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Antimicrobial activity of Epilobium spp. extracts Lucia Battinelli, Beatrice Tita, Maria Grazia Evandri, Gabriela Mazzanti * Department of Pharmacology of Natural Substances and General Physiology, Uni6ersity La Sapienza’, P.le Aldo Moro, 5, 00185 Rome, Italy Received 31 October 2000; accepted 10 January 2001 Abstract
The antimicrobial activity of the Epilobium angustifolium, E. hirsutum, E. palustre, E. tetragonum and E. rosmarinifolium ethanolic extracts was studied in vitro on Gram-positive and Gram-negative bacteria, yeasts and fungi. The cytotoxicity of theextracts was also evaluated using the Artemia salina test. All the extracts showed antimicrobial activity in a range ofconcentrations between 10 and 650 mg/ml of dry extract. E. angustifolium and E. rosmarinifolium had the most broad spectrumof action inhibiting bacteria, yeasts and fungi. The extracts were devoid of toxicity on Artemia salina within the range ofantimicrobial concentrations, suggesting that the action is selective on microorganisms. 2001 E Keywords: Epilobium; Bacteria; Yeasts; Dermatophytes; Artemia salina 1. Introduction
myricetin-3-O-b-D-glucuronide [2–4]. Moreover ethanolic extracts of E. angustifolium and alcoholic The genus name Epilobium derives from the Greek extracts of Willow Herb inhibited the growth of words ‘epi’ (upon) and ‘lobos’ (a pod); the plant is so Staphylococcus aureus, S. albus, Pseudomonas py- called because the flowers are arranged upon long, thin, ocyanea and Candida albicans [1].
pod-like seed cases. The genus Epilobium (Onagraceae) The aim of this work was to evaluate in vitro the antimicrobial activity of the extracts of some Epilobium comprises more than 200 species, among which the species and to establish their selectivity of action by most known are E. angustifolium, E. hirsutum and E.
par6iflorum, perennial herbs generally named WillowHerb, with reference to the willow-like nature of theirleaves [1]. The medicinal parts are the herb and theroots that contain: flavonoids, in particular myricitrin, 2. Materials and methods
guaiaverin, quercitrin, quercetin-3-O-b-D-glucuronide);steroids, in particular beta-sitosterol and its esters; tan- nins [1]. Willow Herb preparations are traditionallyused internally for prostate and gastrointestinal disor- Ethanolic extracts of fresh aerial parts of five species ders and externally as antiphlogistic and antiseptic of Epilobium: E. angustifolium L., E. hirsutum L., E.
remedies, to treat mycoses and to improve the healing palustre L., E. tetragonum L., E. rosmarinifolium of wounds [1]. It has been shown that aqueous extracts Haenke, kindly supplied by Boiron Laboratoires (Lyon, of aerial parts of E. angustifolium have analgesic and France), were used. For biological assays the extracts anti-inflammatory activity and reduce the release of were diluted in order to obtain an alcohol concentra- prostaglandins these effects being due, at least in part, tion that did not interfere with the biological tests. Theconcentrations were expressed as dry extracts prelimi-narily determined by evaporating the extracts under  Giornata di Studio SIPHAR (Italian Society for Pharmacog- vacuum and weighing the dry residues. Tetracycline, nosy), Assisi (Italy), 19 September 2000.
miconazole and podophyllotoxin, used as reference sub- * Correspondence and reprints.
E-mail address: gabriela.mazzanti@uniroma1.it (G. Mazzanti).
stances, were purchased from Sigma (Milan, Italy).
0014-827X/01/$ - see front matter 2001 E ´ ditions scientifiques et me´dicales Elsevier SAS PII: S 0 0 1 4 - 8 2 7 X ( 0 1 ) 0 1 0 4 7 - 3 L. Battinelli et al. / Il Farmaco 56 (2001) 345 – 348 were collected and a suspension of 10 – 15 nauplii (100ml) was placed into each well of a 48-well microplate Standard microorganisms or clinically isolated strains (Kartell, Milan, Italy) containing 900 ml of extract in were used. They were: Gram-positive bacteria (Staphy- artificial sea water; control wells containing artificial sea lococcus aureus two strains, Streptococcus pyogenes water were also prepared. After 24 h of incubation the ATCC 12345, Streptococcus sanguis CDC SS 910, dead nauplii were counted using a Zeiss binocular Bacillus subtilis ATCC 6633, Enterococcus faecalis, Lis- microscope (10 × ), then 200 ml of methanol were added teria monocytogenes ATCC 19111), Gram-negative bac- to each well and 60 min later the total number of nauplii were counted. Epilobium extracts were assayed pneumoniae ATCC 10031, Pseudomonas aeruginosa starting from the concentration of 325 mg/ml; higher ATCC 7853, Shigella flexneri CDC 9767 IAL 1517 and concentrations were not tested because some extracts, Salmonella enteritidis IAL 1132), yeasts (Candida albi- and particularly E. rosmarinifolium, after incubation cans two strains, Candida glabrata two strains and produced a precipitate that hindered the count. Podo- Candida krusei ) and fungi (Microsporum canis, Mi- phyllotoxin, dissolved in artificial sea water, was used crosporum gypseum two strains, Tricophyton rubrum as reference substance. The cytotoxicity, expressed as and Tricophyton mentagrophytes four strains.
with 95% confidence limits, was calculated with the test of Lichtfield and Wilcoxon [7].
