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Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance.

Abstract

There is cumulative resistance against antibiotics of many bacteria. Therefore, the development of new antiseptics and antimicrobial agents for the treatment of skin infections is of increasing interest. We have screened six plant extracts and isolated compounds for antimicrobial effects on bacteria and yeasts with dermatological relevance. The following plant extracts have been tested: Gentiana lutea, Harpagophytum procumbens, Boswellia serrata (dry extracts), Usnea barbata, Rosmarinus officinalis and Salvia officinalis (supercritical carbon dioxide C[O.sub.2] extracts). Additionally, the following characteristic plant substances were tested: usnic acid, carnosol, carnosic acid, ursolic acid, oleanolic acid, harpagoside, boswellic acid and gentiopicroside. The extracts and compounds were tested against 29 aerobic and anaerobic bacteria and yeasts in the agar dilution test. U. barbata-extract and usnic acid were the most active compounds, especially in anaerobic bacteria. Usnea C[O.sub.2]-extract effectively inhibited the growth of several Grampositive bacteria like Staphyloccus aureus (including methicillin-resistant strains--MRSA), Propionibacterium acnes and Corynebacterium species. Growth of the dimorphic yeast Malassezia furfur was also inhibited by Usnea-extract. Besides the Usnea-extract, Rosmarinus-, Salvia-, Boswellia- and Harpagophytum-extracts proved to be effective against a panel of bacteria. It is concluded that due to their antimicrobial effects some of the plant extracts may be used for the topical treatment of skin disorders like acne vulgaris and seborrhoic eczema.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Usnea barbata; Usnic acid; Agar dilution test; Staphylococcus aureus: Propionibacterium acnes; Malassezia furfur; Antimicrobial plant extracts

Introduction

Plants and plant extracts have been used for the treatment of skin disorders for centuries (Augustin and Hoch, 2004; Avalos and Maibach, 2000; Schempp et al., 1999). Because of increasing resistance to antibiotics of many bacteria, plant extracts and plant compounds are of new interest as antiseptics and antimicrobial agents in dermatology (Augustin and Hoch, 2004; Blaschek et al., 2004; Norton, 2000). We screened six plant extracts and 8 isolated compounds for their antimicrobial effects on bacteria and yeasts. The most important selection criterion of the plants was the absence of allergy-inducing compounds. Therefore, pure essential oils have not been included in the screening. We utilized commercially available alcoholic fluid extracts, dry extracts and carbon dioxide (C[O.sub.2])-extracts. The tested fluid extracts contain polar secondary plant substances such as bitter agents (Menyanthes trifoliata L.), steroid saponines (Smilax regelii KILL. et C.V. MORTON), or cucurbitacines (Bryonia cretica L.). The dry extracts are rich in bitter agents (Gentiana lutea L.), harpagoside (Harpagophytum procumbens BURCH. DC.), or boswellic acids (Boswellia serrata ROXB. ex COLEBR.). The supercritical liquid C[O.sub.2]-extraction method enriches lipophilic compounds such as usnic acid (Usnea barbata L.) or diterpene phenols (Salvia officinalis L., Rosmarinus officinalis L.). C[O.sub.2]-extracts usually are viscous and are characterized by a high drug-extract-ratio (10:1-20:1 w/w). Additional extraction of polar compounds is achieved by using entrainers such as isopropylic alcohol. Detailed specification of the extracts used in this study is given in the "Materials and methods" section and in Table 1. If available, we also investigated characteristic compounds isolated from the tested plants. Details on these compounds are given in the "Materials and methods" section.

A broad panel of microbial pathogens associated with various skin infections has been included in the screening: the Gram-positive Staphylococcae and Streptococcae are causing wound infections, furuncles, carbuncles, abscesses, impetigo and erysipelas (Kohler et al., 2001; Madigan et al., 2003). The Gram-negative Enterobacteria are part of the physiological intestinal flora. However, outside the intestine they may cause wound infections and sepsis (Kohler et al., 2001; Madigan et al., 2003). Pseudomonas, another Gram-negative rod, is a frequent pathogen of wound infections. Anaerobic Gram-negative rods may cause skin infections under certain circumstances, i.e. in immunocompromised subjects (Kohler et al., 2001; Madigan et al., 2003). The Gram-positive Corynebacteria and Propionibacteria are part of the physiological skin flora. However, Corynebacteria may cause opportunistic skin infections in immunosuppressed patients. Propionibacterium acnes plays an important role as causative agent in acne vulgaris (Kohler et al., 2001). The yeasts Candida albicans and Candida krusei may occur in low frequency on skin and mucous membranes without causing symptoms. As opportunistic pathogens they may overgrow the normal skin flora and cause skin diseases like intertrigo and candidiasis in diabetics, adipose and immunodeficient subjects. The dimorphic yeast Malassezia furfur that is growing in skin areas rich in sebaceous glands is associated with the pathogenesis of seborrhoic eczema and dundruff (Faergemann, 2004; Grigoriu et al., 1984). Details on the germs and their cultivation are given in the "Materials and methods" section and in Tables 2-5.

