Abstract

In this study, it was determinated the essential oil of cultivated apple mint, Mentha suaveolens Ehrh. composition and in vitro antibacterial activity of against 11 fish pathogen bacteria including Gram-positive (Staphylococcus warneri, Staphylococcus sp., Lactococcus garvieae, Vagococus salmoninarum) and Gram-negative (Aeromonas hydrophila, Aeromonas sobria Aeromonas cavieae, Vibrio anguillarum, Pseudomonas aeroginosa, Yersinia ruckeri, Edwardsiella tarda) by using agar diffusion assay. The main component of M. suaveolence oil was obtained as piperitenone oxide. The essential oil exhibited strong inhibitory activity such as inhibition zone sizes: 30-50mm at 250-1000 µL mL^-1 concentrations against V. anguillarum; 16-20mm at 31.25-125 µL mL^-1 concentrations against P. aeroginosa; 15-18mm at 500-1000 µL mL^-1 concentrations against A. sobria. However, it was found to be moderately effective against E. tarda (8-15 mm), Y. ruckeri (9-12mm), S. warneri (9-10mm), V. salmoninarum (9mm) and Staphylococcus sp. (8-9mm). The essential oil showed weak inhibitory activity against A. cavieae (5-8), A. hydrophila (6-7mm), L. garvieae (5-7mm). Thus, effect of essential oil of M. suaveolens on immune response and disease resistance against Vibrio anguillarum, A. sobria and P. aeroginosa should be investigated in vivo in cultured fish species in future studies.

Key words
Mentha suaveolens; essential oil; bacterial fish pathogens; in vitro antibacterial effect

INTRODUCTION

In aquaculture, antimicrobial agents are widely used to prevent fish diseases caused by infectious agents. It is known that the unconscious use of antimicrobial agents in the treatment of fish diseases causes many negative effects on environment, fish and human health (Schnick et al. 1997SCHNICK RA, ALDERMAN DJ, ARMSTRONG R, LE GOUVELLO R, ISHIHARA S, LACIERDA EC, PERCİVAL S & ROTH M. 1997. World wide aquaculture drug and vaccine registration progress. Bull Eur Assoc Fish Pathol 17(6): 251-260.). Therefore, the use of herbal products to control of fish pathogens in aquaculture is an alternative and current practice.

Phytochemicals such as phenolics, polysaccharides, proteoglycans and flavonoids may play a role in preventing or controlling infectious pathogens (Citarasu 2010CITARASU T. 2010. Herbal biomedicines: a new opportunity for aquaculture industry. Aquacult Int 18(3): 403-414.). Many essential oils and plant extracts have been studied and shown to be effective against fish pathogens (Abutbul et al. 2005ABUTBUL S, GOLAN-GOLDHIRSH A, BARAZANI O, OFIR R & ZILBERG D. 2005. Screening of desert plants for use against bacterial pathogens in fish. Isr J Aquacult/Bamid 57(2): 71-80., Bansemir et al. 2006BANSEMIR A, BLUME M, SCHRÖDER S & LINDEQUIST U. 2006. Screening of cultivated seaweeds for antibacterial activity against fish pathogenic bacteria. Aquacult 252: 79-84., Ekici et al. 2011EKICI S, DILER O, DIDINEN BI & KUBILAY A. 2011. Antibacterial activity of essential oils from medicinal plants against bacterial fish pathogens. Kafkas Univ Vet Fak Derg 17: 47-54., Haniffa & Kavitha 2012HANİFFA MA & KAVİTHA K. 2012. Antibacterial activity of medicinal herbs against the fish pathogen Aeromonas hydrophila. J Agr Tech 8(1): 205-211., Al Laham & Al Fadel 2014AL LAHAM SA & AL FADEL FM. 2014. Antibacterial activity of various plants extracts against antibiotic-resistant Aeromonas hydrophila. Jundishapur J Microbiol 7(7): 1-7., Ontas et al. 2016ONTAS C, BABA E, ERCAN MD, KAPLANER E, KUÇUKAYDIN S & OZTURK M. 2016. Antibacterial activity of Citrus limon peel essential oil and Argania spinosa oil against fish pathogenic bacteria. Kafkas Univ Vet Fak Derg 22(5): 741-749., Diler et al. 2017DILER O, GORMEZ O, DILER I & METIN S. 2017. Effect of oregano (Origanum onites L.) essential oil on growth, lysozyme and antioxidant activity and resistance against Lactacoccus garvieae in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquacult Nutr 23: 844-851., Metin et al. 2017METIN S, DIDINEN BI, MERCIMEK EB & ERSOY AT. 2017. Antibacterial activity of some essential plant oils againts some bacterial fish pathogens. Aquacult Stud 17(1): 59-69.). These compounds may constitute alternative prophylactic and therapeutic agents in aquaculture because of their antibacterial properties (Turker & Yıldırım 2015TURKER H & YILDIRIM AB. 2015. Screening for antibacterial activity of some Turkish plants against fish pathogens: a possible alternative in the treatment of bacterial infections. Biotechnol Biotech Eq 29(2): 281-288.).

