Comparison of total phenolic contents and antioxidant capacities in mint genotypes used as spices

: Objective: Mint ( Mentha spp.) genotypes used as spices are cultivated in Turkey and used for different pur-poses including herbal tea, spices, the cosmetics industry, and are used in folk medicine. While mint species have been used in traditional practices during humanity’s long history, there is limited research on the comparison of their antioxidant capacity and phenolic contents. This aim of the research is to compare antioxidant capacity and phenolic contents in mint clones to determine superior genotypes for herbal and spice usage and cultivation. Methods: Antioxidant capacity was evaluated by ferric reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC). Total phenolic content in clones were determined by Folin-Ciocalteu method. Results: Maximum total phenolic content [28.27±3.95 µg gallic acid equivalent (GAE)/g dry weight (DW)] and FRAP activity 577.09±46.02 µmol Trolox equiv/g DW were obtained from Clone 13 ( M. spicata ), while M. piperita clones were higher for TEAC activity (800.02±1.10 µmol Trolox equiv/g DW). The results were first records for M. villoso nervata . FRAP and TEAC activities selected clones were correlated with total phenolic content (r=0.77; 0.73 respectively). Conclusion: According to the results, it can be suggested that Clone 13, Clone 5, ( M. spicata ) Clone 3 and Clone 8 ( M. piperita ) are more suitable for use as spice, herbal tea, and antioxidant agents. The clones must be selected for commercial cultivation by the grower and they can be used as spices and herbal teas.


Introduction
Edible plants contain biological active components having antioxidant activity in addition to their food values. Antioxidant activities of plants are a result of their phenolic contents [1][2][3]. Phenolics are functional compounds synthesized for defensive metabolites in plants. They have a critical role in the human diet for healthy living. Herbs such as mint are an important source of them [4].
Some mint (Mentha spp) genotypes have been used in the Mediterranean diet as herbal teas and spices [4][5][6][7]. Mentha spicata, M. canadensis, M. piperita etc, have economic importance due to their medicinal and aromatic value [8,9]. Besides essential oil composition, other secondary metabolites composition determines herbal tea and pharmacological properties of the plants [10,11].
While essential oil composition determines quality in cultivated mint, phenolic components and their biological activity (antioxidant, antimicrobial etc.) have vital importance for herbal tea, spice and medical uses of cultivated mint. As results of breeding and characterization research, superior varieties for herb yield and essential oil composition were selected by our group. However, there are no reports on phenol compounds of the selected clones. According to previous reports on Mentha species antioxidant activity of M. piperita and M. spicata species had been carried out by different methodology [12][13][14][15][16][17]. Four of the selected clones belong to M. villoso nervata. Contrary to limited essential oil composition, there are no records on antioxidant capacity of the species, M. villoso nervata. There are also limited reports on comparison of the correlation antioxidant capacity with total phenolic content in Mentha species. Therefore, the aim of the research is to compare antioxidant capacity and phenolic contents in mint clones to determine superior genotypes for herbal and spice uses and cultivation.

Plant material
The selected 13 mint clones of three Turkish cultivated species, Mentha piperita, Mentha spicata and Mentha villoso-nervata, were grown in experimental plot of Agriculture Faculty in Gaziosmanpasa University. Species and origin of selected clones were given Table 1. Rooted samples of each clone were planted experimental plots with three replications. Plants were harvested at the stage of flowering in Mid-July 2009. After, they are dried in drier cabin at 35 o C, the leaves were separated from the aerial parts of the plants. Voucher specimens were deposited at the plants herbarium of Science and Arts Faculty at Gaziosmanpasa University (GOPU). Herbarium numbers of voucher specimens are given in Table 1.

Extraction procedures
Plant material, (100 g) was ground into a fine powder in a mill after 10 minutes of storage in liquid nitrogen. The powdered samples were mixed with 500 mL methanol/ chloroform (3/1) at room temperatures for 24 hour. The residue was re-extracted under same condition until extraction solvents became colourless. The obtained extracts were filtered over Whatman No.1 paper and the filtrate was collected, then, by extraction the solvent mix was removed by a rotary evaporator at 50 o C. The extracts were used for antioxidant capacity and total phenolic compounds content analysis.

Determination of total phenolic content
The total phenolic contents were determined by Folin-Ciocalteu reagent [18]. 0.1 ml of the extract solution (contains 0.1 mg extract) was mixed with water (46 ml). 1 mL of Folin-Ciocalteu reagent was added and mixed thoroughly. 3 mL of Na 2 CO 3 (2%) was added to the mixture. The absorbance was measured at 760 nm. The calibration curve was prepa-red by 0-100 µg/ml solutions of gallic acid in ethanol. The concentration of total phenolic compounds in the extracts was determined as gallic acid equivalent and calculated as µg gallic acid equivalent/g dry weight (DW) of the plant material using the calibration curve (R 2 : 0.99). The data were given as the average of triplicate analyses.

