Research Article Antibacterial Chemical Constituent and ...downloads.hindawi.com/journals/ecam/2013/480509.pdf · the herbal soap produced with S. auriculata demonstrated a high inhibition

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 480509 5 pageshttpdxdoiorg1011552013480509

Research ArticleAntibacterial Chemical Constituent and Antiseptic Herbal Soapfrom Salvinia auriculata Aubl

Samia Lima1 Gaspar Diaz2 and Marisa Alves Nogueira Diaz1

1 Department of Biochemistry and Molecular Biology Federal University of Vicosa 36570-000 Vicosa MG Brazil2 Department of Chemistry Federal University of Minas Gerais 31270-901 Belo Horizonte MG Brazil

Correspondence should be addressed to Marisa Alves Nogueira Diaz marisanogueiraufvbr

Received 14 October 2013 Revised 12 December 2013 Accepted 18 December 2013

Academic Editor Ulysses Paulino de Albuquerque

Copyright copy 2013 Samia Lima et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The bioassay-guided isolation of the active extract of Salvinia auriculata Aubl led to the separation of three main compoundscharacterized as stigmasterone stigmasterol and friedelinol The pure form of diketosteroid presented a potential antibacterialactivity with aminimum inhibitory concentration (MIC) value of 001mgmLminus1 against Staphylococcus aureus isolated from animalswith mastitis infections The active extract also showed a similar result to that previously obtained with pure diketosteroid whentested with the same isolates The present studyrsquos results demonstrate the potential of this plant as an excipient for the productionof antibacterial soaps aimed at controlling bovine mastitis infections especially on small farms

1 Introduction

In recent decades research on plants with antimicrobialproperties has intensified due mainly to the fact that theseplants are considered sources of pharmacologically activecompounds that can become new antibiotics after their pureconstituents or active extracts have been evaluated [1] Plantsthat live in nutrient-rich environments as well as plants livingin environments containing exceptionally high bacterial celldensity (ie aquatic plants) will be overwhelmed by micro-bial biofilms if they lack any means of biofilm control [2 3]For this reason aquatic plants have attracted the interestof researchers and have proven to be promising sources ofantimicrobial agents [4]

Salvinia auriculata Aubl (Salviniaceae) also known asorelha de onca is a native aquatic plant from South Americaand is commonly found in freshwater lakes [5] Currently fewstudies show the potential of S auriculata in the remediationof water sources contaminated with heavy metals [6] and nostudies in the literature report on the antimicrobial agents oftheir chemical constituents and active extracts

In a preliminary study performed with this plant thepresent studyrsquos research group evaluated the antimicrobialpotential of extracts obtained from the roots and leaves ofthis plant against Gram-positive bacteria associated with

bovine mastitis The subsequent results illustrated a strongpotential of this plant to combat Staphylococcus aureus themain etiologically causative agent of bovine mastitis [7] Thisdisease an inflammatory response found in cowsrsquo uddersis the leading infectious disease affecting dairy cattle today[8] According to the Brazilian Department of Agriculturewhile Brazil produces nearly 27 billion gallonsyear of milkto be distributed worldwide the search for the controland treatment of this disease presents a major issue forboth political policies and the Brazilian economy [9] Thusthis work aims to examine the bioassay-guided isolation ofcompounds responsible for antibacterial activity from activeextract and to evaluate the antiseptic potential of an herbalsoap produced with S auriculata

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) Tetramethylsilane(TMS) was used as an internal standard HR-EI-MS spectra

2 Evidence-Based Complementary and Alternative Medicine

were obtained in a microTOFQ II Bruker Daltonics spec-trometer The IR spectra were measured in a Perkin ElmerParagon 1000 FTIR spectrophotometer using potassiumbromide (1 ww) scanning from 400 to 4000 cmminus1

