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the variability of secondary metabolites in mentha × piperita cv . ‘perpeta’ during the development of inflorescence

the variability of secondary metabolites in mentha × piperita cv . ‘perpeta’ during the... Keywords Kúcové slová: Mena × piperita, flavonoidy, fáza kvitnutia, D, polyfenoly, silica INTRODUCTION Mints (Mena L., Lamiaceae) are known and classified as aromatic plants wi high content of essential oil (EO) and are famous medical plants in traditional and conventional medicine all over e world. Mena × piperita, e best known mint, is used chiefly for e treatment of gastrointestinal disorders. Peppermint has also shown strong antimicrobial, antiviral, antioxidant activity and seems to have an antitumour, immunomodulating, antiallergic and chemoprotective potential * fialova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae (Mckay & Blumberg, 2006; Kosálová et al.; 2012). e pharmacological effects of mints are chiefly because of e presence of two main groups of secondary metabolites: EO and phenolic compounds. EO of mints is composed of monoterpenes and sesquiterpenes, e content of which varies from species to species. e main phenolic compounds in mints are phenolic acids (especially rosmarinic acid) and flavonoids (eriodictyol, luteolin, apigenin and eir glycosides). In e Acta Fac. Pharm. Univ. Comen. LXI, 2014 (2), p. 21­25 past, some studies were carried out dealing wi changes in EO composition depending on flowering stage and year/day of harvest (Lawrence, 2007; Felklová et al., 1981; Neugebauerová & Kafková, 2012). e aim of is study is to determine e content of secondary metabolites in leaves of M. × piperita of different harvests according to flowering stage. is study focuses on comparing e quality of peppermint during e development of inflorescence. Massachusetts, USA). e percentage contents were determined at 392 nm and were calculated wi reference to e dried drug and expressed as luteolin-7-O-glucoside (one of e most abundant flavone glycoside in mint family). Total polyphenols (spectrophotometric assay) e content of total polyphenols was determined using a colorimetric meod wi e Folin­Ciocalteau reagent at 760 nm (Spectrophotometer, ermo Electron Corporation Genesys 6, Walam, Massachusetts, USA). e percentage contents were calculated wi reference to e dried drug and expressed as rosmarinic acid. Steam distillation of EO e distillation was carried out immediately after drying of e plant material. 200 ml of water as e distillation liquid was added to 20.0 g of crushed drug in a 500 ml flask. Distillation was carried out at a rate of 3-4 ml/min for 2 h. e volume of isolated EO was measured in e graduated tube. e analysis of EO by GC-MS e EO was analysed using Hewlett Packard GC 5890 Series 2 wi MS 5971 equipped wi INOWAX-MS column (60 m × 0.25 mm, film ickness 0.25 m). e injector and detector temperatures were set at 250 and 280°C, respectively. e column temperature was programmed from 60 to 260°C at a rate of 5°C/min, wi e lower and upper temperatures being held for 3 and 7 min, respectively. e flow rate of e carrier gas (helium) was 1.0 ml/min. 1.0 l of a sample was injected. e composition was reported as a relative percentage of e total peak area. 2. MATERIAL AND MEODS 2.1. Plant material e plants selected for is study were grown in e climatic conditions of e sou-west Slovakia (Bratislava). Variation in e content of substances was followed at weekly intervals, in e period from e beginning of e formation of inflorescence to e phase of overblown flowers. e leaves of M. × piperita cv. `Perpeta' were harvested in during e week period of e two summer mons; during e period from 30.6.2007 to 28.8.2007 (Table 1), on sunny days, morning at 10 am. 2.2. e determination of secondary metabolites Phenol compounds were determined in dry leaves wiin 1 year after e harvest using spectrophotometric meods of e European Pharmacopoeia (Ph. Eur. 6, 2007). Total hydroxycinnamic derivatives (D, Arnow´s assay) e content of D was determined using a colorimetric meod wi e Arnow´s reagent at 505 nm (Spectrophotometer, ermo