antibacterial,antifungal,andantidiabeticeffectsofleaf

Research ArticleAntibacterial Antifungal and Antidiabetic Effects of LeafExtracts from Persea americana Mill (Lauraceae)

Mercy Makopa Benjamin Mangiza Benjamin Banda Winnie Mozirandi Molly Mombeshora and Stanley Mukanganyama

Department of Biochemistry University of Zimbabwe Mount Pleasant Harare Zimbabwe

Correspondence should be addressed to Stanley Mukanganyama smukanganyamamedicuzaczw

Received 13 July 2020 Revised 29 October 2020 Accepted 30 October 2020 Published 11 November 2020

Academic Editor Z Beyir Huyut

Copyright copy 2020MercyMakopa et alis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fruits and leaves of Persia americana are used in traditional medical practices is study was carried out to determine theantibacterial antifungal and antidiabetic effects of the leaf extracts from P americana e antibacterial activities of the leafextracts were evaluated against Klebsiella pneumoniae and Staphylococcus epidermidis while antifungal activities were determinedagainst Candida albicans and Candida tropicalis e antidiabetic potential of the extracts was determined against mammalianα-glucosidase in vitro e broth microdilution method was used to investigate the antibacterial and antifungal susceptibility ofthe microbial strains towards the leaf extracts S epidermidis was the most susceptible microbe out of the tested microorganismse acetone extract was the most potent extract against S epidermidis with a minimum inhibitory concentration (MIC) of 50 μgmL At 100 μgmL the ethanolwater extract 18 of K pneumoniae cells remained viable Cell viability after exposure to thedichloromethane (DCM) and methanol extracts was 28 against C albicans and 8 against C tropicalis respectively e DCMmethanol and acetone extracts caused membrane damage in S epidermidis exhibited by protein leakage Only the acetone extracteffected nucleic acid leakage Screening of extractsrsquo potential to inhibit the activity of α-glucosidase was carried out spectro-photometrically following the production of p-nitrophenol from p-nitrophenol-glucopyranoside (substrate) at a wavelength of405 nm Out of all the tested extracts the methanolic extract showed the best inhibitory activity on α-glucosidase enzyme in atime-dependent and dose-dependent manner Ki and Kinact values were found to be 14mgmL and 24Umin respectively afterincubation for 1 hour It was concluded that the leaf extracts of P americana contain phytochemicals with antibacterial an-tifungal and α-glucosidase inhibitory effects Further studies are required for the identification of the active compounds in the leafextracts responsible for these observed effects

1 Introduction

Plants are known to have naturally occurring chemicals thatare bioactive meaning that they have the ability to interactwith components of living tissue producing an effect [1]eantimicrobial properties exerted by plants have been at-tributed to secondary metabolites [2] Secondarymetabolitesor phytochemicals such as alkaloids tannins flavonoidsterpenoids and glycosides have been studied [3] Secondarymetabolites have resulted in the development of systems thatcan efficiently isolate bioactive compounds in plants [4] withantimicrobial properties Many drugs commonly used todayare of herbal origin about 25 of the prescription drugs

dispensed in the USA contain at least one active ingredientderived from plant material [5]

Persea americana also known as the avocado plant is atree that is indigenous to Central America e avocadoplant belongs to one of the oldest flowering plant familiesknown as Lauraceae [6] Different parts of the plant havereceived remarks for their therapeutic effects against skininfections stomachaches anaemia skin ulcers and diabetes[7] Ajayi et al [8] evaluated the effects of methanolic leafextracts of P americana against clinical strains of Escherichiacoli and Pseudomonas aeruginosa e study showed that theextracts possess potent antibacterial activities e work byAjayi et al [8] attributed the antibacterial effects observed to

HindawiBiochemistry Research InternationalVolume 2020 Article ID 8884300 10 pageshttpsdoiorg10115520208884300

the presence of secondary plant metabolites TraditionallyP americana is widely used as a source of medicine and foodin many parts of the world Leaves of the plant can be used tomake tea and as flavorings in beef as well as bean dishes [5]In developing countries problems are associated with themeans of managing diabetes using antidiabetic drugs due toavailability and affordability Drug candidates have beendeveloped from the phytochemical constituents isolatedfrom medicinal plants A variety of plants are being studiedfor their antidiabetic potential [9]

Candida albicans is an etiological microbe for fungalinfections In a healthy individual the growth of C albicansis regulated by bacteria and other microorganisms that arepart of the natural flora e uncontrolled growth ofC albicans results in candidiasis [10] Reports have docu-mented a shift from C albicans as the cause of the majorityof invasive infections toward non-C albicans speciesC tropicalis is classified as one of the most potent yeasts ofthe non-albicans Candida group [11] Previous studies haveshown that the Candida species are susceptible to plantextracts Extracts from the Combretum molle Piper capenseSolanum aculeastrum Syzygium cordatum and Zanthox-ylum davyi showed antifungal properties against C albicans[12]

Antibacterial properties of medicinal plants are beingincreasingly reported from different parts of the worldStaphylococcus epidermidis is a Gram-positive bacteriumthat is spherically shaped and a facultative anaerobe [13] It ispart of the normal human flora typically skin flora and lessassociated with the mucosal flora in humans [14] ebacterium has been shown to be less pathogenic in humansbut virulence of the strain has been reported in immuno-compromised individuals particularly in nosocomial in-fections [15] K pneumoniae is a Gram-negative rod-shaped non-encapsulated facultative anaerobic and lactosefermenting bacterium e bacterium is responsible forcausing nosocomial infections and has been reported to beresistant to beta lactam antibiotics such as penicillin [16]

e enzyme α-glucosidase is involved in the breakdownof carbohydrates during digestion e enzyme catalysesreactions that result in the release of the terminal alpha-glucose residue from disaccharides and oligosaccharides[17] Avocado leaves contain phytochemicals such as fla-vonoids and phenols in the fruit or the seed and these havebeen reported to have antidiabetic effects [18] A therapeuticapproach to treat diabetes is to decrease postprandialhyperglycaemia Avocado leaves have been used locally tomake avocado tea used as a means of managing diabetes [19]It is therefore important to investigate the effects of the leafextracts on α-glucosidase so as to validate its use as a meansof managing diabetes e aim of the study was to evaluatethe antimicrobial and antidiabetic activity of leaf extractsfrom P americana

2 Materials and Methods

21 Chemicals and Reagents All the chemicals used wereobtained from Sigma Aldrich (Darmstadt Germany) Allsolvents used were of analytical reagent grade these were

acetone methanol n-hexane ethanol ethyl acetatedichloromethane (DCM) and chloroform Dimethyl sulf-oxide (DMSO) was used for dissolving the crude extract aswell as other reagents used Sabouraud dextrose broth(SDB) Sabouraud glucose 2 agar (SGA) Luria Bertanibroth 3-(45-dimethylthiazolyl)-25-diphenyltetrazoliumbromide (MTT) and potassium hydroxide (KOH) edrugs miconazole and fluconazole were obtained fromSigma-Aldrich (Germany) C albicans (NCPF 3255) waspurchased from Sigma Aldrich A clinical strain ofC tropicalis was obtained from the Department of MedicalMicrobiology at Parirenyatwa Hospital in HarareZimbabwe

