phytochemical and in-vitro screening of annona muricata ...njm.com.ng/articles/2018...

Nigerian Journal of Mycology Vol. 10 (2018) 1

PHYTOCHEMICAL AND IN-VITRO SCREENING OFAnnona muricata PLANT AGAINST CLINICAL FUNGI

Ebabhi, A. M*.,Kanife, U. C2.,Bikomo, E.O2., Julius, S. O3., andNnabugwu, E. C2.

1Biology Unit, Science Education, Distance Learning Institute,University of Lagos, Akoka, Lagos Nigeria;

2Department of Biological Science, Yaba College ofTechnology, PMB 2011, Lagos, Nigeria;

3Department of Pharmacy, Faculty of Pharmacy, University ofLagos, Lagos, Nigeria

ABSTRACTThe incidence of fungal infection is on the increase as pathogenic fungi arebecoming resistant to conventional drugs. This research examined the antifungalproperties of the aqueous, N-hexane and ethanol extracts of the leaves, fruits, barkand roots of Annona muricata using the disc diffusion agar assay. The zone ofinhibition varied for the tested dermatophytic organisms- Candida albicans,Trichoderma sp. and Trichophyton mentagrophytes, with respect to the plant partsand type of extract. The ethanol extracts of the test plant parts showed remarkableantifungal effect on the test fungal species. Preliminary phytochemical studies ofthe plant extracts showed that Alkaloid, Cardiac glycosides and phenol were highlypresent in all parts while steroid and phobatanin were presenting low quantity..The quantitative results of various parts of the plant revealed that the highestquantity of 127.98 mg/g was obtained from the ethanol extract of the leaf. Whilethe lowest quantity of 16.61 mg/g was noted in tannin present in the N-hexaneextract of the root. The quantitative analysis was done using ANOVA at 0.05 levelof significance. This findings lay credence to the traditional usage of Annonamuricata for the treatment of infections.

Key words: Annona muricata; Antifungal; dermatophytes; Disc diffusion;Phytochemicals;

ORIGINAL RESEARCH REPORTS NigerJ.mycol Vol.10, 1-16


INTRODUCTIONPlants have remain a pivot support and

sustainer of life on earth. In many parts ofthe world medicinal plants are consideredas the basis for health preservation andcure. In recent years, interests have beengenerated in the development of saferantimicrobial agents from natural plantproducts such as, essential oils and ex-tracts to control diseases like fungal infec-tions. Various plant materials are believedto have antimicrobial activities, as manyessential oils and plant extracts have beenreported to have no side effects on humanand animals (Taveira et al., 2010; Negi2012; Tabassum and Vidyasagar, 2013;Adelakun et al., 2016).

Annona muricata Linn is a member ofthe family of the custard apple tree calledAnnonaceae and a species of the genusAnnona which is known mostly for itsedible fruit. The fruit commonly knownas soursop or graviola, is slightly acidic intaste when ripe(reason for the name‘soursop’). The fruit is juicy, whitish andaromatic with abundant seeds. It is from atypical tropical tree and widely distributedin most of the tropical countries(Moghadamtousi et al., 2015). Thecreamy and delectable flesh of the freshfruit consist of about 67.5 % edible pulp,20 % peel, 8.5 % seeds and 4 % core byweight. Also, contains 80 % water, 1 %protein, 18 % carbohydrates and fairamount of potassium, dietary fiber,vitamin B1, B2 and C. Sugars constitute

about 68 % of the total solids. Soursopprocessed products are much sort after inthe international market. The mostdesirable characteristics of the fruit are itsextremely pleasing fragrance and flavor.Soursop is commonly found in southernpart of Nigeria and it is cultivated mainlyin home gardens. The tree yields up to 10tons per ha and each fruit weighs 0.5 to 2kg (Oyenuga, 1978).

The green leaves are lanceolate, glossyand dark green in colour and had beentraditionally used to treat headaches, hy-pertension, cough, asthma and as anti-spasmodic, sedative and nerving for heartcondition (Ioannis et al., 2015). The fruitis also eaten to elevate a mother’s milkafter child birth while the crushed leafmixture of A. muricata together with A.squamosa and Hibiscus rosa-sinensis isused as a juice on the head to protectagainst fainting (Alothman et al, 2009).

