Antibacterial Activity of Aqueous and Methanol Extracts of Althaea officinalis. and Althaea cannabina. from Turkey

Abstract

The crude methanol and aqueous extracts of aerial parts of Althaea officinalis. L. and Althaea cannabina. L. (Malvaceae) have been investigated for their antibacterial activity by using disk diffusion assays against 137 strains belonging to 52 bacteria species. The minimum inhibitory concentrations (MIC) of single compounds were determined by the microbroth dilution method. The results showed that the aqueous extracts from aerial parts of both species had no antibacterial effects against the test microorganisms, whereas the methanol extracts of both species tested exhibited significant antibacterial activity, especially against Acidovorax facilis., Bacillus. sp., Enterobacter hormachei., and Kocuria rosea.. In comparison with each other, Althaea officinalis. was the most effective against the bacteria used in this study. This is the first report of the antibacterial potency of Althaea officinalis. and Althaea cannabina. on a range of food-borne bacteria. The results provide evidence that Althaea officinalis. and Althaea cannabina. might indeed be potential sources of new antibacterial agents.

Keywords:

• Althaea cannabina
• Althaea officinalis
• antibacterial activity
• plant extracts

Introduction

Many plant families represent a reservoir of effective chemotherapeutics and can provide valuable sources of natural antimicrobials (Balandrin et al., 1985; Satish et al., 1999). Thus, for many thousands of years, plant extracts have been used for a wide variety of purposes (Jones, 1996). In particular, the antimicrobial activity of plant extracts has formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine, and natural therapies (Hammer et al., 1999).

Recently, the acceptance of traditional medicine as an alternative form for health care and the development of microbial resistance to the available antibiotics have led authors to investigate the antimicrobial activity of medicinal plants (Ahmad et al., 1998; Djipa et al., 2000; Scrinivasan et al., 2001).

Althaea. (Malvaceae) is an annual or perennial herb, known vernacularly as “marshmallows”. They have a broad distribution in the western Paleoarctic, being found in North Africa, Mediterranean Europe, France, Britain, the Balkans, Southwest and Central Asia, and southern Russia, but are absent from more northerly latitudes. There are currently five species and one interspecific hybrid placed in the genus (Cullen, 1972).

The flora of Turkey describes four Althaea. species, namely, Althaea officinalis. L., Althaea armeniaca. Ten., Althaea cannabina. L., and Althaea hirsute. L. Althaea officinalis. is a perennial herb growing up to 2 m and widespread in ditches, marshes, and field edges. Althaea cannabina. is a tall perennial regularly exceeding 1 m in height and distributed in open places in Middle and East Anatolia at an altitude of up to 1500 m (Cullen, 1972).

The genus has been used for centuries as both a food and a medicine. Its botanical name comes from the Greek word altho., which means “to cure.” The Romans, Chinese, Egyptians, and Syrians used Althaea. as a source of food, and the Arabs made poultices from its leaves and applied this to the skin to reduce inflammation. The mucilage, or gummy secretion, in the leaves and particularly the root is helpful for soothing sore throats, chapped skin, and minor wounds (Grieve, 1984).

Althaea. is a very useful household medicinal herb in Turkey. The local people who live in the eastern part of Turkey have used it for the treatment of common and minor ailments (e.g., asthma, bronchitis, common cold/sore throat, cough, ulcerative colitis, stomach ulcers, weight loss aid and wound healing) and also as an antitussive, demulcent, diuretic, emollient, laxative, and odontalgic (Baytop, 1996). In this region, it is also used to prepare herby cheese as preservative and aroma source. In addition to Althaea. spp., 25 kinds of herbs can be used to make herby cheese. From these herbs (e.g., Allium. spp., Thymus. spp., Falcaria., etc.), single or mixtures of some herbs can be added to cheese (Ozturk et al., 2000).

Though antimicrobial activity of some Althaea. species has been previously reported in limited studies using a few microorganisms, the activity of Althaea officinalis. and Althaea cannabina. against a wide range of food-borne bacteria has not been studied so far. Hence, the current study is aimed at evaluating the antibacterial effect of crude aqueous and methanol extracts of this herbal plant on food-borne bacteria and comparing the efficiency against conventional antibiotic drugs.

Materials and Methods

Plant material and preparation of extracts

The plant (aerial parts) used for the current study was collected locally in the Van region of Turkey. Plant materials were further identified by senior taxonomist, Avni Ozturk, at the Department of Botany, Yuzuncu Yil University, Van, Turkey, and a voucher specimen was deposited at the herbarium of the Horticulture Department of the Agricultural Faculty of Ataturk University, Erzurum, Turkey. The dried and powdered plant materials (400 g) were extracted successively in a Soxhlet with methanol (MeOH) at 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 obtained were stored in a freezer at −80°C until further tests.

