pyrazinecarboxamides as Potential Elicitors of Flavonolignan and Flavonoid Production in Silybum marianum and Ononis arvensis Cultures In Vitro

The effect of new synthetic pyrazinecarboxamide derivatives as potential elicitors of flavonolignan and flavonoid production in Silybum marianum and Ononis arvensis cultures in vitro was investigated. Both tested elicitors increased the production of flavonolignans in S. marianum callus and suspension cultures and flavonoids in O. arvensis callus and suspension cultures. Compound I, 5-(2-hydroxybenzoyl)-pyrazine-2-carboxamide, has shown to be an effective elicitor of flavonolignans and taxifoline production in Silybum marianum culture in vitro. The maximum content of silydianin (0.11%) in S. marianum suspension culture was induced by 24 h elicitor application in concentration of 1.159 × 10−3 mol/L. The maximum content of silymarin complex (0.08%) in callus culture of S. marianum was induced by 168 h elicitor application of a concentration 1.159 × 10−4 mol/L, which represents contents of silydianin (0.03%), silychristin (0.01%) and isosilybin A (0.04%) compared with control. All three tested concentrations of compound II, N-(2-bromo-3-methylphenyl)-5-tert-butylpyrazin-2-carboxamide increased the flavonoid production in callus culture of O. arvensis in a statistically significant way. The best elicitation effect of all elicitor concentrations had the weakest c3 concentration (8.36 × 10−6 mol/L) after 168 h time of duration. The maximum content of flavonoids (about 5,900%) in suspension culture of O. arvensis was induced by 48 h application of c3 concentration (8.36 × 10−6 mol/L).


Introduction
In vitro cultures have been seen as an alternative source of biologically active compounds [1]. The disadvantage of these cultures is low production of secondary metabolites, therefore new methods for higher production and accumulation of secondary metabolites by cultures in vitro are being constantly evaluated. One of these methods is the method of elicitation.
The elicitor can be regarded as a stress factor involved in the plant-microorganism, plant-pesticide, plant heavy metal or plant-UV irradiation reactions. Due to chemical defensive reactions, signal substances (elicitor) increase the activity of certain enzymatic systems for a short period and these systems catalyse the formation of stress substances similar to the particular secondary metabolites [2].
The objective of this study was to verify other derivatives as elicitors on flavonoid production in callus and suspension culture of Ononis arvensis and on separately substances of silymarin complex in Silybum marianum culture in vitro. Restharrow root from Ononis spinosa or Ononis arvensis L. (Fabaceae) is used as a mild diuretic. The action of the drug has been confirmed in animal experiments, but so far it has not been possible to isolate any active constituents nor have any of its constituents of known structure been tested pharmacologically. In folk medicine restharrow root is also used for gout and rheumatism complaints.
Silymarin from milk thistle (Silybum marianum) is a phytomedicine used for prophylaxis and treatment of liver damage caused by metabolic toxins (alcohol, tissue poisons), after hepatitis, liver cirrhosis and fatty liver. The extract of S. marianum-silymarin is the mixture of flavanolignans, namely silybin, silydianin and silychristin. Most of hepatoprotective properties are attributed to silybin. It has also been found that silybin increases the rate of synthesis of ribosomal nucleic acids through stimulation of the nucleolar polymerase I. This reinforces protein synthesis and accelerates cell-regeneration processes, so that, besides the prophylactic action, there may also be a curative effect. Other components are the 3-desoxy-derivatives of silychristin and silydianin, isosilychristin, isosilybinin and its 3 desoxyderivative silandrin, the 3-desoxycompounds silyhermin, neosilyhermin A and B, 2,3-dehydrosilybin, as well as tri-to pentamers of silybin; other constituents are taxifolin, quercetin, dihydrokaempferol, kaempferol, apigenin, naringenin, eriodyction, chrysoeriol, and 5,7-dihydroxychromone; c.a. 20-30% fixed oil with a high proportion of linoleic acid; sterols-cholesterol, stigmasterol, campesterol, sitosterol [9].
Compounds of the silymarin complex have also other interesting activities, such as anticancer and cancer protective and hypocholesterolemic properties. Proapoptotic activity of silybin in pre-and/or cancerogenic cells and anti-angiogenic activity of silybin show other important activities that bring silymarin preparations closer application in the cancer treatment [10]. Compounds of the silymarin complex show also antioxidant activity [11].

