Antibacterial Activity of Aqueous and Methanol Extracts of Rumex alpinus. and Rumex caucasicus.

Abstract

This study was designed to examine the in vitro. antibacterial activities of Rumex alpinus. L. and Rumex caucasicus. L. (Polygonacea), both known as “kivircik labada” or “evelik” in Turkey, and used to treat constipation, diarrhea, and eczema. Aerial parts of Rumex alpinus. and Rumex caucasicus., extracted with water and methanol, were screened for in vitro. activity by using disk diffusion assays against 121 strains belonging to 52 bacterial species. The minimum inhibitory concentrations (MICs) of single compounds were determined by the microbroth dilution method. The aqueous extract of each species had no antibacterial effect against the test microorganisms, whereas the methanol extract of both species had an inhibitory effect on the growth of microorganisms. Rumex caucasicus. was found to be more effective than Rumex alpinus. on the tested microorganisms. This is the first report of the antibacterial potency of Rumex alpinus. and Rumex caucasicus. on a range of foodborne bacteria. The results provide evidence that Rumex alpinus. and Rumex caucasicus. might be potential sources of new antibacterial agents.

Keywords:

• Antimicrobial activity
• aqueous extract
• methanol extract
• Rumex alpinus
• Rumex caucasicus

Introduction

Food poisoning is still a threat for both consumers and the food industry despite the use of preserving processes. Meanwhile consumers are concerned about the safety of foods containing preservatives. Therefore, there has been a growing interest in new and effective techniques to reduce cases of foodborne illnesses (Alzoreky & Nakahara, 2003).

Many compounds present in plants have been reported to have antimicrobial, antioxidant, antifungal, and antibacterial properties. For centuries, indigenous plants have been used in herbal medicine for curing various diseases (Beuchat & Golden, 1989; Sokmen et al., 1999; Alvarez-Castellanos et al., 2001; Tepe et al., 2005). Wild plants are widely used as medicines by populations that inhabit the rural parts of eastern Anatolia of Turkey. The flowers, stems, leaves, and roots of these plants are usually prepared and consumed to treat a variety of ailments that include pain, edema, digestive problems, arthritis, colds and flu, fevers, and irritability (Baytop, 1996).

The genus Rumex.(Polygonacea) is widespread in the flora of Turkey, represented by 22 species (Cullen, 1972). The most common species of the genus in Turkey are R. patientia. L., R. crispus. L., R. acetosa. L. R. caucasicus. RECH., and R. alpinus. L. (Baytop, 1996). Rumex alpinus. and Rumex caucasicus. are perennial plants distributed in middle and eastern Anatolia at an altitude of 1000–3000 m (Cullen, 1972). The genus has been used extensively in traditional medicine in Turkey to treat a variety of disorders such as constipation, diarrhea, and eczema (Ulukanli et al., 2005). The genus also possesses laxative, diuretic, antipyretic, wound healing, and anti-inflammatory properties (Baytop, 1996; Suleyman et al., 1999). In the eastern part of Turkey, young leaves of Rumex. species are used as a vegetable or for meat stuffed with leaves and also in the preparation of herby cheese as a preservative and aroma source. Along with Rumex. spp., 25 kinds of herbs alone or in a mixture can be added to cheese (Ozturk et al., 2000).

Though the antimicrobial activity of some Rumex. species has been previously reported (Getie et al., 2003; Ulukanli et al., 2005), the activity of Rumex alpinus. and Rumex caucasicus. against food-borne bacteria has not been studied so far. Hence, the current study is aimed at evaluating the antibacterial effect of the aqueous and methanol extracts of this herbal plant on foodborne bacteria and comparing the efficiency against conventional antibiotic drug.

Materials and Methods

Plant material

Plants were collected from northeastern of Turkey and were identified by senior taxonomist, Avni Ozturk, at the Department of Botany, Yuzuncu Yil University, Van-Turkey, and a voucher specimen was deposited in the herbarium of the Horticulture Department of the Agricultural Faculty of Ataturk University, Erzurum, Turkey. The dried and powdered plant material (400 g) was extracted successively in a Soxhlet with methanol (MeOH) for 72 h at a temperature not exceeding the boiling point of the solvent (Lin et al., 1999). The extracts were filtered using Whatman filter paper (no. 1) and then concentrated in vacuo. at 40°C using a rotary evaporator. The residues were stored in freezer at − 80°C until further tests.

Bacterial strains

A total of 121 bacterial strains belonging to 52 bacterial species (listed in Table 1) were used in this study. The bacteria, maintained on nutrient agar (Merck, Darmstadt, Germany), were supplied by the Microbiology Laboratory of the Agricultural Faculty of Ataturk University, Erzurum, Turkey. The food-associated bacteria were selected because they are frequently reported in foods. The identity of the bacteria used in this study was confirmed by the Microbial Identification System in Biotechnology Application and Research Center at Ataturk University.

