Research Article Chemical Constituents and an Alternative...

Research ArticleChemical Constituents and an Alternative Medicinal VeterinaryHerbal Soap Made from Senna macranthera

Flaacutevia Inoue Andrade1 Gislaine Aparecida Purgato1 Thalita de Faria Maia1

Raoni Pais Siqueira1 Sacircmia Lima1 Gaspar Diaz2 and Marisa Alves Nogueira Diaz1

1Departamento de Bioquımica e Biologia Molecular Universidade Federal de Vicosa 36570-000 Vicosa MG Brazil2Departamento de Quımica Universidade Federal de Minas Gerais 31270-901 Belo Horizonte MG Brazil

Correspondence should be addressed to Marisa Alves Nogueira Diaz marisanogueiraufvbr

Received 23 October 2014 Accepted 14 February 2015

Academic Editor Jenny M Wilkinson

Copyright copy 2015 Flavia Inoue Andrade et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Upon undergoing biomonitoring the most active dichloromethane extract retrieved from Senna macranthera roots led to theisolation of three main compounds emodine physione and chrysophanol In this sequence these compounds revealed a potentialantibacterial activity against Staphylococcus aureus strains isolated from animals with mastitis infections with minimum inhibitoryconcentration (MIC) values of 20 90 and 90120583gmLminus1 respectively Therefore an herbal soap was also produced from this sameactive extract This soap was tested in vitro using gloves contaminated by animals with bovine mastitis that had been discardedafter use by milkers and showed similar results to previously tested compounds These results indicate the potential of this plant asan alternative veterinary medicine for the production of antibacterial soaps that aimed at controlling bovine mastitis infections insmall Brazilian farms

1 Introduction

In Brazil the use of medicinal plants contributes significantlyto primary health care These vegetation species are usedin the form of crude extracts infusions or plasters totreat common infections in humans and animals [1] Thealternative use ofmedicinal plants to treat common infectionsin animals is increasing as is knowledge in ethnoveterinarystudies especially in some regions of developing countriesthat have limited access to conventionalmedicines for animalhealth care as they are often unaffordable to poor ruralfarmers [2]

Plants are rich in a wide variety of secondary metabolitessuch as tannins terpenoids alkaloids anthraquinones andflavonoids among other compounds which have demon-strated in vitro antimicrobial properties Of this wide varietyof secondary metabolites the anthraquinones have proven tobe outstanding due to their broader action against severalmicroorganisms [3] These compounds are mainly foundin Senna macranthera (Collad Irwin et Barn) species alsoknown by Brazilian local farmers as pau fava and fedegoso

which belongs to the Fabaceae family It is a native plant fromBrazil that is commonly found in forests and is used as anornamental tree However the Senna genus is also widelydistributed in tropical and subtropical regions throughoutthe world and it has been extensively chemically andpharmacologically investigated [4] Folk medicine in Brazilhas made use of these species (Fedegoso and Senna) as aremedy for various diseases especially to treat infectiousdiseases [2]The aerial parts and roots of S macranthera havebeen used by some farmers to treat bovine mastitis a chronicinfection that produces an inflammatory response in cowrsquosudders and that is caused by Staphylococcus aureus the mainetiologic causative agent of this disease Staphylococci causechronic infections due to their ability to adhere tomany typesof surfaces in turn developing a matrix-encased communityof cells called biofilms which hinders antibiotic action [5]Therefore the search for new antibiotics for the treatment ofbovine mastitis must focus on inhibiting biofilm formationby plants or active compounds Thus this work aimed totest the bioassay-guided isolation compounds responsible forantibacterial activity and to check the effect of plant extracts

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 217598 6 pageshttpdxdoiorg1011552015217598

2 Evidence-Based Complementary and Alternative Medicine

on biofilm formation This study also sought to produce anherbal soap from the active extract of S macranthera andassess its antiseptic potential for alternative uses in veterinarymedicine especially with regard to the treatment of bovinemastitis at small farms in rural areas in Brazil