Materials and methods

Plant extracts and chemicals

The alcoholic fluid extracts of S. regelii, M. trifoliata and Bryonia cretica were purchased from Hetterich (Furth, Germany). G. lutea and H. procumbens dry extracts were from Finzelberg (Andernach, Germany). B. serrata dry extract was provided by HWI (Rheinzabern. Germany). Supercritical C[O.sub.2]-extracts from U. barbata, R. officinalis and S. officinalis were from Flavex (Rehlingen, Germany). Table 1 provides detailed information on drug material, solvent, drug-extract relation and preparation of stock solutions from the extracts. We tempted to obtain highly concentrated water-soluble stock solutions that could easily be incorporated into the aqueous liquid agar medium. On the other hand, it was necessary to dilute toxic solvents like ethanol below a concentration of 1% v/v. The stock solutions differ somewhat with respect to the concentration of the plant extracts. The stock solutions were incorporated into the agar plates at the following concentrations: 100, 20, 10, 2, 1, 0.4 and 0.2 [micro]g/ml.

Most of the isolated plant substances were purchased from Roth (Karlsruhe, Germany) (usnic acid, carnosic acid, oleanolic acid, ursolic acid, harpagoside. gentiopicroside, cucurbitacin E, cucurbitacin I and aucubin). Carnosol was purchased from Alexis (Grunberg, Germany), and 11-keto-boswellic acid was from Phytoplan (Heidelberg, Germany). The substances were dissolved in ethanol 20% v/v and water 80% v/v. Cucurbitacin E was dissolved in dimethyl sulfoxide 20% v/v and water 80% v/v. Stock solutions were prepared at a concentration of 1.28 mg/ml and incorporated into the agar plates according to the two-fold dilution method of the German DIN-standard (Deutsches Institut fur Normung, 2002). The final concentrations were 128, 64, 32, 16, 8, 4, 2 and 1 [micro]g/ml.

Cultivation of bacteria and yeasts

In the present screening we have analyzed 18 aerobic and 9 anaerobic bacteria strains, 2 Candida strains and 1 M. furfur isolate. All strains derived from Type Culture Collections (ATCC and DSM) or patient isolates from the Institute of Medical Microbiology, University of Freiburg (for origin and strain number see Tables 2-5). The susceptibility of all germs to a panel of antibiotics has been described in the ATCC specification sheets or has been tested according to the German Network for Antimicrobial Resistance Surveillance (GENARS, 2004). The test germs were precultivated on appropriate agar plates. Fresh colonies were suspended in Muller-Hinton-Bouillon and the bacterial count was assessed according to the McFarland standard 0.5-1 (Madigan et al., 2003). Yeasts were adjusted to McFarland standard 2 (6 x [10.sup.6] germs/ml). The lipophilic yeast M. furfur was grown on Sabouraud agar containing glucose and olive oil (Grigoriu et al., 1984).

Agar dilution test

Stock solutions of plant extracts or single compounds and their serial dilutions were mixed thoroughly with the liquid agar before solidification to obtain the final concentrations. Subsequently aerobic bacteria were inoculated with a multipoint-inoculator on Muller-Hinton-agar, and anaerobic bacteria on Wilkins-Chalgren-agar. The inoculum of 1 [micro]l of the germ suspensions contained [10.sup.4-5] colony forming units (CFU) for aerobic bacteria and [10.sup.5-6] CFU for anaerobic bacteria. Aerobic bacteria and Candida were incubated at 36 [degrees]C for 24h, and anaerobic bacteria were incubated at 36 [degrees]C for 48 h in an anaerobic jar using Anaerocul[t.sup.R] (90% [N.sub.2], 10% C[O.sub.2]). Malassezia was incubated at 36 [degrees]C for 48 h with 5-10% C[O.sub.2]. The plates were then evaluated visually for the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC). The MIC was defined as the concentration that resulted in no visible growth or less than 10 CFU, corresponding to an inhibition of 99.9% of the inoculum. For the determination of the MBC we used a variant on the agar dilution method. The inoculation spots with no visible growth were cut and top down streaked on Columbia blood agar (aerobes) or yeast-cysteine blood agar (anaerobes) without inhibitory substances The MBC was determined according to the MIC. The lowest concentration without visible growth corresponded with the MBC.

