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Elicitation – a tool to improve secondary metabolites production in Melissa Officinalis L. Suspension cultures / Elicitácia ako nástroj na zlepšenie produkcie sekundárnych metabolitov v suspenzných kultúrach Melissa Officinalis L.

Elicitation – a tool to improve secondary metabolites production in Melissa Officinalis L.... Keywords Kúcové slová: INTRODUCTION The demand for important active natural compound is still high in the field of pharmacy. Isolation from wild-growing or cultivated plants currently often is the only way to obtain appreciable amounts, as chemical synthesis tends to be difficult and non-profitable (Namdeo, 2007). The production of valuable active secondary metabolites used in therapy in medical plants is usually low (usually less than 1% dry weight), as these substances play no fundamental role in the maintenance of plants life processes (Ramakrishna & Ravishankar, 2011). Low yields of natural compounds from * E-mail: balazova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae Urdová J. et al. intact herbs have led to efforts to find new strategies to enhance their biosynthetic capacity. It seems that production of plant secondary metabolites by cell cultures is an attractive alternative to extraction of whole plants (Oksman-Caldentey & Inzé, 2004). Elicitation techniques provide a way to achieve higher yield of plant secondary metabolites due to controlled stress conditions. Lemon balm, Melissa officinalis L. (Lamiaceae), is a medicinal plant of long tradition with a large variety of uses in European traditional medicine. Therapeutic properties of lemon balm are attributed to essential oil that exhibits antioxidative, antibacterial, antiviral, sedative, carminative and antispasmodic effects (Tóth et al., 2003). Phenolic compounds, represented by rosmarinic, caffeic, protocatechuic acids and their methyl esters contribute to many of the activities mentioned as well. Melissa officinalis L. subsp. officinalis is the only of three known subspecies that is used in pharmaceutical preparations nowadays (Marongui et al., 2004). The domestic cultivar `Citra' belongs to frequently studied taxa due to essential oil production (Fialová et al., 2008; Mrlianová et al., 2001, 2002, 2011; Tóth et al., 2003). as rosmarinic acid. Acquired results are the mean of three parallel measurements. RESULTS The effect of four elicitors on Melissa officinalis L. cell suspension cultures production properties was tested in this study. Final concentrations of methyl jasmonate in treated suspension cultures were 10 and 100 mol.l-1, respectively. A Botrytis cinerea hydrolyzate (glucose equivalent 24 g.ml-1) was added to suspension cultures in 1 and 2 ml amounts, respectively. Chlorides salts of cadmium and cobalt (II) were present in media at concentrations 100 and 1 µmol.l-1 each, respectively. The best effect on hydroxycinnamic acid derivatives production was exhibited by the addition of 1 ml of Botrytis cinerea hydrolyzate, among different concentrations of tested biotic elicitors. An increase in the production occurred after 24 h of elicitation and continued up to 72 h (Figure 1). The maximal amount of hydroxycinnamic acid derivatives was approximately triple compared to nontreated suspension cultures. The addition of 2 ml of the Botrytis cinerea hydrolyzate did not show the same effect as with 1 ml of it. Even though the content of hydroxycinnamic acid derivatives increased, total amounts quantified in suspension cultures elicited with 1 ml of hydrolyzate were not achieved. Methyl jasmonate showed a moderate increase in phenolic compounds at concentration 10 mol.l-1. The higher concentration of this elicitor used led rather to a decrease of hydroxycinnamic acid derivatives compared to non-elicited cultures, on the contrary (Figure 2). Chlorides salts of cadmium and cobalt effectively increased phenolic compounds production at concentrations 100 µmol.l-1 each. The highest amount of hydroxycinnamic acid derivatives was quantified after 24 h of treatment with cadmium chloride and the content was almost quadruple compared to non-elicited samples. Prolonging the elicitation time (48, 72 h) led to a decrease in quantity of the studied compounds (Figure 3). Where cadmium chloride stimulated