)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/archive/2015/485469.pdf · Extract of...

Research ArticlePreliminary Investigation of BioactiveCompounds and Bioautographic Studies of Whole PlantExtract of Euphorbia pulcherrima on Escherichia coliStaphylococcus aureus Salmonella typhiand Pseudomonas aeruginosa

H B Sharif1 M D Mukhtar2 Y Mustapha2 and A O Lawal1

1Department of Applied Science College of Science and Technology Kaduna Polytechnic PMB 2021 Kaduna Nigeria2Department of Microbiology Bayero University Kano Nigeria

Correspondence should be addressed to H B Sharif sharifgoshiyahoocom

Received 14 August 2014 Revised 9 December 2014 Accepted 14 December 2014

Academic Editor Barbara R Conway

Copyright copy 2015 H B Sharif 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 aim of this study is to carry out preliminary investigation of bioactive compounds and bioautographic studies of whole plantextract of Euphorbia pulcherrima on Escherichia coli Staphylococcus aureus Salmonella typhi and Pseudomonas aeruginosa TukeyHSD test of hierarchy for the effect of different solvents crude extract on bacterial isolates indicates the methanol extract as themost bioactive The Tukey HSD analysis also showed that the bioactivities of the crude extracts of the various parts of Euphorbiapulcherrima were part dependent and the whole plant was the most bioactive The ethyl acetate fraction of the methanol extract ofthe whole plant of Euphorbia pulcherrima has been shown in this work to contain phytochemicals which have shown remarkableactivities against Escherichia coli Staphylococcus aureus Salmonella typhi and Pseudomonas aeruginosa The bioactivities againstthe test organisms were due to the combined effects of the compounds separated on TLC plates Families of terpenoids flavonoidsalkaloids saponin and steroids that were detected in the extracts were identified by GC-MSThe various classes of phytochemicalsin the E pulcherrima plant provided the antimicrobial potency of the plant

1 Introduction

The search for solutions to the global problems of antibioticresistance in pathogenic microorganisms has often beendriven on the isolation and characterization of new antimi-crobial compounds from a variety of sources includingmedicinal plants [1] Investigations into toxicity of medicinalplants have been reported by [2ndash4] However there is still anincreasing interest in the use of medicinal herbs for meetingthe goal of primary health care delivery worldwide [5 6]

The plant family Euphorbiaceae contains skin irritatingand tumor promoting diterpenoids [7]

However some species are used in folk medicine totreat skin diseases gonorrhea migraine intestinal parasites

and warts [8] The broad range and diversity of biologicalactivities in the Euphorbia genus may be due to the presenceof various components in the plants with different modes ofaction [9 10]

The active principles of many drugs found in plantsare secondary metabolites [11 12] However there is scantyliterature on the bioactive secondary metabolites possessedby Euphorbia pulcherrima that may be responsible for itscurative effect

This present study focused on identification of bioactivecompounds in whole plant extract of Euphorbia pulcherrimaand bioautographic studies of the whole plant extract withEscherichia coli Staphylococcus aureus S typhi and Psaeruginosa as test organisms Bioautography is a microbial

Hindawi Publishing CorporationAdvances in PharmaceuticsVolume 2015 Article ID 485469 14 pageshttpdxdoiorg1011552015485469

2 Advances in Pharmaceutics

detection method hyphenated with planar chromatographytechniques It is based mainly on antimicrobial or antifungalproperties of analyzed substancesThe selection of E pulcher-rima for investigation premised on its use and efficacy in theAfrican ethnopharmacopoeia to treat gastroenteritis relatedailments [13] despite its acclaimed toxicity The medicinalproperties of the plant are also contraindicated in the localtreatment of respiratory tract infection malaria eczemaasthma and wound healing properties

2 Materials and Method

21 Collection and Authentication of Samples of Euphorbiapulcherrima Plant The samples of E pulcherrima werecollected from the bushes along Gwarzo road Kano StateNigeria at different sites between November and December2013 The plants were authenticated at the Herbarium of theDepartment of Biological Sciences Bayero University Kano

22 Preparation of Plant Samples The E pulcherrima wholeplant samples were washed with distilled water and dried atroom temperature in the biological garden at the Departmentof Applied Science Kaduna Polytechnic Dried E pulcher-rima samples were crushed and pulverized using sterilizedmortar and pestle The plant powder was exhaustively andsuccessively extracted with methanol ethanol and water inSoxhlet extractor 500 g of plant was used for the extractionfrom which the crude extract was obtained 100mgmL50mgmL and 25mgmL of the crude extract were used tocarry out the antimicrobial activity evaluation against thetest organisms The methanol extract was used to carry outfurther analyses as it indicated the highest activity againstthe test organisms Preliminary antimicrobial activity studiesconfirmed the methanol extract as the most bioactive againstEscherichia coli Staphylococcus aureus S typhi and Psaeruginosa as test organisms

23 Chromatographic Studies The crude methanol extract ofwhole plant of E pulcherrima (L) was fractionated using ethylacetate The whole plant ethyl acetate fraction was subjectedto thin layer chromatography (TLC) using normal TLCprecoated silica gel G micro slides Various solvent systemsconsisting of hexane and ethyl acetate in the ratios 9 14 1 7 3 3 2 and 1 1 were used at different concentrationsto determine the solvent system that provided maximumseparation of compounds when the slides were sprayed withp-anisaldehyde and visualized under UV light Hexane ethylacetate in ratio 7 3 was used to separate the componentsof the extract and the Rf values were thus calculated Thedeveloped chromatograms were subjected to antimicrobialactivity test by the Agar overlay bioautography method(Onawumi 2000) and observed the zones of inhibition on theTLC plates The ethyl acetate fraction was used in its crudeform

