Phytochemical screening and antibacterial activities of Vernonia ...

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 1 pp. 67ñ73, 2011 ISSN 0001-6837Polish Pharmaceutical SocietyNATURAL DRUGSPHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITIESOF VERNONIA AMBIGUA, VERNONIA BLUMEOIDESAND VERNONIA OOCEPHALA (ASTERACEAE)ABUBAKAR B. ALIYU 1* , ALIYU M. MUSA 2 , MIKHAIL S. ABDULLAHI 3 , HAMISU IBRAHIM 1, 4and ADEBAYO O. OYEWALE 11Department of Chemistry, 2 Department of Pharmaceutical and Medicinal Chemistry,Ahmadu Bello University, Zaria-Nigeria3National Research Institute for Chemical Technology- Basawa, Zaria-Nigeria4School of Chemistry, Faculty of Science and Agriculture, Westville Campus, Private Bag X54001,University of Kwazulu Natal, Durban 4000, South AfricaAbstract: Some Vernonia species (Vernonia ambigua, Vernonia blumeoides and Vernonia oocephala) used inNorthern Nigerian traditional medicine, were subjected to phytochemical screening using standard procedures.The antibacterial activity using the disc diffusion method as outlined by the NCCLS was carried out onKlebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Corynbacterium ulcerans, methicillinresistant Staphylococcus aureus (MRSA), Salmonella typhi, Pseudomonas aeruginosa, Shigella dysentriae,Proteus mirabilis and Pseudomonas fluorescence. The results of the antibacterial activity as indicated by zoneof growth inhibition ranged from 14 to 27 mm for the crude ethanol extracts and chloroform fractions of theVernonia species being studied. The activity of chloroform fraction of V. blumeoides was higher on C. ulceransand K. pneumoniae (27 mm), while the chloroform fractions of V. oocephala and V. ambigua were more activeon P. mirabilis (27 mm) and S. typhi (22 mm), respectively. It is worth of mention that the chloroform fractionsof the three Vernonia species demonstrated activity (20 mm) against MRSA. The minimum inhibitory concentration(MIC) values ranged from 1.25ñ2.5 mg/mL for all the organisms tested. The MIC of 1.25 mg/mL exhibitedby the chloroform fractions on both Gram positive and negative bacteria indicates broad spectrum activityof the Vernonia species being studied. Phytochemical screening of the extracts/fractions revealed the presenceof steroids/terpenes, saponins, flavonoids, alkaloids, tannins and glycosides. The antibacterial activity exhibitedin this study may be attributed to flavonoids, saponinss or sesquiterpene lactones. The overall results indicatethat the extracts/fractions are potent antibacterial preparations at least in vitro. This lends credence to theuse of these plants for the treatment of various infectious diseases.Keywords: phytochemical, antibacterial activities, Vernonia ambigua, Vernonia blumeoides, Vernonia oocephala,AsteraceaeThe use of plant continues to play essentialroles in traditional medicine for the treatment ormanagement of various human diseases, especiallyin rural Africa where infectious diseases are endemicdue to poverty and poor sanitations. In Ghana,Mali, Nigeria and Zambia, the first line of treatmentfor 60% of the children with high fevers, is the useof herbal medicines at home (1). The importance ofplants in medicine remains even of greater relevancewith the current global shift to obtain drugs fromplant sources, as a result of which attention has beengiven to the medicinal value of herbal remedies forsafety, efficacy and economy (2, 3). Plants constitutean important source of active natural productswhich differ widely in terms of structure and therapeuticproperties. The continued investigation intothe secondary plant metabolites for anti-infectiveagents has gained importance in recent yearsbecause of the alarming increase in resistance ofpathogenic microorganisms to existing antibiotics.For instance, the incidence of methicillin resistantStaphylococcus aureus (MRSA) continue toincrease tremendously across the globe and posesenormous therapeutic problems. In Nigeria, Kenyaand Cameroon, the rate of MRSA spread was about21ñ30% in 2003 (4). However, prevalence rate of* Corresponding author: -mail: aliyubabando@gmail.com; phone: +2348057371917, +234809816067467