2 Materials and Methods

21 General Silica gel (70ndash230mesh) and glass columnswereused for column chromatography All of the solvents usedwere of analytical grade The melting point was determinedusing a Thermopan apparatus (C Reichest Optische WerckeAG) 1Hand 13Cnuclearmagnetic resonance (NMR) spectrawere recorded on 300MHz and 75MHzNMR spectrometersrespectively (Mercury 300 spectrometer) Tetra methylsilane(TMS) was used as an internal standard The IR spectra weremeasured in a Perkin Elmer Paragon 1000 FTIR spectropho-tometer using potassium bromide (1ww) scanning from400 to 4000 cmminus1

22 Plant Material S macranthera was collected from Vio-leira a neighborhood located (20∘441015840510158401015840S 40∘5110158402610158401015840W) inVicosa MG Brazil in February 2011 and an authenticatedvoucher (VIC 39644) was deposited in the universityrsquosherbariumThe roots were dried at 40∘C in an air circulationoven Dried roots (800 g) were extracted using n-hexanedichloromethane and ethanol for 5 days each and repeatedat least five times The solvents were concentrated underreduced pressure until they were completely dry and theextracts were stored at 4∘C

23 Phytochemical Studies The active dichloromethaneextract from the S macranthera roots was first subjectedto a chromatography column using dichloromethane as theeluent The eluent polarity was then gradually increased byadding ethyl acetate yielding 50 fractions The fractionsobtained were subsequently submitted to biological assayusing the S aureus strain 3828 (identified by the EmbrapaDairy Cattle from the Milk Microbiology Laboratory) as aninfectious reference microorganism The bioassayed positivefractions were submitted to a preparative thin layer chro-matography (PTLC) eluted with dichloromethaneEtOAc(8 2) allowing for the isolation of three compounds 1 (6mg)2 (4mg) and 3 (5mg) (Figure 1)

24 Bacterial Strains andCultureMedia Thebacterial strainsused in this study which were isolated from animals withmastitis infections were kindly provided by the EmbrapaDairy Cattle from the Milk Microbiology Laboratory (Juizde Fora MG Brazil) Six Staphylococcus aureus strains (38283893 4075 4125 4158 and 4182) and Streptococcus agalactiae(3849) Streptococcus bovis (550) and Escherichia coli (24)bacteria (one strain of each) were used to determine theantimicrobial activity of the extracts fractions and isolatedcompounds Bacteria were routinely cultured on brain heartinfusion (BHI) agar at 37∘C for 16 h before conductingthe experiments The cell concentration was adjusted to106 CFUmLminus1 with an optical density set at 600 nm Stock

cultures were maintained in BHI agar containing 25 glyc-erol at minus80∘C

25 Antibacterial Screening Assay Hole-plate diffusion assaywas initially performed to test the antibacterial activity ofthe fractions obtained from crude extracts of the rootsTo accomplish this the bacteria were cultivated overnightand a suspension containing 106 CFUmLminus1 was spread onplates containing Mueller-Hinton agar (Himedia) Holes ofapproximately 5 times 3mm were made in the agar and filledwith 30 120583L of the fraction stock solutions (50mgmLminus1) and10 120583gmLminus1 for compounds 1 2 and 3 After incubation at37∘C for 24 h inhibition zones were measured in millimetersand compared to the controls The antibiotic ciclopiroxolamine (Uci-Farma) was used as the positive control due toits antibacterial properties [6] Dimethylsulfoxide (DMSO)was used as a negative control Tests were performed twicein triplicate The minimum inhibitory concentration (MIC)of compounds 1 2 and 3was determined by applying a brothmicrodilutionmethod followed by incubation at 37∘C for 24 hand by observingmedia turbidity Tests were performed twicein triplicate

26 Effect of Plant Extracts on Cell Adherence The effectof subinhibitory concentrations of active dichloromethaneextract established biofilms was evaluated according to Nos-tro et al [7] with few modifications Bacterial suspensionswere inoculated on microplates containing 180120583L of BHIuntil reaching the final concentration of 106 CFUmLminus1 andincubated at 37∘C for 24 h The supernatant was withdrawnwells were washed three times with 085 saline and filledagain with BHI containing different concentrations of theactive extracts (MIC 12 MIC 14 MIC 18 MIC and116 MIC) followed by incubation at 37∘C for 24 h Thebiofilm inhibitory concentration (BIC) was defined as theconcentration at which no visible microbial growth wasobserved The assay was performed twice in triplicate