231 Column Chromatography The ethyl acetate extractused for the column chromatography was in crude formThe weight of the extract used was 4 g This was eluted with

200mL of 100 n-hexane first and thenwith varying ratios ofhexane ethyl acetate used 20mL of eluents was collected atintervals and similar compounds were pooled together afterTLC

A glass column of length 30 cm and width 3 cm parkedwith silica gel was used for the partitioning of the ethyl acetatefraction The ethyl acetate fraction was first eluted with200mL of 100 n-hexane and two different sets of fractionswere collected and labeled as A and B The fraction obtainedby eluting the column with 9 1 n-hexane ethyl acetate waslabeled as C n-Hexane ethyl acetate in the ratio 4 1 elutedfractions D E F and G 7 3 n-hexane ethyl acetate mixtureeluted the fraction labeled HThe fractions were further usedto confirm the bioactivity of the ethyl acetate fraction

24 FT-IR and GC-MS Analysis The A B C D E F G andH fractions obtained from the column chromatography wereconcentrated on rotary evaporator and analysed using GC-MS-QP2010 Plus Shimadzu and FT-IR model 8400S scannedin accordance with ATSM1252-98 to determine the probablecompounds responsible for the bioactivity of the fractions

25 Characterization and Authentication of Test OrganismsThe test organismswere typed cultures of E coliATCC 25922Ps aeruginosa ATCC 27853 S aureus ATCC 25923 and theclinical isolates of S typhi obtained from the PharmaceuticalMicrobiology Laboratory Department of PharmaceuticalSciences Ahmadu Bello University Zaria The standardstrains of bacteria were grown on nutrient agar and tested forpurity while the clinical isolates of S typhiwere characterizedas described byCheesebrough [14] by observing their culturalcharacteristics when subcultured on SS agar MacConkeyagar and nutrient agar and the Gram staining reaction forbacteria Biochemical tests for motility citrate and methylred as positive confirmatory tests were performed for S typhi

26 Preparation and Standardization of Inocula McFarlandrsquosstandard method was adopted from Cheesebrough [14] tostandardize the organisms to 10 times 108 cfumL using Genesys20 spectrophotometer for the bacterial test organisms at625 nm optical density

27 Antimicrobial Susceptibility Test The agar well diffusionmethod of the National Committee on Clinical LaboratoryStandard [16] was adopted to test for the antimicrobialactivity of the extracts on the test organisms Sterile mediaplates of nutrient agar were prepared for the bacteria Theseplates were then separately flooded with diluted standardizedovernight cultures and then drained off to remove excessWells of 6mm diameter were made in triplicate in eachplate with a central well for the control using 6mm sterilecork borer The wells were filled with 01mL of dilutedconcentrations of extracts with the aid of sterile pipettesper well 100 50 and 25mgmL of extract were used and1mgmL of the standard antibiotics (ciprofloxacin 500mgand flucamed 50mg) was used as positive controls Steriledistilled water was used as negative control on a separate

Advances in Pharmaceutics 3

Table 1 Rf values for different compounds present in the ethyl acetate fraction

Spots 1 2 3 4 5 6 7 8Rf values 016 042 054 065 071 078 083 089

Table 2 Different fractions of the ethyl acetate fraction partitioned on silica gel column

Serial number Solvent system Fractions eluted Colour Group1 n-Hexane 1 2 3 Black A2 n-Hexane 4 5 6 7 8 9 10 Light green B3 Hexane ethyl acetate (9 1) 11 12 13 14 15 16 17 18 19 20 White C4 Hexane ethyl acetate (4 1) 21 22 23 24 Yellow D5 Hexane ethyl acetate (4 1) 25 26 27 28 29 30 31 32 33 Dark brown E

6 Hexane ethyl acetate (4 1) 34 35 36 37 38 39 40 41 42 43 44 45 4647 48 Dark green F

7 Hexane ethyl acetate (4 1) 49 50 51 52 53 54 55 56 57 58 59 60 6162 Green G

8 Hexane ethyl acetate (7 3) 63 64 65 66 67 68 Brown H

Table 3 FT-IR spectra of the ethyl acetate extract

Serial number Peaks in ethyl acetate extract Motion Functional group

1725268255689307

CndashH bending out of plane Aromatics

2 10416118047

CndashN stretchingCndashOndashC stretch

Aminesesters

3 124991 CndashC(O)ndashC Esters4 137336 CH3 bend Alkanes5 1456 CH3 bend Alkanes

6 161254165112 NndashH stretching Nitrogenous compounds

7 33794 OndashH stretch Alcoholphenol8 10416 CndashO stretch Phenol9 1724 C=O stretching Aliphatic aldehydes

10 285667 Carboxylic acid OndashHstretch Acid

11 292418 CndashH stretching Aliphatic hydrocarbons

plate Diameters of zones inhibition were measured afterincubating the plates at 37∘C for 24 hrs (bacteria) The plateswere replicated in triplicate and the diameter of zones ofinhibition was recorded

28 Bioautographic Studies The agar overlay method wasadopted for the bioautographic studies The agar media wereapplied directly onto the developed TLC plate of the wholeplant ethyl acetate fraction TLC chromatogram 19mL ofmolten nutrient agar was seeded with 1mL of standardizedovernight culture of the susceptible organism This waspoured over the developed chromatogramTLCplate kept in aPetri dishThiswas allowed to solidify and prediffuse for 2 hrsbefore incubation at 37∘C for 24 hrs for the bacterial isolates

