evaluationofthebioactivitiesof rumexcrispus...

Research ArticleEvaluation of the Bioactivities of Rumex crispus L Leaves andRoot Extracts Using Toxicity Antimicrobial andAntiparasitic Assays

Oladayo Amed Idris Olubunmi Abosede Wintola and Anthony Jide Afolayan

Medicinal Plants and Economic Development (MPED) Research Centre Department of Botany University of Fort HareAlice 5700 South Africa

Correspondence should be addressed to Olubunmi Abosede Wintola owintolaufhacza

Received 27 June 2019 Accepted 1 October 2019 Published 30 October 2019

Guest Editor Philip F Uzor

Copyright copy 2019 Oladayo Amed Idris 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

Traditional folks in different parts of the world use Rumex crispus L for the treatment of microbial infections malaria andsleeping sickness in the form of decoction or tincture In the search for a natural alternative remedy this study aimed to evaluatethe antimicrobial antitrypanosomal and antiplasmodial efficacy and the toxicity of R crispus extracts Antimicrobial potency ofthe extracts was evaluated using the agar dilution method to determine the minimum inhibitory concentration (MIC) -eantitrypanosomal activity of the extracts was evaluated with the Trypanosoma brucei brucei model while the antimalaria potencywas tested using Plasmodium falciparum 3D7 strain Toxicity was then tested with brine shrimp assay and cytotoxicity (HeLacells)-e acetone extract of the root (RT-ACE) reveals the highest antimicrobial potency with the lowestMIC value of lt1562mgmL for all bacteria strains and also showed high potent against fungi RT-ACE (IC50 13 μgmL) and methanol extract of the leaf(LF-MEE IC50 15 μgmL) show a strong inhibition of P falciparum -e ethanol extract of the root (RT-ETE IC50 97 μgmL)reveals the highest inhibition of Tb brucei parasite RT-ETE and RT-ACE were found to have the highest toxicity in brine shrimplethality assay (BSLA) and cytotoxicity which correlates in the two assays -is research revealed Rumex crispus has potencyagainst microorganisms Trypanosoma and Plasmodium and could be a potential source for the treatment of these diseases

1 Introduction

Medicinal plants have been used for ages in several ways forantimicrobial hypoglycemic antihelminthic antiulcerogenichepatoprotective antipyretic analgesic insecticidal piscicidepesticide pharmaceutical and cosmetic purposes [1] -ebiological activities and efficacy of medicinal plants have beentested in traditional medicine and in several fields of science[2] -eir potency and curative potentials were documentedindependently across the globe suggesting that plants could bea potential source of novel drugs [2ndash4]-ere has been a rapidincrease in the use of medicinal plants as a curative prototypeand new products are emanating from these natural productsinto the commercial market daily-ese products are believedby the consumers to be safer and effective than the chemo-therapeutic conventional drugs [4 5] It was estimated thatabout 61 of newly developed drugs between 1981 and 2002

have their source from plants and are successful in thetreatment of cancer and several other diseases [2 6] Manymedicinal plants have been screened for antimicrobialantiplasmodial and antitrypanosomal activities and some ofthese plants showed prospect in the treatment of these in-fections [3 7 8] In most cases the medicinal properties of aplant are a function of its phytochemical constituents [9]

11 Resistance of Microorganisms to Drugs -ere have beenreports of multiple drug resistance (MDR) in the humanpathogenic microbial organisms due to indiscriminate use ofcommercially available drugs [4] -e capacity of diseases todrug resistance is the clinical determinants of the patho-genesis of a particular pathogen For instance there havebeen reports of higher resistance to a group of antibiotics inan area where it is regularly misused [10 11] Multiple drug-

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 6825297 13 pageshttpsdoiorg10115520196825297

resistant strains of pathogens and parasites are economicallyimportant and these pose a huge threat to the public healthsystem -is calls for an immediate intervention with newdrugs that use a different mechanism of action in thetreatment of these diseases On the other hand there areseveral scientific evidences to the success of medicinal plantsin treating diseases hence plants could be a novel sourcethat will combat drug resistance [5]

Plants are known to produce complex bioactive organiccompounds that possess several bioactivities -e com-pounds are secreted in various parts of the plants such asseeds fruits flowers leaves roots stems bark and someplant secretions (isothiocyanates plant pigment plant gumphytoalexins essential oils and hydrolytic enzymes) [12]-e bioactive constituent in medicinal plants has showntremendous health benefits For instance the major anti-bacterial bioactive compounds in plants are anthraquinonesand flavonoids which are active against several humanpathogenic bacteria [13] Phenolic compounds flavonoidsand artemisinin are active against malaria causing parasitePlasmodium falciparum [14] It was also reported that fla-vonoids and glycosides in plants inhibit the growth of Africansleeping sickness (Trypanosoma brucei) parasites [15] -eincrease in the occurrence and sustenance of drug-resistantpathogens to drugs has drawn the attention of scientists andpharmaceutical communities towards harnessing the po-tentials of plant-derived substances used successfully in dif-ferent countries by traditional medical practitioners [16]

12 Tests for Toxicity Antimicrobial and Antiparasite-ere is a rapid increase in interest and demand for medicinalplants in various parts of the world because they are con-sidered natural safer and cheaper [17] On the other handthere is a general concern in the scientific communities on thesafety efficacy and pharmacokinetics of medicinal plantsirrespective of their wide acceptance and usage -e possibleside effects of plant products cannot be ruled out regardless oftheir adoption as a novel source of drugs [18 19]

-e source of the toxicity of plants may be due to thecontaminants in the environment or from the plant-secretedphytochemicals [20 21] Different biological models havebeen tested via in vitro and in vivo assays to qualify andquantify the potential toxicity of extracts of medicinal plantsHowever recent studies employed the use of preliminarytoxicity assessment tools using brine shrimp species Arte-mia franciscana Artemia salinaamnocephalus platyurusand Artemia urmiana [22ndash24] which is considered a usefultool to evaluate the potential toxicity of plant extractsCytotoxic screening using HeLa human cervix carcinomacells is an advanced tool to screen substances inducingapoptosis In addition a comparative toxicity study of plantsextract could be tested using in vivo study -e resulthowever showed quite a good correlation in the mean valueof in vitro study lethal concentration (LC50) and the acuteoral lethal dose (LD50) of in vivo study [23 25]

Rumex crispus L (curled dock) is commonly used bytraditional healers for treatment of various diseases andcorrection of disorders such as gastrointestinal tract

disorders antihelminthic diseases anti-inflammatory andarthritis and it is also used as laxative antipyretic anti-oxidant and antimicrobial [5 26] More so there are reportsof the antimicrobial [27ndash29] antitrypanosomal [30] andantiplasmodial [31] properties and toxicity of R crispuswhich were evaluated in this research -is is necessary tovalidate the claim of traditional healer practitioners inEastern Cape South Africa and any other part of the worldthat use R crispus for the treatment of these ailments

2 Materials and Methods

21 Organic Extraction -e leaves and roots of Rumexcrispuswere collected from a fallow damp field University ofFort Hare (32deg47prime123PrimeS 26deg51prime985PrimeE) in the summer of2017 It was identified and a sample of the specimen wasplaced in Giffen herbarium (Idr-Med-201703) Universityof Fort Hare-e leaves and roots of the plant specimen werecarefully sorted and the infected ones were removed -especimen was rinsed dried in an oven at 30degC until a fixedweight and then pulverized with a mechanical grinder(Polymix PX-MFC90D Switzerland)-e pulverized samplewas stored in an airtight container in a refrigerator at 4degCuntil use for extraction

Crude extraction of R crispus was done using standardprocedures -e choice of solvent for the extraction processis an imperative factor that must be considered during theantimicrobial and toxicity tests -e extracting potentials ofdifferent solvents differ therefore extraction was done usingsolvents of low reactivity a technically graded acetonemethanol ethanol and distilled water -e extractant(solvent) and the plant materials used were in the ratio of 1 10 (gmL)-e dried root (RT) and the aerial part (LF) of theplant sample (100 g) were macerated with the extractantsolvents (1000mL) at room temperature -e crude extractof methanol (MEE) ethanol (ETE) and acetone (ACE) wereconcentrated under reduced pressure by using a rotatoryevaporator considering the boiling point of the respectivesolvent and then dried in a desiccator at a mild temperature(40degC) -e filtrate of water samples (WAE) was chilled atminus 40degC with a shell refrigerant (PolyScience AD15R-40-A12E USA) and freeze-dried (Savant vapour trap RV-T41404 USA) for 48 h -e plant crude extracts were storedin sterile bottles and refrigerated at 2 to 4degC until further use

22 Determination of Minimum Inhibitory Concentrations(MICs) of the Extracts Minimum inhibitory concentrations(MICs) were used to determine the lowest concentration ofthe extracts that will inhibit visible microbial growth after anincubation period of 24 h (bacteria) and 48 h (fungi)Usually MICs are diagnostic laboratory tools used toconfirm microbial resistance but are often used in researchlaboratories to determine the in vitro activity of antimi-crobials and its MIC breakpoints [32]

