Quantity and chemical composition of essential oil of peppermint (Mentha × piperita L.) leaves under different drying methods

ABSTRACT This work was aimed at quantitative and qualitative analyses of the essential oil of peppermint leaves under different drying methods. Thin layer drying experiments of the leaves were performed in shade, hot air dryer (at temperatures of 50°C, 60°C, and 70°C), and microwave oven (at power levels of 200, 400, and 800 W). Essential oils of the fresh and dried samples were extracted by hydrodistillation and analyzed using gas chromatography-mass spectrometry (GC/MS). The highest (22.24 g/kg dry matter) and the lowest (1.33 g/kg dry matter) oil yields were obtained from the hot air-dried leaves at temperature of 50°C and microwave-dried leaves at power of 800 W, respectively. In general, increasing drying temperature decreased the essential oil content. The GC/MS analysis of essential oils showed that the chemical compounds belonged mostly to oxygenated monoterpenes class (72.34–86.41%). The chemical compounds group was significantly (p < 0.01) decreased by microwave drying at power levels of 200 and 400 W. The assessed drying methods caused significant (p < 0.05 and/or p < 0.01) variations in the main constituents of the peppermint leaves essential oil including menthol, menthone, menthofuran, 1,8-cineole, and menthyl acetate. The minimum (35.01%) and maximum (47.50%) concentrations of menthol, as the major compound of the oil, were found in hot air-dried leaves at temperature of 50°C and microwave-dried leaves at power of 400 W, respectively. The percentage of menthone, as the second constituent in the essential oil, was significantly lost (p < 0.01) under microwave drying.


Introduction
Botanically, peppermint (Mentha piperita L.) belongs to the Lamiaceae family in the genus Mentha. [1] The herb is mentioned in Chinese traditional medicine and its dried leaves were found in Egyptian pyramids. Peppermint is cultivated mainly for its essential oil obtained from freshly grounded leaves by distillation. Peppermint oil is composed primarily of menthol and menthone as well as several other minor constituents, including menthofuran, 1,8-cineole, and limonene. [2] The chemical composition of peppermint leaves may vary with plant maturity, geographical region, and processing conditions. The plant is normally harvested in full-bloom stage and typically has 0.75-0.85 (g water /g wet matter ) moisture content. [3,4] Due to high moisture contents, safe storage of the fresh medicinal and aromatic plants for long periods is not possible. The water participates in microbial and chemical interplays leading to quality deterioration. Drying, removal of water content by application of heat, increases the shelf life of the plants by slowing microorganism's growth and preventing certain biochemical reactions that might alter the organoleptic characteristics. [5] During this process, depending on the method and conditions of drying and the plant species, several changes occur in the plants that may improve the remediation properties or induce formation of new compounds having remediation properties. [6][7][8] Aromatic and spice plants are most sensitive to drying processes and thus, careful dehydration is a fundamental requirement for achieving a high-quality product.
In the literature, some works have been reported on dehydration influences on oil yield and composition for various medicinal herbs. Blanco et al. dried peppermint leaves at temperatures of 40°C, 60°C, and 80°C, and reported that higher drying temperature sharply decreased the essential oil yield. [9] Omidbaigi et al. studied the influence of different drying methods, including sun, shade, and oven at 40°C, on quantitative and qualitative traits of the essential oil from Roman chamomile (Chamaemelum nobile L. All. var. flora plena). [10] Sefidkon et al. reported that drying of summer savory (Satureja hortensis L.) aerial parts in oven at 45°C resulted in highest oil yield and most concentration of carvacrol. [11] Ghasemi Pirbalouti et al. studied the quantitative and chemical composition of the essential oil of the purple and green basil landraces aerial parts under sunlight, shade, oven, microwave, and freeze-drying treatments and indicated that, for both landraces, the highest oil yields were obtained from shade-dried samples. In addition, in comparison with the fresh samples, significant loss of most monoterpene hydrocarbons of the essential oils were reported. [5] Rahimmalek and Goli dried thyme leaves by using six drying treatments and reported that in spite of relatively low essential oil yield, microwave drying was the best method with the advantages such as shorter drying duration, high color quality, and increased main compounds. [7] Argyropoulos and Müller dried lemon balm leaves in a convective hot air dryer at different temperatures within the range of 30°C and 90°C, and studied the essential oil yield. [12] The main objectives of this study were to (i) assess the effects of different drying methods on quantitative and qualitative characteristics of essential oil of peppermint leaves and (ii) determine the best drying method for preserving the major constituents in the leaves oil.

