Background

Along with the advancement of science and technology, people’s trend to natural and organic materials consumption and plant medicines using instead of chemical medicines has been expanding, so that according to World Health Organization, about 51 percent of the world's people are now using plant medicines (Ahmadi Moghadam et al., 2013). Purple coneflower (Echinacea purpurea L. Moench) is one of the most important and well-known medicinal plants in the world, belonging to Asteraceae family (Manayi et al., 2015) that has been mainly used in chemo-preventive and chemotherapy for infectious diseases in both upper and lower respiratory systems. This species has been traditionally employed for treatment of toothache, bowel pain, snake bite, skin disorders, seizure, chronic arthritis, and cancer (Patel et al., 2008) and its active substances show antioxidant, antibacterial, antiviral and antifungal activities, but many of them are effective on various immune parameters and are currently used in AIDS treatment due to their immune stimulant activity (Mancek and Kreft, 2005). Echinacea purpurea L. Moench, among Echinacea species, is one of the main commercial species that has been introduced and cultivated in some parts of Iran. In addition to being used in some drugs, it is also used in landscape as an ornamental plant (Soudi et al., 2007; Karimian Fariman et al., 2011). 

The most important potential active compounds in the purple coneflower are polysaccharides, caffeic acid derivatives (especially chicoric acid), alkaloids, glycoproteins and essential oils (Bauer and Wagner, 1990). An important group of metabolites found in E. purpurea, with comprehensive pharmacological activity, are phenolic acids (Arceusz et al., 2013). Especially cichoric acid was reported to have immune stimulatory properties, such as promotion of phagocyte activity and exertion of antiviral activity, e.g. HIV-1 integrase and viral replication inhibition (Barrett, 2003).

One of the most important problems in the commercialization of medicinal plants because of secondary metabolites extraction, is low level production and high industrial demands for these compounds, and on the other hand their chemical synthesis is usually complex and costly (Zhao et al., 2005). In medicinal plants, number of secondary metabolites can be increased by various methods such as optimizing cultivation conditions, physical shocks, elicitors or metabolite stimuli (Ahmadi Moghadam et al., 2013). So, one of the most common current methods for secondary metabolites production is the use of elicitors that elicit biosynthesis and accumulation of them by inducing defensive responses.

Elicitors are various chemicals or biological agents that can induce physiological changes and phytoalexins accumulation in plants (Zhao et al., 2005) and can have a profound effect on their physiological and phytochemical reactions (Creelman and Mullet, 1995). Therefore, assessment and obtaining the best cultivation conditions that can lead to plant production with the highest percentage of secondary metabolites is one of the most important aims in research on medicinal plants (Jaafar et al., 2012).

Salicylic acid and methyl jasmonate are phytohormonal elicitors used in most studies, because they biologically elicit important active compounds (Singh and Dwivedi, 2018) and are important phytohormones that actively involved in plant defense mechanisms through octadecanoid and phenylpropanoid pathways, respectively (Shivaji et al., 2010). Salicylic acid is a naturally occurring hormone with chemical formula C₇H₆O₃ and acts as a potential plant regulator with important roles in regulating a number of physiological and biochemical processes. Also, salicylic acid activates systemic resistance system, metabolites and antioxidant enzymes synthesis (Eraslan et al., 2008). These enzymes play an important role in the inactivating oxygen free radicals in plant cells and if detoxification of reactive oxygen is not carried out, serious damages will enter to chlorophylls, proteins, membrane lipids and nuclear acids (Alscher et al., 2002). Manoochehrifar (2011) stated that the activity of enzymes such as catalase, peroxidase, superoxide dismutase, ascorbic peroxidase, glutathione reductase and so on were increased in treated plants by salicylic acid.

Methyl jasmonate with chemical formula C₁₃H₂₀O₃ is a colorless liquid that as a plant hormone is effective in the gene expression and metabolite pathways regulation, defensive responses induction and reproduction (Saisavoey et al., 2014). It also plays an important role in reducing free radicals by enhancing antioxidant enzymes, so that by maintaining high levels of antioxidant enzymes such as catalase and superoxide dismutase, prevents free radicals’ effects from stress on membrane (Wang, 1999). Research have shown that salicylic acid and methyl jasmonate as signaling molecules, like other stimulants, induce the expression of associated genes with secondary metabolites production in plants. In fact, salicylic acid and methyl jasmonate are two plant growth regulators that are a key messenger in activating the plant's defense responses, leading to reduced production of raw materials and biosynthesis and accumulation of a variety of plant secondary compounds (Dong and Zhong, 2001; Yu et al., 2001; Yu et al., 2002) such as phenolic acids and essential oils (Ghasemzadeh et al., 2012).Therefore, the aim of this study was to investigate the effects of different concentrations of salicylic acid and methyl jasmonate elicitors on physiological and phytochemical traits of purple coneflower plant.

1 Materials and Methods

This study was conducted in the research farm of faculty of agriculture, University of Zanjan, Iran, during 2017~2018 and 2018~2019. Meteorological information of experiment years is listed in Table 1.

