antimicrobial activity and total content of …...antimicrobial activity and total content of...

Central European Journal of Biology

* E-mail: [email protected]

Communication

Received 11 March 2010; Accepted 23 June 2010

Keywords: Rheum spp. • R. undulatum L. • Antibacterial activity • Antistaphylococcal activity

1Department of Pharmaceutical Microbiology, Medical University, 20-093 Lublin, Poland

2Department of Pharmaceutical Botany, Medical University, 20-093 Lublin, Poland

Urszula Kosikowska1,*, Helena D. Smolarz2, Anna Malm1

Antimicrobial activity and total content of polyphenols of Rheum L. species growing in Poland

Abstract: In the crude ethanol extracts obtained from the rhizome and roots of Rheum palmatum L., Rheum undulatum L. and Rheum rhaponticum L. growing in Poland concentration of polyphenols ranged from 46.11 to 76.45 mg/g. Concentration of tannins ranged from 7.07% to 8.67%, while anthracene derivatives and anthraquinones varied by species - R. palmatum measured 36.3 and 34 mg/g, while R. undulatum or R. rhaponticum did not exceed 20.4 and 18.1 or 19.8 mg/g and 16.6 mg/g, respectively. Using a broth microdilution method it was found that all of the Rheum spp. extracts were more active against reference strains of Gram-positive bacteria (Staphylococcus spp.) than against those of Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis). The strongest inhibitory effect against Staphylococcus spp. was exerted by R. undulatum extract with MIC = 125-250 μg/mL.The moderate in vitro antibacterial activity of R. undulatum suggests that this plant, often used in the European cuisine to improve flavour, may be also important and useful as an alternative or auxiliary medicine remedy in the treatment of uncomplicated superficial infections caused especially by clinically important staphylococci, potentially pathogenic S. aureus or opportunistic S. epidermidis.

1. IntroductionNowadays there is a need to find naturally occurring substances with antimicrobial activity as an alternative to available antibiotics or chemiotherapeutics due to several serious problems such as growing drug resistance of bacteria or undesirable side effects of drugs. Recently, only a few antibacterial agents have been developed by pharmaceutical or biotechnology companies.

Plants have been shown to be a potential source for multiple antimicrobial agents, as they produce a wide variety of secondary compounds as natural protection against microbial attack [1-6]. The use of plants or plant preparations to treat infections has been a common practice in virtually all cultures for a long time [6,7]. Several plant species have been a subject of scientific investigation for many years in order to learn about the biological activity of their compounds, allowing in the

future development of alternative, natural antimicrobial compounds [1,2,6,8,9].

The Rheum L. genus, belonging to the Polygonaceae family, is represented by about 60 plant species commonly named rhubarb that are endemic or widely distributed over Europe and Asia [10,11]. Plants from Rheum spp., wildly growing or cultivated, are used for therapeutic, cooking or decorative purposes [12].R. palmatum L. (Chinese rhubarb), R. tangaticum Maxim and R. officinale L. have been used mainly in folk Chinese or Tibetan medicine since ancient times [13] due to the therapeutics properties of their dried rhizome and roots, such as obstructive action in the treatment of diarrhea, laxative action in the treatment of constipation, cleansing action upon the gut, and removing debris and then astringing it with antiseptic properties. Rhubarb has been also found to be beneficial for liver, spleen and gallbladder problems and the latest investigation shows also its estrogenic, anti-mitotic, antimicrobial

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Cent. Eur. J. Biol. • 5(6) • 2010 • 814-820DOI: 10.2478/s11535-010-0067-4

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U. Kosikowska et al.

and hypotensive properties [10,12-16]. Rheum species are still used today in herbal medicine in Europe [11,12]. Moreover, the garden rhubarb R. undulatum L. is cultivated usually in various regions of Europe for culinary purposes, and its roots and rhizome are used in folk medicine as a mild laxative, especially suitable for children [12,14,15]. Various species of the genus Rheum contain polyphenol components, such as anthraquinones, anthocyanins, flavonols and stilbenes, which may have bioactive properties [10,12,17-19]. The medicinal use of this species is mainly related to the presence of several secondary metabolites such as anthracenes (emodin, fiscion, chrysophanol, aloe-emodin and rhein derivatives), stilbenes (resveratrol, rhapontigenin, piceatannol and its derivatives), naphtalenes and chromones, in the underground parts of the plants [16-26]. According to the literature [27], several bioactive anthraquinones of rhubarb posses promising anti-cancer properties with a smaller or no cytotoxic effect in several normal cells.

