Research Article Rosmarinic Acid and Its Methyl Ester as...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 604536 11 pageshttpdxdoiorg1011552013604536

Research ArticleRosmarinic Acid and Its Methyl Ester as AntimicrobialComponents of the Hydromethanolic Extract of Hyptisatrorubens Poit (Lamiaceae)

Amin Abedini12 Vincent Roumy12 Seacuteverine Mahieux13 Murielle Biabiany4

Annie Standaert-Vitse15 Ceacuteline Riviegravere12 Sevser Sahpaz12 Franccedilois Bailleul12

Christel Neut13 and Thierry Hennebelle12

1 Univ Lille Nord de France 59000 Lille France2UDSL Laboratoire de Pharmacognosie EA 4481 GRIIOT UFR Pharmacie 59000 Lille France3 UDSL Laboratoire de Bacteriologie INSERM U995 UFR Pharmacie 59000 Lille France4APLAMEDAROM Association des Plantes Medicinales et Aromatiques de Guadeloupe Mompierre 97111 Morne-a-lrsquoeauGuadeloupe France

5 INSERM U1019 CNRS UMR 8204 UDSL Pasteur Institute Center for Infection and Immunity of Lille (CIIL) EA-4547Biology amp Diversity of Emerging Eukaryotic Pathogens (BDEEP) UFR Pharmacie 59010 Lille France

Correspondence should be addressed toThierry Hennebelle thierryhennebelleuniv-lille2fr

Received 27 March 2013 Revised 25 September 2013 Accepted 9 October 2013

Academic Editor Jenny M Wilkinson

Copyright copy 2013 Amin Abedini 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

Primary biological examination of four extracts of the leaves and stems of Hyptis atrorubens Poit (Lamiaceae) a plant speciesused as an antimicrobial agent in Guadeloupe allowed us to select the hydromethanolic extract of the stems for further studiesIt was tested against 46 microorganisms in vitro It was active against 29 microorganisms The best antibacterial activity wasfound against bacteria mostly Gram-positive ones Bioautography enabled the isolation and identification of four antibacterialcompounds from this plant rosmarinic acid methyl rosmarinate isoquercetin and hyperoside The MIC and MBC values ofthese compounds and their combinations were determined against eight pathogenic bacteria The best inhibitory and bactericidalactivity was found for methyl rosmarinate (03mgmL) Nevertheless the bactericidal power of rosmarinic acid was much fasterin the time kill study Synergistic effects were found when combining the active compounds Finally the inhibitory effects of thecompounds were evaluated on the bacterial growth phases at two different temperatures Our study demonstrated for the first timeantimicrobial activity of Hyptis atrorubens with identification of the active compounds It supports its traditional use in FrenchWest Indies Although its active compounds need to be further evaluated in vivo this work emphasizes plants as potent sources ofnew antimicrobial agents when resistance to antibiotics increases dramatically

1 Introduction

Plants have been used for ages to treat human diseases and ithas been estimated that 25 to 50 of currently available drugsare derived from plants [1] One of the families of drugs withthe most urgent need for newmembers is antibiotics In 2011the WHO (World Health Organization) asked for increasedsearch for new drugs as antibiotic resistance increases dra-matically but only few new molecules are in developmentPlants can be a source of new antimicrobial drugs since they

are considered as time-tested and comparatively safe both forhuman use and the environment [2]

Hyptis atrorubens Poit is a herb of the Lamiaceae familythe members of which are frequently used for antimicrobialpurposes [3] This species is native to tropical America Itschemical composition has been little studied except for thevolatile components It has at least two identified essentialoil chemotypes estragole + limonene (chemotype A) orgermacrene D (chemotype B) [4] In the French West Indiesfresh leaves are used topically against dermatitis and athletersquos

2 Evidence-Based Complementary and Alternative Medicine

foot [5] In French Guiana infusion or decoction of aerialparts is used against sore throat and flu [6] The leaves ofH atrorubens showed no anti-HIV activity but displayedcytotoxic activity (IC

50 25 120583gmL) against a culture of human

lymphoblastic leukemia cell line [7] No experimental dataabout the antimicrobial activity of this plant were encoun-tered

The aim of this study was to investigate the antimicrobialactivity ofH atrorubens against 46microorganisms includingbacteria yeasts and dermatophytes to purify and identifyactive compounds and to test the most active ones on thegrowth of selected bacterial strains

2 Materials and Methods

21 Chemical Analysis

211 Materials Optical rotations were measured on aPerkin-Elmer 343 polarimeter UV spectra were recorded ona Biochrom WPA Lightwave II UV-visible spectrometer IRspectra were recorded on a Thermo Nicolet Avatar 320 FT-IR spectrometer NMR spectra (1H 500MHz 13C 125MHz)were recorded on a Bruker Avance 500 spectrometer in theLaboratoire drsquoApplication de RMN (LARMN) Universite deLille 2Mass spectra (ESI-MS2) were performed on a PE SciexAPI 3000 triple quadrupole mass spectrometer equippedwith an ion spray turbo source (Perkin-Elmer Sciex) Chro-matographic separations were carried out by MPLC on aBuchi systemcomposed of aC-605 pumpmodule and aBuchicolumn (Buchi 460 times 35mm packed with Merck LichroprepRP18 25ndash40120583m Darmstadt Germany)TheHPLC apparatuscomprised a Shimadzu LC-10AS pump and SCL-10A detectorwith an analytical and a semipreparative RP18 column (SilicaUpti-prep Strategy 100 A 5 120583m RP 25 times 46 or 10mm Inter-chrom Interchim Montlucon France) TLC was performedon silica gel 60 F

254plates (Merck Darmstadt Germany)

212 PlantMaterials and Extract Preparation The stems andleaves of Hyptis atrorubens were identified and collected inGros Morne Diomar Guadeloupe in June 2011 by MB Avoucher specimen is conserved in the French Institute of theAgricultural Research Centre (INRA) at Petit-bourg (Guade-loupe) with herbarium number 10 473 After a preliminaryantimicrobial screening of various plant parts by bioautogra-phy stems were selected for further studies and purificationof pure compoundsDried andpowdered stems (1 100 g)wereextracted by maceration using four successive solvents (5 L)of increasing polarity petroleum ether dichloromethanemethanol and water-methanol (1 1 vv) The extraction wasrepeated thrice (each time 2 hours)The extracts were filteredand the solvents were removed by evaporation under reducedpressure to give the dry residues The amounts of extractswere 45 g (petroleum ether) 61 g (dichloromethane) 612 g(methanol) and 804 g (water-methanol) respectively

213 Purification and Identification of the Compounds TheH atrorubens stem hydromethanolic extract (804 g) wasdissolved in H

2O and submitted to a H

2O-CH

3OH (0 to

100 CH3OH) gradient on Sephadex LH-20 There were

nine fractions named A to I Bioautography showed B Cand E to be the most active fractions Fraction B (08 g) wassubmitted to a H

2O-CH

3OH (0 to 100 CH

3OH) MPLC

gradient and to semipreparative HPLC [H2OCH

3OH (4 1)]

to yield the pure compound RA (746mg) Fraction C (10 g)was submitted to a H

2O-CH

3OH (0 to 100CH

3OH)MPLC

gradient that afforded a crude mixture of compounds MRand IQ that was submitted to further purification by HPLCusing an increasing amount of CH

3OH in H

2O following

the sequence ( CH3OH) 20 (isocratic 15min) 20 to

40 (linear gradient 5min) 40 (isocratic 10min) andyielded compounds IQ (35mg) and MR (20mg) Fraction E(08 g) was submitted to a H

2O-CH

3OH (0 to 100 CH

3OH)