In the first year of experiment (2017), at first after preparation of the soil according to soil test results (Table 2), purple coneflower transplants at four leaf and 15 cm high stage, were purchased and planted according to planting plan on May 20. Experimental design was conducted as factorial based on complete randomized block design (CRBD) with three replications. In the first year of experiment after completely establishment of plants, different concentrations of salicylic acid and methyl jasmonate were sprayed on plants at 24 hours interval and in two stages (20 days apart), namely, at vegetative growth (6 leaf stage) and onset of reproductive growth stages.Treatments included salicylic acid at four levels of 0 (control), 50, 100 and 150 mM and methyl jasmonate at four levels of 0 (control), 50, 100 and 200 µM. In the second year, treatments were sprayed on remaining plants (four plants per plot) as in the first year. 

Four plants were selected in the first year and four remaining plants in the second year from each plot (totally 8 plants per plot).

1.1 Total protein

Protein concentration was determined using Bradford (1976) method and obtained numbers were used to calculate activity rate of antioxidant enzymes.

1.2 Antioxidant activity based on DPPH

Brand-Williams (1995) method was used to measure antioxidant activity and percentage of DPPH (Di Phenyl Picryl Hydrazyl)) radical inhibition was calculated using the following equation:

I: radical inhibition percentage; A_C: absorbance of control (containing all reactant components without sample); A_S: absorption of sample

Then, results were expressed as IC₅₀ (antioxidant amount that is required to reach 50% of early DPPH concentration).

1.3 Catalase enzyme

Catalase activity was measured according to Bergmeyer (1970) method. 

1.4 Peroxidase enzyme

Herzog and Fahimi (1973) method were used to measure peroxidase enzyme activity.

1.5 Superoxide dismutase enzyme

Superoxide dismutase activity assay was measured according to Beauchamp and Fridovich (1971) method by inhibition monitoring of photochemical reduction of Nitro Blue Tetrazolium (NBT) to purple Formazan. 

1.6 Evaluation of phenolic compounds derivatives 

The amount of Caffeic acid, Chicoric acid, Chlorogenic acid were extracted from phenolic extracts of roots according to Thygesen et al. (2007) method and using high-performance liquid chromatography (HPLC, Model Kanuer, Germany) as mg/g DW.

Data analysis and mean comparison based on Duncan level test were performed using SAS software version 9.1 and MSTAT-C at five percent level.

2 Results 

2.1 Total protein

According to Table 3, it is observed that the effects of salicylic acid and methyl jasmonate were not significant on total protein, while the interaction effect of salicylic acid and methyl jasmonate was significant (p<0.1). According to Figure 1, the highest total protein content (16.31 mg g^-1 FW) was obtained at 50 mM salicylic acid and 50 μM methyl jasmonate concentrations. 

2.2 Antioxidant activity

The results (Table 3) show that the effect of methyl jasmonate and interaction effect of salicylic acid and methyl jasmonate were significant on antioxidant activity (p<0.01). According to Table 4, it is observed that the highest amount of antioxidant activity (57.55%) was obtained in the first year at 100 mM salicylic acid and 100 mM methyl jasmonate concentrations and the highest amount (78.69%) in the second year at 100 mM salicylic acid and 50 mM methyl jasmonate concentrations, respectively.

2.3 Superoxide dismutase enzyme

Based on the results of Table 3, it seems that salicylic acid effect (p< 0.01) and methyl jasmonate effect (p< 0.05) and interaction effect of salicylic acid and methyl jasmonate (p< 0.01) were significant on superoxide dismutase enzyme. The highest amount of superoxide dismutase enzyme was obtained in the first year (88 unit mg^-1) at 100 mM salicylic acid and 100 μM methyl jasmonate concentrations and in the second year (95.74 unit mg^-1) at 100 mM salicylic acid and 200 μM methyl jasmonate concentrations, respectively (Table 4).

2.4 Catalase enzyme

The results of Table 3 show that the effects of salicylic acid and methyl jasmonate and interaction effect of salicylic acid and methyl jasmonate were significant on catalase enzyme (p< 0.01). According to Table 5, it is observed that the highest amount of catalase in the first year (0.629 mM H₂O₂ min^-1 Pro.) at concentrations of 100 mM salicylic acid and 100 µM methyl jasmonate and it’s the highest amount in the second year (1.24 mM H₂O₂ min^-1 Pro.) at concentrations of 100 mM salicylic acid and 50 μM methyl jasmonate were obtained.

Table 5 Important derivatives content of phenolic acids in two years of experiment

2.5 Peroxidase enzyme

According to results (Table 3), it is observed that simple effect of salicylic acid and interaction effect of salicylic acid and methyl jasmonate were significant on peroxidase enzyme (p< 0.01). The results of Table 4 show that the highest peroxidase enzyme were obtained in the first year (2.24 units min^-1 mg^-1 pro.) at 100 mM salicylic acid and 200 μM methyl Jasmonate concentrations and in the second year (2.57 units min^-1 mg^-1 pro.) at 100 mM salicylic acid and 100 μM methyl jasmonate concentrations, respectively.

2.6 Content of important derivatives of phenolic acids

After injecting the standard sample into the HPLC apparatus, the inhibition time (per minute) of Chlorogenic acid, Chicoric acid, and Caffeic acid were respectively obtained (Figure 2). According to chromatogram (Figure 2), amount of chicoric acid was significantly higher than of Chlorogenic acid in all samples of extracts.