The plates were sprayed with aqueous solution of methylthiazolyl tetrazolium (MTT) chloride for the detection ofdehydrogenase activity Zones of inhibition were observed asclear spots against purple background

3 Results

The percentage yield for the E pulcherrimamethanol extractwas 514 with dark green appearance and gummy textureEthanol extract was 455 with dark green appearanceand gummy texture The aqueous extract was 416 andappeared as a dark solid extract The ethyl acetate frac-tion of the methanol extract was consistently giving higher


Table 4 Phytochemicals in E pulcherrima n-hexane fraction of ethyl acetate extract (A)

Peaknumber Name Structure

4 3-Fluorophenyl 2-fluoro-6-(trifluoromethyl)benzoate

F

FF

F

FO

O

14 4-Bromophenyl heptyl phthalate(wax)

O

O

O

O

Br

19

O-(3-(tert-Butyl)cyclohexa-24-dien-1-yl)

(6-methoxypyridin-2-yl)(methyl)carbamothioate

(alkaloid)

O

O

N N S

34Bis(4-methylheptan-3-yl)

phthalate(wax) O

O

O

O

Time (min)

TIC

Figure 1 GC-MS chromatogram for Euphorbia pulcherrima n-hexane fraction of ethyl acetate extract (A)

antimicrobial activity against the test organisms in compari-son to other fractions and so it was used for the study

31 Chromatography Studies The thin layer chromatogramsof the ethyl acetate fraction revealed the presence of eightcompounds with different Rf values as shown in Table 1

The partitioning of the ethyl acetate fraction on silicagel column using 100 n-hexane 9 1 hexane ethyl acetate4 1 hexane ethyl acetate and 7 3 hexane ethyl acetate alsorevealed sixty-eight fractions of different colours that weregrouped into eight using the similarity in their Rf values(Table 2)

Time (min)

TIC

Figure 2 GC-MS chromatogram for Euphorbia pulcherrima n-hexane fraction of ethyl acetate extract (B)

The results of the GC-MS analysis of fractions A B C DE F G and H revealed different phytochemicals (terpenoidsflavonoids alkaloids saponin and steroids) as shown inTables 4ndash11The chromatograms of the extracts are in Figures1ndash8 The presence of the compounds was further confirmedby functional groups such as

OH

OOO

CHC NH2

NH2 H3COCH


Table 5 Phytochemicals in Euphorbia pulcherrima n-hexane fraction of ethyl acetate extract (B)


8 Menthyl acetate

O

O

10 244-Trimethylpentyl 2-ethylbutanoate(wax) O

O

18 226-Trimethyl-7-(prop-1-en-2-yl)cyclooctane-15-dione

O

O

19 Heptan-3-yl isobutyl phthalate(wax)

O

O

O

O

204-(((Tetrahydrofuran-2-yl)methyl)amino)-1-

oxaspiro[45]dec-3-en-2-one(alkaloid)

NH O

O

O

2379-Di-tert-butyl-1-oxaspiro[45]deca-69-diene-28-

dione(flavonoid)

OO

O

25 Butyl heptan-3-yl phthalate(wax)

O

O

O

O

28 2-(tert-Butyl)-6-methyl-4H-13-dioxin-4-one(flavonoid)

O

O

O


Table 5 Continued


32 2-Methyl-2-(3-methyl-2-oxobutyl)cyclohexanone

O

O

34 1235-Tetraisopropylcyclohexane

39 Oleamide(wax)

O

NH2

44 Bis(2-ethylhexyl) phthalate(wax) O

O

OO

Time (min)

TIC

Figure 3 GC-MS chromatogram for Euphorbia pulcherrimahexane ethyl acetate (9 1) fraction of ethyl acetate extract (C)

These functional groups were revealed by the FT-IRanalysis of the ethyl acetate fraction (see Table 3)

32 Bioautography Studies The results of the bioautographytests of ethyl acetate extract against E coli (EC) Ps aerug-inosa (Ps) S typhi (ST) and S aureus (SA) are shown inFigure 9 It revealed that all the eight compounds separatedby TLC had good activities against all the test organismsFigure 9 compares the original TLC plates displaying theseparated components with the developed TLC plate of the

Time (min)

TIC

Figure 4 GC-MS chromatogram for Euphorbia pulcherrimahexane ethyl acetate (4 1) fraction of ethyl acetate extract (D)

whole plant ethyl acetate fraction carrying 19mL of moltennutrient agar which was seeded with 1mL of standard-ized overnight culture of the susceptible organism in Petridishes

4 Discussion

The chromatogram of the Euphorbia pulcherrima extracts AB C D E F G and H obtained from the GC-MS confirmedthe numerous compounds extracted by the ethyl acetate


Table 6 Phytochemicals in Euphorbia pulcherrima hexane ethyl acetate (9 1) fraction of ethyl acetate extract (C)

Peak Name Structure

1 26-Di-tert-butylcyclohexa-25-diene-14-dione(quinone)

O

O

3 24-Di-tert-butylphenol(phenolic)

OH

12 Diisobutyl phthalate(wax)

O

O OO


dione(flavonoid)

OO O

17 Dibutyl phthalate(wax)

O

O

O

O

24

446a6b8a111114b-Octamethyl-12344a566a6b788a910111212a1414a14b-

icosahydropicen-3-ol(steroid)

