Antimicrobial activities of two ethanolic extracts made from fresh and dried leaves of Pelargonium radula (Cav.) L\u27Hérit, were tested against fourteen species of bacteria and fifteen species of fungi. The well-diffusion method indicated the strongest activity against Pseudomonas aeruginosa. The broth dilution method revealed that the most sensitive microorganisms were Bacillus pumilus, Bacillus subtilis, Escherichia coli and Serratia marcescens. Extract prepared from fresh leaves showed significantly higher antimicrobial activity than the extract prepared from dried leaves.Od svježih, odnosno suhih listova Pelargonium radula (Cav.) L’Hérit, pripravljena su dva etanolna ekstrakta. Antimikrobna aktivnost ekstrakata je ispitana na četrnaest bakterijskih vrsta i petnaest vrsta gljivica. Metodom difuzije najjače djelovanje pokazivao je ekstrakt na vrstu Pseudomonas aeruginosa. Najosjetljiviji mikroorganizmi u metodi dilucije bili su Bacillus pumilus, Bacillus subtilis, Escherichia coli i Serratia marcescens. Ekstrakt pripravljen od svježih listova pokazao je znatno veću antimikrobnu aktivnost od ekstrakta priređenog od suhih listova

MARIJANA ZOVKO

STJEPAN PEPELJNJAK

ZDENKA KALOĐERA

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Pelargonium radula (Cav.) L’Hérit (Geraniaceae) is a decorative plant that originatesfrom the Southeast of Africa. In literature, it is frequently found under different synonyms(P. radens H. E. Moore, P. raduloides Hoffmgg., P. multifidum Salisb., P. revolutum Jacq., P.roseum Wild., non Ait., Eckl. & Zeyh.). It is commercially cultivated for its essential oil.The main component of the essential oil is geraniol (up to 50%), while other importantmonoterpenes include citronellol, i-menthone, citronellyl formate, and geranyl formate,their oxides and esters (1).Water and ethanolic extracts of the plant were found to have hypoglycaemic pro-perties (2, 3). There are also indications that the plant extracts possess antimicrobial ac-tivity (4, 5). However, this activity has been investigated only on a small number of mi-croorganisms. In the present study, we have further analyzed the antimicrobial activityof ethanolic extracts prepared from fresh as well as dried plants on 29 microorganisms.409Acta Pharm. 55 (2005) 409–415 Short communicationInvestigation of antimicrobial activity ofPelargonium radula (Cav.) L’HéritSTJEPAN PEPELJNJAK1*ZDENKA KALO\ERA2MARIJANA ZOVKO21 Department of MicrobiologyFaculty of Pharmacy and BiochemistryUniversity of Zagreb, Zagreb, Croatia2 Department of PharmacognosyFaculty of Pharmacy and BiochemistryUniversity of Zagreb, Zagreb, CroatiaReceived May 20, 2005Accepted July 20, 2005Antimicrobial activities of two ethanolic extracts, madefrom fresh and dried leaves of Pelargonium radula (Cav.)L’Hérit, were tested against fourteen species of bacteriaand fifteen species of fungi. The well-diffusion methodindicated the strongest activity against Pseudomonas aeru-ginosa. The broth dilution method revealed that the mostsensitive microorganisms were Bacillus pumilus, Bacillussubtilis, Escherichia coli and Serratia marcescens. Extract pre-pared from fresh leaves showed significantly higher anti-microbial activity than the extract prepared from driedleaves.Keywords: Pelargonium radula, ethanolic extract, well-dif-fusion method, dilution method, antimicrobial activity* Correspondence, e-mail: spep33@yahoo.comEXPERIMENTALExtracts preparationPelargonium radula was cultivated in the Botanical Garden of the Faculty of Phar-macy and Biochemistry in Zagreb. The voucher sample of the plant is deposited in theHerbarium of the Department of Botany, University of Zagreb, Croatia. Plant extractswere obtained by two procedures. Extract I (E1) was obtained from fresh leaves by per-colation with 45% ethanol, and Extract II (E2) by percolation of dried leaves with 90%ethanol. Plant material was macerated for 12 h prior to percolation. For preparation ofboth extracts, 35 g of plant material was taken and 35 g of extract was collected.Thin-layer chromatographic analysisTLC analysis was carried out on Kieselgel 60 F254 (precoated 0.2 mm thick plasticplates, Merck, Germany) using the mobile phase: ethyl acetate/formic acid/acetic acid/water (100:11:11:27, V/V). Visualisation of flavonoids and phenolic acids was achievedby spraying the sheet with 1% methanolic solution of diphenylboric acid aminoethyl es-ter, followed by 5% ethanolic solution of polyethylene glycol 4000. The chromatogramwas evaluated under UV light at 365 nm (6). Apigenin, luteolin, quercitrin, isoquercitrin,rutin and hyperoside and chlorogenic acid (Roth, Germany) were used for referencepurposes.Determination of total phenolics in the extractsThe amount of total phenolic compounds was determined