According to Table 5, it is observed that the highest amount of Chlorogenic acid were obtained in the first year (4.8 mg/g) at 100 mM salicylic acid and 50 μM methyl jasmonate concentrations and in the second year (4.73 mg/g) at 100 mM salicylic acid and 100 μM methyl jasmonate concentrations, respectively. 

Results of Table 5 show that the highest amount of Chicoric acid were obtained in the first year (9.34 mg/g DW) at 100 mM salicylic acid and 100 μM methyl Jasmonate concentrations and in the second year (12.13 mg/g DW) at 100 mM salicylic acid and 100 μM methyl jasmonate concentrations, respectively. 

3 Discussion

3.1 Total protein

Singh and Usha (2003) reported that salicylic acid by increasing of nitrate reductase enzyme activity caused to increase in nitrogen and protein activity of plant. Methyl jasmonate plays a key role in physiological and biochemical reactions and induces accumulation of some intracellular proteins called IIPS (Important Interacting Proteins: thylakoid membrane proteins) that these proteins are very similar to those proteins derived from abscisic acid and salinity stresses, and it is thought that foliar application of these compounds can also be effective as a stress agent.

3.2 Antioxidant activity

Some researchers reported that there is a positive relation between antioxidant activity and phenol content (Divya et al., 2014; Shinde et al., 2016; Choudhury et al., 2017) and mainly antioxidant activity of phenolic compounds in plants is due to their reductive properties and chemical structure that can play an important role in neutralizing reactive oxygen species (ROS) such as free radicals, single and triple oxygen and peroxidases (Zheng and Wang, 2001).

Ghasemi Pirbalouti et al. (2017) stated that low IC₅₀ indicates a stronger ability of extract to act as a DPPH scavenger, while high IC50 is indicator of a lower ability of scavenging activity and a lot of scavenging is needed to achieve 50% of reaction. Zia et al. (2007) stated that the inhibitory effect of DPPH was observed at the highest concentrations in cell cultures treated with salicylic acid and methyl jasmonate (200 μM), in fact the highest antioxidant activity for salicylic acid and methyl jasmonate was at moderate concentrations of 100 µM, therefore, this can be described in terms of inhibitory feedback that occurred in metabolic pathway, it seems that these two elicitors enhance antioxidant levels at lower concentrations and may be due to their positive effects on plant defense signaling.

Mohammad Babar et al. (2007) showed that salicylic acid and methyl jasmonate in treated roots of Ginseng (Panax ginseng) (suspension culture) increase the antioxidant potential, thereby they protect plants against damage and are likely beneficial to human health.

3.3 Effect of salicylic acid and methyl jasmonate elicitors on antioxidant activity of enzymes

The main detoxifying enzymes in oxidative stresses, are catalase, peroxidase and superoxide dismutase and other enzymes that have been shown to play important roles in scavenging of stress-induced reactive oxygen species (ROS) in plants (Ali et al., 2006; Ali et al., 2007).

Considering the effects of salicylic acid and methyl jasmonate on total antioxidant capacity and different antioxidant enzymes as parts of whole antioxidant system, it seems that the effects of these phytochemicals on different parts of antioxidant system appear in different forms (Asghari and Hasanloo, 2015).

3.3.1 Superoxide dismutase enzyme

Superoxide dismutase enzyme belongs to a group of metalloenzymes (Ho et al., 2020) and among antioxidant enzymes, it is the first defense line against oxidizing active species and main scavenger of superoxide (Alscher et al., 2002). Dong et al. (2010) in an experiment showed that whatever salicylic acid concentration elevates, peak of superoxide dismutase activity appears earlier and further. These results indicate that superoxide dismutase reacts rapidly to protect plant from stress-induced damage caused by external salicylic acid. In a study by Agarwal et al. (2005), an external application of salicylic acid increased superoxide dismutase and catalase activity in wheat plants and the highest level was obtained for superoxide dismutase. Also, Darvizheh et al. (2018) showed that antioxidant activity of enzymes in ornamental purple coneflower was increased using salicylic acid. However, this increase is only significant for superoxide dismutase at low concentrations of salicylic acid, so, results show that among tested enzymes, superoxide dismutase showed the highest response to salicylic acid application. 

It has been shown that application of methyl jasmonate causes to increase the activity of superoxide dismutase and catalase enzymes in Japanese Medlar fruits, that leads to decrease in amount of peroxide and hydrogen peroxide ions (Cao et al., 2009). Increasing in superoxide dismutase activity is probably due to re-synthesis (de novo) of two enzymatic proteins and induction of this enzyme’s encoding gene (Verma and Dubey, 2003).

3.3.2 Catalase enzyme

It seems that catalase enzyme reduction is due to increased hydrogen peroxide in peroxisomes as primary message to activate total antioxidant system in plant (Soares et al., 2010) that creates protection against reactive oxygen species (ROS) produced within cells (Kim et al., 2004). 

Alaey et al. (2011) reported that rose flowers treated with salicylic acid showed high water absorption and relative fresh weight and high catalase enzyme activity and salicylic acid delayed reduction in catalase enzyme activity during flowering that is in accordance with results of our study. Therefore, it is reasonable to propose that salicylic acid has a stimulating effect on antioxidant activity and may also acts a reactive oxygen species (ROS) reducer, so, it causes to maintain membrane integrity for a long time.