Electron Corporation Genesys 6, Walam, Massachusetts, USA). e percentage content of D were calculated wi reference to e dried drug and expressed as rosmarinic acid. Flavonoids (spectrophotometric assay) e content of total flavonoids was determined by a spectrophotometric meod using aluminium chloride (Spectrophotometer, ermo Electron Corporation Genesys 6, Walam, 3. RESULTS AND DISCUSSION e examined M. × piperita cv. `Perpeta' (domestic cultivar) was cultivated at a sunlit location in Bratislava. e content of EO and phenolic substances was observed in leaves of 1-yearold plants during e development of floral organs in e week period from 30 June 2007 (beginning of inflorescence Table 1. Developmental stage of plant and air temperatures correspond to harvest time Date of harvest 30 June Average week temperature* (°C/°F) 20.3/68.54 20.3/68.54 19.2/66.56 28.1/82.58 22.6/72.68 19.7/67.46 22.5/72.50 22.9/73.22 23.3/73.94 Developmental stage of floral organ Beginning of inflorescence formation Inflorescence not flowering Before flowering Beginning of flowering Beginning of flowering Full flowering Full flowering End of flowering End of flowering 7 July 14 July 22nd July 31st July 5 August 13 August 21st August 28 August *average week air temperature in e period between each harvest 22 Fialová, S. et al. formation) up to e 28 August 2007 (overblown flowers). Different literatures do not agree on e most appropriate term for e harvest of plant material to be used as samples for studying EO content (Telci et al., 2011; Neugebauerová & Kafková, 2012; Spaldon et al., 1982; Felklová, 1975). erefore, we decided to evaluate and compare e samples at different developmental stage. In is study, we have also examined e proportion of leaves in dry herbal drug (Table 2). e EO was obtained by steam distillation from drug dried for 7 days at 35°C. e contents of EO in e leaves ranged from 23 ml/kg (2.3% V/m) to 36 ml/kg (3.6% V/m) (Table 2). e minimum prescribed content of EO in M. × piperita folium according to Ph. Eur. 6 is 9 ml/kg of EO for e cut drug. is same requirement is also prescribed in e currently available Ph. Eur. 8, 2014. e detected contents of EO during e whole period of development of floral organs for e domestic cultivar `Perpeta' fulfils e Ph. Eur. requirement for e minimal content of EO in drug M. × piperita folium. e quality of e drug M. × piperita folium depends on e constitution of EO, in particular on e presence of desirable compound menol but also on e presence of e ingredients reducing e quality of EO such as menone, menyl acetate and menofuran. e relative percentage content of e menol in EO in our samples ranged from 36% up to 52%, which also meets e requirements of Ph. Eur. for M. × piperita aeeroleum (content of menol 30 ­ 55 %). e lowest value of menol has been detected in e plants at e beginning of inflorescence formation, while e volume of EO reached e highest value (36 ml/kg). Meanwhile, e highest content of menol was detected in e leaves of flowering plants (Fig. 1). By comparing e values of e oer constituents wi respect to e quality of EO, it was found at e high quality EO could be obtained during e stage of full flowering (Fig. 2). Felklová et al. (1981) found e highest content of EO in M. × piperita at e beginning of flowering (Felklová et al., 1981). In our study, EO from e leaves collected at e beginning of flowering had a high amount of menol (52%) and relatively low levels of quality-reducing components menon (8%) and menofuran (2%). In comparison, e highest value of menon has been recorded at e beginning of inflorescence formation. e level of menol and menon has changed rapidly during e investigated period (Fig. 1). e increase of menol could be related to e grow of leaves, which corresponds at e biosynetic level to e processes typical for older leaves and coincides wi e study of several auors of (Gershenson et al., 2000; Croteau & Martinkus, 1979; Brun & Voirin, 1991). It follows at in older leaves, e process of biosynesis predominantly leads to e synesis of menol from its precursor menon (McCaskill & Croteau, 1997). e investigation of phenolic compounds in