27 Effect of Plant Extracts on Biofilm Formation Bacte-rial suspensions were inoculated on microplates containing180 120583L of BHI with different concentrations of the activeextracts (MIC 12 MIC 14 MIC 18 MIC and 116 MIC)until reaching the final concentration of 106 CFUmLminus1 andincubated at 37∘C for 24 h The supernatant was withdrawnand wells were washed three times with 085 saline Theremaining bacterial mass was dried at 37∘C for 15min andstained with 200120583L of crystal violet 01 for 30min Wellswere rewashed and dried as previously described followedby the addition of 300 120583L of ethanol and the measurementof absorbance at 630 nm The test was performed twice intriplicate

28 Production of Herbal Soap The active extract of Smacranthera (250mg)was incorporated into a soap producedaccording to its patent 1005633-5 [8] Later the semi-solidmixture was poured into a mold and allowed to solidifySoap without the extract was also produced to be used as areference product

Evidence-Based Complementary and Alternative Medicine 3

OOH

O

2

H3CO CH3

OH

O

3

CH3

OOH OHOOH

HO

O

1

CH3

OH

Figure 1 Isolated compounds from the most active fraction of the Senna macranthera roots

29 Antibacterial Assay of the Herbal Soap The agar-dilutionmethod was employed in an in vitro evaluation The herbalsoap (10 g) was dissolved in distilled water (50mL) to obtaina 2 suspension The suspension was vigorously shaken todissolve the soap to disperse the foam and to homogenizethe suspension Next 10mL of the soap solution was addedto 20mL of sterile molten culture media in Petri dishes andallowed to set One hundred 120583L of suspension containing106 CFUmLminus1 of a resistant 3828 S aureus strain was thenstreaked on the plates After incubation at 37∘C for 24 hinhibition zones were compared to the control to observe thepresence or absence of microbial growth

Gloves contaminated with S aureus from animals withmastitis infection were used to perform the in vivo evaluation(topical test according to our institutional ethical protocolnumber 773182) The herbal soap (10 g) was dissolved indistilled water (100mL) to obtain a 1 suspension Thissuspension was then vigorously shaken to dissolve the soapto disperse the foam and to homogenize it Thereafter thegloves (12 pairs 6 for each control soap and herbal soaptreatment) were immersed in these solutions for 30 minutesBefore being immersed in the soap the gloves that themilkers had used to milk the cowrsquos udder which had beencontaminated with S aureus were swabbed and the samplewas placed in bottles with sterile normal saline After beingimmersed in both the herbal and control soaps the gloveswere again swabbed and the samples were placed in separatebottles with normal sterile saline solution Aliquots fromthe respective treatments were cultured on an agar plate at37∘C for 24 h to observe the presence or absence of microbialgrowth

3 Results and Discussion

Purification of the most active fraction obtained from thebioassay-guided active dichloromethane extract from Smacranthera roots led to the isolation of three active com-pounds which were characterized as emodine (1) physcione(2) and chrysophanol (3) (Figure 1)

Compound 1 It is an orange amorphous powder fromCHCl

3

mp 261ndash264∘CThe IR spectrum showed absorptionsreferring to a hydroxyl group (3382 cmminus1) and carbonilicgroups (1677 and 1627 cmminus1 resp) The EI mass spectrum of1 displayed a molecular ion [M]+ atmz 270 (100) which isin accordance with the molecular formula C

15

H10

O5

Othermajor fragments were observed atmz 269 and 241 1HNMR(300MHz CDCl

3

) 120575 242 (3H s) 655 (1H d J = 10Hz

H-2) 708 (1H d J = 10Hz H-4) 717 (1H d J = 24 H-7)755 (1H d J = 24Hz H-5) 1208 (1H s H-1) 1228 (1H sH-8) 13C NMR (75MHz CDCl