By methyl jasmonate application, membrane peroxidation increases, and then free radicals enhance and an increase in catalase enzyme activity protects plant from their harmful effects. Also, an increase in catalase activity is due to its antioxidant function, as well, because free radicals’ amount is increased during stress and plants enhance this enzyme activity to escape from free radicals toxicity. Hence, under stress conditions, presence of methyl jasmonate to increase antioxidant enzymes activity and high content of non-enzymatic antioxidants is more essential for plant tolerance against stress to protect it from harmful effects of free radicals (Nasibi, 2010).

3.3.3 Peroxidase enzyme

Peroxidase enzyme plays important roles in plants such as antioxidant capacity increasing and stimulating of some resistance systems against biotic and abiotic stresses (Asghari and Hasanloo, 2015). Cag et al. (2009) reported that peroxidase activity increases by external application of salicylic acid. Asghari and Hasanloo (2015) showed that methyl jasmonate not only increased total antioxidant content of strawberry fruits, but also increased peroxidase activity as an antioxidant and defense enzyme.

Hare and Walling (2006) reported that methyl jasmonate had no effect on peroxidase activity in Datura plant, in this way results of this study indicate that amount of antioxidant activity is not constant and can be changed and it could be for this reason that methyl jasmonate, through production of antioxidant compounds, directly plays a role in free radicals elimination and prevents an increase in antioxidant enzymes activity such as peroxidase (Eskandari-Zanjani et al., 2012) by scavenging these active species and reducing stress effects that is in agreement with results of present study.

It is noteworthy that various experiments result of methyl jasmonate application on antioxidant enzymes are contradictory and what can justify these contradictions is considering time and concentrations of this hormone and type of plant that it sometimes has inhibitory effects (Maciejewska and Kopcewicz, 2002) and sometimes stimulus effects (Wong et al., 2009).

3.4 Content of important derivatives of phenolic acids

Phenylpropanoid is one of the most important pathways in the production of secondary plant metabolites, including phenolic acids. These compounds are also antioxidant molecules, because they involve in free radicals reducing (Pourcel et al., 2007). The key enzymes in the phenylpropanoid pathway are phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL). Also, known Caffeic acid derivatives (Chlorogenic acid, Echinacoside, Cinnarine, and Cichoric acid) are biosynthesized in purple coneflower plant tissues (Shirley, 2001).

Darvizheh et al. (2019) stated that foliar application of salicylic acid can activate phenyl alanine ammonia lyase enzyme activity that leads to an increase in the accumulation of phenolic acid compounds, for this reason, it has increased chlorogenic acid content in the leaves, flowers and roots of purple coneflower and its positive effects have been greater in all plant organs at high concentrations. Research by Ali et al. (2007) showed that salicylic acid can stimulate the phenolic acids accumulation (including chlorogenic acid) in Ginseng roots by altering phenolic synthetic enzymes. Samadi et al. (2016) showed that there is a significant difference between salicylic acid treatment and Chlorogenic acid and Caffeic acid content (p< 0.05) and by increasing of salicylic acid to 588 mM, Chlorogenic acid level increases and then decreases.

Demirci et al. (2017) reported that all parameters of purple coneflower’s roots and stems are reduced according to increasing in methyl jasmonate concentrations, while these treatments increased the Caffeic acid derivatives such as Chlorogenic acid in treated roots and stems with a concentration of 100 μM methyl jasmonate in 45 days after application. As a result, methyl jasmonate may be a promising compound for secondary metabolites production in purple coneflower plants.

There is a positive relationship between application of methyl jasmonate and salicylic acid elicitors and an increase in the phenylalanine ammonia lyase enzyme activity that leads to accumulation of phenolic acid compounds. Therefore, treatment of ginseng roots with methyl jasmonate compared to salicylic acid further stimulates the phenylalanine ammonia lyase enzyme activity, however, content of phenolic acid compounds in treated roots with salicylic acid increases more than treatment with methyl jasmonate (Mohammad Babar et al., 2007).

Among Caffeic acid phenols in purple coneflower, Chicoric acid has been shown the highest antioxidant activity in various laboratory methods (Dalby-Brown et al., 2005) and as a powerful scavenger for reactive oxygen species, it reduces their accumulation under oxidative stress conditions (Schlernitzauer et al., 2013).

Bayram et al. (2015) showed that foliar application of salicylic acid may cause phenolic compounds accumulation, and in confirmation of this, it has been reported that phenolic acid compounds are obtained as a result of pre-treatment with salicylic acid (Radwan, 2012). Kuzel et al. (2009) evaluated the effect of salicylic acid application at different concentrations on purple coneflower for two years and reported that Chicoric acid content of branches was increased by salicylic acid in treated plants.

Also, Darvizheh et al. (2019) reported that the positive effect of salicylic acid on Chicoric acid content is dependent on concentration and the higher effects (150 mg/L) were higher than the lower concentrations (75 mg/L).