23 Antibacterial Activity of the Plant Extracts -e potencyof each extract of R crispus was tested against four strains ofGram-negative (Klebsiella pneumonia ATCC 4352

2 Evidence-Based Complementary and Alternative Medicine

Pseudomonas aeruginosa ATCC 19582 Escherichia coliATCC 8739 and Vibrio cholerae ATCC 14033) bacteria andfour strains of Gram-positive (Bacillus subtilis ATCC 6633Staphylococcus aureus ATCC 43300 Streptococcus pyogenesATCC 19615 and Bacillus cereus ATCC 33019) bacteria-ese strains of bacteria were collected from the Departmentof Microbiology University of Fort Hare South Africa -econcentration of the plant extracts that inhibits the mi-crobial growth after 24 h of incubation was determined byMIC assay as described [33] with modification -e agardilution method was used for the MIC to determine theantibacterial efficiency of R crispus and the resultant efficacywas compared with a commercial antibiotic drug(erythromycin)

-e collected strains of bacteria were subcultured for24 h at 37degC in a sterilized MuellerndashHinton Agar (Oxoidtrade)following the manufacturer preparation instructions -ebacteria were harvested using 5mL of sterilized saline so-lution and the inoculum was adjusted to 106 CFUmL bycomparison with 05McFarland standard 05mL of 0048MBaCl2 (117 wv BaCl2middot2H2O) + 995mL of 018MH2SO4(1 vv) between 106 and 108CFUmL and then the in-oculum was applied to the agar prepared in sterilized Petriplates [13] A dilution range of 1562ndash25mgmL was pre-pared for the plant extracts and concentration range of05ndash8 μgmL of erythromycin was used for the positivecontrol -e plantrsquos extracts were dissolved in 10 dime-thylsulphoxide (DMSO) (except aqueous extracts) anddistilled water and then purified by filtering through Mil-lipore filter paper MuellerndashHinton agar was prepared fol-lowing the manufacturerrsquos instructions a volume of 20mLof the agar was measured in Erlenmeyer flask and auto-claved -ereafter 1mL of the prepared extract was addedswirled and poured into sterile disposable Petri dishes -eagar was allowed to solidify at room temperature -estandardized inoculum was diluted in sterile saline solutionand MuellerndashHinton broth in ratio 1 10 to attain 05McFarland standard between 106 and 108CFUmL Analiquot volume of 01mL of the inoculum was pipetted andtransferred to the agar medium and the plates were in-cubated for 24 h at 37degC

24 Antifungal Screening -e fungi Trichophyton tonsuransATCC 28942 Trichosporon mucoides ATCC 201382 Peni-cillium aurantiogriseum ATCC 16025 Penicillium chrys-ogenum ATCC 10106 Candida glabrata ATCC 66032 andCandida albicans ATCC 32077 were collected from theDepartment of Botany University of Fort Hare and sub-cultured on Sabouraud Dextrose Agar (Oxoidtrade) at 30degC for48 h -e antifungal assay was carried out according to themethod described by Lass-Florl et al [34] withmodification-e medium (Sabouraud Dextrose Agar) was preparedfollowing the manufacturerrsquos instructions and 5mg100mLof chloramphenicol was added to the agar media beforeautoclaving to inhibit bacteria growth -e suspensions ofthe inoculum were transferred into a sterile saline solutionfrom the subculture by carefully collecting the conidia witha sterile cotton swab -e suspension was homogenized for

15 s with a vortex (2000 rpm) and the inoculum was dilutedin MuellerndashHinton broth in ratio 1 10 to obtain a final testinoculum load range between 10times106 and 50times106 CFUmL-e adjusted working inoculum was plated on solidifiedsterilized Sabouraud Dextrose Agar plates with plant ex-tractrsquos dilution range of 0625ndash10mgmL and the controlagar plates was diluted at the range of 1ndash16 μgmL of nys-tatin all plates were incubated for 48 h at 30degC

25 Screening of R crispus against Plasmodium falciparum-e malaria parasites (Plasmodium falciparum sensitivestrain 3D7) were used in blood-stage culture to test theantimalarial efficacy of R crispus extracts through in vitrostudies following the method described by Murugan et al[7] -e parasites were preserved in RPMI 1640 mediumcontaining 2mML glutamine and 25mM Hepes (Lonza)-emediumwas further supplemented with 5 Albumax II20mM glucose 065mM hypoxanthine 60 μgmL genta-mycin and 2ndash4 hematocrite human red blood cells -eparasites were cultured at 37degC under an atmosphere con-dition of 5 CO2 5 O2 and 90 N2 in sealed T75 cultureflasks-e infected erythrocytes were transferred into a freshmedium to propagate the culture and parasitaemia (theconcentration of parasites in the culture) was measured bylight microscopy of Giemsa-stained thin blood smears

In the experiment the tested extracts were prepared in04 dimethyl sulfoxide (DMSO) -en the parasiticidalpotency of tested extracts was done by single concentrationscreening -e single concentration screening was con-ducted by adding 25 μgmL (extracts) to parasite cultures in96-well clear plates and incubated for 48 hours in a CO2incubator at 37degC After 48 hours 20 μL of culture wasremoved from each well and combined with 125 μL of amixture of Malstat and NBTPES solutions in a fresh 96-wellplate -ese solutions were used to measure the activity ofthe parasite lactate dehydrogenase (pLDH) enzyme in thecultures A purple product is formed when pLDH is present[8] and this product is quantified in a Spectramax M3microplate reader at the absorbance of 620 nm For eachextract concentration percentage parasite viability andpLDH activity in extract-treated wells relative to untreatedcontrols were calculated -e extracts were tested in trip-licates and standard deviations (SD) were derived Forcomparative purposes chloroquine (a standard antimalarialdrug) was used as a standard (IC50 values ranging from 001to 005 μM)

26 Antitrypanosomal Activity of R crispus Extracts -esubspecies responsible for sleeping sickness in NaganaTrypanosoma brucei brucei (Tb brucei) is infective tohumans and is commonly used as a drug screening model inthe laboratory -is research adopted the Tb brucei modelas described in [35] with modification -e blood-stageforms of Tb brucei (Strain 427) were grown in IscoversquosModified Dulbeccorsquos Medium (IMDM) enhanced with 10fetal bovine serum and the culture was incubated in 5CO2 at 37degC

Evidence-Based Complementary and Alternative Medicine 3

For primary screening of extractsrsquo trypanocidal activity asingle concentration of 50 μgmL in duplicate was preparedfrom the extract stock in IMDM and added to the culture ofTb brucei in a 96-well plate -e mixture was incubated for48 h and the number of survived parasites was determinedby adding a resazurin-based reagent which would be re-duced to resorufin by living cells Resorufin is a fluorescentcompound (Excitation560Emission590) hence absorbancewas measured in a multiwell fluorescence plate reader(Spectramax M3) to determine viable cells -e percentageparasite viability of each extract was determined by com-paring the resorufin fluorescence in treated wells in relationto untreated controls -e resultant was then plotted againstlog (extract concentration) and the IC50 from the resultingdose response in nonlinear regression was obtained -epercentage parasite viability and response to extracts werethen compared with pentamidine (a drug for trypanoso-miasis) as a positive control

27 Toxicity Test -e toxicity of plant was tested by exposingthe eggs and larvae of Artemia salina to the plant extracts andcomparing their LC50 values with toxic compound (potas-sium dichromate K2CrO7) suitable as a positive control forthis test Further toxicity was done by testing extracts onHeLacells (human cervix adenocarcinoma) All experiments werecarried out in accordance with the guidelines for the Care andUse of Laboratory Animals -e research methodology wasapproved by the University of Fort Hare Animal EthicalCommittee (reference number AFO121SIDR01)

271 Brine Shrimp Hatchability Assay (BSHA) -e BSHAwas carried out as previously described by Otunola et al[36] with modification -e eggs of Artemia salina (brineshrimp) were obtained from aquaculture outlet in East

London South Africa -e eggs of Artemia salina washatched at optimum condition in seawater (salinity 38 pptand pH 85) collected in East London South Africa (32deg 59 S27deg52 E) -e concentration of the extracts and a standarddrug (K2CrO7) were graded (00625 0125 025 05 and10mgmL) by serial dilution in 30mL of filtered seawater insterile Petri dishes Each concentration of extracts andpositive and negative control were tested in triplicate Teneggs of brine shrimp were introduced into the Petri dishes-e hatched nauplii were counted against the ratio of thenumber of eggs introduced at the beginning of the exper-iment at 12 h interval and the level of toxicity was evaluatedusing probit regression (Figure 1)