3

) 120575 212 (C-21015840) 1067 (C-5)1089 (C-7) 1622 (C-1) 1673 (C-6) 1843 (C-10) 1923 (C-9)

Compound 2 It is an orange amorphous powder fromCDCl

3


16

H12

O5

Othermajor fragments were also observed at mz 269 and 240 1HNMR (300MHz CDCl

3

) 120575 242 (3H s CH3

) 393 (3H sOCH3

) 648 (1H s H-2) 658 (1H s H-7) 692 (1H s H-4) 712 (1H s H-5) 1208 (1H s H-1) 1228 (1H s H-8) 13CNMR(75MHzCDCl

3

) 120575 212 (C-21015840) 557 (C-31015840) 1067 (C-5)1089 (C-7) 1622 (C-1) 1673 (C-6) 1843 (C-10) 1908 (C-9)

Compound 3 It is a white amorphous powder from CDCl3

mp 193-194∘CThe IR spectrum showed absorptions referringto a hydroxyl group (3340 cmminus1) and carbonilic groups(1656 and 1619 cmminus1 resp) The EI mass spectrum of 3displayed a molecular ion [M]+ at mz 254 (100) which isin accordance with the molecular formula C

15

H10

O5

Othermajor fragments were also observed at mz 212 197 169 and43 1HNMR (300MHz CDCl

3

) 120575 247 (3H s CH3

) 710 (1Hd J = 11 Hz H-2) 731 (1H dd J = 762 14Hz H-7) 767 (1Hd J = 11 Hz H-4) 770 (1 H t J = 80Hz H-6) 785 (1H ddJ = 752 14Hz H-5) 1203 (1H s H-1) 1214 (1H s H-8)13C NMR (75MHz CDCl

3

) 120575 224 (C-21015840) 1137 (C-13) 1159(C-12) 1199 (C-5) 1214 (C-4) 1214 (C-7) 1245 (C-2) 1333(C-14) 1336 (C-11) 1369 (C-6) 1493 (C-3) 1624 (C-1) 1627(C-8) 1820 (C-10) 1925 (C-9)

All three isolated compounds emodine (1) physcione (2)and chrysophanol (3) are well-known and were identifiedIn addition these compounds also underwent spectroscopicanalysis to compare the reported spectroscopic data Thesecompounds proved to be active against Gram-positive andGram-negative microorganisms [9 10] are very common inthe genus Senna and are known to have several biologicalactivities among them remarkable antimicrobial activity[11] The compounds were tested against S aureus isolatedstrains the main causative agent of bovine mastitis and theobtained results are presented in Table 1

Results from Table 1 are in accordance with the infor-mation provided by local farmers that use this plant totreat bovine mastitis The three isolated compounds showedgood to excellent antimicrobial activities ranging from 20 to


Table 1 MIC (120583gmLminus1) values of compounds isolated from a dichloromethane extract of S macranthera roots against S aureus strains

Microorganisms CompoundsEmodine (1) Physcione (2) Chrysophanol (3) Ciclopirox olamine

S aureus 3828 60 120 190 50S aureus 4125 20 90 90 50S aureus 4158 40 100 190 50

Table 2 Antibacterial activity of the dichloromethane and ethanol extracts from S macranthera roots against Staphylococcus aureus strains

S aureus strains Dichloromethane Ethanol Ciclopirox olamine DMSOInhibition zones (mm plusmn SD)

3828 160 plusmn 058 100 plusmn 058 170 plusmn 071 0003893 130 plusmn 048 120 plusmn 047 180 plusmn 058 0004075 150 plusmn 028 100 plusmn 025 150 plusmn 045 0004125 160 plusmn 016 150 plusmn 023 160 plusmn 035 0004158 150 plusmn 035 70 plusmn 018 160 plusmn 058 0004182 100 plusmn 058 80 plusmn 015 140 plusmn 045 000

190 120583gmLminus1 and are most likely responsible for the antimi-crobial activity observed with the tested dichloromethaneextract

