001) The ALOX 12 (FC = 1500) and PTAFR (FC = 1342) genes were highly expressed among those on the juiceIt was concluded that CPLJ does significantly increase the platelet count in patients with DF and DHF

1 Introduction

Malaysia is blessed with 12000 species of flowering plantsof which 1300 have medicinal properties [1] There is arapidly growing response to the use of medicinal plants bythe Malaysian population WHO estimates that in manycountries 80 of the rural patients seek alternative treatmentusing medicinal plants

Carica papaya is a member of the Caricaceae and isa dicotyledonous polygamous and diploid species [2] Itoriginated from Southern Mexico Central America and thenorthern part of South America It is now cultivated inmany tropical countries such as Bangladesh India IndonesiaSri Lanka the Philippines and the West Indies including

Malaysia Malaysia is known to be one of the top 5 papayaexporting countries [3] The papaya fruit is globally con-sumed either in its fresh form or the form of juices jamsand crystallized dry fruit [4] The ripe fruit is said to bea rich source of vitamin A C and calcium [5] There aremany commercial products derived from the different partsof the C papaya plant the most prominent being papain andchymopapain which is produced from the latex of the youngfruit stem and the leaves C papaya leaves have been usedin folk medicine for centuries Recent studies have shownits beneficial effect as an anti-inflammatory agent [6] for itswound healing properties [7] antitumour as well as immune-modulatory effects [8] and as an antioxidant [9] A toxicitystudy (acute subacute and chronic toxicity) conducted on

2 Evidence-Based Complementary and Alternative Medicine

Sprague Dawley rats administered with Carica papaya leavesjuice (CPLJ) of the sekaki variant revealed that it was safe fororal consumption [10]

Dengue is an arthropod-borne viral disease carried byAedes aegypti as the vector caused by 4 possible viralserotypes namely serotype 1 2 3 and 4 of the Flaviviridaefamily In Malaysia dengue cases have been on the rise since2002 Total of 18371 cases of dengue fever (DF) and denguehaemorrhagic fever (DHF) were reported last year and hadclaimed 33 lives in the same year [11] There is no specificantiviral drug available for the treatment of dengue infectionInfected patients receive supportive management with fluidsblood and blood products complying to the Ministry ofHealth Clinical Practice Guidelines (CPGs) on Managementof Dengue 2010 Each episode of infection is known toinduce a life-long protective immunity to the homologousserotype but confers only partial and transient protectionagainst subsequent infection by the other serotypes Sec-ondary infection is a major risk factor for DHF possiblydue to antibody-dependent enhancement A patient withdengue fever presents typically with fever headache and rashknown as the dengue triadThere are many other nonspecificsigns and symptoms associated with DF and patient canprogress to DHF and typically manifests as abdominal painbleeding and even circulatory collapse The clinical courseof dengue has an abrupt onset followed by three phasesnamely the febrile phase the critical phase and the recoveryphase It is during the critical phase that thrombocytopaeniacharacterized by a decrease in platelet count below 100000 permm3 from the baseline and haemoconcentrationcharacterized by an increase of haematocrit by 20 or moreis detectable before the subsidence of fever and the onset ofshock [12]

Certain genes have been shown to influence plateletproduction and platelet aggregation namely the Arachido-nate 12-lipoxygenase (ALOX 12) also known as the Platelet-type Lipoxygenase as well as the Platelet-Activating FactorReceptor (PTAFR) An increase in activity of these genes isrequired for platelet production and activationThe ALOX 12gene is strongly expressed in megakaryocytes and has beenknown to be responsible for the 12-Hydroxyeicosatetraenoicacid (12-HETE) production of platelets [13]The PTAFR genewas been found to be expressed inmegakaryocytes indicatingthat it could be a precursor for platelet production in additionto its well known role in platelet aggregation [14]

Safety studies based on OECD guidelines for acutesubacute and chronic toxicity were conducted on C papayaextract and showed that it was found to be safe for humanconsumption [10] The present study was conducted todetermine and investigate the traditional claim that CPLJincreases the platelet count in patients with DF and DHF

2 Materials and Method

21 Plant Material and Sample Preparation C papaya leavesof the the sekaki variant were chosen for the study basedon fingerprinting and safety analysis which also containedallowable limit of heavy metals and microbial content For

the purpose of the study a private plantation certified by theMinistry of Agriculture in Semenyih Selangor was identifiedto provide the leaves for the entire duration of the studyto ensure similar source of authenticated raw material usedThe trees in this plantation were kept free of herbicidespesticides and insecticides Juice was prepared fresh from theleaves that were washed thoroughly with an organic vegetablecleaning agent and reverse osmosis water few times Juice wasextracted from 50 grams of fresh leaves using a juice extractorwithout any addition of water under sterile conditions Thefresh juice was aliquoted at a volume of approximately 30mLsin sterile glass vials and transported daily in an icebox andkept at a temperature of below 4∘C to the study site at themale and female dengue wards of Hospital Tengku AmpuanRahimah Klang Selangor

The juice was characterized and standardized using aHigh Performance Liquid Chromatography Diode ArrayDetector according to three markers manghaslin clitorinand rutin The chemical fingerprinting of the leaves wasconsistent throughout the study

22 Subjects An open labeled randomized controlled trialwas conducted to provide clinical data in support of theproposed claimsThe number of patients involved was deter-mined based on the following calculation

With plan to have a continuous response variable fromindependent control and experimental subjects with 1 con-trol(s) per experimental subject expecting the platelet countto increase by 20000 after administration of CPLJ for threeconsecutive days setting the standard deviation for plateletcount at 40000 in a normal population [15] type I error(alpha) = 001 and power of study of 90 the sample sizecalculated by using PS software [16] was 242 with each armof 121 intervention group and control group Anticipatingdropout rate to be 20 the final sample sizewas 290 (145 eachfor intervention and control group)

All the patients meeting the inclusion criteria wererecruited from theDengueWard ofHospital TengkuAmpuanRahimah Klang Selangor Malaysia Subjects were randomlyassigned to either intervention or control group using theblock of 10 method This method was used to ensure that anequal number of participants were allocated to interventionaland control groups Therefore with the block size of tenof every ten consecutively enrolled participants five will beallocated to one interventional group and five to the controlgroup For the randomization 10 identical opaque envelopeswith five envelopes each contained a card labelled as ldquointer-ventional grouprdquo and other 5 envelopes each contained a cardlabelled as ldquocontrol grouprdquo These envelopes were given tothe field investigator during the patient enrolment The fieldinvestigator then blindly selected an envelope for each patientthus assigning the patient to interventional or control groupsThis procedure was repeated until the targeted sample sizewas achieved

The inclusion and exclusion criteria were used to selectpatients who volunteered to be enrolled into the studyInclusion criteria were (a) male and female patients above18 years and below 60 years old (b) all patients who could

Evidence-Based Complementary and Alternative Medicine 3

understand or read Bahasa Malaysia or English irrespectiveof race (c) patients who were confirmed to have DF or DHFgrade I and II (d) patients with a platelet count of less thanor equal to 100000120583L (e) patients with a baseline alaninetransaminase (ALT) level of not more than 3 times of theupper limit of the normal range (not more than 165UL) and(f) patients with a creatinine kinase (CK) value of less than500UL

The exclusion criteria were (a) dengue hemorrhagic fevergrade III and IV (b) pregnant or lactatingwomen (c) patientswho have received blood or blood products transfusionduring the current hospital stay (d) patients with underlyingcomorbids (e) patients who developed Hepatitis with aserum ALT level 3 times higher than the upper limit of thenormal range (gt165UL) and (f) patients with a creatininekinase (CK) value of more than 500UL

The study was conducted in accordance with the ethicalprinciples as outlined in the declaration of Helsinki October2008 following approval from the Medical Review andEthical Committee (MREC) Ministry of Health Malaysiaand was consistent with Good Clinical Practice (GCP) andapplicable regulatory requirements The National MedicalResearch Registry number is NMRR-09-883-4768 Writteninformed consent was obtained from every volunteer priorto clinical trial participation

23 Diagnosis of DF or DHF and Dengue Serotyping Aclinical diagnosis of DF and DHF was made by the clinicianbased on patientsrsquo presentation and blood investigations Inaddition a rapid dengue bedside test (SD Dengue Duo NS1Ag + Ab Combo Standard Diagnostics Korea) was usedto determine dengue status before the subjects were giventhe Carica papaya leave juice This test is able to detect thepresence of NS1 antigen or dengue IgM or IgG antibodiesHowever for ease of interpretation of dengue status onlypatients who had either NS1 or IgM or both detected wereincluded in the studyThe detection of theNS1 antigen or IgMantibodies within the first five to 7 days of onset of symptomsusually indicates a current dengue infection Subsequently allthe samples were subjected tomultiplex real time RT-PCR fordetermination of dengue serotypes

24 Treatment of the Subjects Once a current dengue infec-tion was confirmed a thorough screening of the patientwas conducted Baseline investigations included full bloodcount bleeding profile renal as well as liver function testand cardiac enzymes Patients in the intervention groupreceived fresh juice from 50 grams of C papaya leavesonce daily 15 minutes after breakfast for 3 consecutive dayswhile receiving the standardmanagement as per the NationalClinical Practice Guidelines for the Management of DengueThe controls received the standard management

25 Study Parameters Full blood count was monitored 8hours for the first 48 hours during the study to deter-mine the changes in platelet count and haematocrit levelswhile bleeding profile renal profile and liver function testwere monitored daily to ensure the safety of the juice

The haematological and biochemical tests were conductedby the Pathology Department at Hospital Tengku AmpuanRahimah Klang and the validated results were later tracedand recorded Mean and standard deviation of the baselinehaematological and biochemical parameters were then calcu-lated as shown in Table 1

Blood for RNA extractionwas taken on day 3 after the lastdose of the juice to conduct expression studies on the PTAFRand the ALOX 12 genes RNALater by Ambion (United Statesof America) was added to the whole blood in an EDTA tubeto prevent degradation of RNA

The RNA extraction was carried out at the Institute forMedical research using an RNA extraction kit by AmbionThe RNA concentration and purity were determined usingtheNanodrop 2000 Spectrophotometer byThermo ScientificTheRNAwas then converted to cDNAusing theHighCapac-ity RNA to cDNA kit by Applied Biosystems The cDNAconcentrationwas then determinedusing theNanodrop 2000Spectrophotometer byThermo Scientific

Gene expression was determined using gene expressionassays by Applied Biosystems on the ABI 7500 Fast Systemon 12 RNA samples from the experimental group and 12 RNAsamples from the control group Specific predesigned MGBprobes were used for ALOX 12 and the PTAFR gene The18S Ribosome was used as an endogenous control A 10 120583Lreaction volume was used using the TaqMan Fast UniversalPCR Mastermix (2X) The probe product ID and sequenceused were ALOX 12 (Hs 00167524 M1 NM 000697251015840-FAM-ATTGCCATCCAGCTCAACAAATCC-MGB-31015840)and PTAFR (Hs 00265399 S1 NM 0011647231 51015840-FAM-GCCCGTAATTTATCGCGCTTACTAT MGB-21015840)

26 Statistical Analysis Repeated measure ANCOVA wasused to determine the effect of CPLJ on the mean plateletcount over 48 hours (time effect) Multiple comparisonsof mean platelet count 8 hours after admission with meanplatelet count at the 16 24 32 40 and 48 hours after admis-sion for interventional and control group were performedMultiple paired t-test was conducted to demonstrate if thereis any significant different in mean platelet count for eachcomparison Hence Bonferroni correction was applied toreduce the possibility of rejecting a true null hypotheses(commit a type 1 error) Therefore Bonferroni correctionwas used to adjust the level of significance to 119875 lt 001(005number of comparison = 0055 = 001) The efficacyof treatment of CPLJ (treatment effect) and treatment overtime (time-treatment interaction) in increasing the meanplatelet count was analysed by comparing mean differencein platelet count between interventional and control groupAll the statistical analyses were done using PASW 180 (SPSSInc Chicago USA)The comparative CT methodwas used todetermine the relative quantification of the genes expressed

3 Results

A total of 145 patients were recruited into the interventionalgroup while 145 patients were recruited into the control

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Demographic characteristics and baseline biochemistry investigation of respondents by treatment

Demographic characteristics Interventional group119899 ()

Control group119899 () 120594

2 (df)lowast 119875 value

GenderMen 91 (469) 103 (531) 1644 (1) 0200Women 20 (588) 14 (412)

EthnicityMalay 77 (481) 83 (519)Chinese 4 (571) 3 (429)Indian 11 (500) 11 (500)Others 19 (487) 20 (513)

Age (meanSD) 304 (103)dagger 264 (73)dagger minus3370daggerdagger 0001Type of dengue fever

Classical dengue fever 42 (532) 37 (468) 0971 (1) 0324Dengue haemorrhagic fever 69 (463) 80 (537)

Baseline Haematology investigationPlatelet count (times103120583L) 664 (228)dagger 690 (226)dagger 0847daggerdagger 0398Total white blood cell (times103120583L) 34 (17)dagger 33 (16) minus0297daggerdagger 0766Haemoglobin (g) 139 (15)dagger 142 (14)dagger 1595daggerdagger 0112Haematocrit () 415 (42)dagger 429 (60)dagger 1878daggerdagger 0062Lymphocytes () 418 (142)dagger 409 (148)dagger minus0459daggerdagger 0646Neutrophils () 421 (177)dagger 421 (194)dagger 0017daggerdagger 0987

lowastPearson Chi-Square Df (degree of freedom) daggermean and standard deviation daggerdagger119905-value for independent samples 119879-test

Table 2 Comparison of platelet count in each group based on time

Type of laboratory investigation Interventional group (119899 = 111) Control group (119899 = 117)Platelet count Mean difference (95 CI) 119905 119875 value Mean difference (95 CI) 119905 119875 value8ndash16 hours 0993 (minus1660 3645) 0740 0460 minus1411 (minus3961 1140) minus1094 02768ndash24 hours minus0432 (minus4422 3558) minus0214 0831 2213 (minus0523 4948) 1600 01128ndash32 hours minus2716 (minus7540 2107) minus1114 0267 2775 (minus0796 6347) 1537 01278ndash40 hours minus7890 (minus14472 minus1310) minus2374 0019 0867 (minus3472 5207) 0395 06938ndash48 hours minus16764 (minus24566 minus8964) minus4256 lt0001 minus7703 (minus14055 1351) minus2399 0018Repeated measure ANCOVA within group analysis was applied followed by multiple paired 119905-testslowastBonferroni correction was applied by correcting the level of significance (0055 = 001) Potential covariate (age) was controlled by using repeated measuresANCOVA (ANCOVA controlled for age)

group At the end of the study 111 patients from the interven-tional group and 117 controls were included in the statisticalanalysis Sixty-two patients were excluded from the analysisas 38 patients were lost to followup and 24 patients hadincomplete data (missing results due to sample rejection)

Table 1 shows demographic characteristics and baselinebiochemistry investigation of respondents by treatment Interms of dengue status all patients recruited had eitherdengue NS1 or IgM or both detected while the percentagedistribution of the dengue serotypes among them was DEN1(304) DEN2 (284) DEN 3 (206) andDEN 4 (206)Hence all serotypes were well represented in the study

