Citrus
Antibacterial and Antioxidant Activity of Essential Oils from Citrus spp. S. Frassinetti,* L. Caltavuturo, M. Cini and C. M. Della Croce National Research Council, Institute of Biology and Agricultural Biotechnology (IBBA), Pisa Unit, Research Area of Pisa, Via Moruzzi 1, 56124, Pisa, Italy
B. E. Maserti National Research Council, Institute of Biophysics (IBF), Pisa Unit, Research Area of Pisa, Via Moruzzi 1, 56124, Pisa, Italy Abstract The antibacterial and antioxidant activities of essential oils from Bitter orange, Sweet orange, Lemon and Mandarin were investigated. The antimicrobial capability of these oils was determined against ten strains of Gram-negative and Gram-positive bacteria, including some phytopathogenic strains. The antibacterial activity of the oils was expressed as minimum inhibitory concentrations (MICs). All oils showed good antibacterial activity against both Gram-negative and Gram-positive bacteria. The MICs for selected oils ranged 15–250 μg/mL. The lowest MICs were 15 μg/mL and 20 μg/mL against Xanthomonas citri strains, respectively. The antioxidant and antiradical scavenging properties of the selected oils were tested by means of 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. All examined oils exhibited a free radical scavenging activity, ranging 20–70% of DPPH inhibition. Lemon oil showed the most antioxidant capacity, with DPPH inhibition rate of 70%. Key Word Index Citrus aurantium, Citrus sinensis, Citrus limon, Citrus reticulata, Rutaceae, essential oil composition, antibacterial activity, antioxidant activity.
Introduction Essential oils have been shown to exhibit antibacterial, antifungal and antioxidant properties (1, 2). The concern over the use of essential oils as antimicrobial agents has increased, due to an emergent microbial resistance towards conventional synthetic antimicrobial preservatives. Essential oils are widely used in medicine (2-4), in pharmaceutical and cosmetic industries (5), and in the food industry, where they are used both as flavoring additives and as antioxidants for preservation of stored food crops (6, 7) instead of synthetic chemicals charged to be cytotoxic (8). Among essential oils, those from Citrus plants are particularly interesting, because they could be used in food both as antioxidants (9, 10) and as flavoring compounds. Moreover, Rossi (11) reported the antimicrobial activity of C. sinensis and C. reticulata against both Gram-negative and Gram-positive bacteria. Citrus oils can have antifungal activity, even if their complexity makes it difficult to correlate their action to a specific component. The effects of the volatile component of the Citrus oils and peel extracts against Penicillium have been reported (12, 13); the fungitoxicity of C. sinensis oils against Aspergillus niger has also been described (14). However, there are very
few detailed reports on both antimicrobial and antioxidant properties of Citrus oils. The aim of the present research was to study both antibacterial and antioxidant activities of four Citrus (Bitter and Sweet orange, Lemon and Mandarin) oils, and to test their possible use as preservatives and antioxidant additives in the food industry.
Experimental Essential oils: The essential oils—Bitter orange, Sweet orange, Lemon and Mandarin—used in this work are commercially available from Aboca (Perugia, Italy) The chemical analysis of the oils had been performed by Aboca using gas chromathographic analyses (GC Trace 2000 Termoquest Instrument , according to GC-FID ISO 7609: 1985 method). Aboca kindly provided the authors both the products and the main constituent chemical composition. The plant species and the organs used for oil extraction (by cold-pressing method) are listed in Table I. Microorganisms: Citrus oils were assayed against Gramnegative and Gram-positive bacteria (Table II). Three bacterial strains of phytopathogen Xanthomonas campestris pv. citri
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Table I. Sources of Citrus oils used in this study and main components Plant species
Common name
Product tested
Part of plant
Citrus aurantium L. Orange EO Fruit Citrus sinensis (L.) Osbeck Sweet Orange EO Fruit Citrus limon (L.) N.L. Burm. Lemon EO Fruit Citrus reticulata Blanco Mandarin EO Fruit
Table II. Bacterial strains used in this study Bacteria Gram-negative Xanthomonas campestris pv citri Xanthomonas campestris pv citri Xanthomonas campestris pv citri Escherichia coli Escherichia coli Salmonella choleraesuis Pseudomonas aeruginosa Gram-positive Staphylococcus aureus Enterococcus faecalis Enterobacter aerogenes
