Phytochemical Analysis, In Vitro Evaluation Of Antioxidant .
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 411-426http://www.ijcmas.comOriginal Research ArticlePhytochemical analysis, in vitro evaluation of antioxidant and antimicrobialactivity of methanolic leaf extract of Vernonia amygdalina (bitter leaf) againstStaphylococcus aureus and Pseudomonas aeruginosaMomoh Johnson1*, Odetunde Simeon Kolawole2 and Longe Adeteju Olufunmilayo11Department of Science Laboratory Technology (Biochemistry Unit), School of Pure andApplied Sciences, Lagos State Polytechnic, Ikorodu, Lagos, Nigeria2Department of Science Laboratory Technology (Microbiology Unit), School of Pure andApplied Sciences, Lagos State Polytechnic, Ikorodu, Lagos, Nigeria*Corresponding authorABSTRACTKeywordsAntimicrobialactivity,in osa,Staphylococcusaureus andVernoniaamygdalinaThe study was conducted to determine the phytochemical analysis, in vitroevaluation of antioxidant and antimicrobial activity of methanolic leaf extract ofVernonia amygdalina (bitter leaf) against Staphylococcus aureusandPseudomonas aeruginosa. The qualitative phytochemicals in the extract of V.Ainclude tannin, alkaloids, glycosides, anthraquinone, flavonoids, phlobatanin,saponin etc. The amount of total proanthocyanidins, flavonoids, alkaloids, and totalphenols present in the extract are 0.09 0.03 mg quercetin/g of dry plant material,1.1%, 9.3% and 163.24 mg PE / g DW respectively. The obtained values for Carotene and lycopene were 0.49 0.25 µg and 0.08 0.04 µg/g respectively. Inthe in vitro antioxidant assay, V. amygdalina was found to have DPPH (95.65g/mL) and FRAP (0.708) scavenging activity. Antibacterial activity of the extractby disk diffusion method was characterized by inhibition zones of 20 2.2 mm forS. aureus and 23 1.2 mm for P. aeruginosa. S. aureus was tested against 12standard antimicrobial agents. Of these, pefloxacin, gentamicin, ampiclox,roceptin, zinnacef, amoxicillin, septrin, erythromycin, augumentin and ampicilinwere resistance to the organism while ciprofloxacin and streptomycin weresensitive. P. aeruginosa was sensitive to pefloxacin, gentamicin, amoxicillin,ciprofloxacin, erythromycin, sparfloxacin and tarivid. Resistance to amplicox,roceptin, zinnacef, streptomycin, septrin, augumentin and ampicilin were alsoobserved with the same organism. The minimum inhibitory concentration (MIC)for P. aeruginosa was 30 mg/ml while S. aureus has a value of 70 mg/ml. V.amydalina exhibited a minimum bacteriocidal concentration (MBC) of 60 mg/mlfor P. aeruginosa and 140 mg/ml for S. aureus. The extract exhibited strongpotency against these microorganisms with P. aeruginosa being the mostsusceptible. The results of this study support the use of Vernonia amygdalina asherbal remedies in Nigeria.411
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426chemical compounds such as resins,alkaloids, glycoside, saponins, lactose andessential oils (Soraya, 2011). Many of thesephytochemicals have beneficial effects on along term human health and may be used toeffectively treat human disease (Lai andRoy, 2004).IntroductionStaphylococcus aureus is an importantpathogen of humans and animals and isimplicated in a wide variety of infections. Itis a pathogen of greater concern because ofits virulence (Chambers, 2005), its ability tocause a diverse array of life threateninginfections and its ability to adapt to differentenvironmental conditions (Lowy, 2003).Pseudomonas aeruginosa is a gramnegative, rod shaped, a sporogenous, andmonoflagellatedbacteriumthathasincredible nutritional versatility.Oxidative stress leads to enhancedgeneration of reactive oxygen species (ROS)and has been implicated in the etiology ofover one hundred human diseases includinginflammation, metabolic disorders, cellularaging, atherosclerosis, heart disease, stroke,diabetesmellitus,cancer,malaria,rheumatoid arthritis, HIV / AIDS,Alzheimer s disease, ulcerative colitis andParkinsons disease (Smith et al., 2000;Olukemi et al., 2005 ; Hyun et al., 2006 andAliyu et al.,2008). Antioxidants aresubstance or molecules that are capable ofneutralizing the harmful effects of the ROSthrough the andogenous enzymatic defensesystem. The antioxidant effect of plant ismainly due to phenolic components likeflavonoids, phenolic acids and phenolicditerpenes (Shahidi et al., 1992). Theantioxidantscapacityofphenoliccompounds is mainly due to their redoxproperties, which