MPLC gradient and a subsequent semi-preparative HPLC(H2OCH

3OH (4 1)) enabled the purification of compound

HS (29mg)

214 Data of Isolated Compounds

Compound RA Yellow amorphous powder C18H16O8 ESI-

MS (negative ion)mz 359 [M-H]minus UV 120582max (CH3OH) 221330 nm IR (KBr) (cmminus1) ]max 3382 1697 1606 1522

1H-NMR (CD

3OD) 120575 752 (1H d 119869 = 155Hz H-71015840) 704 (1H

d 119869 = 20Hz H-21015840) 691 (1H dd 119869 = 80 20Hz H-61015840) 675(1H d 119869 = 80Hz H-51015840) 670 (1H d 119869 = 20Hz H-2) 669(1H d 119869 = 80Hz H-5) 657 (1H dd 119869 = 80 20Hz H-6) 626 (1H d 119869 = 155Hz H-81015840) 519 (1H dd 119869 = 10035Hz H-8) 306 (1H dd 119869 = 145 55Hz H-7a) 300 (1Hdd 119869 = 145 55Hz H-7b) 13C-NMR (CD

3OD) 120575 1743 (C-

9) 1694 (C-91015840) 1495 (C-41015840) 1457 (C-71015840) 1450 (C-3) 1449(C-4) 1432 (C- 31015840) 1304 (C-1) 1282 (C-11015840) 1222 (C-6) 1214(C-61015840) 1176 (C-2) 1163 (C-5) 1161 (C-51015840) 1158 (C-81015840) 1153(C-21015840) 778 (C-8) 389 (C-7)

Compound MR Yellow amorphous powder C19H18O8 ESI-

MS (negative ion)mz 373 [M-H]minus UV 120582max (CH3OH) 207217 330 nm IR (KBr) (cmminus1) ]max 3397 1727 1690 1605 15211363 1282 1162 1073 979 1H-NMR (CD

3OD) 120575 752 (1H d

119869 = 155Hz H-71015840) 704 (1H d 119869 = 20Hz H-21015840) 691 (Hdd 119869 = 85 20Hz H-61015840) 675 (1H d 119869 = 85Hz H-51015840) 671(1H d 119869 = 20Hz H-2) 664 (1H d 119869 = 80Hz H-5) 661(1H dd 119869 = 80 20Hz H-6) 627 (1H d 119869 = 155Hz H-81015840) 511 (1H dd 119869 = 75 50Hz H-8) 372 (3H s OCH3)306 (1H dd 119869 = 145 55Hz H-7a) 300 (1H dd 119869 = 14555Hz H-7b) 13C-NMR (CD

3OD) 120575 1721 (C-9) 1682 (C-

91015840) 1501 (C-41015840) 1486 (C-71015840) 1473 (C-31015840) 1461 (C-3) 1457(C-4) 1284 (C-1) 1275 (C-11015840) 1233 (C-61015840) 1228 (C-6) 1179(C-2) 1169 (C-5) 1162 (C-51015840) 1153 (C-21015840) 1142 (C-81015840) 749(C-8) 528 (OCH

3) 380 (C-7)

Compound IQ Light-yellow powder C21H20O12 ESI-MS

(negative ion) mz 463 [M-H]minus UV 120582max (CH3OH) 227

252 nm IR (KBr) (cmminus1) ]max 3310 1665 1607 1507 13781465 1H-NMR (CD

3OD) 120575 770 (1H d 119869 = 20Hz H-21015840)

757 (1H dd 119869 = 75 20Hz H-61015840) 685 (1H d 119869 = 80HzH-51015840) 626 (1H d 119869 = 20Hz H-8) 610 (1H d 119869 = 20HzH-6) 501 (1H d 119869 = 77Hz H-110158401015840) 370 (1H dd 119869 = 12024Hz H-610158401015840a) 356 (1H dd 119869 = 120 54Hz H-610158401015840b) 347

Evidence-Based Complementary and Alternative Medicine 3

(1H dd 119869 = 95 77Hz H-210158401015840) 342 (1H t 119869 = 90Hz H-310158401015840)334 (1H t 119869 = 90Hz H-410158401015840) 321 (1H dd 119869 = 90 24HzH-510158401015840) 13C-NMR (CD

3OD) 120575 1789 (C-4) 1673 (C-7) 1632

(C-5) 1586 (C-9) 1580 (C-2) 1499 (C-31015840) 1496 (C-41015840) 1351(C-3) 1230 (C-11015840) 1228 (C-61015840) 1174 (C-51015840) 1163 (C-21015840) 1052(C-10) 1014 (C-110158401015840) 997 (C-6) 9541 (C-8) 776 (C- 510158401015840) 768(C-310158401015840) 743 (C-210158401015840) 703 (C-410158401015840) 614 (C-610158401015840)

CompoundHS Yellow needles C21H20O12 ESI-MS (negative

ion) mz 463 [M-H]minus UV 120582max (CH3OH) 228 254 nm IR(KBr) (cmminus1) ]max 3300 1660 1610 1510 1376 1460

1H-NMR(CD3OD) 120575 797 (1H d 119869 = 22Hz H-21015840) 754 (1H dd 119869 =

84 22Hz H-61015840) 694 (1H d 119869 = 84Hz H-51015840) 648 (1Hd 119869 = 22Hz H-8) 628 (1H d 119869 = 22Hz H-6) 501 (1Hd 119869 = 77Hz H-110158401015840) 377 (1H dd 119869 = 44 37Hz H-410158401015840)364 (1H dd 119869 = 95 77Hz H-210158401015840) 364 (1H d 119869 = 55HzH-610158401015840b) 360 (1H d 119869 = 55Hz H-610158401015840a) 352 (1H dd 119869 =95 44Hz H-310158401015840) 348 (1H m H-510158401015840) 13C-NMR (CD

3OD)

120575 1776 (C-4) 1641 (C-7) 1612 (C-5) 1592 (C-2) 1563 (C-9)1483 (C-41015840) 1447 (C-31015840) 1337 (C-3) 1218 (C-61015840) 1213 (C-11015840) 1162 (C-51015840) 1153 (C-21015840) 1039 (C-10) 1023 (C-110158401015840) 986(C-6) 935 (C-8) 757 (C- 510158401015840) 734 (C-310158401015840) 713 (C-210158401015840) 681(C-410158401015840) 606 (C-610158401015840)

215 Quantitative Analysis of the Compounds by HPLC Thismethod was used to measure the amount of each compoundin the hydromethanolic extract 20120583L of five concentrationsof each isolated compound and the total extract (5mgmL)were injected successively The mobile phase consisted oftwo components a 005 solution of acetic acid (solventA) and acetonitrile (solvent B) The elution gradient usedwas 17 25 and 50 solvent B at times zero 30 and 40minutes respectively The flow rate was 1mLmin Linearitywas verified by obtaining five point calibration curves overthe concentration range of 0006 to 1mgmL

22 Microbiological Analysis

221 Culture and Preparation of Microorganisms The 36bacteria used in this study (see Table 1) were incubatedovernight at 37∘C in tubes containing slopingMueller-Hinton(MH) agar medium Ten yeasts and dermatophyte clinicalisolates were obtained from the collection of the BDEEPLaboratoryThey were incubated on Sabouraud agarmediumat 37∘C for 48 hours or 30∘C for 2 weeks respectively Thenthe bacteria were dilutedwith Ringerrsquos Cysteine solution (RC)to 106 bacteriamL by means of serial dilution just beforethe antimicrobial assays Final concentration of each bacterialsuspension for MICMBC determination and bioautographywas 104 bacteriamLThe same process was performed for thefungi

