Research Article Received: 23 September 2013

Revised: 18 June 2014

Accepted article published: 25 June 2014

Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6802

Seasonal variability of the main components in essential oil of Mentha × piperita L. Daniela Grulova,a* Laura De Martino,b Emilia Mancini,b Ivan Salamonc and Vincenzo De Feob Abstract BACKGROUND: Mentha × piperita is an important and commonly used flavoring plant worldwide. Its constituents, primarily menthol and menthone, change in the essential oil depending on internal and external factors, of which environmental conditions appear very important. The experiment was established in 2010 for three vegetation season, in order to observe the quantitative changes of the main components of peppermint. The determination of menthol, menthone, limonene, menthyl acetate, menthofuran and 𝜷-caryophyllene was registered. RESULTS: In the experimental season 2011 and 2012 a higher mean temperature than in 2010 and extreme rainfall in July 2011 and 2012 were recorded. Different environmental conditions affected the development of M. × piperita plants and the content and composition of the essential oil. CONCLUSION: Seasonal and maturity variations are interlinked with each other, because the specific ontogenic growth stage differed as the season progressed. Fluctuations in monthly and seasonal temperature and precipitation patterns affected the quality of peppermint essential oil. © 2014 Society of Chemical Industry Keywords: developmental stage; menthol; menthone; peppermint; vegetation season

INTRODUCTION The genus Mentha is the unique source for the production of some of the most economically notable essential oil throughout the world.1 Peppermint (Mentha × piperita L.) is an important and commonly used flavoring agent for foods and beverages, as a fragrance, and as a fungicide or insecticide in many pharmaceutical and industrial products.2,3 Menthol is generally the most abundant component of the essential oil of mature peppermint plants and its amount determines the commercial value of peppermint oil.4,5 Developmental and environmental factors are known to influence greatly the yield and composition of peppermint oils.6 Menthol and menthofuran content are affected by changes in the growing season.4 The oil composition of M. × piperita in natural conditions can also depend on leaf age.7 – 9 The pathway for menthol production is chemically complex. The chemistry and enzymology of this pathway have been well characterized in a few recent papers.10 – 12 The influence of temperature on different species of mint was studied many years ago.13 – 16 Only one study, however, reported the correlation between the qualitative and quantitative changes in essential oil of peppermint and environmental factors, showing that temperature and rainfall changes affect the oil composition.17 On the other hand, a good-quality peppermint oil is characterized by a high content of menthol and low menthofuran at harvest.18 A possible ecological explanation of the quali-quantitative variation of the essential oil is the hypothesis of the functional approach: plants growing at a site are filtered on the basis of traits responding to local environmental factors, and selected plants display effect traits that act on the processes of the ecosystem.19 The production of secondary compounds and J Sci Food Agric (2014)

their storage in plant tissues are energy-intense processes. The proportion of total resources allocated to these processes is difficult to assess, since secondary metabolites are continually being synthesized and metabolized, and allocation varies from one season to another.20 It is well known, in fact, that some environmental factors can influence biochemical pathways and physiological processes that alter plant metabolism.21 The present study was focused on the evaluation of the changes of six main components of the essential oil of peppermint, over 3 years, in controlled conditions in Slovakia.

MATERIAL AND METHODS The study was carried out in the experimental field of the Department of Ecology, University of Prešov, and Laboratories of Excellence Centre, University of Prešov (Slovakia), and in Laboratories of the Department of Pharmacy, University of Salerno (Italy).



Correspondence to: Daniela Grulova, Department of Ecology, Faculty of Humanities and Natural Sciences, University of Prešov, 17 November St, 081 16 Prešov, Slovak Republic, E-mail: [email protected]

a Department of Ecology, Faculty of Humanities and Natural Sciences, University of Prešov, 17 November St, 081 16, Prešov, Slovak Republic b Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132-84084, Fisciano, (Salerno),Italy c Excellence Centre of Animal and Human Ecology, University of Prešov, 17 November St, 081 16, Prešov, Slovak Republic