22 Plant Collection and Preparation P americana leaveswere collected from a tree in Chitungwiza Seke at housenumber 9209 Unit K 177deg Southwest of Chitungwiza Baptist(Greater Harare Zimbabwe) Leaves were taken to theNational Herbarium and Botanical Gardens of Zimbabwefor identification e leaves were washed with tap water toremove dirt and dried in an incubator at 40degC for 72 hrsDried leaves were then ground using traditional pestle andmortar to fine powder weighed and put in a plastic beaker

23 Extraction of Phytochemicals Using Differential Solvents

231 Extraction with DCM Methanol and HydroethanolicWater Solvent (Total Extraction) Two sets of solventmixtures were used (1) DCMmethanol in the ratio 1 1 and(2) ethanolwater in the ratio 1 1e extracts were preparedby adding 20 g of the ground powder to 100mL of thesolvents e mixture of the solvent and the leaf powder wasfiltered to obtain the extract During the filtration processcotton was used first so as to trap the undissolved mattere filtrate obtained was further filtered using a filter paperWhatman no 1 so as to trap the particles that were able topass through the cotton e filtrate was collected and driedunder a fan

232 Serial Exhaustive Extraction Serial exhaustive ex-traction of phytochemicals of the leaf powder fromP americana was carried out serially using solvents of in-creasing polarity hexane dichloromethane ethyl acetateacetone ethanol methanol and water A total mass of 30 gof leaf powder was used in the first serial solvent (hexane)and left for 24 hours for extraction After 24 hours thecontents in the beaker were filterede retained leaf residuewas dried and extracted with another solvent is wasrepeated until all the solvents had been used for extractionUltimately a total of 7 extracts were obtained

24 Screening forAntifungal andAntibacterial Activity of LeafExtracts from P americana

241 Growth of Microorganisms Overnight cultures wereprepared by picking 3 colonies from an agar plate of themicrobe and inoculating into the relevant broth followed by

2 Biochemistry Research International

incubating at 37degC for 24 hrs in a Lab Companion incubator(Jeio Tech Seoul Korea) C tropicalis and C albicans weregrown in SDB while Luria broth was used for the growth ofK pneumoniae and S epidermidis Cells were standardisedusing 05 McFarland standard to give a working concen-tration of 2times106 cfumL

242 Screening for Antifungal and Antibacterial ActivityScreening for antifungal and antibacterial activity of extractswas determined using the broth microdilution method [20]Extracts from serial exhaustive extraction and total ex-traction were tested against C albicans C tropicalisS epidermidis and K pneumoniae Extracts were dissolvedin DMSO and concentrations of 25 50 and 100 μgmL wereprepared by diluting using media A volume of 100 μL ofextract was added to the wells of a 96-well microplate To thetest wells 100 μL of bacterial cells with a concentration of2times106 cfumL was added e wells with media only (SDBLB) were included to check for sterility Wells with cells onlyserved as the negative control Miconazole and levofloxacinwere used as the reference drugs for fungi and bacteriarespectively Pre-incubation readings of the absorbance weremeasured at 590 nm using a Tecan microplate reader (TecanGenios-Pro microplate reader Grodig Austria) e platewas incubated for 24 hrs without shaking at 37degC in a LabCompanion incubator A post-incubation reading of the celldensity was determined A colorimetric determination formetabolically active cells after exposure to the extract wasperformed by adding of 25 μL aliquots of 1mgmL 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) to each of the wells of the 96-well plate and incu-bating the plate at 37degC for 2 hrs In the presence of met-abolically active fungi or bacteria the yellow MTT dye isreduced to a purple formazan [21] Colour intensity wasdetermined by reading absorbance values at 590 nm using aTecan microplate reader where there was partial inhibitionand MIC could not be determined Data are presented aspercentage cell viability Percentage cell viability was ob-tained using the following

cell viability () meanOD sample

meanODpositive controltimes 100 (1)

25 Determination of the Effects of P americana Extracts onNucleic Acid Leakage Propidium iodide is a dye that iscapable of binding to nucleic acidse dye is unable to enterviable cells making it useful for determining the effects ofplant extracts on bacterial membranes To determine theeffect of extracts from P americana on the integrity ofbacterial and fungal membranes the method outlined by El-Nakeeb et al [22] with minor modification was used Anovernight culture of S epidermidis cells was used in theassaye optical density (OD600) of cells was adjusted to 15by diluting with 09 saline solution e negative controlcontained 3mL of cells and 3mLmediae positive controlcontained 3mL of cells and 3mL of 01 sodium dodecylsulphate e cells were exposed to three concentrations of

the most potent extract e exposed cells were incubated at37degC for 30mins After incubation 1mL of the cells wascentrifuged for 1 minute at 11000 rpm using a Geratebaumicrocentrifuge (Engelsdorf Germany) e pellet waswashed with 1mL saline and 3 μL of propidium iodide wasadded to the mixture e mixture was left to stand in thedark for 10 minutes Fluorescence was measured at exci-tation and emission wavelengths of 544 nm and 612 nmrespectively using a f max microplate spectrofluorometer(Molecular Devices Sunnyvale USA)

26 Determination of the Effects of Extracts fromP americanaon Protein Leakage Determination of protein leakage wascarried out according to the method by Du et al [23] withmodifications An overnight culture of S epidermidis cellswas suspended in 09 saline solution to give OD600 15e cell suspension was exposed to plant extracts at con-centrations of half the MIC (12 MIC) MIC and double theMIC (2timesMIC) e samples were incubated at 37degC withshaking (100 rpm) for 2 hrs Cell suspension aliquots of500 μL were centrifuged at 4000 rpm for 4minutes To 50 μLof the supernatant 950 μL of Bradford reagent was added todetermine the protein content using Bradfordrsquos method [24]e controls used were 3 DMSO 01 SDS and untreatedcells Bovine serum albumin (BSA) was used as a standardprotein in the Bradford assay e colour was allowed todevelop for 10min before the absorbance was measured at590 nm using a Tecan GeniosPro microplate reader

27 Effects of Extracts from P americana on α-Glucosidasefrom Male SpraguendashDawley Rats

271 Preparation of α-Glucosidase from male Spra-guendashDawley rats e study was approved by Departmentalof Board of Biochemistry (Paper HBC 470 7092018) Fivemale SpraguendashDawley rats weighing 90ndash160 g purchasedfrom the Animal House at the University of Zimbabwe wereused as a source of α-glucosidase Animals were maintainedand handled according to the recommendations of the goodlaboratory practice and animal handling (NIH) guidance forthe care and use of laboratory animals Publication No85ndash23 1985 e rats were fed with certified food pelletsrodent combroids (National foods (PVT) Ltd HarareZimbabwe) ad libitum and allowed free access to drinkingwater After 22 hrs of fasting the rats were sacrificed bycervical dislocation e abdominal cavity was opened bymeans of dissection Parts of the small intestine just belowthe duodenum and above the ceacum were collected fromthe animals e collected intestines were cleaned thor-oughly using 09 saline Small pieces of the intestines wereminced on ice e liver and kidney samples were cut intosuitable pieces and homogenized in 10mM PBS pH 74containing 1 Triton X-100 with a motor driven Potter-Elvehjem homogenizer type R2R (Heidolph Elektro KGKelheim Germany) e homogenate was centrifuged at12000 rpm for 15mins Butanol was added in the ratio 1 1 tothe supernatant fraction e tubes were capped and a post-mitochondria supernatant fraction was prepared by

