Antimicrobial activity of wax and hexane extracts from Citrus spp. peels

ARTICLES

Antimicrobial activity of wax and hexane extracts from Citrus spp. peels

Susana JohannI, ⁺; Vetúria Lopes de Oliveira^I; Moacir G Pizzolatti^II; Jan Schripsema^III; Raimundo Braz-Filho^III; Alexsandro Branco^IV; Artur Smânia Jr^I

^IDepartamento de Microbiologia e Parasitologia

^IIDepartamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil

^IIISetor de Química de Produtos Naturais, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brasil

^IVDepartamento de Saúde, Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brasil

ABSTRACT

Antibacterial and antifungal properties of wax and hexane extracts of Citrus spp. peels were tested using bioautographic and microdilution techniques against three plant pathogenic fungi (Penicillium digitatum, Curvularia sp., and Colletotrichum sp.), two human pathogens (Trichophyton mentagrophytes and Microsporum canis), and two opportunistic bacteria (Escherichia coli and Staphylococcus aureus). Two polymethoxylated flavonoids and a coumarin derivative, were isolated and identified from peel extracts, which presented antimicrobial activity especially against M. canis and T. mentagrophytes: 4',5,6,7,8-pentamethoxyflavone (tangeritin) and 3',4',5,6,7,8-hexamethoxyflavone (nobiletin) from C. reticulata; and 6,7-dimethoxycoumarin (also known as escoparone, scoparone or scoparin) from C. limon.

Key words: polymethoxylated flavonoids - antibiosis - pathogens - fungi - bacteria

Citrus spp. are considered an important source of polymethoxylated flavonoids (PMF), a class of secondary plant metabolites (Afek et al. 1986). PMF's are generally found bound to sugar moieties (glycosides) or in exceptional circumstances as free aglycones (Robards et al. 1997). They are often used to establish phylogenetic relationships among plants (Mizuno et al. 2001). However, flavonoids are known to have physiological effects on other organisms. They have been shown to reduce erythrocyte aggregation and sedimentation rates in human blood (Robbins 1976), as well as to exhibit antiviral (Brinkworth et al. 2002), antimutagenic (Iwasse et al. 2001), and antimicrobial properties (Afek et al. 1986, Cushinie & Lamb 2005). Some studies have indicated that foods containing high amounts of flavonoids may reduce the risk of heart diseases, due to their antioxidant properties (Young et al. 1999, Arts et al. 2001, Tripoli et al. 2007).

Studies of the antimicrobial activities of flavonoids have become important because of the increasing occurrence of opportunistic systemic mycosis, as well as the rising prevalence of drug resistance in human pathogenic bacteria (Afek et al. 1986). Drug-resistant bacteria and fungi have complicated the treatment of infectious diseases in immunocompromised AIDS and cancer patients. The evolution of multiple drug resistant human pathogenic microorganisms, has driven the search for new sources of antimicrobial substances, including plant metabolites (Nostro et al. 2002).

The dramatic increase in the resistance of plant pathogens to chemical fungicides has led to the use of repeated applications of chemical fungicides to control plant diseases. As a consequence, public concern has increased the demand for safer and less environmentally harmful agrochemicals (Wedge & Nagle 2000). New antifungal and antibacterial agents are necessary to address this situation.

PMFs with their lipophylic properties, are usually present in vacuoles or in the wax cuticle of plants. Several studies have detected their presence in the wax epicuticular layer on plants where they play a role in protecting these regions against UV radiation and microbial pathogens (Yousef & Tawil 1980, Fang et al. 2001).

In addition, Hamed and Hetta (2005) recorded the ability of C. reticulata to reduce the hazardous effect of Schistosoma mansoni and reduction of worm burden and ova count. In a previous study we described the isolation and antifungal activity of two new polymethoxylated flavonoids from C. aurantifolia (Johann et al. 2007).

