Scholarly article on topic 'Antimicrobial properties of Teucrium polium against some clinical pathogens'

Antimicrobial properties of Teucrium polium against some clinical pathogens Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Esmaeil Darabpour, Hossein Motamedi, Seyyed Mansour Seyyed Nejad

Abstract Objective To explore the antibacterial effect of the alcoholic extracts of aerial parts of Teucrium polium, native in Iran on some pathogenic bacteria. Methods Antibacterial activity of ethanolic extract (50 to 400 mg/mL) and methanolic extract (400 and 600 mg/mL) was evaluated by disc diffusion method. Results The ethanolic extract results showed that Bacillus anthracis was the most sensitive species, while Escherichia coli and Proteus mirabilis were more resistant than others. In the case of the methanolic extract, Bordetella bronchiseptica was the most sensitive and Proteus mirabilis and Arcanobacterium pyogenes were the most resistant species. The hydroalcoholic extract of Teucrium polium had a relatively satisfactory effect on Salmonella typhi. The minimal inhibitory concentration (MIC) of Staphylococcus aureus and Salmonella typhi was 40 mg/mL and Bordetella bronchiseptica and Bacillus anthracis was 10 mg/mL. The minimal bactericidal concentration (MBC) against Bacillus anthracis was 10 mg/mL while against other species were not found (>200 mg/mL). The methanolic extract had also synergistic effect with methicillin, vancomycin against Staphylococcus aureus and with novobiocin against Salmonella typhi. Conclusions These results suggest that this plant contains relatively good antibacterial activity and it can be used as a source of antiseptic compounds for medicinal uses.

Academic research paper on topic "Antimicrobial properties of Teucrium polium against some clinical pathogens"

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Asian Pacificjournal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

Document heading

Antimicrobial properties of Teucriumpolium against some clinical pathogens

Esmaeil Darabpour , Hossein Motamedi, Seyyed Mansour Seyyed Nejad

Department of Biology, Faculty of Science, Shahid Chamran University, Ahvaz, Iran

ARTICLE INFO

Article history:

Received 7 December 2009

Received in revised form 30 December2009

Accepted 10 January 2010

Available online 20 February 2010

Keywords: Teucriumpolium Antibiotic Herbal medicine

ABSTRACT

Objective: To explore the antibacterial effect of the alcoholic extracts of aerial parts of Teucrium polium, native in Iran on some pathogenic bacteria. Methods: Antibacterial activity of ethanolic extract (50 to 400 mg/mL) and methanolic extract (400 and 600 mg/mL) was evaluated by disc diffusion method. Results: The ethanolic extract results showed that Bacillus anthracis was the most sensitive species, while Escherichia coli and Proteus mirabilis were more resistant than others. In the case of the methanolic extract, Bordetella bronchiseptica was the most sensitive and Proteus mirabilis and Arcanobacterium pyogenes were the most resistant species. The hydroalcoholic extract of Teucriumpolium had a relatively satisfactory effect on Salmonella typhi. The minimal inhibitory concentration (MIC) of Staphylococcus aureus and Salmonella typhi was 40 mg/mL and Bordetella bronchiseptica and Bacillus anthracis was 10 mg/mL. The minimal bactericidal concentration (MBC) against Bacillus anthracis was 10 mg/mL while against other species were not found (>200 mg/mL). The methanolic extract had also synergistic effect with methicillin, vancomycin against Staphylococcus aureus and with novobiocin against Salmonella typhi. Conclusions: These results suggest that this plant contains relatively good antibacterial activity and it can be used as a source of antiseptic compounds for medicinal uses.

1. Introduction

In recent years, due to alarming increase in the rate of infections with antibiotic resistant microorganisms and side effects of some synthetic antibiotics, there is increasing interest in medicinal plants as a natural alternate to synthetic drugs. Plants are known to produce a variety of compounds to protect themselves against a variety of their own pathogens and therefore can serve as antimicrobial substances[i>2], Teucrium polium (T. polium) locally named "Kalpooreh" has been known as an important traditional medicinal plant in Khuzestan, South West of Iran. T.polium is a member of the Lamiaceae family, a grass plant, durable, with 10-30 cm in height and callous white exterior that ordinarily have dispersal in rocky and sandy area of Europe zones, North of Africa and South West of Asia like IranP]. Medical reputation of this plant was noticed in traditional medicine by Socrates and JalinousP]. Researchers showed that this plant have anti-diabetic, anti-spasmodic, antiinflammatory, analgesic and anti-oxidant effects[4-io].

