Simultaneous determination of six active metabolites in Astragalus mongholicus (Fisch.) Bge. under salt stress by ultra-pressure liquid chromatography with tandem mass spectrometry

Radix Astragali, A widely used Chinese Herbal Medicine, is derived from the dried roots of Astragalus mongholicus (Fisch.) Bge. (Zu et al. 2009). A. mongholicus generally is mixed with other ingredients to make some medicated food in its edible aspect, which it has also traditionally been utilized in cosmetics. Pharmacological studies and clinical practice have demonstrated that A. mongholicus possess various biological activities including tonic, immunostimulant, hepatoprotective, diuretic, antidiabetic, cardioprotective, anti-oxidative and anti-tumor properties (Sun et al. 2008; Boye et al. 2015; Chen et al. 2015). More than 100 chemical constituents of A. mongholicus have been isolated and identified (Lv et al. 2015; Napolitano et al. 2013). Isoflavonoids and triterpene saponins have been considered as two types of the major bioactive metabolites found in A. mongholicus. Formononetin, ononin, calycosin and calycosin-7-O-?-d-glucoside, which boost energy, strengthen the immune system, and promote health activities and skin growth, are the major isoflavonoids in A. mongholicus (Xiao et al. 2004; Krasteva et al. 2015). Astrageloside IV has protective effects on cardiovascular system, immune, digestive and nervous system (Ren et al. 2013). Cycloastragenol is the synthetic precursor compound of astragaloside IV. And all of them could be the marker compounds for the chemical evaluation of A. mongholicus (Yesilada et al. 2005; Pu et al. 2015). In addition, formononetin, ononin, calycosin and calycosin-7-O-?-d-glucoside, are the most important metabolites in isoflavonoids biosynthesis pathway (Xu et al. 2011); astragaloside IV and cycloastragenol, are the essential metabolites of triterpene saponin biosynthesis pathway (Park et al. 2015). Synthesis or decomposition of these compounds has important implications for the quality of A. mongholicus as medicines (Zheng et al. 2015; Liu et al. 2015a).

Salinity is possibly the most imperative ecological restriction that causes extensive crop yield losses all over the world, and its threat is escalating day by day (Peng et al. 2011; AbdElgawad et al. 2016). The only way to control the soil salinization process and to maintain the sustainability of landscape and agricultural fields is to combat the salinization problems by environmentally safe and clean techniques, such as: use of salt-tolerant species (Hasanuzzaman et al. 2014; Moore 1984). A. mongholicus has been proved to be a salt-tolerant plant (Wdowiak-Wrobel et al. 2013), it was one of the potential species to control the soil salinization. So it is extremely critical to search for the salt-tolerance and the ability of physiological adaptation of A. mongholicus. When plants are exposed to salt stresses, they display intricate regulatory mechanisms to enhance their response, including cellular changes and metabolic responses (Molinier et al. 2006; Bruce et al. 2007; Chen et al. 2007). Importantly, we must focus on the content of major bioactive metabolites in A. mongholicus. under salt stress to ensure the medicinal value of A. mongholicus, then we can explore the possibility of A. mongholicus to be used as potential salinity species. As two types of the major metabolites and active components in A. mongholicus, the change in isoflavonoids and triterpene saponins level needs to be observed carefully. So we developed and applied the rapid and sensitive UPLC-MS method for simultaneous determination of astragaloside IV, cycloastragenol, calycosin-7-O-?-d-glucoside, calycosin, ononin and formononetin in A. mongholicus under different levels of salt stresses.

Because of the trace amounts of compounds in the seedlings, their quantitative analysis in the seedlings was difficult. In attempts to improve the determination of these compounds in A. mongholicus, several studies have been reported. The current method for the determination of these compounds mainly uses high-performance liquid chromatography (HPLC) coupled with DAD or ESI–MS Detection (Kwon and Park 2012; Gong et al. 2015). Being the common analytical tool for various compounds, HPLC and LC–MS both are being more and more widely applied in biological research, and have been used to quantify the marked compounds in biological samples such as A. mongholicus seedlings (Liu et al. 2015a; b). However, the threshold of sensitivities of these HPLC methods for detecting these compounds in A. mongholicus seedling is high. Furthermore, the major weakness of these LC–MS methods mainly includes chromatographic running time, which at more than 18 min is considered too long. This is time consuming and not suitable for analyzing large number of sample. Even though some experiments were investigated for determination of some compounds in A. mongholicus (Lv et al. 2011; Wu et al. 2005; Qi et al. 2006), there is no established sensitive method regarding of simultaneous determination of astragaloside IV, cycloastragenol, calycosin-7-O-?-d-glucoside, calycosin, ononin and formononetin in seedlings was published.

Therefore, this study aimed at developing a sensitive and validated ultra-pressure liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI–MS) method with a short time for simultaneous determination of astragaloside IV, cycloastragenol, calycosin-7-O-?-d-glucoside, calycosin, ononin and formononetin in A. mongholicus under different levels of salt stress.