222 MIC Determination (Solid Media) The minimuminhibitory concentration (MIC) was studied using MH agarin Petri dishes seeded by a multiple inoculator [8] Thehydromethanolic extract was tested at six final concentrations(10 5 25 12 06 and 03mgmL) against 46 microorgan-isms The agar plates were incubated for 24 hours at 37∘C

Control

Figure 1 Comparison between the positive control and thehydromethanolic extract of stems at 12mgmL and 36 bacteria after24 hours of incubation

The activity was then estimated visually by the presence orabsence of colonies (Figure 1) MIC values were recorded asthe lowest concentrations of compounds showing no growthSolvents used were checked for absence of antibacterialactivity Positive controls were used for bacteria (see Table 1)

223 Bioautography To identify the compounds responsiblefor the antibacterial activity we adopted an immersion bioau-tographymethod [9] After thin layer chromatography (TLC)(silica gel 60 F

254 Merck) the plates were covered by MH

agar containing a Staphylococcus epidermidis 5001 suspensionin square Petri dishes After incubation (24 h at 37∘C)growth was revealed by iodonitrotetrazolium chloride (INT)(2mgmL) and growth inhibition zones were measured

224 MICMBC Determinations by Broth MicrodilutionMethod A serial dilution technique using 96-well microtiterplates was used to determine theMIC of the pure compoundsagainst sensitive bacteria selected by low MIC values [10]Nine concentrations of each compound from 25mgmL to093 times 10

minus2mgmL were used They were serially twofolddiluted with RC in nine wells Two wells were representedas bacteria culture control (positive control) and mediumsterility control (negative control) Then the wells wereloaded with MH liquid medium and bacterial suspension(104 bacteriamL) giving a final volume of 200 120583L The plateswere incubated overnight at 37∘C Bacterial growth wasindicated visually and then by direct spray of 02mgmL INTto each well and the plates were incubated at 37∘C for atleast 30min Bacterial growth in the wells was indicated by areddish-pink colorMICvalueswere determined as the lowestconcentrations of compounds showing clear wells

The minimal bactericidal concentration (MBC) wasdetermined by the subculture of 100 120583L from samples withno visible bacterial growth before INT spray After 24 hours ofincubation the lowest concentration subcultured showing nogrowth was considered as the MBC The isolated substanceswere also tested together (at equal concentrations) to searchfor synergy which was evaluated by the FIC (fractionalinhibitory concentration) indexThe combined effectwas cal-culated by the following formula and results were interpretedas synergy (S FIC le 05) addition (A 05 lt FIC lt 1)


Table 1 Antimicrobial activity ofHyptis atrorubens hydromethanolic extracts (stems and leaves) against 29 microorganisms (lowastno growth)

Strains Stem extract (mgmL) Antibiotics10 5 25 12 06 03 G V A

Gram-negative bacteria

Acinetobacter baumannii 9010 lowast lowast S R RAcinetobacter baumannii 9011 lowast lowast lowast R R R

Proteus mirabilis 11060 lowast lowast lowast S R RProteus mirabilis 11061 lowast lowast S R R

Providencia stuartii 11038 lowast S R SStenotrophomonas maltophilia lowast lowast lowast lowast lowast lowast S R RPseudomonas aeruginosa 8131 lowast lowast lowast S R R

Pseudomonas aeruginosa ATCC 27583 lowast lowast lowast lowast R R R

Gram-positive bacteria

Mycobacterium smegmatis 5003 lowast lowast lowast lowast lowast S S SStaphylococcus aureus 8146 lowast lowast lowast lowast S S SStaphylococcus aureus 8147 lowast lowast lowast lowast S S S

Staphylococcus epidermidis 10282 lowast lowast lowast lowast lowast lowast S S SStaphylococcus epidermidis 5001 lowast lowast lowast lowast lowast lowast S S S

Corynebacterium T25-17 lowast lowast lowast lowast lowast S S SEnterococcus sp 8152 lowast lowast lowast lowast I S SEnterococcus sp 8153 lowast lowast lowast lowast R S S

Enterococcus faecalis C159-6 lowast lowast lowast lowast lowast lowast R R RStaphylococcus lugdunensis T26A3 lowast lowast lowast lowast lowast S S SStaphylococcus warneri T12A12 lowast lowast lowast lowast lowast S R S

Yeast

Candida krusei lowast lowast lowast lowast lowast mdash mdash mdashCandida glabrata lowast mdash mdash mdashCandida kefyr lowast lowast mdash mdash mdash

Candida albicans lowast mdash mdash mdashCandida parapsilosis lowast lowast lowast mdash mdash mdash

Dermatophyte

Microsporum canis lowast lowast lowast lowast mdash mdash mdashTrichophyton rubrum lowast lowast lowast lowast lowast mdash mdash mdash

Trichophyton mentagrophytes lowast lowast lowast lowast lowast mdash mdash mdashTrichophyton soudanense lowast lowast lowast mdash mdash mdashTrichophyton tonsurans lowast lowast lowast lowast lowast mdash mdash mdash

Microorganisms that were resistant to all extracts are not shown Positive controls MIC (120583gmL) gentamicin (G) S le4 R gt8 vancomycin (V) S le4 R gt16amoxicillin (A) S le4 R gt16

indifference (I 1 lt FIC lt 2) and antagonism (AN FIC ge 2)[11]

FIC = (MIC of A in combinationMIC of A alone) + (MICof B in combinationMIC of B alone)

225 Time Kill Study To determine the time-dependent rateof reduction of the bacterial population by active compoundsat room temperature 24-well microtiter plates were used [12]The surviving population was counted after exposure to twoconcentrations of pure compound (MIC and 4 timesMIC) afterfive different delays (0 15 and 60 minutes and 4 and 24hours) and compared to controls Three tubes were prepared(controlMIC and 4 timesMIC) each containing 8mL of RC For1198790 1 mL of bacterial suspension (106 bacteriamL) was added

to every tube Then 1mL of RC was added in the controltube whereas 1mL of the corresponding concentration of

compound was used for the other tubes 100 120583L of tenfolddilutions in RC diluent were plated on MH agar after thedifferent delays indicated above Colonies were counted after24 hours of incubation at 37∘C Results are expressed as logCFUmL

226 Growth Curves In order to follow the growth of bacte-ria at subinhibitory concentrations of the active compoundsat 37∘C [13] 4 tubes (control MIC MIC2 and MIC4)were prepared each containing 8mL of Brain Heart liquidmedium (BH) 1mL of bacterial suspension and 1mL ofthe corresponding compound Tubes were incubated at 37∘C100 120583L of each tube were sampled after 2 4 5 6 7 8 and 24hours 100 120583L of tenfold dilutions were plated on MH agarColonies are counted after incubation at 37∘C for 24 hoursand counts are expressed as log CFUmL


227 Comparison of Growth Curves at 4∘C and 37∘C Growthcurves were also performed at 4∘C using the samemethod asdescribed above to compare the action of the active productson bacteria in growth or stationary phase

3 Results

31 MIC Determination of Crude Extracts Antimicrobialassays were performed on the petroleum ether methylenechloride methanolic and hydromethanolic extracts fromthe stems or leaves of H atrorubens Thus eight extractswere screened in order to select an extract with both goodactivity and a reasonable probability of recovering sufficientamounts of active compounds for further assays Resultsobtained with the stem hydromethanolic extract which wasthemost active on a broad spectrumof bacteria are presentedin Table 1 It was active at different degrees against 29 outof 46 microbial strains Staphylococcus epidermidis 10282Staphylococcus epidermidis 5001 Enterococcus faecalis C159-6 and Stenotrophomonas maltophilia were the most sensitivestrains TheMIC of this extract against these four strains wasat least 03mgmL

