Research ArticleEvaluating Antiplasmodial and Antimalarial Activities of Soybean(Glycine max) Seed Extracts on P falciparum Parasite Culturesand P berghei-Infected Mice

Kevin Nyandwaro 1 Job Oyweri 2 Francis Kimani3 and Amos Mbugua 1

1Department of Medical Laboratory Science Jomo Kenyatta University of Agriculture and Technology NairobiPO Box 90420ndash80100 Nairobi Kenya2Kenya Medical Research Institute Centre for Biotechnology Research and Development PO Box 54840ndash0002 Nairobi Kenya3Department of Pure and Applied Sciences Technical University of Mombasa PO Box 90420ndash80100 Mombasa Kenya

Correspondence should be addressed to Kevin Nyandwaro nyandwaroteyagmailcom

Received 14 November 2019 Revised 12 January 2020 Accepted 24 January 2020 Published 18 February 2020

Academic Editor Patrizia Messi

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

Background Plasmodium parasite resistance to artemisinin-based combination therapies (ACTs) calls for development of newaffordable safe and effective antimalarial drugs Studies conducted previously on soybean extracts have established that theypossess antimicrobial anti-inflammatory anticancerous and antioxidant properties )e activity of such extracts on Plasmodiumparasites has not been potentially exploited Objectives )e aim of this study was to determine the antiplasmodial activity ofsoybean extracts using Plasmodium falciparum cultures followed by an in vivo evaluation of safety and antimalarial activity of theextracts in Plasmodium berghei ANKA strain-infected mice Method Aqueous methanol and peptide extracts of soybean seedswere prepared An in vitro evaluation of the extracts for antiplasmodial activity was carried out using two P falciparum strains D6a chloroquine-sensitive Sierra Leone 1 strain and W2 a chloroquine-resistant Indochina 1 strain Following the in vitro as-sessment two active extracts (peptide and methanol) were selected for in vivo assay with mice infected with P berghei ANKAstrain )e two extracts were tested for their therapeutic potential (curative test) )e peptide extract was further assessed todetermine whether it could prevent the establishment of a P berghei infection (prophylactic test) For the curative tests methanoland peptide extracts were separately administered orally to three groups of five P berghei-infected Swiss albino mice for four daysat three dosage levels 800 400 and 200mgkgday In the prophylactic test the similar dosage regimen was applied at baseline to 3groups of uninfected mice using the peptide extract which was administered orally for 4 days Results Peptide and methanolextracts showed good activity with IC50 of 1997plusmn 257 μgml and 1014plusmn 904 μgml respectively against the D6 strain )e IC50values for the peptide and methanol extracts were 2861plusmn 132 μgml and 1487plusmn 343 μgml respectively against the W2 strainMethanol and peptide extracts exhibited high parasite-suppressive (therapeutic) activity of 729 and 719 respectively usingthe 800mgkg dose In the prophylactic test the peptide extract exhibited suppressive activity of 647 upon use of 800mgkgNotably there was a significant decrease (Plt 0001) in suppression with lower doses Conclusion )e results show the presence ofantimalarial properties in soybean extracts with higher curative activity when compared to the prophylactic activity Howevermore research needs to be conducted on this plant to possibly establish lead compounds

1 Introduction

Globally malaria cases are approximately 300 million eachyear with about one million deaths [1] A majority of themalaria cases and deaths are found in sub-Saharan Africa

where mortality is highest among infants and children below5 years [2] )e use of effective drugs to treat malaria cases isa key intervention However antimalarial drug treatmentssuch as artemisinin-based combination therapies (ACTs)face the growing threat of drug resistance [3] Drug

HindawiJournal of PathogensVolume 2020 Article ID 7605730 8 pageshttpsdoiorg10115520207605730

resistance parasites develop following prolonged exposure toantimalarial drugs )is drug pressure facilitates emergenceof parasite strains that possess molecular mechanisms thatcompromise or circumvent drug efficacy Such is the casewith ACT-resistant P falciparum parasites that delay par-asite clearance and have been identified in South-East Asia)e growing threat of drug resistance to ACTs and othercurrently administered drugs necessitates the developmentof novel products for malaria treatment [4]

About 122 drugs have been produced from 94 species ofplants through discovery of ethno botanical leads [5] Amajority of traditional medicinal plants are thought to besafe based on the ethno medical knowledge available Drugshave been generated from plants because of their availabilityefficacy and their mode of action [6] Nonetheless morestudies are necessary to discover evaluate and validate newdrugs from plants sources [7]

Soybeans (Glycine max) belong to the large botanicalfamily Leguminosae and they grow in tropical subtropicaland temperate climatic regions )eir seeds measure 8 to10mm in diameter and grow within a pod similar to that ofpeas [8] )ey contain 20 oil content and are rich inphytochemicals which have health benefits hence makingthem neutraceuticals or functional food crops [9] Tradi-tionally Glycine max leaves roots and seeds are used fortreatment of wide range of ailments including malaria [10])e plant has been demonstrated to possess significant bi-ological activities such as antioxidant [11] anti-inflamma-tory [12] antidiabetic [13] antidepressants [14] andantimicrobial [15] Soybeans have a healing potential againstatherosclerosis osteoporosis and various types of cancersincluding those affecting the prostate breast and uterus[15] )ey also contain isoflavones which are thought to be amajor contributor to the antioxidative activity of this legume[16] A previous study on antimalarial properties of soybeanfat emulsions showed that Glycine max exhibited a prom-ising inhibitory activity against 3D7 strain of P falciparumwith IC50 values ranging from 807plusmn 213 to 1302plusmn 235 μgml [17] )e soybean lipid extracts used in this study byDeharo et al were Intralipidreg and Ivelipreg which arecommercial products used for intravenous lipid supple-mentation )e study demonstrated the antimalarial po-tential of soybeans even though radical cure could not beestablished )e current study set out to assess anti-plasmodial and antimalarial activities of soybeans seed ex-tracts besides the lipid extracts Additionally a toxicity studywas conducted to determine the lethal dose of the extracts

2 Overview of Materials and Methodology ofStudy Design

)e study was a laboratory-based experimental investigationof the potential antiplasmodial and antimalarial activity ofsoybean (Glycine max) extract (SE) )e antiplasmodialactivity of crude SE was accomplished through evaluation ofantiplasmodial activity of crude SE (in vitro) on P falci-parum cultures and a Plasmodium berghei mouse malariamodel (in vivo) evaluation )e study was carried out at theCentre for Biotechnology Research and development

(CBRD) and Animal House in the Kenya Medical ResearchInstitute (KEMRI) respectively

21 Sourcing of Glycine max Seeds Soybean seeds variety SB19 were collected and packed in plastic paper bags fromKenya Agriculture and Livestock Research Organization(KALRO) in Nairobi Kenya in October 2018 )e seedswere air-dried for 3 weeks at room temperature and pul-verized using laboratory mills

22 Preparation of Soybean Extracts )ree extracts (watermethanol and peptide) were prepared from the milledsoybean seeds

221 Aqueous and Methanol Extract Preparation 100 g ofSoybean powder was macerated in 1 litre of respectivesolvents (water or methanol) After dissolution the solutionsof samples were shaken in an incubator shaker at a moderatetemperature of 37degCplusmn 2 for 7 days )e samples were takenout of shaker afterwards and filtered initially with muslincloth and then with Whatman filter paper 01 so that atransparent solution was obtained )e aqueous andmethanol filtrates were next placed in an open water bath forthe solvent to evaporate at 35degCplusmn 2 for 6-7 hours daily untildried extracts are obtained )e dried extracts were trans-ferred into vials and stored at minus 20degC for further analysis andfuture use

222 Peptide Extract Preparation To prepare the peptideextract 10 g of soybean powder was added to a 100ml coldextraction buffer (10mM Na2HPO4 15mM NaH2PO4100m MKCl and 15 EDTA) at pH 5 )e solution wasincubated for 3 hours at 4degC)e precipitate was obtained bysaturation and centrifuged at 2500 revolutions per minute)e crude peptide pellets were air-dried and stored at minus 20degCfor further analysis and future use A single extract wasprepared for use in both in vitro and in vivo experimentsDespite prolonged usage of Glycine max extract for monthsit still demonstrated continued activity therefore possiblinglong shelf life

23 Preliminary Phytochemical Screening Screening forphytochemicals of soybean extracts was carried out usingstandard methods with some modifications [18]

24 In Vitro Culturing of P falciparum Chloroquine- (CQ-)sensitive (D6) and CQ-resistant (W2) P falciparum strainswere used to assess the antiplasmodial activity of soybeanextract and fraction of erythrocytic stages in vitro )ecultures were maintained at the Centre for BiotechnologyResearch and Development (CBRD) at the Kenya MedicalResearch Institute (KEMRI))e P falciparum cultures weremaintained according to the method described by Tragerand Jensen [18] with minor modifications P falciparum (D6and W2) cultures were maintained in fresh human eryth-rocytes suspended at 4 hematocrit in RPMI 1640 (sigma)

2 Journal of Pathogens

containing 02 sodium bicarbonate 05 albumax and45 μgL hypoxanthine and 50 μgL gentamicin and incu-bated at 37degC under a gas mixture of 5 oxygen 5 carbondioxide and 90 nitrogen Infected erythrocytes weretransferred into fresh complete medium to propagate theculture daily In P falciparum (INDO strain) culture me-dium albumax was replaced by 10 pooled human serumParasitemia was determined qualitatively and quantitativelyby light microscopy using (Giemsa staining)

25 In Vitro Antiplasmodial Assays Using Soybean ExtractsTo assess the antiplasmodial activity of the SE extracts anin vitro serial microdilution assay technique that measuresthe ability of extracts to inhibit the incorporation of [G3H]-hypoxanthine by P falciparum was used [19] )e exper-iments were conducted on 96-well plates First 25 μl ofculture medium was added to all the wells of a 96-well flat-bottomedmicrotiter plate except those on the 2nd row 50 μlaliquots of the three extract solutions (aqueous methanoland peptide) were added in duplicate to the wells of thesecond row A Titertek motorized hand diluter (Flowlaboratories Uxbridge UK) was used to make two-foldserial dilution of each soybean extract over a 64-foldconcentration range )e stock solutions (100 μgml) of thethree soybean extracts were diluted two-fold down until aconcentration of 156 μgml was reached )e susceptibilitytest was performed using an initial parasitemia of 04 byadding 200 μl of 15 hematocrit P falciparum culture toeach well on the 96-well plate with the exception of the last4 wells of the first row Parasitized and nonparasitizederythrocytes were added into all test wells in such a way that04 parasitemia and 15 hematocrit was maintainedBefore each antiplasmodial test parasite cultures weresynchronized in a ring stage which were obtained followingserial treatment with chloroquine )e first row acted aspositive control and was therefore not treated)e first fourwells served as negative control for normal growth andcontained 200 μl of Plasmodium blank red blood cellsChloroquine substituted the extract as reference drugs(positive controls) for standardizing the experiment Afterthe plates were incubated at 37degC (3 CO2 5 O2 and 92NO2) for 48 hrs radio-labelled hypoxanthine was thenadded to each well (05 μl in 25 μl of the culture medium))e plates were further incubated for 18 hours to allow itsuptake by surviving parasites After the second incubationthe plates were frozen overnight at minus 20degC to stop thegrowth )e plates were thawed at room temperature for15 hours before harvesting Each well was harvested usingBetaplate TM cell harvester (Wallace Zurich Switzerland)which transferred the red blood cells onto a glass fibre filterwhere they were washed with distilled water )e driedfilters were then inserted into a plastic foil with 10ml ofscintillation fluid and counted in a Betaplate TM liquidscintillation counter (Wallac micro beta Trilux) )e re-sults were recorded as counts per minute (cpm) per well ateach concentration Data were transferred to MS Excelsoftware and expressed as a percentage of the untreatedcontrols )e drug concentration capable of inhibiting 50

of the P falciparum (IC50) was determined by logarithmictransformation of drug concentration and radioactivecounts per minutes (cpm) using the formula

IC50 antiloglogX1 + logY50 minus logY1( 1113857(logX2 minus logX1)

(logY2 minus logY1)1113890 1113891

(1)

where Y50 was the cpm value midway between parasitizedand nonparasitized control cultures X1 and X2 were theconcentrations and Y1 and Y2 were the cpm values re-spectively for the data points above and below the cpmmidpoints [20] )e determined SE IC50 values are classifiedin Table 1 as per criteria provided by [21]

26 Animals Handling Parasites Inoculation and Antima-larial Effect of Soybean Extracts Female Swiss albino miceaging 6ndash8 weeks old were randomly selected from KEMRIanimal facility Nairobi )ey were weighed to achieve20plusmn 2 g and housed in well-labeled standard microlon typeII cages in the experimental rooms )e relative humidity inthe rooms was maintained at 60ndash70 Additionally ap-propriate ventilation and room temperature were observed)e animals were fed with viable rodent feed and providedwith water ad libitum Each cage accommodated five miceper test sample Plasmodium berghei ANKA parasites werethe candidates for evaluation of parasite reduction in mice[21] )e parasites were obtained from Kenya Medical Re-search Institute (KEMRI) Nairobi and maintained by sub-passage in mice Blood infected with P berghei strain ANKAwas harvested from donor mice by heart puncture into 15mlcentrifuge tubes containing 1 (wv) heparin )e para-sitized blood was diluted to attain roughly 108 parasitizedred blood cells (RBCs) per ml )e experimental animalswere intraperitoneally infected with 02ml (2times107 para-sitized RBCs) and randomly grouped

27 Drugs and Administration )e soybean extracts PBSand chloroquine were administered orally to the test groupsnegative group and the positive group respectively Astainless metallic feeding cannula was used during admin-istration [22]

28 Assessment of Prophylactic Activity of Soybean Extract)e prophylactic activity of SE and chloroquine wereassessed by using the method described by Ryley and Peters[23] )e mice were randomly divided into 5 groups of 5mice each in a cage Groups 1 to 3 were orally administeredwith 200 400 and 800mgkgday of SE respectively Group4 mice were administered with 5mgkgday of chloroquine(positive control) while group 5 mice received a placeboconsisting of 3 dimethyl sulfoxide and 10 Tween 80 inPBS (negative control) Administration of the SE continuedconsecutively for 3 days ie from day 0 to day 3 On thefourth day the mice were inoculated with P berghei(ANKA) )e parasitemia level of each mouse was assessedby blood smear after 72 hours )e survival rate wasmonitored on a daily basis in all groups for 28 days

