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Phenolic compounds variation in Mentha L. Species in the course of a four-years period / Kolísanie fenolových látok v rôznych druhoch Mentha L. Počas 4 ročného obdobia

Phenolic compounds variation in Mentha L. Species in the course of a four-years period /... Keywords Kúcové slová: 1. INTRODUCTION The species of the genus Mentha L. of the family Lamiaceae belong to the most conventional medicinal plants worldwide. Lawrence et al. (2007) have reviewed the published literature about mints. The genus comprises 18 species and about 11 hybrids placed in 4 sections, of which, the most popular and used are the members of the section Mentha, especially the hybrid M. × piperita. Peppermint has been reported to possess these biological activities: digestive, cholekinetic, choleretic, antispasmodic, antibacterial, antiviral, fungicidal, antioxidant, anti-inflammatory, expectorant, myorelaxant, analgesic as well as insecticidal, aphrodisiac, local anaesthetic, antiemetic, antiulcer, astringent, vasodilating, etc. (Duke et al., 2002; Mckay & Blumberg, 2006; Kosálová et al., 2012). Spearmint (M. spicata) leaves are more commonly used for culinary purposes, but they possess antimicrobial, antioxidant, stimulant, antispasmodic and carminative effects (Lawrence et al., 2007). An antimicrobial and antioxidant effect was proven within many different mints (Gulluce et al., 2007; Fialová et al., 2008; Fialová et al., 2012). The pharmacological effects of mints are chiefly bound to the presence of two main groups of secondary metabolites: essential oil and phenolic compounds. The essential oil of mints is composed of monoterpenes and sesquiterpenes, and the content proportion of these groups' compounds varies from species to species. The main phenolic compounds in mints are phenolic acids (especially rosmarinic acid) and flavonoids (eriodictyol, luteolin, apigenin and their glycosides). * E-mail: fialova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae Recently, several studies dealing with changes in essential oil composition depending on flowering stage and year/day of harvest were carried out and published (Lawrence, 2007; Felklová et al., 1981; Neugebauerová & Kafková, 2012). The aim of this study is to quantify the content of phenolic compounds in leaves of different Mentha species during a 4-year period. RESULTS AND DISCUSSION From a commercial point of view, the most famous mint is peppermint, which is the reason why this species was the frequent subject of research. Mentha × piperita is industrially cultivated worldwide. However, it is kept at one locality for 3­4 years only, as later, it gives a small yield of leaves and essential oil and becomes overgrown with weeds. Considering the length of cultivation at the same place, mints were assessed particularly for the essential oil content and quality (Telci & Shahbaz, 2005; Vaverková et al., 2009), but there are not similar investigations of other secondary metabolites. In our study, we examined the contents of phenolic compounds of different Mentha species. For the quantification experiments, we selected our domestic cultivar Mentha × piperita cv. `Perpeta' and M. spicata, in spite of their high susceptibility to contamination by mint rust (Puccinia menthae), which can completely destroy an entire crop of mint in the second vegetation year already. Therefore, we paid special attention to select healthy plant material only. When comparing the quality of the leaves of plants of different age (1­4 years), the analyses were done after 6 months of storage. Furthermore, the content of phenolic compounds was compared after 1­4 years of storage. We suggest that the losses of phenolic compounds caused by storage (1­4 years) will be minimal. As it was presented in available literature already, the decrease of phenolic compounds in Lamiaceae plants should be minimal, provided correct storage conditions. The decrease of the chosen polar compounds (e.g. tannins) after 3 years storage was about 5% only (Murko et al., 1974). The loss on drying of the investigated Mentha taxa ranged from 8 to 10%, which corresponds to the requirements of the European Pharmacopoeia for leaves drugs (Ph. Eur. 7, 2011). Ph. Eur. 7 requires expressing the content of phenolic compounds in dried drugs. For mints (peppermint), the pharmacopoeias prescribe essential oil quantification, but it is known that plants belonging to the family Lamiaceae subfamily Nepetoideae are, in addition to the essential oil, rich in phenolic constituents with valuable antioxidant activity. The most prominent group of phenolics are phenylpropanoic acid (hydroxycinnamic derivatives), in particular, rosmarinic acid (Petersen & Simmonds, 2003). Lamaison et al. (1991) described the determination of THD in different French Mentha species (3.1­6.5%) using Arnow's