The stem hydromethanolic extract was mainly activeagainst Gram-positive bacteria No antimicrobial activity wasfound against the following 17 strains Citrobacter freundii11041 11042 11043 Enterobacter aerogenes 9004 Enterobactercloacae 11050 11051 11053 Escherichia coli 8137 8138 8157Escherichia coli ATCC 25922 Klebsiella pneumoniae 1101611017 Salmonella sp 11033 11037 Serratia marcescens 1105611057 which are all Gram-negative bacteria belonging to theEnterobacteriaceae familyThe in vitro antifungal activity wasalso assessed on 5 yeast strains and 5 dermatophytesThe bestresults were obtained for three dermatophytes (Trichophytonrubrum Trichophytonmentagrophytes andTrichophyton ton-surans) Yeasts were less sensitive than dermatophytes Giventhe relatively weak antifungal activity and time-consumingexperiments required for dermatophytes we focused ourstudy only on antibacterial activity for the following steps ofthe work

32 Purification Isolation and Identification According tothese preliminary results (Table 1) and the high extractionefficiency the hydromethanolic extract of stems was selectedand used for further phytochemical studies The bioautogra-phy analysis was performed using Staphylococcus epidermidis5001 one of the most sensitive microorganisms The resultsshowed optimal activity for fractions B and C and a milderactivity for fraction E (Table 2)

These fractions led to pure compounds via biogu-ided fractionations (see Section 2) Four compounds weredetected as responsible for themajor part of the antimicrobialactivity of H atrorubens Their structures were establishedby spectroscopic analysis (mono- and bidimensional NMRMS) and direct comparison with published data [14 15]as rosmarinic acid (RA 093 yield) methyl rosmari-nate (MR 002) quercetin-3-O-glucoside (isoquercetinIQ 004) and quercetin-3-O-galactoside (hyperoside HS003) (Figure 2) These compounds are known but to the

Table 2 Results of bioautography test of fractions ldquoArdquo to ldquoIrdquo

FractionsS

epidermidis10282

Sepidermidis

5001

E faecalisC159-6

Smaltophilia

A ++ + + ++B +++ +++ ++ +++C +++ +++ ++ +++D minus minus minus minus

E + + + minus

F + + ++ +G + + + minus

H + ++ + minus

I + ++ + ++(minus) no effect (+) significant effect rated from + (1 cm inhibition zone) to+++ (gt3 cm inhibition zone)

best of our knowledge this is the first report of their presencein the species Hyptis atrorubens

33 Quantitative Analysis In order to enable a correctcharacterization of the assayed extract all four majoractive compounds were quantified in H atrorubens stemhydromethanolic extract The results of quantitative analysisare shown in Table 3 The compound RA (rosmarinic acid)was found in a far higher amount (560) than the threeothers which makes Hyptis atrorubens a good source of RA

34 Antibacterial Activity of the Compounds The antibacte-rial compounds were evaluated using microdilution assaysagainst eight bacterial strains to measure the MIC and MBC(Table 4) The MICs ranged from 03mgmL to the limitconcentration of 25mgmL At a glance phenylpropanoidsRA andMR clearly display a stronger activity than flavonoidsIQ and HS MIC values of both later compounds were neverlt12mgmL and they were often at the highest assayed con-centration of 25mgmL or above MR was considered to bethe most active compound because it displayed significantlylower MIC and MBC values in most cases but RA was morebactericidal in 2 cases Staphylococcus epidermidis 5001 andStenotrophomonas maltophilia The three strains Staphylo-coccus epidermidis 5001 Stenotrophomonas maltophilia andEnterococcus faecalis C159-6 displayed the highest sensitivityin this test

35 Synergistic Effects between the Compounds As MICvalues of isolated compoundswere not so different from thoseof the crude extract MICs were also established for mixturesof two or more of these compounds using the determinationof the FIC index (Table 5) to reveal possible synergisticmixtures Such synergism although rare was encountered ina few cases

Association of both flavonoids proved at best indifferentA mixture of both phenylpropanoids could result in indif-ferent or additive effects in most cases Synergistic actionswere mainly observed for the association of at least one


O

OO

O

COOH

OH

OH

OH

OHOH

OHOHOH

OH

OH

OHOH

OH OH

OH

OH

COOCH3

OO

O

O O

O

O O

RA MR

IQ HS

1

2

34

5

6

78

9

1

2

34

5

6

78

9

2

34

5

6 6

5

43

277

8 8

9 9

1010

HO

HOHO

HO

HO

HOHO

HO

1998400

2998400

3998400

4998400

5998400

6998400

1998400

2998400

3998400

4998400

5998400

6998400

7998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400 7

998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400

1998400998400

1998400998400

2998400998400

2998400998400 3

9984009984003998400998400

4998400998400

4998400998400

5998400998400

5998400998400

6998400998400

6998400998400

Figure 2 Chemical structure of four active compounds rosmarinic acid (RA) methyl rosmarinate (MR) isoquercetin (IQ) and hyperoside(HS)

Table 3 Results of quantitative analysis of the four compounds by HPLC and linearity of calibration

Compounds Retention time(min)

Linear range(mgmL)

y = ax + b(linear model)

Correlation coefficient(1198772)

Percent in total extract()

RA 2067 plusmn 045 0125ndash1000 5898776 = (2 times 10minus7)119909 + 243823 09996 560

MR 3877 plusmn 052 0030ndash1000 705509 = (2 times 10minus7)119909 + 38893 09995 003

IQ 1538 plusmn 096 0006ndash0500 103118 = (2 times 10minus7)119909 minus 31067 09999 008

HS 1788 plusmn 085 0006ndash0500 218757 = (2 times 10minus7)119909 minus 57439 09995 006

119910 peak area of compound in total extract 119909 concentration of compound in total extract

phenylpropanoid with at least one flavonoidThe lowest MIC(007mgmL) was reached with an association of all fourcompounds on Enterococcus faecalis C159-6

36 Bactericidal Curves (Time Kill Study) In order to estab-lish the time-dependency of the bactericidal action wemeasured the rate of reduction of the bacterial population bythe two most active compounds (RA and MR) against twobacterial strains Staphylococcus epidermidis 5001 (Figure 3)and Stenotrophomonas maltophilia (Figure 4)

361 Staphylococcus epidermidis 5001 Staphylococcus epider-midis 5001 was selected as a model bacterium for Gram-positives Although Enterococcus faecalis C159-6 could beconsidered as more representative (because it was moresensitive to MR as was the general case) and more sensitivewe preferred to use Staphylococcus epidermidis 5001 whichprovokes skin infections that aremore in accordance with thetraditional use of the plant and which is particularly sensitiveto RA (the major component of the assayed extract)

A concentration of 125mgmL (4 timesMIC) of RA reducedthe bacterial population by 5 log in 4 hours reaching thedetection threshold (lt100 bacteriamL) quickly whereas MRonly reached this threshold after 24 hours At 03mgmL

(MIC) a similar reduction was observed for both phenyl-propanoids after 4 hours but the decrease in the bacterialpopulation continued only with RA to reach a 3 log reductionafter 24 hours

362 Stenotrophomonas maltophilia In the case of Stenot-rophomonas maltophilia a Gram-negative bacterium in spiteof similarMIC andMBCvalues of RA andMR in comparisonwith Staphylococcus epidermidis 5001 the difference betweenthe two phenylpropanoids was greaterMR did not reduce thebacterial population by more than 15 log at any concentra-tion In contrast using RA at 125mgmL (4 timesMIC) bacteriawere below the detection threshold (lt100 bacteriamL) after15 minutes (Figure 4)

37 Growth Curves

371 Growth Curves at 37∘C The three concentrations of RAand MR were tested for their effect on the growth phasesof Staphylococcus epidermidis 5001 at 37∘C The results arepresented in Figure 5