Phytochemical screening and antibacterial activities of Veronia ambigua... 69Test for free anthraquinones (Bontragerís test)Small portion of the extract was shaken with10 mL of benzene and filtered. Five milliliters of10% NH 3 solution was added to the filtrate andstirred. The production of a pink-red or violet colorindicates the presence of free anthraquinones.Test for saponins (Frothing test)Small quantity of the extract was dissolved in10 mL of distilled water. This was then shaken vigorouslyfor 30 s and was allowed to stand for 30min. A honey comb formed for more than 30 minindicates saponins.Test for steroids and triterpenes (Lieberman-Burchardís test)Equal volume of acetic anhydride was added tothe extract. One milliliter of conc. H 2 SO 4 was addeddownside the tube and the color change wasobserved immediately and later. Red, pink or purplecolor indicates the presence of triterpenes while blueor blue-green indicates steroids.Test for flavonoids (Shinoda test)About 0.5 g of the extract was dissolved in 1.5mL of 50% methanol and warmed on steam bath.Metallic magnesium and 5 drops of concentratedhydrochloric acid were added. A red or orange colorindicates the presence of flavonoids aglycone.Test for tannins (Ferric chloride test)About 0.5 mL of extract was dissolved in 10mL of distilled water and then filtered. Few drops offerric chloride solution were added to the filtrate.Formation of a blue-black precipitate indicateshydrolyzable tannins and green precipitate indicatesthe presence of condensed tannins.Test for alkaloids (Dragendoffís test)To about 0.5 g of each extract, 1% diluted HCl(20 mL) was added in a conical flask, heated on asteam bath and then filtered. The filtrate was madealkaline with 28% NH 3 solution and then extractedwith chloroform (3 ◊ 5 cm 3 ). The combined CHCl 3extracts were concentrated and treated with equalvolume of 1% HCl. Dragendorffís reagents (2 mL)were added and occurence of orange-red precipitateindicated the presence of alkaloids.Test organismsTen clinical bacterial strains: Klebsiella pneumoniae,Streptococcus pyogenes, Staphylococcusaureus, Corynbacterium ulcerans, methicillin resistantStaphylococcus aureus (MRSA), Salmonellatyphi, Pseudomonas aeruginosa, Shigella dysentriae,Proteus mirabilis and Pseudomonas fluorescencewere obtained from the Department ofMicrobiology, Ahmadu Bello University TeachingHospital (ABUTH) Shika. The isolates were purifiedon nutrient agar (OXOID) plates and characterizedusing standard microbiological and biochemicalprocedures (26, 27). The MRSA strains used inthis study were clinical isolates from urethral swab,seminal fluid, urine, high virginal swab, blood, skinand sputum of patients with symptoms of S. aureusassociateddiseases. The isolates were identified bystandard method (26). The disc diffusion methodoutlined by the NCCLS (28) was used with 1 µgoxacillin disk (Oxoid). The zones of inhibition weremeasured after incubation at 35 O C for 24 h. Isolateswith zones diameter = 10 mm were consideredmethicillin resistant. The organisms were maintainedon agar slope at 4 O C and subcultured for 24 hbefore use.Determination of antibacterial activityThe disc diffusion method was used (29).Stock solution (100 mg/mL) of each extract andfractions were prepared using the extractants. Discs(6 mm diameter) were prepared using Whatman filterpaper and sterilized by autoclaving. The blanksterile discs were placed on the inoculated MuellerHinton Agar (MHA) surface and impregnated with15 µL of stock solutions (1500 µg/dics). Antibioticdiscs of ampiclox (75 µg/dics) and streptomycin (30µg/dics) were used as positive control, whereas discsof the extracting solvents were used as negative control.The plates were incubated at 37 O C for 24 h. Alltests were performed in triplicate and the antibacterialactivities were expressed as the mean diameter ofinhibition zones (mm) produced by the plantextracts.Minimum inhibitory concentration (MIC)The minimum inhibitory concentration (MIC)was determined using micro-broth dilution methodsas outlined by NCCLS (30). Dilutions (1ñ9mg/mL) of concentrations of extract and fractionsthat exhibited sensitivity against the test organismswere prepared using test tubes containing 9 mL ofdouble strength broth. The test tubes were inoculatedwith the suspension of the standardized inocula.These were incubated at 37 O C for 24 h andobserved for growth. The minimum inhibitory concentrations(MICs) of the extracts/fractions foreach test organism were regarded as the lowestconcentration that inhibited visible growth of thetest organisms.