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www.soci.org Locality and climatic characteristics Mentha × piperita L. was grown at the experimental field of the Department of Ecology, University of Prešov, in the eastern part of Slovakia (49∘ 59′ N, 21∘ 13′ E). The area is located in the temperate climatic zone in the northern part of the Košice Basin and passes to the North Spiš-Šariš intermediate mass. The altitude is 244 m above sea level. The annual average temperature in the area ranges between 8 and 9 ∘ C. The coldest month is January, with an average temperature of −3.5 ∘ C; the warmest month is August, with an average temperature of 19 ∘ C. The annual precipitation is about 600.0 mm. The considered parameters, such as the meteorological data of monthly average air temperature and precipitation, were acquired from the nearest hydrometeorological station – Research and Breeding Station Mal´y Šariš – which is about 2 km as the crow flies. All data were compared with the long-term normal temperature and precipitation, which were evaluated during the period 1961–1990, recorded by the institution mentioned. Each year, soil samples were taken to obtain current data on their chemical characteristics: P, K and Mg concentration, pH, Cox and percent humus content. Plant material The M. × piperita cultivar ‘Kristínka’ has been cultivated in an experimental field of the Department of Ecology, University of Prešov, since the year 2006. This cultivar was registered as new in 2013. Its characteristic is an high content of menthol. Plant material was collected in one day during the first week of each month (from April to September) throughout the experiment (Table 1). Five random samples from different parts of the experimental field were harvested. Drying of samples Fresh plant material was arranged in a thin layer on metal trays and placed in a Binder dryer. The drying process was carried out for 36–48 h at 30 ∘ C until constant dry mass weight. Isolation of essential oil Fifteen grams of each sample of peppermint were grounded in a blender and then subjected to hydrodistillation in a Clevenger-type apparatus for 2 h in order to extract the oil. The oils were solubilized in n-hexane and stored under N2 at +4 ∘ C in the dark until analysis. The plant materials yielded reddish-yellow oils. Samples for analysis by gas chromatography–mass spectrometry (GC-MS) were diluted 1:1000 in n-hexane. GC-MS analyses and identification of components GC-MS analyses were carried out on a Varian 450-GC connected with a Varian 220-MS. Separation was achieved using a FactorFourTM capillary column VF 5 ms (30 m × 0.25 mm i.d., 0.25 μm film thickness). Injector type 1177 was heated to a temperature of 220 ∘ C. Injection mode was splitless (1 μL of a 1:1000 n-hexane solution). Helium was used as a carrier gas at a constant column flow rate of 1.2 mL min−1 . Column temperature was programmed: initial temperature was 50 ∘ C for 10 min, then increased to 100 ∘ C at 3 ∘ C min−1 , maintained isothermal for 5 min and then increased to 150 ∘ C at 10 ∘ C min−1 . The total time for analysis was 46.67 min. The mass spectrometer trap was heated to 200 ∘ C, manifold 50 ∘ C and transfer line 270 ∘ C. Mass spectra were scanned every 1 s in the range 40–650 m/z. Components were identified by comparison of their mass spectra with those stored in NIST 02

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(software library) or with mass spectra from the literature22,23 and a home-made library, as well as by comparison of their retention indices with standards and calculated Kovats indices. Statistical analysis The data were subjected to statistical analysis following analysis of variance (ANOVA) using the statistics software Statgraphic 5.0. The method used was ANOVA – Type III sum of squares. The significance of differences between any two treatment means was determined by comparing with least significant difference (LSD) values at 5% level of probability (P < 0.05). LSD values were computed from error variance and table ‘t’ values at 5% probability level. Influence of year, month and repetition was compared.