reagent. THD content expressed as rosmarinic acid for M. × piperita was 6.1%, M. longifolia 5.1% and M. spicata 6.5%. The species M. × villosa was not included to their study. When monitoring the contents of phenolics in 1- up to 4-year-old plants, the highest content of THD was recorded in leaves of M. × piperita cv.'Perpeta' (from 7.13% to 11.32%) with the height in 3rd year. In other investigated samples, we detected the highest content of THD in 4th year. In other investigated species, the highest content of THD was recorded in the 4th year. In general, we can conclude that the content of THD in MATERIAL AND METHODS Plant material The plants [M. × piperita cv. `Perpeta' (MP), M. spicata subsp. spicata (MS), M. spicata var. crispa (MSC), M. × villosa cv. `Snezná' (MV), M. longifolia ssp. longifolia (ML), M. longifolia var. lavanduliodora (MLL)] were cultivated at the climate conditions of South-West Slovakia, in the Medicinal Plants Garden, Faculty of Pharmacy in Bratislava. Cultures were planted on a light sand-loam soil in a sunny location. The leaves were harvested in June/July, in the plant flowering phase on sunny days, morning at 11 am. The plants were dried at 32­35 °C and stored at room temperature. Voucher specimens were deposited at the Department of Pharmacognosy and Botany, Faculty of Pharmacy, Comenius University in Bratislava. We analysed the 1- to 4-year-old material at the same time. Secondary metabolites quantification Phenolic compounds were determined in dry leaves using spectrophotometric methods of the European Pharmacopoeia (Ph. Eur. 7, 2011). Total hydroxycinnamic derivatives (THD, Arnow's assay) THD content was quantified using a colorimetric method with the Arnow's reagent at 505 nm (Spectrophotometer Genesys 6, Thermo Electron Corp. UK). The THD percentage was calculated with reference to the dried drug and expressed as rosmarinic acid. Flavonoids Total flavonoids content was quantified by a spectrophotometric method using aluminium chloride (Spectrophotometer Genesys 6, UK). The percentage was calculated using the external standard method (calibration curve at 392 nm) with reference to the dried drug and expressed as luteolin-7-O-glucoside ( = 392 nm) and quercetin ( = 420). Total polyphenols and tannins Total polyphenols content was quantified using a colorimetric method with Folin­Ciocalteu's reagent at 760 nm (Spectrophotometer Genesys 6, UK). Tannins content was quantified using the pharmacopoeial hide-powder method. The percentage was calculated with reference to the dried drug and expressed as rosmarinic acid. Measurements were performed in triplicate at least. Acta Fac. Pharm. Univ. Comen. LXII, 2015 (Suppl IX): 2-7. Phenolic compounds variation in Mentha L. species in the course of a four-years... Figure 1. THD expressed as rosmarinic acid ( = 505 nm) in dry leaves of 1- to 4­year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. Lavanduliodora, THD = total hydroxycinnamic derivatives Figure 2. Total polyphenols expressed as rosmarinic acid ( = 760 nm) in dry leaves of 1- to 4-year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. lavanduliodora Mentha species is higher in older growth, 3rd and 4th year of vegetation (Fig. 1). The contents of phenolic compounds in drugs are commonly determined using Folin­Ciocalteu reagent. By this method with hide powder could be determined the content of tannins as well. Tannins in Lamiaceae family represent a special group of polyphenols, which named `Lamiaceae tannins'. It is due to their chemical structure, esters, where the glycoside part is replaced by polyhydroxy acid. The European Pharmacopoeia requires expressing polyphenols and tannins as pyrogallol. However, we expressed them as rosmarinic acid, the most abundant polyphenol compound in mints (Figs. 2 and 3). The determination of total polyphenols and tannins resemble results of THD determination. The highest content of total polyphenols was detected in 3-year-old M. × piperita cv.'Perpeta' (12.74%) and the highest content of tannins in 4-year-old peppermint. The content of total polyphenols in M. longifolia and M. spicata var. crispa was highest in 3rd Figure 3. Tannins expressed as rosmarinic acid ( = 760 nm) in dry leaves of 1- to 4-year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. lavanduliodora Figure 4. Total flavonoids [%] expressed as luteolin-7-O-glucoside ( = 392 nm) in dry leaves of 1- to 4-year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. lavanduliodora vegetation year (11.98% and 9.44%, respectively). M. spicata and M. × villosa cv. `Snezná' have shown the highest content of polyphenols in 4th year of vegetation (11.53 and 8.25%, respectively). Only M. longifolia var. lavanduliodora had higher content of polyphenols in first 2 years of vegetation. Very similar results brought the determination of tannins (Fig. 3). With