Journal of Pathogens 3

postinoculation Giemsa-stained thin blood smears wereprepared from the tail of each animal to determine para-sitemia and percentage inhibition

29 Assessment of Curative Effect of Soybean Extract )emethod to evaluate the schizontocidal activity of SE inestablished infection as described by Tona et al [24] wasused 02ml of P berghei ANKA (2times107parasitized RBCs)was injected intraperitoneally into 25 mice on the first dayTwo hours later the mice were divided randomly into fivegroups of 5 mice each)e experimental groups were treatedorally with a single 02ml dose of SE at one of the threedosage levels of 200 400 and 800mgkg in groups 1 to 3respectively For the positive control group (group 4) 5mlkgday of chloroquine was administered Group 5 whichwas the negative control received the placebo (vehicle 3dimethyl sulfoxide 10 tween 80 in PBS) )e SE and drugswere administered daily for 3 (days 0 to 3) days )in smearsof Giemsa were prepared from blood obtained from the tailcollected on each day of treatment for the purpose ofmonitoring the infection Parasitemia was determined after4 days (24 hrs after last treatment) by microscopic exami-nation by counting parasites in 4 fields of covering 100erythrocytes per view of thin blood film sampled from thetail of experiment mouse stained with 10 Giemsa solution)e difference between the mean number of parasites perview in negative control group (100) and those of ex-perimental groups was calculated and expressed as per-centage parasitemia suppression (chemo suppression) foreach group according to Tona et al [24]

parasitemia suppression(PS) (A minus B)

A1113890 1113891 times 100 (2)

where Amean parasitemia in negative control on day 4and B corresponding parasitemia in the test group

Percentage parasitemia was calculated as parasitized noof erythrocytes per 100 erythrocytes while chemo sup-pression percentage was taken as inhibition of parasitemultiplication relative to the control expressed in percent-age )e survival rate was monitored on a daily basis in allgroups for 28 days postinoculation Parasites in the negativecontrol group were assumed to have experienced 1 chemosuppression (potency of the drug to inhibit parasitemultiplication)

210 Determination of Extract Toxicity )e median lethaldose (LD50) was determined for estimating acute toxicity ofthe soybean extract using the method of Lorke [25] )emethod involved intraperitoneal administration of differentdoses (1500 2500 3500 and 5000mgkg) of SE to the four

groups of Swiss albino mice Afterwards mortality andmanifestation of physical signs in mice for possible toxicitywas monitored

211 Data and Statistical Analysis Analyzed data was pre-sented as meanplusmn standard deviation In vitro tests wereperformed in duplicate and data were evaluated byMicrosoft Excel 2016 with the aid of nonlinear regressionanalysis to determine IC50 Analysis for the in vivo work wasachieved using SPSS version 25 Within groups the statis-tical significance for contrast of parasitemia reduction andsurvival time was attained using one-way analysis of vari-ance (ANOVA) and Tukeyrsquos Honest Significant Differencepost hoc test

212 Ethical Consideration Permission to carry out thestudy was obtained from Scientific and Ethics Review Unit(SERU) (study SERU no 3795) )e experiments werecarried out in compliance with Animal Care and UseCommittee (ACUC) Regulations of KEMRI In addition theinternationally established values for laboratory animal useand care as per WHO recommendations were followedIntraperitoneal injection was conducted using gauge 23needles Death during experimental ends was done in achamber of carbon (IV) oxide gas followed by incineration

3 Results

31 Phytochemical Analysis Eight qualitative chemical testswere performed on the three soybean extracts to determinethe presence of phenols flavonoids tannins steroids al-kaloids saponins glycosides and terpenoids )e resultsshowed presence of steroids alkaloids and saponins in theaqueous extract and methanol extract (Table 2) Flavanoidssteroids glycosides and tannins were present in the peptideextract

32 Acute Toxicity Study )e results of acute toxicityshowed that the extract caused no mortality within the doserange of 1500ndash5000mgkg in the initial 24 hrs as well aswithin the successive 14 days)is was a clear indication thatdoses used were safe No physical and behavioral signs ofovertoxicity such as decreased motor activity decreasedbodylimb tone writhing respiration and death amongstothers were observed )is proposes that LD50 of the extractis greater than 5000mgKg

33 In Vitro Assessment of Antiplasmodium Activity of SE)e activities of the three different extracts of Glycine maxagainst two strains of P falciparum ranged from inactive forthe aqueous extract to moderately active for both themethanol and peptide extracts (Table 3) Methanol andpeptide extracts showed good activity against D6 and W2strains However methanol extract showed the best activityfollowed by peptide extract

Water extract had IC50gt 200 μgml thus exhibiting noactivity against the two strains of P falciparum

Table 1 Adopted classification of antiplasmodial activity

IC50 value (μgml) Category of activitygt100 Inactive50ndash100 Low10ndash50 Moderatele10 High

4 Journal of Pathogens

34 In Vivo Antimalarial Curative Activity of Methanol andPeptide Extracts of Glycine max Two soybean extracts thatdisplayed activity (methanol and peptide) in the in vitrostudies were next tested for antimalarial activity against Pberghei ANKA-infected Swiss albino mice )is was ac-complished using the 4-day suppressive method for meth-anol and peptide extracts )e results are presented inTable 4 Chemosuppresion was established in a dose-de-pendent manner after four days of antimalarial treatmentusing SE extracts (200ndash800mgkg) )e mean parasitemia ingroups treated with methanol ranged from 348plusmn 037 to599plusmn 018 while that of animals treated with peptide extractvaried from 361plusmn 027 to 587plusmn 025 )e mean parasitemiain the negative control group was 1287plusmn 026 )ere was asignificant percentage parasitemia difference between thetest groups when compared with the untreated controlgroup (Plt 0001) At 800mgkg the extracts demonstratedthe highest chemosuppresion Remarkably there was a slightdifference in chemosuppresion between the positive controland the two extracts )e extracts were able to prolongsurvival of the animals after treatment termination incomparison with the negative control

35 In Vivo Antimalarial Prophylactic Activity of PeptideExtract ofGlycinemax In prophylactic assay parasitemia inthe negative control group was significantly higher than inany of the test group (Plt 0001) All the animals in thepositive group displayed suppression of parasitemia of8309 )e peptide extracts group suppression of para-sitemia was 6466 5712 and 4314 for doses of 800400 and 200mgkg )e mean parasitemia in groups treatedwith peptide ranged from 394 + 046 to 635 + 022 )eresults are presented in Table 5 )e peptide extract was ableto prolong survival of the animals after treatment termi-nation in comparison with the negative control

4 Discussion

Many antimalarial drugs currently available on the markethave been developed from plants and natural products [25]Plasmodium falciparum resistance to the existing antima-larials necessitates the development of improved drug in-terventions [26] )e best solution to this challenge remainsto be probably therapeutic plants [27 28] Artemisinin

derivatives have been used to manage malaria for long [29]Quinine which is widely used for treatment of malaria wasobtained from Cinchona ofcinalis plants [30] Most people inAfrica have greatly relied on traditional remedies due toaffordability [30] )is study investigated antimalarial ac-tivities and safety properties of soybean (Glycine max) inorder to determine their potential as a source of a novel andcheaper antimalarial agent

)e study clearly demonstrates the potential activity ofthe Glycine max seeds extract against malaria Phyto-chemical analysis of methanol and peptide extract revealedthe presence of flavanoids alkaloids steroids and glycosidesunlike in aqueous extract which had only steroids and al-kaloids It has been reported that oral administration ofphytochemicals such as saponins tannins and phenolspossess ability to suppress cellular immunity [31] Previousresearch has shown that the amount of secondary metab-olites available in the plants under examination play agreater role in drug research Formerly alkaloids saponinsflavanoids and tannins have been demonstrated to aidantimicrobial and antimalarial activities of selected medic-inal plants [32] Notably our results were in conformity withthose of Arora [14] who reported presence of alkaloidsflavonoids tannins and saponins when determining anti-microbial effects ofGlycine max)erefore the results of thisstudy may have been influenced by single or combination ofthe mentioned phytochemical ingredients in the crude ex-tracts in exerting activity against malaria

An ideal antimalarial should be safe without any adverseeffects We ran toxicity study to evaluate the suitability of theuse of this plant extract )e outcomes indicated that theconcentrations of the extracts applied on the in vivo ex-periments were harmless with no animal death noted withinthe initial 24 h and successive 14 days at 1500ndash5000mgkgdosage Importantly animals stayed alive for the entire fourdays of the experiments indicating safety as suggested bySatayavivad et al [33] in their work

Our findings agree with those of Deharo et al [17] whocarried out the evaluation of antiplasmodial and antimalarialproperties of soybean fat emulsions )ey recorded anti-plasmodial activity of IC50 of 1302plusmn 235mgmiddotmlminus 1 upon useof Ivelip test sample indicating quick parasite inhibition Inthis study in vitro assays of peptide and methanol extractsshowed activity with IC50 of 1997plusmn 257 μgml and1014plusmn 904 μgml against D6 strain and 2861plusmn 132 μgmland 1487plusmn 343 μgml against W2 strain respectivelyHowever we differed on the in vivo assay whereby theyreported lower percentage parasite suppression upon use ofIvelip test sample ie at 32 gmiddotkgminus 1 a reduction of 35plusmn 26was recorded )e difference in the results may be possiblydue to difference of the samples used Nevertheless theirfinding and ours provide a clear picture on the potential useof this plant for malaria management In our study the invivo assay showed good activity of significant reduction inpercentage parasitemia on the test groups compared to thenegative control group (Plt 0001) In vivo antimalarialactivity falls into 3 categories of classification moderategood and very good if the extract displayed parasitemiasuppression percentage equal to or greater than 50 [34] In

Table 2 Results of the phytochemical screening of the three ex-tracts of soybean extract

Phytochemical Aqueousextract

Methanolextract

Peptideextract

Phenols ndash ndash ndashFlavonoids ndash + +Tannins ndash ndash +Steroids + + +Alkaloids + + +Saponins + + ndashGlycosides ndash + +Terpenoids ndash ndash ndashPhytochemicals are indicated as present (+) or absent (ndash)

Journal of Pathogens 5

the present study very good results were obtained in thecurative test with methanol and peptide extracts exhibitingover 50 chemosuppresion Methanol and peptide extractsexhibited high suppressive activity of 729 and 719 using800mgkg dose respectively Notably there was significantdecrease (Plt 0001) in activity with lower doses to 647and 649 at 400mgkg and 534 and 544 at 200mgkgrespectively in the curative test A maximum parasitesuppression of 729 and 719 was produced by methanoland peptide in the highest dose of 800mgkg and longestsurvival time compared to other doses )is might be due tothe fact that active compounds responsible for the anti-malarial activity mostly occur in low levels in naturalproducts and activity may not be detected in lower doses[35] Likewise in the prophylactic test the peptide extractexhibited suppressive activity of 647 at 800mgkg and571 at 400mgkg and 431 at 200mgkg Consistent withthe above exhibited results Bonkian et al [36] used twoSahelian plant extracts that showed a dose-dependent ac-tivity)ey recorded parasite reduction of 575 359 and449 at doses of 100 250 and 500mg extractbody weightof Guiera senegalensis extract respectively On the contraryBauhinia rufescens extract demonstrated parasite

suppression activity of 506 222 and 257 ElsewhereMenard et al [37] worked on induction of artemisinin re-sistance )ey were able to isolate a resistant phenotype after32 successive drug pressure passage cycles An illustration byBlasco et al [38] shows that chloroquine was the main stayfor malaria eradication after its discovery until parasiteresistance undermined its use within a short period callingfor an immediate withdrawal )is vividly explains that theuse of crude extracts have a chance to delay resistance unlikesingle compounds Additionally crude extracts act as apreliminary step for isolation of pure effective antimalarialagents Studies conducted previously on Glycine maxestablished indeed that it has antioxidant properties [11] Ithas been reported that antioxidant activity can inhibit hemepolymerization as heme as to be oxidized before polymer-ization and the unpolymerized heme is very toxic to malaria[39] )erefore this can also be assumed to be one of thefactors that lead to presence of antimalarial activity inGlycine max )e chemosuppresion data showed that par-asite clearance was much more pronounced on the fourthday)is may be attributed to high drug concentration in theblood due to repeated dosing Interestingly the parasitereduction activity exhibited on the fourth day suggests that

Table 3 In vitro antiplasmodial activity of three extracts of soybeans Glycine max

Treatment Extract yield () D6ndashIC50 (meanplusmn SD) (μgml) W2ndashIC50 (meanplusmn SD) (μgml)Aqueous extract 990 gt200 gt200Methanol extract 740 10142plusmn 9043 14867plusmn 3439Peptide extract 5750 19967plusmn 2517 28613plusmn 1324Control (CQ) 0011plusmn 3120 0091plusmn 0031

Table 4 In vivo antimalarial activities of crude extract of Glycine max in curative test on day 4 and animal survival time

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppression of parasite Mean survivaltime (days)

Methanol extract200 599plusmn 018 5345 1050plusmn 058400 454plusmn 022 6467 1125plusmn 096800 348plusmn 037 7293 1625plusmn 096

Peptide extract200 587plusmn 025 5439 1100plusmn 082400 452plusmn 013 6489 1240plusmn 114800 361plusmn 027 7190 1560plusmn 152

CQ 119plusmn 036 9072 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10tween 80 in PBS) 1287plusmn 026 500plusmn 082

lowast)e results are expressed as meanplusmn SD

Table 5 In vivo antimalarial activities of crude extract ofGlycine max in prophylactic test on day 4 postparasite exposure and animal survivaltime

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppressionof parasite

Mean survivaltime (days)

Peptide extract200 635plusmn 022 4314 750plusmn 058400 478plusmn 030 5712 925plusmn 096800 394plusmn 046 6466 1025plusmn 096

CQ 189plusmn 016 8309 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10 Tween 80 in PBS) 1116plusmn 115 500plusmn 082lowast)e results are expressed as meanplusmn SD