Table 2 presents the multiple comparisons of meanplatelet count 8 hours after admission with mean platelet

count at 16 24 32 40 and 48 hours after admission forinterventional and control group Multiple paired t-test wasconducted to demonstrate if there was any significant dif-ference in mean platelet count for each comparison HenceBonferroni correction was applied to reduce the possibility ofrejecting a true null hypothesis (committing a type 1 error)Based on the number of patients recruitedwith complete data(111 patients from the intervention group and 117 control)the power of study was 870 (standard deviation of plateletcount of 40000 type I error probability of 001 and thetrue difference in mean platelet count of 20000 betweenthe intervention and control group) Overall there was asignificant increase in mean platelet count over 40 hours inboth groups (Wilkrsquos Lambda = 0939 119875 = 0015 effect size

Evidence-Based Complementary and Alternative Medicine 5

646644

625

644 631

608623

586

708

607

796

694

50

55

60

65

70

75

80

85

90

Experimental control (hours)

Mea

n pl

atel

et co

unt

8 16 24 32 40 48

Figure 1 Comparison of mean platelet count between intervention and control group based on time (time-treatment effect)

Table 3 Comparison of platelets count between experiment andcontrol group (treatment effect regardless of time)

Type of lab Investigation Mean difference (95 CI) 119875 valuePlatelet count 2349 (minus5151 9850) 0538Repeated measure ANCOVA between group analyses was applied Level ofsignificance was set at 005 (two-tailed)

= 006 and power = 840) after adjusting for age Furtheranalysis by using multiple paired t-test on each of the groupsshowed that there was a significant increase in mean plateletcount at 40 hours compared to 8 hours after intervention inthe intervention group (119905 = minus4256 119875 le 0001) but not inthe control group (119905 = minus2399 119875 = 0018) after adjustment ofBonferroni correction (119875 = 0055 = 001)

Table 3 presents the overall efficacy of CPLJ supplementa-tion by comparing the mean platelet count of interventionaland control group regardless the time period Study on thetreatment effect of CPLJ on platelet count regardless of timedid not show any significant difference inmean platelet countbetween intervention and control group (119865 = 1128 119875 =0289) However analysis of the effect of CPLJ over the studyperiod (time and treatment effect) showed that there was asignificant interaction between treatment groups and time(Wilkrsquos Lambda = 0934 effect size = 006 and power =870)

Figure 1 shows the time-treatment effect of CPLJ Theintervention group had a significantly higher mean plateletcount than control group at 40 hours and 48 hours ofintervention

Data from the gene expression study were analyzed usingthe ldquoPCR Array Data Analysis versus 33rdquo software recom-mended by Applied Biosystems and showed that ALOX12(ΔCT mean = 1602 FC = 1500) and PTAFR genes (ΔCTmean= 1487 FC= 1342)were found to be highly expressed inthe intervention group when compared to the control group

4 Discussion

The study conducted shows that there is a rationale behindthe use of CPLJ in the treatment of some of DF and DHFIt is definitely worth investigating this plant for its potentialmedicinal benefits With rapid urbanization and global travelleading to drastic demographic changes dengue is a threatto almost 40 of the worldrsquos population There is still nospecific treatment for dengue Previous attempts to identify apotential antiviral for the treatment of dengue has been facedwith several challenges such as the presence of four distinctviral serotypes which frequently undergo mutations findingan appropriate model for infection and protective action ofa given drug as well as yield interesting therapeutic avenuesfor tailored response modifier drugs The current availablemouse model (AG129) available has its limitations such aslow viral load and a short period of viraemia The journeyto drug discovery through the study of immune-modulatoryeffects against dengue infection lies on the research of genericcompounds and natural products [17]

Research groups around Asia have attempted to studythe efficacy of CPLJ in rapidly increasing platelet counts inDF as well as DHF induced thrombocytopenia but therehas been no conclusive evidence drawn from those studiesDengue is generally a self-limiting disease and the diseaseinduced thrombocytopenia usually reverses itself after takinga slight dip during the phase of defervescence However asignificant number of patients succumb to the disease duringthe thrombocytopenic period Many mechanisms come intoplay during the critical phase of the disease to help reversethe disease state at this point Animal studies in elucidatingsafety data have been conducted on normal Sprague Dawleyrats using freeze dried CPLJ however no significant increasein platelet count was observed among the rats given thejuice and the rats kept as control [10] This was probablydue to the fact that the juice was freeze dried and certainessential compounds could have been lost during the processof freeze drying or perhaps the right disease model was not

6 Evidence-Based Complementary and Alternative Medicine

used for the study Haematocrit level which is an importantparameter which is usually monitored to determine the rateof improvement in haemoconcentration was found to besignificantly reduced in both groups of people White Bloodcell count which is found to be reduced in viral infections wasalso found to increase in both groups

The RNA was extracted from the blood of the patientsrecruited and gene expression of two genes namely theALOX 12 and the PTAFR which were conducted so farThere was a 15-fold increase in the ALOX 12 gene activityamong the patients in the experimental group as comparedto those in the control group at the end of the 3 days ALOX12 is known to be associated with increased megakaryocyteproduction as well as its conversion to platelets through 12-HETE mediated pathway which in turn leads to increasedplatelet production A study was conducted at the RoyalCollege of Surgeons Ireland to determine the platelet specificgenes The Alox 12 gene was highly expressed in platelets andfound to be a platelet specific gene byMcRedmond et al [18]A study conducted in Temple University School of MedicinePhiladelphia provided evidence that ALOX12 is a directtarget of transcription factor RUNX1 in megakaryocytes andplatelets RUNX1 is a transcription factor that regulates theexpression of haemopoietic-specific genes When there isRUNX1 haplodeficiency it affects overall haemopoiesis andhence ALOX 12 expression in platelets is decreased Therewas also an agonist-induced decreased 12-HETE productionin platelets with the decrease in ALOX 12 expression Thisprovides further evidence that platelet production is associ-ated with ALOX 12 expression [13]

This finding supports the claim that the juice consump-tion during the course of dengue infection has the potentialto induce the rapid production of platelets This was clearlydemonstrated by the significant increase in the mean plateletcount after 40 hours and 48 hours of juice consumptionThe PTAFR gene which is known to be responsible forincreased platelet production and aggregation was expressed1342-folds among the patients who consumed the juice ascompared to the control group indicating that the juicehad played an important role in addressing the arresting ofbleeding tendencies among these patients A study conductedin Brazil showed that injection of Platelet Activating Factor(PAFPTAFR) in mice induced an increase in platelet countHowever after a certain level further administration of PAFfailed to induce platelet production indicating autosensitiza-tion These findings show that PAFPTAFR can induce therelease of platelets which may be relevant to thrombocytosis[19] We are currently investigating many other genes todetermine other roles of theCPLJ other than its role in plateletproduction and activation

As all plants C papaya leaves are rich in compounds ofdifferent properties Further studies need to be conductedbefore determining the inflammatory pathways affected bythe juice unopposed However it can be concluded thatthe administration of CPLJ in DF and DHF is safe anddoes induce the rapid increase in platelet count It mayplay a valuable role in the management of DF in the nearfuture

Conflict of Interests

Theresearchwas conducted in the absence of any commercialor financial relationships that could be construed as a poten-tial conflict of interests

Acknowledgments

The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and the Director of theInstitute forMedical Research for giving them the permissionto publish this paper This research was funded by theMinistry ofHealthMalaysia (project code JPP-IMR09-003)They would like to thank Dr Nor Asiah biostatistician ofIMR for reviewing their statistical analysis the Director ofHospital Tengku Ampuan Rahimah for allowing them touse the hospital facilities and the staff of The Departmentof Medicine and the Department of Pathology for theirguidance support and cooperation during the study

References

[1] I H BurkillADictionary of the Economic Products of theMalayPeninsula vol 1 The Crown Agents for the Colonies LondonUK 1935

[2] K Arumuganathan and E D Earle ldquoNuclear DNA contentof some important plant speciesrdquo Plant Molecular BiologyReporter vol 9 no 3 pp 208ndash218 1991

[3] FAOSTATPapaya marketing-general information GFruit Website 2012 httpwwwitfnetorggfruitTemplates20Englishpapayamarketinfohtm

[4] V N Villegas ldquoCarica papaya Lrdquo in Plant Resources of South-East Asia 2 Edible Fruits and Nuts E M W Verheij and R ECoronel Eds vol 2 pp 223ndash225 PROSEA Wageningen TheNetherlands 1997

[5] H Y Nakasone and R E Paull Tropical Fruits CAB Interna-tional Walllingford NY USA 1998

[6] B V Owoyele O M Adebukola A A Funmilayo and A OSoladoye ldquoAnti-inflammatory activities of ethanolic extract ofCarica papaya leavesrdquo Inflammopharmacology vol 16 no 4 pp168ndash173 2008

[7] S Gurung and N Skalko-Basnet ldquoWound healing propertiesof Carica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[8] N Otsuki N H Dang E Kumagai A Kondo S Iwata andCMorimoto ldquoAqueous extract of Carica papaya leaves exhibitsanti-tumor activity and immunomodulatory effectsrdquo Journal ofEthnopharmacology vol 127 no 3 pp 760ndash767 2010

[9] N A Imaga G O Gbenle V I Okochi et al ldquoPhytochemicaland antioxidant nutrient constituents of Carica papaya andparquetina nigrescens extractsrdquo Scientific Research and Essaysvol 5 no 16 pp 2201ndash2205 2010

[10] S Z Halim N R Abdullah Z Afzan B A Abdul RashidI Jantan and Z Ismail ldquoAcute toxicity of Carica papaya leafextract in Sprague Dawley ratsrdquo Journal of Medicinal PlantsResearch vol 5 no 10 pp 1867ndash1872 2011

[11] Ministry of Health of Malaysia Vector borne disease controlunit report 2010

[12] World Health Organization Clinical diagnosis 16 2011

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

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Behavioural Neurology

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Volume 2014

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Disease Markers

BioMed Research International

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Oxidative Medicine and Cellular Longevity

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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 616737 7 pageshttpdxdoiorg1011552013616737

Research ArticleCarica papaya Leaves Juice Significantly Acceleratesthe Rate of Increase in Platelet Count among Patients withDengue Fever and Dengue Haemorrhagic Fever

Soobitha Subenthiran1 Tan Chwee Choon2 Kee Chee Cheong3

Ravindran Thayan4 Mok Boon Teck1 Prem Kumar Muniandy1

Adlin Afzan1 Noor Rain Abdullah1 and Zakiah Ismail1

1 Bioassay Unit Herbal Medicine Research Center Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia2 Department of Internal Medicine Tengku Ampuan Rahimah Hospital Jalan Langat 41200 Klang Malaysia3 Epidemiology and Biostatistics Unit Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia4Virology Unit Infectious Disease Research Center Institute for Medical Research Jalan Pahang 50588 Kuala Lumpur Malaysia

Correspondence should be addressed to Soobitha Subenthiran drsoobihotmailcom

Received 7 January 2013 Revised 21 March 2013 Accepted 21 March 2013

Academic Editor Martin Kohlmeier

Copyright copy 2013 Soobitha Subenthiran et al This 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 is properlycited

The study was conducted to investigate the platelet increasing property ofCarica papaya leaves juice (CPLJ) in patients with denguefever (DF) An open labeled randomized controlled trial was carried out on 228 patients with DF and dengue haemorrhagic fever(DHF) Approximately half the patients received the juice for 3 consecutive days while the others remained as controls and receivedthe standard management Their full blood count was monitored 8 hours for 48 hours Gene expression studies were conductedon the ALOX 12 and PTAFR genes The mean increase in platelet counts were compared in both groups using repeated measureANCOVA There was a significant increase in mean platelet count observed in the intervention group (119875 lt 0001) but not inthe control group 40 hours since the first dose of CPLJ Comparison of mean platelet count between intervention and controlgroup showed that mean platelet count in intervention group was significantly higher than control group after 40 and 48 hours ofadmission (119875 lt 001) The ALOX 12 (FC = 1500) and PTAFR (FC = 1342) genes were highly expressed among those on the juiceIt was concluded that CPLJ does significantly increase the platelet count in patients with DF and DHF

1 Introduction

Malaysia is blessed with 12000 species of flowering plantsof which 1300 have medicinal properties [1] There is arapidly growing response to the use of medicinal plants bythe Malaysian population WHO estimates that in manycountries 80 of the rural patients seek alternative treatmentusing medicinal plants

Carica papaya is a member of the Caricaceae and isa dicotyledonous polygamous and diploid species [2] Itoriginated from Southern Mexico Central America and thenorthern part of South America It is now cultivated inmany tropical countries such as Bangladesh India IndonesiaSri Lanka the Philippines and the West Indies including

Malaysia Malaysia is known to be one of the top 5 papayaexporting countries [3] The papaya fruit is globally con-sumed either in its fresh form or the form of juices jamsand crystallized dry fruit [4] The ripe fruit is said to bea rich source of vitamin A C and calcium [5] There aremany commercial products derived from the different partsof the C papaya plant the most prominent being papain andchymopapain which is produced from the latex of the youngfruit stem and the leaves C papaya leaves have been usedin folk medicine for centuries Recent studies have shownits beneficial effect as an anti-inflammatory agent [6] for itswound healing properties [7] antitumour as well as immune-modulatory effects [8] and as an antioxidant [9] A toxicitystudy (acute subacute and chronic toxicity) conducted on

2 Evidence-Based Complementary and Alternative Medicine

Sprague Dawley rats administered with Carica papaya leavesjuice (CPLJ) of the sekaki variant revealed that it was safe fororal consumption [10]

Dengue is an arthropod-borne viral disease carried byAedes aegypti as the vector caused by 4 possible viralserotypes namely serotype 1 2 3 and 4 of the Flaviviridaefamily In Malaysia dengue cases have been on the rise since2002 Total of 18371 cases of dengue fever (DF) and denguehaemorrhagic fever (DHF) were reported last year and hadclaimed 33 lives in the same year [11] There is no specificantiviral drug available for the treatment of dengue infectionInfected patients receive supportive management with fluidsblood and blood products complying to the Ministry ofHealth Clinical Practice Guidelines (CPGs) on Managementof Dengue 2010 Each episode of infection is known toinduce a life-long protective immunity to the homologousserotype but confers only partial and transient protectionagainst subsequent infection by the other serotypes Sec-ondary infection is a major risk factor for DHF possiblydue to antibody-dependent enhancement A patient withdengue fever presents typically with fever headache and rashknown as the dengue triadThere are many other nonspecificsigns and symptoms associated with DF and patient canprogress to DHF and typically manifests as abdominal painbleeding and even circulatory collapse The clinical courseof dengue has an abrupt onset followed by three phasesnamely the febrile phase the critical phase and the recoveryphase It is during the critical phase that thrombocytopaeniacharacterized by a decrease in platelet count below 100000 permm3 from the baseline and haemoconcentrationcharacterized by an increase of haematocrit by 20 or moreis detectable before the subsidence of fever and the onset ofshock [12]

Certain genes have been shown to influence plateletproduction and platelet aggregation namely the Arachido-nate 12-lipoxygenase (ALOX 12) also known as the Platelet-type Lipoxygenase as well as the Platelet-Activating FactorReceptor (PTAFR) An increase in activity of these genes isrequired for platelet production and activationThe ALOX 12gene is strongly expressed in megakaryocytes and has beenknown to be responsible for the 12-Hydroxyeicosatetraenoicacid (12-HETE) production of platelets [13]The PTAFR genewas been found to be expressed inmegakaryocytes indicatingthat it could be a precursor for platelet production in additionto its well known role in platelet aggregation [14]