OH

34 Lup-20(29)-en-3-ol acetate(saponin)

H

H

H

OO

H

35

4-(3-Hydroxy-3-methylpentyl)-34a88-tetramethyldecahydronaphthalen-1-ol

(saponin)

OH

OH


Table 7 Phytochemicals in Euphorbia pulcherrima hexane ethyl acetate (4 1) fraction of ethyl acetate extract (D)

Peak Name Structure

5 12-Dimethyl-3-(prop-1-en-2-yl)cyclopentane


O

O

O

O


O

O

O

O

20



OH

22

446a6b8a111114b-Octamethyl-12344a566a6b788a9101112b131414a14b-


HO

24

(8S9S10R13R14S17R)-17-((2R5R)-5-Ethyl-6-methylheptan-2-yl)-1013-dimethyl-2347891011121314151617-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl

nonadecanoate(steroid) O

HH

H

O

25 4a88a-Trimethyl-2-(prop-1-en-2-yl)-12344a568a-octahydronaphthalene

303a-(Hydroxymethyl)-5a5b8811a-pentamethyl-1-(prop-1-en-

2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-ol(saponin)

OH

HO


Table 7 Continued

Peak Name Structure

322a5a88-Tetramethyl-3-(6-methylheptan-2-

yl)hexadecahydrocyclopenta[a]cyclopropa[e]phenanthrene(steroid)

Table 8 Phytochemicals in Euphorbia pulcherrima hexane ethyl acetate (4 1) fraction of ethyl acetate extract (E)

Peak Name Structure


O

O OO

26

5-Methyl-5-(4812-trimethyltridecyl)dihydrofuran-2(3H)-

one(wax) O O


OO

O

34(E)-12-Bis(5-methylbenzo[d]oxazol-2-

yl)ethene(alkaloid) O

N O

N

Time (min)

TIC

Figure 5 GC-MS chromatogram for Euphorbia pulcherrimahexane ethyl acetate (4 1) fraction of ethyl acetate extract (E)

Time (min)

TIC

Figure 6 GC-MS chromatogram for Euphorbia pulcherrimahexane ethyl acetate (4 1) fraction of ethyl acetate extract (F)


Table 9 Phytochemicals in Euphorbia pulcherrima hexane ethyl acetate (4 1) fraction of ethyl acetate extract (F)

Peak Name Structure

1 2-Phenylacetic acid(wax)

OH

O

3 4-(2-Hydroxyethyl)-2-methoxyphenol(phenolic)

OH

O

HO

4 24-Di-tert-butylphenol(terpenoid)

OH

5447a-Trimethyl-5677a-tetrahydrobenzofuran-

2(4H)-one(steroid)

OO

9 Triethyl 2-hydroxypropane-123-tricarboxylate(wax)

OO

OO

OOOH

32

(E)-17-(5-Ethyl-6-methylhept-3-en-2-yl)-1013-dimethylhexadecahydro-1H-

cyclopenta[a]phenanthren-3-yl acetate(steroid)

OO

solution However not all of the compounds represented bythe peaks may be of relevance in the light of antimicrobialactivity The useful antimicrobial phytochemicals includeterpenoids flavonoids alkaloids saponin and steroidsThesecategories of phytochemicals have been detected in theextracts and their names are presented in Tables 4ndash11 Theantimicrobial activities of the plant may be attributable tothe presence of the identified phytochemicals in the extractsTerpenes or terpenoids are active against bacteria [17ndash26]Flavonoids which are hydroxylated phenolic substances butoccur as C6-C3 units linked to an aromatic ring were presentin all extracts Since they are known to be synthesized byplants in response tomicrobial infection [27] it should not be

surprising that they have been found to be effective antimi-crobial substances against microorganisms Their activity isprobably due to their ability to complex with extracellularand soluble proteins and to complex with bacterial cell wallssimilar to quinones More lipophilic flavonoids may also dis-rupt microbial membranes [28] The Euphorbia pulcherrimafractions A B C D E F G and H have been found tobe largely similar in composition but fraction H obtainedfrom hexane ethyl acetate (7 3) appears to contain morealkaloids and terpenoids than other fractions Generally themixtures of hexane and ethyl acetate weremore effective thanonly hexane in the extraction of the phytochemicals from theEuphorbia pulcherrima


Table 10 Phytochemicals in Euphorbia pulcherrima hexane ethyl acetate (4 1) fraction of ethyl acetate extract (G)

Peak Name Structure

1 24-Di-tert-butylphenyl 5-hydroxypentanoate(saponin) O

O

HO

2 1198731-dodecyl-1198732-(thiazol-2-yl)oxalamide

(alkaloid)

N

S NH

OHN

O

6 79-Di-tert-butyl-1-oxaspiro[45]deca-69-diene-28-dione(flavonoid)

OO O

19



OH

Time (min)

TIC

Figure 7 GC-MS chromatogram for Euphorbia pulcherrimahexane ethyl acetate (4 1) fraction of ethyl acetate extract (G)

All the compoundsA B C D E F G andH showed goodactivity against the test organisms with zones of inhibitionsranging between 18 and 35mm at the highest concentrationlevel (100mgmL)

The bioautography results confirmed the presence of dif-ferent antimicrobial compounds in the ethyl acetate fractionAll the compounds A B C D E F G and H showed goodactivity against the test organisms with zones of inhibitionsevident from the clearing of the spots from the TLC plates

Time (min)

TIC

Figure 8 GC-MS chromatogram for Euphorbia pulcherrimahexane ethyl acetate (7 3) fraction of ethyl acetate extract (H)