the production of hydroxycinnamic acid derivatives 24 h after elicitation, cobalt (II) chloride exhibited different time course accumulation of phenolic compounds. Maximum amount of hydroxycinnamic acid derivatives was observed in suspension cultures exposed to cobalt (II) chloride during 72 h (Figure 4). Lower concentrations of both metal ions showed a similar time course accumulation of hydroxycinnamic acid derivatives in Melissa officinalis L. suspension cultures as higher concentrations. MATERIAL AND METHODS Plant material Melissa officinalis L. cv. `Citra' suspension cultures prepared from long-term calli were used in our experiment, grown in 25 ml of Murashige and Skoog liquid medium supplemented with kinetine (0.1 mg.l-1), 2,4-dichlorphenoxyacetic acid (1 mg.l-1) and sucrose (30 g.l-1). Cultures were cultivated at 23°C, in 60 lux light (12 h per day) on a rotary shaker (150 rpm). Elicitor treatment Four elicitors were tested, each at two different concentrations. Suspension cultures of Melissa officinalis L. were elicited with 1 and 2 ml of Botrytis cinerea hydrolyzate (glucose equivalent 24 g.ml-1). Commercial methyl jasmonate (Sigma, St. Louis, USA) was present in growth media at concentrations 10 and 100 mol.l-1, respectively. Cadmium and cobalt (II) chlorides were tested at final concentrations 1 and 100 mol.l-1. For control samples, elicitors were omitted. Elicitation was performed on the 14th day of sub-cultivation under aseptic conditions in a sterile air cabinet. Subsequently, elicited suspension cultures were maintained under the same conditions mentioned above and harvested 24, 48 and 72 h after elicitation. Determination of total hydroxycinnamic derivates according to Arnow's method acid DISCUSSION Elicitors have been chosen based on previous work performed at the Department of Cellular and Molecular Biology of Drugs, Faculty of Pharmacy in Bratislava. The Botrytis cinerea hydrolyzate is a successful elicitor, the stimulating effect on secondary metabolites production of which has been Hydroxycinnamic acid derivatives content was determined using Arnow's method (Arnow, 1937) as adopted in the Ph. Eur. 8 (European Pharmacopoeia, 2014) and expressed Acta Fac. Pharm. Univ. Comen. LXII, 2015 (Suppl IX): 46-50. Figure 1. Changes in total hydroxycinnamic acid derivatives content [%] in Melissa officinalis L. suspension cultures after elicitation with Botrytis cinerea hydrolyzate Figure 2. Changes in total hydroxycinnamic acid derivatives content [%] in Melissa officinalis L. suspension cultures after elicitation with methyl jasmonate (MJ) Figure 3. Changes in total hydroxycinnamic acid derivatives content [%] in Melissa officinalis L. suspension cultures after elicitation with cadmium chloride (CdCl2) Urdová J. et al. Figure 4. Changes in total hydroxycinnamic acid derivatives content [%] in Melissa officinalis L. suspension cultures after elicitation with cobalt chloride (CoCl2) confirmed in plant in vitro cultures of Papaveraceae family plants before (Balazová et al., 2011; Bilka et al., 2012). The outcomes of this study are in agreement with the results of these mentioned works. The optimum dosage of Botrytis cinerea for the Melissa officinalis L. suspension cultures elicitation was found to be 1 ml of hydrolyzate, glucose equivalent 24 g.ml-1. The concentration of 100 mol.l-1 methyl jasmonate was frequently reported in literature as a successful tretment to elicit secondary metabolites in various plant species (Ketchum et al., 1999; Mizukami et al., 1993). We expected similar effects at this concentration on the phenolic compounds formation in Melissa officinalis L. suspension cultures. The results of experiments have not confirmed our expectations, however. The total hydroxycinnamic acid derivatives content in elicited suspension cultures was lower than in non-elicited samples. We assume that such a high dosage of methyl jasmonate could induce a hypersensitive response leading to cell death and leakage of cell content into the medium. The presence of methyl jasmonate at 10 mol.l-1 concentration increased the content of hydroxycinnamic acid derivatives during 72-h long elicitation only slightly. The relation between methyl jasmonate dosage and secondary metabolites production has been investigated in many plants (Lee-Parson et al., 2004; Ram et al. 2013). These works reported that methyl jasmonate effectively increased the production of secondary metabolites at concentrations below 100 mol.l-1. Recently, elicitation with heavy metals was studied to reveal their effect on secondary metabolites production in plant suspension cultures (Bhuvaneswari et al. 2012; Cai et al. 2013). The stimulation of secondary metabolites accumulation by metals salts has been proven in cell cultures of several plants, such as Eschscholtzia californica Cham. (Balazová et al. 2008), Papaver somniferum L. (Bilková et al. 2006), Catharanthus roseus L. (Srivastava & Srivastava, 2010), Vitis vinifera L. (Cai et al., 2013). On the other hand, heavy metals can negatively affect biological processes in plant organisms (López-Millán et al., 2009). Cadmium chloride at concentration 100 µmol.l-1 increased the content of hydroxycinnamic acid derivatives in Melissa officinalis L. suspension cultures almost quadruple over non-elicited samples 24 h after elicitor treatment. Compared to cadmium, cobalt (II) chloride showed different time course accumulation of hydroxycinnamic acid derivatives. The maximal amount of phenolic compounds in suspension cultures was reached 72 h after elicitation with cobalt (II) chloride at concentration 100 µmol.l-1. The hydroxycinnamic acid derivatives content has exhibited similar time course accumulation in our study as the triterpenoid saponins content in Gymnema sylvestre suspension cultures elicited with cadmium and cobalt salts (Bhuvaneswari et al., 2012). CONCLUSION Among the tested elicitors, cadmium chloride and Botrytis cinerea hydrolyzate have been proven to be the best in enhancing hydroxycinnamic acid derivatives accumulation in Melissa officinalis L. suspension cultures. Successful stimulation of secondary metabolites production in cell suspension cultures of medicinal plants by elicitation particularly depends on the type of elicitor, its dosage, and exposure time. Acta Fac. Pharm. Univ. Comen. LXII, 2015 (Suppl IX): 46-50. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

Elicitation – a tool to improve secondary metabolites production in Melissa Officinalis L. Suspension cultures / Elicitácia ako nástroj na zlepšenie produkcie sekundárnych metabolitov v suspenzných kultúrach Melissa Officinalis L.

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Publisher
de Gruyter
Copyright
Copyright © 2015 by the
ISSN
1338-6786
eISSN
1338-6786
DOI
10.1515/afpuc-2015-0012
Publisher site
See Article on Publisher Site

Abstract

Keywords Kúcové slová: INTRODUCTION The demand for important active natural compound is still high in the field of pharmacy. Isolation from wild-growing or cultivated plants currently often is the only way to obtain appreciable amounts, as chemical synthesis tends to be difficult and non-profitable (Namdeo, 2007). The production of valuable active secondary metabolites used in therapy in medical plants is usually low (usually less than 1% dry weight), as these substances play no fundamental role in the maintenance of plants life processes (Ramakrishna & Ravishankar, 2011). Low yields of natural compounds from * E-mail: balazova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae Urdová J. et al. intact herbs have led to efforts to find new strategies to enhance their biosynthetic capacity. It seems that production of plant secondary metabolites by cell cultures is an attractive alternative to extraction of whole plants (Oksman-Caldentey & Inzé, 2004). Elicitation techniques provide a way to achieve higher yield of plant secondary metabolites due to controlled stress conditions. Lemon balm, Melissa officinalis L. (Lamiaceae), is a medicinal plant of long tradition with a large variety of uses in European traditional medicine. Therapeutic properties of lemon balm are attributed to essential oil that exhibits antioxidative, antibacterial, antiviral, sedative, carminative and antispasmodic effects (Tóth et al., 2003). Phenolic compounds, represented by rosmarinic, caffeic, protocatechuic acids and their methyl esters contribute to many of the activities mentioned as well. Melissa officinalis L. subsp. officinalis is the only of three known subspecies that is used in pharmaceutical preparations nowadays (Marongui et al., 