Results

Aerobic bacteria and Candida

Table 2 summarizes the results obtained with plant extracts in aerobic bacteria and Candida. The C[O.sub.2]-extracts and Harpagophytum were the most active plant extracts. Usnea C[O.sub.2]-extract inhibited the growth of all tested Gram-positive bacteria including penicillin-resistant Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), and Enterococcus faecalis. Rosmarinus-extract inhibited the growth of all tested aerobic bacteria, with the exception of Klebsiella pneumoniae. Salvia-extract showed a similar inhibition spectrum compared to Rosmarinus-extract, but with higher MIC and MBC and some gaps in the Gram-negative spectrum. Harpagophytum-extract was effective in all tested germs, but generally at higher concentrations compared to Rosmarinus-extract. The Boswellia-extract was effective against Streptococcus pyogenes, Corynebacterium amycolatum and E. faecalis. Menyanthesextract inhibited the growth of some Gram-positive and Gram-negative bacteria at a higher concentration (mostly at 100 [micro]g/ml). G. lutea, Bryonia cretica and S. regelii did not display any activity, except against S. pyogenes. Candida was only inhibited by Rosmarinus-and Harpagophytum-extracts. Testing of the single compounds revealed that only (+)-usnic acid and carnosic acid display significant activity in aerobic bacteria (negative data not shown). Both substances inhibited the growth of Gram-positive bacteria and of E. faecalis and Pseudomonas maltophilia, (+)-usnic acid being much more active than carnosic acid. The compounds were not active against C. albicans and C. krusei. The MIC and MBC data of (+)-usnic acid and carnosic acid are summarized in Table 3.

Anaerobic bacteria and Malassezia furfur

Usnea-extract was effective at low concentrations against all anaerobic bacteria except Veillonella parvula. Usnea was also tested in M. furfur that was inhibited at a concentration of 10 [micro]g/ml (Table 4). Salvia- and Rosmarinus-extracts were effective in most of the anaerobic bacteria, but at higher concentrations compared to Usnea-extract. Boswellia inhibited the growth of Clostridium perfringens, P. acnes and Porphyromonas gingivalis at low concentrations. Harpagophytum, in contrast to its activity in aerobic bacteria, was not effective in anaerobic bacteria. The other plant extracts did not display marked effectivity in anaerobic bacteria (Table 4). (+)-Usnic acid and carnosic acid were effective in all anaerobic bacteria except V. parvula, (+)-usnic acid being superior to carnosic acid (Table 5). The other purified compounds were not active against anaerobic bacteria (not shown).

Discussion

We have tested nine plant extracts for antimicrobial activity differing in their production method and drug-extract relation. The C[O.sub.2]-extracts from U. barbata, R. officinalis and S. officinalis were the most active compounds with respect to the inhibition panel and the MTC and MBC concentrations. Antibacterial effects of lichen species have been described recently (Gulluce et al., 2006; Rowe et al., 1999), but antimicrobial effects of U. barbata have poorly been investigated. In contrast, broad antimicrobial activity of usnic acid has been reported by several authors (Cocchietto et al., 2002; De Battisti et al., 1991; Dobrescu et al., 1993; Ingolfsdottir, 2002; Lauterwein et al., 1995). In addition to these effects, we have described here for the first time antimicrobial activity of (+)-usnic acid against P. acnes, Corynebacterium pseudodiphtericum and M. furfur. P. acnes and M. furfur are associated with the pathogenesis of acne, seborrhoic eczema and dundruff (Bolognia et al., 2003; Faergemann, 2004). Therefore, Usnea-extract may be useful in the topical treatment of these skin disorders. Both Usnea-extract and (+)-usnic acid additionally inhibited the growth of aerobic Gram-positive bacteria including Streptooccae, Corynebacteria and MRSA. The inhibition spectrum and MIC/MBC of U. barbata-extract and (+)-usnic acid are similar, suggesting that the antimicrobial activity of Usnea-extract is mainly mediated by usnic acid. Candida-yeasts were inhibited neither by Usnea-extract nor by (+)-usnic acid.