All parts of A. muricata plant are usedin natural medicine in the tropics, butlittle information is available with respectto antifungal activities of A. muricataleaf, bark, fruit and root extracts. The barkand root have been used to treat diseaseconditions such as diabetics (Adeyemi etal., 2008; 2010) and arthritis. Other re-ported medicinal uses include its antican-cer (Oberlies et al., 1997), antibacterialand antifungal actions as well as, it anti-nociceptive and anti-inflammatory effect(de Sousa et al., 2010). Coria-Téllez et al.(2016) also gave a comprehensive review

Phytochemical and in-vitro Screening of Annona muricata Plant against Clinical Fungi


on the ethnobotanical studies of A. muri-cata. Reports on resistance to antifungaldrug shave posed strong barrier to effec-tive treatment of fungal infections andalso due to economic crises some of theseantifungal drugs have been unreachableespecially to the masses. The purpose ofthis study was to investigate the antifun-gal activity of the root, bark, leaf and fruitextracts of Annona muricata on threeclinical fungi i.e. Candida albicans,Trichoderma spp. and Trichophyton men-tagrophytes and to make available trado-medicinal means of treating fungaldiseases at an affordable price.

MATERIALS AND METHODSCollection and identification of plantmaterials

The healthy root, bark, leaf and fruitsof A. muricata were collected locally atUmuhu-Okabia, Orsu Local Govern-ment area, Imo state, Nigeria from Oc-tober to November, 2016. These weretransported in ziplock transparent bagsand authenticated at the Lagos Univer-sity Herbarium. This medicinal plantparts were chosen based on their tradi-tional medicinal uses and reported bio-logical activities.

Sample PreparationsA.muricata root, bark, and leaf were

washed thoroughly under running tapwater, air dried, and then homogenizedto fine powder. These were stored in

sterile air tight container for the experi-mented work. The fruits were washedunder a running tap water, seeds andpericaps were carefully removed andpulp was lypolized for further use. Twohundred grams of the milled A. murica-ta plant parts (leaves, bark, root andfruit) were each extracted with threedifferent solvents: aqueous, ethanol andhexane at 1:2 w/v. The method of ex-traction follows what was described bySilva et al. (2014).

Test for Purity of the Organic ExtractsEach extract obtained was tested to

ensure its purity via spreading a drop onsterile plates containing potato dextroseagar. The plates were incubated andexamined for possible growth ofcontaminants, the absence ofmicroorganism growth confirmed thepurity of the test extracts (Okigbo et al.2009).

Sampling and Isolation of fungiScrapings were collected from the

Nigerian Institute of Medical Research(NIMR), Yaba, Lagos, Nigeria fromdifferent active zones of the lesion onpatients’ bodies (Samuel et al. (2014).All the samples were routinely grown onSabouraud dextrose agar (SDA) (Oxoid,Basingstoke, England) for 12 - 18 daysat 30 °C. Pure culture of isolatedorganisms were certified as fungi usingtheir cultural, morphological character-

Ebabhi, Kanife, Bikomo, Julius, and Nnabugwu


istics as well as comparing them withconfirmed representatives of differentspecies in relevant texts like Ellis et al.(2007).

Fungal suspensionFungal suspensions were prepared

from each pure fungus culture grown onSDA by adding ten millilitres ofsterilized distilled water to colonies offresh young growing culture of eachfungus, and the suspensions were madeby gently probing the colony with the tipof a sterilised Pasteur pipette. Afterwhich each resulting mixture of conidiaand hyphal fragments were asepticallytransferred into sterile tubes.

Preparation of Disc for fungicidalassay

The discs for fungicidal activity wereprepared as described by Adekunle andIkumapayi (2006). The discs (Filterpapers Whatman No. 1) were sterilizedin an autoclave at 120oC for 15 min anddried in an oven at 60oC for 30 min. Thediscs were later soaked into extracts at0.025glint, 0.1glint and 0.2glint for 24hours prior to use.