Mentha, a genus of the Lamiaceae family, is represented by about 31 species and 13 natural hybrids, mainly perennial herbs, growing wildly in damp or wet places throughout Europe, Asia, Africa, Australia and North America (Kumar et al. 2011KUMAR P, MİSHRA S, MALİK A & SATYA S. 2011. Insecticidal properties of Mentha species: a review. Ind Crops Prod 34(1): 802-817.). Mints are fast growing, invasive and generally tolerate a wide range of agro-climatic conditions (Božović et al. 2015BOŽOVIĆ M, PİROLLİ A & RAGNO R. 2015. Mentha suaveolens Ehrh.(Lamiaceae) essential oil and its main constituent piperitenone oxide: biological activities and chemistry. Molecules 20(5): 8605-8633.). The most common and popular mints for cultivation are peppermint (M. piperita), spearmint (Mentha spicata) and apple mint (M. suaveolens) (Chauhan & Agarwal 2013CHAUHAN SS & AGARWAL R. 2013. Evaluation of antibacterial activity of volatile oil from Mentha spicata L. J Drug Deliv Ther 3(4): 120-121.). Mentha suaveolens is an aromatic herb and has a spearmint flavor, which is used in the Mediterranean areas in the traditional medicine. It has an extensive range of biological activities, including cytotoxic, antimicrobial, antioxidant, anti-inflammatory, hypotensive, analgesic, sedative and insecticidal properties (Moreno et al. 2002MORENO L, BELLO R, PRİMO-YÚFERA E & ESPLUGUES J. 2002. Pharmacological properties of the methanol extract from Mentha suaveolens Ehrh. Phytother Res 16(S1): 10-13., El-Kashoury et al. 2014EL-KASHOURY ESA, EL-ASKARY HI, KANDİL ZA, EZZAT SM, SALEM MA & SLEEM AA. 2014. Chemical and biological study of Mentha suaveolens Ehrh. cultivated in Egypt. J Med Plant Res 8(20): 747-755., Božović et al. 2015BOŽOVIĆ M, PİROLLİ A & RAGNO R. 2015. Mentha suaveolens Ehrh.(Lamiaceae) essential oil and its main constituent piperitenone oxide: biological activities and chemistry. Molecules 20(5): 8605-8633.).