272 Brine Shrimp Lethality Assay (BSLA) In the studyBSLA was carried out adopting the method as described in[37] with slight modifications -e brine shrimp eggs wereincubated at the optimum condition in a glass jar for 48 h Acount of 10 nauplii with an inverted microscope waspipetted into different Petri dishes with a graded concen-tration of extracts potassium dichromate and controlseawater -e lethality of larvae was observed with aninverted microscope at an interval of 12 h and the totallethality was computed using the Standardized LethalityIndex (SLI) -e condition of the exposed larvae wasevaluated with SLI as used by Agrafioti et al [38] Based onthe index each experimental nauplius was ranked from 0 to3 to measure the intensity of the response In the scaleresponses 0 normal nauplii responses 1 nauplii wereknocked down but were able to swim for short intervalsresponses 2 nauplii were knocked down and unable to swimbut with visible movement and responses 3 nauplii weredead (Table 1 Figures 2ndash4) -us the lethality index wascalculated with the equation

lethality index 11139444

n0

N0 times W1( 1113857 + N1 times W2( 1113857 + N2 times W3( 1113857 + N3 times W4( 1113857

Nt times Nob times Maxw

1113890 1113891 times 100 (1)

whereN0 number of nauplii at rank 0N1 number of naupliiat rank 1 N2 number of nauplii at rank 2 N3 number ofnauplii at rank 3Nt total number of naupliiNob number ofobservations (three days six observations) Maxw maxi-mum lethality coefficient and lethality coefficients W1 00W2 017 W3 033 W4 050 Wi i (0 + 1 + 2 + 3 )

i 0 1 2 3

273 Cytotoxicity Assay -e cytotoxicity of the extracts ofR crispus was estimated using HeLa (human cervix ade-nocarcinoma) cells as described by Larayetan et al [8] -estock solutions were prepared in 1 DMSO to attain a finalconcentration of 50 μgmL-e HeLa cells were incubated ata fixed concentration (50 μgml) of the extracts in 96-wellplates for 48 h -e viability of the cells after treatment withextracts was determined by adding resazurin-based reagent

and reading resorufin fluorescence in a microplate reader(Spectramax M3) -e results were expressed as percentageviability in resorufin fluorescence-treated extracts per wellsrelative to the untreated wells (controls) -e cytotoxicity ofthe extracts was done in duplicate and the positive controlstandard drug (emetine) was used for comparative reference

28 Statistical Analysis -e mean of the triplicates of theresults for the assay was expressed as meanplusmn SD -e datawere analyzed statistically by one-way analysis of varianceand differences between data were extrapolated by FisherrsquosLeast Significant Difference (LSD) test where it is applicable-e relationship between the response to the stimulus of theindependent variables (concentration) and the quantal (deador alive all or none) was analyzed by probit regression toestimate the LC50 and LC99 using statistical software (SPSS)


3 Results

31 Antimicrobial Activity of Plant Extracts -e antibacte-rial and antifungal potency of the extracts is given in Tables 2and 3 -e result shows that all extracts of R crispus have avarying degree of antimicrobial activities with a variation inthe level of potency

311 Minimal Inhibitory Concentration (MIC) of PlantExtracts -e bactericidal properties of R crispus wasconducted using a graded concentration of the plant ex-tracts to find theminimal inhibitory concentration (MIC) ofthe extracts against predispose bacterial strains (Gram-negativeK pneumoniae P aeruginosa E coli andV cholera

Gram-positive B subtilis S aureus S pyogenes and B ce-reus) -e concentration effect of the plant extracts andstandard drug is reported in Table 2 -e MIC was confirmedby the absence of the growth of tested bacterial strains on thesurface of the agar which shows the potential bactericidalactivity of the plantrsquos extracts against the tested bacteria

-e acetone extract of the root (RT-ACE) registered thehighest antimicrobial potency with the lowest MIC value oflt1562mgmL in all the experimental strains of bacteria Alsothe ethanol extract of the root (RT-ETE) andmethanol extract ofthe root (RT-MEE) show high inhibitory capacity of the bac-teria On the contrary theMIC of water extract of the root (RT-WAE) water extract of the leaf (LF-WAE) and acetone extractof the leaf (LF-ACE) was low -e methanol extract of the leaf(LF-MEE) and ethanol extract of the leaf (LF-ETE) showed amoderate but an undulating MIC on the bacterial strains -emean MIC value of R crispus root extracts proved to havesignificant effects against bacterial strains more than the leafextracts which aligns with previous research as reported byMostafa et al [33] on how extracts of the underground parts ofmany plants could be more active than the aerial parts

312 Antifungal Activity of Plant Extracts Antifungalsusceptibility testing remains less developed compared toantibacterial testing However it is an adoption of antibac-terial testing -e knowledge of the mechanism of fungalresistance to drugs through the MIC method has beenvaluable in identifying and measuring resistance in in vitrosystems [34] In the study the endpoint was read visually witha viewing mirror and the growth level (MIC) was recorded(Table 3) -e highest level of MDR was recorded in

00625mgmL 0125mgmL 025mgmL 05mgmL 1mgmL

RT-MEERT-ETERT-ACE

RT-WAELF-MEELF-ETE

LF-ACELF-WAEK2CrO7

a b

c

a b

aa

ba

a b

aa

ba

b

a

da

bb

cb

c d

a b

cb

c d

c d

b b b

b

b

a a

aa

a b

b c

ca

a b

a b

a ac c

d c d

db

cc

da

a b

ad0

10

20

30

40

50

60

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80

h

atch

abili

ty

Figure 1 Percentage hatchability of A salina cysts incubated for 72 h in different solvent extracts and five concentrations -e values aremeans of hatchability potential for R crispus extractscontrolplusmn SD of three replicates -e bars within a concentration with different lettersare significantly different mean values of agt bgt cgt d RT-MEE methanol extract of root RT-ETE ethanol extract of root RT-ACE acetoneextract of root RE-WAE water extract of root LF-MEE methanol extract of leaf LF-ETE ethanol extract of leaf LF-ACE acetone extract ofleaf and LF-WAE water extract of leaf

Table 1 LC50 and LC99 (mgmL) and the confidence limits of thedose of solvent extraction of root and leaf of R crispus using brineshrimp lethality assay for an exposure period of 72 h

Plant extracts LC50 (mgmL) LC99 (mgmL)RT-MEE 0491 (0269ndash0606) 1548 (1051ndash8588)RT-ETE 0275 (0174ndash0357) 1196 (0808ndash2997)RT-ACE 0363 (0241ndash0440) 0872 (0652ndash2282)RT-WAE 0612 (0328ndash0989) 3467 (3126ndash6950)LF-MEE 2734 (1535ndash3120) 5678 (5385ndash12120)LF-ETE 4658 (2211ndash5399) 17239 (14094ndash22399)LF-ACE 1824 (2543ndash3099) 28566 (22204ndash134009)LF-WAE 2990 (1309ndash3908) 6854 (4767ndash153999)K2CrO7 0101 (ndash0048ndash0187) 0941 (0706ndash1547)LC lethal concentration RT-MEE methanol extract of root RT-ETEethanol extract of root RT-ACE acetone extract of root RE-WAE waterextract of root LF-MEE methanol extract of leaf LF-ETE ethanol extractof leaf LF-ACE acetone extract of leaf LF-WAE water extract of leaf


C glabrata for all the extracts (gt10mgmL) as recorded inTable 3 High resistance was also recorded in P chrysogenumand C albicans across the extracts and the positive control Itwas established by Lass-Florl et al [34] that some fungi (mold)are considered resistant to some antifungal drugs (itraco-nazole) at MICs ge8 μgmL but susceptible to MICs of0125ndash05 μgml -e MICs of the extracts against testedfungal extracts showed varying concentrations as shown inTable 3 -e overall MIC of leaf extracts showed a higher

antifungal potency contrary to the lesser MIC of leaf in theantibacterial test -is may be due to the slight differencesin the phytochemical composition of the leaf and root ofR crispus

32 Antiplasmodial Activity of R crispus Extracts -e sol-vent extracts of R crispus were subjected to an in vitroscreening of malaria-causing parasite (P falciparum strain

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

K 2Cr

O7

lethality hatchability

0

10

20

30

40

50

60

70

80

90

100

h

atch

abili

ty an

d le

thal

ity

Figure 2 Overall percentage hatchability and lethality of the extracts of R crispus and the standard K2CrO7 after 72 h incubation period-evalues are means of hatchability and lethality potential for all the concentrations of the extractscontrolplusmn SD of replicates RT-MEEmethanol extract of root RT-ETE ethanol extract of root RT-ACE acetone extract of root RE-WAE water extract of root LF-MEEmethanol extract of leaf LF-ETE ethanol extract of leaf LF-ACE acetone extract of leaf and LF-WAE water extract of leaf


le

thal

ity o

f roo

t ext

ract

s

RT-MEERT-ETERT-ACE

RT-WAEK2CrO7

cb

cc

ba

cb

cc

ba

cb

cb

ca

c d

bc

da a b a

bc

a

0

10

20

30

40

50

60

70

80

Figure 3 Percentage mortality of Artemia salina larva in different concentrations of the root extracts of R crispus and control in a testperiod of 72 h -e values are mean of the triplicates of mortality in each plant extracts (meanplusmn SD) -e bars within a concentration withdifferent letters are significantly different mean values of agt bgt cgt d RT-MEE methanol extract of root RT-ETE ethanol extract of rootRT-ACE acetone extract of root RE-WAE water extract of root LF-MEE methanol extract of leaf LF-ETE ethanol extract of leaf LF-ACEacetone extract of leaf LF-WAE water extract of leaf