Plant material
By cutting the herb manually, aerial parts of the Mentha × piperita were harvested before flowering at the early bloom from farm lands of Isfahan, Southwest Iran. Plant identity was confirmed by Dr Shrimardi placed in the Herbarium of the Research Center for Medicinal Plants, Shahrekord Branch, I. A.U., Iran. The harvested plants were stored in a refrigerator at 4 ± 1°C until the drying experiments were started. To determine the initial moisture content of the peppermint leaves, 4 batches (50 g) of the leaves were placed in an oven at 105°C for 24 h and the following equation was used [13] : where M 0 is the initial moisture content of samples (g water /g wet matter ), and W 0 and W d are masses (g) of the fresh and dry matter, respectively.

Drying experiments
Different drying methods and conditions listed in Table 1 were selected to dry the leaves. Before the experiments, the plants were taken out of the refrigerator and the leaves were separated accurately from the stems. To perform microwave drying, a domestic microwave oven cavity (LG MG-4012, Korea) at the three different power levels of 200, 400, and 800 W was used. For convective drying, a laboratory scale dryer was employed. The leaves were dried at a constant air flow rate of 0.4 m/s and temperatures of 50°C, 60°C, and 70°C while relative humidity of the drying air remained at the environment relative humidity level (approximately 40%). In case of shade drying, the leaves were spread in the shade under natural air flow and ambient temperature (mean value of 22°C).
For each drying treatment, 150 g of the leaves was used as a thin layer with an approximate thickness of 10 mm. During the experiments, the samples lot was weighed and instantaneous moisture content of the leaves was computed using Eq. (2): where M is the instantaneous moisture content at each time (g water /g wet matter ) and W is the samples mass at each time (g). The drying experiments were replicated three times and all of them were continued until the samples lot reached to final moisture content of about 0.12 g water /g wet matter , which is suitable for safe storage and also for essential oil extraction.

Chemicals
Homologous series of C 5 -C 24 n-alkanes used for identification (by calculation of their retention indexes) was purchased from Sigma-Aldrich Co. (Steineheim, Germany). Anhydrous sodium sulfate (Na 2 SO 4 ) was bought from Merck & Co. (Darmstadt, Germany).

Samples preparation
Dried and fresh leaves were subjected to hydrodistillation (1000 mL distillated water) for 3 h using a Clevenger-type apparatus. The extracted essential oil contents were determined on the basis of dry matter in triplicates and kept in amber glass vials at 4 ± 1°C until analysis.

Gas chromatography-mass spectrometry
Gas chromatography/mass spectrometry (GC/MS) analyses of essential oil samples of the peppermint leaves were performed on an Agilent Technologies 7890 gas chromatograph coupled to Agilent 5975 C mass selective detector and quadrupole EI mass analyzer (Agilent Technologies, Palo Alto, CA, USA). An HP-5MS 5% column (coated with methyl silicone) (30 m × 0.25 mm, 0.25-µm film thicknesses) was used as the stationary phase. Helium was used as the carrier gas at 0.8 mL/min flow rate. The temperature was programmed from 60°C to 280°C at 4°C/min ramp rate. The injector and the GC/MS interface temperatures were maintained at 290°C and 300°C, respectively. Mass spectra were recorded at 70 eV. Mass range was from m/z 50 to 550. The ion source and the detector temperatures were maintained at 250°C and 150°C, respectively.

Identification of the oil constituents
Constituents were identified by comparison of their retention index (RI) relative to C 5 -C 24 n-alkanes obtained on a nonpolar HP-5MS column by comparison of the RI, provided in the literature, by comparison of the mass spectra with those recorded by the NIST 08 (National Institute of Standards and Technology) and Willey (ChemStation data system). The individual constituents were identified by retention indices and compared with constituents known from the literature. [14] The peak area percentages were computed from HP-5MS column without the use of flame ionization detector (FID) response factors.

Statistical analyses
One-way ANOVA by SPSS (19.0) was used for statistical analysis of the data, and Duncan's multiple range test was applied to compare the means of main constituents for essential oils.

