ANTIMICROBIAL ACTIVITY OF PLANT EXTRACTS FROM SERBIA

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JOURNAL No:
Volume 36, Issue 1-2
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1-6
KEYWORDS
Antimicrobial activity, plant extract, Rhamnus frangula L., Carum carvi L., Petroselinum crispum (Mill.) A.W. Nym. ex Hill, Mentha x piperita L.,  Betula pendula Roth.
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Šarić Ć. Ljubiša1*, Čabarkapa S. Ivana1, Beljkaš M. Bojana1, Mišan Č. Aleksandra1, Sakač B. Marijana1, Plavšić V. Dragana1
1 Institute for Food Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia

ABSTRACT

Abstract:

The aim of this work was to evaluate antimicrobial properties of ethanolic extracts of plants (Mentha x piperita L., Carum carvi L., Petroselinum crispum (Mill.) A.W. Nym. ex Hill, Betula pendula Roth. and  Rhamnus frangula L.) originated from Serbia. The antimicrobial activity was tested by paper disc diffusion method and by microdilution technique against six pathogenic bacteria (Bacillus Cereus ATCC 10876, Enterococcus Faecalis ATCC 14506, Salmonella Choleraesuis ATCC 10708, Staphylococcus Aureus ATCC 11632, Proteus Mirabilis ATCC 12453 and Escherichia Coli ATCC 10536).
B. cereus was the most susceptible to the extracts of M. piperita, R. frangula and B. pendula among tested microorganisms. Ethanolic extracts of Betula pendula Roth., Mentha x piperita L. and Rhamnus frangula L.  have been shown to possess the strongest antimicrobial activity against Bacillus Cereus, where the minimum inhibitory concentrations were  10 mg·ml-1 (Betula pendula Roth.) and 50 mg·ml-1 (Mentha x piperita L. and Rhamnus frangula L.)
The highest antibacterial potential was exibited by ethanolic extract of Mentha x piperita L., followed by Rhamnus frangula L. and Betula pendula Roth. Contrary to this, the extracts of Carum carvi L. and Petroselinum crispum (Mill.) A.W. Nym. ex Hill did not show significant antimicrobial effects towards investigated bacteria.

INTRODUCTION

The application and research for drugs and food and feed supplements obtained from plants have increased in recent years (Frankič, 2009). Spices and herbs and their constituents are generally recognized to be safe, either because of their traditional use without any documented detrimental impact or because of dedicated toxicological studies (Frankič, 2009). Being a rich source of secondary biomolecules which exhibit significant pharmacological effects, spices and herbs appeal to many consumers who question the safety of synthetic food additives (Craig, 1999).
Plant extracts and essential oils have been used as alternatives to antibiotic due to their antimicrobial activities and the favorable effect on the animal intestinal system (Al-Kassien, 2009). Spices and herbs can have a great influence on the function and reactivity of the immune system of the farm animals (Craig, 1999). The growth promoting active feed suplements improve stability of feed and auspicious impact the digestive micropopulation mostly through inhibition of pathogenic microorganisms growth. In consideration of promoted health status of intestinal tract, farm animals are less exposed to the toxins produced by different microorganisms (Frankič et al., 2009). Windisch et al. (2008) reported that spices and herbs have beneficially effect the stress resistance of the animals and amplify the absorption of essential nutrient. There is also, one other important advantage of using plant extract of herbs and spices or their essential oils instead synthetic drugs in feed: synthetic drugs residues in animal meat and eggs can cause health problems in people who consume them, esspecialy due to increasing resistance of pathogens present in the human body as a result of prolonged use of synthetic drugs (Barbour et al., 2010).
The objective of this work was to evaluate and compare the antibacterial effects of  ethanolic extracts of Mentha x piperita L.,  Carum carvi L., Petroselinum crispum (Mill.) A.W. Nym. ex Hill, Betula pendula Roth. and  Rhamnus frangula L. against six pathogenic bacteria.

