Research Article Phytochemical Evaluation, Antimicrobial ...downloads.hindawi.com/journals/bmri/2014/497606.pdf · have been isolated from various parts of the plant, namely phenols,

Research ArticlePhytochemical Evaluation Antimicrobial Activityand Determination of Bioactive Components from Leaves ofAegle marmelos

Farina Mujeeb Preeti Bajpai and Neelam Pathak

Department of Biosciences Integral University Kursi Road Lucknow 226026 India

Correspondence should be addressed to Neelam Pathak pathak neelamyahoocom

Received 29 January 2014 Revised 19 April 2014 Accepted 23 April 2014 Published 11 May 2014

Academic Editor Paul M Tulkens

Copyright copy 2014 Farina Mujeeb et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The therapeutic value of Aegle marmelos Correa (Rutaceae) commonly known as ldquoBaelrdquo has been recognized as a component oftraditional medication for the treatment of various human ailments The plant though being highly explored still lacks sufficientevidences for the best variety possessing the highest degree of medicinal values The present study is focused on phytochemicalscreening of aqueous and methanolic leaf extracts of 18 varietiesaccessions of A marmelos The crude extracts of A marmelosrevealed the presence of several biologically active phytochemicals with the highest quantity of alkaloids flavonoids and phenolsin Pant Aparna variety The antibacterial efficacy was investigated against pathogenic bacterial strains and the highest inhibitoryactivity of aqueous extract was obtained against S epidermidis whereas methanolic extract was found to be most potent against Saureus at 40mgmL concentration However in aqueous ethanol the best results were observed against E aerogenes followed byKpneumonia and S epidermidis The MIC of aqueous and methanol extract of Aegle marmelos ranged from 10mgmL to 40mgmLwhereas in aqueous ethanol it ranged between 40mgmL and 160mgmL The GC-MS analysis revealed the presence of manybioactive compounds such as flavonoids alcohols aldehydes aromatic compounds fatty acid methyl esters terpenoids phenolicsand steroids that can be postulated for antibacterial activity

1 Introduction

India is widely known as the botanical garden of theworld since it is the largest producer of medicinal herbs[1] Medicinal plants act as an indigenous source of newcompounds possessing therapeutic value and can also be usedin drug development 80 of the population of developingcountries depend on traditional medicines mostly naturalplant products for their primary health care needs as esti-mated by WHO [2] Because of the growing recognition ofnatural products the demand for medicinal plants has beenincreasing all over the world They have minimal toxicityare cost effective and pharmacologically active and providean easy remedy for many human ailments as compared tothe synthetic drugs which are a subject of adulteration andside effects [3] The alarming increase in the rate of infectionby antibiotic-resistant microorganisms has urged scientiststo search for compounds which have potential antimicrobial

activity [4] The ability to synthesize compounds by sec-ondarymetabolism possessing antimicrobial potentialmakesplants an invaluable source of pharmaceutical and thera-peutic products [5] The effectiveness of plant extracts onmicroorganism has been studied worldwide [6ndash9]

Bael (Aegle marmelos) has been known to be one ofthe most important medicinal plants of India since Charak(1500 BC) [10] More than 100 phytochemical compoundshave been isolated from various parts of the plant namelyphenols flavonoids alkaloids cardiac glycosides saponinsterpenoids steroids and tannins These compounds are wellknown to possess biological and pharmacological activityagainst various chronic diseases such as cancer and cardio-vascular and gastrointestinal disorders [11ndash14] Antioxidantantiulcer antidiabetic anticancer antihyperlipidaemic anti-inflammatory antimicrobial antispermatogenic effects havealso been reported on various animal models by the crudeextracts of this plant [14ndash22] Every part of Aegle marmelos

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 497606 11 pageshttpdxdoiorg1011552014497606

2 BioMed Research International

plant such as its fruits stem bark and leaves possessesmedicinal property and is used for treating various eye andskin infections [23] Leaf is considered to be one of thehighest accumulatory parts of the plant containing bioactivecompounds which are synthesized as secondary metabolites[24] The present study was therefore aimed at evaluatingthe phytochemical potential and antibacterial activity ofAeglemarmelos aqueous and methanolic leaf extracts

2 Materials and Methods

21 Collection and Identification of Plant Material The freshleaves of Aegle marmelos from 18 varietiesaccessions werecollected from the orchard of Narendra Deva University ofAgriculture andTechnologyKumarganj Faizabad IndiaThetaxonomy of the plant was authenticated

22 Extract Preparation The leaves of the plantwere properlywashed in tap water and rinsed in distilled water The rinsedleaves were hot air-dried for 3 days The dried leaves ofeach plant were pulverized using pestle mortar to obtain apowdered form which was stored in airtight glass containersat 4∘C until used 10 g of powdered sample was soaked indistilled water andmethanol (200mL and 100mL) separatelyfor 12 hrs at room temperatureThe extracts were then filteredand concentrated to a final volume of 50mL and subjected tophytochemical analysis

For antibacterial screening the leaf extracts were preparedfrom Pant Aparna variety by following the protocol ofHarborne [25] in Soxhlet apparatus using 180mL of distilledwater and methanol For aqueous ethanol 50 130 volume ofdistilled water and ethanol were taken Every extraction wascarried out for 24 hrs and the extract was then dried weighedand stored in refrigerator at 4∘C

23 Phytochemical Analysis Qualitative phytochemical anal-yses of both the extracts were performed by following theprotocol of Adetuyi and Popoola [26] Trease and Evans [27]and Sofowora [28]

Tannins 200mg of plant material was boiled in 10mLdistilled water and few drops of FeCl

3were added to the

filtrate a blue-black precipitate indicated the presence ofTannins

Alkaloids 200mg plant material was boiled in 10mLmethanol and filtered 1HCl was added followed by 6 dropsof Dragendorff reagent and brownish-red precipitate wastaken as evidence for the presence of alkaloids

Saponins (Frothing test) 5mL distilled water was added to200mg plant material 05mL filtrate was diluted to 5mLwith distilled water and shaken vigorously for 2 minutesFormation of stable foam indicates the presence of saponins

Cardiac Glycosides (Keller-Kiliani test) 2mL filtrate wastreated with 1mL glacial acetic acid containing few drops ofFeCl3Conc H

2SO4was added to the above mixture giving

green-blue colour depicting the positive results for presenceof cardiac glycosides

Steroids (Liebermann-Burchard reaction) 200mg plant mate-rial was added in 10mL chloroform Acetic anhydride wasadded in the ratio of 1 1 which resulted into the formationof blue-green ring pointing towards the presence of steroids

Terpenoids (Salkowski test) To 200mg plant material 2mL ofchloroform (CHCl

3) and 3mL of concentrated sulphuric acid

(H2SO4) were carefully added A reddish brown colouration

signified the presence of terpenoids

Flavonoids To the aqueous filtrate 5mL of dilute ammoniasolution was added followed by concentrated H

2SO4 A

yellow colouration indicated the presence of flavonoids

PhlobatanninsThedeposition of a red precipitate denoted thepresence of phlobatanninswhen 200mg of plantmaterial wasdissolved in 10mL of aqueous extract and few drops of 1HCl were added in the boiling tube

Anthraquinones 500mg of dried plant leaves were boiled in10 HCl for 5mins and filtrate was allowed to cool Equalvolume of CHCl

3with few drops of 10 NH

3was added to

2mL filtrate The formation of rose-pink colour implies thepresence of Anthraquinones