70 ABUBAKAR B. ALIYU et al.Table 1. Phytochemical screening of extracts.Plant constituentVernonia ambigua Vernonia blumeoides Vernonia oocephalaEE CF EE CF EE CFAlkaloids + + + + + +Flavonoids + + + + + +Saponins + ñ + ñ + ñTannins + - + ñ + ñSteroids/terpenes + + + + + +Anthraquinones ñ ñ ñ ñ ñ ñGlycosides + ñ + ñ + ñEE = ethanol extract, CF = chloroform fraction, + = positive, and ñ = negativeTable 2. Antibacterial susceptibility test.Zone of inhibition of growth (mm)V. ambigua V. blumeoides V. oocephalaTest organismsEE CF EE CF EE CFAmpiclox(75µg/dics)Streptomycin(30 µg/disc)S. aureus 18 20 22 24 18 20 20 27MRSA 14 20 17 20 20 20 NT NTS. pyogenes 16 18 16 18 16 24 0 22C. ulcerans 16 19 24 27 20 22 NT NTS. typhi 20 22 18 20 0 0 0 0S. dysenteriae 0 0 14 18 0 0 NT NTP. mirabilis 0 0 0 0 22 27 NT NTP. aeruginosa 18 20 0 0 0 0 26 0P. fluorescence 0 0 0 0 0 0 NT NTK. pneumoniae 16 19 22 27 20 25 0 20EE = ethanol extract, CF = chloroform fraction, MRSA = methicillin resistant Staphylococcus aureus, NT = not testedRESULTSPhytochemical screening revealed the presenceof alkaloids, flavonoids, saponins, tannins,steroids/terpenes and glycosides in the crudeextracts/fractions of the Vernonia samples.However, saponins, tannins and glycosides werefound absent in the chloroform fractions of all thesamples. Anthraquinones were absent in theextracts and fractions of all the three Vernoniaplants being investigated (Table 1). The results ofthe antibacterial activities showed that the plantextracts/fractions exhibit remarkable activityagainst the test organisms (C. ulcerans, K. pneumonia,Staphylococcus aureus, P. mirabilis, S. typhi, S.dysentriae, MRSA and P. aeruginosa) with zone ofgrowth inhibition ranging from 14 to 27 mm. Thesusceptibilities of P. aeruginosa, S. dysentriae andP. mirabilis were only found in each case to theextract/fraction of V. ambigua, V. blumeoides and V.oocephala, respectively. The MIC values rangedfrom 1.25ñ2.5 mg/mL for all the organisms tested(Table 3). P. fluorescence was, however, resistant tothe extracts/fractions of all the Vernonia samples(Table 2).DISCUSSION AND CONCLUSIONPhytochemical screening of plant products orextracts aimed to evaluate the antibacterial propertiesmay be of immense importance in the discoveryof new therapeutic agents, especially at this timewhen the scientific community is preoccupied withsearching for alternative treatment to combat the

Phytochemical screening and antibacterial activities of Veronia ambigua... 71Table 3. Determination of minimum inhibitory concentration (MIC).Minimum inhibitory concentration (mg/mL)Test organisms Vernonia ambigua Vernonia bluemoides Vernonia oocephalaEE CF EE CF EE CFS. aureus 2.5 1.25 1.25 1.25 2.5 1.25MRSA 2.5 1.25 2.5 1.25 1.25 1.25S. pyogenes 2.5 2.5 2.5 2.5 2.5 1.25C. ulcerans 2.5 2.5 1.25 1.25 1.25 1.25S. typhi 1.25 1.25 2.5 1.25 NT NTS. dysenteriae NT NT 2.5 2.5 NT NTP. mirabilis NT NT NT NT 1.25 1.25P. aeruginosa 1.25 1.25 NT NT NT NTK. pneumoniae 2.5 2.5 1.25 1.25 1.25 1.25EE = ethanol extract, CF = chloroform fraction, MRSA = methicillin resistant Staphylococcus aureusincreasing threat of drug resistant