Microsoft Word - 17-Agra_13942.doc 790 Original Article Biosci. J., Uberlândia, v. 28, n. 5, p. 790-798, Sept./Oct. 2012 SEASONAL VARIATION OF VEGETATIVE GROWTH, ESSENTIAL OIL YIELD AND COMPOSITION OF MENTHOL MINT GENOTYPES AT SOUTHERN BRAZIL VARIAÇÃO SAZONAL DO CRESCIMENTO VEGETATIVO, PRODUTIVIDADE E COMPOSIÇÃO DE ÓLEO ESSENCIAL EM GENÓTIPOS DE MENTA NO SUDESTE BRASILEIRO Vera Maria Carvalho Silva SANTOS 1 ; Marco Antônio Silva PINTO 2 ; Humberto BIZZO 3 ; Cícero DESCHAMPS 4 1. Professora, Doutora, Instituto Federal Catarinense - IFC, Araquari, SC, Brasil. veramcss@ifc-araquari.edu.br 2. Pesquisador, Doutor, Embrapa Agroindústria de Alimentos, Rio de Janeiro, RJ, Brasil. 3. Analista Embrapa Agroindústria de Alimentos, Rio de Janeiro, RJ, Brasil. 4. Professor, Doutor, Departamento de Fitotecnia e Fitossanitarismo, Universidade Federal do Paraná - UFPR, Curitiba, PR, Brasil. ABSTRACT: Menthol has economic importance to the flavor, food and pharmaceutical industries. Ten menthol mint (Mentha spp) genotypes were assessed for essential oil content and composition at Southern Brazil environmental conditions at two harvest times (February and May). The experimental design was in completely randomized blocks with a 10 x 2 factorial for genotypes and harvest time. The essential oil was obtained by hydrodistillation in a Clevenger apparatus. The essential oil content varied from 0.8 to 5.3% and was greater in February for all the investigated genotypes. The main constituents identified in the essential oil samples were menthol (12 - 92.7%), mentone (2.2 - 56.9%), and neomenthol (2.9 - 12.1%). Menthol levels were superior in May and showed a negative correlation with mentone and neomenthol, which in turn were higher in February. Menthol levels were positively correlated with menthyl acetate. Pulegone, 1.8 cineol, and limonene were also detected in lower concentrations in some genotypes. Thirteen other essential oil constituents were identified as trace elements in essential oil. Mentha canadensis L. showed the highest essential oil content (5.3 % - February and 3.5% - May) as well as the highest menthol content (89.6% - February, 92.7% - May) in both harvests. From the analyzed results, Southern Brazil local environmental conditions are appropriated for menthol production, with two harvests and M. canadensis L. can be recommended as a promising genetic source. The summer harvest (February) favored oil yield, although with a slight decrease in menthol content. The challenge of achieving higher essential oil and menthol yields depends on strategies to increase herb yield by developing innovative agronomic practices. KEYWORD: Lamiaceae. Mentha. Genetic resources. Biomass. Menthol. Menthone. INTRODUCTION The mint family is composed by different species that are grown worldwide to explore their fresh or dried leaves, as well as their essential oils, as flavoring or spices in a wide variety of food (MORRIS, 2007). India, China and United States are the main mint producer countries. Although Brazil has been the world’s leading menthol producer in the early 1970's, it now imports annually approximately US$ 10 million of mint essential oil (BIZZO et al., 2009). The major p- menthane monoterpene compound is (-)-menthol, which is important to the pharmaceutical, food and confectionery industries. Peltate glandular trichomes have been identified as the sites of essential oil production and storage in mint (DESCHAMPS et al., 2006; CROTEAU et al., 2005). The eight steps of the menthol