Assay of fungicidal activity of Annonamuricata plant extracts

The disc diffusion method byAdekunle and Ikumapayi (2006) wasadopted for the antifungal assay. Thetests were conducted with authenticated

pure cultures of the fungi to determinetheir respective tolerance of the extracts.A 0.5 ml aliquot of the stockedsuspension of each fungus was droppedaseptically using a dropper over theentire agar surface of freshly preparedSDA plates and evenly spread using aspreader. This was done to ensure aneven distribution of the spores and laterdried at room temperature for 15 minprior to the application of the discs onthe agar plates. The discs (4) which havebeen soaked in different extracts atdifferent concentrations were placed atthe four edges on the surface of the agarplate with the aid of a sterilized forceps.The inoculated plates were later placedin an incubator at 28oC.The plates wereobserved for zones of inhibition after48hr. The extent of inhibition wasdetermined by measuring the diameterof the inhibition zone using a transparenthalf (1/2) metre rule (Booths 1971). Themean zones of inhibition of the threereplicates were expressed in millimetres.The discs were also soaked in equivalentvolume of ketocronazol and mycoteneand used respectively as positive control.

Qualitative and QuantitativePhytochemical Screening

The presence and amount ofphytochemicals in the aqueous, hexaneand ethanol extracts of Annona muricatawere determined according to standardmethods of Harbone (1973); Edeoga et



al (2005); Sofowora (2006); Trease andEvans (2009) and Prohp and Onoagbe(2012). The phytochemicals tested forinclude Alkaloids, saponnin, tannin,flavonoids, phlobatannins, steroids,phenol, terpenoids and cardiacglycosides

Data AnalysisThe data were expressed as mean ±

S.E.M. and were statistically analysedusing one-way analysis of variance(ANOVA). Means were separated by theDuncan new multiple range test. Valueswere considered significant at p≤-0.05

RESULTSQualitative analysis of Annonamuricata plant parts

The phytochemical analysis ofAnnona muricata plant parts showed thepresence of various compounds. Thequalitative results proved that alkaloids,cardiac glycoside and phenol werepresent in all parts using ethanol, n-hexane and aqueous as solvents. Whilesteroid and phlobatanin obtained at alower quantity as depicted in Table 1.Tannin was only noted in the aqueousextracts of the plant parts. The presenceof steroid was not noted in the plantparts. Various parts of the plant extractsshowed the presence of flavonoid withthe exception of the aqueous extract ofthe root.

Quantitative phytochemicals presentin Annona muricata plant parts

The quantitative assay of A. muricatawas achieved using the ethanol, n-hexane and aqueous solvents of thevarious plant parts of leaves, fruit, barkand root. It was noted that flavonoid wasthe dominant phytochemical present inthe plant parts. The highest quantity of127.98 mg/g was obtained from theethanol extract of the leaf. The lowestquantity of 16.61 mg/g was noted intannin present in the N-hexane extractof the root. It was also noted that the leafextracts from all solvents contain thelargest amount of each phytochemical.These results are depicted in Figures 1a,1b and 1c.

Antifungal assay of the plant partsFrom the in-vitro antifungal assay it

was shown that the leaf extracts from thevarious solvents at 0.1 g/ml was potentagainst Trichoderma sp with the highestzone of inhibition given at 12.17±0.48mm in the ethanol extract. The aqueousextracts of the leaf at variousconcentration also showed significantzones of inhibition against Trichodermasp while the N-hexane extract of theplant parts were not significantcompared to the controls. The root andfruit extracts at the differentconcentrations did not show any level ofsignificance against Trichoderma sp asthe mean zone of inhibitions were less



than 10 mm. This is with the exceptionof the aqueous extract of the root at 0.1g/ml with a mean value of 11.20±1.11mm. However, the aqueous extract ofthe bark at 0.1 g/ml and 0.2 g/mlsignificantly inhibited the growth of thetest organism

For the ethanol extract of Annonamuricata plant parts inhibition ofCandida albicans in the leaf, bark andfruit were potent at varyingconcentration when compared with thecontrols. It was noted that with theexception of the root extract all otherplant parts had significant level ofinhibition on C. albicans at 0.1 g/mlconcentration. The aqueous extract ofleaf at 0.2 g/ml was shown to inhibit thegrowth of C. albicans with mean zoneinhibition at 12.48±0.52 mm. Therewere no significant difference at P<0.5between the extracts of the leaf at 0.1mg/g with the control used in this studyas depicted in Figure 3.