The aim of this study was to investigate the effects of wax and hexane extracts of Citrus spp. peels on the growth of three common pathogens, Penicillium digitatum, Curvularia sp., and Colletotrichum sp., which are responsible for serious losses in Citrus plantations (Rosseti et al. 1993). The extracts and the compounds isolated were also tested against the dermatophyte fungi, Trichophyton mentagrophytes and Microsporum canis, and the opportunistic bacteria, Escherichia coli and Staphylococcus aureus.

MATERIALS AND METHODS

Plant material - Fruits from three Citrus species: C. sinensis, C. limon, and C. reticulata were obtained from markets in Florianópolis, state of Santa Catarina, Brazil.

Extraction and isolation - Fruit peels of each species were removed manually and 750 g C. sinensis, 520 g C. limon, and 495 g C. reticulata were separately macerated in hexane (1000 ml) at room temperature for 40 s. The extracts were then filtered and the peels retained. The extracts were concentrated under reduced pressure and the respective wax extracts were collected; further 1000 ml of hexane were added to the peels and they were kept at room temperature for 72 h; once again the hexane extracts were concentrated under reduced pressure.

Purification of the extracts was achieved by re-crystalization, providing a mixture of the flavones (210 mg), tangeritin (1) and nobiletin (2), from C. reticulata, and a cumarine derivade, escoparone (120 mg, 3) from C. limon (Figure).

Analyses of the compounds were performed using thin-layer chromatography (TLC), on silica gel SiF₂₅₄ plates (Merck, Darmstadt, Germany). Plates were developed with toluene/acetic acid (4/1) as the eluent and visualized under ultra-violet light (254/366 nm). Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for ¹H and 100 MHz for ¹³C on a JEOL Eclipse + 400 spectrometer, using TMS as the internal standard or by reference to solvent signals. GC-EIMS spectra were run at 70 eV on a Shimadzu QP-2000 spectrometer.

Mixture of the flavones 1 and 2 - IR n 2940, 1712, 1646, 1590, 1518, 1410 cm^-1. GC-EIMS 70 eV m/z (rel. int.): 1 [R_t 16.7 min], 372 ([M]^.+, 29), 357 ([M-CH₃]⁺; 2 [R_t 19.2 min], 402 ([M]+, 32), 387 ([M-CH₃]⁺, 1) d_H 7.88 (d, J = 8.8 Hz, H-2' and 6'), 7.02 (d, J = 8.8, H-3' and 5'), 6.63 (s, H-3), 4.11 (s, CH₃O-7), 4.02 (s, CH₃O-8), 3.94 (s, CH₃O-6 and 5), 3.89 (s, CH₃O-4'); 2), 7.57 (dd, J = 1.8; 8.8 Hz, H-6'), 7.42 (d, J = 1.8, H-2'), 6.99 (d, 8.4, H-5'), 6.66 (s, H-3), 4.12 (s, CH₃O-7), 4.03 (s, CH₃O-8), 3.95 (s, CH₃O-6 and 5), 3.98 (s, CH₃O-3'), 3.96 (s, CH₃O-4').¹³C NMR (100 MHz, CDCl₃): 1) 161.18 (C-2), 177.39 (C-4), 148.30 (C-5), 144.00 (C-6), 151.33 (C-7), 137.94 (C-8), 147.66 (C-9), 114.72 (C-10), 123.72 (C-1'), 162.23 (C-4'), 106.56 (CH-3), 127.99 (CH-2'), 114.44 (CH-3'), 114.44 (CH-5'), 127.99 (CH-6'), 62.02 (CH₃O-5), 62.21 (CH₃O-6), 61.71 (CH₃O-7), 62.04 (CH₃O-8), 55.43 (CH₃O-4'); 2) 161.04 (C-2), 177.30 (C-4), 148.34 (C-5), 144.00 (C-6), 151.39 (C-7), 137.94 (C-8), 147.66 (C-9), 114.72 (C-10), 123.91 (C-1'), 149.23 (C-3'), 151.89 (C-4'), 106.76 (CH-3), 108.52 (CH-2'), 111.18 (CH-5'), 119.58 (CH-6'), 62.02 (CH₃O-5), 62.21 (CH₃O-6), 61.71 (CH₃O-7), 62.04 (CH₃O-8), 56.14 (CH₃O-3'), 56.03 (CH₃O-4').

