Antibacterial, antifungal, and antimycobacterial activity of Ilex aquifolium leaves

Abstract

The objective of this study was to examine antibacterial, antifungal, and antimycobacterial properties of Ilex aquifolium L. (Aquifoliaceae) growing in Turkey. The ethanol, ethyl acetate, chloroform, and n-hexane extracts prepared from the leaves of I. aquifolium were tested against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Proteus vulgaris, Salmonella typhimurium, and Candida albicans for antibacterial and antifungal evaluation using the microdilution broth susceptibility assay. In addition, antimycobacterial activity of the crude extracts of I. aquifolium was evaluated by microplate Alamar blue assay. The results showed that the extracts tested, except n-hexane, possessed moderate antibacterial and antifungal activity varying from 62.5 to 250 μg/mL. On the other hand, the ethanol extract of the leaves exhibited a minimum inhibitory concentration value (MIC) against Mycobacterium tuberculosis H₃₇Ra strain of 200 μg/mL.

Keywords::

• Antibacterial activity
• Aquifoliaceae
• Ilex aquifolium

Introduction

Tuberculosis (TB) still remains among serious public health problems. In the world, TB ranks second among the leading infectious diseases, with ca. 2–3 million deaths in 7–8 million new cases of active TB each year (World Health Organization, 2004). The presence of a number of highly effective medications for treating TB has increased in the last three decades, yet the morbidity and mortality of TB continue to increase, especially when coupled with immunodeficiency virus (HIV). Furthermore, multidrug resistant TB strains have led to the search for new classes of potent antimycobacterial drugs, including natural products, against this infectious disease (Duncan, 2003).

Ilex aquifolium L. (Aquifoliaceae) (European or English holly), one of the most well known species of genus Ilex, is a dioecious broad-leaved evergreen perennial shrub or tree with red drupes (Harder et al., 1965; Yaltırık, 1966). The genus has two species growing in Turkey, namely I. aquifolium and I. colchica Poj. (Yaltırık, 1966). Today, hollies are grown comm ercially for decorative purposes during Christmas because of their colored berries and shiny green leaves (Grieve, 1982).

I. aquifolium leaves have been used in folk medicine for several purposes, such as in intermittent fevers and rheumatisms, for their antipyretic properties, and as astringent, diuretic, and expectorant agents (Grieve, 1982). Muller et al. (1998) reported the inhibitory effect of I. aquifolium against lipid peroxidation. There have been several studies on the chemical composition of I. aquifolium which reported that anthocyanins, flavonoids, terpenoids, sterols, amino acids, alkaloids, fatty acids, alcohols, carbohydrates, carotenoids, cyanogenic glucoside, phenols, and phenolic acids were present in the plant (Valadon et al., 1975; Knights & Smith 1977; Catalano et al., 1978; Budzikiewicz & Thomas, 1980; Alikaridis, 1987; Nahrstedt & Wray 1990; Van Genderen & Jaarsma, 1990; Erdemoglu et al., 2000; Sun et al., 2000).

As a part of our ongoing research on the screening of biologically active Turkish plants, we have investigated the antibacterial and antifungal activity of crude extracts of the leaves of I. aquifolium, as well as tested for antimycobacterial activity against Mycobacterium tuberculosis H₃₇Ra strain.

Materials and methods

Plant material

Ilex aquifolium was collected from the vicinity of Amasya, Turkey, in February 1998. Plant material was collected by one of us (N.E.) and identified by Prof. Dr. Mecit Vural (Department of Biology, Faculty of Science, Gazi University, Ankara). A voucher specimen (2302) is kept in the Herbarium of the Faculty of Pharmacy, Gazi University, Ankara, Turkey.

Preparation of crude extracts

Air-dried and powdered leaves of Ilex aquifolium were accurately weighed (10 g) and separately extracted with 96% ethanol, ethyl acetate (Merck), chloroform (Merck), and n-hexane (Merck) using maceration at room temperature, in duplicate (× 50 mL). After filtration, the combined extracts were evaporated to dryness in vacuo and the crude EtOH, EtOAc, CHCl₃, and n-hexane extracts were obtained. The percentage yields of the extracts are given in Table 2.

Antimicrobial assay

Microorganisms were stored at +4°C on agar slants. Standard strains of the following bacteria, namely Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 27853), Enterobacter aerogenes (NRRL 3567), Proteus vulgaris (NRRL B-123), and Salmonella typhimurium (NRRL B-4420) for the determination of antibacterial activity, and standard strains of Candida albicans (clinical isolate; Osmangazi University, Faculty of Medicine, Eskisehir, Turkey) for the determination of antifungal activity were used.

A microdilution broth susceptibility assay (Koneman et al., 1997; Zgoda & Porter, 2001) was used for antimicrobial evaluation of the extracts. Stock solution was prepared in dimethylsulfoxide (DMSO; Carlo-Erba, France). Dilution series using sterile distilled water were prepared from 4 to 0. 007 mg/mL in micro-test tubes (Eppendorf), and were transferred to 96-well microtiter plates. Overnight grown bacterial and C. albicans suspensions in Mueller-Hinton broth were standardized (for bacteria and C. albicans app. 10⁸ and 10⁶ cfu/mL, respectively) using McFarland No. 0.5 standard solution. Each microorganism suspension was then added into the wells. The last well column with medium and microorganism served as a positive growth control. After incubation at 37°C for 18–24 h, the first well without turbidity was determined as the minimal inhibitory concentration (MIC). Chloramphenicol was used as the standard antibacterial agent whereas ketoconazole was used as the antifungal.

