Antimicrobial Activities of Some Plants from the Eastern Anatolia Region of Turkey

ABSTRACT

Crude extracts obtained from the roots and aerial parts of Rumex crispus.L. and Acinos rotundifolius.Pers. and the whole plants of Ajuga chamaepitys. L. and of Lycopus europaeus. L. were evaluated for in vitro. antimicrobial activity against five Gram-positive bacteria including Staphylococcus aureus., Micrococcus luteus, Mycobacterium smegmatis, Bacillus subtilis, Bacillus subtilis. var. niger., and three Gram-negative bacteria including Aeromonas hydrophila, Klebsiella pneumoniae, Pseudomonas aeruginosa., and the yeast Candida albicans.. The inhibition zone diameter was determined for each extract using the agar well diffusion method at a concentration of 12.5 mg/ml. The acetone extracts of the roots of R. crispus. and A. rotundifolius. demonstrated significant inhibitory effects against most microorganisms under test.

Keywords:

• Agar well diffusion method
• antimicrobial activity
• Lamiaceae
• plant extracts
• Polygonaceae

Introduction

Bioprospecting is described as tapping the potential of bioresources for the welfare of humanity and to improve the quality of life in a sustainable manner. It is an environment-friendly exploitation of biological resources and also described as the search for valuable compounds in nature (Baser, 2002). Plant-derived medicines have been part of traditional health care in most parts of the world for thousands of years (Palambo & Semple, 2001). Infectious diseases have been treated with herbal remedies throughout the history of mankind (Franzblau & Cross, 1986). The treatment of infectious diseases with antimicrobial agents continues to present problems in modern-day medicine with many studies showing significant increase in the incidence of bacterial resistance to several antibiotics (Kumarasamy et al., 2002). Due to increased resistance of many microorganisms toward established antibiotics, much recent attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine (Essawi & Srour, 2000). Therefore, it has been of great interest to carry out a screening of plants used in folk medicine in order to discover and develop new and effective therapeutic agents (Paz et al., 1995; Awadh Ali et al., 2001).

Several records are available on the studies of the folk medicine in some provinces of Turkey. It seems that traditional medicine is common practice in Turkey like in many developing countries (Sezik et al., 199119972001; Tabata et al., 1994; Yesilada et al., 19951999; Fujita et al., 1995; Honda et al., 1996). Of the approximately 10,000 species of flowering plants in Turkey, only 25% of the medicines used for people's health have been obtained from these plants so far (Sener et al., 1996). Studies on the folk medicine of several medicinal plants has increased, and the antimicrobial activities of several plants used in Turkish traditional medicine have been screened recently by numerous investigators (Digrak et al., 1999ab; Sokmen et al., 1999; Sagdic et al., 2002; Sagdic & Ozcan, 2003).

The genus Rumex. is widespread in the flora of Turkey, which is represented by 22 species. Rumex crispus. L. (Polygonaceae) is known as kivircik labada. or evelik.. In Anatolia, leaves of R. crispus. have been commonly used either as vegetables or for meat stuffed with leaves (Baytop, 1999). The roots of some Rumex. species are reported to contain compounds such as anthracene derivatives (0.2–1.7%) and catechic tannin (6–12%). As it contains no toxic substances, it can be used as a safe natural medicine source (Baytop, 1999). In Turkish folk medicine, the roots of the Rumex. species are used to treat constipation, diarrhoea, eczema, and are also used for the maturation of abcess, tonic, and blood purifier (Baytop, 1999; Yesilada, 1999).

In Turkey, the genus Ajuga. comprises 10 species. Ajuga chamaepitys. L. (Lamiaceae) is called yercami, basirotu, soguklama otu, or yer selvisi. (Honda et al., 1996; Baytop, 1999). Previous chemical studies of A. chamaepitys. aerial parts reported α.-pinene (16.1%), β.-pinene (34.3%), and germacrene-D (5.6%). Aerial parts of this species also includes resins and bitter substances (Baytop, 1999; Azizan et al., 2002). Since medieval times, it has been used as a diuretic, tonic, for perspiration, menser remover, wound healing, and against scorpion and snake bites and it has been used also as a remedy for haemorrhoids, stomachache, and common colds (Honda et al., 1996; Baytop, 1999).