leaves harvested at different developmental stages of inflorescence of M. × piperita has been provided by spectrophotometrical meods (Ph. Eur. 6, 2007); ese meods also find mention in e currently available Ph. Eur. 8, 2014. In e available literature, no relative study about e content of phenolic compounds in M. × piperita during development of inflorescence can be found. e contents of secondary metabolites contributing to erapeutic effect of mint respond for e quality of drug. Contrary to e yield of EO, which decreases during e development of inflorescence by up to 40 %, e content of phenolic compound increases until e beginning of flowering. ese observations are especially evident in e content of flavonoids, e production of which conspicuously depends on UV light (Dolzhenko et al., 2010). At e beginning of flowering, we detected e content of D was 2.91%, e content of flavonoids expressed as luteolin-7-O-glucoside was 1.48 % Fig. 1. e variation of major monoterpenes in EO of Mena × piperita cv. `Perpeta' during development of inflorescence Acta Fac. Pharm. Univ. Comen. LXI, 2014 (2), p. 21­25 Table 2. e mass proportion of leaves in dry herb [%] and e content of EO [%, V/m] in dry leaves of Mena × piperita cv. `Perpeta' during development of inflorescence Date of harvest 30 June 7 July 14 July Developmental stage of inflorescence Beginning of inflorescence formation Inflorescence not flowering Before flowering Beginning of flowering Beginning of flowering Full flowering Full flowering End of flowering End of flowering e proportion of leaves in dry herb [%]a) 61.0 64.7 61.9 50.3 46.6 36.9 34.0 33.3 30.7 e content of EO [% V/m]b) 3.6 3.4 2.9 2.9 2.7 2.5 2.3 2.3 2.5 22nd July 31 July st 5 August 13 August 21st August 28 August a) n = 100, b) n = 3 and e content of total polyphenols was 10.06 %. Considering phenolic compounds, we detected e best quality drug at e beginning of flowering (Table 3, Fig. 2). 4. CONCLUSION Our results show e best time for e harvest of M. × piperita cv. `Perpeta' leaves considered e best quality drug depending on e contents of secondary metabolites. e optimal period for e harvest has been determined as e stage at e beginning of flowering. During is period, we can obtain from e leaves e EO wi highest content of menol. Besides it, e content of phenolic compounds in leaves such as flavonoids or D are e highest during is period as well. Our work confirmed and complements e earlier observations on EO in peppermint. ACKNOwLEDGEMENTS is study was partially supported by VEGA 1/0059/11 (Screening of biological activities of selected plant species and e identification of eir active constituents). Fig. 2. Secondary metabolites in leaves of M. × piperita cv. `Perpeta' during development of inflorescence. e contents of total polyphenols expressed as rosmarinic acid (content [%]*0.5), total hydroxycinnamic derivatives (D) expressed as rosmarinic acid [%], flavonoids expressed as luteolin-7-O-glucoside [%] and EO [%]. Fialová, S. et al. Table 3. e content of D expressed as rosmarinic acid [%], e content of flavonoids expressed as luteolin-7-O-glucoside [%] and e content of total polyphenols expressed as rosmarinic acid [%] in dry leaves of Mena × piperita cv. `Perpeta' during development of inflorescence Date of harvest 30 June 7 July 14 July Developmental stage of inflorescence Beginning of inflorescence formation Inflorescence not flowering Before flowering Beginning of flowering Beginning of flowering Full flowering Full flowering End of flowering End of flowering D [%]* 2.51 ± 0.09 2.60 ± 0.08 2.90 ± 0.12 2.91 ± 0.07 2.75 ± 0.07 2.72 ± 0.07 2.40 ± 0.10 2.68 ± 0.05 2.51 ± 0.06 Flavonoids [%]* 0.84 ± 0.06 1.12 ± 0.09 1.11 ± 0.03 1.08 ± 0.06 1.48 ± 0.11 1.02 ± 0.08 0.92 ± 0.03 1.18 ± 0.04 1.35 ± 0.07 Total polyphenols [%]* 5.75 ± 0.02 5.96 ± 0.02 5.14 ± 0.05 7.92 ± 0.08 10.06 ± 0.08 7.67 ± 0.37 6.23 ± 0.14 7.08 ± 0.32 7.03 ± 0.08 22nd July 31st July 5 August 13 August 21st August 28 August * Values [%] are presented as means ± standard deviation, n = 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