biosynthesis pathway and the specific enzymes and gene properties have been described (CHANG et al., 2010; GERSHERZON et al., 2000). The understanding of the regulation of gene expression distributed in four subcellular compartments and the influence of plant ontogeny on oil production have begun to be explained (CHANG et al., 2010; RIOS – ESTEPA et al., 2008; CROTEAU et al., 2005; GERSHERZON et al., 2000). However, the correlation between genotypic acquirements with ecological interactions in physiological plant regulation plays a decisive role in essential oil modulation. (GOBBO-NETO; LOPES, 2007; SANGWAN et al., 2001). The genotypic variability of the genus Mentha is favored by cytomixis and consequent polyploidy, transgressive segregation and the ease of hybridization, resulting in significant chemical polymorphism (TUCKER; NACZI, 2007). In addition to genotypic traits, it has been demonstrated that genetic expression for oil production is also affected by plant ontogeny and environmental regulation, such as soil and seasonal variations (RIOS-ESTEPA et al., 2008; SANGWAN et al., 2001). Due to the economic importance of menthol, great emphasis has been Received: 12/11/11 Accepted: 03/05/12 791 Seasonal variation... SANTOS, V. M. C. S. Biosci. J., Uberlândia, v. 28, n. 5, p. 790-798, Sept./Oct. 2012 given to the conservation of mint genetic materials as a valuable bioresource to increase essential oil yield and quality, allowing economic production of mint-related commodities (KHANUJA et al., 2000; FRANZ, 2010). This work is part of a research program directed to mint production in Brazil which includes germplasm evaluation in different edaphoclimatic conditions throughout the country. The main objective of this work was to evaluate the vegetative growth, essential oil yield and composition of different mint genotypes at the environmental conditions of Southern Brazil. MATERIAL AND METHODS Plant material and experimental conditions Ten mint genotypes were obtained from the germplasm bank of Embrapa Genetic Resources and Biotechnology, Brazil (Table 1), by vegetative propagation using stem cuttings with 5 to 7 cm in length, Plantmax HT® as substrate, and grown under greenhouse conditions at Federal University of Parana (UFPR), on styrofoam trays with three intermittent mist irrigations during the day. Plants were transferred to field in November 2009, at the Catarinense Federal Institute, Campus Araquari (S 26º23’56’’, W 48º44’30’’, 4m), on sandy Espodossolo soil (EMBRAPA, 1999). They were harvested twice, in February (90 days after planting) and May 2010 (180 days after planting). The experimental design was in completely randomized blocks with a 10 x 2 factorial, four replications and sixteen plants each. Before transferring the plants to the field, the experimental area was fertilized with 40 kg/ha of nitrogen, 30 kg/ha of phosphorus and 30 kg/ha of potassium, with two additional nitrogen fertilization (20 kg/ha) 30 days after transplanting and after the first harvest. Plants were spaced by 60 cm between rows and 30 cm between each other. The total area of the experimental unit was 4.2 m2 and the samples were collected from 0.72 m2 of this area. Plant height, fresh and dry weights were determined. The samples were dried at 65ºC in a FANEN (São Paulo, Brazil) model 320SE circulation oven until constant weight. The essential oil was obtained by hydrodistillation of 100 g of fresh leaves in a Clevenger apparatus for 2 hours. Table 1. Identification of genotype