The genus Acinos. (Lamiaceae) is represented by five species in the flora of Turkey. A. rotundifolius. Pers. is the most widespread Acinos. species in Turkey (Kaya et al., 1999a). There has been a significant research interest toward the assay of composition of various Acinos. essential oils (Kaya et al., 1999abc). The composition of the essential oil of A. rotundifolius. includes mainly germacrene-D, hexadecanoic acid, menthol, spathulenol, bicyclogermacene, caryophyllene oxide, and α.-cadinol (Kaya et al., 1999a). Some species of the genus are strongly or slightly odorous and used for medicinal purposes such as sedative, antiinflammatory, melancholy, for shortness of breath, and for improving digestion and treating bruises, toothache, sciatica, and neuralgia (Kaya et al., 1999a).

The genus Lycopus. is represented by only one species, namely Lycopus europaeus. L. (Lamiaceae) and is called kurtayagi. in Turkey (Baytop, 1999). It contains compounds such as isopimarane diterpenes and straight-chain aliphatic precursors of cyclic diterpenes (Jeremic et al., 1985; Hussein & Rodriguez, 2000; Gibbons et al., 2003). Traditionally, L. europaeus. is reported to be for the treatment of constipation, for blood stopping, and for its anti-inflammatory properties (Baytop, 1999). This plant is also scientifically known to have antithyreotropic and antigonadotropic activities due to phenolic compounds (Bucar & Kartnig, 1995).

In the Turkish Eastern region, the people generally use herbal remedies for the treatment of common and minor ailments (e.g., gastrointestinal disorders, cold, flu, cough and other respiratory problems, rheumatic pain, kidney stone, hemorrhoids, eye diseases, etc.) and for minor skin troubles. The use of medicinal plants is usually the first choice of treatment among the rural people in this region (Tabata et al., 1994). The screening of plant extracts and natural products for antimicrobial activity has shown that higher plants represent a potential source of new anti-infective agents as well as serving drug discovery from natural products for primary lead compounds. As a continuing research directed at the antimicrobial screening of plants grown in Kars (Far Eastern Anatolia), this paper describes the results of the preliminary investigation on the antimicrobial activity of the hexane, acetone, and methanol extracts from four different plant species used in Turkish folk medicine.

Materials and Methods

Plant material

A total of four plants, Rumex crispus. (roots, aerial parts), Ajuga chamaepitys. (whole plant), Acinos rotundifolius. (roots, aerial parts), and Lycopus europaeus. (whole plant), were collected from Kars region, Eastern Turkey. The plants were identified by Dr. A. Ilcim, and voucher specimens were deposited at the Herbarium of the Department of Biology, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey.

Preparation of extracts

All plant material was air-dried at room temperature before grinding to powder with a Waring blender. After grinding, plant material was extracted by using a Soxhlet extractor with solvents of increasing polarity beginning with hexane, followed by acetone and methanol. Extraction with each solvent was carried out for 8 to 10 h. The solvent was removed in a rotary vacuum evaporator at 40°C. The percentage yield was determined for each extract, and the different extracts of the plants were dissolved in the extraction solvents at a concentration of 12.5 mg/ml. Each solution was sterilized with a 0.22-µm membrane filter. The extracts were then kept under refrigerated conditions until further use.

Microorganisms used

The following strains of bacteria were used: Staphylococcus aureus. Cowan 1, Micrococcus luteus. LA 2971, Mycobacterium smegmatis. CCM 2067, Bacillus subtilis. IGM 22, Bacillus subtilis. var. niger. ATCC 10, Aeromonas hydrophila. ATCC 7966, Klebsiella pneumoniae. FMC 5, and Pseudomonas aeruginosa. ATCC 27853. The yeast strain used in this study was Candida albicans. ATCC 10231.