Antimycobacterial assay

Antimycobacterial bioassay was performed using the microplate Alamar blue assay (MABA) (Collins & Franzblau, 1997). Suspensions of Mycobacterium tuberculosis H₃₇Ra strain were prepared at approximately 10⁵ cells/mL concentration. Samples were dissolved in dimethylsulfoxide (DMSO) at 20 mg/mL in sterile conditions and subsequently diluted in sterile Middlebrook liquid medium 7H9 broth to 200 μg/mL. Two-fold dilutions were prepared. The assay was performed in clear 96-well sterile microplates. One hundred microliters of bacterial suspension were added to each well of the microtiter plate together with the plant extracts in Middlebrook 7H9 medium to a final volume of 200 μL, and the final concentrations of the plant extracts were 50, 100, and 200 μg/mL. Following incubation for 7 days at 37°C, one control without sample was developed with 20 μL of ×10 Alamar blue solution and 12 μL of sterile 10% Tween 80. The plates were reincubated at 37°C for 24 h. After this incubation period, if the dye turned pink, indicating bacterial growth, Alamar blue and Tween solutions were then added to all remaining wells in the plate. The results were read the following day and the MIC values of the extracts were calculated. Rifampin, isoniazid, and kanamycin were used as references. The MIC ranges of the references were 0.0047–0.0095, 0.05–0.1, and 2.5–5.0 μg/mL, respectively.

Results and discussion

Four extracts prepared from the leaves of Ilex aquifolium were screened against the standard strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Proteus vulgaris, Salmonella typhimurium, and Candida albicans for their antibacterial and antifungal evaluation using the microdilution broth susceptibility assay, and the results compared to those obtained with reference agents (Table 1). The microorganisms were inhibited with moderate activity, having MIC values of 62.5–250 μg/mL, lower than those of the antimicrobial standard agents. However, the EtOH extract showed stronger inhibition against Proteus vulgaris at a concentration of 62.5 μg/mL. None of the extracts showed inhibitory effects on Pseudomonas aeruginosa.

Table 1.  Antibacterial and antifungal activities of Ilex aquifolium leaf extracts.

Microorganism	Source	MIC (μg/mL)
		EtOH extract	EtOAc extract	CHCl3 extract	n-Hexane extract	Std
Bacteria
Escherichia coli	NRRL B-3008	—	250	—	—	31.25*
Staphylococcus aureus	ATCC 6538	250	250	—	—	3.9*
Pseudomonas aeruginosa	ATCC 27853	—	—	—	—	62.5*
Enterobacter aerogenes	NRLL B-3567	—	—	250	—	62.5*
Proteus vulgaris	NRLL B-123	62.5	—	250	250	15.6*
Salmonella typhimurium	NRRL B-4420	—	—	250	—	31.25*
Fungi
Candida albicans	Clinically isolated	250	250	125	—	62.5**

Tests were done in triplicate. Test concentration of extracts 2 mg/mL in DMSO.

MIC, minimum inhibitory concentration; —, no inhibition; Std: *chloramphenicol; **ketoconazole.

On the other hand, the crude extracts obtained from the leaves of Ilex aquifolium were also tested for evaluation of the minimum inhibitory concentration to Mycobacterium tuberculosis H₃₇Ra strain with a microplate technique using Alamar blue. Active extracts exhibiting varying degrees of inhibition in the in vitro primary screen test at 200 μg/mL were retested at a lower concentration to determine the actual MIC values. The results of antimycobacterial activity assay of these extracts are summarized in Table 2. The antimycobacterial activity of the extracts was compared with rifampin, isoniazid, and kanamycin. The ethanol extract of the leaves showed activity at a concentration of 200 μg/mL. The rest of the extracts were found to be inactive.

Table 2.  Antimycobacterial activity results for extracts of Ilex aquifolium against Mycobacterium tuberculosis H₃₇Ra.

Extract	Yield (w/w, %)	Results against TB at 200 μg/mL	MIC value (μg/mL)
EtOH extract	18.3	Active	200
EtOAc extract	7.4	Inactive	—
CHCl3 extract	8.4	Inactive	—
n-Hexane extract	6.4	Inactive	—

In recent years, the crude extracts of many plants, especially those with ethnomedicinal uses, have been assessed for antimicrobial potency using in vitro assays. These studies showed that a number of antifungal, antibacterial, and antimycobacterial natural compounds have been isolated (Cowan, 1999; Cantrell et al., 2001; Rios & Recio, 2005). In fact, the triterpenes, widely distributed in I. aquifolium, especially ursolic and oleanolic acid derivatives (Knights & Smith 1977; Catalano et al., 1978; Van Genderen & Jaarsma, 1990), have been also reported to illustrate antimycobacterial (Cantrell et al., 2001; Vik et al., 2007) and antifungal (Cowan, 1999) activities. In this study, the ethanol extract was subjected to thin layer chromatography (TLC) for investigation of the chemical composition of the plant. Spraying of TLC plates with 30% H₂SO₄ solution, followed by heating at 105°C for 1–2 min, showed some regions corresponding to triterpene type compounds that were visualized as red spots with UV light (254 nm). Therefore, it can be stated that triterpenes in the ethanol extract could be responsible for antimicrobial activity. To the best of our knowledge, this is the first study describing antibacterial, antifungal, and antimycobacterial activities of I. aquifolium.

Declaration of interest: The authors report no conflicts of interest.