Demirci et al. (2017) reported that application of methyl jasmonate increased the Caffeic acid derivatives in roots and stems of purple coneflower compared to control and the highest amount of Chicoric acid (one of the most important Caffeic acid derivatives in roots and stems) is obtained 45 days after 100 μM methyl jasmonate application.

The use of methyl jasmonate and salicylic acid as elicitors, increase abundance of phenolic acid compounds such as Chicoric acid (Hassini et al., 2017) and these elicitors can increase the productivity of plant cells by expressing the encoding genes of involved enzymes in the secondary metabolites’ biosynthesis (Yousefzadi et al., 2010).

Authors’ contributions

Mahmood Mohebby and Seyed Najmeddin Mortazavi are the main experimental designers of this research work. The paper has written by Mahmood Mohebby and read by all authors. Azizollah Kheiry and Jalal Saba were performed as advisors of the research work study and Jalal Saba checked the analysis as well. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by Department of Horticultural Sciences, University of Zanjan, Iran. The authors are also grateful for worthy supports provided by Dr. Shekari and Dr. Zangani (Department of Genetic Engineering and Plant Production).

References

Agarwal S., Sairam K.R., Srivastava G.C., Aruna T., and Meena C.R., 2005, Role of ABA, salicylic acid, calcium and hydrogen peroxide on antioxidant enzyme induction in wheat seedlings, Plant Science, 169: 559-570

https://doi.org/10.1016/j.plantsci.2005.05.004

Ahmadi Moghadam Y., Piri K.H., Bahramnejad B., and Habibi P., 2013, Methyl Jasmonate and Salicylic acid effects on the dopamine production in hairy cultures of Portulaca oleracea (purslan), Bulletin on Environmental Pharmacology Life Science, 2(6): 89-94

Alaey M., Babalar M., Naderi R. and Kafi M., 2011, Effect of pre- and postharvest salicylic acid treatment on physio-chemical attributes in relation to vase life of rose cut flowers, Postharvest Biology and Technology, 61: 91-94

https://doi.org/10.1016/j.postharvbio.2011.02.002

Ali M., Yu K.W., Hahn E.J., and Paek K.Y., 2006, Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and nonenzymatic antioxidant in suspension culture of Panax ginseng roots in bioreactors, Plant Cell Reports, 25:613-620

https://doi.org/10.1007/s00299-005-0065-6

PMid:16463159

Ali M.B., Hahn E.J., and Paek K.Y., 2007, Methyl jasmonate and salicylic acid induced oxidative stress and accumulation of phenolics in Panax ginseng bioreactor root suspension cultures, Molecules, 12:607-621

https://doi.org/10.3390/12030607

PMid:17851415 PMCid:PMC6149333

Alscher R.G., Erturk N., and Health L.S, 2002, Role of superoxide dismutases (SODs) in controlling oxidative stress in plants, Journal of Experimental Botany, 53:1331-1341

https://doi.org/10.1093/jexbot/53.372.1331

PMid:11997379

Arceusz A., Wesolowski M., and Konieczynski P., 2013, Methods for extraction and determination of Phenolic Acids in Medicinal Plants: A Review, Natural Product Communications, 8(12): 1821-29

https://doi.org/10.1177/1934578X1300801238

Asghari M., and Hasanloo A.R., 2015, Interaction effects of salicylic acid and methyl jasmonate on total antioxidant content, catalase and peroxidase enzymes activity in“Sabrosa” strawberry fruit during storage, Scientia Horticulturae, 197: 490-495

https://doi.org/10.1016/j.scienta.2015.10.009

Barrett B., 2003, Medicinal properties of Echinacea: a critical review, Phytomedicine, 10: 66-86

https://doi.org/10.1078/094471103321648692

PMid:12622467

Bauer R., and Wagner H., 1990, Echinacea, Handbuch fur Ärzte, Apotheker und Andere Naturwissenschaftler, Wiss- enschaftliche Verlagsgessellschaft GmbH, Stuttgart, 182

Bayram D., Yigit E., and Akbulut G. B., 2015, The effects of salicylic acid on Helianthus annuus L. exposed to quizalofop-P-ethyl, American Journal of Plant Sciences, 6: 2412–2425

https://doi.org/10.4236/ajps.2015.614244

Beauchamp C., and Fridovich I., 1971, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Analytical Biochemistry, 44: 276-87

https://doi.org/10.1016/0003-2697(71)90370-8

Bergmeyer H.U., 1970, Methoden der Enzymatischen Analyse, Akademie-Verlag Berlin, 1:636-647

Bradford M.M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding, Analytical Biochemistry, 72: 248-254

https://doi.org/10.1016/0003-2697(76)90527-3

Brand-Williams W., Cuvelier M.E., and Berset C., 1995, Use of a free radical method to evaluation antioxidant activity, Food Science Technology, Lebensmittel-Wissenschaft und Technologie, 28: 25-30

https://doi.org/10.1016/S0023-6438(95)80008-5

Cag S., Cevahiroz G., Sarsag M., and Saglam, N. G., 2009, Effect of salicylic acid on pigment, Protein content and peroxidase activity in excised sunflower cotyledons, Pakistan Journal of Botany, 41(5): 2297-2303