one exception (M. longifolia var. lavanduliodora), we shall also conclude that the content of polyphenols and tannins increases with the plant age. Flavonoids were examined using the method with aluminium chloride. From the drug, they were extracted with acetone. Aglycones were shaken out into ethyl acetate. Two different types of flavonoids could be detected in mints. First group represents flavonoids that after reaction with AlCl3 absorb UV around 392 nm (luteolin type) and second group flavonoids with maximum absorbance at = 420 nm (quercetin type). The highest contents of flavonoids expressed as luteolin-7-Oglycoside (Fig. 4) in M. spicata var. crispa, M. × villosa and M. longifolia and M. longifolia var. lavanduliodora were detected in 3-year-old plants (0.84; 1.66; 2.11 and 0.69%, respectively). M. × piperita and M. spicata have the highest content of flavonoids of luteolin type in the 1st year. Acta Fac. Pharm. Univ. Comen. LXII, 2015 (Suppl IX): 2-7. Phenolic compounds variation in Mentha L. species in the course of a four-years... Figure 5. Total flavonoids [%] expressed as quercetin ( = 420 nm) in dry leaves of 1- to 4-year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. lavanduliodora The highest content of flavonoids expressed as quercetin (Fig. 5) was recorded in 3-year-old plants only in M. spicata var. crispa (1.18%). In other investigated species, we detected highest levels of flavonoids express as quercetin in the 1st year of vegetation. Anyway, the differences in flavonoid levels could be better explained by the influence of external factors (long-term weather before harvesting time, intensity of sunlight, pathogens and time of the day of harvesting). As mentioned above, only little is known about differences in secondary metabolites content in mints depending on the age of plant. In the period of 4 years (1998­2001), Telci and Shabhaz (2005) investigated the content and composition of essential oil of Mentha × piperita L., from Turkey (Gaziantep and Adana). The highest content of essential oil was recorded in the 2nd harvest of 2nd year (2.8%), while the minimal levels were detected in the 1st harvest of 3rd year (1.7 and 1.6%, respectively) (Telci & Shabhaz, 2005). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

Phenolic compounds variation in Mentha L. Species in the course of a four-years period / Kolísanie fenolových látok v rôznych druhoch Mentha L. Počas 4 ročného obdobia

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Publisher
de Gruyter
Copyright
Copyright © 2015 by the
ISSN
1338-6786
eISSN
1338-6786
DOI
10.1515/afpuc-2015-0013
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See Article on Publisher Site

Abstract

Keywords Kúcové slová: 1. INTRODUCTION The species of the genus Mentha L. of the family Lamiaceae belong to the most conventional medicinal plants worldwide. Lawrence et al. (2007) have reviewed the published literature about mints. The genus comprises 18 species and about 11 hybrids placed in 4 sections, of which, the most popular and used are the members of the section Mentha, especially the hybrid M. × piperita. Peppermint has been reported to possess these biological activities: digestive, cholekinetic, choleretic, antispasmodic, antibacterial, antiviral, fungicidal, antioxidant, anti-inflammatory, expectorant, myorelaxant, analgesic as well as insecticidal, aphrodisiac, local anaesthetic, antiemetic, antiulcer, astringent, vasodilating, etc. (Duke et al., 2002; Mckay & Blumberg, 2006; Kosálová et al., 2012). Spearmint (M. spicata) leaves are more commonly used for culinary purposes, but they possess antimicrobial, antioxidant, stimulant, antispasmodic and carminative effects (Lawrence et al., 2007). An antimicrobial and antioxidant effect was proven within many different mints (Gulluce et al., 2007; Fialová et al., 2008; Fialová et al., 2012). The pharmacological effects of mints are chiefly bound to the presence of two main groups of secondary metabolites: essential oil and phenolic compounds. The essential oil of mints is composed of monoterpenes and sesquiterpenes, and the content proportion of these groups' compounds varies from species to species. The main phenolic compounds in mints are phenolic acids (especially rosmarinic acid) and flavonoids (eriodictyol, luteolin, apigenin and their glycosides). * E-mail: fialova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae Recently, several studies dealing with changes in essential oil composition depending on flowering stage and year/day of harvest were carried out and published (Lawrence, 2007; Felklová et al., 1981; Neugebauerová & Kafková, 2012). The aim of this study is to quantify the content of phenolic compounds in leaves of different Mentha species during a 4-year period. RESULTS AND DISCUSSION From a commercial point of view, the most famous mint is