At the MIC concentration the two compounds loweredslowly the number of viable bacteria in accordance withthe time kill study At subinhibitory concentrations the


Table 4 MIC and MBC values in the microdilution assay for the active compounds expressed in mgmL

Bacteria RA MR IQ HSMIC MBC MIC MBC MIC MBC MIC MBC

Staphylococcus epidermidis 5001 03 03 03 06 12 12 25 gt25Stenotrophomonas maltophilia 03 03 03 06 12 25 12 12Enterococcus faecalis C159-6 03 06 03 03 12 25 25 gt25Staphylococcus lugdunensis T26A3 06 12 06 06 25 25 25 gt25Pseudomonas aeruginosa ATCC 27583 25 gt25 12 25 12 gt25 25 gt25Corynebacterium T25-17 25 gt25 12 12 12 gt25 25 gt25Mycobacterium smegmatis 5003 12 25 06 06 25 gt25 25 gt25Staphylococcus warneri T12A12 12 25 03 03 12 gt25 25 gt25

Table 5Antibacterial activities indicated by Fractional InhibitoryConcentrations of combined compounds against selectedmicroorganisms

Bacteria MIC (mgmL)-FICRA + MR RA + IQ RA + HS MR + IQ MR + HS IQ + HS RA + MR + IQ + HS

Staphylococcus epidermidis 5001 03-(I) 12-(AN) 03-(I) 03-(I) 03-(I) 250-(I) 015-(I)Stenotrophomonas maltophilia 03-(I) 06-(I) 06-(I) 03-(I) 06-(I) 250-(A) 015-(I)Enterococcus faecalis C159-6 015-(A) 06-(I) 03-(I) 015-(A) 03-(I) 125-(I) 007-(S)Staphylococcus lugdunensis T26A3 06-(I) 03-(A) 06-(I) 015-(S) 06-(I) 250-(I) 03-(I)Pseudomonas aeruginosa ATCC 27583 12-(I) 12-(I) 12-(A) 12-(I) 12-(I) 250 -(I) 06-(I)Corynebacterium T25-17 03-(S) 06-(A) 06-(A) 015-(S) 06-(A) 125 -(I) 03-(A)Mycobacterium smegmatis 5003 03-(A) 06-(A) 03-(S) 03-(A) 03-(A) 250-(I) 03-(A)Staphylococcus warneri T12A12 015-(A) 06-(A) 03-(S) 03-(I) 03-(I) 250-(I) 03-(I)Synergy (S FIC le 05) addition (A 05 lt FIC lt 1) indifference (I 1 lt FIC lt 4) and antagonism (AN FIC ge 4) (ratio 1 1)

phase of latency was longer and the bacterial level did notreach the control values after 24 hours Thus subinhibitoryconcentrations retained a strong effect on bacterial growthThey delayed the entry of bacteria into the exponential phase2 hours atMIC4 (75mgL) and 3 hours atMIC2 (150mgL)for RA In addition their stationary phases were one andtwo log under control respectively In Figure 5(b) similaralthough slightly weaker results are presented for MR

372 Growth Curves at 4∘C At this temperature when thebacteria remained in stationary phase subinhibitory concen-trations had no intense effects on bacterial populations (themaximal reduction observed was by 05 log) However ascan be observed in Figure 6(a) the MIC concentration of RA(03mgmL) reduced the number of bacteria by one log after24 hours This result as can be seen by comparing Figures5 and 6 is very close to that obtained at 37∘C which meansthat the antibacterial activity does not need multiplication ofthe bacteria and is present at the same level in bacteria instationary phase

4 Discussion

Biological in vitro activities of plants should always be submit-ted to caution Many studies use very high nonphysiologicalconcentrations of drugs which will never be achieved inhuman tissue [16] The research of antibacterial activity isoften only done by diffusion tests which cannot be quantifiedMoreover with that method different products cannot becompared because the diffusion depends on the molecular

size and can be very different from one product to anotheror be of uncertain significance in crude extracts containing amixture of different potentially active compounds

Antimicrobial studies often concern only a few strainsfrom one or two species We chose to determine the MICagainst a large choice of microorganisms which enables us todiscover extractswith a large-scale activityMost of the strainshave been recently isolated from human infections Forcomparison we also included some reference strains from theAmerican Type Culture Collection (ATCC) These strainshowever have often been isolated years ago and subculturedon laboratory culturemediumonly and no longer reflect whatis encountered today in hospitals

The aim of our study was not only to test crude extractsbut also to purify chemically defined compounds Theirbiological activities were further characterized The firstapproach was done by time kill study which exhibited theirtime dependent action Our main compound RA decreasedthe viable count by more than 5 log in a very short time Thisaction can be compared to that of disinfectants and is muchfaster than most antibiotics

Then growth curves were established showing thatconcentrations below the MIC could still slow the growthof the bacterial strains Even when the concentration wasbelow the MIC level a biological effect (bacteriostatic effectlasting some hours) can still be present The growth curveswere established by using the time and material consumingmethod of viable counts Optical density (OD) is oftenpreferred [17] but many bacterial strains (including Staphylo-coccus andPseudomonas ones) form extracellular compounds


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)

ControlMIC4 times MIC

(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)

Figure 3 Killing curve of Staphylococcus epidermidis 5001 for RA (a) and MR (b)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)

Figure 4 Killing curve of Stenotrophomonas maltophilia for RA (a) and MR (b)

increase the OD without changes in viable count At MIC2and MIC4 our growth curves show both a lag phasebefore reaching the exponential phase and a lower finalconcentration Even after 24 hours the count remains belowthe control level (Figure 5)

The antimicrobial activity of Lamiaceae species is oftenattributed to volatile components from their essential oilAlthough these compounds have been more thoroughlystudied it is often difficult to correlate in vitro findingswith ethnopharmacological observations as their presencein traditional modes of preparation are often uncertain due

to high variability and inadequate extraction protocols Thecase of thyme (Thymus vulgaris L) is well known withat least 7 chemotypes among which only one has thymolas the major compound [18] Similarly the genus Hyptiscomprises several species that have at least two chemotypes[4] Consequently it seemed interesting to test nonvolatilefractions the composition of which is often less variableleading to more trustful results RA is a therapeutic andcosmetic secondary metabolite with very low toxicity It hasalready been characterized for many biological activities [19]but its antimicrobial activity has been neither systematically


MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)

Figure 5 Growth curve of Staphylococcus epidermidis 5001 for RA (a) and MR (b) at 37∘C

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


addressed nor quantified Its mechanism of action is notclearly known in spite of scanning electron micrographsthat show a damaged cell surface under RA treatment [20]The results of our study confirmed and measured the rateof inhibitory and bactericidal activities of this compoundagainst eight pathogenic bacteria three of which are oftenconcerned by resistance to available antibiotics (Staphylo-coccus epidermidis 5001 Stenotrophomonas maltophilia andEnterococcus faecalis C159-6)

The methyl ester of RA (methyl rosmarinate) displayssimilar biological activities as the free acid [21] A recentpaper afforded preliminary (MIC on five species) data aboutits antimicrobial activity [22] Our study demonstrates that

this compound is more inhibitory and bactericidal in com-parison with RA

Two other compounds IQ and HS (3-O-glycosides of theflavonol quercetin) are largely present in medicinal plantsand food In spite of strong structural similarity they differ intheir biological activity and bioavailability because they havedistinct sugar moieties [23] These compounds have beenstudied for numerous activities among them antiviral [24]and antimicrobial [25 26]

We tried to measure the antibacterial activity of all fourcompoundswithmicrobiological indicators (MICMBC andFIC) Although these indicators did not exhibit remark-able activity for either compound (the MIC of RA for


instance was rather disappointing in comparison with thatof the total extract) they did display synergistic effectsstronger inhibitory and bactericidal activity were obtainedfor mixtures of compounds in six cases and the lowestMIC (70 120583gmL) was achieved for a combination of all fourcompounds which is very close to MICs of antibiotics