the variability of secondary metabolites in mentha × piperita cv . ‘perpeta’ during the development of inflorescence

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de Gruyter
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1338-6786
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Abstract

Keywords Kúcové slová: Mena × piperita, flavonoidy, fáza kvitnutia, D, polyfenoly, silica INTRODUCTION Mints (Mena L., Lamiaceae) are known and classified as aromatic plants wi high content of essential oil (EO) and are famous medical plants in traditional and conventional medicine all over e world. Mena × piperita, e best known mint, is used chiefly for e treatment of gastrointestinal disorders. Peppermint has also shown strong antimicrobial, antiviral, antioxidant activity and seems to have an antitumour, immunomodulating, antiallergic and chemoprotective potential * fialova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae (Mckay & Blumberg, 2006; Kosálová et al.; 2012). e pharmacological effects of mints are chiefly because of e presence of two main groups of secondary metabolites: EO and phenolic compounds. EO of mints is composed of monoterpenes and sesquiterpenes, e content of which varies from species to species. e main phenolic compounds in mints are phenolic acids (especially rosmarinic acid) and flavonoids (eriodictyol, luteolin, apigenin and eir glycosides). In e Acta Fac. Pharm. Univ. Comen. LXI, 2014 (2), p. 21­25 past, some studies were carried out dealing wi changes in EO composition depending on flowering stage and year/day of harvest (Lawrence, 2007; Felklová et al., 1981; Neugebauerová & Kafková, 2012). e aim of is study is to determine e content of secondary metabolites in leaves of M. × piperita of different harvests according to flowering stage. is study focuses on comparing e quality of peppermint during e development of inflorescence. Massachusetts, USA). e percentage contents were determined at 392 nm and were calculated wi reference to e dried drug and expressed as luteolin-7-O-glucoside (one of e most abundant flavone glycoside in mint family). Total polyphenols (spectrophotometric assay) e content of total polyphenols was determined using a colorimetric meod wi e Folin­Ciocalteau reagent at 760 nm (Spectrophotometer, ermo Electron Corporation Genesys 6, Walam, Massachusetts, USA). e percentage contents were calculated wi reference to e dried drug and expressed as rosmarinic acid. Steam distillation of EO e distillation was carried out immediately after drying of e plant material. 200 ml of water as e distillation liquid was added to 20.0 g of crushed drug in a 500 ml flask. Distillation was carried out at a rate of 3-4 ml/min for 2 h. e volume of isolated EO was measured in e graduated tube. e analysis of EO by GC-MS e EO was analysed using Hewlett Packard GC 5890 Series 2 wi MS 5971 equipped wi INOWAX-MS column (60 m × 0.25 mm, film ickness 0.25 m). e injector and detector temperatures were set at 250 and 280°C, respectively. e column temperature was programmed from 60 to 260°C at a rate of 5°C/min, wi e lower and upper temperatures being held for 3 and 7 min, respectively. e flow rate of e carrier gas (helium) was 1.0 ml/min. 1.0 l of a sample was injected. e composition was reported as a relative percentage of e total peak area. 2. MATERIAL AND MEODS 2.1. Plant material e plants selected for is study were grown in e climatic conditions of e sou-west Slovakia (Bratislava). Variation in e content of substances was followed at weekly intervals, in e period from e beginning of e formation of inflorescence to e phase of overblown flowers. e leaves of M. × piperita cv. `Perpeta' were harvested in during e week period of e two summer mons; during e period from 30.6.2007 to 28.8.2007 (Table 1), on sunny days, morning at 10 am. 2.2. e determination of secondary metabolites Phenol compounds were determined in dry leaves wiin 1 year after e harvest using spectrophotometric meods of e European Pharmacopoeia (Ph. Eur. 6, 2007). Total hydroxycinnamic derivatives (D, Arnow´s assay) e content of D was determined using a colorimetric meod wi e Arnow´s reagent at 505 nm (Spectrophotometer, ermo Electron