from mint collection (MC) evaluated in this study GENOTYPE DENOMINATION CIENTIFIC NAME ORIGIN MC* 3 Chocolate mint Mentha x piperita L. Purdue University - USA MC 23 Pepermint Mentha x piperita L. Purdue University - USA MC 34 EMATER 2 Mentha sylvestris L. UnB - Brazil MC 37 EMATER 3 Mentha canadensis L. UnB - Brazil MC 43 Hortelã 560 Mentha piperita L. CPQBA - Brazil MC 57 IAC 9 Mentha sp. IAC - Brazil MC 69 UFC 5 Mentha x piperita L. UFC - Brazil MC 75 Hortelã – PR1 Mentha arvensis L. PR1 Brazil MC 76 Hortelã – PR2 Mentha arvensis L. PR2 Brazil MC 78 Hortelã – PR3 Mentha arvensis L. PR3- Brazil EMATER – Empresa de Assistência técnica e Extensão Rural,CPQBA - Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas - CPQBA , Instituto Agronômico de Campinas - IAC - , Universidade Federal do Ceará – UFC, Universidade de Brasília - UnB, Genótipos de produtores do Paraná (PR1, PR2 e PR3).MC* = Mint collection from Embrapa Genetic Resources and Biotechnology Chemicals Dichloromethane spectroscopic grade (Tedia, Fairfield, USA) was used as solvent. The standards were purchased from Aldrich (Milwaukee, USA). Analysis of the essential oils The oils were analyzed in an Agilent (Palo Alto, USA) 6890N gas chromatograph fitted with a 5% phenyl - 95% methylsilicone (HP5, 25m X 0.32mm X 0.25µm) fused silica capillary column. The oven temperature was programmed from 60ºC to 240ºC at 3ºC/min, and hydrogen was used as carrier gas (1.5 mL/min). The oils were diluted to 1% in dichloromethane and 1.0µL of this solution was injected in split mode (1:100). The injector was kept at 250ºC and the detector (FID) at 280ºC. Mass spectra were obtained in an Agilent 5973N system operating in electronic ionization mode (EI) at 70 eV, with scan mass range of 40-500 m/z. The sampling rate was 3.15 scans/s. The ion source was kept at 230ºC, mass analyzer at 150ºC and transfer line at 260ºC. The mass detector was coupled to an Agilent 6890 gas chromatograph fitted with a low bleeding 5% phenyl - 95% 792 Seasonal variation... SANTOS, V. M. C. S. Biosci. J., Uberlândia, v. 28, n. 5, p. 790-798, Sept./Oct. 2012 methylsilicone (HP-5MS, 30m X 0.25mm X 0.25µm) fused silica capillary column. The injection procedure and oven temperature program were the same as above. Helium was the carrier gas, at 1.0 mL/min. Linear retention indices (LRI) were measured (DOOL; KRATZ, 1963) by injecting a series of n-alkanes (C7-C26) in the same column and conditions indicated above for GC analyses. Identification of the oil components was based on a computer search using the Wiley 6th ed. library of mass spectral data and by comparison of their calculated linear retention indices with data from the literature (ADAMS, 2007). Standard solutions of menthol and menthone were injected for confirmation. Statistical Analysis For each genotype and harvest time the average oil content and the percentage of the identified compounds were calculated using the average mean of the four blocks of replications. MSTAT-C (NISSEN, 1993) was used to verify mean homogeneity with a Barlett Test and data was analyzed using the analysis of variance (ANOVA) of the factorial design by the randomization method. The main plot treatments were the ten genotypes and the subplots consisted of the two harvest times. A Tukey honestly significance difference test at p<0.05 compared means of biomass yield, oil content and yield as well as the percentage of the identified constituents. RESULTS AND DISCUSSION The M. arvensis genotypes exhibited the highest