Safety studies based on OECD guidelines for acutesubacute and chronic toxicity were conducted on C papayaextract and showed that it was found to be safe for humanconsumption [10] The present study was conducted todetermine and investigate the traditional claim that CPLJincreases the platelet count in patients with DF and DHF

2 Materials and Method

21 Plant Material and Sample Preparation C papaya leavesof the the sekaki variant were chosen for the study basedon fingerprinting and safety analysis which also containedallowable limit of heavy metals and microbial content For

the purpose of the study a private plantation certified by theMinistry of Agriculture in Semenyih Selangor was identifiedto provide the leaves for the entire duration of the studyto ensure similar source of authenticated raw material usedThe trees in this plantation were kept free of herbicidespesticides and insecticides Juice was prepared fresh from theleaves that were washed thoroughly with an organic vegetablecleaning agent and reverse osmosis water few times Juice wasextracted from 50 grams of fresh leaves using a juice extractorwithout any addition of water under sterile conditions Thefresh juice was aliquoted at a volume of approximately 30mLsin sterile glass vials and transported daily in an icebox andkept at a temperature of below 4∘C to the study site at themale and female dengue wards of Hospital Tengku AmpuanRahimah Klang Selangor

The juice was characterized and standardized using aHigh Performance Liquid Chromatography Diode ArrayDetector according to three markers manghaslin clitorinand rutin The chemical fingerprinting of the leaves wasconsistent throughout the study

22 Subjects An open labeled randomized controlled trialwas conducted to provide clinical data in support of theproposed claimsThe number of patients involved was deter-mined based on the following calculation

With plan to have a continuous response variable fromindependent control and experimental subjects with 1 con-trol(s) per experimental subject expecting the platelet countto increase by 20000 after administration of CPLJ for threeconsecutive days setting the standard deviation for plateletcount at 40000 in a normal population [15] type I error(alpha) = 001 and power of study of 90 the sample sizecalculated by using PS software [16] was 242 with each armof 121 intervention group and control group Anticipatingdropout rate to be 20 the final sample sizewas 290 (145 eachfor intervention and control group)

All the patients meeting the inclusion criteria wererecruited from theDengueWard ofHospital TengkuAmpuanRahimah Klang Selangor Malaysia Subjects were randomlyassigned to either intervention or control group using theblock of 10 method This method was used to ensure that anequal number of participants were allocated to interventionaland control groups Therefore with the block size of tenof every ten consecutively enrolled participants five will beallocated to one interventional group and five to the controlgroup For the randomization 10 identical opaque envelopeswith five envelopes each contained a card labelled as ldquointer-ventional grouprdquo and other 5 envelopes each contained a cardlabelled as ldquocontrol grouprdquo These envelopes were given tothe field investigator during the patient enrolment The fieldinvestigator then blindly selected an envelope for each patientthus assigning the patient to interventional or control groupsThis procedure was repeated until the targeted sample sizewas achieved

The inclusion and exclusion criteria were used to selectpatients who volunteered to be enrolled into the studyInclusion criteria were (a) male and female patients above18 years and below 60 years old (b) all patients who could

Evidence-Based Complementary and Alternative Medicine 3

understand or read Bahasa Malaysia or English irrespectiveof race (c) patients who were confirmed to have DF or DHFgrade I and II (d) patients with a platelet count of less thanor equal to 100000120583L (e) patients with a baseline alaninetransaminase (ALT) level of not more than 3 times of theupper limit of the normal range (not more than 165UL) and(f) patients with a creatinine kinase (CK) value of less than500UL

The exclusion criteria were (a) dengue hemorrhagic fevergrade III and IV (b) pregnant or lactatingwomen (c) patientswho have received blood or blood products transfusionduring the current hospital stay (d) patients with underlyingcomorbids (e) patients who developed Hepatitis with aserum ALT level 3 times higher than the upper limit of thenormal range (gt165UL) and (f) patients with a creatininekinase (CK) value of more than 500UL

The study was conducted in accordance with the ethicalprinciples as outlined in the declaration of Helsinki October2008 following approval from the Medical Review andEthical Committee (MREC) Ministry of Health Malaysiaand was consistent with Good Clinical Practice (GCP) andapplicable regulatory requirements The National MedicalResearch Registry number is NMRR-09-883-4768 Writteninformed consent was obtained from every volunteer priorto clinical trial participation

23 Diagnosis of DF or DHF and Dengue Serotyping Aclinical diagnosis of DF and DHF was made by the clinicianbased on patientsrsquo presentation and blood investigations Inaddition a rapid dengue bedside test (SD Dengue Duo NS1Ag + Ab Combo Standard Diagnostics Korea) was usedto determine dengue status before the subjects were giventhe Carica papaya leave juice This test is able to detect thepresence of NS1 antigen or dengue IgM or IgG antibodiesHowever for ease of interpretation of dengue status onlypatients who had either NS1 or IgM or both detected wereincluded in the studyThe detection of theNS1 antigen or IgMantibodies within the first five to 7 days of onset of symptomsusually indicates a current dengue infection Subsequently allthe samples were subjected tomultiplex real time RT-PCR fordetermination of dengue serotypes

24 Treatment of the Subjects Once a current dengue infec-tion was confirmed a thorough screening of the patientwas conducted Baseline investigations included full bloodcount bleeding profile renal as well as liver function testand cardiac enzymes Patients in the intervention groupreceived fresh juice from 50 grams of C papaya leavesonce daily 15 minutes after breakfast for 3 consecutive dayswhile receiving the standardmanagement as per the NationalClinical Practice Guidelines for the Management of DengueThe controls received the standard management

25 Study Parameters Full blood count was monitored 8hours for the first 48 hours during the study to deter-mine the changes in platelet count and haematocrit levelswhile bleeding profile renal profile and liver function testwere monitored daily to ensure the safety of the juice

The haematological and biochemical tests were conductedby the Pathology Department at Hospital Tengku AmpuanRahimah Klang and the validated results were later tracedand recorded Mean and standard deviation of the baselinehaematological and biochemical parameters were then calcu-lated as shown in Table 1

Blood for RNA extractionwas taken on day 3 after the lastdose of the juice to conduct expression studies on the PTAFRand the ALOX 12 genes RNALater by Ambion (United Statesof America) was added to the whole blood in an EDTA tubeto prevent degradation of RNA

The RNA extraction was carried out at the Institute forMedical research using an RNA extraction kit by AmbionThe RNA concentration and purity were determined usingtheNanodrop 2000 Spectrophotometer byThermo ScientificTheRNAwas then converted to cDNAusing theHighCapac-ity RNA to cDNA kit by Applied Biosystems The cDNAconcentrationwas then determinedusing theNanodrop 2000Spectrophotometer byThermo Scientific

Gene expression was determined using gene expressionassays by Applied Biosystems on the ABI 7500 Fast Systemon 12 RNA samples from the experimental group and 12 RNAsamples from the control group Specific predesigned MGBprobes were used for ALOX 12 and the PTAFR gene The18S Ribosome was used as an endogenous control A 10 120583Lreaction volume was used using the TaqMan Fast UniversalPCR Mastermix (2X) The probe product ID and sequenceused were ALOX 12 (Hs 00167524 M1 NM 000697251015840-FAM-ATTGCCATCCAGCTCAACAAATCC-MGB-31015840)and PTAFR (Hs 00265399 S1 NM 0011647231 51015840-FAM-GCCCGTAATTTATCGCGCTTACTAT MGB-21015840)

26 Statistical Analysis Repeated measure ANCOVA wasused to determine the effect of CPLJ on the mean plateletcount over 48 hours (time effect) Multiple comparisonsof mean platelet count 8 hours after admission with meanplatelet count at the 16 24 32 40 and 48 hours after admis-sion for interventional and control group were performedMultiple paired t-test was conducted to demonstrate if thereis any significant different in mean platelet count for eachcomparison Hence Bonferroni correction was applied toreduce the possibility of rejecting a true null hypotheses(commit a type 1 error) Therefore Bonferroni correctionwas used to adjust the level of significance to 119875 lt 001(005number of comparison = 0055 = 001) The efficacyof treatment of CPLJ (treatment effect) and treatment overtime (time-treatment interaction) in increasing the meanplatelet count was analysed by comparing mean differencein platelet count between interventional and control groupAll the statistical analyses were done using PASW 180 (SPSSInc Chicago USA)The comparative CT methodwas used todetermine the relative quantification of the genes expressed

3 Results

A total of 145 patients were recruited into the interventionalgroup while 145 patients were recruited into the control

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Demographic characteristics and baseline biochemistry investigation of respondents by treatment

Demographic characteristics Interventional group119899 ()

Control group119899 () 120594

2 (df)lowast 119875 value

GenderMen 91 (469) 103 (531) 1644 (1) 0200Women 20 (588) 14 (412)

EthnicityMalay 77 (481) 83 (519)Chinese 4 (571) 3 (429)Indian 11 (500) 11 (500)Others 19 (487) 20 (513)

Age (meanSD) 304 (103)dagger 264 (73)dagger minus3370daggerdagger 0001Type of dengue fever

Classical dengue fever 42 (532) 37 (468) 0971 (1) 0324Dengue haemorrhagic fever 69 (463) 80 (537)

Baseline Haematology investigationPlatelet count (times103120583L) 664 (228)dagger 690 (226)dagger 0847daggerdagger 0398Total white blood cell (times103120583L) 34 (17)dagger 33 (16) minus0297daggerdagger 0766Haemoglobin (g) 139 (15)dagger 142 (14)dagger 1595daggerdagger 0112Haematocrit () 415 (42)dagger 429 (60)dagger 1878daggerdagger 0062Lymphocytes () 418 (142)dagger 409 (148)dagger minus0459daggerdagger 0646Neutrophils () 421 (177)dagger 421 (194)dagger 0017daggerdagger 0987

lowastPearson Chi-Square Df (degree of freedom) daggermean and standard deviation daggerdagger119905-value for independent samples 119879-test

Table 2 Comparison of platelet count in each group based on time

Type of laboratory investigation Interventional group (119899 = 111) Control group (119899 = 117)Platelet count Mean difference (95 CI) 119905 119875 value Mean difference (95 CI) 119905 119875 value8ndash16 hours 0993 (minus1660 3645) 0740 0460 minus1411 (minus3961 1140) minus1094 02768ndash24 hours minus0432 (minus4422 3558) minus0214 0831 2213 (minus0523 4948) 1600 01128ndash32 hours minus2716 (minus7540 2107) minus1114 0267 2775 (minus0796 6347) 1537 01278ndash40 hours minus7890 (minus14472 minus1310) minus2374 0019 0867 (minus3472 5207) 0395 06938ndash48 hours minus16764 (minus24566 minus8964) minus4256 lt0001 minus7703 (minus14055 1351) minus2399 0018Repeated measure ANCOVA within group analysis was applied followed by multiple paired 119905-testslowastBonferroni correction was applied by correcting the level of significance (0055 = 001) Potential covariate (age) was controlled by using repeated measuresANCOVA (ANCOVA controlled for age)

group At the end of the study 111 patients from the interven-tional group and 117 controls were included in the statisticalanalysis Sixty-two patients were excluded from the analysisas 38 patients were lost to followup and 24 patients hadincomplete data (missing results due to sample rejection)

Table 1 shows demographic characteristics and baselinebiochemistry investigation of respondents by treatment Interms of dengue status all patients recruited had eitherdengue NS1 or IgM or both detected while the percentagedistribution of the dengue serotypes among them was DEN1(304) DEN2 (284) DEN 3 (206) andDEN 4 (206)Hence all serotypes were well represented in the study

Table 2 presents the multiple comparisons of meanplatelet count 8 hours after admission with mean platelet

count at 16 24 32 40 and 48 hours after admission forinterventional and control group Multiple paired t-test wasconducted to demonstrate if there was any significant dif-ference in mean platelet count for each comparison HenceBonferroni correction was applied to reduce the possibility ofrejecting a true null hypothesis (committing a type 1 error)Based on the number of patients recruitedwith complete data(111 patients from the intervention group and 117 control)the power of study was 870 (standard deviation of plateletcount of 40000 type I error probability of 001 and thetrue difference in mean platelet count of 20000 betweenthe intervention and control group) Overall there was asignificant increase in mean platelet count over 40 hours inboth groups (Wilkrsquos Lambda = 0939 119875 = 0015 effect size

Evidence-Based Complementary and Alternative Medicine 5

646644

625

644 631

608623

586

708

607

796

694

50

55

60

65

70

75

80

85

90

Experimental control (hours)

Mea

n pl

atel

et co

unt

8 16 24 32 40 48

Figure 1 Comparison of mean platelet count between intervention and control group based on time (time-treatment effect)

Table 3 Comparison of platelets count between experiment andcontrol group (treatment effect regardless of time)

Type of lab Investigation Mean difference (95 CI) 119875 valuePlatelet count 2349 (minus5151 9850) 0538Repeated measure ANCOVA between group analyses was applied Level ofsignificance was set at 005 (two-tailed)

= 006 and power = 840) after adjusting for age Furtheranalysis by using multiple paired t-test on each of the groupsshowed that there was a significant increase in mean plateletcount at 40 hours compared to 8 hours after intervention inthe intervention group (119905 = minus4256 119875 le 0001) but not inthe control group (119905 = minus2399 119875 = 0018) after adjustment ofBonferroni correction (119875 = 0055 = 001)

Table 3 presents the overall efficacy of CPLJ supplementa-tion by comparing the mean platelet count of interventionaland control group regardless the time period Study on thetreatment effect of CPLJ on platelet count regardless of timedid not show any significant difference inmean platelet countbetween intervention and control group (119865 = 1128 119875 =0289) However analysis of the effect of CPLJ over the studyperiod (time and treatment effect) showed that there was asignificant interaction between treatment groups and time(Wilkrsquos Lambda = 0934 effect size = 006 and power =870)

Figure 1 shows the time-treatment effect of CPLJ Theintervention group had a significantly higher mean plateletcount than control group at 40 hours and 48 hours ofintervention

Data from the gene expression study were analyzed usingthe ldquoPCR Array Data Analysis versus 33rdquo software recom-mended by Applied Biosystems and showed that ALOX12(ΔCT mean = 1602 FC = 1500) and PTAFR genes (ΔCTmean= 1487 FC= 1342)were found to be highly expressed inthe intervention group when compared to the control group

4 Discussion

The study conducted shows that there is a rationale behindthe use of CPLJ in the treatment of some of DF and DHFIt is definitely worth investigating this plant for its potentialmedicinal benefits With rapid urbanization and global travelleading to drastic demographic changes dengue is a threatto almost 40 of the worldrsquos population There is still nospecific treatment for dengue Previous attempts to identify apotential antiviral for the treatment of dengue has been facedwith several challenges such as the presence of four distinctviral serotypes which frequently undergo mutations findingan appropriate model for infection and protective action ofa given drug as well as yield interesting therapeutic avenuesfor tailored response modifier drugs The current availablemouse model (AG129) available has its limitations such aslow viral load and a short period of viraemia The journeyto drug discovery through the study of immune-modulatoryeffects against dengue infection lies on the research of genericcompounds and natural products [17]