6 Journal of Pathogens

the bioavailability of the chemical components present in thecrude extracts is not possibly affected by biotransformationand physiological factors )e mechanism of action ofGlycine max is not yet known However existing literaturestudies have shown that some plants and seeds exhibitantiplasmodial activity either by causing red blood cellsoxidation [40] or by inhibitory protein synthesis [40]depending on their phytochemical constituents Flavonoidsare known to exert antiplasmodial activity by chelating withnucleic acid base pairing of the parasite [36] )erefore it ispossible that the antiplasmodial activity exhibited by Glycinemax could have been as a result of the abovementioned waysor by yet a different unknown mechanism )e limitation ofthe study is that identification of specific compounds re-sponsible for antimalarial activity was not accomplishedbecause this was beyond the scope of the present study)erefore we recommend that further analysis should becarried out on Glycine max seeds to identify the specificantimalarial compounds present

5 Conclusion

)e results of this study provide evidence-based activity ofGlycine max against the malaria parasite )erefore itprovides room for future exploitation of the plant in theaforementioned therapeutic effect

)e possibility of Glycine max incorporated in foodsubstances should be considered in a wake to determine ifthe body can develop immune to malaria )e use of Glycinemax in combination with other herbs for effective treatment[41ndash48]

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

KN and AM designed the study KN involved in data col-lection data analysis and preparation of the manuscriptdraft AM FK KK and JO contributed to analysis andinterpretation of data All authors contributed equally to thiswork

Acknowledgments

)e authors are sincerely thankful to the AFRICA-ai-JAPANProject for sponsoring this study )e authors are grateful toJomo Kenyatta University of Agriculture and Technology forallowing progress of the work)e authors thank the KEMRIadministration in particular the director of Centre forBiotechnology and Research Development (CBRD) and theKEMRI Animal House for provision of space in thelaboratories

Supplementary Materials

)e supplementary material consists of an excel spread sheetwhich has all the in vivo work data It includes the summaryof different extract doses negative and positive controls andhow they affected red blood cells in mice)e data were usedto determine different parameters used to measure the effectof the extract such as parasitemia average parasitemiachemosuppresion and mean survival days which are allexplained in the article (Supplementary Materials)

References

[1d] D A Bundy ldquoInvestment in child and adolescent health anddevelopment key messages from Disease Control Prioritiesrdquoe Lancet vol 391 no 10121 pp 687ndash699 2018

[2] M N Aminake and G Pradel ldquoAntimalarial drugs resistancein Plasmodium falciparum and the current strategies toovercome themrdquo in Microbial pathogens and strategies forcombating them science technology and education pp 1ndash774no 1 Formatex Research Center Badajoz Spain 2013

[3] J Recht A M Siqueira W M Monteiro S M HerreraS Herrera and M V Lacerda ldquoMalaria in Brazil ColombiaPeru and Venezuela current challenges inmalaria control andeliminationrdquo Malaria Journal vol 16 no 1 p 273 2017

[4] E A Ashley and A P Phyo ldquoDrugs in development formalariardquo Drugs vol 78 no 9 pp 861ndash879 2018

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquo)erelevance of higher plants in lead compound discoveryprogramsrdquo Journal of Natural Products vol 74 no 6pp 1539ndash1555 2011

[6] R G Ridley ldquoMedical need scientific opportunity and thedrive for antimalarial drugsrdquo Nature vol 415 no 6872pp 686ndash693 2002

[7] H Ginsburg and E Deharo ldquoldquoA call for using naturalcompounds in the development of new antimalarial treat-mentsndashan introductionrdquoMalaria Journal vol 10 no 1 p S12011

[8] P Smỳkal ldquoLegume crops phylogeny and genetic diversity forscience and breedingrdquo Critical Reviews in Plant Sciencesvol 34 no 1ndash3 pp 43ndash104 2015

[9] S A El Sohaimy ldquoFunctional foods and nutraceuticals-modern approach to food sciencerdquo World Applied SciencesJournal vol 20 no 5 pp 691ndash708 2012

[10] E C Okeke H N Eneobong A O UzuegbunamA O Ozioko and H Kuhnlein ldquoIgbo traditional food systemdocumentation uses and research needsrdquo Pakistan Journal ofNutrition vol 7 no 2 pp 365ndash376 2008

[11] D Malencic M Popovic and J Miladinovic ldquoPhenoliccontent and antioxidant properties of soybean (Glycine max(L) Merr) seedsrdquoMolecules vol 12 no 3 pp 576ndash581 2007

[12] M Y Kim G Y Jang N S Oh et al ldquoCharacteristics and invitro anti-inflammatory activities of protein extracts frompre-germinated black soybean [Glycine max (L)] treated withhigh hydrostatic pressurerdquo Innovative Food Science ampEmerging Technologies vol 43 pp 84ndash91 2017

[13] E Nuntildeez B Lopez-Corcuera J Vazquez C Gimenez andC Aragon ldquoDifferential effects of the tricyclic antidepressantamoxapine on glycine uptake mediated by the recombinantGLYT1 and GLYT2 glycine transportersrdquo British Journal ofPharmacology vol 129 no 1 pp 200ndash206 2000

[14] M Arora S Singh and R Kaur ldquoPhytochemical analysisprotein content amp antimicrobial activities of selected samples

Journal of Pathogens 7

containing 02 sodium bicarbonate 05 albumax and45 μgL hypoxanthine and 50 μgL gentamicin and incu-bated at 37degC under a gas mixture of 5 oxygen 5 carbondioxide and 90 nitrogen Infected erythrocytes weretransferred into fresh complete medium to propagate theculture daily In P falciparum (INDO strain) culture me-dium albumax was replaced by 10 pooled human serumParasitemia was determined qualitatively and quantitativelyby light microscopy using (Giemsa staining)

25 In Vitro Antiplasmodial Assays Using Soybean ExtractsTo assess the antiplasmodial activity of the SE extracts anin vitro serial microdilution assay technique that measuresthe ability of extracts to inhibit the incorporation of [G3H]-hypoxanthine by P falciparum was used [19] )e exper-iments were conducted on 96-well plates First 25 μl ofculture medium was added to all the wells of a 96-well flat-bottomedmicrotiter plate except those on the 2nd row 50 μlaliquots of the three extract solutions (aqueous methanoland peptide) were added in duplicate to the wells of thesecond row A Titertek motorized hand diluter (Flowlaboratories Uxbridge UK) was used to make two-foldserial dilution of each soybean extract over a 64-foldconcentration range )e stock solutions (100 μgml) of thethree soybean extracts were diluted two-fold down until aconcentration of 156 μgml was reached )e susceptibilitytest was performed using an initial parasitemia of 04 byadding 200 μl of 15 hematocrit P falciparum culture toeach well on the 96-well plate with the exception of the last4 wells of the first row Parasitized and nonparasitizederythrocytes were added into all test wells in such a way that04 parasitemia and 15 hematocrit was maintainedBefore each antiplasmodial test parasite cultures weresynchronized in a ring stage which were obtained followingserial treatment with chloroquine )e first row acted aspositive control and was therefore not treated)e first fourwells served as negative control for normal growth andcontained 200 μl of Plasmodium blank red blood cellsChloroquine substituted the extract as reference drugs(positive controls) for standardizing the experiment Afterthe plates were incubated at 37degC (3 CO2 5 O2 and 92NO2) for 48 hrs radio-labelled hypoxanthine was thenadded to each well (05 μl in 25 μl of the culture medium))e plates were further incubated for 18 hours to allow itsuptake by surviving parasites After the second incubationthe plates were frozen overnight at minus 20degC to stop thegrowth )e plates were thawed at room temperature for15 hours before harvesting Each well was harvested usingBetaplate TM cell harvester (Wallace Zurich Switzerland)which transferred the red blood cells onto a glass fibre filterwhere they were washed with distilled water )e driedfilters were then inserted into a plastic foil with 10ml ofscintillation fluid and counted in a Betaplate TM liquidscintillation counter (Wallac micro beta Trilux) )e re-sults were recorded as counts per minute (cpm) per well ateach concentration Data were transferred to MS Excelsoftware and expressed as a percentage of the untreatedcontrols )e drug concentration capable of inhibiting 50

of the P falciparum (IC50) was determined by logarithmictransformation of drug concentration and radioactivecounts per minutes (cpm) using the formula

IC50 antiloglogX1 + logY50 minus logY1( 1113857(logX2 minus logX1)

(logY2 minus logY1)1113890 1113891

(1)

where Y50 was the cpm value midway between parasitizedand nonparasitized control cultures X1 and X2 were theconcentrations and Y1 and Y2 were the cpm values re-spectively for the data points above and below the cpmmidpoints [20] )e determined SE IC50 values are classifiedin Table 1 as per criteria provided by [21]

26 Animals Handling Parasites Inoculation and Antima-larial Effect of Soybean Extracts Female Swiss albino miceaging 6ndash8 weeks old were randomly selected from KEMRIanimal facility Nairobi )ey were weighed to achieve20plusmn 2 g and housed in well-labeled standard microlon typeII cages in the experimental rooms )e relative humidity inthe rooms was maintained at 60ndash70 Additionally ap-propriate ventilation and room temperature were observed)e animals were fed with viable rodent feed and providedwith water ad libitum Each cage accommodated five miceper test sample Plasmodium berghei ANKA parasites werethe candidates for evaluation of parasite reduction in mice[21] )e parasites were obtained from Kenya Medical Re-search Institute (KEMRI) Nairobi and maintained by sub-passage in mice Blood infected with P berghei strain ANKAwas harvested from donor mice by heart puncture into 15mlcentrifuge tubes containing 1 (wv) heparin )e para-sitized blood was diluted to attain roughly 108 parasitizedred blood cells (RBCs) per ml )e experimental animalswere intraperitoneally infected with 02ml (2times107 para-sitized RBCs) and randomly grouped

27 Drugs and Administration )e soybean extracts PBSand chloroquine were administered orally to the test groupsnegative group and the positive group respectively Astainless metallic feeding cannula was used during admin-istration [22]

28 Assessment of Prophylactic Activity of Soybean Extract)e prophylactic activity of SE and chloroquine wereassessed by using the method described by Ryley and Peters[23] )e mice were randomly divided into 5 groups of 5mice each in a cage Groups 1 to 3 were orally administeredwith 200 400 and 800mgkgday of SE respectively Group4 mice were administered with 5mgkgday of chloroquine(positive control) while group 5 mice received a placeboconsisting of 3 dimethyl sulfoxide and 10 Tween 80 inPBS (negative control) Administration of the SE continuedconsecutively for 3 days ie from day 0 to day 3 On thefourth day the mice were inoculated with P berghei(ANKA) )e parasitemia level of each mouse was assessedby blood smear after 72 hours )e survival rate wasmonitored on a daily basis in all groups for 28 days

Journal of Pathogens 3

postinoculation Giemsa-stained thin blood smears wereprepared from the tail of each animal to determine para-sitemia and percentage inhibition

29 Assessment of Curative Effect of Soybean Extract )emethod to evaluate the schizontocidal activity of SE inestablished infection as described by Tona et al [24] wasused 02ml of P berghei ANKA (2times107parasitized RBCs)was injected intraperitoneally into 25 mice on the first dayTwo hours later the mice were divided randomly into fivegroups of 5 mice each)e experimental groups were treatedorally with a single 02ml dose of SE at one of the threedosage levels of 200 400 and 800mgkg in groups 1 to 3respectively For the positive control group (group 4) 5mlkgday of chloroquine was administered Group 5 whichwas the negative control received the placebo (vehicle 3dimethyl sulfoxide 10 tween 80 in PBS) )e SE and drugswere administered daily for 3 (days 0 to 3) days )in smearsof Giemsa were prepared from blood obtained from the tailcollected on each day of treatment for the purpose ofmonitoring the infection Parasitemia was determined after4 days (24 hrs after last treatment) by microscopic exami-nation by counting parasites in 4 fields of covering 100erythrocytes per view of thin blood film sampled from thetail of experiment mouse stained with 10 Giemsa solution)e difference between the mean number of parasites perview in negative control group (100) and those of ex-perimental groups was calculated and expressed as per-centage parasitemia suppression (chemo suppression) foreach group according to Tona et al [24]

parasitemia suppression(PS) (A minus B)

A1113890 1113891 times 100 (2)

where Amean parasitemia in negative control on day 4and B corresponding parasitemia in the test group

Percentage parasitemia was calculated as parasitized noof erythrocytes per 100 erythrocytes while chemo sup-pression percentage was taken as inhibition of parasitemultiplication relative to the control expressed in percent-age )e survival rate was monitored on a daily basis in allgroups for 28 days postinoculation Parasites in the negativecontrol group were assumed to have experienced 1 chemosuppression (potency of the drug to inhibit parasitemultiplication)

210 Determination of Extract Toxicity )e median lethaldose (LD50) was determined for estimating acute toxicity ofthe soybean extract using the method of Lorke [25] )emethod involved intraperitoneal administration of differentdoses (1500 2500 3500 and 5000mgkg) of SE to the four

groups of Swiss albino mice Afterwards mortality andmanifestation of physical signs in mice for possible toxicitywas monitored

211 Data and Statistical Analysis Analyzed data was pre-sented as meanplusmn standard deviation In vitro tests wereperformed in duplicate and data were evaluated byMicrosoft Excel 2016 with the aid of nonlinear regressionanalysis to determine IC50 Analysis for the in vivo work wasachieved using SPSS version 25 Within groups the statis-tical significance for contrast of parasitemia reduction andsurvival time was attained using one-way analysis of vari-ance (ANOVA) and Tukeyrsquos Honest Significant Differencepost hoc test

212 Ethical Consideration Permission to carry out thestudy was obtained from Scientific and Ethics Review Unit(SERU) (study SERU no 3795) )e experiments werecarried out in compliance with Animal Care and UseCommittee (ACUC) Regulations of KEMRI In addition theinternationally established values for laboratory animal useand care as per WHO recommendations were followedIntraperitoneal injection was conducted using gauge 23needles Death during experimental ends was done in achamber of carbon (IV) oxide gas followed by incineration

3 Results

31 Phytochemical Analysis Eight qualitative chemical testswere performed on the three soybean extracts to determinethe presence of phenols flavonoids tannins steroids al-kaloids saponins glycosides and terpenoids )e resultsshowed presence of steroids alkaloids and saponins in theaqueous extract and methanol extract (Table 2) Flavanoidssteroids glycosides and tannins were present in the peptideextract

