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«ISSN 0974 – 5211 Journal of Natural Products Volume 7 (2014) Research Paper Antibacterial activity and modulation ...»

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Nadia M., et al., /Journal of Natural Products, Vol. 7(2014): 131-140

ISSN 0974 – 5211

Journal of Natural Products

Volume 7 (2014)


Research Paper

Antibacterial activity and modulation of antibiotic resistance by

Crataegus azarolus extracts

Nadia M.1,2, Imen M.1, Mounira K.1*, Fadwa C. 1, Zied G. 1, Kamel G.1, Thierry

H. 2, Leila C. G.1,3

Division of Pharmacognosy and Molecular Biology, Faculty of Pharmacy at Monastir,

Laboratoire de Pharmacognosie, E.A. 1043, Université de Lille 2, Faculté de Pharmacie B.P.

83, 59006 Lille cedex, France Laboratory Cellular and Molecular Biology, Faculty of Dental Medicine at Monastir, Rue Avicenne, 5000, Monastir, Tunisia *Corresponding Author (Received 20 April 2014; Revised 20 May - 08 July 2014; Accepted 19 July 2014) ABSTRACT Antibiotic resistance among bacterial pathogens is a serious problem for human and veterinary medicine, which necessitates the development of novel therapeutics and antimicrobial strategies. The aim of the present study was to investigate the antibacterial activity of Crataegus azarolus leaves extracts against both Grampositive and Gram-negative bacteria and against four multidrug-resistant strains of E.

coli. Minimum inhibitory concentration (MIC) values of tested extracts as well as of some antibiotics were determined by the standard broth microdilution method. All extracts exhibited antibacterial effect against reference strains; Staphylococcus aureus, Enterococcus faecalis, Salmonella enteritidis and Salmonella typhimurium. It appears that ethyl acetate and TOF (Total oligomer flavonoids) enriched extracts have the greatest antibacterial activity against these reference strains. Besides, as these two active extracts revealed the best antibacterial effect against multiresistant strains of E.

coli, we decided to test the effect of each, combined to the antibiotic against which the strains were resistant. In the interaction study, the tested extracts acted in synergy to lower the susceptibility of two strains of multidrug-resistant E. coli (EC 6574 and EC 6228), to amoxicillin and ofloxacin with TOF enriched extract and to ofloxacin and cefotaxim with ethyl acetate extract, but no synergy was observed for the remaining strains. These results indicate that extracts from C. azarolus could be used as a source of natural products when administered in combination with beta-lactam antibiotics to combat bacterial infections caused by resistant E. coli.

Keywords: Crataegus azarolus; Antibacterial activity; Antibiotic resistance; Synergism.


Escherichia coli are one of the principal causes of infectious diseases in humans.

These bacteria are known to produce enterotoxins whose properties and role in diarrheal disease have been widely investigated. The activity of cytotoxins and their Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved Nadia M., et al., /Journal of Natural Products, Vol. 7(2014): 131-140 role in human infection have been identified, mainly in infections of the urinary tract (Hughes, et al., 1982; Scotland, et al., 1980). In relation to pathogenic bacteria, a growing and worrisome problem is the increase in bacterial resistance to antibiotics (Georgopapadakou, 2005; Nostro, et al., 2004). Acquired multi drug resistance to antimicrobial agents creates an extensive trouble in case of the management of intra and extra intestinal infections caused by E. coli, which are a major source of illness, death, and increased healthcare costs (Gupta, et al., 2001). According to the development of antimicrobial resistance, a considerable scientific research has focused on the antibacterial properties of plant products. In fact, medicinal plants have been the source of many medications that are now applied in clinical practice. The use of extracts as antimicrobial agents shows a low risk of increasing resistance to their action, because they are complex mixtures, making microbial adaptability very difficult (Daferera, et al., 2003; Gibbons, 2004).

Hawthorn (Crataegus oxyacantha and Crataegus laevigata) leaves, flowers and berries are recognize for along for their effects on heart health (treatment of chronic heart failure, high blood pressure, and arrhythmia). However, little information is available on their antimicrobial effect. Indeed, Kostic, et al. (2012) evaluated antimicrobial activity of Crataegus oxyacantha against selected test microorganisms: Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella abony and revealed that tested extracts from hawthorn fruit exhibited good antimicrobial activity. But Tadic et al. (2008), showed that hawthorn berries ethanol extract revealed a moderate bactericidal activity, especially against gram-positive bacteria: Micrococcus flavus, Bacillus subtilis, and Lysteria monocytogenes.

