Evaluation of the antibacterial and antifungal activity of citrus fruit essential oil

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ORIGINAL ARTICLE

RODRIGUES, Amanda Sabino [1], SILVA, Michelli Aparecida Bertolazo da [2]

RODRIGUES, Amanda Sabino. SILVA, Michelli Aparecida Bertolazo da. Evaluation of the antibacterial and antifungal activity of citrus fruit essential oil. Revista Científica Multidisciplinar Núcleo do Conhecimento. Year 03, Ed. 06, vol. 03, pp. 106-118, June 2018. ISSN: 0959-2448, Access link: https://www.nucleodoconhecimento.com.br/health/citrus-fruits

ABSTRACT

Essential oils of citrus fruits represent an important class of chemicals secreted by plants, showing several benefits for human health, plants that hold great interest generating antimicrobial activity in the pros biotechnology therefore many microorganisms exhibit resistance to antibiotics. In this context, evidence of biological activity in extracts allows the development of new products. The objective of this work to evaluate the antibacterial and antifungal activity of the essential oils of Tahiti lime (Citrus aurantifolia) and Pear orange (Citrus sinensis) against the microorganisms Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus epidermidis (ATCC 14990) and  Candida albicans (ATCC 289065). The method used for extraction of essential oils was distillation by steam, using the solvents hexane and dichloromethane for separation of fractions. The evaluation of the results was carried out by measuring the diameter of the inhibition halo on the growing front discs soaked with 10 µ l of each fraction. The results for both are promising, requiring the completion of new reviews for confirmation of the efficacy of the product.

Keywords: Essential oil, Pear orange, Tahiti lime, Antibacterial activity, Antifungal activity.

1. INTRODUCTION

The Brazilian production of oranges and lemons in the 2013 harvest, according to the IBGE – Brazilian Institute of Geography and Statistics, was 18.5 million tons. Most of the juice production is in the state of São Paulo, which generates a considerable amount of industrial waste. Concern for the environment has motivated researchers to find a use for industrial waste, as these, after being generated, need a suitable destination to avoid generating environmental impacts, harming biodiversity. Therefore, there is a growing interest of Biotechnology in studies proposing alternative uses for biologically active agro-industrial residues, reusing them and adding value to substances that would be discarded. The development of projects that use industrial waste, especially in the development of new drugs, food, energy generation and prevention of environmental pollution, are of great value for sustainable development (PELIZER et al, 2007; REZZADORI et al., 2009; SCHENBERG, 2010).

The number of cases of bacterial and fungal infections, as well as the resistance acquired by these microorganisms against antimicrobials has drawn the attention of the community and researchers. In this context, the search for new active substances in infection control is extremely necessary. If we consider that new plant species are frequently discovered, it is estimated that only 1% of the existing biodiversity has been used. Therefore, there still seem to be many sources to be explored in search of new antibiotics and antifungals (CAUMO et al, 2010).

In view of the above, the present work aims to preliminarily evaluate the antibacterial and antifungal activity of the essential oil of the Pear Orange (Citrus sinensis) and Tahiti Lime (Citrus aurantifolia) peel, aiming at the future development of a product with antimicrobial activity.

2. MATERIALS AND METHODS

To carry out the experiments, Pear Oranges (Citrus sinensis) and Tahiti Lime (Citrus aurantifolia) were obtained ready for consumption in a Supply Center of Paraná S/A – CEASA/PR.

2.1 PEAR ORANGE ESSENTIAL OIL EXTRACTION

The extraction of the essential oil of Pear orange was conducted according to the methodology of Trancoso (2013): initially, isolated the part of the flavedo is (outside of the peel) that were chopped in a 4 mm diameter using 128 grams of bark for each extraction. Three extractions were performed with Pear orange. The method used was the hidrodestilação in Clevenger apparatus (distillation by steam drag) with addition of 500 ml of distilled water with duration of 5 hours in constant temperature of 96 degrees C.

2.2 TAHITI LIME ESSENTIAL OIL EXTRACTION

Two extractions were carried out with 56 grams of chopped Tahiti lime peel in a diameter of 4 mm, with 200 ml of distilled water for hydrodistillation in a Clevenger apparatus (steam drag distillation) lasting three hours at a temperature of 96 degrees Ç.

2.3 SOLVENT EXTRACTION

The extracts obtained were subjected to separation with dichloromethane and hexane, using 5 ml of solvent for each 100 ml of oil/water solution in a separatory funnel. The solutions obtained were then stored in beakers and left to rest for 24 hours for the total volatilization of the organic solvents, thus obtaining only the essential oil required.