32 Acute Toxicity Study )e results of acute toxicityshowed that the extract caused no mortality within the doserange of 1500ndash5000mgkg in the initial 24 hrs as well aswithin the successive 14 days)is was a clear indication thatdoses used were safe No physical and behavioral signs ofovertoxicity such as decreased motor activity decreasedbodylimb tone writhing respiration and death amongstothers were observed )is proposes that LD50 of the extractis greater than 5000mgKg

33 In Vitro Assessment of Antiplasmodium Activity of SE)e activities of the three different extracts of Glycine maxagainst two strains of P falciparum ranged from inactive forthe aqueous extract to moderately active for both themethanol and peptide extracts (Table 3) Methanol andpeptide extracts showed good activity against D6 and W2strains However methanol extract showed the best activityfollowed by peptide extract

Water extract had IC50gt 200 μgml thus exhibiting noactivity against the two strains of P falciparum

Table 1 Adopted classification of antiplasmodial activity

IC50 value (μgml) Category of activitygt100 Inactive50ndash100 Low10ndash50 Moderatele10 High

4 Journal of Pathogens

34 In Vivo Antimalarial Curative Activity of Methanol andPeptide Extracts of Glycine max Two soybean extracts thatdisplayed activity (methanol and peptide) in the in vitrostudies were next tested for antimalarial activity against Pberghei ANKA-infected Swiss albino mice )is was ac-complished using the 4-day suppressive method for meth-anol and peptide extracts )e results are presented inTable 4 Chemosuppresion was established in a dose-de-pendent manner after four days of antimalarial treatmentusing SE extracts (200ndash800mgkg) )e mean parasitemia ingroups treated with methanol ranged from 348plusmn 037 to599plusmn 018 while that of animals treated with peptide extractvaried from 361plusmn 027 to 587plusmn 025 )e mean parasitemiain the negative control group was 1287plusmn 026 )ere was asignificant percentage parasitemia difference between thetest groups when compared with the untreated controlgroup (Plt 0001) At 800mgkg the extracts demonstratedthe highest chemosuppresion Remarkably there was a slightdifference in chemosuppresion between the positive controland the two extracts )e extracts were able to prolongsurvival of the animals after treatment termination incomparison with the negative control

35 In Vivo Antimalarial Prophylactic Activity of PeptideExtract ofGlycinemax In prophylactic assay parasitemia inthe negative control group was significantly higher than inany of the test group (Plt 0001) All the animals in thepositive group displayed suppression of parasitemia of8309 )e peptide extracts group suppression of para-sitemia was 6466 5712 and 4314 for doses of 800400 and 200mgkg )e mean parasitemia in groups treatedwith peptide ranged from 394 + 046 to 635 + 022 )eresults are presented in Table 5 )e peptide extract was ableto prolong survival of the animals after treatment termi-nation in comparison with the negative control

4 Discussion

Many antimalarial drugs currently available on the markethave been developed from plants and natural products [25]Plasmodium falciparum resistance to the existing antima-larials necessitates the development of improved drug in-terventions [26] )e best solution to this challenge remainsto be probably therapeutic plants [27 28] Artemisinin

derivatives have been used to manage malaria for long [29]Quinine which is widely used for treatment of malaria wasobtained from Cinchona ofcinalis plants [30] Most people inAfrica have greatly relied on traditional remedies due toaffordability [30] )is study investigated antimalarial ac-tivities and safety properties of soybean (Glycine max) inorder to determine their potential as a source of a novel andcheaper antimalarial agent

)e study clearly demonstrates the potential activity ofthe Glycine max seeds extract against malaria Phyto-chemical analysis of methanol and peptide extract revealedthe presence of flavanoids alkaloids steroids and glycosidesunlike in aqueous extract which had only steroids and al-kaloids It has been reported that oral administration ofphytochemicals such as saponins tannins and phenolspossess ability to suppress cellular immunity [31] Previousresearch has shown that the amount of secondary metab-olites available in the plants under examination play agreater role in drug research Formerly alkaloids saponinsflavanoids and tannins have been demonstrated to aidantimicrobial and antimalarial activities of selected medic-inal plants [32] Notably our results were in conformity withthose of Arora [14] who reported presence of alkaloidsflavonoids tannins and saponins when determining anti-microbial effects ofGlycine max)erefore the results of thisstudy may have been influenced by single or combination ofthe mentioned phytochemical ingredients in the crude ex-tracts in exerting activity against malaria

An ideal antimalarial should be safe without any adverseeffects We ran toxicity study to evaluate the suitability of theuse of this plant extract )e outcomes indicated that theconcentrations of the extracts applied on the in vivo ex-periments were harmless with no animal death noted withinthe initial 24 h and successive 14 days at 1500ndash5000mgkgdosage Importantly animals stayed alive for the entire fourdays of the experiments indicating safety as suggested bySatayavivad et al [33] in their work

Our findings agree with those of Deharo et al [17] whocarried out the evaluation of antiplasmodial and antimalarialproperties of soybean fat emulsions )ey recorded anti-plasmodial activity of IC50 of 1302plusmn 235mgmiddotmlminus 1 upon useof Ivelip test sample indicating quick parasite inhibition Inthis study in vitro assays of peptide and methanol extractsshowed activity with IC50 of 1997plusmn 257 μgml and1014plusmn 904 μgml against D6 strain and 2861plusmn 132 μgmland 1487plusmn 343 μgml against W2 strain respectivelyHowever we differed on the in vivo assay whereby theyreported lower percentage parasite suppression upon use ofIvelip test sample ie at 32 gmiddotkgminus 1 a reduction of 35plusmn 26was recorded )e difference in the results may be possiblydue to difference of the samples used Nevertheless theirfinding and ours provide a clear picture on the potential useof this plant for malaria management In our study the invivo assay showed good activity of significant reduction inpercentage parasitemia on the test groups compared to thenegative control group (Plt 0001) In vivo antimalarialactivity falls into 3 categories of classification moderategood and very good if the extract displayed parasitemiasuppression percentage equal to or greater than 50 [34] In

Table 2 Results of the phytochemical screening of the three ex-tracts of soybean extract

Phytochemical Aqueousextract

Methanolextract

Peptideextract

Phenols ndash ndash ndashFlavonoids ndash + +Tannins ndash ndash +Steroids + + +Alkaloids + + +Saponins + + ndashGlycosides ndash + +Terpenoids ndash ndash ndashPhytochemicals are indicated as present (+) or absent (ndash)

Journal of Pathogens 5

the present study very good results were obtained in thecurative test with methanol and peptide extracts exhibitingover 50 chemosuppresion Methanol and peptide extractsexhibited high suppressive activity of 729 and 719 using800mgkg dose respectively Notably there was significantdecrease (Plt 0001) in activity with lower doses to 647and 649 at 400mgkg and 534 and 544 at 200mgkgrespectively in the curative test A maximum parasitesuppression of 729 and 719 was produced by methanoland peptide in the highest dose of 800mgkg and longestsurvival time compared to other doses )is might be due tothe fact that active compounds responsible for the anti-malarial activity mostly occur in low levels in naturalproducts and activity may not be detected in lower doses[35] Likewise in the prophylactic test the peptide extractexhibited suppressive activity of 647 at 800mgkg and571 at 400mgkg and 431 at 200mgkg Consistent withthe above exhibited results Bonkian et al [36] used twoSahelian plant extracts that showed a dose-dependent ac-tivity)ey recorded parasite reduction of 575 359 and449 at doses of 100 250 and 500mg extractbody weightof Guiera senegalensis extract respectively On the contraryBauhinia rufescens extract demonstrated parasite

suppression activity of 506 222 and 257 ElsewhereMenard et al [37] worked on induction of artemisinin re-sistance )ey were able to isolate a resistant phenotype after32 successive drug pressure passage cycles An illustration byBlasco et al [38] shows that chloroquine was the main stayfor malaria eradication after its discovery until parasiteresistance undermined its use within a short period callingfor an immediate withdrawal )is vividly explains that theuse of crude extracts have a chance to delay resistance unlikesingle compounds Additionally crude extracts act as apreliminary step for isolation of pure effective antimalarialagents Studies conducted previously on Glycine maxestablished indeed that it has antioxidant properties [11] Ithas been reported that antioxidant activity can inhibit hemepolymerization as heme as to be oxidized before polymer-ization and the unpolymerized heme is very toxic to malaria[39] )erefore this can also be assumed to be one of thefactors that lead to presence of antimalarial activity inGlycine max )e chemosuppresion data showed that par-asite clearance was much more pronounced on the fourthday)is may be attributed to high drug concentration in theblood due to repeated dosing Interestingly the parasitereduction activity exhibited on the fourth day suggests that

Table 3 In vitro antiplasmodial activity of three extracts of soybeans Glycine max

Treatment Extract yield () D6ndashIC50 (meanplusmn SD) (μgml) W2ndashIC50 (meanplusmn SD) (μgml)Aqueous extract 990 gt200 gt200Methanol extract 740 10142plusmn 9043 14867plusmn 3439Peptide extract 5750 19967plusmn 2517 28613plusmn 1324Control (CQ) 0011plusmn 3120 0091plusmn 0031

Table 4 In vivo antimalarial activities of crude extract of Glycine max in curative test on day 4 and animal survival time

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppression of parasite Mean survivaltime (days)

Methanol extract200 599plusmn 018 5345 1050plusmn 058400 454plusmn 022 6467 1125plusmn 096800 348plusmn 037 7293 1625plusmn 096

Peptide extract200 587plusmn 025 5439 1100plusmn 082400 452plusmn 013 6489 1240plusmn 114800 361plusmn 027 7190 1560plusmn 152

CQ 119plusmn 036 9072 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10tween 80 in PBS) 1287plusmn 026 500plusmn 082

lowast)e results are expressed as meanplusmn SD

Table 5 In vivo antimalarial activities of crude extract ofGlycine max in prophylactic test on day 4 postparasite exposure and animal survivaltime

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppressionof parasite

Mean survivaltime (days)

Peptide extract200 635plusmn 022 4314 750plusmn 058400 478plusmn 030 5712 925plusmn 096800 394plusmn 046 6466 1025plusmn 096

CQ 189plusmn 016 8309 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10 Tween 80 in PBS) 1116plusmn 115 500plusmn 082lowast)e results are expressed as meanplusmn SD

6 Journal of Pathogens

the bioavailability of the chemical components present in thecrude extracts is not possibly affected by biotransformationand physiological factors )e mechanism of action ofGlycine max is not yet known However existing literaturestudies have shown that some plants and seeds exhibitantiplasmodial activity either by causing red blood cellsoxidation [40] or by inhibitory protein synthesis [40]depending on their phytochemical constituents Flavonoidsare known to exert antiplasmodial activity by chelating withnucleic acid base pairing of the parasite [36] )erefore it ispossible that the antiplasmodial activity exhibited by Glycinemax could have been as a result of the abovementioned waysor by yet a different unknown mechanism )e limitation ofthe study is that identification of specific compounds re-sponsible for antimalarial activity was not accomplishedbecause this was beyond the scope of the present study)erefore we recommend that further analysis should becarried out on Glycine max seeds to identify the specificantimalarial compounds present

5 Conclusion

)e results of this study provide evidence-based activity ofGlycine max against the malaria parasite )erefore itprovides room for future exploitation of the plant in theaforementioned therapeutic effect

)e possibility of Glycine max incorporated in foodsubstances should be considered in a wake to determine ifthe body can develop immune to malaria )e use of Glycinemax in combination with other herbs for effective treatment[41ndash48]

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

KN and AM designed the study KN involved in data col-lection data analysis and preparation of the manuscriptdraft AM FK KK and JO contributed to analysis andinterpretation of data All authors contributed equally to thiswork

Acknowledgments

)e authors are sincerely thankful to the AFRICA-ai-JAPANProject for sponsoring this study )e authors are grateful toJomo Kenyatta University of Agriculture and Technology forallowing progress of the work)e authors thank the KEMRIadministration in particular the director of Centre forBiotechnology and Research Development (CBRD) and theKEMRI Animal House for provision of space in thelaboratories

Supplementary Materials

)e supplementary material consists of an excel spread sheetwhich has all the in vivo work data It includes the summaryof different extract doses negative and positive controls andhow they affected red blood cells in mice)e data were usedto determine different parameters used to measure the effectof the extract such as parasitemia average parasitemiachemosuppresion and mean survival days which are allexplained in the article (Supplementary Materials)

References

[1d] D A Bundy ldquoInvestment in child and adolescent health anddevelopment key messages from Disease Control Prioritiesrdquoe Lancet vol 391 no 10121 pp 687ndash699 2018

[2] M N Aminake and G Pradel ldquoAntimalarial drugs resistancein Plasmodium falciparum and the current strategies toovercome themrdquo in Microbial pathogens and strategies forcombating them science technology and education pp 1ndash774no 1 Formatex Research Center Badajoz Spain 2013

[3] J Recht A M Siqueira W M Monteiro S M HerreraS Herrera and M V Lacerda ldquoMalaria in Brazil ColombiaPeru and Venezuela current challenges inmalaria control andeliminationrdquo Malaria Journal vol 16 no 1 p 273 2017

[4] E A Ashley and A P Phyo ldquoDrugs in development formalariardquo Drugs vol 78 no 9 pp 861ndash879 2018

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquo)erelevance of higher plants in lead compound discoveryprogramsrdquo Journal of Natural Products vol 74 no 6pp 1539ndash1555 2011

[6] R G Ridley ldquoMedical need scientific opportunity and thedrive for antimalarial drugsrdquo Nature vol 415 no 6872pp 686ndash693 2002

[7] H Ginsburg and E Deharo ldquoldquoA call for using naturalcompounds in the development of new antimalarial treat-mentsndashan introductionrdquoMalaria Journal vol 10 no 1 p S12011

[8] P Smỳkal ldquoLegume crops phylogeny and genetic diversity forscience and breedingrdquo Critical Reviews in Plant Sciencesvol 34 no 1ndash3 pp 43ndash104 2015

[9] S A El Sohaimy ldquoFunctional foods and nutraceuticals-modern approach to food sciencerdquo World Applied SciencesJournal vol 20 no 5 pp 691ndash708 2012

