Research Article Phytochemical Analysis and …downloads.hindawi.com/journals/bri/2015/493879.pdfResearch Article Phytochemical Analysis and Biological Activities of Cola nitida Bark

Research ArticlePhytochemical Analysis and Biological Activities ofCola nitida Bark

Durand Dah-Nouvlessounon1 Hubert Adoukonou-Sagbadja2

Nafan Diarrassouba3 Haziz Sina1 Adolphe Adjanohoun4 Mariam Inoussa1

Donald Akakpo1 Joachim D Gbenou5 Simeon O Kotchoni6

Mamoudou H Dicko7 and Lamine Baba-Moussa1

1Laboratoire de Biologie et de Typage Moleculaire en Microbiologie Faculte des Sciences et TechniquesUniversite drsquoAbomey-Calavi 05 BP 1604 Cotonou Benin2Laboratoire des Ressources Genetiques et drsquoAmelioration des Especes Departement de Genetique et des BiotechnologiesFAST Universite drsquoAbomey-Calavi BP 526 Cotonou Benin3Universite Peleforo Gon Coulibaly de Korhogo UFR des Sciences Biologiques BP 1328 Korhogo Cote drsquoIvoire4Centre de Recherches Agricoles Sud Institut National des Recherches Agricoles du Benin Attogon BP 884 Cotonou Benin5Laboratoire de Pharmacognosie et des Huiles Essentielles FSSUAC 01 BP 4521 Cotonou Benin6Department of Biology and Center for Computational and Integrative Biology Rutgers University 315 Penn StreetCamden NJ 08102 USA7Laboratoire de Biochimie Alimentaire Enzymologie Biotechnologie Industrielle et BioinformatiqueUFRSVT Universite de Ouagadougou 03 BP 7021 Ouagadougou 03 Burkina Faso

Correspondence should be addressed to Lamine Baba-Moussa laminesaidyahoofr

Received 20 December 2014 Revised 11 January 2015 Accepted 11 January 2015

Academic Editor Tzi Bun Ng

Copyright copy 2015 Durand Dah-Nouvlessounon et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Kola nut is chewed in many West African cultures and is used ceremonially The aim of this study is to investigate some biologicaleffects of Cola nitidarsquos bark after phytochemical screeningThe bark was collected dried and then powdered for the phytochemicalscreening and extractions Ethanol and ethyl acetate extracts of C nitida were used in this study The antibacterial activity wastested on ten reference strains and 28 meat isolated Staphylococcus strains by disc diffusion methodThe antifungal activity of threefungal strains was determined on the Potato-Dextrose Agar medium mixed with the appropriate extract The antioxidant activitywas determined by DPPH and ABTS methods Our data revealed the presence of various potent phytochemicals For the referenceand meat isolated strains the inhibitory diameter zone was from 175 plusmn 07mm (C albicans) to 95 plusmn 07mm (P vulgaris) TheMICranged from 0312mgmL to 5000mgmL and the MBC from 0625mgmL to gt20mgmL The highest antifungal activity wasobserved with F verticillioides and the lowest one with P citrinum The two extracts have an excellent reducing free radical activityThe killing effect of A salina larvae was perceptible at 104mgmL The purified extracts of Cola nitidarsquos bark can be used to holdmeat products and also like phytomedicine

1 Introduction

The African continent has a huge biodiversity with a highnumber of plants used for medicinal [1] food and in tradi-tional ceremonies In recent years there has been a gradualrevival of interest in the use of medicinal plants in developingnations [2] Despite the existence of hospitals in developing

countries and laws that consider traditional medicine as anillegal act the valorization of the traditional medicine hasbecome an early criterion of identity as well as rights of healthand education [3] In the same way it was reported that about80 of Africans use medicinal plants to treat various diseases[4] Among themedicinal plants cola nut (Cola nitida) highlyspreads in Africa and particularly in Benin

Hindawi Publishing CorporationBiochemistry Research InternationalVolume 2015 Article ID 493879 12 pageshttpdxdoiorg1011552015493879

2 Biochemistry Research International

Indeed C nitida is a plant native to tropical WestAfrica and belongs to Sterculiaceae family [5] This plant isnowadays cultivated from Senegal to Nigeria in the WestIndies and South America [6] In West Africanrsquos forest areascola is perhaps second in importance to the palm tree as anindigenous cash crop [7] Cola nut has been an importantarticle of international trade in many parts of Africa [8]The nuts of C nitida contain about two percent of caffeineand are chewed by many people as a stimulant It is a veryspecial and important item used in social and ceremonialactivities by Africans The nuts of cola also have industrialusage for the production of drugs soft drinks wines candiesand beverages [9] such as Coca-Cola and Pepsi-Cola [10]It has many pharmacological properties and contains someactive principles it prevents sleep thirst and hunger and actsas an antidepressant [7] The cola nuts are source of antiox-idants and contain a wide array of complex secondary plantmetabolites such as theobromine d-catechin L-epicatechinand kolatin [11] The use of the plant in the treatment ofcertain diseases has been reported by several authors [12 13]

Nowadays there exist more than 250 types of infectionscaused by bacteria and fungi [14 15] Among these microor-ganisms we find the Pseudomonas Escherichia coli [16] andbacteria of the genus Staphylococcus that are known to beone of the main elements of human physiological flora [17]and are responsible of many diseases [18ndash20] In the rankof fungi the moulds producing toxins are mainly of genusAspergillus Penicillium and Fusarium [21] With the adventof modern medicine and for the treatment of infectionsabusive and often uncontrolled use of antibiotics brings upa phenomenon of resistance in most of the bacteria andfungi Beyond infectious diseases oxidative stress is a veryserious phenomenon that can trigger molecular and cellularevents in the body The consequences are multiple such ascancer [22] cerebral and cardiovascular diseases diabetesand hypertension [23]

To face these health problems (resistance of microorgan-isms and natural phenomenon of production in the body ofthe free radicals) the track of medicinal plants deserves to beexplored In this direction in Benin several ethnobotanicalstudies [24ndash26] have focused for several years on identifyingmedicinal species Others have demonstrated the efficacy ofmedicinal plants in the fight against certain fungal strains[27] and pathogenic Gram-positive and Gram-negative bac-teria involve several pathologies [28] Furthermore in Beninvery little study has been conducted on C nitida and thereferences which relate to its phytochemical composition andits biological properties are quasi-nonexistent In the sameway the work realized on the species in other countriesconcerns seed those concerning species bark are rare andthe results are disparate The aim of this study was tomake the phytochemical screening of C nitida bark and toinvestigate in vitro some biological (antimicrobial antifungaland cytotoxic) activities of its extracts

2 Material and Methods

21 Collection of Plant Material The bark of C nitida wascollected in the village of Aglogbe (commune of Adjarra

6∘241015840010158401015840N 2∘121015840010158401015840E)Department ofOueme southernBeninThe plants materials were air-dried at 25∘Cndash30∘C for twoweeks ground and sieved into a bark powder The smoothpowder was stored in airtight glassware and kept in darknessat minus20∘C until use

22 Phytochemical Profiling The phytochemical profiling ofthe bark of C nitida to determine the major constituents(nitrogenous polyphenolic and terpenic compound andglycosides) was done according to Houghton and Raman[29]

23 Preparation of Ethanol and Ethyl Acetate Extracts Theseextracts were made using an adapted method of the onedescribed by Sanogo et al [30] and NrsquoGuessan et al [31]Thismethod consisted of macerating 50 g of C nitida powders in500mL of 96 ethanol for 72 hoursThe obtained extract wasfiltered thrice using Whatman filter paper Half of the filtratewas directly dried at 40∘C to obtain the ethanolic extract ofCnitida To the second half of the filtrate 200mL of H

2O and

100mL of ethyl acetate were added The solution was gentlymixed and left settled until we obtain two phases (about45min)The lower phasewas collected and dried as describedpreviously to obtain the ethyl acetate extract The alcoholicand ethyl acetate extracts were stored in labeled sterile bottlesand kept at minus20∘C until further use