3D7) -e percentage viability of the parasite is equivalent toplasmodium lactate dehydrogenase (pLDH) enzyme re-leased -e extracts RT-ACE (3572plusmn 208) and LF-MEE(1711plusmn 382) caused a significant decrease in the level ofpLDH causing viability of the parasite to be less than 50(Figure 5) Due to the bioactive of RT-ACE and LF-MEEthey were put forward for pLDH IC50 (50 inhibitory

concentration) screening and compared with the positivecontrol (chloroquine) -eir percentage viabilities were thenplotted against the logarithm of extract concentration (250to 011 μgmL) a 3-fold dilution but chloroquine was in theconcentration of 001ndash005 μM -e IC50 values were ob-tained from the resulting dose-response curve by nonlinearregression IC50 values of RT-ACE LF-MEE and


LF-MEELF-ETE

LF-ACELF-WAE

aa

aa

aa

aa a

aa

a

aa

bb

a b

ab

ca

b

le

thal

ity o

f lea

f ext

ract

s

0

10

20

30

40

50

60

Figure 4 Percentagemortality ofArtemia salina larva in different concentrations of the leaf extracts of R crispus in a test period of 72 h-evalues are mean of the triplicates of mortality in each plant extracts (meanplusmn SD) -e bars within a concentration with different letters aresignificantly different mean values of agt bgt cgt d RT-MEE methanol extract of root RT-ETE ethanol extract of root RT-ACE acetoneextract of root RE-WAE water extract of root LF-MEE methanol extract of leaf LF-ETE ethanol extract of leaf LF-ACE acetone extract ofleaf LF-WAE water extract of leaf

Table 2 Minimal inhibitory concentration (MIC) of tested bacterial organisms against R crispus extracts

R crispus extracts (mgmL)Gram-negative bacteria Gram-positive bacteria

K pneumoniae P aeruginosa E coli V cholerae B subtilis S aureus S pyogenes B cereusRT-MEE lt1562 625 25 lt1562 lt1562 lt1562 lt1562 lt1562RT-ETE 1562 1562 25 lt1562 lt1562 lt1562 lt1562 lt1562RT-ACE lt1562 lt1562 lt1562 lt1562 lt1562 lt1562 lt1562 lt1562RT-WAE gt25 gt25 gt25 gt25 gt25 125 gt25 gt25LF-MEE lt1562 25 25 lt1562 125 125 125 3125LF-ETE 25 gt25 gt25 25 25 25 25 25LF-ACE gt25 gt25 gt25 gt25 gt25 gt25 gt25 gt25LF-WAE gt25 gt25 gt25 gt25 gt25 gt25 gt25 gt25Erythromycin (μgmL) 8 gt8 gt8 8 gt8 2 lt05 1RT-MEE methanol extract of root RT-ETE ethanol extract of root RT-ACE acetone extract of root RE-WAE water extract of root LF-MEE methanolextract of leaf LF-ETE ethanol extract of leaf LF-ACE acetone extract of leaf LF-WAE water extract of leaf

Table 3 Minimal inhibitory concentration (MIC) of tested fungal organisms against R crispus extracts and standard drug

R crispus extracts (mgmL) T tonsurans T mucoides P aurantiogriseum P chrysogenum C glabrata C albicansRT-MEE 25 25 25 gt10 gt10 5RT-ETE 25 lt0625 lt0625 gt10 gt10 5RT-ACE 125 125 125 10 gt10 25RT-WAE 10 gt10 10 gt10 gt10 gt10LF-MEE 5 25 25 gt10 gt10 10LF-ETE 0625 0625 125 gt10 gt10 gt10LF-ACE 0625 0625 25 gt10 gt10 5LF-WAE 125 0625 0625 gt10 gt10 gt10Nystatin (μgmL) 1 1 1 4 32 2RT-MEE methanol extract of root RT-ETE ethanol extract of root RT-ACE acetone extract of root RE-WAE water extract of root LF-MEE methanolextract of leaf LF-ETE ethanol extract of leaf LF-ACE acetone extract of leaf LF-WAE water extract of leaf


chloroquine were evaluated 13 μgmL 15 μgmL and10 μM respectively as shown in Figures 6 and 7

33 Antitrypanosomal Activity of R crispus Extracts -epotential of R crispus extracts was tested on Tb bruceiparasite causative agents of African sleeping sickness -eethanol extract of the root of R crispus (RT-ETE) has thehighest potency among the extracts acting against theparasite and reducing the viability to 4327plusmn 292 as shownin Figure 8 RT-ETE was thereafter put forward for furtherIC50 screening and the outcome was compared to the IC50 ofpentamidine (standard control) IC50 97 μgmL and IC500017 μM respectively-e IC50 was obtained from the dose-response curve extrapolated by nonlinear regression usingpercentage viabilities plotted against logarithm (Figures 9and 10)

34 Toxicity Test

341 Brine Shrimp Hatchability -e percentage inhibitionof the hatchability of the eggs of Artemia salina after 72 hexposure to the plant extracts and standard drug at optimumincubation conditions is shown in Figure 1 -e entire testedextract composite shows some degree of inhibition of therate of hatching of A salina eggs compared to the control-e highest percentage of successfully hatched eggs intolarvae in the least concentration used (00625mgmL) wasobserved in the extracts RT-ETE (57) RT-WAE (58)LF-ETE (5467) and LF-WAE (5667) -is could be dueto the polarity of the extraction solvents (ethanol and water)which brings about the fair inhibition of hatchability of thebrine shrimp eggs -e hatching success of the cysts in RT-ACE has no significant difference compared to the positivecontrol (K2CrO7) At the concentration 0125mgmL of thetested samples there was an overall more successful hatch ofthe cyst with the highest percentage in the extract of RT-

0

10

20

30

40

50

60

70

80

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity

Figure 5 Bar graph shows the percentage parasite (P falciparum)viabilityplusmn SD tested against R crispus extracts RT-MEE methanolextract of root RT-ETE ethanol extract of root RT-ACE acetoneextract of root RE-WAE water extract of root LF-MEE methanolextract of leaf LF-ETE ethanol extract of leaf LF-ACE acetoneextract of leaf LF-WAE water extract of leaf

v

iabi

lity

0

20

40

60

80

100

0 1 2ndash1Log(microgmL)

RT-ACE

Figure 6 Dose-response plots and viability of parasite to RT-ACE (acetone extract of the root) IC50 13 μgmL

v

iabi

lity

Chloroquine

0

20

40

60

80

100

ndash2 ndash1 0ndash3Log(microM)

Figure 7 Dose-response plots and viability of parasite tochloroquine standard IC50 10 μM

0102030405060708090

100110

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity

Figure 8 Percentage viability of Tb brucei obtained from the testagainst extracts of R crispus RT-MEE methanol extract of rootRT-ETE ethanol extract of root RT-ACE acetone extract of rootRE-WAE water extract of root LF-MEE methanol extract of leafLF-ETE ethanol extract of leaf LF-ACE acetone extract of leafand LF-WAE water extract of leaf


MEE (70) while the least was in RT-ACE (1267) At thehighest concentration of 1mgmL the toxicity of the extractsexpressed their potency as postulated -e extracts of theroot of R crispus showed higher toxicity compared to the leafextracts and hence the success of hatchability was very low(Figure 1) -e overall percentage hatchability of A salinacyst against the extracts is shown in Figure 2 -e waterextracts of the root (724) and leaf (722) are not sig-nificantly different they have the highest hatchability andhence less toxic compared to other extracts -e acetoneextract of the root (RT-ACE) has the lowest percentagehatchability and it is not significantly different from thestandard drug (K2CrO7) with an inhibition percentage of164 and 218 respectively (Figure 2)

342 Brine Shrimp Lethality -e exposure of A salina totoxins could have led to an elevation of glutathioneS-transferase (sGST) activity in the phase II detoxicationsystem of the larva -e depletion of glutathione levels andintracellular thiols which act against oxidative stress in thisorganism by these toxins leads to the death of the larva [39]In most cases the difference between toxins and nontoxic isdose or concentration -e lethal concentration (LC50 andLC99) of the extracts and the standard toxic drug wasevaluated with probit regression and recorded as shown inTable 1 In the study the level of toxicity of the extracts andthe control in descending order is as follows K2CrO7 (LC500101mgmL) RT-ETE (LC50 0275mgmL) RT-ACE (LC500363mgmL) RT-MEE (LC50 0491mgmL) RT-WAE(LC50 0612mgmL) LF-ACE (LC50 1824mgmL) LF-MEE(LC50 2734mgmL) LF-WAE (LC50 2990mgmL) and LF-ETE (LC50 4658mgmL) (Table 1) -e overall percentagemortality of all concentrations of the extracts of R crispus in72 h as shown in Figure 6 indicated that there was no sig-nificant difference between the toxicity of the extracts of RT-MEE (43) RT-ACE (428) RT-WAE (456) and LF-MEE (458) -e highest toxicity among the extracts wasRT-ETE (586) while K2CrO7 has the mortality of 892 asthe control -e acetone extract of the leaf (LF-ACE) has thelowest mortality of 216 among all tested samples contraryto the acetone extract of the root (RT-ACE)