Reducing Sugars To the 10mL of aqueous extract a few dropsof Fehlingrsquos solution A and B were added an orange redprecipitate suggests the presence of reducing sugars

24 Quantitative Estimation of Phytochemicals

241 Determination of Alkaloids Alkaloids content wasmeasured by following the protocol described by Harborne[29] A suspension was prepared by dispersing 5 gm of thedried leaves in 10 acetic acid solution in ethanol andkept at 28∘C for 4 hrs which was further filtered throughWhatmanNumber 42Thereafter alkaloidwas precipitated byconcentrating the filtrate to one quarter of its original volumeand drops of conc aqueous NH

4OH were added Finally the

precipitate was washed with 1 ammonia solution and driedat 80∘C in the oven The content of alkaloid was calculatedand expressed as mggm of sample

242 Determination of Flavonoids The flavonoids contentwas also determined by Harborne [29] method Briefly 5 gmof leaves was boiled in 2M HCl for 30mins under refluxand filtered after cooling Equal volume of ethyl acetate wasthen added drop wise in filtrate The weight of precipitatedflavonoid was determined and reported as mgg

243 Determination of Tannins The quantitative estimationof tannins was performed by the method of Swain [30] withminor modifications in our lab Finely powdered leaves ofAegle marmelos were kept in a beaker containing 20mL of50methanol covered with parafilm and then heated at 80∘Cin water bath for 1 hr with continuous stirring The extract

BioMed Research International 3

was quantitatively filtered using a double layered WhatmanNumber 1 filter paper and rinsed by 50 methanol 1mLof sample extract was treated with 20mL distilled water25mL Folin-Denis reagent and 10mL of 17 Na

2CO3for

the development of a bluish-green colour and was allowedto stand for 20 minutes The absorbance was measured at760 nm and amount of tannin was calculated by comparingit with standard curve prepared in the range of 0ndash10 ppm

244 Determination of Saponins Saponin analysis was per-formed according to the method described by Brunner [31]100mL Isobutyl alcohol was added to 1 g of finely powderedsample and stirred for 5 hrs 20mL of 40 saturated solutionof Magnesium carbonate was added to the mixture andfiltered 2mL of 5 FeCl

3solution and 50mL volume of

distilled water was added to 1mL of colourless solution andkept for 30min for colour (blood red) development Theabsorbance of the samples along with the standard wereread at 380 nm and calculated in mggm Standard saponinsolution was prepared in the reference range of 0ndash10 ppm

245 Determination of Total Phenols Five grams of thepowdered leaves was boiled with 50mL of ether for 15minsand distributed in the ratio 1 2 (extract distilledwater) 2mLof ammonium hydroxide followed with 5mL of pentanol wasadded to it and incubated at the room temperature for 30minutes The absorbance was read at 505 nm as described byObodoni and Ochuko [32]

25 Procuring of Bacterial Strain Pure cultures of five testorganisms namely S aureus (NCIM 2079) S epidermidis(NCIM 2493) B cereus (NCIM 2156) E aerogenes (NCIM5139) and K pneumoniae (NCIM 2957) were procured fromNational Chemical Laboratory Pune Stock cultures weremaintained at 4∘C on agar slants of nutrient media Activecultures for experiment were prepared by transferring a loopfull of microorganism from the stock cultures to 50mL ofsterile nutrient broth

26 Antibacterial Activity The extracts mentioned abovewere tested against five pathogenic bacterial strains threegram-positive bacteria (B cereus S epidermidis and Saureus) and two gram-negative bacteria (E aerogens Kpneumoniae) Antibacterial screening was done using agarwell diffusion method [33] For this 20mL of sterile Mueller-Hinton Agar (Hi-media) was poured in sterile autoclavedpetri plates After solidification the sterile cotton swab wasdipped into the bacterial culture The entire agar surface ofeach plate was evenly inoculated by swabbing The sevenuniform wells were prepared with the help of sterile 6mmdiameter cork-borer Each well was filled with the variousconcentrations of both the aqueous andmethanol extract (1020 25 30 and 40mgmL) respectively whereas in case ofaqueous ethanol (40 80 100 and 120mgmL) concentra-tions were used and allowed for diffusion for 45 minutesThe plates were then incubated at 37∘C for 24 hrs Triplicateplates were prepared for each treatment and the average zoneof inhibition excluding well was recorded 9 DMSO was

used as negative control Turbidity of bacterial culture wasmaintained up to 1 times 108 CFUmLThe antibacterial potentialof extracts was compared with standard antibiotic Ampicillin(10 120583gdisc) with paper disc (Hi-media) method

27 Gas Chromatography Mass Spectrometry Analysis Thegas chromatography-mass spectrometry (GC-MS) analysisof methanolic extract of leaves of Aegle marmelos (var PantAparna)was performed using a GC-MS (Model QP 2010Plus Shimadzu Tokyo Japan) equippedwith aVF-5ms fusedsilica capillary column of 30m length 025mmdiameter and025 120583m film thickness The column oven temperature wasprogrammed from 80∘C to 310∘C for 2∘C minminus1 Ionizationof the sample components was performed in electron impactmode (EI 70 eV) The temperature of the injector was fixedto 270∘C and detector to 230∘C Helium (999995 purity)was the carrier gas fixed with a flow rate of 121mLminminus1The mass range from 40ndash650 mz was scanned at a rate of30 scanss 20 120583Lof themethanolic extract ofAeglemarmeloswas injected with aHamilton syringe to the GC-MSmanuallyfor total ion chromatographic analysis in split injectiontechnique Total running time of GC-MS was 56min Therelative percentage of each extract constituent was expressedas percentage with peak area normalization

The bioactive compounds of methanol extract wereidentified by comparing their retention indices and patternsof mass spectra with reference to Wiley Registry of MassSpectral Datarsquos New York (Wiley 8) and Fatty Acid MethylEsters Library version 10 (FAME library) sources

3 Result and Discussion

31 Phytochemical Profiling The present study was carriedon aqueous and methanolic extracts of Aegle marmelos toinvestigate the presence of medicinally important phyto-chemicals in the leaves of different varietiesaccessions Boththe extracts revealed the presence of various phytochemicalssuch as tannins saponins flavonoids alkaloids terpenoidscarotenoids cardiac glycosides and reducing sugars in allthe varieties and accessions while phlobatannins and antho-cyanins were absent (Table 1) The presence of differentphytochemicals and the antimicrobial activity of ethanolicpetroleum ether chloroform and methanolic extract of asingle unidentified variety of Aegle marmelos have beenpreviously reported [34 35] however our study is first everreport to the best of our knowledge on qualitative and quan-titative comparative analysis of various varietiesaccessionsavailable in India The findings conclude that amongst 18varieties and accessions the variety Pant Aparna was foundto be the best The antibacterial activity of this variety usingmethanol aqueous and aqueous ethanol as the solvent hasbeen investigated The activity was further confirmed byperforming GC-MS which revealed the presence of differentphytochemicals

The quantitative phytochemical estimation specifies thatthe leaves of all varieties contain a significant amount ofalkaloid flavonoids phenolic saponins and tannin content


Table 1 Qualitative analysis of phytochemicals in aqueous and methanol extract of different varietiesaccessions of Bael (Aegle marmelos)leaves

