Research Article Moringa oleifera Flower Pod Extract as ...downloads.hindawi.com/journals/bmri/2016/7584318.pdf · Moringa has more iron than spinach, more calcium than ... Estimation

Research ArticleUse of Moringa oleifera Flower Pod Extract asNatural Preservative and Development of SCAR Marker forIts DNA Based Identification

Iram Gull1 Attia Javed1 Muhammad Shahbaz Aslam1

Roohi Mushtaq2 and Muhammad Amin Athar2

1 Institute of Biochemistry and Biotechnology University of the Punjab Quaid-i-Azam Campus Lahore 54590 Pakistan2Department of Biochemistry and Biotechnology University of Sargodha Lahore Campus Lahore 53800 Pakistan

Correspondence should be addressed to Iram Gull iramgull86yahoocom

Received 24 February 2016 Revised 13 May 2016 Accepted 22 May 2016

Academic Editor Patricia Morales

Copyright copy 2016 Iram Gull et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The use ofMoringa oleifera as natural food preservative has been evaluated in the present study In addition for quality assurancethe study has also been focused on the shelf life of product to authenticate the identification of plant by development of DNA basedmarker Among the different extracts prepared from flower pods ofMoringa oleifera methanol and aqueous extract exhibited highantibacterial and antioxidant activity respectively The high phenolic contents (535 plusmn 0169mgGAEg) and flavonoid contents(109plusmn0094mgQEg) were also recorded inmethanol and aqueous extract respectively Due to instability of bioactive compoundsin aqueous extract methanol extract is considered as potent natural preservativeThe shelf life of methanol extract was observed fortwo months at 4∘C under dark conditionsThe developed SCAR primers (MOF

217317

MOR317

) specifically amplified a fragment of317 bp fromDNA ofMoringa oleifera samples collected from different regions of Punjab province of PakistanThemethanol extractofMoringa oleifera flower pods has great potential to be used as natural preservative and nutraceutical in food industry

1 Introduction

Foodborne diseases are global issue with significant impacton human health [1] According to theWorld Health Organi-zation report (2015) sim1 in 10 people in the world fall ill afterconsuming contaminated food annually [2] Food protectionfrommicrobial and chemical deterioration is essential in foodindustry Food borne pathogens such as E coli S aureus Styphi Shigella and P aeruginosa not only cause foodborneillnesses but also spoil food products [3] Generally chemi-cally synthesized preservatives including butylated hydroxylanisole (BHA) butylated hydroxyl toluene (BHT) tertiarybutylated hydroquinone (TBHQ) and propyl gallate (PG)are used in food industry to avoid microbial and oxidativedeterioration of foodproducts [4]However serious concernshave been raised against the use of synthetic preservatives dueto the health risk issue and toxicity [5]

With increase in consumer consciousness about safety offood additives there is a growing need to search safer alterna-tives [6] Plants are the richest biosource ofnatural compoundshaving antioxidant [7] antimicrobial [8] and antiviral activ-ity [9] Plant extracts are safe natural substitute to chemicalfood additives to avoidmicrobial and oxidative food spoilageBefore large scale production and use of plant extracts it isimportant to consider the stability of plant extracts over theperiod of time under different storage conditions [10]

Additionally authentic identification of therapeuticallyimportant plants to prevent advertent or inadvertent adulter-ation by low quality plants is a serious issue [11] Substitutionor adulteration of a medicinal plant with low quality inherenttoxic herb by fraudulent actions can negatively affect the qual-ity ofmedicinal plants [12] Generally for standardization andquality control macroscopic microscopic (morphology andhistology) and chemical profiling (HPLC TLC and GC) of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7584318 12 pageshttpdxdoiorg10115520167584318

2 BioMed Research International

medicinal plants are in practice for their authentication [13ndash15] Microscopic and macroscopic studies need to be con-ducted by expert personnel Further the chemical composi-tion of plants is affected by change of growth stage environ-mental factors storage conditions and harvesting processThe molecular authentication procedures are considered asmore precise specific and suitable for medicinal plant iden-tification in comparison to above discussed methods becauseDNAbased techniques are independent of growth stage envi-ronmental factors andphysical formof plantmaterial [16 17]

Moringa oleifera is a plant of familyMoringaceae Variousparts of the plant are used as nutritious food commodityin many countries that is Pakistan India Philippines andHawaii and in some parts of Africa [18] In underdevelopedcountries (Senegal and Haiti) powder of leaves of Moringaoleifera has also been used to treat malnutrition in childrenpregnant women and nursing mothers [19] The leaves ofMoringa has more iron than spinach more calcium thanmilk more potassium than banana andmore vitamin C thanoranges and the protein quality rivals the egg andmilk protein[20] For a long time it has also been used as traditionalmedi-cine for treatment of many diseases [21ndash23]

Therefore the objectives of the present study are (i)to evaluate the antimicrobial and antioxidant activity ofMoringa oleiferaflower pods and (ii) to develop SCARmarkerusing leaves as plant material for authentic identification ofthis medicinal plant

2 Materials and Methods

21 Collection of Plant Material and Preparation of CrudeExtracts Fresh flower pods of Moringa oleifera were pur-chased from the local market of Lahore Pakistan in themonth of March and April 2014 and authenticated mor-phologically from Department of Botany University of thePunjab Lahore Pakistan The flower pods (500 g) were com-pletely air-dried and pulverized mechanically into powderusing electric grinder The powder (5 g) was extracted bysoaking in 50mL of each solvent (methanol ethanol waterchloroform and acetone) separately in screw capped 250mLErlenmeyer flasks for 24 hours with shaking at 160 rpm inorbital shaker at ambient temperature The extracts werefiltered through eight layers of muslin cloth and then cen-trifuged at 8000timesg for 15 minutes The clear supernatant wascollected and dried at 45∘C in rotary evaporator (Laborota4000-efficient Heidolph) All the dried residues were dis-solved in Dimethylsulfoxide (DMSO) except aqueous extractresidues which were dissolved in water to known concentra-tions The extracts were stored at 4∘C till further use

22 Bacterial Strains Six bacterial strains (E coli S typhiShigella S aureus and B subtilis) isolated from different foodsamples after biochemical characterizations [24] were used inthe present study

23 Antibacterial Activity Assay The antibacterial activityassay was performed by disc diffusion method [25] Sterilenutrient agar plates were seeded with 107 CFUmL of each

bacterial strain under aseptic conditions The sterile discsof Whatman Number 1 filter paper (5mm) were dipped inextracts for 5 minutes and placed on the surface of seededplates and pressed gentlyTheplateswere incubated overnightat 37∘C The disc soaked in DMSO was used as negativecontrolThe test was performed in triplicate for each bacterialstrain The antibacterial activity was recorded as mean ofdiameter of zone of inhibition (mm)

24 Minimum Inhibitory Concentration (MIC) The mini-mum inhibitory concentration (MIC) was determined asdescribed by Bag and Chattopadhyay [26] MIC assay wasperformed in 96-well microtitre plate Each well containing90 120583L of nutrient brothwas inoculatedwith 10120583L of each bac-terial culture (5 times 105 CFUmL) The extracts (100 120583L) withconcentrations ranging from900mgmL to 625mgmLwereadded in each well separately After overnight incubationof plate at 37∘C 40 120583L of 04mgmL p-iodonitrotetrazoliumviolet (INT) was added in each well and incubated again for 6hours After incubation the plates were observed for changein color from yellow to red to purple for viable bacteria Thelowest concentration of plant extract with no color changewas recorded as MIC The experiment was performed intriplicate

25 Time-Kill Kinetic Analysis The bacterial killing rate wasdetermined by time-kill kinetic assay at 1x MIC as describedin our earlier study [27] The cultures collected at differenttime (0ndash24hours)were seriallydilutedandused to determinedviable cell count The results were recorded as logarithm ofCFU against incubation time

26 Storage Stability Assay The stability of Moringa oleiferaflower pod extracts was evaluated over the period of timewith and without their exposure to light while being stored atdifferent temperatures (minus20∘C 4∘C and room temperature)At the end of each storage time the stability of antimicrobialcompounds in crude extracts was determined by antimicro-bial assay as described previously

27 Antioxidant Assay

271 Ferric Reducing Power Assay (FRPA) Ferric reducingpower assay was performed as described by Mohamed et al[28] Briefly 100 120583L of each extract was mixed with 25mL of200mMphosphate buffer pH 66 and 25mL of 1 potassiumferricyanide [K

3Fe(CN)

6] After incubation at 50∘C for 20

minutes 25mL of 10 trichloroacetic acid was added tomixture and centrifuged at 10000 rpm for 10 minutes Theupper layer of solution (5mL) was mixed with 5mL of dis-tilled water and 1mL of 01 ferric chloride The absorbancewas measured at 700 nm Ascorbic acid was used as standardand results were expressed by plotting absorbance againstconcentration

28 Phytochemical Studies The extracts of Moringa oleiferawere screened for the presences of major phytochemicalssuch as phenolics flavonoids alkaloids terpenoids tannins

BioMed Research International 3

steroids glycosides and anthraquinone using standard pro-cedures as described in Trease and Evans [29] and Sofowora[30]