Some Rheum species, such as R. palmatum or R. officinale, have been reported to possess potential antibacterial activity against a wide spectrum of bacteria, both Gram-positive and Gram-negative microorganisms [10,15,28-31]. The aim of our study was to screen the effectiveness of the crude ethanol extracts obtained from the root parts of R. palmatum, R. undulatum and R. rhaponticum, growing in Poland for the growth inhibition of potentially pathogenic or opportunistic microorganisms. The in vitro activity of the extracts against the reference strains of bacteria was assessed using the broth microdilution method. The total content of polyphenols, anthracene derivatives, anthraquinones and tannins in the extracts was also estimated.

2. Experimental Procedures2.1 Plant material The plant material used in the research was three-year-old rhizome and roots (in this study named roots) of the following Rheum species cultivated in Poland: Rheum palmatum L., obtained from the company Herbapol S.A. (Lublin, Poland), and Rheum rhaponticum L. or Rheum undulatum L. collected in autumn in the Botanical Garden of University of Maria Curie-Skłodowska in Lublin in 2007. The plant material was dried for 15 days at room temperature in the absence of light in a well-ventilated place and then stored at room temperature in air-tight glass jar protected from light. The voucher specimens (collection number 451-453) have been deposited in the Department of Pharmaceutical Botany, Medical University in Lublin.

2.2 Preparation of ethanol extractsCut up (0.5 cm pieces), air-dried and finally powdered roots were extracted twice (15 min for each extraction) with ethanol and next with 50% ethanol in ultrasonic bath at room temperature. The combined extracts were concentrated under reduced pressure at 40oC and the residue was dried. Samples of the dry plant extracts were dissolved in dimethyl sulphoxide (DMSO) and were kept under refrigerated conditions.

2.3 Antimicrobial testingThe crude ethanol extracts obtained from the root of R. palmatum, R. undulatum and R. rhaponticum were screened for in vitro antimicrobial activity using the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) [32] against a panel of reference strains of aerobic bacteria including Gram-positive (Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228) or Gram-negative (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453) microorganisms. All these microorganisms came from American Type Culture Collection (ATCC), routinely used for the evaluation of antimicrobials. All bacterial cultures were first grown on nutrient agar plates at 35oC for 18 - 24 h. Microbial suspensions were prepared in sterile saline (0.85% NaCl) with an optical density of McFarland standard 0.5 - 150 x 106 CFU (Colony Forming Units)/mL and then diluted to a 1:10 solution. The 96-well microplates with Mueller-Hinton broth containing from 31.25 to 500 μg/mL of the tested extracts were used. All stock solutions of the tested extracts were dissolved in DMSO. 20 µL of each bacterial suspension was added per each well; total volume was 200 µL. It was found that DMSO at the final concentration used had no influence on the growth of the tested microorganisms. After incubation (35oC for 18 h), optical density (OD600) was measured for bacterial cultures in the presence and absence (control culture) of the tested extracts. The MIC (minimal inhibitory concentration), defined as the lowest concentration of the test samples preventing visible growth of bacteria, was estimated. The MIC values for control antimicrobial agents such as antibiotics (gentamicin, cefuroxim) and rhein, an anthracene derivative, were also estimated.

2.4 Determination of total phenol contents Total phenol content was determined by the colorimetric procedure adapted from Singleton and Rossi [33]. A 1 g portion of powdered plant sample was twice extracted with 70% methanol (35 mL) for 1 h in boiling bath water. The obtained extracts were

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Antimicrobial activity and total content of polyphenols of Rheum L. speciesgrowing in Poland

transferred to a 100 mL volumetric flash, which was filled up to its volume with extraction solvent. 0.1 mL of each extract solution was added to 4 mL distilled water and 1 mL of Folin-Ciocalteu reagent. After 3 min, 4 mL of Na2CO3 (70 g/L) were added. The solution was mixed and left to stand for 30 min. The absorption was measured at 660 nm (UV-VIS spectrophotometer, Carl Zeis, Jena). The concentration of polyphenols in the samples was estimated on the basis of the standard curve of gallic acid. Linearity of the detection response for the standard compounds was confirmed between 10-120 µg/mL. Results are expressed as mg gallic acid equivalents (GAE) per 1 g of dry material. All samples were analysed in triplicate.