2.4 EVALUATION OF ANTIBACTERIAL ACTIVITY

For the evaluation of the antibacterial effect of the fractions obtained in extraction of essential oil, used bacterial species: Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus epidermidis (ATCC 14990). Microorganisms were sown in sterile Petri dishes with agar Mueller-Hinton KASVI ®.

Were used for development of inhibition halos sterile filter paper discs soaked in 10 μL of each fraction obtained essential oil in solvent extraction. To control were used, amikacin, antibiotics clindamycin and Vancomycin DME ®. The plates were stored in an oven at 37 degrees C for 48 hours. The halos of inhibition were then measured for preliminary analysis of antimicrobial activity. All tests were performed in triplicate.

2.5 EVALUATION OF ANTIFUNGAL ACTIVITY

For the evaluation of the antibacterial effect of the fractions obtained in extraction of essential oil, fungal species was used Candida albicans (ATCC 289065), sown in sterile Petri dishes with agar Sabouraud KASVI ®.

Sterile filter paper discs soaked in 10 μL of essential oil of each fraction obtained in the solvent extraction were used to verify the development of inhibition halos. For control, sterile filter paper discs soaked in Nystatin cream 25,000 IU/g (NEO QUÍMICA®) were used. The plates were stored at 25 degrees C for 96 hours. Inhibition halos were then measured for preliminary analysis of antimicrobial activity. All tests were performed in triplicate.

3. RESULTS AND DISCUSSION

3.1 EVALUATIONS OF ANTIBACTERIAL ACTIVITY

3.1.1 EVALUATION OF ANTIBACTERIAL ACTIVITY: PEAR ORANGE (CITRUS SINENSIS)

Table 1 shows the result of the average diameter of inhibition obtained from the evaluations of the antibacterial activity of the essential oil of Pear orange in extractions with solvent dichloromethane and hexane. It can be observed that there was no presence of halos of inhibition for the fractions tested essential oil. However, this does not prevent new research being carried out regarding the potential of the antibacterial effect in front of S. aureus and S. epidermidis, testing other extraction methods.

The antibiotics Vancomycin, Clindamycin and Amikacin were used as positive control in this test. Amikacin presented the best result for the two microorganisms, and for S. aureus the average of the 25.4 mm was halos and for S. epidermidis was 32.1 mm average.

Table 01. The average diameter of inhibition of the fractions of essential oil of Pear orange and controls for Staphylococcus aureus and Staphylococcus epidermidis.

Antimicrobial/Fraction Average value of the halo of inhibition (mm)

S. aureus

Average value of the halo of inhibition (mm)

S. epidermidis

Clindamycin 17.4 27.8
Vancomycin 15.6 19.3
Amikacin 25.4 32.1
Dichloromethane fraction 0 0
Hexane fraction 0 0

3.1.2 EVALUATION OF ANTIBACTERIAL ACTIVITY: TAHITI LIME (CITRUS AURANTIFOLIA)

Are expressed in table 2 the values of the average diameter of inhibition obtained from the evaluations of the antibacterial activity of the essential oil of Tahiti lime in extractions with solvents dichloromethane and hexane. You can see that there has been development of inhibition halos for all micro-organisms tested with the dichloromethane fraction. Taking into consideration the hexane fraction, there was development of inhibition halos for S. epidermidis and P. aeruginosa.

Table 2. The average diameter of inhibition of the fractions of Tahiti lime essential oil and controls for Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa.

Antimicrobial/Fraction Average value of the halo of inhibition (mm)

S. aureus

Average value of the halo of inhibition (mm)

S. epidermidis

Average value of the halo of inhibition (mm)

P. aeruginosa

Amikacin 25.45 28.5 37
Vancomycin 0 0 19
Clindamycin 23.15 23.15 22.6
Dichloromethane fraction 7.3 12.3 14
Hexane fraction 0 12.6 10.3

The Tahiti Lime (Citrus aurantifolia) has 70% of d-limonene in your shell, where the essential oil is extracted (JUNIOR et al., 2007). According to Oliveira (2013) d-limonene, has extensive industrial application in the cosmetic area as fragrance and food area as antimicrobial agent that might be observed in this study.

The result obtained in plates with micro-organisms P. aeruginosa and S. epidermidis was shown to be significant, with diameter of halos above 10 mm showing some sensitivity of microorganisms in relation to essential oil. These results were similar to those demonstrated by Soares (2008), which reported development of inhibition halos of 14 mm.

Subramenium et al., (2014) explored the effectiveness of d-limonene as potential antibiotics against species of the genus Streptococcus and concluded that the Limonene has approximately 75-95% of antibiotic activity against the pathogens tested.