24 Microorganismrsquos Cultures The tested microorganismsinclude ten references twenty height Staphylococcus meatisolated strains and three fungal strains (Penicillium cit-rinum Aspergillus tamarii and Fusarium verticillioides) Thethree fugal strains were part of the microorganisms isolatedin the Beninese traditional cheese wagashi by Sessou et al[27]The reference strains were Escherichia coliATCC 25922Staphylococcus aureus ATCC 29213 Staphylococcus epider-midisT22695 Pseudomonas aeruginosaATCC 27853 Proteusmirabilis A24974 Micrococcus luteus ATCC 10240 Proteusvulgaris A25015 Streptococcus oralis Enterococcus faecalisATCC 29212 andCandida albicansMHMRThe Staphylococ-cus strains used in this study were those isolated from threedifferentmeat products in IvoryCoast byAttien et al [32] andstored in the Laboratory of Biology and Molecular Typing inMicrobiology (University of Abomey-Calavi Benin)

25 Antimicrobial Activity

251 Sensitivity Test The disc diffusion method [33] wasused to screen the antimicrobial activity Briefly two tothree sterile paper discs (6mm as diameter) were lodgedunder aseptic conditions on Mueller Hinton agar Petri dishpreviously flooded with the appropriate bacterial culture(adjusted to 05 McFarland standard) The discs were asep-tically impregnated with 25120583L of C nitida extract solution(20mgmL) These dishes were kept for 15ndash30min at roomtemperature before incubation at 37∘C for 24 and 48 hours

After the incubation period the dishes were examinedfor inhibitory zones [34] Each sample was used in triplicatefor the determination of antibacterial and antifungal activity

Biochemistry Research International 3

Blank disc impregnated with solvent was used as negativecontrol

252 Determination of Minimum Inhibitory Concentrations(MIC) The minimum inhibitory concentrations (MIC) ofcrude extract of plants were performed by macrodilutionmethod [35] First the extracts were diluted in sterilizeddistilled water to the highest concentration of 20 000120583gmLand then nine dilutions were performed to obtain theconcentrations of 10 000 120583gmL 5 000120583gmL 2 500120583gmL1 250120583gmL 625120583gmL 3125 120583gmL 15625 120583gmL7812 120583gmL and 3906 120583gmL in screw tube To 1mL ofthe above concentrations was added 1mL of the bacteriainoculum (106 UFCml) to obtain 2mL as a final volumeCulture medium without samples and others withoutmicroorganisms were used in the tests as control Tubeswere incubated at 37∘C for 18ndash24 hours and growth wasindicated by turbidity The MIC is the lowest concentrationof the compound at which the microorganism tested doesnot demonstrate visible growth (turbidity)

253 Minimum Bactericidal Concentration (MBC) Theminimum bactericidal concentration (MBC) of the testedmicroorganisms was determined by subculturing the testdilutions onto a fresh solid medium and further incubationfor 18ndash24 h The highest dilution that yielded no bacterialgrowth on solid medium was taken as MBC [36]

26 Evaluation of the Cytotoxicity Activity of Cola nitidarsquos BarkExtracts The cytotoxic effect of the extracts was evaluatedaccording to an adaptation of the method described byKawsar et al [37] The tests are carried out twice on 72 hlarvae ofArtemia salina Briefly a test was constituted of 16Asalina larvae in a 2mL solution containing 1mL of the extracttested concentration and 1mL of sea water The number ofsurviving larvae is counted after incubation (24 h) and theDL50was calculated using the regression line obtained from

the surviving larvae in function of the extracts concentrationrepresentation

27 Antifungal Activity The in vitro antifungal activity of theextracts was evaluated according to the method previouslydescribed byKumar et al [38] andDohou et al [39]The assaywas performed on the Potato-DextroseAgarmedium Brieflythe extracts (20mgmL) used for the antifungal activity weredissolved with sterilized distillated water or if necessary witha water-ethanol mixture (60 40) One mL of the dissolvedextract (20mgmL) was thoroughly mixed with 10mL ofthe sterilized Potato-Dextrose Agar medium before it wastransferred to sterile Petri dishes for solidification After themedium solidification a sterile 6mmdisc treated with fungalstrain was placed in each Petri plate Each treatment wasreplicated twice Plates were incubated at 25 plusmn 1∘C for 5days Fungal radial growth was measured by averaging thetwo diameters taken from each colony Percentage growth

inhibition of the fungal colonies was calculated using theformula

Inhibition Percentage ()

=Controlrsquos growth minus Treatmentrsquos growth

Controlrsquos growthtimes 100

(1)

28 Antioxidant Activity Determinations The antioxidantactivity was measured using both DPPH (22-diphenyl-1-picrylhydrazyl) and ABTS [221015840-azinobis(3-ethylbenzothia-zoline-6-sulfonic acid)] methods

The ABTS assay was conducted according to the methoddescribed by Re et al [40] The working solution of ABTS+(10mg of ABTS 26mL of deionized water and 172mg ofpotassium persulphate) was left to stand at room temperaturefor 12 h in the dark before use This solution was diluted withethanol until obtaining an absorbance of 070plusmn002 at 734 nmTwenty 120583L of each extract sample (1mgmL) was diluted witha fresh prepared ABTS solution to a total volume of 1mLAll the assays were performed in triplicate the absorbancewas read after 15min in dark at 734 nm and the referencemolecule was ascorbic acidThe concentration of compoundswith a capability to reduce ABTS+ radical cation is expressedas 120583mol equivalent ascorbic acid (120583mol EqAA) per gram ofdry extract using the following formula used by Guenne et al[41]

The DPPH method was conducted by adaptation asdescribed by Scherer andGodoy [42] Equal volumes (100120583L)of DPPH (50120583M) and plant extracts (200120583gsdotmLminus1) weremixed in a 96-well microplate and allowed to stand indarkness for 20ndash30min at room temperature Then theabsorbance was read at 517 nm and the blank was a mixtureof methanol and DPPH (v v) The inhibitory percentage ofDPPH radical indicating the antioxidant activity of extractsand quercetol gallic acid was obtained using the formulaestablished by Schmeda-Hirschmann et al [43]

The concentration providing 50 inhibition (IC50) was

determined graphically using a calibration curve in thelinear range by plotting the extract concentration and thecorresponding scavenging effect Antioxidant activity index(AAI) was calculated according to the formula used byScherer and Godoy [42]

29 Statistical Analysis All experiment was done in triplicateand data thus obtained were reported as a mean plusmn standarddeviation (SD) The data were analyzed using GraphPadPrism 5 software Differences of 119875 lt 005 were consideredsignificant

3 Results

31 Phytochemical Screening The result of phytochemicalscreening of C nitidarsquos bark powder revealed the presence ofvarious potent phytochemicals such as tannins saponins andflavonoids (Table 1)

32 Antibacterial Activity The results of antibacterial activ-ity using ethanol and ethyl acetate extract of C nitida


Table 1 Phytochemical composition of Cola nitidarsquos bark powder

Chemical compound Cola nitidarsquos barkAlkaloids minus

Tannins +Saponosides (MI) + (167)Anthocyanins +Flavonoids +Steroids minus

Triterpenes minus

Coumarin minus

Reducing compound minus

Glycosides +Cyanogenic derivate minus

+ = presence minus = absence MI Moss Index

Ethanol Ethyl acetate0

20

40

60

80

100

Sensitive strainsResistant strains

Extraction solvent

()

Figure 1 Antimicrobial effect ofC nitida extracts on Staphylococcusstrains isolated from meat

(20mgmL) showed a various effect on reference and meatisolated strains Indeed among the reference strains weobserved that there was an antimicrobial activity on all thestrains except E coli (Table 2) Then 90 (910) of the testedreference strains were sensitive toC nitidarsquos ethanol and ethylacetate extracts

Concerning the meat isolated strains our data displaysthat 7857 (2228) of the tested strains were sensitive to ethylacetate extracts against 6785 (1928) for the ethanol extract(Figure 1)

321 Susceptibility The inhibitory diameter zones of thesensitive strains vary according to species and the kind ofextract Thus for the reference strains Figure 2 indicatedthat there was not a significant variation of the diameterof inhibitory zones according to the time (119875 gt 005) withboth ethanol extract (Figure 2(a)) and ethyl acetate extract(Figure 2(b)) There was not also any significant difference

comparing the inhibition diameters of the meat isolatedStaphylococcus strains (Figures 2(c) and 2(d))