play an important role inabsorbing and neutralizing free radicals,quenching singlet and triplet oxygen anddecomposing peroxides (Osawa, 1994).Antibiotics are naturally occurring orsynthetic organic compounds which inhibitor destroy selective bacteria, generally atlow concentrations (Brooks et al., 2007). Inthe last two decades, antibiotic resistance isemerging as a serious problem worldwide(Walsh, 2000 and Cohen, 2002). This haslead to the exploration for alternative, safeand efficient antimicrobial compounds fromnatural resources like plants. The increasingresistance of bacteria and fungi to currentlymarketed antimicrobial agents is becoming aworld-wide medical problem (World HealthOrganization, 2004). In recent years manybacteria have developed antimicrobial drugresistance, these include but not limited toStaphylococcus aureus and most of theEnterobacteriaceae, such as Klebsiellapneumonia (WHO, 2004). Statistics indicatethat more than 70% of the bacteria causinginfections are resistant to at least one of thedrugs most commonly used to treat them(WHO, 2004).Vernonia amygdalina (V.A) is a shrub thatgrows predominantly in the tropical Africa.Leaves from this plant serve as foodvegetable and culinary herb in soup(Argheore et al., 1998). Vernoniaamygdalina commonly known as bitter leafis a shrub or small tree of 25 m tall,belonging to the family Asteraceae. It haspetiolate leaves of about 6 mm diameter andelliptic shape. The leaves are green with acharacteristic odour and a bitter taste. Thetaxonomic classification of VernoniaMedicinal plants have formed the basis ofhealth care throughout the world since theearliest days of humanity and have remainedrelevant in both developing and thedeveloped nations of the world for variouschemotherapeutic purposes. Plants haveability to synthesize a wide variety of412
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426amygdalina is as follows: Kingdom: plantae,Division: Angiosperms, Order : Asterales,Family: Asteraceae, Genius: Vernonia,Species: V. amygdalina, Botanical Name:Vernonia amygdalina. It is commonly calledBitter leaf in English language, Onugbuin Igbo language, it is called Etidot , inEfik, Ijaw and Ibibio, Ewuro in Yorubalanguage, Oriwo in Edo and Chusa-dokiin Hausa (Egedigwe, 2010). In many partsof Nigeria, the plant has been domesticated.Studies on the nutritional composition of thebitter leaf are numerous (Nimenibo-Uadia,2003). V. amygdalina has been found to berich in minerals, especially phosphorus,calcium, potassium, magnesium, zinc, ironand some vitamins like vitamin A, C and E.VA extracts have been shown to exhibitprofound ethnomedical and pharmacologicalproperties viz, anti-diabetic (Momoh et al.,2014), antimalarial (Abort and Raserika,2003), antihelminthic and antibioticproperties (Farombi, 2003). The presentstudy investigated the in vitro antioxidantand antibacterial activity of methanolic leafextract of Vernonia amygdalina againstStaphylococcus aureus and PseudomonasaeruginosaBiochemistry Laboratory, pulverised tocoarse power using blender. Extraction wascarried out by dispersing 200g of thegrounded Vernonia amygdalina plantmaterial in 1L of 80% methanol and shakingwas done with GFL shaker for 72 hours.This was followed by vacuum filtration andconcentrated by rotary evaporator at atemperature not exceeding 40oC. Theconcentrated extract was dried to completedryness in an aerated oven at 40oC for 48hours. The extract was latter stored in arefrigerator at 4oC.Qualitative phytochemical analysis ofVernonia amygdalinaPhytochemical analysis for phytochemicalconstituents were carried out on themethanolic extract of Vernonia amygdalinausing standard phytochemical procedures bySofowora (1993), Harborne (1973), Treaseand Evans (1989).Quantitative phytochemical analysis ofthe methanolic leaf extract of VernoniaamygdalinaDetermination of total proanthocyanidinscontent of the extractMaterials and MethodsCollection and identification of plantextractDetermination of proanthocyanidin wasbased on the procedure reported by Sun etal., (1998). A volume of 0.5 ml of 0.1 mg/mlof extract solution was mixed with 3 ml of4% vanillin-methanol solution and 1.5 mlhydrochloric acid; the mixture was allowedto stand for 15 min. The absorbance wasmeasured at 500 nm. Extract samples wereevaluated at a final concentration of 0.1mg/ml. Total proanthocyanidin content wereexpressed as catechin equivalents (mg/g)using the following equation based on thecalibration curve:y 0.5825x, R2 0.9277, where x was theabsorbance and y is the catechin equivalent(mg/g).The leaves of Vernonia amygdalina wereobtained from Ikorodu in Lagos State,Nigeria. The plant was authenticated by MrsShokefun a botanist from l biology Unit), Lagos StatePolytechnic, Ikorodu.Preparation of methanolic leaf extract ofVernonia amygdalinaThe leaves of Vernonia amygdalina werewashed, air dried under shade in the413