6,7-Dimethoxycoumarin (3) - IR n 3402, 1712, 1610, 1496 cm-1. EIMS 70 eV m/z (rel. int.): 206 ([M]+, 100), 178 ([M-CO]+, 94), 163 ([M-CH3-CO]+, 62), 135 (35). 1H NMR (200 MHz, CDCl3): dH 7.96 (d, J = 9.6 Hz, H-4), 6.41 (br s), 6.29 (br s), 6.15 (d, J = 9.6 Hz, H-3), 3.89 (s, CH₃O-6), 3.85 (s, CH₃O-7). 13C NMR (50 MHz, CDCl3): dC 164.4 (C-2), 162.2 (C-7), 157.6 (C-6), 157.5 (C-9), 139.4 (C-4), 111.6 (C-3), 104.7 (C-10), 95.5 (C-5), 93.5 (C-8), 56.6 (CH₃O-7), 56.5 (CH₃O-6).

Microorganisms, growth conditions, and preparation of inoculum - Fungi studied were Colletotrichum sp., P. digitatum, and Curvularia sp., isolated from Citrus (Johann 2003), and T. mentagrophytes and M. canis (clinical isolates). They were maintained on potato dextrose agar (PDA) at 4ºC before being tested. Experiments were carried out on PDA and nutrient agar. Inoculum was prepared in order to attain a fungal suspension of 5.10⁵ spores/ml, in nutrient broth (Espinel Ingroff et al. 1993).

Bacterial species were E. coli ATCC 25922, and S. aureus ATCC 2593. The bacteria were maintained in Brain Heart Infusion medium at -20ºC and tested in Müeller-Hinton broth. Inoculum was a bacterial suspension adjusted to 10⁸ c.f.u./ml (Robbins 1976).

All media were purchased from Difco Laboratories.

Bioautography tests - Extracts and isolated compounds from Citrus spp. were dissolved in hexane or dimethylsulfoxide (DMSO) (Merck) depending on the polarity of the sample, to provide concentrations of 100 µg/ml; 50 µl were then applied to silica gel TLC plates of 60F₂₅₄ (Merck) with graduated micropipettes (Rahalison et al. 1994). The plates were submerged twice in one of the bacterial or fungal suspensions for 5 min, and then transferred to sterile Petri dishes and incubated for 24 h at 37ºC for bacteria, and 72 h at 30-35ºC for fungi, in a hermetic bell-jar. Inoculated plates were then sprayed with an aqueous solution of p-Iodonitrotetrazolium violet (INT, Sigma®) (1 mg/ml) and incubated for a further 4 h at 36 ± 1ºC. Inhibition was observed as clear zones against a rose-red colored background. The diameters of the zones were measured. Solvents (hexane and DMSO) were used as negative controls, and tetracycline (Sigma Chemical Co, US) and fluconazol (Sigma) (1.60 µg/ml) were used as positive antibacterial and antifungal controls, respectively. Aliquots of the fungal and bacterial suspensions were grown on culture media to verify that the microorganisms remained viable. All tests were performed in duplicate.

Minimum inhibitory concentration (MIC) - The MIC was determined through a standard two-fold microdi-lution technique (Souza et al. 2005). Nutrient broth was used for fungi and Muller Hinton broth for bacteria. Susceptibility was determined by the microbroth dilution method performed in sterile flat-bottom 96-well micro-plates (Difco Laboratories, Detroit, MI, US). Extracts and fractions were dissolved in DMSO after the addition of appropriate culture media. Serial dilutions were then performed maintaining a constant volume of 1000 µl per tube. The natural products were tested at eight concentrations from 1000 to 7.8 µg/ml. Tetracycline and fluconazol were also included, at an initial concentration of 0.8 mg/ml.

After inoculation with the microorganisms, plates were incubated at 37ºC for 24 h for bacteria, and 35ºC for 72 h for fungi. The endpoints were determined visually by comparison with drug-free growth in control wells. The MICs, expressed in µg/ml were defined as the lowest extract concentration for which the well was optically clear. Tests were performed in duplicate.