Figure 3 shows that inmost of the cases the susceptibilityof the tested reference strains varied depending on the kindof solvent used for the extraction but their effect was notstatistically different (119875 gt 005) Globally in both referencestrains (Figure 3(a)) and meat isolated ones (Figure 3(b))the ethyl acetate extracts were more efficient Thus in thereference strains we observed the highest diameter on Calbicans (175 plusmn 07mm) and the lowest on P vulgaris (95 plusmn07mm) But only with S oralis the same diameters wereobtained with both ethanol and ethyl acetate extracts (155 plusmn07mm) With the meat isolated Staphylococcus strains thehighest diameter was observed on S lentus using both ethylacetate extract (1819plusmn104mm) and ethanol extract (1633plusmn115mm)

322 Determination of Minimum Inhibitory Concentrations(MIC) Table 3 shows the minimum inhibitory concentra-tions (MIC) of C nitidarsquos bark extracts on ten referencestrains and on twenty height strains of nine Staphylococcusspecies isolated from meat products

Considering the reference strains the mean concentra-tion values widely vary depending on the tested strains andranged from 0312mgmL to 5000mgmL For ethyl acetateextract the lowest MIC was 0312mgmL with Candidaalbicans Five strains (Staphylococcus aureus Pseudomonasaeruginosa Proteus mirabilis Proteus vulgaris and Ente-rococcus faecalis) display MIC of 125mgmL whereas thehighest concentration (25mgmL) value was recorded withMicrococcus luteus Considering the ethanol extract thelargest MIC was observed on Micrococcus luteus (5mgmL)while the most sensitive strains displaying the lowest MIC(0312mgmL) were Staphylococcus aureus Streptococcusoralis and Enterococcus faecalis Three other strains (Proteusmirabilis Staphylococcus epidermidis and Proteus vulgaris)had 125mgmL as MIC value

For the meat isolated strains it globally appears thatthe mean values ranged from 0078mgmL to 1250mgmLFor ethyl acetate extract the lowest MIC was 0078mgmLwith three species of Staphylococcus (S equorum S sapro-phyticus and S lentus) and the largest MIC (0625mgmL)was observed with S haemolyticus Considering the ethanolextract the largest MIC (125mgmL) was observed on Saureus and S xylosus while the most sensitive strain display-ing the lowest MIC (0078mgmL) was S haemolyticus Theother values for the MIC were 0156mgmL (S equorum andS lentus) 0312mgmL (S simulans and S saprophyticus) and0625mgmL (S sciuri and S cohnii)

323 Determination of the Minimum Bactericidal Concentra-tion (MBC) Table 4 presents the MBC of Cola nitidarsquos barkextract on ten reference strains and on twenty height meatisolated Staphylococcus strains

For the reference strains results show that the MBCsvaried (from 125mgmL to gt20mgmL) according to thebacterial strains and the kind of extract With the ethanolextract the lowest MBC was 25mgmL (S aureus P aerugi-nosa S epidermidis E faecalis and C albicans) whereas the


Table2Re

sults

ofagar

disc

diffu

sionassays

show

ingthea

ntibacteria

lactivity

ofsometestedmicroorganism

sinthep

resenceo

fextracts

References

trains

2

1998400

2998400

2998400

Staphylococcus

aureus

Streptococcuso

ralis

Staphylococcus

epidermidis

Escherich

iacoli

Pseudomonas

aeruginosa

MeatisolatedStaphylococcus

Staphylococcus

saprophyticus

Staphylococcus

lentus

Staphylococcus

haem

olyticu

sStaphylococcus

equorum

Staphylococcus

simulan

s11015840con

trol2ethano

lextractsand21015840ethylacetatee

xtracts


48h24h

0

5

10

15

20

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

Strains

Refe

renc

e str

ains

Inhi

bito

ry d

iam

eter

(mm

)

(a) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

(b) Ethyl acetate

48h24h

0

5

10

15

20

Mea

t iso

late

d str

ains

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

Strains

Inhi

bito

ry d

iam

eter

(mm

)

(c) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(d) Ethyl acetate

Figure 2 Medium inhibitory diameter zone of C nitida extracts on reference and meat isolated Staphylococcus strains after 24 h and 48 h ofincubation Reference strains are the following S aur Staphylococcus aureusM lutMicrococcus luteus S epi Staphylococcus epidermidisS ora Streptococcus oralis Ps aer Pseudomonas aeruginosa E foe Enterococcus faecalis P vul Proteus vulgaris E coli Escherichia coliC alb Candida albicans P mir Proteus mirabilis meat isolated strains are the following S sci S sciuri S aur S aureus S sim S simulansS xyl S xylosus S coh S cohnii S equ S equorum S sap S saprophyticus S hae S haemolyticus and S len S lentus

highest was gt20mgmL (M luteus) With the ethyl acetateextract the highest MBCwas gt20mgmL (M luteus) and thelowest (125mgmL)was recorded on S aureus andE faecalis

Considering the meat isolated strains our data dis-plays that the MBC of the tested extracts varied from0625mgmL to 5mgmL depending on the tested Staphy-lococcus species Using ethanol extract the lowest MBC(0625mgmL) was observed on S equorumwhile the highestMBC (5mgmL) was obtained with this extract on S cohniiand S xylosus With the ethyl acetate extract the largestMBC (5mgmL) was obtained on S xylosus while the lowestMBC (0625mgmL) was obtained on S sciuri S equorum Ssaprophyticus and S lentus

324 Evaluation of Bactericidal and Bacteriostatic Effects ofCola nitida Bark Extracts The ratio MBCMIC was calcu-lated to evaluate the kind of effect exerted by the Cola nitidabark extracts on the tested strains Our data displays that theextracts have both bactericidal and bacteriostatic effects on

reference and meat isolated strains Thus with the referencestrains the ethyl acetate extract has bactericidal effect onS aureus P aeruginosa S epidermidis and E faecalis Theethanol extract had a bactericidal effect on only S epidermidis(Table 5) With the meat isolated Staphylococcus strains weobserved a bactericidal effect on S sciuri (with ethyl acetateextract) and S aureus (with ethanol extract) while all theother strains show the bacteriostatic effect in presence of thetested extracts (Table 5)

33 Antifungal Activity of Cola nitida Bark Extracts Figure 4indicated that the antifungal activity using ethanol and ethylacetate extracts of C nitida (18mgmL) was statisticallyvariable in regard of the used fungal strains (119875 = 00016)The inhibitory rate varies from 20 to 467 Moreover theantifungal effect varies according to the kind of extract(119875 = 0 007) Indeed the interaction between the strainsand the ethyl acetate extract displays a difference of actionconsidering F verticillioides and A tamari (119875 lt 001)


S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

0

5

10

15

20

EthanolEthyl acetate

Strains

Inhi

bito

ry d

iam

eter

(mm

)

Reference strains

lowastlowastlowast

(a)

0

5

10

15

20

Ethyl acetateEthanol

Strains

Inhi

bito

ry d

iam

eter

(mm

)

Meat isolated strains

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(b)

Figure 3 Comparison of medium inhibitory diameter of C nitidarsquos ethyl acetate extract and ethanol extract on reference strains andmeat isolated Staphylococcus strains Reference strains are the following S aur Staphylococcus aureus M lut Micrococcus luteus S epiStaphylococcus epidermidis S ora Streptococcus oralis Ps aer Pseudomonas aeruginosa P vul Proteus vulgaris E coli Escherichia coli Calb Candida albicans P mir Proteus mirabilis meat isolated strains are the following S sci S sciuri S aur S aureus S sim S simulansS xyl S xylosus S coh S cohnii S equ S equorum S sap S saprophyticus S hae S haemolyticus and S len S lentus lowast119875 lt 005lowastlowast

119875 lt 001

000

1000

2000

3000

4000

5000

6000

Fusariumverticillioides

Aspergillustamarii

Penicilliumcitrinum

Inhi

bitio

n ra

te (

)

Fungal strains Ethanol extractEthyl acetate extract

Low activity (resistant)

Fair activity (halfway between

resistant and sensitive)

NS lowastlowast lowastlowast

NS P gt 005lowastlowast P lt 001

Figure 4 Inhibition rate of Cola nitidarsquos extracts in the fungal growth

and then F verticillioides and P citrinum (119875 lt 0001)With the ethanol extract there was no statistical differenceindependently of the strains (119875 gt 005)

34 Antioxidant Activity of Cola nitida Bark Extracts Table 6shows the radical scavenging activity by DPPH Our data

reveal that the IC50of the ethanol extract (900plusmn173 120583gsdot120583Lminus1)

was about two times higher than the one observed withthe ethyl acetate extract (453 plusmn 098 120583gsdot120583Lminus1) Moreoverit appears that the IC