2004). The domestic cultivar `Citra' belongs to frequently studied taxa due to essential oil production (Fialová et al., 2008; Mrlianová et al., 2001, 2002, 2011; Tóth et al., 2003). as rosmarinic acid. Acquired results are the mean of three parallel measurements. RESULTS The effect of four elicitors on Melissa officinalis L. cell suspension cultures production properties was tested in this study. Final concentrations of methyl jasmonate in treated suspension cultures were 10 and 100 mol.l-1, respectively. A Botrytis cinerea hydrolyzate (glucose equivalent 24 g.ml-1) was added to suspension cultures in 1 and 2 ml amounts, respectively. Chlorides salts of cadmium and cobalt (II) were present in media at concentrations 100 and 1 µmol.l-1 each, respectively. The best effect on hydroxycinnamic acid derivatives production was exhibited by the addition of 1 ml of Botrytis cinerea hydrolyzate, among different concentrations of tested biotic elicitors. An increase in the production occurred after 24 h of elicitation and continued up to 72 h (Figure 1). The maximal amount of hydroxycinnamic acid derivatives was approximately triple compared to nontreated suspension cultures. The addition of 2 ml of the Botrytis cinerea hydrolyzate did not show the same effect as with 1 ml of it. Even though the content of hydroxycinnamic acid derivatives increased, total amounts quantified in suspension cultures elicited with 1 ml of hydrolyzate were not achieved. Methyl jasmonate showed a moderate increase in phenolic compounds at concentration 10 mol.l-1. The higher concentration of this elicitor used led rather to a decrease of hydroxycinnamic acid derivatives compared to non-elicited cultures, on the contrary (Figure 2). Chlorides salts of cadmium and cobalt effectively increased phenolic compounds production at concentrations 100 µmol.l-1 each. The highest amount of hydroxycinnamic acid derivatives was quantified after 24 h of treatment with cadmium chloride and the content was almost quadruple compared to non-elicited samples. Prolonging the elicitation time (48, 72 h) led to a decrease in quantity of the studied compounds (Figure 3). Where cadmium chloride stimulated the production of hydroxycinnamic acid derivatives 24 h after elicitation, cobalt (II) chloride exhibited different time course accumulation of phenolic compounds. Maximum amount of hydroxycinnamic acid derivatives was observed in suspension cultures exposed to cobalt (II) chloride during 72 h (Figure 4). Lower concentrations of both metal ions showed a similar time course accumulation of hydroxycinnamic acid derivatives in Melissa officinalis L. suspension cultures as higher concentrations. MATERIAL AND METHODS Plant material Melissa officinalis L. cv. `Citra' suspension cultures prepared from long-term calli were used in our experiment, grown in 25 ml of Murashige and Skoog liquid medium supplemented with kinetine (0.1 mg.l-1), 2,4-dichlorphenoxyacetic acid (1 mg.l-1) and sucrose (30 g.l-1). Cultures were cultivated at 23°C, in 60 lux light (12 h per day) on a rotary shaker (150 rpm). Elicitor treatment Four elicitors were tested, each at two different concentrations. Suspension cultures of Melissa officinalis L. were elicited with 1 and 2 ml of Botrytis cinerea hydrolyzate (glucose equivalent 24 g.ml-1). Commercial methyl jasmonate (Sigma, St. Louis, USA) was present in growth media at concentrations 10 and 100 mol.l-1, respectively. Cadmium and cobalt (II) chlorides were tested at final concentrations 1 and 100 mol.l-1. For control samples, elicitors were omitted. Elicitation was performed on the 14th day of sub-cultivation under aseptic conditions in a sterile air cabinet. Subsequently, elicited suspension cultures were maintained under the same conditions mentioned above and harvested 24, 48 and 72 h after elicitation. Determination of total hydroxycinnamic derivates according to Arnow's method acid DISCUSSION Elicitors have been chosen based on previous work performed at the Department of Cellular and Molecular Biology of Drugs, Faculty of Pharmacy in Bratislava. The Botrytis cinerea hydrolyzate is a successful elicitor, the stimulating effect on secondary metabolites production of which has been Hydroxycinnamic acid derivatives content was determined using Arnow's method (Arnow, 1937) as