The antimicrobial activity was evaluated by deter- 3. Results
mining the minimum inhibitory concentration (MIC)and the minimum cytocidal concentration (MCC). The The Epilobium extracts tested showed antimicrobial MIC values (minimum concentration that inhibits the activity with a different spectrum of action (Table 1).
inoculum growth) of Epilobium extracts against bacteria All the extracts inhibited the growth of Gram-positive and yeasts was determined on 96-well culture plates by and Gram-negative bacteria with MIC values between a microdilution method using Mueller – Hinton broth 81 and 650 mg/ml; the action was mostly bactericidal.
(Becton – Dickinson, Milan, Italy). The experiments on Only E. rosmarinifolium and E. angustifolium were ac- dermatophytes were carried out in tubes using Tryp- tive against yeasts; their MIC values were between 162 tone Soya broth (Oxoid, Unipath S.p.A., Milan, Italy) and 325 mg/ml. Finally, all the Epilobium extracts inhib- plus 5% Peptone (Sigma, Milan, Italy). Eight two-fold ited the growth of the fungi, generally between 81 and dilutions of the extracts were carried out starting from 650 mg/ml; E. hirsutum and E. angustifolium, instead, the concentration of 650 mg/ml (about 2% of ethanol).
showed MIC values very low, corresponding to 10 All preparations were sterilized with a 0.22-mm filter.
mg/ml, against Microsporum canis. The action was al- The wells were inoculated with a microorganism sus- pension at a density of 105 cells/ml. The plates were In Artemia salina test none of the Epilobium extracts incubated at 37°C for 24 h (bacteria) or 48 h (yeasts).
showed cytotoxicity at 325 mg/ml, the maximal concen- The inoculum concentration of dermatophytes was ap- proximately 4 × 104 spores/ml and the tubes were incu- used as reference substance, was 9.5 mg/ml (C.L. 6.4– bated for two weeks. After incubation the plates or tubes were observed in order to determine the MICs.
The cultures that did not present growth were used toinoculate plates of solid medium (Mueller – Hinton 4. Discussion
Agar for bacteria and Sabouraud Agar for yeasts andfungi) in order to determine the MCC (minimum con- In our experiments the extracts of Epilobium spp.
centration that kills the inoculum). Tetracycline and tested possess antimicrobial activity. E. angustifolium miconazole, solubilized in 3% ethyl alcohol, were used and E. rosmarinifolium seem to have the most broad as reference antibiotics. Proper blanks were prepared spectrum of action inhibiting bacteria, yeasts and fungi, simultaneously; samples were tested in triplicate.
generally between 81 and 650 mg/ml. These MIC valuesappear high in comparison with those of standard antibiotics; however, it has to be underlined that theraw extracts are constituted by a mixture of chemical The cytotoxicity was evaluated on Artemia salina compounds in which the active principles are generally Leach according to the method of Mongelli et al. [5], contained in low percentage. The activity of E. hirsutum slightly modified by Renzini et al. [6]. Brine shrimp eggs and E. angustifolium against Microsporum canis that is (Euroaquarium S.p.A., Bologna, Italy) were hatched in inhibited at a concentration as low as 10 mg/ml appears artificial sea water; after 48 h the phototrophic nauplii to be particularly interesting. The present results on one L. Battinelli et al. / Il Farmaco 56 (2001) 345 – 348 Table 1Minimum inhibitory concentration (MIC) and minimum cytocidal concentration (MCC) of Epilobium spp. extracts L. monocytogenes ATCC 19111 S. flexneri CDC 9767 IAL 1517 T. mentagrophytes (4 strains) a Concentrations of Epilobium extracts are referred to dry extract; blank cell denotes no effect; ATCC, American Type Colture Collection; CDC, Collecione de Coltura; IAL Istituto Alberto Luz.
b Tetracycline for bacteria, miconazole for yeasts and fungi.
Table 2Cytotoxicity of Epilobium spp. extracts on Artemia salina Leach a a Concentrations of Epilobium extracts are referred to the dry extract; values are expressed as M9S.E. of at least three replications; blank cell hand confirm the antimicrobial activity observed in E.
crobial concentrations. A deeper study in order to angustifolium and reveal a similar activity in other identify the active principles would be of interest.
species of Epilobium, and on the other hand, they offera scientific basis to the traditional use of Willow Herbpreparations as antiseptic and antimycotic remedies to References
treat eczema, seborrhea, psoriasis and other skin condi-tions. Moreover, the antimicrobial activity appears to [1] Epilobium species, PDR for Herbal Medicines, 1st ed., Medical be selective on microorganisms because all the extracts Economics Company, Montvale, New Jersey, 1998, pp. 828 – are devoid of cytotoxicity within the range of antimi- L. Battinelli et al. / Il Farmaco 56 (2001) 345 – 348 [2] A. Hiermann, H. Juan, W. Sametz, Influence of Epilobium ex- tracts on prostaglandin biosynthesis and carragenin induced oedema of the rat paw, J. Ethnopharmacol. 17 (1986) 161 – [5] E. Mongelli, V. Martino, J. Coussio, G. Ciccia, Screening of Argentine plants using the brine shrimp microwell cytotoxicity [3] A. Hiermann, M. Reidlinger, H. Juan, W. Sametz, Isolation of assay, Int. J. Pharmacogn. 34 (1996) 249 – 254.
the antiphlogistic principle from Epilobium angustifolium, Planta [6] G. Renzini, F. Scazzocchio, M. Lu, G. Salvatore, G. Mazzanti, Antibacterial and cytotoxic activity of Hyssopus officinalis L. oils, [4] B. Tita, A. Vitalone, F. Bordi, C. Baldazzi, G. Mazzanti, L. Saso, J. Essent. Oil Res. 11 (1999) 649 – 654.
Attivita` antiproliferativa di Epilobium angustifolium su cellule [7] R.J. Tallarida, R.B. Murray, Manual of Pharmacologic Calcula-

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