peppermint, which is the reason why this species was the frequent subject of research. Mentha × piperita is industrially cultivated worldwide. However, it is kept at one locality for 3­4 years only, as later, it gives a small yield of leaves and essential oil and becomes overgrown with weeds. Considering the length of cultivation at the same place, mints were assessed particularly for the essential oil content and quality (Telci & Shahbaz, 2005; Vaverková et al., 2009), but there are not similar investigations of other secondary metabolites. In our study, we examined the contents of phenolic compounds of different Mentha species. For the quantification experiments, we selected our domestic cultivar Mentha × piperita cv. `Perpeta' and M. spicata, in spite of their high susceptibility to contamination by mint rust (Puccinia menthae), which can completely destroy an entire crop of mint in the second vegetation year already. Therefore, we paid special attention to select healthy plant material only. When comparing the quality of the leaves of plants of different age (1­4 years), the analyses were done after 6 months of storage. Furthermore, the content of phenolic compounds was compared after 1­4 years of storage. We suggest that the losses of phenolic compounds caused by storage (1­4 years) will be minimal. As it was presented in available literature already, the decrease of phenolic compounds in Lamiaceae plants should be minimal, provided correct storage conditions. The decrease of the chosen polar compounds (e.g. tannins) after 3 years storage was about 5% only (Murko et al., 1974). The loss on drying of the investigated Mentha taxa ranged from 8 to 10%, which corresponds to the requirements of the European Pharmacopoeia for leaves drugs (Ph. Eur. 7, 2011). Ph. Eur. 7 requires expressing the content of phenolic compounds in dried drugs. For mints (peppermint), the pharmacopoeias prescribe essential oil quantification, but it is known that plants belonging to the family Lamiaceae subfamily Nepetoideae are, in addition to the essential oil, rich in phenolic constituents with valuable antioxidant activity. The most prominent group of phenolics are phenylpropanoic acid (hydroxycinnamic derivatives), in particular, rosmarinic acid (Petersen & Simmonds, 2003). Lamaison et al. (1991) described the determination of THD in different French Mentha species (3.1­6.5%) using Arnow's reagent. THD content expressed as rosmarinic acid for M. × piperita was 6.1%, M. longifolia 5.1% and M. spicata 6.5%. The species M. × villosa was not included to their study. When monitoring the contents of phenolics in 1- up to 4-year-old plants, the highest content of THD was recorded in leaves of M. × piperita cv.'Perpeta' (from 7.13% to 11.32%) with the height in 3rd year. In other investigated samples, we detected the highest content of THD in 4th year. In other investigated species, the highest content of THD was recorded in the 4th year. In general, we can conclude that the content of THD in MATERIAL AND METHODS Plant material The plants [M. × piperita cv. `Perpeta' (MP), M. spicata subsp. spicata (MS), M. spicata var. crispa (MSC), M. × villosa cv. `Snezná' (MV), M. longifolia ssp. longifolia (ML), M. longifolia var. lavanduliodora (MLL)] were cultivated at the climate conditions of South-West Slovakia, in the Medicinal Plants Garden, Faculty of Pharmacy in Bratislava. Cultures were planted on a light sand-loam soil in a sunny location. The leaves were harvested in June/July, in the plant flowering phase on sunny days, morning at 11 am. The plants were dried at 32­35 °C and stored at room temperature. Voucher specimens were deposited at the Department of Pharmacognosy and Botany, Faculty of Pharmacy, Comenius University in Bratislava. We analysed the 1- to 4-year-old material at the same time. Secondary metabolites quantification Phenolic compounds were determined in dry leaves using spectrophotometric methods of the European Pharmacopoeia (Ph. Eur. 7, 2011). Total hydroxycinnamic derivatives (THD, Arnow's assay) THD content was quantified using a colorimetric method with the Arnow's reagent at 505 nm (Spectrophotometer Genesys 6, Thermo Electron Corp. UK). The THD percentage was calculated with reference to the dried drug and expressed as rosmarinic acid. Flavonoids Total flavonoids content was quantified by a spectrophotometric method using aluminium chloride (Spectrophotometer Genesys 6, UK). The percentage was calculated using the external standard method (calibration curve at 392 nm) with reference to the dried drug and expressed as luteolin-7-O-glucoside ( = 392 nm) and quercetin ( = 420). Total polyphenols and tannins Total polyphenols content was quantified using a colorimetric method with Folin­Ciocalteu's reagent at 760 nm (Spectrophotometer Genesys 6, UK). Tannins content was quantified using the pharmacopoeial