The essential oil of M. suaveolens includes piperitone oxide and piperitenone oxide as major components. Other chemotypes of this species showed high percentage of alcohols (menthol) or ketones (pulegone, piperitenone and dihydrocarvone) (Oumzil et al. 2002OUMZİL H, GHOULAMİ S, RHAJAOUİ M, ILİDRİSSİ A, FKİH-TETOUANİ S, FAİD M & BENJOUAD A. 2002. Antibacterial and antifungal activity of essential oils of Mentha suaveolens. Phytother Res 16(8): 727-731., Sutour et al. 2008SUTOUR S, BRADESİ P, DE ROCCA-SERRA D, CASANOVA J & TOMİ F. 2008. Chemical composition and antibacterial activity of the essential oil from Mentha suaveolens ssp. insularis (Req.) Greuter. Flavour Frag J 23(2): 107-114., El-Kashoury et al. 2014EL-KASHOURY ESA, EL-ASKARY HI, KANDİL ZA, EZZAT SM, SALEM MA & SLEEM AA. 2014. Chemical and biological study of Mentha suaveolens Ehrh. cultivated in Egypt. J Med Plant Res 8(20): 747-755., Božović et al. 2015BOŽOVIĆ M, PİROLLİ A & RAGNO R. 2015. Mentha suaveolens Ehrh.(Lamiaceae) essential oil and its main constituent piperitenone oxide: biological activities and chemistry. Molecules 20(5): 8605-8633.).

There are a few studies on chemical the antibacterial and anti-fungal effects of M. suaveolens (Oumzil et al. 2002OUMZİL H, GHOULAMİ S, RHAJAOUİ M, ILİDRİSSİ A, FKİH-TETOUANİ S, FAİD M & BENJOUAD A. 2002. Antibacterial and antifungal activity of essential oils of Mentha suaveolens. Phytother Res 16(8): 727-731., Sutour et al. 2008SUTOUR S, BRADESİ P, DE ROCCA-SERRA D, CASANOVA J & TOMİ F. 2008. Chemical composition and antibacterial activity of the essential oil from Mentha suaveolens ssp. insularis (Req.) Greuter. Flavour Frag J 23(2): 107-114., El-Kashoury et al. 2014EL-KASHOURY ESA, EL-ASKARY HI, KANDİL ZA, EZZAT SM, SALEM MA & SLEEM AA. 2014. Chemical and biological study of Mentha suaveolens Ehrh. cultivated in Egypt. J Med Plant Res 8(20): 747-755., Božović et al. 2015BOŽOVIĆ M, PİROLLİ A & RAGNO R. 2015. Mentha suaveolens Ehrh.(Lamiaceae) essential oil and its main constituent piperitenone oxide: biological activities and chemistry. Molecules 20(5): 8605-8633.). Antibacterial effects of ethanolic extract and fractions of M. suaveolens against human pathogenic bacteria Staphylococcus aureus (ATCC12600), Streptococcus faecalis (ATCC19433), Bacillus subtilis (ATCC6051), Escherichia coli (ATCC11775), Neisseria gonorrhoeae (ATCC19424), Pseudomonas aeruginosa (ATCC10145) (El-Kashoury et al. 2014EL-KASHOURY ESA, EL-ASKARY HI, KANDİL ZA, EZZAT SM, SALEM MA & SLEEM AA. 2014. Chemical and biological study of Mentha suaveolens Ehrh. cultivated in Egypt. J Med Plant Res 8(20): 747-755.) and major components of Mentha suaveolens essential oils against Staphylococcus aureus, Staphylococcus simulans, Staphylococcus saprophyticus, Enterococcus sp., Bacillus anthracis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Proteus mirabilis, Enterobacter avium, Citrobacter freundii (Oumzil et al. 2002) has been reported in the previous studies. Moreover, there is no study on antimicrobial activity of M. suaveolens against fish pathogens. Thus, in this study was identified the major constituents of essential oil of M. suaveolens and investigated antibacterial effects against fish pathogens.

MATERIALS AND METHODS

Plant material

Essential oil of “apple mint”, a variety of Mentha suaveolence Ehrh., was used in the research. Cuttings from rhizomes of the cultivar were planted in experimental pilots of TARUM in Isparta University of Applied Sciences. Fertilizer was applied after planting at the rate of 50 kg ha−¹ nitrogen, and 50 kg ha−¹ P₂O₅. Plots were watered with drip irrigation system and kept weed free by hand hoeing. Plants were harvested at floral initiation (in mid-July). Fresh material after harvest was dried at 35ºC in drier cabin for essential oil distillation.