RESULTS AND DISCUSSION

Compounds isolated in the present study were tested for their antimicrobial activity against human pathogenic fungi and bacteria. Besides microorganisms of medical importance, plant pathogenic microorganisms were also tested. They were P. digitatum, Colletotrichum sp., and Curvularia sp. These fungi were isolated from the same lots of C. sinensis fruits used for the extraction of the tested compounds.

Results obtained by bioautography tests against fungi and bacteria are given in Table I. In this assay, bacteria were more sensitive to the citrus extracts than were the fungi. Comparing the bioautographic test results (Table I), for each specific substance, with the MIC test results (Table II), shows that the higher inhibition diameter does not always correspond to the minor value obtained with the MIC test. Similar observations have been reported (Yousef & Tawil 1980). It is possible that the presence of different moieties in the basic structures of the substances were present in the solutions and may have interfered with the diffusion rates of the compounds on the silica gel plates.

Comparing the MICs of the wax extracts with the hexane extracts it shows them to have very similar antimicrobial activities, excepting those of C. limon whose hexane extract exhibited a stronger activity against T. mentagrophytes and M. canis than did the wax extract (Table II). As previously mentioned, plant pathogenic fungi tested in this work were isolated from the same lots of fruits that the extracts were obtained from. This fact partially justifies the low activity of the compounds against the plant pathogenic fungi and the lower activity against the human pathogenic fungi.

Antimicrobial activity of flavonoids obtained from plants extracts other than the Citrus fruits has been studied by other authors. Leaf extracts from Helichrysum italicum, flower extracts from Nepeta cataria (Nostro et al. 2000) and epicuticular wax extracts from Arrabidaea brachypoda (Alcerito et al. 2002) contained flavonoids that exhibited antimicrobial activity against S. aureus when submitted to the bioautographic technique.

The wax and crude hexane extracts from Citrus spp. peels were initially analyzed by TLC. By comparing their R_f, the presence of different compounds were observed in the wax and crude hexane extracts, due to the presence of different banding patterns. Three lipophylic substances were obtained (Figure). Peels of C. reticulata fruits yielded the already described flavones tangeretin and nobiletin (Tripoli et al. 2007, Wang et al. 2007). The compound escoparone was isolated from peels of C. limon. No compound was isolated from peels of C. sinensis.

Some studies have related the stage of growth to the production of phenolic compounds by fruits. Ortunõ et al. (1999) studied different species of Citrus and reported the presence of nobiletin, sinesetin, and tangeretin at the exponential growth phase and quercetogetin and heptamethoxyflavone during the stationary phase. According to these authors, compounds observed at the exponential phase could be the precursors of those observed during the stationary phase. In the present study, nobiletin and tangeretin were detected in peels of fruits at exponential phase. The presence of 6,7-dimethoxy-coumarin in citrus fruits after 6 days of storage was observed by Tatum and Berry (1997). These authors suggested that this compound is produced when fruits are either under stress conditions or at their senescent stage.

A correlation between the production of compounds and stress has been noticed during the course of plant infection by microorganisms. The gradual increase of 6,7-dimethoxy-coumarin content in C. aurantium, C. limon, C. paradise, C. sinensis, Ponctirus trifoliata, and Troyer citrange was observed during the course of infection by Phytophthora citrophtora, indicating that this process is part of the plant's responses to invading pathogens (Afek et al. 1986).

Our results indicate that the peels of the Citrus species present substantial antimicrobial properties. Antifungal activity of some of the extracts and compounds was of such a level that it would probably be therapeutically useful, and it is possible that some of the extracts may be clinically applicable for the treatment of dermatophyte infections caused by M. canis.

ACKNOWLEDGMENT

To Dr Geoff Carr, London, for reviewing the manuscript.

Received 20 December 2006

Accepted 16 July 2007

Financial suport: CNPq, Capes

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Publication Dates

• Publication in this collection
  31 Aug 2007
• Date of issue
  Sept 2007

History

• Accepted
  16 July 2007
• Received
  20 Dec 2006