22 Plant Material S auriculata was collected from a pondlocated in Recanto das Cigarras (20∘4510158402710158401015840S 45∘5110158404610158401015840W)Federal University of Vicosa Minas Gerais Brazil fromDecember 2010 to February 2011 An authenticated voucher(VIC 32122) was deposited in the universityrsquos herbariumAfter the material has been exhaustively washed in water theroots and leaves were separatedThe plant parts were dried at40∘C for 24 h in an air circulation oven and 800 g of roots wasextracted using n-hexane for 2 days and repeated at least fivetimes The solvent was concentrated under reduced pressureuntil it was completely dry and stored at 4∘C

23 Phytochemical Studies Thecrude extract of the roots wasdissolved in a minimum amount of hexane and adsorbedon silica gel (70ndash230mesh) The extract was subjected to achromatography column using n-hexane as a mobile phaseThe eluent polarity was then gradually increased by addingCH2

Cl2

yielding 58 fractions which subsequently under-went biological assay using the S aureus strain 4127 (iden-tified by Embrapa Dairy CattlemdashMilk Microbiology Labora-tory) as a reference microorganismThe positive fraction wassubmitted to a preparative thin layer chromatography (PTLC)eluted with petrolEtOAc 8 2 allowing for the isolation of 1(65mg) 2 (270mg) and 3 (55mg) compounds

24 Bacterial Strains andCultureMedia Thebacterial strainsused in this study which were isolated from animals withmastitis infections were kindly provided by the EmbrapaDairy Cattle mdashMilk Microbiology Laboratory (Juiz de ForaMinas Gerais Brazil) Five S aureus strains (582 680 22214006 and 4127) were used to determine the antimicrobialactivity of the fractions and isolated compounds Bacteriawere routinely cultured on brain heart infusion (BHI) agarat 37∘C for 16 h before conducting the experiments The cellconcentration was adjusted to 106 CFUmLminus1 with an opticaldensity set at 600 nm Stock cultures were maintained in BHIagar containing 25 glycerol 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 5times3mmweremade in the agar and filled with30 120583L of the fraction stock solutions (50mgmLminus1) and with10 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 [10] Dimethylsulfoxide (DMSO)was used as a negative control Tests were performed twice intriplicate The minimum inhibitory concentration (MIC) of

compound 1 was determined by applying a broth microdilu-tion method followed by incubation at 37∘C for 24 h and byobservingmedia turbidity [11] Tests were performed twice intriplicate

26 Production of Herbal Soap The active extract of Sauriculata (250mg) was incorporated into a soap producedaccording to its patent 1005633-5 [12] 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

27 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 4127 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

3 Results and Discussion

Purification of the active fraction obtained from the bioassay-guided active extract of S auriculata roots led to the isolationof two steroids and one triterpeneThese isolated compoundswere characterized as stigmasterone (1) stigmasterol (2) andfriedelinol (3) (Figure 1)

Compound 1was obtained as colorless needlessmp 187ndash189∘C The molecular formula of C

29

H46

O2

was determinedby HR-EI-MS [M+H]+mz = 4266740 The fragment at119898119911 = 245 suggests the presence of two carbonyl groups[13] while the IV spectrum confirms the presence of carbonylgroups with absorption at 1715 cmminus1 Careful analysis of 1Hnuclearmagnetic resonance (NMR) spectrum allowed for theobservation of two double doublets at 120575 520 (dd 119869 = 15184Hz H-22) and 508 (dd 119869 = 151 84Hz H-23) whichcorrespond to typical olefinic protons of steroid side chainsThe nature of the double bond was assumed to be trans basedon the value of the coupling constant 119869 = 1510Hz fromH-22H-23 In the COSY spectrum H-2120572 and H-2120573 showedcross peaks at 120575 162 and 120575 21 with 2H-1 whereas H-4120572 andH-4120573 presented cross correlations with H-5 at 120575 26 Bothprotons at C-7 presented the same cross peak at 120575 186 (H-8) In the 13C nuclear magnetic resonance (NMR) spectrumtwo signals at 120575 1381 and 1298 could be observed confirmingthe presence of olefinic carbons C-22 and C-23 respectivelyIn addition signals could be observed at 120575 2117 and 1205752095 which represent the characteristics of two quaternarycarbons of carbonyl groups as defined by the DistortionlessEnhancement by Polarization Transfer (DEPT) spectrumAccording to the present studyrsquos data and compared to reportsfound in the literature [13 14] compound 1 is in fact thestigmast-22-ene-36-dione MP 187ndash189∘C IR (KBr) 29252853 1715 cmminus1 1H NMR (300MHz CDCl