^Corresponding author: Esmaeil Darabpour, Department of Biology, Faculty of Science, Shahid Chamran University, Ahvaz, Iran Tel: 0098)611-3331045 Fax: 0098)611-3331045 E-mail: Ismal dar@yahoo.com

The antimicrobial effects of T. polium have been rarely studied by research. The aim of this study was to explore antibacterial properties of ethanolic and methanolic extracts of T.polium against some ofclinical pathogens.

2. Materials and methods

2.1. Plant collection and identification

The plants used in this study were collected in April, 2008 from hills around of Behbahan (south east of Khuzestan, Iran). The taxonomic identification of this plant was done by Herbarium in Department of Agricultural College, Shahid Chamran University.

2.2. Preparation ofplant extract

The aerial parts of T. polium were dried at room temperature for ten days and then ground to a fine powder. One gram of powder was extracted by using 10 mL of alcohol (ethanol or methanol)-distilled water solution (8 : 2 v/v), centrifugation (3 000 rpm) for 15 mins and collecting of the supernatants. This process was repeated for three times. Solvents were then removed by evaporation[iU2].

2.3.Bacterial strains

Twelve bacterial species were used in this study. The gram-positive species were Bacillus pumilus (B. pumilus), Bacillus anthracis (B. anthracis), Bacillus licheniformis (B. licheniformis), Bacillus cereus (B.cereus), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis) and Gram negative species bacteria were Yersinia enterocolitica (Y. enterocolitica), Escherichia coli (E. coli), Salmonella typhi (S. typhi), Bordetella bronchiseptica (B. bronchiseptica), Proteus mirabilis (P. mirabilis), Arcanobacteriumpyogenes (A. pyogenes). These species were originally isolated from patients and identified by standard biochemical reactions.

2.4. Antibacterial susceptibility testing

The isolates were grown in trypone soya broth (TSB) medium at 37 °C for 22 hrs. Final inoculums bacterial numbers were adjusted to 10s cfu mL_1 with reference to the Mc Farland turbidometry[i3,i4]. One milliliter of the test tubes suspension was added to each plate containing Muller Hinton Agar (MHA, Merck) by sterile cotton swab and allowed to remain in contact for 1 min. Four concentrations of ethanolic extract (50, 100, 200 and 400 mg/mL) and two concentrations of methanolic extract (400, 600 mg/mL) were prepared. The sterile filter paper discs (6 mm diameter) were saturated by addition of 50 |xL portions of different concentrations of both extracts[i5,i6]. Then discs were placed on cultured plates. The plates were incubated at 37 °C for 24 hrs and the inhibition zone around each disc was measured. As positive controls, discs containing different concentrations of eight antibiotics including nafcillin 1 meg, colistin lmcg, tetracycline 30 meg, novobiocin 30 meg, penicillin 10 meg, vancomycin 30 meg, methicillin 5 meg and oxacillin lmcg were used. All these synthetic antibiotics were produced by Difco.

2.5. MIC and MBC Determination

Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of ethanolic and methanolic extracts of T. polium were determined against four species including, S. aureus for ethanolic extract and B. bronchiseptica, B. anthracis and S. typhi for methanolic extract (somewhat important and more sensitive bacteria). MIC was determined by macro broth dilution assay method[i7,i8]. In the tube dilution assay, standard bacterial suspension was added to tubes containing 1 mL Muller Hinton Broth (MHB, Merck) and different concentrations of extracts (5, 10, 20, 40, 80, 160, 200 mg/mL). The tubes were incubated at 37 °C for 24 hrs. The first tube in the above series with no sign of visible growth was reported as the MIC. MBC was determined by culture done by one loop of the culture medium from those tubes (in MIC assay) showing no apparent growth on MHA and subsequent incubation at 37 °C for 24 hrs. The least concentration that showed no colony formation on agar was assumed as MBC for the extracts.

2.6. Study ofthe synergistic effect

To determine the synergistic effect of the T. polium with synthetic antibiotics against S. aureus, S. Typhi and B. anthracis, methanolic extract at 400 mg/mL was added to the discs containing antibiotics including novobiocin 30 meg, methicillin 5 meg and vancomycin 30 mcg[i9].