Biochemistry Research International 3

centrifugation at 7500 rpm for 30min at 2ndash4degC in a BeckmanOptima LE-80 k ultracentrifuge (Beckman Instruments IncCalifornia USA) e resultant mixture was centrifuged at20000 rpm for 30mins e aqueous layer was collected andstored as the crude enzymee crude enzyme was separatedinto 1mL aliquots and frozen at minus80degC All enzyme prep-arations were carried out at 4degC Protein content in thecytosolic fractions was determined using the Lowry method[25]

272 Determining the Effects of Extracts from P americanaon α-Glucosidase Activity e assay was carried out byslightly modifying the method used by Braunlich et al [26]e α-glucosidase was dissolved in 100mM phosphatebuffer pH 68 and used as the enzyme extract p-Nitro-phenyl-α-D-glucopyranoside (pNPG) was used as thesubstrate Leaf extracts were used in the concentrationranging within 0ndash100 μgmL No plant extract was added tothe samples treated as controls Equal volumes (50 μL) ofPBS pH 74 enzyme and extract were pre-incubated for10mins at 37degC Further 1 hr incubation was allowed afteradding 50 μL of the substrate 5mM pNPG Acarbose wasused as the reference inhibitor Absorbance was read at405 nm using a Stat fax 2100 micro-plate reader (AwarenessTechnologies Inc Westport USA)

273 Determination of Time-Dependent Effects of Extractsfrom P americana on α-Glucosidase Inactivation ofα-glucosidase was determined using modified methods ofKim et al [27] as well as by Chelladurai and Chinnachamy[28] e enzyme and extract were incubated at 37degC Ali-quots were withdrawn at 60min intervals for 5 hrs andα-glucosidase activity was determined as described beforee inactivation parameters Kinact and Ki were obtained byplotting graphs of the percentage of remaining enzymeactivity over time To investigate concentration-dependentinactivation incubations were set up for time-dependentinactivation with varying concentrations of leaf extracts at50 500 1000 1500 and 2000 μgmL

28 Statistical Analysis Data analyses were performedusing GraphPad Prism 5 for Windows version 503Software (GraphPad Prism Inc San Diego CA USA)Levels of significance were determined using one-wayANOVA with the Dunnet multiple comparison posttestAll data were expressed as mean plusmn standard deviationand ple 005 values were considered as statisticallysignificant

3 Results

31 Effects of Extracts from P americana on the Growth ofK pneumoniae S epidermidis C albicans and C tropicalise extracts from P americana were tested for their anti-microbial activities against 2 bacterial strains and 2 fungalstrainse results are reported in Table 1e ethanolwaterextract resulted in cell viability of less than 50 against all 4

test strains Maximal activity of the ethanolwater extract wasobserved for S epidermidis with total inhibition at 100 μgmL of the extract Out of the tested extracts K pneumoniaeand S epidermidis were most susceptible to the acetoneextract while C albicans and C tropicalis were most sus-ceptible to the DCM and methanol extracts respectively Asresults of MIC determinations the extracts fromP americana leaf inhibited the growth of S epidermidis withtheMICs from 50 to 100 μgmL (Table 1)e best activity ofall extracts against all the test strains was obtained with theacetone extract against S epidermidis showing the lowestMIC value of 50 μgmL It was noted that the Gram-positiveS epidermidis showed greater susceptibility (total inhibition30 cell viability) towards the extracts from P americanacompared to the Gram-negativeK pneumoniae (18ndash77 cellviability)

For the fungal strains C tropicalis showed greatersusceptibility towards the extracts from P americana with aminimum cell viability of 8 when exposed to the methanolextract compared to C albicans with a minimum cell via-bility of 28 when exposed to the DCM extract MIC forreference drug was 625 μgmL levofloxacin againstK pneumoniae 156 μgmL levofloxacin againstS epidermidis 125 μgmL miconazole against C albicansand 625 μgmL miconazole against C tropicalis Based onthe results that the ethanolwater DCMmethanol and ac-etone extracts from P americana were the most potent ininhibiting the growth of S epidermidis the effects of thethree extracts on the integrity of the membrane of thebacterial were determined

32 Effect of P Americana Leaf Extracts on Membrane In-tegrity of S epidermidis e ability and the extent of theethanolwater DCMmethanol and acetone to disrupt themembrane of S epidermidis as a mode of action wereassessed by investigating the effects of extracts on the leakageof proteins and nucleic acids Protein content was estimatedafter S epidermidis was separately exposed to the 3 extractsand the results are shown in Figure 1

All the 3 extracts showed significant protein leakage at2timesMIC At concentrations of 12 MIC and MIC proteinleakage was not significantly different from that of untreatedcells

For the nucleic acid results (Figure 2) membrane dis-ruption was evidenced by an increased uptake of propidiumiodide by S epidermidis cells exposed to 2timesMIC of theacetone extract in comparison to the unexposed cells(plt 005) (Figure 2(c)) At concentrations of 12 MIC andMIC the acetone extract and the rest of the extracts were notable to cause significant nucleic acid leakage fromS epidermidis cells when compared to the control

33 Effects of Extracts from P americana on α-GlucosidaseActivity e effects of extracts obtained by serial exhaustiveextraction from P americana on the activity of α-glucosidaseenzyme are shown in Figure 3 e standard inhibitoracarbose was observed to inhibit the activity of crude en-zyme Increase in concentration of inhibitor resulted in a


Table 1 Effects of extracts from P americana on the cell viability of K pneumoniae S epidermidis C albicans and C tropicalis

ExtractCell viability ()

K pneumoniae S epidermidis C albicans C tropicalisEthanolwater 18 MIC 100 μgmL 39 14DCMmethanol 34 MIC 100 μgmL 67 92Hexane mdash mdash 73 84DCM mdash mdash 28 52Ethyl acetate 26 30 77 mdashAcetone 25 MIC 50 μgmL 70 76Ethanol mdash mdash 59 32Methanol mdash mdash 55 8Water 77 mdash 63 91(mdash) cell viability not determined in bold cell viability of less than 50

Concentration of ethanol water extract (μgmL)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

0

20

40

60

80

100

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowast

lowastlowastlowast

(a)

0

20

40

60

80

100

Concentration of DCM methanol extract (μgmL)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowastlowast

lowastlowastlowast

(b)

0

20

40

60

80

100

Concentration of acetone extract (μgmL)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowastlowast

lowastlowastlowast

(c)