Table 1. Antibacterial activity of Rumex alpinus. and Rumex caucasicus. methanol and aqueous extracts against bacteria

		Inhibition zone diameter around test disk									Inhibition zone diameter around test disk
		Methanol extracts (300 µg/disk)		Aqueous extract		Negative control (MeOH)					Methanol extracts (300 µg/disk)		Aqueous extract		Negative control (MeOH)
Bacterial species	Number of strains	RA	RC	RA	RC	RA	RC	Standard antibiotic disk	Bacterial species	Number of strains	RA	RC	RA	RC	RA	RC	Standard antibiotic disk
Acidovorax facilis.	1	–	–	–	–	–	–	28 mm (OFX)	Enterobacter hormaechei.	1	12 mm	10 mm	–	–	–	–	22 (OFX)
Arthrobacter agilis.	1	–	–	–	–	–	–	31 mm (SCF)	Enterobacter intermedius.	2	8 mm	8 mm	–	–	–	–	16 (SCF)
Arthrobacter atrocyaneus.	1	8 mm	–	–	–	–	–	15 mm (OFX)	Enterobacter sakazakii.	2	–	–	–	–	–	–	21 (NET)
Arthrobacter ilicis.	1	8 mm	–	–	–	–	–	20 mm (OFX)	Erwinia carotovora.	3	–	9 mm	–	–	–	–	20 (NET)
Arthrobacter protophormiae.	5	–	–	–	–	–	–	21 mm (NET)	Erwinia chrysanthemi.	1	7 mm	10 mm	–	–	–	–	17 (SCF)
Bacillus cereus.	5	–	–	–	–	–	–	21 mm (NET)	Exiguobacterium acetylicum.	1	8 mm	11 mm	–	–	–	–	20 (OFX)
Bacillus dipsauri.	3	–	–	–	–	–	–	26 mm (OFX)	Flavimonas oryzihabitans.	1	10 mm	8 mm	–	–	–	–	30 (OFX)
Bacillus flexus.	2	8 mm	12 mm	–	–	–	–	27 mm (SCF)	Kocuria kristinae.	2	9 mm	–	–	–	–	–	24 (NET)
Bacillus lentimorbus.	3	–	–	–	–	–	–	30 mm (OFX)	Kocuria rosea.	1	–	–	–	–	–	–	15 (OFX)
Bacillus lichemiformis.	4	–	–	–	–	–	–	29 mm (OFX)	Micrococcus luteus.	4	–	–	–	–	–	–	28 (OFX)
Bacillus marinus.	3	–	–	–	–	–	–	14 mm (OFX)	Micrococcus lylae.	3	–	–	–	–	–	–	30 (OFX)
Bacillus megaterium.	6	–	–	–	–	–	–	26 mm (OFX)	Moraxella catarrhalis.	1	–	10 mm	–	–	–	–	18 (OFX)
Bacillus psychrosaccharolyticus.	1	8 mm	8 mm	–	–	–	–	15 mm (OFX)	Neisseria subflava.	3	–	–	–	–	–	–	24 (OFX)
Bacillus pumilus.	2	–	–	–	–	–	–	24 mm (SCF)	Paenibacillus apiarius.	2	9 mm	8 mm	–	–	–	–	30 (OFX)
Bacillus sphaericus.	4	–	8 mm	–	–	–	–	21 mm (OFX)	Paenibacillus macerans.	2	10 mm	11 mm	–	–	–	–	30 (OFX)
Bacillus. sp.	2	15 mm	16 mm	–	–	–	–	20 mm (SCF)	Paenibacillus polymyxa.	2	–	–	–	–	–	–	10 (OFX)
Bacillus subtilis.	1	12 mm	–	–	–	–	–	29 mm (OFX)	Pantoea agglomerans.	1	7 mm	8 mm	–	–	–	–	30 (OFX)
Brevibacillus agri.	5	–	–	–	–	–	–	27 mm (SCF)	Proteus vulgaris.	4	–	12 mm	–	–	–	–	20 (OFX)
Brevibacillus brevis.	3	–	–	–	–	–	–	32 mm (NET)	Pseudomonas putida.	2	7 mm	10 mm	–	–	–	–	17 (OFX)
Brevibacterium linens.	2	–	–	–	–	–	–	22 mm (SCF)	Pseudomonas syringae.	1	11 mm	–	–	–	–	–	15 (OFX)
Chryseomonas luteola.	2	11 mm	8 mm	–	–	–	–	30 mm (OFX)	Pychrobacter–immobilis.	2	–	9 mm	–	–	–		20 (OFX)
Citrobacter amalonaticus.	1	10 mm	9 mm	–	–	–	–	23 mm (NET)	Salmonella typhimurium.	3	–	–	–	–	–	–	28 (OFX)
Corynebacterium ammoniagenes.	1	13 mm	11 mm	–	–	–	–	20 mm (OFX)	Serratia liquefaciens.	1	8 mm	8 mm	–	–	–	–	30 (OFX)
Corynebacterium cystitidis.	4	–	–	–	–	–	–	18 mm (OFX)	Shigella dysenteriae.	4	–	–	–	–	–	–	21 (NET)
Corynebacterium flavescens.	1	11 mm	11 mm	–	–	–	–	24 mm (OFX)	Staphylococcus cohnii.	2	–	9 mm	–	–	–	–	12 (OFX)
Enterococcus faecalis.	4	–	–	–	–	–	–	10 mm (SCF)	Xanthomonas arb. corylina.	2	9 mm	10 mm	–	–	–	–	22 (OFX)
Total 52 bacterial species	121 strains