spectrophotometricallyaccording to Swain and Hills (7), and the amount of tannins was calculated according tothe method of Schneider (8). Briefly, 2 mL of plant extract, previously diluted with meth-anol, was pipetted into a flask containing 10 mL of sodium acetate buffer and 8 mL ofdistilled water (Solution 1). To 10 mL of solution 1, 50 mg of casein was added, and themixture was then vortexed for 45 minutes and filtrated (Solution 2). To 1 mL of each so-lution, 0.5 mL of Folin and Ciocalteu’s phenol reagent was added, and the solutionswere diluted to 10 mL with saturated sodium carbonate solution. Absorbance measure-ments were recorded at 725 nm and tannic acid was used in the construction of the stan-dard curve. The amount of tannins was calculated as the difference in the amount ofphenolic compounds before and after adsorption to casein. Estimation was carried outin triplicate.Analysis of the flavonoid content in extractsFlavonoid content was determined spectrophotometrically, according to the methodof Christ and Müller (9). In short, 10 mL of acetone, 2 mL of 25% hydrochloric acid and1 mL of 0.5% solution of hexamethylenetetramine was added to 10 mL of the extracts.After heating at 56 C for 30 minutes, samples were cooled and diluted to 100 mL. 20 mLof each extract was diluted with water, and extracted three times with ethyl acetate. Or-ganic layers were combined, washed twice with water, dried over anhydrous sodium410S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.sulfate, and diluted to 50 mL. To 10 mL of each extract, 2 mL of 2% aluminum chloridesolution in 5% methanolic solution of acetic acid was added. The samples were dilutedto 25 mL with 5% methanolic solution of acetic acid. Absorbances (A) were read after 30minutes at 425 nm. An extract solution prepared in the same manner but without addi-tion of aluminum chloride solution served as a blank. Experiments were performed intriplicate.Flavonoid content (x) expressed as quercetin percent was calculated from the equationx =Ab0 772.where b is the mass of the extract, in grams.Microbial strainsSix Gram-positive (Bacillus cereus ATCC 11778, Bacillus pumilus NCTC 8241, B. sub-tilis NCTC 8236, Sarcina lutea ATCC 9341, Staphylococcus aureus ATCC 6538P, Enterococcusfaecalis ATCC 29212) and eight Gram-negative (Escherichia coli 923 MFBF, Klebsiella oxytocaMFBF, Salmonella sp. 1993 MFBF, Shigella sonnei MFBF, Serratia marcescens MFBF, Citro-bacter freundii 051 MFBF, Proteus rettgeri MFBF, Pseudomonas aeruginosa ATCC 27895) bacte-rial strains were used in the study. Furthermore, acivity against eight yeast strains (Can-dida albicans MFBF, C. apicola MFBF, C. guilliermondii MFBF, C. krusei MFBF, C. parapsilosisMFBF, C. kefyr MFBF, C. tropicalis MFBF, Hansenula anomala MFBF), five moulds (Asper-gillus flavus 3769 MFBF, A. niger 259 MFBF, A. ochraceus MFBF, Penicillium sp. MFBF) aswell as three dermatophytes (Microsporum gypseum 539 MFBF, Trichophyton mentagrophytes981 MFBF, Epidermophytom floccosum 951 MFBF) was tested. All tested species denotedwith MFBF were from the collection of the Department of Microbiology, Faculty of Phar-macy and Biochemistry, University of Zagreb, Zagreb, Croatia.Diffusion methodFresh cultures of bacterial strains were used for inoculum preparation. Before pre-paration, bacterial strains were cultured on Müller-Hinton agar (Merck, Germany) for 18h at 37 C with physiological saline. Density of inoculums used was 106 cells mL–1 ascompared to MacFarland’s standard solution of BaSO4 (0.1 mL of 1% BaCl2 + 9.9 mL of1% H2SO4). Yeasts were cultivated on Sabouraud 2%-dextrose agar (Biolife, Italy) withthe addition of chloramphenicol (50 mg L–1) for 5 days at 25 C for suppression of bacte-rial growth. One mL of inoculum was mixed with approximately 16 mL of melted agar,previously cooled down to 45 C. Müller-Hinton or Sabouraud 2%-dextrose agar wereused for bacterial or yeast strains, respectively. Inoculated agars were then cooled toroom temperature. The wells (6 mm in diameter) were cut from the agar with stainlesssteel cylinders and 0.2 mL of extract solution (E1 or E2) or 70% ethanol was deliveredinto them. To ensure enough time for diffusion of samples into agar, plates were incu-bated at 4 C for 1 h. They were then incubated at either 37 °C for bacteria or 25 °C foryeasts for appropriate time periods under aerobic conditions. The diameter of the inhibi-tion zone around each hole was measured and recorded.411S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.Dilution methodFor determination of minimum inhibitory concentration (MIC) and minimum mic-robicidal concentration (MMC), the broth twofold macrodilution method in Müller-Hint-on or Sabouraud broth (Merck, Germany) was applied. In short, test strains