Figure 1 Effect of leaf extracts from P americana on protein leakage from S epidermidis cells at 12 MIC MIC and 2 MIC (a) Waterethanol extract (b) DCMwater extract and (c) acetone extract e values shown are for meanplusmn standard deviation for n 3 Untreatedcells were the negative control and SDS (sodium dodecyl sulphate) was the positive control e asterisks indicate a significant differencefrom the control with lowastlowastplt 001 and lowastlowastlowastplt 0001


decrease in the activity of the enzyme Complete inhibitionwas achieved at a concentration above 100 μgmL of acar-bose With respect to extracts prepared using non-polarsolvents a slight decrease in the activity of α-glucosidaseactivity was observed with an increase in the concentrationof the extract up to 100 μgmL Activity of the enzyme wasuninhibited by the DCM extract (Figure 3(a)) A decrease inactivity of the enzyme with respect to hexane extract wasobserved at extract concentration between 0 and 50 μgmLAt concentrations above 50 μgmL enzyme activity wasobserved to increase with increase in concentration ofhexane extract e change in activity of the enzyme in thepresence of the non-polar solvent extracts was not

significant Extracts of intermediate polarity ethyl-acetateand acetone had a slight inhibitory potential towards theenzyme but however the change in activity was not sig-nificant (Figure 3(a)) e polar solvents presented with adecrease in activity of the enzyme with increase in polarity ofthe solvent of extraction e methanolic extract was themost potent inhibitor inhibiting about 25 of the enzyme(Figure 3(b)) However all extracts did not cause a signif-icant change in the activity of α-glucosidase e initialscreening using all extracts was to determine the extract withthe most potent inhibitory activitye extract with the mostpotent inhibitory activity the methanol extract was used insubsequent assays

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Concentration of ethanol waterextract (μgmL)

6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(a)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Concentration of DCMmethanolextract (μgmL)

6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(b)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Concentration of acetone extract(μgmL)

6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(c)

Figure 2 Effect of leaf extracts from P americana on nucleic acid leakage from (S) epidermidis cells at 12 MIC MIC and 2 MIC(a) Ethanolwater extract (b) DCMmethanol extract and (c) acetone extract e values shown are for meanplusmn standard deviation for n 3Untreated cells were the negative control and SDS (sodium dodecyl sulphate) was the positive control e asterisks indicate a significantdifference from the control with lowastplt 005 and lowastlowastlowastplt 0001

0

50

100

150

Extract key

100 200 300 400 5000Concentration of extract (μgmL)

Activ

ity (

)

AcetoneHexaneDCM

EthylacetateAcarboseNegative control

(a)

Extract key


0

50

100

150

Activ

ity (

)

EthanolMethanolWater

Ethanol waterAcarboseNegative control

(b)

Figure 3 e effects of extracts of intermediate polarity and non-polar extracts (a) and polar extracts (b) from P americana on the activityon α-glucosidase


34 Inactivation of α-Glucosidase by the Methanol Extract ofP americana As the most potent extract that inhibited theactivity of alpha-glucosidase shown in Figure 3(b) the time-dependence effects on α-glucosidase activity of the methanolextract were determined e methanol extract inactivatedα-glucosidase in a time-dependent manner (Figure 4(a))

e concentration-dependent inactivation of the activityof α-glucosidase by the methanol extract was determined Ageneral decrease in activity was observed with increase intime at all concentrations of extract (Figure 4(b))

e inactivation parameters Kinact and inhibition con-stant Ki of the methanol extract with α-glucosidase wereobtained by analysing the data using the following

In EE0( 11138571113858 1113859

t minus Κinact middot

[l]

[l]+ Ki1113888 1113889 (2)

Data from the concentration-dependent effects wasreplotted (Figure 5) to obtain values for Kinact and Ki andthese were found to be 14mgmL and 24Uminrespectively

4 Discussion

e study aimed to determine the antimicrobial activities ofleaf extracts from P americana against K pneumoniaeS epidermidis C albicans and C tropicalis as well as theantidiabetic effects of the extracts Studies have shown thatplants are a source of drug development [29] Plants containvarious active compounds that can be used to developsynthetic products used in the food cosmetics and phar-maceutical industries Previous studies have shown thatP americana possesses a variety of phytochemicals that arebeneficial to mankind [5]

e ethanolwater extract was the most potent against all4 test strains High potency of the hydroethanolic extractshas been reported elsewhere against Candida [30] andbacteria [31] Maximal activity of the ethanolwater extractwas observed against S epidermidis with total inhibition at100 μgmL of the extract e hydroethanolic media ofextraction are known to solubilise alkaloids often possessingcompounds that have significant physiological and thera-peutic effects [32] K pneumoniae and S epidermidis weremost susceptible to the acetone extract Acetone extractshave shown noteworthy reduction in the growth of variousmicrobes [33 34] In other studies [8] the acetone extractwas shown to particularly extract tannins flavonoids ter-penoids alkaloids and saponins from P americana leaveswhich may be attributed to the antibacterial activity ob-served in this study e antimicrobial activity of extractsfrom P americanawas evaluated against both Gram-positive(S epidermidis) and Gram-negative (K pneumoniae) bac-teria S epidermidis showed greater susceptibility towardsthe majority of the extracts from P americana compared toK pneumoniae (Table 1) As Gram-negative bacteriaK pneumoniae tend to be less susceptible to antimicrobialagents than Gram-positive S epidermidis because of thepresence of the extra protection given by the outer mem-brane [35] e water extract showed limited reduction in

the viability of all tested strains (63ndash91) Similar resultswere obtained in the study by Korukluoglu et al [36] Intheir study they showed that the aqueous extract of oliveleaves had reduced antibacterial effect against several Gram-positive and Gram-negative bacteria Nevertheless somestudies support that aqueous extracts possess antimicrobialactivity against pathogenic bacteria [37 38] For the fungalstrains C tropicalis extract showed susceptibility towardsthe extracts from P americana as indicated by reduced cellviability especially towards the waterethanol (14) ethanol(32) and methanol (8) extracts Susceptibility ofC tropicalis has been reported in other studies [39 40]Susceptibility of microbes to extracts K pneumoniae (45ndash80) S epidermidis (44ndash100) C albicans (29ndash22)and C tropicalis (39ndash33) generally showing that bacteriawere more susceptible to extracts compared to fungi In astudy on the antimicrobial activity of herbal extracts byKhan et al [40] similar results were obtained where Ter-minalia arjuna and Eucalyptus globulus showed greateractivity towards bacteria in comparison to fungi Whencompared to fungi bacteria whether Gram-positive orGram-negative usually respond differently to antimicro-bials e underlying reasons for these varied responses arenot yet fully understood at present but the chemicalcomposition of outer cellular layers is the most probablefactor of prime importance [41] e fact that the cell wall ofbacteria is made up of peptidoglycan [35] while that of fungiis made up of chitin [42] may be one of the explanations ifthe antimicrobial targets cell wall synthesis However itshould be noted that with other antimicrobials some degreeof ldquocross-activityrdquo occurs pertaining to bacteria and fungiSuch differences in susceptibility to extracts bring thequestion of the mode of action of the extracts showingbioactivity