RESULTS The average monthly air temperatures (∘ C) and the sum of precipitation (mm) during the experiment are presented in Table 2. The temperature and precipitation in three vegetation seasons varied. The mean temperature in the 6 months (from April to September) in 2010 reached 14.8 ∘ C and the sum of the rainfall in the same period was 427.2 mm. In 2011, in the same period, the mean temperature rose to 16.27 ∘ C and the sum of rainfall decreased to 364.1 mm. The last year of the experiment (2012) was the warmest in the vegetation season (16.93 ∘ C), with the sum of precipitation being 408.3 mm. The mean temperature from the beginning to the end of seasons 2011 and 2012 was higher than in 2010. Air temperature increased until July in 2010 and 2012 and decreased in September. Extreme rainfall was recorded in July 2011 and 2012 which were respectively 66% and 47% higher in comparison to the long-term period data. Consequently, plant growth varied. However, the soil condition during the 3-year experiment was stable. The pH was 7.2, Cox 1.75% and humus content reached 3%; content of P was 138 mg kg−1 , K 92 mg kg−1 and Mg 92 mg kg−1 . The quantities of total essential oil (Table 3) in the month of April in each of the 3 years were different: these differences reflect the mean temperature in this month in different years. Higher temperature causes higher evaporation of volatile components of the essential oil. The highest amount was noted in June 2010 (0.84 ± 0.1%), whereas in 2011 and 2012 the highest amount was noted at the end of the growing season (0.46 ± 0.03% and 0.42 ± 0.01%, respectively). Significant differences were noted in the quantity of the five main components in comparison to the quantity in the three different vegetation seasons (Figs 1 and 2). An example of a GC-MS chromatogram of M. × piperita essential oil is reported in Fig. 3. The sum of menthol, menthone, limonene, menthyl acetate, menthofurane and 𝛽-caryophyllene represented 97.1–98.7% of the total essential oil (Table 3). The quantity of limonene in each season decreased from 10.81 ± 0.83% (April 2010) to 7.98 ± 0.24% (April 2012) and from 3.19 ± 0.96% (September 2010) to 2.34 ± 0.86% (September 2012). The highest amount of this component was noted in 2010 (5.46 ± 0.63%) and the lowest in 2012 (4.29 ± 0.51%) (Fig. 1). In 2010, menthone amount increased from April to August and decreased to 14.12 ± 1.05% in September. The amounts in 2011 and 2012 differed. In fact, the highest content of menthone was noted at the beginning of the season (April 2011) and in May 2012 and until September decreased in 2011 and 2012. The highest content of menthone was noted in 2012 (16.60 ± 0.74%) (Fig. 2). Menthol, the major component of peppermint essential oil, increased in the first and third years from April until July, and

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Seasonal changing in Mentha × piperita L. essential oil

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Table 1. Harvest date of the plant material and the developmental stages Day of harvest

Experimental year 2010 2011 2012

Developmental stage 2 April 2 May 4 June 1 July 5 August 3 September Before flowering Before flowering Flowering: early bloom Flowering: full bloom Flowering: full bloom Flowering: late bloom 4 April 5 May 1 June 1 July 1 August 3 September Before flowering Before flowering Flowering: full bloom Flowering: full bloom Flowering: late bloom After flowering 3 April 5 May 2 June 3 July 1 August 4 September Before flowering Before flowering Flowering: full bloom Flowering: full bloom Flowering: late bloom After flowering

Table 2. Average temperatures and sum of precipitations in the studied period and in long-term period (1961–1990) Year 2010 Month

(∘ C)

April May June July August September

8.59 13.87 16.89 18.58 17.85 13.43

2011 (mm) 51.2 75.1 82.0 92.2 75.3 51.4

(∘ C) 10.62 14.35 18.25 18.44 19.76 16.22

decreased in September. In 2011 this compound also increased from April to August. Figure 2 shows that menthol reached the highest amount in 2012 (71.35%), with comparable amounts in 2010 (67.42%) and 2011 (67.12%). Menthyl acetate reached the lowest amount in July 2011 and in August 2010 and 2012 The highest amount of this compound (6.65 ± 0.41%) was registered in 2011 (Fig. 1). 𝛽-Caryophyllene, after a transient increase in May and June of the first two years (in 2010 from 2.87 ± 0.44% to 9.23 ± 0.07% and in 2011 from 3.23 ± 0.02% to 6.45 ± 1.16%), decreased until the end of the year; in 2012 the amount of 𝛽-caryophyllene recorded was 3.11 ± 0.15% to 1.75 ± 0.52%. Figure 1 shows the annual differences. Menthofuran was also measured as a component of interest for the evaluation of quality of essential oil. It was the only component present in amounts less than 0.5% during all three years. ANOVA was used to confirm the potential influence of the factors month, year and repetition. No significant differences were observed between the amount of analyzed components in the samples collected at the same time, but remarkable differences were reported in comparing the amounts of the six constituents in different years and months (Table 3).

DISCUSSION Productivity of plants is associated with several characteristics of the species, which interact in a complex manner with the environment.24 Mentha × piperita L. shows a wide adaptation ability under different climate and soil conditions, but a temperate climate is important for its quality. Green25 reported that the best quality of peppermint essential oil was obtained from plants cultivated in areas situated north of the 40th parallel. However, other Authors have reported that the plant could be grown successfully up to the 34th parallel.26 Our study area is located directly on the 49th parallel line. Peppermint grows throughout the year, J Sci Food Agric (2014)

2012 (mm) 14.8 56.8 98.7 165.0 18.4 10.4

(∘ C) 9.89 15.6 18.85 21.8 19.73 15.71

1961–1990 (mm) 48.1 69.0 76.9 147.0 29.3 38.3

(∘ C)