plant height (64.17 to 64.83 cm - Table 2). M. x piperita (MC 3 and MC 23), cultivated in Santa Catarina lead to higher plant heights (28.25 and 21.92cm), when compared to those from Brasilia (18.3 and 15.6 cm), respectively (GRIZI et al., 2006). M. canadensis (MC 37) in this study presented lower plant height than the cultivars Cornmint and Sakhalin mint studied in Finland (AFLATUNI, 2005). The leaf and herb dry yield were affected by both genotypes and harvest time (Table 2). Plants harvested in February showed higher vegetative growth than those harvested in May despite the fact that the vegetation period was very similar (95 and 90 days). The environmental conditions of temperature and precipitation, probably affected the vegetative growth. In February, the average temperature was 6.9ºC higher with a long-day photoperiod of 14 hours, increasing carbon assimilation and biomass production. In addition, precipitation rates lower than 60 mm in April and May could also have limited plant growth in the second harvest. Similar results have been reported for mint species in Curitiba, Brazil, with superior biomass production in summer than autumn (MONTEIRO, 2009; DESCHAMPS et al., 2008). Table 2. Plant height, dry leaf yield and total dry yield (herb yield) of mint species at two harvests Mint genotypes Height (cm) Leaf dry yield kg/ha Total dry yield (herb yield) kg/ha Feb May Total Feb May Total MC 3 28.25 2163 Aab 585 Bab 2748 a 2905 Aab 1119 Bab 4024 b MC 23 21.92 1177 Ac 257 Bb 1434 c 1566 Ac 475 Bb 2041 d MC 34 28.83 1532 Aabc 548 Bab 2080 b 2128 Abc 967Bab 3095 c MC 37 38.50 1829 Aabc 1219 Ba 3048 a 2395 Abc 1867 Ba 4262 b MC 43 28.08 1749 Aabc 286 Bb 2035 b 2374 Abc 549 Bb 2923 c MC 57 27.92 1632 Aabc 692 Bab 2324 b 2198 Abc 1673 Bab 3871 bc MC 69 19.25 1463 Aabc 512 Bab 1975 b 1929 Abc 984 Bab 2913 c MC 75 64.25 2261 Aa 972 Bab 3233 a 3735 Aa 2161 Ba 5896 a MC 76 64.83 1656 Aabc 1306 Ba 2962 a 2657 Aabc 1810 Ba 4467 b MC 78 64.17 1736 Aabc 1243 Ba 2979 a 2690 Aabc 2053 Ba 4743 b Average 1720 762 2458 1366 Coefficient of Variation –CV Leaf dry yield =29.82%. Total dry yield CV= 26.33%; Means followed by the same capital letters on lines and the same lowercase letters on columns are not significantly different for Tukey test at the 0.05 level. Highest herb and leaf yield were observed in M. arvensis (MC 75, MC 76, MC 78), M. canadensis and Mentha x piperita L. (MC 3) genotypes. The M. arvensis genotypes and M. canadensis (MC 37) were harvested at full bloom, when plant structure is commonly changed due to leaf senescence. This did not occur with M. canadensis, as demonstrated by the leaf-herb ratios 793 Seasonal variation... SANTOS, V. M. C. S. Biosci. J., Uberlândia, v. 28, n. 5, p. 790-798, Sept./Oct. 2012 (Table 3). In spite of the leaf senescence, the correlation with essential oil production was positive due to the total herb yield, justifying harvesting at full bloom (ROHLOFF et al., 2005). M. piperita L. (MC 69) in turn exhibited the lowest herb and leaf yield due to its dwarf characteristics and prostrate growth, evidenced by the plant height and the leaf-herb ratio. Although M. arvensis cultivars achieved the highest values of dry herb yield in this study, they are significantly lower than those obtained commercially in India, which ranged from 5.14 to 8.25 t/ha (SRIVASTAVA, et al., 2002). Table 3. Means of leaf/herb ratio of mint species at two harvests Mint genotypes Dry leaf / dry herb Feb May Average MC 3 0,73 Aa 0,50 Bab 0,62 ab MC 23 0,74 Aa 