Bacterial strains

A total of 121 bacterial strains belonging to 52 bacterial species (which are listed in Table 1) were used in this study. The bacteria, maintained on Nutrient Agar (UA; 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. 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 Althaea officinalis. and Althaea cannabina. methanol and aqueous extracts against bacteria.

		Inhibition zone diameter around test disk									Inhibition zone diameteraround 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	AC	AO	AC	AO	AC	AO	Standard antibiotic disk	Bacterial species	Number of strains	AC	AO	AC	AO	AC	AO	Standard antibiotic disk
Acidovorax facilis.	1	10 mm	11 mm	–	–	–	–	28 mm (OFX)	Enterobacter hormaechei.	1	11 mm	9 mm	–	–	–	–	22 (OFX)
Arthrobacter agilis.	1	–	8 mm	–	–	–	–	31 mm (SCF)	Enterobacter intermedius.	2	–	–	–	–	–	–	16 (SCF)
Arthrobacter atrocyaneus.	4	–	–	–	–	–	–	15 mm (OFX)	Enterobacter sakazakii.	2	–	–	–	–	–	–	21 (NET)
Arthrobacter ilicis.	4	–	–	–	–	–	–	20 mm (OFX)	Erwinia carotovora.	3	8 mm	–	–	–	–	–	20 (NET)
Arthrobacter protophormiae.	5	–	–	–	–	–	–	21 mm (NET)	Erwinia chrysanthemi.	4	–	–	–	–	–	–	17 (SCF)
Bacillus cereus.	5	–	–	–	–	–	–	21 mm (NET)	Exiguobacterium acetylicum.	1	9 mm	8 mm	–	–	–	–	20 (OFX)
Bacillus dipsauri.	3	–	–	–	–	–	–	26 mm (OFX)	Flavimonas oryzihabitans.	3	–	–	–	–	–	–	30 (OFX)
Bacillus flexus.	2	–	–	–	–	–	–	30 mm (SCF)	Kocuria kristinae.	2	9 mm	–	–	–	–	–	15 (OFX)
Bacillus lentimorbus.	3	–	–	–	–	–	–	33 mm (OFX)	Kocuria rosea.	1	11 mm	11 mm	–	–	–	–	15 (OFX)
Bacillus lichemiformis.	4	–	–	–	–	–	–	29 mm (OFX)	Micrococcus luteus.	4	–	8 mm	–	–	–	–	15 (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.	4	–	–	–	–	–	–	30 (OFX)
Bacillus sphaericus.	4	–	–	–	–	–	–	21 mm (OFX)	Paenibacillus macerans.	2	–	–	–	–	–	–	30 (OFX)
Bacillus. spp.	2	10 mm	12 mm	–	–	–	–	20 mm (SCF)	Paenibacillus polymyxa.	2	–	–	–	–	–	–	10 (OFX)
Bacillus subtilis.	1	–	11 mm	–	–	–	–	29 mm (OFX)	Pantoea agglomerans.	1	9 mm	–	–	–	–	–	30 (OFX)
Brevibacillus agri.	2	–	–	–	–	–	–	27 mm (OFX)	Proteus vulgaris.	4	–	–	–	–	–	–	20 (OFX)
Brevibacillus brevis.	3	–	–	–	–	–	–	32 mm (NET)	Pseudomonas putida.	2	8 mm	7 mm	–	–	–	–	17 (OFX)
Brevibacterium linens.	2	–	–	–	–	–	–	22 mm (SCF)	Pseudomonas syringae.	3	–	7 mm	–	–	–	–	15 (OFX)
Chryseomonas luteola.	2	8 mm	10 mm	–	–	–	–	30 mm (OFX)	Pychrobacter immobilis.	2	9 mm	9 mm	–	–	–	–	20 (OFX)
Citrobacter amalonaticus.	3	–	–	–	–	–	–	23 mm (NET)	Salmonella typhimurium.	3	–	–	–	–	––	–	28 (OFX)
Corynebacterium ammoniagenes.	1	–	12 mm	–	–	–	–	20 mm (OFX)	Serratia liquefaciens.	3	–	–	–	–	–	–	30 (OFX)
Corynebacterium cystitidis.	4	–	–	–	–	–	–	18 mm (OFX)	Shigella dysenteriae.	4	–	–	–	–	–	–	21 (NET)
Corynebacterium flavescens.	1	–	7 mm	–	–	–	–	24 mm (OFX)	Staphylococcus cohnii.	2	9 mm	8 mm	–	–	–	–	12 (OFX)
Enterococcus faecalis.	4	–	–	–	–	–	–	10 mm (SCF)	Xanthomonas arb. corylina.	2	–	–	–	–	–	–	22 (OFX)
Total 52 bacterial species	137 strains

OFX, ofloxacin (5 µg/disk); SCF, sulbactam (30 µg) + cefoperazona (75 µg) (105 µg/disk); NET, netilmicin (30 µg/disk) were used as positive reference standards antibiotic disks (Oxoid). AC: A. cannabina.; AO: A. officinalis..