OFX, ofloxacin (5 µg/disk); SCF, sulbactam (30 µg) + cefoperazona (75 µg) (105 µg/disk); and NET, netilmicin (30 µg/disk) were used as positive reference standard antibiotic disks (Oxoid).

RA, Rumex alpinus.; RC, Rumex caucasicus..

Antibacterial activity test

The antibacterial activity of the extracts was carried out by disk diffusion test (Kim et al., 1995) using 100 µL of suspension containing 10⁸ CFU/mL of bacteria spread on nutrient agar (NA) medium. Sterile 6-mm-diameter filter paper disks were impregnated with 300 µg of sterile test material and placed onto nutrient agar. Negative controls were prepared using the same solvents employed to dissolve the plant extracts. Ofloxacin (5 µg/disk), sulbactam (30 µg) + cefoperazona (75 µg) (105 µg/disk), and/or netilmicin (30 µg/disk) were used as positive reference standards to determine the sensitivity of one strain in each bacterial species tested. The inoculated plates with food-associated bacteria were incubated at 27°C for 24 h. The diameter of the clear zone around the disk was measured and expressed in millimeters as its antibacterial activity. Five disks per plate and three plates were used, and each test was run in triplicate (Djipa et al., 2000).

Microdilution assays

The minimum inhibitory concentration (MIC) values were also studied for the bacteria that were determined as sensitive to the extracts in disk diffusion assay. The inocula of bacteria were prepared from 12-h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. Rumex alpinus. and Rumex caucasicus. extracts dissolved in 0.5% dimethylsulfoxide (DMSO) were first diluted to the highest concentration (500 µg/mL) to be tested, and then serial two-fold dilutions were made in a concentration range from 7.80 to 500 µg/mL in 10 mL sterile test tubes containing nutrient broth. MIC values of Rumex alpinus. and Rumex caucasicus. extracts against bacterial strains were determined based on a microwell dilution method (Swanson et al., 1992). The 96-well plates were prepared by dispensing into each well 95 µL of nutrient broth and 5 µL of the inoculum. A 100 µL aliquot from Rumex alpinus. and Rumex caucasicus. extracts initially prepared at the concentration of 500 µg/mL was added into the first wells. Then, 100 µL from their serial dilutions was transferred into six consecutive wells. The last well containing 195 µL of nutrient broth without compound and 5 µL of the inoculum on each strip was used as negative control. The final volume in each well was 200 µL. Maxipime (Bristol-Myers Squibb, New York, USA) at the concentration range of 500–7.8 µg/mL was prepared in nutrient broth and used as standard drug for positive control. Contents of each well were mixed on a plate shaker at 300 rpm for 20 s and then incubated at appropriate temperatures for 24 h. Microbial growth was determined by absorbance at 600 nm using the ELx 800 universal microplate reader (Biotek Instrument Inc., Highland Park, VT, USA) and confirmed by plating 5-µL samples from clear wells on nutrient agar medium. The extract tested in this study was screened two-times against each organism. The MIC of each extract was taken as the lowest concentration that showed no growth (Ozturk & Ercisli, 2006).