0,56 Bab 0,65 ab MC 34 0,72 Aa 0,59 Bab 0,65 ab MC 37 0,76 Aa 0,66 Ba 0,71 a MC 43 0,73 Aa 0,49 Bab 0,61 ab MC 57 0,75 Aa 0,42 Bb 0,58 b MC 69 0,74 Aa 0,51 Bab 0,63 ab MC 75 0,60 Aa 0,54 Bab 0,57 b MC 76 0,63 Aa 0,59 Bab 0,61 ab MC 78 0,63 Aa 0,60 Bab 0,61 ab Average 0,70 0,55 Dry leaf / dry herb CV=13.89%. Means followed by the same capital letters on lines and the same lowercase letters on columns are not significantly different for Tukey test at the 0.05 level. Significant differences were observed in essential oil content among harvest times and genotypes (Table 4). All of the genotypes had relatively high essential oil contents (0.8 to 5.3%). The essential oil content was higher in February (2.7%) than in May (1.5%). Summer environmental conditions (February), such as high temperature and long days favored essential oil biosynthesis. This is explained by the increase in biomass associated to plant modulation of photosynthetic carbon production into the metabolic machinery of monoterpene biosynthesis (KHANUJA et al., 2000). Similar results have been reported at high altitude conditions in southern Brazil, where a 50% decrease in essential oil content among seven mint genotypes was observed when plants were harvest in winter compared to summer (DESCHAMPS et al., 2008). Table 4. Essential oil content (%) and essential oil yield (L/ha) of mint genotypes at two harvests time Mint genotypes Essential oil content (%) Essential oil yield (L/ha) Feb May Feb May Average Total MC 3 2.8 Abc 1.2 Bbc 81.9 Ab 13.9 Bbc 47.6 b 95.1 MC 23 2.1 Abcd 1.3 Bbc 31.8 Ade 5.8 Bc 18.8 c 37.6 MC 34 2.5 Abc 1.4 Bc 51.5 Acd 12.0 Bbc 31.8 bc 63.5 MC 37 5.3 Aa 3.5 Ba 125.8 Aa 61.1 Ba 93.45 a 186.9 MC 43 2.9 Abc 2.2 Bab 67.4 Abc 11.4 Bbc 39.4 bc 78.8 MC 57 3.4 Ab 0.8 Bc 72.9 Abc 14.8 Bbc 43.8 bc 87.7 MC 69 1.6 Acd 0.9 Bc 27.6 Ae 8.1 Bc 17.8 c 35.7 MC 75 2.1 Abc 1.2 Bc 81.9 Ab 23.3 Bbc 52.6 bc 105.2 MC 76 2.2 Abc 1.5 Bbc 56.8 Ac 33.6 Bb 45.6 b 90.4 MC 78 2.2 Abc 1.4 Bbc 57.4 Ac 28.1 Bbc 42.8 bc 85.4 Average 2.7 1.5 65.4 21.2 Essential oil content CV= 25.33%. Essential oil yield CV=23.13%. Means followed by the same capital letters on lines and the same lowercase letters on columns are not significantly different for Tukey test at the 0.05 level. In the northern hemisphere, M. spicata also presented higher essential oil during the summer months from July to September (13). Study on photoperiodic influence in mint have correlated long days to flowering induction (SANGWAN et al., 2001). In fact, in February, all of the mint genotypes 794 Seasonal variation... SANTOS, V. M. C. S. Biosci. J., Uberlândia, v. 28, n. 5, p. 790-798, Sept./Oct. 2012 were at bloom stage, while in May only the M. arvensis and M. canadensis had attained the stage of bud formation. Besides environmental conditions, senescence of older leaves in February due to flowering induced a reduction of leaf area, with greater oil gland density resulting in higher oil accumulation. Mentha canadensis L. (MC 37), presented the highest essential oil content in both harvests (5.3 % in February and 3.5% in May). When studied in Curitiba, (940 m) Brazil, this genotype had lower oil content (2.8%) (MONTEIRO, 2009). The effect of the altitude correlated to higher UV radiation in secondary metabolites is described for M. piperita, increasing oil content from UV-A radiation during the day, while at night a decrease was observed, (MAFFEI et al., 2000) which may indicate that essential oil biosynthesis can be negatively affected by the altitude