[10] E C Okeke H N Eneobong A O UzuegbunamA O Ozioko and H Kuhnlein ldquoIgbo traditional food systemdocumentation uses and research needsrdquo Pakistan Journal ofNutrition vol 7 no 2 pp 365ndash376 2008

[11] D Malencic M Popovic and J Miladinovic ldquoPhenoliccontent and antioxidant properties of soybean (Glycine max(L) Merr) seedsrdquoMolecules vol 12 no 3 pp 576ndash581 2007

[12] M Y Kim G Y Jang N S Oh et al ldquoCharacteristics and invitro anti-inflammatory activities of protein extracts frompre-germinated black soybean [Glycine max (L)] treated withhigh hydrostatic pressurerdquo Innovative Food Science ampEmerging Technologies vol 43 pp 84ndash91 2017

[13] E Nuntildeez B Lopez-Corcuera J Vazquez C Gimenez andC Aragon ldquoDifferential effects of the tricyclic antidepressantamoxapine on glycine uptake mediated by the recombinantGLYT1 and GLYT2 glycine transportersrdquo British Journal ofPharmacology vol 129 no 1 pp 200ndash206 2000

[14] M Arora S Singh and R Kaur ldquoPhytochemical analysisprotein content amp antimicrobial activities of selected samples

Journal of Pathogens 7

postinoculation Giemsa-stained thin blood smears wereprepared from the tail of each animal to determine para-sitemia and percentage inhibition

29 Assessment of Curative Effect of Soybean Extract )emethod to evaluate the schizontocidal activity of SE inestablished infection as described by Tona et al [24] wasused 02ml of P berghei ANKA (2times107parasitized RBCs)was injected intraperitoneally into 25 mice on the first dayTwo hours later the mice were divided randomly into fivegroups of 5 mice each)e experimental groups were treatedorally with a single 02ml dose of SE at one of the threedosage levels of 200 400 and 800mgkg in groups 1 to 3respectively For the positive control group (group 4) 5mlkgday of chloroquine was administered Group 5 whichwas the negative control received the placebo (vehicle 3dimethyl sulfoxide 10 tween 80 in PBS) )e SE and drugswere administered daily for 3 (days 0 to 3) days )in smearsof Giemsa were prepared from blood obtained from the tailcollected on each day of treatment for the purpose ofmonitoring the infection Parasitemia was determined after4 days (24 hrs after last treatment) by microscopic exami-nation by counting parasites in 4 fields of covering 100erythrocytes per view of thin blood film sampled from thetail of experiment mouse stained with 10 Giemsa solution)e difference between the mean number of parasites perview in negative control group (100) and those of ex-perimental groups was calculated and expressed as per-centage parasitemia suppression (chemo suppression) foreach group according to Tona et al [24]

parasitemia suppression(PS) (A minus B)

A1113890 1113891 times 100 (2)

where Amean parasitemia in negative control on day 4and B corresponding parasitemia in the test group

Percentage parasitemia was calculated as parasitized noof erythrocytes per 100 erythrocytes while chemo sup-pression percentage was taken as inhibition of parasitemultiplication relative to the control expressed in percent-age )e survival rate was monitored on a daily basis in allgroups for 28 days postinoculation Parasites in the negativecontrol group were assumed to have experienced 1 chemosuppression (potency of the drug to inhibit parasitemultiplication)

210 Determination of Extract Toxicity )e median lethaldose (LD50) was determined for estimating acute toxicity ofthe soybean extract using the method of Lorke [25] )emethod involved intraperitoneal administration of differentdoses (1500 2500 3500 and 5000mgkg) of SE to the four

groups of Swiss albino mice Afterwards mortality andmanifestation of physical signs in mice for possible toxicitywas monitored

211 Data and Statistical Analysis Analyzed data was pre-sented as meanplusmn standard deviation In vitro tests wereperformed in duplicate and data were evaluated byMicrosoft Excel 2016 with the aid of nonlinear regressionanalysis to determine IC50 Analysis for the in vivo work wasachieved using SPSS version 25 Within groups the statis-tical significance for contrast of parasitemia reduction andsurvival time was attained using one-way analysis of vari-ance (ANOVA) and Tukeyrsquos Honest Significant Differencepost hoc test

212 Ethical Consideration Permission to carry out thestudy was obtained from Scientific and Ethics Review Unit(SERU) (study SERU no 3795) )e experiments werecarried out in compliance with Animal Care and UseCommittee (ACUC) Regulations of KEMRI In addition theinternationally established values for laboratory animal useand care as per WHO recommendations were followedIntraperitoneal injection was conducted using gauge 23needles Death during experimental ends was done in achamber of carbon (IV) oxide gas followed by incineration

3 Results

31 Phytochemical Analysis Eight qualitative chemical testswere performed on the three soybean extracts to determinethe presence of phenols flavonoids tannins steroids al-kaloids saponins glycosides and terpenoids )e resultsshowed presence of steroids alkaloids and saponins in theaqueous extract and methanol extract (Table 2) Flavanoidssteroids glycosides and tannins were present in the peptideextract

32 Acute Toxicity Study )e results of acute toxicityshowed that the extract caused no mortality within the doserange of 1500ndash5000mgkg in the initial 24 hrs as well aswithin the successive 14 days)is was a clear indication thatdoses used were safe No physical and behavioral signs ofovertoxicity such as decreased motor activity decreasedbodylimb tone writhing respiration and death amongstothers were observed )is proposes that LD50 of the extractis greater than 5000mgKg

33 In Vitro Assessment of Antiplasmodium Activity of SE)e activities of the three different extracts of Glycine maxagainst two strains of P falciparum ranged from inactive forthe aqueous extract to moderately active for both themethanol and peptide extracts (Table 3) Methanol andpeptide extracts showed good activity against D6 and W2strains However methanol extract showed the best activityfollowed by peptide extract

Water extract had IC50gt 200 μgml thus exhibiting noactivity against the two strains of P falciparum

Table 1 Adopted classification of antiplasmodial activity

IC50 value (μgml) Category of activitygt100 Inactive50ndash100 Low10ndash50 Moderatele10 High

4 Journal of Pathogens

34 In Vivo Antimalarial Curative Activity of Methanol andPeptide Extracts of Glycine max Two soybean extracts thatdisplayed activity (methanol and peptide) in the in vitrostudies were next tested for antimalarial activity against Pberghei ANKA-infected Swiss albino mice )is was ac-complished using the 4-day suppressive method for meth-anol and peptide extracts )e results are presented inTable 4 Chemosuppresion was established in a dose-de-pendent manner after four days of antimalarial treatmentusing SE extracts (200ndash800mgkg) )e mean parasitemia ingroups treated with methanol ranged from 348plusmn 037 to599plusmn 018 while that of animals treated with peptide extractvaried from 361plusmn 027 to 587plusmn 025 )e mean parasitemiain the negative control group was 1287plusmn 026 )ere was asignificant percentage parasitemia difference between thetest groups when compared with the untreated controlgroup (Plt 0001) At 800mgkg the extracts demonstratedthe highest chemosuppresion Remarkably there was a slightdifference in chemosuppresion between the positive controland the two extracts )e extracts were able to prolongsurvival of the animals after treatment termination incomparison with the negative control

35 In Vivo Antimalarial Prophylactic Activity of PeptideExtract ofGlycinemax In prophylactic assay parasitemia inthe negative control group was significantly higher than inany of the test group (Plt 0001) All the animals in thepositive group displayed suppression of parasitemia of8309 )e peptide extracts group suppression of para-sitemia was 6466 5712 and 4314 for doses of 800400 and 200mgkg )e mean parasitemia in groups treatedwith peptide ranged from 394 + 046 to 635 + 022 )eresults are presented in Table 5 )e peptide extract was ableto prolong survival of the animals after treatment termi-nation in comparison with the negative control

4 Discussion

Many antimalarial drugs currently available on the markethave been developed from plants and natural products [25]Plasmodium falciparum resistance to the existing antima-larials necessitates the development of improved drug in-terventions [26] )e best solution to this challenge remainsto be probably therapeutic plants [27 28] Artemisinin

derivatives have been used to manage malaria for long [29]Quinine which is widely used for treatment of malaria wasobtained from Cinchona ofcinalis plants [30] Most people inAfrica have greatly relied on traditional remedies due toaffordability [30] )is study investigated antimalarial ac-tivities and safety properties of soybean (Glycine max) inorder to determine their potential as a source of a novel andcheaper antimalarial agent

)e study clearly demonstrates the potential activity ofthe Glycine max seeds extract against malaria Phyto-chemical analysis of methanol and peptide extract revealedthe presence of flavanoids alkaloids steroids and glycosidesunlike in aqueous extract which had only steroids and al-kaloids It has been reported that oral administration ofphytochemicals such as saponins tannins and phenolspossess ability to suppress cellular immunity [31] Previousresearch has shown that the amount of secondary metab-olites available in the plants under examination play agreater role in drug research Formerly alkaloids saponinsflavanoids and tannins have been demonstrated to aidantimicrobial and antimalarial activities of selected medic-inal plants [32] Notably our results were in conformity withthose of Arora [14] who reported presence of alkaloidsflavonoids tannins and saponins when determining anti-microbial effects ofGlycine max)erefore the results of thisstudy may have been influenced by single or combination ofthe mentioned phytochemical ingredients in the crude ex-tracts in exerting activity against malaria

An ideal antimalarial should be safe without any adverseeffects We ran toxicity study to evaluate the suitability of theuse of this plant extract )e outcomes indicated that theconcentrations of the extracts applied on the in vivo ex-periments were harmless with no animal death noted withinthe initial 24 h and successive 14 days at 1500ndash5000mgkgdosage Importantly animals stayed alive for the entire fourdays of the experiments indicating safety as suggested bySatayavivad et al [33] in their work

Our findings agree with those of Deharo et al [17] whocarried out the evaluation of antiplasmodial and antimalarialproperties of soybean fat emulsions )ey recorded anti-plasmodial activity of IC50 of 1302plusmn 235mgmiddotmlminus 1 upon useof Ivelip test sample indicating quick parasite inhibition Inthis study in vitro assays of peptide and methanol extractsshowed activity with IC50 of 1997plusmn 257 μgml and1014plusmn 904 μgml against D6 strain and 2861plusmn 132 μgmland 1487plusmn 343 μgml against W2 strain respectivelyHowever we differed on the in vivo assay whereby theyreported lower percentage parasite suppression upon use ofIvelip test sample ie at 32 gmiddotkgminus 1 a reduction of 35plusmn 26was recorded )e difference in the results may be possiblydue to difference of the samples used Nevertheless theirfinding and ours provide a clear picture on the potential useof this plant for malaria management In our study the invivo assay showed good activity of significant reduction inpercentage parasitemia on the test groups compared to thenegative control group (Plt 0001) In vivo antimalarialactivity falls into 3 categories of classification moderategood and very good if the extract displayed parasitemiasuppression percentage equal to or greater than 50 [34] In

Table 2 Results of the phytochemical screening of the three ex-tracts of soybean extract

Phytochemical Aqueousextract

Methanolextract

Peptideextract

Phenols ndash ndash ndashFlavonoids ndash + +Tannins ndash ndash +Steroids + + +Alkaloids + + +Saponins + + ndashGlycosides ndash + +Terpenoids ndash ndash ndashPhytochemicals are indicated as present (+) or absent (ndash)

Journal of Pathogens 5

the present study very good results were obtained in thecurative test with methanol and peptide extracts exhibitingover 50 chemosuppresion Methanol and peptide extractsexhibited high suppressive activity of 729 and 719 using800mgkg dose respectively Notably there was significantdecrease (Plt 0001) in activity with lower doses to 647and 649 at 400mgkg and 534 and 544 at 200mgkgrespectively in the curative test A maximum parasitesuppression of 729 and 719 was produced by methanoland peptide in the highest dose of 800mgkg and longestsurvival time compared to other doses )is might be due tothe fact that active compounds responsible for the anti-malarial activity mostly occur in low levels in naturalproducts and activity may not be detected in lower doses[35] Likewise in the prophylactic test the peptide extractexhibited suppressive activity of 647 at 800mgkg and571 at 400mgkg and 431 at 200mgkg Consistent withthe above exhibited results Bonkian et al [36] used twoSahelian plant extracts that showed a dose-dependent ac-tivity)ey recorded parasite reduction of 575 359 and449 at doses of 100 250 and 500mg extractbody weightof Guiera senegalensis extract respectively On the contraryBauhinia rufescens extract demonstrated parasite

suppression activity of 506 222 and 257 ElsewhereMenard et al [37] worked on induction of artemisinin re-sistance )ey were able to isolate a resistant phenotype after32 successive drug pressure passage cycles An illustration byBlasco et al [38] shows that chloroquine was the main stayfor malaria eradication after its discovery until parasiteresistance undermined its use within a short period callingfor an immediate withdrawal )is vividly explains that theuse of crude extracts have a chance to delay resistance unlikesingle compounds Additionally crude extracts act as apreliminary step for isolation of pure effective antimalarialagents Studies conducted previously on Glycine maxestablished indeed that it has antioxidant properties [11] Ithas been reported that antioxidant activity can inhibit hemepolymerization as heme as to be oxidized before polymer-ization and the unpolymerized heme is very toxic to malaria[39] )erefore this can also be assumed to be one of thefactors that lead to presence of antimalarial activity inGlycine max )e chemosuppresion data showed that par-asite clearance was much more pronounced on the fourthday)is may be attributed to high drug concentration in theblood due to repeated dosing Interestingly the parasitereduction activity exhibited on the fourth day suggests that

Table 3 In vitro antiplasmodial activity of three extracts of soybeans Glycine max

Treatment Extract yield () D6ndashIC50 (meanplusmn SD) (μgml) W2ndashIC50 (meanplusmn SD) (μgml)Aqueous extract 990 gt200 gt200Methanol extract 740 10142plusmn 9043 14867plusmn 3439Peptide extract 5750 19967plusmn 2517 28613plusmn 1324Control (CQ) 0011plusmn 3120 0091plusmn 0031

Table 4 In vivo antimalarial activities of crude extract of Glycine max in curative test on day 4 and animal survival time

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppression of parasite Mean survivaltime (days)

Methanol extract200 599plusmn 018 5345 1050plusmn 058400 454plusmn 022 6467 1125plusmn 096800 348plusmn 037 7293 1625plusmn 096