Results and Discussion

The in vitro. antibacterial activities of Rumex alpinus. and Rumex caucasicus. methanol and aqueous extracts against examined microorganisms in the current study and their potency were quantitatively assessed by the presence or absence of inhibition zones and zone diameters (Table 1) and MIC values (Table 2). According to our results, the aqueous extracts of both Rumex alpinus. and Rumex caucasicus. had no antimicrobial activity against any of the bacterial species tested in the current study. However, methanol extracts of Rumex alpinus. and Rumex caucasicus. showed inhibition effects on the growth of 23 of 52 and 26 of 52 bacterial species, respectively (Table 1). These results confirmed the evidence reported in previous studies that methanol is the better solvent for more consistent extraction of antimicrobial substances from medical plants compared with water (Ahmad et al., 1998; Lin et al., 1999). When compared with each other, it was found that Rumex alpinus. was more effective than Rumex caucasicus. methanol extracts in terms of inhibition of a number of bacteria–species. The most pronounced activity with inhibition zones of more than 10 mm was shown by the methanol extracts of Rumex caucasicus. against Bacillus flexus., Bacillus. sp., Corynebacterium ammoniagenes., Corynebacterium flavescens., Exiguobacterium acetylicum., Paenibacillus macerans., and Proteus vulgaris., and the methanol extract of Rumex alpinus. against Bacillus. sp., Bacillus subtilis., Chryseomonas luteola., Corynebacterium ammoniagenes., Corynebacterium flavescens., Enterobacter hormaechei., and Pseudomonas syringae syringae. (Table 1).

Table 2. The MIC values (µg/mL) of Rumex alpinus. and Rumex caucasicus. methanol and aqueous extracts against bacteria tested in a microdilution assay

Bacterial species	Rumex alpinus.	Rumex caucasicus.	Standard drug Maxipime (µg/mL)
Arthrobacter atrocyaneus.	500	–	62.50
Arthrobacter ilicis.	125	62.50	7.81
Bacillus flexus.	125	125	62.50
Bacillus psychrosaccharolyticus.	31.25	31.25	7.81
Bacillus sphaericus.	–	250	31.25
Bacillus. sp.	250	250	7.81
Bacillus subtilis.	500	–	7.81
Chryseomonas luteola.	250	250	31.25–62.50
Citrobacter amalonaticus.	500	500	7.81
Corynebacterium ammoniagenes.	500	–	7.81
Corynebacterium flavescens.	250	500	7.81
Enterobacter hormaechei.	250	250	7.81
Enterobacter intermedius.	62.50–250	62.50–250	31.25
Erwinia carotovora.	–	500	31.25
Erwinia chrysanthemi.	250	500	15.60
Exiguobacterium acetylicum.	250	250	7.81
Flavimonas oryzihabitans.	500	500	15.60
Kocuria kristinae.	500	15.60	15.60
Moraxella catarrhalis.	–	500	7.81
Paenibacillus apiarius.	250	250	62.50
Paenibacillus macerans.	500	500	250–500
Pantoea agglomerans.	250	62.50	7.81
Proteus vulgaris.	–	250	62.50
Pseudomonas putida.	500	500	250–500
Pseudomonas syringae syringae.	500	–	250
Psychrobacter immobilis.	–	500	15.60
Serratia liquefaciens.	250	250	62.50
Staphylococcus cohni cohn.	–	250	7.81
Xanthomonas arboricola corylina.	500	500	250–500

In previous studies, Gram-negative bacteria were not susceptible to plant extracts when compared with Gram-positive bacteria (Brantner et al., 1996; Ojala et al., 2000). However, in our study, plant extracts did not show any selective activity against Gram-negative and Gram-positive bacteria.

Maximum inhibition zones (MIC) values for microorganisms sensitive to the methanol extract of Rumex alpinus. and Rumex caucasicus. were in the range 7–16 mm, 31.25–500 µg/mL, respectively (Tables 1 and 2).

The results obtained in the course of the current study are in agreement to a certain degree with the traditional uses of Rumex alpinus. and Rumex caucasicus. (Baytop, 1996). As far as our literature survey could ascertain, several studies were carried out with antibacterial activity of some Rumex. species such as Rumex crispus. (Yildirim et al., 2001; Ulukanli et al., 2005), Rumex nervosus., and Rumex abyssinicus. (Getie et al., 2003), and species demonstrated significant inhibitory effects against most microorganisms under test.

According to a literature search, nothing is known about the antibacterial properties and uses of Rumex alpinus. and Rumex caucasicus. against a number of food-borne bacteria. Therefore, this may be the first report providing data that the extracts of Rumex alpinus. and Rumex caucasicus. evaluated against a wide range of bacteria possess potential antibacterial activities. Based on these results, it is possible to conclude that aerial parts of Rumex alpinus. and Rumex caucasicus. have a stronger and broader spectrum of antibacterial activity against a number of foodborne bacteria, and an extract of these species can be used as an antimicrobial agent for preservative in food products.

The ability of edible plant extracts to inhibit pathogenic bacteria in food matrices and the impact of plant materials on organoleptic properties of food require further studies.