Ferric reducing antioxidant power (FRAP)
Reducing power of mint samples was measured by method of Oyaizu [19] with a slight modification [20]. According to this method the reduction of Fe 3+ to Fe 2+ was determined by  measuring absorbance of the Perl's Prussian blue complex. This method is based on the reduction of (Fe 3+ ) ferricyanide in stoichiometric excess relative to the antioxidants. For positive control Trolox was used. Absorbance of these mixtures was measured at 700 nm using a UV spectrophotometer (Hitachi U-2900). Increased absorbance indicates ferric reducing power capability of the samples [21]. Results were given as µmol trolox equiv/g DW. The data were given as the average of triplicate analyses.

Trolox equivalent antioxidant capacity (TEAC)
ABTS •+ radical scavenging activity was determined using a modified method of Re et al. [22]. The stock solutions included 5 mL of 7 mM ABTS •+ solution and 88 µL of 140 mM K 2 S 2 O 8 . The working solution was allowed to react for 16 hours at room temperature in the dark. The ABTS solution was adjusted with distilled water to an absorbance of 0.650±0.050 at 734 nm using the UV-visible spectrophotometer. Fresh ABTS •+ solution was prepared for each assay. 0.1 mL of Mentha species extract was allowed to react with 2.9 mL of the ABTS •+ solution in the dark at room temperature for 10 minutes then the absorbance was measured at 734 nm. The data were given as the average of triplicate analyses. Results were given as µmol trolox equiv/g DW [22].

Statistical analysis
The numerical data of total phenolic content and antioxidant activity are presented as mean±standard deviation calculated from triplicate. The values were subject to analysis of variance (ANOVA) using randomized block design. The mean data being significant in variance analysis were grouped with Duncan multiple test. The relationship between total phenolic content and antioxidant activities were also determined by correlation values. The significance threshold was set at 0.01. All statistical calculation were performed using the SPSS 20 statistical software [23].

Results and Discussion
Several methods have been developed to evaluate total antioxidant capacity of food and dietary supplements, herbal extracts, and pure compounds. Nevertheless, few of them have been used widely due to the difficulty of measuring total antioxidant capacity as result of limited methodological protocol and free radical sources [24]. In this study, the antioxidant capacities of the selected clones extracts were evaluated with TEAC and FRAP in vitro tests (Table 2). Total phenolic contents were also evaluated and a direct correlation was observed between the total phenolic content of the extracts and their antioxidant capacities ( Table 2 and Figure 1).

Total phenolic contents
The amount of total phenolic compound ranged from 9.39 (clone 15) to 28.27 (clone 13) µg GAE/g DW (   [13,15,[25][26][27]. The content in M. spicata was found as 334 mg CAE/g plant, 84 mg GAE/g plant, 80 mg GAE/g plant, 27 mg GAE/g plant and 22.43 mg GAE/g fresh plant by Husseinimehr et al. [28], Elmastas et al. [12], Naidu et al. [29], Conforti et al. [30] and Derakhshani et al. [31] respectively. No investigation of total phenolic content in M. villoso nervata has been included in the literature. Phenolic compounds that are synthesized for defending and protecting purpose and have an important role in the human diet varies according to genetic structure, growing, and climate conditions [32]. In the research the change in the phenolic content is due to genetic differences of the clones because the used clones are grown in the same ecologic and field conditions. The total phenolic content of M. piperita in this work was lower than that of some reports [13,15,26] and higher than that of others [27,33]. Total phenolic amounts of M. spicata clones were generally higher than that of Ninckavar's result [13] except two clones (Clone 15 and 11). Total phenolic contents in all clones of M. spicata in this study were lower than a previous study [12,28,34]. Total phenolic contents in M. spicata clones were in agreement with results reported by Derakhshani et al. except clone-11, clone-12 and clone-15 [31]. Those three clone phenolic contents were lower than results of Derakhshani et al. [31]. Total phenolic contents in M. spicata clones were higher than that of Naidu et al's result [29]. Although, there are no records on total phenolic amount of M. villoso nervata, total phenolic content of the species was similar to the records of M. spicata and lower than that of other mentha species such as M. piperita, M. longifolia, M. pulegium, M. roduntifolia [13]. Even though there was significant variation statistically (p<0.01) between clones of the spearmint (M. spicata and M. villoso nervata) species, M. piperita Clones (two clones) had similar data, statistically. Total phenolic of the clones was 23.99 (µg GAE/g DW) in Clone 3 and 21.56 (µg GAE/g DW) in Clone 8. There is dramatic variation in total phenolic amount of M. spicata and M. villoso nervata (Table 2).