2.5 Colorimetric estimation of anthracene derivatives and anthraquinones

The chemical assay is a classical one based on the Bornträger’s reaction of anthraquinones. The procedure was carried out in accordance with Polish Pharmacopoeia IV [34]. In the first step, the total glycoside forms of anthracene in the dried and powdered plant samples were hydrolyzed with acetic acid and extracted with diethyl ether. These extracts were alkalized with NaOH solution, then freed and liberated by hydrolysis anthraquinones were determined colorimetrically at 530 nm. In the next step, the reduced forms of anthracene from the same alkaline extracts were transformed into the oxidative forms by heating in a water bath. The content of anthracene derivatives was determinated colorometrically at 530 nm. Isticin (1,8-dihydroxyanthraquinone) was used as a standard in both cases. Linearity of the detection response for the standard compound was confirmed between 5-25 µg mL. All samples were analysed in triplicate. The results was calculated as mg anthraquinones per 1 g of plant dry weight.

2.6 Determination of tannins The content of tannins was determined by the titrimerically–weight method described in Polish Pharmacopoeia VI [35]. The tannins were precipitated from water extract and examined as insoluble copper salts.

2.7 StatisticsThe GraphPad InStad 3 software was used for the statistical analyses. We evaluated the relationship between the total content of the various derivatives in Rheum spp. extracts and their activity against reference bacterial strains with Spearman rank correlation coefficient (r). Values of P<0.05 were considered statistically significant.

3. Results The Rheum root ethanol extracts were analyzed for the total content of polyphenols, anthracene derivatives, anthraquinones and tannins as the main biologically active components of species from Polygonaceae family [12-16,18-21]. As shown in Table 1, the total content of polyphenols ranged from 46.11 to 76.45 mg/g in the tested dry material. Among the extracts, R. undulatum and R. palmatum extracts contained the highest amount of phenolics; their low levels were found in R. rhaponticum extract. The content of tannins was similar and ranged from 7.07% to 8.67%. The distinguishing mark of R. palmatum extract was the content of anthracene derivatives - 36.3 mg/g of anthracene derivatives including 34 mg/g of anthraquinones. The total content of anthracene derivatives and anthraquinones in R. undulatum or R. rhaponticum did not exceed 20.4 and 18.1 or 19.8 mg/g and 16.6 mg/g, respectively.

According to our results presented in Figure 1, R. undulatum extract showed the strongest inhibitory effect on the reference strains of Staphylococcus spp. (S. aureus ATCC 25923, S. aureus ATCC 6538, S. epidermidis ATCC 12228) with MIC values ranging from 125 to 250 μg/mL; at lower extract concentrations, about 20 to 95% inhibition of the bacterial growth was observed. R. palmatum extract exerted partial inhibitory effect on the growth of the reference strains of S. aureus at a rate ranging from about 40 to 95% inhibition; total growth inhibition was not observed at the maximal extract concentration used (MIC >500 μg/mL). However, in the case of S. epidermidis ATCC 12228, MIC was 250 μg/mL; below this concentration about

Species Total polyphenols mg/g

Total anthracene derivatives

mg/g

Total anthraquinones mg/g

Tanins (%)

Rheum undulatum 76.45 ± 0.81 20.4 ± 0.60 18.1 ± 0.80 7.60 ± 0.35

Rheum palmatum 75.34 ± 0.77 36.3 ± 0.80 34.0 ± 1.2 8.67 ± 0.36

Rheum rhaponticum 46.11 ± 0.81 19.8 ± 0.60 16.6 ± 0.50 7.07 ± 0.25

Table 1. The content of polyphenols, anthracene derivatives, anthraquinones and tannins in the crude ethanol extracts from the roots of Rheum species.

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Figure 1. The influence of ethanol extracts obtained from the roots of Rheum undulatum (a), Rheum palmatum (b) and Rheum rhaponticum(c) on the growth of the bacterial reference strains. Sa 25923, Staphylococcus aureus ATCC 25923 (OD600=0.374), Sa 6538, S. aureus ATCC 6538 (OD600=0.456), Se 12228, S. epidermidis ATCC 12228 (OD600=0.566), Ec 3521, Escherichia coli ATCC 3521 (OD600=0.616),Kp 13883, Klebsiella pneumoniae ATCC 13883 (OD600=0.702), Pm 12453, Proteus mirabilis ATCC 12453 (OD600=0.681).