Similarly as can be seen in Table 2 both dichloromethaneand ethanol extracts showed good activity in combatting Saureus the main causative agent of bovine mastitis Likewisethe same extracts also showed positive activity against othermicroorganisms such as Streptococcus agalactiae Strepto-coccus bovis and Escherichia coli (Table 3) which are alsocausative agents of this disease

According to Table 3 the dichloromethane extract can beconsidered more active than an ethanol extract against thetested bacteria except for E coli whose activity was lower

As the two extracts showed significant antibacterial activ-ity against the three tested bacteria the MIC have beenmade with all of them (Table 4) The attained MIC values ofthe most active dichloromethane extract against the testedbacteria proved to be better when compared to that previ-ously found from other extracts of plants with antimicrobialactivities [12 13] According to Aligiannis criteria [14] thedichloromethane extract can be considered strong inhibitorsfor S aureus and S agalactiae and moderate for S bovisHence the ethanol extract can be considered moderate forthe three tested microorganisms (Table 4)

Several studies have shown that biofilm formation is akey factor in the establishment and persistence of infectionscaused by S aureus and S agalactiae in animals with mastitisinfection [15] Currently no vaccines or immunotherapieshave been approved to treat these Staphylococcal infectionsThis area of research has peaked considerable interest mainlyfor the use of small molecules to combat staphylococcalbiofilms [16]Thus the dichloromethane and ethanol extractswere tested in subinhibitory concentrations of MIC values(12 14 18 and 116) to evaluate BIC and adhesion onpreformed biofilms against thesemicroorganismsThe resultsshowed that the dichloromethane extract presented the low-est BIC values for S aureus and S agalactiae equal to 18 and12 respectively of the obtained MIC values For the ethanol

extract the BIC values were 14 and 12 on S aureus and Sagalactiae respectively (Table 5)

According to Table 5 the best BIC value (0063mgmLminus1)obtained for the dichloromethane extract against S aureuscan indicate that this extract can inhibit bacterial filmformation in the initial phase of adhesion and formation ofbiofilms This value was similar to those found for antibioticsubstances reported in prior literature [8]

Due to the promising results concerning the antibacterialactivities of active extracts from S macranthera roots andcompounds isolated through biomonitoring according to theabove discussion the authors of this study were encouragedto prepare an herbal soap containing the active extract Asa result the herbal soaps should represent a new alternativethat is easily accessible for use by farmers from small farmsin the rural areas of Brazil who already use this plant in anattempt to control bovine mastitis For this purpose a newprocedure was devised for antibacterial testsThe herbal soapwas tested in vivo using gloves that had been contaminatedby animals with bovine mastitis and that had been discardedafter having been used by the milkers of the universityrsquosbovine culture sector

Figure 2 showed that the herbal soap obtained from themost active dichloromethane extract of the S macrantheraroots reduces the bacterial load to 790 plusmn 40CFUThis resultis in agreement with those observed by small farmers

Figure 3 shows the results observed in the in vivo evalua-tionwith themilkersrsquo discarded gloves immersed or not in the1 suspension of herbal soap together with an active extractof S macranthera Figure 3(a) showed the microbial growthin the Petri dishes in which the gloves were not immersed inthe herbal soap suspension By contrast Figure 3(b) showedno microbial growth in the Petri dishes after the milkersrsquodiscarded gloves had been immersed for 30min in the herbalsoap suspension

The results obtained from in vivo tests (Figure 3) sug-gested that the active extract of this plant can be incorporated


Table 3 Antibacterial activity of the dichloromethane and ethanol extracts against Streptococcus agalactiae (3849) Streptococcus bovis (550)and Escherichia coli (24) strains

ExtractsMicroorganisms

S agalactiae (3849) S bovis (550) E coli (24)Inhibition zones plusmn SD (mm)

Dichloromethane 150 plusmn 028 80 plusmn 020 13 plusmn 030Ethanol 100 plusmn 052 40 plusmn 036 20 plusmn 028Ciclopirox olaminelowast 150 plusmn 038 180 plusmn 020 180 plusmn 047DMSOlowastlowast 000 000 000lowastPositive control lowastlowastNegative control