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426500 l of the F-C reagent. After shaking, themixture was incubated for 3 min at roomtemperature. Then 2000 l of 20 % Na2CO3solution was added. The volume obtainedwas mixed vigorously, and held for 60 minin the dark at ambient temperature. Theabsorbance of the solution was thenmeasured at 650 nm against a blank in aspectrophotometer. The sample wasanalysed in triplicate and the averagecontent was noted for each measurement.The total phenolic content, expressed as mgof pyrrocatechol equivalents (PE) per g ofdry weight of plant material (mg PE / gDW), was calculated through the calibrationcurve obtained using the equation givenbellow:Flavonoid content determinationOne hundred millilitres of 80% aqueousmethanol was used to repeatedly extract 1 gof the defatted sample at room temperature.The solution was then filtered throughWhatman filter paper. The filtrate wasevaporated to dryness in a crucible over awater bath and weighed to a constant weightusing the method of Oseni et al (2014).Alkaloid content determinationTo about 1 g of the defatted sample ofVernonia amygdalina, 80 ml of 10% aceticacid in ethanol was added. The beaker wascovered and then allowed to stand for 4hours. The suspension was then filtered andthe extract concentrated on a water bath toone-quarter of the original volume.Concentrated ammonium hydroxide wasadded drop wise to the extract until theprecipitation was completed. The precipitatewas collected and washed with diluteammonium hydroxide and then filtered toobtain the alkaloid residue. This was driedand weighed using method of Oseni et al(2014).Absorbance 0.0828 x C, R2 0.9993where C was the concentration in mg/l.Determinationof-Carotenelycopene content of the extractand-Carotene and lycopene were determinedby the method of Barros et al (2007). Thedried extract of Vernonia amygdalina (100mg) was vigorously shaken with acetonehexane mixture (4:6, 10 mL) for 5 min andfiltered through a disposable filter (0.45 m,Millipore). The absorbance of the filtratewas measured at 453, 505, and 663 nm.Contents of -carotene and lycopene werecalculated according to the followingequations:Total phenolic content of the extractThe quantitative determination of totalphenolic content using Folin-Ciocalteu (FC) reagent involves oxidation in alkalinesolution of phenols by the yellowmolybdotungstophosphoric heteropolyanionreagent and colorimetric measurement of theresultant molybdotungstophosphateblueaccording to the method of Singleton andRosi (1965) and modified by Dogÿan et al.,(2005). The polyphenol fraction (extractcorresponding to 1 g of dry plant material)was dissolved in 5 ml of double distilledwater. An aliquot of 100 l of this solutionwas diluted with double distilled water to 3ml. Afterward, the obtained solution wasadded to 300 l of double distilled water and-carotene (mg/100 mL) 0.216 (A663)0.304 (A505) 0.452 (A453);lycopene (mg/100 mL) 0.0458 (A663) 0.372 (A505) 0.0806 (A453).The assays were carried out in triplicates,the results were mean SD and expressed asg of carotenoid/g of the extract.414
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426In-vitro plant antioxidantVernonia amygdalinaassay(2.5 ml, 1% w/v ). After incubation at 50 Cfor 20 min, 2.5 ml of trichloroacetic acid(10 % w/v) was then added to the mixturefollowed by centrifuging at 3000 rpm for 10min. Consequently, 5 ml of the upper layerwas mixed with 2.5 ml of distilled water and0.5 ml of ferric chloride (0.1 % w/v). After30 min of incubation at room temperature inthe dark, absorbance of the resultingsolution was measured at 700 nm using aspectrophotometer. The ferric reducingpower capacities of the plant extracts andstandard antioxidants were expressedgraphically by plotting absorbance againstconcentration. Samples for the assay wereprepared in triplicateofDPPH Radical Scavenging Activity Assay.1,1-Diphenyl-2-picrylhydrazyl (DPPH) freeradical scavenging activity was measuredaccording