e mechanism of action of the 3 most potent extractsfrom P americana (Table 1) was determinede three mostpotent extracts against S epidermidiswere the ethanolwaterDCMmethanol and the acetone extracts Antibacterialagents generally act on the membranes of microbes to affectdisruption and permeabilization [43] e ability and theextent of plant extracts to permeabilise S epidermidismembrane were assessed by the protein (Figure 1) andnucleic acid (Figure 2) assays Exposure of cells to all 3extracts at a concentration of 2timesMIC caused an increase inprotein leakage indicating membrane permeabilizationViable bacterial cells are impermeable to propidium iodide afluorescent dye but upon membrane disruption or per-meabilization the dye can enter the cells Significant fluo-rescence was observed in bacterial cells treated with theacetone extract at a concentration of 2timesMIC (Figure 2(c))suggesting inner membrane permeabilization ese resultsare somewhat in agreement with previous studies conductedon waterethanol extracts of Cissus welwitschii against Ba-cillus cereus byMoyo andMukanganyama [44] In the studythe authors observed protein and nucleic acid leakage effectsof the extracts Other studies have reported protein leakagemechanisms and elimination of reactive oxidation speciesinduced by phytochemicals [45] which can be used to ex-plain the result obtained in this study where protein leakage


effects by the ethanolwater and DCMmethanol extractsagainst S epidermidis were noted but nucleic acid leakagewas absent Results could be explained by the assumptionthat there was disruption of cell membrane which led to theleakage of proteins [46] but the extracts were not able to acton nuclear membrane which surrounds nucleic acid ma-terial hence no nucleic acid leakage occurred [47]

e effects of leaf extracts from P americana on theactivity of α-glucosidase were investigated Diabetes is acommon metabolic disease characterised by abnormallyhigh plasma glucose levels leading to major complicationssuch as diabetic neuropathy retinopathy and cardiovasculardiseases [48] One therapeutic approach to treat diabetes isto retard the absorption of glucose via the inhibition ofenzymes such as α-glucosidase in the digestive organs [49]It has been shown that diabetes can be managed by drinking

tea made from leaves of P americana [19] Hence this studydetermined the effect of leaf extracts from P americana onthe activity of the enzyme α-glucosidase [17] e methanolextract was found to have the most potent effect on theinhibition of the activity of α-glucosidase (Figure 3(b))Increase in percentage inhibition as a function of time isindicative of irreversible inhibition [50] Irreversible inhi-bition involves the formation of a covalent linkage betweenthe enzyme and the enzyme molecule is interaction re-sults in inactivation of the enzyme [51] Accordingly themethanolic extract exhibited irreversible inhibition on al-pha-glucosidase Kinact value of 14mgmL observed in thisstudy with respect to the methanolic extract may indicateweak binding of the inhibitor to the enzyme molecule emagnitude of the value indicates the binding affinity of theinhibitor to the enzyme e smaller the value of Kinact thebetter the affinity and the better the binding [52]e activityof the enzyme with respect to various extract concentrationswas observed to decrease with the increase in the concen-tration of the plant extract indicating that more enzymemolecules were inactivated at high extract concentrationse value of Ki may help to determine the time required foradministration of a dose to achieve effective inhibition invivo

5 Conclusions

P americana leaf extracts were shown to have antibacterialactivity againstK pneumonia and S epidermidis and extractshave fungistatic activity against C albicans and C tropicalisDisruption of membrane integrity as shown by proteinleakage and nucleic acid leakage may be some of themechanisms of action of the potent extracts Extracts fromP americana leaves also inhibited the activity of α-gluco-sidase P americanamay serve as sources of lead compoundsthat may be exploited as potential therapeutic agents to treat

0

50

100 R2 = 096T12 = 373 microgmL

500 1000 1500 2000 25000[MeOH] microgmL

Perc

enta

ge ac

tivity

rem

aini

ng

(a)

[MeOH extract] microgmL

100 200 300 4000Time (min)

0

50

100

Perc

enta

ge ac

tivity

of c

ontro

l

0250500

100015002000

(b)

Figure 4 e effects of increasing the concentration of the methanolic (MeOH) extract from P americana on the activity of α-glucosidaseenzyme over time (a) and the concentration-dependent effects of the methanol extracts (b)

500 1000 1500 2000 25000Concentration of methanol extract (μgmL)

00

05

10

15

ln (E

E0)

Figure 5 A plot of the natural logarithm of the percentageremaining activity of α-glucosidase against the pre-incubationtimes at different concentrations of the methanol extract con-centration from P americana


bacterial and fungal infections in humans as well as partiallymanage diabetes Isolation of the bioactive components ofthe extracts has been suggested for future studies as this mayconcentrate the active constituent and enhance the effec-tiveness of the extracts

Data Availability

e datasets generated and analysed during the currentstudy are available from the corresponding author uponreasonable request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors acknowledge the assistance of Mr SimbarasheSithole for the technical assistance Support from theSwedish International Development Agency (SIDA)through the International Science Programmes (ISP) (ISPIPICS ZIM01 Uppsala University Uppsala Sweden) isacknowledged ISP IPICSZIM01 supported the researchunder the title ldquoBiomolecular Interactions Analysesrdquo Sup-port from the Alliance for Global Health and Science(University of California Berkeley) is also acknowledged

References

[1] Y Liu F Chan H Sun et al ldquoResveratrol protects humankeratinocytes HaCaTcells fromUVA-induced oxidative stressdamage by downregulating Keap1 expressionrdquo EuropeanJournal of Pharmacology vol 650 no 1 pp 130ndash137 2011

[2] A Upadhyay I Upadhyaya A Kollanoor-Johny andK Venkitanarayanan ldquoCombating pathogenic microorgan-isms using plant-derived antimicrobials a minireview of themechanistic basisrdquo BioMed Research International vol 2014Article ID 761741 18 pages 2014

[3] A G Atanasov B Waltenberger E-M Pferschy-Wenziget al ldquoDiscovery and resupply of pharmacologically activeplant-derived natural products a reviewrdquo BiotechnologyAdvances vol 33 no 8 pp 1582ndash1614 2015

[4] J B Harborne Phytochemical Methods A Guide to ModernTechniques of Plant Analysis Springer Science amp BusinessMedia New York NY USA 2nd edition 2012

[5] M Yasir S Das and M Kharya ldquoe phytochemical andpharmacological profile of Persea americana millrdquo Pharma-cognosy Reviews vol 4 no 7 pp 77ndash84 2010

[6] A B S Siqueira L R N D A Rodriguez R K B Santos et alldquoAntifungal activity of propolis against Candidaspecies iso-lated from cases of chronic periodontitisrdquo Brazilian OralResearch vol 29 no 1 pp 1ndash6 2015

[7] T P Cushni and A Lamb ldquoRecent advances in understandingantibacterial properties of flavonoidsrdquo International Journalof Antimicrobial Agents vol 38 no 2 pp 99ndash107 2011

[8] O Ajayi S Awala O Olalekan and O Alabi ldquoEvaluation ofantimicrobial potency and phytochemical screening of Perseaamericana leaf extracts against selected bacterial and fungalisolates of clinical importancerdquoMicrobiology Research JournalInternational vol 20 no 1 pp 1ndash11 2017