50values of the reference molecules

(quercetin and gallic acid) are lower than the tested extractsThe ethyl acetate extract of C nitida had an AAI value


Table 3 Minimum inhibitory concentrations of C nitidarsquos barkextract on reference strains andmeat isolated Staphylococcus strains

Strains

Minimum inhibitoryconcentrations (mgml)Ethanolextract

Ethyl acetateextract

Reference strainsStaphylococcus aureus 0312 125Pseudomonas aeruginosa 0625 125Proteus mirabilis 125 125Micrococcus luteus 05 25Staphylococcus epidermidis 125 0625Proteus vulgaris 125 125Streptococcus oralis 0312 0625Enterococcus faecalis 0312 125Escherichia coli mdash mdashCandida albicans 0625 0312

Meat isolated Staphylococcus strainsS sciuri 0625 0312S aureus 125 0312S simulans 0312 0152S cohnii 0625 0312S xylosus 125 0312S equorum 0156 0078S saprophyticus 0312 0078S haemolyticus 0078 0625S lentus 0156 0078

(1102 plusmn 149 120583gsdot120583Lminus1) higher than the one observed with theethanol extract (571 plusmn 123 120583gsdot120583Lminus1)

Table 6 shows also the antioxidant activity of Cola nitidabark extracts as the ability to reduce ABTS∙+ cation Thisactivity was determined from a linear regression curve (119910 =minus0001119909 + 05721 1198772 = 09715) Data in Table 6 indicatethat the extracts of Cola nitida have more significant activitythan the one obtained with ascorbic acid used as reference(3502 plusmn 073 120583molEqAAsdotgminus1) In addition our data indicatethat the ethanol extract reduces more the ABTS∙+ cation thanthe DPPH one Nevertheless independently of the methodsthe two Cola nitida extracts follow the same efficacy order

35 Cytotoxicity Assay of Cola nitidarsquos Bark Extracts Thebioassay to determine the lethality effect of Cola nitidarsquosbark extracts on Artemia salina was used to evaluate thecytotoxicity of our extracts Thus Figure 5 indicates theevolution of mortality according to the tested concentrationsof our extracts Indeed for the ethanol extracts the mortalityof the A salina larvae was observed from the concentrationof 052mgsdotmLminus1 whereas the killing effect started to beperceptible at 104mgsdotmLminus1

Table 4 Minimum bactericidal concentrations of C nitidarsquos barkextract on reference strains andmeat isolated Staphylococcus strains

Strains

Minimum bactericidalconcentrations (mgml)Ethanolextract


Reference strainsStaphylococcus aureus 25 125Pseudomonas aeruginosa 25 25Proteus mirabilis 10 05Micrococcus luteus gt20 gt20Staphylococcus epidermidis 25 25Proteus vulgaris 10 05Streptococcus oralis 05 25Enterococcus faecalis 25 125Escherichia coli mdash mdashCandida albicans 25 25


0102030405060708090

100

003 006 013 026 052 104 208 416

Surv

ival

()

EtOH EcEAC Ec

EtOH Ec ethanol extractsEAC Ec ethyl acetate extract

Extract concentration (mgmiddotmLminus1)

DL50

Figure 5 Variation of A salina larval mortality according to Cnitida extracts concentration

4 Discussion

The qualitative screening of Cola nitidarsquos bark extractsrevealed the presence of various phytochemical componentssuch as tannins flavonoids and saponins (Table 1) The pres-ence of tannins in our tested extracts suggests the probablebiological activities Indeed tannins are reported not only


Table 5 Bactericidal and bacteriostatic effects of Cola nitidaextracts on reference strains and meat isolated strains

StrainsCMBCMI

Ethanolextract


Reference strainsStaphylococcus aureus 801 1lowast

Pseudomonas aeruginosa 4 2lowast

Proteus mirabilis 8 4Micrococcus luteus gt4 gt8Staphylococcus epidermidis 2lowast 2lowast

Proteus vulgaris 8 4Streptococcus oralis 1602 4Enterococcus faecalis 801 1lowast

Escherichia coli mdash mdashCandida albicans 4 801

Meat isolated Staphylococcus strainsS sciuri 8 2lowast

S aureus 2lowast 801S simulans 801 1644S cohnii 4 1602S xylosus 8 801S equorum 401 801S saprophyticus 801 801S haemolyticus 3205 4S lentus 1602 801

With lowast = bactericidal effects without lowast = bacteriostatic effects

Table 6 Parameters of free radical scavenging activity by DPPHradical and ABTS methods

DPPH ABTSIC50 AAI 119862

(120583gsdot120583lminus1) (120583molEqAAsdotgminus1)Ethanol extract 900 plusmn 173 571 plusmn 123 4972 plusmn 035Ethyl acetateextract 453 plusmn 098 1102 plusmn 149 5339 plusmn 00

Quercetin 451 plusmn 035 1111 plusmn 085 mdashGallic acid 073 plusmn 012 6274 plusmn 554 mdashAscorbic acid mdash mdash 3502 plusmn 073

to promote tissue regeneration in case of superficial burninjury but also to have antibacterial antiviral antifungaland antioxidant effects [44] The presence of flavonoids inthe extracts indicates their potentiality to reduce in vitrocholesterol agents and to induce an antifungal activity [4445] Flavonoids are known to inhibit120572-amylase activitywhichregulates the amount of glucose in the blood therefore theextracts of C nitida can be used as an antidiabetic Thepresence of flavonoids and saponins has earlier been reportedin 2009 by NrsquoGuessan et al [46] in Cote drsquoIvoire during theirwork on the same plants Nevertheless we observed in ourstudy the absence of triterpene and steroid and the presence

of tannins whereas triterpene and steroid were observedwithout tannins in the same organ of the plant [46] and theother parts of the same plant [13 47] These observationsmay be probably due in the case of the same organ tothe collection conditions such as origin of the plants theconditions and the periods of harvesting organs We shouldnotice that the environment may influence the synthesis andexpression of phytochemical components in the plant [48ndash52] Some plant physiologists went further saying that plantscomponents can be produced only at a certain time andorin a determined condition For the same plant species thereis an unequal distribution of secondary metabolites throughthe organs

Among the ten reference strains at the unique concen-tration of 20mgmL the ethanol and ethyl acetate extractshave inhibited the growth of yeast and Gram + and Gram minusbacteria (Table 2) An antimicrobial effect was not observedon E coli at the used concentration This observation on thesusceptibility of E coli is different from those observed in2011 by Indabawa and Arzai [53] during their study on theantibacterial activity on the seed of C nitida Indeed thoseauthors found that C nitidarsquos seed aqueous extract inhibitsthe growth of E coli at the concentration of 500 120583gmL Thatdifference can be explained by the fact that we do not use thesame organs

Concerning the meat isolated Staphylococcus strains ourresults indicate that the solvent plays a role in the extractionof active principles (Figure 1) The ethanol extracts are lesseffective than ethyl acetate extracts (119875 = 0028) on meatisolated Staphylococcus strains These results are similar tothose of Bolou et al [54] obtained during their study onTerminalia glaucescens when they demonstrated that theethyl acetate extract was more effective than the ethanolextract at the same dose on certain microorganisms Thepossible explanation to the difference of activity betweenthe two extracts may be the ability of solvent to solubilizeand extract some phytomoleculesThus according to Cowan[55] during the liquid-liquid extraction phytomolecules aredistributed between the solvents according to their polarityand solubility It can be thus concluded that the activeantimicrobial compounds contained in the bark of C nitidaaremore soluble in the ethyl acetate solvent than ethanol oneThe active antistaphylococcal principles contained in the barkof C nitida are more concentrate by ethyl acetate

Analyzing Figure 2 it appears that the inhibition zonediameters were not significantly different independently ofthe extract solvent regardless of duration (24 h and 48 h)on both reference (Figures 2(a) and 2(b)) and food isolatedstrains (Figures 2(c) and 2(d)) Our results are different fromthose of Arekemase et al [56] in their study when theyobserved a significant difference of inhibition diameters inthe time We can notice that Arekemase et al [56] used 10times higher concentration to the one we used that may thusbe one of the reasons Indeed with the highest concentrationit is possible to have an increase of the inhibition diameterbecause the active antimicrobial substance is in excess