Research groups around Asia have attempted to studythe efficacy of CPLJ in rapidly increasing platelet counts inDF as well as DHF induced thrombocytopenia but therehas been no conclusive evidence drawn from those studiesDengue is generally a self-limiting disease and the diseaseinduced thrombocytopenia usually reverses itself after takinga slight dip during the phase of defervescence However asignificant number of patients succumb to the disease duringthe thrombocytopenic period Many mechanisms come intoplay during the critical phase of the disease to help reversethe disease state at this point Animal studies in elucidatingsafety data have been conducted on normal Sprague Dawleyrats using freeze dried CPLJ however no significant increasein platelet count was observed among the rats given thejuice and the rats kept as control [10] This was probablydue to the fact that the juice was freeze dried and certainessential compounds could have been lost during the processof freeze drying or perhaps the right disease model was not

6 Evidence-Based Complementary and Alternative Medicine

used for the study Haematocrit level which is an importantparameter which is usually monitored to determine the rateof improvement in haemoconcentration was found to besignificantly reduced in both groups of people White Bloodcell count which is found to be reduced in viral infections wasalso found to increase in both groups

The RNA was extracted from the blood of the patientsrecruited and gene expression of two genes namely theALOX 12 and the PTAFR which were conducted so farThere was a 15-fold increase in the ALOX 12 gene activityamong the patients in the experimental group as comparedto those in the control group at the end of the 3 days ALOX12 is known to be associated with increased megakaryocyteproduction as well as its conversion to platelets through 12-HETE mediated pathway which in turn leads to increasedplatelet production A study was conducted at the RoyalCollege of Surgeons Ireland to determine the platelet specificgenes The Alox 12 gene was highly expressed in platelets andfound to be a platelet specific gene byMcRedmond et al [18]A study conducted in Temple University School of MedicinePhiladelphia provided evidence that ALOX12 is a directtarget of transcription factor RUNX1 in megakaryocytes andplatelets RUNX1 is a transcription factor that regulates theexpression of haemopoietic-specific genes When there isRUNX1 haplodeficiency it affects overall haemopoiesis andhence ALOX 12 expression in platelets is decreased Therewas also an agonist-induced decreased 12-HETE productionin platelets with the decrease in ALOX 12 expression Thisprovides further evidence that platelet production is associ-ated with ALOX 12 expression [13]

This finding supports the claim that the juice consump-tion during the course of dengue infection has the potentialto induce the rapid production of platelets This was clearlydemonstrated by the significant increase in the mean plateletcount after 40 hours and 48 hours of juice consumptionThe PTAFR gene which is known to be responsible forincreased platelet production and aggregation was expressed1342-folds among the patients who consumed the juice ascompared to the control group indicating that the juicehad played an important role in addressing the arresting ofbleeding tendencies among these patients A study conductedin Brazil showed that injection of Platelet Activating Factor(PAFPTAFR) in mice induced an increase in platelet countHowever after a certain level further administration of PAFfailed to induce platelet production indicating autosensitiza-tion These findings show that PAFPTAFR can induce therelease of platelets which may be relevant to thrombocytosis[19] We are currently investigating many other genes todetermine other roles of theCPLJ other than its role in plateletproduction and activation

As all plants C papaya leaves are rich in compounds ofdifferent properties Further studies need to be conductedbefore determining the inflammatory pathways affected bythe juice unopposed However it can be concluded thatthe administration of CPLJ in DF and DHF is safe anddoes induce the rapid increase in platelet count It mayplay a valuable role in the management of DF in the nearfuture

Conflict of Interests

Theresearchwas conducted in the absence of any commercialor financial relationships that could be construed as a poten-tial conflict of interests

Acknowledgments

The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and the Director of theInstitute forMedical Research for giving them the permissionto publish this paper This research was funded by theMinistry ofHealthMalaysia (project code JPP-IMR09-003)They would like to thank Dr Nor Asiah biostatistician ofIMR for reviewing their statistical analysis the Director ofHospital Tengku Ampuan Rahimah for allowing them touse the hospital facilities and the staff of The Departmentof Medicine and the Department of Pathology for theirguidance support and cooperation during the study

References

[1] I H BurkillADictionary of the Economic Products of theMalayPeninsula vol 1 The Crown Agents for the Colonies LondonUK 1935

[2] K Arumuganathan and E D Earle ldquoNuclear DNA contentof some important plant speciesrdquo Plant Molecular BiologyReporter vol 9 no 3 pp 208ndash218 1991

[3] FAOSTATPapaya marketing-general information GFruit Website 2012 httpwwwitfnetorggfruitTemplates20Englishpapayamarketinfohtm

[4] V N Villegas ldquoCarica papaya Lrdquo in Plant Resources of South-East Asia 2 Edible Fruits and Nuts E M W Verheij and R ECoronel Eds vol 2 pp 223ndash225 PROSEA Wageningen TheNetherlands 1997

[5] H Y Nakasone and R E Paull Tropical Fruits CAB Interna-tional Walllingford NY USA 1998

[6] B V Owoyele O M Adebukola A A Funmilayo and A OSoladoye ldquoAnti-inflammatory activities of ethanolic extract ofCarica papaya leavesrdquo Inflammopharmacology vol 16 no 4 pp168ndash173 2008

[7] S Gurung and N Skalko-Basnet ldquoWound healing propertiesof Carica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[8] N Otsuki N H Dang E Kumagai A Kondo S Iwata andCMorimoto ldquoAqueous extract of Carica papaya leaves exhibitsanti-tumor activity and immunomodulatory effectsrdquo Journal ofEthnopharmacology vol 127 no 3 pp 760ndash767 2010

[9] N A Imaga G O Gbenle V I Okochi et al ldquoPhytochemicaland antioxidant nutrient constituents of Carica papaya andparquetina nigrescens extractsrdquo Scientific Research and Essaysvol 5 no 16 pp 2201ndash2205 2010

[10] S Z Halim N R Abdullah Z Afzan B A Abdul RashidI Jantan and Z Ismail ldquoAcute toxicity of Carica papaya leafextract in Sprague Dawley ratsrdquo Journal of Medicinal PlantsResearch vol 5 no 10 pp 1867ndash1872 2011

[11] Ministry of Health of Malaysia Vector borne disease controlunit report 2010

[12] World Health Organization Clinical diagnosis 16 2011

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

Sprague Dawley rats administered with Carica papaya leavesjuice (CPLJ) of the sekaki variant revealed that it was safe fororal consumption [10]

Dengue is an arthropod-borne viral disease carried byAedes aegypti as the vector caused by 4 possible viralserotypes namely serotype 1 2 3 and 4 of the Flaviviridaefamily In Malaysia dengue cases have been on the rise since2002 Total of 18371 cases of dengue fever (DF) and denguehaemorrhagic fever (DHF) were reported last year and hadclaimed 33 lives in the same year [11] There is no specificantiviral drug available for the treatment of dengue infectionInfected patients receive supportive management with fluidsblood and blood products complying to the Ministry ofHealth Clinical Practice Guidelines (CPGs) on Managementof Dengue 2010 Each episode of infection is known toinduce a life-long protective immunity to the homologousserotype but confers only partial and transient protectionagainst subsequent infection by the other serotypes Sec-ondary infection is a major risk factor for DHF possiblydue to antibody-dependent enhancement A patient withdengue fever presents typically with fever headache and rashknown as the dengue triadThere are many other nonspecificsigns and symptoms associated with DF and patient canprogress to DHF and typically manifests as abdominal painbleeding and even circulatory collapse The clinical courseof dengue has an abrupt onset followed by three phasesnamely the febrile phase the critical phase and the recoveryphase It is during the critical phase that thrombocytopaeniacharacterized by a decrease in platelet count below 100000 permm3 from the baseline and haemoconcentrationcharacterized by an increase of haematocrit by 20 or moreis detectable before the subsidence of fever and the onset ofshock [12]

Certain genes have been shown to influence plateletproduction and platelet aggregation namely the Arachido-nate 12-lipoxygenase (ALOX 12) also known as the Platelet-type Lipoxygenase as well as the Platelet-Activating FactorReceptor (PTAFR) An increase in activity of these genes isrequired for platelet production and activationThe ALOX 12gene is strongly expressed in megakaryocytes and has beenknown to be responsible for the 12-Hydroxyeicosatetraenoicacid (12-HETE) production of platelets [13]The PTAFR genewas been found to be expressed inmegakaryocytes indicatingthat it could be a precursor for platelet production in additionto its well known role in platelet aggregation [14]

Safety studies based on OECD guidelines for acutesubacute and chronic toxicity were conducted on C papayaextract and showed that it was found to be safe for humanconsumption [10] The present study was conducted todetermine and investigate the traditional claim that CPLJincreases the platelet count in patients with DF and DHF

2 Materials and Method

21 Plant Material and Sample Preparation C papaya leavesof the the sekaki variant were chosen for the study basedon fingerprinting and safety analysis which also containedallowable limit of heavy metals and microbial content For

the purpose of the study a private plantation certified by theMinistry of Agriculture in Semenyih Selangor was identifiedto provide the leaves for the entire duration of the studyto ensure similar source of authenticated raw material usedThe trees in this plantation were kept free of herbicidespesticides and insecticides Juice was prepared fresh from theleaves that were washed thoroughly with an organic vegetablecleaning agent and reverse osmosis water few times Juice wasextracted from 50 grams of fresh leaves using a juice extractorwithout any addition of water under sterile conditions Thefresh juice was aliquoted at a volume of approximately 30mLsin sterile glass vials and transported daily in an icebox andkept at a temperature of below 4∘C to the study site at themale and female dengue wards of Hospital Tengku AmpuanRahimah Klang Selangor

The juice was characterized and standardized using aHigh Performance Liquid Chromatography Diode ArrayDetector according to three markers manghaslin clitorinand rutin The chemical fingerprinting of the leaves wasconsistent throughout the study

22 Subjects An open labeled randomized controlled trialwas conducted to provide clinical data in support of theproposed claimsThe number of patients involved was deter-mined based on the following calculation

With plan to have a continuous response variable fromindependent control and experimental subjects with 1 con-trol(s) per experimental subject expecting the platelet countto increase by 20000 after administration of CPLJ for threeconsecutive days setting the standard deviation for plateletcount at 40000 in a normal population [15] type I error(alpha) = 001 and power of study of 90 the sample sizecalculated by using PS software [16] was 242 with each armof 121 intervention group and control group Anticipatingdropout rate to be 20 the final sample sizewas 290 (145 eachfor intervention and control group)

All the patients meeting the inclusion criteria wererecruited from theDengueWard ofHospital TengkuAmpuanRahimah Klang Selangor Malaysia Subjects were randomlyassigned to either intervention or control group using theblock of 10 method This method was used to ensure that anequal number of participants were allocated to interventionaland control groups Therefore with the block size of tenof every ten consecutively enrolled participants five will beallocated to one interventional group and five to the controlgroup For the randomization 10 identical opaque envelopeswith five envelopes each contained a card labelled as ldquointer-ventional grouprdquo and other 5 envelopes each contained a cardlabelled as ldquocontrol grouprdquo These envelopes were given tothe field investigator during the patient enrolment The fieldinvestigator then blindly selected an envelope for each patientthus assigning the patient to interventional or control groupsThis procedure was repeated until the targeted sample sizewas achieved

The inclusion and exclusion criteria were used to selectpatients who volunteered to be enrolled into the studyInclusion criteria were (a) male and female patients above18 years and below 60 years old (b) all patients who could

Evidence-Based Complementary and Alternative Medicine 3

understand or read Bahasa Malaysia or English irrespectiveof race (c) patients who were confirmed to have DF or DHFgrade I and II (d) patients with a platelet count of less thanor equal to 100000120583L (e) patients with a baseline alaninetransaminase (ALT) level of not more than 3 times of theupper limit of the normal range (not more than 165UL) and(f) patients with a creatinine kinase (CK) value of less than500UL

The exclusion criteria were (a) dengue hemorrhagic fevergrade III and IV (b) pregnant or lactatingwomen (c) patientswho have received blood or blood products transfusionduring the current hospital stay (d) patients with underlyingcomorbids (e) patients who developed Hepatitis with aserum ALT level 3 times higher than the upper limit of thenormal range (gt165UL) and (f) patients with a creatininekinase (CK) value of more than 500UL

The study was conducted in accordance with the ethicalprinciples as outlined in the declaration of Helsinki October2008 following approval from the Medical Review andEthical Committee (MREC) Ministry of Health Malaysiaand was consistent with Good Clinical Practice (GCP) andapplicable regulatory requirements The National MedicalResearch Registry number is NMRR-09-883-4768 Writteninformed consent was obtained from every volunteer priorto clinical trial participation

23 Diagnosis of DF or DHF and Dengue Serotyping Aclinical diagnosis of DF and DHF was made by the clinicianbased on patientsrsquo presentation and blood investigations Inaddition a rapid dengue bedside test (SD Dengue Duo NS1Ag + Ab Combo Standard Diagnostics Korea) was usedto determine dengue status before the subjects were giventhe Carica papaya leave juice This test is able to detect thepresence of NS1 antigen or dengue IgM or IgG antibodiesHowever for ease of interpretation of dengue status onlypatients who had either NS1 or IgM or both detected wereincluded in the studyThe detection of theNS1 antigen or IgMantibodies within the first five to 7 days of onset of symptomsusually indicates a current dengue infection Subsequently allthe samples were subjected tomultiplex real time RT-PCR fordetermination of dengue serotypes

24 Treatment of the Subjects Once a current dengue infec-tion was confirmed a thorough screening of the patientwas conducted Baseline investigations included full bloodcount bleeding profile renal as well as liver function testand cardiac enzymes Patients in the intervention groupreceived fresh juice from 50 grams of C papaya leavesonce daily 15 minutes after breakfast for 3 consecutive dayswhile receiving the standardmanagement as per the NationalClinical Practice Guidelines for the Management of DengueThe controls received the standard management

25 Study Parameters Full blood count was monitored 8hours for the first 48 hours during the study to deter-mine the changes in platelet count and haematocrit levelswhile bleeding profile renal profile and liver function testwere monitored daily to ensure the safety of the juice

The haematological and biochemical tests were conductedby the Pathology Department at Hospital Tengku AmpuanRahimah Klang and the validated results were later tracedand recorded Mean and standard deviation of the baselinehaematological and biochemical parameters were then calcu-lated as shown in Table 1

Blood for RNA extractionwas taken on day 3 after the lastdose of the juice to conduct expression studies on the PTAFRand the ALOX 12 genes RNALater by Ambion (United Statesof America) was added to the whole blood in an EDTA tubeto prevent degradation of RNA

The RNA extraction was carried out at the Institute forMedical research using an RNA extraction kit by AmbionThe RNA concentration and purity were determined usingtheNanodrop 2000 Spectrophotometer byThermo ScientificTheRNAwas then converted to cDNAusing theHighCapac-ity RNA to cDNA kit by Applied Biosystems The cDNAconcentrationwas then determinedusing theNanodrop 2000Spectrophotometer byThermo Scientific

Gene expression was determined using gene expressionassays by Applied Biosystems on the ABI 7500 Fast Systemon 12 RNA samples from the experimental group and 12 RNAsamples from the control group Specific predesigned MGBprobes were used for ALOX 12 and the PTAFR gene The18S Ribosome was used as an endogenous control A 10 120583Lreaction volume was used using the TaqMan Fast UniversalPCR Mastermix (2X) The probe product ID and sequenceused were ALOX 12 (Hs 00167524 M1 NM 000697251015840-FAM-ATTGCCATCCAGCTCAACAAATCC-MGB-31015840)and PTAFR (Hs 00265399 S1 NM 0011647231 51015840-FAM-GCCCGTAATTTATCGCGCTTACTAT MGB-21015840)