microorganisms.The result of antibacterial evaluation of the aerialpart of Vernonia ambigua, Vernonia blumeoides andVernonia oocephala showed that the chloroformfractions were more active than the crude ethanolextracts against the test organisms (Table 2).Difference in polarity of solvents is perhaps responsiblefor the difference in solubility of plant activeprinciples. It means that the antibacterial agent(s)have concentrated in the chloroform fractions hencevariation in degree of activity. The susceptibility ofC. ulcerans and K. pneumonia to chloroform fractionof V. blumeoides demonstrate higher (27 mm)antibacterial activity in terms of growth inhibition ofthe test organisms. It is interesting to note that theethanol extract and chloroform fraction of V.oocephala, as well as the chloroform fractions of V.ambigua and V. blumeoides, demonstrated goodantibacterial activity against the MRSA. The significanceof this outcome is the efficacy at which thefractions demonstrate the observed activity, whichmay be a milestone in the continued search anddevelopment of newer drugs or phytotherapeuticagents against the MRSA.The ethanol extracts and chloroform fractionsof the Vernonia species demonstrated activityagainst both Gram negative (P. aeruginosa, S. typhi,S. dysentriae and P. mirabilis) and Gram positivebacteria (S. aureus, C. ulcerans and S. pyogenes)(Table 2). The susceptibility of P. aeruginosa to onlythe extract and fraction of Vernonia ambigua may bea pointer to its potential as a drug against this organism.Infections caused by Pseudomonas speciessuch as mastitis are often difficult to combat (31).The fact that P. mirabilis was susceptible to only theextract/fraction of V. oocephala indicates the potencyof the plant against diseases caused by the organism(Table 2). Proteus mirabilis is a pathogenicGram-negative bacterium that frequently causes kidneyinfections, typically established by ascendingcolonization of the urinary tract (32, 33). The organismproduces a variety of unique virulence factorsthat contribute to its pathogenicity and persistence inthe human host (34).Low MIC indicates the minimum inhibitoryconcentration required to inhibits the growth of thetest organism, which translate to high potency of theextract or fraction. The MIC of 1.25 mg/mL exhibitedby the chloroform fractions on both Gram positiveand negative bacteria indicates broad spectrumactivity of the Vernonia species being studied. Theorder of potency was: V. oocephala > V. blumeoides> V. ambigua (Table 3). The results of our findingscompared or even surpassed the report of antibacterialactivity of bark extracts of V. tenoreana whichexhibits MICs of 10, 15 and 20 mg/mL for varioustest organisms (35). Similar report revealed the antibacterialpotency of active leaf extract of V. amygdalinawith MICs of 22.5ñ26.0 mg/mL and19.8ñ26.4 mg/mL for the Gram-negative and Grampositiveisolates tested (36). Owing to their popularuse as remedies for many infectious diseases, plantswith secondary metabolites such as alkaloids,saponins, terpenoids, flavonoids, sesquiterpene lactonesand steroids have been found to have antimicrobialproperties in vitro (37ñ39). Thus higher antibacterialactivity exhibited by the chloroform fractionsin this study may be attributed to flavonoids orsteroids/terpenes. These major chemical constituentshave been identified and characterized