281 Phenolic Compounds To 1mL of extract add 4 dropsof ethanol and 3 drops of 01 FeCl

3 The appearance of red

color indicated the presence of phenolic compounds

282 Anthraquinone To 2mL of plant extract 1mL ofdiluted 10 ammonia was added The pink red colorationin ammoniacal (lower) layer showed the presence of anthra-quinone

283 Flavonoids 1mL of extract was diluted with water to5mLThen 2mL of 10 NaOH was added to develop yellowcolor The change from yellow color to colorless solution bythe addition of diluted HCL indicated the presence of flavon-oids

284 Alkaloids 1mL of extract was diluted with 1 HCL upto 5mL To the 1mL of the diluted extract few drops of Dra-gendorff rsquos reagent were added The orange red precipitatesindicated the presence of alkaloids

285 Terpenoids To 1mL of extract 2mL of chloroformwasadded Then 2mL of H

2SO4was added The reddish brown

color at interface confirmed the presence of terpenoids inextract

286 Tannins Few drops of 1 FeCl3were added to the

1mL of extract The presence of brownish green precipitatesindicated the presence of tannins

287 Glycoside To 1mL of extract 2mL of glacial acetic acidand a drop of 5 FeCl

3were added The development of

brown ring indicated the presence of glycoside

288 Steroids To 1mL of extract few drops of concentratedH2SO4were addedThe appearance of red color indicated the

presence of steroids

29 Estimation of Total Phenolic Contents Total phenoliccontents were estimated in extracts using Folin Ciocalteureagent based assay as described by Barku et al [31] withslightmodifications Briefly 150120583L of extract wasmixed thor-oughly with 750120583L of Folin Ciocalteu reagent (diluted 10times with distilled water) and then added 600120583L of 75 gLNa2CO3 The mixture was placed at room temperature for 30

minutes for color developmentThe absorbance of developedblue color was recorded at 765 nm Gallic acid was used asstandard for calibration curve (Figure 1(a)) The total pheno-lic contents were expressed as milligram (mg) of gallic acidequivalent (GAE)gram (g) of dry weight

210 Estimation of Total Flavonoids Total flavonoid contentswere estimated as described by Manian et al [32] Briefly150 120583L of each plant extract was diluted up to 1mL with

minus005

0

005

01

015

02

025

03

0 20 40 60 80 100 120Concentration (120583gmL)

Standard curve for gallic acid

Abso

rban

ce at

765

nm

y = 00027x minus 00014

R2 = 1

(a)

minus0010

001002003004005006007

0 50 100 150 200 250

Standard curve for Quercetin

Concentration (120583gmL)Ab

sorb

ance

at510

nm

y = 00003x minus 00003

R2 = 09995

(b)

Figure 1 (a) Standard curve of gallic acid (b) standard curve ofQuercetin

distilled water followed by addition of 45120583L of 5 sodiumnitrite After incubation for 5 minutes at room temperature65 120583L of 10 aluminium chloride was added and allowedto stand for 6 minutes at room temperature After that300 120583L of 1N NaOH was added and absorbance was taken at510 nm Quercetin was used as standard for calibration curve(Figure 1(b)) Total flavonoid contentswere expressed asmggof Quercetin equivalent

211 Statistical Analysis Each experiment was performed intriplicate The mean standard error and one way ANOVAwere performed by SPSS version 210 software The pairwisesignificant difference between the means of treatment levelswas assessed by Duncanrsquos multiple range test at 119901 lt 005

212 SCAR Marker Development Leaf samples of Moringaoleifera were collected from different localities of PunjabPakistan Leaves were washed and stored at minus80∘C DNA wasisolated using method described by J J Doyle and J L Doyle[33] Random Amplification of Polymorphic DNA (RAPD)PCR was performed to develop DNA finger prints using17 random decamers (Table 1) The PCR reaction mixture(25 120583L) included 05120583L ofMoringa oleiferaDNA (100 ng120583L)25 120583L of 10 120583M random decamer 05 120583L of 10mM dNTPs2 120583L of MgCl

2 25 120583L of Taq buffer and 025120583L of Taq DNA

polymerase (5 u120583L) and volume was made up to 25 120583L withsterilized water The amplification reactions were performedby initial denaturation at 95∘C for 1 minute followed by 30cycles of denaturation at 95∘C for 1 minute annealing at 40∘C


Table 1 Random decamers used for RAPD fingerprints ofMoringaoleifera

OPA-01 51015840-CAG GCC CTT C-31015840

OPA-02 51015840-TGC CGA GCT G-31015840

OPA-03 51015840-AGT CAG CCA C-31015840

OPA-04 51015840-AAT CGG GCT G-31015840

OPA-05 51015840-AGG GGT CTT G-31015840

OPA-06 51015840-GGT CCC TGA C-31015840

OPA-07 51015840-GAA ACG GGTG-31015840

OPA-08 51015840-GTG ACG TAG G-31015840

OPA-09 51015840-GGG TAA CGC C-31015840

OPA-10 51015840-GTG ATC GCA G-31015840

OPA-11 51015840-CAA TCG CCG T-31015840

OPA-12 51015840-TCG GCG ATA G-31015840

OPA-13 51015840-CAG CAC CCA C-31015840

OPA-14 51015840-TCT GTG CTG G-31015840

OPA-15 51015840-TTC CGA ACC C-31015840

OPA-16 51015840-AGC CAG CGA A-31015840

OPA-17 51015840-GAC CGC TTG T-31015840

for 45 seconds and extension at 72∘C for 1 minute RAPD-PCR patterns were analyzed on 1 agarose gel electrophore-sis The monomorphic band in all samples was excised fromgel purified ligated into pTZ57RT vector and finally usedfor transformation of E coli DH5120572 competent cells Recom-binant clones were selected by blue white screening [34] Theplasmid was isolated from white colonies and subjected todigestion with xbaI The linearized plasmid size was equal tothe size of vector pTZ57RT and the size of the insert whichconfirmed successful cloning of DNA fragment of interestThe cloned DNA fragment was sequenced commerciallyThenucleotide sequence of DNA fragment was used to designedSCAR primer pair (MOF

271371and MOR

271 MOR

371) The

amplicon usingMOF271371

andMOR371

was used as templatefor PCR amplification using MOF

271371and MOR

271SCAR

primersThe SCAR primer pair was used to amplifyMoringaoleiferaDNA isolated fromdifferent localities of Punjab usingsamePCRconditions except 25120583Lof each 10 uMMOF

271371

MOR271

and MOR371

SCAR primer instead of randomdecamer in reaction mixture and annealing at 66∘C Theamplification was analyzed by 1 agarose gel electrophoresis

3 Results and Discussion

In food industry plant antimicrobial compounds have greatpotential to be used as biopreservative In addition theantioxidant activity of plant extracts prevents the oxidativedegradation of food stuff Plants are the natural source of anti-microbial and antioxidant compounds Use of plant extractshaving both activities improves food quality and alsoincreases the acceptability of plant extracts as a replacementto the synthetic preservatives [35]

31 Antibacterial Activity The extracts of flower pods ofMoringa oleifera prepared in solvents of different polarity

were examined for antibacterial activity against food bornepathogens (Bacillus subtilisE coli S aureus Salmonella typhiand Shigella) using disc diffusion method The extractsshowed broad spectrum of activity against these pathogensThe results are shown in Table 2 All the extracts showed anti-microbial activity against the used bacterial strainsThe high-est antibacterial activity against all used strains was recordedwith methanol extract while the lowest was observed withaqueous extractThe descending order of antimicrobial activ-ity of different extracts against tested bacterial strains wasas follows methanol extract gt acetone extract gt chloroformextract gt ethanol extract gt aqueous extract The descendingorder of sensitivity of bacterial strains for a different extractswas as follows methanol extract Shigella gt E coli gt B subti-lis gt S typhi ethanol extract S typhi gt B subtilis gt E coli gtShigella chloroform extract S typhi gt E coli gt Shigella gtB subtilis aqueous extract E coli gt B subtilis gt Shigellaand S typhi The negligible antibacterial activity of aqueousextract of Moringa is in agreement with the results of thestudy reported by [36] It is also reported in many studiesthat different parts of Moringa oleifera have been used toinvestigate their antimicrobial activity However sensitivityof pathogens to Moringa extracts prepared in polar tononpolar solvents is variable Some pathogens are sensitiveto ethyl acetateacetone extracts prepared from root bark[37 38] whereas other pathogens showed high sensitivity tomethanol extract prepared from stem bark [39] Chloroformextract from seeds and ethanol extract of Moringa leaveshas also been reported as effective against pathogens [40]In our study methanol extract of Moringa oleifera flowerpods is observed as most effective against all tested foodborne pathogens It is concluded that difference in sensitivityof extracts prepared fromdifferent parts ofMoringa is directlyrelated to the concentration of antibacterial compounds indifferent parts of the same plant Furthermore the growthstage of plant environmentalgeographical factors storageconditions and difference in procedures of extract prepara-tion also influence the chemical profile of plant and the con-centration of antimicrobial compounds in extracts of samepart of same plant

32 Minimum Inhibitory Concentration (MIC) The resultsare presented in Table 3 The lowest MIC value was recordedwith methanol extract against all investigated food patho-gens The lowest MIC value for S typhi was 125mgmLfollowed by 175mgmL for E coli and 25mgmL forB subtilisand ShigellaThe highMIC values were observed for aqueousextract (916mgmL) The MIC values clearly indicated thatamong different extract of flower pods of Moringa oleiferamethanol extract renders promising antibacterial activityagainst number of food borne and food spoiling bacteriaIn the study of Bukar et al [40] Shigella was resistant toethanol and chloroform extract prepared from leaves andseeds of Moringa oleifera while E coli was found to besensitive to ethanol extract at concentration of 200mgmLand 500mgmL respectively Despite following the same pro-cedure to prepare the extracts the variation in sensitivity offood pathogens can be noticed against extracts prepared fromdifferent parts ofMoringa oleifera It leads to a conclusion that


Table 2 Antibacterial activity of flower pods extractsMoringa oleifera

Zone of inhibition in (mm)Methanol extract(220mgmL)

Acetone extract(200mgmL)

Chloroform extract(430mgmL)

Ethanol extract(959mgmL)

Aqueous extract(916mgmL)

Shigella 193 plusmn 0981a 14 plusmn 1247b 126 plusmn 0544bc 11 plusmn 0471bc 10 plusmn 0471c

E coli 183 plusmn 1186a 153 plusmn 0720b 133 plusmn 1186bc 116 plusmn 0981bc 103 plusmn 054c

B subtilis 17 plusmn 0471a 13 plusmn 0942b 13 plusmn 0942b 106 plusmn 0720b 103 plusmn 0720b

S typhi 17 plusmn 0942a 14 plusmn 0720ab 14 plusmn 1247ab 13 plusmn 0942bc 10 plusmn 0471c

Values are mean of three independent experiments Mean values followed by the different letters in the same row differ significantly (119901 lt 005) according toDuncanrsquos multiple range test

Table 3 Minimum inhibitory concentration (MIC) of flower pods extracts ofMoringa oleifera

Minimum inhibitory concentration (mgmL)Methanol extract Acetone extract Chloroform extract Ethanol extract Aqueous extract

Shigella 25 plusmn 000 275 plusmn 000 531 plusmn 000 119 plusmn 000 916 plusmn 000E coli 175 plusmn 000 25 plusmn 000 531 plusmn 000 119 plusmn 000 916 plusmn 000B subtilis 25 plusmn 000 275 plusmn 000 268 plusmn 000 119 plusmn 000 916 plusmn 000S typhi 125 plusmn 000 175 plusmn 000 107 plusmn 000 119 plusmn 000 916 plusmn 000Values are mean of three independent experiments

sensitivity of food pathogens varies from extract to extractprepared from different parts of the same plant

33 Time-Kill Kinetic Analysis Time-kill kinetic assay wasperformed with methanol extract to determine the timeduration during which the bacteria under investigation arekilled The results are shown in Figure 2 It was observed thatthe viable count of bacteria was significantly decreased aftereach specified interval of time After 2 hours 50 decrease inlog CFU was recorded for E coli 34 for B subtilis and 44for S typhi and Shigella After 12 hours decrease in logCFUwas achieved up to 99 for E coli 75 for B subtilis and95 for S typhi and Shigella after 12 hours No viable countwas recorded after 24 hours It was depicted from results thatantimicrobial effect of extracts is increasing proportionatelyto time duration against the pathogen under investiga-tion The significant reduction (3 log

10

CFU) was observedbetween 6 and 8 hours of bacterial exposure to extract

34 Phytochemical Analysis The plant extracts with knownantimicrobial activity are of great importance in food preser-vation The bioactive chemical substances (polyphenolsflavonoids alkaloids terpenoids tannins and others) aremainly responsible for rendering a definite action on micro-bial and chemical quality of food [41] The phytochemicalsanalysis of different extracts of Moringa oleifera was alsoconducted (Table 4)The preliminary phytochemical analysisshowed that flavonoids phenol tannins and glycosides werepresent in all extracts which indicated that these are themajor secondary metabolites in flower pods of Moringaoleifera However quantity of compounds is commensuratewith the polarity of solvent and the part of plant under study[42] The variation in composition of secondary metabo-lites in different parts (leaves seeds and bark) of Moringaoleifera has also been reported [43ndash46] In present study

002040608

112141618

2

0 2 4 6 8 12 24Incubation time (hours)

E coliBacillus subtilis

ShigellaSalmonella typhi

log

CFU(c

olon

y fo

rmin

g un

it)

Figure 2 Time-kill kinetic assay for tested bacterial strains at 1xMIC Values are mean of three independent experiments

methanol extract contained all the tested phytochemicalsexcept anthraquinone The anthraquinone was not detectedin any of the extracts of Moringa oleifera flower pods Inaqueous and chloroform extracts all tested phytochemicalswere present except steroids and anthraquinone In ethanoland acetone extract alkaloids and terpenoids were also notdetectedThe results of antibacterial assay of different extractsand their phytochemical analysis showed that combinationof different compounds may be responsible for bioactivityof plant extracts The diversity of phytochemicals in plantextracts provides good opportunity to effectively control themicrobial growth as some microorganisms are not killed bysinglepure antimicrobial compound [47]


Table 4 Phytochemical analysis of flower pods extracts ofMoringa oleifera

Methanol extract Ethanol extract Chloroform extract Acetone extract Aqueous extractAlkaloid Wagnerrsquos test

+

minus + minus +Anthraquinone Borntragers test minus minus minus minus minus

Flavonoid Alkaline reagent test + + + + +Terpenoids Salkowski test (modified) + minus + minus +Tannins Braemerrsquos test + + + + +Glycoside Keller Kiliani test + + + + +Phenols FeCl

3

test + + + + +Steroids Salkowski test + + minus minus minus

The sign ldquo+rdquo indicates ldquodetectionrdquo and the sign ldquominusrdquo indicates ldquono detectionrdquo of compound in the extracts

Table 5 Total phenolic and flavonoid contents in flower pod extractofMoringa oleifera

Total phenoliccontents (mgGAEg)

Total flavonoids(mgQEg)

Methanol extract 538 plusmn 0169a 913 plusmn 0021b

Ethanol extract 2593 plusmn 0151c 870 plusmn 0085c

Chloroform extract 141 plusmn 0945d 341 plusmn 0006e

Acetone extract 277 plusmn 0124c 405 plusmn 0026d

Aqueous extract 424 plusmn 0094b 109 plusmn 0094a

Values are mean of three independent experiments Mean values followed bydifferent letters in same column differ significantly (119901 lt 005) according toDuncanrsquos multiple range test

341 Total Phenolic Contents (TPC) The total phenolic con-tents ofMoringa oleifera flower pods are presented in Table 5The results revealed significant variation in total phenoliccontents in different extracts The highest TPC was presentin methanol extract (538 plusmn 0169mgGAEg) while lowestin chloroform extract (141mgGAEg) The results of ourstudy corroborate the results of other studies discussed belowSohaimy et al [48] showed that among the different extractsof theMoringa oleifera leaves methanol extract had the high-est amount of TPC (4835mgGAEg) Similarly Abdulka-dir et al [49] also reported high TPC (4804mgGAEg)in methanol extract Alhakmani et al [50] reported lowTPC (1931mgGAEg) in ethanol extract of Moringa oleiferaflower pods The comparable results were also recordedin the present study as low TPC (2593mgGAEg) inethanol extract was noticed compared to methanol extract(4835mgGAEg) The slight difference in TPC might bedue to the difference in geographical origin of plant Boththe high TPC and high antimicrobial activity of methanolextract represent that phenolic compounds may be respon-sible for antimicrobial activity of methanol extract It ispertinent to mention that methanol extracts contained mostof the secondary metabolites from plants [28] Therefore thebioactivity of methanol extracts can be explained in termsof high TPC The aqueous extract contained high TPC butshowed negligible antibacterial activity that might be due toinstability of active compounds in water

342 Total Flavonoid Contents (TFC) The flavonoids con-stitute major component of plant phenolic compound and

0002004006008

01012014016018

02

Methanol Acetone Ethanol Chloroform AqueousFlower pod extracts

a

b

c cd

Abso

rban

ce at

750

nm

Figure 3 Antioxidant activity of different extracts ofMoringa oleif-era flower pods by Ferricyanide Reducing Power Assay (FRPA)Mean values having the same letter do not differ significantly (119901 lt005) according to Duncanrsquos multiple range test

exert positive effect on human healthThe flavonoid contentsin different extracts prepared from flower pods of Moringaoleifera are summarized in Table 5 Total flavonoid contentsranged from 109plusmn0094mgQEg to the 341plusmn0006mgQEgThe highest contents were recorded in aqueous extract(109 plusmn 0094mgQEg) Hence water was most effective inextraction of flavonoids from flower pods ofMoringa Akhtaret al [51] reported 69 plusmn 20mgQEg in methanol extract and91plusmn35mgQEg in aqueous extract prepared from leaves andbark of Moringa oleifera which are lower than the flavonoidcontents of methanol extract (913mgQEg) and aqueousextract (109mgQEg) prepared from flower pods that wererecorded in the present study Therefore for acquisition ofhigh flavonoid contents aqueous extract of Moringa oleiferaflower pods is potent natural biosource

35 Ferricyanide Reducing Power Assay (FRPA) Antioxidantactivity of plant extracts prevents the cell and tissue damagecaused by reactive oxygen species (ROS) Reducing powerof plant extract indicates its antioxidant activity [52] Thebioactive compounds in plant extracts convert the ROS intomore stable products by donation of electrons [53]

In the present study the reducing power of Moringaoleifera flower pod extracts was investigated by reducing Fe+3ions to Fe+2 ionsThe results displayed in Figure 3 showed thatthe aqueous extract had the highest reducing power followedby methanol extract The chloroform extract showed the


0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

A

B B CD DE

DEE

AB B

CD DE

DDE E

D

Storage duration at 4∘C (weeks)DarkLight

(a)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

AB C

DDE

EF

G G

AB

CD

EF F

GG

DarkLight

Storage duration at minus20∘C (weeks)

(b)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

Storage duration at room temperature (weeks)

A

B CD

E

F

G HI

A

BC

DE

FG

DarkLight

(c)

Figure 4 Stability of methanol extract at different storage temperatures for the period of two months with and without exposure of light (a)Stability of extract at 4∘C (b) at minus20∘C (c) at room temperature Mean values having the same letter do not differ significantly (119901 lt 005)according to Duncanrsquos multiple range test

lowest reducing powerThe high reducing power ofmethanoland aqueous extract can be correlated to the high phenolicand flavonoid contents estimated in these extracts Phenoliccompounds are considered as effective hydrogen donor thatmakes them good antioxidant [54] The results of the presentstudy corroborate the results of previous studies where ithas been reported that plant phenolic compounds governantioxidant activity of plant extracts [28 55ndash57] Thereforeaqueous extract of Moringa oleifera flower pods can beconsidered as a potent source of natural antioxidants

High flavonoids and TPC in aqueous and methanolextract might be responsible for its high bioactivity (antibac-terial and antioxidant activity) However stability of com-pounds in methanol extract makes it appropriate choice asnatural preservative with promising antibacterial and antiox-idant activity

36 Storage Stability of Moringa oleifera Extracts Takinginto consideration the pharmaceutical and nutraceuticalimportance of herbal extracts it is also important to validatetheir stability and functionality over the period of time It hasbeen reported that functional products in plant extracts aresensitive to many factors that is light storage temperatureand storage duration [10] In the present study stabilityof Moringa oleifera extracts was determined to ensure thestability and functionality of extract over the period of timeThe extract was stored at three different temperatures (minus20∘C4∘C and room temperature) with and without exposure tolight for the period of two months The results shown inFigure 4 indicated that sim5 decrease in bioactivity of extractswas observed with extracts exposed to light compared toextracts stored under dark conditions Decrease in bioactivityof extract was observed over the period of time Further rapid


1000 bp750bp500bp250bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Figure 5 1 agarose gel electrophoretic analysis of RAPD pattern ofMoringa oleifera DNA using 17 random decamers OPA 1ndashOPA 17 LaneM DNA size marker Lane 1ndash17 RAPD pattern ofMoringa oleifera using OPA 1ndashOPA 17 OPA 9 gave reproducible same banding pattern withDNA sample ofMoringa oleifera collected from different localities of Punjab province Arrow indicates the selected monomorphic fragment(750 bp) for cloning and sequencing

and significant decrease in bioactivity of extract was recordedduring storage at room temperature (Figure 4(c)) Afterstorage of one week at room temperature 40 decrease inbioactivity was observed and this decrease reached to 60after four weeks The total bioactivity was lost after storageof six weeks at room temperature At low temperatures theextracts were quite stable At 4∘C extract maintained itsactivity up to 75 after one month storage and showed 70activity even after two months (Figure 4(a)) The extract lostits bioactivity up to 60 after storage of twomonths at minus20∘C(Figure 4(b)) Therefore it is recommended to store theMoringa oleifera extracts at 4∘C in brown bottles The shelflife of the extract is 2 months at this storage temperature

37 SCARMarker forMoringa oleifera With revival of herbalmedicine in modern world it has utterly become indispens-able to deal with the issues related to the quality purityauthentication and standardization of raw plant materialMoringa oleifera has great potential as a natural substitute tochemical food preservatives and as a natural antioxidant Inaddition to this authentic identification of this valuable plantis also highly crucial Generally used organoleptic anatom-ical and analytical techniques have several drawbacks andlimitations such as being time-consuming being expensivebeing affected by environmental factors and requirementof botanical experts [58 59] Keeping in view the limita-tions and drawbacks of these techniques the present studyfocused on molecular authentication of Moringa oleifera byconverting RAPD (Random Amplification of PolymorphicDNA)marker into SCAR (SequenceCharacterizedAmplifiedRegion) marker The DNA isolated from Moringa oleiferaleaves samples collected from different regions of PunjabPakistan was amplified to develop RAPD pattern OPA 9primer showed reproducible banding pattern (Figure 5) Thefragment of sim750 bp (indicated by arrow) monomorphic inall samples was excised from gel purified and cloned Thepositive transformants were sequenced using universal M13primers To ensure the accuracy of sequence both (sense andantisense) strands of fragment were sequenced The reversecomplement sequence of antisense strand was compared

Table 6 SCAR primers forMoringa oleifera

MOF217317

51015840 GGGTACGCCTCTGTAAATGGAGCTACG-GTGAG 31015840

MOR217

51015840 ATTTGATTTGGCAATGGACAACTGTAG-CTCACACCAG GTT 31015840

MOR317

51015840 CCAAAGAGATGTAACGCAAGCCAGCCT-GTATAGTAAAAC 31015840

with sequence of sense strand using freely available onlineprogram ClustalW The sequence is shown in Figure 6 Thesequence was submitted to Genbank and Accession numberis KU247867The sequence was used to design SCAR primers(MOF

271317 MOR

217 and MOR

317) The primers were syn-

thesized commercially The expected size of amplicon usingMOF271317

andMOR317

was 317 bp To increase the precisionof developed SCAR markers the 317 bp amplicon was usedas template for primer pair MOF

271317and MOR

217 The

expected size of amplicon was 217 bp The developed SCARprimers (Table 6) were used to amplify DNA from Moringaoleifera samples randomly collected from different localitiesof Punjab province A fragment of 317 bp was successfullyamplified from all samples using developed MOF

217317and

MOR317

SCAR primers (Figure 7) In second round ofPCR using 317 bp amplicon as template withMOF

217317and

MOR217

also successfully amplified a fragment of 217 bp inall samples (Figure 7) The conversion of RAPD marker intoSCARmarker simplifies the identification procedure Insteadof complex banding pattern and lack of reproducibility ofRAPD SCAR marker relies on amplification of a singleband [60] Two rounds of PCR increase the accuracy andreliability of developed SCAR markers SCAR marker basedidentification is more simple and straightforward than othermolecular techniques that is intersimple sequence repeat(ISSR) Amplified Fragment Length Polymorphisms (AFLP)Simple Sequence Repeats (SSR) and RAPD In a simple PCRreaction using long primers a specific band can be amplifiedThe SCAR marker developed in the present study simplifiesthe identification of Moringa oleifera samples collected fromof Punjab Pakistan just by a PCR reaction


Moringa sense strand NNNTNATAAACTCGCGATGCATCTAGATTGGGTAACGCCTCTGTAAATGG 50Reverse complement of antisense strand ---NNNNNNNCTCGCGATGCATCTAGATTGGGTAACGCCTCTGTAAATGG 47

Moringa sense strand AGCTACGGTGAGGAGGAGAGTCGCGGGGAAGGCTCTTTCGATTACGCGAG

Reverse complement of antisense strand AGCTACGGTGAGGAGGAGAGTCGCGGGGAAGGCTCTTTCGATTACGCGAG

Moringa sense strand CGCTTCACCTTGACTAAAGAGTCTGTGCTCGTGGTCTCAGTAGAGCTGTT

Reverse complement of antisense strand CGCTTCACCTTGACTAAAGAGTCTGTGCTCGTGGTCTCAGTAGAGCTGTT

Moringa sense strand GCGGCTGTTGTGTGAGATTTTGGCCAGTTCAAACTGTGAGACAGTTATTG

Reverse complement of antisense strand GCGGCTGTTGTGTGAGATTTTGGCCAGTTCAAACTGTGAGACAGTTATTG

Moringa sense strand AAAAAGGAGGAGAGAGGGAAGAGGGTCACGGCATGATGATGATGATGATG

Reverse complement of antisense strand AAAAAGGAGGAGAGAGGGAAGAGGGTCACGGCATGATGATGATGATGATG

Moringa sense strand ATGAGAGAGTGGTTTTACTATACAGGCTGGCTTGCGTTACATCTCTTTGG 300Reverse complement of antisense strand ATGAGAGAGTGGTTTTACTATACAGGCTGGCTTGCGTTACATCTCTTTGG 297

Moringa sense strand CCATGTGTATATATAGTCCTGAGCACTGTCTTTCTTTGTGTTTGTATGGA

Reverse complement of antisense strand CCATGTGTATATATAGTCCTGAGCACTGTCTTTCTTTGTGTTTGTATGGA

Moringa sense strand TTAATTGCGCAACCTGGTGTGAGCTACAGTTGTCCATTGCCAAATCAAAT 400Reverse complement of antisense strand TTAATTGCGCAACCTGGTGTGAGCTACAGTTGTCCATTGCCAAATCAAAT 397

10097

150147

200197

250247

350347

lowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowast







MOR271

MOR371

MOF271371

Figure 6 Comparison of sense strand and reverse complement of antisense strand sequence of selected fragment of Moringa oleifera fromRAPD pattern of OPA 9 using ClustalW program MOF

271371

highlighted by red color is indicating the region selected for forward primerMOR

271

and MOR371

highlighted by red color are indicating the region selected for designing reverse primer The symbol lowast indicates thehomology between the sequences

M 1 2

500bp400bp300bp200 bp

100 bp

Figure 7 1 agarose electrophoresis analysis of PCR products amplified with developed SCAR primers forMoringa oleifera Lane M DNAsize marker Lane 1 PCR product of 317 bp using SCAR primer MOF

217317

MOR317

Lane 2 PCR product of 217 bp using 317 bp amplicon astemplate with MOF

217317

and MOR217

SCAR primers


Moringa oleifera has gained widespread popularity dueto potent antibacterial [37 61 62] antifungal [63 64]antioxidant [65] antiproliferative [66] antidiabetic [67] anti-inflammatory [68] cholesterol lowering [69] and hepatopro-tective [70] activities of its extracts prepared from differentparts (root bark stem leaves seeds fruit and flower) Thesephytochemicals have been reported to be involved treatmentof diseases The phenols and flavonoids act as antioxidantsthat prevent oxidative damage and control degenerative dis-eases [71] Alkaloids are known for antibacterial and antifun-gal activities [72] whereas glycosides have been reported forregulation of heart beat and treatment of congestive heartfailure [73] Tannins have been reported for treatment ofdiabetes [74]Themethanol extract ofMoringa oleifera flowerpods contains number of phytochemicals (phenolic com-pounds flavonoids alkaloid terpenoids and tannins) andhigh total phenolic and flavonoid contents as recorded in thepresent study These findings show the potential of methanolextract of Moringa oleifera flower pods for its use as naturalpreservative and nutraceutical in food industry

4 Conclusion

The methanol extract of Moringa oleifera flower pods hasvast potential as a nutraceutical and a natural substitute tosynthetic food preservatives Further research is necessary forreal application of these extracts in food as extrapolation ofresults from in vitro studies to food products is not straight-forward due to complex nature of food and different intercon-necting environments The identification at molecular levelwith the developed SCARmarker ofMoringa oleifera is usefulfor authentication and quality assurance of this miracle plant

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

All authors equally participated in designing experimentsacquisition analysis and interpretation of data Professor DrMuhammad Amin Athar critically revised and approved thefinal version of paper All authors read and approved the finalpaper

Acknowledgments

The authors are very thankful to the Department of BotanyUniversity of the Punjab Lahore Pakistan for identificationof plant material used in the present study The study fundedby the research budget of Institute of Biochemistry andBiotechnology University of the Punjab Lahore Pakistan

References

[1] T Hintz K K Matthews and R Di ldquoThe use of plant antimi-crobial compounds for food preservationrdquo BioMed ResearchInternational vol 2015 Article ID 246264 12 pages 2015

[2] WHO WHO Estimates of the Global Burden of Foodborne Dis-eases FoodborneDiseases Burden Epidemiology Reference Group2007ndash2015 World Health Organization Geneva Switzerland2015

[3] G W Gould ldquoBiodeterioration of foods and an overview ofpreservation in the food and dairy industriesrdquo InternationalBiodeterioration and Biodegradation vol 36 no 3-4 pp 267ndash277 1995

[4] S Roller ldquoThe quest for natural antimicrobials as novel meansof food preservation status report on a European researchprojectrdquo International Biodeterioration and Biodegradation vol36 no 3-4 pp 333ndash345 1995

[5] B Sun and M Fukuhara ldquoEffects of co-administration of buty-lated hydroxytoluene butylated hydroxyanisole and flavonoidson the activation of mutagens and drug-metabolizing enzymesin micerdquo Toxicology vol 122 no 1-2 pp 61ndash72 1997

[6] S U Tavasalkar H NMishra and SMadhavan ldquoEvaluation ofantioxidant efficacy of natural plant extracts against syntheticantioxidants in sunflower oilrdquo Scientific Reports vol 1 p 5042012

[7] F Shahidi and Y Zhong ldquoNovel antioxidants in food qualitypreservation and health promotionrdquo European Journal of LipidScience and Technology vol 112 no 9 pp 930ndash940 2010

[8] K A Hammer C F Carson and T V Riley ldquoAntimicrobialactivity of essential oils and other plant extractsrdquo Journal ofApplied Microbiology vol 86 no 6 pp 985ndash990 1999

[9] J-Y Pang K-J Zhao J-B Wang Z-J Ma and X-H XiaoldquoGreen tea polyphenol epigallocatechin-3-gallate possesses theantiviral activity necessary to fight against the hepatitis B virusreplication in vitrordquo Journal of Zhejiang University SCIENCE Bvol 15 no 6 pp 533ndash539 2014

[10] W Qu I A P Breksa Z Pan H Ma and T H McHughldquoStorage stability of sterilized liquid extracts from pomegranatepeelrdquo Journal of Food Science vol 77 no 7 pp C765ndashC772 2012

[11] U Kiran S Khan K JMirzaM Ram andM Z Abdin ldquoSCARmarkers a potential tool for authentication of herbal drugsrdquoFitoterapia vol 81 no 8 pp 969ndash976 2010

[12] R A Barthelson P Sundareshan D W Galbraith and R LWoosley ldquoDevelopment of a comprehensive detection methodfor medicinal and toxic plant speciesrdquo American Journal ofBotany vol 93 no 4 pp 566ndash574 2006

[13] K Chan ldquoSome aspects of toxic contaminants in herbalmedicinesrdquo Chemosphere vol 52 no 9 pp 1361ndash1371 2003

[14] Y L Siow Y Gong K K W Au-Yeung C W H Woo P CChoy and O Karmin ldquoEmerging issues in traditional Chinesemedicinerdquo Canadian Journal of Physiology and Pharmacologyvol 83 no 4 pp 321ndash334 2005

[15] HWagner R Bauer DMelchart P G Xiao and A StaudingerChromatographic Fingerprint Analysis of Herbal MedicinesThin-Layer and High Performance Liquid Chromatography ofChinese Drugs vol 1-2 Springer Vienna Austria 2nd edition2011

[16] M Li H Cao P P-H But and P-C Shaw ldquoIdentification ofherbal medicinal materials using DNA barcodesrdquo Journal ofSystematics and Evolution vol 49 no 3 pp 271ndash283 2011

[17] M Li K Y-B Zhang P P-H But and P-C Shaw ldquoForensicallyinformative nucleotide sequencing (FINS) for the authentica-tion of Chinese medicinal materialsrdquo Chinese Medicine vol 6article 42 2011

[18] F Anwar M Ashraf and M I Bhanger ldquoInterprovenancevariation in the composition of Moringa oleifera oilseeds from


Pakistanrdquo Journal of the American Oil Chemistsrsquo Society vol 82no 1 pp 45ndash51 2005

[19] I Oduro W O Ellis and D Owusu ldquoNutritional potentialof two leafy vegetables Moringa oleifera and Ipomoea batatasleavesrdquo Scientific Research and Essays vol 3 no 2 pp 57ndash602008

[20] W J Fahey ldquoMoringa oleifera a review of the medical evidencefor its nutitional therapeutic and prophylactic propertiesrdquoTreesfor Life Journal vol 1 pp 1ndash5 2005

[21] L J FuglieTheMiracle TreeMoringa oleifera Natural Nutritionfor The Tropics Church World Service Dakar Senegal 1999

[22] A G Bakre A O Aderibigbe and O G Ademowo ldquoStudieson neuropharmacological profile of ethanol extract ofMoringaoleifera leaves in micerdquo Journal of Ethnopharmacology vol 149no 3 pp 783ndash789 2013

[23] S Fakurazi I Hairuszah and U Nanthini ldquoMoringa oleiferaLam prevents acetaminophen induced liver injury throughrestoration of glutathione levelrdquo Food and Chemical Toxicologyvol 46 no 8 pp 2611ndash2615 2008

[24] N Sherman and J G Cappuccino Microbiology A LaboratoryManual vol 81 6th edition 2005

[25] P R Murray E J Baron M A Pfaller F C Tenover and RH YolkeMannual of Clinical Microbiology ASM WashingtonDC USA 6th edition 1995

[26] A Bag and R R Chattopadhyay ldquoEvaluation of synergisticantibacterial and antioxidant efficacy of essential oils of spicesand herbs in combinationrdquo PLoS ONE vol 10 no 7 Article IDe131321 2015

[27] I Gull M Sohail M S Aslam and M A Athar ldquoPhytochem-ical toxicological and antimicrobial evaluation of Lawsoniainermis extracts against clinical isolates of pathogenic bacteriardquoAnnals of Clinical Microbiology and Antimicrobials vol 12article 36 2013

[28] A A Mohamed S I Ali and F K El-Baz ldquoAntioxidant andantibacterial activities of crude extracts and essential oils ofSyzygium cumini leavesrdquo PLoS ONE vol 8 no 4 Article IDe60269 2013

[29] G E Trease andW C Evans Pharmacognosy Bailliere TindallLondon UK 13th edition 1989

[30] A Sofowora Medicinal Plants and Traditional Medicinal inAfrica Screening Plants for Bioactive Agents Spectrum BooksIbadan Nigeria 2nd edition 1993

[31] V Y A Barku Y Opoku-Boahen E Owusu-Ansah N T KD Dayie and F E Mensah ldquoIn-vitro assessment of antioxidantand antimicrobial activities of methanol extracts of six woundhealing medicinal plantsrdquo Journal of Natural Sciences Researchvol 3 pp 74ndash80 2013

[32] R Manian N Anusuya P Siddhuraju and S Manian ldquoTheantioxidant activity and free radical scavenging potential of twodifferent solvent extracts of Camellia sinensis (L) O KuntzFicus bengalensis L and Ficus racemosa Lrdquo Food Chemistry vol107 no 3 pp 1000ndash1007 2008

[33] J J Doyle and J L Doyle ldquoA rapid isolation procedure for smallquantities of fresh leaf tissuerdquoPhytochemical Bulletin vol 19 pp11ndash15 1987

[34] J Sambrook and D Russell Molecular Cloning A LaboratoryManual Cold Spring Harbor Press Cold Spring Harbor NYUSA 3rd edition 2001

[35] I Gulcin S Beydemir H A Alici M Elmastas and M EBuyukokuroglu ldquoIn vitro antioxidant properties of morphinerdquoPharmacological Research vol 49 no 1 pp 59ndash66 2004

[36] BMoyo P JMasika andVMuchenje ldquoAntimicrobial activitiesof Moringa oleifera Lam leaf extractsrdquo African Journal ofBiotechnology vol 11 no 11 pp 2797ndash2802 2012

[37] G Dewangani K M Koley V P Vadlamudi A Mishra APoddar and S D Hirpurkar ldquoAntibacterial activity ofMoringaoleifera (drumstick) root barkrdquo Journal of Chemical and Phar-maceutical Research vol 2 pp 424ndash428 2010

[38] J R Anitha K G Velliyur A Y Sangilimuthu and D Sun-darsanam ldquoAntimicrobial activity of Moringa oleifera (Lam)root extractrdquo Journal of Pharmacy Research vol 4 no 5 pp1426ndash1427 2011

[39] C Bolin andG Salyabart ldquoAntibacterial activities of themetha-nolic extract of stem bark of Spondias pinnata Moringa oleiferaand Alstonia scholarisrdquo Asian Journal of Traditional Medicinesvol 6 pp 163ndash167 2011

[40] A Bukar A Uba and T Oyeyi ldquoAntimicrobial profile ofmoringa oleifera lam Extracts against some foodmdashbornemicroorganismsrdquo Bayero Journal of Pure and Applied Sciencesvol 3 no 1 pp 43ndash48 2010

[41] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005

[42] F Anwar S Latif M Ashraf and A H Gilani ldquoMoringaoleifera a food plant with multiple medicinal usesrdquo Phytother-apy Research vol 21 no 1 pp 17ndash25 2007

[43] O A Akingbade A A Akinjinmi U S Ezechukwu et alldquoAntibacterial effect of Moringa oleifera on multidrug resistantPseudomonas aeruginosa isolates from wound infections inAbeokuta Ogun state NigeriardquoWorld Rural Observations vol5 no 3 pp 6ndash10 2013

[44] M E Abalaka S Y Daniyan S B Oyeleke and S OAdeyemo ldquoThe antibacterial evaluation ofmoringaOleifera leafextracts on selected bacterial pathogensrdquo Journal of Microbiol-ogy Research vol 2 no 2 pp 1ndash4 2012

[45] A Kawo B Abdullahi A Halilu Z Gaiya M Dabai and MDakare ldquoPreliminary phytochemical screening proximate andelemental composition of Moringa oleifera lam seed powderrdquoBayero Journal of Pure and Applied Sciences vol 2 no 1 2009

[46] H P N Sholapur and B M Patil ldquoPharmacognostic andphytochemical investigations on the bark of Moringa oleiferaLamrdquo Indian Journal of Natural Products and Resources vol 4no 1 pp 96ndash101 2013

[47] L R Beuchat ldquoControl of foodborne pathogens and spoilagemicroorganisms by naturally occurring microorganismsrdquo inMicrobial Food Contamination C LWilson and S Droby Edspp 149ndash169 CRC Press London UK 2007

[48] S A Sohaimy G M Hamad S E Mohamed M H Amarand R R Al-Hindi ldquoBiochemical and functional properties ofMoringa oleifera leaves and their potential as a functional foodrdquoGARJAS vol 4 pp 188ndash199 2015

[49] A R Abdulkadir D D B Zawawi A K Yonusa and M SJahan ldquoIn-vitro antioxidant potential total phenolic contentand total flavovoid content of methanolic flower and seed andseed extract of Miracle tree Moringa oleifera Lamrdquo AustralianJournal of Basic and Applied Sciences vol 9 pp 27ndash31 2015

[50] F Alhakmani S Kumar and S A Khan ldquoEstimation of totalphenolic content in-vitro antioxidant and anti-inflammatoryactivity of flowers of Moringa oleiferardquo Asian Pacific Journal ofTropical Biomedicine vol 3 no 8 pp 623ndash627 2013

[51] N Akhtar I Haq and B Mirza ldquoPhytochemical analysis andcomprehensive evaluation of antimicrobial and antioxidant


properties of 61 medicinal plant speciesrdquo Arabian Journal ofChemistry 2015

[52] P Jayanthi and P Lalitha ldquoReducing power of the solventextracts of Eichhornia crassipes (Mart) solmsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 3 no 3pp 126ndash128 2011

[53] R L Prior X Wu and K Schaich ldquoStandardized methodsfor the determination of antioxidant capacity and phenolicsin foods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[54] C A Rice-Evans N J Miller P G Bolwell PM Bramley and JB Pridham ldquoThe relative antioxidant activities of plant-derivedpolyphenolic flavonoidsrdquo Free Radical Research vol 22 no 4pp 375ndash383 1995

[55] L G Roy and A Urooj ldquoAntioxidant potency ph and heatstability of selected plant extractsrdquo Journal of Food Biochemistryvol 37 no 3 pp 336ndash342 2013

[56] N Saeed M R Khan and M Shabbir ldquoAntioxidant activitytotal phenolic and total flavonoid contents of whole plantextracts Torilis leptophylla Lrdquo BMC Complementary and Alter-native Medicine vol 12 article 221 2012

[57] L Jing H Ma P Fan R Gao and Z Jia ldquoAntioxidant potentialtotal phenolic and total flavonoid contents of Rhododendronanthopogonoides and its protective effect on hypoxia-inducedinjury in PC12 cellsrdquo BMC Complementary and AlternativeMedicine vol 15 article 287 2015

[58] J Barnes ldquoQuality efficacy and safety of complementary medi-cines fashions facts and the futuremdashpart I Regulation andqualityrdquo British Journal of Clinical Pharmacology vol 55 no 3pp 226ndash233 2003

[59] A Vlietinck L Pieters and S Apers ldquoLegal requirements forthe quality of herbal substances and herbal preparations for themanufacturing of herbal medicinal products in the EuropeanUnionrdquo Planta Medica vol 75 no 7 pp 683ndash688 2009

[60] L Yang S Fu M A Khan W Zeng and J Fu ldquoMolecularcloning and development of RAPD-SCAR markers for Dimo-carpus longan variety authenticationrdquo SpringerPlus vol 2 no 1article 501 pp 1ndash8 2013

[61] P A Okiki B D Balogun I A Osibote and S AsosoldquoAntibacterial activity of methanolic extract ofMoringa oleiferaLam Leaf on ESBL producing bacterial isolates from urineof patients with urinary tract infectionsrdquo Journal of BiologyAgriculture and Healthcare vol 5 pp 124ndash132 2015

[62] A O Oluduro ldquoEvaluation of antimicrobial properties andnutritional potentials of Moringa oleifera Lam leaf in South-Western NigeriardquoMalaysian Journal of Microbiology vol 8 no2 pp 59ndash67 2012

[63] M A Sayeed M S Hossain M E Chowdhury and MHaque ldquoIn vitro antimicrobial activity of methanolic extractofMoringa oleifera Lam fruitsrdquo Journal of Pharmacognosy andPhytochemistry vol 1 pp 94ndash98 2012

[64] P-H Chuang C-W Lee J-Y Chou M Murugan B-J Shiehand H-M Chen ldquoAnti-fungal activity of crude extracts andessential oil of Moringa oleifera Lamrdquo Bioresource Technologyvol 98 no 1 pp 232ndash236 2007

[65] B Moyo S Oyedemi P J Masika and V Muchenje ldquoPolyphe-nolic content and antioxidant properties of Moringa oleiferaleaf extracts and enzymatic activity of liver from goats supple-mented withMoringa oleifera leavessunflower seed cakerdquoMeatScience vol 91 no 4 pp 441ndash447 2012

[66] S Sreelatha A Jeyachitra and P R Padma ldquoAntiproliferationand induction of apoptosis by Moringa oleifera leaf extract on

human cancer cellsrdquo Food and Chemical Toxicology vol 49 no6 pp 1270ndash1275 2011

[67] R Gupta M Mathur V K Bajaj et al ldquoEvaluation ofantidiabetic and antioxidant activity of Moringa oleifera inexperimental diabetesrdquo Journal ofDiabetes vol 4 no 2 pp 164ndash171 2012

[68] I C Ezeamuziea A W Ambakederemoa F O Shodeb and SC Ekwebelema ldquoAntiinflammatory effects of Moringa oleiferaroot extractrdquo International Journal of Pharmacognosy vol 34pp 207ndash212 2008

[69] L K Mehta R Balaraman A H Amin P A Bafna andO D Gulati ldquoEffect of fruits of Moringa oleifera on the lipidprofile of normal and hypercholesterolaemic rabbitsrdquo Journal ofEthnopharmacology vol 86 no 2-3 pp 191ndash195 2003

[70] K Ruckmani S Kavimani R Anandan and B Jaykar ldquoEffectofMoringa oleifera Lam on paracetamol induced hepatoxicityrdquoIndian Journal of Pharmaceutical Sciences vol 60 pp 33ndash351998

[71] D E Okwu ldquoPhytochemicals and vitamin contents indigenousspices of South Eastern Nigeriardquo Journal of Sustainable Agricul-ture and the Environment vol 6 pp 30ndash34 2004

[72] D E Okwu and M E Okwu ldquoChemical composition ofSpondias mombin Linn plants partsrdquo Journal of SustainableAgriculture and the Environment vol 6 no 2 pp 140ndash147 2004

[73] G Leverin and H McMatron ldquoAlkaloids and glycosidesrdquoClinical Microbiology Reviews vol 11 pp 156ndash250 1999

[74] M Kumari and S Jain ldquoScreening of potential sources of tanninand its therapeutic applicationrdquo International Journal of Nutri-tion and Food Sciences vol 4 pp 26ndash29 2015

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

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Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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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


medicinal plants are in practice for their authentication [13ndash15] Microscopic and macroscopic studies need to be con-ducted by expert personnel Further the chemical composi-tion of plants is affected by change of growth stage environ-mental factors storage conditions and harvesting processThe molecular authentication procedures are considered asmore precise specific and suitable for medicinal plant iden-tification in comparison to above discussed methods becauseDNAbased techniques are independent of growth stage envi-ronmental factors andphysical formof plantmaterial [16 17]

Moringa oleifera is a plant of familyMoringaceae Variousparts of the plant are used as nutritious food commodityin many countries that is Pakistan India Philippines andHawaii and in some parts of Africa [18] In underdevelopedcountries (Senegal and Haiti) powder of leaves of Moringaoleifera has also been used to treat malnutrition in childrenpregnant women and nursing mothers [19] The leaves ofMoringa has more iron than spinach more calcium thanmilk more potassium than banana andmore vitamin C thanoranges and the protein quality rivals the egg andmilk protein[20] For a long time it has also been used as traditionalmedi-cine for treatment of many diseases [21ndash23]

Therefore the objectives of the present study are (i)to evaluate the antimicrobial and antioxidant activity ofMoringa oleiferaflower pods and (ii) to develop SCARmarkerusing leaves as plant material for authentic identification ofthis medicinal plant

2 Materials and Methods

21 Collection of Plant Material and Preparation of CrudeExtracts Fresh flower pods of Moringa oleifera were pur-chased from the local market of Lahore Pakistan in themonth of March and April 2014 and authenticated mor-phologically from Department of Botany University of thePunjab Lahore Pakistan The flower pods (500 g) were com-pletely air-dried and pulverized mechanically into powderusing electric grinder The powder (5 g) was extracted bysoaking in 50mL of each solvent (methanol ethanol waterchloroform and acetone) separately in screw capped 250mLErlenmeyer flasks for 24 hours with shaking at 160 rpm inorbital shaker at ambient temperature The extracts werefiltered through eight layers of muslin cloth and then cen-trifuged at 8000timesg for 15 minutes The clear supernatant wascollected and dried at 45∘C in rotary evaporator (Laborota4000-efficient Heidolph) All the dried residues were dis-solved in Dimethylsulfoxide (DMSO) except aqueous extractresidues which were dissolved in water to known concentra-tions The extracts were stored at 4∘C till further use

22 Bacterial Strains Six bacterial strains (E coli S typhiShigella S aureus and B subtilis) isolated from different foodsamples after biochemical characterizations [24] were used inthe present study

23 Antibacterial Activity Assay The antibacterial activityassay was performed by disc diffusion method [25] Sterilenutrient agar plates were seeded with 107 CFUmL of each

bacterial strain under aseptic conditions The sterile discsof Whatman Number 1 filter paper (5mm) were dipped inextracts for 5 minutes and placed on the surface of seededplates and pressed gentlyTheplateswere incubated overnightat 37∘C The disc soaked in DMSO was used as negativecontrolThe test was performed in triplicate for each bacterialstrain The antibacterial activity was recorded as mean ofdiameter of zone of inhibition (mm)

24 Minimum Inhibitory Concentration (MIC) The mini-mum inhibitory concentration (MIC) was determined asdescribed by Bag and Chattopadhyay [26] MIC assay wasperformed in 96-well microtitre plate Each well containing90 120583L of nutrient brothwas inoculatedwith 10120583L of each bac-terial culture (5 times 105 CFUmL) The extracts (100 120583L) withconcentrations ranging from900mgmL to 625mgmLwereadded in each well separately After overnight incubationof plate at 37∘C 40 120583L of 04mgmL p-iodonitrotetrazoliumviolet (INT) was added in each well and incubated again for 6hours After incubation the plates were observed for changein color from yellow to red to purple for viable bacteria Thelowest concentration of plant extract with no color changewas recorded as MIC The experiment was performed intriplicate

25 Time-Kill Kinetic Analysis The bacterial killing rate wasdetermined by time-kill kinetic assay at 1x MIC as describedin our earlier study [27] The cultures collected at differenttime (0ndash24hours)were seriallydilutedandused to determinedviable cell count The results were recorded as logarithm ofCFU against incubation time

26 Storage Stability Assay The stability of Moringa oleiferaflower pod extracts was evaluated over the period of timewith and without their exposure to light while being stored atdifferent temperatures (minus20∘C 4∘C and room temperature)At the end of each storage time the stability of antimicrobialcompounds in crude extracts was determined by antimicro-bial assay as described previously

27 Antioxidant Assay

271 Ferric Reducing Power Assay (FRPA) Ferric reducingpower assay was performed as described by Mohamed et al[28] Briefly 100 120583L of each extract was mixed with 25mL of200mMphosphate buffer pH 66 and 25mL of 1 potassiumferricyanide [K

3Fe(CN)

6] After incubation at 50∘C for 20

minutes 25mL of 10 trichloroacetic acid was added tomixture and centrifuged at 10000 rpm for 10 minutes Theupper layer of solution (5mL) was mixed with 5mL of dis-tilled water and 1mL of 01 ferric chloride The absorbancewas measured at 700 nm Ascorbic acid was used as standardand results were expressed by plotting absorbance againstconcentration

28 Phytochemical Studies The extracts of Moringa oleiferawere screened for the presences of major phytochemicalssuch as phenolics flavonoids alkaloids terpenoids tannins






















minus005

0

005

01

015

02

025

03



Abso

rban

ce at

765

nm

y = 00027x minus 00014

R2 = 1

(a)

minus0010

001002003004005006007

0 50 100 150 200 250



sorb

ance

at510

nm

y = 00003x minus 00003

R2 = 09995

(b)









OPA-01 51015840-CAG GCC CTT C-31015840

OPA-02 51015840-TGC CGA GCT G-31015840

OPA-03 51015840-AGT CAG CCA C-31015840

OPA-04 51015840-AAT CGG GCT G-31015840

OPA-05 51015840-AGG GGT CTT G-31015840

OPA-06 51015840-GGT CCC TGA C-31015840

OPA-07 51015840-GAA ACG GGTG-31015840

OPA-08 51015840-GTG ACG TAG G-31015840

OPA-09 51015840-GGG TAA CGC C-31015840

OPA-10 51015840-GTG ATC GCA G-31015840

OPA-11 51015840-CAA TCG CCG T-31015840

OPA-12 51015840-TCG GCG ATA G-31015840

OPA-13 51015840-CAG CAC CCA C-31015840

OPA-14 51015840-TCT GTG CTG G-31015840

OPA-15 51015840-TTC CGA ACC C-31015840

OPA-16 51015840-AGC CAG CGA A-31015840

OPA-17 51015840-GAC CGC TTG T-31015840


271371and MOR

271 MOR

371) The


andMOR371


271371and MOR

271SCAR


271371

MOR271

and MOR371
























10



002040608

112141618

2




log

CFU(c

olon

y fo

rmin

g un

it)






+



3














0002004006008

01012014016018

02


a

b

c cd

Abso

rban

ce at

750

nm






0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

A

B B CD DE

DEE

AB B

CD DE

DDE E

D


(a)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

AB C

DDE

EF

G G

AB

CD

EF F

GG

DarkLight


(b)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity


A

B CD

E

F

G HI

A

BC

DE

FG

DarkLight

(c)






1000 bp750bp500bp250bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17





MOF217317


MOR217


MOR317



271317 MOR

217 and MOR



andMOR317


271317and MOR

217 The


217317and

MOR317


217317and

MOR217
















10097

150147

200197

250247

350347









MOR271

MOR371

MOF271371


271371


271

and MOR371


M 1 2

500bp400bp300bp200 bp

100 bp


217317

MOR317


217317

and MOR217

SCAR primers



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















minus005

0

005

01

015

02

025

03



Abso

rban

ce at

765

nm

y = 00027x minus 00014

R2 = 1

(a)

minus0010

001002003004005006007

0 50 100 150 200 250



sorb

ance

at510

nm

y = 00003x minus 00003

R2 = 09995

(b)









OPA-01 51015840-CAG GCC CTT C-31015840

OPA-02 51015840-TGC CGA GCT G-31015840

OPA-03 51015840-AGT CAG CCA C-31015840

OPA-04 51015840-AAT CGG GCT G-31015840

OPA-05 51015840-AGG GGT CTT G-31015840

OPA-06 51015840-GGT CCC TGA C-31015840

OPA-07 51015840-GAA ACG GGTG-31015840

OPA-08 51015840-GTG ACG TAG G-31015840

OPA-09 51015840-GGG TAA CGC C-31015840

OPA-10 51015840-GTG ATC GCA G-31015840

OPA-11 51015840-CAA TCG CCG T-31015840

OPA-12 51015840-TCG GCG ATA G-31015840

OPA-13 51015840-CAG CAC CCA C-31015840

OPA-14 51015840-TCT GTG CTG G-31015840

OPA-15 51015840-TTC CGA ACC C-31015840

OPA-16 51015840-AGC CAG CGA A-31015840

OPA-17 51015840-GAC CGC TTG T-31015840


271371and MOR

271 MOR

371) The


andMOR371


271371and MOR

271SCAR


271371

MOR271

and MOR371
























10



002040608

112141618

2




log

CFU(c

olon

y fo

rmin

g un

it)






+



3














0002004006008

01012014016018

02


a

b

c cd

Abso

rban

ce at

750

nm






0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

A

B B CD DE

DEE

AB B

CD DE

DDE E

D


(a)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

AB C

DDE

EF

G G

AB

CD

EF F

GG

DarkLight


(b)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity


A

B CD

E

F

G HI

A

BC

DE

FG

DarkLight

(c)






1000 bp750bp500bp250bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17





MOF217317


MOR217


MOR317



271317 MOR

217 and MOR



andMOR317


271317and MOR

217 The


217317and

MOR317


217317and

MOR217
















10097

150147

200197

250247

350347









MOR271

MOR371

MOF271371


271371


271

and MOR371


M 1 2

500bp400bp300bp200 bp

100 bp


217317

MOR317


217317

and MOR217

SCAR primers



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



OPA-01 51015840-CAG GCC CTT C-31015840

OPA-02 51015840-TGC CGA GCT G-31015840

OPA-03 51015840-AGT CAG CCA C-31015840

OPA-04 51015840-AAT CGG GCT G-31015840

OPA-05 51015840-AGG GGT CTT G-31015840

OPA-06 51015840-GGT CCC TGA C-31015840

OPA-07 51015840-GAA ACG GGTG-31015840

OPA-08 51015840-GTG ACG TAG G-31015840

OPA-09 51015840-GGG TAA CGC C-31015840

OPA-10 51015840-GTG ATC GCA G-31015840

OPA-11 51015840-CAA TCG CCG T-31015840

OPA-12 51015840-TCG GCG ATA G-31015840

OPA-13 51015840-CAG CAC CCA C-31015840

OPA-14 51015840-TCT GTG CTG G-31015840

OPA-15 51015840-TTC CGA ACC C-31015840

OPA-16 51015840-AGC CAG CGA A-31015840

OPA-17 51015840-GAC CGC TTG T-31015840


271371and MOR

271 MOR

371) The


andMOR371


271371and MOR

271SCAR


271371

MOR271

and MOR371
























10



002040608

112141618

2




log

CFU(c

olon

y fo

rmin

g un

it)






+



3














0002004006008

01012014016018

02


a

b

c cd

Abso

rban

ce at

750

nm






0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

A

B B CD DE

DEE

AB B

CD DE

DDE E

D


(a)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

AB C

DDE

EF

G G

AB

CD

EF F

GG

DarkLight


(b)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity


A

B CD

E

F

G HI

A

BC

DE

FG

DarkLight

(c)






1000 bp750bp500bp250bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17





MOF217317


MOR217


MOR317



271317 MOR

217 and MOR



andMOR317


271317and MOR

217 The


217317and

MOR317


217317and

MOR217
















10097

150147

200197

250247

350347









MOR271

MOR371

MOF271371


271371


271

and MOR371


M 1 2

500bp400bp300bp200 bp

100 bp


217317

MOR317


217317

and MOR217

SCAR primers



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















10



002040608

112141618

2




log

CFU(c

olon

y fo

rmin

g un

it)






+



3














0002004006008

01012014016018

02


a

b

c cd

Abso

rban

ce at

750

nm






0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

A

B B CD DE

DEE

AB B

CD DE

DDE E

D


(a)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

AB C

DDE

EF

G G

AB

CD

EF F

GG

DarkLight


(b)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity


A

B CD

E

F

G HI

A

BC

DE

FG

DarkLight

(c)






1000 bp750bp500bp250bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17





MOF217317


MOR217


MOR317



271317 MOR

217 and MOR



andMOR317


271317and MOR

217 The


217317and

MOR317


217317and

MOR217
















10097

150147

200197

250247

350347









MOR271

MOR371

MOF271371


271371


271

and MOR371


M 1 2

500bp400bp300bp200 bp

100 bp


217317

MOR317


217317

and MOR217

SCAR primers



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




+



3














0002004006008

01012014016018

02


a

b

c cd

Abso

rban

ce at

750

nm






0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

A

B B CD DE

DEE

AB B

CD DE

DDE E

D


(a)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

AB C

DDE

EF

G G

AB

CD

EF F

GG

DarkLight


(b)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity


A

B CD

E

F

G HI

A

BC

DE

FG

DarkLight

(c)






1000 bp750bp500bp250bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17





MOF217317


MOR217


MOR317



271317 MOR

217 and MOR



andMOR317


271317and MOR

217 The


217317and

MOR317


217317and

MOR217
















10097

150147

200197

250247

350347









MOR271

MOR371

MOF271371


271371


271

and MOR371


M 1 2

500bp400bp300bp200 bp

100 bp


217317

MOR317


217317

and MOR217

SCAR primers



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

A

B B CD DE

DEE

AB B

CD DE

DDE E

D


(a)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity

AB C

DDE

EF

G G

AB

CD

EF F

GG

DarkLight


(b)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

an

tibac

teria

l act

ivity


A

B CD

E

F

G HI

A

BC

DE

FG

DarkLight

(c)






1000 bp750bp500bp250bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17





MOF217317


MOR217


MOR317



271317 MOR

217 and MOR



andMOR317


271317and MOR

217 The


217317and

MOR317


217317and

MOR217
















10097

150147

200197

250247

350347









MOR271

MOR371

MOF271371


271371


271

and MOR371


M 1 2

500bp400bp300bp200 bp

100 bp


217317

MOR317


217317

and MOR217

SCAR primers



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1000 bp750bp500bp250bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17





MOF217317


MOR217


MOR317



271317 MOR

217 and MOR



andMOR317


271317and MOR

217 The


217317and

MOR317


217317and

MOR217
















10097

150147

200197

250247

350347









MOR271

MOR371

MOF271371


271371


271

and MOR371


M 1 2

500bp400bp300bp200 bp

100 bp


217317

MOR317


217317

and MOR217

SCAR primers



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















10097

150147

200197

250247

350347









MOR271

MOR371

MOF271371


271371


271

and MOR371


M 1 2

500bp400bp300bp200 bp

100 bp


217317

MOR317


217317

and MOR217

SCAR primers



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion


Competing Interests




Acknowledgments


References























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































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 Moringa oleifera Flower Pod Extract as ...downloads.hindawi.com/journals/bmri/2016/7584318.pdf · Moringa has more iron than spinach, more calcium than ... Estimation