Antioxidant activities Ferric reducing antioxidant power (FRAP)
FRAP activities varied between 280.73 and 577.09 µmol Trolox equiv/g DW in M. spicata. The lowest and the highest FRAP activity of M. piperita were 317.6 and 558.33 µmol Trolox equiv/g DW, while 314.76 and 374.83 µmol Trolox equiv/g DW in M. villoso nervata ( Table 2). Different studies have indicated that the electron donation capacity of bioactive compounds is associated with antioxidant activity [35,36]. The presence of reductants in the samples causes the reduction of the Fe 3+ / ferricyanide complex to the ferrous form. Therefore, Fe 2+ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm [37]. There are a number of assays designed to measure overall antioxidant activity [38]. FRAP assay take advantage of an electron transfer reaction in which a ferric salt is used as an oxidant [39].
In this assay, the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of antioxidant samples. The reducing capacity of an extract may serve as a significant indicator of its potential antioxidant activity. The outcome of the reducing reaction is to terminate the radical chain reactions that may otherwise be very damaging [40].
Reduction power of Mentha species extracts ranged from 280.7 to 577.1 µmol Trolox equiv./g DW. The highest activity was seen in Mentha spicata Clone 13 and the lowest in Mentha spicata Clone 11 ( Table 2). The second highest FRAP activity of this study was observed M. piperita Clone 3. FRAP result of M. piperita Clone 3 was agreement with some previous reported results [13,15,16]. According to the results in this study, FRAP activity differences in Mentha clones were correlated (r=0.77) with total phenolic content (Figure 1a). Chrpova et al. [15] studied antioxidant activities of some herbs belongs Lamiaceae, including M. piperita, used for medicinal purposes, and they reported that M. piperita had strong free radical scavenging activity and good correlation (r=0.96) with total phenolic contents [15].
Studies conducted on free radical scavenging or antioxidant activities of medicinal plant reveal that the efficiency of plant species depends on assay methodology, reflecting the complexity of the mechanisms involved in total antioxidant capacity [41][42][43].

Trolox equivalent antioxidant capacity (TEAC)
Results of cation radical scavenging activities were given in Table 2. As seen in Table 2, TEAC activity of Mentha species extracts ranged from 375.1 to 800.02 µmol Trolox equiv/g DW. The highest activity was seen in Mentha piperita clone 3 while the lowest in Mentha spicata Clone 15 (Table 2).
Assays based upon the use of DPPH • and ABTS •+ radicals are among the most popular spectrophotometric methods for determination of the antioxidant capacity of food, beverages, plant extracts and pure compounds [44,45]. Both chromogen radical compounds can directly react with antioxidants. DPPH • and ABTS •+ scavenging methods are used to evaluate the antioxidant activity of compounds due to the simple, rapid, sensitive, and reproducible procedure [40].
The reaction of the preformed radical with free-radical scavengers can be easily monitored by following the decay of the sample absorbance at 734 nm. ABTS •+ radicals are more reactive than DPPH radicals and unlike the reactions with DPPH • radical which involve H atom transfer, the reactions with ABTS •+ radicals involve electron transfer process [46]. Lopez et al. studied antioxidant activities of 5 Mentha species. According to their report ABTS •+ radical scavenging activities of M. piperita showed the best activity when compared to other species [14]. Lopez et al's results also agree with our result ( Table 2). Previous antioxidant activity reports on Mentha species clearly showed antioxidant activities of M. piperita and M. spicata. [12][13][14][15][16].
TEAC activity differences in Mentha clones were correlated with total phenolic content (r=0.73). It can be said that phenolic compounds are responsible for TEAC activities of Mentha species.

Conclusion
Results obtained in the research is summarized as following: 1. Different methods and units in antioxidant activity caused difficulty in comparison of the results. So, a standard unit should be used by researchers for accurate evaluation of antioxidant results in Mentha species in future researches. 2. There is a positive correlation with total antioxidant capacity and total phenolic content in Mentha clones. In future studies the relationship between antioxidant capacity and phenolic contents on Mentha species should be based on antioxidant activities and individual phenolic compound contents in Mentha species. 3. According to antioxidant capacity and total phenolic content results it can be suggested that Clone 13, Clone 5 (M. spicata) Clone3 and Clone 8 (M. piperita) are more suitable for use as spice and herbal tea. The results contain valuable data for breeding studies too. 4. The result of M. villoso nervata is the first record on both antioxidant capacity and phenolic content. 5. In future studies the relationship between antioxidant capacity and specific phenolic components of Mentha species should be studied in Mentha species.