-e percentage sensitivity of the larva to extracts pereach concentration used was tested and the effect of thevarious concentrations of the plant extracts used on themortality of the larvae is shown in Figures 3 and 4 At thelowest concentration (00625mgmL) of the root extractsfollowed by 0125mgmL the toxicity of the extracts and thecontrol drug was not fully expressed -e percentage le-thality of all the extracts at these concentrations (00625mgmL and 0125mgmL) as shown in Figure 3 was low and lessthan 50 of the shrimp were knocked down At the con-centration 1mgmL there is no significant difference(Plt 005) between the toxicity of RT-MEE (673) RT-ETE(726) and K2CrO7 (753) which is highly toxicHowever the toxicity of the aqueous extract (RT-WAE) wasmild at the mortality of 48 of nauplii (Figure 11) InFigure 4 the concentrations of the leaf extracts used rangefrom 025 to 4mgmL which is slightly higher than the

concentration used for the root extracts due to its lesstoxicity as confirmed by the preliminary lethal concentrationtest done -ere is no significant difference between themortality of shrimp larva in the concentrations 025ndash2mgmL of the leaf extracts of R crispus -e toxicity of the leafextract was fully expressed at the concentration of 4mgmLLF-MEE has the highest percentage mortality of 50 fol-lowed by LF-WAE (426) while the LF-ACE has the lowestmortality of 12 -e toxicity of LF-ACE is the leastcompared to other extracts of the leaf contrary to thetoxicity of RT-ACE which has the highest toxicity in theroot extract (Figures 3 and 4)

343 Cytotoxicity Activity -e extracts were tested againstHeLa (human cervix adenocarcinoma) cells at a concentrationof 50μgmL-e result (Figure 12) revealed all extracts are notcytotoxic except RT-ETE (4655plusmn 484) and RT-ACE(3731plusmn 569) which reduce the viability of HeLa cells tobelow 50

4 Discussion

-e potential of phytomedicine for developing antimicrobialand antiparasitic drugs seems rewarding becausemost plantsthat have been tested against microbes and parasites showenormous therapeutic outcome with presumed fewer sideeffects but these potentials are yet untapped [40 41] -egenus Rumex has been reported and verified scientifically tobe effective against a broad range of microorganisms[28 29 41 42] and parasites [43] -e verification of theantimicrobial and antiparasitic potential of R crispus ex-tracts in this research shows that it could be a potentialsource for new antibacterial antifungal antitrypanosomaland antiplasmodial drugs

-is study revealed extracts of Rumex crispus were ef-fective against bacterial growth at different levels of con-centrations and there were significant positive correlationsbetween extract concentration and antimicrobial activitythrough the test for theMIC of bacteria (Table 2) andMIC of

RT-ETE

v

iabi

lity

0

20

40

60

80

100

0 1 2ndash1Log(μgmL)

Figure 9 Percentage viability and dose-response curve of Tbbrucei to RT-ETE (ethanol extract of root) IC50 97 μgmL


fungi (Table 3) In this research the acetone extract of theroot shows a tremendous inhibition power against bac-terial growth at the concentration of lt1562mgmLcompared to other extracts However the overall anti-bacterial potency of the extracts of the root was signifi-cantly higher than the extracts of the leaf which supportthe previous research done by Orban-Gyapai et al [29]Contrary to the antibacterial potency of the leaf extracts ofR crispus the overall MIC of the extracts shows a higherantifungal potency compared to the root extracts Fur-thermore specific in vivo studies are recommended todetermine the efficacy of these extracts in the treatment ofantimicrobial infections

-e antiplasmodial activity of R crispus extracts was testedand compared with the standard antimalarial drug (chloro-quine) Generally a compound is considered inactive againstPlasmodium when it shows an IC50gt 200μM low activitywhen IC50 is 100ndash200μM moderately active when IC50 is20ndash100μM good activity when IC50 is 1ndash20μM and excellentor potent antiplasmodial activity when IC50 is lt1μM [44] Fornatural products or extracts in which themolarmass is difficultto determine they are categorized as inactive moderate goodand excellent antiplasmodials if the IC50 values were gt5μgmL1ndash5μgmL 01ndash1μgmL and lt01μgmL respectively [45]RT-ACE and LF-MEE have the highest potency against thePlasmodium parasite with an IC50 of 13 μgmL and 15μgmLrespectively However these two extracts brought about re-duction in plasmodium lactate dehydrogenase (pLDH) activityto less than 50 as shown in Figure 1

-e antitrypanosomal activity of the extracts of R crispuswas examined using Trypanosoma brucei assay It was foundthat only RT-ETE reduced the viability of Tb brucei below50 hence RT-ETE was put forward for IC50 screening andthe IC50 value was found to be 97 ugmL Larayetan et al [8]confirmed the IC50 value of compounds on Trypanosomale20 μgmL are considered good IC50 between 20 and 60 μgmL are considered moderate while IC50gt 100 μgmL istermed not active

-e test for toxicity using brine shrimp assay is a viabletool for preliminary evaluation of the toxicity of samplesespecially plant extracts [46 47] -e toxicity of R crispuswas evaluated using hatchability of cysts and lethality of thelarvae of Artemia salina with varying concentrations InTable 1 and Figures 2ndash4 10 shows the lethal concentration(LC) and mean toxicity of the root of R crispus are almosttwice as that of the leaf -is could be due to the activephytochemical content of the root which was confirmed tobe roughly more than in the leaf [5] -e aqueous extractsalso show a mean value of lesser toxicity compared to othersolvent extracts except for aqueous extracts of the root thatshow slightly higher toxicity hence the aqueous extracts arepresumed to be less toxic when compared to other solventextracts

Bastos et al [48] interpreted the toxicity of brine shrimpin accordance with Meyer toxicity index that LC50 valuesgt 1mgmL are considered nontoxic and LC50 valuesge 05mgmL but LC50 values lt1mgmL are considered mildtoxic while LC50 valueslt 05mgmL are considered highlytoxic [48] Nevertheless it was found in this study that RT-

v

iabi

lity

Pentamidine

0

20

40

60

80

100


Figure 10 Percentage viability and dose-response curve of Tbbrucei to pentamidine (standard control) IC50 0017 μM

v

iabi

lity

LF-MEE

0

20

40

60

80

100


Figure 11 Dose-response plots and viability of parasite to LF-MEE (methanol extract of the leaf) IC50 15 μgmL

0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity

Figure 12 Percentage HeLa cell viabilityplusmn SD obtained aftertesting on R crispus extracts RT-MEE methanol extract of rootRT-ETE ethanol extract of root RT-ACE acetone extract of rootRE-WAE water extract of root LF-MEE methanol extract of leafLF-ETE ethanol extract of leaf LF-ACE acetone extract of leaf LF-WAE water extract of leaf


ETE has the lowest LC50 value (LC50 0275mgmL) followedby the LC50 value of RT-ACE (LC50 0363mgmL) -eresponse of the larva to LF-ETE (LC50 4658mgmL) LF-MEE (LC50 2734mgmL) LF-WAE (LC50 2990mgmL)and LF-ACE (LC50 1824mgmL) thus reveal the extracts arenontoxic according to Bastos et al [48]

To further ascertain the toxicity level of R crispus ex-tracts cytotoxicity of the extracts was conducted using HeLa(human cervix adenocarcinoma) cells at a concentration of50 μgmL -e study shows that RT-MEE RT-WAE LF-MEE LF-ETE LF-ACE and LF-WAE did not reduce thepercentage viability of HeLa cells below 50 hence samplesdid not cause significant cytotoxic effects which denote theirsafety when used as drugs On the contrary RT-ETE andRT-ACE reveal some degree of toxicity and reduce the vi-ability of HeLa cells to 4655plusmn 484 and 3731plusmn 569respectively (Figure 12) However it is worthy of note thatthere is a correlation between the outcome of brine shrimplethality assay (BSLA) and cytotoxicity assay using HeLacells both assays revealed that RT-ETE and RT-ACE aremost toxic among the extracts

5 Conclusion

Based on the results of the research the extracts of R crispushave therapeutic potentials against some microbes (fungaland bacteria) and parasites (Trypanosoma and Plasmodium)It was confirmed that the antimicrobial activity of the ex-tracts does not dependent on Gram-negative or Gram-positive bacteria and hypha or single-celled-form fungi -elevel of toxicity and potency of the extracts correlate with theinhibition of the viability of HeLa cells and Artemia salina-e results therefore further confirms the use of Rumexcrispus by traditional healers and recognized the potential ofmedicinal plants as the prime source of novel compounds inthe search of new treatment for African sleeping sicknessmalaria and microbial infections