adopted in the Ph. Eur. 8 (European Pharmacopoeia, 2014) and expressed Acta Fac. Pharm. Univ. Comen. LXII, 2015 (Suppl IX): 46-50. Figure 1. Changes in total hydroxycinnamic acid derivatives content [%] in Melissa officinalis L. suspension cultures after elicitation with Botrytis cinerea hydrolyzate Figure 2. Changes in total hydroxycinnamic acid derivatives content [%] in Melissa officinalis L. suspension cultures after elicitation with methyl jasmonate (MJ) Figure 3. Changes in total hydroxycinnamic acid derivatives content [%] in Melissa officinalis L. suspension cultures after elicitation with cadmium chloride (CdCl2) Urdová J. et al. Figure 4. Changes in total hydroxycinnamic acid derivatives content [%] in Melissa officinalis L. suspension cultures after elicitation with cobalt chloride (CoCl2) confirmed in plant in vitro cultures of Papaveraceae family plants before (Balazová et al., 2011; Bilka et al., 2012). The outcomes of this study are in agreement with the results of these mentioned works. The optimum dosage of Botrytis cinerea for the Melissa officinalis L. suspension cultures elicitation was found to be 1 ml of hydrolyzate, glucose equivalent 24 g.ml-1. The concentration of 100 mol.l-1 methyl jasmonate was frequently reported in literature as a successful tretment to elicit secondary metabolites in various plant species (Ketchum et al., 1999; Mizukami et al., 1993). We expected similar effects at this concentration on the phenolic compounds formation in Melissa officinalis L. suspension cultures. The results of experiments have not confirmed our expectations, however. The total hydroxycinnamic acid derivatives content in elicited suspension cultures was lower than in non-elicited samples. We assume that such a high dosage of methyl jasmonate could induce a hypersensitive response leading to cell death and leakage of cell content into the medium. The presence of methyl jasmonate at 10 mol.l-1 concentration increased the content of hydroxycinnamic acid derivatives during 72-h long elicitation only slightly. The relation between methyl jasmonate dosage and secondary metabolites production has been investigated in many plants (Lee-Parson et al., 2004; Ram et al. 2013). These works reported that methyl jasmonate effectively increased the production of secondary metabolites at concentrations below 100 mol.l-1. Recently, elicitation with heavy metals was studied to reveal their effect on secondary metabolites production in plant suspension cultures (Bhuvaneswari et al. 2012; Cai et al. 2013). The stimulation of secondary metabolites accumulation by metals salts has been proven in cell cultures of several plants, such as Eschscholtzia californica Cham. (Balazová et al. 2008), Papaver somniferum L. (Bilková et al. 2006), Catharanthus roseus L. (Srivastava & Srivastava, 2010), Vitis vinifera L. (Cai et al., 2013). On the other hand, heavy metals can negatively affect biological processes in plant organisms (López-Millán et al., 2009). Cadmium chloride at concentration 100 µmol.l-1 increased the content of hydroxycinnamic acid derivatives in Melissa officinalis L. suspension cultures almost quadruple over non-elicited samples 24 h after elicitor treatment. Compared to cadmium, cobalt (II) chloride showed different time course accumulation of hydroxycinnamic acid derivatives. The maximal amount of phenolic compounds in suspension cultures was reached 72 h after elicitation with cobalt (II) chloride at concentration 100 µmol.l-1. The hydroxycinnamic acid derivatives content has exhibited similar time course accumulation in our study as the triterpenoid saponins content in Gymnema sylvestre suspension cultures elicited with cadmium and cobalt salts (Bhuvaneswari et al., 2012). CONCLUSION Among the tested elicitors, cadmium chloride and Botrytis cinerea hydrolyzate have been proven to be the best in enhancing hydroxycinnamic acid derivatives accumulation in Melissa officinalis L. suspension cultures. Successful stimulation of secondary metabolites production in cell suspension cultures of medicinal plants by elicitation particularly depends on the type of elicitor, its dosage, and exposure time. Acta Fac. Pharm. Univ. Comen. LXII, 2015 (Suppl IX): 46-50.

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Jun 1, 2015

References