Another C[O.sub.2]-extract with a broad antimicrobial spectrum was the Rosmarinus-extract. The growth of 28 out of 29 germs was inhibited by Rosmarinus-extract, but usually at higher concentrations compared to the Usnea-extract. Remarkably, Rosmarinus-extract was the only extract that also inhibited the growth of both Candida strains. Rosmarinus-extract was most effective against C. perfringens and P. gingivalis (MIC and MBC < 1 [micro]g/ml). In the literature, only sparse information on antimicrobial activities of Rosmarinus is available. Most of the published work refers to essential oil from Rosmarinus (Blaschek et al., 2004; Mangena and Muyima, 1999). Here we have used a C[O.sub.2]-extract from Rosmarinus leaves that contains other secondary metabolites compared to the essential oil (Del Bano et al., 2003). Major components of the essential oil are campher, [alpha]-pinene and 1,8-cineol (Blaschek et al., 2004). In contrast, major compounds of the leaf-extract are diterpene phenols (carnosol and carnosic acid), and the triterpenoids oleanolic acid and ursolic acid (Del Bano et al., 2003). Additionally, the C[O.sub.2] leaf-extract contains 1-2% (v/w) essential oil according to manufacturers specification (Flavex Naturextrakte, Rehlingen, Germany). To our knowledge, there are no comparable studies that investigated Rosmarinus C[O.sub.2] leaf-extract in the germs tested here. Solely an inhibitory effect of an ethanolic Rosmarinus leaf-extract on the growth of Clostridium botulinum has been reported (Blaschek et al., 2004), providing a rationale for the preservation of sausage with Rosmarinus. Here we have found inhibitory activity of Rosmarinus-extract on C. perfringens (MIC and MBC < 1 [micro]g/ml).

S. officinalis C[O.sub.2]-extract showed a similar inhibition spectrum as Rosmarinus-extract, but generally with higher MIC/MBC concentrations. It has been shown in the past that essential oil from S. officinalis inhibits the growth of bacteria, yeasts and dermatophytes (Jalsenjak et al., 1978; Janssen et al., 1989). An ethanolic extract from S. officinalis leaves was highly effective against P. gingivalis (Osawa et al., 1991).

B. serrata gum resin and boswellic acids have recently gained new interest because of the rediscovery of its antiinflammatory properties (Ammon et al., 1991). Antimicrobial effects of B. serrata have not been reported so far. Boswellia has been used in traditional medicine for the treatment of thrush that is caused by Candida species (Blaschek et al., 2004). We could not find antimicrobial activity of Boswellia dry extract or 11-keto-[beta]-boswellic acid against C. albicans or C. krusei. However, the extract was highly effective against selected aerobic and anaerobic bacteria such as Streptococcae, Corynebacteria, C. perfringens and P. acnes. Because 11-keto-[beta]-boswellic acid was not effective in these germs, we suggest that the effective components are other boswellic acids or essential oils contained in the extract.

H. procumbens-extract is known for its antiinflammatory properties (Fiebich et al., 2001). In the present screening, Harpagophytum-extract inhibited the growth of all aerobic bacteria, C. krusei and two anaerobic bacteria strains. The characteristic compound of Harpagophytum, harpagoside, was not effective in the screening, suggesting that other compounds in the extract represent the active principle. Bitter agents of G. lutea have been described to possess antifungal activity (Guerin and Reveillere, 1985). In the screening presented here, Gentiana dry extract did not display significant antimicrobial activity against bacteria and yeasts.

Bryonia, Smilax and Menyanthes did not display pronounced antimicrobial effects at the concentrations tested.

The most interesting extract is the supercritical C[O.sub.2]-extract from U. barbata with a defined content of (+)-usnic acid. Although usnic acid is a lichen acid with a weak sensitizing potential, this should not be a problem in clinical practice because the MIC/MBC concentrations of Usnea-extract are clearly below the sensitizing concentrations (Hausen et al., 1993). Because of its high efficacy against P. acnes, Corynebacteria, Fusobacterium and M. furfur, Usnea-extract may be effective in the treatment of acne, rosacea, seborrhoic eczema and dundruff (Bolognia et al., 2003). These skin conditions are associated with superinfection of the skin with the above-mentioned germs. Topical standard treatment modalities for these skin disorders are problematic in some aspects (Leyden, 2004a). Benzoyl peroxide and retinoids are skin irritating. Frequently used topical antibiotics such as macrolides or tetracyclines may cause resistance of bacteria that prevents the use of these substances in severe infections (Leyden, 2004b). Therefore, it would be interesting to perform clinical studies with Usnea-extract in these skin disorders.