Cao S., Zheng Y., Wang K., Jin P., and Rui, H., 2009, Methyl jasmonate reduces chilling injury and enhances antioxidant enzyme activity in postharvest loquat fruit, Food Chemistry, 115:1458–1463

https://doi.org/10.1016/j.foodchem.2009.01.082

Choudhury J., Majumdar S., Roy S., and Chakraborty U., 2017, Antioxidant activity and phytochemical screening of two edible wetland pteridophytes Diplazium esculentum (Retz.) Sw. and Marsilea minuta L.- a comparative study, World Journal of Pharmaceutical and Medical Research, 3(9):195-203

Creelman R.A., and Mullet J.E., 1995, Jasmonic acid distribution and action in plants:Regu- lation during development and response to biotic and abiotic stress, Proceedings of the National Academy of Sciences, 92: 4114-19

https://doi.org/10.1073/pnas.92.10.4114

PMid:11607536 PMCid:PMC41895

Dalby-Brown L., Barsett H., Landbo A. R., Meyer A. S., and Molgaard P., 2005, Synergistic antioxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from Echinacea purpurea on in Vitro oxidation of human low-density lipoproteins, Journal of Agricultural and Food Chemistry, 53: 9413-9423

https://doi.org/10.1021/jf0502395

PMid:16302756

Darvizheh H., Zahedi M., Abaszadeh B., and Razmjoo J., 2018, Changes in some antioxidant enzymes and physiological indices of purple coneflower (Echinacea purpurea L.) in response to water deficit and foliar application of salicylic acid and spermine under field condition, Scientia Horticulturae, 247: 390-399

https://doi.org/10.1016/j.scienta.2018.12.037

Darvizheh H., Zahedi M., Abbaszadeh B., and Razmjoo J., 2019, Effects of foliar application of salicylic acid and spermine on the phenological stages and caffeic acid derivatives yield of purple coneflower (Echinacea purpurea L.) under drought stress, Iranian Journal of Medicinal and Aromatic Plants, 35(5): 705-720

Demirci S., Van Dijk A.D.J., Sanchez Perez G., Aflitos S.A., De Ridder D., and Peters S.A., 2017, Distribution, position and genomic characteristics of crossovers in tomato recombinant inbred lines derived from an interspecific cross between Solanum lycopersicum and Solanum pimpinellifolium, Plant Journal, 89, 554-564

https://doi.org/10.1111/tpj.13406

PMid:27797425

Divya P., Puthusseri B., and Neelwarne B., 2014, The effect of plant regulators on the concentration of carotenoids and phenolic compounds in foliage of coriander, Food Science and Technology, 56: 101-110

https://doi.org/10.1016/j.lwt.2013.11.012

Dong H.D., and Zhong J.J., 2001, Significant improvement of taxane production in suspension cultures of Taxus chinensis by combining elicitation with sucrose feed, BiochemicalEngineering, 8:145-50

https://doi.org/10.1016/S1369-703X(01)00096-1

Dong J., Wan G., and Liang Z., 2010, Accumulation of salicylic acid-induced phenolic compounds and raised activities of secondary metabolic and antioxidative enzymes in Salvia miltiorrhiza cell culture, Journal of Biotechnology, 148: 99-104

https://doi.org/10.1016/j.jbiotec.2010.05.009

PMid:20576504

Eraslan F., Inal A., David J., and Gunes, A., 2008, Interactive effects of salicylic acid and silicon on oxidative damage and antioxidant activity in spinach (Spinacia oleracea L.cv. Matado) grown under boron toxicity and salinity, Plant Growth Regulation, 33: 292-210

Eskandari-Zanjani K., Shiranirad A. M., Moradi-Aghdam  A., and Taherkhani T., 2012, Effect of salicylic acid application in salt stress conditions on physiological and morphological characteristics of Artemisia (Artemisia annua L.), Ecophysiology of Crop Plants, 4(24): 428-415

Ghasemi Pirbalouti A., Malekpoor F., Salimi A., Golparvar A., and Hamedi B., 2017, Effects of foliar of the application chitosan and reduced irrigation on essential oil yield, total phenol content and antioxidant activity of extracts from green and purple basil, Acta Scientiarum Polonorum Hortorum Cultus, 16(6): 177-186

https://doi.org/10.24326/asphc.2017.6.16

Ghasemzadeh A., Jaafar H.Z.E., Karimi E., and Ibrahim M.H., 2012, Combined effect of CO2 enrichment and foliar application of salicylic acid on the production and antioxidant activities of anthocyanin, flavonoids and isoflavonoids from ginger, BMC Complementary and Alternative Medicine, 12:229-239

https://doi.org/10.1186/1472-6882-12-229

PMid:23176249 PMCid:PMC3545976

Hare J. D., and Walling L. L., 2006, Constitutive and jasmonate-inducible traits of Datura Wrightii, Journal of chemical Ecology, 32: 29-45

https://doi.org/10.1007/s10886-006-9349-8

PMid:16525868

Hassini I., Martinez-Ballesta M.C., Boughanmi N., Diego A., Moreno D.A., Carvajal M., 2017, Improvement of broccoli sprouts (Brassica oleracea L. var. italica) growth and quality by KCl seed priming and methyl jasmonate under salinity stress, Scientia Horticulturae, 226:141-151