strain NCPPB 3236 NCPPB 3562 NCPPB 3832 ATCC 10536 ATCC 25922 ATCC 14028 ATCC 27853
ATCC 25923 ATCC 19433 ATCC 13048
NCPPB: National Collection of Plant Pathogenic Bacteria, Central Science Laboratory, York, U.K. ATCC: American Type Culture Collection, Rockville, MD
from the NCCPB (National Collection of Plant Pathogenic Bacteria, Central Science Laboratory, York, UK) and seven strains of bacteria from the ATCC (American Type Culture Collection, Rockville, MD) were used. The bacterial strains and their ATCC numbers are listed in Table II. Subcultures were obtained by growing bacteria for 24 h in Oxoid Nutrient Broth (NB) at 37°C. Antimicrobial Activity Assay: The Broth microdilution assay method (15,16) was used for the determination of antimicrobial activity of the oils, with some modifications, as follows. Bacterial cultures grown over 16 h were diluted with sterile physiological saline solution with reference to the McFarland standard (bioMèrieux, Mary l’Etoile, France) to achieve inoculums of approximately 105 cell/mL; these cultures (100 µL) were inoculated in 2 mL of Mueller Hinton Broth medium (MHB; Oxoid, Basingstoke, UK). Then, 100 µL of different dilutions of the oils in DMSO (Sigma) were added to triplicate test tubes, to give final concentration ranging 10–500 µg/mL. The cultures were incubated at 37°C for 24 h. Control samples and blank samples were incubated under the same conditions. The final concentration of DMSO did not exceed 2% v/v and did not affect the bacterial growth. The bacterial growth in the presence of oil samples (at different concentrations) was compared visually with the growth of control cultures. Determination of minimum inhibitory concentration (MIC): The optical density (750 nm) of the bacterial cultures, performed as described above, was successively determined by 28/Journal of Essential Oil Research
Main constituent d-limonene 93% myrcene 1.85% d-limonene 95% myrcene 1.88% d-limonene 70%
g-terpinene 9.5%
d-limonene 67%
g-terpinene 17%
a Perkin-Elmer spectrophotometer. The backgrounds for each sample and the growth of control cultures were also measured. All assays were repeated three times. From these results, the antibacterial activity was expressed as MIC (17). MIC was defined as the lowest EO concentration giving a reduction of > 90% in the observed absorbance (16). Antioxidant Activity Assay/DPPH radical scavenging assay: The antioxidant activity was measured using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical (Sigma-Aldrich) reduction assay, according to the method of Brand-Williams (18). The free radical-scavenging activity of each oil was determined according to the method of Tepe (19), with some modification. Aliquots 0.1 mL of the oil solutions in methanol 80% (ranging 50–1000 µg/mL) were mixed with 1.9 mL of DPPH solution (0.2 mM in methanol 80%). The mixture was shaken and left at room temperature for 30 min in the dark. The absorbance of the solution was measured at 517 nm in a Perkin-Elmer spectrophotometer. The radical scavenging activities, expressed as percentage inhibition of DPPH, were obtained from the following equation: Scavenging effect (%) = {[A0 – (A –Ab)] /A0 ] } where A0 was the control (Absorbance 517 of DPPH solution), Ab was the blank (Absorbance 517 of essential oil solutions), and A was the sample absorbance (Absorbance 517 of the oils with DPPH). Trolox (Sigma-Aldrich), a water-soluble analogue of vitamin E, showing a potent antioxidant activity, was used as a standard reference. A Trolox standard calibration curve for DPPH radical was measured at the concentration range of 10–300 μm. The curve equation was: y = 12.6 + 0.17x r = 0.96. Each oil sample was tested in triplicate. Data were expressed as a mean of three independent experiments ± standard deviation, and analyzed by the student’s “t” test. Differences are considered significant when p = 0.05.
Results and Discussion Four different, commercially available Citrus oils were tested for their antimicrobial activities against ten strains of Gram-negative and Gram-positive bacteria, including some phytopathogenic strains (Table II). The antimicrobial activities have been determined by measuring the minimum inhibitory concentration (MIC) (Table III). All of the Citrus oils tested showed a great potential against the examined species. The MIC for selected Vol. 23, January/February 2011
Citrus
Figure I. Antibacterial activity of the Citrus essential oils against the Gram-negative Escherichia coli ATCC 10536. Growth on MHB medium at 37°C for 24 h. Data were expressed as means ± sd of three different experiments.