MATERIAL AND METHODS

Test bacteria

Antibacterial investigations were carried out against E. Coli ATCC 10536, S. Choleraesuis ATCC 10708, Staph. Aureus ATCC 11632, P. Mirabilis ATCC 12453,  E. Faecalis ATCC 14506 and B. Cereus ATCC 10876.

Plant samples

Herbal drugs used in this study (Table 1)are the products of the Institute for Research of Medicinal Plants "Dr Josif Pančić" from Belgrade. Herbal drugs were in the form of powder with granulation of up to 3 mm.
Table 1.Plants used in experiments.

Botanical name

Sample

Drug

Mentha x piperita L.

Mint leaves

Menthae piperitae folium

Carum carvi L.

Caraway fruits

Carvi fructus

Petroselinum crispum

 

 

(Mill.) A.W. Nym. ex Hill

Parsley fruits

Petroselini fructus

Rhamnus frangula L.

Buckthorn bark

Frangulae cortex

Betula pendula

Birch leaves

Betula pendula folium

Preparation of plant extracts

Crude plant extracts were obtained by maceration with ethanol/water mixture (80:20, v/v), with the ratio of raw materials to ethanol solution of 1:10, for 24 h at room temperature and subsequently extracted in a ultrasonic bath at room temperature for 10 min. After filtration through a filter paper (Whatman, Grade 4 Chr, UK) and vacuum-evaporation of the solvent at 40 °C, yield of the extract was measured The combined extracts were stored at -4 °C until further use.

Testing of antibacterial susceptibility

The paper disc diffusion method was used to screen the antibacterial activity of plant extracts and performed by using Mueller Hinton agar (MHA) obtained by Torlak, Serbia. The experiment was carried out according to the National Committee for Clinical Laboratory Standards Guidelines (NCCLS, 1999). Test Bacteria were grown in Mueller Hinton broth (Torlak, Serbia). Bacterial suspension was diluted with sterile physiological solution to 108 cfu· ml-1 (turbidity = McFarland standard 0.5). One hundred microlitres of bacterial suspension were swabbed uniformly on surface
of MHA and the inoculums was allowed to dry for 5 minutes. Sterilized filter paper discs (Whatman, 6 mm in diameter) were placed on the surface of the MHA and soaked with 20 µl of a solution of each plant extracts (500 mg·ml-1, 250 mg·ml-1, 100 mg·ml-1 and 50 mg·ml-1) in 80% ethanol (Merck-Darmstadt, Germany). 80% ethanol was used as a negative control. The inoculated plates were stored at 4 °C for 2 h and then incubated at 37 °C for 24 h in the inverted position. The diameters (mm) of the inhibition zones were measured (diameter of paper disc, 6 mm is included). Studies were performed in triplicate and the results are expressed as means along with the standard deviaton (SD) of three parallel measurements.

Determination of minimum inhibitory concentration (MIC)

A broth microdilution susceptibility assay was performed using National Committee for Clinical Laboratory Standards Guidelines methods for the determination of the MIC (NCCLS, 2000). Stock solution of the dry plant extract was prepared in 80% ethanol and then serial dilutions were made with sterile physiological solution in a concentration range from 5.0 to 50.0 mg·ml-1. The 96-well plates were prepared by dispensing into each well 160 μl of Mueller Hinton broth (MHB), 20 μl of the plant extract and 20 μl of the inoculum. The inoculum of microorganisms was prepared using 24 h cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. The final volume in each well was 200 μl. A positive control (containing 20 μl inoculum and 180 μl MHB) and negative control (containing 20 μl of plant extract and 180 μl MHB without inoculum) were included on each microplate. The contents of the wells were mixed and the micro-plates were incubated at 37 °C for 24 h. The MIC was defined as the lowest concentration of the compounds to inhibit the growth of microorganisms. The experiment was carried out in triplicate.

Minimum bactericidal concentration (MBC) of plant extracts

MBC was determined by subculturing the 5 µl of test dilution from each well on to a nutrient agar (Torlak, Belgrade, Serbia) plates and incubating further at 37 °C for 24 h. The complete absence of growth at applied concentration was considered as the minimum bactericidal concentration.