Diff Bael varietyaccessions aTA bPHL cSAP dTER eFLA fCAR gANTH hCAR iRED jALKNB-17 + minus + + + + minus + + +Pant Aparna + minus + + + + minus + + +NB-9 + minus + + + + minus + + +NB-5 + minus + + + + minus + + +AM-4 + minus + + + + minus + + +NB-7 + minus + + + + minus + + +AM-7 + minus + + + + minus + + +AM-3 + minus + + + + minus + + +NB-1 + minus + + + + minus + + +Kaghzi + minus + + + + minus + + +NB-4 + minus + + + + minus + + +NB-16 + minus + + + + minus + + +PSujana + minus + + + + minus + + +PSujata + minus + + + + minus + + +AM-1 + minus + + + + minus + + +AM-2 + minus + + + + minus + + +AM-6 + minus + + + + minus + + +AM-8 + minus + + + + minus + + +aTA tannins bPHL phlobatannins cSAP saponins dTER terpenoids eFLA flavonoids fCAR cardiac glycosides gANTH combined anthraquinones hCARcarotenoids iRED reducing sugar jALK alkanoids + present minus absent

Table 2 Quantitative estimation of phytochemicals (mgg)

Different Baelvarietyaccessions Alkaloids Flavonoids Phenols Saponins Tannins

NB-17 862 plusmn 010 262 plusmn 0065 228 plusmn 0004 765 plusmn 014 453 plusmn 015Pant Aparna 1608 plusmn 005lowastlowast 639 plusmn 0061lowastlowast 294 plusmn 0004lowastlowast 1198 plusmn 020 832 plusmn 040NB-9 107 plusmn 015 198 plusmn 0058 1022 plusmn 0032 536 plusmn 015 378 plusmn 035NB-5 956 plusmn 010 232 plusmn 0055 126 plusmn 0005 657 plusmn 025 643 plusmn 005AM-4 116 plusmn 014 239 plusmn 0053 114 plusmn 0007 423 plusmn 044 497 plusmn 010NB-7 656 plusmn 025 185 plusmn 0051 100 plusmn 0005 465 plusmn 010 345 plusmn 025AM-7 428 plusmn 030 391 plusmn 0049 196 plusmn 0045 1340 plusmn 030 576 plusmn 035AM-3 1434 plusmn 020 289 plusmn 0048 1778 plusmn 0079 1276 plusmn 020 724 plusmn 015NB-1 378 plusmn 015 104 plusmn 0047 58 plusmn 0085 421 plusmn 045 320 plusmn 025Kaghzi 594 plusmn 015 174 plusmn 0046 114 plusmn 0004 585 plusmn 035 531 plusmn 018NB-4 1057 plusmn 030 124 plusmn 0043 96 plusmn 0004 750 plusmn 024 360 plusmn 042NB-16 580 plusmn 025 104 plusmn 0007 76 plusmn 0007 320 plusmn 015 435 plusmn 036PSujana 745 plusmn 045 164 plusmn 0070 1582 plusmn 0007 602 plusmn 030 662 plusmn 028PSujata 1198 plusmn 025 239 plusmn 0088 1364 plusmn 0008 853 plusmn 025 643 plusmn 008AM-1 1279 plusmn 020 235 plusmn 0225 1522 plusmn 0082 789 plusmn 055 405 plusmn 024AM-2 843 plusmn 035 124 plusmn 0045 86 plusmn 0084 59 plusmn 034 532 plusmn 031AM-6 1503 plusmn 040 269 plusmn 0108 2022 plusmn 0010 1094 plusmn 028 1054 plusmn 005AM-8 1336 plusmn 025 266 plusmn 0078 1838 plusmn 0078 925 plusmn 055 729 plusmn 018lowastlowast

refers to significant values (119875 lt 001)

However the variety called Pant Aparna contains the high-est amount of alkaloids flavonoids and phenol (Table 2)The alkaloids content was quantitatively estimated and wasfound in the range of 378 plusmn 015ndash1608 plusmn 005mggm indifferent varieties of Bael leaves but the maximum content

was observed in Pant Aparna (1608 plusmn 005mggm) Simi-larly all the varieties exhibited good quantity of flavonoidsand phenols starting from 104 plusmn 0047mgmL and 58 plusmn0085mgmL respectively in variety NB-1 reaching up to639 plusmn 0061mgmL and 294 plusmn 0004 in Pant Aparna


Table 3 Zone of Inhibition by aqueous extract

Aqueous extractConc (mgmL) 10 15 20 25 30 40 Standard ControlName of bacteria Zone of inhibition (mm) Ampicillin (10mcgdisc) DMSOK pneumoniae 733 plusmn 003 833 plusmn 003 85 plusmn 002 65 plusmn 002 983 plusmn 004 106 plusmn 003 213 plusmn 0088 0B cereus 733 plusmn 003 833 plusmn 003 900 900 900 plusmn 005 933 plusmn 005lowastlowast 103 plusmn 0088 0E aerogenes 800 800 833 plusmn 003 900 900 900lowastlowast 113 plusmn 0088 0S aureus 866 plusmn 003 966 plusmn 003 106 plusmn 003 116 plusmn 003 126 plusmn 003 130 406 plusmn 0120 0S epidermidis 966 plusmn 003 110 120 130 136 plusmn 003 143 plusmn 006lowast 206 plusmn 0145 0Values are expressed as mean plusmn SEM and analyzed by one-way analysis of variance (ANOVA) followed by Dennettrsquos 119905 test lowast119875 lt 005 lowastlowast119875 lt 001

Table 4 Zone of inhibition by methanol extract

Methanol extractConc (mgmL) 10 15 20 25 30 40 Standard ControlName of bacteria Zone of inhibition (mm) Ampicillin (10mcgdisc) DMSOK pneumoniae 93 plusmn 003 96 plusmn 003 106 plusmn 003 113 plusmn 003 110 plusmn 005 110 plusmn 005lowast 213 plusmn 0088 0B cereus 86 plusmn 003 100 106 plusmn 003 113 plusmn 003 106 plusmn 003 120 plusmn 005lowastlowast 103 plusmn 0088 0E aerogenes mdash mdash mdash mdash mdash mdash 113 plusmn 0088 0S aureus 100 110 110 123 plusmn 003 120 130 406 plusmn 0120 0S epidermidis 80 plusmn 005 93 plusmn 006 100 103 plusmn 003 110 120lowast 206 plusmn 0145 0Values are expressed as mean plusmn SEM and analyzed by one-way analysis of variance (ANOVA) followed by Dennettrsquos 119905 test lowast119875 lt 005 lowastlowast119875 lt 001

Earlier report on quantitative yield also revealed that Aeglemarmelos contained highest quantity of alkaloids flavonoidsand tannins as compared to other medicinal plants [36]The highest content of saponins was found in variety AM-7(1340plusmn030) followed by Pant Aparna and AM-6 Among allthe varieties analysed Pant Aparna was found to be the mostpromising one which prompted us to project Pant Aparna forfurther studies

32 Antibacterial Study The pharmacological action of theplant cannot be ascertained by the result of phytochemicalstudies onlyThus the antibacterial activity against pathogenicbacteria was also evaluated The present investigation showsthe efficacy of all the extracts against the selected pathogenicbacteria (Tables 3 4 and 5)