and consequent higher UV exposure. Two M. canadensis evaluated in Finland (AFLATUNI, 2005) achieved essential oil content ranging from 1.7 to 2.8% and two other M. canadensis studied in Brasilia, Brazil, presented 2.03 and 4.17% of oil content during the dry season (GRACINDO et al., 2006). The Indian reports on oil and menthol yield for M. arvensis are greatly superior to the values obtained here. Srivastava et al. (2002) describe oil yields of 99.5 to 165 kg/ha and menthol yields of 72.5 to 101 kg/ha. However, their oil content levels ranged from 0.7 to 0.9 and menthol content from 66.7 to 76% which are lower than those found in this study. Therefore, the component responsible for the limitation of oil and menthol yield here was the herb yield, which was two to three times lower than in India. Essential oil (Table 4) and menthol yield (Table 5) data were also influenced by genotype and harvest time. For the material harvested in February, both yields are increased. M. canadensis L. (MC 37) presented the highest oil and menthol yield values. The reports of Monteiro (2009) with the same cultivar were higher for oil (149.4 L/ha) and menthol (126.7 L/ha) yield, although oil content (2,7%) and menthol content (85.6%) were lower. This means that the difference was a consequence of a higher herb yield. In Finland, Cornmint and Sakhalin mint, both M. canadensis L, achieved oil yields ranging from 10 to 51 Kg/ha at three different harvest times. In this case, lower oil content and herb yield limited the values of oil yield (AFLATUNI, 2005). Mentha x piperita L. (MC 3) and Mentha sp. (MC 57) showed potential for essential oil production, however, their menthol yield was low. Table 5. Menthol yield (L/ha) means of mint genotypes at two harvests time Genotype Feb May Average Total MC 3 32.8 Acde 9.3 Bcd 21.1 cd 42.1 MC 23 12.7 Ag 3.3 Bd 7.9 e 16.0 MC 34 24.5 Aef 5.6 Bd 15.1 de 30.1 MC 37 110.2 Aa 56.6 Ba 83.4 a 166.8 MC 43 28.7 Ade 5.5 Bd 17.1 de 34.2 MC 57 8.7 Ag 5.5 Bd 7.6 e 14.2 MC 69 13.9 Afg 4.2 Bd 9.1 e 18.1 MC 75 62.2 Ab 17.9 Bbc 46.1 b 80.1 MC 76 40.5 Ac 25.1 Bb 32.8 b 65.6 MC 78 38.8 Acd 20.1 Bbc 29.5 bc 58.9 Average 37.3 A 15.3 B Menthol yield CV= 19.89%. Means followed by the same capital letters on lines and the same lowercase letters on columns are not significantly different for Tukey test at the 0.05 level. The main components identified in the oil are presented in Table 6. The highest menthol contents were obtained at the second harvest (May) for all genotypes, except M. sylvestris L., while its direct precursor, menthone and the isomer neomenthol were lower. In contrast, characteristics of essential oil in February can be described as “immature” with lower menthol and higher menthone levels (WILDUNG; CROTEAU, 2005) The enzyme menthone-reductase is responsible for converting menthone into menthol (CROTEAU et al. 2005). This enzymatic reaction occurs during the late leaf development (15-55 days) while high menthone content is typical at the early stages of leaf development (RIOS – ESTEPA et al., 2008; WILDUNG; CROTEAU, 2005). This might explain the inverse relationship between menthol/menthone results since at the February harvest a significant senescence of the older leaves 795 Seasonal variation... SANTOS, V. M. C. S. Biosci. J., Uberlândia, v. 28, n. 5, p. 790-798, Sept./Oct. 2012 occurred with concomitant flowering, reducing the ratio of old (mature) to young (immature) leaves (data not presented). The negative correlation of menthol content with menthone