Data Availability

On request the authors can provide the underlying data forthe research article

Conflicts of Interest

-e authors declare no conflicts of interest

Authorsrsquo Contributions

Oladayo Amed Idris designed experiments performed theexperiments analyzed data and wrote the manuscript Allauthors contributed to the proofreading and editing of themanuscript

Acknowledgments

-e authors are grateful to Dr Larayetan Rotimi De-partment of Pure and Applied Chemistry University of FortHare for the extensive discussions interactions and

assistance with antitrypanosomal antiplasmodial and cy-totoxicity parts of this article

References

[1] M S Amjad M Arshad and R Qureshi ldquoEthnobotanical in-ventory and folk uses of indigenous plants from Pir NasooraNational Park Azad Jammu and KashmirrdquoAsian Pacific Journalof Tropical Biomedicine vol 5 no 3 pp 234ndash241 2015

[2] N Bhalodia and V Shukla ldquoAntibacterial and antifungalactivities from leaf extracts of Cassia fistula l an ethno-medicinal plantrdquo Journal of Advanced Pharmaceutical Tech-nology amp Research vol 2 no 2 p 104 2011

[3] A J Afolayan ldquoExtracts from the shoots of Arctotis arcto-toides inhibit the growth of bacteria and fungirdquo Pharma-ceutical Biology vol 41 no 1 pp 22ndash25 2003

[4] T Selvamohan V Ramadas and S S Kishore ldquoAntimicrobialactivity of selected medicinal plants against some selectedhuman pathogenic bacteriardquo Pelagia Research Library Ad-vances in Applied Science Research vol 3 no 5 pp 3374ndash3381 2012

[5] O A Idris O A Wintola and A J Afolayan ldquoPhytochemicaland antioxidant activities of Rumex crispus L in treatment ofgastrointestinal helminths in Eastern Cape province SouthAfricardquo Asian Pacific Journal of Tropical Biomedicine vol 7no 12 pp 1071ndash1078 2017

[6] G M Cragg and D J Newman ldquoBiodiversity a continuingsource of novel drug leadsrdquo Pure and Applied Chemistryvol 77 no 1 pp 7ndash24 2005

[7] K Murugan N Aarthi K Kovendan et al ldquoMosquitocidaland antiplasmodial activity of Senna occidentalis (Cassiae)and Ocimum basilicum (Lamiaceae) fromMaruthamalai hillsagainst Anopheles stephensi and Plasmodium falciparumrdquoParasitology Research vol 114 no 10 pp 3657ndash3664 2015

[8] R Larayetan M O Ojemaye O O Okoh and A I OkohldquoSilver nanoparticles mediated by Callistemon citrinus ex-tracts and their antimalaria antitrypanosoma and antibac-terial efficacyrdquo Journal of Molecular Liquids vol 273pp 615ndash625 2019

[9] J Srivastava H Chandra A R Nautiyal and S J S KalraldquoAntimicrobial resistance (AMR) and plant-derived antimi-crobials (PDAms) as an alternative drug line to control in-fectionsrdquo 3 Biotech vol 4 no 5 pp 451ndash460 2014

[10] D Dubey S Rath M C Sahu N Nayak N K Debata andR N Padhy ldquoStatus of multidrug resistance in tubercle ba-cillus and phytochemicals for the controlrdquo Journal of PublicHealth vol 21 no 1 pp 115ndash119 2013

[11] M P Mishra S Rath S S Swain G Ghosh D Das andR N Padhy ldquoIn vitro antibacterial activity of crude extracts of9 selected medicinal plants against UTI causing MDR bac-teriardquo Journal of King Saud University-Science vol 29 no 1pp 84ndash95 2017

[12] J R Borchardt D L Wyse C C Sheaffer et al ldquoJournal ofmedicinal plant researchrdquo Academic Journals vol 2 no 42008

[13] H A M Mostafa A A Elbakry and A A Eman ldquoEvaluationof antibacterial and antioxidant activities of different plantparts of Rumex vesicarius L (polygonaceae)rdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 3no 2 pp 109ndash118 2011

[14] H A Martinez-Correa R G Bitencourt A C A V KayanoP M Magalhatildees F T M Costa and F A Cabral ldquoIntegratedextraction process to obtain bioactive extracts of Artemisia


annua L leaves using supercritical CO2 ethanol and waterrdquoIndustrial Crops and Products vol 95 pp 535ndash542 2017

[15] F Nardella J-B Galle M Bourjot B Weniger andC Vonthron-Senecheau ldquoAntileishmanial and anti-trypanosomal activities of flavonoidsrdquo in Sustainable Devel-opment and Biodiversity pp 163ndash194 Springer BerlinGermany 2018

[16] D Savoia ldquoPlant-derived antimicrobial compounds alter-natives to antibioticsrdquo Future Microbiology vol 7 no 8pp 979ndash990 2012

[17] M Kuria P K Njenga and V Ngumi ldquoEthnobotanicalstudies of Strychnos henningsii in five (Gilg) natural habitatsin Kenyardquo International Journal of Medicinal Plants Researchvol 1 no 6 pp 2169ndash2303 2012

[18] O A Idris O AWintola and A J Afolayan ldquoHelminthiasesprevalence transmission host-parasite interactions re-sistance to common synthetic drugs and treatmentrdquo Heliyonvol 5 no 1 article e01161 2019

[19] J B Githiori S Athanasiadou and S M -amsborg ldquoUse ofplants in novel approaches for control of gastrointestinalhelminths in livestock with emphasis on small ruminantsrdquoVeterinary Parasitology vol 139 no 4 pp 308ndash320 2006

[20] T H Bello and O A Idris ldquo-e effect of antioxidant (gallicacid) on the testes of lead acetate induced Wistar ratrdquo Tox-icology and Environmental Health Sciences vol 10 no 5pp 261ndash267 2018

[21] O Idris O Wintola and A Afolayan ldquoComparison of theproximate composition vitamins (ascorbic acid α-tocopheroland retinol) anti-nutrients (phytate and oxalate) and the GC-MS analysis of the essential oil of the root and leaf of Rumexcrispus Lrdquo Plants vol 8 no 3 p 51 2019

[22] P Mayorga K R Perez S M Cruz and A CaceresldquoComparison of bioassays using the anostracan crustaceansArtemia salina and -amnocephalus platyurus for plant ex-tract toxicity screeningrdquo Revista Brasileira de Farmacognosiavol 20 no 6 pp 897ndash903 2010

[23] M R Hamidi B Jovanova and T Kadifkova PanovskaldquoToxicological evaluation of the plant products using brineshrimp (Artemia salina L) modelrdquo Macedonian Pharma-ceutical Bulletin vol 60 no 1 pp 9ndash18 2014

[24] T Veni and T Pushpanathan ldquoComparison of the Artemiasalina and Artemia fransiscana bioassays for toxicity of Indianmedicinal plantsrdquo Journal of Coastal Life Medicine vol 2no 6 pp 453ndash457 2014

[25] A L Parra R S Yhebra I G Sardintildeas and L I BuelaldquoComparative study of the assay of Artemia salina L and theestimate of the medium lethal dose (LD50 value) in mice todetermine oral acute toxicity of plant extractsrdquo Phytomedi-cine vol 8 no 5 pp 395ndash400 2001

[26] A Vasas O Orban-Gyapai and J Hohmann ldquo-e genusRumex review of traditional uses phytochemistry andpharmacologyrdquo Journal of Ethnopharmacology vol 175pp 198ndash228 2015

[27] I Coruh A Gormez S Ercisli andM Sengul ldquoTotal phenoliccontent antioxidant and antibacterial activity of Rumexcrispus grown wild in Turkeyrdquo Pharmaceutical Biologyvol 46 no 9 pp 634ndash638 2008

[28] M Wegiera U Kosikowska A Malm and H SmolarzldquoAntimicrobial activity of the extracts from fruits of Rumex Lspeciesrdquo Open Life Sciences vol 6 no 6 pp 1036ndash1043 2011

[29] O Orban-Gyapai E Liktor-Busa N Kusz et al ldquoAntibac-terial screening of Rumex species native to the CarpathianBasin and bioactivity-guided isolation of compounds fromRumex aquaticusrdquo Fitoterapia vol 118 pp 101ndash106 2017

[30] NWorku AMossie A Stich et al ldquoEvaluation of the in vitroefficacy of Artemisia annua Rumex abyssinicus and Cathaedulis Forsk extracts in cancer and Trypanosoma brucei cellsrdquoISRN Biochemistry vol 2013 Article ID 910308 10 pages2013

[31] K H Lee and K-H Rhee ldquoAntimalarial activity of nepodinisolated from Rumex crispusrdquo Archives of Pharmacal Re-search vol 36 no 4 pp 430ndash435 2013

[32] J M Andrews ldquoDetermination of minimum inhibitoryconcentrationsrdquo Journal of Antimicrobial Chemotherapyvol 48 no 1 pp 5ndash16 2001