The aqueous extract showed highest antibacterial activ-ity against S epidermidis (143mm) followed by S aureus(13mm) and K pneumonia (106mm) The maximum zoneof inhibition was observed at the concentration of 40mgmLVarying degree of antibacterial activity by leaf extractsof Aegle marmelos against various tested bacterial specieshas been reported [37] The presence of the compoundsCuminaldehyde and Eugenol may be responsible for theantibacterial activity of leaf extracts against various bacterialstrains [38] The aqueous acetone and petroleum etherextracts of A marmelos were found to be effective against Bcoagulans B subtilis B thuringiensis P aeruginosa and Saureus as reported earlier [39]The dried fruit extract ofAeglemarmelos showed potential antibacterial activity against Paeruginosa S flexneri E coli B subtilis S epidermidis andS aureus [40]

On the other hand the methanol extract exhibited amuch better antibacterial activity as compared to aqueous

extract against K pneumoniae B cereus and S aureus Themethanol extract was most potent against S aureus showingthe maximum zone of inhibition at the concentration of40mgmL The highest sensitivity of S aureus may be dueto its cell wall structure and outer membrane [41] Noactivity was seen against E aerogenes A few reports havealso mentioned significant in vitro antimicrobial activity bythe methanol extracts of Aegle marmelos leaves and flowers[35 42] The MIC value of both aqueous and methanolextract was significant at 10mgmL however for aqueousethanol it was 40mgmL which is analogous to the MICvalues reported using crude extracts of other medicinalplants The antimicrobial activity of papaya seed extract withMIC value of 118mgmL was found to be effective againstS typhi by Yismaw et al [43] In Zizyphus sp MIC valuesof 25mgmL [44] have been reported against S aureusSimilarly in case of F religiosa the final MIC values were2ndash4-fold higher than initial MIC which ranged from 47 to188mgmL and from 24 to 188mgmL for resistant and forsensitive isolates respectively MIC values (25mgmL) werereported by Valsaraj et al [44] while higher concentrationof 150mgmL was reported by Ahmad and Beg [45] againstS aureus In yet another study the antibacterial activity ofwhole plant of Borreria hispida (Linn) was evaluated againstvarious organisms where the MIC of methanolic extractof Borreria hispida was found to range from 250 120583gmLto 50mgmL Moreover higher MIC values ranging from0125 to 32mgmL were also reported against at least one ofthe test microorganisms from nine ethnobotanically Indianmedicinal plants [46]

The high antibacterial activity in the methanolic extractmay be due to the presence of tannins flavonoids andterpenoids These medicinally bioactive components exert


Table 5 Zone of inhibition by aqueous ethanol extract

Aqueous ethanolConc (mgmL) 40 80 120 160Name of bacteria Zone of inhibition (mm)K pneumonia 90 plusmn 005 110 plusmn 005 123 plusmn 003 146 plusmn 006lowast

B cereus 90 100 110 113 plusmn 003E aerogenes 100 150 160 183 plusmn 003lowastlowast

S aureus 103 plusmn 003 116 plusmn 003 120 130S epidermidis 103 plusmn 003 123 plusmn 003 133 plusmn 006 146 plusmn 003lowast

Values are expressed as mean plusmn SEM and analyzed by one-way analysis of variance (ANOVA) followed by Dennettrsquos 119905 test lowast119875 lt 005 lowastlowast119875 lt 001 The standardantibiotic and control are the same as that of aqueous and methanolic extract

antimicrobial action through different mechanism Tan-nins cause inhibition in the cell wall synthesis by formingirreversible complexes with prolene rich protein [47] Thesaponins have the ability to cause leakage of proteins andcertain enzymes from the cell [48] Terpenoids are respon-sible for dissolution of the cell wall of microorganism byweakening the membranous tissue [49] Flavonoids whichhave been found to be effective antimicrobial substancesagainst a wide array of microorganisms in vitro are knownto be synthesized in response to microbial infection byplants They have the ability to complex with extracellularand soluble proteins and to complex with bacterial cell walls[50] Furthermore steroids are known for their antibacterialactivity specifically associated with membrane lipids andcause leakage from liposomes [51]

In the case of aqueous ethanol the maximum antibac-terial activity was seen against E aerogenes followed byS epidermidis and K pneumonia (Table 5) At the highestconcentration of 160mgmL it showed 183mm zone ofinhibition The antibiotic susceptibility showed that amongall the bacterial strains S aureus was found to be moresusceptible to ampicillin followed by K pneumonia

33 GC-MS Analysis In all thirty-three compoundswere identified from the GC-MS analysis of methanolicextract of Bael leaves exhibiting various phytochemicalactivities and were predominantly responsible for theantimicrobial activity found in the extract against thepathogenic bacteria The retention time and percentage peakof various bioactive compounds are presented in Table 6The major phytoconstituents present in the leaf extractwere 1-Dodecanol (483) 4H-Pyran-4-one 23-dihydro-35-dihydroxy-6-methyl-(111) 23Dioxabicyclo[222]oct-5-ene1-Methyl-4-(1-Methylethyl)-(Limonene dioxide 1) occupyingtwo peak areas that is (053) and (040) Bicyclo[311]heptane-23-diol 266-trimethyl (23-Pinanediol)(097) 2-Cyclohexen-1-one 4-hydroxy-3-methyl-6-(1-methylethyl)-(063 041 017) Phenol 26-bis(11-dimethylethyl)-4-methyl-(BHT) (087) Tetradecanoic acid (Myristic acid)(200) 2(4H)-Benzofuranone 5677A-Tetrahydro-6-hydroxy-447a-trimethyl13-cyclohexadiene2-methyl-5-(1methylethyl)-(1-Phellandrene) 2-Propenoic acid3-(4-hydroxy-3-methoxyphenyl)- methyl ester (Cinnamic

acid 4-hydroxy-3-methoxy- methyl ester) 371115-Tetramethyl-2-hexadecen-1-ol commonly known as phytola diterpene has significant antimicrobial properties againstmany bacterial strains [52] 91215-Octadecatrienoicacid methyl ester (Linolenic acid methyl ester) showingantibacterial and anticandidal activity 2-Hexadecen-1-ol371115-Tetramethyl (Phytol isomer) (637) Octadecanoicacid (Stearic acid) (407) Benzene 12-dimethoxy-4-[[(4methylphenyl) sulfonyl]methyl (1076) fatty alcohols suchas Ergost-5-en-3-ol (3beta) (campesterol) Stigmasta-5 22-dien-3-ol Stigmast-5-en-3-ol (3beta)- may besynergistically responsible for the antimicrobial activity

The different phyto compounds responsible for bioactiv-ity have been identified and characterized in different medic-inal plants 4H-Pyran-4-one 23-dihydro-3 5-dihydroxy-6-methyl- a potent anti-inflammatory and antioxidant com-pound possessed antibacterial activity in Barleria prionitis(Linn) rhizomeA long-chain fatty alcohol 1-Dodecanol wasreported with highest antibacterial activity against Staphy-lococcus aureus [53] Phenol 26-bis(11-dimethylethyl)-4-methyl commonly known as Butylated hydroxytoluene(BHT) an antioxidant has also demonstrated markedantimicrobial activity inhibiting or decreasing the growthof gram-positive bacteria at a higher degree than thegram-negative bacteria belonging to the family Enter-obacteriaceae [54] Derivatives of cinnamic acid such asesters amides acids and hydrazides have been reportedto have antibacterial antifungal and antiviral proper-ties [55] 2(4H)-Benzofuranone 5677a-tetrahydro-447a-trimethyl a bioactive compound possessing the propertiessuch as analgesic antidiabetic antibacterial and antifun-gal was identified from the methanolic fractions of theAzadirachta indica [56] The extract contained (023) of13-Cyclohexadiene 2-methyl-5-(1-methylethyl) commonlyknown as 1-phellandrene Stigmasterol has been reportedearlier as a strong antioxidant having antibacterial activityagainst multidrug resistant mycobacteria [57 58]