Results and discussion
Effect of drying methods on the essential oil yield The essential oils from the fresh and dried peppermint leaves were found to be a yellow liquid. The average amounts of extracted essential oils are shown in Fig. 1. Average oil yield of the fresh leaves was determined to be 16.15 g/kg dry matter , which is comparable with the results reported by some researchers for fresh peppermint leaves. Chi et al. verified an average oil yield of about 8 mL/kg dry matter . [15] Moghaddam et al. determined the essential oil content to be 13.8 g/kg dry matter . [16] Scavroni et al. reported a mean oil yield of about 13.9 mL/kg dry matter . [17] The differences among the four reported values could be attributed to the environmental and experimental conditions to which plants were submitted. Essential oil contents from the leaves dried in convective dryer at temperatures of 50°C, 60°C, and 70°C were obtained to be 22.24, 19.55, and 15.26 g/kg dry matter , respectively. The results showed that, compared to the fresh leaves, hot air drying at temperature of 70°C led to an insignificant (p ≤ 0.05) decrement in the oil yield while temperatures of 50°C and 60°C significantly increased it. The observation is in agreement with those reported by Blanco et al. for peppermint where they determined essential oil content of the leaves dried at temperatures of 40°C, 60°C, and 80°C to be 1.0, 0.14, and 0.12 (%, V/W), respectively. [9] Roholf et al. found that increasing drying temperature from 30°C to 70°C caused a continuous decrement in essential oil yield of peppermint harvested at different growing stages including early bloom, full bloom, and late bloom. [4] Chi et al. dried peppermint leaves using air temperatures in the range of 40-80°C and reported that increasing drying temperatures in the considered range resulted in decreased essential oil contents. [15] Nozad et al. found the same results for dried spearmint leaves in hot air dryer at temperatures of 30°C, 40°C , and 50°C. [18] Similar observation was also reported by Saeidi et al. for Mentha longifolia (L.) Hudson dried in oven at 40°C and 80°C, [19] Argyropoulos and Müller for convective drying of lemon balm, [12] Shahhoseini et al. for oven-dried lemon verbena (Lippia citriodora), [20] and Hamrouni Sellami et al. for oven and infrared drying of Laurus nobilis L. leaves. [21] It is worth noting that, unlike the findings reported before, some researchers indicated that an increment in essential oil yield of aromatic plants such as Satureja hortensis could be induced by increasing drying temperature. [11] These opposite findings may arise from differences in the plants species, secretory structures as well as essential oil composition.
Significant differences (p ≤ 0.05) among oil yields extracted from the leaves dried by microwave power and the other samples were observed (Fig. 1). According to the results, the peppermint leaves were extra sensitive to microwave power, whereas the essential oil content of the microwave-dried samples was very little in comparison with the other considered samples. These results are in agreement with those reported for basil landraces [5] and for sage (Salvia officinalis L.). [22] The observation indicates that the biological structure of the oil glands of peppermint leaves is compromised, resulting in a loss of essential oil yield. [5] Dìaz-Maroto et al. in anatomical assessments of fresh and dried leaves of spearmint found that epithelial cells in the dried samples can collapse and split open. [3] As the results show, no significant differences were found among the applied microwave power levels where the oil yields from the leaves dried at the powers of 200, 400, and 800 W were obtained as 2.23, 1.43, and 1.33 (g/kg dry matter ), respectively. At the beginning of medicinal and aromatic plants drying process, moisture is transferred by diffusion from inner regions to surfaces of the materials and drags the essential oil with it. Therefore, since the diffusion is more pronounced at higher microwave powers, the proposed mechanism explains the reason for the significant loss of the essential oil.
Average essential oil content obtained from the shade-dried leaves was determined to be 18.89 g/kg dry matter , which is not statistically different with the oven drying at 60°C. Also, in comparison with fresh leaves, shade drying resulted in significant increment in the essential oil yield. Hamrouni Sellami et al. found that the total essential oil content of L. nobilis was increased after natural air drying at ambient temperature. [21] Ghasemi Pirbalouti et al. dried two basil landraces using different drying methods and reported that for both landraces, in comparison with fresh and dried samples, shade-dried plants resulted the most essential oil yield. Ghasemi Pirbalouti et al. reported that shade drying caused an increment in essential oil yield of fresh Bakhtiari savory (Satureja bachtiarica Bunge.). [23] The obtained results from statistical analyses indicated that essential oil yield was significantly affected by different drying methods (p ≤ 0.05). As shown in Fig. 1, drying of the leaves at air temperature of 70°C and microwave power of 800 W led to the highest (22.24 g/kg dry matter ) and the lowest (1.33 g/kg dry matter ) oil yields, respectively. It is obvious that drying of leaves resulted in both increment and decrement in essential oil quantity. Some authors have reported similar results in the open literature for different medicinal and aromatic plants. [5,22,23] Generally, according to Hamrouni Sellami et al., [21] essential oil yield of medicinal and aromatic plants is influenced by drying, whereas depending on the process time and temperature as well as the method, the oil yield could be increased and/or decreased. Furthermore, changes in the oil content are dependent on the plant species. The obtained results in the present work indicated that the studied peppermint leaves were ultrasensitive to microwaves where the oil yield was approximately spoiled in this dehydration method.