26 Statistical Analysis Repeated measure ANCOVA wasused to determine the effect of CPLJ on the mean plateletcount over 48 hours (time effect) Multiple comparisonsof mean platelet count 8 hours after admission with meanplatelet count at the 16 24 32 40 and 48 hours after admis-sion for interventional and control group were performedMultiple paired t-test was conducted to demonstrate if thereis any significant different in mean platelet count for eachcomparison Hence Bonferroni correction was applied toreduce the possibility of rejecting a true null hypotheses(commit a type 1 error) Therefore Bonferroni correctionwas used to adjust the level of significance to 119875 lt 001(005number of comparison = 0055 = 001) The efficacyof treatment of CPLJ (treatment effect) and treatment overtime (time-treatment interaction) in increasing the meanplatelet count was analysed by comparing mean differencein platelet count between interventional and control groupAll the statistical analyses were done using PASW 180 (SPSSInc Chicago USA)The comparative CT methodwas used todetermine the relative quantification of the genes expressed

3 Results

A total of 145 patients were recruited into the interventionalgroup while 145 patients were recruited into the control

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Demographic characteristics and baseline biochemistry investigation of respondents by treatment

Demographic characteristics Interventional group119899 ()

Control group119899 () 120594

2 (df)lowast 119875 value

GenderMen 91 (469) 103 (531) 1644 (1) 0200Women 20 (588) 14 (412)

EthnicityMalay 77 (481) 83 (519)Chinese 4 (571) 3 (429)Indian 11 (500) 11 (500)Others 19 (487) 20 (513)

Age (meanSD) 304 (103)dagger 264 (73)dagger minus3370daggerdagger 0001Type of dengue fever

Classical dengue fever 42 (532) 37 (468) 0971 (1) 0324Dengue haemorrhagic fever 69 (463) 80 (537)

Baseline Haematology investigationPlatelet count (times103120583L) 664 (228)dagger 690 (226)dagger 0847daggerdagger 0398Total white blood cell (times103120583L) 34 (17)dagger 33 (16) minus0297daggerdagger 0766Haemoglobin (g) 139 (15)dagger 142 (14)dagger 1595daggerdagger 0112Haematocrit () 415 (42)dagger 429 (60)dagger 1878daggerdagger 0062Lymphocytes () 418 (142)dagger 409 (148)dagger minus0459daggerdagger 0646Neutrophils () 421 (177)dagger 421 (194)dagger 0017daggerdagger 0987

lowastPearson Chi-Square Df (degree of freedom) daggermean and standard deviation daggerdagger119905-value for independent samples 119879-test

Table 2 Comparison of platelet count in each group based on time

Type of laboratory investigation Interventional group (119899 = 111) Control group (119899 = 117)Platelet count Mean difference (95 CI) 119905 119875 value Mean difference (95 CI) 119905 119875 value8ndash16 hours 0993 (minus1660 3645) 0740 0460 minus1411 (minus3961 1140) minus1094 02768ndash24 hours minus0432 (minus4422 3558) minus0214 0831 2213 (minus0523 4948) 1600 01128ndash32 hours minus2716 (minus7540 2107) minus1114 0267 2775 (minus0796 6347) 1537 01278ndash40 hours minus7890 (minus14472 minus1310) minus2374 0019 0867 (minus3472 5207) 0395 06938ndash48 hours minus16764 (minus24566 minus8964) minus4256 lt0001 minus7703 (minus14055 1351) minus2399 0018Repeated measure ANCOVA within group analysis was applied followed by multiple paired 119905-testslowastBonferroni correction was applied by correcting the level of significance (0055 = 001) Potential covariate (age) was controlled by using repeated measuresANCOVA (ANCOVA controlled for age)

group At the end of the study 111 patients from the interven-tional group and 117 controls were included in the statisticalanalysis Sixty-two patients were excluded from the analysisas 38 patients were lost to followup and 24 patients hadincomplete data (missing results due to sample rejection)

Table 1 shows demographic characteristics and baselinebiochemistry investigation of respondents by treatment Interms of dengue status all patients recruited had eitherdengue NS1 or IgM or both detected while the percentagedistribution of the dengue serotypes among them was DEN1(304) DEN2 (284) DEN 3 (206) andDEN 4 (206)Hence all serotypes were well represented in the study

Table 2 presents the multiple comparisons of meanplatelet count 8 hours after admission with mean platelet

count at 16 24 32 40 and 48 hours after admission forinterventional and control group Multiple paired t-test wasconducted to demonstrate if there was any significant dif-ference in mean platelet count for each comparison HenceBonferroni correction was applied to reduce the possibility ofrejecting a true null hypothesis (committing a type 1 error)Based on the number of patients recruitedwith complete data(111 patients from the intervention group and 117 control)the power of study was 870 (standard deviation of plateletcount of 40000 type I error probability of 001 and thetrue difference in mean platelet count of 20000 betweenthe intervention and control group) Overall there was asignificant increase in mean platelet count over 40 hours inboth groups (Wilkrsquos Lambda = 0939 119875 = 0015 effect size

Evidence-Based Complementary and Alternative Medicine 5

646644

625

644 631

608623

586

708

607

796

694

50

55

60

65

70

75

80

85

90

Experimental control (hours)

Mea

n pl

atel

et co

unt

8 16 24 32 40 48

Figure 1 Comparison of mean platelet count between intervention and control group based on time (time-treatment effect)

Table 3 Comparison of platelets count between experiment andcontrol group (treatment effect regardless of time)

Type of lab Investigation Mean difference (95 CI) 119875 valuePlatelet count 2349 (minus5151 9850) 0538Repeated measure ANCOVA between group analyses was applied Level ofsignificance was set at 005 (two-tailed)

= 006 and power = 840) after adjusting for age Furtheranalysis by using multiple paired t-test on each of the groupsshowed that there was a significant increase in mean plateletcount at 40 hours compared to 8 hours after intervention inthe intervention group (119905 = minus4256 119875 le 0001) but not inthe control group (119905 = minus2399 119875 = 0018) after adjustment ofBonferroni correction (119875 = 0055 = 001)

Table 3 presents the overall efficacy of CPLJ supplementa-tion by comparing the mean platelet count of interventionaland control group regardless the time period Study on thetreatment effect of CPLJ on platelet count regardless of timedid not show any significant difference inmean platelet countbetween intervention and control group (119865 = 1128 119875 =0289) However analysis of the effect of CPLJ over the studyperiod (time and treatment effect) showed that there was asignificant interaction between treatment groups and time(Wilkrsquos Lambda = 0934 effect size = 006 and power =870)

Figure 1 shows the time-treatment effect of CPLJ Theintervention group had a significantly higher mean plateletcount than control group at 40 hours and 48 hours ofintervention

Data from the gene expression study were analyzed usingthe ldquoPCR Array Data Analysis versus 33rdquo software recom-mended by Applied Biosystems and showed that ALOX12(ΔCT mean = 1602 FC = 1500) and PTAFR genes (ΔCTmean= 1487 FC= 1342)were found to be highly expressed inthe intervention group when compared to the control group

4 Discussion

The study conducted shows that there is a rationale behindthe use of CPLJ in the treatment of some of DF and DHFIt is definitely worth investigating this plant for its potentialmedicinal benefits With rapid urbanization and global travelleading to drastic demographic changes dengue is a threatto almost 40 of the worldrsquos population There is still nospecific treatment for dengue Previous attempts to identify apotential antiviral for the treatment of dengue has been facedwith several challenges such as the presence of four distinctviral serotypes which frequently undergo mutations findingan appropriate model for infection and protective action ofa given drug as well as yield interesting therapeutic avenuesfor tailored response modifier drugs The current availablemouse model (AG129) available has its limitations such aslow viral load and a short period of viraemia The journeyto drug discovery through the study of immune-modulatoryeffects against dengue infection lies on the research of genericcompounds and natural products [17]

Research groups around Asia have attempted to studythe efficacy of CPLJ in rapidly increasing platelet counts inDF as well as DHF induced thrombocytopenia but therehas been no conclusive evidence drawn from those studiesDengue is generally a self-limiting disease and the diseaseinduced thrombocytopenia usually reverses itself after takinga slight dip during the phase of defervescence However asignificant number of patients succumb to the disease duringthe thrombocytopenic period Many mechanisms come intoplay during the critical phase of the disease to help reversethe disease state at this point Animal studies in elucidatingsafety data have been conducted on normal Sprague Dawleyrats using freeze dried CPLJ however no significant increasein platelet count was observed among the rats given thejuice and the rats kept as control [10] This was probablydue to the fact that the juice was freeze dried and certainessential compounds could have been lost during the processof freeze drying or perhaps the right disease model was not

6 Evidence-Based Complementary and Alternative Medicine

used for the study Haematocrit level which is an importantparameter which is usually monitored to determine the rateof improvement in haemoconcentration was found to besignificantly reduced in both groups of people White Bloodcell count which is found to be reduced in viral infections wasalso found to increase in both groups

The RNA was extracted from the blood of the patientsrecruited and gene expression of two genes namely theALOX 12 and the PTAFR which were conducted so farThere was a 15-fold increase in the ALOX 12 gene activityamong the patients in the experimental group as comparedto those in the control group at the end of the 3 days ALOX12 is known to be associated with increased megakaryocyteproduction as well as its conversion to platelets through 12-HETE mediated pathway which in turn leads to increasedplatelet production A study was conducted at the RoyalCollege of Surgeons Ireland to determine the platelet specificgenes The Alox 12 gene was highly expressed in platelets andfound to be a platelet specific gene byMcRedmond et al [18]A study conducted in Temple University School of MedicinePhiladelphia provided evidence that ALOX12 is a directtarget of transcription factor RUNX1 in megakaryocytes andplatelets RUNX1 is a transcription factor that regulates theexpression of haemopoietic-specific genes When there isRUNX1 haplodeficiency it affects overall haemopoiesis andhence ALOX 12 expression in platelets is decreased Therewas also an agonist-induced decreased 12-HETE productionin platelets with the decrease in ALOX 12 expression Thisprovides further evidence that platelet production is associ-ated with ALOX 12 expression [13]

This finding supports the claim that the juice consump-tion during the course of dengue infection has the potentialto induce the rapid production of platelets This was clearlydemonstrated by the significant increase in the mean plateletcount after 40 hours and 48 hours of juice consumptionThe PTAFR gene which is known to be responsible forincreased platelet production and aggregation was expressed1342-folds among the patients who consumed the juice ascompared to the control group indicating that the juicehad played an important role in addressing the arresting ofbleeding tendencies among these patients A study conductedin Brazil showed that injection of Platelet Activating Factor(PAFPTAFR) in mice induced an increase in platelet countHowever after a certain level further administration of PAFfailed to induce platelet production indicating autosensitiza-tion These findings show that PAFPTAFR can induce therelease of platelets which may be relevant to thrombocytosis[19] We are currently investigating many other genes todetermine other roles of theCPLJ other than its role in plateletproduction and activation

As all plants C papaya leaves are rich in compounds ofdifferent properties Further studies need to be conductedbefore determining the inflammatory pathways affected bythe juice unopposed However it can be concluded thatthe administration of CPLJ in DF and DHF is safe anddoes induce the rapid increase in platelet count It mayplay a valuable role in the management of DF in the nearfuture

Conflict of Interests

Theresearchwas conducted in the absence of any commercialor financial relationships that could be construed as a poten-tial conflict of interests

Acknowledgments

The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and the Director of theInstitute forMedical Research for giving them the permissionto publish this paper This research was funded by theMinistry ofHealthMalaysia (project code JPP-IMR09-003)They would like to thank Dr Nor Asiah biostatistician ofIMR for reviewing their statistical analysis the Director ofHospital Tengku Ampuan Rahimah for allowing them touse the hospital facilities and the staff of The Departmentof Medicine and the Department of Pathology for theirguidance support and cooperation during the study

References

[1] I H BurkillADictionary of the Economic Products of theMalayPeninsula vol 1 The Crown Agents for the Colonies LondonUK 1935

[2] K Arumuganathan and E D Earle ldquoNuclear DNA contentof some important plant speciesrdquo Plant Molecular BiologyReporter vol 9 no 3 pp 208ndash218 1991

[3] FAOSTATPapaya marketing-general information GFruit Website 2012 httpwwwitfnetorggfruitTemplates20Englishpapayamarketinfohtm

[4] V N Villegas ldquoCarica papaya Lrdquo in Plant Resources of South-East Asia 2 Edible Fruits and Nuts E M W Verheij and R ECoronel Eds vol 2 pp 223ndash225 PROSEA Wageningen TheNetherlands 1997

[5] H Y Nakasone and R E Paull Tropical Fruits CAB Interna-tional Walllingford NY USA 1998

[6] B V Owoyele O M Adebukola A A Funmilayo and A OSoladoye ldquoAnti-inflammatory activities of ethanolic extract ofCarica papaya leavesrdquo Inflammopharmacology vol 16 no 4 pp168ndash173 2008

[7] S Gurung and N Skalko-Basnet ldquoWound healing propertiesof Carica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[8] N Otsuki N H Dang E Kumagai A Kondo S Iwata andCMorimoto ldquoAqueous extract of Carica papaya leaves exhibitsanti-tumor activity and immunomodulatory effectsrdquo Journal ofEthnopharmacology vol 127 no 3 pp 760ndash767 2010

[9] N A Imaga G O Gbenle V I Okochi et al ldquoPhytochemicaland antioxidant nutrient constituents of Carica papaya andparquetina nigrescens extractsrdquo Scientific Research and Essaysvol 5 no 16 pp 2201ndash2205 2010

[10] S Z Halim N R Abdullah Z Afzan B A Abdul RashidI Jantan and Z Ismail ldquoAcute toxicity of Carica papaya leafextract in Sprague Dawley ratsrdquo Journal of Medicinal PlantsResearch vol 5 no 10 pp 1867ndash1872 2011

[11] Ministry of Health of Malaysia Vector borne disease controlunit report 2010

[12] World Health Organization Clinical diagnosis 16 2011

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

understand or read Bahasa Malaysia or English irrespectiveof race (c) patients who were confirmed to have DF or DHFgrade I and II (d) patients with a platelet count of less thanor equal to 100000120583L (e) patients with a baseline alaninetransaminase (ALT) level of not more than 3 times of theupper limit of the normal range (not more than 165UL) and(f) patients with a creatinine kinase (CK) value of less than500UL

The exclusion criteria were (a) dengue hemorrhagic fevergrade III and IV (b) pregnant or lactatingwomen (c) patientswho have received blood or blood products transfusionduring the current hospital stay (d) patients with underlyingcomorbids (e) patients who developed Hepatitis with aserum ALT level 3 times higher than the upper limit of thenormal range (gt165UL) and (f) patients with a creatininekinase (CK) value of more than 500UL

The study was conducted in accordance with the ethicalprinciples as outlined in the declaration of Helsinki October2008 following approval from the Medical Review andEthical Committee (MREC) Ministry of Health Malaysiaand was consistent with Good Clinical Practice (GCP) andapplicable regulatory requirements The National MedicalResearch Registry number is NMRR-09-883-4768 Writteninformed consent was obtained from every volunteer priorto clinical trial participation