Results and Discussion
Successful elicitation is subject to many factors that are specific for each elicitor and for each explant culture. This work focused on the elicitor types, elicitor concentration and time duration of elicitor´s effect. The results (Tables 1 and 2) indicate that the elicitor used during the production of flavonolignans in S. marianum callus and suspension cultures influenced the production favourably. Table 1. Content of silymarin complex substances (%) and taxifolin (%) in Silybum marianum callus culture afer elicitor treatment.

Silybum marianum Suspension Culture
Elicitor at c 1 concentration increased the content of silydianin at all tested treatment time points: (0.02%) after 6 and 12 h of elicitor application and (0.01%) after 48 and 168 h. The maximum content of silydianin (0.11%) was induced by 24-hours application of c 1 concentration. The higher levels of other flavonolignans such as isosilybin A (0.01%) after 48 h and isosilybin B (0.01%) after 72 h of c 1 concentration were determined ( Table 2). Elicitor at c 2 concentration had no noticeable effect on flavonolignan production. This concentration increased only taxifoline content (0.01%) after 48h of duration and silybine A (0.01%) after 12 h of duration. Similar results with c 3 concentration of elicitor were found ( Table 2).

Flavonolignans in Nutrient Medium
The content of flavonolignans was not only determined in the callus and suspension cultures but also in the nutrient media in which S. marianum cultures in vitro were cultivated. Only taxifolin, silydianin and silychristin were eliminated into the nutrient medium. The highest level of taxifolin in comparison with content in suspension culture (threefold higher) was released into medium. The highest production of taxifolin (0.06%) after application of c 2 concentration and 72 h duration and (0.05%) after after c 1 and 24 h duration was determined (Table 3). Sampling at concentration c 2 after 168 h was not evaluated for contamination of nutrient medium. Table 3. Content of taxifolin, silychristin and silydianin (%) in 100 mL of suspension culture nutrient medium. The newly synthetized pyrazinecarboxamide derivatives were also tested for their abiotic effects in previous experiments with Silybum marianum culture in vitro. The compound 5-tert-butyl-N-mtolylpyrazine-2-carboxamide at a concentration 3.71 × 10 −7 mol/L and within 72 h of elicitation increased flavonolignan production by 893% in suspension culture versus control. The flavonolignan production in callus culture after elicitation with N-(5-bromo-2-hydroxyphenyl)-5-tert-butyl-6chloropyrazine-2-carboxamide was also increased by about 1039% (24 h elicitation and concentration of 2.59 × 10 −4 mol/L) [6].

Ononis arvensis Callus Culture
All three tested concentrations of compound II increased the flavonoid production in callus culture in a statistically significant manner ( Table 4). The best elicitation effect of all elicitor concentrations was at the weakest c 3 concentration (8.36 × 10 −6 mol/L) after 168 h. This was a statistically important increase in the flavonoid production (about 1,506%) in comparison with control. Higher flavonoid production was also reached after 24 and 48 h treatment with c 1 concentration and after 6-and 12 h treatment with c 3 concentration (Table 4).

Ononis arvensis Suspension Culture
The maximum content of flavonoids (about 5,800%) was induced by 48 h application of c 3 concentration. Elicitor concentration c 2 had no influence on flavonoid production at any time of elicitor treatment (Table 5). Concentration c 2 on the contrary decreased flavonoid production in the studied times. Ring substituted pyrazinecarboxamides were also tested for their abiotic effects in previous studies. These compounds were able to increase the secondary metabolites production in plant cultures in vitro. Flavonoid production in O. arvensis culture in vitro was increased after 6, 12, 72 and 168 h elicitation with 6-chloro-N-(4-chloro-3-methylphenyl)-pyrazine-2-carboxamide. The amount of flavonoids released into nutriet medium was not studied.

Conclusions
The results of the performed experiments show that successful elicitation is subject to many factors that are specific to each elicitor and for each explant culture. An important part of successful elicitation is the type of elicitor, its concentration and the time of its action. All results clearly indicate that the tested compound 5-(2-hydroxybenzoyl)-pyrazine-2-carboxamide (I) has shown to be an effective elicitor of flavonolignans and taxifolin production in Silybum marianum culture in vitro. The compound N-(2-bromo-3-methylphenyl)-5-tert-butylpyrazine-2-carboxamide (II) also seems to be an effective elicitor for flavonoid production in Ononis arvensis culture in vitro. These newly synthesized chemical compounds were proven to be promising elicitors for the induction of secondary metabolism in explant cultures.