(Figure 9) The bioactivity against the test organisms wasobserved to be attributable to the combined effects of thecompounds separated on the TLC plate This is because allthe spots on the TLC plates were cleared consequently uponactivity against the test organisms It is indicative of thesynergistic effects of the components of the extract againstthe test organisms This finding corroborates the works ofEsimone et al [29] The identified compounds might havefound their way into the cells of the test organisms and


Table 11 Phytochemicals in Euphorbia pulcherrima hexane ethyl acetate (7 3) fraction of ethyl acetate extract (H)

Peak Name Structure

4

O

O

O

(E)-5-(1-(Cyclohexa-15-dien-1-yl)ethylidene)-3-methoxy-4-methylhexahydro-1H-cyclopenta[c]furan-1-one

(terpenoid)

O

O

O

5 236-Trimethylnaphthalene

7 3-(2-Methylprop-1-en-1-yl)-1H-indene(terpenoid)


13 2-Phenylbicyclo[321]octa-26-diene

14 Undecan-5-ylbenzene

18 (455-Trimethylcyclopenta-13-dien-1-yl)benzene

21 (2-Methyl-[111015840-biphenyl]-3-yl)methanolOH

31 Phenanthrene

39 4-Methylnaphtho[12-b]thiophene

S


Table 11 Continued

Peak Name Structure


OO O

49 5-Methyl-33-diphenyl-3H-pyrazole(alkaloid)

NN

Figure 9 Bioautography results of ethyl acetate extract against E coli (EC) Ps aeruginosa (Ps) S typhi (ST) and S aureus (SA)

ensured the production of efflux pump inhibitors (resistancemodifying agents) whichmay have facilitated the penetrationof more phytochemicals into the microbial cells especially ofthe multidrug resistant organisms Researches have shownthat plant medicinal compounds have resistance modifyingactivities in vitro [1] Similarly Tegos et al [30] reportedthat two MDR inhibitors (IN

271and MC

207110) from Berber-

ine plant were found to have increased the effectivenessof 13 plants antimicrobial compounds against both Gram-positive and Gram-negative bacteria including those knownto express efflux pumps

5 Conclusion

Theethyl acetate fraction of themethanol extract of thewholeplant of Euphorbia pulcherrima contains phytochemicalswhich have shown remarkable activities against Escherichiacoli Staphylococcus aureus S typhi and Ps aeruginosa Thebioactivities against the test organisms were due to thecombined effects of the compounds separated on the TLCplates Preparative TLC of the various hexane ethyl acetatefractions are therefore suggested This should lead to theisolation of the individual bioactive agent and investigationof their independent bioactivity

Conflict of Interests

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

Acknowledgments

The authors are grateful to Benedict O Odjobo AnalyticalUnit Federal Institute of Industrial Research Oshodi forthe GC-MS analysis and the National Research Institute forChemical Technology Zaria for the FT-IR

References

[1] T Sibanda and A I Okoh ldquoThe challenges of overcomingantibiotic resistance plant extracts as potential sources ofantimicrobial and resistance modifying agentsrdquoAfrican Journalof Biotechnology vol 6 no 25 pp 2886ndash2896 2007

[2] A A Adedapo M O Abatan and O O Olorunsogo ldquoToxiceffects of some plants in the genus Euphorbia on haematologicaland biochemical parameters of ratsrdquoVeterinary Archive vol 74no 1 pp 33ndash63 2004

[3] E U Etuk B M Agaie P A Onyeyili and C U OttahldquoToxicological studies of aqueous stem bark extract of Boswelliadalzielii in albino ratsrdquo Indian Journal of Pharmacology vol 38no 5 pp 359ndash360 2006

[4] N A Siddique M Mujeeb A K Najmi and M AkramldquoEvaluation of antioxidant activity quantitative estimation ofphenols and flavonoids in different parts of Aegle marmelosrdquoAfrican Journal of Plant Science vol 4 no 1 pp 1ndash5 2010

[5] K Kalimuthu S Vijayakumar and R Senthilkumar ldquoAntimi-crobial activity of the biodiesel plant Jatropha curcas LrdquoInternational Journal of Pharma and Bio Sciences vol 1 no 3article 29 2010


[6] F O Ekundayo C A Adeboye and E A Ekundayo ldquoAntimi-crobial activities and phytochemical screening of pignut (Jat-rophas curcas Linn) on some pathogenic bacteriardquo Journal ofMedicinal Plants Research vol 5 no 7 pp 1261ndash1264 2011

[7] S Evans and S Taylor ldquoPro-inflamatory tumor promotingand antitumor diterpene of the plant families Euphorbiaceaeand Thymelaeacaeaerdquo in Progress in the Chemistry of OrganicNatural Products W Herz H Griscbach and GW Kirby Edsvol 44 pp 1ndash99 1983

[8] A K Singla and K Pathak ldquoPhytoconstituents of Euphorbiaspeciesrdquo Fitoterapia vol 61 no 6 pp 483ndash516 1990

[9] A Correa R J Strasser and M A Martins-Loucao ldquoResponseof plants to ectomycorrhizae in N-limited conditions whichfactors determine its variationrdquo Mycorrhiza vol 18 no 8 pp413ndash427 2008

[10] A Kumar L K Sharma and G Agarwal ldquoMedicinal plants forskin and hair carerdquo Indian Journal of Traditional Knowledge vol2 no 1 pp 62ndash68 2003