were grownin a nutrition medium containing progressively lower dilutions of the test extract andincubated at 37 °C for bacteria or 25 °C for yeasts. The last two tubes were free of test ex-tract and served as growth control in broth and 70% ethanol. After incubation, approxi-mately the 10 L of content of each test-tube was transfered with a loop onto MüllerHinton or Sabouraud agar. Agar plates were incubated for an appropriate time underaerobic conditions at 37 °C or 25 °C for bacteria or yeasts, respectively. MIC was definedas the lowest concentration of extract that allows no more than 20% bacterial growth,and MMC as the lowest extract concentration from which the microorganisms did notrecover and grow when transferred to fresh medium.RESULTS AND DISCUSSIONThe presence of flavonoids quercitrin, isoquercitrin, rutin and hyperoside as well aschlorogenic acid in both extracts was confirmed by the TLC method. The results of spectro-metric determination showed that ethanolic extract differed in the amount of phenolic com-pounds. Extract prepared from fresh leaves with 45% ethanol (E1) contained 0.453 ± 0.001%(mean ± SD, n = 3) and the extract prepared from dried leaves (E2) 0.228 ± 0.011% (mean ±SD, n = 3) of phenols. The amount of tannins in E1 was found to be 0.206 ± 0.005% (mean ±SD, n = 3), while E2 contained 0.080 ± 0.015% (mean ± SD, n = 3) of these compounds. Thecontent of flavonoids was also different. E1 contained 0.018 ± 0.001% (mean ± SD, n = 3),while E2 contained 0.028 ± 0.001% (mean ± SD, n = 3) of flavonoids.Ethanolic extracts of Pelargonium radula were screened for antimicrobial activityagainst 14 bacterial, 8 yeast, 5 mould and 3 dermatophyte strains by the agar diffusionmethod (Table I). 70% ethanol, which was used as the control, showed no zones of inhi-bition. Extract E1 was active against all the bacterial strains tested. Among the microor-ganisms tested, Pseudomonas aeruginosa was the most susceptible to the extract, inhibi-tion zone being 22 mm. E1 was active against all the bacterial strains included in testing.Inhibition zones ranged from 11–15 mm for a vast majority of bacteria. The activityagainst yeasts and moulds was of somewhat reduced spectrum. Only Candida guillier-mondii, C. parapsilosis and C. kefyr were moderately sensitive to E1. On the other hand, E1exerted activity against two of the three dermatophytes included in investigation. Incontrast to E1, which exhibited activity of a relatively broad spectrum, extract E2 was in-active against the majority of the microorganisms tested. The bacterium most susceptibleto extract E1 was Pseudomonas aeruginosa. The inhibition zone around the well was 15 mm.The results of the broth twofold macrodilution method for E1 are presented in TableII. The procedure was performed only on microorganisms that were found to be sensitiveto the extracts in the diffusion method. All the tested bacteria were inhibited by extractconcentrations ranging from 3.13 to 60%. Among the Gram-positive bacteria, the strongesteffect of the extract (MIC 3.13%) was observed against Bacillus pumilus and Bacillus subtilis.As regards Gram-negative strains, E1 demonstrated the lowestMIC against Escherichia coli412S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.and Serratia marcescens, while the growth of Klebsiella oxytoca, Salmonella sp., Shigella sonneiand Citrobacter freundii, was inhibited by the 6.25% concentration. The only yeast tested bythe dilution method, Candida guilliermondii was inhibited by E1 concentration of 6.25%,while Microsporum gypseum and Trichophyton mentagrophytes were inhibited by the concen-tration of 12.5%. The lowest MMC (6.25%) among all the microorganisms included in thedilution method was observed for Bacillus pumilus, Bacillus subtilis, Escherichia coli andSerratia marcescens. Table II shows the results of the dilution method for E2. In addition toits limited spectrum of activity, extract E2 demonstrated relatively high MICs for all themicroorganisms included in the dilution method. The most sensitive one was Staphylococ-cus aureus (MIC 20%). For the other microorganisms testedMICs ranged from 25 to 50%.413S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.Table I. Results of the well-diffusion method for E1 and E2MicroorganismInhibition zone (mm)E1 E2Bacillus cereus ATCC 11778 14 12Bacillus pumilus NCTC 8241 11 –Bacillus subtilis NCTC 8236 14 11Sarcina lutea ATCC 9341 8 8Staphylococcus aureus ATCC 6538P 12 12Enterococcus faecalis ATCC 29212 12 –Escherichia coli 923 MFBF 7 –Klebsiella oxytoca MFBF 12 –Salmonella sp. 1993 MFBF 14 –Shigella sonnei MFBF 15 –Serratia marcescens MFBF 12 –Citrobacter freundii 051 MFBF 15 –Proteus rettgeri MFBF 14 –Pseudomonas aeruginosa ATCC 27895 22 15Candida albicans MFBF – –Candida apicola MFBF – –Candida guilliermondii MFBF 10 –Candida krusei MFBF – –Candida parapsilosis MFBF 8 –Candida kefyr MFBF – –Candida tropicalis MFBF 7 –Hansenula anomala MFBF – –Aspergillus flavus 3769 MFBF – –Aspergillus niger 259 MFBF – –Aspergillus ochraceus MFBF – –Penicillium sp. MFBF – 11Microsporum gypseum 539 MFBF 7 –Trichophyton mentagrophytes 981 MFBF 6 –Epidermophytom floccosum 951 MFBF – –ATCC – American Type Culture Collection, Rockville, USA; NCTC – National Collection of Type Cultures,London, Great Britain; MFBF – collection of microorganisms of the Department of Microbiology, Faculty ofPharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.CONCLUSIONSWhile the efficacy of the dried-leaves extract was found to be moderate, the fresh--leaves extract demonstrated significant antimicrobial activity. Comparison of the flavonoidcontent of the extracts shows that E2 is richer in these compounds than E1. Therefore, it414S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.Table II. Results of the dilution method for E1MicroorganismConcentration (%)MICa MMCbBacillus cereus ATCC 11778 25 50Bacillus pumilus NCTC 8241 3.13 6.25Bacillus subtilis NCTC 8236 3.13 6.25Sarcina lutea ATCC 9341 50 60Staphylococcus aureus ATCC 6538P 20 25Enterococcus faecalis ATCC 29212 12.5 25Escherichia coli 923 MFBF 3.13 6.25Klebsiella oxytoca MFBF 6.25 12.5Salmonella sp. 1993 MFBF 6.25 12.5Shigella sonnei MFBF 6.25 12.5Serratia marcescens MFBF 3.13 6.25Citrobacter freundii 051 MFBF 6.25 12.5Proteus rettgeri MFBF 10 12.5Pseudomonas aeruginosa ATCC 27895 25 50Candida guilliermondii MFBF 6.25 12.5Microsporum gypseum 539 MFBF 12.5 25Trichophyton mentagrophytes 981 MFBF 12.5 25a Minimum inhibitory concentration.b Minimum microbicidal (bactericidal or fungicidal) concentration.Table III. Results of the dilution method for E2MicroorganismConcentration (%)MICa MMCbBacillus cereus ATCC 11778 25 50Bacillus subtilis NCTC 8236 30 50Sarcina lutea ATCC 9341 50 60Staphylococcus aureus ATCC 6538P 20 25Pseudomonas aeruginosa ATCC 27895 25 50Penicillium sp. MFBF 50 60a Minimum inhibitory concentrationb Minimum microbicidal (bactericidal or fungicidal) concentrationmight be concluded that flavonoids are not the main active compounds of the extracts,but rather other phenolic compounds and/or essential oil. Results reported here contrib-ute to the knowledge of the antimicrobial efficacy of Pelargonium radula.REFERENCES1. Z. Kalo|era, N. Bla`evi} and M. Volenec, Composition of the essential oil of Pelargonium radula(Cav.) L’Herit: A study of its vegetative cycle, Acta Pharm. 51 (2001) 153157.2. M. Sljep~evi}, Z. Kalo|era and M. Had`ija, Hypoglycaemic effect of Pelargonium radula (Cav.)L’Herit, Acta Pharm. 47 (1997) 101–108.3. Z. Kalo|era, M. Had`ija, M. Slijep~evi} and P. Theiss, Stimulation of DNA synthesis in isolatedLangerhans islets treated by Pelargonium radula (Cav.) L’Herit. Extracts with hypoglycaemic effi-cacy, Acta Pharm. 49 (1999) 181–188.4. H. Wollmann, G. Habicht, I. Lau and I. Schultz, Various properties of the volatile oils of Pelar-gonium roseum Willd. from indigenous cultivation, Pharmazie 28 (1973) 56–58.5. M. Lis-Balchin, G. Buchbauer, K. Ribisch and M.-T. Wenger, Comparative antibacterial effects ofnovel Pelargonium essential oils and solvent extracts, Lett. Appl. Microbiol. 27 (1998) 135–141.6. H. Wagner, S. Bladt and E. M. Zgainski, Drogenanalyse, Springer Verlag, Berlin 1983, pp. 163– 165.7. T. Swain and W. E. Hills, The phenolic constituents of Prunus domestica I. – The quantitativeanalysis of phenolic constituents, J. Sci. Food Agric. 10 (1959) 63–68.8. G. Schneider, Zur Bestimmung der Gerbstoffe mit Casein, Arch. Pharm. 309 (1976) 38–44.9. B. Christ and K. H. Müller, Zur serienmäßigen Bestimmung des Gehaltes an Flavonol-Derivat-en in Drogen, Arch. Pharm. 293 (1960) 1033–1042.S A @ E T A KIspitivanje antimikrobne aktivnosti Pelargonium radula (Cav.) L’HéritSTJEPAN PEPELJNJAK, ZDENKA KALO\ERA i MARIJANA ZOVKOOd svje`ih, odnosno suhih listova Pelargonium radula (Cav.) L’Hérit, pripravljena sudva etanolna ekstrakta. Antimikrobna aktivnost ekstrakata je ispitana na ~etrnaest bak-terijskih vrsta i petnaest vrsta gljivica. Metodom difuzije najja~e djelovanje pokazivao jeekstrakt na vrstu Pseudomonas aeruginosa. Najosjetljiviji mikroorganizmi u metodi dilu-cije bili su Bacillus pumilus, Bacillus subtilis, Escherichia coli i Serratia marcescens. Ekstraktpripravljen od svje`ih listova pokazao je znatno ve}u antimikrobnu aktivnost od eks-trakta prire|enog od suhih listova.Klju~ne rije~i: Pelargonium radula, etanolni ekstrakt, metoda difuzije, metoda dilucije, antimikrobnaaktivnostFarmaceutsko-biokemijski fakultet Sveu~ili{ta u Zagrebu, Zagreb415S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.