72 ABUBAKAR B. ALIYU et al.from most Vernonia species (40ñ44). Although themechanisms of action of bioactive constituents ofthe Vernonia species being studied may be difficultto speculate; however, many antibacterial agentsmay exhibit their action through inhibition of nucleicacid, protein and membrane phospholipidsbiosynthesis (45). It is probable that the antibacterialagent(s) in the fractions of the Vernonia might actvia some of the above mechanisms.The overall results indicate that the extractsand fractions are potent antibacterial preparations atleast in vitro. However, in vivo evaluation may berequired to ascertain that active concentrations ofthe extract when absorbed may remain bioactive forthe time to completely kill the pathogens. Furtherphytochemical and pharmacological studies arechallenging task in order to better understand theeffects of these important pharmaceutical resources.Plants of the Vernonia genus (Asteraceae) mayprove to be a rich source of compounds with potentialantimicrobial activities. Bioactivity guided isolation,purification, characterization and structuralelucidation of the active constituents from theVernonia species is on-going in our laboratory.AcknowledgmentThe authors acknowledged the contribution ofthe authority of Ahmadu Bello University, Zaria, forproviding the facilities for conducting this research.REFERENCES1. World Health Organization (2003) TraditionalMedicine. http://www.who.int/mediacentre/factsheets/fs134/en/print.html2. Glombitza K.W., Mahran G.H., Mirhom Y.W.,Michael K.G. Motawi T.K.: Planta Med. 60,244 (1993).3. Mahabir D., Gulliford M.C.: Rev. Panam. SaludPublica. 1, 1 (1997).4. Kesah C., Ben-Redjeb S., Odugbemi T.O.,Boye C.S.B., Dosso M., Ndinya-Achola J.O.,Koulla-Shiro S. et al.: Clin. Microb. Infect. 9,153 (2003).5. Onanuga A., Oyi A.R., Onaolapo J.A.: Afr. J.Biotechnol. 4, 1321 (2005).6. Mirza S.H., Beeching N.J., Hart C.A.: J. Med.Microbiol. 44, 317 (1996).7. Mulvey M.R., Bryce E., Boyd D., Ofner-Agostini M., Christianson S., Simor A.E., PatonS.: Antimicrob. Agents Chemother. 48, 1204(2004).8. Rhomberg P.R., Fritsche T.R., Sader H.S.,Jones R.N.: Microbiol. Infect. Dis. 56, 57(2006).9. Hoban D.J.: Antimicrob. Agents Chemother.52, 1430 (2008).10. Wilson C.O., Gisvold O., Doerge R.F.: Textbookof Organic Medicinal and PharmaceuticalChemistry. 6 th edn., pp. 34ñ37, Lippincot Co.,Philadelphia 1971.11. Nwaogu L.A., Alis C.S., Ibegbulem C.O., IgweC.U.: Afr. J. Biotechnol. 6, 890 (1971).12. Akah P.A.; Okafor C.L.: Phytother. Res. 6, 171(1992).13. Akah P.A., Ekekwe R.K.: Fitoterapia 66, 352(1995).14. Akinpelu D.A.: Fitoterapia 70, 432 (1999).15. Iwalokun B.A.: Afr. Health Sci. 8, 25 (2008).16. Rasoanaivo P., Petitjean A., Ratsinanmanga-Urvers S., Rakoto-Ratsimamanga A.: J.Ethnopharmacol. 