to the method of Barros et al(2007) with slight modification on the basisof the method of Blois (1958). Differentconcentrations of ethanol dilutions ofsamples were mixed with 2.0 volume of 6.5 10 5M solution of DPPH. The resultingsolutions were thoroughly mixed andabsorbance was measured at 517 nm afterkeeping the tubes in dark for 30 minutes.The scavenging activity was determined bycomparing the absorbance with that ofcontrol containing equal volumes of DPPHsolution and ethanol. The radical scavengingactivity was obtained by the followingequation:Preparation of methanolicimpregnated paper discsextractThe methanolic extract impregnated paperdiscs were prepared as described byEkundayo and Ezeogu (2006). WhatmanNo. 1 filter paper was cut into discs of 6 mmdiameter using an office perforator. Thediscs were placed in glass Petri dish andsterilized in hot air oven at 160 C for 1hour. Each disc was impregnated with 20 lportion of stock solution of the methanolicextract of Vernonia amygdalina (100mg/ml). The discs were dried in an incubatorat 35-37 C for 2 hours.Radical scavening activity (%) Acontrol AsampleAcontrol 100The IC50 was defined as the concentration(in g/mL) of the extract required to depletethe amount of DPPH radical by 50%. Gallicacid (GA) and butylated hydroxytoluene(BHT) were used as positive controlFerric reducing antioxidant power assay(FRAP)Test organismsThe two bacterial strains used in thisinvestigationwereobtainedfromMicrobiology Department, University ofLagos, Nigeria. The organisms areStaphylococcus aureus, a Gram-positivebacteria and Pseudomonas aeruginosa aGram-negativebacteria.Themicroorganisms were maintained at 4 C onNutrient Agar slant in the Department ofScienceLaboratoryTechnology(Microbiology Unit), School of Pure andThe reducing power assay was conducted aspreviously described by Wang et al. (2008)and Oyaizu (1986) with ascorbic acid (AA)and tert-butyl-4-hydroxyanisole (BHA)being used as the positive controls. In brief,2.5 ml of individual deionized water dilutedVernonia amygdalina extract (ranged from0.1 to 1 mg/ml) was sequentially mixed withequal volume of phosphate buffer (2.5 ml,0.2 M, pH 6.6) and potassium ferricyanide415
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-426Applied Science, Lagos State Polytechnic,Ikorodu, Lagos, Nigeria and freshsubcultures were made before use.Antibiotic susceptibility testingSusceptibility of organisms to differentantibiotics were tested using disk diffusionmethod on freshly prepared Mueller Hintonagar and standardized by the method ofNational Committee for Clinical LaboratoryStandard using some selected antibioticsnamely: Amoxicillin (30ug), Ciprofloxacin(5ug), Gentamicin (10ug), Ampicillin(25ug), Streptomycin (25ug) etc. For eachcombination of the antibiotics and thebacterial strains, the experiment wasperformed in triplicate. The bacteria with aclear zone of inhibition of more than 17 mmwere considered to be sensitiveInoculum preparationA loopful of isolated colonies wasinoculated into 4 ml of peptone water,incubated at 37 C for 4 hours. This activelygrowing bacterial suspension was thenadjusted with peptone water so as to obtain aturbidity visually comparable to that of 0.5McFarland standard prepared by mixing 0.5ml of 1.75% (w/v) barium chloridedehydrate (BaCl2. 2H2O) with 99.5 ml of1% (v/v) tetraoxosulphate (vi) acid 0 colony forming units perml (CFU/ml).Minimum inhibitory concentration (MIC)MIC is defined as the lowest extractconcentration that inhibited the growth ofthe test organisms as indicated by absence ofvisible turbidity in the tube compared withthe control tubes. The minimum inhibitoryconcentration (MIC) of the extracts wasdetermined using the tube serial dilutionmethod. Extract (100 mg/ml) was dissolvedin water and diluted with Nutrient broth intwo fold serial dilutions in test tubes. Anovernight broth culture of the test organismwas adjusted to McFarland turbiditystandard and 50 l (0.05 ml) of the cellsuspension added to each of the tubes. Thetubes were incubated aerobically at 37 C for18 hours.Determination of antibacterial activity bydisk diffusion methodSensitivity of Staphylococcus aureus andPseudomonasaeruginosastrainstomethanolic leaf extracts of VernoniaAmygdalina were measured in terms of zoneof inhibition using disk diffusion method asdescribed byKirby-Bauer diffusiontechnique (Cheesbrough, 2002). Here thediscs were soaked in the leaf extract asdescribed