Gerhardt et al., (2012) conducted the evaluation of antibacterial activity of alcoholic extracts from bark of citrus bergamot-ponkan, pomelo and lemon-bergamot, as the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). In this study, the extract of lemon-bergamot presented the best result and the bacteria more sensitive to the extracts was P. aeruginosa.

3. 2 EVALUATION OF ANTIFUNGAL ACTIVITY

3. 2.1 EVALUATION OF ANTIFUNGAL ACTIVITY: PEAR ORANGE (CITRUS SINENSIS)

In table 3 can observe the average values of diameter of inhibition obtained from the evaluations of the antifungal activity of the essential oil of Pear orange in extractions with solvent dichloromethane and hexane solvent. One can observe relevant sensitivity of the fungus in front of the dichloromethane fraction.

Table 3. The average diameter of inhibition of the fractions of essential oil of Pear orange and control for C. albicans.

Antimicrobial/Fraction Average value of the halo of inhibition (mm)

C. albicans

Nystatin 13
Dichloromethane Fraction 17
Hexane Fraction 0

Extraction with dichloromethane showed the best result, where the diameter of the halo was 17 mm. Oliveira et al. (2007) showed a comparison of the antioxidant and antifungal activity of lemon, orange, apple, eggplant, potato, rice grains and wheat on the fungus Aspergillus flavus. Among all the fruits, orange showed 80% and lemon 95% of antifungal activity, showing the highest percentages of inhibition.

Similar results were obtained by Cavalcanti et al., (2012) in which conducted a research of antifungal activity of essential oils of Orange, lemon, Mandarin, pindaíba and guabiroba about strains of C. albicans (ATCC 289065), C. tropicalis ( ATCC 40042), C. krusei (ATCC 40147) of the oral cavity, which leads to the development of oral candidiasis. In this research, the Pear orange presented 14.5 mm halo to C. albicans, C. tropicalis 16.5 mm and 40.0 mm to C. krusei.

According to Martos et al., (2007) in their study on the effect of Lemon, Tangerine, Grapefruit and Orange essential oils on A. niger, A. flavus, P. chrysogenum and P. verrucosum, the Orange essential oil was the most effective against A. niger.

3.2.2 EVALUATION OF ANTIFUNGAL ACTIVITY: TAHITI LIME (CITRUS AURANTIFOLIA)

In table 4 can be observed the values of average diameter of inhibition obtained from the evaluations of the antifungal activity of the essential oil of Tahiti lime in extractions with solvent dichloromethane and hexane. You can see some sensitivity of the fungus in front of the dichloromethane fraction.

Table 4. The average diameter of inhibition of the fractions of Tahiti lime essential oil and control for C. albicans.

Antimicrobial/Fraction Average value of the halo of inhibition (mm)

C. albicans

Nystatin 13.5
Dichloromethane Fraction 8.3
Hexane Fraction 0

According to Bhagavani et al., (1992), 60% of essential oils possess antifungal activity and 35% exhibit antibacterial activity. Candidiasis has been a very common infection and Candida albicans is considered one of the major causative agents of this (LIMA et al., 2006).

It was possible to observe the presence of significant diameter inhibition halos on extraction with dichloromethane, showing the result of the average inhibition halo to 8.3 mm. Lima et al. (2006) showed an equivalent result, in that the essential oil of lemon (C. limon) inhibited 42% of strains of different species of Candida which were used, with halos of inhibition up to 10 mm.

Araújo et al., (2005) presented the in vitro antifungal effect of lemon peel extract on C. albicans, C. tropicalis, C. stellatoidea and C. krusei, the lemon extract showed antifungal activity against C. albicans, C. tropicalis and C. krusei with inhibition zones of 11 mm to 18 mm.

CONCLUSION

The results obtained show that:

The essential oil of Pear orange showed no antibacterial activity against microorganisms S. aureus  and S. epidermidis.

Tahiti lime essential oil showed antibacterial activity against the strains of S. aureus, S.epidermidis and P. aeruginosa, extraction with dichloromethane exhibited halos with larger diameter compared to extraction with hexane.

Evaluation of antifungal activity, essential oils of Tahiti lime and Pear orange showed activity in the extraction with dichloromethane with emphasis on the essential oil of Pear orange showing greatest potential inhibitor.

There is a need for more research in an attempt to analyze the antimicrobial activity of the oils used, considering other variables such as: different solvents extraction techniques, micro-organisms, toxicity, as well as use of methods of analysis of income, isolation and purification of active principles, seeking the applicability of components in the treatment of infections.

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[1] Academic degree in Biotechnology from the University Tuiuti do Paraná (Curitiba, PR).

[2] Pharmacist Profa. Doctoral candidate at the University Tuiuti do Paraná (Curitiba, PR).

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