Ispitivanje antimikrobne aktivnosti Pelargonium radula (Cav.) L’Hérit

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Pelargonium radula (Cav.) L’Hérit (Geraniaceae) is a decorative plant that originatesfrom the Southeast of Africa. In literature, it is frequently found under different synonyms(P. radens H. E. Moore, P. raduloides Hoffmgg., P. multifidum Salisb., P. revolutum Jacq., P.roseum Wild., non Ait., Eckl. & Zeyh.). It is commercially cultivated for its essential oil.The main component of the essential oil is geraniol (up to 50%), while other importantmonoterpenes include citronellol, i-menthone, citronellyl formate, and geranyl formate,their oxides and esters (1).Water and ethanolic extracts of the plant were found to have hypoglycaemic pro-perties (2, 3). There are also indications that the plant extracts possess antimicrobial ac-tivity (4, 5). However, this activity has been investigated only on a small number of mi-croorganisms. In the present study, we have further analyzed the antimicrobial activityof ethanolic extracts prepared from fresh as well as dried plants on 29 microorganisms.409Acta Pharm. 55 (2005) 409–415 Short communicationInvestigation of antimicrobial activity ofPelargonium radula (Cav.) L’HéritSTJEPAN PEPELJNJAK1*ZDENKA KALO\ERA2MARIJANA ZOVKO21 Department of MicrobiologyFaculty of Pharmacy and BiochemistryUniversity of Zagreb, Zagreb, Croatia2 Department of PharmacognosyFaculty of Pharmacy and BiochemistryUniversity of Zagreb, Zagreb, CroatiaReceived May 20, 2005Accepted July 20, 2005Antimicrobial activities of two ethanolic extracts, madefrom fresh and dried leaves of Pelargonium radula (Cav.)L’Hérit, were tested against fourteen species of bacteriaand fifteen species of fungi. The well-diffusion methodindicated the strongest activity against Pseudomonas aeru-ginosa. The broth dilution method revealed that the mostsensitive microorganisms were Bacillus pumilus, Bacillussubtilis, Escherichia coli and Serratia marcescens. Extract pre-pared from fresh leaves showed significantly higher anti-microbial activity than the extract prepared from driedleaves.Keywords: Pelargonium radula, ethanolic extract, well-dif-fusion method, dilution method, antimicrobial activity* Correspondence, e-mail: spep33@yahoo.comEXPERIMENTALExtracts preparationPelargonium radula was cultivated in the Botanical Garden of the Faculty of Phar-macy and Biochemistry in Zagreb. The voucher sample of the plant is deposited in theHerbarium of the Department of Botany, University of Zagreb, Croatia. Plant extractswere obtained by two procedures. Extract I (E1) was obtained from fresh leaves by per-colation with 45% ethanol, and Extract II (E2) by percolation of dried leaves with 90%ethanol. Plant material was macerated for 12 h prior to percolation. For preparation ofboth extracts, 35 g of plant material was taken and 35 g of extract was collected.Thin-layer chromatographic analysisTLC analysis was carried out on Kieselgel 60 F254 (precoated 0.2 mm thick plasticplates, Merck, Germany) using the mobile phase: ethyl acetate/formic acid/acetic acid/water (100:11:11:27, V/V). Visualisation of flavonoids and phenolic acids was achievedby spraying the sheet with 1% methanolic solution of diphenylboric acid aminoethyl es-ter, followed by 5% ethanolic solution of polyethylene glycol 4000. The chromatogramwas evaluated under UV light at 365 nm (6). Apigenin, luteolin, quercitrin, isoquercitrin,rutin and hyperoside and chlorogenic acid (Roth, Germany) were used for referencepurposes.Determination of total phenolics in the extractsThe amount of total phenolic compounds was determined spectrophotometricallyaccording to Swain and Hills (7), and the amount of tannins was calculated according tothe method of Schneider (8). Briefly, 2 mL of plant extract, previously diluted with meth-anol, was pipetted into a flask containing 10 mL of sodium acetate buffer and 8 mL ofdistilled water (Solution 1). To 10 mL of solution 1, 50 mg of casein was added, and themixture was then vortexed for 45 minutes and filtrated (Solution 2). To 1 mL of each so-lution, 0.5 mL of Folin and Ciocalteu’s phenol reagent was added, and the solutionswere diluted to 10 mL with saturated sodium carbonate solution. Absorbance measure-ments were recorded at 725 nm and tannic acid was used in the construction of the stan-dard curve. The amount of tannins was calculated as the difference in the amount ofphenolic compounds before and after adsorption to casein. Estimation was carried outin triplicate.Analysis