Antimicrobial activity

Antimicrobial activity of the plant extracts was determined by a modified agar well diffusion method (Vlietinck et al., 1995). Bacteria were cultured at 30°C for 24 h in Mueller Hinton broth (MHB, Difco) and the yeast studied was incubated in potato dextrose broth (PDA, Difco) for 48 h. An inoculum consisting of 10⁶ CFU/ml for bacteria and 10⁴ CFU/ml for yeast was pipetted onto the MHA for bacteria and the PDA for yeast and spread well onto the agar. After inoculation of the media, four wells, 12 mm in diameter, were removed with a sterile cork borer and positioned one per quadrant of the 100-mm Petri dishes. Four wells were aseptically filled up with 0.1 ml of each plant extract. Controls (hexane, acetone, and methanol) without the test compound were carried out in parallel. The plates were kept at room temperature for 1 h to allow diffusion of extract into agar. Afterwards, the Petri dishes were inverted and left to incubate at 37°C for 18–24 h for bacteria and at 30°C for 24–48 h for the yeast. The diameters of the inhibition zones were measured in millimeters at the end of the incubation times. Tests were performed in duplicate.

Results and Discussion

A total of 18 extracts representing four plant species distributed among two families were investigated. The results of the antimicrobial screening of plant extracts are presented in Table 1. The plant species investigated differed considerably in their activity against the test microorganisms. The solvent controls did not show any activities against the microorganisms.

Table 1.. Results of the screening of four plant extracts by means of the agar well diffusion method.

Details of plant extracts			Bacteria tested [Inhibition zone diameter (mm)]
Plant name Voucher no.	Plant part^a.	Extract type^b. Yield (%)	S. aureus.	M. luteus.	M. smegmatis.	B. subtilis.	B. subtilis. var. niger.	A. hydrophila.	K. pneumoniae.	P. aeruginosa.	C. albicans.
R.. crispus. L.	R	H (0.64)	—	15	15	22	16	17	16	18	—
1416
	R	A (1.94)	23	22	21	24	24	18	20	16	17
	R	M (12.65)	17	16	15	16	15	—	15	—	—
R.. crispus. L.	AP	H (1.65)	—	—	14	16	15	—	—	—	—
1416
	AP	A (1.68)	—	14	16	16	—	16	15	—	—
	AP	M (12.72)	—	—	—	—	—	—	—	15	—
Acinos. rotundifolius. Pers.	R	H (0.89)	—	14	—	16	—	—	—	—	—
1216
	R	A (3.44)	24	27	21	28	40	23	22	23	14
	R	M (12.50)	14	16	15	18	29	14	16	—	—
Acinos. rotundifolius.	AP	H (1.95)	—	13	14	14	—	13	13	13	—
1216
	AP	A (1.89)	13	17	16	16	21	13	16	—	13
	AP	M (7.51)	14	16	14	—	34	—	16	13	—
L.. europaeus. L.	WP	H (1.94)	—	14	14	—	—	15	15	—	—
1336
	WP	A (1.87)	—	—	13	—	—	—	15	—	—
	WP	M (20.97)	—	15	—	—	—	—	14	—	—
Ajuga. chamaepitys. L.	WP	H (1.42)	14	15	16	17	16	14	16	—	—
1321
	WP	A (5.51)	15	16	16	15	—	—	16	—	—
	WP	M (16.49)	—	—	14	—	—	—	16	14	—

^a.Plant part: RT, root; AP, aerial parts; WP, whole plant.

^b.Extract type: H, hexane; A, acetone; M, methanol.

As shown in Table 1, all the extracts of the whole plants of L. europaeus, A. chamaepitys., and the leaves of R.. crispus. had no antiyeast activity. The acetone extract of R.. crispus. roots and the acetone extracts of A.. rotundifolius. roots and aerial parts exhibited inhibitory action against C.. albicans..

The hexane extract of the root of R.. crispus. showed only inactivity against S.. aureus.. In addition, the methanol extract of the root did not inhibit the growth of A.. hydrophila. and P.. aeruginosa.. On the other hand, the acetone extract showed activity against all the Gram-positive and Gram-negative bacteria tested. As can be seen in Table 1, it seems that the antimicrobial activity against Gram-positive bacteria was more pronounced than against Gram-negative bacteria. These differences may be attributed to the fact that the cell wall in Gram-positive bacteria consists of a single layer, whereas the Gram-negative cell wall is a multilayer structure and quite complex (Essawi & Srour, 2000).