(a) Control (b) Soap of S macranthera 790 plusmn 40 UFC

Figure 2 In vitro antibacterial activities of herbal soap produced with the most active extract of S macranthera Tests were performed intriplicate

(a) Result without immersing milkersrsquo discardedgloves in herbal soup

(b) Result after milkersrsquo discarded gloves were im-mersed in S macranthera herbal soap

Figure 3 In vivo antibacterial activities of herbal soap with the active extract of Senna macranthera and the milkersrsquo discarded gloves

into herbal soaps that aimed at cleaning the animalsrsquo uddersbefore milking especially in small farms

4 Conclusion

In summary the inhibitory effects of the active extract ofS macranthera against S aureus strains can be attributed toanthraquinone compounds 1 2 and 3 found in the roots ofthis plant The activity of the dichloromethane extract maywell be associated with the synergism among the isolatedcompounds and other compounds that have not yet been iso-lated from this plant Nevertheless the herbal soap produced

with an active extract of S macranthera demonstrated a highinhibition against S aureus when the milkersrsquo discardedgloves were immersed in the 1 suspension of herbal soapindicating the potential of this plant as an excipient in theproduction and use of antiseptic soaps to control bovinemastitis infections especially in small farms

Conflict of Interests

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


Table 4 MIC (mgmLminus1) values of activity extracts from S macran-thera roots against S aureus S agalactiae and S bovis

Microorganisms Extract MIC

S aureus (3828)Dichloromethane 05

Ethanol 10Positive control 005

S agalactiae (3849)Dichloromethane 06


S bovis (550)Dichloromethane 08


Table 5 BIC (mgmLminus1) values of the dichloromethane and ethanolextracts against S aureus and S agalactiae bacteria

Extracts Microorganisms BIC

DichloromethaneS aureus (3828) 0063

S agalactiae (3849) 03Positive control 0025

EthanolS aureus (3828) 025


Acknowledgments

The authors are grateful to CAPES for scholarships awardedto Flavia Andrade Inoue andThalita de Faria Maia to CNPqfor their financial support (4701532011-3) and EmbrapaGado do Leite Juiz de Fora Minas Gerais who kindlyprovided the bacterial strains

References

[1] C C Rossi A P Aguilar M A N Diaz and A D O BRibon ldquoAquatic plants as potential sources of antimicrobialcompounds active against bovine mastitis pathogensrdquo AfricanJournal of Biotechnology vol 10 no 41 pp 8023ndash8030 2011

[2] F B Holetz G L Pessini N R Sanches D A G Cortez C VNakamura and B P Dias Filho ldquoScreening of some plants usedin the Brazilian folk medicine for the treatment of infectiousdiseasesrdquoMemorias do Instituto Oswaldo Cruz vol 97 no 7 pp1027ndash1031 2002

[3] K S Almeida and F L C Freitas ldquoEtnoveterinaria a fitoterapiana visao do futuro profissional veterinariordquo Revista Verde vol1 no 1 pp 67ndash74 2006

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

[5] C Viegas Jr A de Rezende D H S Silva et al ldquoAspectosquımicos biologicos e etnofarmacologicos do genero CassiardquoQuımica Nova vol 29 no 6 pp 1279ndash1286 2006

[6] A K Gupta and T Plott ldquoCiclopirox a broad-spectrum anti-fungal with antibacterial and anti-inflammatory propertiesrdquoInternational Journal of Dermatology vol 43 no 1 pp 3ndash82004

[7] A Nostro A S Roccaro G Bisignano et al ldquoEffects of oreganocarvacrol and thymol on Staphylococcus aureus and Staphylococ-cus epidermidis biofilmsrdquo Journal of Medical Microbiology vol56 no 4 pp 519ndash523 2007

[8] M A N Diaz ldquoComposicoes domissaneantes a base de oleode macauba e extratos de Salvinia auriculata e seus derivadoscom acao terapeutica e seu uso para prevencao eou controle demastite bovinardquo BR PI1103394-0 Patent 2012