23 Diagnosis of DF or DHF and Dengue Serotyping Aclinical diagnosis of DF and DHF was made by the clinicianbased on patientsrsquo presentation and blood investigations Inaddition a rapid dengue bedside test (SD Dengue Duo NS1Ag + Ab Combo Standard Diagnostics Korea) was usedto determine dengue status before the subjects were giventhe Carica papaya leave juice This test is able to detect thepresence of NS1 antigen or dengue IgM or IgG antibodiesHowever for ease of interpretation of dengue status onlypatients who had either NS1 or IgM or both detected wereincluded in the studyThe detection of theNS1 antigen or IgMantibodies within the first five to 7 days of onset of symptomsusually indicates a current dengue infection Subsequently allthe samples were subjected tomultiplex real time RT-PCR fordetermination of dengue serotypes

24 Treatment of the Subjects Once a current dengue infec-tion was confirmed a thorough screening of the patientwas conducted Baseline investigations included full bloodcount bleeding profile renal as well as liver function testand cardiac enzymes Patients in the intervention groupreceived fresh juice from 50 grams of C papaya leavesonce daily 15 minutes after breakfast for 3 consecutive dayswhile receiving the standardmanagement as per the NationalClinical Practice Guidelines for the Management of DengueThe controls received the standard management

25 Study Parameters Full blood count was monitored 8hours for the first 48 hours during the study to deter-mine the changes in platelet count and haematocrit levelswhile bleeding profile renal profile and liver function testwere monitored daily to ensure the safety of the juice

The haematological and biochemical tests were conductedby the Pathology Department at Hospital Tengku AmpuanRahimah Klang and the validated results were later tracedand recorded Mean and standard deviation of the baselinehaematological and biochemical parameters were then calcu-lated as shown in Table 1

Blood for RNA extractionwas taken on day 3 after the lastdose of the juice to conduct expression studies on the PTAFRand the ALOX 12 genes RNALater by Ambion (United Statesof America) was added to the whole blood in an EDTA tubeto prevent degradation of RNA

The RNA extraction was carried out at the Institute forMedical research using an RNA extraction kit by AmbionThe RNA concentration and purity were determined usingtheNanodrop 2000 Spectrophotometer byThermo ScientificTheRNAwas then converted to cDNAusing theHighCapac-ity RNA to cDNA kit by Applied Biosystems The cDNAconcentrationwas then determinedusing theNanodrop 2000Spectrophotometer byThermo Scientific

Gene expression was determined using gene expressionassays by Applied Biosystems on the ABI 7500 Fast Systemon 12 RNA samples from the experimental group and 12 RNAsamples from the control group Specific predesigned MGBprobes were used for ALOX 12 and the PTAFR gene The18S Ribosome was used as an endogenous control A 10 120583Lreaction volume was used using the TaqMan Fast UniversalPCR Mastermix (2X) The probe product ID and sequenceused were ALOX 12 (Hs 00167524 M1 NM 000697251015840-FAM-ATTGCCATCCAGCTCAACAAATCC-MGB-31015840)and PTAFR (Hs 00265399 S1 NM 0011647231 51015840-FAM-GCCCGTAATTTATCGCGCTTACTAT MGB-21015840)

26 Statistical Analysis Repeated measure ANCOVA wasused to determine the effect of CPLJ on the mean plateletcount over 48 hours (time effect) Multiple comparisonsof mean platelet count 8 hours after admission with meanplatelet count at the 16 24 32 40 and 48 hours after admis-sion for interventional and control group were performedMultiple paired t-test was conducted to demonstrate if thereis any significant different in mean platelet count for eachcomparison Hence Bonferroni correction was applied toreduce the possibility of rejecting a true null hypotheses(commit a type 1 error) Therefore Bonferroni correctionwas used to adjust the level of significance to 119875 lt 001(005number of comparison = 0055 = 001) The efficacyof treatment of CPLJ (treatment effect) and treatment overtime (time-treatment interaction) in increasing the meanplatelet count was analysed by comparing mean differencein platelet count between interventional and control groupAll the statistical analyses were done using PASW 180 (SPSSInc Chicago USA)The comparative CT methodwas used todetermine the relative quantification of the genes expressed

3 Results

A total of 145 patients were recruited into the interventionalgroup while 145 patients were recruited into the control

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Demographic characteristics and baseline biochemistry investigation of respondents by treatment

Demographic characteristics Interventional group119899 ()

Control group119899 () 120594

2 (df)lowast 119875 value

GenderMen 91 (469) 103 (531) 1644 (1) 0200Women 20 (588) 14 (412)

EthnicityMalay 77 (481) 83 (519)Chinese 4 (571) 3 (429)Indian 11 (500) 11 (500)Others 19 (487) 20 (513)

Age (meanSD) 304 (103)dagger 264 (73)dagger minus3370daggerdagger 0001Type of dengue fever

Classical dengue fever 42 (532) 37 (468) 0971 (1) 0324Dengue haemorrhagic fever 69 (463) 80 (537)

Baseline Haematology investigationPlatelet count (times103120583L) 664 (228)dagger 690 (226)dagger 0847daggerdagger 0398Total white blood cell (times103120583L) 34 (17)dagger 33 (16) minus0297daggerdagger 0766Haemoglobin (g) 139 (15)dagger 142 (14)dagger 1595daggerdagger 0112Haematocrit () 415 (42)dagger 429 (60)dagger 1878daggerdagger 0062Lymphocytes () 418 (142)dagger 409 (148)dagger minus0459daggerdagger 0646Neutrophils () 421 (177)dagger 421 (194)dagger 0017daggerdagger 0987

lowastPearson Chi-Square Df (degree of freedom) daggermean and standard deviation daggerdagger119905-value for independent samples 119879-test

Table 2 Comparison of platelet count in each group based on time

Type of laboratory investigation Interventional group (119899 = 111) Control group (119899 = 117)Platelet count Mean difference (95 CI) 119905 119875 value Mean difference (95 CI) 119905 119875 value8ndash16 hours 0993 (minus1660 3645) 0740 0460 minus1411 (minus3961 1140) minus1094 02768ndash24 hours minus0432 (minus4422 3558) minus0214 0831 2213 (minus0523 4948) 1600 01128ndash32 hours minus2716 (minus7540 2107) minus1114 0267 2775 (minus0796 6347) 1537 01278ndash40 hours minus7890 (minus14472 minus1310) minus2374 0019 0867 (minus3472 5207) 0395 06938ndash48 hours minus16764 (minus24566 minus8964) minus4256 lt0001 minus7703 (minus14055 1351) minus2399 0018Repeated measure ANCOVA within group analysis was applied followed by multiple paired 119905-testslowastBonferroni correction was applied by correcting the level of significance (0055 = 001) Potential covariate (age) was controlled by using repeated measuresANCOVA (ANCOVA controlled for age)

group At the end of the study 111 patients from the interven-tional group and 117 controls were included in the statisticalanalysis Sixty-two patients were excluded from the analysisas 38 patients were lost to followup and 24 patients hadincomplete data (missing results due to sample rejection)

Table 1 shows demographic characteristics and baselinebiochemistry investigation of respondents by treatment Interms of dengue status all patients recruited had eitherdengue NS1 or IgM or both detected while the percentagedistribution of the dengue serotypes among them was DEN1(304) DEN2 (284) DEN 3 (206) andDEN 4 (206)Hence all serotypes were well represented in the study

Table 2 presents the multiple comparisons of meanplatelet count 8 hours after admission with mean platelet

count at 16 24 32 40 and 48 hours after admission forinterventional and control group Multiple paired t-test wasconducted to demonstrate if there was any significant dif-ference in mean platelet count for each comparison HenceBonferroni correction was applied to reduce the possibility ofrejecting a true null hypothesis (committing a type 1 error)Based on the number of patients recruitedwith complete data(111 patients from the intervention group and 117 control)the power of study was 870 (standard deviation of plateletcount of 40000 type I error probability of 001 and thetrue difference in mean platelet count of 20000 betweenthe intervention and control group) Overall there was asignificant increase in mean platelet count over 40 hours inboth groups (Wilkrsquos Lambda = 0939 119875 = 0015 effect size

Evidence-Based Complementary and Alternative Medicine 5

646644

625

644 631

608623

586

708

607

796

694

50

55

60

65

70

75

80

85

90

Experimental control (hours)

Mea

n pl

atel

et co

unt

8 16 24 32 40 48

Figure 1 Comparison of mean platelet count between intervention and control group based on time (time-treatment effect)

Table 3 Comparison of platelets count between experiment andcontrol group (treatment effect regardless of time)

Type of lab Investigation Mean difference (95 CI) 119875 valuePlatelet count 2349 (minus5151 9850) 0538Repeated measure ANCOVA between group analyses was applied Level ofsignificance was set at 005 (two-tailed)

= 006 and power = 840) after adjusting for age Furtheranalysis by using multiple paired t-test on each of the groupsshowed that there was a significant increase in mean plateletcount at 40 hours compared to 8 hours after intervention inthe intervention group (119905 = minus4256 119875 le 0001) but not inthe control group (119905 = minus2399 119875 = 0018) after adjustment ofBonferroni correction (119875 = 0055 = 001)

Table 3 presents the overall efficacy of CPLJ supplementa-tion by comparing the mean platelet count of interventionaland control group regardless the time period Study on thetreatment effect of CPLJ on platelet count regardless of timedid not show any significant difference inmean platelet countbetween intervention and control group (119865 = 1128 119875 =0289) However analysis of the effect of CPLJ over the studyperiod (time and treatment effect) showed that there was asignificant interaction between treatment groups and time(Wilkrsquos Lambda = 0934 effect size = 006 and power =870)

Figure 1 shows the time-treatment effect of CPLJ Theintervention group had a significantly higher mean plateletcount than control group at 40 hours and 48 hours ofintervention

Data from the gene expression study were analyzed usingthe ldquoPCR Array Data Analysis versus 33rdquo software recom-mended by Applied Biosystems and showed that ALOX12(ΔCT mean = 1602 FC = 1500) and PTAFR genes (ΔCTmean= 1487 FC= 1342)were found to be highly expressed inthe intervention group when compared to the control group

4 Discussion

The study conducted shows that there is a rationale behindthe use of CPLJ in the treatment of some of DF and DHFIt is definitely worth investigating this plant for its potentialmedicinal benefits With rapid urbanization and global travelleading to drastic demographic changes dengue is a threatto almost 40 of the worldrsquos population There is still nospecific treatment for dengue Previous attempts to identify apotential antiviral for the treatment of dengue has been facedwith several challenges such as the presence of four distinctviral serotypes which frequently undergo mutations findingan appropriate model for infection and protective action ofa given drug as well as yield interesting therapeutic avenuesfor tailored response modifier drugs The current availablemouse model (AG129) available has its limitations such aslow viral load and a short period of viraemia The journeyto drug discovery through the study of immune-modulatoryeffects against dengue infection lies on the research of genericcompounds and natural products [17]

Research groups around Asia have attempted to studythe efficacy of CPLJ in rapidly increasing platelet counts inDF as well as DHF induced thrombocytopenia but therehas been no conclusive evidence drawn from those studiesDengue is generally a self-limiting disease and the diseaseinduced thrombocytopenia usually reverses itself after takinga slight dip during the phase of defervescence However asignificant number of patients succumb to the disease duringthe thrombocytopenic period Many mechanisms come intoplay during the critical phase of the disease to help reversethe disease state at this point Animal studies in elucidatingsafety data have been conducted on normal Sprague Dawleyrats using freeze dried CPLJ however no significant increasein platelet count was observed among the rats given thejuice and the rats kept as control [10] This was probablydue to the fact that the juice was freeze dried and certainessential compounds could have been lost during the processof freeze drying or perhaps the right disease model was not

6 Evidence-Based Complementary and Alternative Medicine

used for the study Haematocrit level which is an importantparameter which is usually monitored to determine the rateof improvement in haemoconcentration was found to besignificantly reduced in both groups of people White Bloodcell count which is found to be reduced in viral infections wasalso found to increase in both groups

The RNA was extracted from the blood of the patientsrecruited and gene expression of two genes namely theALOX 12 and the PTAFR which were conducted so farThere was a 15-fold increase in the ALOX 12 gene activityamong the patients in the experimental group as comparedto those in the control group at the end of the 3 days ALOX12 is known to be associated with increased megakaryocyteproduction as well as its conversion to platelets through 12-HETE mediated pathway which in turn leads to increasedplatelet production A study was conducted at the RoyalCollege of Surgeons Ireland to determine the platelet specificgenes The Alox 12 gene was highly expressed in platelets andfound to be a platelet specific gene byMcRedmond et al [18]A study conducted in Temple University School of MedicinePhiladelphia provided evidence that ALOX12 is a directtarget of transcription factor RUNX1 in megakaryocytes andplatelets RUNX1 is a transcription factor that regulates theexpression of haemopoietic-specific genes When there isRUNX1 haplodeficiency it affects overall haemopoiesis andhence ALOX 12 expression in platelets is decreased Therewas also an agonist-induced decreased 12-HETE productionin platelets with the decrease in ALOX 12 expression Thisprovides further evidence that platelet production is associ-ated with ALOX 12 expression [13]

This finding supports the claim that the juice consump-tion during the course of dengue infection has the potentialto induce the rapid production of platelets This was clearlydemonstrated by the significant increase in the mean plateletcount after 40 hours and 48 hours of juice consumptionThe PTAFR gene which is known to be responsible forincreased platelet production and aggregation was expressed1342-folds among the patients who consumed the juice ascompared to the control group indicating that the juicehad played an important role in addressing the arresting ofbleeding tendencies among these patients A study conductedin Brazil showed that injection of Platelet Activating Factor(PAFPTAFR) in mice induced an increase in platelet countHowever after a certain level further administration of PAFfailed to induce platelet production indicating autosensitiza-tion These findings show that PAFPTAFR can induce therelease of platelets which may be relevant to thrombocytosis[19] We are currently investigating many other genes todetermine other roles of theCPLJ other than its role in plateletproduction and activation

As all plants C papaya leaves are rich in compounds ofdifferent properties Further studies need to be conductedbefore determining the inflammatory pathways affected bythe juice unopposed However it can be concluded thatthe administration of CPLJ in DF and DHF is safe anddoes induce the rapid increase in platelet count It mayplay a valuable role in the management of DF in the nearfuture

Conflict of Interests

Theresearchwas conducted in the absence of any commercialor financial relationships that could be construed as a poten-tial conflict of interests

Acknowledgments

The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and the Director of theInstitute forMedical Research for giving them the permissionto publish this paper This research was funded by theMinistry ofHealthMalaysia (project code JPP-IMR09-003)They would like to thank Dr Nor Asiah biostatistician ofIMR for reviewing their statistical analysis the Director ofHospital Tengku Ampuan Rahimah for allowing them touse the hospital facilities and the staff of The Departmentof Medicine and the Department of Pathology for theirguidance support and cooperation during the study

References

[1] I H BurkillADictionary of the Economic Products of theMalayPeninsula vol 1 The Crown Agents for the Colonies LondonUK 1935

[2] K Arumuganathan and E D Earle ldquoNuclear DNA contentof some important plant speciesrdquo Plant Molecular BiologyReporter vol 9 no 3 pp 208ndash218 1991

[3] FAOSTATPapaya marketing-general information GFruit Website 2012 httpwwwitfnetorggfruitTemplates20Englishpapayamarketinfohtm