3. Results

Table 1 showed the results of antibacterial assays of ethanolic and methanolic extracts of T. polium against various bacterial species. The results showed that antibacterial effects of both extracts of T. polium against B. Pumilus, B. anthracis, B. licheniformis, B. cereus, S. aureus, S. epidermidis, Y. enterocolitica, E. coli, S. typhi, B. bronchiseptica at lower concentrations were decreased. However, antibacterial effect of T. polium ethanolic extract agaist A. pyogenes was increased with decreased concentrations. Also, antibacterial effect of ethanolic extracts against P. mirabilis was observed only atlO mg/mL. These results suggest that ethanolic extract was effective against all of bacterial species even at one concentration.

A. pyogenes and P. mirabilis were resistant to methanolic extract even at highest concentration. However, B. anthracis and B. bronchiseptica were most susceptible microorganism to ethanolic and methanolic extracts, respectively. E. coli and P. mirabilis were the species most resistant to both extracts. Furthermore, results showed anti-staphylococcal effect of ethanolic extract of T. polium was better than methanolic extracts. MIC and MBC results showed that MIC of ethanolic extract against S. aureus was 40 mg/mL and for methanolic extract against B. bronchiseptica was 10 mg/mL, but MBC against both bacteria were not found. Also MIC value for methanolic extract against S. typhi and B. anthracis was 40 mg/mL and 10 mg/mL, respectively. MBC value against S. typhi was not found (>200 mg/mL) while against

B. anthracis was 10 mg/mL. The study of synergistic effect of T.polium with antibiotic discs showed that the methanolic extract of this plant in combination with novobiocin against S. typhi and in combination with methicillin and vancomycin against S. aureus have synergism while the combination of this extract with methicillin against B. anthracis didn't have change (Table 2).

4. Discussion

Nowadays, increased resistance of pathogenic microorganisms to synthetic antibiotics has caused a concern in universal health and pharmaceutical industries which are looking for new way for production of novel antibacterial products. Medicinal plants are good choices, because these natural resources have fewer side effects, with low-cost and satisfactory effect on broad spectrum of antibiotic resistant microorganisms. In many parts of the world, the extracts of medicinal plants are used for their antibacterial,

Table 1

Antibacterial activity of ethanolic and methanolic extracts from the aerial part of T.polium against some pathogenic bacteria.

Bacterial species Different concentrations ofextract (mg/mL) Ethanolic Methanolic Antibiotic

50 100 200 400 400 600 VA TE P NF NB CL MT OX

B. anthracis 10 11 14 18 20 22 21 25 R R - - 23 R

B.pumilus 8 9 10 10 12 14 18 14 R R - - 16 R

B. licheniformis R R 7 8 10 13 16 29 R R - - 16 R

B. cereus R R 9 10 11 15 - - - - - - R R

S. aureus 8 9 11 12 8 9 22 R R R - - R R

S. epidermidis 7 7 10 13 9 10 - - - - - - R R

A. Pyogenes 11 11 9 R R R - - - - - - - -

E. coli R R R 9 R 9 - - R 26 10 R R

S. typhi 9 10 10 13 14 16 - - R 12 R R R

P. mirabilis R 11 R R R R - - R 30 12 R R

B.bronehiseptica 9 9 10 11 22 26 - - R 24 R R R

Y.enterocolitica 7 7 9 9 10 11 - - - - - - -

R: Resistant, -: Not used;

YA: Vancomycin 30 meg; TE: Tetracycline 30 meg; P: Penicillin 10 meg; NF: Nafcillin 1 meg; NB: Novobiocin 30 meg; CL: Colistin 10 meg; ME: Methicillin 5 meg; OX: Oxacillin 1 meg.

Table 2

Results of the study of synergistic effect of T.polium methanolic extract with antibiotic discs.

Result_Db (mm)_Da (mm)_Antibiotic Disc_Bacterial species

(Syn) 16 0 Methicillin S. aureus (Syn) 27 22 Vancomycin S. aureus (Syn) 18 12 Novobiocin S. typhi (-)_23_23_Methicillin_B. anthracis_

Da: Antibiotic, Db: Antibiotic+Extract, Syn: Synergism, (-): Not enhanced.