Acknowledgments

This work was supported by a grant from the Manfred und Ursula Muller-Stiftung under the auspices of the 'Stifterverband fur die Deutsche Wissenschaft' (Grant T159/11925). These studies were part of the research program "medical plants in dermatology", conducted at the Department of Dermatology, University of Freiburg.

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S. Weckesser (a), K. Engel (a), B. Simon-Haarhaus (a), A. Wittmer (b), K. Pelz (b), C.M. Schempp (a,*)

(a) Department of Dermatology, University of Freiburg, Hauptstrasse 7, 79104 Freiburg, Germany

(b) Institute of Medical Microbiology, University of Freiburg, Freiburg, Germany

Received 6 April 2006; accepted 13 October 2006

[star] Parts of this study were presented at the 4th congress on phytopharmaceuticals and phytotherapy, October 6-8, 2005, Berlin, Germany.

*Corresponding author. Tel.: + 49 761 270 6701; fax: +49 761 270 6655.

E-mail address: christoph.schempp@uniklinik-freiburg.de (C.M. Schempp).
Table 1. Specification of the tested extracts and characteristic
compounds

 Drug material Solvent

Usnea barbata L. Usneae barbatae lichen Carbon dioxide
Rosmarinus Rosmarini officinalis Carbon dioxide/isopropyl
 officinalis L. folium alcohol
Salvia officinalis L. Salviae officinalis Carbon dioxide/isopropyl
 folium alcohol
Boswellia serrata Boswellia serrata Ethanol (70% v/v)
 ROXB. ex COLEBR. resin, Indian origin
Harpagophytum Harpagophyti radix [H.sub.2]O
 procumbens
 (BURCH.) DC.
Gentiana lutea L. Gentianae radix [H.sub.2]O
Menyanthes Menyanthidis folium Ethanol (55% v/v)
 trifoliata L.
Bryonia cretica L. Bryoniae radix Ethanol (70% v/v)
Smilax regelii Sarsaparillae radix Ethanol (70% v/v)
 KILL. et C.V.
 MORTON

 Drug-extract Extract form
 relation

Usnea barbata L. 3-4:1 (w/w) Suspension
Rosmarinus 14-17:1 (w/w) Viscous paste
 officinalis L.
Salvia officinalis L. 14-20:1 (w/w) Viscous paste
Boswellia serrata 3.8-4.2:1 (w/w) Powder
 ROXB. ex COLEBR.
Harpagophytum 1.5-2.5:1 (w/w) Powder
 procumbens
 (BURCH.) DC.
Gentiana lutea L. 2-4:1 (w/w) Powder
Menyanthes 1:1 (w/v) Fluid extract (dry substance
 trifoliata L. 23%)
Bryonia cretica L. 1:1 (w/v) Fluid extract (dry substance
 10%)
Smilax regelii 1:1 (w/v) Fluid extract (dry substance
 KILL. et C.V. 10%)
 MORTON

 Characteristic Preparation of
 compounds stock solutions

Usnea barbata L. Usnic acid (4% w/w) 250 mg/ml in [H.sub.2]O
Rosmarinus Diterpene phenols 250 mg/ml in [H.sub.2]O
 officinalis L. (14% w/w)
Salvia officinalis L. Diterpene phenols 10 mg/ml in 20% ethanol
 (35% w/w)
Boswellia serrata Boswellic acids 10 mg/ml in 20%
 ROXB. ex COLEBR. (80% w/w) DMSO/10% [H.sub.2]O
Harpagophytum Harpagoside 200 mg/ml in PBS
 procumbens (2.6% w/w)
 (BURCH.) DC.
Gentiana lutea L. Secoiridoide bitter 400 mg/ml in PBS
 agents
Menyanthes Secoiridoide bitter 1:7 (v/v) in [H.sub.2]O
 trifoliata L. agents
Bryonia cretica L. Curcurbitacines 1:7 (v/v) in [H.sub.2]O
Smilax regelii Saponines 1:5.5 (v/v) in [H.sub.2]O
 KILL. et C.V.
 MORTON

 Tested single compounds

Usnea barbata L. (+)-usnic acid
Rosmarinus Carnosol, carnosic acid, ursolic acid, oleanolic
 officinalis L. acid
Salvia officinalis L. Carnosol, carnosic acid, ursolic acid, oleanolic
 acid
Boswellia serrata 11-keto-[beta]-boswellic acid
 ROXB. ex COLEBR.
Harpagophytum Harpagoside
 procumbens
 (BURCH.) DC.
Gentiana lutea L. Gentiopicroside
Menyanthes Aucubin
 trifoliata L.
Bryonia cretica L. Curcurbitacin E cucurbitacin I
Smilax regelii
 KILL. et C.V.
 MORTON