[9] R M Coopoosamy and M L Magwa ldquoAntibacterial activityof chrysophanol isolated from Aloe excelsa (Berger)rdquo AfricanJournal of Biotechnology vol 5 no 16 pp 1508ndash1510 2006

[10] YMKimCH LeeHG Kim andH S Lee ldquoAnthraquinonesisolated from Cassia tora (Leguminosae) seed show an anti-fungal property against phytopathogenic fungirdquo Journal ofAgricultural and Food Chemistry vol 52 no 20 pp 6096ndash61002004

[11] M A N Diaz C C Rossi V R Mendonca et al ldquoScreeningof medicinal plants for antibacterial activities on Staphylococcusaureus strains isolated from bovine mastitisrdquo Brazilian Journalof Pharmacognosy vol 20 no 5 pp 24ndash28 2010

[12] M C T Duarte G M Figueira B Pereira P M Magalhaesand C Delarmelina ldquoAtividade antimicrobiana do extrato deAnacardium occidentale Linn em amostras multiresistentes deStaphylococcus aureusrdquo Revista Brasileira de Farmacognosiavol 14 no 1 pp 6ndash8 2004

[13] S Virtuoso A Davet J F G Dias et al ldquoEstudo preliminar daatividade antibacteriana das cascas de Erythrina velutinaWilldFabaceae (Leguminosae)rdquo Brazilian Journal of Pharmacognosyvol 15 no 2 pp 137ndash142 2005

[14] N Aligiannis E Kalpoutzakis S Mitaku and I B ChinouldquoComposition and antimicrobial activity of the essential oilsof two Origanum speciesrdquo Journal of Agricultural and FoodChemistry vol 49 no 9 pp 4168ndash4170 2001

[15] Y K Ghiorghi E Mairey A Mallet et al ldquoDual role forpilus in adherence to epithelial cells and biofilm formation inStreptococcus agalactiaerdquo PLoS Pathogens vol 5 pp 412ndash4222009

[16] M Garcıa-Castillo M I Morosini A Valverde et al ldquoDif-ferences in biofilm development and antibiotic susceptibilityamong Streptococcus pneumoniae isolates from cystic fibro-sis samples and blood culturesrdquo Journal of AntimicrobialChemotherapy vol 59 no 2 pp 301ndash304 2007

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


on biofilm formation This study also sought to produce anherbal soap from the active extract of S macranthera andassess its antiseptic potential for alternative uses in veterinarymedicine especially with regard to the treatment of bovinemastitis at small farms in rural areas in Brazil

2 Materials and Methods

21 General Silica gel (70ndash230mesh) and glass columnswereused for column chromatography All of the solvents usedwere of analytical grade The melting point was determinedusing a Thermopan apparatus (C Reichest Optische WerckeAG) 1Hand 13Cnuclearmagnetic resonance (NMR) spectrawere recorded on 300MHz and 75MHzNMR spectrometersrespectively (Mercury 300 spectrometer) Tetra methylsilane(TMS) was used as an internal standard The IR spectra weremeasured in a Perkin Elmer Paragon 1000 FTIR spectropho-tometer using potassium bromide (1ww) scanning from400 to 4000 cmminus1

22 Plant Material S macranthera was collected from Vio-leira a neighborhood located (20∘441015840510158401015840S 40∘5110158402610158401015840W) inVicosa MG Brazil in February 2011 and an authenticatedvoucher (VIC 39644) was deposited in the universityrsquosherbariumThe roots were dried at 40∘C in an air circulationoven Dried roots (800 g) were extracted using n-hexanedichloromethane and ethanol for 5 days each and repeatedat least five times The solvents were concentrated underreduced pressure until they were completely dry and theextracts were stored at 4∘C