DISCUSSIONMany plant based foods and herbs

contain powerful phytochemicalsubstances that can improve the qualityof our health and protect us against manydiet related diseases. The costing andeffective eradication of fungal diseasesis becoming unreachable for an averagecitizen in the developing countries. Thisis in addition to poor diagnostic practicein most of the health care facilities which

results in wrong prescriptions. Our studyshowed the phytochemicals present inAnnona muricata plant parts and also theantifungal activities of the extracts ofthese plant parts. The qualitativephytochemical screening of A. muricatashowed the presence of alkaloids,flavonoid, anthraquinone and phenols inalmost all plant extracts. It has beenreported that flavonoids and phenolicsare free radical scavengers that preventoxidative cell damage, and have stronganticancer activities (Pourmorad et al.,2008). They might induce mechanismthat affect cancer cells and inhibit tumorinvasion (Rafat et al., 2008). Theseactivities could be attributed to theirability to neutralize and quench freeradicals (Pourmorad et al., 2008; Omaleand Okafor, 2008). It was noted thattannin was present in all the aqueousextracts of the plant parts except for thefruit extract. Herbs that have tannins astheir component are astringent in natureand are used for the treatment ofintestinal disorders such as diarrhoeaand dysentery (Bajai, 2001). The plantkingdom has proved to be most usefulin the treatment of diseases and theyprovide an important source of all theworld’s pharmaceuticals. The mostimportant of these bioactive constituentsof plants are steroids, terpenoids,flavanoids, alkaloids, tannins andglycosides.

The result of our present study



revealed that the selected plant partspossess almost all important bioactivecompounds like cardiac glycosideswhich are cardioactive compoundsbelonging to triterpenoids class ofcompounds. Tannins are inhibitory tofungi, yeasts and bacteria. Severaltannins or related phenolic compoundshave been reported to possess antiviralactivity. For example, corilagin,previously isolated in the studies fromAcalypha species, has been reported toinhibit HIV reverse transcriptase (Singhet al, 2010). They have also been usedfor treating intestinal disorders such asdiarrhea and dysentery (Dharmananda,2003). Flavonoids extracted have shownto exhibit wide range of biologicalactivities like antimicrobial, anti-inflammatory, anti-allergic, anti-analgesic cytostatic, and anti-oxidantproperties. Efforts to produce largequantities of active secondarycompounds by plant tissue culturetechniques have been developed for therapid, large scale production of cells andtheir secondary compounds (Hussain etal., 2012 and Filová, 2014). These activecomponents can be isolated throughcallus without exploiting the plants fromnatural resources.

The use of medicinal plants forprevention, treatment and eradication ofdiseases by the locals of everycommunity around the globe cannot beover emphasized. The ascertained pure

extract devoid of contamination in ourpresent study agrees with the works ofOkigbo et al. (2009) and Anukworji etal. (2012).The antifungal activities ofthe root, bark, leaf and fruit extracts ofA.muricata in vitro on the growth ofthree clinical organisms of Candidaalbicans, Trichoderma spp. andTrichophyton mentagrophytes wasconfirmed in this study. This studydiscovered that A.muricata extractspossess a broad spectrum of activityagainst the test organisms at varyingconcentration for different solvents. Thefungicidal effect of the leaf against theclinical pathogens correlates theestablishment it efficacy onmicroorganisms in several studies likeMithunPai et al. (2016). The aqueousleaf extract showed higher efficacyagainst Trichophyton mentagrophytesthan the other solvents. However, notmuch have been established on otherplant parts of A. muricata.