and neomenthol observed in February, has been reported as a result of increased photoperiodic influence of long day in M. arvensis (SANGWAN et al., 2001). Menthyl acetate is described as an undesirable compound because it promotes changes in organoleptic properties and oil oxidation (PAULUS et al., 2007). This compound is also used as a commercial indicator of an “over mature” oil (WILDUNG; CROTEAU, 2005). This second stage of oil maturation occurs at late bloom increasing the menthol and menthyl acetate levels as occurred during the the harvest of May (RIOS – ESTEPA et al., 2008; ROHLOFF et al., 2005). The higher menthyl acetate contents in May might suggest that an earlier harvest can decrease the level of mentyl acetate and improve oil quality. In the case of Mentha x piperita L. (MC 69) the higher levels of menthyl acetate seems due to a specific genotypic characteristic. Table 6. Qualitative and quantitative composition of the essential oil from ten mint genotypes at two harvests Genotype February Menthol Mentone Neomenthol Menthyl Acetate Pulegone 1.8 cineol Limonene MC* 3 40.4 d 35.9 b 9.0 ab 1.44 b 2.80 c 4.20 a 0.46 bcd MC 23 39.7 d 32.9 bc 12.0 a 1.82 b - 4.22 a - MC 34 47.6 d 26.5 c 8.9 ab 1.57 b 4.97 b 3.58 a - MC 37 89.6 a 2.6 e 3.0 c - - - 0.51 abcd MC 43 42.6 d 29.9 bc 10.9 ab 1.52 b 3.39 bc 3.80 a 0.50 abcd MC 57 12.0 e 56.8 a 8.6 ab 1.75 b 14.01 a 3.64 a 0.11 d MC 69 50.5 cd 13.0 d 8.4 bc 9.92 a 2.51 c 1.95 a 0.85 abc MC 75 75.9 ab 10.4 de 6.4 bc 3.47 b - - 0.24 cd MC 76 71.2 b 16.2 d 7.2 abc 2.35 b - - 0.63 abc MC 78 67.6 bc 17.1 d 8.9 ab 3.07 b - - 0.94 ab May Genotype Menthol Mentone Neomenthol Menthyl Acetate Pulegone 1.8 cineol Limonene MC* 3 66.93 bcd 15.36 b 5.57 b 1.95 e 0.30 c 3.28 a 1.15 a MC 23 57.53 cde 14.80 bc 3.30 b 10.35 bc - 1.20 a - MC 34 46.70 ef 10.73 bcd 6.03 ab 10.93 b 4.04 a 2.40 a - MC 37 92.69 a 2.20 e 2.95 b - - - 0.31 bc MC 43 48.44 ef 11.56 bcd 10.41 a 9.80 bc 3.59 a 2.69 a 0.92 ab MC 57 37.33 f 26.00 a 4.57 b 4.70 de 5.84 a 2.34 a 0.09 c MC 69 51.78 ef 3.63 de 5.89 ab 25.92 a 1.28 b 1.90 a 1.13 ab MC 75 76.76 ab 4.98 de 6.86 ab 9.07 bcd - - 0.60 ab MC 76 74.59 bc 6.69 cd 7.23 ab 5.76 cd - - 0.36 bc MC 78 71.93 bc 6.30 cde 7.09 ab 9.50 bc - - 0.62 ab CV% 13.88 20.95 25.81 15.43 13.60 18.39 24.75 Means followed by the letters are not significantly different for Tukey test at the 0.05 level. Low contents of pulegone and 1,8-cineol were observed in the essential oil of M. piperita genotypes and these compounds were absent in M. canadensis and M. arvensis genotypes. Pulegone along with menthofuran (not present) are produced and accumulated according to the level of environmental stress (WILDUNG; CROTEAU, 2005). Therefore it is reasonable to presume that the M. canadensis and M. arvensis genotypes exhibited good adaptation to local environmental conditions. Limonene, an earlier precursor of the menthol biosynthesis pathway appears in low levels (approximately 1%) probably due to the plants ontogenic stage at harvest time (RIOS – ESTEPA et al., 2008). Other constituents identified at amounts lower than 0.3% in oil analysis were: isomenthol, isopulegone, isomenthone, linalool, α-pinene, β- pinene, sabinene, mircene, piperitone, 3-octanol, (E)-caryophyllene, β-germacrene and bicyclogermacrene. The genotype M. canadensis L. (MC 37) showed the highest menthol content at both harvests (89.6 and 92.7). The results were higher than those reported by Monteiro, (2009) with the same genotype (85.6%) in Curitiba, Brazil and also