This last observation underlines the importance of theanalysis of mixtures of compounds Synergy studies areseldom done in ethnopharmacology but seem as a sensibleapproach as several different compounds are always foundin plants and these associations can contribute to an antimi-crobial defense scheme This needs to be explored [27] Inthe crude extract of H atrorubens probably more than thefour chemically defined compounds are present In Table 2various activities are observed for the fractions E to I whichare not further studied here These fractions will be furtheranalysed to isolate their active compounds

Work on the isolated compounds needs to continuein order to reveal their mechanism of action Electronmicroscopy can help to visualize cell damage after contact ofthe bacteriawith the identified compounds Proteomics of thebacterial membranewill revealmodifications after contact Invivo infectious animal models can also be a helpful tool inapproaching the practical use of the products These animalmodels should focus on infections with frequent implicationsof Gram-positive microorganisms as Gram-negatives likeenterobacteria are not covered by the identified compoundsSkin infections seem as a convenient target as staphylococciare the most frequently implicated and it is consistent withsome traditional uses of the species

In the case of RA the presence of a carboxylic acidicgroup makes it possible that the ionisability of this chemicalfunction andor the formation of salts with mineral cations isinvolved as was observed with antibiotics such as polymyx-ins [28] bacitracin [29] and daptomycin [30] This couldcontribute to explaining discrepancies between the respectiveactivities of this compound and its less acidic methyl ester

In conclusion our research suggests the presence of upto now underestimated substances with antimicrobial actionin natural productsThis action is evidenced by different con-cordant tests likeMICMBC determination time kill growthcurves and synergy tests Plants have evolved throughout thetime in the presence of bacteria and show good resistanceagainst bacterial invasion They constitute a potential sourceto strengthen the fight of humans against bacteria that aremore and more resistant to antibiotics

A part of the results have been published as a poster inthe International Congress on Natural Products Research inNew York City (June 2012) [31]

References

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

[2] A C Abreu A J McBain and M Simoes ldquoPlants as sources ofnew antimicrobials and resistance-modifying agentsrdquo NaturalProduct Reports vol 29 pp 1007ndash1021 2012

[3] P K Lai and J Roy ldquoAntimicrobial and chemopreventiveproperties of herbs and spicesrdquo Current Medicinal Chemistryvol 11 no 11 pp 1451ndash1460 2004

[4] O G S Maia and L H A Andrade ldquoDatabase of the amazonaromatic plants and their essential oilsrdquo Quimica Nova vol 32no 3 pp 595ndash622 2009

[5] J-L Longuefosse and E Nossin ldquoMedical ethnobotany surveyinMartiniquerdquo Journal of Ethnopharmacology vol 53 no 3 pp117ndash142 1996

[6] P Grenand C Moretti H Jacquemin and M F PrevostPharmacopees Traditionnelles en Guyane Institut de Recherchepour le Developpement Editions Paris France 2nd edition2004

[7] M D Antoun E Martinez R Caballero et al ldquoEvaluation ofthe flora of Puerto Rico for in vitro cytotoxic and anti-HIVactivitiesrdquo Pharmaceutical Biology vol 37 no 4 pp 277ndash2801999

[8] R S Pereira T C Sumita M R Furlan A O C Jorge and MUeno ldquoAntibacterial activity of essential oils onmicroorganismsisolated from urinary tract infectionrdquo Revista de Saude Publicavol 38 no 2 pp 326ndash328 2004

[9] L Rahalison M Hamburger K Hostettmann M Monodand E Frenk ldquoA bioautographic agar overlay method forthe detection of antifungal compounds from higher plantsrdquoPhytochemical Analysis vol 2 pp 199ndash203 1991

[10] L J McGaw T Rabe S G Sparg A K Jager J N Eloff andJ Van Staden ldquoAn investigation on the biological activity ofCombretum speciesrdquo Journal of Ethnopharmacology vol 75 no1 pp 45ndash50 2001

[11] M C Berenbaum ldquoCriteria for analyzing interactions betweenbiologically active agentsrdquo Advances in Cancer Research vol 35pp 269ndash335 1981

[12] N Supardy I Darah S Shaida Fariza and A Z Nurul ldquoInhi-bition of Klebsiella pneumoniae ATCC 13883 cells by hexaneextract of Halimeda discoidea (Decaisne) and the identificationof its potential bioactive compoundsrdquo Journal of Microbiologyand Biotechnology vol 22 pp 872ndash888 2012

[13] A Castillo J Liebana E Lopez et al ldquoInterference of antibioticsin the growth curves of oral streptococcirdquo International Journalof Antimicrobial Agents vol 27 no 3 pp 263ndash266 2006

[14] N Al-Musayeib S Perveen I FatimaMNasir andAHussainldquoAntioxidant anti-glycation and anti-inflammatory activities ofphenolic constituents from Cordia sinensisrdquo Molecules vol 16no 12 pp 10214ndash10226 2011

[15] E C Tatsis S Boeren V Exarchou A N Troganis J Vervoortand I P Gerothanassis ldquoIdentification of themajor constituentsof Hypericum perforatum by LCSPENMR andor LCMSrdquoPhytochemistry vol 68 no 3 pp 383ndash393 2007

[16] V Butterweck and A Nahrstedt ldquoWhat is the best strategy forpreclinical testing of botanicals A critical perspectiverdquo PlantaMedica vol 78 pp 747ndash754 2012

[17] S Vijayarathna Z Zakaria Y Chen L Y Latha J R Kanwarand S Sasidharan ldquoThe antimicrobial efficacy of Elaeis guineen-sis characterization in vitro and in vivo studiesrdquoMolecules vol17 pp 4860ndash4877 2012

[18] K Keefover-Ring J D Thompson and Y B Linhart ldquoBeyondsix scents defining a seventh Thymus vulgaris chemotypenew to southern France by ethanol extractionrdquo Flavour andFragrance Journal vol 24 no 3 pp 117ndash122 2009

[19] V P Bulgakov Y V Inyushkina and S A Fedoreyev ldquoRos-marinic acid and its derivatives biotechnology and applicationrdquoCritical Reviews in Biotechnology vol 32 pp 203ndash217 2012


[20] H P Bais T SWalker H P Schweizer and JM Vivanco ldquoRootspecific elicitation and antimicrobial activity of rosmarinic acidin hairy root cultures of Ocimum basilicumrdquo Plant Physiologyand Biochemistry vol 40 no 11 pp 983ndash995 2002

[21] E-R Woo and S P Mei ldquoAntioxidative constituents fromLycopus lucidusrdquo Archives of Pharmacal Research vol 27 no 2pp 173ndash176 2004

[22] L Lin D Zhu L Zou B Yang and M Zhao ldquoAntibacterialactivity-guided purification and identification of a novel C-20oxygenated ent-kaurane from Rabdosia serra (Maxim) HarardquoFood Chemistry vol 139 pp 902ndash909 2013

[23] A R Gohari S Saeidnia M Malmir M Yazdanpanah and YAjani ldquoSterols and flavonoids of Lomatopodium staurophyllumrdquoJournal of Medicinal Plants vol 10 no 39 pp 43ndash48 2011

[24] L-LWuX-B Yang Z-MHuangH-Z Liu andG-XWu ldquoInvivo and in vitro antiviral activity of hyperoside extracted fromAbelmoschus manihot (L) medikrdquo Acta Pharmacologica Sinicavol 28 no 3 pp 404ndash409 2007