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 NA medium. Sterile 6-mm-diameter filter paper disks were impregnated with 300 µg of the sterile test material and placed onto NA. 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 minimal inhibitory concentration (MIC) values were also studied for the bacteria that were determined as sensitive to the extracts in the disk diffusion assay. The inocula of bacteria were prepared from 12-h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. Althaea officinalis. and Althaea cannabina. extracts dissolved in 0.5% dimethylsulfoxide (DMSO) were first diluted to the highest concentration (500 µg/mL) to be tested, and then serial twofold 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 radish 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 align of from Althaea officinalis. and Althaea cannabina. 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 a 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, Vermont, USA) and confirmed by plating 5-µL samples from clear wells on NA 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

Table 1 shows antibacterial activities of methanol and aqueous extracts of Althaea officinalis. and Althaea cannabina. against 121 strains of 52 microorganism species examined in the current study. Their antibacterial potency was quantitatively assessed by the presence or absence of inhibition zones and zone diameters (Table 1) and MIC values (Table 2).

Table 2.. The MIC values (µg/mL) of Althaea cannabina. and Althaea officinalis. methanol and aqueous extracts against bacteria tested in the microdilution assay.

	MIC (µg/mL)
Bacterial species	Althaea cannabina.	Althaea officinalis.	Standard drug Maxipime
Acidivorax facilis.	500	250	250
Arthrobacter agilis.	–	500	7.81
Bacillus psychrosaccharolyticus.	62.50	31.25	7.81
Bacillus. sp.	62.50	62.50	7.81
Bacillus subtilis.	–	125	7.81
Chryseomonas luteola.	250–500	125–250	31.25–62.50
Corynebacterium ammoniagenes.	–	250	7.81
Corynebacterium flavescens.	–	250	7.81
Enterobacter hormaechei.	62.50–125	250	7.81
Erwinia carotovora.	250	–	31.25
Exiguobacterium acetylicum.	250	125	7.81
Kocuria kristinae.	250–500	–	15.60
Kocuria rosea.	125	250	31.25
Micrococcus luteus.	–	250	62.50
Moraxella catarrhalis.	–	250	7.81
Pantoea agglomerans.	500	–	7.81
Pseudomonas putida.	250–500	250	250–500
Pseudomonas syringae syringae.	–	250	250
Psychrobacter immobilis.	500	500	15.60
Staphylococcus cohni cohn.	125	250	7.81

As can be seen from Table 1, the aqueous extracts of both Althaea officinalis. and Althaea hirsute. did not show antibacterial activity against any of the bacterial species tested in the current study. However, methanol extracts of Althaea officinalis. and Althaea cannabina. were found to have inhibition effects on the growth of 17 of 52 and 13 of 52 bacteria 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).

Iauk et al. (2003) and Bonjar et al. (2004) reported that methanol extracts of Althaea officinalis. had broad-spectrum antibacterial effects against Porphyromonas gingivalis., Prevotella. spp., and Pseudomonas fluorescents.. Our findings concur with these studies, despite the different bacterial species used. When compared with each other, it was found that Althaea officinalis. is more effective than Althaea cannabina. in terms of inhibition of a number of bacterial species.

The most pronounced activity with inhibition zones of more than 9 mm was shown by the methanol extracts of Althaea officinalis. against Acidovorax facilis., Bacillus. sp., Bacillus subtilis., Chryseomonas luteola., Corynebacterium ammoniagenes., Enterobacter hormaechei., Kocuria rosea., and Moraxella catarrhalis. and methanol extract of Althaea cannabina. against Acidovorax facilis., Bacillus. sp., Bacillus subtilis., Enterobacter hormaechei., and Kocuria rosea. (Table 1). Maximum inhibition zone (MIC) values for microorganisms sensitive to methanol extract of Althaea officinalis. and Althaea hirsute. were in the range of 7 to 12 mm; 31.25 to 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 Althaea officinalis. and Althaea cannabina. (Baytop, 1996). According to literature searched, nothing is known about the antibacterial properties and uses of Althaea officinalis. and Althaea cannabina. against a number of food-borne bacteria. Therefore, this may be the first report to provide data that the extracts of Althaea officinalis. and Althaea cannabina. evaluated against a wide range of bacteria possess potential antibacterial activities.

The data summarized above indicate that the two Althaea. species under study showed antibacterial activity. The combination of antibacterial action and low toxicity of some extracts may indicate their potential as antibacterial herbal remedies. However, further phytochemical studies are required to determine the types of compounds responsible for the antibacterial effects of these species.