Substituted Pyrazinecarboxamides as Abiotic Elicitors of Flavolignan Production in Silybum marianum (L.) Gaertn Cultures in Vitro

Substituted pyrazinecarboxamides markedly influenced production of flavonolignans in Silybum marianum callus and suspension cultures. In this study the effect of two compounds, N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide (1) and N-(3-iodo-4-methylphenyl)-5-tert-butyl-pyrazine-2-carboxamide (2), as abiotic elicitors on flavono-lignan production in callus culture of S. marianum was investigated. Silymarin complex compounds have hepatoprotective, anticancer and also hypocholesterolemic activity. In vitro flavonolignan concentration in cells is very low and the elicitation is one of the methods to increase production. Elicitors were tested at three concentrations and at different culture times. In the case of elicitation with 1, the greatest increase of flavonolignan and taxifoline production was observed at concentration c1a after 6-hours of elicitation and after 24 and 72-hours at concentration c1b. However, increased production of silychristin, one of the compounds in the silymarin complex, was achieved after only 6-hours elicitation with c1a (2.95 × 10-4 mol/L). The content of silychristin was 2-times higher compared to the control sample. An increased production of silychristin was reached with compound 2 at the concentration c2 (2.53 × 10-3 mol/L) after 72 h of elicitation. The production of silychristin in this case was increased 12-times compared to control.


Introduction
Many polycyclic compounds of biological or industrial significance have the pyrazine ring in their structure. There is a high interest in these compounds due to the widespread occurrence of pyrazines in Nature, especially in the flavor compounds of many food systems, their effectiveness at very low concentrations, as well as the still increasing number of applications of synthetic pyrazines in the flavor, fragrance and pharmaceutical industries. In plants or insects, some pyrazines play roles as attractants, pheromones and signal substances, and similar substances are found in food [1].
Silymarin from Silybum marianum is a phytomedicine traditionally used in the treatment of liver disorders and is now included as a complementary and alternative medicine for liver diseases. The active extract of S. marianum, known as silymarin, is a mixture of flavanolignans, namely silybin, silydianin, and silychristine. Although the whole plant is used as a medicinal, the seeds contain the highest content of silymarin (1.5-3.0%). Most of its hepatoprotective properties are attributed to the presence of silybin, which is the main constituent (60-70%) of silymarin. The mechanism of action of silymarin is not well understood. Reports indicate that it acts in multiple ways. Silymarin stabilizes the membrane structure of hepatocytes and thus prevents toxins from entering the cell through enterohepatic recirculation. It promotes liver regeneration by stimulating nucleolar polymerase A and by increasing ribosomal protein synthesis. Silymarin is one of the most successful examples of development of a modern drug from a traditional medicine. However, standardization of silymarin in its various formulations and effective dosages is still lacking [7]. Compounds of the silymarin complex have other interesting activities, e.g. anticancer and cancer protective and also hypocholesterolemic activity. These effects were demonstrated in a large variety of illnesses of different organs, e.g. prostate, lungs, CNS, kidneys, pancreas and also in skin protection. Proapoptotic activity of silybin in pre-and/or cancerogenic cells and anti-angiogenic activity of silybin are other important findings that bring silymarin preparations closer to application in the cancer treatment [8]. Silybin inhibits expression of HIF-1 alpha through suppression of protein translation in prostate cancer cells [9]. Kim determined the effect of silibinin on TNF-α-induced MMP-9 expression (tumor necrosis factor (TNF)α) in gastrin cancer cells. [10] Silibinin possesses strong antioxidant activity and also modulates many molecular changes caused by xenobiotics and ultraviolet radiation to protect the skin [11].
Two ring-substituted (alkylated, brominated) phenylamides of pyrazinecarboxylic acid markedly influenced production of flavonolignans in callus and suspension culture of S. marianum [12]. Additionally the effects of foliar or soil fertilization of 5-tert-butyl-N-m-tolylpyrazine-2-carboxamide on the vegetative and reproductive growth, some physiological parameters, seed yield and silymarin content of field grown milk thistle (S. marianum) plants were studied and the accumulation of flavonoids and silymarin compounds in the seeds was positively influenced. Treatments with foliar fertilizer and 5-tert-butyl-N-m-tolylpyrazine-2-carboxamide resulted in improvement of plant biomass, nutrient accumulation, flowering rate and seed yield and quality [13,14].

