Antimicrobial activity of Pelargonium endlicherianum Fenzl. (Geraniaceae) roots against some microorganisms

Pelargonium endlicherianum Fenzl. (Geraniaceae) is one of the important species of Pelargonium growing in Turkey. The purpose of this study was to evaluate the antimicrobial activities of P. endlicherianum roots on medically important microorganisms. The antimicrobial activities of 11% ethanol and 70% methanol P. endlicherianum extracts against various bacteria and yeast were studied using the agar dilution method at concentrations ranging from 0.375 to 30.00 mg/ml. The minimum inhibitory concentrations (MICs) of P. endlicherianum extracts against some microorganisms was determined. The highest inhibition was exhibited against Staphylococcus aureus by the 70% methanol extract of P. endlicherianum at 1.38 ± 0.36 mg/ml. The results of this study suggest that P. endlicherianum extracts possess antimicrobial activity against some of the tested microorganisms.


INTRODUCTION
Pelargonium endlicherianum Fenzl.(Geraniaceae) belongs to the family Geraniaceae which has about 750 species scattered widely around the world growing as annuals.Members of the family occur mostly in temperate and subtropical climates and have many important medicinal features (Brendler and van Wyk, 2008).A standardized extract obtained from P. sidoides DC. roots is used and prescribed by physicians in the treatment of some illnesses such as acute rhinosinusitis and tonsillopharyngitis (Bachert et al., 2009;Bereznoy et al., 2003).The liquid preparation of this extract, which is known as UMCA ® (Abdi Ibrahim, Turkey), is used for treating upper respiratory tract infections and coughs especially for children.Much scientific research had been conducted on the anti infective and immunomodulatory effects of Pelargonium.sidoides both in vitro and in vivo (Conrad and Frank, 2008;Conrad et al., 2007;Kim et al., 2009;Kolodziej H, 2008).In vitro studies of P. sidoides have investigated its antibacterial, antimycobacterial, antifungal, immunomodulator activities and effects on the mucosiliary system (Lewu et al., 2006;Lis-Balchin et al., 1998;Neugebauer et al., 2005;Janecki et al., 2007;Kayser et al., 2001;Kayser et al., 2003;Kolodziej et al., 2003).The crude extract and compounds derived from P. sidoides, Pelargonium reniforme and Pelargonium radula have been previously tested against Gram positive, Gram negative bacteria and yeasts (Kayser and Kolodziej, 1997;Papeljnjak et al., 2005).
Turkey has rich flora, and the Pelargonium species are being grown both in the wild and as cultivars (Davis, 1967).One of the wild species, P. endlicherianum (local name "solucanotu") and the decoction prepared from the roots and fresh flowers are used against intestinal parasites (Bozan et al., 1999).However, until now, few studies have been conducted on the roots of P. endlicherianum.The aim of this study was to reveal the antimicrobial activity of P. endlicherianum collected from Turkey.

MATERIALS AND METHODS
Plant material and extract preparation P. endlicherianum were collected in the month of March 2009 from Eskisehir, Turkey.The botanical identification of the plant was done at Department of Pharmaceutical Botany, Erciyes University.The roots were washed and dried at 40°C and powdered in a hammermill.The dry powdered root was extracted by soaking with 11% ethanol and 70% methanol for 24 h at 25°C in a shaking water bath.After this procedure, the extracts were filtered, and the residue was extracted by same procedure once more.The extracts were then evaporated in a vacuum room temperature.These extracts were sterilized by filtration through a membrane filter with a 0.45 µm diameter.They were then dissolved in water, and the final concentrations of the extracts were obtained in the medium ranging from 0.375 to 30.00 mg/ml.

Determination of total phenolics
Total phenols were estimated as gallic acid equivalents (GAE), expressed as mg gallic acid/g extract (Singleton et al., 1999).To ca.6.0 ml H2O, 100 µl sample was transferred in a 10.0 ml volumetric flask, to which 500 µl undiluted Folin-Ciocalteu reagent was added subsequently.After 1 min, 1.5 ml 20% (w/v) Na2CO3 was added and the volume was made up to 10.0 ml with H2O.After 2 h incubation at 25°C, the absorbance was measured at 760 nm and compared to a gallic acid calibration curve.The data are presented as the average of triplicate analyses.

Microorganisms and antimicrobial activity test
The 14 bacterial cultures and 2 yeast cultures used for screening were: Staphylococcus aureus American type culture collection Antimicrobial activity testing was performed by the agar dilution method according to CLSI protocol standards (CLSI M7-A7., 2006).By adjusting density standards (Phoenix Spec Nephelometer, Becton Dickinson, USA), the microorganism suspensions were prepared to equal the density of a 0.5 McFarland standard (1×10 8 CFU/ml for bacteria, 1 to 5 × 10 6 CFU/ml for yeast).Proper media for the microorganisms were prepared by mixing them with sterile Mueller Hinton Agar to give final extract concentrations of between 0.375 and 30.00 mg/ml.Blood was added for some microorganisms such as S. sanguinis, S. pneumoniae, S. agalactia, S. mutans, S. pyogenes, E. faecalis and L. acidophilus in medium to optimum growth conditions.The microorganisms were inoculated on the plate which included different concentrations of P. endlicherianum extracts.Inoculations were performed by means of spots containing a 1×10 4 CFU/spot from each of adjusted microorganism inoculums, and the plates were incubated under the proper conditions for each microorganism.Control samples included: growth control and solvent control.Streptomycin and fluconazole were used as positive reference standards for bacterial and yeasts strains, respectively.The MIC was defined as the lowest concentration of plant extract that did not result in any visible growth of the microorganisms when compared with growth control.All determinations were performed in six times.