3

) 120575 074 (3H s

Evidence-Based Complementary and Alternative Medicine 3

O

O

22

23

3 6HO

HO

22

23

3

61 2

3

323

30 29

24

2528

27

26

1717

Figure 1 Isolated compounds from the active fraction of Salvinia auriculata roots

Table 1 Antibacterial activity of hexane extract from Salvinia auriculata roots

S aureus Hexane extract Ciclopirox olamine DMSOInhibition zones (mm plusmn SD)

2221 2067 plusmn 033 2167 plusmn 067 0003008 2067 plusmn 088 2133 plusmn 088 0004072 2000 plusmn 058 2300 plusmn 058 0003007 2100 plusmn 033 2100 plusmn 058 0004163 2000 plusmn 058 2100 plusmn 058 0004347 2300 plusmn 058 2100 plusmn 058 000

18-CH3

) 082 (3H s 26-CH3

) 084 (3H s 29-CH3

) 089 (3Hs 27-CH

3

) 099 (3H s 19-CH3

) 107 (3H s 21-CH3

) 120(1H m 28-H) 146 (1H m 28-H) 156 (1H m 24-H) 160(1H m 25-H) 208 (1H m 20-H) 508 (1H dd 119869 = 151284Hz 23-H) 520 (1H dd 119869 = 1510 84 Hz 22-H) 13CNMR (75MHz CDCl

3

) 120575 1220 (C-18) 1256 (C-29) 1893(C-26) 2140 (C-21) 2189 (C-27) 2620 (C-28) 3264 (C-25) 4068 (C-20) 5147 (C-24) 12980 (C-23) 13810 (C-22)20950 (C-3) 21170 (C-6) MS (EI 70 eV)119898119911() = 397 (7)303 (3) 315 (13) 259 (3) 245 (10) 217 (5) 190 (7) 175 (3)147 (8) 137 (6) 119 (83) HRMS-FAB 119898119911[M + H+] calcd forC29

H46

O2

4266743 found 4266740 Compounds 2 and 3 arealso known compounds andwere identified by comparing thespectroscopic data with the reported values All compoundswere isolated from S auriculata for the first time

Compound 1 can only be detected in the final stages ofplant development and is considered a rare plant constituent[15] Steroids containing 36-dione groups within their struc-tures present prominent biological activities including anti-inflammatory antiallergic and allelopathy [16 17] Resultsconcerning the antimicrobial activity of compound 1 are

in accordance with findings from the literature regardingantimicrobial activity for this compound

In a prior study [7] the hexane extract from S auriculataroots presented a high activity against S aureus strainsisolated from animals with mastitis infection as shown inTable 1

Results from Table 1 are in accordance with those dis-played for compound 1 (see Table 2) which exhibited inhi-bition zones as large as the hexane extract and even greaterthan the positive control confirming that this compoundmaywell be responsible for the activity of the extract Compounds2 and 3 showed no activity against tested bacteria

Table 3 shows the MIC value for compound 1 and thebiofilm inhibitory concentration (BIC) value for the activeextract as previously published [7]

The MIC values obtained for the active extract arelower than some values previously found in extracts withantimicrobial activity [18 19] Based on theAligiannis criteriawithin MIC values (extracts can present strong (005 to05mgmLminus1)moderate (06 to 15mgmLminus1) or weak activity(gt15mgmLminus1)) compound 1 as well as the active extract


Table 2 Antibacterial activity of compounds isolated from Salvinia auriculata roots

S aureus Compound 1 Compound 2 Compound 3 Ciclopirox olamine DMSOInhibition zones (mm plusmn SD)