Table 2. Effects of plant extracts on aerobic bacteria and Candida
species in the agar dilution test

 Usnea Salvia
 MIC MBC MTC MBC

Staphylococcus aureus (ATCC 25923) 2 100 100 100
Staphylococcus aureus, Pen. res. (E 12431/98) 2 100 100 100
Staphylococcus aureus, MRSA (RV5/98) 10 100 100 100
Staphylococcus epidermidis (DSM 1798) 1 100 20 100
Staphylococcus lugdunensis (NG 3406/94) 1 100 20 100
Streptococcus pyogenes gr. A (E 12449/98) 1 1 10 100
Streptococcus agalactiae gr. B (DSM 2134) 1 1 20 100
Enterococcus faecalis (ATCC 19212) 1 100 100 100
Escherichia coli (ATCC 25922) -- -- -- --
Morganella morganii (E 10679/93) -- -- 100 100
Klebsiella pneumoniae (ATCC 27736) -- -- -- --
Pseudomonas aeruginosa (ATCC 27853) -- -- 100 100
Serratia marcescens (E 8382/92) -- -- -- --
Pseudomonas maltophilia (D 141/92) -- -- 100 100
Bacillus subtilis (ATCC 6633) 1 20 20 20
Micrococcus luteus (DSM 348) 1 1 100 100
Corynebacterium amycolatum (ATCC 494368) 1 10 20 100
Corynebact. pseudodiphtericum (RV 2/95) 1 1 10 20
Candida albicans (ATCC 90028) -- -- -- --
Candida krusei (ATCC 6258) -- -- -- --

 Rosmarinus Boswellia
 MIC MBC MIC MBC

Staphylococcus aureus (ATCC 25923) 10 10 100 --
Staphylococcus aureus, Pen. res. (E 12431/98) 10 10 100 --
Staphylococcus aureus, MRSA (RV5/98) 10 10 -- --
Staphylococcus epidermidis (DSM 1798) 10 10 100 100
Staphylococcus lugdunensis (NG 3406/94) 10 10 -- --
Streptococcus pyogenes gr. A (E 12449/98) 10 10 1 100
Streptococcus agalactiae gr. B (DSM 2134) 10 10 2 20
Enterococcus faecalis (ATCC 19212) 20 20 2 100
Escherichia coli (ATCC 25922) 100 100 -- --
Morganella morganii (E 10679/93) 10 20 -- --
Klebsiella pneumoniae (ATCC 27736) -- -- -- --
Pseudomonas aeruginosa (ATCC 27853) 20 20 -- --
Serratia marcescens (E 8382/92) 100 100 -- --
Pseudomonas maltophilia (D 141/92) 10 10
Bacillus subtilis (ATCC 6633) 10 10 2 2
Micrococcus luteus (DSM 348) 10 10 100 n.a.
Corynebacterium amycolatum (ATCC 494368) 2 10 1 20
Corynebact. pseudodiphtericum (RV 2/95) 2 2 10 20
Candida albicans (ATCC 90028) 20 100 -- --
Candida krusei (ATCC 6258) 20 100 -- --

 Harpagophytum Gentiana
 MIC MBC MIC MBC

Staphylococcus aureus (ATCC 25923) 10 20 -- --
Staphylococcus aureus, Pen. res. (E 12431/98) 10 10 -- --
Staphylococcus aureus, MRSA (RV5/98) 10 20 -- --
Staphylococcus epidermidis (DSM 1798) 10 100 -- --
Staphylococcus lugdunensis (NG 3406/94) 10 100 -- --
Streptococcus pyogenes gr. A (E 12449/98) 100 100 100 n.e.
Streptococcus agalactiae gr. B (DSM 2134) 100 100 -- --
Enterococcus faecalis (ATCC 19212) 100 100 -- --
Escherichia coli (ATCC 25922) 100 100 -- --
Morganella morganii (E 10679/93) 20 20 -- --
Klebsiella pneumoniae (ATCC 27736) 100 100 -- --
Pseudomonas aeruginosa (ATCC 27853) 100 100 -- --
Serratia marcescens (E 8382/92) 100 100 -- --
Pseudomonas maltophilia (D 141/92) 20 100 -- --
Bacillus subtilis (ATCC 6633) 100 100 -- --
Micrococcus luteus (DSM 348) 100 100 -- --
Corynebacterium amycolatum (ATCC 494368) 10 20 10 n.e.
Corynebact. pseudodiphtericum (RV 2/95) 20 100 100 n.e.
Candida albicans (ATCC 90028) 100 -- -- --
Candida krusei (ATCC 6258) 100 100 -- --

