Mentha longifolia ( L . ) ssp . longifolia Essential Oil : Source of Natural Antioxidant and Antimutagen as Food Additive

This research was performed to control the antioxidant activity, mutagenicity and antimutagenic effect of Mentha longifolia (L.) ssp. longifolia essential oil (EO), which is considered as a possible ingredient when producing healthy food. The antiradical activity was established using DPPH (2,2-diphenyl-1-picrylhydrazyl radical) and β -carotene/linoleic acid bleaching assays. The total phenolic content in the EO was evaluated by Folin Ciocalteau method (FCR).  Ames Salmonella/microsome mutagenicity assay was applied todetect possible mutagenic and antimutagenic behavior. Our observations reveal that the IC 50 value for DPPH radicals was 5.27 ± 0.13 mg/mL. The total antioxidant efficiency increased with an increase in the concentration of the EO, and IC 50 value 11.7 ± 0.21 mg/mL. The total of phenolics was 186 ± 8.9 mg/g gallic acid equivalent/EO. Also, any concentrations of the EO used did not show mutagenic action but exhibited strong antimutagenic effects at 10.0-4.0 µg/plate concentrations. This research proposes that because of the antioxidant and antimutagenic characteristics, the EO is very advantageous and significant to the company’s manufacturing food additives.


Introduction
Lipid oxidation is a major cause for food quality deterioration and generation of off odours and off flavours, decreasing shelf life, altering texture and colour, and decreasing the nutritional value of food [1].It can also generate potential toxic compounds through the activity of free radicals [2].It has also been acknowledged that these toxic compounds endanger health and cause ailments related to digestion, aging, cancer, mutagenesis and cardiovascular disorders.The oxidative degradation of lipids is also known to harm biological membranes, enzymes and proteins, which will ultimately affect the human health [3].
Addition of antioxidants is considered the most efficient, suitable and inexpensive technique out of all the approaches used to regulate the lipid oxidation [4].Due to their exclusive characteristics that help in enhancing the shelf-life of food items without causing any harmful impacts on the physical or nutritional characteristics, the antioxidants are recognized as a crucial part of the food additives [5].But the synthetic antioxidants and/or food preservatives are still consumed in far less in the edible food products, owing to the severe health and safety concerns that appear to have hazardous outcomes and to be potential carcinogens [6].Hence, there is a requirement to discover different supplied of safe, efficient and suitable natural preservers [7].
Owing to their antifungal/antimicrobial behavior, their antioxidant attributes or antimutagenic/antigenotoxic abilities, the EO, that are the secondary metabolites of plants, have always played their part in the packing of eatables [8,9,10].In this perspective, EO could play a promising role either individually or as co-adjuvant in the development of food preservatives [11].

Plant collection and EO isolation
M. longifolia (L.) Hudson ssp.longifolia were collected from wild populations growing locality in Adana (Turkey), in June 2012 and was identified by Dr. Olcay Ceylan, Department of Biology, University of Mugla Sıtkı Kocman, Mugla, Turkey.The dried and powdered samples were submitted to waterdistillation method.

DPPH radical scavenging activity
The ability of the EO to scavenge DPPH free radicals was measured by the method of Ebrahimabadi et al. [18] with a little modification.One milliliters amount containing diverse dilutions of EO was combined with 1 mL DPPH solution (0.2 mM).The mixture was stirred and left in a dark at 25ºC for half an hour.The absorbance was later measured at 517 nm.Butylated hydroxytoluene (BHT) and ascorbic acid were the two chemicals utilized as standard antioxidants.Free radical scavenging activity was calculated using the equation below: Here, RSA is radical scavenging activity, Absc is the optical density of the DPPH solution, and Abss is the optical density of the sample formulations.The oil concentration containing 50% radical inhibition activity (IC50) was evaluated from the graph concerning the free radical scavenging action (%) with against the EO concentration.The results were demonstrated as IC50 ± standard deviation.