It was also found that the hexane extracts of the aerial parts of R.. crispus. showed only antibacterial activity against M.. smegmatis., B.. subtilis., and B.. subtilis. var. niger.. However, the acetone extracts of the aerial parts were ineffective on the growth of S.. aureus., B.. subtilis. var. niger., and P.. aeruginosa.. The methanol extracts of the aerial parts of R.. crispus. showed no activity against all the bacteria except P.. aeruginosa..

In a previous study, Yildirim et al. (2001) found that ether, ethanol, and hot water extracts of the leaves of R.. crispus. were active against S. aureus. and B.. subtilis. and inactive against Escherichia coli., P.. aeruginosa., and C.. albicans.. The result is not generally in concordance with that of our study. This may be the consequence of different types of strain, different solvent extracts, and different assay methods.

Comparison of the inhibitory zone values of all extracts of the root and aerial parts of R.. crispus. reveals that both extracts have no similar activities against the bacteria. It seems that acetone extract of the root of the R.. crispus. is more active than that of the hexane and methanol extracts. It may be that the potentially active compounds in R.. crispus. resided in an acetonic extract of the roots or it is possible that the species, which showed less inhibitory activity at the test concentration, might show inhibition of bacterial growth if used in higher concentrations, as the actual compounds present in these extracts may be of minute quantities.

As can be seen from Table 1, the acetone extracts of the root of A.. rotundifolius. showed activity against all the bacteria used, and the maximum activity was observed against B.. subtilis. var. niger., having an inhibition zone of 40 mm. The hexane extracts from A.. rotundifolius. root were active against two Gram-positives, namely, M.. luteus. and M.. smegmatis.. The methanol extracts of the A.. rotundifolius. root were only inactive against P.. aeruginosa.. It is noted that the methanol extract of the root inhibited the growth of B.. subtilis. var. niger., with an inhibition zone of 29 mm.

The results obtained with the hexane extracts of the aerial parts of A.. rotundifolius. revealed no growth inhibition against S.. aureus. and B.. subtilis. var. niger.. It was also observed that methanol extracts were ineffective in inhibiting B.. subtilis. and A.. hydrophila., but it was highly effective against B.. subtilis., with an inhibition zone of 34 mm. The acetone extracts of the aerial parts of A.. rotundifolius. inhibited the growth of all test bacteria used in this study, with the exception of P.. aeruginosa..

Comparing the antimicrobial activities of the extracts of the roots and the aerial parts of A.. rotundifolius. were not equally active against the microorganisms. The most potent compounds are possibly found in an acetone extracts of the roots of A.. rotundifolius.. Some differences are thought to derive from different chemical compounds in the plant extract types.

All solvent extracts of the L.. europaeus. demonstrated no activity against S.. aureus.. In a previous study, five organic solvents including hexane, chloroform, ethyl acetate, acetone, and methanol of the crude extracts of the L.. europaeus. were tested against S.. aureus. (Gibbons et al., 2003) and reported to be not active against S.. aureus.. Data from L.. europaeus. in our study was not in accordance with this study. The hexane extracts of the whole plants of L.. europaeus. showed only antibacterial efficacy against four bacteria. No activity was observed against six bacteria with the acetone and methanol extracts. In general, the results of the antibacterial test indicated that L.. europaeus. has a less inhibitory activity against the microorganisms.

Similar zones of inhibition were obtained for S.. aureus., M.. luteus., M.. smegmatis., B..subtilis., K.. pneumoniae., and P.. aeruginosa. with the hexane and acetone extracts of the A.. chamaepitys., whereas the methanol extracts demonstrated insignificant activity against the test microorganisms.

To summarize the results, the hexane, acetone, and methanol extracts of four plant species showed antimicrobial activity against one or more microorganisms. However, the activity of the acetone extracts of the roots of R.. crispus. and A.. rotundifolius. revealed promising results against mostly Gram-positive pathogens, and this indicates the presence of potent antimicrobial agents in the crude extracts of these plants. Therefore, further purification and isolation work is necessary to determine the types of agents responsible for the antimicrobial effects of these medicinal plants.