 Bryania Sarsaparilla
 MIC MBC MIC MBC

Staphylococcus aureus (ATCC 25923) -- -- -- --
Staphylococcus aureus, Pen. res. (E 12431/98) -- -- -- --
Staphylococcus aureus, MRSA (RV5/98) -- -- -- --
Staphylococcus epidermidis (DSM 1798) -- -- -- --
Staphylococcus lugdunensis (NG 3406/94) -- -- -- --
Streptococcus pyogenes gr. A (E 12449/98) 100 100 20 100
Streptococcus agalactiae gr. B (DSM 2134) -- -- -- --
Enterococcus faecalis (ATCC 19212) -- -- -- --
Escherichia coli (ATCC 25922) -- -- -- --
Morganella morganii (E 10679/93) -- -- -- --
Klebsiella pneumoniae (ATCC 27736) -- -- -- --
Pseudomonas aeruginosa (ATCC 27853) -- -- -- --
Serratia marcescens (E 8382/92) -- -- -- --
Pseudomonas maltophilia (D 141/92) -- -- -- --
Bacillus subtilis (ATCC 6633) -- -- -- --
Micrococcus luteus (DSM 348) -- -- -- --
Corynebacterium amycolatum (ATCC 494368) -- -- -- --
Corynebact. pseudodiphtericum (RV 2/95) -- -- -- --
Candida albicans (ATCC 90028) -- -- -- --
Candida krusei (ATCC 6258) -- -- -- --

 Menyanthes
 MIC MBC

Staphylococcus aureus (ATCC 25923) 100 100
Staphylococcus aureus, Pen. res. (E 12431/98) 100 100
Staphylococcus aureus, MRSA (RV5/98) 100 100
Staphylococcus epidermidis (DSM 1798) 100 100
Staphylococcus lugdunensis (NG 3406/94) 100 100
Streptococcus pyogenes gr. A (E 12449/98) 20 100
Streptococcus agalactiae gr. B (DSM 2134) 100 --
Enterococcus faecalis (ATCC 19212) -- --
Escherichia coli (ATCC 25922) -- --
Morganella morganii (E 10679/93) 100 100
Klebsiella pneumoniae (ATCC 27736) -- --
Pseudomonas aeruginosa (ATCC 27853) -- --
Serratia marcescens (E 8382/92) -- --
Pseudomonas maltophilia (D 141/92) 100 100
Bacillus subtilis (ATCC 6633) 100 --
Micrococcus luteus (DSM 348) -- --
Corynebacterium amycolatum (ATCC 494368) 20 100
Corynebact. pseudodiphtericum (RV 2/95) 20 100
Candida albicans (ATCC 90028) -- --
Candida krusei (ATCC 6258) -- --

MIC and MBC of stock solutions are indicated in [micro]g/ml.
--, no inhibition; n.e., not evaluable.

Table 3. Effects of (+)-usnic acid and carnosic acid on aerobic bacteria
and Candida species in the agar dilution test

 Carnosic
 (+)-Usnic acid acid
 MIC MBC MIC MBC

Staphylococcus aureus (ATCC 25923) 16 64 64 64
Staphylococcus aureus, Pen. res. (E 12431/98) 16 64 128 128
Staphylococcus aureus, MRSA (RV5/98) 16 64 -- --
Staphylococcus epidermidis (DSM 1798) 4 32 64 128
Staphylococcus lugdunensis (NG 3406/94) 16 64 128 128
Streptococcus pyogenes group A (E 12449/98) 8 64 64 128
Streptococcus agalactiae group B (DSM 2134) 16 64 64 n.e.
Enterococcus faecalis (ATCC 19212) 4 64 -- --
E. coli (ATCC 25922) -- -- -- --
Morganella morganii (E 10679/93) -- -- -- --
Klebsiella pneumoniae (ATCC 27736) -- -- -- --
Pseudomonas aeruginosa (ATCC 27853) -- -- -- --
Serratia marcescens (E 8382/92) -- -- -- --
Pseudomonas maltophilia (D 141/92) 64 128 -- --
Bacillus subtilis (ATCC 6633) 8 8 64 128
Micrococcus luteus (DSM 348) 8 32 -- --
Corynebacterium amycolatum (ATCC 494368) 8 32 64 128
Corynebacterium pseudodiphtericum (RV 2/95) 4 8 32 128
Candida albicans (ATCC 90028) -- -- -- --
Candida krusei (ATCC 6258) -- -- -- --

MIC and MBC of compounds are indicated in [micro]g/ml.
--, no inhibition: n.e., not evaluable.