In our study, across board the ethanolextract of A. muricata showed maximumantifungal activity than aqueous andN-hexane extracts. This maybe becauseethanol penetrates more into the matrixof plants thereby extracting more activecomponents than other solvents. Thiscould also be due to the fact that highquantities of phytochemicals likeflavonoids were extracted more with theethanol extract. Flavonoids have beenshown to have broad inhibitory spectrum



against a range of fungi as flavonoidinhibits spore germination of plantpathogens. For instance, flavonoids fromthe shrub-Eysenhardtia texana possessactivity against the human opportunisticpathogen Candida albicans (Wachter etal., 1999). The activity of Propolisagainst dermatophytic fungi andCandida spp. had also been attributed toits high flavonoids content by Yaghoubiet al. (2007).

The activities of the extracts againstthe pathogens showed that thefungistatic activity of the aqueous andhexane extracts were quite ineffectivewhich agrees with Kalidindi et al. (2015)who reported that the effect of aqueousextract of Annona squamosal did notshow any significant inhibition on testorganisms used. Earlier, Oluremi et al.(2010) had stated that some organismsare not susceptible to the extract’sactivity and the resistance toantimicrobial agents cannot beeliminated but curtailed since someorganisms are intrinsically resistant. Theless potency of hexane and aqueousextracts also correlates the works ofEl-Mahmood (2010).The absence ofactivity in the hexane and aqueousextracts might either be due to lack ofsolubility of the active principles(Parekh and Chanda, 2007 and Francoiset al., 2015) or insufficient quantities ofactive components in the crude extracts(Taylor et al., 2001) as shown in the

phytochemical screening. The antifungal drugs (mycotene and

ketocronazol) used as control showedmore potency at 0.1 mg/g than theextracts of the plants at the variousconcentrations. This may be due to thefact that the drugs contain specificpurified active components in definiteconcentration as against the crudebioactive components from the extractsof A. muricata leaf, root, bark and fruitused in this study. This substantiatesMithunPai et al. (2016) in which despitethe fact that the aqueous leaf extracts ofsoursop showed high fungicidal effectsagainst the oral pathogens tested thesewere less effective compared to thepositive controls used.

CONCLUSIONIn conclusion, the present study

demonstrates that Annona muricata hassome antifungal activity against humanpathogenic fungi. This result thusprovides the rational for the continueduse of the plant in traditional folkloremedicine. This review is hoped to be asource of enlightenment and motivationfor researchers to further perform in vivochemical investigations on the biologicalactivities of A. muricata to gain insightinto developing new agricultural andpharmaceutical agents. The use of thisplant parts is validated scientifically.



Table 1: The qualitative phytoconstituents of Annona muricata parts indifference solvents

Aq=Aqueous; Eth=Ethanol; Hex=N-Hexane; += presence; -=absence of compound

Plant part Leaf Fruit Bark Root

Extract / Phyto Aq Eth Hex Aq Eth Hex Aq Eth Hex Aq Eth Hex

Alkaloids + + + + + + + + + + + +

Flavonoid + + + + + + + + + - + +

Tannin + - - - - - + - - + - -

Cardiac glycoside + + + + + + + + + + + +

Phenol + + + + + + + + + - + +

Saponin + + + + + - + + - + + +

Trepenoid + + + + + + + + + - + +

Combine

Anthraquinone

- + + + + + + + + + + +

Phlobatanin + - - + - - + - - + - +

Steroid - - - + - - - - - + +

Fig.1a: Phytochemical composition of Annona muricata ethanolic extract.



Fig.1b: Phytochemical composition of Annona muricata N-hexane extract.

Fig.1c: Phytochemical composition of Annona muricata aqueous extract.



Fig. 2: The zone of inhibition induced by A. muricata extracts on Trichoderma spp

Fig. 3: The zone of inhibition induced by A. muricata extracts on C. albicans Ethanolextract of the leaf, root, bark, and fruit of Annona muricata plant parts on T.mentagrophytes shows comparison with control A at varying concentrations. Whilelevel of significance varies from ethanol > aqueous > N-hexane.



Fig. 4: The zone of inhibition induced by A. muricata extracts on T. mentagrophytes

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