Results and Discussion
The condensation of chlorides of substituted pyrazinecarboxylic acids with ring substituted anilines yielded several substituted pyrazinecarboxylic acid amides [15]. Two newly synthesized substituted amides of pyrazinecarboxylic acid, N- (3- Figure 1) were investigated as abiotic elicitors of flavonolignan production in callus culture of S. marianum. Both studied compounds were designed in preference with the lipophilic and/or electron-withdrawing substituents on the benzene moiety.  (1) Compound 1 at concentration c 1 (Table 1) had no important effect on S. marianum calluses, since there is no statistically significant increase or decrease of flavonolignan and taxifolin production. As shown in the Figure 2 there are two decreases in flavonolignan and taxifolin production, after 12 and 48-hours elicitation, and two increases, after 72 and 168-hours elicitation. However, these results are not statistically significant and therefore is was not appropriate to make any conclusions from this fact.

Elicitation with N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide
The effect of compound 1 at concentrations c 1a and c 1b was greater. There was one statistically significant increase in flavonolignan and taxifolin production with c 1a , after 6-hours elicitation (7-times control value), and two with c 1b , after 24 (13-times control value) and 72-hours (10-times control value) elicitation. The detailed HPLC analysis shows that in case of 6-hours' elicitation with compound 1 at concentration c 1a half of the detected content corresponded to taxifolin (0.04%), while the other half was the flavonolignan silychristin (0.04%), whereas after 24 and 72-hours of elicitation with concentration c 1b almost the whole detected content was attributed to taxifolin. Since the uses of S. marianum in therapy are based on the pharmacological activities of silymarin, a component of which is also silychristin, the important effect of elicitation with compound 1 in our study is after 6-hours with concentration c 1a = 10 mg/100 mL. Moreover, with concentration c 1b after 168-hours elicitation there is a decrease in content of flavonolignans and taxifolin, however, the decrease is not statistically significant. In Figure 2 the dependence of the content of flavonolignans and taxifolin related to the elicitor exposure times compared between different concentrations of compound 1 and between controls is presented. All experimental analyses were carried out in a minimum of three independent samples for each elicitation period and each concentration of elicitor.  (2) Results listed in Table 2 show one statistically significant increase in flavonolignan and taxifolin production at each concentration of compound 2; after 72-hours elicitation at c 2 (12-times control value) and after 6-hours elicitation at c 2a (13-times control value) and c 2b (4-times control value). The detailed HPLC analysis of these three results shows that with the c 2 concentration the main detected compound was the flavonolignan silychristin, whereas with c 2a and c 2b concentrations only taxifolin was detected. As shown in Figure 3, declines in the flavonolignans and taxifolin contents were observed. These effects were found after 6, 12 and 72-hours elicitor treatment at concentration c 2 , and after 24-hours elicitor treatment at concentration c 2b . An increase in flavonolignan and taxifolin production after 24-hours elicitation at concentration c 2a was noted.

Exposure time (hours)
c1=100 mg/100 mL c1a=10 mg/100 mL c1b=1 mg/100 mL Results mentioned above are also shown in Figure 3, which shows the effect of elicitation after different exposure times in comparison between three different concentrations of compound 2.

Exposure time (hours)
c2=100 mg/100 mL c2a=10 mg/100 mL c2b=1 mg/100 mL An increased flavonolignan production in S. marianum callus and suspension cultures after treatment with similar compounds, i.e., the 3-methylamide of 5-tert-butylpyrazine-2-carboxylic acid and the 5-bromo-2-hydroxyphenylamide of 5 tert-butyl-6-chloropyrazine-2-carboxylic acid were also observed in our previous studies [12]. The production of flavonolignans in S. marianum callus cultures was generally very low. To clarify this phenomenon we propose the following scenario. The used callus cultures were old (72 nd -74 th passages) and therefore the production of secondary metabolites was to poor. It could be possible that ethanol which was used as a solvent for elicitors and was also added to control samples, caused poorer production of examined secondary metabolites. Preferable for this experiment would be the application of suspension cultures.
Suspension cultures are morphologically more homogenous than callus tissue, thus, likely to show a more uniform biochemical response to elicitor action than the callus tissue. Homogeneous contact between cells in the callus and the culture medium is lacking, making reproducibility of experiments extremely difficult if the size or thickness of callus is slightly changed.