Peptide extract200 587plusmn 025 5439 1100plusmn 082400 452plusmn 013 6489 1240plusmn 114800 361plusmn 027 7190 1560plusmn 152

CQ 119plusmn 036 9072 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10tween 80 in PBS) 1287plusmn 026 500plusmn 082

lowast)e results are expressed as meanplusmn SD

Table 5 In vivo antimalarial activities of crude extract ofGlycine max in prophylactic test on day 4 postparasite exposure and animal survivaltime

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppressionof parasite

Mean survivaltime (days)

Peptide extract200 635plusmn 022 4314 750plusmn 058400 478plusmn 030 5712 925plusmn 096800 394plusmn 046 6466 1025plusmn 096

CQ 189plusmn 016 8309 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10 Tween 80 in PBS) 1116plusmn 115 500plusmn 082lowast)e results are expressed as meanplusmn SD

6 Journal of Pathogens

the bioavailability of the chemical components present in thecrude extracts is not possibly affected by biotransformationand physiological factors )e mechanism of action ofGlycine max is not yet known However existing literaturestudies have shown that some plants and seeds exhibitantiplasmodial activity either by causing red blood cellsoxidation [40] or by inhibitory protein synthesis [40]depending on their phytochemical constituents Flavonoidsare known to exert antiplasmodial activity by chelating withnucleic acid base pairing of the parasite [36] )erefore it ispossible that the antiplasmodial activity exhibited by Glycinemax could have been as a result of the abovementioned waysor by yet a different unknown mechanism )e limitation ofthe study is that identification of specific compounds re-sponsible for antimalarial activity was not accomplishedbecause this was beyond the scope of the present study)erefore we recommend that further analysis should becarried out on Glycine max seeds to identify the specificantimalarial compounds present

5 Conclusion

)e results of this study provide evidence-based activity ofGlycine max against the malaria parasite )erefore itprovides room for future exploitation of the plant in theaforementioned therapeutic effect

)e possibility of Glycine max incorporated in foodsubstances should be considered in a wake to determine ifthe body can develop immune to malaria )e use of Glycinemax in combination with other herbs for effective treatment[41ndash48]

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

KN and AM designed the study KN involved in data col-lection data analysis and preparation of the manuscriptdraft AM FK KK and JO contributed to analysis andinterpretation of data All authors contributed equally to thiswork

Acknowledgments

)e authors are sincerely thankful to the AFRICA-ai-JAPANProject for sponsoring this study )e authors are grateful toJomo Kenyatta University of Agriculture and Technology forallowing progress of the work)e authors thank the KEMRIadministration in particular the director of Centre forBiotechnology and Research Development (CBRD) and theKEMRI Animal House for provision of space in thelaboratories

Supplementary Materials

)e supplementary material consists of an excel spread sheetwhich has all the in vivo work data It includes the summaryof different extract doses negative and positive controls andhow they affected red blood cells in mice)e data were usedto determine different parameters used to measure the effectof the extract such as parasitemia average parasitemiachemosuppresion and mean survival days which are allexplained in the article (Supplementary Materials)

References

[1d] D A Bundy ldquoInvestment in child and adolescent health anddevelopment key messages from Disease Control Prioritiesrdquoe Lancet vol 391 no 10121 pp 687ndash699 2018

[2] M N Aminake and G Pradel ldquoAntimalarial drugs resistancein Plasmodium falciparum and the current strategies toovercome themrdquo in Microbial pathogens and strategies forcombating them science technology and education pp 1ndash774no 1 Formatex Research Center Badajoz Spain 2013

[3] J Recht A M Siqueira W M Monteiro S M HerreraS Herrera and M V Lacerda ldquoMalaria in Brazil ColombiaPeru and Venezuela current challenges inmalaria control andeliminationrdquo Malaria Journal vol 16 no 1 p 273 2017

[4] E A Ashley and A P Phyo ldquoDrugs in development formalariardquo Drugs vol 78 no 9 pp 861ndash879 2018

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquo)erelevance of higher plants in lead compound discoveryprogramsrdquo Journal of Natural Products vol 74 no 6pp 1539ndash1555 2011

[6] R G Ridley ldquoMedical need scientific opportunity and thedrive for antimalarial drugsrdquo Nature vol 415 no 6872pp 686ndash693 2002

[7] H Ginsburg and E Deharo ldquoldquoA call for using naturalcompounds in the development of new antimalarial treat-mentsndashan introductionrdquoMalaria Journal vol 10 no 1 p S12011

[8] P Smỳkal ldquoLegume crops phylogeny and genetic diversity forscience and breedingrdquo Critical Reviews in Plant Sciencesvol 34 no 1ndash3 pp 43ndash104 2015

[9] S A El Sohaimy ldquoFunctional foods and nutraceuticals-modern approach to food sciencerdquo World Applied SciencesJournal vol 20 no 5 pp 691ndash708 2012

[10] E C Okeke H N Eneobong A O UzuegbunamA O Ozioko and H Kuhnlein ldquoIgbo traditional food systemdocumentation uses and research needsrdquo Pakistan Journal ofNutrition vol 7 no 2 pp 365ndash376 2008

[11] D Malencic M Popovic and J Miladinovic ldquoPhenoliccontent and antioxidant properties of soybean (Glycine max(L) Merr) seedsrdquoMolecules vol 12 no 3 pp 576ndash581 2007

[12] M Y Kim G Y Jang N S Oh et al ldquoCharacteristics and invitro anti-inflammatory activities of protein extracts frompre-germinated black soybean [Glycine max (L)] treated withhigh hydrostatic pressurerdquo Innovative Food Science ampEmerging Technologies vol 43 pp 84ndash91 2017

[13] E Nuntildeez B Lopez-Corcuera J Vazquez C Gimenez andC Aragon ldquoDifferential effects of the tricyclic antidepressantamoxapine on glycine uptake mediated by the recombinantGLYT1 and GLYT2 glycine transportersrdquo British Journal ofPharmacology vol 129 no 1 pp 200ndash206 2000

[14] M Arora S Singh and R Kaur ldquoPhytochemical analysisprotein content amp antimicrobial activities of selected samples

Journal of Pathogens 7

34 In Vivo Antimalarial Curative Activity of Methanol andPeptide Extracts of Glycine max Two soybean extracts thatdisplayed activity (methanol and peptide) in the in vitrostudies were next tested for antimalarial activity against Pberghei ANKA-infected Swiss albino mice )is was ac-complished using the 4-day suppressive method for meth-anol and peptide extracts )e results are presented inTable 4 Chemosuppresion was established in a dose-de-pendent manner after four days of antimalarial treatmentusing SE extracts (200ndash800mgkg) )e mean parasitemia ingroups treated with methanol ranged from 348plusmn 037 to599plusmn 018 while that of animals treated with peptide extractvaried from 361plusmn 027 to 587plusmn 025 )e mean parasitemiain the negative control group was 1287plusmn 026 )ere was asignificant percentage parasitemia difference between thetest groups when compared with the untreated controlgroup (Plt 0001) At 800mgkg the extracts demonstratedthe highest chemosuppresion Remarkably there was a slightdifference in chemosuppresion between the positive controland the two extracts )e extracts were able to prolongsurvival of the animals after treatment termination incomparison with the negative control

35 In Vivo Antimalarial Prophylactic Activity of PeptideExtract ofGlycinemax In prophylactic assay parasitemia inthe negative control group was significantly higher than inany of the test group (Plt 0001) All the animals in thepositive group displayed suppression of parasitemia of8309 )e peptide extracts group suppression of para-sitemia was 6466 5712 and 4314 for doses of 800400 and 200mgkg )e mean parasitemia in groups treatedwith peptide ranged from 394 + 046 to 635 + 022 )eresults are presented in Table 5 )e peptide extract was ableto prolong survival of the animals after treatment termi-nation in comparison with the negative control

4 Discussion

Many antimalarial drugs currently available on the markethave been developed from plants and natural products [25]Plasmodium falciparum resistance to the existing antima-larials necessitates the development of improved drug in-terventions [26] )e best solution to this challenge remainsto be probably therapeutic plants [27 28] Artemisinin

derivatives have been used to manage malaria for long [29]Quinine which is widely used for treatment of malaria wasobtained from Cinchona ofcinalis plants [30] Most people inAfrica have greatly relied on traditional remedies due toaffordability [30] )is study investigated antimalarial ac-tivities and safety properties of soybean (Glycine max) inorder to determine their potential as a source of a novel andcheaper antimalarial agent

)e study clearly demonstrates the potential activity ofthe Glycine max seeds extract against malaria Phyto-chemical analysis of methanol and peptide extract revealedthe presence of flavanoids alkaloids steroids and glycosidesunlike in aqueous extract which had only steroids and al-kaloids It has been reported that oral administration ofphytochemicals such as saponins tannins and phenolspossess ability to suppress cellular immunity [31] Previousresearch has shown that the amount of secondary metab-olites available in the plants under examination play agreater role in drug research Formerly alkaloids saponinsflavanoids and tannins have been demonstrated to aidantimicrobial and antimalarial activities of selected medic-inal plants [32] Notably our results were in conformity withthose of Arora [14] who reported presence of alkaloidsflavonoids tannins and saponins when determining anti-microbial effects ofGlycine max)erefore the results of thisstudy may have been influenced by single or combination ofthe mentioned phytochemical ingredients in the crude ex-tracts in exerting activity against malaria

An ideal antimalarial should be safe without any adverseeffects We ran toxicity study to evaluate the suitability of theuse of this plant extract )e outcomes indicated that theconcentrations of the extracts applied on the in vivo ex-periments were harmless with no animal death noted withinthe initial 24 h and successive 14 days at 1500ndash5000mgkgdosage Importantly animals stayed alive for the entire fourdays of the experiments indicating safety as suggested bySatayavivad et al [33] in their work

Our findings agree with those of Deharo et al [17] whocarried out the evaluation of antiplasmodial and antimalarialproperties of soybean fat emulsions )ey recorded anti-plasmodial activity of IC50 of 1302plusmn 235mgmiddotmlminus 1 upon useof Ivelip test sample indicating quick parasite inhibition Inthis study in vitro assays of peptide and methanol extractsshowed activity with IC50 of 1997plusmn 257 μgml and1014plusmn 904 μgml against D6 strain and 2861plusmn 132 μgmland 1487plusmn 343 μgml against W2 strain respectivelyHowever we differed on the in vivo assay whereby theyreported lower percentage parasite suppression upon use ofIvelip test sample ie at 32 gmiddotkgminus 1 a reduction of 35plusmn 26was recorded )e difference in the results may be possiblydue to difference of the samples used Nevertheless theirfinding and ours provide a clear picture on the potential useof this plant for malaria management In our study the invivo assay showed good activity of significant reduction inpercentage parasitemia on the test groups compared to thenegative control group (Plt 0001) In vivo antimalarialactivity falls into 3 categories of classification moderategood and very good if the extract displayed parasitemiasuppression percentage equal to or greater than 50 [34] In

Table 2 Results of the phytochemical screening of the three ex-tracts of soybean extract

Phytochemical Aqueousextract

Methanolextract

Peptideextract

Phenols ndash ndash ndashFlavonoids ndash + +Tannins ndash ndash +Steroids + + +Alkaloids + + +Saponins + + ndashGlycosides ndash + +Terpenoids ndash ndash ndashPhytochemicals are indicated as present (+) or absent (ndash)

Journal of Pathogens 5

the present study very good results were obtained in thecurative test with methanol and peptide extracts exhibitingover 50 chemosuppresion Methanol and peptide extractsexhibited high suppressive activity of 729 and 719 using800mgkg dose respectively Notably there was significantdecrease (Plt 0001) in activity with lower doses to 647and 649 at 400mgkg and 534 and 544 at 200mgkgrespectively in the curative test A maximum parasitesuppression of 729 and 719 was produced by methanoland peptide in the highest dose of 800mgkg and longestsurvival time compared to other doses )is might be due tothe fact that active compounds responsible for the anti-malarial activity mostly occur in low levels in naturalproducts and activity may not be detected in lower doses[35] Likewise in the prophylactic test the peptide extractexhibited suppressive activity of 647 at 800mgkg and571 at 400mgkg and 431 at 200mgkg Consistent withthe above exhibited results Bonkian et al [36] used twoSahelian plant extracts that showed a dose-dependent ac-tivity)ey recorded parasite reduction of 575 359 and449 at doses of 100 250 and 500mg extractbody weightof Guiera senegalensis extract respectively On the contraryBauhinia rufescens extract demonstrated parasite

suppression activity of 506 222 and 257 ElsewhereMenard et al [37] worked on induction of artemisinin re-sistance )ey were able to isolate a resistant phenotype after32 successive drug pressure passage cycles An illustration byBlasco et al [38] shows that chloroquine was the main stayfor malaria eradication after its discovery until parasiteresistance undermined its use within a short period callingfor an immediate withdrawal )is vividly explains that theuse of crude extracts have a chance to delay resistance unlikesingle compounds Additionally crude extracts act as apreliminary step for isolation of pure effective antimalarialagents Studies conducted previously on Glycine maxestablished indeed that it has antioxidant properties [11] Ithas been reported that antioxidant activity can inhibit hemepolymerization as heme as to be oxidized before polymer-ization and the unpolymerized heme is very toxic to malaria[39] )erefore this can also be assumed to be one of thefactors that lead to presence of antimalarial activity inGlycine max )e chemosuppresion data showed that par-asite clearance was much more pronounced on the fourthday)is may be attributed to high drug concentration in theblood due to repeated dosing Interestingly the parasitereduction activity exhibited on the fourth day suggests that

Table 3 In vitro antiplasmodial activity of three extracts of soybeans Glycine max

Treatment Extract yield () D6ndashIC50 (meanplusmn SD) (μgml) W2ndashIC50 (meanplusmn SD) (μgml)Aqueous extract 990 gt200 gt200Methanol extract 740 10142plusmn 9043 14867plusmn 3439Peptide extract 5750 19967plusmn 2517 28613plusmn 1324Control (CQ) 0011plusmn 3120 0091plusmn 0031

Table 4 In vivo antimalarial activities of crude extract of Glycine max in curative test on day 4 and animal survival time

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppression of parasite Mean survivaltime (days)