[9] A S Chhipa and S S Sisodia ldquoIndian medicinal plants withantidiabetic potentialrdquo Journal of Drug Delivery and Bera-peutics vol 9 no 1 pp 257ndash265 2019

[10] D A Soloviev S Jawhara and W A Fonzi ldquoRegulation ofinnate immune response toCandida albicansInfections byαMβ2-Pra1p interactionrdquo Infection and Immunity vol 79no 4 pp 1546ndash1558 2011

[11] R J Kothavade M M Kura A G Valand andM H Panthaki ldquoCandida tropicalis its prevalence patho-genicity and increasing resistance to fluconazolerdquo Journal ofMedical Microbiology vol 59 no 8 pp 873ndash880 2010

[12] V Steenkamp A C Fernandes and C E J V RensburgldquoScreening of Venda medicinal plants for antifungal activityagainst Candida albicansrdquo South African Journal of Botanyvol 73 no 2 pp 256ndash258 2007

[13] D Chessa G Ganau and V Mazzarello ldquoAn overview ofStaphylococcus epidermidis and Staphylococcus aureus with afocus on developing countriesrdquo Be Journal of Infection inDeveloping Countries vol 9 no 6 pp 547ndash550 2015

[14] P D Fey Staphylococcus epidermidis Methods and ProtocolsVol 1106 Humana Press Ohio NJ USA 5th edition 2016

[15] T H Nguyen M D Park and M Otto ldquoHost response toStaphylococcus epidermidis colonization and infectionsrdquoFrontiers in Cellular and Infection Microbiology vol 7 p 902017

[16] C Chen M Kielhlbauch M Waters and A Kallen ldquoPan-resistant New Delhi metallo-beta-lactamase-producingKlebsiella pneumoniaemdashWashoe County Nevada 2016rdquoMorbidity and Mortality Weekly Report vol 1 p 33 2016

[17] S S Nair V Kavrekar and A Mishra ldquoIn vitro studies onalpha amylase and alpha glucosidase inhibitory activities ofselected plant extractsrdquo European Journal of ExperimentalBiology vol 3 no 1 pp 128ndash132 2013

[18] U Arukwe B A Amadi M K C Duru et al ldquoChemicalcomposition of Persea americana leaf fruit and seedrdquo In-ternational Journal of Recent Research and Applied Studiesvol 11 no 2 pp 346ndash349 2012

[19] B Antia J Okokon and P Okon ldquoHypoglycemic activity ofaqueous leaf extract of Persea americana millrdquo Indian Journalof Pharmacology vol 37 no 5 pp 325-326 2005

[20] EUCAST (European Committee for Antimicrobial Suscep-tibility Testing) ldquoDetermination of minimum inhibitoryconcentrations (MICs) of antibacterial agents by broth di-lutionrdquo Clinical Microbiology Infectious Diseases vol 9pp 1ndash7 2003

[21] T Arun and M Rabeeth ldquoGenotoxic effect of paracetamolcontaining tablets in cultured human lymphocytesrdquo Inter-national Journal of Biomedical Research vol 1 no 2pp 21ndash30 2010

[22] M A El-Nakeeb H M Abou-Shleib A M KhalilH G Omar and O M El-Halfawy ldquoMembrane permeabilityalteration of some bacterial clinical isolates by selected an-tihistaminicsrdquo Brazilian Journal of Microbiology vol 42no 3 pp 992ndash1000 2011

[23] W Du C Sun Z Liang Y Han and J Yu ldquoAntibacterialactivity of hypocrellin A against Staphylococcus aureusrdquoWorld Journal of Microbiology and Biotechnology vol 28no 11 pp 3151ndash3157 2012

[24] M M Bradford ldquoA rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of proteinndashdye bindingrdquo Analytical Biochemistryvol 72 no 1-2 pp 248ndash254 1976


[25] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin- phenol reagentrdquoJournal of Biological Chemistry vol 193 pp 265ndash275 1951

[26] M Braunlich R Slimestad H Wangensteen C BredeK Malterud and H Barsett ldquoExtracts anthocyanins andprocyanidins from Aronia melanocarpa as radical scavengersand enzyme inhibitorsrdquo Nutrients vol 5 no 3 pp 663ndash6782013

[27] Y-M Kim Y-K Jeong M-H Wang W-Y Lee andH-I Rhee ldquoInhibitory effect of pine extract on α-glucosidaseactivity and postprandial hyperglycemiardquo Nutrition vol 21no 6 pp 756ndash761 2005

[28] G R M Chelladurai and C Chinnachamy ldquoAlpha amylaseand Alpha glucosidase inhibitory effects of aqueous stemextract of Salacia oblonga and its GC-MS analysisrdquo BrazilianJournal of Pharmaceutical Sciences vol 54 no 1 pp 1ndash102018

[29] N Kumar Z A Wani and S Dhyani ldquoEthnobotanical studyof the plants used by the local people of Gulmarg and its alliedareas Jammu amp Kashmir Indiardquo International Journal ofCurrent Research in Bioscience and Plant Biology vol 2 no 9pp 16ndash23 2015

[30] B Moyo and S Mukanganyama ldquoe anticandidal andtoxicity properties of Lampranthus franciscirdquo Journal ofMycology vol 2015 Article ID 898202 15 pages 2015

[31] D M Oliveira F G Melo S O Balogun et al ldquoAntibacterialmode of action of the hydroethanolic extract of Leonotisnepetifolia (L) R Br involves bacterial membrane pertur-bationsrdquo Journal of Ethnopharmacology vol 172 pp 356ndash363 2015

[32] R Irchhaiya A Kumar A Yadav et al ldquoMetabolites in plantsand its classificationrdquo World Journal of Pharmacy andPharmaceutical Sciences vol 4 no 1 pp 287ndash305 2015

[33] M Obeidat M Shatnawi M Al-lawi et al ldquoAntimicrobialactivity of crude extracts of some plant leavesrdquo ResearchJournal of Microbiology vol 7 no 1 pp 59ndash67 2012

[34] A Borges H Jose V Homem and M Simotildees ldquoComparisonof techniques and solvents on the antimicrobial and anti-oxidant potential of extracts from Acacia dealbata and Oleaeuropaeardquo Antibiotics vol 9 no 48 pp 1ndash19 2020

[35] R M Epand C Walker R F Epand and N A MagarveyldquoMolecular mechanisms of membrane targeting antibioticsrdquoBiochimica et Biophysica Acta (BBA)mdashBiomembranesvol 1858 no 5 pp 980ndash987 2016

[36] M Korukluoglu Y Sahan A Yigit E T Ozer and S GucerldquoAntibacterial activity and chemical constitutions of Oleaeuropaea L leaf extractsrdquo Journal of Food Processing andPreservation vol 34 no 3 pp 383ndash396 2010

[37] Z A Zakaria M L Zakaria Z Amom and M N M DesaldquoAntimicrobial activity of the aqueous extract of selectedMalaysian herbsrdquo African Journal of Microbiology Researchvol 5 no 30 pp 5379ndash5383 2011