Corporation Genesys 6, Walam, Massachusetts, USA). e percentage content of D were calculated wi reference to e dried drug and expressed as rosmarinic acid. Flavonoids (spectrophotometric assay) e content of total flavonoids was determined by a spectrophotometric meod using aluminium chloride (Spectrophotometer, ermo Electron Corporation Genesys 6, Walam, 3. RESULTS AND DISCUSSION e examined M. × piperita cv. `Perpeta' (domestic cultivar) was cultivated at a sunlit location in Bratislava. e content of EO and phenolic substances was observed in leaves of 1-yearold plants during e development of floral organs in e week period from 30 June 2007 (beginning of inflorescence Table 1. Developmental stage of plant and air temperatures correspond to harvest time Date of harvest 30 June Average week temperature* (°C/°F) 20.3/68.54 20.3/68.54 19.2/66.56 28.1/82.58 22.6/72.68 19.7/67.46 22.5/72.50 22.9/73.22 23.3/73.94 Developmental stage of floral organ Beginning of inflorescence formation Inflorescence not flowering Before flowering Beginning of flowering Beginning of flowering Full flowering Full flowering End of flowering End of flowering 7 July 14 July 22nd July 31st July 5 August 13 August 21st August 28 August *average week air temperature in e period between each harvest 22 Fialová, S. et al. formation) up to e 28 August 2007 (overblown flowers). Different literatures do not agree on e most appropriate term for e harvest of plant material to be used as samples for studying EO content (Telci et al., 2011; Neugebauerová & Kafková, 2012; Spaldon et al., 1982; Felklová, 1975). erefore, we decided to evaluate and compare e samples at different developmental stage. In is study, we have also examined e proportion of leaves in dry herbal drug (Table 2). e EO was obtained by steam distillation from drug dried for 7 days at 35°C. e contents of EO in e leaves ranged from 23 ml/kg (2.3% V/m) to 36 ml/kg (3.6% V/m) (Table 2). e minimum prescribed content of EO in M. × piperita folium according to Ph. Eur. 6 is 9 ml/kg of EO for e cut drug. is same requirement is also prescribed in e currently available Ph. Eur. 8, 2014. e detected contents of EO during e whole period of development of floral organs for e domestic cultivar `Perpeta' fulfils e Ph. Eur. requirement for e minimal content of EO in drug M. × piperita folium. e quality of e drug M. × piperita folium depends on e constitution of EO, in particular on e presence of desirable compound menol but also on e presence of e ingredients reducing e quality of EO such as menone, menyl acetate and menofuran. e relative percentage content of e menol in EO in our samples ranged from 36% up to 52%, which also meets e requirements of Ph. Eur. for M. × piperita aeeroleum (content of menol 30 ­ 55 %). e lowest value of menol has been detected in e plants at e beginning of inflorescence formation, while e volume of EO reached e highest value (36 ml/kg). Meanwhile, e highest content of menol was detected in e leaves of flowering plants (Fig. 1). By comparing e values of e oer constituents wi respect to e quality of EO, it was found at e high quality EO could be obtained during e stage of full flowering (Fig. 2). Felklová et al. (1981) found e highest content of EO in M. × piperita at e beginning of flowering (Felklová et al., 1981). In our study, EO from e leaves collected at e beginning of flowering had a high amount of menol (52%) and relatively low levels of quality-reducing components menon (8%) and menofuran (2%). In comparison, e highest value of menon has been recorded at e beginning of inflorescence formation. e level of menol and menon has changed rapidly during e investigated period (Fig. 1). e increase of menol could be related to e grow of leaves, which corresponds at e biosynetic level to e processes typical for older leaves and coincides wi e study of several auors of (Gershenson et al., 2000; Croteau & Martinkus, 1979; Brun & Voirin, 1991). It follows at in older leaves, e process of biosynesis predominantly leads to e synesis of menol from its precursor menon (McCaskill & Croteau, 1997). e investigation of phenolic compounds in leaves