Preparation of essential oil

The essential oil was obtained with the distillation process using a Clevenger apparatus. Distilled water (2000 mL) was used for the distillation of dried plant samples (100 g). Distillation time was approximately 2 h at boiling point. The oil phase was separated and stored in dark glass bottle at 4 °C in fridge until both antibacterial studies and Gas chromatography-mass spectroscopy (GC-MS) analysis.

Gas chromatography-mass spectroscopy analysis of the essential oil

The GC-MS TIC (total ion chromatogram) analysis was performed using a Hewlett-Packard 6890 series gas chromatograph (Perkin Elmer (PE) Auto System XL, USA), fitted with a flame ionization detector (FID). The PE Auto System XL gas chromatograph was employed under the following conditions: capillary column, CPWax 52CB (50 m x 0.32 mm; film thickness ¼ 0.25 Im); oven temperature programmed, 60–220°C raised at a rate of 2 °C min^-1 and then held at 220 °C for 20 min; injector and detector temperatures, 240°C; carrier gas, helium at flow rate of 40 mL min^-1 ; and split ratio, 1/20 mL min^-1. Relative percentage amounts were calculated from chromatograms by the Turbo Crom Navigator computer program (Baydar et al. 2004BAYDAR H, SAĞDİÇ O, ÖZKAN G & KARADOĞAN T. 2004. Antibacterial activity and composition of essential oils from Origanum, Thymbra and Satureja species with commercial importance in Turkey. Food Control 15(3): 169-172.).

In vitro antibacterial activity

Fish pathogen bacteria were obtained from the culture collection in Isparta University of Applied Sciences, Microbiology Laboratory of Fisheries Faculty. Bacteria strains; Staphylococcus warneri, Staphylococcus sp., Lactococcus garvieae, Vagococus salmoninarum, Vibrio anguillarum from rainbow trout; Aeromonas sobria from yellow tail cichlid; Aeromonas hydrophila ATCC 7966, Aeromonas cavieae ATCC 15468, Pseudomonas aeroginosa ATCC27953, Yersinia ruckeri, Edwardsiella tarda DSMZ 300052 were used in the test.

Agar disc diffusion method was performed for the antibacterial screening of the essential oil of M. suaveolens Ehrh. For the test, bacteria were grown in Tryptic Soy Broth for 24 h at 25 °C and then 100 μL of each culture transferred into 100 mL cooled (to 45°C) Tryptic Soy Agar. After solidifying and drying for 15–20 minute, wells were punched (diameter = 3 mm) and 25 μL of different concentrations prepared with 96% ethanol (the test concentrations: 1000, 500, 250, 125, 62.5, 31.25 µL mL^-1) added to wells in triplicates. Controls were prepared using 96% ethanol. Plates were incubated at 25°C for 24h and observed for clearing zones around the wells (Andrews 2004ANDREWS JM. 2004. BSAC standardized disc susceptibility testing method (version 3). J Antimicrob Chemother 53(5): 713-728.). The antibacterial activity of plant extracts was interpreted as proposed by Bansemir et al. (2006)BANSEMIR A, BLUME M, SCHRÖDER S & LINDEQUIST U. 2006. Screening of cultivated seaweeds for antibacterial activity against fish pathogenic bacteria. Aquacult 252: 79-84.. Inhibition zones >15 mm were categorized as strong activity, from 8 to 15 mm as moderate activity, and from 1 to 8 mm as weak activity.

