Production of rare cycloartane saponins from Astragalus thracicus (Griseb) compared to Astragalus membranaceus (Fisch.) Bunge – native and biotechnological sources

compared


Introduction
The proven immunomodulatory, antiviral, and antitumor effects of representatives of the Astragalus genus are mainly due to the triterpene saponins of the cycloartane type contained in them. Due to the complexity of their structure, they are still most efficiently obtained from native plants. Variations in the quantity and quality of the plant material, a long period of development before starting saponin production (Astragalus roots), and the excessive collection of endangered species for pharmaceutical pur-poses (A. membranaceus) are just some of the problems associated with obtaining these natural products. Therefore, the discovery of new plant species (A. thracicus) that contain them is essential for the pharmaceutical industry. Thus, comparative metabolomic analysis between endangered Asia species A. membranaceus and Bulgaria endemic species A. thracicus could provide important information about possible alternative sources. In addition, biotechnological methods could serve as an opportunity to protect those endemic species and to increase the amount of produced saponins.
Astragalus L. Fabaceae (Leguminosae) is the largest genus of herbaceous plants in the pea and bean family it includes approximately 3000 species, distributed across Asia, Africa, Europe, South, and North America, although the center of origin and biodiversity of plants of the genus Astragalus is Eurasia, and in particular, the mountainous parts of Southwest Asia (Podlech 2008).
The most used species of the genus is A. membranaceus Bunge var. mongholicus (Bunge), (syn. A. mongholicus var. mongholicus), the drug used to be the dried whole or thinly sliced roots. The plant substance, due to its wide use, has been included in the editions of the European Pharmacopoeia after 2012 (Ph. Eur. monograph, as well as in the Chinese Materia medica. Phytochemical studies on Astragalus species have been conducted due to their effects as immunostimulants or as anticancer agents (Ionkova et al. 2014;Krasteva et al. 2016). In particular, the chemical composition of the dried roots of Astragalus spp. protects the heart, brain, kidneys, intestines, liver, and lungs from various diseases associated with oxidative stress (Hong et al. 1992;Shahzad et al. 2016). Various isolated components of Astragalus spp. show significant antiproliferative activity. The highest activity against T-cell leukemia (SKW-3) cells was registered for rhamnocitrin 4'-D-galactopyranoside isolated from A. hamosus (Krasteva et al. 2008).
Biotechnological techniques and approaches are an extremely attractive alternative to over-exploited wild species. Some of the advantages of the in vitro techniques are the propagation of the plants in aseptic controlled conditions and their large-scale production in a year-round system without seasonal constraints (Isah et al. 2018). The plant cell techniques provide some high-efficiency methods for isolation and extraction of the secondary metabolites within a short time compared to the wild plant populations and the simplicity of the methods from in vitro-produced tissues makes it suitable for commercial application (Kolewe et al. 2008). Apart from these advantages, some metabolites can be produced by in vitro cultures but are generally not found in intact plants (Pavlov et al. 2005).

Plant material
The in vitro cultures of A. thracicus were successfully established and maintained in our lab. The native A. thracicus roots were carefully collected from their natural environment, while native A. membranaceus roots were delivered by HerbaSinica Hilsdorf GmbH (Ch.-B. 160601H004).

General experimental procedures
All solvents were at least of analytical grade, whereas solvents used for semi-preparative HPLC analysis, i.e., EtOAc and MeOH were HPLC grade and were purchased from Fischer Scientific (Loughborough, UK). The following reference substances of cycloartane saponins were used: Astragaloside I (≥ 95.0%) delivered by Cayman chemical company; Astragaloside II (≥ 99.8%) obtained from Sigma-Aldrich and Astragaloside IV (≥ 98.0%) purchased from Tokyo chemical industry Co., LTD.

Analysis and quantification of cycloartane saponins
Astragaloside I (1), Astragaloside II (2), and Astragaloside IV (3) were used as external standards for the quantitative analysis of saponins. Each of the standards was dissolved in 50% MeOH and then subsequently diluted to reach 5 concentration levels used to build the calibration curves for quantitative assay covering the concentration range of 1.35-21.60 µg/mL for Astragaloside I; 1.20-19.00 µg/ mL for Astragaloside II and 2.00-27 µg/mL for Astragaloside IV, respectively. All solutions were stored in the refrigerator at 4 °C.