of the flavonoid content in extractsFlavonoid content was determined spectrophotometrically, according to the methodof Christ and Müller (9). In short, 10 mL of acetone, 2 mL of 25% hydrochloric acid and1 mL of 0.5% solution of hexamethylenetetramine was added to 10 mL of the extracts.After heating at 56 C for 30 minutes, samples were cooled and diluted to 100 mL. 20 mLof each extract was diluted with water, and extracted three times with ethyl acetate. Or-ganic layers were combined, washed twice with water, dried over anhydrous sodium410S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.sulfate, and diluted to 50 mL. To 10 mL of each extract, 2 mL of 2% aluminum chloridesolution in 5% methanolic solution of acetic acid was added. The samples were dilutedto 25 mL with 5% methanolic solution of acetic acid. Absorbances (A) were read after 30minutes at 425 nm. An extract solution prepared in the same manner but without addi-tion of aluminum chloride solution served as a blank. Experiments were performed intriplicate.Flavonoid content (x) expressed as quercetin percent was calculated from the equationx =Ab0 772.where b is the mass of the extract, in grams.Microbial strainsSix Gram-positive (Bacillus cereus ATCC 11778, Bacillus pumilus NCTC 8241, B. sub-tilis NCTC 8236, Sarcina lutea ATCC 9341, Staphylococcus aureus ATCC 6538P, Enterococcusfaecalis ATCC 29212) and eight Gram-negative (Escherichia coli 923 MFBF, Klebsiella oxytocaMFBF, Salmonella sp. 1993 MFBF, Shigella sonnei MFBF, Serratia marcescens MFBF, Citro-bacter freundii 051 MFBF, Proteus rettgeri MFBF, Pseudomonas aeruginosa ATCC 27895) bacte-rial strains were used in the study. Furthermore, acivity against eight yeast strains (Can-dida albicans MFBF, C. apicola MFBF, C. guilliermondii MFBF, C. krusei MFBF, C. parapsilosisMFBF, C. kefyr MFBF, C. tropicalis MFBF, Hansenula anomala MFBF), five moulds (Asper-gillus flavus 3769 MFBF, A. niger 259 MFBF, A. ochraceus MFBF, Penicillium sp. MFBF) aswell as three dermatophytes (Microsporum gypseum 539 MFBF, Trichophyton mentagrophytes981 MFBF, Epidermophytom floccosum 951 MFBF) was tested. All tested species denotedwith MFBF were from the collection of the Department of Microbiology, Faculty of Phar-macy and Biochemistry, University of Zagreb, Zagreb, Croatia.Diffusion methodFresh cultures of bacterial strains were used for inoculum preparation. Before pre-paration, bacterial strains were cultured on Müller-Hinton agar (Merck, Germany) for 18h at 37 C with physiological saline. Density of inoculums used was 106 cells mL–1 ascompared to MacFarland’s standard solution of BaSO4 (0.1 mL of 1% BaCl2 + 9.9 mL of1% H2SO4). Yeasts were cultivated on Sabouraud 2%-dextrose agar (Biolife, Italy) withthe addition of chloramphenicol (50 mg L–1) for 5 days at 25 C for suppression of bacte-rial growth. One mL of inoculum was mixed with approximately 16 mL of melted agar,previously cooled down to 45 C. Müller-Hinton or Sabouraud 2%-dextrose agar wereused for bacterial or yeast strains, respectively. Inoculated agars were then cooled toroom temperature. The wells (6 mm in diameter) were cut from the agar with stainlesssteel cylinders and 0.2 mL of extract solution (E1 or E2) or 70% ethanol was deliveredinto them. To ensure enough time for diffusion of samples into agar, plates were incu-bated at 4 C for 1 h. They were then incubated at either 37 °C for bacteria or 25 °C foryeasts for appropriate time periods under aerobic conditions. The diameter of the inhibi-tion zone around each hole was measured and recorded.411S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.Dilution methodFor determination of minimum inhibitory concentration (MIC) and minimum mic-robicidal concentration (MMC), the broth twofold macrodilution method in Müller-Hint-on or Sabouraud broth (Merck, Germany) was applied. In short, test strains were grownin a nutrition medium containing progressively lower dilutions of the test extract andincubated at 37 °C for bacteria or 25 °C for yeasts. The last two tubes were free of test ex-tract and served as growth control in broth and 70% ethanol. After incubation, approxi-mately the 10 L of content of each test-tube was transfered with a loop onto MüllerHinton or Sabouraud agar. Agar plates were incubated for an appropriate time underaerobic conditions at 37 °C or 25 °C for bacteria or yeasts, respectively. MIC was definedas the lowest concentration of extract that allows no more than 20% bacterial growth,and MMC as the lowest extract concentration from