[25] S Sujatha and S RoselineKantida ldquoCytotoxicity and antimicro-bial studies of quercetin-3-O-galactoside (hyperoside) isolatedfrom the leaves of Psidium guajava (Linn)rdquoNovus InternationalJournal of Pharmaceutical Technology vol 1 pp 30ndash37 2012

[26] S Li Z Zhang A Cain B Wang M Long and J TaylorldquoAntifungal activity of camptothecin trifolin and hyperosideisolated from Camptotheca acuminatardquo Journal of Agriculturaland Food Chemistry vol 53 no 1 pp 32ndash37 2005

[27] J Gertsch ldquoBotanical drugs synergy and network pharmacol-ogy forth and back to intelligent mixturesrdquo Planta Medica vol77 no 11 pp 1086ndash1098 2011

[28] A P Zavascki L Z Goldani J Li and R L Nation ldquoPolymyxinB for the treatment of multidrug-resistant pathogens a criticalreviewrdquo Journal of Antimicrobial Chemotherapy vol 60 no 6pp 1206ndash1215 2007

[29] K J Stone and J L Strominger ldquoMechanism of action ofbacitracin complexation with metal ion and C 55 -isoprenylpyrophosphaterdquo Proceedings of the National Academy of Sciencesof the United States of America vol 68 no 12 pp 3223ndash32271971

[30] J N Steenbergen J Alder G M Thorne and F P TallyldquoDaptomycin a lipopeptide antibiotic for the treatment ofserious Gram-positive infectionsrdquo Journal of AntimicrobialChemotherapy vol 55 no 3 pp 283ndash288 2005

[31] A Abedini V Roumy C Neut et al ldquoAntibacterial activityof the methanolic extract of Hyptis atrorubens (Lamiaceae)rdquoPlanta Medica vol 78 p 1103 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


foot [5] In French Guiana infusion or decoction of aerialparts is used against sore throat and flu [6] The leaves ofH atrorubens showed no anti-HIV activity but displayedcytotoxic activity (IC

50 25 120583gmL) against a culture of human

lymphoblastic leukemia cell line [7] No experimental dataabout the antimicrobial activity of this plant were encoun-tered

The aim of this study was to investigate the antimicrobialactivity ofH atrorubens against 46microorganisms includingbacteria yeasts and dermatophytes to purify and identifyactive compounds and to test the most active ones on thegrowth of selected bacterial strains

2 Materials and Methods

21 Chemical Analysis

211 Materials Optical rotations were measured on aPerkin-Elmer 343 polarimeter UV spectra were recorded ona Biochrom WPA Lightwave II UV-visible spectrometer IRspectra were recorded on a Thermo Nicolet Avatar 320 FT-IR spectrometer NMR spectra (1H 500MHz 13C 125MHz)were recorded on a Bruker Avance 500 spectrometer in theLaboratoire drsquoApplication de RMN (LARMN) Universite deLille 2Mass spectra (ESI-MS2) were performed on a PE SciexAPI 3000 triple quadrupole mass spectrometer equippedwith an ion spray turbo source (Perkin-Elmer Sciex) Chro-matographic separations were carried out by MPLC on aBuchi systemcomposed of aC-605 pumpmodule and aBuchicolumn (Buchi 460 times 35mm packed with Merck LichroprepRP18 25ndash40120583m Darmstadt Germany)TheHPLC apparatuscomprised a Shimadzu LC-10AS pump and SCL-10A detectorwith an analytical and a semipreparative RP18 column (SilicaUpti-prep Strategy 100 A 5 120583m RP 25 times 46 or 10mm Inter-chrom Interchim Montlucon France) TLC was performedon silica gel 60 F

254plates (Merck Darmstadt Germany)

212 PlantMaterials and Extract Preparation The stems andleaves of Hyptis atrorubens were identified and collected inGros Morne Diomar Guadeloupe in June 2011 by MB Avoucher specimen is conserved in the French Institute of theAgricultural Research Centre (INRA) at Petit-bourg (Guade-loupe) with herbarium number 10 473 After a preliminaryantimicrobial screening of various plant parts by bioautogra-phy stems were selected for further studies and purificationof pure compoundsDried andpowdered stems (1 100 g)wereextracted by maceration using four successive solvents (5 L)of increasing polarity petroleum ether dichloromethanemethanol and water-methanol (1 1 vv) The extraction wasrepeated thrice (each time 2 hours)The extracts were filteredand the solvents were removed by evaporation under reducedpressure to give the dry residues The amounts of extractswere 45 g (petroleum ether) 61 g (dichloromethane) 612 g(methanol) and 804 g (water-methanol) respectively

213 Purification and Identification of the Compounds TheH atrorubens stem hydromethanolic extract (804 g) wasdissolved in H

2O and submitted to a H

2O-CH

3OH (0 to

100 CH3OH) gradient on Sephadex LH-20 There were

nine fractions named A to I Bioautography showed B Cand E to be the most active fractions Fraction B (08 g) wassubmitted to a H

2O-CH

3OH (0 to 100 CH

3OH) MPLC

gradient and to semipreparative HPLC [H2OCH

3OH (4 1)]

to yield the pure compound RA (746mg) Fraction C (10 g)was submitted to a H

2O-CH

3OH (0 to 100CH

3OH)MPLC

gradient that afforded a crude mixture of compounds MRand IQ that was submitted to further purification by HPLCusing an increasing amount of CH

3OH in H

2O following

the sequence ( CH3OH) 20 (isocratic 15min) 20 to

40 (linear gradient 5min) 40 (isocratic 10min) andyielded compounds IQ (35mg) and MR (20mg) Fraction E(08 g) was submitted to a H

2O-CH

3OH (0 to 100 CH

3OH)

MPLC gradient and a subsequent semi-preparative HPLC(H2OCH

3OH (4 1)) enabled the purification of compound

HS (29mg)

214 Data of Isolated Compounds

Compound RA Yellow amorphous powder C18H16O8 ESI-

MS (negative ion)mz 359 [M-H]minus UV 120582max (CH3OH) 221330 nm IR (KBr) (cmminus1) ]max 3382 1697 1606 1522

1H-NMR (CD

3OD) 120575 752 (1H d 119869 = 155Hz H-71015840) 704 (1H

d 119869 = 20Hz H-21015840) 691 (1H dd 119869 = 80 20Hz H-61015840) 675(1H d 119869 = 80Hz H-51015840) 670 (1H d 119869 = 20Hz H-2) 669(1H d 119869 = 80Hz H-5) 657 (1H dd 119869 = 80 20Hz H-6) 626 (1H d 119869 = 155Hz H-81015840) 519 (1H dd 119869 = 10035Hz H-8) 306 (1H dd 119869 = 145 55Hz H-7a) 300 (1Hdd 119869 = 145 55Hz H-7b) 13C-NMR (CD

3OD) 120575 1743 (C-

9) 1694 (C-91015840) 1495 (C-41015840) 1457 (C-71015840) 1450 (C-3) 1449(C-4) 1432 (C- 31015840) 1304 (C-1) 1282 (C-11015840) 1222 (C-6) 1214(C-61015840) 1176 (C-2) 1163 (C-5) 1161 (C-51015840) 1158 (C-81015840) 1153(C-21015840) 778 (C-8) 389 (C-7)

Compound MR Yellow amorphous powder C19H18O8 ESI-

MS (negative ion)mz 373 [M-H]minus UV 120582max (CH3OH) 207217 330 nm IR (KBr) (cmminus1) ]max 3397 1727 1690 1605 15211363 1282 1162 1073 979 1H-NMR (CD