RESULTS AND DISCUSSION

Paper disc difusion method

The effects of three different concentrations of five plant extracts on the growth of test microorganisms by the paper disc diffusion method are presented in Tables 2 – 7.
Table 2. Antibacterial properties of plant extractsagainst B. Cereus ATCC 10876  at three dilutions (inhibition zone diameter in mm; diameter of paper disc, 6 mm, included).

Plant extracts

Dilution of plant extracts (mg.ml-1)

500

250

100

50

Control
(80% ethanol)

M.  piperita

20.3 ± 0.6

20.0 ± 0.0

18.7 ± 1.15

18.7 ± 1.15

n.d.

R. frangula

20.3 ± 0.6

19.0 ± 1.0

18.3 ± 0.6

15.3 ± 0.6

n.d.

B.  pendula

23.5 ± 0.7

17.3 ± 2.1

17.0 ± 2.0

14.5 ± 0.7

n.d.

P.  crispum

n.d.

n.d.

n.d.

n.d.

n.d.

C.  carvi

n.d.

n.d.

n.d.

n.d.

n.d.

Note:  n.d. – not detected
Table 3.Antibacterial properties of plant extractsagainst E. Faecalis ATCC 14506  at three dilutions (inhibition zone diameter in mm; diameter of paper disc, 6 mm, included).

Plant extracts

Dilution of plant extracts (mg.ml-1)

500

250

100

50

Control
(80% ethanol)

M.  piperita

18.3 ± 0.6

18.3 ± 0.6

17.3 ± 1.52

17.0 ± 2.0

n.d.

R. frangula

18.3 ± 2.1

11.0 ± 1.0

10.3 ± 0.6

10.3 ± 0.6

n.d.

B.  pendula

15.0 ± 0.0

13.7 ± 1.5

12.3 ± 2.5

12.3 ± 1.5

n.d.

P.  crispum

n.d.

n.d.

n.d.

n.d.

n.d.

C.  carvi

n.d.

n.d.

n.d.

n.d.

n.d.

Note:  n.d. – not detected
Table 4.Antibacterial properties of plant extractsagainst S. Aureus ATCC 11632  at three dilutions (inhibition zone diameter in mm; diameter of paper disc, 6 mm, included).

Plant extracts

Dilution of plant extracts (mg.ml-1)

500

250

100

50

Control
(80% ethanol)

R. frangula

18.3 ± 2.1

15.7 ± 0.6

12.7 ± 0.6

12.3 ± 0.6

n.d.

B.  pendula

11.3 ± 1.2

10.7 ± 1.2

10.3 ± 1.2

9.3 ± 0.6

n.d.

P.  crispum

10.7 ± 0.6

9.7 ± 0.6

10.0 ± 1.0

n.d.

n.d.

M.  piperita

n.d.

n.d.

n.d.

n.d.

n.d.

C.  carvi

n.d.

n.d.

n.d.

n.d.

n.d.

Note:  n.d. – not detected
Table 5.Antibacterial properties of plant extractsagainst P. Mirabilis ATCC 12453  at three dilutions (inhibition zone diameter in mm; diameter of paper disc, 6 mm, included)

Plant extracts

Dilution of plant extracts (mg.ml-1)

500

250

100

50

Control
(80% ethanol)

R. frangula

15.3 ± 0.6

14.7 ± 0.6

14.0 ± 0.0

12.3 ± 0.6

n.d.

M.  piperita

14.0 ± 0.0

13.7 ± 0.6

11.3 ± 1.15

n.d.

n.d.

B.  pendula

12.3 ± 2.5

12.7 ± 1.2

10.3 ± 0.6

10.3 ± 0.6

n.d.

C.  carvi

15.0 ± 0.0

n.d.

n.d.

n.d.

n.d.

P.  crispum

10.7 ± 1.6

n.d.

n.d.

n.d.

n.d.