Based on the foregoing, it can be said that C. azarolus was not studied for such activity. And to our knowledge, we investigated for the first time the effectiveness of extracts from C. azarolus leaves against multidrug-resistant strains of E. coli as well as their modulating effect on bacterial resistance to the target antibiotics.


Bacterial material: Five bacterial strains were used as reference, Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212), and the Gram-negative bacteria (Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076 and Salmonella typhimurium NRRLB 4420). Synergetic activities of C. azarolus leaf extracts were tested against four multidrug-resistant strains of E. coli with the resistance profile described in Table 1. All these strains were kindly provided from the clinical bacterial collection of CHU Fattouma Bourguiba, Monastir-Tunisia. The antibiotics used are the Cefotaxim, the Amoxicilin, the Piperacilin, the Ofloxacin and the tetracycline (Sigma-Aldrich, Canada Ltd., Oakville, ON).

Preparation of plant extracts: C. azarolus’s leaves were collected from Oued Hatem in Sousse, a region situated in the coast center of Tunisia, in September 2011.

Identification was carried out by Prof. Harzallah Skhiri Fethia (Higher Biotechnology Institute of Monastir, University of Monastir, Tunisia) according to the Flora of Tunisia (Pottier-Alapetite, 1979). A voucher specimen (C. a-09-11) has been kept in our laboratory for future reference.

The fresh leaves of C. azarolus, were dried at room temperature and reduced to coarse powder. The powdered leaves were extracted by boiling water for 15 to 20min. The crude extract obtained was filtered and lyophilized (aqueous extract). The Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved Nadia M., et al., /Journal of Natural Products, Vol. 7(2014): 131-140 residue was dissolved in water. In order to obtain an extract enriched with Total Oligomer Flavonoids (TOF), powder was macerated in water/acetone mixture (1v/2v), for 24h with continuous stirring. The extract was filtered and the acetone was evaporated under low pressure in order to obtain an aqueous phase. Tannins in the aqueous phase were precipitated with an excess of NaCl for 24h at 5°C. The mixture was then filtered and the filtrate solution was recovered. This latter was extracted with ethyl acetate, concentrated and precipitated with an excess of chloroform. The precipitate was separated and yielded TOF extract (Ghedira, et al., 1991). Hexane (Hex), chloroform (Chl), ethyl acetate (EA), and butanol (BuOH) extracts were obtained by liquid-liquid extraction with solvents increasing polarity after ten days maceration in methanol. These four types of extracts, with different polarities, were concentrated to dryness and each residue was kept at 4°C. Then each extract was resuspended in the adequate solvent. The same solvent was used in the corresponding negative control.

Quantitative and qualitative analysis of extracts Determination of total polyphenol and flavnoid contents: The polyphenol content of C. azarolus leaves extracts was quantified by the Folin–Ciocalteau reagent (Kumar and Chattopadhyay, 2007). Aliquots of test samples (100µl) were mixed with 2ml of 2% Na2CO3 and incubated at room temperature for 2min. After addition of 100µl 50% Folin–Ciocalteau phenol reagent, the reaction tube was further incubated for 30min at room temperature, and finally absorbance was read at 720nm. Gallic acid was used as a standard. A known volume of each extract was placed in a 10 ml volumetric flask to estimate flavonoid content according to the modified method of Zhishen, et al., (1999). After addition of 75µl of NaNO2 (5%), 150µl of freshly prepared AlCl3 (10%), and 500µl of NaOH (1N) solutions, the volume was adjusted with distilled water until 2.5ml. After 5 min incubation, the total absorbance was measured at 510nm. Quercetin was used as a standard for constructing a calibration curve.

Determination of tannin content: According to Nwabueze (2007), quantification of tannins in the samples was achieved by dissolving 5g of extract in 50ml of distilled water in a conical flask, allowing the mixture to stand 30min with shaking the flask at 10min intervals and then centrifuging it at 5000xg to obtain a supernatant (tannin extract). The extract was diluted to 100ml in a standard flask using distilled water.