Inhibition of β-carotene/linoleic acid bleaching assay
The β-carotene bleaching potential of the EO was determined according to by Rauter et al. [19].In short, 1 ml of β-carotene was combined with 200 mg of Tween 60 and 25 µl of linoleic acid.The combination was stirred and the chloroform was evaporated.Then distilled water (100 ml) was included to the combination and agitated.From this emulsion, 2.5 ml transferred into test tubes and 0.5 ml of the EO was added.The initial optical density of samples was determined after 1 min of vortexing at 470 nm.After incubation at 50 °C, the absorbance of every sample at 470 nm was measured after every 15 minutes, for the next 180 minutes.The antioxidant behavior of the oil was contrasted against the activity of BHT and ascorbic acid.The antioxidant behavior of the samples was assessed by applying the formula mentioned below: Here, R is the bleaching rate in the slope of ln (Abs) vs. time line, which can be assessed with the help of a linear regression, where t is used to denote the time in minutes.
Here, AA is antioxidant activity, Absc is the optical density of the β-carotene solution of dimethyl sulfoxide (DMSO) and Abss signifies the optical density of the β-carotene suspension of sample preparations.The results were illustrated as the IC50 ± standard deviation.

Determination of total phenolic content
As per Singleton et al. [20], FCR technique was applied to determine the total phenol content of the EO.Roughly, around 100 µl of FC reagents (0.2 N) was added to 200 µl of a suspension of EO (1 mg/mL), vortexed for 1 min, and after 3 min, the mixture was neutralized with 2 mL of 5% aqueous Na2CO3 solution.The optical density of the samples was recorded to be at 760 nm after 2 hours.The same technique was implemented on the standards solution of Gallic acid to acquire a Standard curve.The total phenol content was articulated as milligram of Gallic acid equivalent per gram of the oil (mg/g GAE oil).

Mutagenicity and antimutagenicity assay
The possible mutagenic/antimutagenic influences offered by EO were assessed on two histidinedependant (His−) mutant sample strains of Salmonella typhimurium TA98 and TA100.Before the experiment, the strains were analyzed for spontaneous reversion and genetic integrity including UV sensitivity, ampicillin resistance, histidine requirement, crystal violet sensitivity, and dose-dependent cytotoxic effects were observed of the EO as presented by Mortelmans and Zeiger [21].
The tube containing DMSO was used to determine spontaneous reversion.After incubating for 72 h at 37 °C, the His + revertant colonies were determined.
The mutagenicity of 4-NPD and NaN3 as a positive control in the absence of the EO was described as 100% mutagenicity.The percentage inhibition was computed by applying the formula given below: Here, A is the number of revertants present in each plate whilst mutagen and test samples are present, and B is the number of revertants in each plate in the positive control, while C is the total number of spontaneous revertants present in each plate.

Statistical analysis
All determinations of antioxidant activity were performed in triplicate.Each dose of mutagenic and antimutagenic activity was tested using triplicate plates in two independent experiments.The results were presented as the average and Standard deviation.The comparison of average values of each samples were analyzed with one-way ANOVA, followed by Tukey's test.

Antioxidant activity and total phenolic content
The antioxidant action and the phenol content of EO, in this research were assessed by methods DPPH, βcarotene/linoleic acid bleaching and FCR assays.The results obtained from the EO displayed that the antiradical activity against DPPH free radical and IC50 values of 5.27 ± 0.13 mg/mL, while the ascorbic acid and BHT were 0.01 ± 0.03 mg/mL and 0.184 ± 0.01 mg/mL, respectively (Table 1).
The antioxidant action by β-carotene/linoleate model system of EO was examined and contrasted against ascorbic acid and BHT (Table 1).It was found that the IC50 of the EO was 11.7 ± 0.21 mg/mL, while those of BHT and ascorbic acid were 0.05 ± 0.12 mg/mL and 0.020 ± 0.2 mg/mL, respectively.The total phenolic content in EO was 186 ± 8.9 mg Gallic acid equivalent/g EO (Table 1).

