Glucosinolate degradation products of Aurinia leucadea (Guss.) K.Koch and Lepidium draba L. from Croatia

Abstract Two wild-growing Brassicaceae plants of Croatian origin, Aurinia leucadea (Guss.) K. Koch and Lepidium draba L., were investigated to uncover glucosinolates via GC/MS analysis of their degradation products. The main constituents of Aurinia leucadea (Guss.) K.Koch distillate were hex-5-enenitrile (28.8%) and but-3-enyl isothiocyanate (18.8%), while 4,5-epithiopentanenitrile (50%) and 5,6-epithiohexanenitrile (18.5%) were the main volatile compounds in autolysate. 4-(Methylsulfanyl)butyl isothiocyanate (96.4%) constituted almost the entire Lepidium draba L. distillate, while the autolysate was characterized by 4-(methylsulfinyl)butyl isothiocyanate (57.3%). So, regarding the glucosinolate degradation products, the main glucosinolates of A. leucadea were glucobrassicanapin and gluconapin, and of L. draba glucoerucin and glucoraphanin. Graphical abstract


Introduction
Glucosinolates, a group of phytochemicals particularly abundant in plants belonging to Brassicaceae family, are interesting as a source of volatile degradation products possessing various biological activities (Venditti and Bianco 2020).Aurinia leucadea (Guss.)K. Koch and Lepidium draba L. are insufficiently explored Brassicaceae plants.As far as we know, there are two reports on glucosinolate degradation products or glucosinolates of A. leucadea (Bla zevi c et al. 2011;2013).As regards L. draba, there are some reports on glucosinolate degradation products or intact glucosinolates (Daxenbichler et al. 1991;Fahey et al. 2001;Fr echard et al. 2002;Senatore et al. 2003;Powell et al. 2005;Afsharypuor and Jamali 2006;Radoni c et al. 2011;Bayan 2016).Regardless of the fact that these plants are investigated to some extent, it seems to us that it is of interest to continue our research of volatile compounds, especially glucosinolate degradation products, from these Brassicaceae plants.In order to provide more complete insight into composition and content of A. leucadea and L. draba glucosinolate degradation products, the methods used in this research were somewhat different in comparison to our previous reports.

Results and discussion
According to the degradation products, the main glucosinolates in both isolates of A. leucadea were glucobrassicanapin and gluconapin, with glucobrassicanapin dominating in distillate and gluconapin in autolysate (Table 1).The dominating compounds in both isolates were nitriles, representing 52.5% of the distillate and 70.8% of the autolysate.Thus, hex-5-enenitrile was the main compound in distillate, while 4,5-epithiopentanenitrile was the main compound in autolysate.In general, isolate obtained by endogenous myrosinase hydrolysis followed by solvent extraction at room temperature (autolysate) is characterized by high percentage of epithionitriles, 68.5%, in contrast to 12.2% identified in distillate.It is well-known that the formation of these nitriles is affected by the presence of epithiospecifier protein.Our results are in accordance with the findings of Matusheski et al. (2004) who reported that this protein is heat-sensitive.
In our previous paper, two approaches were used, autolysis followed by hydrodistillation and exogenous myrosinase hydrolysis followed by solvent extraction (Bla zevi c et al. 2011).The dominating compounds in both volatile extracts were isothiocyanates, while the content of nitriles was much smaller (0.9-4.8%).In this research, epithionitriles were the major compounds, while in our previous research their content was almost insignificant (68.5% as opposed to 0.4%).These differences could be caused by different isolation of volatile glucosinolate degradation products after autolysis, namely hydrodistillation (at 100 C) as opposed to solvent extraction at room temperature, and the fact that the epithiospecifier protein is heat-sensitive.Bla zevi c et al. ( 2013) investigated the glucosinolates of seed and root of A. leucadea of Croatian origin too and reported that the volatile fractions obtained after exogenous enzyme hydrolysis contained mostly isothiocyanates.Such qualitative and quantitative variations in glucosinolate degradation products, and indirectly glucosinolates, were probably caused by different methods used for the preparation of plant material and extracts.
According to the chemical and percentage composition of the glucosinolate degradation products in distillate and autolysate, L. draba is characterized by the presence of two major glucosinolates, glucoerucin and glucoraphanin.Glucoerucin degradation product, 4-(methylsulfanyl)butyl isothiocyanate constituted almost the entire distillate (96.4%).The degradation product of glucoraphanin, 4-(methylsulfinyl)butyl isothiocyanate (57.3%), was the main constituent of autolysate.In previous article we reported that glucoerucin degradation products were the main compounds in the distillate, while the glucoraphanin degradation products were the main compounds in isolate obtained by exogenous myrosinase hydrolysis (Radoni c et al. 2011).The main difference between this (fresh plant material) and our previous research (dried plant material) was the quantity of the glucoerucin degradation products found in distillates, that is, 41.8% versus 96.4%.Knowing that 4-(methylsulfanyl)butyl isothiocyanate (erucin) is the reduced analogue of the sulforaphane, and could be formed not only from glucoerucin degradation, but also by in vivo reduction of sulforaphane (Melchini and Traka 2010), it is possible that lower quantity of erucin in distillate of dried plant material was caused by its oxidation to sulforaphane during drying of plant material.

Conclusion
The results of this study showed that the main volatile compounds of A. leucadea (Guss.)K. Koch.and L. draba L., regardless of the isolation method, were glucosinolate degradation products.Different approaches to the glucosinolate degradation, that is, thermally or enzymatically, and isolation of liberated volatile products, that is, hydrodistillation or solvent extraction, provides more complete insight on composition and content of glucosinolate degradation products and, indirectly, enables conclusion about glucosinolates present in these plants.

Table 1 .
Glucosinolates and their degradation products (%) identified in isolates of A. leucadea and L. draba.