(mm)

8.72 13.95 16.87 18.67 18.11 13.4

49.74 78.4 85.9 99.0 78.8 55.2

so adequate nutrients are needed. The above-ground biomass grew rapidly during the late spring until it reached full canopy in July–August, and maturation of the plants clearly depends on climatic conditions. Clark and Menary8 reported very important relationships between vegetation and soil but not all properties of soils act directly and immediately on the vegetation. As mentioned above, the pathways of the main components of essential oil of peppermint are known.10 Voirin and Bayet9 reported the results of detailed analyses of monoterpene metabolic steps, i.e. reduction of menthone to menthol, acetylation of menthol and disappearance of limonene in the leaves at different maturity stages from plants harvested in June. Maffei17 compared the quantity of the main components in an essential oil of peppermint in two different vegetation season. He described the increased quantity of limonene, menthone and menthyl acetate in the season with lower temperatures and high sum of precipitation along with the decrease of the main component, menthol. Menthone reached a higher amount in the warmer vegetation season and the quantities of menthyl acetate were comparable in warmer and in colder vegetation seasons. Environmental conditions seem not to affect the amounts of menthofuran. Our data partially agree with this report, in particular for limonene and menthol amounts. However, our data disagree for menthone: in the cited paper the highest amount of menthone was reached in the year with cooler temperature and higher precipitation; our observation showed the highest menthone amount in the warmer season. Also Dai27 generally mentioned that changes in temperature and humidity affect the quality of essential oil. The data from our study may provide complementary knowledge about the relationship between biodiversity and ecosystem functioning,28 and knowledge about the variability in the main components of essential oil production influenced by environmental conditions seems to be necessary to predict the yield and harvest time of M. × piperita.

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Table 3. Percentage of the six main components of the essential oil of peppermint during the study Main components (%) ± SD Month

Year

Limonene

Menthone

April

2010 2011 2012 2010 2011 2012 2010 2011 2012 2010 2011 2012 2010 2011 2012 2010 2011 2012

10.81 ± 0.83∗ 8.36 ± 0.05 7.98 ± 0.24 3.85 ± 0.05 6.17 ± 0.23∗ 4.72 ± 0.87 5.72 ± 1.01∗ 4.90 ± 0.87 4.05 ± 0.23 4.64 ± 0.83 4.07 ± 0.90 3.45 ± 0.02∗ 4.56 ± 0.12 5.19 ± 0.77 3.25 ± 0.87 3.19 ± 0.96 3.46 ± 0.08 2.34 ± 0.86∗

15.29 ± 1.82∗ 19.68 ± 0.15 18.82 ± 0.34 7.19 ± 0.41 11.54 ± 0.43 19.97 ± 0.23∗ 19.79 ± 0.40 16.34 ± 0.85 18.26 ± 1.25 18.29 ± 4.03 18.26 ± 2.19 18.43 ± 0.64 20.44 ± 0.43 13.37 ± 0.59∗ 17.23 ± 0.72 14.12 ± 1.05∗ 3.62 ± 0.10 6.89 ± 1.23

May

June

July

August

September

Menthofuran 0.10 ± 0.00∗ 0.31 ± 0.01 0.42 ± 0.01 0.11 ± 0.00 0.10 ± 0.00 0.10 ± 0.00 0.20 ± 0.00 0.21 ± 0.10 0.13 ± 0.00∗ 0.22 ± 0.00 0.20 ± 0.01 0.20 ± 0.00 0.31 ± 0.01 0.45 ± 0.00∗ 0.32 ± 0.01 0.27 ± 0.00 0.34 ± 0.01 0.21 ± 0.01

Menthol

Total EO yield

Menthyl acetate

62.15 ± 1.93 58.79 ± 0.23 60.17 ± 0.65 68.78 ± 0.78 64.19 ± 2.36∗ 68.84 ± 1.02 65.06 ± 3.84 68.56 ± 2.97 72.01 ± 1.87 70.52 ± 3.51 69.70 ± 1.30 76.72 ± 1.52∗ 69.83 ± 0.24 71.27 ± 2.87 75.45 ± 0.89∗ 68.20 ± 0.35 70.26 ± 0.98 74.95 ± 0.75∗