[38] F G Mugweru D W Nyamai M W Arika et al ldquoAnti-microbial activity of aqueous extracts of Maytemus putter-lickoides Senna spectabilis and Olinia usambarensis onselected diarrhea-causing bacteriardquo Journal of Bacteriologyand Parasitology vol 7 no 2 p 270 2016

[39] S Salari T Bakhshi F Sharififar A Naseri andP G N Almani ldquoEvaluation of antifungal activity of stan-dardized extract of Salvia rhytidea benth (Lamiaceae) againstvarious Candida isolatesrdquo Journal de Mycologie Medicalevol 26 no 4 pp 323ndash330 2016

[40] R Khan B Islam M Akram et al ldquoAntimicrobial activity offive herbal extracts against multi drug resistant (MDR) strains

of bacteria and fungus of clinical originrdquo Molecules vol 14no 2 pp 586ndash597 2009

[41] A D Russel ldquoSimilarities and differences in the responses ofmicroorganisms to biocidesrdquo Journal of Antimicrobial Che-motherapy vol 52 pp 750ndash763 2003

[42] M D Lenardon C A Munro and N A Gow ldquoChitinsynthesis and fungal pathogenesisrdquo Current Opinion in Mi-crobiology vol 13 no 4 pp 416ndash423 2010

[43] K Saritha A Rajesh K Manjulatha O H Setty andS Yenugu ldquoMechanism of antibacterial action of the alcoholicextracts of Hemidesmus indicus (L) R Br ex Schult Leucasaspera (Wild) Plumbago zeylanica L and Tridax procumbens(L) R Br ex Schultrdquo Frontiers in Microbiology vol 6 pp 1ndash92015

[44] B Moyo and S Mukanganyama ldquoAntibacterial effects ofCissus welwitschii and Triumfetta welwitschii extracts againstEscherichia coli and Bacillus cereusrdquo International Journal ofBacteriology vol 2015 Article ID 162028 10 pages 2015

[45] Z Ling Z Xu W Liang J Mei and H Wang ldquoAntibacterialactivity and mode of action Mentha arvensis ethanol extractsagainst multidrug resistant Acinetobacter baumaniirdquo TropicalJournal of Pharmaceutical Research vol 11 pp 2099ndash21062015

[46] C Fimognari Dietary and Non-dietary Phytochemicals andCancer MDPI New York NY USA 1st edition 2018

[47] A Banerjee and T Majumder Be DNA Intercalators ofEthidium Bromide and Propidium Iodide also Bind to His-tones CRC Press New Delhi India 2006

[48] V Kumar O Prakash S Kumar and S Narwal ldquoα-gluco-sidase inhibitors from plants a natural approach to treatdiabetesrdquo Pharmacognosy Reviews vol 5 no 9 pp 19ndash292011

[49] A Kimura J-H Lee I-S Lee et al ldquoTwo potent competitiveinhibitors discriminating α-glucosidase family I from familyIIrdquo Carbohydrate Research vol 339 no 6 pp 1035ndash10402004

[50] G C Adam B F Cravatt and E J Sorensen ldquoProfiling thespecific reactivity of the proteome with non-directed activity-based probesrdquo Chemistry amp Biology vol 8 no 1 pp 81ndash952001

[51] T S Maurer and H-L Fung ldquoComparison of methods foranalyzing kinetic data from mechanism-based enzyme inac-tivation application to nitric oxide synthaserdquo AAPSPharmSci vol 2 no 1 pp 68ndash77 2000

[52] G Scapin ldquoStructural biology and drug discoveryrdquo CurrentPharmaceutical Design vol 12 no 17 pp 2087ndash2097 2005


the presence of secondary plant metabolites TraditionallyP americana is widely used as a source of medicine and foodin many parts of the world Leaves of the plant can be used tomake tea and as flavorings in beef as well as bean dishes [5]In developing countries problems are associated with themeans of managing diabetes using antidiabetic drugs due toavailability and affordability Drug candidates have beendeveloped from the phytochemical constituents isolatedfrom medicinal plants A variety of plants are being studiedfor their antidiabetic potential [9]

Candida albicans is an etiological microbe for fungalinfections In a healthy individual the growth of C albicansis regulated by bacteria and other microorganisms that arepart of the natural flora e uncontrolled growth ofC albicans results in candidiasis [10] Reports have docu-mented a shift from C albicans as the cause of the majorityof invasive infections toward non-C albicans speciesC tropicalis is classified as one of the most potent yeasts ofthe non-albicans Candida group [11] Previous studies haveshown that the Candida species are susceptible to plantextracts Extracts from the Combretum molle Piper capenseSolanum aculeastrum Syzygium cordatum and Zanthox-ylum davyi showed antifungal properties against C albicans[12]

Antibacterial properties of medicinal plants are beingincreasingly reported from different parts of the worldStaphylococcus epidermidis is a Gram-positive bacteriumthat is spherically shaped and a facultative anaerobe [13] It ispart of the normal human flora typically skin flora and lessassociated with the mucosal flora in humans [14] ebacterium has been shown to be less pathogenic in humansbut virulence of the strain has been reported in immuno-compromised individuals particularly in nosocomial in-fections [15] K pneumoniae is a Gram-negative rod-shaped non-encapsulated facultative anaerobic and lactosefermenting bacterium e bacterium is responsible forcausing nosocomial infections and has been reported to beresistant to beta lactam antibiotics such as penicillin [16]

e enzyme α-glucosidase is involved in the breakdownof carbohydrates during digestion e enzyme catalysesreactions that result in the release of the terminal alpha-glucose residue from disaccharides and oligosaccharides[17] Avocado leaves contain phytochemicals such as fla-vonoids and phenols in the fruit or the seed and these havebeen reported to have antidiabetic effects [18] A therapeuticapproach to treat diabetes is to decrease postprandialhyperglycaemia Avocado leaves have been used locally tomake avocado tea used as a means of managing diabetes [19]It is therefore important to investigate the effects of the leafextracts on α-glucosidase so as to validate its use as a meansof managing diabetes e aim of the study was to evaluatethe antimicrobial and antidiabetic activity of leaf extractsfrom P americana

2 Materials and Methods

21 Chemicals and Reagents All the chemicals used wereobtained from Sigma Aldrich (Darmstadt Germany) Allsolvents used were of analytical reagent grade these were

acetone methanol n-hexane ethanol ethyl acetatedichloromethane (DCM) and chloroform Dimethyl sulf-oxide (DMSO) was used for dissolving the crude extract aswell as other reagents used Sabouraud dextrose broth(SDB) Sabouraud glucose 2 agar (SGA) Luria Bertanibroth 3-(45-dimethylthiazolyl)-25-diphenyltetrazoliumbromide (MTT) and potassium hydroxide (KOH) edrugs miconazole and fluconazole were obtained fromSigma-Aldrich (Germany) C albicans (NCPF 3255) waspurchased from Sigma Aldrich A clinical strain ofC tropicalis was obtained from the Department of MedicalMicrobiology at Parirenyatwa Hospital in HarareZimbabwe