Note:  n.d. – not detected
Table 6. Antibacterial properties of plant extractsagainst S. Choleraesuis ATCC 10708  at three dilutions (inhibition zone diameter in mm; diameter of paper disc, 6 mm, included)

Plant extracts

Dilution of plant extracts (mg.ml-1)

500

250

100

50

Control
(80% ethanol)

B.  pendula

14.7 ± 0.6

12.7 ± 1.2

12.0 ± 2.0

10.7 ± 0.6

n.d.

M.  piperita

15.7 ± 1.15

16.3 ± 1.15

16.0 ± 1.0

13.3 ± 1.5

n.d.

R. frangula

15.3 ± 0.6

13.0 ± 1.0

11.7 ± 0.6

11.0 ± 1.0

n.d.

C.  carvi

n.d.

n.d.

n.d.

n.d.

n.d.

P.  crispum

n.d.

n.d.

n.d.

n.d.

n.d.

Note:  n.d. – not detected
Table 7. Antibacterial properties of plant extractsagainst E. Coli ATCC 10536  at three dilutions (inhibition zone diameter in mm; diameter of paper disc, 6 mm, included)

Plant extracts

Dilution of plant extracts (mg.ml-1)

500

250

100

50

Control
(80% ethanol)

M.  piperita

14.7 ± 0.6

14.0 ± 1.7

13.3 ± 1.5

13.3 ± 0.6

n.d.

R. frangula

14.0 ± 1.7

12.0 ± 1.0

11.3 ± 0.6

10.7 ± 0.6

n.d.

B.  pendula

11.7 ± 1.2

11.3 ± 1.5

11.3 ± 1.15

9.7 ± 0.6

n.d.

C.  carvi

n.d.

n.d.

n.d.

n.d.

n.d.

P.  crispum

n.d.

n.d.

n.d.

n.d.

n.d.

Note:  n.d. – not detected

According to the results of antimicrobial screening given in Tables 2-7, the strongest antimicrobial activities against tested microorganisms were obtained for extracts of M.  piperita and R.  frangula. The ethanolic extract of B. pendula has been shown to possess the strongest antimicrobial effect against B. Cereus. This plant extract exhibited moderate antimicrobial activity towards the others investigated bacteria, except in the case of E. Faecalis and S. Choleraesuis, where at the higest working concentration (500 mg·ml-1) it has been shown to possess the intermediate effect. The extracts of the C. carvi and P. crispum at the concentration of 500 mg·ml-1 exhibited intermediate and moderate antibacterial effects, respectively.  C. carvi and P. crispum did not show anti-bacterial activity towards tested bacteria at lower concentrations (250 mg·ml-1, 100 mg·ml-1 and 50 mg·ml-1). B. cereus was the most susceptible to the extracts of M.  piperita, R.  frangula and B. pendula among tested micro-organisms.
MIC of the extract of B. pendula varied from 10 mg·ml-1 (B. cereus) to 50 mg·ml-1 (other tested bacteria), while the MBC was from 25 mg·ml-1 (B. cereus) to 50 mg·ml-1 (other tested bacteria). MIC of the extracts of M. piperita and R. frangula were 50 mg.ml-1 (B. cereus) and 50 mg·ml-1 (other tested bacteria). MBC of the extracts of M. piperita and R. frangula were 50 mg·ml-1 for other investigated strains. Obtained results of the investigations of the antimicrobial activity of mint EOs are in accordance with literature data (Deans & Baratta, 1998,  Bupesh et al., 2007, Sharafi et al., 2010). This results point out that EOs of mint from Serbia could be useful in controling the development of tested bacteria (B. Cereus, E. Faecalis) in different food and feed. The biological activity of B. pendula against Bac. Coagulans reported by Lindberg et al., 2004. Izhaki (2002) noticed the antibacterial effect of R. frangula. According to Ulate-Rodriquez, 1997, ethanolic extracts of dried parsley can decrease the number of viable micropopulations of E. Coli, but in this investigation ethanolic extract of parsley did not showed antibacterial activity. There is no literature data of the application of B. pendula and R. frangula in food and feed.

ACKNOWLEDGMENTS

The paper is part of the investigation realized in the Project (TR 20068) supported by the Ministry of Science and Technological Development, Republic of Serbia.



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