Screening of mutagenic and antimutagenic activity
The EO in the nontoxic dose range of 10.0 -2.0 µg/plate was observed for signs of mutagenicity and it was discovered that when different concentrations of EO were present, it did not alter considerably, in the number of revertants when compared to spontaneous mutation incidences (data not shown).
Therefore, the M. longifolia ssp.longifolia EO seems to be non-genotoxic against TA98 and TA100 strains.
The EO in the nontoxic dose variety range of 10.0 -2.0 µg/plate were also measured for antimutagenicity of two dissimilar types of genotoxic compounds on S. typhimurium TA98 and TA100 (Table 2).The report revealed that in the TA100 strain, the EO was strong efficient in decreasing the influence of NaN3 genotoxicity, inhibition percentages range between 45.6 and 52.5% with respect to the observed level of the positive control.Maximum inhibition of TA98 was observed with concentrations of 10.0-4.0 µg/plate (46.7%-53.7%)against 4-NPD genotoxicity (Table 2).The EO displayed moderate antimutagenic action (30.8%) in opposition to 4-NPD genotoxicity at 2.0 µg/plate dose on the TA 98 strain.

Discussion and Conclusion
To get over the problems concerning stability, like fats and oils, synthetic antioxidants are commonly combined to avoid the oxidative degradation of lipids inside of foodstuffs [23].But presently, their use in foods has heavily criticized due to potential health risk corresponding to toxic effects and to be potential carcinogens [6,24].As a result, natural antioxidants gained from plants are received a great deal of interest, as they can be biologically active constituent with various health endorsing influences [7].
According to the β-carotene bleaching tests the IC50 values of the EO were found to be 11.7 ± 0.21 mg/mL (Table 1).In other studies, IC50 values of M. longifolia EO against DPPH free radical is determined as 10.700 μg/mL [12] and >8000 mg/L [13].Similarly in a study, according to the β-carotene bleaching test the EO of M. longifolia ssp.longifolia was displayed only 24% inhibition at 2 mg/ml [12].
The antioxidative efficiency of natural resources was mainly caused because of the phenolic compounds [25].Phenolic compounds were recounted to be of great importance in inhibiting autoxidation of oils [26].M. longifolia ssp.longifolia EO can be declared very beneficial and a good resource of phenol antioxidants.The total phenolic content in EO was 186 ± 8.9 mg Gallic acid equivalent/g EO (Table 1).
Products of lipid oxidation have been implicated as having toxic, carcinogenic, mutagenic properties [24].For this reason, in this study we researched mutagenic potential and antimutagenic activity of EO.
In this study, EO of M. longifolia ssp.longifolia did not display mutagenic activity.In a study M. longifolia extract has shown the ability to counteract the 2-amino-3-methylimidazo-quinolidine (IQ) and nitrosopiperidine (NP) mutagenic effects [27].
In the present experimental conditions, the EO was effective antimutagens against two different types of genotoxic compounds.The current results reveal that the antimutagenic action in Ames test which might be ascribed in portion to influential radical scavenger related to the EO.
The antimutagenic activity of the M. longifolia was previously assayed by scrutinizing the organic extracts from the various parts of the plant [27,30].The M. longifolia extracts revealed an effectual antimutagenic action regarding NP, IQ [27] and NaN3 [30].In another study, luteolin [31] and apigenin derivatives [28,29] were isolated from M. longifolia extracts show significant antimutagenic activity.But the antimutagenic action of the M. longifolia EO has not been researched.
Even though the food industry having variety preservers, it is still facing a shortage of powerful food preservers to maintain the quality of the foodsand make them safety.This EO could possibly be consumed as a food component to substitute the synthetic food antioxidants and as mutagenprotective food component.It can also help in encouraging health welfare, and reduce the chance of acquiring cancer and similar severe ailments that are usually linked with lipid metabolism problem.

Table 1 .
DPPH radical scavenging activity, total antioxidant activity and total phenol content of the M. longifolia ssp.longifolia EO a IC50 values represent the means ± standard deviation.b Reference compounds.c Not studied.Significance compared to control at p<0.000

Table 2 .
Antimutagenicity of the EO of M. longifolia ssp.longifolia against S. typhimurium TA98 and TA100