The minimum inhibitory concentration (MIC) was vari-able depending on the strains and extracts (Table 3) Ourfound concentrations were higher than those reported by


Dahake et al [57] when they proved that S aureus andBacillus subtilisweremore sensitive to the ethanol leaf extractof Anacardium occidentale with MIC = 1562120583gmL Thedifference may probably be due to the divergent extractionmethods and the different origins of strains used Indeeddepending on the extraction methods the antimicrobialagents extractedmay have different concentrations Also thisindicates that C nitidarsquos bark extracts were less active atlowest concentration compared with those of A occidentale

The minimum bactericidal concentrations (MBCs) werevariable according not only to the strains but also to thetype of extracts (Table 4) Our results were not the sameas those obtained by Sika et al [28] reporting a range of0078 to 0625mgmL (for reference strains) and from 0078to 125mgmL (for meat isolated Staphylococcus strains) asMBCs during an in vitro test on A occidentale extracts Asthe used strains are from the same origin the difference maybe due to the phytochemical composition of the extracts

The ratio between the MIC and MBC shows thataccording to the strains extracts have both bactericidaland bacteriostatic effects on reference and food isolatedstrains (Table 5) The two kinds of activities were alreadyreported in previous studies on some plants such as Erythrinasenegalensis [58] and A occidentale [28]

The antifungal activity of Cola nitidarsquos bark extracts(Figure 3) was statistically variable in regard of the usedfungal strains (119875 = 00016) Independently of the extractsA tamarii was the most resistant strain Indeed the limitsestablished by Reyes et al [59] allow concluding that ourextracts have antifungal activity against the three testedstrains In comparison with studies performed using Cnitidarsquos seeds on one hand onAspergillus niger andAspergillusfumigatus in Nigeria [60] and on the other hand in CotedrsquoIvoire against Fusarium oxysporum [61] we can concludethat the bark ofC nitida has an interesting antifungal activityIndeed in their study in Nigeria (80mgmL) and CotedrsquoIvoire (304mgmL) these authors reported extremely highconcentration in comparison to the 18mgmLwe used in ourstudy

The antioxidant activity of different extracts by bothmethods (DPPH and ABTS) is reported in Table 6 Ourresults with the DPPH method corroborate those reportedin Cameroon by Momo et al [62] on the stems of Cola nitidaextracts (ethanol and aqueous extract) and in Nigeria on theseed of C nitida [12] Indeed Momo et al [62] found outthat the ethanol extract of C nitida stems has a high DPPHreduction percentage whereas the aqueous extract does nothave any reduction activity of DPPH radical This resultsuggests that the extraction solvent plays an important rolein the scavenging of free radical In their studies Ayebe et al[12] and Momo et al [62] reported the highest IC

50values

in comparison to ours The difference of values may be dueto the used organs bark in our study seed in Nigeria [12]and stem in Cameroon [62] To end we can also notice thatthe reduction of free radical ability varies from a referencemolecule to another and from an extract to another thisvariation may be due to the concentration of antiradicalmolecules and assays conditions Therefore considering theAntiradical Indexes Activity [41] we can conclude that

the Cola nitida bark ethanol and ethyl acetate extracts have astrong free radical scavenging activity (AAI gt 2) Combiningthe two methods (DPPH and ABTS) we observe that Colanitidarsquos bark extracts have an excellent reducing free radicalactivity

Referring to the values reported by Mousseux [63] thetwo extracts of C nitidarsquos bark we tested in our studies(Figure 5) are not toxic at the tested doses Our resultcorroborates those found in Nigeria by Ayebe et al [12]during their study testing the toxicity of Cola nitidarsquos seedsaqueous extract on the rat

5 Conclusion

Through the obtained results we can say that Cola nitidarsquosbark contains many secondary metabolites dominated bypolyphenol compounds The presence of those compoundsconfers to C nitidarsquos bark through the ethanol and theethyl acetate extract some important biological activitiesThetested extract displays more bactericidal effect on referencestrains than on meat isolated Staphylococcus strains Formost of the investigated biological activity the ethyl acetateextract is more effective than the ethanol extract The morepurified extracts of C nitidarsquos bark can be useful both in foodconservation and in human medicine

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors thankUEMOA for the financial support throughthe project LBTMM-PAES-UEMOA-2012 The authors alsothank International Foundation for Science (IFS) for financialsupport

References

[1] A Sofowora Medicinal Plants and Traditional Medicine inAfrica Spectrum Books Limited Sunshine House IbadanNigeria 2nd edition 1993

[2] O M Iniaghe S O Malomo and J O Adebayo ldquoProximatecomposition and phytochemical constituents of leaves of someAcalypha speciesrdquo Pakistan Journal of Nutrition vol 8 no 3 pp256ndash258 2009

[3] A M Diallo Etude des plantes medicinales de niafunke (regionTombouctou) Phytochimie et pharmacologie de Maeruacrassi-foliaForsk (Capparidacee) [These de Doctorat] Universite deBamako Faculte de Medecine de Pharmacie et dOdonto-Stomatologie 2005

[4] WHO Strategie de lOMS pour la medicine traditionnelle pour2002ndash2005 2002

[5] A AkoegninouW J van der Burg and L J G van derMaesenFlore Analytique du Benin edited by V Adjakidje J P EssouB Sinsin H Yedomonhan Wagening University Papers 062Backhus Publishers 2006

[6] K Kouame and M Scande ldquoCola nitida (vent) Schott amp EndlrdquoSeed Leaflet Forest amp Landscape no 111 pp 1ndash2 2006


[7] F C Mokwunye Functional characterisation of kola nut powderfor beverage production [MS thesis] University of AgricultureAbeokuta Nigeria 2009

[8] O Nzekwu ldquoKola nutrdquo Nigeria Magazine vol 71 pp 298ndash3051961

[9] C O Jayeola ldquoPreliminary studies on the use of kolanuts (Colanitida) for soft drink productionrdquo Journal of Food Technology inAfrica vol 6 no 1 pp 25ndash26 2001

[10] G Javies ldquoThe rise and fall of cocaine colardquo httpwwwunzorgPubLewRockwell 2002may-00039

[11] S T Lowor P C Aculey and M K Assuah ldquoAnalysis of somequality indicators in cured Cola nitida (Vent)rdquo Agriculture andBiology Journal of North America vol 1 no 6 pp 1206ndash12142010

[12] E K Ayebe H F Yapi A A Edjeme et al ldquoIn vivo in vitroantioxidant activity assessment amp acute toxicity of aqueousextract ofCola nitida (Sterculiaceae)rdquoAsian Journal of Biochem-ical and Pharmaceutical Research vol 2 no 4 pp 144ndash155 2012

[13] S Muhammad and A Fatima ldquoStudies on phytochemical eval-uation and antibacterial properties of two varieties of kolanut(Cola nitida) in Nigeriardquo Journal of Biosciences and Medicinesvol 2 no 3 pp 37ndash42 2014

[14] K J Ryan andCGRay SherrisMedicalMicrobiologyMcGraw-Hill 4th edition 2004

[15] F VincenotM Saleh andG Prevost ldquoLes facteurs de virulencede Staphylococcus aureusrdquo Revue Francophone des Laboratoiresvol 38 no 407 pp 61ndash69 2008

[16] S Chang D M Sievert J C Hageman et al ldquoInfectionwith vancomycin-resistant Staphylococcus aureus containingthe vanA resistance generdquoTheNewEngland Journal ofMedicinevol 348 no 14 pp 1342ndash1347 2003

[17] L Malıkova I Sedlacek D Novakova and M Nemec ldquoRibo-typing and biotyping of Staphylococcus epidermidis isolatedfrom hospital environmentrdquo Folia Microbiologica vol 52 no 4pp 375ndash380 2007

[18] K Zhang J Sparling B L Chow et al ldquoNew quadriplex PCRassay for detection of methicillin and mupirocin resistanceand simultaneous discrimination of Staphylococcus aureus fromcoagulase-negative staphylococcirdquo Journal of ClinicalMicrobiol-ogy vol 42 no 11 pp 4947ndash4955 2004

[19] P Morellion Y A Que and M P Glauser ldquoStaphylococcusaureusrdquo in Principles and Practice of Infectious Diseases GL Mandel J E Bennett and R Dolin Eds pp 2321ndash2351Churchill Livindstone Philadelphia Pa USA 2005