Five milliliters of the diluted extract and 5ml of standard tannic acid (0.01g/L) were measured into different 50ml volumetric flasks. One milliliter of Folin-Denis reagent was added to each flask, followed by 2.5ml of saturated sodium carbonate solution.

The solutions were made up to the 50ml mark with distilled water and incubated at room temperature (20–30°C) for 90min. The absorption of each solution was measured against that of the reagent blank (containing 5ml of distilled water in place of extract or standard tannic acid solution) in a Genesys 10 UV scanning spectrophotometer at a 760nm wavelength. Tannin content (tannic acid equivalents)

was calculated in triplicate, using the following formula:

Sample reading − blank Tannin content = Standard reading − blank Determination of proanthocyanidins content: The proanthocyanidins were determined by UV spectrophotometry method based on acid hydrolysis and colour formation. The HCl/butan-1-ol assay was used to quantify the total proanthocyanidins (Porter, et al., 1986). One milligram of each extract was dissolved in 1ml of methanol.

0.25ml of this solution was added 3ml of a 95% solution of n-butanol/HCl (95/5; v/v) Copyright © 2014, Journal of Natural Products, INDIA, Dr. Sudhanshu Tiwari, All rights reserved Nadia M., et al., /Journal of Natural Products, Vol. 7(2014): 131-140 in stoppered test tubes followed by addition of 0.1ml of a solution of NH4 Fe (SO4)2, 12 H2O in HCl [2M] (0.2%; w/v). The tubes were incubated for 40min at 95°C. For a control sample, 0.25ml of methanol was used. After incubation, the samples were cooled and analyzed by measuring absorbance at 540nm. Cyanidin chloride was used as a standard.

High Performance Liquid Chromatography “HPLC” conditions: HPLC analyses were performed with Shimadzu HPLC system which consisted of valve injector, SCLA VP model system controller, SPD-10A model UV/VIS detector and LC-10AS model pump. The chromatographic column was Novapak C18 (4µm, 150 × 4mm i.d.) (Waters). For the analysis of triterpenic acids, eluent A and B were acetonitrile/H2O (20:80; v/v) and 100% acetonitrile, respectively. The gradient elution program was as follows: 0min = 75% B, 15min = 85% B. Before injecting the next sample, the column was equilibrated with the initial mobile phase for 10min. The flow rate was constant at 1ml/min. For the analysis of flavonoids, eluent A and B were acetic acid 0.5% in water and 100% acetonitrile, respectively. Gradient elution was carried out according to the following program: solvent B was increased from 17 to 25% in the first 30min, then increased more from 25 to 50% from 30 to 60min and then returned to 17% in 5min. The flow rate was constant at 1ml/min in both two analyses and injection volume was 20µl. UV detection was performed at 254 and 360nm.

Concentrations of injected solutions were 5mg/ml for crude extract and 1mg/1ml for pure standard in methanol.

Determination of the minimum inhibitory concentration (MIC): MIC values were determined by standard broth microdilution method (Clinical and Laboratory Standards Institute (CLSI), 2002; Palanippan and Holley, 2010). Nutrient broth (Pronadisa, Hispanlab, S.A, Spain) was used for bacterial growth. Antibiotic solutions and natural antimicrobials were prepared at different concentrations for each bacteria and serially diluted in the microwell plates. Test inoculums were prepared from the fresh bacterial cultures by serial dilutions to yield 5 × 105cfu/ml in each well (CLSI

2002) and incubated for 16h at 37°C in a 96 wells plate. The MIC was defined as the lowest antibiotic or antimicrobial concentration which prevented visible growth (Palanippan and Holley, 2010). 40µl of 2-[4-iodophenyl]-3-[4-dinitrophenyl]-5phenyltetrazolium chloride (INT) (Sigma, St. Louis, MO, USA) dissolved in ethanol (0.2mg/ml) was added to each well, and plates were incubated for 1 to 2h at 37°C to allow detection of cell viability (Palanippan and Holley, 2010). The electron transport system of respiring organisms reduces INT to INT-formazan and absorbance was measured directly at 450nm (Grare, et al., 2008). Absence of microbial growth is translated by the absence of culture medium coloring, when incubated with INT. The determination of MIC values was made in triplicate (CLSI, 2002).

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