[11] A Ghani Introduction to Pharmacognosy Ahmadu Bello Uni-versity Press Zaria Nigeria 1990

[12] I N DobelisMagic and Medicine of Plants The Readers DigestAssociation Inc 1993

[13] A I Yakubu andM DMukhtar ldquoIn vitro antimicrobial activityof some phytochemical fractions of Euphorbia pulcherima L(Poinsettia)rdquo Journal of Medicinal Plants Research vol 5 no 12pp 2470ndash2475 2011

[14] M Cheesebrough Medical Laboratory Manual for TropicalCountries ELBS 2002

[15] S Murugan R Anand P Uma Devi N Vidhya and K ARajesh ldquoEfficacy of Euphorbia milli and Euphorbia pulcherrimaon aflatoxin producing fungi (Aspergillus flavus and Aspergillusparasiticus)rdquo African Journal of Biotechnology vol 6 no 6 pp718ndash719 2007

[16] National Committee for Clinical Laboratory Standards ldquoPer-formance for antimicrobial susceptibility testingrdquo Tech RepStandard M10-S5 NCCLC Villanova Pa USA 1994

[17] A A Ahmed A AMahmoud H JWilliams A Ian Scott J HReibenspies and T J Mabry ldquoNew sesquiterpene 120572-methylenelactones from the Egyptian plant Jasonia candicansrdquo Journal ofNatural Products vol 56 no 8 pp 1276ndash1280 1993

[18] J A Amaral A Ekins S R Richards and R Knowles ldquoEffect ofselected monoterpenes on methane oxidation denitrificationand aerobic metabolism by bacteria in pure culturerdquo Appliedand Environmental Microbiology vol 64 no 2 pp 520ndash5251998

[19] J T Barre B F Bowden J C Coll et al ldquoA bioactive triterpenefrom Lantana camarardquo Phytochemistry vol 45 no 2 pp 321ndash324 1997

[20] S Habtemariam A I Gray and P G Waterman ldquoA newantibacterial sesquiterpene from Premna oligotrichardquo Journalof Natural Products vol 56 no 1 pp 140ndash143 1993

[21] M Himejima K R Hobson T Otsuka D L Wood and IKubo ldquoAntimicrobial terpenes from oleoresin of ponderosapine tree Pinus ponderosa a defense mechanism against micro-bial invasionrdquo Journal of Chemical Ecology vol 18 no 10 pp1809ndash1818 1992

[22] I Kubo H Muroi and M Himejima ldquoAntibacterial activity oftotarol and its potentiationrdquo Journal of Natural Products vol 55no 10 pp 1436ndash1440 1992

[23] L Mendoza M Wilkens and A Urzua ldquoAntimicrobial studyof the resinous exudates and of diterpenoids and flavonoids

isolated from some Chilean Pseudognaphalium (Asteraceae)rdquoJournal of Ethnopharmacology vol 58 no 2 pp 85ndash88 1997

[24] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[25] C C Tassou E H Drosinos and G J E Nychas ldquoEffectsof essential oil from mint (Mentha piperita) on Salmonellaenteritidis and Listeria monocytogenes in model food systems at4∘ and 10∘Crdquo Journal of Applied Bacteriology vol 78 no 6 pp593ndash600 1995

[26] R S L Taylor F Edel N P Manandhar and G H N Tow-ers ldquoAntimicrobial activities of southern Nepalese medicinalplantsrdquo Journal of Ethnopharmacology vol 50 no 2 pp 97ndash1021996

[27] R A Dixon P M Dey and C J Lamb ldquoPhytoalexinsenzymology and molecular biologyrdquo Advances in Enzymologyand Related Areas of Molecular Biology vol 55 pp 1ndash69 1983

[28] H Tsuchiya M Sato T Miyazaki et al ldquoComparative studyon the antibacterial activity of phytochemical flavanonesagainst methicillin-resistant Staphylococcus aureusrdquo Journal ofEthnopharmacology vol 50 no 1 pp 27ndash34 1996

[29] CO EsimoneMUAdikwu BUzuegbu andAUdeogaranyaldquoThe effect of ethylenediamine tetraacetic acid on the antimi-crobial properties of benzoic acidrdquo Journal PharmaceuticalResearch and Development vol 4 pp 1ndash8 1999

[30] G Tegos F R Stermitz O Lomovskaya and K Lewis ldquoMul-tidrug pump inhibitors uncover remarkable activity of plantantimicrobialsrdquo Antimicrobial Agents and Chemotherapy vol46 no 10 pp 3133ndash3141 2002

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


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BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


detection method hyphenated with planar chromatographytechniques It is based mainly on antimicrobial or antifungalproperties of analyzed substancesThe selection of E pulcher-rima for investigation premised on its use and efficacy in theAfrican ethnopharmacopoeia to treat gastroenteritis relatedailments [13] despite its acclaimed toxicity The medicinalproperties of the plant are also contraindicated in the localtreatment of respiratory tract infection malaria eczemaasthma and wound healing properties

2 Materials and Method

21 Collection and Authentication of Samples of Euphorbiapulcherrima Plant The samples of E pulcherrima werecollected from the bushes along Gwarzo road Kano StateNigeria at different sites between November and December2013 The plants were authenticated at the Herbarium of theDepartment of Biological Sciences Bayero University Kano