[4] V N Villegas ldquoCarica papaya Lrdquo in Plant Resources of South-East Asia 2 Edible Fruits and Nuts E M W Verheij and R ECoronel Eds vol 2 pp 223ndash225 PROSEA Wageningen TheNetherlands 1997

[5] H Y Nakasone and R E Paull Tropical Fruits CAB Interna-tional Walllingford NY USA 1998

[6] B V Owoyele O M Adebukola A A Funmilayo and A OSoladoye ldquoAnti-inflammatory activities of ethanolic extract ofCarica papaya leavesrdquo Inflammopharmacology vol 16 no 4 pp168ndash173 2008

[7] S Gurung and N Skalko-Basnet ldquoWound healing propertiesof Carica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[8] N Otsuki N H Dang E Kumagai A Kondo S Iwata andCMorimoto ldquoAqueous extract of Carica papaya leaves exhibitsanti-tumor activity and immunomodulatory effectsrdquo Journal ofEthnopharmacology vol 127 no 3 pp 760ndash767 2010

[9] N A Imaga G O Gbenle V I Okochi et al ldquoPhytochemicaland antioxidant nutrient constituents of Carica papaya andparquetina nigrescens extractsrdquo Scientific Research and Essaysvol 5 no 16 pp 2201ndash2205 2010

[10] S Z Halim N R Abdullah Z Afzan B A Abdul RashidI Jantan and Z Ismail ldquoAcute toxicity of Carica papaya leafextract in Sprague Dawley ratsrdquo Journal of Medicinal PlantsResearch vol 5 no 10 pp 1867ndash1872 2011

[11] Ministry of Health of Malaysia Vector borne disease controlunit report 2010

[12] World Health Organization Clinical diagnosis 16 2011

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Demographic characteristics and baseline biochemistry investigation of respondents by treatment

Demographic characteristics Interventional group119899 ()

Control group119899 () 120594

2 (df)lowast 119875 value

GenderMen 91 (469) 103 (531) 1644 (1) 0200Women 20 (588) 14 (412)

EthnicityMalay 77 (481) 83 (519)Chinese 4 (571) 3 (429)Indian 11 (500) 11 (500)Others 19 (487) 20 (513)

Age (meanSD) 304 (103)dagger 264 (73)dagger minus3370daggerdagger 0001Type of dengue fever

Classical dengue fever 42 (532) 37 (468) 0971 (1) 0324Dengue haemorrhagic fever 69 (463) 80 (537)

Baseline Haematology investigationPlatelet count (times103120583L) 664 (228)dagger 690 (226)dagger 0847daggerdagger 0398Total white blood cell (times103120583L) 34 (17)dagger 33 (16) minus0297daggerdagger 0766Haemoglobin (g) 139 (15)dagger 142 (14)dagger 1595daggerdagger 0112Haematocrit () 415 (42)dagger 429 (60)dagger 1878daggerdagger 0062Lymphocytes () 418 (142)dagger 409 (148)dagger minus0459daggerdagger 0646Neutrophils () 421 (177)dagger 421 (194)dagger 0017daggerdagger 0987

lowastPearson Chi-Square Df (degree of freedom) daggermean and standard deviation daggerdagger119905-value for independent samples 119879-test

Table 2 Comparison of platelet count in each group based on time

Type of laboratory investigation Interventional group (119899 = 111) Control group (119899 = 117)Platelet count Mean difference (95 CI) 119905 119875 value Mean difference (95 CI) 119905 119875 value8ndash16 hours 0993 (minus1660 3645) 0740 0460 minus1411 (minus3961 1140) minus1094 02768ndash24 hours minus0432 (minus4422 3558) minus0214 0831 2213 (minus0523 4948) 1600 01128ndash32 hours minus2716 (minus7540 2107) minus1114 0267 2775 (minus0796 6347) 1537 01278ndash40 hours minus7890 (minus14472 minus1310) minus2374 0019 0867 (minus3472 5207) 0395 06938ndash48 hours minus16764 (minus24566 minus8964) minus4256 lt0001 minus7703 (minus14055 1351) minus2399 0018Repeated measure ANCOVA within group analysis was applied followed by multiple paired 119905-testslowastBonferroni correction was applied by correcting the level of significance (0055 = 001) Potential covariate (age) was controlled by using repeated measuresANCOVA (ANCOVA controlled for age)

group At the end of the study 111 patients from the interven-tional group and 117 controls were included in the statisticalanalysis Sixty-two patients were excluded from the analysisas 38 patients were lost to followup and 24 patients hadincomplete data (missing results due to sample rejection)

Table 1 shows demographic characteristics and baselinebiochemistry investigation of respondents by treatment Interms of dengue status all patients recruited had eitherdengue NS1 or IgM or both detected while the percentagedistribution of the dengue serotypes among them was DEN1(304) DEN2 (284) DEN 3 (206) andDEN 4 (206)Hence all serotypes were well represented in the study

Table 2 presents the multiple comparisons of meanplatelet count 8 hours after admission with mean platelet

count at 16 24 32 40 and 48 hours after admission forinterventional and control group Multiple paired t-test wasconducted to demonstrate if there was any significant dif-ference in mean platelet count for each comparison HenceBonferroni correction was applied to reduce the possibility ofrejecting a true null hypothesis (committing a type 1 error)Based on the number of patients recruitedwith complete data(111 patients from the intervention group and 117 control)the power of study was 870 (standard deviation of plateletcount of 40000 type I error probability of 001 and thetrue difference in mean platelet count of 20000 betweenthe intervention and control group) Overall there was asignificant increase in mean platelet count over 40 hours inboth groups (Wilkrsquos Lambda = 0939 119875 = 0015 effect size

Evidence-Based Complementary and Alternative Medicine 5

646644

625

644 631

608623

586

708

607

796

694

50

55

60

65

70

75

80

85

90

Experimental control (hours)

Mea

n pl

atel

et co

unt

8 16 24 32 40 48

Figure 1 Comparison of mean platelet count between intervention and control group based on time (time-treatment effect)

Table 3 Comparison of platelets count between experiment andcontrol group (treatment effect regardless of time)

Type of lab Investigation Mean difference (95 CI) 119875 valuePlatelet count 2349 (minus5151 9850) 0538Repeated measure ANCOVA between group analyses was applied Level ofsignificance was set at 005 (two-tailed)

= 006 and power = 840) after adjusting for age Furtheranalysis by using multiple paired t-test on each of the groupsshowed that there was a significant increase in mean plateletcount at 40 hours compared to 8 hours after intervention inthe intervention group (119905 = minus4256 119875 le 0001) but not inthe control group (119905 = minus2399 119875 = 0018) after adjustment ofBonferroni correction (119875 = 0055 = 001)

Table 3 presents the overall efficacy of CPLJ supplementa-tion by comparing the mean platelet count of interventionaland control group regardless the time period Study on thetreatment effect of CPLJ on platelet count regardless of timedid not show any significant difference inmean platelet countbetween intervention and control group (119865 = 1128 119875 =0289) However analysis of the effect of CPLJ over the studyperiod (time and treatment effect) showed that there was asignificant interaction between treatment groups and time(Wilkrsquos Lambda = 0934 effect size = 006 and power =870)

Figure 1 shows the time-treatment effect of CPLJ Theintervention group had a significantly higher mean plateletcount than control group at 40 hours and 48 hours ofintervention

Data from the gene expression study were analyzed usingthe ldquoPCR Array Data Analysis versus 33rdquo software recom-mended by Applied Biosystems and showed that ALOX12(ΔCT mean = 1602 FC = 1500) and PTAFR genes (ΔCTmean= 1487 FC= 1342)were found to be highly expressed inthe intervention group when compared to the control group

4 Discussion

The study conducted shows that there is a rationale behindthe use of CPLJ in the treatment of some of DF and DHFIt is definitely worth investigating this plant for its potentialmedicinal benefits With rapid urbanization and global travelleading to drastic demographic changes dengue is a threatto almost 40 of the worldrsquos population There is still nospecific treatment for dengue Previous attempts to identify apotential antiviral for the treatment of dengue has been facedwith several challenges such as the presence of four distinctviral serotypes which frequently undergo mutations findingan appropriate model for infection and protective action ofa given drug as well as yield interesting therapeutic avenuesfor tailored response modifier drugs The current availablemouse model (AG129) available has its limitations such aslow viral load and a short period of viraemia The journeyto drug discovery through the study of immune-modulatoryeffects against dengue infection lies on the research of genericcompounds and natural products [17]

Research groups around Asia have attempted to studythe efficacy of CPLJ in rapidly increasing platelet counts inDF as well as DHF induced thrombocytopenia but therehas been no conclusive evidence drawn from those studiesDengue is generally a self-limiting disease and the diseaseinduced thrombocytopenia usually reverses itself after takinga slight dip during the phase of defervescence However asignificant number of patients succumb to the disease duringthe thrombocytopenic period Many mechanisms come intoplay during the critical phase of the disease to help reversethe disease state at this point Animal studies in elucidatingsafety data have been conducted on normal Sprague Dawleyrats using freeze dried CPLJ however no significant increasein platelet count was observed among the rats given thejuice and the rats kept as control [10] This was probablydue to the fact that the juice was freeze dried and certainessential compounds could have been lost during the processof freeze drying or perhaps the right disease model was not

6 Evidence-Based Complementary and Alternative Medicine

used for the study Haematocrit level which is an importantparameter which is usually monitored to determine the rateof improvement in haemoconcentration was found to besignificantly reduced in both groups of people White Bloodcell count which is found to be reduced in viral infections wasalso found to increase in both groups

The RNA was extracted from the blood of the patientsrecruited and gene expression of two genes namely theALOX 12 and the PTAFR which were conducted so farThere was a 15-fold increase in the ALOX 12 gene activityamong the patients in the experimental group as comparedto those in the control group at the end of the 3 days ALOX12 is known to be associated with increased megakaryocyteproduction as well as its conversion to platelets through 12-HETE mediated pathway which in turn leads to increasedplatelet production A study was conducted at the RoyalCollege of Surgeons Ireland to determine the platelet specificgenes The Alox 12 gene was highly expressed in platelets andfound to be a platelet specific gene byMcRedmond et al [18]A study conducted in Temple University School of MedicinePhiladelphia provided evidence that ALOX12 is a directtarget of transcription factor RUNX1 in megakaryocytes andplatelets RUNX1 is a transcription factor that regulates theexpression of haemopoietic-specific genes When there isRUNX1 haplodeficiency it affects overall haemopoiesis andhence ALOX 12 expression in platelets is decreased Therewas also an agonist-induced decreased 12-HETE productionin platelets with the decrease in ALOX 12 expression Thisprovides further evidence that platelet production is associ-ated with ALOX 12 expression [13]

This finding supports the claim that the juice consump-tion during the course of dengue infection has the potentialto induce the rapid production of platelets This was clearlydemonstrated by the significant increase in the mean plateletcount after 40 hours and 48 hours of juice consumptionThe PTAFR gene which is known to be responsible forincreased platelet production and aggregation was expressed1342-folds among the patients who consumed the juice ascompared to the control group indicating that the juicehad played an important role in addressing the arresting ofbleeding tendencies among these patients A study conductedin Brazil showed that injection of Platelet Activating Factor(PAFPTAFR) in mice induced an increase in platelet countHowever after a certain level further administration of PAFfailed to induce platelet production indicating autosensitiza-tion These findings show that PAFPTAFR can induce therelease of platelets which may be relevant to thrombocytosis[19] We are currently investigating many other genes todetermine other roles of theCPLJ other than its role in plateletproduction and activation

As all plants C papaya leaves are rich in compounds ofdifferent properties Further studies need to be conductedbefore determining the inflammatory pathways affected bythe juice unopposed However it can be concluded thatthe administration of CPLJ in DF and DHF is safe anddoes induce the rapid increase in platelet count It mayplay a valuable role in the management of DF in the nearfuture

Conflict of Interests

Theresearchwas conducted in the absence of any commercialor financial relationships that could be construed as a poten-tial conflict of interests

Acknowledgments

The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and the Director of theInstitute forMedical Research for giving them the permissionto publish this paper This research was funded by theMinistry ofHealthMalaysia (project code JPP-IMR09-003)They would like to thank Dr Nor Asiah biostatistician ofIMR for reviewing their statistical analysis the Director ofHospital Tengku Ampuan Rahimah for allowing them touse the hospital facilities and the staff of The Departmentof Medicine and the Department of Pathology for theirguidance support and cooperation during the study

References

[1] I H BurkillADictionary of the Economic Products of theMalayPeninsula vol 1 The Crown Agents for the Colonies LondonUK 1935

[2] K Arumuganathan and E D Earle ldquoNuclear DNA contentof some important plant speciesrdquo Plant Molecular BiologyReporter vol 9 no 3 pp 208ndash218 1991

[3] FAOSTATPapaya marketing-general information GFruit Website 2012 httpwwwitfnetorggfruitTemplates20Englishpapayamarketinfohtm

[4] V N Villegas ldquoCarica papaya Lrdquo in Plant Resources of South-East Asia 2 Edible Fruits and Nuts E M W Verheij and R ECoronel Eds vol 2 pp 223ndash225 PROSEA Wageningen TheNetherlands 1997

[5] H Y Nakasone and R E Paull Tropical Fruits CAB Interna-tional Walllingford NY USA 1998

[6] B V Owoyele O M Adebukola A A Funmilayo and A OSoladoye ldquoAnti-inflammatory activities of ethanolic extract ofCarica papaya leavesrdquo Inflammopharmacology vol 16 no 4 pp168ndash173 2008

[7] S Gurung and N Skalko-Basnet ldquoWound healing propertiesof Carica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[8] N Otsuki N H Dang E Kumagai A Kondo S Iwata andCMorimoto ldquoAqueous extract of Carica papaya leaves exhibitsanti-tumor activity and immunomodulatory effectsrdquo Journal ofEthnopharmacology vol 127 no 3 pp 760ndash767 2010

[9] N A Imaga G O Gbenle V I Okochi et al ldquoPhytochemicaland antioxidant nutrient constituents of Carica papaya andparquetina nigrescens extractsrdquo Scientific Research and Essaysvol 5 no 16 pp 2201ndash2205 2010

[10] S Z Halim N R Abdullah Z Afzan B A Abdul RashidI Jantan and Z Ismail ldquoAcute toxicity of Carica papaya leafextract in Sprague Dawley ratsrdquo Journal of Medicinal PlantsResearch vol 5 no 10 pp 1867ndash1872 2011

[11] Ministry of Health of Malaysia Vector borne disease controlunit report 2010

[12] World Health Organization Clinical diagnosis 16 2011

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

646644

625

644 631

608623

586

708

607

796

694

50

55

60

65

70

75

80

85

90

Experimental control (hours)

Mea

n pl

atel

et co

unt

8 16 24 32 40 48

Figure 1 Comparison of mean platelet count between intervention and control group based on time (time-treatment effect)

Table 3 Comparison of platelets count between experiment andcontrol group (treatment effect regardless of time)

Type of lab Investigation Mean difference (95 CI) 119875 valuePlatelet count 2349 (minus5151 9850) 0538Repeated measure ANCOVA between group analyses was applied Level ofsignificance was set at 005 (two-tailed)

= 006 and power = 840) after adjusting for age Furtheranalysis by using multiple paired t-test on each of the groupsshowed that there was a significant increase in mean plateletcount at 40 hours compared to 8 hours after intervention inthe intervention group (119905 = minus4256 119875 le 0001) but not inthe control group (119905 = minus2399 119875 = 0018) after adjustment ofBonferroni correction (119875 = 0055 = 001)