37, 117 (1993).17. Olorode O.: Taxanomy of West African FloweringPlants, 1 st edn., pp. 98-100, Longman,London 1984.18. Krishna Kumari G.N., Masilamani S., GaneshM.R., Aravind S., Sridhar S.R., Zaluzanin D.:Fitoterapia 74, 479 (2003).19. Kuo Y.H., Kuo Y.J., Yu A.S., Wu M.D., OngC.W., Yang Kuo L.M. et al.: Chem. Pharm.Bull. 51, 425 (2003).20. Koul J.L., Koul S., Singh C., Taneja S.C.,Shanmugavel M., Kampasi H. et al.: PlantaMed. 69, 164 (2003).21. Huang Y., Ding Z.H., Liu J.K.: Z. Naturforsch.[C] 58, 3470 (2003).22. Tchinda A.T., Tane P., Ayafor J.F., ConnollyJ.D.: Phytochemistry 63, 841 (2003).23. Nergard C.S., Diallo D., Michaelsen T.E.,Malterud K.E., Kiyohara H., Matsumoto T. etal.: J. Ethnopharmacol. 9, 141 (2004).24. Sofowora A.: Medicinal Plants and TraditionalMedicine in Africa. p. 289, Spectrum BooksLtd., Ibadan 1993.25. Evans W.C.: Trease and Evans Pharmacognosy.15 th edn., pp. 191ñ193, 513ñ540, W.B. Saunders,Sydney/Toronto 2002.26. Cowan S.T., Steel K.F.: Manual forIdentification of Medical Bacteria. 2 nd edn., pp.97ñ115, Cambridge University Press, London1974.27. McFaddin J.F.: Biochemical Tests forIdentification of Medical Bacteria. pp.392ñ452, Williams and Wilkins Co., Baltimore1977.28. NCCLS: Performance Standards AntimicrobialDisc Susceptibility Tests. Approved Standard,

Phytochemical screening and antibacterial activities of Veronia ambigua... 73Fifth edn., NCCLS Document M2-A5,Villanova, PA, USA 1993.29. Nostro A., Germano M.P., DíAngelo V.,Marino A., Cannattelli M.A.: Lett. Appl.Microbiol. 30, 379 (2000).30. NCCLS: Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria That GrowAerobically; Approved Standard Fifth edn.,NCCLS Document M7-A5, NCCLS, Wayne,PA, USA 2000.31. Salie F., Eagles P.F.K., Leng H.J.M.: J.Ethnopharmacol. 52, 27 (1996).32. Rubin, R. H., Tolkoff-Rubin, N. E., CotranR.S.: In The Kidney (Brenner, B.M., Rector,F.C. Eds. , pp. 1085ñ1141, W. B. Saunders Co.,Philadelphia 1986.33. Rauprich O., Matsushita M., Weijer C.J.,Siegert F., Esipov S.E., Shapiro J.A.: J.Bacteriol. 178, 6525 (1996).34. Coker C., Poore C.A., Xin L., Mobley H.L.T.:Microbes Infect. 2, 1497 (2000).35. Ogundare A.O., Adetuyi F.C., AkinyosoyeF.A.: Afr. J. Biotechnol. 5, 1663 (2006).36. Iwalokun B.A., Bamiro S.B., Durojaiye O.O.:West Afr. J. Pharmacol. Drug Res. 19, 9 (2003).37. Cowan M.M.: Clin. Microbiol. Rev. 12, 564(2002).38. Sibanda T., Okoh A.I.: Afr. J. Biotechnol. 6,2886 (2007).39. Cartagena E., Montanaro S., BardÛn A.: Rev.Latinoamer. QuÌm. 36, 43 (2008).40. Oligashi H., Jisaka M., Takagaki, T., Tada, T.,Huffman, MA, Nishida T. et al.: Agric. Biol.Chem. 55, 1201 (1991).41. Jisaka, M., Oligashi, H., Tagekawa, K.,Huffman, MA, Koshimizu, K.: Biosci. Biotech.Biochem. 57, 833 (1993).42. Igede, G.O., Oleszek, W., Jurzysta, M., Burda,S., Fanfunso, M., Fasanmade, A.A.: J. Agric.Food Chem. 42, 2445-2448 (1994).43. Geraldo de Carvalho M., Miranda da Costa P.,Abreub H.: J. Braz. Chem. Soc. 10, 163 (1999).44. Kuo Y., Kuo Y., Yu A., Wu M., Ong C., Yangkuo L., Huang J., Chen C., Li S.: Chem. Pharm.Bull. 51, 425 (2003).45. Fraklin T.J., Snow G.A., Barrett-Bee K.J.,Nolan R.D.: Biochemistry of antimicrobialaction. Fourth edn., pp. 71-73, 112, Chapmanand Hall, London 1987.Received: 4. 08. 2009

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