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40 to 94% of growth inhibition was noted, depending on the extract concentration. Similarly, R. rhaponticum extract also showed partial inhibitory effect on the growth of the reference strains of Staphylococcus spp., reaching about 15 to 95% inhibition depending on the extract concentration; no total growth inhibition was observed at the maximal extract concentration (MIC >500 μg mL). For comparison, MIC values for reference strains of Staphylococcus spp. were 0.12 to 0.49 μg/mL for gentamicin and 0.49 to 0.98 μg/mL for cefuroxime. Rhein, an anthraquinone derivative present in Rheum species [24], inhibited the growth of staphylococcal strains with MIC=15.62-62.5 mg/mL.

As shown in Figure 1, the growth of the reference strains of the Enterobacteriaceae family (E. coli ATCC 25922, K. pneumoniae ATCC 13883, P. mirabilis ATCC 12453) was not inhibited totally even at the maximal concentration of the extracts of Rheum spp. (MIC >500 μg/mL); only partial growth inhibition at a rate ranging from about 10 to 90% was noted. Besides, slight stimulation of bacterial growth to about 120% was observed at lower concentrations of R. undulatum and R. rhaponticum extracts.

Our results indicate that under comparable conditions, all of the Rheum spp. extracts were more active against Gram-positive bacteria, represented by Staphylococcus spp. than against Gram-negative bacteria of the Enterobacteriaceae family. There was no significant relationship between antibacterial activity and the total content of polyphenols, anthracene derivatives, anthraquinones and tannins in the ethanol extracts of the roots of Rheum species (Spearman r was from -0.5293 to -0.2126; P from 0.1080 to 0.5846).

4. DiscussionMedicinal plants, which have been used in virtually all cultures for centuries, may be regarded as a reservoir of biologically active compounds, including those possessing antimicrobial activity [1-7]. According to our results, the crude ethanol extracts obtained from the roots of Rheum palmatum, Rheum undulatum and Rheum rhaponticum growing in Poland inhibited the growth of staphylococcal reference strains at concentrations ranging from 125 to >500 mg/mL; the

R. undulatum extract exerted the strongest inhibitory effect against staphylococci with MICs ranging from 125 to 250 μg/mL, suggesting its moderate in vitro bioactivity. These MIC values are comparable to those in the literature concerning the in vitro effect of Rheum spp. on the growth of other Gram-positive bacteria, e.g. Streptococcus mutans and S. sobrinus [9]. It is worth noting that the reported MIC value of plant extracts is often in the range of 100 – 1000 mg/mL [30,36].

Considering literature data on antimicrobial activity of the Rheum spp. [28,31], it should be noted that the main constituents of R. palmatum, responsible for inhibitory action against staphylococci appear to be anthraquinone derivatives, including aloe-emodin, emodin and rhein; antistaphylococcal activity may be due to the inhibition of respiratory chain [25,30]. Rhein seems to be the main compound of rhubarb responsible for antibacterial activity [30]. As shown by other authors [31], rhein has been reported to act against clinical isolates of S. aureus with MICs ranging from 4 to 16 μg/mLand the MIC value of S. aureus ATCC 25923 equal to 16 μg/mL. The MIC values of rhein presented in this paper for the three reference staphylococci species were similar or somewhat higher (15.62-62.5 μg/mL).

It is well-known that staphylococci are the most common etiologic agents of bacterial skin diseases, usually endogenous in origin [37,38]. The main reservoir of staphylococci for these diseases is the normal microflora of skin [39-41]. Some predisposing conditions (e.g. immunosuppression, genetic predisposition, systemic or chronic diseases, antibacterial therapy, hospitalization), create an “opportunity” for the bacteria without or with limited pathogenic capacity (referred to as “opportunists”) to infect and to cause disease [42-46].Bacterial skin infections may often cause serious clinical problems; eradication of infecting bacteria requires prolonged antibiotic therapy or even surgical intervention [39,40,41,43]. Our data suggest that R. undulatum, similarly to R. palmatum [36], may be also useful as an alternative or auxiliary medicine remedy due to its antimicrobial effect. This could be useful especially in the treatment of uncomplicated skin or soft tissue infections or those located within oral cavity, caused by staphylococci such as potentially pathogenic S. aureus or opportunistic S. epidermidis, and by other Gram-positive bacteria.

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