RESULTS AND DISCUSSION

M. suaveolens essential oil include piperitone oxide and piperitenone oxide as major components (Božović et al. 2015BOŽOVIĆ M, PİROLLİ A & RAGNO R. 2015. Mentha suaveolens Ehrh.(Lamiaceae) essential oil and its main constituent piperitenone oxide: biological activities and chemistry. Molecules 20(5): 8605-8633.). Other chemotypes of this species showed high percentage of alcohols (menthol) or ketones (pulegone, piperitenone and dihydrocarvone) (Oumzil et al. 2002OUMZİL H, GHOULAMİ S, RHAJAOUİ M, ILİDRİSSİ A, FKİH-TETOUANİ S, FAİD M & BENJOUAD A. 2002. Antibacterial and antifungal activity of essential oils of Mentha suaveolens. Phytother Res 16(8): 727-731., Sutour et al. 2008SUTOUR S, BRADESİ P, DE ROCCA-SERRA D, CASANOVA J & TOMİ F. 2008. Chemical composition and antibacterial activity of the essential oil from Mentha suaveolens ssp. insularis (Req.) Greuter. Flavour Frag J 23(2): 107-114., El-Kashoury et al. 2014EL-KASHOURY ESA, EL-ASKARY HI, KANDİL ZA, EZZAT SM, SALEM MA & SLEEM AA. 2014. Chemical and biological study of Mentha suaveolens Ehrh. cultivated in Egypt. J Med Plant Res 8(20): 747-755., Božović et al. 2015BOŽOVIĆ M, PİROLLİ A & RAGNO R. 2015. Mentha suaveolens Ehrh.(Lamiaceae) essential oil and its main constituent piperitenone oxide: biological activities and chemistry. Molecules 20(5): 8605-8633.). In the present study, the chemical composition of M. suaveolence essential oil was characterized by one hundred different components and were determined 10 volatile constituents, representing 87.68% of the total composition (Table I). The main component of M. suaveolence oil was piperitenone oxide (66.23%).

The antimicrobial activities of the essential oil of M. suaveolence against fish pathogens were represented in Table II. The essential oil exhibited strong inhibitory activity against V. anguillarum (30-50mm) at 250-1000 µL mL^-1 concentrations against; P. aeroginosa (16-20mm) at 31.25-125 µL mL^-1 concentrations against; A. sobria (15-18mm) at 500-1000 µL mL^-1 concentrations. In addition, the essential oil was found to be moderately effective against E.tarda (8-15 mm at all concentrations), Y. ruckeri (9-12mm at 125-1000 µL mL^-1), S. warneri (9-10mm at 125-1000 µL mL^-1), V. salmoninarum (9mm at 250 and 500 µL mL^-1), Staphylococcus sp. (8-9mm at 250-1000 µL mL^-1) in the present study. Similarly, in the previous studies noted Mentha piperita essential oil had moderate effect against Y. ruckeri and V. salmoninarum (Adel et al. 2016ADEL M, SAFARI R, GHİTANCHİ AH & ZORRİEHZAHRA MJ. 2016. Chemical composition and in vitro antimicrobial activity of some Iranian medical herbs against Yersinia ruckeri. Iran J Fish Sci 15(3): 1108-1123., Metin et al. 2017METIN S, DIDINEN BI, MERCIMEK EB & ERSOY AT. 2017. Antibacterial activity of some essential plant oils againts some bacterial fish pathogens. Aquacult Stud 17(1): 59-69.).

In the present study, M. suaveolence essential oil showed weak inhibitory activity against A. hydrophila. In contrast, Birinci Yıldırım & Türker (2018) noted strong antibacterial activity of M. piperita against A. hydrophila.

CONCLUSION

In conclusion, it was found that the main component of M. suaveolence essential oil was piperitenone oxide (66.23%) in this study. M. suaveolence essential oil exhibited strong antibacterial effect against V. anguillarum (30-50mm), P. aeroginosa (16-20mm), A. sobria (15-18mm); moderately antibacterial effect against E.tarda (8-15 mm), Y. ruckeri (9-12mm), S. warneri (9-10mm), V. salmoninarum (9mm), Staphylococcus sp. (8-9mm) and weak antibacterial effect against A. cavieae (5-8), A. hydrophila (6-7mm), L. garvieae (5-7mm). However, effect on immune response and disease resistance against Vibrio anguillarum, A. sobria and P. aeroginosa of essential oil of M. suaveolens should be investigated in vivo in cultured fish species in future studies.

REFERENCES

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