Several bioactive compounds namely Marmin [59] andMarmelosin coumarin derivatives [60] andAegeline an alka-loid have been previously reported fromAeglemarmelos [61]However the flavonoids and phenolics present in significantamount in this plant are still unexplored The preferentialquantity of these compounds in the methanolic extract ofPant Aparna as revealed by the present study directed to focus


Table 6 Activity of the phytocomponents identified from methanolic leaf extract of Aegle marmelos

S no RT Compound name Molecularformula MW Peak

area Compound

naturelowastlowastActivity

1 7178 4H-Pyran-4-one23-dihydro-35-dihydroxy-6-methyl- C6H8O4 144 111 Flavonoid fraction

Antimicrobialanti-inflammatoryantiproliferative

2 6555 1-Butanol 3-methyl- acetate C7H14O2 130 532 Alcoholiccompound Antimicrobial

3 120923 Dioxabicyclo[222]oct-5-ene1-methyl-4-(1-methylethyl)-(Limonenedioxide 1)

C10H16O2 168 053 Terpene antimicrobialactivity

4 13385Bicyclo[311]heptane-23-diol266-trimethyl(23-Pinanediol)


4 13762 2-Cyclohexen-1-one4-hydroxy-3-methyl-6-(1-methylethyl)- C10H16O2 168 063 antibacterial

5 15203 1-Dodecanol C12H26O 186 483 long-chain fattyalcohol antibacterial

6 16143Phenol26-bis(11-dimethylethyl)-4-methyl(BHT)

C15H24O 220 087 Antimicrobial antioxidantactivity

7 16457 Benzoic acid 4-ethoxy- ethyl ester C11H14O3 194 033 Aromaticacid ester Antimicrobial Preservative

8 181012-Propanol 111015840-[(1-methyl-12-ethanediyl)bis(oxy)]bis-(Tripropyleneglycol)

C9H20O4 192 087 Antimicrobialactivity

9 20189 1-Tetradecanol acrylate C17H32O2 268 146 Fatty acid esters Anti-inflammatoryantimicrobial

10 205231345-Tetrahydroxy-cyclohexanecarboxylic acid(Quinic acid)

C7H12O6 192 039 Aromatic acid antimicrobialactivity anti-inflammatory

11 21839 Tetradecanoic acid(Myristic acid) C14H28O2 228 200 Fatty acid

Antifungal Antioxidantcancer preventivenematicidehypercholesterolemicLubricant

12 22070 2(4H)-Benzofuranone 5677a-tetrahydro-6-hydroxy-447a-trimethyl C11H16O3 196 026 Triterpene antimicrobial

13 22262 1-Heptadecanol(1-Eicosanol) C17H36O 256 033 Aliphatic alcohol Antimalarial antifungal

Antioxidant

14 22409 13-Cyclohexadiene 2-methyl-5-(1-methylethyl)-(1-Phellandrene) C10H16 136 023 Monoterpene Antibacterial

15 22736 16-Octadiene 7-methyl-3-methylene(beta-myrcene) C10H16 136 013 Monoterpene Antibacterial

16 23404

2-Propenoic acid3-(4-hydroxy-3-methoxyphenyl)- methylester(Cinnamic acid 4-hydroxy-3-methoxy-methyl ester )

C11H12O4 208 066 Aromatic methylesters

Antimicrobialantioxidant antiviral

17 23819 Pentadecanoic acid C15H30O2 242 008 Fatty acid Antibacterial

18 24897 371115-Tetramethyl-2-hexadecen-1-ol(Phytol) C20H40O 296 032 Diterpene

Antimicrobial anticanceranti-inflammatoryanti-diuretic


Table 6 Continued


area Compound


19 24986 hexadecanoic acid methyl ester(Palmitic acid methyl ester) C17H34O2 270 072 Fatty acid methyl

ester

Antioxidanthypocholesterolemicnematicide pesticideantiandrogenic flavorhemolytic 5-Alpha reductaseinhibitor

20 24951 Pentadecanoic acid C15H30O2 242 799 Saturated fatty acid Antimicrobial

21 27266 9-Octadecenoic acid C18H34O2 282 031 Unsaturated fattyacid antibacterial

22 27664 Heptadecanoic acid C17H34O2 270 039 Saturated fatty acid Antimicrobial

23 2829891215-Octadecatrienoic acid methylester(Linolenic acid methyl ester)

C19H32O2 292 107 Fatty acid methylester

Antibacterial and anticandidalAntiinflammatoryhypocholesterolemic cancerpreventivehepatoprotective nematicideinsectifuge antihistaminicantiarthritic anticoronaryantieczemic antiacne 5-Alphareductase inhibitorAntiandrogenic

24 28554 2-Hexadecen-1-ol 371115-tetramethyl(Phytol isomer) C20H40O 296 637 Diterpene

AntimicrobialAnti-inflammatoryAnticancer Diuretic

25 29227 Cis-9-Hexadecenal C16H30O 238 1140 Aldehyde Antimicrobial

26 29573 Octadecanoic acid(Stearic acid) C18H36O2 284 406 Fatty acid Antimicrobial

27 41240 Benzene 12-dimethoxy-4-[[(4methylphenyl)sulfonyl]methyl C16H18O4S 306 1076 Aromatic sulfur

compound Antimicrobial

28 44155 Cholest-5-en-3-ol (3beta)- C27H46O 386 024 Steroidal Antibacterial

29 45238 Ergost-5-en-3-ol (3beta)- C27H46O 386 050 SteroidalAntimicrobialanti-inflammatoryeffects

30 45509 Stigmasta-522-dien-3-ol C29H48O 412 063 Steroidal

Antioxidantantibacterial activityantiinflammatory antiarthriticantiasthma diuretic

31 46038 Stigmast-5-en-3-ol (3beta)- C29H50O 414 308 SteroidalAntimicrobial antioxidantantiinflammatory antiarthriticantiasthma diuretic

32 47033 Vitamin E C29H50O2 430 058

Antioxidant andAntimicrobial activityAnalgesicAntidiabaticAntiinflammatoryAntidermatiticAntileukemicAntitumorAnticancerHepatoprotectiveAntispasmodic


Table 6 Continued


area Compound


33 44454 alpha-Tocopherol C29H50O2 430 026

Anti-inflammatoryantioxidant antimicrobialradical scavengingantispasmodic

lowastlowastSource Dr Dukersquos Phytochemical and Ethnobotanical Databases

towards the purification and characterization of potentialcompound from this variety

4 Conclusion

Medicines derived from plants have made immense con-tribution towards the betterment of human health and actas a source of inspiration for novel drug compounds Fromthe above research it can be concluded that this plant hasimmense potential to be used in the area of pharmacologyand as a prospective source of valuable drugs Due to thepresence of various compounds that are essential for goodhealth it can also be used to improve the health status ofsocietyThe extracts showed a significantly high antibacterialactivity against the microorganisms The data clearly depictsthe presence of compounds used for treating various bacterialdiseases indicating its use in the traditional system ofmedicine since ancient times Further the broad spectrumactivity of aqueous methanol and aqueous ethanol extractsproves to be encouraging in the development of novelantimicrobial formulation in the near future