hide-powder method. The percentage was calculated with reference to the dried drug and expressed as rosmarinic acid. Measurements were performed in triplicate at least. Acta Fac. Pharm. Univ. Comen. LXII, 2015 (Suppl IX): 2-7. Phenolic compounds variation in Mentha L. species in the course of a four-years... Figure 1. THD expressed as rosmarinic acid ( = 505 nm) in dry leaves of 1- to 4­year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. Lavanduliodora, THD = total hydroxycinnamic derivatives Figure 2. Total polyphenols expressed as rosmarinic acid ( = 760 nm) in dry leaves of 1- to 4-year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. lavanduliodora Mentha species is higher in older growth, 3rd and 4th year of vegetation (Fig. 1). The contents of phenolic compounds in drugs are commonly determined using Folin­Ciocalteu reagent. By this method with hide powder could be determined the content of tannins as well. Tannins in Lamiaceae family represent a special group of polyphenols, which named `Lamiaceae tannins'. It is due to their chemical structure, esters, where the glycoside part is replaced by polyhydroxy acid. The European Pharmacopoeia requires expressing polyphenols and tannins as pyrogallol. However, we expressed them as rosmarinic acid, the most abundant polyphenol compound in mints (Figs. 2 and 3). The determination of total polyphenols and tannins resemble results of THD determination. The highest content of total polyphenols was detected in 3-year-old M. × piperita cv.'Perpeta' (12.74%) and the highest content of tannins in 4-year-old peppermint. The content of total polyphenols in M. longifolia and M. spicata var. crispa was highest in 3rd Figure 3. Tannins expressed as rosmarinic acid ( = 760 nm) in dry leaves of 1- to 4-year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. lavanduliodora Figure 4. Total flavonoids [%] expressed as luteolin-7-O-glucoside ( = 392 nm) in dry leaves of 1- to 4-year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. lavanduliodora vegetation year (11.98% and 9.44%, respectively). M. spicata and M. × villosa cv. `Snezná' have shown the highest content of polyphenols in 4th year of vegetation (11.53 and 8.25%, respectively). Only M. longifolia var. lavanduliodora had higher content of polyphenols in first 2 years of vegetation. Very similar results brought the determination of tannins (Fig. 3). With one exception (M. longifolia var. lavanduliodora), we shall also conclude that the content of polyphenols and tannins increases with the plant age. Flavonoids were examined using the method with aluminium chloride. From the drug, they were extracted with acetone. Aglycones were shaken out into ethyl acetate. Two different types of flavonoids could be detected in mints. First group represents flavonoids that after reaction with AlCl3 absorb UV around 392 nm (luteolin type) and second group flavonoids with maximum absorbance at = 420 nm (quercetin type). The highest contents of flavonoids expressed as luteolin-7-Oglycoside (Fig. 4) in M. spicata var. crispa, M. × villosa and M. longifolia and M. longifolia var. lavanduliodora were detected in 3-year-old plants (0.84; 1.66; 2.11 and 0.69%, respectively). M. × piperita and M. spicata have the highest content of flavonoids of luteolin type in the 1st year. Acta Fac. Pharm. Univ. Comen. LXII, 2015 (Suppl IX): 2-7. Phenolic compounds variation in Mentha L. species in the course of a four-years... Figure 5. Total flavonoids [%] expressed as quercetin ( = 420 nm) in dry leaves of 1- to 4-year-old mints. Values are presented as means ± standard deviation. MP = M. × piperita cv. `Perpeta', MS = M. spicata, MSC = M. spicata var. crispa, MV = M. × villosa cv. `Snezná', ML = M. longifolia, MLL = M. longifolia ssp. lavanduliodora The highest content of flavonoids expressed as quercetin (Fig. 5) was recorded in 3-year-old plants only in M. spicata var. crispa (1.18%). In other investigated species, we detected highest levels of flavonoids express as quercetin in the 1st year of vegetation. Anyway, the differences in flavonoid levels could be better explained by the influence of external factors (long-term weather before harvesting time, intensity of sunlight, pathogens and time of the day of harvesting). As mentioned above, only little is known about differences in secondary metabolites content in mints depending on the age of plant. In the period of 4 years (1998­2001), Telci and Shabhaz (2005) investigated the content and composition of essential oil of Mentha × piperita L., from Turkey (Gaziantep and Adana). The highest content of essential oil was recorded in the 2nd harvest of 2nd year (2.8%), while the minimal levels were detected in the 1st harvest of 3rd year (1.7 and 1.6%, respectively) (Telci & Shabhaz, 2005).

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Jun 1, 2015

References