582 230 plusmn 058 000 000 170 plusmn 058 000680 230 plusmn 060 000 000 180 plusmn 060 0002221 210 plusmn 033 000 000 170 plusmn 058 0004006 200 plusmn 067 000 000 170 plusmn 058 0004127 200 plusmn 058 000 000 170 plusmn 050 000

Table 3 MIC and BIC values obtained from compound 1 and the active extract respectively on Staphylococcus aureus

MIC (mgmLminus1) BIC (mgmLminus1)Active extract 03 0075Compound 1 001 mdashCiclopirox olamine 005 0025

of S auriculata with MIC values of 001 and 03mgmLminus1respectively can be considered strong inhibitors [20] Thevalues determined for BIC were similar to those found forantibiotic substances reported in the literature [21 22]

By contrast the herbal soap produced using the activeextract of S auriculata showed a high antimicrobial activityagainst the tested S aureus According to the results observedin the in vitro evaluation no microbial growth could beobserved on the plate containing the herbal soap of Sauriculata in relation to the control (ciclopirox olamine)Thisconfirms that this extract does in fact have antimicrobialactivity and could be used as an excipient for soap antiseptics

4 Conclusion

In summary the strong inhibitory effects of the active extractof S auriculata against S aureus strains can be attributedto compound 1 a 36-dioxygenated steroid found primarilyin aquatic plants [10] Moreover this activity may well beassociated with a synergism with other compounds that havenot yet been isolated from the active extract Neverthelessthe herbal soap produced with S auriculata demonstrateda high inhibition against an S aureus infection of cowsrsquoudders indicating the potential of the plant as an excipientin the production of antiseptic soap aimed at cleansing theanimalsrsquo udders before milking in turn controlling bovinemastitis infections especially on small farms Therefore itcan be concluded that these findings are of high economicindustrial and veterinary significance

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors are grateful to CAPES for scholarships awardedto Samia Lima to FAPEMIG for their financial support(CEX-APQ-00454-11) and to MAVPB (EmbrapaCNPGLJuiz de Fora Minas Gerais) who kindly provided the bacte-rial strains

References

[1] P A Melendez and V A Capriles ldquoAntibacterial properties oftropical plants from Puerto Ricordquo Phytomedicine vol 13 no 4pp 272ndash276 2006

[2] J-F Hu E Garo M G Goering et al ldquoBacterial biofilminhibitors from Diospyros dendordquo Journal of Natural Productsvol 69 no 1 pp 118ndash120 2006

[3] D A Vattem K Mihalik S H Crixell and R J C McLeanldquoDietary phytochemicals as quorum sensing inhibitorsrdquo Fitoter-apia vol 78 no 4 pp 302ndash310 2007

[4] H Ozbay and A Alim ldquoAntimicrobial activity of some waterplants from the northeastern Anatolian region of TurkeyrdquoMolecules vol 14 no 1 pp 321ndash328 2009

[5] H Lorenzi Plantas Daninhas do Brasil Terrestres AquaticasParasitas e Toxicas Plantarum Sao Paulo Brazil 1st edition2000

[6] D C F Soares E F de Oliveira G D de Fatima Silva LP Duarte V J Pott and S A V Filho ldquoSalvinia auriculataaquatic bioindicator studied by instrumental neutron activationanalysis (INAA)rdquo Applied Radiation and Isotopes vol 66 no 5pp 561ndash564 2008

[7] 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

[8] S J LeBlanc K D Lissemore D F Kelton T F Duffield and KE Leslie ldquoMajor advances in disease prevention in dairy cattlerdquoJournal of Dairy Science vol 89 no 4 pp 1267ndash1279 2006

[9] Secretaria de Estado e Agricultura ldquoPecuaria e Abastecimentode Minas Geraisrdquo 2012 httpwwwagriculturamggovbr

[10] S G Jue G W Dawson and R N Brogden ldquoCiclopiroxolamine 1 cream a preliminary review of its antimicrobialactivity and therapeutic userdquo Drugs vol 29 no 4 pp 330ndash3411985