[20] F Z Akcam G B Tinaz O Kaya A Tigli E Ture and SHosoglu ldquoEvaluation of methicillin resistance by cefoxitin diskdiffusion and PBP2a latex agglutination test in mecA-positiveStaphylococcus aureus and comparison of mecA with femAfemB femX positivitiesrdquo Microbiological Research vol 164 no4 pp 400ndash403 2009

[21] M Zohra Etude Phytochimique et Activites Biologiques dequelques Plantes medicinales de la Region Nord et Sud-Ouestde lAlgerie [These de doctorat] Universite Abou Bekr Belkaid2013

[22] D Bagchi C K Sen M Bagchi and M Atalay ldquoAnti-angiogenic antioxidant and anti-carcinogenic properties of anovel anthocyanin-rich berry extract formulardquo Biochemistryvol 69 no 1 pp 75ndash80 2004

[23] O Patthamakanokporn P Puwastien A Nitithamyong andP P Sirichakwal ldquoChanges of antioxidant activity and totalphenolic compounds during storage of selected fruitsrdquo Journal

of Food Composition and Analysis vol 21 no 3 pp 241ndash2482008

[24] N Sokpon and C Ouinsavi ldquoUtilisation de Khaya senegalensisen medecine traditionnelle au Beninrdquo Revue de Medecine etPharmacopee Africaines vol 16 pp 9ndash19 2002

[25] B Biecke ldquoEthnobotanishestudie van geneeskrachtigeplntenin Manigri en Igbere Beninrdquo Bioingenieur Universiteit Gentinhetland En BosBeheer 2004

[26] E Adjanohoun V Adjakidje M R A Ahyi et al Contributionaux Etudes Ethnobotaniques et Floristiques en Republique Popu-laire du Benin ACCT Paris France 2nd edition 1989

[27] P Sessou S Farougou P Azokpota et al ldquoIn-vitro antifungalactivity of essential oil of Pimenta racemosa against fungalisolates from wagashi a traditional cheese produced in BeninrdquoInternational Journal of Natural and Applied Sciences vol 8 no1 pp 25ndash34 2012

[28] C K Sika H Sina H Adoukonou-Sagbadja et al ldquoAntimi-crobial activity of Anacardium occidentale L leaves and barksextracts on pathogenic bacteriardquo African Journal of Microbiol-ogy Research vol 8 no 25 pp 2458ndash2467 2014

[29] P J Houghton and A Raman Laboratory Hand Book for theFractionation of Natural Extracts Chapman and Hall LondonUK 1998

[30] R Sanogo D Diallo S Diarra C Ekoumou and DBougoudogo ldquoActivite antibacterienne et antalgique de deuxrecettes traditionnelles utilisees dans le traitement des infec-tions urinaires et la cystite au Malirdquo Mali Medical vol 21 pp18ndash24 2006

[31] J D NrsquoGuessan A P Bidie B N Lenta B Weniger P Andreand F Guede-Guina ldquoIn vitro assays for bioactivity-guidedisolation of anti salmonella and antioxidant compounds inThonningia sanguinea flowersrdquoAfrican Journal of Biotechnologyvol 6 no 14 pp 1685ndash1689 2007

[32] P Attien H Sina W Moussaoui et al ldquoPrevalence and antibi-otic resistance of Staphylococcus strains isolated from meatproducts sold in Abidjan streets (Ivory Coast)rdquo African Journalof Microbiology Research vol 7 no 26 pp 3285ndash3293 2013

[33] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoTheAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[34] S Gupta M Ali and M S Alam ldquoA naphthoquinone fromLawsonia inermis stem barkrdquo Phytochemistry vol 33 no 3 pp723ndash724 1993

[35] A Saha and M S Rahman ldquoAntimicrobial activity of crudeextract from Calycopteris floribunsardquo Bangladesh Journal ofMicrobiology vol 25 no 2 pp 137ndash139 2008

[36] N N Farshori MM Al-Oqail E S Al-Sheddi M A Siddiquiand A Rauf ldquoAntimicrobial potentiality of Polyalthia longifoliaseed oil against multi drug resistant (MDR) strains of bacteriaand fungus of clinical originrdquo African Journal of MicrobiologyResearch vol 19 no 7 pp 1977ndash1982 2013

[37] SMAKawsar EHuq andNNahar ldquoCytotoxicity assessmentof the aerial parts ofMacrotyloma uniflorum linnrdquo InternationalJournal of Pharmacology vol 4 no 4 pp 297ndash300 2008

[38] S Kumar A Thomas A Sahgal A Verma T Samuel andM K K Pillai ldquoEffect of the synergist piperonyl butoxideon the development of deltamethrin resistance in yellow fevermosquito Aedes aegypti L (Diptera Culicidae)rdquo Archives ofInsect Biochemistry and Physiology vol 50 no 1 pp 1ndash8 2002


[39] N Dohou K Yamni A Badoc and A Douira ldquoActiviteantifongique drsquoextraits de Thymelaea lythroides sur troischampignons pathogenes du rizrdquo Bulletin de la Societe depharmacie de Bordeaux vol 143 no 1ndash4 pp 31ndash38 2004

[40] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[41] S Guenne N Ouattara A Hilou J F Millogo and OG Nacoulma ldquoAntioxidant enzyme inhibition activities andpolyphenol contents of threeAsteraceae species used in BurkinaFaso traditionally medicinerdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 3 no 5 pp 524ndash528 2011

[42] R Scherer and H T Godoy ldquoAntioxidant activity index (AAI)by the 22-diphenyl-1-picrylhydrazyl methodrdquo Food Chemistryvol 112 no 3 pp 654ndash658 2009

[43] G Schmeda-Hirschmann J A Rodriguez C Theoduloz S LAstudillo G E Feresin and A Tapia ldquoFree-radical scavengersand antioxidants from Peumus boldus Mol (lsquoBoldorsquo)rdquo FreeRadical Research vol 37 no 4 pp 447ndash452 2003

[44] J Bruneton Les Tanins Editions Medicales InternationalesParis France 1999

[45] AOrtuno A Baidez PGomez et al ldquoCitrus paradisi andCitrussinensis flavonoids their influence in the defence mechanismagainst Penicillium digitatumrdquo Food Chemistry vol 98 no 2pp 351ndash358 2006

[46] K NrsquoGuessan B Kadja G N Zirihi D Traore and L Ake-AssildquoScreening phytochimique de quelques plantes medicinalesivoiriennes utilisees en pays Krobou (Agboville Cote-drsquoIvoire)rdquoSciences amp Nature vol 6 no 1 pp 1ndash15 2009

[47] M A Sonibare M O Soladoye O O Esan and O OSonibare ldquoPhytochemical and antimicrobial studies of fourspecies of Cola Schott amp Endl (Sterculiaceae)rdquo African Journalof Traditional Complementary and AlternativeMedicines vol 6no 4 pp 518ndash525 2009

[48] W J Cybulski III W T Peterjohn and J H Sullivan ldquoTheinfluence of elevated ultraviolet-B radiation (UV-B) on tissuequality and decomposition of loblolly pine (Pinus taeda L)needlesrdquo Environmental and Experimental Botany vol 44 no3 pp 231ndash241 2000

[49] M R Braga M P M Aidar M A Marabesi and J R L deGodoy ldquoEffects of elevated CO

2

on the phytoalexin productionof two soybean cultivars differing in the resistance to stemcanker diseaserdquo Environmental and Experimental Botany vol58 no 1ndash3 pp 85ndash92 2006

[50] H Tsukaya R Tsujino M Ikeuchi et al ldquoMorphological vari-ation in leaf shape in Ainsliaea apiculata with special referenceto the endemic characters of populations on Yakushima IslandJapanrdquo Journal of Plant Research vol 120 no 3 pp 351ndash3582007

[51] A Folkers K Huve C Ammann et al ldquoMethanol emissionsfrom deciduous tree species dependence on temperature andlight intensityrdquo Plant Biology vol 10 no 1 pp 65ndash75 2008

[52] H Shen Y Tang H Muraoka and I Washitani ldquoCharacteris-tics of leaf photosynthesis and simulated individual carbon bud-get in Primula nutans under contrasting light and temperatureconditionsrdquo Journal of Plant Research vol 121 no 2 pp 191ndash2002008