higher than the 18 genotypes of the mint collection studied in India by Shasany et al. (2010) when the highest menthol content was 83.8%. At Brasilia, Brazil 796 Seasonal variation... SANTOS, V. M. C. S. Biosci. J., Uberlândia, v. 28, n. 5, p. 790-798, Sept./Oct. 2012 another M. canadensis L. (MC 20) was characterized with 65% of menthol (GRACINDO et al., 2006). These data express the potential of this M. canadensis L. genotype for menthol production under the local conditions. The result of menthol content of the M. arvensis (MC 75, 76 and 78) genotypes was also high. These values are consistent with that reported by Srivastava et al. (2002) for three cultivars at ten locations, varying from 66.7% to 76%, of menthol content but were lower than that described by Anwar et al.(2010) ranging from 77.6. to 82.4 on six cultivars, in India. Gracindo et al. (2006) also studied the oil composition of Mentha x piperita L. (MC 3 and MC 23) in Brasilia, and found that the levels of menthol varied from 38.0 to 43.0%, menthone from 11.4 to 20.6% and neomenthol from 1.9 to 4.7%, respectively. In contrast, they also detected 16.2% of carvone in MC 3 and 1.0% of limonene in MC 23, which were not detected in this work. CONCLUSIONS From the results it can be stated that the environmental conditions in Southern Brazil are viable for menthol production, with two harvests. The summer harvest (February) favored oil yield, although with a slight decrease in menthol content. M. canadensis L. (MC 37) can be recommended for local menthol production. The challenge of achieving higher essential oil and menthol yields depends on strategies to increase herb yield by developing innovative agronomic practices. ACKNOWLEDGEMENTS The authors acknowledge Dr. Roberto F. Vieira, from Embrapa Genetic Resources and Biotechnology, for providing the plant material for the experiment. RESUMO: O mentol, constituinte majoritário do óleo essencial de menta é usado nas indústrias farmacêutica, alimentícia e de aromas. Onze genótipos de Mentha sp. foram estudados em relação ao desenvolvimento vegetativo, rendimento, produtividade e composição de óleo essencial nas condições edafoclimáticas do litoral Norte Catarinense, em duas épocas (fevereiro e maio). O trabalho foi conduzido em delineamento experimental de blocos ao acaso, em esquema fatorial 10 x 2 para genótipos e épocas de colheita. A extração do óleo essencial foi realizada por hidrodestilação em aparelho graduado Clevenger. O teor de óleo essencial variou entre 0,8 e 5,3%, sendo maior em fevereiro para todos os genótipos. Os constituintes majoritários identificados foram mentol (12 – 92,7%), mentona (2,2 – 56,9%), e neomentol (2,9 – 12,1). Os maiores teores de mentol foram observados em maio, apresentando correlação negativa com mentona e neomentol, os quais foram superiores em fevereiro. Os teores de acetato de metila apresentaram correlação positiva com os de mentol. Pulegona, 1,8 cineol, e limoneno foram identificados em menores concentrações e outros treze constituintes foram detectados como elementos traço em alguns genótipos. Mentha canadensis L. apresentou os maiores teores de óleo essencial, (5,3 % - Fevereiro e 3,5% - Maio) e mentol (89,6% - Fevereiro, 92,7% - Maio) em ambas as colheitas. Os resultados obtidos permitem concluir que as condições edafoclimáticas do litoral Norte Catarinense são adequadas para a produção de mentol, com duas colheitas, recomendando-se o genótipo M. canadensis L. 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