[33] A A Mostafa A A Al-Askar K S Almaary T M DawoudE N Sholkamy and M M Bakri ldquoAntimicrobial activity ofsome plant extracts against bacterial strains causing foodpoisoning diseasesrdquo Saudi Journal of Biological Sciencesvol 25 no 2 pp 253ndash258 2018

[34] C Lass-Florl M Cuenca-Estrella D W Denning andJ L Rodriguez-Tudela ldquoAntifungal susceptibility testing inAspergillusspp according to EUCASTmethodologyrdquoMedicalMycology vol 44 no s1 pp 319ndash325 2006

[35] S Jain M Jacob LWalker and B Tekwani ldquoScreening NorthAmerican plant extracts in vitro against Trypanosoma bruceifor discovery of new antitrypanosomal drug leadsrdquo BMCComplementary and Alternative Medicine vol 16 no 1p 131 2016

[36] G A Otunola J O Unuofin and A J Afolayan ldquoToxicityevaluation of Vernonia mespilifolia less (a South Africamedicinal plant) using brine shrimprdquo Journal of Pharma-cology and Toxicology vol 12 no 2 pp 103ndash110 2017

[37] W O Abosede A Sunday and A A Jide ldquoToxicologicalinvestigations of Aloe ferox Mill extracts using brine shrimp(Artemia salina L) assayrdquo Pakistan Journal of PharmaceuticalSciences vol 28 no 2 pp 635ndash640 2015

[38] P Agrafioti C G Athanassiou T N Vassilakos G Vlontzosand F H Arthur ldquoUsing a lethality index to assess suscep-tibility of Tribolium confusum and Oryzaephilus sur-inamensis to insecticidesrdquo PLoS One vol 10 no 11 Article IDe0142044 2015

[39] M M Milhem A S Al-Hiyasat and H Darmani ldquoToxicitytesting of restorative dental materials using brine shrimplarvae (Artemia salina)rdquo Journal of Applied Oral Sciencevol 16 no 4 pp 297ndash301 2008

[40] M Wink ldquoMedicinal plants a source of anti-parasitic sec-ondary metabolitesrdquo Molecules vol 17 no 11pp 12771ndash12791 2012

[41] M S Maryam S Parvin and M Mahboobeh ldquoEvaluation ofantibacterial activity of extract of Rumex alveollatus leafagainst Staphylococcus aureus and Pseudomonas aerugi-nosardquo Zahedan Journal of Research in Medical Sciencesvol 15 pp 58ndash61 2013

[42] S Yadav S Kumar P Jain et al ldquoAntimicrobial activity ofdifferent extracts of roots of Rumex nepalensis Sprengrdquo In-dian Journal of Natural Products and Resources vol 2 no 1pp 65ndash69 2011

[43] I E Cock M I Selesho and S F Van Vuuren ldquoA review ofthe traditional use of Southern African medicinal plants forthe treatment of selected parasite infections affectinghumansrdquo Journal of Ethnopharmacology vol 220 pp 250ndash264 2018

[44] C R Nogueira and L M X Lopes ldquoAntiplasmodial naturalproductsrdquo Molecules vol 16 no 3 pp 2146ndash2190 2011

[45] M T Lemma A M Ahmed M T Elhady et al ldquoMedicinalplants for in vitro antiplasmodial activities a systematic


review of literaturerdquo Parasitology International vol 66 no 6pp 713ndash720 2017

[46] O Wintola and A Afolayan ldquoAn inventory of indigenousplants used as anthelmintics in Amathole district municipalityof the Eastern Cape province South Africardquo African Journalof Traditional Complementary and Alternative Medicinesvol 12 no 4 pp 112ndash121 2015

[47] A J Afolayan F U Ohikhena and O A Wintola ldquoToxicityassessment of different solvent extracts of the medicinal plantPhragmanthera capitata (Sprengel) Balle on brine shrimp(Artemia salina)rdquo International Journal of Pharmacologyvol 12 no 7 pp 701ndash710 2016

[48] M Bastos M Lima L M Conserva V S AndradeE M Rocha and R P Lemos ldquoStudies on the antimicrobialactivity and brine shrimp toxicity of Zeyheria tuberculosa(Vell) Bur (Bignoniaceae) extracts and their main constit-uentsrdquo Annals of Clinical Microbiology and Antimicrobialsvol 8 no 1 p 16 2009


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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Volume 2018

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Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

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Research and TreatmentAIDS


Gastroenterology Research and Practice


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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

resistant strains of pathogens and parasites are economicallyimportant and these pose a huge threat to the public healthsystem -is calls for an immediate intervention with newdrugs that use a different mechanism of action in thetreatment of these diseases On the other hand there areseveral scientific evidences to the success of medicinal plantsin treating diseases hence plants could be a novel sourcethat will combat drug resistance [5]

Plants are known to produce complex bioactive organiccompounds that possess several bioactivities -e com-pounds are secreted in various parts of the plants such asseeds fruits flowers leaves roots stems bark and someplant secretions (isothiocyanates plant pigment plant gumphytoalexins essential oils and hydrolytic enzymes) [12]-e bioactive constituent in medicinal plants has showntremendous health benefits For instance the major anti-bacterial bioactive compounds in plants are anthraquinonesand flavonoids which are active against several humanpathogenic bacteria [13] Phenolic compounds flavonoidsand artemisinin are active against malaria causing parasitePlasmodium falciparum [14] It was also reported that fla-vonoids and glycosides in plants inhibit the growth of Africansleeping sickness (Trypanosoma brucei) parasites [15] -eincrease in the occurrence and sustenance of drug-resistantpathogens to drugs has drawn the attention of scientists andpharmaceutical communities towards harnessing the po-tentials of plant-derived substances used successfully in dif-ferent countries by traditional medical practitioners [16]

12 Tests for Toxicity Antimicrobial and Antiparasite-ere is a rapid increase in interest and demand for medicinalplants in various parts of the world because they are con-sidered natural safer and cheaper [17] On the other handthere is a general concern in the scientific communities on thesafety efficacy and pharmacokinetics of medicinal plantsirrespective of their wide acceptance and usage -e possibleside effects of plant products cannot be ruled out regardless oftheir adoption as a novel source of drugs [18 19]

-e source of the toxicity of plants may be due to thecontaminants in the environment or from the plant-secretedphytochemicals [20 21] Different biological models havebeen tested via in vitro and in vivo assays to qualify andquantify the potential toxicity of extracts of medicinal plantsHowever recent studies employed the use of preliminarytoxicity assessment tools using brine shrimp species Arte-mia franciscana Artemia salinaamnocephalus platyurusand Artemia urmiana [22ndash24] which is considered a usefultool to evaluate the potential toxicity of plant extractsCytotoxic screening using HeLa human cervix carcinomacells is an advanced tool to screen substances inducingapoptosis In addition a comparative toxicity study of plantsextract could be tested using in vivo study -e resulthowever showed quite a good correlation in the mean valueof in vitro study lethal concentration (LC50) and the acuteoral lethal dose (LD50) of in vivo study [23 25]

Rumex crispus L (curled dock) is commonly used bytraditional healers for treatment of various diseases andcorrection of disorders such as gastrointestinal tract

disorders antihelminthic diseases anti-inflammatory andarthritis and it is also used as laxative antipyretic anti-oxidant and antimicrobial [5 26] More so there are reportsof the antimicrobial [27ndash29] antitrypanosomal [30] andantiplasmodial [31] properties and toxicity of R crispuswhich were evaluated in this research -is is necessary tovalidate the claim of traditional healer practitioners inEastern Cape South Africa and any other part of the worldthat use R crispus for the treatment of these ailments

2 Materials and Methods

21 Organic Extraction -e leaves and roots of Rumexcrispuswere collected from a fallow damp field University ofFort Hare (32deg47prime123PrimeS 26deg51prime985PrimeE) in the summer of2017 It was identified and a sample of the specimen wasplaced in Giffen herbarium (Idr-Med-201703) Universityof Fort Hare-e leaves and roots of the plant specimen werecarefully sorted and the infected ones were removed -especimen was rinsed dried in an oven at 30degC until a fixedweight and then pulverized with a mechanical grinder(Polymix PX-MFC90D Switzerland)-e pulverized samplewas stored in an airtight container in a refrigerator at 4degCuntil use for extraction

Crude extraction of R crispus was done using standardprocedures -e choice of solvent for the extraction processis an imperative factor that must be considered during theantimicrobial and toxicity tests -e extracting potentials ofdifferent solvents differ therefore extraction was done usingsolvents of low reactivity a technically graded acetonemethanol ethanol and distilled water -e extractant(solvent) and the plant materials used were in the ratio of 1 10 (gmL)-e dried root (RT) and the aerial part (LF) of theplant sample (100 g) were macerated with the extractantsolvents (1000mL) at room temperature -e crude extractof methanol (MEE) ethanol (ETE) and acetone (ACE) wereconcentrated under reduced pressure by using a rotatoryevaporator considering the boiling point of the respectivesolvent and then dried in a desiccator at a mild temperature(40degC) -e filtrate of water samples (WAE) was chilled atminus 40degC with a shell refrigerant (PolyScience AD15R-40-A12E USA) and freeze-dried (Savant vapour trap RV-T41404 USA) for 48 h -e plant crude extracts were storedin sterile bottles and refrigerated at 2 to 4degC until further use