Abiotic elicitors in Silybum marianum L. callus culture
A study was made about effect of several compounds on the production of silymarin in S. marianum cultures. Yeast extract stimulated the accumulation and production of silymarin, chitin and chitosan stimulated only silymarin accumulation. Furthermore, jasmonic acid potentiated the yeast extract effect, and methyljasmonate strongly promoted the accumulation of silymarin by acting on number of steps of the metabolic pathway. [12] There was testing the effect of two substituted amides of pyrazinecarboxylic acid and the results showed that they markedly influenced production of flavonolignans in an in vitro culture. [8]

Material and methods
Callus culture was derived from the germinating seeds of plant Silybum marianum (L.) Gaertn. (Asteraceae). Calluses were cultivated on MS (Murashige and Skoog) medium [16] containing NAA (naphtaleneacetic acid) as growth regulator at a concentration of 5.4 × 10 -5 mol/L. Callus cultures were cultivated on paper bridges in Erlenmeyer flasks. These cultures were incubated in growth chambers at laboratory temperature under a 16 hours photoperiod. As elicitors compounds 1 (N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide, C 12  samples into which 96% ethanol (1 mL) was pipetted. Every 6, 12, 24, 48, 72 and 168 hours we withdrew the calluses, controls were withdrawn after 24 and 168 hours. At each time interval and concentration we withdrew four flasks. After withdrawal, calluses were dried on filter paper at laboratory temperature (25 °C) and pulverized, weighed and used for determination of flavonolignans.

Flavonolignan analysis
Flavonolignan analysis was done by high-performance liquid chromatography (HPLC). A sample of of callus tissues (0.100 g, dry mass) was extracted twice (in a water bath under reflux condenser) with 80 % V/V aqueous methanol (10 mL) for 10 min. The extract was filtrated and then concentrated to 10 mL by vacuum distillation. Approximately 1.7 mL of distillate was placed in vials (after filtration through a 0.45 µm microfilter) and injected into the HPLC system.
HPLC analysis was performed on a Jasco chromatography system (pump PU-2089, detector MD-2015, autosampler AS-2055) equipped with a LiChrospher RP-18 250x4 (5 μm) column. Detection was performed by diode array detector with a wave-length span from 190 -450 nm. The wave-length at 288 nm was used. Volume of injection was 20 µL. Mobile phase was composed of two phases: phase A -methanol solution of o-phosphoric acid (0.15% W/V), and phase B-Water solution of o-phosphoric acid (0.15% W/V). The elution profile was changing through time. At first elution was linear gradient with 100% of phase B at time t = 0 min and 50% of each phase (V/V) at time t = 5 min. Then elution was isocratic with 50% of each phase (V/V) until t = 25 min. The flow was 1.4 mL/min. As standards silymarin and taxifolin were used. [12] To determine whether there was difference between values of samples one-way analysis of variance (ANOVA) was applied. Values of p ≤ 0.05 were considered as significantly different. The differences between means were determined using Turkey´s multiple comparison test.

Conclusions
In the case of elicitation with compound 1 (N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide) on callus cultures of S. marianum the greatest increase of flavonolignan and taxifolin production was observed after 6-hours of elicitation at concentration c 1a and 24 and 72-hours of elicitation at concentration c 1b . However, the increased production of silychristin, one of the compounds in silymarin complex, was reached only after 6-hours elicitation at c 1a = 10 mg/100 mL (2.95 × 10 -4 mol/L). The content of silychristin was 3-times higher compared to control sample. The elicitation with compound 2 (N-(3-iodo-4-methylphenyl)-5-tert-butylpyrazine-2-carboxamide) on callus cultures of S. marianum was higher after 72-hours exposure time at c 2 and after 6-hours at c 2a and c 2b concentrations. The increased production of silychristin was reached only after 72-hours elicitation at concentration c 2 = 100 mg/100 mL (2.53 × 10 -3 mol/L). The production of silychristin was increased 12-times compared to control sample. Achieved results will be the subject of future studies?