antifungal and antiviral activitiesPO], T. polium is one of the most important medicinal plants that extensively used in traditional medicine in Iran. On the basis of this study, ethanolic and methanolic extracts of T. polium aerial parts are effective against both gram-positive and gram-negative bacteria. Ethanolic extract at 200 and 400 mg/mL and methanolic extract at 400 and 600 mg/mL doses showed high antibacterial activity, so that these concentrations had growth inhibition on 6 out of 7 gram positive bacteria. Furthermore, ethanolic extract at 400 mg/mL and methanolic extract in 600 mg/mL extract were growth inhibitory on 4 out of 5 gram-negative bacteria. In general, considering diameter of inhibition zones showed that hydroalcoholic extract of T. polium was more effective against grampositive bacteria than gram-negative bacteria. T.polium was effective only atlO mg/mL concentration of ethanolic extract against P. mirabilis. This observed resistance could be due to cell membrane permeability or other genetic factors. The diameter of inhibition zones around the more active extracts in particular methanolic extract were comparable with standard antibiotics used as positive controls. All of the gram-positive and gram-negative bacteria were resistant to nafcillin and oxacillin. More of the bacterial species were resistant to methicillin and penicillin. Antibacterial effect of methanolic extract against B. bronchiseptica was more comparable to standards. DNA hybridization results suggest that B. bronchiseptica, B. pertussis and B. parapertussis are

closely related (72%-94%), hence it is presumable that methanolic extract of T. polium may be effective against B. pertussis and B. parapertussis that cause pertussisPi], The MBC for B. anthracis was found at 10 mg/mL and for the other species, S. aureus, S. typhi and B. bronchiseptica, didn't show bactericidal activity even at 200 mg/mL (MBC >200 mg/mL). This result may be due to high bacteriostatic effect of T. polium extracts; we can name these extracts as bacteriostatic agents as they can inhibit bacterial growth but generally do not kill them[i7]. Exceptionally the MIC and MBC values for B. anthracis were the same. It is reported that for bactericidal antimicrobials the MIC and MBC are often near or aquiline values, so, it can be said that this extract have bactericidal activity against this species[22]. The methanolic extract had significant synergistic effect in combination with methicillin against S. aureus compare to methicilin alone. This could be new hope for treatment of methicillin resistant S. aureus (MRSA) infections and restricting resistant strains distribution. The synergism between this extract and methicilin isn't against B. anthracis that may be due to the same target site for these compounds in this species. The compounds found in this plant includes alkaloids, triterpenes, flavonoids, glycosides, sterols, tannin and saponin[8,23-25]. Flavonoides are one of the broadest groups of phenolic compounds[26]. Phenolic compounds with capacity to chelate transition metals, reduce reactivity of metal ion by forming an inert metal-ligand chelation

of transition metals (such as iron) and thus could reduce their bioavailability for bacteria^]. We speculate that plant tannine have antimicrobial activity because non-live central wood of many trees has a high concentration of Tannines that assist in preventing bacterial and fungal decay[26]. On the other hand, in the essential oil of this plant, there are compounds including a -pinene, P -myrcene, cadinol, myrtenal, limonene and presumably a -pinene could play an important role in antibacterial activity of T. poliumizs]. By studying the diameter of inhibition zones of ethanolic and methanolic extracts of T. polium against bacteria, in particular against B. bronchiseptica, we can truly realize differential ability of these alcohols in extraction of plant bioactive constituents. Hence, more researches with choice of a suitable solvent indicate antibacterial activity of T. polium against a specific pathogen. With additional research, we study antibacterial activity of T. polium with more accuracy by purification of bioactive constituents from different parts of this plant.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

The authors wish to thank the vice chancellor for research of Shahid Chamran University for the research grant.

References

[lJNimri LF, Meqdam MM, Alkofahi A. Antimicrobial activity of Jordanian medicinal plants. JPharm Biol 1999; 37(3): 169-201.

[2]Grayer RJ, Harborne JB. A survey of antifungal compounds from higher plants. JPhytochcm 1994; 37(1): 19-42.

[3]Zargari A. Medicinal plants. 6th ed. Tehran: Tehran University Publication; 1994.

[4]Shahraki MR, Arab MR, Mirimokaddam E, Jey-Palan M. The effect of Teucrium polium (Calpoureh) on liver function, serum lipids and Glucose in diabetic male rats. Ir Biomed J 2006; 11(1): 65-8.

[5]Esmaeili MA, Yazdanparast R. Hypoglycemic effect of Teucrium polium: Studies with rat pancreatic islets. J Ethnopharmacol 2004; 95(1): 27-30.

[6]Panovska TK, Kulevanova S, Gjorgoski I, Bogdanova M, Petrushevska G. Hepatoprotective effect of the ethyl acetate extract of Teucrium polium L. against carbontetrachloride-induced hepatic injury in rats. Acta Pharm 2007; 57(2): 241-8.