22 Preparation of Plant Samples The E pulcherrima wholeplant samples were washed with distilled water and dried atroom temperature in the biological garden at the Departmentof Applied Science Kaduna Polytechnic Dried E pulcher-rima samples were crushed and pulverized using sterilizedmortar and pestle The plant powder was exhaustively andsuccessively extracted with methanol ethanol and water inSoxhlet extractor 500 g of plant was used for the extractionfrom which the crude extract was obtained 100mgmL50mgmL and 25mgmL of the crude extract were used tocarry out the antimicrobial activity evaluation against thetest organisms The methanol extract was used to carry outfurther analyses as it indicated the highest activity againstthe test organisms Preliminary antimicrobial activity studiesconfirmed the methanol extract as the most bioactive againstEscherichia coli Staphylococcus aureus S typhi and Psaeruginosa as test organisms

23 Chromatographic Studies The crude methanol extract ofwhole plant of E pulcherrima (L) was fractionated using ethylacetate The whole plant ethyl acetate fraction was subjectedto thin layer chromatography (TLC) using normal TLCprecoated silica gel G micro slides Various solvent systemsconsisting of hexane and ethyl acetate in the ratios 9 14 1 7 3 3 2 and 1 1 were used at different concentrationsto determine the solvent system that provided maximumseparation of compounds when the slides were sprayed withp-anisaldehyde and visualized under UV light Hexane ethylacetate in ratio 7 3 was used to separate the componentsof the extract and the Rf values were thus calculated Thedeveloped chromatograms were subjected to antimicrobialactivity test by the Agar overlay bioautography method(Onawumi 2000) and observed the zones of inhibition on theTLC plates The ethyl acetate fraction was used in its crudeform

231 Column Chromatography The ethyl acetate extractused for the column chromatography was in crude formThe weight of the extract used was 4 g This was eluted with

200mL of 100 n-hexane first and thenwith varying ratios ofhexane ethyl acetate used 20mL of eluents was collected atintervals and similar compounds were pooled together afterTLC

A glass column of length 30 cm and width 3 cm parkedwith silica gel was used for the partitioning of the ethyl acetatefraction The ethyl acetate fraction was first eluted with200mL of 100 n-hexane and two different sets of fractionswere collected and labeled as A and B The fraction obtainedby eluting the column with 9 1 n-hexane ethyl acetate waslabeled as C n-Hexane ethyl acetate in the ratio 4 1 elutedfractions D E F and G 7 3 n-hexane ethyl acetate mixtureeluted the fraction labeled HThe fractions were further usedto confirm the bioactivity of the ethyl acetate fraction

24 FT-IR and GC-MS Analysis The A B C D E F G andH fractions obtained from the column chromatography wereconcentrated on rotary evaporator and analysed using GC-MS-QP2010 Plus Shimadzu and FT-IR model 8400S scannedin accordance with ATSM1252-98 to determine the probablecompounds responsible for the bioactivity of the fractions

25 Characterization and Authentication of Test OrganismsThe test organismswere typed cultures of E coliATCC 25922Ps aeruginosa ATCC 27853 S aureus ATCC 25923 and theclinical isolates of S typhi obtained from the PharmaceuticalMicrobiology Laboratory Department of PharmaceuticalSciences Ahmadu Bello University Zaria The standardstrains of bacteria were grown on nutrient agar and tested forpurity while the clinical isolates of S typhiwere characterizedas described byCheesebrough [14] by observing their culturalcharacteristics when subcultured on SS agar MacConkeyagar and nutrient agar and the Gram staining reaction forbacteria Biochemical tests for motility citrate and methylred as positive confirmatory tests were performed for S typhi

26 Preparation and Standardization of Inocula McFarlandrsquosstandard method was adopted from Cheesebrough [14] tostandardize the organisms to 10 times 108 cfumL using Genesys20 spectrophotometer for the bacterial test organisms at625 nm optical density

27 Antimicrobial Susceptibility Test The agar well diffusionmethod of the National Committee on Clinical LaboratoryStandard [16] was adopted to test for the antimicrobialactivity of the extracts on the test organisms Sterile mediaplates of nutrient agar were prepared for the bacteria Theseplates were then separately flooded with diluted standardizedovernight cultures and then drained off to remove excessWells of 6mm diameter were made in triplicate in eachplate with a central well for the control using 6mm sterilecork borer The wells were filled with 01mL of dilutedconcentrations of extracts with the aid of sterile pipettesper well 100 50 and 25mgmL of extract were used and1mgmL of the standard antibiotics (ciprofloxacin 500mgand flucamed 50mg) was used as positive controls Steriledistilled water was used as negative control on a separate



Spots 1 2 3 4 5 6 7 8Rf values 016 042 054 065 071 078 083 089








1725268255689307


2 10416118047


Aminesesters









3 Results






F

FF

F

FO

O


O

O

O

O

Br

19



(alkaloid)

O

O

N N S


phthalate(wax) O

O

O

O

Time (min)

TIC





Time (min)

TIC



OH

OOO

CHC NH2

NH2 H3COCH




8 Menthyl acetate

O

O


O


O

O


O

O

O

O



NH O

O

O


dione(flavonoid)

OO

O


O

O

O

O


O

O

O


Table 5 Continued



O

O


39 Oleamide(wax)

O

NH2


O

OO

Time (min)

TIC




Time (min)

TIC



4 Discussion




Peak Name Structure


O

O


OH


O

O OO


dione(flavonoid)

OO O


O

O

O

O

24



OH


H

H

H

OO

H

35


(saponin)

OH

OH



Peak Name Structure



O

O

O

O


O

O

O

O

20



OH

22



HO

24



HH

H

O




OH

HO


Table 7 Continued

Peak Name Structure




Peak Name Structure


O

O OO

26


one(wax) O O


OO

O



N O

N

Time (min)