Table 4. Effects of plant extracts on anaerobic bacteria and Malassezia
in the agar dilution test

 Usnea Salvia Rosmarinus
 MIC MBC MIC MBC MIC MBC

Clostridium perfringens 0.4 0.4 10 10 1 1
 (ATCC 13124)
Propionibactrium acnes 0.4 1 10 100 10 100
 (FR 024/12-10)
Fusobacterium nucleatum 0.4 1 -- -- 10 10
 (ATCC 25586)
Bacteroides fragilis (ATCC 25285) 0.4 0.4 10 20 10 10
Bacteroides vulgatus (ATCC 29327) 0.4 0.4 10 10 2 10
Prevotella intermedia (NCTC 9336) 0.4 1 20 20 10 10
Veillonella parvula (A 11-9) -- -- 100 -- 10 20
Porphyromonas gingivalis (W 83) 0.2 0.2 2 10 1 1
Peptococcus magnus (D385-8/94) 0.4 0.4 100 100 10 20
Malassezia furfur (HK 001) 10 10 n.e. n.e. n.e. n.e.

 Boswellia Harpagophytum Gentiana
 MIC MBC MIC MBC MIC MBC

Clostridium perfringens 1 10 -- -- -- --
 (ATCC 13124)
Propionibactrium acnes 1 10 100 -- -- --
 (FR 024/12-10)
Fusobacterium nucleatum -- -- 100 100 100 n.a.
 (ATCC 25586)
Bacteroides fragilis (ATCC 25285) -- -- -- -- -- --
Bacteroides vulgatus (ATCC 29327) 20 -- -- -- -- --
Prevotella intermedia (NCTC 9336) -- -- -- -- -- --
Veillonella parvula (A 11-9) -- -- -- -- -- --
Porphyromonas gingivalis (W 83) 1 20 100 100 100 n.a.
Peptococcus magnus (D385-8/94) 20 100 -- -- -- --
Malassezia furfur (HK 001) n.e. n.e. n.e. n.e. n.e. n.e.

 Bryonia Sarsaparilla Menyanthes
 MIC MBC MIC MBC MIC MBC

Clostridium perfringens 100 100 -- -- 20 100
 (ATCC 13124)
Propionibactrium acnes -- -- 100 100 100 100
 (FR 024/12-10)
Fusobacterium nucleatum 100 100 -- -- 100 100
 (ATCC 25586)
Bacteroides fragilis (ATCC 25285) -- -- -- -- -- --
Bacteroides vulgatus (ATCC 29327) -- -- -- -- -- --
Prevotella intermedia (NCTC 9336) -- -- -- -- -- --
Veillonella parvula (A 11-9) -- -- -- -- -- --
Porphyromonas gingivalis (W 83) 20 100 10 20 20 20
Peptococcus magnus (D385-8/94) -- -- 100 100 20 20
Malassezia furfur (HK 001) n.e. n.e. n.e. n.e. n.e. n.e.

MIC and MBC of stock solutions are indicated in [micro]g/ml.
--, no inhibition; n.e., not evaluable.

Table 5. Effects of (+)-usnic acid and carnosic acid on anaerobic
bacteria and Malassezia in the agar dilution test

 (+)-Usnic acid Carnosic acid
 MIC MBC MIC MBC

Clostridium perfringens (ATCC 13124) 2 8 16 16
Propionibacterium acnes (FR 024/12-10) 1 4 16 32
Fusobacterium nucleatum (ATCC 25586) 2 4 128 128
Bacteroides fragilis (ATCC 25285) 1 8 64 64
Bacteroides vulgatus (ATCC 29327) 2 4 32 32
Prevotella intermedia (NCTC 9336) 2 2 64 64
Veillonella parvula (A 11-9) 64 -- -- --
Porphyromonas gingivalis (W 83) 1 2 16 32
Peptococcus magnus (D 385-8/94) 4 16 32 64
Malassezia furfur (HK 001) 64 64 n.e. n.e.

MIC and MBC of compounds are indicated in [micro]g/ml.
--, no inhibition; n.e., not evaluated.
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Author:Weckesser, S.; Engel, K.; Simon-Haarhaus, B.; Wittmer, A.; Pelz, K.; Schempp, C.M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:4EUGE
Date:Aug 1, 2007
Words:5436
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