[53] I I Indabawa andAHArzai ldquoAntibacterial activity ofGarciniakola and Cola nitida seed extractsrdquo Bayero Journal of Pure andApplied Sciences vol 4 no 1 pp 52ndash55 2011

[54] G E K Bolou B Attioua A C NrsquoGuessan A Coulibaly J DNrsquoGuessan and A J Djaman ldquoEvaluation in vitro de lrsquoactiviteantibacterienne des extraits de Terminalia glaucescens planchsur Salmonella typhi et Salmonella typhimuriumrdquo Bulletin de laSociete Royale des Sciences de Liege vol 80 pp 772ndash790 2011

[55] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[56] MOArekemaseG POyeyiola andMBAliyu ldquoAntibacterialactivity ofAnacardiumoccidentaleon some enterotoxin produc-ing bacteriardquo Internationnal Journal of Biology vol 3 no 4 pp392ndash399 2011

[57] A P Dahake V D Joshi and A B Joshi ldquoAntimicrobialscreening of different extract of Anacardium occidentale Linnleavesrdquo International Journal of ChemTech Research vol 1 no 4pp 856ndash858 2009

[58] S Dramane K W Mamidou and K Kagoyire ldquoEvalua-tion des activites antimicrobiennes et anti-radicaux libres dequelques taxons bioactifs de Coterdquo European Journal of Scien-tific Research vol 40 no 2 pp 307ndash317 2010

[59] C R Reyes V R I Quiroz E M Jimenez A Navarro-Ocana and H J Cassani ldquoAntifungal activity of selected plantsecondary metabolites against Coriolus versicolorrdquo Journal ofTropical Forest Products vol 3 no 1 pp 110ndash113 1997

[60] A I Kanoma I Muhammad I D Ibrahim K Shehu HM Maishanu and A D Isah ldquoPhytochemical screening ofvarious species of cola nut extracts for antifungal activity againstphytopathogenic fungirdquo The American Journal of Biology LifeSciences vol 2 no 1 pp 18ndash23 2014

[61] D Kone C Brahima B J Odjochounou et al ldquoFungicides andbiological products activities towards fungi causing diseases onbanana and vegetable in Cote drsquoIvoirerdquo in Fungicides OdileCarisse Ed InTech 2010

[62] C E N Momo A F Ngwa G I F Dongmo and J E ObenldquoAntioxidant properties and 120572-amylase inhibition of Terminaliasuperba Albizia sp Cola nitida Cola odorata and Harun-gana madagascarensis used in the management of diabetes inCameroonrdquo Journal of Health Science vol 55 no 5 pp 732ndash7382009

[63] M Mousseux Test de toxicite sur larves de Artemia salinaentretien dun elevage de balanes DEUST Aquaculture Univer-site Francaise de Pacifique Centre Universitaire de NouvelleCaledonie 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Indeed C nitida is a plant native to tropical WestAfrica and belongs to Sterculiaceae family [5] This plant isnowadays cultivated from Senegal to Nigeria in the WestIndies and South America [6] In West Africanrsquos forest areascola is perhaps second in importance to the palm tree as anindigenous cash crop [7] Cola nut has been an importantarticle of international trade in many parts of Africa [8]The nuts of C nitida contain about two percent of caffeineand are chewed by many people as a stimulant It is a veryspecial and important item used in social and ceremonialactivities by Africans The nuts of cola also have industrialusage for the production of drugs soft drinks wines candiesand beverages [9] such as Coca-Cola and Pepsi-Cola [10]It has many pharmacological properties and contains someactive principles it prevents sleep thirst and hunger and actsas an antidepressant [7] The cola nuts are source of antiox-idants and contain a wide array of complex secondary plantmetabolites such as theobromine d-catechin L-epicatechinand kolatin [11] The use of the plant in the treatment ofcertain diseases has been reported by several authors [12 13]

Nowadays there exist more than 250 types of infectionscaused by bacteria and fungi [14 15] Among these microor-ganisms we find the Pseudomonas Escherichia coli [16] andbacteria of the genus Staphylococcus that are known to beone of the main elements of human physiological flora [17]and are responsible of many diseases [18ndash20] In the rankof fungi the moulds producing toxins are mainly of genusAspergillus Penicillium and Fusarium [21] With the adventof modern medicine and for the treatment of infectionsabusive and often uncontrolled use of antibiotics brings upa phenomenon of resistance in most of the bacteria andfungi Beyond infectious diseases oxidative stress is a veryserious phenomenon that can trigger molecular and cellularevents in the body The consequences are multiple such ascancer [22] cerebral and cardiovascular diseases diabetesand hypertension [23]

To face these health problems (resistance of microorgan-isms and natural phenomenon of production in the body ofthe free radicals) the track of medicinal plants deserves to beexplored In this direction in Benin several ethnobotanicalstudies [24ndash26] have focused for several years on identifyingmedicinal species Others have demonstrated the efficacy ofmedicinal plants in the fight against certain fungal strains[27] and pathogenic Gram-positive and Gram-negative bac-teria involve several pathologies [28] Furthermore in Beninvery little study has been conducted on C nitida and thereferences which relate to its phytochemical composition andits biological properties are quasi-nonexistent In the sameway the work realized on the species in other countriesconcerns seed those concerning species bark are rare andthe results are disparate The aim of this study was tomake the phytochemical screening of C nitida bark and toinvestigate in vitro some biological (antimicrobial antifungaland cytotoxic) activities of its extracts

2 Material and Methods

21 Collection of Plant Material The bark of C nitida wascollected in the village of Aglogbe (commune of Adjarra

6∘241015840010158401015840N 2∘121015840010158401015840E)Department ofOueme southernBeninThe plants materials were air-dried at 25∘Cndash30∘C for twoweeks ground and sieved into a bark powder The smoothpowder was stored in airtight glassware and kept in darknessat minus20∘C until use

22 Phytochemical Profiling The phytochemical profiling ofthe bark of C nitida to determine the major constituents(nitrogenous polyphenolic and terpenic compound andglycosides) was done according to Houghton and Raman[29]

23 Preparation of Ethanol and Ethyl Acetate Extracts Theseextracts were made using an adapted method of the onedescribed by Sanogo et al [30] and NrsquoGuessan et al [31]Thismethod consisted of macerating 50 g of C nitida powders in500mL of 96 ethanol for 72 hoursThe obtained extract wasfiltered thrice using Whatman filter paper Half of the filtratewas directly dried at 40∘C to obtain the ethanolic extract ofCnitida To the second half of the filtrate 200mL of H

2O and

100mL of ethyl acetate were added The solution was gentlymixed and left settled until we obtain two phases (about45min)The lower phasewas collected and dried as describedpreviously to obtain the ethyl acetate extract The alcoholicand ethyl acetate extracts were stored in labeled sterile bottlesand kept at minus20∘C until further use

24 Microorganismrsquos Cultures The tested microorganismsinclude ten references twenty height Staphylococcus meatisolated strains and three fungal strains (Penicillium cit-rinum Aspergillus tamarii and Fusarium verticillioides) Thethree fugal strains were part of the microorganisms isolatedin the Beninese traditional cheese wagashi by Sessou et al[27]The reference strains were Escherichia coliATCC 25922Staphylococcus aureus ATCC 29213 Staphylococcus epider-midisT22695 Pseudomonas aeruginosaATCC 27853 Proteusmirabilis A24974 Micrococcus luteus ATCC 10240 Proteusvulgaris A25015 Streptococcus oralis Enterococcus faecalisATCC 29212 andCandida albicansMHMRThe Staphylococ-cus strains used in this study were those isolated from threedifferentmeat products in IvoryCoast byAttien et al [32] andstored in the Laboratory of Biology and Molecular Typing inMicrobiology (University of Abomey-Calavi Benin)

25 Antimicrobial Activity

251 Sensitivity Test The disc diffusion method [33] wasused to screen the antimicrobial activity Briefly two tothree sterile paper discs (6mm as diameter) were lodgedunder aseptic conditions on Mueller Hinton agar Petri dishpreviously flooded with the appropriate bacterial culture(adjusted to 05 McFarland standard) The discs were asep-tically impregnated with 25120583L of C nitida extract solution(20mgmL) These dishes were kept for 15ndash30min at roomtemperature before incubation at 37∘C for 24 and 48 hours