3OD) 120575 752 (1H d

119869 = 155Hz H-71015840) 704 (1H d 119869 = 20Hz H-21015840) 691 (Hdd 119869 = 85 20Hz H-61015840) 675 (1H d 119869 = 85Hz H-51015840) 671(1H d 119869 = 20Hz H-2) 664 (1H d 119869 = 80Hz H-5) 661(1H dd 119869 = 80 20Hz H-6) 627 (1H d 119869 = 155Hz H-81015840) 511 (1H dd 119869 = 75 50Hz H-8) 372 (3H s OCH3)306 (1H dd 119869 = 145 55Hz H-7a) 300 (1H dd 119869 = 14555Hz H-7b) 13C-NMR (CD

3OD) 120575 1721 (C-9) 1682 (C-

91015840) 1501 (C-41015840) 1486 (C-71015840) 1473 (C-31015840) 1461 (C-3) 1457(C-4) 1284 (C-1) 1275 (C-11015840) 1233 (C-61015840) 1228 (C-6) 1179(C-2) 1169 (C-5) 1162 (C-51015840) 1153 (C-21015840) 1142 (C-81015840) 749(C-8) 528 (OCH

3) 380 (C-7)

Compound IQ Light-yellow powder C21H20O12 ESI-MS

(negative ion) mz 463 [M-H]minus UV 120582max (CH3OH) 227

252 nm IR (KBr) (cmminus1) ]max 3310 1665 1607 1507 13781465 1H-NMR (CD

3OD) 120575 770 (1H d 119869 = 20Hz H-21015840)

757 (1H dd 119869 = 75 20Hz H-61015840) 685 (1H d 119869 = 80HzH-51015840) 626 (1H d 119869 = 20Hz H-8) 610 (1H d 119869 = 20HzH-6) 501 (1H d 119869 = 77Hz H-110158401015840) 370 (1H dd 119869 = 12024Hz H-610158401015840a) 356 (1H dd 119869 = 120 54Hz H-610158401015840b) 347



3OD) 120575 1789 (C-4) 1673 (C-7) 1632






3OD)






Control






















Yeast



Dermatophyte












3 Results






FractionsS

epidermidis10282

Sepidermidis

5001

E faecalisC159-6

Smaltophilia


E + + + minus

F + + ++ +G + + + minus

H + ++ + minus








O

OO

O

COOH

OH

OH

OH

OHOH

OHOHOH

OH

OH

OHOH

OH OH

OH

OH

COOCH3

OO

O

O O

O

O O

RA MR

IQ HS

1

2

34

5

6

78

9

1

2

34

5

6

78

9

2

34

5

6 6

5

43

277

8 8

9 9

1010

HO

HOHO

HO

HO

HOHO

HO

1998400

2998400

3998400

4998400

5998400

6998400

1998400

2998400

3998400

4998400

5998400

6998400

7998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400 7

998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400

1998400998400

1998400998400

2998400998400

2998400998400 3

9984009984003998400998400

4998400998400

4998400998400

5998400998400

5998400998400

6998400998400

6998400998400




Linear range(mgmL)















37 Growth Curves












4 Discussion







100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)






MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)














References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3OD) 120575 1789 (C-4) 1673 (C-7) 1632






3OD)






Control






















Yeast



Dermatophyte












3 Results






FractionsS

epidermidis10282

Sepidermidis

5001

E faecalisC159-6

Smaltophilia


E + + + minus

F + + ++ +G + + + minus

H + ++ + minus








O

OO

O

COOH

OH

OH

OH

OHOH

OHOHOH

OH

OH

OHOH

OH OH

OH

OH

COOCH3

OO

O

O O

O

O O

RA MR

IQ HS

1

2

34

5

6

78

9

1

2

34

5

6

78

9

2

34

5

6 6

5

43

277

8 8

9 9

1010

HO

HOHO

HO

HO

HOHO

HO

1998400

2998400

3998400

4998400

5998400

6998400

1998400

2998400

3998400

4998400

5998400

6998400

7998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400 7

998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400

1998400998400

1998400998400

2998400998400

2998400998400 3

9984009984003998400998400

4998400998400

4998400998400

5998400998400

5998400998400

6998400998400

6998400998400




Linear range(mgmL)















37 Growth Curves












4 Discussion







100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)






MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)














References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Yeast



Dermatophyte












3 Results






FractionsS

epidermidis10282

Sepidermidis

5001

E faecalisC159-6

Smaltophilia


E + + + minus

F + + ++ +G + + + minus

H + ++ + minus








O

OO

O

COOH

OH

OH

OH

OHOH

OHOHOH

OH

OH

OHOH

OH OH

OH

OH

COOCH3

OO

O

O O

O

O O

RA MR

IQ HS

1

2

34

5

6

78

9

1

2

34

5

6

78

9

2

34

5

6 6

5

43

277

8 8

9 9

1010

HO

HOHO

HO

HO

HOHO

HO

1998400

2998400

3998400

4998400

5998400

6998400

1998400

2998400

3998400

4998400

5998400

6998400

7998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400 7

998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400

1998400998400

1998400998400

2998400998400

2998400998400 3

9984009984003998400998400

4998400998400

4998400998400

5998400998400

5998400998400

6998400998400

6998400998400




Linear range(mgmL)















37 Growth Curves












4 Discussion







100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)






MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)














References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Results






FractionsS

epidermidis10282

Sepidermidis

5001

E faecalisC159-6

Smaltophilia


E + + + minus

F + + ++ +G + + + minus

H + ++ + minus








O

OO

O

COOH

OH

OH

OH

OHOH

OHOHOH

OH

OH

OHOH

OH OH

OH

OH

COOCH3

OO

O

O O

O

O O

RA MR

IQ HS

1

2

34

5

6

78

9

1

2

34

5

6

78

9

2

34

5

6 6

5

43

277

8 8

9 9

1010

HO

HOHO

HO

HO

HOHO

HO

1998400

2998400

3998400

4998400

5998400

6998400

1998400

2998400

3998400

4998400

5998400

6998400

7998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400 7

998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400

1998400998400

1998400998400

2998400998400

2998400998400 3

9984009984003998400998400

4998400998400

4998400998400

5998400998400

5998400998400

6998400998400

6998400998400




Linear range(mgmL)















37 Growth Curves












4 Discussion







100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)






MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)














References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

OO

O

COOH

OH

OH

OH

OHOH

OHOHOH

OH

OH

OHOH

OH OH

OH

OH

COOCH3

OO

O

O O

O

O O

RA MR

IQ HS

1

2

34

5

6

78

9

1

2

34

5

6

78

9

2

34

5

6 6

5

43

277

8 8

9 9

1010

HO

HOHO

HO

HO

HOHO

HO

1998400

2998400

3998400

4998400

5998400

6998400

1998400

2998400

3998400

4998400

5998400

6998400

7998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400 7

998400

8998400

9998400

1998400

2998400

3998400

4998400

5998400

6998400

1998400998400

1998400998400

2998400998400

2998400998400 3

9984009984003998400998400

4998400998400

4998400998400

5998400998400

5998400998400

6998400998400

6998400998400




Linear range(mgmL)















37 Growth Curves












4 Discussion







100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)






MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)














References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























4 Discussion







100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)






MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)














References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)


100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(a)

100E + 00

100E + 01

100E + 02

100E + 03

100E + 04

100E + 05

100E + 06

100E + 07

0 200 400 600 800 1000 1200 1400 1600

Log

CFU

mL

Time (min)


(b)






MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)














References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MIC2MIC4

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

(a)

100E + 10

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)


100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(a)

100E + 09

100E + 04

100E + 05

100E + 06

100E + 07

100E + 08

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Log

CFU

mL

Time (hours)

ControlMIC

MIC2MIC4

(b)














References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Rosmarinic Acid and Its Methyl Ester as...

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Transcript of Research Article Rosmarinic Acid and Its Methyl Ester as...