23 Phytochemical Studies The active dichloromethaneextract from the S macranthera roots was first subjectedto a chromatography column using dichloromethane as theeluent The eluent polarity was then gradually increased byadding ethyl acetate yielding 50 fractions The fractionsobtained were subsequently submitted to biological assayusing the S aureus strain 3828 (identified by the EmbrapaDairy Cattle from the Milk Microbiology Laboratory) as aninfectious reference microorganism The bioassayed positivefractions were submitted to a preparative thin layer chro-matography (PTLC) eluted with dichloromethaneEtOAc(8 2) allowing for the isolation of three compounds 1 (6mg)2 (4mg) and 3 (5mg) (Figure 1)

24 Bacterial Strains andCultureMedia Thebacterial strainsused in this study which were isolated from animals withmastitis infections were kindly provided by the EmbrapaDairy Cattle from the Milk Microbiology Laboratory (Juizde Fora MG Brazil) Six Staphylococcus aureus strains (38283893 4075 4125 4158 and 4182) and Streptococcus agalactiae(3849) Streptococcus bovis (550) and Escherichia coli (24)bacteria (one strain of each) were used to determine theantimicrobial activity of the extracts fractions and isolatedcompounds Bacteria were routinely cultured on brain heartinfusion (BHI) agar at 37∘C for 16 h before conductingthe experiments The cell concentration was adjusted to106 CFUmLminus1 with an optical density set at 600 nm Stock

cultures were maintained in BHI agar containing 25 glyc-erol at minus80∘C

25 Antibacterial Screening Assay Hole-plate diffusion assaywas initially performed to test the antibacterial activity ofthe fractions obtained from crude extracts of the rootsTo accomplish this the bacteria were cultivated overnightand a suspension containing 106 CFUmLminus1 was spread onplates containing Mueller-Hinton agar (Himedia) Holes ofapproximately 5 times 3mm were made in the agar and filledwith 30 120583L of the fraction stock solutions (50mgmLminus1) and10 120583gmLminus1 for compounds 1 2 and 3 After incubation at37∘C for 24 h inhibition zones were measured in millimetersand compared to the controls The antibiotic ciclopiroxolamine (Uci-Farma) was used as the positive control due toits antibacterial properties [6] Dimethylsulfoxide (DMSO)was used as a negative control Tests were performed twicein triplicate The minimum inhibitory concentration (MIC)of compounds 1 2 and 3was determined by applying a brothmicrodilutionmethod followed by incubation at 37∘C for 24 hand by observingmedia turbidity Tests were performed twicein triplicate

26 Effect of Plant Extracts on Cell Adherence The effectof subinhibitory concentrations of active dichloromethaneextract established biofilms was evaluated according to Nos-tro et al [7] with few modifications Bacterial suspensionswere inoculated on microplates containing 180120583L of BHIuntil reaching the final concentration of 106 CFUmLminus1 andincubated at 37∘C for 24 h The supernatant was withdrawnwells were washed three times with 085 saline and filledagain with BHI containing different concentrations of theactive extracts (MIC 12 MIC 14 MIC 18 MIC and116 MIC) followed by incubation at 37∘C for 24 h Thebiofilm inhibitory concentration (BIC) was defined as theconcentration at which no visible microbial growth wasobserved The assay was performed twice in triplicate

27 Effect of Plant Extracts on Biofilm Formation Bacte-rial suspensions were inoculated on microplates containing180 120583L of BHI with different concentrations of the activeextracts (MIC 12 MIC 14 MIC 18 MIC and 116 MIC)until reaching the final concentration of 106 CFUmLminus1 andincubated at 37∘C for 24 h The supernatant was withdrawnand wells were washed three times with 085 saline Theremaining bacterial mass was dried at 37∘C for 15min andstained with 200120583L of crystal violet 01 for 30min Wellswere rewashed and dried as previously described followedby the addition of 300 120583L of ethanol and the measurementof absorbance at 630 nm The test was performed twice intriplicate

28 Production of Herbal Soap The active extract of Smacranthera (250mg)was incorporated into a soap producedaccording to its patent 1005633-5 [8] Later the semi-solidmixture was poured into a mold and allowed to solidifySoap without the extract was also produced to be used as areference product


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Acknowledgments


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OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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4 Conclusion




















Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































4 Conclusion




















Acknowledgments


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































Acknowledgments


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 Chemical Constituents and an Alternative...

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