After the incubation period the dishes were examinedfor inhibitory zones [34] Each sample was used in triplicatefor the determination of antibacterial and antifungal activity












(1)







3 Results







Triterpenes minus

Coumarin minus





20

40

60

80

100


Extraction solvent

()













Table2Re

sults

ofagar

disc

diffu

sionassays

show

ingthea

ntibacteria

lactivity


sinthep

resenceo

fextracts

References

trains

2

1998400

2998400

2998400

Staphylococcus

aureus

Streptococcuso

ralis

Staphylococcus

epidermidis

Escherich

iacoli

Pseudomonas

aeruginosa


Staphylococcus

saprophyticus

Staphylococcus

lentus

Staphylococcus

haem

olyticu

sStaphylococcus

equorum

Staphylococcus

simulan

s11015840con

trol2ethano


xtracts


48h24h

0

5

10

15

20

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

Strains

Refe

renc

e str

ains

Inhi

bito

ry d

iam

eter

(mm

)

(a) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

(b) Ethyl acetate

48h24h

0

5

10

15

20

Mea

t iso

late

d str

ains

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

Strains

Inhi

bito

ry d

iam

eter

(mm

)

(c) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(d) Ethyl acetate








S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)

Reference strains

lowastlowastlowast

(a)

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)


S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(b)


119875 lt 001

000

1000

2000

3000

4000

5000

6000


Aspergillustamarii

Penicilliumcitrinum

Inhi

bitio

n ra

te (

)
















Strains









Strains





0102030405060708090

100

003 006 013 026 052 104 208 416

Surv

ival

()

EtOH EcEAC Ec



DL50


4 Discussion




StrainsCMBCMI

Ethanolextract


























50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












(1)







3 Results







Triterpenes minus

Coumarin minus





20

40

60

80

100


Extraction solvent

()













Table2Re

sults

ofagar

disc

diffu

sionassays

show

ingthea

ntibacteria

lactivity


sinthep

resenceo

fextracts

References

trains

2

1998400

2998400

2998400

Staphylococcus

aureus

Streptococcuso

ralis

Staphylococcus

epidermidis

Escherich

iacoli

Pseudomonas

aeruginosa


Staphylococcus

saprophyticus

Staphylococcus

lentus

Staphylococcus

haem

olyticu

sStaphylococcus

equorum

Staphylococcus

simulan

s11015840con

trol2ethano


xtracts


48h24h

0

5

10

15

20

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

Strains

Refe

renc

e str

ains

Inhi

bito

ry d

iam

eter

(mm

)

(a) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

(b) Ethyl acetate

48h24h

0

5

10

15

20

Mea

t iso

late

d str

ains

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

Strains

Inhi

bito

ry d

iam

eter

(mm

)

(c) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(d) Ethyl acetate








S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)

Reference strains

lowastlowastlowast

(a)

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)


S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(b)


119875 lt 001

000

1000

2000

3000

4000

5000

6000


Aspergillustamarii

Penicilliumcitrinum

Inhi

bitio

n ra

te (

)
















Strains









Strains





0102030405060708090

100

003 006 013 026 052 104 208 416

Surv

ival

()

EtOH EcEAC Ec



DL50


4 Discussion




StrainsCMBCMI

Ethanolextract


























50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Triterpenes minus

Coumarin minus





20

40

60

80

100


Extraction solvent

()













Table2Re

sults

ofagar

disc

diffu

sionassays

show

ingthea

ntibacteria

lactivity


sinthep

resenceo

fextracts

References

trains

2

1998400

2998400

2998400

Staphylococcus

aureus

Streptococcuso

ralis

Staphylococcus

epidermidis

Escherich

iacoli

Pseudomonas

aeruginosa


Staphylococcus

saprophyticus

Staphylococcus

lentus

Staphylococcus

haem

olyticu

sStaphylococcus

equorum

Staphylococcus

simulan

s11015840con

trol2ethano


xtracts


48h24h

0

5

10

15

20

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

Strains

Refe

renc

e str

ains

Inhi

bito

ry d

iam

eter

(mm

)

(a) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

(b) Ethyl acetate

48h24h

0

5

10

15

20

Mea

t iso

late

d str

ains

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

Strains

Inhi

bito

ry d

iam

eter

(mm

)

(c) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(d) Ethyl acetate








S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)

Reference strains

lowastlowastlowast

(a)

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)


S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(b)


119875 lt 001

000

1000

2000

3000

4000

5000

6000


Aspergillustamarii

Penicilliumcitrinum

Inhi

bitio

n ra

te (

)
















Strains









Strains





0102030405060708090

100

003 006 013 026 052 104 208 416

Surv

ival

()

EtOH EcEAC Ec



DL50


4 Discussion




StrainsCMBCMI

Ethanolextract


























50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Re

sults

ofagar

disc

diffu

sionassays

show

ingthea

ntibacteria

lactivity


sinthep

resenceo

fextracts

References

trains

2

1998400

2998400

2998400

Staphylococcus

aureus

Streptococcuso

ralis

Staphylococcus

epidermidis

Escherich

iacoli

Pseudomonas

aeruginosa


Staphylococcus

saprophyticus

Staphylococcus

lentus

Staphylococcus

haem

olyticu

sStaphylococcus

equorum

Staphylococcus

simulan

s11015840con

trol2ethano


xtracts


48h24h

0

5

10

15

20

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

Strains

Refe

renc

e str

ains

Inhi

bito

ry d

iam

eter

(mm

)

(a) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

(b) Ethyl acetate

48h24h

0

5

10

15

20

Mea

t iso

late

d str

ains

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

Strains

Inhi

bito

ry d

iam

eter

(mm

)

(c) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(d) Ethyl acetate








S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)

Reference strains

lowastlowastlowast

(a)

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)


S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(b)


119875 lt 001

000

1000

2000

3000

4000

5000

6000


Aspergillustamarii

Penicilliumcitrinum

Inhi

bitio

n ra

te (

)
















Strains









Strains





0102030405060708090

100

003 006 013 026 052 104 208 416

Surv

ival

()

EtOH EcEAC Ec



DL50


4 Discussion




StrainsCMBCMI

Ethanolextract


























50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


48h24h

0

5

10

15

20

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

Strains

Refe

renc

e str

ains

Inhi

bito

ry d

iam

eter

(mm

)

(a) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

(b) Ethyl acetate

48h24h

0

5

10

15

20

Mea

t iso

late

d str

ains

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

Strains

Inhi

bito

ry d

iam

eter

(mm

)

(c) Ethanol

48h24h

0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(d) Ethyl acetate








S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)

Reference strains

lowastlowastlowast

(a)

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)


S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(b)


119875 lt 001

000

1000

2000

3000

4000

5000

6000


Aspergillustamarii

Penicilliumcitrinum

Inhi

bitio

n ra

te (

)
















Strains









Strains





0102030405060708090

100

003 006 013 026 052 104 208 416

Surv

ival

()

EtOH EcEAC Ec



DL50


4 Discussion




StrainsCMBCMI

Ethanolextract


























50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


S a

ur

Ps a

er

P m

ir

M l

ut

S ep

i

P vu

l

S o

ra

E fo

e

E co

li

C a

lb

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)

Reference strains

lowastlowastlowast

(a)

0

5

10

15

20


Strains

Inhi

bito

ry d

iam

eter

(mm

)


S sc

i

S a

ur

S si

m

S x

yl

S co

h

S eq

uo

S sa

p

S h

ae

S le

n

(b)


119875 lt 001

000

1000

2000

3000

4000

5000

6000


Aspergillustamarii

Penicilliumcitrinum

Inhi

bitio

n ra

te (

)
















Strains









Strains





0102030405060708090

100

003 006 013 026 052 104 208 416

Surv

ival

()

EtOH EcEAC Ec



DL50


4 Discussion




StrainsCMBCMI

Ethanolextract


























50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Strains









Strains





0102030405060708090

100

003 006 013 026 052 104 208 416

Surv

ival

()

EtOH EcEAC Ec



DL50


4 Discussion




StrainsCMBCMI

Ethanolextract


























50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



StrainsCMBCMI

Ethanolextract


























50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







50values




5 Conclusion




Acknowledgments


References





















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













2























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Phytochemical Analysis and …downloads.hindawi.com/journals/bri/2015/493879.pdfResearch Article Phytochemical Analysis and Biological Activities of Cola nitida Bark