Effect of drying methods on the essential oil composition
The extracted essential oils from the fresh and dried peppermint leaves were analyzed by GC/MS, and the obtained results are presented in Table 2. As shown, in fresh peppermint leaves, 36 components were identified in total that accounting for 93.84% of the total volatiles. In the fresh samples, the principal components were menthol (44.39%), menthone (15.36%), menthofuran (10.27%), 1,8-cineole (5.81%), menthyl acetate (4.78%), neoisomenthol (2.37%), and limonene (1.87%). According to Pino et al., [24] the major constituents in peppermint grown in Jalisco (Italy) were identified to be menthol (35.4%), menthofuran (18.2%), menthone (15.4%), and menthyl acetate (12.4%). Scavroni et al. studied the effect of harvesting time (including 90, 110, and 120 days after planting) on quality of essential oil of peppermint and identified menthyl acetate (35.01-55.68), menthol (11.28-42.32), menthofuran (4.56-17.45), and 1,8-cineole (2.93-5.56%,) as the main compounds in the oil. [17] The differences among the results reported in the open literature for chemical compounds of peppermint, and generally for medicinal and aromatic plants, could be attributed to the geographic origin of the plant, genetic, and harvesting time as well as to the extraction method.
The effects of the considered drying methods and conditions on the chemical compositions of peppermint leaves could be discussed according to Table 2. The results demonstrated that the drying treatments could significantly change (p ≤ 0.05 or p ≤ 0.01) the chemical profiles of essential oils from the leaves where some of the essential oil compounds have been lost and/or increased due to the formation of new constituents by oxidation, glycoside hydrolysis, esterification, and other processes. [23] In addition, there were significant differences between the applied drying methods and fresh sample in terms of menthol (p ≤ 0.05), menthone (p ≤ 0.01), menthyl acetate (p ≤ 0.05), menthofuran (p ≤ 0.01), neoisomenthol (p ≤ 0.01), 1,8-cineole (p ≤ 0.01), viridiflorol (p ≤ 0.01), germacrene-D (p ≤ 0.01), and β-caryophyllene (p ≤ 0.05) ( Table 2).
The percentage of menthol, as the main compound in the oil, increased by drying the fresh leaves at microwave power levels of 800 and 400 W while the other drying treatments decreased the component. Statistical analyses results showed that, in comparison with the fresh samples, the relative quantity of menthol in the oil decreased significantly under hot air drying at temperature of 50°C. According to the results, the highest (47.50%) and the lowest (35.01%) percentages of menthol were obtained in the leaves dried at air temperature of 50°C and microwave power of 800 W, respectively. Drying of the peppermint leaves under considered dehydration methods decreased the percentage of menthone as the second main constituent identified in the essential oils. According to the statistical analyses, menthone percentage was decreased significantly (p ≤ 0.05 and/or p ≤ 0.01) under microwave treatments, whereas differences among the fresh, hot air-dried, and shade-dried leaves were not significant. Hot air drying at temperature of 50°C and shade drying increased and decreased slightly percentage of menthofuran, respectively, while the other applied dehydration methods caused significant decrement (p ≤ 0.01) in the component compared to the fresh leaves. The rates of 1,8-cineole increased in the cases of hot air and shade drying, whereas the constituent decreased significantly (p ≤ 0.01) under microwave drying technique. Drying of the peppermint leaves resulted in significant increments (p ≤ 0.05 and/or p ≤ 0.01) in certain volatiles such as menthyl acetate, β-caryophyllene, germacrene-D, and others.
In general, microwave drying reduced the concentrations of menthone, menthofuran, and 1,8cineole (Table 2). These constituents seem to have more affinity to the water fraction contained in leaves and, therefore, they were lost with the water removal during the drying process. At high temperatures, the biological structure of the oil glands of medicinal and aromatic plants can be affected and the epithelial cells in the dried samples of some sensitive plants can be collapsed. [5,7]      Results of this study indicated that the highest percentages of menthol (as the major constituent) and menthyl acetate were obtained from the samples dried by the microwave ( Table 2). Quality of the essential oil from peppermint is described by high menthol and low menthone levels. [4] Therefore, the obtained results revealed that, among the assessed drying methods, microwave power had the best performance from the oil quality aspect.