Methanol extract200 599plusmn 018 5345 1050plusmn 058400 454plusmn 022 6467 1125plusmn 096800 348plusmn 037 7293 1625plusmn 096

Peptide extract200 587plusmn 025 5439 1100plusmn 082400 452plusmn 013 6489 1240plusmn 114800 361plusmn 027 7190 1560plusmn 152

CQ 119plusmn 036 9072 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10tween 80 in PBS) 1287plusmn 026 500plusmn 082

lowast)e results are expressed as meanplusmn SD

Table 5 In vivo antimalarial activities of crude extract ofGlycine max in prophylactic test on day 4 postparasite exposure and animal survivaltime

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppressionof parasite

Mean survivaltime (days)

Peptide extract200 635plusmn 022 4314 750plusmn 058400 478plusmn 030 5712 925plusmn 096800 394plusmn 046 6466 1025plusmn 096

CQ 189plusmn 016 8309 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10 Tween 80 in PBS) 1116plusmn 115 500plusmn 082lowast)e results are expressed as meanplusmn SD

6 Journal of Pathogens

the bioavailability of the chemical components present in thecrude extracts is not possibly affected by biotransformationand physiological factors )e mechanism of action ofGlycine max is not yet known However existing literaturestudies have shown that some plants and seeds exhibitantiplasmodial activity either by causing red blood cellsoxidation [40] or by inhibitory protein synthesis [40]depending on their phytochemical constituents Flavonoidsare known to exert antiplasmodial activity by chelating withnucleic acid base pairing of the parasite [36] )erefore it ispossible that the antiplasmodial activity exhibited by Glycinemax could have been as a result of the abovementioned waysor by yet a different unknown mechanism )e limitation ofthe study is that identification of specific compounds re-sponsible for antimalarial activity was not accomplishedbecause this was beyond the scope of the present study)erefore we recommend that further analysis should becarried out on Glycine max seeds to identify the specificantimalarial compounds present

5 Conclusion

)e results of this study provide evidence-based activity ofGlycine max against the malaria parasite )erefore itprovides room for future exploitation of the plant in theaforementioned therapeutic effect

)e possibility of Glycine max incorporated in foodsubstances should be considered in a wake to determine ifthe body can develop immune to malaria )e use of Glycinemax in combination with other herbs for effective treatment[41ndash48]

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

KN and AM designed the study KN involved in data col-lection data analysis and preparation of the manuscriptdraft AM FK KK and JO contributed to analysis andinterpretation of data All authors contributed equally to thiswork

Acknowledgments

)e authors are sincerely thankful to the AFRICA-ai-JAPANProject for sponsoring this study )e authors are grateful toJomo Kenyatta University of Agriculture and Technology forallowing progress of the work)e authors thank the KEMRIadministration in particular the director of Centre forBiotechnology and Research Development (CBRD) and theKEMRI Animal House for provision of space in thelaboratories

Supplementary Materials

)e supplementary material consists of an excel spread sheetwhich has all the in vivo work data It includes the summaryof different extract doses negative and positive controls andhow they affected red blood cells in mice)e data were usedto determine different parameters used to measure the effectof the extract such as parasitemia average parasitemiachemosuppresion and mean survival days which are allexplained in the article (Supplementary Materials)

References

[1d] D A Bundy ldquoInvestment in child and adolescent health anddevelopment key messages from Disease Control Prioritiesrdquoe Lancet vol 391 no 10121 pp 687ndash699 2018

[2] M N Aminake and G Pradel ldquoAntimalarial drugs resistancein Plasmodium falciparum and the current strategies toovercome themrdquo in Microbial pathogens and strategies forcombating them science technology and education pp 1ndash774no 1 Formatex Research Center Badajoz Spain 2013

[3] J Recht A M Siqueira W M Monteiro S M HerreraS Herrera and M V Lacerda ldquoMalaria in Brazil ColombiaPeru and Venezuela current challenges inmalaria control andeliminationrdquo Malaria Journal vol 16 no 1 p 273 2017

[4] E A Ashley and A P Phyo ldquoDrugs in development formalariardquo Drugs vol 78 no 9 pp 861ndash879 2018

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquo)erelevance of higher plants in lead compound discoveryprogramsrdquo Journal of Natural Products vol 74 no 6pp 1539ndash1555 2011

[6] R G Ridley ldquoMedical need scientific opportunity and thedrive for antimalarial drugsrdquo Nature vol 415 no 6872pp 686ndash693 2002

[7] H Ginsburg and E Deharo ldquoldquoA call for using naturalcompounds in the development of new antimalarial treat-mentsndashan introductionrdquoMalaria Journal vol 10 no 1 p S12011

[8] P Smỳkal ldquoLegume crops phylogeny and genetic diversity forscience and breedingrdquo Critical Reviews in Plant Sciencesvol 34 no 1ndash3 pp 43ndash104 2015

[9] S A El Sohaimy ldquoFunctional foods and nutraceuticals-modern approach to food sciencerdquo World Applied SciencesJournal vol 20 no 5 pp 691ndash708 2012

[10] E C Okeke H N Eneobong A O UzuegbunamA O Ozioko and H Kuhnlein ldquoIgbo traditional food systemdocumentation uses and research needsrdquo Pakistan Journal ofNutrition vol 7 no 2 pp 365ndash376 2008

[11] D Malencic M Popovic and J Miladinovic ldquoPhenoliccontent and antioxidant properties of soybean (Glycine max(L) Merr) seedsrdquoMolecules vol 12 no 3 pp 576ndash581 2007

[12] M Y Kim G Y Jang N S Oh et al ldquoCharacteristics and invitro anti-inflammatory activities of protein extracts frompre-germinated black soybean [Glycine max (L)] treated withhigh hydrostatic pressurerdquo Innovative Food Science ampEmerging Technologies vol 43 pp 84ndash91 2017

[13] E Nuntildeez B Lopez-Corcuera J Vazquez C Gimenez andC Aragon ldquoDifferential effects of the tricyclic antidepressantamoxapine on glycine uptake mediated by the recombinantGLYT1 and GLYT2 glycine transportersrdquo British Journal ofPharmacology vol 129 no 1 pp 200ndash206 2000

[14] M Arora S Singh and R Kaur ldquoPhytochemical analysisprotein content amp antimicrobial activities of selected samples

Journal of Pathogens 7

the present study very good results were obtained in thecurative test with methanol and peptide extracts exhibitingover 50 chemosuppresion Methanol and peptide extractsexhibited high suppressive activity of 729 and 719 using800mgkg dose respectively Notably there was significantdecrease (Plt 0001) in activity with lower doses to 647and 649 at 400mgkg and 534 and 544 at 200mgkgrespectively in the curative test A maximum parasitesuppression of 729 and 719 was produced by methanoland peptide in the highest dose of 800mgkg and longestsurvival time compared to other doses )is might be due tothe fact that active compounds responsible for the anti-malarial activity mostly occur in low levels in naturalproducts and activity may not be detected in lower doses[35] Likewise in the prophylactic test the peptide extractexhibited suppressive activity of 647 at 800mgkg and571 at 400mgkg and 431 at 200mgkg Consistent withthe above exhibited results Bonkian et al [36] used twoSahelian plant extracts that showed a dose-dependent ac-tivity)ey recorded parasite reduction of 575 359 and449 at doses of 100 250 and 500mg extractbody weightof Guiera senegalensis extract respectively On the contraryBauhinia rufescens extract demonstrated parasite

suppression activity of 506 222 and 257 ElsewhereMenard et al [37] worked on induction of artemisinin re-sistance )ey were able to isolate a resistant phenotype after32 successive drug pressure passage cycles An illustration byBlasco et al [38] shows that chloroquine was the main stayfor malaria eradication after its discovery until parasiteresistance undermined its use within a short period callingfor an immediate withdrawal )is vividly explains that theuse of crude extracts have a chance to delay resistance unlikesingle compounds Additionally crude extracts act as apreliminary step for isolation of pure effective antimalarialagents Studies conducted previously on Glycine maxestablished indeed that it has antioxidant properties [11] Ithas been reported that antioxidant activity can inhibit hemepolymerization as heme as to be oxidized before polymer-ization and the unpolymerized heme is very toxic to malaria[39] )erefore this can also be assumed to be one of thefactors that lead to presence of antimalarial activity inGlycine max )e chemosuppresion data showed that par-asite clearance was much more pronounced on the fourthday)is may be attributed to high drug concentration in theblood due to repeated dosing Interestingly the parasitereduction activity exhibited on the fourth day suggests that

Table 3 In vitro antiplasmodial activity of three extracts of soybeans Glycine max

Treatment Extract yield () D6ndashIC50 (meanplusmn SD) (μgml) W2ndashIC50 (meanplusmn SD) (μgml)Aqueous extract 990 gt200 gt200Methanol extract 740 10142plusmn 9043 14867plusmn 3439Peptide extract 5750 19967plusmn 2517 28613plusmn 1324Control (CQ) 0011plusmn 3120 0091plusmn 0031

Table 4 In vivo antimalarial activities of crude extract of Glycine max in curative test on day 4 and animal survival time

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppression of parasite Mean survivaltime (days)

Methanol extract200 599plusmn 018 5345 1050plusmn 058400 454plusmn 022 6467 1125plusmn 096800 348plusmn 037 7293 1625plusmn 096

Peptide extract200 587plusmn 025 5439 1100plusmn 082400 452plusmn 013 6489 1240plusmn 114800 361plusmn 027 7190 1560plusmn 152

CQ 119plusmn 036 9072 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10tween 80 in PBS) 1287plusmn 026 500plusmn 082

lowast)e results are expressed as meanplusmn SD

Table 5 In vivo antimalarial activities of crude extract ofGlycine max in prophylactic test on day 4 postparasite exposure and animal survivaltime

Treatment Dose (mgkg) Meanplusmn SD parasitemia () suppressionof parasite

Mean survivaltime (days)

Peptide extract200 635plusmn 022 4314 750plusmn 058400 478plusmn 030 5712 925plusmn 096800 394plusmn 046 6466 1025plusmn 096

CQ 189plusmn 016 8309 2825plusmn 150Vehicle (3 dimethyl sulfoxide and 10 Tween 80 in PBS) 1116plusmn 115 500plusmn 082lowast)e results are expressed as meanplusmn SD

6 Journal of Pathogens

the bioavailability of the chemical components present in thecrude extracts is not possibly affected by biotransformationand physiological factors )e mechanism of action ofGlycine max is not yet known However existing literaturestudies have shown that some plants and seeds exhibitantiplasmodial activity either by causing red blood cellsoxidation [40] or by inhibitory protein synthesis [40]depending on their phytochemical constituents Flavonoidsare known to exert antiplasmodial activity by chelating withnucleic acid base pairing of the parasite [36] )erefore it ispossible that the antiplasmodial activity exhibited by Glycinemax could have been as a result of the abovementioned waysor by yet a different unknown mechanism )e limitation ofthe study is that identification of specific compounds re-sponsible for antimalarial activity was not accomplishedbecause this was beyond the scope of the present study)erefore we recommend that further analysis should becarried out on Glycine max seeds to identify the specificantimalarial compounds present

5 Conclusion

)e results of this study provide evidence-based activity ofGlycine max against the malaria parasite )erefore itprovides room for future exploitation of the plant in theaforementioned therapeutic effect

)e possibility of Glycine max incorporated in foodsubstances should be considered in a wake to determine ifthe body can develop immune to malaria )e use of Glycinemax in combination with other herbs for effective treatment[41ndash48]

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

KN and AM designed the study KN involved in data col-lection data analysis and preparation of the manuscriptdraft AM FK KK and JO contributed to analysis andinterpretation of data All authors contributed equally to thiswork

Acknowledgments

)e authors are sincerely thankful to the AFRICA-ai-JAPANProject for sponsoring this study )e authors are grateful toJomo Kenyatta University of Agriculture and Technology forallowing progress of the work)e authors thank the KEMRIadministration in particular the director of Centre forBiotechnology and Research Development (CBRD) and theKEMRI Animal House for provision of space in thelaboratories

Supplementary Materials

)e supplementary material consists of an excel spread sheetwhich has all the in vivo work data It includes the summaryof different extract doses negative and positive controls andhow they affected red blood cells in mice)e data were usedto determine different parameters used to measure the effectof the extract such as parasitemia average parasitemiachemosuppresion and mean survival days which are allexplained in the article (Supplementary Materials)

References

[1d] D A Bundy ldquoInvestment in child and adolescent health anddevelopment key messages from Disease Control Prioritiesrdquoe Lancet vol 391 no 10121 pp 687ndash699 2018

[2] M N Aminake and G Pradel ldquoAntimalarial drugs resistancein Plasmodium falciparum and the current strategies toovercome themrdquo in Microbial pathogens and strategies forcombating them science technology and education pp 1ndash774no 1 Formatex Research Center Badajoz Spain 2013

[3] J Recht A M Siqueira W M Monteiro S M HerreraS Herrera and M V Lacerda ldquoMalaria in Brazil ColombiaPeru and Venezuela current challenges inmalaria control andeliminationrdquo Malaria Journal vol 16 no 1 p 273 2017

[4] E A Ashley and A P Phyo ldquoDrugs in development formalariardquo Drugs vol 78 no 9 pp 861ndash879 2018

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquo)erelevance of higher plants in lead compound discoveryprogramsrdquo Journal of Natural Products vol 74 no 6pp 1539ndash1555 2011

[6] R G Ridley ldquoMedical need scientific opportunity and thedrive for antimalarial drugsrdquo Nature vol 415 no 6872pp 686ndash693 2002

[7] H Ginsburg and E Deharo ldquoldquoA call for using naturalcompounds in the development of new antimalarial treat-mentsndashan introductionrdquoMalaria Journal vol 10 no 1 p S12011

[8] P Smỳkal ldquoLegume crops phylogeny and genetic diversity forscience and breedingrdquo Critical Reviews in Plant Sciencesvol 34 no 1ndash3 pp 43ndash104 2015

[9] S A El Sohaimy ldquoFunctional foods and nutraceuticals-modern approach to food sciencerdquo World Applied SciencesJournal vol 20 no 5 pp 691ndash708 2012