A spectrum of compounds showing strong antibacterialantioxidant and anti-inflammatory activities was revealedby the GC-MS analysis of the methanolic extract of Aeglemarmelos

Antimicrobials derived from plants possess vast curativeproperties since they have fewer side effects as compared tosynthetic antimicrobials drugs Aegle marmelos is of utmostimportance for ethnobotanical purposes and it has beenplaced in the priority list of thirty-two medicinal plants byThe National Medicinal Plants Board of Govt of India [62]The present study contributes to the current knowledge ofpresence of various phytochemical active compounds in 18varietiesaccessions of Aegle marmelos possessing significantbroad spectrum antibacterial efficacy Further fractionationand purification will elucidate the potential compoundwhich is a pressing need because of the upcoming resistanceof the currently available antibiotics

Conflict of Interests

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

Acknowledgments

The authors would like to gratefully acknowledge UGC NewDelhi for its financial support in the form of Maulana Azad

National Fellowship and Honrsquoble Vice Chancellor of ourIntegral University

References

[1] N Shariff M S Sudarshana S Umesha and P HariprasadldquoAntimicrobial activity of Rauvolfia tetraphylla and Physalisminima leaf and callus extractsrdquo African Journal of Biotechnol-ogy vol 5 no 10 pp 946ndash950 2006

[2] G Vines ldquoHerbal harvests with a future towards sustainablesources for medicinal plantsrdquo 2004 Plant life Internationalhttpwwwplantlifeorguk

[3] B L Saet H C Kwang N K Su et al ldquoThe antimicrobialactivity of essential oil from Dracocephalum foetidum againstpathogenic microorganismsrdquo Journal of Microbiology vol 45no 1 pp 53ndash57 2007

[4] P H Davis Flora of Turkey and East Eagean Island vol 7Edinburg University Press Edinburg Tex USA 1982

[5] M Lis-Balchin and S G Deans ldquoBioactivity of selected plantessential oils against Listeria monocytogenesrdquo Journal of AppliedMicrobiology vol 82 no 6 pp 759ndash762 1997

[6] D A Ates and O T Erdogrul ldquoAntimicrobial activities of vari-ous medicinal and commercial plant extractrdquo Turkish Journal ofBiology vol 27 pp 157ndash162 2003

[7] H R El-Seedi T Ohara N Sata and S Nishiyama ldquoAntimicro-bial diterpenoids from Eupatorium glutinosum (Asteraceae)rdquoJournal of Ethnopharmacology vol 81 no 2 pp 293ndash296 2002

[8] R Rojas B Bustamante J Bauer I Fernandez J Alban andO Lock ldquoAntimicrobial activity of selected Peruvian medicinalplantsrdquo Journal of Ethnopharmacology vol 88 no 2-3 pp 199ndash204 2003

[9] V Duraipandiyan M Ayyanar and S Ignacimuthu ldquoAntimi-crobial activity of some ethnomedicinal plants used by Paliyartribe from Tamil Nadu Indiardquo BMC Complementary andAlternative Medicine vol 6 article 35 2006

[10] Chemexcil Selected Medicinal Plants of India Basic Chemi-cals Pharmaceutical and Cosmetic Export Promotion CouncilBombay India 1992

[11] L Badam S S Bedekar K B Sonawane and S P Joshi ldquoIn vitroantiviral activity of bael (Aegle marmelos Corr) upon humancoxsackieviruses B1-B6rdquo Journal of Communicable Diseases vol34 no 2 pp 88ndash99 2002

[12] Y L Chew J K Goh and Y Y Lim ldquoAssessment of invitro antioxidant capacity and polyphenolic composition ofselected medicinal herbs from Leguminosae family in Penin-sular Malaysiardquo Food Chemistry vol 116 no 1 pp 13ndash18 2009

[13] A K Gupta andN TandonReviews on IndianMedicinal Plantsvol 1 Indian Council of Medicinal Research New Delhi India2004


[14] N Kamalakkannan and P Stanely Mainzen Prince ldquoAnti-hyperlipidaemic effect of Aegle marmelos fruit extract instreptozotocin-induced diabetes in ratsrdquo Journal of the Scienceof Food and Agriculture vol 85 no 4 pp 569ndash573 2005

[15] V Arul S Miyazaki and R Dhananjayan ldquoStudies on the anti-inflammatory antipyretic and analgesic properties of the leavesofAeglemarmelosCorrrdquo Journal of Ethnopharmacology vol 96no 1-2 pp 159ndash163 2005

[16] G C Jagetia P Venkatesh and M S Baliga ldquoAegle marmelos(L) Correa inhibits the proliferation of transplanted Ehrlichascites carcinoma in micerdquo Biological and Pharmaceutical Bul-letin vol 28 no 1 pp 58ndash64 2005

[17] N Kamalakkannan and P S M Prince ldquoHypoglycaemic effectof water extracts of Aegle marmelos fruits in streptozotocindiabetic ratsrdquo Journal of Ethnopharmacology vol 87 no 2-3 pp207ndash210 2003

[18] M Rajadurai M Padmanabhan and P S M Prince ldquoEffect ofAegle marmelos leaf extract and alpha-tocopherol on lipid per-oxidation and antioxidants in isoproterenol-induced myocar-dial infarction in ratsrdquo Cardiology vol 1 pp 40ndash45 2005

[19] M C Sabu and R Kuttan ldquoAntidiabetic activity of Aeglemarmelos and its relationship with its antioxidant propertiesrdquoIndian Journal of Physiology and Pharmacology vol 48 no 1pp 81ndash88 2004

[20] K Saradha Jyothi and B Subba Rao ldquoAntibacterial activityof extracts from Aegle marmelos against standard pathogenicbacterial strainsrdquo International Journal of PharmTech Researchvol 2 no 3 pp 1824ndash1826 2010

[21] T K Sur S Pandit and T Pramanik ldquoAntispermatogenicactivity of leaves of Aegle marmelos corr in albino rats apreliminary reportrdquo Biomedicine vol 19 no 3 pp 199ndash2021999

[22] G C Yen P D Duh and C L Tsai ldquoRelationship betweenantioxidant activity and maturity of peanut hullsrdquo Journal ofAgricultural and Food Chemistry vol 41 no 5 pp 67ndash70 1993

[23] C Kingston S Jeeva G M Jeeva S Kiruba B P Mishra andD Kannan ldquoIndigenous knowledge of usingmedicinal plants intreating skin diseases in Kanyakumari District Southern IndiardquoIndian Journal of Traditional Knowledge vol 8 no 2 pp 196ndash200 2009

[24] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[25] J B Harborne Phytochemical Methods Chapman amp Hall NewYork NY USA 11th edition 1984

[26] A O Adetuyi and A V Popoola ldquoExtraction and dyes abilitypotential studies of the colourant in zanthoxylum zanthoxy-loides plant on cotton fabricrdquo Journal of Science EngineeringTechnology vol 8 no 2 pp 3291ndash3299 2001

[27] G E Trease and W C Evans Pharmacognosy Brailliar TiridalCan Macmillian Publishers 11th edition 1989

[28] A SofoworaMedicinal Plants and Traditional Medicine inWestAfrica John Wiley and Sons New York NY USA 1982

[29] J B Harborne Phytochemical Methods A Guide to ModernTechniques of Plant Analysis Chapman amp Hall Ltd LondonUK 1973