[7]Tariq M, Ageel AM, Al-Siad MS. Anti-inflammatory activiy of T. polium. Int J Tissue React 1989; 11(4): 185-8.

[8]Heidari MR, Karaminezhad Ranjbar, Davvand E, Jalali S. Evaluation of the analgesic effect of Teucrium polium extract in mice.

Daru 2001; 24(4): 277-85.

[9]Panovska TK, Kulevanova S, Stefova M. In vitro antioxidant activity of some Teucrium species (lamiaceae). Acta Pharm 2005; 55(2): 207-

[10]Hasani P, Yasa N, Vosough-Ghanbari S, Mohammadirad A, Dehghan G, Abdollahi M. In vivo antioxidant potential of Teucrium polium, as compared to a -tocopherol. Acta Pharm 2007; 57(11): 123-9.

[llJSeyyednejad M, Ebrahimzadeh H, Talaie A. Carbohydrate content in olive zard c.v. and alternate bearing pattern. J Int Sugar 2001; 103(1226): 84-7.

[12]Moazedi AA, Mirzaie-Damabi N, Seyyednejad SM, Zadkarami MR, Amirzargar A. Spasmolytic effect of Petroselinum crispum (parsley) on rat's ileum at different calcium chloride concentrations.

Pak J Biol Sci 2007; 10(22): 4036-42.

[13]Burt AS, Reinders RD. Antibacterial activity of selected plant essential oils against Esherichia coli 0157: H7. lett Applid Microbial 2003; 36(3):162-7.

[14]Zakaria Z, Sreenivasan S, Mohammad M. Antimicrobial activity of Piper ribesoides root extract against Staphylococcus aureus. JABS 2007; 1(3): 87-90.

[15]Cermelli C, Fabio A, Fabio G, Quaglio P. Effect of essential oil on respiratory bacteria and viruses. CurrMicrobial 2008; 56(1): 89-92.

[16]Hsieh PC, Mau JL, Huang SH. Antimicrobial effect of various combinations ofplant extracts. FoodMicrobial 2001; 18(l):35-43.

[17]Forbes BA, Suhm DF, Wissfeld AS. Baily & Scott's diagnostic microbiology. 10th ed. USA: Mobsy, Inc.; 1998.

[18]National Committee for Clinical Laboratory Standards. Methods for dilution in antimicrobial susceptibility test, Approved Standard,M2-M5. National Committee for Clinical Laboratory Standards, Villanova, PA; 1993.

[19]Mahboobi M, Shahcheraghi F, Feizabadi MM. Bactericidal effects of essential oils from clove, lavender and geranium on multi-drug resistant isolates of Pseudomonas aeruginosa. Inform J Biol 2006; 4(2): 137-40.

[20]Hassawi D, Kharma A. Antimicrobial activity of some medical plants against Candida albicans. JBiol Sci 2006; 6(1): 109-14.

[21]Brooks GF, Butel J, Morse SA. Jawetz, Melnick & Adelberg's medical microbiology. 23th ed. USA: Mc Graw-Hill Companies Inc. ; 2004.

[22]Reuben KD, Abdulrahman FI, Akan JC, Usman H, Sodipo OA, Egwu GO. Phytochemical screening and in vitro antimicrobial investigation of the methanolic extract of Croton zambesicus Muell ARG stem bark. Euro JSci Res 2008; 23(l):134-40.

[23]Wassel GM, Ahemed SS. Chemical composition of the wild egyption plant Teucriumpolium. JPharmzie 1974; 29(8): 540-1.

[24]Vokou D, Bessiere JM. Volatile constituents of Teucrium polium. JNat Prod 1985; 48(3): 498-9.

[25]Hassan MM, Muhtadi FJ, Al-Badr AA. GLC mass spectrometry of Teucriumpolium oil. JPharm Sci 1979; 68(6): 800-1.

[26]Taiz L, Zeiger E. Secondary methabolites and plant defence. In: Plantphysiology. 3rd ed. USA: Sinauer Associates; 2002.

[27]Wong PYY, Kitts DD. Studies on the dual antioxidant and antibacterial properties of parsley (Petroselinum crispum) and cilantro (Coriandrum sativum) extracts. Food Chemistry 2006; 97(3):505-15.

[28]Kabouche Z, Boutaghane N, Laggoune S, Kabouche A, Ait-Kaki Z, Benlabed K. Comparative antibacterial activity of five lamiaceae essential oils from Algeria. IntJAromatherapy 2005; 15(3): 129-33.