[10] E C Okeke H N Eneobong A O UzuegbunamA O Ozioko and H Kuhnlein ldquoIgbo traditional food systemdocumentation uses and research needsrdquo Pakistan Journal ofNutrition vol 7 no 2 pp 365ndash376 2008

[11] D Malencic M Popovic and J Miladinovic ldquoPhenoliccontent and antioxidant properties of soybean (Glycine max(L) Merr) seedsrdquoMolecules vol 12 no 3 pp 576ndash581 2007

[12] M Y Kim G Y Jang N S Oh et al ldquoCharacteristics and invitro anti-inflammatory activities of protein extracts frompre-germinated black soybean [Glycine max (L)] treated withhigh hydrostatic pressurerdquo Innovative Food Science ampEmerging Technologies vol 43 pp 84ndash91 2017

[13] E Nuntildeez B Lopez-Corcuera J Vazquez C Gimenez andC Aragon ldquoDifferential effects of the tricyclic antidepressantamoxapine on glycine uptake mediated by the recombinantGLYT1 and GLYT2 glycine transportersrdquo British Journal ofPharmacology vol 129 no 1 pp 200ndash206 2000

[14] M Arora S Singh and R Kaur ldquoPhytochemical analysisprotein content amp antimicrobial activities of selected samples

Journal of Pathogens 7

the bioavailability of the chemical components present in thecrude extracts is not possibly affected by biotransformationand physiological factors )e mechanism of action ofGlycine max is not yet known However existing literaturestudies have shown that some plants and seeds exhibitantiplasmodial activity either by causing red blood cellsoxidation [40] or by inhibitory protein synthesis [40]depending on their phytochemical constituents Flavonoidsare known to exert antiplasmodial activity by chelating withnucleic acid base pairing of the parasite [36] )erefore it ispossible that the antiplasmodial activity exhibited by Glycinemax could have been as a result of the abovementioned waysor by yet a different unknown mechanism )e limitation ofthe study is that identification of specific compounds re-sponsible for antimalarial activity was not accomplishedbecause this was beyond the scope of the present study)erefore we recommend that further analysis should becarried out on Glycine max seeds to identify the specificantimalarial compounds present

5 Conclusion

)e results of this study provide evidence-based activity ofGlycine max against the malaria parasite )erefore itprovides room for future exploitation of the plant in theaforementioned therapeutic effect

)e possibility of Glycine max incorporated in foodsubstances should be considered in a wake to determine ifthe body can develop immune to malaria )e use of Glycinemax in combination with other herbs for effective treatment[41ndash48]

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

KN and AM designed the study KN involved in data col-lection data analysis and preparation of the manuscriptdraft AM FK KK and JO contributed to analysis andinterpretation of data All authors contributed equally to thiswork

Acknowledgments

)e authors are sincerely thankful to the AFRICA-ai-JAPANProject for sponsoring this study )e authors are grateful toJomo Kenyatta University of Agriculture and Technology forallowing progress of the work)e authors thank the KEMRIadministration in particular the director of Centre forBiotechnology and Research Development (CBRD) and theKEMRI Animal House for provision of space in thelaboratories

Supplementary Materials

)e supplementary material consists of an excel spread sheetwhich has all the in vivo work data It includes the summaryof different extract doses negative and positive controls andhow they affected red blood cells in mice)e data were usedto determine different parameters used to measure the effectof the extract such as parasitemia average parasitemiachemosuppresion and mean survival days which are allexplained in the article (Supplementary Materials)

References

[1d] D A Bundy ldquoInvestment in child and adolescent health anddevelopment key messages from Disease Control Prioritiesrdquoe Lancet vol 391 no 10121 pp 687ndash699 2018

[2] M N Aminake and G Pradel ldquoAntimalarial drugs resistancein Plasmodium falciparum and the current strategies toovercome themrdquo in Microbial pathogens and strategies forcombating them science technology and education pp 1ndash774no 1 Formatex Research Center Badajoz Spain 2013

[3] J Recht A M Siqueira W M Monteiro S M HerreraS Herrera and M V Lacerda ldquoMalaria in Brazil ColombiaPeru and Venezuela current challenges inmalaria control andeliminationrdquo Malaria Journal vol 16 no 1 p 273 2017

[4] E A Ashley and A P Phyo ldquoDrugs in development formalariardquo Drugs vol 78 no 9 pp 861ndash879 2018

[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquo)erelevance of higher plants in lead compound discoveryprogramsrdquo Journal of Natural Products vol 74 no 6pp 1539ndash1555 2011

[6] R G Ridley ldquoMedical need scientific opportunity and thedrive for antimalarial drugsrdquo Nature vol 415 no 6872pp 686ndash693 2002

[7] H Ginsburg and E Deharo ldquoldquoA call for using naturalcompounds in the development of new antimalarial treat-mentsndashan introductionrdquoMalaria Journal vol 10 no 1 p S12011

[8] P Smỳkal ldquoLegume crops phylogeny and genetic diversity forscience and breedingrdquo Critical Reviews in Plant Sciencesvol 34 no 1ndash3 pp 43ndash104 2015

[9] S A El Sohaimy ldquoFunctional foods and nutraceuticals-modern approach to food sciencerdquo World Applied SciencesJournal vol 20 no 5 pp 691ndash708 2012

[10] E C Okeke H N Eneobong A O UzuegbunamA O Ozioko and H Kuhnlein ldquoIgbo traditional food systemdocumentation uses and research needsrdquo Pakistan Journal ofNutrition vol 7 no 2 pp 365ndash376 2008

[11] D Malencic M Popovic and J Miladinovic ldquoPhenoliccontent and antioxidant properties of soybean (Glycine max(L) Merr) seedsrdquoMolecules vol 12 no 3 pp 576ndash581 2007

[12] M Y Kim G Y Jang N S Oh et al ldquoCharacteristics and invitro anti-inflammatory activities of protein extracts frompre-germinated black soybean [Glycine max (L)] treated withhigh hydrostatic pressurerdquo Innovative Food Science ampEmerging Technologies vol 43 pp 84ndash91 2017

[13] E Nuntildeez B Lopez-Corcuera J Vazquez C Gimenez andC Aragon ldquoDifferential effects of the tricyclic antidepressantamoxapine on glycine uptake mediated by the recombinantGLYT1 and GLYT2 glycine transportersrdquo British Journal ofPharmacology vol 129 no 1 pp 200ndash206 2000

[14] M Arora S Singh and R Kaur ldquoPhytochemical analysisprotein content amp antimicrobial activities of selected samples

Journal of Pathogens 7

of Glycine max Linnrdquo International Journal of Research inEngineering and Technology vol 2 no 11 pp 570ndash574 2013

[15] G Paliyath M Bakovic and K Shetty Functional FoodsNutraceuticals and Degenerative Disease Prevention JohnWiley amp Sons Hoboken NJ USA 2011

[16] P Pushpangadan ldquoFunctional foods and nutraceuticals withspecial focus on mother and child carerdquo Annals of Phyto-medicine vol 3 no 1 pp 4ndash24 2014

[17] E Deharo M Krugliak D Baccam and H GinsburgldquoAntimalarial properties of soy-bean fat emulsionsrdquo Inter-national Journal for Parasitology vol 25 no 12 pp 1457ndash1462 1995

[18] W Trager and J Jensen ldquoHuman malaria parasites in con-tinuous culturerdquo Science vol 193 no 4254 pp 673ndash675 1976

[19] D G Sixsmith H C Spencer J D Chulay andWMWatkinsldquoIn vitro antimalarial activity of tetrahydrofolate dehydroge-nase inhibitorsrdquoeAmerican Journal of TropicalMedicine andHygiene vol 33 no 5 pp 772ndash776 1984

[20] J W Gathirwa G M Rukunga E N M Njagi et al ldquo)e invitro anti-plasmodial and in vivo anti-malarial efficacy ofcombinations of some medicinal plants used traditionally fortreatment of malaria by the Meru community in KenyardquoJournal of Ethnopharmacology vol 115 no 2 pp 223ndash2312008

[21] A L Ager ldquoRodent malaria modelsrdquo in Antimalarial Drugs Ipp 225ndash264 Springer Berlin Germany 1984

[22] W Peters ldquoDrug resistance in Plasmodium berghei IChloroquine resistancerdquo Experimental Parasitology vol 17no 1 pp 80ndash89 1965

[23] J F Ryley and W Peters ldquo)e antimalarial activity of somequinolone estersrdquo Annals of Tropical Medicine amp Parasitologyvol 64 no 2 pp 209ndash222 1970

[24] L Tona KMesia N PNgimbi et al ldquoIn-vivo antimalarial activityof Cassia occidentalism Morinda morindoidesandPhyllanthusnirurirdquo Annals of Tropical Medicine amp Parasitology vol 95 no 1pp 47ndash57 2001

[25] M Heinrich ldquoEthnobotany and its role in drug developmentrdquoPhytotherapy Research vol 14 no 7 pp 479ndash488 2000

[26] M S Mueller I B Karhagomba HM Hirt and EWemakorldquo)e potential of Artemisia annua L as a locally producedremedy for malaria in the tropics agricultural chemical andclinical aspectsrdquo Journal of Ethnopharmacology vol 73 no 3pp 487ndash493 2000

[27] M Gasquet ldquoEvaluation in vitro and in vivo of a traditionalantimalarial ldquoMalarial 5rdquordquo Fitoterapia vol 64 p 423 1993

[28] C W Wright and J D Phillipson ldquoNatural products and thedevelopment of selective antiprotozoal drugsrdquo PhytotherapyResearch vol 4 no 4 pp 127ndash139 1990

[29] L Zhang F Jing F Li et al ldquoDevelopment of transgenicArtemisia annua (Chinese wormwood) plants with an en-hanced content of artemisinin an effective anti-malarial drugby hairpin-RNA-mediated gene silencingrdquo Biotechnology andApplied Biochemistry vol 52 no 3 pp 199ndash207 2009

[30] A B CunninghamAfricanMedicinal Plants UNESCO ParisFrance 1993

[31] M O Nafiu T A Abdulsalam and M A Akanji ldquoPhyto-chemical analysis and antimalarial activity aqueous extract ofLecaniodiscus cupanioides rootrdquo Journal of Tropical Medi-cine vol 2013 Article ID 605393 4 pages 2013

[32] P Ghosh A Mandal P Chakraborty M RasulM Chakraborty and A Saha ldquoTriterpenoids from Psidiumguajava with biocidal activityrdquo Indian Journal of Pharma-ceutical Sciences vol 72 no 4 p 504 2010

[33] J Satayavivad S Noppamas S Aimon and T YodhathaildquoToxicological and antimalaria activity of Eurycoma longifoliaJack extracts in micerdquo e Journal of Phytopharmacologyvol 5 pp 14ndash27 1998

[34] P A Tarkang F A Okalebo L S Ayong G A Agbor andA N Guantai ldquoAntimalarial activity of a polyherbal product(Nefang) during early and established Plasmodium infectionin rodent modelsrdquoMalaria Journal vol 13 no 1 p 456 2014

[35] R Batista A De Jesus Silva Junior and A De Oliveira ldquoPlant-derived antimalarial agents new leads and efficient phyto-medicines Part II Non-alkaloidal natural productsrdquo Mole-cules vol 14 no 8 pp 3037ndash3072 2009

[36] L N Bonkian R S Yerbanga B Koama et al ldquoIn Vivoantiplasmodial activity of two sahelian plant extracts onPlasmodium berghei ANKA infected NMRI micerdquo Evidence-Based Complementary and Alternative Medicine vol 2018Article ID 6859632 4 pages 2018

[37] S Menard T Ben Haddou A P Ramadani et al ldquoInductionof multidrug tolerance inPlasmodium falciparumby extendedartemisinin pressurerdquo Emerging Infectious Diseases vol 21no 10 pp 1733ndash1741 2015

[38] B Blasco D Leroy and D A Fidock ldquoAntimalarial drugresistance linking Plasmodium falciparum parasite biology tothe clinicrdquo Nature Medicine vol 23 no 8 pp 917ndash928 2017

[39] D Monti B Vodopivec N Basilico P Olliaro andD Taramelli ldquoA novel endogenous antimalarial Fe(II)-Protoporphyrin IXα (heme) inhibits hematin polymerizationto β-hematin (malaria pigment) and kills malaria parasitesdaggerrdquoBiochemistry vol 38 no 28 pp 8858ndash8863 1999

[40] B Baragantildea ldquoA novel multiple-stage antimalarial agent thatinhibits protein synthesisrdquo Nature vol 522 no 7556 p 3152015

[41] F Ariey B Witkowski C Amaratunga et al ldquoA molecularmarker of artemisinin-resistant Plasmodium falciparummalariardquo Nature vol 505 no 7481 pp 50ndash55 2014

[42] H A Madziga S Sanni and U K Sandabe ldquoPhytochemicaland elemental analysis of Acalypha wilkesiana leafrdquo Journal ofAmerican Science vol 6 no 11 pp 510ndash514 2010

[43] R E Desjardins C J Canfield J D Haynes and J D ChulayldquoQuantitative assessment of antimalarial activity in vitro by asemiautomated microdilution techniquerdquo AntimicrobialAgents and Chemotherapy vol 16 no 6 pp 710ndash718 1979

[44] J Okokon K Ofodum K Ajibesin B Danladi andK Gamaniel ldquoPharmacological screening and evaluation ofantiplasmodial activity of Croton zambesicus against Plas-modium berghei berghei infection in micerdquo Indian Journal ofPharmacology vol 37 no 4 p 243 2005

[45] D Lorke ldquoA new approach to practical acute toxicity testingrdquoArchives of Toxicology vol 54 no 4 pp 275ndash287 1983

[46] S Saxena N Pant D C Jain and R S Bhakuni ldquoAntimalarialagents from plant sourcesrdquo Current Science vol 85 no 9pp 1314ndash1329 2003

[47] G L Salvagno F Sanchis-Gomar A Picanza and G LippildquoRed blood cell distribution width a simple parameter withmultiple clinical applicationsrdquo Critical Reviews in ClinicalLaboratory Sciences vol 52 no 2 pp 86ndash105 2015

[48] J E Okokon N B Augustine and D MohanakrishnanldquoAntimalarial antiplasmodial and analgesic activities of rootextract ofAlchornea laxiflorardquo Pharmaceutical Biologyvol 55 no 1 pp 1022ndash1031 2017

8 Journal of Pathogens