https://doi.org/10.1016/j.scienta.2017.08.030

Herzog V., and Fahimi H. D., 1973, Determination of the activity of peroxidase, Analytical Biochemistry, 55: 554-562

https://doi.org/10.1016/0003-2697(73)90144-9

Ho T.T., Murthy H.N., and Park S.Y., 2020, Methyl Jasmonate Induced Oxidative Stress and Accumulation of Secondary Metabolites in Plant Cell and Organ Cultures, International Journal of Molecular Sciences, 21(716): 1-18

https://doi.org/10.3390/ijms21030716

PMid:31979071 PMCid:PMC7037436

Jaafar H.Z.E., Ibrahim M.H., and Mohamad Fakri N.F., 2012, Impact of Soil Field Water Capacity on Secondary Metabolites, Phenylalanine Ammonia-lyase (PAL), Maliondialdehyde (MDA) and Photosynthetic Responses of Malaysian Kacip Fatimah (Labisia pumila Benth), Molecules, 17: 7305-7322

https://doi.org/10.3390/molecules17067305

PMid:22695235 PMCid:PMC6268701

Karimian Fariman Z., Azizi M., and Noori S., 2011, Seed Germination and Dormancy Breaking Techniques for Echinacea purpurea L., Journal of Biological and Environmental Sciences,5(13):7-107

Kim Y.H., Kim Y., Cho E., Kwak S., Kwon S., Bae J., and Huh G.H., 2004, Alterations in intracellular and extracellular activities of antioxidant enzymes during suspension culture of sweet potato, Phytochemistry, 65: 2471-2484

https://doi.org/10.1016/j.phytochem.2004.08.001

PMid:15381411

Kuzel S., Vydra J., Triska J., Vrchotova N., Hruby M., and Cigler P., 2009, Elicitation of pharmacologically active substances in an intact medical plant. Journal of Agricultural and Food Chemistry, 57: 7907-7911

https://doi.org/10.1021/jf9011246

PMid:19663425

Maciejewska B., and Kopcewicz J., 2002, Inhibitory effect of methyl jasmonate on flowering and elongation growth in Pharbitis nil, Journal of Plant growth Regulators, 21: 216-223

https://doi.org/10.1007/s003440010061

Manayi A., Vazirian M., and Saeidnia S., 2015, Echinacea purpurea: Pharmacology, phytochemistry and analysis methods, Pharmacogn Review, 9(17): 63-72

https://doi.org/10.4103/0973-7847.156353

PMid:26009695 PMCid:PMC4441164

Mancek B., and Kreft S., 2005, Determination of Cichoric Acid Content in Dried Press Juice of Purple Coneflower (Echinacea Purpurea) with Capillary Electrophoresis, Talanta, 66(5):1094-1097

https://doi.org/10.1016/j.talanta.2005.01.028

PMid:18970094

Manoochehrifar P., Lari Yazdi H., and Zaji B., 2011, The effect of salicylic acid on activities of catalase and peroxidase in 7 days seedlings of Zea mays under drought stress. Proceedings of the 1st National Conference on New Concepts in Agriculture, Islamic Azad University, Saveh Branch

Mohammad Babar A., Eun-Joo H., and Kee-Yoeup P., 2007, Methyl Jasmonate and Salicylic Acid Induced Oxidative Stress and Accumulation of Phenolics in Panax ginseng Bioreactor Root Suspension Cultures, Molecules, 12: 607-621

https://doi.org/10.3390/12030607

PMid:17851415 PMCid:PMC6149333

Nasibi F., 2010, Effect of different concentrations of sodium nitroprusside (SNP) pretreatment on oxidative damages induced by drought stress in tomato plant, Plant Biology, 9: 36-74

Patel T., Crouch A., Dowless K., and Freier D., 2008, Acute effects of oral administration of a glycerol extract of Echinacea purpurea on peritoneal exudate cells in female swiss mice, Brain, Behavior, and Immunity, 22:39

https://doi.org/10.1016/j.bbi.2008.04.124

Pourcel L., Routaboul J.M., and Cheynier V., 2007, Flavonoid oxidation in plants: from biochemical properties to physiological functions, Trends in Plant Science, 12: 29-36

https://doi.org/10.1016/j.tplants.2006.11.006

PMid:17161643

Radwan D.E.M., 2012, Salicylic acid induced alleviation of oxidative stress caused by clethodim in maize (Zea mays L.) leaves, Pesticide Biochemistry and Physiology, 102: 182-188

https://doi.org/10.1016/j.pestbp.2012.01.002

Saisavoey T., Thongchul N., Sangvanich P. and Karnchanatat A., 2014, Effect of methyl jasmonate on isoflavonoid accumulation and antioxidant enzymes in Pueraria mirifica cell suspension culture, Journal of Medicinal Plant Research. 8(9): 401-407

https://doi.org/10.5897/JMPR2013.5363

Samadi S., Ghasemnezhad A., Alizadeh M., and Alami M., 2016, Influence of elicitors on photosynthesis pigments, caffeic acid, chlorogenic acid and proline content of Cynara scolymus callus, Iranian Journal of Horticultural Science and Technology, 47:435-443

