Evaluation of Antibacterial and antifungal Activity of the essential oil from citrus fruits

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Evaluation of Antibacterial and antifungal Activity of the essential oil from citrus fruits
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RODRIGUES, Amanda Sabino [1], SILVA, Michelli Aparecida Bertolazo da [2]

RODRIGUES, Amanda Sabino; SILVA, Michelli Aparecida Bertolazo da. Evaluation of Antibacterial and antifungal Activity of the essential oil from citrus fruits. Multidisciplinary Core scientific journal of knowledge. 03 year, Ed. 06, vol. 03, pp. 106-118, June 2018. ISSN: 0959-2448

Summary

Essential oils of citrus fruits represent an important class of chemicals secreted by plants, showing several benefits for human health, plants that holds 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 orange (Citrus sinensis) Pear 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 drag 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, Orange, pear, Tahiti lime, antibacterial activity, Antifungal Activity.

1. Introduction

The brazilian production of Orange and lemon in 2013, according to the IBGE – Brazilian Institute of geography and statistics, was 18.5 million tonnes. Most of the production is in the State of São Paulo to the production of juice, which generates a considerable amount of industrial waste. The concern for the environment has motivated researchers to find a utility to industrial waste, as these, once generated, need an appropriate destination not to generate environmental impacts, harming biodiversity. Given this, there is a growing interest in biotechnology studies proposing alternative uses for the agroindustrial waste biologically active, reusing and adding value to substances that would otherwise be discarded. The development of projects using industrial waste, especially in the development of new drugs, food, power 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 resistance acquired by these microorganisms antimicrobial has called attention to front the community and researchers. In this context, the research of new active substances in the control of infections if makes it extremely necessary. If we consider that new species are discovered frequently, it is estimated that only 1% of the scope of existing biodiversity has been used. Thus, there seem to be many sources yet to be explored in search of new antibiotics and antifungals (MICHAEL CASE et al., 2010).

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

2. Material and methods

For conducting the experiments were obtained Pear Oranges (Citrus sinensis) and Thaiti Lemons (Citrus aurantifolia) ready-to-eat in a Supply Center of Paraná S/A-CEASA/PR.

2.1 Orange essential oil Extractions Pera

The extraction of the essential oil of Orange Pear 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 orange pear. 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 Extracts

There were two extractions with 56 grams of Tahiti minced lemon peel in a 4 mm diameter, with 200 ml of distilled water to hidrodestilação in Clevenger apparatus (distillation by drag the value) with duration of 3 hours at temperature of 96 degrees C.

2.3 solvent extraction

The extracts obtained were split 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 stand for 24 hours to complete the volatilization organic solvents, thus, 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 ®.

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 disks were used sterile filter paper soaked in Nystatin cream 25,000 IU/g (NEO ® CHEMISTRY), the plates were stored at a temperature of 25 degrees C for 96 hours. The halos of inhibition were then measured for preliminary analysis of antimicrobial activity. All tests were performed in triplicate.

3. Result and discussion

3.1 evaluation of antibacterial activity

antibacterial activity Assessment 3.1.1 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 Orange Pear 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 researches are 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 Orange Pear 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

 

antibacterial activity Assessment 3.1.2 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 Lemon Thaiti 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 lemon essential oil Thaiti 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 (JAIN et al., 2007). According to Oliveira (2013) d-limonene, has extensive industrial application in 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 Smith (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 (CBM) . 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

antifungal activity Evaluation 3.2.1 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 Orange Pear 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 Orange Pear 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

 

The extraction with dichloromethane exhibited the best result, where the diameter of the halo was 17 mm. Oliveira et al. (2007) showed a comparison of antifungal and antioxidant activity of fruits lemon, Orange, Apple, eggplant, potatoes, grains of rice and wheat on the fungus Aspergillus flavus. Among all the fruits, the Orange and the lemon 80% exhibited 95% antifungal activity, featuring the largest percentages of inhibition.

Similar results were obtained by Chandra 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 Orange PEAR 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 your study on the effect of the essential oils of lemon, Tangerine, Grapefruit and orange on the niger, a. flavus …, p. chrysogenum, and p. verrucosum, Orange essential oil was more effective against a. niger.

Evaluation of antifungal activity 3.2.2 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 lemon Thaiti 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 lemon essential oil Thaiti 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) showed the in vitro antifungal effect of the extract of lemon peels on c. albicans, c. tropicalis, c. stellatoidea, c. krusei, lemon extract presented antifungal activity on c. albicans, c. tropicalis and c. krusei with halos of inhibition of 11 mm to 18 mm.