Effect of drying methods on the main chemical groups of the essential oil components
In general, the chemical compounds could be categorized into four main chemical groups: (1) oxygenated monoterpenes, (2) monoterpene hydrocarbons, (3) sesquiterpene hydrocarbons, and (4) oxygenated sesquiterpenes. Average concentrations of the main chemical groups for the peppermint essential oil, as expressed in percentage, are presented in Fig. 2. The results revealed that chemical composition of the fresh and dried leaves is characterized mainly by the presence of oxygenated monoterpenes (72.34-86.41%). Low levels of monoterpene hydrocarbons (1.09-8.75%), sesquiterpene hydrocarbons (2.07-10.72%), and oxygenated sesquiterpenes (0.36-5.49%) are also presented.
Based on the obtained results (Fig. 2), except hot air drying at temperature of 70°C, dehydration of the fresh leaves decreased oxygenated monoterpenes in the essential oil. The minimum (72.34%) value of oxygenated monoterpenes was obtained in the microwave-dried leaves at power level of 200 W while the group concentration in the fresh leaves was determined to be 86.19%. The decrement was mainly due to decreases in the values of the main constituents of the group such as menthofuran and menthone (Table 2). Furthermore, it is worth noticing that microwave drying at power levels of 200 and 400 W resulted in significant loss in the most oxygenated monoterpenes. Ghasemi Pirbalouti et al. found a significant decrement in oxygenated monoterpenes in basil landraces dried in an oven at temperature of 60°C and at microwave power of 500 W. [5] Compared to the fresh samples, the concentration of the monoterpene hydrocarbons was significantly (p ≤ 0.05 and/or p ≤ 0.01) increased in the cases of hot air (at temperatures of 50°C, 60°C, and 70°C) and shade drying where the contents were obtained to be 5.01%, 8.75%, 6.85%, 7.10%, and 7.46%, respectively. A work performed by Shahhoseini  the results of the current work show that the constituents group content decreased significantly (p ≤ 0.01) in the case of microwave drying and was determined as 1.09%, 1.79%, and 2.24% in the leaves dried at the powers of 200, 400, and 800 W, respectively. [20] Figure 2 shows that the percentage of sesquiterpene hydrocarbons of the essential oils increased significantly (p ≤ 0.01) in the microwave-dried leaves where the class contents for fresh and dried leaves at the powers of 200, 400, and 800 W were determined as 2.19%, 10.72%, 8.91%, and 6.14%, respectively. Similarly, Jerković et al., [25] Bartley and Jacobs, [26] Díaz-Maroto et al., [3] and Ghasemi Pirbalouti et al. [5] reported increment in sesquiterpenes concentration of the essential oils for different herbs after drying by microwave power. In addition, values of the chemical group in the oils of leaves dried at temperatures of 50°C, 60°C, 70°C, and shade-dried leaves were obtained to be 4.08%, 6.29%, 2.07%, and 2.94%, respectively. A trend similar to that of sesquiterpene hydrocarbons was observed for oxygenated sesquiterpenes where the components class contents were determined to be 0.36%, 0.59%, 0.71%, 0.29%, 5.49%, 3.99%, 2.16%, and 0.52% for the samples presented in Fig. 2 (from left to right), respectively. Growth in sesquiterpenes after drying has been reported by Dìaz-Maroto et al., [3] Hamrouni Sellami et al., [22] and Ghasemi Pirbalouti et al. [5]

Conclusion
In the present study, influences of different drying methods on essential oil quantity and chemical composition of peppermint leaves were investigated. In summary, the obtained results demonstrated that the changes in essential oil yield and constituents of the leaves depend on the drying method, drying duration, and drying temperature. Quantitative and qualitative analyses of the essential oil revealed statistically significant differences among the studied treatments. The results also showed that microwave drying at power levels of 200, 400, and 800 W affects significantly the concentration of the main essential oil components. Shade, hot air (at 50°C, 60°C, and 70°C), and the microwave drying resulted in small losses of the volatile compounds as compared with fresh leaves. In addition, in some cases, the microwave method improved the yield of some bioactive compounds, such as menthol, menthyl acetate, viridiflorol, germacrene-D, and β-caryophyllene. In final, microwave power was found to be the best option to dry peppermint leaves. This conclusion was based on the following facts: (i) a relatively short time was required to dry the samples (4 min in microwave 800 W), and (ii) the high concentrations of menthol, menthyl acetate, viridiflorol, germacrene-D, and β-caryophyllene were found. Furthermore, drying of the peppermint leaves at ambient air as well as hot air temperatures of 50°C, 60°C, and 70°C can be recommended for high oil yield and resulted in a better quality product in terms of menthofuran, neoisomenthol, and 1,8-cineole.