[30] T Swain ldquoTannins and ligninsrdquo inHerbivoresTheir Interactionswith Plant Metabolites G A Rosenthal and D H Janzen EdsAcademic Press New York NY USA 1979

[31] J H Brunner ldquoDirect spectrophotometric determination ofsaponinsrdquo Analytical Chemistry vol 34 pp 1314ndash1326 1984

[32] B O Obodoni and P O Ochuko ldquoPhytochemical studies andcomparative efficacy of the crude extracts of some Homostaticplants in Edo andDelta States of NigeriardquoGlobal Journal of PureApplied Science vol 8 no 3 pp 203ndash208 2001

[33] POOlutiolaO Famurewa andHG SonntagAn Introductionto General MicrobiologymdashA Practical Approach 2nd edition1991

[34] D Venkatesan C M Karrunakarn S Selva Kumar and P TPalani Swamy ldquoIdentification of phytochemical constituents ofAegle marmelos responsible for antimicrobial activity againstselected pathogenic organismsrdquo Ethnobotanical Leaflets vol 13pp 1362ndash1372 2009

[35] S Kothari V Mishra S Bharat and S D Tonpay ldquoAntimicro-bial activity and phytochemical screening of serial extracts fromleaves ofAegle marmelos (Linn)rdquoActa Poloniae Pharmaceuticavol 68 no 5 pp 687ndash692 2011

[36] R Dhandapani and B Sabna ldquoPhytochemical constituents ofsome Indian medicinal plantsrdquo Ancient Science of Life vol 27no 4 pp 1ndash8 2008

[37] R P Rastogi and B N Mehrotra ldquoAncient-modern concor-dance in Ayurvedic plantsrdquo inCompendium of IndianMedicinalPlants R P Rastogi Ed vol 3 Central Drug Research InstituteLucknow India Information Directorate New Delhi India1993

[38] J A Duke Handbook of Biologically Active Phytochemicals andTheir Activities CRC Press 1992

[39] V Mohan A G Molly and A N Eldo ldquoAegle marmelosextract as a potent bactericiderdquo Asian Journal of MicrobiologyBiotechnology and Environmental Sciences vol 7 no 4 pp 639ndash644 2005

[40] D Supriya S Ashish S Ankit G Nirmala and R C AgrawalldquoEvaluation of in vitro antibacterial potential of ripe fruitsof Aegle marmelosrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 179ndash181 2012

[41] L L Zaika ldquoSpices and herbs their antimicrobial activity and itsdeterminationrdquo Journal of Food Safety vol 9 no 2 pp 97ndash1181988

[42] K Suresh P K Senthilkumar and B Karthikeyan ldquoAntimi-crobial activity of Aegle marmelos against clinical pathogensrdquoJournal of Phytology vol 1 no 5 pp 323ndash327 2009

[43] G Yismaw B Tessema A Mulu and M Tiruneh ldquoThe invitro assessment of antibacterial effect of papaya seed extractagainst bacterial pathogens isolated from urine wound andstoolrdquo Ethiopian Medical Journal vol 46 no 1 pp 71ndash77 2008

[44] R Valsaraj P Pushpangadan U W Smitt A Adsersen and UNyman ldquoAntimicrobial screening of selected medicinal plantsfrom Indiardquo Journal of Ethnopharmacology vol 58 no 2 pp75ndash83 1997

[45] I Ahmad and A Z Beg ldquoAntimicrobial and phytochemicalstudies on 45 Indian medicinal plants against multi-drugresistant human pathogensrdquo Journal of Ethnopharmacology vol74 no 2 pp 113ndash123 2001

[46] S Maji P Dandapat D Ojha et al ldquoIn vitro antimicrobialpotentialities of different solvent extracts of ethnomedicinalplants against clinically isolated human pathogensrdquo Journal ofPhytology vol 2 no 4 pp 57ndash64 2010

[47] B Mamtha K Kavitha K K Srinivasan and P G ShivanandaldquoAn in vitro study of the effect of Centella asiatica [Indian pen-nywort] on enteric pathogensrdquo Indian Journal of Pharmacologyvol 36 no 1 p 41 2004


[48] R M Zablotowicz R E Hoagland and S C Wagner ldquoEffectof saponins on the growth and activity of rhizosphere bacteriardquoAdvances in Experimental Medicine and Biology vol 405 pp83ndash95 1996

[49] N EHemandezM L Tereschuk andL RAbdala ldquoAntimicro-bial activity of flavonoids inmedicinal plants fromTafı del Valle(Tucuman Argentina)rdquo Journal of Ethnopharmacology vol 73no 1-2 pp 317ndash322 2000

[50] C Marjorie ldquoPlant products as antimicrobial agentsrdquo ClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[51] R F Epand P B Savage and R M Epand ldquoBacteriallipid composition and the antimicrobial efficacy of cationicsteroid compounds (Ceragenins)rdquo Biochimica et BiophysicaActa Biomembranes vol 1768 no 10 pp 2500ndash2509 2007

[52] V Bharathy B Maria Sumathy and F Uthayakumari ldquoDeter-mination of phytocomponents by GC-MS in leaves of Jatrophagossypifolia Lrdquo Science Research Reporter vol 2 no 3 pp 286ndash290 2012

[53] N Togashi A Shiraishi M Nishizaka et al ldquoAntibacterialactivity of long-chain fatty alcohols against StaphylococcusaureusrdquoMolecules vol 12 no 2 pp 139ndash148 2007

[54] P Turcotte and S A Saheb ldquoAntimicrobial activity of phenolicantioxidantsrdquo Canadian Journal of Microbiology vol 24 no 11pp 1306ndash1320 1978

[55] M Sova ldquoAntioxidant and antimicrobial activities of cinnamicacid derivativesrdquo Mini Reviews in Medicinal Chemistry vol 12no 8 pp 749ndash767 2012

[56] V Moorthy and M Boominathan ldquoThe antimicrobial activ-ities of crude extracts and fraction of Psidium guajava andAzadirachta indica against Staphylococcus aureus in chronicdisease affected patientsrdquo International Journal of UniversalPharmacy and Life Sciences vol 1 no 2 pp 2249ndash6793 2011

[57] D Hamdan M Z El-Readi A Tahrani et al ldquoSecondarymetabolites of ponderosa lemon (Citrus pyriformis) andtheir antioxidant anti-inflammatory and cytotoxic activitiesrdquoZeitschrift fur Naturforschung C vol 66 no 7-8 pp 385ndash3932011

[58] V M Navarro-Garcıa J Luna-Herrera M G Rojas-BribiescaP Alvarez-Fitz and M Y Rıos ldquoAntibacterial activity of aris-tolochia brevipes against multidrug-resistant MycobacteriumtuberculosisrdquoMolecules vol 16 no 9 pp 7357ndash7364 2011

[59] A E Nugroho S Riyanto M A Sukari and K MaeyamaldquoAnti-allergic effects of Marmin a coumarine isolated fromAegle marmelos Correa in vitro studyrdquo International Journal ofPhytomedicine vol 3 no 1 pp 84ndash97 2011

[60] P P Ram G Varun S Balram C Deepak R Veermaand B Anil ldquoExtraction and isolation of marmelosin fromAegle marmelos synthesis and evaluation of their derivative asantidiabetic agentrdquoDer Pharmacia Lettre vol 4 no 4 pp 1085ndash1092 2012