22 Plant Collection and Preparation P americana leaveswere collected from a tree in Chitungwiza Seke at housenumber 9209 Unit K 177deg Southwest of Chitungwiza Baptist(Greater Harare Zimbabwe) Leaves were taken to theNational Herbarium and Botanical Gardens of Zimbabwefor identification e leaves were washed with tap water toremove dirt and dried in an incubator at 40degC for 72 hrsDried leaves were then ground using traditional pestle andmortar to fine powder weighed and put in a plastic beaker

23 Extraction of Phytochemicals Using Differential Solvents

231 Extraction with DCM Methanol and HydroethanolicWater Solvent (Total Extraction) Two sets of solventmixtures were used (1) DCMmethanol in the ratio 1 1 and(2) ethanolwater in the ratio 1 1e extracts were preparedby adding 20 g of the ground powder to 100mL of thesolvents e mixture of the solvent and the leaf powder wasfiltered to obtain the extract During the filtration processcotton was used first so as to trap the undissolved mattere filtrate obtained was further filtered using a filter paperWhatman no 1 so as to trap the particles that were able topass through the cotton e filtrate was collected and driedunder a fan

232 Serial Exhaustive Extraction Serial exhaustive ex-traction of phytochemicals of the leaf powder fromP americana was carried out serially using solvents of in-creasing polarity hexane dichloromethane ethyl acetateacetone ethanol methanol and water A total mass of 30 gof leaf powder was used in the first serial solvent (hexane)and left for 24 hours for extraction After 24 hours thecontents in the beaker were filterede retained leaf residuewas dried and extracted with another solvent is wasrepeated until all the solvents had been used for extractionUltimately a total of 7 extracts were obtained

24 Screening forAntifungal andAntibacterial Activity of LeafExtracts from P americana

241 Growth of Microorganisms Overnight cultures wereprepared by picking 3 colonies from an agar plate of themicrobe and inoculating into the relevant broth followed by
















3 Results













SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

0

20

40

60

80

100

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowast

lowastlowastlowast

(a)

0

20

40

60

80

100


SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowastlowast

lowastlowastlowast

(b)

0

20

40

60

80

100


SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowastlowast

lowastlowastlowast

(c)





SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(a)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(b)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(c)


0

50

100

150

Extract key


Activ

ity (

)

AcetoneHexaneDCM


(a)

Extract key


0

50

100

150

Activ

ity (

)



(b)






In EE0( 11138571113858 1113859


[l]

[l]+ Ki1113888 1113889 (2)


4 Discussion









5 Conclusions


0

50

100 R2 = 096T12 = 373 microgmL

500 1000 1500 2000 25000[MeOH] microgmL

Perc

enta

ge ac

tivity

rem

aini

ng

(a)


100 200 300 4000Time (min)

0

50

100

Perc

enta

ge ac

tivity

of c

ontro

l

0250500

100015002000

(b)



00

05

10

15

ln (E

E0)




Data Availability




Acknowledgments


References






































































3 Results













SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

0

20

40

60

80

100

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowast

lowastlowastlowast

(a)

0

20

40

60

80

100


SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowastlowast

lowastlowastlowast

(b)

0

20

40

60

80

100


SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowastlowast

lowastlowastlowast

(c)





SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(a)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(b)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(c)


0

50

100

150

Extract key


Activ

ity (

)

AcetoneHexaneDCM


(a)

Extract key


0

50

100

150

Activ

ity (

)



(b)






In EE0( 11138571113858 1113859


[l]

[l]+ Ki1113888 1113889 (2)


4 Discussion









5 Conclusions


0

50

100 R2 = 096T12 = 373 microgmL

500 1000 1500 2000 25000[MeOH] microgmL

Perc

enta

ge ac

tivity

rem

aini

ng

(a)


100 200 300 4000Time (min)

0

50

100

Perc

enta

ge ac

tivity

of c

ontro

l

0250500

100015002000

(b)



00

05

10

15

ln (E

E0)




Data Availability




Acknowledgments


References




























































SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

0

20

40

60

80

100

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowast

lowastlowastlowast

(a)

0

20

40

60

80

100


SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowastlowast

lowastlowastlowast

(b)

0

20

40

60

80

100


SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC

Prot

ein

conc

entr

atio

n (μ

gm

L)

lowastlowast

lowastlowastlowast

(c)





SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(a)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(b)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(c)


0

50

100

150

Extract key


Activ

ity (

)

AcetoneHexaneDCM


(a)

Extract key


0

50

100

150

Activ

ity (

)



(b)






In EE0( 11138571113858 1113859


[l]

[l]+ Ki1113888 1113889 (2)


4 Discussion









5 Conclusions


0

50

100 R2 = 096T12 = 373 microgmL

500 1000 1500 2000 25000[MeOH] microgmL

Perc

enta

ge ac

tivity

rem

aini

ng

(a)


100 200 300 4000Time (min)

0

50

100

Perc

enta

ge ac

tivity

of c

ontro

l

0250500

100015002000

(b)



00

05

10

15

ln (E

E0)




Data Availability




Acknowledgments


References


























































SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(a)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(b)

SDS

Con

trol

DM

SO

12

MIC

MIC

2 M

IC


6

4

2

0

Fluo

resc

ence

inte

nsity

(AU

)

(c)


0

50

100

150

Extract key


Activ

ity (

)

AcetoneHexaneDCM


(a)

Extract key


0

50

100

150

Activ

ity (

)



(b)






In EE0( 11138571113858 1113859


[l]

[l]+ Ki1113888 1113889 (2)


4 Discussion









5 Conclusions


0

50

100 R2 = 096T12 = 373 microgmL

500 1000 1500 2000 25000[MeOH] microgmL

Perc

enta

ge ac

tivity

rem

aini

ng

(a)


100 200 300 4000Time (min)

0

50

100

Perc

enta

ge ac

tivity

of c

ontro

l

0250500

100015002000

(b)



00

05

10

15

ln (E

E0)




Data Availability




Acknowledgments


References



























































In EE0( 11138571113858 1113859


[l]

[l]+ Ki1113888 1113889 (2)


4 Discussion









5 Conclusions


0

50

100 R2 = 096T12 = 373 microgmL

500 1000 1500 2000 25000[MeOH] microgmL

Perc

enta

ge ac

tivity

rem

aini

ng

(a)


100 200 300 4000Time (min)

0

50

100

Perc

enta

ge ac

tivity

of c

ontro

l

0250500

100015002000

(b)



00

05

10

15

ln (E

E0)




Data Availability




Acknowledgments


References



























































5 Conclusions


0

50

100 R2 = 096T12 = 373 microgmL

500 1000 1500 2000 25000[MeOH] microgmL

Perc

enta

ge ac

tivity

rem

aini

ng

(a)


100 200 300 4000Time (min)

0

50

100

Perc

enta

ge ac

tivity

of c

ontro

l

0250500

100015002000

(b)



00

05

10

15

ln (E

E0)




Data Availability




Acknowledgments


References

























































Data Availability




Acknowledgments


References
























































antibacterial,antifungal,andantidiabeticeffectsofleaf

Documents