harvested at different developmental stages of inflorescence of M. × piperita has been provided by spectrophotometrical meods (Ph. Eur. 6, 2007); ese meods also find mention in e currently available Ph. Eur. 8, 2014. In e available literature, no relative study about e content of phenolic compounds in M. × piperita during development of inflorescence can be found. e contents of secondary metabolites contributing to erapeutic effect of mint respond for e quality of drug. Contrary to e yield of EO, which decreases during e development of inflorescence by up to 40 %, e content of phenolic compound increases until e beginning of flowering. ese observations are especially evident in e content of flavonoids, e production of which conspicuously depends on UV light (Dolzhenko et al., 2010). At e beginning of flowering, we detected e content of D was 2.91%, e content of flavonoids expressed as luteolin-7-O-glucoside was 1.48 % Fig. 1. e variation of major monoterpenes in EO of Mena × piperita cv. `Perpeta' during development of inflorescence Acta Fac. Pharm. Univ. Comen. LXI, 2014 (2), p. 21­25 Table 2. e mass proportion of leaves in dry herb [%] and e content of EO [%, V/m] in dry leaves of Mena × piperita cv. `Perpeta' during development of inflorescence Date of harvest 30 June 7 July 14 July Developmental stage of inflorescence Beginning of inflorescence formation Inflorescence not flowering Before flowering Beginning of flowering Beginning of flowering Full flowering Full flowering End of flowering End of flowering e proportion of leaves in dry herb [%]a) 61.0 64.7 61.9 50.3 46.6 36.9 34.0 33.3 30.7 e content of EO [% V/m]b) 3.6 3.4 2.9 2.9 2.7 2.5 2.3 2.3 2.5 22nd July 31 July st 5 August 13 August 21st August 28 August a) n = 100, b) n = 3 and e content of total polyphenols was 10.06 %. Considering phenolic compounds, we detected e best quality drug at e beginning of flowering (Table 3, Fig. 2). 4. CONCLUSION Our results show e best time for e harvest of M. × piperita cv. `Perpeta' leaves considered e best quality drug depending on e contents of secondary metabolites. e optimal period for e harvest has been determined as e stage at e beginning of flowering. During is period, we can obtain from e leaves e EO wi highest content of menol. Besides it, e content of phenolic compounds in leaves such as flavonoids or D are e highest during is period as well. Our work confirmed and complements e earlier observations on EO in peppermint. ACKNOwLEDGEMENTS is study was partially supported by VEGA 1/0059/11 (Screening of biological activities of selected plant species and e identification of eir active constituents). Fig. 2. Secondary metabolites in leaves of M. × piperita cv. `Perpeta' during development of inflorescence. e contents of total polyphenols expressed as rosmarinic acid (content [%]*0.5), total hydroxycinnamic derivatives (D) expressed as rosmarinic acid [%], flavonoids expressed as luteolin-7-O-glucoside [%] and EO [%]. Fialová, S. et al. Table 3. e content of D expressed as rosmarinic acid [%], e content of flavonoids expressed as luteolin-7-O-glucoside [%] and e content of total polyphenols expressed as rosmarinic acid [%] in dry leaves of Mena × piperita cv. `Perpeta' during development of inflorescence Date of harvest 30 June 7 July 14 July Developmental stage of inflorescence Beginning of inflorescence formation Inflorescence not flowering Before flowering Beginning of flowering Beginning of flowering Full flowering Full flowering End of flowering End of flowering D [%]* 2.51 ± 0.09 2.60 ± 0.08 2.90 ± 0.12 2.91 ± 0.07 2.75 ± 0.07 2.72 ± 0.07 2.40 ± 0.10 2.68 ± 0.05 2.51 ± 0.06 Flavonoids [%]* 0.84 ± 0.06 1.12 ± 0.09 1.11 ± 0.03 1.08 ± 0.06 1.48 ± 0.11 1.02 ± 0.08 0.92 ± 0.03 1.18 ± 0.04 1.35 ± 0.07 Total polyphenols [%]* 5.75 ± 0.02 5.96 ± 0.02 5.14 ± 0.05 7.92 ± 0.08 10.06 ± 0.08 7.67 ± 0.37 6.23 ± 0.14 7.08 ± 0.32 7.03 ± 0.08 22nd July 31st July 5 August 13 August 21st August 28 August * Values [%] are presented as means ± standard deviation, n = 3

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Dec 30, 2014

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