TIC


Time (min)

TIC




Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Spots 1 2 3 4 5 6 7 8Rf values 016 042 054 065 071 078 083 089








1725268255689307


2 10416118047


Aminesesters









3 Results






F

FF

F

FO

O


O

O

O

O

Br

19



(alkaloid)

O

O

N N S


phthalate(wax) O

O

O

O

Time (min)

TIC





Time (min)

TIC



OH

OOO

CHC NH2

NH2 H3COCH




8 Menthyl acetate

O

O


O


O

O


O

O

O

O



NH O

O

O


dione(flavonoid)

OO

O


O

O

O

O


O

O

O


Table 5 Continued



O

O


39 Oleamide(wax)

O

NH2


O

OO

Time (min)

TIC




Time (min)

TIC



4 Discussion




Peak Name Structure


O

O


OH


O

O OO


dione(flavonoid)

OO O


O

O

O

O

24



OH


H

H

H

OO

H

35


(saponin)

OH

OH



Peak Name Structure



O

O

O

O


O

O

O

O

20



OH

22



HO

24



HH

H

O




OH

HO


Table 7 Continued

Peak Name Structure




Peak Name Structure


O

O OO

26


one(wax) O O


OO

O



N O

N

Time (min)

TIC


Time (min)

TIC




Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





F

FF

F

FO

O


O

O

O

O

Br

19



(alkaloid)

O

O

N N S


phthalate(wax) O

O

O

O

Time (min)

TIC





Time (min)

TIC



OH

OOO

CHC NH2

NH2 H3COCH




8 Menthyl acetate

O

O


O


O

O


O

O

O

O



NH O

O

O


dione(flavonoid)

OO

O


O

O

O

O


O

O

O


Table 5 Continued



O

O


39 Oleamide(wax)

O

NH2


O

OO

Time (min)

TIC




Time (min)

TIC



4 Discussion




Peak Name Structure


O

O


OH


O

O OO


dione(flavonoid)

OO O


O

O

O

O

24



OH


H

H

H

OO

H

35


(saponin)

OH

OH



Peak Name Structure



O

O

O

O


O

O

O

O

20



OH

22



HO

24



HH

H

O




OH

HO


Table 7 Continued

Peak Name Structure




Peak Name Structure


O

O OO

26


one(wax) O O


OO

O



N O

N

Time (min)

TIC


Time (min)

TIC




Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




8 Menthyl acetate

O

O


O


O

O


O

O

O

O



NH O

O

O


dione(flavonoid)

OO

O


O

O

O

O


O

O

O


Table 5 Continued



O

O


39 Oleamide(wax)

O

NH2


O

OO

Time (min)

TIC




Time (min)

TIC



4 Discussion




Peak Name Structure


O

O


OH


O

O OO


dione(flavonoid)

OO O


O

O

O

O

24



OH


H

H

H

OO

H

35


(saponin)

OH

OH



Peak Name Structure



O

O

O

O


O

O

O

O

20



OH

22



HO

24



HH

H

O




OH

HO


Table 7 Continued

Peak Name Structure




Peak Name Structure


O

O OO

26


one(wax) O O


OO

O



N O

N

Time (min)

TIC


Time (min)

TIC




Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Table 5 Continued



O

O


39 Oleamide(wax)

O

NH2


O

OO

Time (min)

TIC




Time (min)

TIC



4 Discussion




Peak Name Structure


O

O


OH


O

O OO


dione(flavonoid)

OO O


O

O

O

O

24



OH


H

H

H

OO

H

35


(saponin)

OH

OH



Peak Name Structure



O

O

O

O


O

O

O

O

20



OH

22



HO

24



HH

H

O




OH

HO


Table 7 Continued

Peak Name Structure




Peak Name Structure


O

O OO

26


one(wax) O O


OO

O



N O

N

Time (min)

TIC


Time (min)

TIC




Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Peak Name Structure


O

O


OH


O

O OO


dione(flavonoid)

OO O


O

O

O

O

24



OH


H

H

H

OO

H

35


(saponin)

OH

OH



Peak Name Structure



O

O

O

O


O

O

O

O

20



OH

22



HO

24



HH

H

O




OH

HO


Table 7 Continued

Peak Name Structure




Peak Name Structure


O

O OO

26


one(wax) O O


OO

O



N O

N

Time (min)

TIC


Time (min)

TIC




Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Peak Name Structure



O

O

O

O


O

O

O

O

20



OH

22



HO

24



HH

H

O




OH

HO


Table 7 Continued

Peak Name Structure




Peak Name Structure


O

O OO

26


one(wax) O O


OO

O



N O

N

Time (min)

TIC


Time (min)

TIC




Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Table 7 Continued

Peak Name Structure




Peak Name Structure


O

O OO

26


one(wax) O O


OO

O



N O

N

Time (min)

TIC


Time (min)

TIC




Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Peak Name Structure


OH

O


OH

O

HO


OH


2(4H)-one(steroid)

OO


OO

OO

OOOH

32



OO





Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Peak Name Structure


O

HO


(alkaloid)

N

S NH

OHN

O


OO O

19



OH

Time (min)

TIC




Time (min)

TIC





Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Peak Name Structure

4

O

O

O


(terpenoid)

O

O

O








31 Phenanthrene


S


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Table 11 Continued

Peak Name Structure


OO O


NN



271and MC



5 Conclusion




Acknowledgments


References





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/archive/2015/485469.pdf · Extract of...

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