Extraction and purification of crude extracts
The dried plant material from in vitro cultures of A. thracicus, native A. thracicus roots, and A. membranaceus roots were exhaustively extracted with 80% MeOH. The extracts were filtered and concentrated under reduced pressure and after that fractionated by solid phase extraction (SPE) using C18 cartridges (500 mg) to obtain H 2 O, EtOAc, and MeOH fractions. Further separation of the fractions was achieved by blotting the samples onto Diaion HP 20, and subsequently eluted with 100 mL 40% and 90% MeOH for each of the EtOAc fractions and 30% and 90% MeOH respectively for each of the MeOH fractions, which resulted in 4 fractions for each initial extract (Scheme 1). Each of the fractions was subjected to UHPLC-HRESI-MS analysis.

In vitro cultivation
Shoot culture from A. thracicus was derived from MS medium, while the roots were obtained by cultivation in the dark regimen of cultivation using modified MS medium supplemented with 2 mg/L NAA (Ms-Li). The suspension cultures were initiated when cultivated on MS medium supplemented with 2 mg/L kinetin, 0.2 mg/L IAA, 0.1 mg/L 2,4-D, and 1 g/L casein (G48) and cultivated in a dark and light regimen of cultivation (Ionkova et al. 2010).

Identification of cycloartane saponins
Within UHPLC-HRESI-MS analysis astragaloside I, II, and IV were determined at negative ion mode as adducts with HCOOH. For astragaloside I (1) (Fig. 1). The compounds in samples were identified according to the described above retention times, m/z ratios and spectrum fragmentations. The three important cycloartane metabolites (1-3) were found in all analyzed samples from A. thracicus and A. membranaceus.

Calibration model
A visual evaluation of the linear regression line plot showed that the method was linear for all of the standards (Fig. 2). The determination coefficient for compound 1 was r 2 = 0.9858, for compound 2 r 2 = 0.9932 and compound 3 r 2 = 0.9960, respectively. After the linearity was investigated the regression analysis was performed. The determination coefficients and regression equations are presented in Table 1.

Quantification of cycloartane saponins
The amount of the individual saponins was calculated due to the calibration equation formula for Astragaloside I y = 7E+06x-1E+07; Astragaloside II y = 6E+06x-7E+06 and Astragaloside IV y = 1E+07x-1E+07 within each of the derived fractions from in vitro cultures of A. thracicus, native roots of A. thracicus and native roots of A. membranaceus. In general, the highest amount of compounds 1-3 was found in EtOAc 90% fractions, except compound 3, which eluted in large amounts also in MeOH 90% (Suppl. material 1: figs S4-S6). This unexpected chromatographic behavior may be related to the absence of an acetylene residue in the sugar moiety of the molecule, which probably makes it more polar. This can also be confirmed by the retention times from the LC-HRESI-MS analysis of a mixture including the three saponins (Fig. 1).
Cycloartane saponins were proved in the highest amount (1.50 mg/g DW (1), 1.01 mg/g DW (2), and 0.91 mg/g DW (3)) in in vitro root cultures from A. thracicus. Even for astragaloside II and IV, the amount was two folds higher than the amount in native roots (0.47 mg/g DW (2), 0.40 mg/g DW (3)). In vitro shoot cultures of A. thracicus also produce a higher amount of astragaloside II and IV (0.80 mg/g DW (2), 0.48 mg/g DW (3)) than the native source provides. Аn expected low amount of cycloartane saponins was observed in suspension culture cultivated on G48 medium such as those grown in the dark could produce a higher amount of astragaloside I (0.34 mg/g DW), while astragaloside II and IV are observed in higher amounts (0.25 mg/g DW and 0.35 mg/g DW) when suspension cultures are cultivated at light regimen. In all, in vitro cultures of A. thracicus the observed metabolites were in higher abundance than in native roots of A. membranaceus (0.23 mg/g DW (1), 0.18 mg/g DW (2) and 0.05 mg/g DW (3)) (Fig. 3).

Conclusion
The comparative LC-HRESI-MS analysis showed for the first time that the endemic species A. thracicus biosynthesized identical rare cycloartane saponins as A. membranaceus. In the present study, a new biotechnological platform was also created that provides a higher production of cycloartane saponins-astragaloside I, II, and IV compared to the wild species. The in vitro root cultures of A. thracicus reached two folds higher amounts of saponins than native root of A. thracicus, and up to six times higher when compared with A. membranaceus. In addition, our results provide a fast LC-HRESI-MS protocol for the identification of cycloartane saponins.