which the microorganisms did notrecover and grow when transferred to fresh medium.RESULTS AND DISCUSSIONThe presence of flavonoids quercitrin, isoquercitrin, rutin and hyperoside as well aschlorogenic acid in both extracts was confirmed by the TLC method. The results of spectro-metric determination showed that ethanolic extract differed in the amount of phenolic com-pounds. Extract prepared from fresh leaves with 45% ethanol (E1) contained 0.453 ± 0.001%(mean ± SD, n = 3) and the extract prepared from dried leaves (E2) 0.228 ± 0.011% (mean ±SD, n = 3) of phenols. The amount of tannins in E1 was found to be 0.206 ± 0.005% (mean ±SD, n = 3), while E2 contained 0.080 ± 0.015% (mean ± SD, n = 3) of these compounds. Thecontent of flavonoids was also different. E1 contained 0.018 ± 0.001% (mean ± SD, n = 3),while E2 contained 0.028 ± 0.001% (mean ± SD, n = 3) of flavonoids.Ethanolic extracts of Pelargonium radula were screened for antimicrobial activityagainst 14 bacterial, 8 yeast, 5 mould and 3 dermatophyte strains by the agar diffusionmethod (Table I). 70% ethanol, which was used as the control, showed no zones of inhi-bition. Extract E1 was active against all the bacterial strains tested. Among the microor-ganisms tested, Pseudomonas aeruginosa was the most susceptible to the extract, inhibi-tion zone being 22 mm. E1 was active against all the bacterial strains included in testing.Inhibition zones ranged from 11–15 mm for a vast majority of bacteria. The activityagainst yeasts and moulds was of somewhat reduced spectrum. Only Candida guillier-mondii, C. parapsilosis and C. kefyr were moderately sensitive to E1. On the other hand, E1exerted activity against two of the three dermatophytes included in investigation. Incontrast to E1, which exhibited activity of a relatively broad spectrum, extract E2 was in-active against the majority of the microorganisms tested. The bacterium most susceptibleto extract E1 was Pseudomonas aeruginosa. The inhibition zone around the well was 15 mm.The results of the broth twofold macrodilution method for E1 are presented in TableII. The procedure was performed only on microorganisms that were found to be sensitiveto the extracts in the diffusion method. All the tested bacteria were inhibited by extractconcentrations ranging from 3.13 to 60%. Among the Gram-positive bacteria, the strongesteffect of the extract (MIC 3.13%) was observed against Bacillus pumilus and Bacillus subtilis.As regards Gram-negative strains, E1 demonstrated the lowest MIC against Escherichia coli412S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.and Serratia marcescens, while the growth of Klebsiella oxytoca, Salmonella sp., Shigella sonneiand Citrobacter freundii, was inhibited by the 6.25% concentration. The only yeast tested bythe dilution method, Candida guilliermondii was inhibited by E1 concentration of 6.25%,while Microsporum gypseum and Trichophyton mentagrophytes were inhibited by the concen-tration of 12.5%. The lowest MMC (6.25%) among all the microorganisms included in thedilution method was observed for Bacillus pumilus, Bacillus subtilis, Escherichia coli andSerratia marcescens. Table II shows the results of the dilution method for E2. In addition toits limited spectrum of activity, extract E2 demonstrated relatively high MICs for all themicroorganisms included in the dilution method. The most sensitive one was Staphylococ-cus aureus (MIC 20%). For the other microorganisms tested MICs ranged from 25 to 50%.413S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.Table I. Results of the well-diffusion method for E1 and E2MicroorganismInhibition zone (mm)E1 E2Bacillus cereus ATCC 11778 14 12Bacillus pumilus NCTC 8241 11 –Bacillus subtilis NCTC 8236 14 11Sarcina lutea ATCC 9341 8 8Staphylococcus aureus ATCC 6538P 12 12Enterococcus faecalis ATCC 29212 12 –Escherichia coli 923 MFBF 7 –Klebsiella oxytoca MFBF 12 –Salmonella sp. 1993 MFBF 14 –Shigella sonnei MFBF 15 –Serratia marcescens MFBF 12 –Citrobacter freundii 051 MFBF 15 –Proteus rettgeri MFBF 14 –Pseudomonas aeruginosa ATCC 27895 22 15Candida albicans MFBF – –Candida apicola MFBF – –Candida guilliermondii MFBF 10 –Candida krusei MFBF – –Candida parapsilosis MFBF 8 –Candida kefyr MFBF – –Candida tropicalis MFBF 7 –Hansenula anomala MFBF – –Aspergillus flavus 3769 MFBF – –Aspergillus niger 259 MFBF – –Aspergillus ochraceus MFBF – –Penicillium sp. MFBF – 11Microsporum gypseum 539 MFBF 7 –Trichophyton mentagrophytes 981 MFBF 6 –Epidermophytom floccosum 951 MFBF – –ATCC – American Type Culture