7.31 ± 0.82 7.45 ± 0.16 7.35 ± 0.56 6.07 ± 0.03 6.83 ± 0.14 2.95 ± 0.02∗ 1.55 ± 0.03∗ 2.52 ± 0.11 2.15 ± 0.12 1.25 ± 0.72 1.42 ± 0.85 2.05 ± 0.03∗ 0.79 ± 0.04 4.49 ± 0.78∗ 0.69 ± 0.01 12.18 ± 0.52∗ 17.24 ± 0.41 15.78 ± 0.76

𝛽-Caryophyllene 2.87 ± 0.44 3.23 ± 0.02 3.11 ± 0.15 9.23 ± 0.07 6.45 ± 1.16 2.41 ± 0.54∗ 4.12 ± 1.56 5.03 ± 0.65 2.38 ± 0.22∗ 3.33 ± 0.44 3.79 ± 0.55 1.74 ± 0.01∗ 1.93 ± 0.05 2.81 ± 0.76∗ 0.86 ± 0.12 2.43 ± 0.32 2.41 ± 0.06 1.75 ± 0.52∗

(%) 0.15 ± 0.01 0.13 ± 0.02 0.08 ± 0.03 0.29 ± 0.02∗ 0.12 ± 0.01 0.11 ± 0.01 0.84 ± 0.10∗ 0.14 ± 0.02 0.25 ± 0.01 0.54 ± 0.10 0.18 ± 0.02∗ 0.38 ± 0.01 0.30 ± 0.02 ∗ 0.43 ± 0.01 0.41 ± 0.02 0.25 ± 0.01∗ 0.46 ± 0.02 0.42 ± 0.02

Values are the mean of five replicates ± SD. ∗ Statistics: ANOVA at 95% level (P < 0.05).

8 7 6

%

5

2010

4

2011

3

2012

2 1 0

menthyl acetate

limonene

beta-caryophyllene

Figure 1. Percentage of the mean quantity of menthyl acetate, limonene and 𝛽-caryophyllene in three vegetation seasons (2010–2012). Values are the mean ± SD.

Generally, the yield and essential oil composition of aromatic plants depend to a large extent on various extrinsic and intrinsic factors, such as ecological and climatic conditions, geographical location, season at the time of collection, stage of development, plant ontogenesis phase, ecological conditions, harvest methods, postharvest processing of plant materials and method of oil extraction;29 differences are also due to genetic and chemotypic variations, as observed in other aromatic species.30,31 It is often difficult to segregate these factors, since many are interdependent and influence one another. One of the most important characteristics of oil accumulation and composition is its dependence on the developmental/growth stage of plants.29 The peppermint oil yield increased from early to full bloom and late bloom stages, because there could be a preferential

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accumulation of highest essential oil during the flowering stage of plants.29 Environmental factors could result in biochemical and physiological alterations in plants modifying the quantity and quality of the essential oil, particularly in view of the rapidly expanding areas in different geographic and seasonal situations of the globe where they grow. Therefore, it appears pertinent to examine the spectrum of variations, including those exerted by air temperature and precipitation changes, on the yield and composition of the essential oil. Our data showed that the impact of the plant developmental stage on essential oil composition differs: some compounds, such as menthol and menthone, increase from the period before flowering to the full-bloom flowering; other constituents,

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Seasonal changing in Mentha × piperita L. essential oil

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80 70 60

%

50

2010 2011

40

2012

30 20 10 0

menthol

menthon

Figure 2. Percentage of the mean quantity of menthol and menthon in three vegetation seasons (2010–2012). Values are the mean ± SD.

Figure 3. A GC-MS chromatogram of Mentha × piperita.

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www.soci.org such as limonene and menthyl acetate, decrease over the same period. Verma et al.32 reported the changes in essential oil composition of Majorana hortensis Moench. during plant ontogeny: they showed considerable variations in the qualitative composition of the oils obtained from plants of different ages. The oils were dominated by oxygenated monoterpenes with the maximum at 90 (late vegetative stage) and 120 days (flower initiation). On the other hand, monoterpene hydrocarbons were found to be increased with advancement of age. Therefore, environmental variations in the essential oil content and composition of M. hortensis might be due to the variation in enzyme levels and their pool sizes in response to changing weather conditions during different months.32 The monoterpene accumulation in peppermint leaves has been shown to be restricted to leaves not older than 3 weeks with low catabolic losses (

Seasonal variability of the main components in essential oil of Mentha × piperita L.

Mentha × piperita is an important and commonly used flavoring plant worldwide. Its constituents, primarily menthol and menthone, change in the essenti...
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