RESULTS
Two crude extracts used in this study were obtained from the roots of P. endlicherianum.The extractions were carried out using 11% ethanol and 70% methanol solvents.The ethanol and methanol extracts of P. endlicherianum roots gave a high yield of 1.5 and 7.2% (% concentration w/v) respectively (Table 1).The results of our investigations showed that the 11% ethanol and 70% methanol extracts of P. endlicherianum roots exhibited antimicrobial activities against some of the test microorganisms.The results of the general screening for antimicrobial activity are shown in Table 2.In this study, the highest inhibition was exhibited by the 70% methanol P. endlicherianum extract against S. aureus, the lowest inhibition was exhibited by the 11% ethanol P. endlicherianum extract against L. acidophilus.In addition, the concentrations of both P. endlicherianum extracts also showed no effect against microorganisms such as K. pneumoniae, P. mirabilis, C. albicans, C. glabrata.This study showed that P. endlicherianum has broad-spectrum antibacterial activity but no antifungal activity.

DISCUSSION
P. sidoides is traditionally used to cure tuberculosis in areas of Southern Africa (Brendler and van Wyk, 2008).The antimicrobial activities of P. sidoides extract are known, and a commercialized extract derived from the roots of P. sidoides is also used to treat acute bronchitis  (Agbabiaka et al., 2008).P. sidoides does not grow in Turkey.However, P. endlicherianum is among the species of Pelargonium growing in Turkey (Sezik et al., 2001).In this study, we investigated the antimicrobial activities of P. endlicherianum because of its similarities to P. sidoides.
It had been shown that aqueous alcohol is a better extraction solvent for plant than water to extraction phenolic compounds (Ahmad et al., 1998).Standardized extract of P. sidoides, commonly known as EPs 7630, is produced by the extraction of milled roots with 11% (w/w) aqueous ethanol (Schoetz et al., 2008).However, in this study, we also used 11% ethanol extract and 70% methanol extract which is a standard method (Naczk and Shahidi, 2004).
The phenolic compounds often cited as antimicrobial and their antimicrobial effects may be associated with the presence of phenols in plant extracts as had been shown similarly in other studies (Cowan et al., 1999).In this study, analysis of these extracts showed the presence of phenols and the methanol extract of P. endlicherianum displayed a higher antimicrobial activity against some microorganisms.The reason for this activity could be due to the presence of large amounts of phenolic compounds in 70% metanol extract P. endlicherianum.Kayser et al. reported the antibacterial activity of P. sidoides against microorganisms such as S. aureus, S. pneumoniae, E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa, H. influenza, and the MICs were found to be between 200 to 2000 µg/ml (Kayser et al., 1997).The dried roots of P. reniforme and P. sidoides have been Taylor, 2004).Pepeljnjak et al. (2005) demonstrated the antimicrobial activity of P. radula.Until now, no detailed research regarding P. endlicherianum had been conducted (Pepeljnjak et al., 2005).We find only one study on the antimicrobial activity of P. endlicherianum.Cakal et al. (2009) showed that the antimicrobial activities of the 11% ethanol and 70% methanol extracts of P. endlicherianum were effective against some microorganisms such as E. coli, S. aureus, P. aeruginosa, S. epidermidis.In our study, antimicrobial activity of P. endlicherianum extracts against E. coli, S. aureus, P. aeruginosa, S. epidermidis, S. pyogenes, S. agalactia, S. pneumoniae, S. mutans, S. sanguinis, S. caprae and L. acidophilus was found.These microorganisms are medically important and responsible for human illness.This is the first report showing the antimicrobial efficacy of P. endlicherianum against bacteria such as S. pyogenes, S. agalactia, S. pneumoniae, S. mutans, S. sanguinis, S. caprae and L. acidophilus.
The results of this study suggest that P. endlicherianum extracts possess anti-bacterial activities against some of the tested microorganisms which are significant pathogens in humans.P. endlicherianum may to be a potential source of antimicrobial compounds which could be used to treat infections caused by these bacteria.

Table 1 .
The yields and amount of total phenol of P. endlicherianum.

Table 2 .
MIC values (mg/ml) of the extracts of P. endlicherianum RD: Reference drugs (streptomycin for bacteria, fluconazole for fungi).Values are mean± standard deviation of six experiments in replicate.