[11] N Caetano A Saraiva R Pereira D Carvalho M C BPimentel and M B S Maia ldquoDeterminacao da atividadeantimicrobiana de extratos de plantas de uso popular comoanti-inflamatoriordquo Brazilian Journal of Pharmacognosy vol 12supplement 1 pp 132ndash135 2002

[12] M A N Diaz ldquoComposicoes domissaneantes a base de oleode macauba e extratos de Salvinia auriculata e seus derivados


com acao terapeutica e seu uso para prevencao eou controle demastite bovinardquo BR PI1103394-0 patent 2012

[13] P Georges M Sylvestre H Ruegger and P Bourgeois ldquoKetos-teroids and hydroxyketosteroids minor metabolites of sugar-cane waxrdquo Steroids vol 71 no 8 pp 647ndash652 2006

[14] M Isabel Fernandez J R Pedro and E Seoane ldquoConstituentsof a hexane extract of Phoenix dactyliferardquo Phytochemistry vol22 no 9 pp 2087ndash2088 1983

[15] N S Radulovic and N D Dordevic ldquoSteroids from poisonhemlock (Conium maculatum L) a GC-MS analysisrdquo Journalof the Serbian Chemical Society vol 76 no 11 pp 1471ndash14832011

[16] G Aliotta P Monaco G Pinto A Pollio and L PreviteraldquoPotential allelochemicals from Pistia stratiotes Lrdquo Journal ofChemical Ecology vol 17 no 11 pp 2223ndash2234 1991

[17] P-L Tsai J-P Wang C-W Chang S-C Kuo and P-D LeeChao ldquoConstituents and bioactive principles of Polygonumchinensisrdquo Phytochemistry vol 49 no 6 pp 1663ndash1666 1998

[18] M C T Duarte G M Figueira B Pereira P M Magalhaesand C Delarmelina ldquoAtividade antimicrobiana de extratoshidroalcoolicos de especies da colecao de plantas medicinaisrdquoBrazilian Journal of Pharmacognosy vol 14 no 1 pp 6ndash8 2004

[19] 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

[20] 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

[21] 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

[22] 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

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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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

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


were obtained in a microTOFQ II Bruker Daltonics spec-trometer The IR spectra were measured in a Perkin ElmerParagon 1000 FTIR spectrophotometer using potassiumbromide (1 ww) scanning from 400 to 4000 cmminus1

22 Plant Material S auriculata was collected from a pondlocated in Recanto das Cigarras (20∘4510158402710158401015840S 45∘5110158404610158401015840W)Federal University of Vicosa Minas Gerais Brazil fromDecember 2010 to February 2011 An authenticated voucher(VIC 32122) was deposited in the universityrsquos herbariumAfter the material has been exhaustively washed in water theroots and leaves were separatedThe plant parts were dried at40∘C for 24 h in an air circulation oven and 800 g of roots wasextracted using n-hexane for 2 days and repeated at least fivetimes The solvent was concentrated under reduced pressureuntil it was completely dry and stored at 4∘C

23 Phytochemical Studies Thecrude extract of the roots wasdissolved in a minimum amount of hexane and adsorbedon silica gel (70ndash230mesh) The extract was subjected to achromatography column using n-hexane as a mobile phaseThe eluent polarity was then gradually increased by addingCH2

Cl2

yielding 58 fractions which subsequently under-went biological assay using the S aureus strain 4127 (iden-tified by Embrapa Dairy CattlemdashMilk Microbiology Labora-tory) as a reference microorganismThe positive fraction wassubmitted to a preparative thin layer chromatography (PTLC)eluted with petrolEtOAc 8 2 allowing for the isolation of 1(65mg) 2 (270mg) and 3 (55mg) compounds