Table 3 presents the overall efficacy of CPLJ supplementa-tion by comparing the mean platelet count of interventionaland control group regardless the time period Study on thetreatment effect of CPLJ on platelet count regardless of timedid not show any significant difference inmean platelet countbetween intervention and control group (119865 = 1128 119875 =0289) However analysis of the effect of CPLJ over the studyperiod (time and treatment effect) showed that there was asignificant interaction between treatment groups and time(Wilkrsquos Lambda = 0934 effect size = 006 and power =870)

Figure 1 shows the time-treatment effect of CPLJ Theintervention group had a significantly higher mean plateletcount than control group at 40 hours and 48 hours ofintervention

Data from the gene expression study were analyzed usingthe ldquoPCR Array Data Analysis versus 33rdquo software recom-mended by Applied Biosystems and showed that ALOX12(ΔCT mean = 1602 FC = 1500) and PTAFR genes (ΔCTmean= 1487 FC= 1342)were found to be highly expressed inthe intervention group when compared to the control group

4 Discussion

The study conducted shows that there is a rationale behindthe use of CPLJ in the treatment of some of DF and DHFIt is definitely worth investigating this plant for its potentialmedicinal benefits With rapid urbanization and global travelleading to drastic demographic changes dengue is a threatto almost 40 of the worldrsquos population There is still nospecific treatment for dengue Previous attempts to identify apotential antiviral for the treatment of dengue has been facedwith several challenges such as the presence of four distinctviral serotypes which frequently undergo mutations findingan appropriate model for infection and protective action ofa given drug as well as yield interesting therapeutic avenuesfor tailored response modifier drugs The current availablemouse model (AG129) available has its limitations such aslow viral load and a short period of viraemia The journeyto drug discovery through the study of immune-modulatoryeffects against dengue infection lies on the research of genericcompounds and natural products [17]

Research groups around Asia have attempted to studythe efficacy of CPLJ in rapidly increasing platelet counts inDF as well as DHF induced thrombocytopenia but therehas been no conclusive evidence drawn from those studiesDengue is generally a self-limiting disease and the diseaseinduced thrombocytopenia usually reverses itself after takinga slight dip during the phase of defervescence However asignificant number of patients succumb to the disease duringthe thrombocytopenic period Many mechanisms come intoplay during the critical phase of the disease to help reversethe disease state at this point Animal studies in elucidatingsafety data have been conducted on normal Sprague Dawleyrats using freeze dried CPLJ however no significant increasein platelet count was observed among the rats given thejuice and the rats kept as control [10] This was probablydue to the fact that the juice was freeze dried and certainessential compounds could have been lost during the processof freeze drying or perhaps the right disease model was not

6 Evidence-Based Complementary and Alternative Medicine

used for the study Haematocrit level which is an importantparameter which is usually monitored to determine the rateof improvement in haemoconcentration was found to besignificantly reduced in both groups of people White Bloodcell count which is found to be reduced in viral infections wasalso found to increase in both groups

The RNA was extracted from the blood of the patientsrecruited and gene expression of two genes namely theALOX 12 and the PTAFR which were conducted so farThere was a 15-fold increase in the ALOX 12 gene activityamong the patients in the experimental group as comparedto those in the control group at the end of the 3 days ALOX12 is known to be associated with increased megakaryocyteproduction as well as its conversion to platelets through 12-HETE mediated pathway which in turn leads to increasedplatelet production A study was conducted at the RoyalCollege of Surgeons Ireland to determine the platelet specificgenes The Alox 12 gene was highly expressed in platelets andfound to be a platelet specific gene byMcRedmond et al [18]A study conducted in Temple University School of MedicinePhiladelphia provided evidence that ALOX12 is a directtarget of transcription factor RUNX1 in megakaryocytes andplatelets RUNX1 is a transcription factor that regulates theexpression of haemopoietic-specific genes When there isRUNX1 haplodeficiency it affects overall haemopoiesis andhence ALOX 12 expression in platelets is decreased Therewas also an agonist-induced decreased 12-HETE productionin platelets with the decrease in ALOX 12 expression Thisprovides further evidence that platelet production is associ-ated with ALOX 12 expression [13]

This finding supports the claim that the juice consump-tion during the course of dengue infection has the potentialto induce the rapid production of platelets This was clearlydemonstrated by the significant increase in the mean plateletcount after 40 hours and 48 hours of juice consumptionThe PTAFR gene which is known to be responsible forincreased platelet production and aggregation was expressed1342-folds among the patients who consumed the juice ascompared to the control group indicating that the juicehad played an important role in addressing the arresting ofbleeding tendencies among these patients A study conductedin Brazil showed that injection of Platelet Activating Factor(PAFPTAFR) in mice induced an increase in platelet countHowever after a certain level further administration of PAFfailed to induce platelet production indicating autosensitiza-tion These findings show that PAFPTAFR can induce therelease of platelets which may be relevant to thrombocytosis[19] We are currently investigating many other genes todetermine other roles of theCPLJ other than its role in plateletproduction and activation

As all plants C papaya leaves are rich in compounds ofdifferent properties Further studies need to be conductedbefore determining the inflammatory pathways affected bythe juice unopposed However it can be concluded thatthe administration of CPLJ in DF and DHF is safe anddoes induce the rapid increase in platelet count It mayplay a valuable role in the management of DF in the nearfuture

Conflict of Interests

Theresearchwas conducted in the absence of any commercialor financial relationships that could be construed as a poten-tial conflict of interests

Acknowledgments

The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and the Director of theInstitute forMedical Research for giving them the permissionto publish this paper This research was funded by theMinistry ofHealthMalaysia (project code JPP-IMR09-003)They would like to thank Dr Nor Asiah biostatistician ofIMR for reviewing their statistical analysis the Director ofHospital Tengku Ampuan Rahimah for allowing them touse the hospital facilities and the staff of The Departmentof Medicine and the Department of Pathology for theirguidance support and cooperation during the study

References

[1] I H BurkillADictionary of the Economic Products of theMalayPeninsula vol 1 The Crown Agents for the Colonies LondonUK 1935

[2] K Arumuganathan and E D Earle ldquoNuclear DNA contentof some important plant speciesrdquo Plant Molecular BiologyReporter vol 9 no 3 pp 208ndash218 1991

[3] FAOSTATPapaya marketing-general information GFruit Website 2012 httpwwwitfnetorggfruitTemplates20Englishpapayamarketinfohtm

[4] V N Villegas ldquoCarica papaya Lrdquo in Plant Resources of South-East Asia 2 Edible Fruits and Nuts E M W Verheij and R ECoronel Eds vol 2 pp 223ndash225 PROSEA Wageningen TheNetherlands 1997

[5] H Y Nakasone and R E Paull Tropical Fruits CAB Interna-tional Walllingford NY USA 1998

[6] B V Owoyele O M Adebukola A A Funmilayo and A OSoladoye ldquoAnti-inflammatory activities of ethanolic extract ofCarica papaya leavesrdquo Inflammopharmacology vol 16 no 4 pp168ndash173 2008

[7] S Gurung and N Skalko-Basnet ldquoWound healing propertiesof Carica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[8] N Otsuki N H Dang E Kumagai A Kondo S Iwata andCMorimoto ldquoAqueous extract of Carica papaya leaves exhibitsanti-tumor activity and immunomodulatory effectsrdquo Journal ofEthnopharmacology vol 127 no 3 pp 760ndash767 2010

[9] N A Imaga G O Gbenle V I Okochi et al ldquoPhytochemicaland antioxidant nutrient constituents of Carica papaya andparquetina nigrescens extractsrdquo Scientific Research and Essaysvol 5 no 16 pp 2201ndash2205 2010

[10] S Z Halim N R Abdullah Z Afzan B A Abdul RashidI Jantan and Z Ismail ldquoAcute toxicity of Carica papaya leafextract in Sprague Dawley ratsrdquo Journal of Medicinal PlantsResearch vol 5 no 10 pp 1867ndash1872 2011

[11] Ministry of Health of Malaysia Vector borne disease controlunit report 2010

[12] World Health Organization Clinical diagnosis 16 2011

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

used for the study Haematocrit level which is an importantparameter which is usually monitored to determine the rateof improvement in haemoconcentration was found to besignificantly reduced in both groups of people White Bloodcell count which is found to be reduced in viral infections wasalso found to increase in both groups

The RNA was extracted from the blood of the patientsrecruited and gene expression of two genes namely theALOX 12 and the PTAFR which were conducted so farThere was a 15-fold increase in the ALOX 12 gene activityamong the patients in the experimental group as comparedto those in the control group at the end of the 3 days ALOX12 is known to be associated with increased megakaryocyteproduction as well as its conversion to platelets through 12-HETE mediated pathway which in turn leads to increasedplatelet production A study was conducted at the RoyalCollege of Surgeons Ireland to determine the platelet specificgenes The Alox 12 gene was highly expressed in platelets andfound to be a platelet specific gene byMcRedmond et al [18]A study conducted in Temple University School of MedicinePhiladelphia provided evidence that ALOX12 is a directtarget of transcription factor RUNX1 in megakaryocytes andplatelets RUNX1 is a transcription factor that regulates theexpression of haemopoietic-specific genes When there isRUNX1 haplodeficiency it affects overall haemopoiesis andhence ALOX 12 expression in platelets is decreased Therewas also an agonist-induced decreased 12-HETE productionin platelets with the decrease in ALOX 12 expression Thisprovides further evidence that platelet production is associ-ated with ALOX 12 expression [13]

This finding supports the claim that the juice consump-tion during the course of dengue infection has the potentialto induce the rapid production of platelets This was clearlydemonstrated by the significant increase in the mean plateletcount after 40 hours and 48 hours of juice consumptionThe PTAFR gene which is known to be responsible forincreased platelet production and aggregation was expressed1342-folds among the patients who consumed the juice ascompared to the control group indicating that the juicehad played an important role in addressing the arresting ofbleeding tendencies among these patients A study conductedin Brazil showed that injection of Platelet Activating Factor(PAFPTAFR) in mice induced an increase in platelet countHowever after a certain level further administration of PAFfailed to induce platelet production indicating autosensitiza-tion These findings show that PAFPTAFR can induce therelease of platelets which may be relevant to thrombocytosis[19] We are currently investigating many other genes todetermine other roles of theCPLJ other than its role in plateletproduction and activation

As all plants C papaya leaves are rich in compounds ofdifferent properties Further studies need to be conductedbefore determining the inflammatory pathways affected bythe juice unopposed However it can be concluded thatthe administration of CPLJ in DF and DHF is safe anddoes induce the rapid increase in platelet count It mayplay a valuable role in the management of DF in the nearfuture

Conflict of Interests

Theresearchwas conducted in the absence of any commercialor financial relationships that could be construed as a poten-tial conflict of interests

Acknowledgments

The authors would like to thank the Director General ofHealth Ministry of Health Malaysia and the Director of theInstitute forMedical Research for giving them the permissionto publish this paper This research was funded by theMinistry ofHealthMalaysia (project code JPP-IMR09-003)They would like to thank Dr Nor Asiah biostatistician ofIMR for reviewing their statistical analysis the Director ofHospital Tengku Ampuan Rahimah for allowing them touse the hospital facilities and the staff of The Departmentof Medicine and the Department of Pathology for theirguidance support and cooperation during the study

References

[1] I H BurkillADictionary of the Economic Products of theMalayPeninsula vol 1 The Crown Agents for the Colonies LondonUK 1935

[2] K Arumuganathan and E D Earle ldquoNuclear DNA contentof some important plant speciesrdquo Plant Molecular BiologyReporter vol 9 no 3 pp 208ndash218 1991

[3] FAOSTATPapaya marketing-general information GFruit Website 2012 httpwwwitfnetorggfruitTemplates20Englishpapayamarketinfohtm

[4] V N Villegas ldquoCarica papaya Lrdquo in Plant Resources of South-East Asia 2 Edible Fruits and Nuts E M W Verheij and R ECoronel Eds vol 2 pp 223ndash225 PROSEA Wageningen TheNetherlands 1997

[5] H Y Nakasone and R E Paull Tropical Fruits CAB Interna-tional Walllingford NY USA 1998

[6] B V Owoyele O M Adebukola A A Funmilayo and A OSoladoye ldquoAnti-inflammatory activities of ethanolic extract ofCarica papaya leavesrdquo Inflammopharmacology vol 16 no 4 pp168ndash173 2008

[7] S Gurung and N Skalko-Basnet ldquoWound healing propertiesof Carica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[8] N Otsuki N H Dang E Kumagai A Kondo S Iwata andCMorimoto ldquoAqueous extract of Carica papaya leaves exhibitsanti-tumor activity and immunomodulatory effectsrdquo Journal ofEthnopharmacology vol 127 no 3 pp 760ndash767 2010

[9] N A Imaga G O Gbenle V I Okochi et al ldquoPhytochemicaland antioxidant nutrient constituents of Carica papaya andparquetina nigrescens extractsrdquo Scientific Research and Essaysvol 5 no 16 pp 2201ndash2205 2010

[10] S Z Halim N R Abdullah Z Afzan B A Abdul RashidI Jantan and Z Ismail ldquoAcute toxicity of Carica papaya leafextract in Sprague Dawley ratsrdquo Journal of Medicinal PlantsResearch vol 5 no 10 pp 1867ndash1872 2011

[11] Ministry of Health of Malaysia Vector borne disease controlunit report 2010

[12] World Health Organization Clinical diagnosis 16 2011

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

[13] G Kaur G Jalagadugula G Mao and A K Rao ldquoRUNX1corebinding factor A2 regulates platelet 12-lipoxygenase gene(ALOX12) studies in human RUNX1 haplodeficiencyrdquo Bloodvol 115 no 15 pp 3128ndash3135 2010

[14] I C Macaulay M R Tijssen D C Thijssen-Timmer et alldquoComparative gene expression profiling of in vitro differentiatedmegakaryocytes and erythroblasts identifies novel activatoryand inhibitory platelet membrane proteinsrdquo Blood vol 109 no8 pp 3260ndash3269 2007

[15] A M Butkiewicz H Kemona V Dymicka-Piekarska JMatowicka-Karma P Radziwon and A Lipska ldquoPlatelet countmean platelet volume and thrombocytopoietic indices inhealthy women and menrdquo Thrombosis Research vol 118 no 2pp 199ndash204 2006

[16] W D Dupont andW D Plummer ldquoPower and sample size cal-culations a review and computer programrdquo Controlled ClinicalTrials vol 11 no 2 pp 116ndash128 1990

[17] B Selisko H Dutartre J C Guillemot et al ldquoComparativemechanistic studies of de novo RNA synthesis by flavivirusRNA-dependent RNA polymerasesrdquoVirology vol 351 no 1 pp145ndash158 2006

[18] J P McRedmond S D Park D F Reilly et al ldquoIntegrationof proteomics and genomics in platelets a profile of plateletproteins and platelet-specific genesrdquo Molecular amp CellularProteomics vol 3 no 2 pp 133ndash144 2004

[19] M A Martins P M Martins H C Faria Neto et al ldquoIntra-venous injections of PAF-acether induce platelet aggregation inratsrdquoEuropean Journal of Pharmacology vol 149 no 1-2 pp 89ndash96 1988

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

PPARRe sea rch

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom