research article antibacterial attributes of apigenin...

Research ArticleAntibacterial Attributes of Apigenin Isolated fromPortulaca oleracea L

Hanumantappa B Nayaka Ramesh L Londonkar Madire K Umesh and Asha Tukappa

Department of Post Graduate Studies and Research in Biotechnology Gulbarga University Gulbarga Karnataka 585106 India

Correspondence should be addressed to Ramesh L Londonkar londonkarramesh53gmailcom

Received 29 November 2013 Revised 14 April 2014 Accepted 14 April 2014 Published 13 May 2014

Academic Editor Gary Dykes

Copyright copy 2014 Hanumantappa B Nayaka et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The flavonoid apigenin was isolated from aerial part of P oleracea L The dried sample of plant was powdered and subjected tosoxhlet extractor by adding 80mL of ethanol water (70 30) The extract was centrifuged at 11000 rpm for 30min supernatantwas taken for further use The fraction was concentrated and subjected to PTLC The 119877119891 value of isolated apigenin was calculated(082) Purified material was also subjected to its IR spectra LC-MS NMR and HPLC for structural elucidation The apigeninso-obtained was subjected to antibacterial activity on five pathogenic bacterial strains like Pseudomonas aeruginosa Salmonellatyphimurium Proteus mirabilis Klebsiella pneumoniae and Enterobacter aerogenes among all the bacterial strains Salmonellatyphimurium (1736 plusmn 018) andProteusmirabilis (1912 plusmn 001)have shownmaximumdiameter of inhibition zone for flavonoid andremaining bacterial strains have shownmoderate diameter of inhibition zone when compared with control values 1456 plusmn 021 and1168 plusmn 013 respectively The minimum inhibitory concentration (MIC) of the flavonoid isolated from P oleracea L was tested atthe concentration ranging from undiluted sample to 10mg permL of concentrationTheminimum inhibition concentration (MIC)for the flavonoid for all tested bacterial strains was found to be gt4mg per mL Hence the apigenin has antibacterial property andcan be used to develop antibacterial drugs

1 Introduction

Plants have potent biochemical components which arephytomedicine Plant based natural constituents can bederived from any part of the plant like bark leaves flowersroots fruits seeds and so forth and used as medicineby the man since the time immemorial [1] The benefi-cial medicinal effects of plant materials are typically theresult of combinations of secondary products present inthe plant The medicinal values of plants are unique toparticular plant species or groups and are consistent withthis concept as the combination of secondary products ina particular plant is taxonomically distinct [2] Flavonoidsare group of about 4000 naturally occurring polyphenolcompounds found universally in all the plants [3] Theseare primarily recognized as the pigments responsible forthe colors of leaves especially in autumn Flavonoids arewidely distributed in fruits vegetables nuts seeds herbsspices stems and flowers as well as tea and red wine

They are usually subdivided according to their substituentspresent in flavanols (kaempferol quercetin) anthocyaninsflavones flavonones and chalcones These flavonoids displaya remarkable array of biochemical and pharmacologicalactions namely anti-inflammatory antioxidant antiallergichepatoprotective antithrombotic antiviral and anticarcino-genic activities [4] Phytocompounds appear to play a vitalrole in defense mechanism against pathogens and predatorsand contribute to physiological functions of plant such as seedmaturation and dormancy [5] They are synthesized fromphenyl propanoid and acetate derived precursors Flavonoidsare important for humanbeings due to their antioxidative andradical scavenging effects as well as their potential as estro-genic and anticancer activities [6] In recent years antibioticresistance has become a global concern and this problem ismore important in developing country because the infectiousdiseases are still an important cause of morbidity and mor-tality among humans Plants rudely synthesize substances fortheir defense against insects herbivores andmicroorganisms

Hindawi Publishing CorporationInternational Journal of BacteriologyVolume 2014 Article ID 175851 8 pageshttpdxdoiorg1011552014175851

2 International Journal of Bacteriology

[7] Nowadays multiple drug resistance is developed dueto the indiscriminate use of drugs which are commonlyused in the treatment of infectious diseases [8] In additionto this problem antibiotics are sometimes associated withadverse effect on the host including hypersensitivity immunesuppression and allergic reaction [9] Because of side effectand resistance that the pathogenic microorganism developedagainst antibiotics recently much attention has been paidto extraction of biologically active compounds from plantsPlant based antimicrobials represent a vast updated sourceof medicine Antibacterials of plant origin have enormoustherapeutic potential They are effective in the treatment ofinfectious diseases while simultaneously mitigating many ofthe side effects that are often associatedwith synthetic antimi-crobials The plant systematic classification is as followsKingdommdashPlantae (Plants) SubkingdommdashTracheobionta(vascular plants) DivisionmdashSpermatophyte (seed plant)SubdivisionmdashAngiosperm ClassmdashDicotyledoneae OrdermdashCaryophyllales FamilymdashPortulacaceae GenusmdashPortulacaSpeciesmdasholeracea The plant P oleracea L (Purslane) is com-monly known as Porsulane an herbaceous weed This plantis an annual succulent prostrate herb stem is about 1530 cmlong reddish swollen at the nodes and quite glabrousLeaves are fresh subsessile 625mm long and alternateor subopposite Flowers are sessile axillary and terminalfew-flowered heads Microscopic analysis of the leaf powderinvariably shows spherical mineral crystals sieve plantstracheas with spiral annular and scalar form thickeningand vessels with bordered pits [10] The present study dealswith the isolation purification and identification of flavonoidapigenin from P oleracea L (Figure 6) and also determinesantibacterial activity on pathogenic bacteria

2 Materials and Methods

21 Plant Material Healthy aerial part of the plant of Poleracea L was collected from around Gulbarga Universitycampus during the month of June 2012 The plant materialwas identified and authenticated from the Department ofBotany Gulbarga University Gulbarga Karnataka (India)voucher specimen (numberHGUG-5013) has been depositedin herbarium of the same department

22 Chemicals Methanol ethanol ethyl acetate petroleumether diethyl ether H2SO4 chloroform HCl KOH hexanesilica gel 60ndash120 mesh tween 80 phosphate buffer salineFCR reagent all the chemicals solvents and reagents wereanalytical grade and were obtained from Hi media

23 Isolation of Total Flavonoids by Soxhlet ExtractionMethodBefore extraction P oleracea L was crushed into powderby versatile plant pulverize The powder of the sample wasdegreased by soxhlet extractor with petroleum ether until thecolor of elute becomes colorless The same powder samplewas accurately weighed and placed in soxhlet extractor byadding 80mL of ethanol water (70 30) solvent followed bythe extraction for up to 5 h and then extract solution was

concentrated The extract was centrifuged at 11000 rpm for30min supernatant was taken for further use [11]

24 Qualitative Test for Flavonoids Two methods were usedto determine the presence of flavonoids in the extract 5mLof dilute ammonia solution was added to a portion oftotal flavonoid extracts followed by addition of concentratedH2SO4 [12 13]

25 Determination of Total Flavonoid Content by UV-Spectrophotometric Method Firstly 2mL of the sample solu-tion was accurately removed in a volumetric flask (10mL) byadding 06mL of NaNO2 (5) solution shaking up and thenstanding for 6min Secondly 05mL of the Al(NO3)3 (10)solution was added to the volumetric flask shaken and left tostand for 6min Finally 30mL of the NaOH (43) solutionwas added to the volumetric flask followed by additionof water to the scale shaken and left to stand for 15minbefore determination Using the sample solution withoutcoloration as reference solution and 500 nm as determinationwavelength the coloration method was used to determinethe content of flavonoids in the sample by ultraviolet-visibledetector [11]

26 Separation of Bioactive Compounds by PTLC Glass plates(20 times 20 cm) thickly coated (04ndash05 nm) with silica gel ldquoGrdquo(45 g80mL water) and activated at 100∘C for 30 minutes andcooled at room temperature were used for preparative thinlayer chromatography (PTLC) The extract of flavonoid andstandard apigenin were applied on plate and developed inn-butanol-water-acetic acid solvent system (mobile phase)12 2 1 vvvThe chromatogramwas air-dried and visualizedunder visible and UV light and also in iodine chamber Thespots were marked and the 119877119891 values were calculated [14]

27 Separation of Flavonoids by Column ChromatographyThe total flavonoids which are isolated can be purified andseparated by column chromatography separation methodThe 45 cm length and 3 cm width of the column were usedand it is filled with the slurry of silica gel-H of mesh size60ndash120120583 (Hi media Mumbai) to 13 portion using n-hexaneCare should be taken to avoid the air bubble formationduring column packing Set the column by the solvent n-hexane 10 g of total flavonoid extract was bound with silicagel and loaded on the top of the column The column waseluted with gradient solvent system of n-butanol-water-aceticacid system 12 2 1 vvv until the color of the column iscolorless

28 Physicochemical Structure Elucidation of Flavonoids bySpectral Analysis The pure compound of P oleracea Lisolated was subjected to IR NMR LC-MS and HPLCstudies to obtain spectral data for the detection of functionalgroup number of protons molecular mass of the compoundand purity respectively [15]

International Journal of Bacteriology 3

29 Antimicrobial Susceptibility Test

291 Microorganisms The bacterial strains employed in thecurrent study were procured from Institute of MicrobialTechnology (IMTECH) Chandigarh (India) which includePseudomonas aeruginosa (MTCC 424) Klebsiella pneu-moniae (MTCC109) Salmonella typhimurium (MTCC98)Proteus mirabilis (MTCC425) and Enterobacter aerogenes(MTCC111)These species were originally isolated from clini-cal samples and identified by standard biochemical reactions

292 Media Nutrient broth (Hi Media M002) containspeptic digest of animal tissue (5 gL) yeast extract (150 gL)and beef extract (15 gL) were used for the growth of bacterialcultures Antibiotic assay media No 11 (Hi Media MM004)containing Peptic digest of Animal tissue (6 gL) Caseinenzyme hydrolysed (4 gL) Yeast extract (150 gL) Dextrose(100 gL) Agar (1500 gL) was used for antibacterial activity

293 Disc Diffusion Assay The antimicrobial activity ofapigenin was evaluated using a slightly modified agar discdiffusion method [16] A bacterial culture grown for 18 h wasserially diluted in 9mLof 01peptone to obtain 105 CFUmLand 100 120583L spread on the surface of Mueller Hinton (MH)agar in Petri plates An aliquot (10 120583L) of apigenin waspipetted on a sterile paper disc (Whatman No 1 55mmpaper disc) on the agar surface A disc impregnated with analiquot (10 120583L) of streptomycin (Fluka Switzerland) servedas a positive control on the same plate The plates wereinverted and incubated for 18 h at 37∘C Microbial inhibitionwas determined by measuring the diameter of the clear zoneof inhibition of growth around each disc and recorded asdiameter of inhibition zone (DIZ) in millimeter All assayswere performed using a randomized complete block designwith two replicates

294 Broth Dilution Assay To determine the minimalinhibitory concentration (MIC) quantitative serial dilutionsof apigenin were tested against five pathogenic bacterialstrains with some modifications of the method describedby Hufford and Clark [17 18] Twofold serial dilution ofapigenin was made with MH broth After adding 20120583L ofapigenin to the first tube containing 1mL of MH broth serialtransfers were made through to the fourth tube A 05mLaliquot (5 times 105 CFUmL) of test microorganism was addedto each tube A control tube contained 10 120583L of streptomycinin MH broth and microorganism was maintained Tubeswere subsequently incubated at 37∘CThe tubes were visuallyexamined for the lowest concentration of apigenin thatshowed inhibition of microbial growth (indicated by a clearsolution) after 24 h and 48 h The concentration in the lowestserial dilution of the apigenin at which growth did not occuron broth was recorded as the MIC Finally the pH valueof the inoculated broth containing the MIC of apigeninwas measured Buffering of broth containing apigenin of Poleracea L was done using NaOH and subjected to brothdilution assay as the above to determine the MIC values

Table 1 119877119891 values of isolated bioactive compound and standardapigenin

Sl number Samples Solvent system(12 2 1 vvv) 119877119891 values

1 Standardapigenin

n-butanol-water-aceticacid 082

2 Isolatedcompound


3 Results

31 Qualitative Test for Flavonoids The yellow colorationis observed after adding H2SO4 and it disappeared onstanding Few drops of 1 aluminum solution were added toa portion of flavonoid extract and again yellow colorationwasobserved this indicates the presence of flavonoids

32 Separation of Bioactive Compound by PTLC When thedeveloped plates were sprayedwith 5 ethanol ferric chloridesolution and placed in iodine chamber they showed a spotwhich has a brownish color The 119877119891 value (082) of apigeninisolated from the P oleracea L coincided with the 119877119891 value ofstandard apigenin The results of PTLC 119877119891 values are shownin Table 1

33 Determination of Total Flavonoid Content by UV-SpectrophotometricMethod Thebasic structure of flavonoidswas presented in Figure 1(a) and most of the flavonoidspresent in P oleracea L will be having 3101584041015840-dihydroxy-substituted structure as shown in Figure 1(b) Flavonoidswith3101584041015840-dihydroxy-substituted structure have shown a specialcolor by reacting with the system of NaNO2-Al(NO3)3-NaOH The color reaction of flavonoids and chromogenicsystem is presented in Figure 1(c) This method is basedon the reaction of aluminum ion with flavonoid at alkalinemedium forming red chelates By measuring the absorptionof such red chelates it is possible to determine the flavonoids

34 Separation of Flavonoids by Column ChromatographyThe total flavonoids of ethanol extract of P oleracea L ofabout 20 g were fractionated on a silica gel-H (60ndash120 mesh)column at room temperature and pressure 260C 1 bar Thetotal 32 fractions of 100mL of each were collected Each ofthe elutes was then crystallized with chloroformThe purifiedcompoundwas subjected to its spectral analysis for structuralelucidation

35 Physicochemical Structure Elucidation of Flavonoids bySpectral Analysis

FT-IR It can be resolved from the IR spectra that thereare intensive bands in the 110ndash1000 cmminus1 span that arecharacteristics of glycoside bonds and not bands of anysugar type There are bonds of VCH vibration of CH2group at approximately 2952 cmminus1 and 2842 cmminus1 and 120575CHvibration at approximately 1451 cmminus1 In the spectrum of VOH


O1

2

3

45

6

7

81998400

2998400 3998400

4998400

59984006998400

OH

1

OH

45

6

7

8O

3

2 1998400

2998400 3998400

4998400

59984006998400

(a)

(b)

O

O

OH

Oxidation

Orange

N

O

O

O

O

ONitrosylation

OO

N

O

O

O

Quino-structure

HN

(c)Red

2Al3+3

2Al3+32Al3+3

NaNO2H+

HNO2

+NaOH

Ominus

OH + NaNO2

+13Al3+ + HNO2

Figure 1 The color reaction of flavonoids and chromogenic system

3612705350

3103525542

2952385996

1842066534

2133617287

1647575925

1451747293

1407817236

1110487515

1011926585

100

90

80

70

60

50

40

30

20

0

10

minus10

minus20

4000

3600

3200

3800

2400

2000

1800

1600

1400

1200

1000

800

600

400

(cmminus1)

T(

)

Figure 2 FT-IR spectrum of isolated compound apigenin from Poleracea L

vibration there are bonds which are found at approximately3612 cmminus1 and others at approximately 3103 cmminus1 that aremost probably the result of VOH vibration of phenol OHgroups The intensive band at approximately 1647 cmminus1 ismost probably the result of VC=O vibration of C=O group

from central heterocyclic ring while the VCminusO vibrationoccurs at approximately 1110 cmminus1 With these above spectralcharacteristics it is indicated that the probable compound isapigenin (Figure 2)

1H NMR The 1H NMR spectrum shows two singlets at 1032and 1745 120575 (ppm) resonated tomultiple of two protons of aro-matic systemsThe compound singlet peak at 120575 799 ppm (1H)was indicated for aromatic proton of phenolic hydroxyl Theidentification of purified compound was further confirmedby 1H spectrum data available in literature (Figure 3)

LC-MS The mass spectrum that displayed a molecular ion(M+) peak atmz 26919 indicates themolecular weight of thecompound corresponds to the molecular weight of apigeninand also corresponds to molecular formula C15H10O5 Thisfurther confirms that the structure of the isolated compoundis apigenin (Figure 4)

HPLC Chromatographic system and conditions are the LCsystem consisted of a Waters 600 solvent delivery pumpa Waters 2487 UV-visible spectrophotometric detector anEmpower system controller (Waters USA) and HPLC ana-lytical column (Diamonsil C 18 Column 46 times 200mm id5 120583M particle size Dikma) connected with a guard columnfilled with the same chromatographic stationary phase Themobile phase for HPLC analysis consisting ofMethanol-02Phosphoric acid (45 55 vv) were filtered under reduced


10 9 8 7 6 5 4 3 2 1

30624

31985

228825

5600

6629

12274

7997

1032

1745

(ppm)

HBNAYAK

Sample A

1H in CD3OD

New Avance 300MHz

Figure 3 NMR spectrum of isolated compound apigenin from P oleracea L

pressure and degassed by ultrasonic before use HPLC anal-ysis with UV detection at 350 nm was performed at a flowrate of 10mLmin HPLC column temperature was 35∘CThe sample injection volumewas 10120583LThe chromatographicpeak of the apigenin is shown in the chromatogram inFigure 5

36 Antibacterial Susceptibility Test

361 Disc Diffusion Assay The mean diameters of theinhibition zones of apigenin against five pathogenic bacterialstrains are shown in Table 2 The results from disc diffusionassay showed that among all bacterial strains S typhimurium(1736 plusmn 018) and P mirabilis (1912 plusmn 001) have shownsignificant (119875 lt 005) zone of inhibition where remainingbacterial strains have shown less significant (119875 lt 005) zoneof inhibition when compared with control values (1456 plusmn021) and (1168 plusmn 013) respectively

362 Broth Dilution Assay The results of the MIC valuesof apigenin of P oleracea L obtained at 24 h and 48 hare presented in Table 3 The present experimental datademonstrated that the apigenin of P oleracea L has displayedthe antibacterial activity with MIC value gt 4mgmL againstall pathogenic bacterial strains which were subjected in thepresent study The MIC values did not change after 48 h andalso did not correlate well with the diameter of inhibitionzones from the disc diffusion assay

4 Discussion

The present study is conducted to extract flavonoids of Poleracea L by modified method of previous study [11] Thismethod of extraction was the best which had the highestcontent of quercetin rutin and apigenin which are glycol-flavones with a low impurity More than 2000 flavonoidshave been reported among woody and nonwoody plants [19]PTLC UV and IR spectral studies have provided a newdimension to the chemistry of flavonoids to such an extentthat their presence has become important in taxonomic study[20] Presence of flavonoids has been reported in manyplant species like Lycium barbarum Passiflora palmer Cassiaangustifolia and Jatropha curcas L [21ndash24] Apigenin hasbeen reported in plant species like Bellis perennis L andAdinandra nitida [15 25] It is well known that apigenin hasan antioxidant anticarcinogenic and spasmolytic activitiesand can reduce high blood pressure That is why leavesof P oleracea L are a good source of apigenin whichcan be added to food as a kind of functional ingredient orused as a vegetable which have many beneficial effects onhuman health It can also be used in medicine in standardforms of administration such as capsules tablets and oralsuspensions [26] The extraction isolation purification andcharacterization of this compound from P oleracea L andstructural elucidation of this compound have been studiedIn the present study the results of qualitative tests (specificfor flavonoids) of flavonoids can be comparedwith the resultsof previous studies conducted on phytotoxicity and antimi-crobial activities of flavonoids in Ocimum gratissimum [27]


200 250 300 350 400 450 500 550 600 650 700 750 8000102030405060708090100

Rela

tive a

bund

ance

21422

26919

25232

27039

52006

33010

39122

40988

27919

31020

21526

42025

46501

33113

52118

37411

50304

60546

31128

66076

77085

43617

67098

47412

61599

71560

59306

54908

56406

79718

74612

(mz)

HBNayaka EWF 481ndash533 RT 1179ndash1315 AV 53 NL 216E6F +c ESI Full ms [20000ndash200000]

Figure 4 LC-MS spectrum of isolated compound apigenin from P oleracea L

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

1037

1386

1222

1437

1508

2668

1655

1836

2936

3005

2016

2200

2527

3555

718

3918

3144

3655

4148

5997

5793

5615

5430

5286

5060

4941

4549

4457

686

461

325

100

Time (min)

NL129E6SpectrumMaximumnm = 2000ndash8000 PDAHBNayakaEWF

RT 000ndash6000

(120583AU

)

01 times 1052 times 1053 times 1054 times 1055 times 1056 times 1057 times 1058 times 1059 times 10510 times 10511 times 10512 times 105

Figure 5 HPLC chromatogram of isolated compound apigenin from P oleracea L

OH

OHOH

OH

OH

OH

O

O

O O

Figure 6 Structure of flavonoid apigenin isolated from P oleracea L

Table 2 Antimicrobial activity (diameter of inhibition zone) of apigenin of P oleracea L on pathogenic bacteria

Bacterial strains MTCC number Diameter of inhibition zone (mm)Streptomycin as control (10120583L) Apigenin (10120583L)

P aeruginosa MTCC424 1742 plusmn 054 1224 plusmn 041

K pneumoniae MTCC109 2133 plusmn 033 1052 plusmn 038

S typhimurium MTCC98 1456 plusmn 021 1736 plusmn 018lowastlowast

P mirabilis MTCC425 1168 plusmn 013 1912 plusmn 001lowastlowastlowast

E aerogenes MTCC111 1966 plusmn 027 1402 plusmn 003

lowastlowastModerate significant lowastlowastlowastmost significant at 119875 le 005 Values are expressed as mean plusmn SD


Table 3 Antibacterial activity (minimum inhibition concentrationafter 24 h and 48 h mgmL) of apigenin of P oleracea L

Bacterial strains MTCC number Apigenin of P oleracea LP aeruginosa MTCC424 gt4K pneumoniae MTCC109 gt4S typhimurium MTCC98 gt4P mirabilis MTCC425 gt4E aerogenes MTCC111 gt4

In the present study the flavonoids contents are determinedbyNaNO2-Al(NO3)3-NaOH colorimetricmethodThe resultindicates that theP oleracea Lhas containedmore flavonoidsBasic structure of flavonoidswas presented in Figure 1(a) andmost of the flavonoids in P oleracea L have 3101584041015840-dihydroxy-substituted structure as shown in Figure 1(b) Flavonoidswith3101584041015840-dihydroxy-substituted structure can show special colorby reacting with the system of NaNO2-Al(NO3)3-NaOHThe color reaction of flavonoids and chromogenic systemis presented in Figure 1 As shown in Figure 1 this methodis based on the reaction of aluminum ion with flavonoidat alkaline medium forming red chelates By measuring theabsorption of such red chelates it is possible to determinethe flavonoids [11] The structural elucidation of apigenin ispossible because of spectral data obtained In the presentstudy the IR spectrum has vibration bonds at 3612 cmminus1and 3103 cmminus1 approximate which are most probably theresult of VOH vibration of phenol OH groups The intensiveband at approximately 1647 cmminus1 is most probably the resultof VC=O vibration of C=O group from central heterocyclicring Similar results were obtained in the study conductedfor extraction of apigenin from the sage(Salvia officinalis L)from Jordan [14] The LC-MS spectrum that displayed themolecular ion (M+) peak at mz 26919 in the present studyindicates the molecular weight of the apigenin compoundthis result is similar to the LC-MS spectrum study conductedby [15] in the plant Adinandra nitida For the confirmation ofpurity the compound is again subjected to HPLC analysisthe results of HPLC chromatogram showed single basepeak at 103 AU it is again conformed with the compoundapigenin Nowadays multiple drug resistance developed dueto the indiscriminate use of drugs commonly used in thetreatment of infectious diseases Unfortunately bacteria havegenetic ability to transmit and acquire resistance to drugsand chemicals [28] Based on the results of antibacterialstudies shown in Table 2 the apigenin had an antibacterialactivity more significant on S typhimurium and P mirabiliswhen compared with control group This may be due to thechemical nature of apigenin cell membrane permeabilityand other factors In general composition of the inhibitionzones diameter showed that apigenin was more effectiveagainst both gram positive and gram negative bacteria Thisdifference may be due to several possible reasons such aspermeability barrier provided by the presence of cell wallwith multilayer structure in gram negative bacteria or themembrane accumulationmechanism or presence of enzymesin periplasmic space which are able to break down foreign

molecules introduced from outside [29] The MIC value(Table 3) for apigenin isolated from P oleracea L against allthe pathogenic bacterial strains which were subjected in thepresent study was gt4mgmL it is reported that for bacterialantimicrobials the MIC was often near or Aquila values [30]

5 Conclusion

Theflavonoids of P oleracea Lwere isolated by employing theextraction with ethanol water (70 30) solution The resultof qualitative test which is specific for flavonoids confirmedthat the isolated compound was flavonoid The results ofPTLC HPLC LC-MS FT-IR and NMR further confirmedthat the compound is apigenin The antibacterial results ofthe present study will suggest that the isolated apigeninbioactive compound is also having potential antibacterialactivity indicating that it can be used for development ofantibacterial drugs for the treatment of diseases associatedwith these pathogenic bacteria

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful to University Grant CommissionNew Delhi (India) for providing financial support to carryout this research work under the scheme of Rajeev GandhiNational Fellowship (UGC-RGNF) The authors are alsothankful to Gulbarga University Gulbarga for providing allthe necessary facilities to carry out this research

References

[1] M C Gordon and J N David ldquoNatural product drug discoveryin the next millenniumrdquo Pharmaceutical Biology vol 39 pp 8ndash17 2001

[2] M Wink ldquoIntroduction biochemistry role and biotechnologyof secondary productsrdquo in Biochemistry of Secondary ProductMetabolism M Wink Ed pp 1ndash16 CRC Press Boca RatomFla USA 1999

[3] J B Harborne ldquoNature distribution and function of plantflavonoidsrdquo Progress in Clinical and Biological Research vol 213pp 15ndash24 1986

[4] E Middleton and C Kandaswami ldquoThe impact of plantflavonoids on mammalian biology implications for immunityinflammation and cancer in the flavonoidsrdquo in Advances inResearch Science I R Harborne Ed pp 619ndash645 Chapmanand Hall London UK 1993

[5] B Winkel-Shirley ldquoBiosynthesis of flavonoids and effects ofstressrdquo Current Opinion in Plant Biology vol 5 no 3 pp 218ndash223 2002

[6] K Springob and K Saito ldquoMetabolic engineering of plantsecondary metabolism promising approach to the productionof pharmaceuticalsrdquo Science and Culture vol 68 pp 76ndash852002


[7] O O Aboaba S I Smith and F O Olude ldquoAntibacterialeffect of edible plant extract on Escherichia coli 0157 H7 partrdquoPakistan Journal of Nutrition vol 5 no 4 pp 325ndash327 2006

[8] J Davies ldquoInactivation of antibiotics and the dissemination ofresistance genesrdquo Science vol 264 no 5157 pp 375ndash382 1994

[9] I Ahmad ZMehmood and FMohammad ldquoScreening of someIndian medicinal plants for their antimicrobial propertiesrdquoJournal of Ethnopharmacology vol 62 no 2 pp 183ndash193 1998

[10] G Banerjee and A Mukherjee ldquoAntibacterial activity of acommon weed Portulaca oleracea Lrdquo Geobios vol 30 pp 143ndash144 2003

[11] H Zhu Y Wang Y Liu Y Xia and T Tang ldquoAnalysis offlavonoids in Portulaca oleracea L by UV-vis spectrophotom-etry with comparative study on different extraction technolo-giesrdquo Food Analytical Methods vol 3 no 2 pp 90ndash97 2010

[12] A Sofowara Medicinal Plants and Traditional Medicine inAfrica Spectrum Books Ibadan Nigeria 1993

[13] J B Harbone Phytochemical Methods pp 49ndash188 Chapmanand Hall London UK 1973

[14] D Sinisa C Milorad and A Salameh ldquoThe extraction ofapigenin and luteolinfrom the sage Salvia officinalis L fromJordanrdquoThe Scientific Journal Factauniversities Series Workingand Living Environmental Protection vol 1 no 5 pp 87ndash932000

[15] B Liu Z Ning J Goo and K Xu ldquoPreparing apigenin fromleaves of Adinandranitidardquo Food Technology and Biotechnologyvol 46 no 1 pp 111ndash115 2008

[16] A L Barry The Antimicrobial Suseptabalitytest Principale andPractices Lea and Febiger Philadelphia Pa USA 1976

[17] C D Hufford and A M Clark ldquoDiscovery and development ofnew drugs for systemic opportunistic infectionrdquo in Studyies inNatural Products Chemistry A-U Rahman Ed pp 421ndash452Elsevier 1988

[18] N S Weerakkody N Caffin M S Turner and G A DykesldquoIn vitro antimicrobial activity of less-utilized spice and herbextracts against selected food-borne bacteriardquoFoodControl vol21 no 10 pp 1408ndash1414 2010

[19] J B Harborne ldquoPlant phenolicrdquo in Secondary Plant ProductsE A Bell and B V Charlewood Eds p 320 Springer BerlinGermany 1980

[20] E B Smith Prospective in Phytochemistry J B Harborne and TSwain Eds Academic Press London UK 1969

[21] M L Harsh T N Nag and S Jain ldquoArid zone plants ofRajasthan a source of Antimicrobialsrdquo Communication in Phys-iological Ecology vol 8 pp 129ndash131 1983

[22] A Ulubelen T J Mabry G Dellamonica and J ChopinldquoFlavonoids from Passiflora palmerirdquo Journal of Natural Prod-ucts vol 47 no 2 pp 384ndash385 1984

[23] A Goswami and A Reddi ldquoAntimicrobial activity of flavonoidsof medicinallyimportant plant Cassia angustifolia in vivo and invitrordquo Journal of Phytological Research vol 17 pp 179ndash181 2004

[24] S Saxena R Sharma S Rajore and A Batra ldquoIsolation andidentification of flavonoid ldquoVitexinrdquo from Jatrophacurcas LrdquoJournal of Plant Sciences Research vol 21 pp 116ndash117 2005

[25] J Nazaruk and J Gudej ldquoApigenin glycosides from the flow-ers of Bellis perennis Lrdquo Acta Poloniae PharmaceuticamdashDrugResearch vol 57 no 2 pp 129ndash130 2000

[26] C Engelmann E Blot Y Panis et al ldquoApigeninmdashstrongcytostatic and anti-angiogenic action in vitro contrasted by lackof efficacy in vivordquo Phytomedicine vol 9 no 6 pp 489ndash4952002

[27] O Ighodaro A S Macdonald and A O Oludare ldquoPhytotoxicand anti-microbial activities of flavonoids in Ocimum gratissi-mumrdquo Life Science Journal vol 7 no 3 pp 45ndash48 2010

[28] G G F Nascimento J Locatelli P C Freitas and G L SilvaldquoAntibacterial activity of plant extracts and phytochemicals onantibiotic-resistant bacteriardquo Brazilian Journal of Microbiologyvol 31 no 4 pp 247ndash256 2000

[29] J Parekh and S Chanda ldquoAntibacterial and phytochemicalstudies on twelve species of Indian medicinal plantsrdquo AfricanJournal of Biomedical Research vol 10 pp 175ndash181 2007

[30] K D Reuben F I Abdulrahman J C Akan H Usman OA Sodipo and G O Egwu ldquoPhytochemical screening and invitro antimicrobial investigation of the Methanolic extract ofCroton zambesicus muell ARG stem barkrdquo European Journalof Scientific Research vol 23 no 1 pp 134ndash140 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


[7] Nowadays multiple drug resistance is developed dueto the indiscriminate use of drugs which are commonlyused in the treatment of infectious diseases [8] In additionto this problem antibiotics are sometimes associated withadverse effect on the host including hypersensitivity immunesuppression and allergic reaction [9] Because of side effectand resistance that the pathogenic microorganism developedagainst antibiotics recently much attention has been paidto extraction of biologically active compounds from plantsPlant based antimicrobials represent a vast updated sourceof medicine Antibacterials of plant origin have enormoustherapeutic potential They are effective in the treatment ofinfectious diseases while simultaneously mitigating many ofthe side effects that are often associatedwith synthetic antimi-crobials The plant systematic classification is as followsKingdommdashPlantae (Plants) SubkingdommdashTracheobionta(vascular plants) DivisionmdashSpermatophyte (seed plant)SubdivisionmdashAngiosperm ClassmdashDicotyledoneae OrdermdashCaryophyllales FamilymdashPortulacaceae GenusmdashPortulacaSpeciesmdasholeracea The plant P oleracea L (Purslane) is com-monly known as Porsulane an herbaceous weed This plantis an annual succulent prostrate herb stem is about 1530 cmlong reddish swollen at the nodes and quite glabrousLeaves are fresh subsessile 625mm long and alternateor subopposite Flowers are sessile axillary and terminalfew-flowered heads Microscopic analysis of the leaf powderinvariably shows spherical mineral crystals sieve plantstracheas with spiral annular and scalar form thickeningand vessels with bordered pits [10] The present study dealswith the isolation purification and identification of flavonoidapigenin from P oleracea L (Figure 6) and also determinesantibacterial activity on pathogenic bacteria

2 Materials and Methods

21 Plant Material Healthy aerial part of the plant of Poleracea L was collected from around Gulbarga Universitycampus during the month of June 2012 The plant materialwas identified and authenticated from the Department ofBotany Gulbarga University Gulbarga Karnataka (India)voucher specimen (numberHGUG-5013) has been depositedin herbarium of the same department

22 Chemicals Methanol ethanol ethyl acetate petroleumether diethyl ether H2SO4 chloroform HCl KOH hexanesilica gel 60ndash120 mesh tween 80 phosphate buffer salineFCR reagent all the chemicals solvents and reagents wereanalytical grade and were obtained from Hi media

23 Isolation of Total Flavonoids by Soxhlet ExtractionMethodBefore extraction P oleracea L was crushed into powderby versatile plant pulverize The powder of the sample wasdegreased by soxhlet extractor with petroleum ether until thecolor of elute becomes colorless The same powder samplewas accurately weighed and placed in soxhlet extractor byadding 80mL of ethanol water (70 30) solvent followed bythe extraction for up to 5 h and then extract solution was

concentrated The extract was centrifuged at 11000 rpm for30min supernatant was taken for further use [11]

24 Qualitative Test for Flavonoids Two methods were usedto determine the presence of flavonoids in the extract 5mLof dilute ammonia solution was added to a portion oftotal flavonoid extracts followed by addition of concentratedH2SO4 [12 13]

25 Determination of Total Flavonoid Content by UV-Spectrophotometric Method Firstly 2mL of the sample solu-tion was accurately removed in a volumetric flask (10mL) byadding 06mL of NaNO2 (5) solution shaking up and thenstanding for 6min Secondly 05mL of the Al(NO3)3 (10)solution was added to the volumetric flask shaken and left tostand for 6min Finally 30mL of the NaOH (43) solutionwas added to the volumetric flask followed by additionof water to the scale shaken and left to stand for 15minbefore determination Using the sample solution withoutcoloration as reference solution and 500 nm as determinationwavelength the coloration method was used to determinethe content of flavonoids in the sample by ultraviolet-visibledetector [11]

26 Separation of Bioactive Compounds by PTLC Glass plates(20 times 20 cm) thickly coated (04ndash05 nm) with silica gel ldquoGrdquo(45 g80mL water) and activated at 100∘C for 30 minutes andcooled at room temperature were used for preparative thinlayer chromatography (PTLC) The extract of flavonoid andstandard apigenin were applied on plate and developed inn-butanol-water-acetic acid solvent system (mobile phase)12 2 1 vvvThe chromatogramwas air-dried and visualizedunder visible and UV light and also in iodine chamber Thespots were marked and the 119877119891 values were calculated [14]

27 Separation of Flavonoids by Column ChromatographyThe total flavonoids which are isolated can be purified andseparated by column chromatography separation methodThe 45 cm length and 3 cm width of the column were usedand it is filled with the slurry of silica gel-H of mesh size60ndash120120583 (Hi media Mumbai) to 13 portion using n-hexaneCare should be taken to avoid the air bubble formationduring column packing Set the column by the solvent n-hexane 10 g of total flavonoid extract was bound with silicagel and loaded on the top of the column The column waseluted with gradient solvent system of n-butanol-water-aceticacid system 12 2 1 vvv until the color of the column iscolorless

28 Physicochemical Structure Elucidation of Flavonoids bySpectral Analysis The pure compound of P oleracea Lisolated was subjected to IR NMR LC-MS and HPLCstudies to obtain spectral data for the detection of functionalgroup number of protons molecular mass of the compoundand purity respectively [15]









1 Standardapigenin


2 Isolatedcompound


3 Results








O1

2

3

45

6

7

81998400

2998400 3998400

4998400

59984006998400

OH

1

OH

45

6

7

8O

3

2 1998400

2998400 3998400

4998400

59984006998400

(a)

(b)

O

O

OH

Oxidation

Orange

N

O

O

O

O

ONitrosylation

OO

N

O

O

O

Quino-structure

HN

(c)Red

2Al3+3

2Al3+32Al3+3

NaNO2H+

HNO2

+NaOH

Ominus

OH + NaNO2

+13Al3+ + HNO2


3612705350

3103525542

2952385996

1842066534

2133617287

1647575925

1451747293

1407817236

1110487515

1011926585

100

90

80

70

60

50

40

30

20

0

10

minus10

minus20

4000

3600

3200

3800

2400

2000

1800

1600

1400

1200

1000

800

600

400

(cmminus1)

T(

)








10 9 8 7 6 5 4 3 2 1

30624

31985

228825

5600

6629

12274

7997

1032

1745

(ppm)

HBNAYAK

Sample A

1H in CD3OD

New Avance 300MHz






4 Discussion



200 250 300 350 400 450 500 550 600 650 700 750 8000102030405060708090100

Rela

tive a

bund

ance

21422

26919

25232

27039

52006

33010

39122

40988

27919

31020

21526

42025

46501

33113

52118

37411

50304

60546

31128

66076

77085

43617

67098

47412

61599

71560

59306

54908

56406

79718

74612

(mz)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

1037

1386

1222

1437

1508

2668

1655

1836

2936

3005

2016

2200

2527

3555

718

3918

3144

3655

4148

5997

5793

5615

5430

5286

5060

4941

4549

4457

686

461

325

100

Time (min)


RT 000ndash6000

(120583AU

)



OH

OHOH

OH

OH

OH

O

O

O O















5 Conclusion




Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









1 Standardapigenin


2 Isolatedcompound


3 Results








O1

2

3

45

6

7

81998400

2998400 3998400

4998400

59984006998400

OH

1

OH

45

6

7

8O

3

2 1998400

2998400 3998400

4998400

59984006998400

(a)

(b)

O

O

OH

Oxidation

Orange

N

O

O

O

O

ONitrosylation

OO

N

O

O

O

Quino-structure

HN

(c)Red

2Al3+3

2Al3+32Al3+3

NaNO2H+

HNO2

+NaOH

Ominus

OH + NaNO2

+13Al3+ + HNO2


3612705350

3103525542

2952385996

1842066534

2133617287

1647575925

1451747293

1407817236

1110487515

1011926585

100

90

80

70

60

50

40

30

20

0

10

minus10

minus20

4000

3600

3200

3800

2400

2000

1800

1600

1400

1200

1000

800

600

400

(cmminus1)

T(

)








10 9 8 7 6 5 4 3 2 1

30624

31985

228825

5600

6629

12274

7997

1032

1745

(ppm)

HBNAYAK

Sample A

1H in CD3OD

New Avance 300MHz






4 Discussion



200 250 300 350 400 450 500 550 600 650 700 750 8000102030405060708090100

Rela

tive a

bund

ance

21422

26919

25232

27039

52006

33010

39122

40988

27919

31020

21526

42025

46501

33113

52118

37411

50304

60546

31128

66076

77085

43617

67098

47412

61599

71560

59306

54908

56406

79718

74612

(mz)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

1037

1386

1222

1437

1508

2668

1655

1836

2936

3005

2016

2200

2527

3555

718

3918

3144

3655

4148

5997

5793

5615

5430

5286

5060

4941

4549

4457

686

461

325

100

Time (min)


RT 000ndash6000

(120583AU

)



OH

OHOH

OH

OH

OH

O

O

O O















5 Conclusion




Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


O1

2

3

45

6

7

81998400

2998400 3998400

4998400

59984006998400

OH

1

OH

45

6

7

8O

3

2 1998400

2998400 3998400

4998400

59984006998400

(a)

(b)

O

O

OH

Oxidation

Orange

N

O

O

O

O

ONitrosylation

OO

N

O

O

O

Quino-structure

HN

(c)Red

2Al3+3

2Al3+32Al3+3

NaNO2H+

HNO2

+NaOH

Ominus

OH + NaNO2

+13Al3+ + HNO2


3612705350

3103525542

2952385996

1842066534

2133617287

1647575925

1451747293

1407817236

1110487515

1011926585

100

90

80

70

60

50

40

30

20

0

10

minus10

minus20

4000

3600

3200

3800

2400

2000

1800

1600

1400

1200

1000

800

600

400

(cmminus1)

T(

)








10 9 8 7 6 5 4 3 2 1

30624

31985

228825

5600

6629

12274

7997

1032

1745

(ppm)

HBNAYAK

Sample A

1H in CD3OD

New Avance 300MHz






4 Discussion



200 250 300 350 400 450 500 550 600 650 700 750 8000102030405060708090100

Rela

tive a

bund

ance

21422

26919

25232

27039

52006

33010

39122

40988

27919

31020

21526

42025

46501

33113

52118

37411

50304

60546

31128

66076

77085

43617

67098

47412

61599

71560

59306

54908

56406

79718

74612

(mz)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

1037

1386

1222

1437

1508

2668

1655

1836

2936

3005

2016

2200

2527

3555

718

3918

3144

3655

4148

5997

5793

5615

5430

5286

5060

4941

4549

4457

686

461

325

100

Time (min)


RT 000ndash6000

(120583AU

)



OH

OHOH

OH

OH

OH

O

O

O O















5 Conclusion




Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


10 9 8 7 6 5 4 3 2 1

30624

31985

228825

5600

6629

12274

7997

1032

1745

(ppm)

HBNAYAK

Sample A

1H in CD3OD

New Avance 300MHz






4 Discussion



200 250 300 350 400 450 500 550 600 650 700 750 8000102030405060708090100

Rela

tive a

bund

ance

21422

26919

25232

27039

52006

33010

39122

40988

27919

31020

21526

42025

46501

33113

52118

37411

50304

60546

31128

66076

77085

43617

67098

47412

61599

71560

59306

54908

56406

79718

74612

(mz)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

1037

1386

1222

1437

1508

2668

1655

1836

2936

3005

2016

2200

2527

3555

718

3918

3144

3655

4148

5997

5793

5615

5430

5286

5060

4941

4549

4457

686

461

325

100

Time (min)


RT 000ndash6000

(120583AU

)



OH

OHOH

OH

OH

OH

O

O

O O















5 Conclusion




Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


200 250 300 350 400 450 500 550 600 650 700 750 8000102030405060708090100

Rela

tive a

bund

ance

21422

26919

25232

27039

52006

33010

39122

40988

27919

31020

21526

42025

46501

33113

52118

37411

50304

60546

31128

66076

77085

43617

67098

47412

61599

71560

59306

54908

56406

79718

74612

(mz)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

1037

1386

1222

1437

1508

2668

1655

1836

2936

3005

2016

2200

2527

3555

718

3918

3144

3655

4148

5997

5793

5615

5430

5286

5060

4941

4549

4457

686

461

325

100

Time (min)


RT 000ndash6000

(120583AU

)



OH

OHOH

OH

OH

OH

O

O

O O















5 Conclusion




Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusion




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

research article antibacterial attributes of apigenin...

Documents