24 Bacterial Strains andCultureMedia Thebacterial strainsused in this study which were isolated from animals withmastitis infections were kindly provided by the EmbrapaDairy Cattle mdashMilk Microbiology Laboratory (Juiz de ForaMinas Gerais Brazil) Five S aureus strains (582 680 22214006 and 4127) were used to determine the antimicrobialactivity of the fractions and isolated compounds Bacteriawere routinely cultured on brain heart infusion (BHI) agarat 37∘C for 16 h before conducting the experiments The cellconcentration was adjusted to 106 CFUmLminus1 with an opticaldensity set at 600 nm Stock cultures were maintained in BHIagar containing 25 glycerol 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 5times3mmweremade in the agar and filled with30 120583L of the fraction stock solutions (50mgmLminus1) and with10 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 [10] Dimethylsulfoxide (DMSO)was used as a negative control Tests were performed twice intriplicate The minimum inhibitory concentration (MIC) of

compound 1 was determined by applying a broth microdilu-tion method followed by incubation at 37∘C for 24 h and byobservingmedia turbidity [11] Tests were performed twice intriplicate

26 Production of Herbal Soap The active extract of Sauriculata (250mg) was incorporated into a soap producedaccording to its patent 1005633-5 [12] 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

27 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 4127 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

3 Results and Discussion

Purification of the active fraction obtained from the bioassay-guided active extract of S auriculata roots led to the isolationof two steroids and one triterpeneThese isolated compoundswere characterized as stigmasterone (1) stigmasterol (2) andfriedelinol (3) (Figure 1)

Compound 1was obtained as colorless needlessmp 187ndash189∘C The molecular formula of C

29

H46

O2

was determinedby HR-EI-MS [M+H]+mz = 4266740 The fragment at119898119911 = 245 suggests the presence of two carbonyl groups[13] while the IV spectrum confirms the presence of carbonylgroups with absorption at 1715 cmminus1 Careful analysis of 1Hnuclearmagnetic resonance (NMR) spectrum allowed for theobservation of two double doublets at 120575 520 (dd 119869 = 15184Hz H-22) and 508 (dd 119869 = 151 84Hz H-23) whichcorrespond to typical olefinic protons of steroid side chainsThe nature of the double bond was assumed to be trans basedon the value of the coupling constant 119869 = 1510Hz fromH-22H-23 In the COSY spectrum H-2120572 and H-2120573 showedcross peaks at 120575 162 and 120575 21 with 2H-1 whereas H-4120572 andH-4120573 presented cross correlations with H-5 at 120575 26 Bothprotons at C-7 presented the same cross peak at 120575 186 (H-8) In the 13C nuclear magnetic resonance (NMR) spectrumtwo signals at 120575 1381 and 1298 could be observed confirmingthe presence of olefinic carbons C-22 and C-23 respectivelyIn addition signals could be observed at 120575 2117 and 1205752095 which represent the characteristics of two quaternarycarbons of carbonyl groups as defined by the DistortionlessEnhancement by Polarization Transfer (DEPT) spectrumAccording to the present studyrsquos data and compared to reportsfound in the literature [13 14] compound 1 is in fact thestigmast-22-ene-36-dione MP 187ndash189∘C IR (KBr) 29252853 1715 cmminus1 1H NMR (300MHz CDCl

3

) 120575 074 (3H s


O

O

22

23

3 6HO

HO

22

23

3

61 2

3

323

30 29

24

2528

27

26

1717





18-CH3

) 082 (3H s 26-CH3

) 084 (3H s 29-CH3

) 089 (3Hs 27-CH

3

) 099 (3H s 19-CH3

) 107 (3H s 21-CH3


3


H46

O2
















4 Conclusion




Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

O

22

23

3 6HO

HO

22

23

3

61 2

3

323

30 29

24

2528

27

26

1717





18-CH3

) 082 (3H s 26-CH3

) 084 (3H s 29-CH3

) 089 (3Hs 27-CH

3

) 099 (3H s 19-CH3

) 107 (3H s 21-CH3


3


H46

O2
















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

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Antibacterial Chemical Constituent and ...downloads.hindawi.com/journals/ecam/2013/480509.pdf · the herbal soap produced with S. auriculata demonstrated a high inhibition