Figure II. Antibacterial activity of the Citrus essential oils against the Gram-positive Staphylococcus aureus ATCC 25293. Growth on MHB medium at 37°C for 24 h. Data were expressed as means ± sd of three different experiments.
oils ranged 15–230 μg/mL. In particular, C. limon showed the lowest MIC values of 15 μg/mL and 20 μg/mL, respectively, against Xanthomonas citri strains. This antimicrobial activity might be attributed to the presence of limonene and gamma Vol. 23, January/February 2011
terpinene (Table I), which have been reported to exert their toxic effects through the disruption of bacterial or fungal membrane integrity and the inhibition of respiration and ion transport processes (20). Among the examined bacteria, both Journal of Essential Oil Research/29
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Table III. Minimum inhibitory concentrations (MICs) of Citrus oils against tested bacteria
Citrus aurantium Bitter Orange
Citrus sinensis Sweet Orange
Citrus limon Lemon
Citrus reticulata Mandarin
Gram-negative Xanthomonas campestris pv citri NCPPB 3236 20 20 15 Xanthomonas campestris pv citri NCPPB 3562 20 20 15 Xanthomonas campestris pv citri NCPPB 3832 25 25 20 Escherichia coli ATCC 10536 35 35 40 Escherichia coli ATCC 25922 25 25 30 Salmonella cholerasuis ATCC 14028 50 50 50 Pseudomonas aeruginosa ATCC 27853 75 70 70 Gram-positive Staphylococcus aureus ATCC 25923 200 200 200 Enterococcus faecalis ATCC 29212 150 150 150 Enterobacter aerogenes ATCC 13048 100 100 130
20 20 25 40 35 55 70
230 150 120
Figure III. Free radical-scavenging activity of Citrus essential oils evaluated by the DPPH assay, and comparison with that of the reference (Trolox 100 μg/mL). Data were expressed as means ± sd of three different experiments.
Gram-positive and Gram-negative bacteria were sensitive to the Citrus oils, but the Gram-positive strains were more resistant, showing the highest MIC values. For the better elucidation of the antibacterial activity of Citrus oils, the bacterial growth of representative Gram-negative (Escherichia coli ATCC 10536), and Gram-positive strain (Staphylococcus aureus ATCC 25293) are shown in Figures I and II. The Gram-negative strains showed a very high sensitivity to the Citrus oils; in particular Pseudomonas aeruginosa was inhibited at 70–75 μg/mL (MIC), while E. coli strains were most susceptible, showing MIC values ranging 25–45 μg/mL (Table III). The Gram-positive strains Enterococcus faecalis, Enterobacter aerogenes and S. aureus were inhibited at oils concentrations ranging 100–250 μg/mL (MIC). Although Gram-negative organisms are generally reported to be more resistant to active antimicrobial compounds (21), 30/Journal of Essential Oil Research
other studies have found Gram-positive bacteria less sensitive to essential oils than Gram-negative strains (22, 23). The results obtained in the present study are in agreement with the latter literature data. Moreover, other authors suggested that the simple relation involving cell structure and microbial sensitivity to essential oils is not yet well established, and possible antagonistic or synergistic effects among the various active constituents of the oils should be taken into consideration (24). The use of natural antioxidants are of great interest in the food industry, since their possible use as natural additives emerged from a growing tendency to replace synthetic antioxidants with natural ones, because most antioxidants currently employed, such as BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene) can be cytotoxic and increase the development of cancerous cells (8, 10). Hence in this work the antioxidant properties of Citrus oils Vol. 23, January/February 2011
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were also tested. Free radical generation is directly related to oxidation in foods and biological systems; therefore, the determination of free radical scavenging in food is important (25). Among the numerous of methods that can be used, TEAC, DPPH, and PCL are functional for determining the activity of both hydrophilic and lipophilic species, thus ensuring a better comparison of the results and proposing a wider range of possible applications. Taking this into account, the antioxidant activity was determined by the DPPH test, because this method is simple, reproducible and is widely used in the food industry (26). All the tested Citrus oils showed a good antioxidant activity depending on the concentration. Lemon oil showed the best antioxidant capacity, with an inhibition rate of 70%, comparable to that of Trolox, a potent synthetic antioxidant and also comparable with those found in Oregano oil by Kulisic et al. (27). The present work’s results are also in agreement with those obtained by Wei and Shibamoto, demonstrating the scavenging abilities ranging 39–90% at a level of 200 μg/mL of various essential oils (28). The significant antioxidant activity showed by all the tested Citrus oils might be correlated to the presence of monoterpenes, particularly g-terpinene and limonene, which are the most abundant compounds in the oils examined (Table I) and have been reported to posses good antioxidant activity (28, 29). In conclusion, our results provide a further confirmation that the oils of Citrus spp. may be used as a potential natural antioxidant and antimicrobial agents in the food industry. Acknowledgements:
This study was part of the project Programma Internazionale Pic Interreg Iii- A Italia-Francia “Isole” (2000-2006): Il Citrus come sistema modello per l’area mediterranea: studio varietale per resistenza a stress biotici e abiotici. Le Citrus comme modèle de système pour la zone méditerrranéenne: étude variétale de résistance aux Stress biotiques et abiotiques. References 1.
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