Schlernitzauer A., Oiry C., Hamad R., Galas S., Cortade F., Chabi B., Casas F., Pessemesse1 L., Fouret G., FeilletCoudray1 C., Cros G., Cabello G., Magous R., and Wrutniak-Cabello C., 2013, Chicoric Acid Is an Antioxidant Molecule That Stimulates AMP Kinase Pathway in L6 Myotubes and Extends Lifespan in Caenorhabditis elegans, PLOS One, 8(11): 1-11

https://doi.org/10.1371/journal.pone.0078788

PMid:24244361 PMCid:PMC3823992

Shinde S.S., Patil S.M., Rane N.R., Adsul A.A., Gholve A.R., Pawarl P.K., Yadav S.R., and Govindwar S.P., 2016, Comprehensive investigation of free radical quenching potential, total phenol, flavonoid and saponin content, and chemical profiles of twelve Chlorophytum Ker Gawl, Species. Indian Journal of Natural Products and Resources, 7(2): 125-134

Shirley B.W., 2001, Flavonoid biosynthesis: a control model for genetics, biochemistry, cell biology, and biotechnology, Plant Physiology, 126:485-493

https://doi.org/10.1104/pp.126.2.485

PMid:11402179 PMCid:PMC1540115

Shivaji R., Camas A., Ankala A., Engelberth J., Tumlinson J.H., and Williams W.P., 2010, Plants on constant alert: elevated levels of jasmonic acid and jasmonate-induced transcripts in caterpillar-resistant maize, Journal of Chemical Ecology, 36:179-191

https://doi.org/10.1007/s10886-010-9752-z

PMid:20148356

Singh A., and Dwivedi P., 2018, Methyl-jasmonate and salicylic acid as potent elicitors for secondary metabolite production in medicinal plants, Journal of Pharmacognosy and Phytochemistry, 7(1): 750-757

Singh B., and Usha K., 2003, Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress, Plant Growth Regulation, 39: 137-141

https://doi.org/10.1023/A:1022556103536

Soares A.M.S., Souza T.F., Jacinto T., and Machado O.L.T., 2010, Effect of methyl jasmonate on antioxidative enzyme activities and on the contents of ROS and H₂O₂ in Ricinus communisleaves, Brazil Journal of Plant Physiology, 22: 151-158

https://doi.org/10.1590/S1677-04202010000300001

Soudi S., Hashemi S.M., Hosseini A.Z., Ghaemi A., and Jafarabadi M. A., 2007, Anti leishmanial Effect of Echinacea purpurea Root Extract Cultivated in Iran. Iranian Journal of Pharmaceutical Research, 6(2): 147-149

Thygesen L., Thulin J., Mortensen A., Skibsted L.H., and Molgaard P., 2007, Antioxidant activity of cichoric acid and alkamides from Echinacea purpurea alone and in combination, Food Chemistry, 101:74-81

https://doi.org/10.1016/j.foodchem.2005.11.048

Verma S., and Dubey R. S., 2003, Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants, Plant Science, 164: 645- 655

https://doi.org/10.1016/S0168-9452(03)00022-0

Wang S.Y., 1999, Methyl Jasmonate reduces water stress in strawberry, Journal of Plant Growth Regulation, 18: 127-134

https://doi.org/10.1007/PL00007060

PMid:10594248

Wong C.E., Singh M. B., and Bhalla P.L., 2009, Floral initiation process at the soybean shoot apical meristem may involve multiple hormonal pathways, Plant Signal and Behavior, 7: 648-651

https://doi.org/10.4161/psb.4.7.8978

PMCid:PMC2710565

Yousefzadi M., Sharifi M., Behmanesh M. Moyano E., and Palazon, J, 2010, “Salicylic acid improves podophyllotoxin production in cell cultures of Linum album by increasing the expression of genes related with its biosynthesis”., Biotechnology Letter, 32:1739-1743

https://doi.org/10.1007/s10529-010-0343-4

PMid:20607358

Yu K.W., Gao W., Hahn E.J., and Paek K.Y., 2002, Jasmonic acid improving ginsenoside accumulation in adventitious root culture of Panax ginseng C.A Meyer, Biochemical Engineering Journal, 11:211-215

https://doi.org/10.1016/S1369-703X(02)00029-3

Yu L.J., Lan W.Z., Qin W.M., and Xu H.B., 2001, Effects of salicylic acid on fungal elicitor induced membrane-lipid peroxidation and Taxol production in cell suspension cultures of Taxus chinensis, Process Biochemical, 37: 477 482

https://doi.org/10.1016/S0032-9592(01)00243-6

Zhao J.L., Davis C., and Verpoorte R., 2005, Elicitor signal transduction leading to production of plant secondary metabolites., Biotechnology Advances, 23: 283-333

https://doi.org/10.1016/j.biotechadv.2005.01.003

PMid:15848039

Zheng W., and Wang S.Y., 2001, Antioxidant activity and phenolic compounds in selected herbs, Journal of Agricultural Food Chemistry, 49: 5165-5170

https://doi.org/10.1021/jf010697n

PMid:11714298

Zia M., Mannan A., and Chaudhary M. F., 2007, Effect of growth regulators and amino acids on artemisinin production in the callus of Artemisia absinthium, Pakistan Journal of Botany, 39: 799-805