above before use.The plates containing Muller Hinton weresmear with 0.1ml of the inoculums usingswab stick. This was followed by mountingof the impregnated paper disc. Theinoculated Staphylococcus aureusandPseudomonas aeruginosa isolates wereincubated at 37 C for 24 hours and thediameter of any resultant zone of inhibitionwas measured. For each combination ofextract and the bacterial strains, theexperiment was performed in triplicate. Thebacteria with a clear zone of inhibition ofmore than 17 mm were considered to he MBC of the methanolic leaf extract ofVernonia amygdalinawas prepared bymodification of the method of Spencer andSpencer (2004). Here 0.1ml, aliquots ofsamples taken from the non-turbid tubes ofthe MIC assay tube was sub-cultured ontoMueller Hinton agar plates. The resultingplates were then incubated aerobically at416
Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 411-42637 C for 24 hrs. The lowest concentration ofthe extract at which no colonies ofStaphylococcus aureus and Pseudomonasaeruginosa were seen was taken as theMBC. The results were compared with thatof control using sterilized distilled water.The experiment was performed in triplicate.The MBC was taken as the concentration ofthe extract that did not show any growth ona new set of agar plates.lower when compared to the standardcompounds used in this study.Data analysisFigure I below shows the DPPH radicalscavengingactivitiesofVernoniaamygdalina. Figure 2 below shows thereducing power activities of eand-Carotene and lycopene were found insmall amounts. The obtained values for Carotene and lycopene were 0.49 0.25 µgand 0.08 0.04 µg/g respectively.All analyses were carried out in triplicateand results expressed as mean SEM. Thedata analysis was done using the Graph Padprism computer software version 6.Student s -test and one-way analysis ofvariance (ANOVA) were used forcomparison. A P-value 0.05 wasconsidered significant. The IC50 values werecalc
The study was conducted to determine the phytochemical analysis, in vitro evaluation of antioxidant and antimicrobial activity of methanolic leaf extract of Vernonia amygdalina (bitter leaf) against Staphylococcus aureus and Pseudomonas aeruginosa. The qualitative phytochemicals in the extract of V.A include tannin, alkaloids, glycosides, anthraquinone, flavonoids, phlobatanin, saponin etc .
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Phytochemical analysis for major classes of metabolites is an important first step in pharmacological evaluation of plant extracts. Some journals require that pharmacological studies be accompanied by a comprehensive phytochemical analysis. Details of such analysis are found in several text books (Harborne, 1984; Evans, 2009). The main secondary metabolite classes include flavonoids .
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Phytochemical analysis and comparison of in-vitro 36 microorganism. 40µl of the extract was placed in the well with the use of pasture pipette. The plates were left on the bench for an hour at room temperature to allow diffusion of the extract into the agar. The plates were incubated at 37 oC for 24hrs for bacteria and 30 C for 48hrs for fungi.
Phytochemical analysis and in vitro anti-proliferative activity of Viscum album ethanolic extracts Carla Holandino1,2*, Michelle Nonato de Oliveira Melo1,3, Adriana Passos Oliveira1, João Vitor da Costa Batista1, Marcia Alves Marques Capella4, Rafael Garrett3, Mirio Grazi2, Hartmut Ramm2, Claudia Dalla Torre2, Gerhard Schaller2, Konrad Urech2, Ulrike Weissenstein2 and Stephan Baumgartner2,5,6 .
Phytochemical Analysis and In vitro Antioxidant Activity of Jojoba Oil Suhas Manoharan I BDS Student, Saveetha dental college and hospitals No.162 , PH road, Chennai – 600077 Vishnupriya.V Associate professor Department of Biochemistry Saveetha dental college and hospitals No.162 , PH road, Chennai – 600077 Gayathri.R Assistant professor Department of Biochemistry Saveetha dental college .
In vitro antioxidant activity and phytochemical analysis of ethanolic extract of Lentinus connatus L. Sushila Devi 1, Mayukh Chakraborty 2, Chandrima Chakraborty 3, S. K. Borthakur 1 and N. Irabanta Singh 4* 1Botany Department, Gauhati University, Guwahati 2Department of Biotechnology, St. Xavier’s College, Kolkata 3Department of Botany, University of Calcutta, Kolkata 4Centre of Advanced .
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