[61] A E Nugroho S Riyanto M A Sukari and K MaeyamaldquoEffects of aegeline a main alkaloid of Aegle marmelos Correaleaves on the histamine release from mast cellsrdquo PakistanJournal of Pharmaceutical Sciences vol 24 no 3 pp 359ndash3672011

[62] C P Kala ldquoEthnobotany and ethnoconservation of Aeglemarmelos (L) Correardquo Indian Journal of Traditional Knowledgevol 5 no 4 pp 537ndash540 2006

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


plant such as its fruits stem bark and leaves possessesmedicinal property and is used for treating various eye andskin infections [23] Leaf is considered to be one of thehighest accumulatory parts of the plant containing bioactivecompounds which are synthesized as secondary metabolites[24] The present study was therefore aimed at evaluatingthe phytochemical potential and antibacterial activity ofAeglemarmelos aqueous and methanolic leaf extracts

2 Materials and Methods

21 Collection and Identification of Plant Material The freshleaves of Aegle marmelos from 18 varietiesaccessions werecollected from the orchard of Narendra Deva University ofAgriculture andTechnologyKumarganj Faizabad IndiaThetaxonomy of the plant was authenticated

22 Extract Preparation The leaves of the plantwere properlywashed in tap water and rinsed in distilled water The rinsedleaves were hot air-dried for 3 days The dried leaves ofeach plant were pulverized using pestle mortar to obtain apowdered form which was stored in airtight glass containersat 4∘C until used 10 g of powdered sample was soaked indistilled water andmethanol (200mL and 100mL) separatelyfor 12 hrs at room temperatureThe extracts were then filteredand concentrated to a final volume of 50mL and subjected tophytochemical analysis

For antibacterial screening the leaf extracts were preparedfrom Pant Aparna variety by following the protocol ofHarborne [25] in Soxhlet apparatus using 180mL of distilledwater and methanol For aqueous ethanol 50 130 volume ofdistilled water and ethanol were taken Every extraction wascarried out for 24 hrs and the extract was then dried weighedand stored in refrigerator at 4∘C

23 Phytochemical Analysis Qualitative phytochemical anal-yses of both the extracts were performed by following theprotocol of Adetuyi and Popoola [26] Trease and Evans [27]and Sofowora [28]

Tannins 200mg of plant material was boiled in 10mLdistilled water and few drops of FeCl

3were added to the

filtrate a blue-black precipitate indicated the presence ofTannins

Alkaloids 200mg plant material was boiled in 10mLmethanol and filtered 1HCl was added followed by 6 dropsof Dragendorff reagent and brownish-red precipitate wastaken as evidence for the presence of alkaloids

Saponins (Frothing test) 5mL distilled water was added to200mg plant material 05mL filtrate was diluted to 5mLwith distilled water and shaken vigorously for 2 minutesFormation of stable foam indicates the presence of saponins

Cardiac Glycosides (Keller-Kiliani test) 2mL filtrate wastreated with 1mL glacial acetic acid containing few drops ofFeCl3Conc H

2SO4was added to the above mixture giving

green-blue colour depicting the positive results for presenceof cardiac glycosides

Steroids (Liebermann-Burchard reaction) 200mg plant mate-rial was added in 10mL chloroform Acetic anhydride wasadded in the ratio of 1 1 which resulted into the formationof blue-green ring pointing towards the presence of steroids

Terpenoids (Salkowski test) To 200mg plant material 2mL ofchloroform (CHCl

3) and 3mL of concentrated sulphuric acid

(H2SO4) were carefully added A reddish brown colouration

signified the presence of terpenoids

Flavonoids To the aqueous filtrate 5mL of dilute ammoniasolution was added followed by concentrated H

2SO4 A

yellow colouration indicated the presence of flavonoids

PhlobatanninsThedeposition of a red precipitate denoted thepresence of phlobatanninswhen 200mg of plantmaterial wasdissolved in 10mL of aqueous extract and few drops of 1HCl were added in the boiling tube

Anthraquinones 500mg of dried plant leaves were boiled in10 HCl for 5mins and filtrate was allowed to cool Equalvolume of CHCl

3with few drops of 10 NH

3was added to

2mL filtrate The formation of rose-pink colour implies thepresence of Anthraquinones

Reducing Sugars To the 10mL of aqueous extract a few dropsof Fehlingrsquos solution A and B were added an orange redprecipitate suggests the presence of reducing sugars

24 Quantitative Estimation of Phytochemicals

241 Determination of Alkaloids Alkaloids content wasmeasured by following the protocol described by Harborne[29] A suspension was prepared by dispersing 5 gm of thedried leaves in 10 acetic acid solution in ethanol andkept at 28∘C for 4 hrs which was further filtered throughWhatmanNumber 42Thereafter alkaloidwas precipitated byconcentrating the filtrate to one quarter of its original volumeand drops of conc aqueous NH

4OH were added Finally the

precipitate was washed with 1 ammonia solution and driedat 80∘C in the oven The content of alkaloid was calculatedand expressed as mggm of sample

242 Determination of Flavonoids The flavonoids contentwas also determined by Harborne [29] method Briefly 5 gmof leaves was boiled in 2M HCl for 30mins under refluxand filtered after cooling Equal volume of ethyl acetate wasthen added drop wise in filtrate The weight of precipitatedflavonoid was determined and reported as mgg

243 Determination of Tannins The quantitative estimationof tannins was performed by the method of Swain [30] withminor modifications in our lab Finely powdered leaves ofAegle marmelos were kept in a beaker containing 20mL of50methanol covered with parafilm and then heated at 80∘Cin water bath for 1 hr with continuous stirring The extract



2CO3for

















































area Compound























Antioxidant



16 23404








Table 6 Continued


area Compound



ester



















32 47033 Vitamin E C29H50O2 430 058



Table 6 Continued


area Compound






4 Conclusion






Acknowledgments



References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2CO3for

















































area Compound























Antioxidant



16 23404








Table 6 Continued


area Compound



ester



















32 47033 Vitamin E C29H50O2 430 058



Table 6 Continued


area Compound






4 Conclusion






Acknowledgments



References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































area Compound























Antioxidant



16 23404








Table 6 Continued


area Compound



ester



















32 47033 Vitamin E C29H50O2 430 058



Table 6 Continued


area Compound






4 Conclusion






Acknowledgments



References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























area Compound























Antioxidant



16 23404








Table 6 Continued


area Compound



ester



















32 47033 Vitamin E C29H50O2 430 058



Table 6 Continued


area Compound






4 Conclusion






Acknowledgments



References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















area Compound























Antioxidant



16 23404








Table 6 Continued


area Compound



ester



















32 47033 Vitamin E C29H50O2 430 058



Table 6 Continued


area Compound






4 Conclusion






Acknowledgments



References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




area Compound























Antioxidant



16 23404








Table 6 Continued


area Compound



ester



















32 47033 Vitamin E C29H50O2 430 058



Table 6 Continued


area Compound






4 Conclusion






Acknowledgments



References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 6 Continued


area Compound



ester



















32 47033 Vitamin E C29H50O2 430 058



Table 6 Continued


area Compound






4 Conclusion






Acknowledgments



References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 6 Continued


area Compound






4 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
























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 Phytochemical Evaluation, Antimicrobial ...downloads.hindawi.com/journals/bmri/2014/497606.pdf · have been isolated from various parts of the plant, namely phenols,