Conclusion

The results obtained show that:

The essential oil of Orange PEAR showed no antibacterial activity against micro-organisms 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 orange Pear showed activity in the extraction with dichloromethane with emphasis on the essential oil of Orange Pear 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.

References

AGUILAR, C. R. F.; PERE IRA, M. S. V.; HAILU, J. S.; PEREIRA, J. V.; MALHOTRA, a. b. in vitro antifungal Effect of Citrus limon linn. about Candidas. Brazilian Dental Journal. Vol. 63, no. 4, p. 227-229. 2005.

BHAVANANI N. M.; BALLOW C. H. New agents for Gram-positive bacteria. Curr Opin Microbiol. Vol. 13. p. 528. Oct. 2000.

BIZZO, H. R. Essential oils in Brazil: General aspects, development and perspectives. New Chemistry. Vol. 32. No. 3. p. 591. 2009.

MICHAEL CASE, K.; DUARTE, M.; CARGNIN, S. T.; RAJ, V.; TASCA, T.;  MACEDO, a. j. bacterial resistance in the environment and implications in the hospital clinic. Liberato magazine. New Hamburg, vol. 11, n. 16, p. 184-188.  Jul/dez. 2010.

CHANDRA, Y. W.; ALMEIDA, L. F. D.; Padilha, w. w. n. antifungal Activity of essential oils on three strains of Candida. Brazilian Dental Journal Central. Vol. 20. No 52. 2011.

GERHARDT, c. use of citrus peel in the perspective of food: prospection of antibacterial activity. Braz. J. Food Technology. Vol. IV. p. 11-17. May. 2012.

JAIN, M. R. M.; PASTORE, g. m. biotransformation of Limonene: a review of the main metabolic routes. New chemistry, vol. 30, no. 2, p. 382-386. 2007.

LIMA, I.; OLIVEIRA, A. G.; LIMA, E.; FARIAS, N. M. P.; Souza, e. l. antifungal Activity of essential oils on Candida species. Brazilian Journal of Pharmacognosy.  Vol. 16, no. 2, p. 197-201. Apr 2006/Jun..

MARTOS, M. V.; NAVAJAS, Y. R.; LOPEZ, j. f. Antifungal activity of lemon (Citrus lemon l.), mandarin (Citrus reticulata l.), grapefruit (Citrus paradisi l.) and orange (Citrus sinensis l.) essential oils. Food Control. Vol. 19, no. 12, p. 1130-1138. Dec. 2008.

OLIVEIRA, M. S.; DORS, G. C.; SMITH, L. A. S.; FURLONG, e. b. antioxidant and antifungal Activity of plant extracts.  Nutr. Araraquara. Vol. 18, no. 3, p. 268-273,/set.. 2007.

OLIVEIRA, s. Biodegradation and bioconversão of d-limonene by bacteria isolated from domestic sewage. 2013. Dissertation (master in development of biotechnological processes)-Faculty of chemical engineering. State University of Campinas, Campinas-SP.

PELIZER, L. H.; PONTIERI M. H.; MATHEW i.. agro-industrial wastes utilization in biotechnological processes as environmental impact reduction perspective. Journal of Technology Management & Innovation. Vol. 2, no. 1. P. 119-120. 2007.

REZZADORI, K.; BENEDETTI, s. Propositions for recovery of waste from the processing of orange juice. International Workshop on Advances in Cleaner Production. Federal University of Santa Catarina, Florianópolis, p. 1-3. May. 2009.

SCHENBERG, a. c. g. Biotechnology and sustainable development. Advanced Studies. Vol. 24. No. 70. p. 7-13. 2010.

SMITH, G. D.; OLIVEIRA, B. C.; DRUMOND, S. R. M.; TARIQ, B. D.; SILVA, A. C.; P, N. W. W. Antibacterial activity of tropical plant dyes on microorganisms of the oral cavity. Dental magazine of Araçatuba. Aracatuba, vol. 29. No 01. p. 20-24, jan/jun.2008.

SOBRAMENIUM, G.; VIJAYAKUMAR, K.; PANDIAN n. k. Limonene inhibits streptococcal biofilm formation by targeting surface-associated virulence factors. J. Med. Microbiol. Vol. 62. No. 8, p. 879-890. Aug. 2015.

TRANCOSO, M. D., design: essential oils extraction, importance and applications in everyday life. Revista Praxis. Year 5 No. 09. p. 90-92. Jun 2013.

[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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