Collection, Rockville, USA; NCTC – National Collection of Type Cultures,London, Great Britain; MFBF – collection of microorganisms of the Department of Microbiology, Faculty ofPharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.CONCLUSIONSWhile the efficacy of the dried-leaves extract was found to be moderate, the fresh--leaves extract demonstrated significant antimicrobial activity. Comparison of the flavonoidcontent of the extracts shows that E2 is richer in these compounds than E1. Therefore, it414S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.Table II. Results of the dilution method for E1MicroorganismConcentration (%)MICa MMCbBacillus cereus ATCC 11778 25 50Bacillus pumilus NCTC 8241 3.13 6.25Bacillus subtilis NCTC 8236 3.13 6.25Sarcina lutea ATCC 9341 50 60Staphylococcus aureus ATCC 6538P 20 25Enterococcus faecalis ATCC 29212 12.5 25Escherichia coli 923 MFBF 3.13 6.25Klebsiella oxytoca MFBF 6.25 12.5Salmonella sp. 1993 MFBF 6.25 12.5Shigella sonnei MFBF 6.25 12.5Serratia marcescens MFBF 3.13 6.25Citrobacter freundii 051 MFBF 6.25 12.5Proteus rettgeri MFBF 10 12.5Pseudomonas aeruginosa ATCC 27895 25 50Candida guilliermondii MFBF 6.25 12.5Microsporum gypseum 539 MFBF 12.5 25Trichophyton mentagrophytes 981 MFBF 12.5 25a Minimum inhibitory concentration.b Minimum microbicidal (bactericidal or fungicidal) concentration.Table III. Results of the dilution method for E2MicroorganismConcentration (%)MICa MMCbBacillus cereus ATCC 11778 25 50Bacillus subtilis NCTC 8236 30 50Sarcina lutea ATCC 9341 50 60Staphylococcus aureus ATCC 6538P 20 25Pseudomonas aeruginosa ATCC 27895 25 50Penicillium sp. MFBF 50 60a Minimum inhibitory concentrationb Minimum microbicidal (bactericidal or fungicidal) concentrationmight be concluded that flavonoids are not the main active compounds of the extracts,but rather other phenolic compounds and/or essential oil. Results reported here contrib-ute to the knowledge of the antimicrobial efficacy of Pelargonium radula.REFERENCES1. Z. Kalo|era, N. Bla`evi} and M. Volenec, Composition of the essential oil of Pelargonium radula(Cav.) L’Herit: A study of its vegetative cycle, Acta Pharm. 51 (2001) 153157.2. M. Sljep~evi}, Z. Kalo|era and M. Had`ija, Hypoglycaemic effect of Pelargonium radula (Cav.)L’Herit, Acta Pharm. 47 (1997) 101–108.3. Z. Kalo|era, M. Had`ija, M. Slijep~evi} and P. Theiss, Stimulation of DNA synthesis in isolatedLangerhans islets treated by Pelargonium radula (Cav.) L’Herit. Extracts with hypoglycaemic effi-cacy, Acta Pharm. 49 (1999) 181–188.4. H. Wollmann, G. Habicht, I. Lau and I. Schultz, Various properties of the volatile oils of Pelar-gonium roseum Willd. from indigenous cultivation, Pharmazie 28 (1973) 56–58.5. M. Lis-Balchin, G. Buchbauer, K. Ribisch and M.-T. Wenger, Comparative antibacterial effects ofnovel Pelargonium essential oils and solvent extracts, Lett. Appl. Microbiol. 27 (1998) 135–141.6. H. Wagner, S. Bladt and E. M. Zgainski, Drogenanalyse, Springer Verlag, Berlin 1983, pp. 163– 165.7. T. Swain and W. E. Hills, The phenolic constituents of Prunus domestica I. – The quantitativeanalysis of phenolic constituents, J. Sci. Food Agric. 10 (1959) 63–68.8. G. Schneider, Zur Bestimmung der Gerbstoffe mit Casein, Arch. Pharm. 309 (1976) 38–44.9. B. Christ and K. H. Müller, Zur serienmäßigen Bestimmung des Gehaltes an Flavonol-Derivat-en in Drogen, Arch. Pharm. 293 (1960) 1033–1042.S A @ E T A KIspitivanje antimikrobne aktivnosti Pelargonium radula (Cav.) L’HéritSTJEPAN PEPELJNJAK, ZDENKA KALO\ERA i MARIJANA ZOVKOOd svje`ih, odnosno suhih listova Pelargonium radula (Cav.) L’Hérit, pripravljena sudva etanolna ekstrakta. Antimikrobna aktivnost ekstrakata je ispitana na ~etrnaest bak-terijskih vrsta i petnaest vrsta gljivica. Metodom difuzije najja~e djelovanje pokazivao jeekstrakt na vrstu Pseudomonas aeruginosa. Najosjetljiviji mikroorganizmi u metodi dilu-cije bili su Bacillus pumilus, Bacillus subtilis, Escherichia coli i Serratia marcescens. Ekstraktpripravljen od svje`ih listova pokazao je znatno ve}u antimikrobnu aktivnost od eks-trakta prire|enog od suhih listova.Klju~ne rije~i: Pelargonium radula, etanolni ekstrakt, metoda difuzije, metoda dilucije, antimikrobnaaktivnostFarmaceutsko-biokemijski fakultet Sveu~ili{ta u Zagrebu, Zagreb415S. Pepeljnjak et al.: Investigation of antimicrobial activity of Pelargonium radula (Cav.) L’Hérit, Acta Pharm. 55 (2005) 409–415.

Ispitivanje antimikrobne aktivnosti Pelargonium radula (Cav.) L’Hérit

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