22 Determination of Minimum Inhibitory Concentrations(MICs) of the Extracts Minimum inhibitory concentrations(MICs) were used to determine the lowest concentration ofthe extracts that will inhibit visible microbial growth after anincubation period of 24 h (bacteria) and 48 h (fungi)Usually MICs are diagnostic laboratory tools used toconfirm microbial resistance but are often used in researchlaboratories to determine the in vitro activity of antimi-crobials and its MIC breakpoints [32]

23 Antibacterial Activity of the Plant Extracts -e potencyof each extract of R crispus was tested against four strains ofGram-negative (Klebsiella pneumonia ATCC 4352
















n0



1113890 1113891 times 100 (1)


i 0 1 2 3





3 Results







RT-MEERT-ETERT-ACE

RT-WAELF-MEELF-ETE

LF-ACELF-WAEK2CrO7

a b

c

a b

aa

ba

a b

aa

ba

b

a

da

bb

cb

c d

a b

cb

c d

c d

b b b

b

b

a a

aa

a b

b c

ca

a b

a b

a ac c

d c d

db

cc

da

a b

ad0

10

20

30

40

50

60

70

80

h

atch

abili

ty








RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

K 2Cr

O7


0

10

20

30

40

50

60

70

80

90

100

h

atch

abili

ty an

d le

thal

ity



le

thal

ity o

f roo

t ext

ract

s

RT-MEERT-ETERT-ACE

RT-WAEK2CrO7

cb

cc

ba

cb

cc

ba

cb

cb

ca

c d

bc

da a b a

bc

a

0

10

20

30

40

50

60

70

80






LF-MEELF-ETE

LF-ACELF-WAE

aa

aa

aa

aa a

aa

a

aa

bb

a b

ab

ca

b

le

thal

ity o

f lea

f ext

ract

s

0

10

20

30

40

50

60










34 Toxicity Test


0

10

20

30

40

50

60

70

80

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity


v

iabi

lity

0

20

40

60

80

100


RT-ACE


v

iabi

lity

Chloroquine

0

20

40

60

80

100



0102030405060708090

100110

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity








4 Discussion



RT-ETE

v

iabi

lity

0

20

40

60

80

100









v

iabi

lity

Pentamidine

0

20

40

60

80

100



v

iabi

lity

LF-MEE

0

20

40

60

80

100



0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity





5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

































n0



1113890 1113891 times 100 (1)


i 0 1 2 3





3 Results







RT-MEERT-ETERT-ACE

RT-WAELF-MEELF-ETE

LF-ACELF-WAEK2CrO7

a b

c

a b

aa

ba

a b

aa

ba

b

a

da

bb

cb

c d

a b

cb

c d

c d

b b b

b

b

a a

aa

a b

b c

ca

a b

a b

a ac c

d c d

db

cc

da

a b

ad0

10

20

30

40

50

60

70

80

h

atch

abili

ty








RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

K 2Cr

O7


0

10

20

30

40

50

60

70

80

90

100

h

atch

abili

ty an

d le

thal

ity



le

thal

ity o

f roo

t ext

ract

s

RT-MEERT-ETERT-ACE

RT-WAEK2CrO7

cb

cc

ba

cb

cc

ba

cb

cb

ca

c d

bc

da a b a

bc

a

0

10

20

30

40

50

60

70

80






LF-MEELF-ETE

LF-ACELF-WAE

aa

aa

aa

aa a

aa

a

aa

bb

a b

ab

ca

b

le

thal

ity o

f lea

f ext

ract

s

0

10

20

30

40

50

60










34 Toxicity Test


0

10

20

30

40

50

60

70

80

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity


v

iabi

lity

0

20

40

60

80

100


RT-ACE


v

iabi

lity

Chloroquine

0

20

40

60

80

100



0102030405060708090

100110

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity








4 Discussion



RT-ETE

v

iabi

lity

0

20

40

60

80

100









v

iabi

lity

Pentamidine

0

20

40

60

80

100



v

iabi

lity

LF-MEE

0

20

40

60

80

100



0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity





5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















3 Results







RT-MEERT-ETERT-ACE

RT-WAELF-MEELF-ETE

LF-ACELF-WAEK2CrO7

a b

c

a b

aa

ba

a b

aa

ba

b

a

da

bb

cb

c d

a b

cb

c d

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b

b

a a

aa

a b

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a b

a b

a ac c

d c d

db

cc

da

a b

ad0

10

20

30

40

50

60

70

80

h

atch

abili

ty








RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

K 2Cr

O7


0

10

20

30

40

50

60

70

80

90

100

h

atch

abili

ty an

d le

thal

ity



le

thal

ity o

f roo

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ract

s

RT-MEERT-ETERT-ACE

RT-WAEK2CrO7

cb

cc

ba

cb

cc

ba

cb

cb

ca

c d

bc

da a b a

bc

a

0

10

20

30

40

50

60

70

80






LF-MEELF-ETE

LF-ACELF-WAE

aa

aa

aa

aa a

aa

a

aa

bb

a b

ab

ca

b

le

thal

ity o

f lea

f ext

ract

s

0

10

20

30

40

50

60










34 Toxicity Test


0

10

20

30

40

50

60

70

80

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity


v

iabi

lity

0

20

40

60

80

100


RT-ACE


v

iabi

lity

Chloroquine

0

20

40

60

80

100



0102030405060708090

100110

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity








4 Discussion



RT-ETE

v

iabi

lity

0

20

40

60

80

100









v

iabi

lity

Pentamidine

0

20

40

60

80

100



v

iabi

lity

LF-MEE

0

20

40

60

80

100



0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity





5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

K 2Cr

O7


0

10

20

30

40

50

60

70

80

90

100

h

atch

abili

ty an

d le

thal

ity



le

thal

ity o

f roo

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ract

s

RT-MEERT-ETERT-ACE

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cb

cc

ba

cb

cc

ba

cb

cb

ca

c d

bc

da a b a

bc

a

0

10

20

30

40

50

60

70

80






LF-MEELF-ETE

LF-ACELF-WAE

aa

aa

aa

aa a

aa

a

aa

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a b

ab

ca

b

le

thal

ity o

f lea

f ext

ract

s

0

10

20

30

40

50

60










34 Toxicity Test


0

10

20

30

40

50

60

70

80

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity


v

iabi

lity

0

20

40

60

80

100


RT-ACE


v

iabi

lity

Chloroquine

0

20

40

60

80

100



0102030405060708090

100110

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

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LF-A

CE

LF-W

AE

v

iabi

lity








4 Discussion



RT-ETE

v

iabi

lity

0

20

40

60

80

100









v

iabi

lity

Pentamidine

0

20

40

60

80

100



v

iabi

lity

LF-MEE

0

20

40

60

80

100



0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity





5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















LF-MEELF-ETE

LF-ACELF-WAE

aa

aa

aa

aa a

aa

a

aa

bb

a b

ab

ca

b

le

thal

ity o

f lea

f ext

ract

s

0

10

20

30

40

50

60










34 Toxicity Test


0

10

20

30

40

50

60

70

80

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity


v

iabi

lity

0

20

40

60

80

100


RT-ACE


v

iabi

lity

Chloroquine

0

20

40

60

80

100



0102030405060708090

100110

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity








4 Discussion



RT-ETE

v

iabi

lity

0

20

40

60

80

100









v

iabi

lity

Pentamidine

0

20

40

60

80

100



v

iabi

lity

LF-MEE

0

20

40

60

80

100



0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity





5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















34 Toxicity Test


0

10

20

30

40

50

60

70

80

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity


v

iabi

lity

0

20

40

60

80

100


RT-ACE


v

iabi

lity

Chloroquine

0

20

40

60

80

100



0102030405060708090

100110

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity








4 Discussion



RT-ETE

v

iabi

lity

0

20

40

60

80

100









v

iabi

lity

Pentamidine

0

20

40

60

80

100



v

iabi

lity

LF-MEE

0

20

40

60

80

100



0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity





5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























4 Discussion



RT-ETE

v

iabi

lity

0

20

40

60

80

100









v

iabi

lity

Pentamidine

0

20

40

60

80

100



v

iabi

lity

LF-MEE

0

20

40

60

80

100



0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity





5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























v

iabi

lity

Pentamidine

0

20

40

60

80

100



v

iabi

lity

LF-MEE

0

20

40

60

80

100



0102030405060708090

100110120

RT-M

EE

RT-E

TE

RT-A

CE

RT-W

AE

LF-M

EE

LF-E

TE

LF-A

CE

LF-W

AE

v

iabi

lity





5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















5 Conclusion


Data Availability






Acknowledgments



References


























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















evaluationofthebioactivitiesof rumexcrispus...

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